Cell production of pure iron oxide nanoparticles

Abstract

The invention relates to cell production of pure iron oxide nanoparticles. Specifically, the present invention discloses a method for preparing high purity iron oxide nanoparticles using nanoparticle-producing cells, comprising: a) a pre-growth stage comprising the amplification of nanoparticle-producing cells in a pre-growth and / or fed batch culture medium, and b) a growth stage comprising the amplification of nanoparticle-producing cells in a fed batch culture medium, comprising expanding nanoparticle-producing cells derived from a pre-growth stage in a growth and / or fed-batch culture medium wherein per kilogram or per liter of pre-growth and / or growth and / or fed-batch culture medium in the pre-growth and / or growth and / or fed-batch culture medium: i) no higher than 0.005 g of a yeast extract, and ii) no more than 0.001 g of a CMR agent selected from the group consisting of boric acid and nitrilotriacetic acid.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of October 8, 2019, application number 201910962115.1, and invention name “Cellular production of pure iron oxide nanoparticles”. Technical Field

[0002] The field of the invention is the biological production of nanoparticles containing low levels of impurities. Background Art

[0003] Nanoparticle-producing bacteria, such as magnetotactic bacteria, are known to accumulate impurities in their crystalline structures. For example, when magnetotactic bacteria are grown in the presence of cobalt, they produce magnetosomes containing iron oxide and cobalt (S. Staniland et al, Nature nanotechnology, V.3, p.158 (2008)). For medical applications, it is desirable that nanoparticles contain low levels of toxic impurities, such as cobalt. Summary of the Invention

[0004] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle production cells, comprising:

[0005] a) a pre-growth phase comprising expanding the nanoparticle-producing cells in a pre-growth and / or fed-batch culture medium (media), preferably such that the nanoparticle-producing cells produce substantially no nanoparticles, and

[0006] b) a growth phase comprising expanding the nanoparticle-producing cells derived from the pre-growth phase in a growth and / or fed-batch medium, preferably allowing the nanoparticle-producing cells to produce nanoparticles,

[0007] wherein the pre-growth and / or growth and / or fed-batch culture medium comprises per kilogram or per liter:

[0008] i) not more than 5.10 5 , 5.10 3 , 50, 5, 0.5, 0.005, 0.0005, 0.00005 or 5·10 -10 g yeast extract (yeast extract, yeast extract, yeast extract), and / or

[0009] ii) not more than 10 5 , 10 3 , 10, 1, 0.1, 0.001, 10 -5 or 10 -10g CMR reagent, preferably selected from the group consisting of boric acid and nitrilotriacetic acid,

[0010] Wherein, when a fed-batch medium is present, a fed-batch medium is preferably used as the medium to supplement the pre-growth and / or growth medium,

[0011] The nanoparticles produced in the growth stage are more than those in the pre-growth stage, preferably at least 0, 0.1, 0.5, 2, 5, 10, 10 3 , 10 5 or 10 10 times, wherein the multiple is preferably Q2 / Q1, wherein Q1 and Q2 are preferably nanoparticles produced in the pre-growth stage and the growth stage or sub-stage, respectively,

[0012] and / or

[0013] It is preferred that the pre-growth and / or growth and / or fed-batch medium comprises at least one further compound, which is preferably involved in bacterial growth and / or magnetosome production or synthesis, wherein preferably the further compound is a carbon source, a nitrogen source, a calcium source, a vitamin source, an oxygen source, an iron source, a phosphate source, a phosphorus source and / or a magnesium source. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : TGA-DSC analysis of whole magnetotactic bacteria and magnetosomes extracted from magnetotactic bacteria according to lysis condition 2. (a) Change in weight percentage as a function of temperature and the derivative of this change as a function of temperature for a sample containing 3 mg of freeze-dried magnetotactic bacteria (MSR-1) (carbon = 44%), heated between 20°C and 600°C at a rate of 6°C / min. (b) Heat flux (mW) generated by a sample containing 3 mg of freeze-dried magnetotactic bacteria (MSR-1) (carbon = 44%), heated between 20°C and 600°C at a rate of 6°C / min. (c) Change in weight percentage as a function of temperature and the derivative of this change as a function of temperature for a sample containing 3 mg of freeze-dried magnetotactic bacteria (MSR-1) (carbon = 44%), extracted from whole bacteria according to lysis condition 2 (condition n°2), heated between 20°C and 600°C at a rate of 6°C / min. (d) Heat flux (mW) generated as a function of temperature for a sample containing 3 mg of freeze-dried magnetosomes (magnetosome chains, carbon = 7%) extracted from whole bacteria according to lysis condition 2 (condition n° 2) when the sample is heated at a rate of 6°C / min between 20°C and 600°C. As for (a) and (c), the y-axis can be replaced by mass percentage to obtain the same curve.

[0015] Figure 2 : TGA-DSC analysis of magnetosomes extracted from magnetotactic bacteria according to Condition 3 and chemically synthesized SIGMA nanoparticles. (a) Change in weight percentage as a function of temperature and the derivative of this change as a function of temperature for a sample containing 3 mg of freeze-dried magnetosomes (magnetosomes, carbon = 4%) extracted from magnetotactic bacteria according to Condition 3 (Condition n°3). (b) Heat flux (mW) generated by a sample containing 3 mg of freeze-dried magnetosomes (magnetosomes, carbon = 4%) extracted from magnetotactic bacteria according to Condition 3 (Condition n°3) as a function of temperature. (c) Change in weight percentage as a function of temperature and the derivative of this change as a function of temperature for a sample containing 3 mg of freeze-dried SIGMA nanoparticles (carbon = 0.3%). (d) Heat flux (mW) generated by a sample containing 3 mg of freeze-dried SIGMA nanoparticles (carbon = 0.3%) as a function of temperature. For (a) and (c), the y-axis can be replaced by mass percentage to obtain the same curves.

[0016] Figure 3 An illustrative example of the method according to the invention can be used, which utilizes pre-growth and growth phases for the amplification of magnetotactic bacteria and the production of magnetosomes. The method according to the invention follows a series of pre-growth phases 1, i and i+1, wherein the pre-growth phase is preferably achieved by PGS1 The nanoparticle producing cells are usually expanded in this volume for about 7 days, and then the nanoparticle producing cells are expanded from V PGS1 Transfer to V PGSi Medium (V PGSi Typically about 500 ml), the nanoparticle production cells are typically expanded in this volume for about 3 days, and then the nanoparticle production cells are removed from the V PGSi Transfer to V PGSi+1 Medium (V PGSi+1 Nanoparticle production cells are typically expanded in this volume for about 3 days until the optical density (OD) is typically equal to 1, and then transferred to V GS0 in a volume (typically 45 liters) where, in the presence of oxygen bubbled through the growth medium, V GS0 During a growth phase of preferably about 5 days, the culture medium is fed in batches (usually 5-10 liters, with ammonia (2.76.10 -1 M, 4.77g); lactic acid (1.1M, 100g); K2HPO4 (1.72.10 -2 M, 3g); MgSO4(1.95.10 -3 M, 0.48g); FeCl3(7.4.10 -3M, 2 g); thiamine (1.2.10 -6 M, 0.0004 g); CaCl2 (1.3.10 -3 M, 0.2 g)) is supplemented to bring the optical density of bacterial growth to typically 5 to 40, and the number of magnetosomes produced per liter of growth medium to 5 to 500 mg, where the pre-growth medium (per liter: NH4Cl (7.41.10 -3 M, 0.4 g); sodium lactate (2.3.10 -2 M, 2.6 g); K2HPO4 (5.74.10 -4 M, 0.1 g); MgSO4 (4.1.10 -4 M, 0.1 g); FeCl3 (1.8.10 -6 M, 0.0005 g); thiamine (1.2.10 -7 M, 0,00004 g); CaCl2 (0.9.10 -4 M, 0.015 g)) preferably contains a restricted concentration of iron or an iron source (preferably <2 μM) to prevent nanoparticle production; the growth medium (per liter: NH4Cl (4.13.10 -3 M, 0.22 g); sodium lactate (1.4.10<{ -2 M, 1.3 g); K2HPO4 (1.55.10 -4 M, 0.027 g); MgSO4 (1.1.10 -4 M, 0.027 g); FeCl3 (1.8.10 -6 M, 0.0005 g); thiamine (1.2.10 -7 M, 0,00004 g); CaCl2 (0.9.10 -4 M, 0.015 g)) preferably contains an iron or an iron source (preferably >2 μM) to promote nanoparticle production. In the pre-growth stage: bacteria multiply abundantly and basically do not produce nanoparticles; in the growth stage: bacteria multiply abundantly and produce a large number of nanoparticles. In the pre-growth stage: air / oxygen bubbled into the growth medium: none or <2 **

[0017] Figure 4 ​: An illustrative example of the pre-growth stage, showing the concentrations C1 and C2 of the carbon source, nitrogen source, and iron source at the start and end of the pre-growth stage. At the start of the pre-growth stage: C2 is approximately equal to 0 g / L; C 总量 is approximately equal to C1. At the end of the pre-growth stage: C1 < C2. C1 represents the content of carbon, nitrogen, and iron in the pre-growth medium without bacteria per liter of the pre-growth medium; C2 represents the content of carbon, nitrogen, and iron in the pre-growth medium with bacteria per liter of the pre-growth medium (Note: C2 can also represent the quantity of carbon, nitrogen, and iron consumed by bacteria per liter of the pre-growth medium).

[0018] Figure 5 : An illustrative example of the growth stage (between T = 0 h and T = 20 h), showing the concentrations C1 and C2 of the carbon source, nitrogen source, and iron source at the start of the growth stage and 20 hours after the start. At the start of the growth stage (T = 0 h): C2 is approximately equal to 0 g / L; C 总量 is approximately equal to C1. At 20 hours after the start of the growth stage: C 1碳 ≥ C 2碳 ; C 1氮 ≥ C 2氮 ; C 1氮 = C 2氮 ; C 1氮 ≤ C 2氮 ; C 1铁 ≥ C 2铁 . C1 represents the content of carbon, nitrogen, and iron in the growth medium without bacteria per liter of the growth medium; C2 represents the content of carbon, nitrogen, and iron in the pre-growth medium with bacteria per liter of the growth medium (Note: C2 can also represent the quantity of carbon, nitrogen, and iron consumed by bacteria per liter of the growth medium).

[0019] Figure 6 : An illustrative example of the growth stage (between T = 10 h and T = 40 h), showing the concentrations C1 and C2 of the carbon source, nitrogen source, and iron source 40 hours after the start of the growth stage and more than 40 hours after the start. 20 hours after the start of the growth stage: C 1碳 ≤ C 2碳 ; C 1氮 ≥ C 2氮 ; C 1氮 = C 2氮 ; C 1氮 ≤ C 2氮 ; C 1铁 ≤ C 2铁 ; More than 40 hours after the start of the growth stage: C 1碳 ≤ C 2碳 ; C 1氮 ≥ C 2氮 ; C 1氮 = C 2氮 ; C1氮 ≤C 2氮 ; C 1铁 ≤C 2铁 C1 represents the carbon, nitrogen, and iron content of the pre-growth medium without bacteria per liter of growth medium; C2 represents the carbon, nitrogen, and iron content of the pre-growth medium with bacteria per liter of growth medium. DETAILED DESCRIPTION

[0020] In one embodiment of the present invention, the nanoparticle-producing cells are preferably, when they are injected into the nanoparticles containing at least one compound preferably at a concentration greater than 10 -6 , 1, or 10 6 A eukaryotic or prokaryotic cell having the ability or function to produce or synthesize nanoparticles when expanded in a culture medium containing 1 μM nanoparticles or a culture medium containing 1 μM nanoparticles.

[0021] In one embodiment of the present invention, the nanoparticle-producing cells do not produce substantially any nanoparticles, wherein the cells produce less than 10 nanoparticles per liter of pre-growth and / or growth and / or fed-batch culture medium. 50 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -5 mg nanoparticles, or wherein the nanoparticles cells preferably comprised in the pre-growth and / or growth and / or fed-batch culture medium comprise less than 10 50 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -5 mg of nanoparticles per liter of pre-growth and / or growth and / or fed-batch medium.

[0022] In one embodiment of the present invention, nanoparticle producing cells produce nanoparticles, wherein the cells produce more than 0, 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 or 10 5 mg nanoparticles per liter of pre-growth and / or growth and / or fed-batch culture medium, or wherein preferably the nanoparticles cells contained in the pre-growth and / or growth and / or fed-batch culture medium contain less than 0, 10 -50 , 10 -20 , 10 -10 , 10-5 , 10 -1 , 1, 10, 10 3 or 10 5 mg of nanoparticles per liter of pre-growth and / or growth and / or fed-batch medium.

[0023] In one embodiment of the present invention, the number of nanoparticles produced by the nanoparticle cell during the pre-growth phase is equal to Q2-Q1, where Q1 and Q2 are the number of nanoparticles produced at times t1 and t2 during the pre-growth phase, wherein t2 is greater than t1, preferably t2 / t1 is greater than 1, 2, 5, 10 or 10 3 , preferably t2 is the end time of the pre-growth phase, and t1 is the start time of the pre-growth phase.

[0024] In one embodiment of the present invention, the number of nanoparticles produced by the nanoparticle cell during the growth phase is equal to Q2'-Q1', where Q1' and Q2' are the number of nanoparticles produced at times t1' and t2' during the growth phase, wherein t2' is greater than t1', preferably t2' / t1' is greater than 1, 2, 5, 10 or 10 3 , preferably t2' is the end time of the growth phase, and t1' is the start time of the growth phase.

[0025] Preferably Q2'-Q1' is greater than Q2-Q1, preferably by a factor of at least 0, 10 -10 , 10 -1 , 1, 5, 10, 10 3 or 10 5 .

[0026] Preferably, the total number of nanoparticles produced by the nanoparticle-producing cells is equal to Q 总量 =Q2'-Q1'+Q2-Q1.

[0027] In some cases, Q 总量 Can be greater than 10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 0, 1, 5, 10 or 100 mg of nanoparticles, preferably greater than 10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 0, 1, 5, 10 or 100 mg of iron contained in the nanoparticles, preferably per liter of pre-growth and / or growth medium.

[0028] In some cases, Q 总量Can be less than 10 50 , 10 10 , 10 5 , 10 3 , 100, 50, 10 or 1 mg of nanoparticles, preferably less than 10 50 , 10 10 , 10 5 , 10 3 , 100, 50, 10 or 1 mg of iron contained in the nanoparticles, preferably per liter of pre-growth and / or growth medium.

[0029] In one embodiment of the invention, the yeast extract is an element selected from the group consisting of: i) whole yeast extract, ii) culture medium comprising greater than 1, 10, 50 or 90% of compounds derived from whole yeast extract, and iii) chemical equivalents of yeast extract.

[0030] In one embodiment of the invention, the peptone is an element selected from the group consisting of: i) intact peptone, ii) culture medium comprising more than 1, 10, 50 or 90% of compounds derived from intact peptone, and iii) chemical equivalents of peptone.

[0031] The present invention relates to a method for preparing high-purity iron oxide nanoparticles using nanoparticle-producing cells, preferably comprising a pre-growth phase comprising expanding the nanoparticle-producing cells, preferably in a pre-growth medium, while preferably substantially no nanoparticles are produced; preferably followed by a growth phase comprising expanding the nanoparticle-producing cells from the pre-growth phase, preferably in a growth medium, while producing nanoparticles, wherein the growth phase preferably distinguishes itself from the pre-growth phase by at least one characteristic selected from the group consisting of:

[0032] i)C FeGS / C FePGS The ratio is greater than 0, 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where C FeGS and C FePGS are the concentrations of iron or iron source in the growth medium and pre-growth medium, respectively, ii) C CGS / C CPGS The ratio is greater than 0, 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where C CGS and C CPGSare the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively; iii) C NGS / C NPGS The ratio is greater than 0, 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where C NGS and C NPGS where ΔpH is the concentration of nitrogen or nitrogen source in the growth medium and pre-growth medium, respectively. GS / ΔpH PGS The ratio is less than 0, 10 10 , 10 5 , 10 3 , 10 2 , 1, 0.5 or 0.1, where ΔpH GS and ΔpH PGS are the changes in pH in the growth medium and pre-growth medium,

[0033] v)Q GGS / Q GPGS The ratio is greater than 0, 10 10 , 10 5 , 10 3 , 10 2 , 1, 0.5 or 0.1, where Q GGS and Q GPGS is the amount of gas, oxygen or air introduced or bubbled into the growth medium and pre-growth medium, respectively,

[0034] vi)N SSGS / N SSPGS The ratio is less than 0, 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where N SSGS and N SSPGS are the number of sub-stages of the growth stage and the number of sub-stages of the pre-growth stage, respectively, where the two sub-stages are separated by transferring nanoparticle-producing cells from the first sub-stage to the second sub-stage, and

[0035] vii) the growth medium is supplemented with fed-batch medium and the pre-growth medium is not supplemented with fed-batch medium, and / or

[0036] Wherein, preferably, the pre-growth, growth and / or fed-batch medium does not include:

[0037] I) at least one compound or combination of compounds contained in or derived from a yeast extract, wherein the at least one compound or combination of compounds is selected from the group consisting of:

[0038] I.1) Higher than 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 Grams of protein per liter of pre-growth and / or growth and / or fed-batch medium;

[0039] I.2) Higher than 0, 10 -10 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 Grams of nucleic acid per liter of pre-growth and / or growth and / or fed-batch medium;

[0040] I.3) Higher than 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 grams of glutathione per liter of pre-growth and / or growth and / or fed-batch medium;

[0041] I.4) Higher than 0, 10 -10 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 grams of at least one compound per liter of pre-growth and / or growth and / or fed-batch medium, wherein the at least one compound is selected from the group consisting of dextran, mannan, trehalose, flavor nucleotides, B vitamins, biotin and volatile aromatic compounds,

[0042] I.5) higher than 0, 10 -10 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 grams of calcium per liter of pre-growth and / or growth and / or fed-batch medium,

[0043] I.6) higher than 0, 10 -10 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 grams of phosphorus per liter of pre-growth and / or growth and / or fed-batch medium,

[0044] I.7) higher than 0, 10 -10 , 10 -8 , 10 -1, 1, 10 3 or 10 5 grams of zinc per liter of pre-growth and / or growth and / or fed-batch medium,

[0045] I.8) higher than 0, 10 -10 , 10 -1 , 1, 10 3 or 10 5 grams of chromium per liter of pre-growth and / or growth and / or fed-batch medium,

[0046] I.9) higher than 0, 10 -10 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 grams of potassium per liter of pre-growth and / or growth and / or fed-batch medium,

[0047] I.10) higher than 0, 10 -10 , 10 -1 , 1, 10 3 or 10 5 grams of cobalt per liter of pre-growth and / or growth and / or fed-batch medium,

[0048] I.11) higher than 0, 10 -10 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 grams of manganese per liter of pre-growth and / or growth and / or fed-batch medium,

[0049] I.12) higher than 0, 10 -10 , 10 -1 , 1, 10 3 or 10 5 grams of strontium per liter of pre-growth and / or growth and / or fed-batch medium,

[0050] I.13) higher than 0, 10 -10 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 grams of magnesium per liter of pre-growth and / or growth and / or fed-batch medium,

[0051] I.14) higher than 0, 10 -10 , 10 -4 , 10 -1 , 1, 10 3 or 10 5 grams of yeast extract per liter of pre-growth and / or growth and / or fed-batch medium, and

[0052] I.15) greater than 0, 1, 5, 10, 15, 20 or 50 different components derived from yeast extract;

[0053] II) at least one compound or combination of compounds contained in or derived from peptone, wherein the at least one compound or combination of compounds is selected from the group consisting of:

[0054] II.1) Higher than 0, 10 -10 , 10 -4 , 10 -1 , 1, 10 3 or 10 5 grams of ash per liter of pre-growth and / or growth and / or fed-batch medium,

[0055] II.2) Higher than 0, 10 -10 , 10 -3 , 10 -1 , 1, 10 3 or 10 5 Grams of protein per liter of pre-growth and / or growth and / or fed-batch medium,

[0056] II.3) Higher than 0, 10 -10 , 10 -4 , 10 -1 , 1, 10 3 or 10 5 grams of sucrose per liter of pre-growth and / or growth and / or fed-batch medium,

[0057] II.4) Higher than 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 grams of raffinose per liter of pre-growth and / or growth and / or fed-batch medium,

[0058] II.5) Higher than 0, 10 -10 , 10 -4 , 10 -1 , 1, 10 3 or 10 5 grams of neutral detergent fiber per liter of pre-growth and / or growth and / or fed-batch medium,

[0059] II.6) Higher than 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 or 10 5grams of ethereal extract per liter of pre-growth and / or growth and / or fed-batch medium,

[0060] II.7) Higher than 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 grams of peptone per liter of pre-growth and / or growth and / or fed-batch medium, and

[0061] II.8) greater than 0, 1, 5, 10, 15, 20, or 50 different components from peptone; III) comprising at least one compound or combination of compounds from or derived from Wolfe's minerals or mineral elixirs, wherein at least one compound or combination of compounds is selected from the group consisting of:

[0062] III.1) Higher than 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 moles of nitrilotriacetic acid per liter of pre-growth and / or growth and / or fed-batch medium,

[0063] III.2) Higher than 0, 10 -20 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 moles of magnesium sulfate per liter of pre-growth and / or growth and / or fed-batch medium,

[0064] III.3) Higher than 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 moles of sodium chloride per liter of pre-growth and / or growth and / or fed-batch medium,

[0065] III.4) Higher than 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 moles of manganese sulfate per liter of pre-growth and / or growth and / or fed-batch medium,

[0066] III.5) Higher than 0, 10 -20 , 10 -6 , 10-1 , 1, 10 3 or 10 5 moles of ferrous sulfate per liter of pre-growth and / or growth and / or fed-batch medium,

[0067] III.6) Higher than 0, 10 -20 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 moles of cobalt nitrate per liter of pre-growth and / or growth and / or fed-batch medium,

[0068] III.7) Higher than 0, 10 -20 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 moles of calcium chloride per liter of pre-growth and / or growth and / or fed-batch medium,

[0069] III.8) Higher than 0, 10 -20 , 10 -6 , 10 -1 , 1, 10 3 or 10 5 moles of zinc sulfate per liter of pre-growth and / or growth and / or fed-batch medium,

[0070] III.9) Higher than 0, 10 -20 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 moles of copper sulfate per liter of pre-growth and / or growth and / or fed-batch medium,

[0071] III.10) higher than 0, 10 -20 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 moles of potassium aluminum sulfate derived from Wolfe minerals or mineral elixirs per liter of pre-growth and / or growth and / or fed-batch medium,

[0072] III.11) Higher than 0, 10 -20 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 moles of boric acid per liter of pre-growth and / or growth and / or fed-batch medium,

[0073] III.12) Higher than 0, 10-20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of sodium molybdate per liter of pre-growth and / or growth and / or fed-batch medium,

[0074] III.13) Higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of sodium selenite per liter of pre-growth and / or growth and / or fed-batch medium,

[0075] III.14) Higher than 0, 10 -20 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 moles of sodium tungstate per liter of pre-growth and / or growth and / or fed-batch medium,

[0076] III.15) Higher than 0, 10 -20 , 10 -7 , 10 -1 , 1, 10 3 or 10 5 moles of nickel chloride per liter of pre-growth and / or growth and / or fed-batch medium,

[0077] III.16) Higher than 0, 10 -20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of Wolfe minerals or mineral elixirs per liter of pre-growth and / or growth and / or fed-batch medium, and

[0078] III.17) more than 0,10 different ingredients from Wolfe minerals or mineral elixirs; IV) more than 0,10 -20 , 10 -5 , 10 -1 , 1, 10 3 or 10 5 moles of EDTA per liter of pre-growth and / or growth and / or fed-batch medium;

[0079] and / or

[0080] V) at least one compound or combination of compounds contained in or derived from Wolff's vitamins, wherein the at least one compound or combination of compounds is selected from the group consisting of: V.1) greater than 0, 10-20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of biotin per liter of pre-growth and / or growth and / or fed-batch medium,

[0081] V.2) higher than 0, 10 -20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of calcium pantothenate per liter of pre-growth and / or growth and / or fed-batch medium,

[0082] V.3) higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of folic acid per liter of pre-growth and / or growth and / or fed-batch medium,

[0083] V.4) higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of inositol per liter of pre-growth and / or growth and / or fed-batch medium,

[0084] V.5) is higher than 0, 10 -20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of niacin per liter of pre-growth and / or growth and / or fed-batch medium,

[0085] V.6) is higher than 0, 10 -20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of p-aminobenzoic acid per liter of pre-growth and / or growth and / or fed-batch medium,

[0086] V.7) is higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of pyridoxine hydrochloride per liter of pre-growth and / or growth and / or fed-batch medium,

[0087] V.8) is higher than 0, 10-20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of riboflavin per liter of pre-growth and / or growth and / or fed-batch medium,

[0088] V.9) is higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 moles of thiamine hydrochloride per liter of pre-growth and / or growth and / or fed-batch medium,

[0089] V.10) is higher than 0, 10 -20 , 10 -8 , 10 -1 , 1, 10 3 or 10 5 moles of lipoic acid per liter of pre-growth and / or growth and / or fed-batch medium,

[0090] V.11) is higher than 0, 10 -20 , 10 -9 , 10 -1 , 1, 10 3 or 10 5 Moore's At least one ingredient of Wolfe's Vitamins, and

[0091] V.12) greater than 0, 1, 5, 10, 15 or 20 different Wolff vitamins; and / or

[0092] Preferably, wherein measured per milligram or per gram or per milliliter or per liter of pre-growth, growth and / or fed-batch medium / culture medium, the pre-growth, growth and / or fed-batch medium preferably does not comprise more than:

[0093] 1) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 or 1 gram of protein preferably derived from yeast extract,

[0094] 2) 0, 10 -20 , 10 -10 , 10 -6 , 10 -3 or 1 gram of nucleic acid preferably derived from yeast extract,

[0095] 3) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3or 1 gram of glutathione preferably derived from yeast extract,

[0096] 4) 0, 10 -20 , 10 -10 , 10 -7 , 10 -1 or 1 gram of at least one compound preferably derived from yeast extract, wherein the at least one compound is selected from the group consisting of glucans, mannans, trehalose, flavor nucleotides, B vitamins, biotin and volatile aroma compounds,

[0097] 5)0, 10 -20 , 10 -10 , 10 -7 , 10 -1 or 1 gram of calcium preferably derived from yeast extract,

[0098] 6)0, 10 -20 , 10 -10 , 10 -6 , 10 -1 or 1 gram of phosphorus preferably derived from yeast extract,

[0099] 7)0, 10 -20 , 10 -10 , 10 -8 , 10 -1 or 1 gram of zinc, preferably from yeast extract,

[0100] 8)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 gram of chromium, preferably from yeast extract,

[0101] 9)0, 10 -20 , 10 -10 , 10 -7 , 10 -5 , 10 -1 or 1 gram of potassium, preferably from yeast extract,

[0102] 10)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 gram of cobalt preferably derived from yeast extract,

[0103] 11)0, 10 -20 , 10 -9 , 10 -5 , 10 -1 or 1 gram of manganese, preferably from yeast extract,

[0104] 12)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 gram of strontium preferably derived from yeast extract,

[0105] 13)0, 10 -20 , 10 -10 , 10 -7 , 10 -3 , 10 -1 or 1 gram of magnesium, preferably from yeast extract,

[0106] 14)0, 10 -20 , 10 -10 , 10 -4 , 10 -1 or 1 gram of yeast extract,

[0107] 15) 0, 1, 5, 10 or 15 different ingredients from yeast extract,

[0108] 16)0, 10 -20 , 10 -10 , 10 -4 , 10 -1 or 1 gram of ash preferably derived from peptone,

[0109] 17)0, 10 -20 , 10 -10 , 10 -3 , 10 -1 or 1 gram of protein, preferably derived from peptone,

[0110] 18)0, 10 -20 , 10 -10 , 10 -4 , 10 -1 or 1 gram of sucrose, preferably derived from peptone,

[0111] 19)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 gram of raffinose, preferably derived from peptone,

[0112] 20)0, 10 -20 , 10 -10 , 10 -4 , 10 -1 or 1 gram of neutral detergent fiber preferably derived from peptone,

[0113] 21)0, 10 -20 , 10 -10 , 10 -5 , 10-1 or 1 gram of an ether extract preferably derived from peptone,

[0114] 22)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 g of peptone,

[0115] 23) 0, 1, 5, 7, 10 or 15 different components from peptone,

[0116] 24)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 mol of nitrilotriacetic acid preferably derived from wolf minerals or mineral elixirs,

[0117] 25)0, 10 -20 , 10 -10 , 10 -6 , 10 -1 or 1 mol of magnesium sulfate preferably derived from wolf minerals or mineral elixirs,

[0118] 26)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 mol of sodium chloride preferably derived from wolf minerals or mineral elixirs,

[0119] 27)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 mol of manganese sulfate preferably derived from wolf minerals or mineral elixirs,

[0120] 28)0, 10 -20 , 10 -10 , 10 -6 , 10 -1 or 1 mol of ferrous sulfate preferably derived from wolf minerals or mineral elixirs,

[0121] 29)0, 10 -20 , 10 -10 , 10 -6 , 10 -1 or 1 mol of cobalt nitrate preferably derived from wolf minerals or mineral elixirs,

[0122] 30)0, 10 -20 , 10 -10 , 10 -6 , 10 -1or 1 mol of calcium chloride preferably derived from Wolfe minerals or mineral elixirs,

[0123] 31)0, 10 -20 , 10 -10 , 10 -6 , 10 -1 or 1 mol of zinc sulfate preferably derived from wolf minerals or mineral elixirs,

[0124] 32)0,10 -20 , 10 -10 , 10 -7 , 10 -1 or 1 mol of copper sulfate preferably derived from wolf minerals or mineral elixirs,

[0125] 33)0,10 -20 , 10 -10 , 10 -7 , 10 -1 or 1 mol of potassium aluminum sulfate preferably derived from wolf minerals or mineral elixirs,

[0126] 34)0, 10 -20 , 10 -10 , 10 -7 , 10 -1 or 1 mol of boric acid preferably derived from wolf minerals or mineral elixirs,

[0127] 35)0, 10 -20 , 10 -10 , 10 -8 , 10 -3 , 10 -1 or 1 mol of sodium molybdate preferably derived from wolf minerals or mineral elixirs,

[0128] 36)0,10 -20 , 10 -15 , 10 -9 , 10 -3 , 10 -1 or 1 mol of sodium selenite preferably derived from wolf minerals or mineral elixirs,

[0129] 37)0,10 -20 , 10 -7 , 10 -3 , 10 -1 or 1 mol of sodium tungstate preferably derived from wolf minerals or mineral elixirs,

[0130] 38)0,10 -20 , 10 -7 , 10 -3 , 10 -1or 1 mol of nickel chloride preferably derived from wolf minerals or mineral elixirs,

[0131] 39)0, 10 -20 , 10 -8 , 10 -3 , 10 -1 or 1 mol of Wolf minerals or mineral elixir,

[0132] 40) 0, 1, 5, 10 or 20 different ingredients of Wolfe minerals or mineral elixirs,

[0133] 41)0, 10 -20 , 10 -10 , 10 -5 , 10 -1 or 1 mol of EDTA,

[0134] 42)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of biotin preferably derived from Wolff vitamins,

[0135] 43)0,10 -50 , 10 -30 , 10 -8 , 10 -3 , 10 -1 or 1 mol of calcium pantothenate preferably derived from Wolff vitamins,

[0136] 44)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of folic acid preferably derived from Wolff vitamins,

[0137] 45)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of inositol preferably derived from Wolff vitamins,

[0138] 46)0, 10 -50 , 10 -30 , 10 -10 , 10 -8 , 10 -3 , 10 -1 or 1 mol of niacin, preferably derived from Wolff vitamins,

[0139] 47)0, 10-50 , 10 -30 , 10 -8 , 10 -3 , 10 -1 or 1 mol of p-aminobenzoic acid preferably derived from Wolff vitamins,

[0140] 48)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of pyridoxine hydrochloride preferably derived from Wolff vitamins,

[0141] 49)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of riboflavin preferably derived from Wolff vitamins,

[0142] 50)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of thiamine hydrochloride preferably derived from Wolff vitamins,

[0143] 51)0, 10 -50 , 10 -30 , 10 -8 , 10 -3 , 10 -1 or 1 mol of lipoic acid preferably derived from Wolff vitamins,

[0144] 52)0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 or 1 mol of at least one component of Wolff's vitamins,

[0145] 53) 0, 1, 5, 10 or 20 different Wolf vitamins,

[0146] 54) 0, 1, 2, 3, 6, 10 or 100 different vitamins,

[0147] 55)0, 10 -50 , 10 -20 , 10 -9 , 10 -8 , 10 -7 , 10 -5 , 10 -3, 10 -1 or 1 mol of at least one vitamin,

[0148] 56)0, 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -4 , 10 -2 , 10 -1 , 1, 10, or 10 3 g of yeast extract,

[0149] 57)0,10 -50 , 10 -20 , 10 -9 , 10 -3 , 10 -1 , 1, 5 or 10 mol of at least one component of yeast extract,

[0150] 58) 0, 1, 2, 5, 10 or 100 different components of yeast extract,

[0151] 59)0, 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10, or 10 3 g of peptone,

[0152] 60)0, 10 -50 , 10 -9 , 10 -5 , 10 -3 , 10 -1 , 1 or 10 mol of at least one component of peptone,

[0153] 61) 0, 1, 2, 5, 10 or 100 different components of peptone,

[0154] 62) 0, 1, 2, 5, 10 or 100 different CMR reagents,

[0155] 63)0,10 -50 , 10 -9 , 10 -5 , 0.05, 10 -1 , 1, 10, 10 3 or 10 6 mg of at least one CMR reagent,

[0156] 64) 0, 1, 2, 5, 10 or 100 different chelating agents,

[0157] 65)0, 10 -50 , 10-20 , 10 -9 , 10 -8 , 10 -3 , 10 -1 , 1, 5, 10, or 10 3 mol at least one chelating agent,

[0158] 66) 0, 1, 2, 5, 10 or 100 different amino acids,

[0159] 67)0,10 -50 , 10 -10 , 10 -5 , 10 -3 , 1, 10, 10 3 , 10 5 or 10 10 mg of at least one amino acid,

[0160] 68) 0, 1, 2, 5, 10 or 100 different toxic or cytotoxic compounds,

[0161] 69)0, 10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 mg of at least one toxic or cytotoxic compound,

[0162] 70) 0, 1, 3 or 7 heavy metals other than iron,

[0163] 71)0, 10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 mg of at least one heavy metal other than iron,

[0164] 72) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 metals or chemical elements selected from cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper,

[0165] 73) 1 mg of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and / or copper,

[0166] 74)0, 10 -50 , 10 -10 , 0.5, 1, 5, 10, 10 3 or 10 6 ml or 10-50 , 10 -30 , 10 -10 , 10 -8 , 10 -5 , 10 -3 , 1, 10, 10 3 or 10 6 mol Wolf vitamins,

[0167] 75)10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 mol at least one Wolf Vitamin ingredient,

[0168] 76) 0, 1, 2, 5, 10 or 100 different ingredients of Wolff vitamins,

[0169] 77)0,10 -50 , 10 -10 , 0.5, 1, 5, 10, 10 3 or 10 6 mL or 10 -50 , 10 -30 , 10 -10 , 10 -8 , 10 -5 , 10 -3 , 1, 10, 10 3 or 10 6 mol of Wolf minerals,

[0170] 78)10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 mol of at least one Wolf mineral component,

[0171] 79) 0, 1, 2, 5, 7, 10, 15 or 100 different compositions of Wolf minerals,

[0172] 80)10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 mol mineral elixir,

[0173] 81)10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 mol of at least one ingredient of a mineral elixir, and / or

[0174] 82) 0, 1, 2, 5, 10, 14 or 100 different ingredients of mineral elixirs.

[0175] The present invention also relates to a method according to the present invention, wherein the fed-batch medium comprises at least one compound selected from the group consisting of iron, an iron source, carbon, a carbon source, nitrogen, a nitrogen source and combinations thereof, and the concentration of the at least one compound in the fed-batch medium is greater than 10 -6 μM, 1 μM and / or 10 6 μM.

[0176] In one embodiment of the present invention, the at least one vitamin component of the Wolfe vitamin or vitamin solution is selected from the group consisting of folic acid, folate, pyridoxine, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, niacin, pantothenic acid, vitamin B12, aminobenzoic acid, lipoic acid, all-trans retinol, retinal, alternative provitamin A-functionalized carotenoids including all-trans beta-carotene, niacin, niacinamide, niacinamide, ribosides, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherols, tocotrienols, theoquinone, menadione, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 , Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i (where V can be any letter from A to Z and i can be any integer from 1 to 100), and their derivatives.

[0177] In another embodiment of the present invention, at least one component of the wolf mineral is selected from the group consisting of nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate heptahydrate, hydrated copper sulfate, potassium aluminum sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate dihydrate, nickel chloride, and derivatives thereof.

[0178] In another embodiment of the present invention, the at least one component of the yeast extract is selected from the group consisting of: i) at least one protein, ii) at least one nucleic acid, iii) at least one functional peptide, iv) glutathione, v) glucan, vi) mannan, vii) trehalose, viii) flavor nucleotides, ix) B vitamins, x) biotin, x) at least one volatile aroma compound, xi) calcium, xii) phosphorus, xiii) zinc, xiv) iron, xv) chromium, xvi) potassium, xvii) cobalt, xviii) manganese, xix) strontium, xx) magnesium, and xxi) derivatives thereof.

[0179] In another embodiment of the present invention, at least one ingredient of the mineral elixir is selected from the group consisting of nitrilotriacetic acid, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3 and their derivatives.

[0180] In one embodiment of the present invention, the following two statements are equivalent: the pre-growth, growth and / or fed-batch medium does not contain at least one compound above a certain amount, concentration or amount and the pre-growth, growth and / or fed-batch medium contains at least one compound below a certain amount, concentration or amount.

[0181] In one embodiment of the present invention, the nanoparticle producing cells are expanded or grown in a pre-growth and / or growth and / or fed-batch medium, preferably in a pre-growth and / or growth medium rather than in a fed-batch medium.

[0182] In one embodiment of the present invention, the growth and / or pre-growth medium comprises a growth and / or pre-growth medium: i) without the need to inject a fed-batch medium into the growth and / or pre-growth medium or before the injection of a fed-batch medium into the growth and / or pre-growth medium; or ii) with the need to inject a fed-batch medium into the growth and / or pre-growth medium or after the injection of a fed-batch medium into the growth and / or pre-growth medium.

[0183] In one embodiment of the present invention, the fed-batch medium is a fed-batch medium or a medium before it is injected into a growth and / or pre-growth medium.

[0184] In another embodiment of the present invention, the fed-batch medium is part of the pre-growth and / or pre-growth medium after it is injected into the growth and / or pre-growth medium.

[0185] In one embodiment of the present invention, the parameter C FeGS 、CFePGS 、C CGS 、C CPGS 、C NGS 、C NPGS , ΔpH GS and / or ΔpH PGS Present in or measured in pre-growth, growth and / or fed-batch media.

[0186] In one embodiment of the present invention, the parameter Q GGS , Q GPGS 、N SSGS and N SSPGS , and the case where the growth medium is supplemented with fed-batch medium but the pre-growth medium is not supplemented with fed-batch medium, measured at the beginning or end of the pre-growth and / or growth phase.

[0187] In one embodiment of the present invention, the derivative is a derivative of at least one first compound selected from the group consisting of: i) a second compound that differs from the at least one first compound by at least one different atom or functional group, preferably derived from at least one first compound after transformation of the at least one first compound, preferably the second compound has at least one common atom or functional group with the at least one first compound, ii) a non-hydrated form of at least one first compound, iii) a hydrated form of at least one first compound, iv) a reduced form of at least one first compound, v) an oxidized form of at least one first compound, vi) an acidic form of at least one first compound, v) a basic form of at least one first compound, vi) a crystalline or solid form of at least one first compound, vii) a soluble or dissolved form of at least one first compound, and x) a salt of at least one first compound.

[0188] The present invention relates to a method according to the invention, wherein the growth stage distinguishes itself from the pre-growth stage by at least one characteristic selected from the group consisting of:

[0189] i)C FeGS / C FePGS The ratio is greater than 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where C FeGS and C FePGS are the concentrations of iron or iron source in the growth medium and pre-growth medium, respectively, ii) C CGS / C CPGS The ratio is greater than 10 -5 , 10 -3 , 1, 10, 10 3 or 105 , where C CGS and C CPGS are the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively; iii) C NGS / C NPGS The ratio is greater than 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where C NGS and C NPGS where ΔpH is the concentration of nitrogen or nitrogen source in the growth medium and pre-growth medium, respectively. GS / ΔpH PGS The ratio is less than 10 10 , 10 5 , 10 3 , 10 2 , 1, 0.5 or 0.1, where ΔpH GS and / ΔpH PGS are the changes in pH in the growth medium and pre-growth medium,

[0190] v)Q GGS / Q GPGS The ratio is greater than 10 10 , 10 5 , 10 3 , 10 2 , 1, 0.5 or 0.1, where Q GGS and Q GPGS is the amount of gas, oxygen or air introduced or bubbled into the growth medium and pre-growth medium, respectively,

[0191] vi)N SSGS / N SSPGS The ratio is less than 10 -5 , 10 -3 , 1, 10, 10 3 or 10 5 , where N SSGS and N SSPGS the number of sub-stages of the growth stage and the number of sub-stages of the pre-growth stage, respectively, wherein the two sub-stages are separated by transferring the nanoparticle-producing cells from a first sub-stage (preferably associated with the growth of the nanoparticle-producing cells in the first volume) to a second sub-stage (preferably associated with the growth of the nanoparticle-producing cells in the second volume), and

[0192] vii) The growth medium is supplemented by the fed-batch medium, whereas the pre-growth medium is not supplemented by the fed-batch medium.

[0193] The present invention also relates to a method according to the invention, wherein the pre-growth and / or growth and / or fed-batch medium preferably comprises per kilogram or per liter of pre-growth and / or growth and / or fed-batch medium less than:

[0194] i) by mass or volume 10 3 %, or 1% by mass or volume, or 10% by mass or volume -2 %, or by mass or volume 5·10 -3 %, or 10 3 g, or 10 2 g, or 10g, or 10 10 mL, or 10 5 mL, or 10 3 mL, or 10mL, or 5mL, or 1mL, or 0.5mL, or 10 -5 mL, or 10 3 mol, or 10mol, or 1mol, or 10 -5 mol, or 10 -8 mol, or 10 -9 mol, or 10 -10 mol of vitamins, preferably Wolff vitamins, or chemical components selected from the group consisting of folic acid, folate, pyridoxine, pyridoxine hydrochloride, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine hydrochloride, niacin, pantothenic acid, calcium pantothenate, inositol, p-aminobenzoic acid, aminobenzoic acid, lipoic acid, all-trans retinol, retinal, alternative provitamin A-functionalized carotenoids including all-trans beta-carotene, niacin, niacinamide, niacinamide, nucleosides, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherols, tocotrienols, theoquinone, menadione, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 , Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i and their derivatives, where V can be any letter from A to Z, and i can be any integer from 1 to 100,

[0195] ii) 1, 5, 6, 10 or 20 different vitamins, preferably Wolff vitamins, or chemical components selected from the group consisting of folic acid, folate, pyridoxine, pyridoxine hydrochloride, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine hydrochloride, niacin, pantothenic acid, calcium pantothenate, inositol, p-aminobenzoic acid, aminobenzoic acid, lipoic acid, all-trans retinol, retinal, alternative provitamin A-functionalized carotenoids including all-trans beta-carotene, niacin, niacinamide, niacinamide, nucleosides, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherols, tocotrienols, theoquinone, menadione, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 , Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i and their derivatives, where V can be any letter from A to Z, and i can be any integer from 1 to 100,

[0196] iii) by mass or volume 10 3 %, or 10% by mass or volume, or 1% by mass or volume, or 10% by mass or volume -2 %, or 10 by mass or volume -5 %, or 10 3 g, or 10g, or 1g, or 10 5 mL, or 10 3 mL, or 10mL, or 1mL, or 10 -3 mL, or 10 3 mol, or 10mol, or 1mol, or 10 -3 mol, or 10 -7 mol, or 10 -8 mol, or 10 - 10 mol of a mineral, preferably a Wolf mineral or a mineral elixir, or a chemical component selected from the group consisting of nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, hydrated copper sulfate, potassium aluminum sulfate, potassium aluminum sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3 and derivatives thereof,

[0197] iv) 1, 3, 7, or 10 different components of minerals, preferably Wolf minerals or mineral elixirs, or chemical components selected from the group consisting of nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, hydrated copper sulfate, potassium aluminum sulfate, potassium aluminum sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and derivatives thereof,

[0198] v)10 -50 g, or 10 -10 g, or 10 -5 g, or 0.005g, or 10 -1 g, or 1g, or 10g or 10 5 g, or 10 -50 M, or 10 -8 M, or 10 -3 M, or 10 -1 M, or 1M or 10 3 M is at least one component of a yeast extract or at least one compound derived from a yeast extract group, selected from the group consisting of: at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, glucan, mannan, trehalose, flavoring nucleotides, B vitamins, biotin, at least one volatile aroma compound, calcium, phosphorus, zinc, iron, chromium, potassium, cobalt, manganese, strontium, magnesium and derivatives thereof,

[0199] vi) 1, 2, 3, 5, 10, 15, 20 or 50 different components of a yeast extract or different compounds derived from a yeast extract, selected from the group consisting of at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, glucan, mannan, trehalose, flavor nucleotides, B vitamins, biotin, at least one volatile aroma compound, calcium, phosphorus, zinc, iron, chromium, potassium, cobalt, manganese, strontium, magnesium and derivatives thereof,

[0200] vii)10 -50 g, or 10 -10 g, or 10 -3 g, or 0.01g, or 1g, or 5g, or 10g or 10 5 g, or 10 -50 M, or 10 - 20 M, or 10 -8M, or 10 -3 M, or 10 -1 M, or 1M, or 10M or 10 3 M at least one component of peptone or at least one compound derived from peptone, selected from the group consisting of ash, protein, sucrose, stachyose, raffinose, neutral detergent fiber, ether extract (etherate, ether extract, Ethereal Extract) and derivatives thereof,

[0201] viii) 1, 3, 5, 10, 20 or 50 different components of peptone or different components of compounds derived from peptone, selected from the group consisting of ash, protein, sucrose, stachyose, raffinose, neutral detergent fiber, ether extracts and derivatives thereof,

[0202] ix)10 -50 , 10 -10 , 10 -5 , 0.001, 10 -1 , 1, 10, 10 3 or 10 5 g EDTA,

[0203] x)10 -50 , 10 -10 , 10 -5 , 0.001, 10 -1 , 1, 10, 10 3 or 10 5 grams of at least one amino acid,

[0204] xi) 1, 3, 5, 10, 20 or 50 different amino acids,

[0205] xii) 1, 5, 7, 12, 15, 20 or 50 different CMR, toxic or cytotoxic compounds selected from the group consisting of nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride and their derivatives,

[0206] xiii) 1, 2, 5, 10, 50 or 100 chemical elements or heavy metals selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper and their derivatives,

[0207] xiv)10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 or 10 5a chemical element or heavy metal selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper and their derivatives,

[0208] xv)10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 or 10 5 at least one CMR, toxic or cytotoxic compound selected from the group consisting of nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride and any derivatives thereof, and / or

[0209] xvi)10 -50 , 10 -10 , 10 -5 , 0.01, 10 -1 , 1, 10, 10 3 or 10 5 grams of peptone.

[0210] The present invention also relates to a method according to the invention, wherein the concentration of the at least one compound in the pre-growth and / or growth medium is C2 or C 总量 =C1+C2, where:

[0211] - C1 is the concentration of at least one compound in the pre-growth and / or growth medium that is not consumed by the nanoparticle-producing cells,

[0212] - C2 is the concentration of at least one compound in the pre-growth and / or growth medium consumed by the nanoparticle-producing cells,

[0213] as well as,

[0214] - preferably, C1 and / or C2 are measured, separated or differentiated by using a method capable of separating at least one compound consumed by the nanoparticle-producing cells from at least one compound not consumed by the nanoparticle-producing cells (e.g. centrifugation or tangential filtration), and

[0215] - Preferably, C1 and / or C2 are measured or taken into account at the beginning, during or at the end of the pre-growth and / or growth phase.

[0216] In one embodiment of the invention, a compound in the pre-growth and / or growth medium consumed by the nanoparticle-producing cells is, preferably, a compound contained within the nanoparticle-producing cells when the nanoparticle-producing cells consume such compound.

[0217] In another embodiment of the invention, the one compound in the pre-growth and / or growth medium which is not consumed by the nanoparticle-producing cells is, preferably, a compound which is contained outside the nanoparticle-producing cells when the nanoparticle-producing cells do not consume such compound.

[0218] In one embodiment, a method is used to separate consumed and unconsumed compounds. The method preferably separates whole bacteria from a pre-growth and / or growth medium that does not contain such bacteria. The method can be centrifugation or filtration, preferably tangential filtration, or a method capable of separating whole bacteria from a liquid culture medium, based on the separation of low-size and / or low-weight elements contained in the pre-growth and / or growth medium from large-size and / or large-weight elements in the whole bacteria.

[0219] The present invention also relates to a method according to the invention, wherein the concentration of at least one compound contained in the fed-batch medium, preferably iron, an iron source, carbon, a carbon source, nitrogen and / or a nitrogen source, is greater than 10 -6 μM, 1 μM and / or 10 6 μM.

[0220] The present invention also relates to a method according to the invention, wherein the pre-growth, growth and / or fed-batch growth medium does not comprise at least one compound in a concentration that influences the growth of the nanoparticle-producing cells and / or the production of nanoparticles, and / or wherein the pre-growth, growth and / or fed-batch growth medium is substantially free of at least one compound, wherein the at least one compound is selected from the group consisting of: 1) Wolffian vitamins or a medium containing more than half the total number of different components of Wolffian vitamins, 2) a component of Wolffian vitamins, 3) folic acid, 4) pyridoxine, 5) riboflavin, 6) biotin, 7) thiamine, 8) niacin, 9) pantothenic acid, 10) vitamin B12 12, 11) aminobenzoic acid, 12) lipoic acid, 13) Wolff minerals or a culture medium containing more than half the total number of different components of Wolff minerals, 14) nitrilotriacetic acid, 15) magnesium sulfate, 16) sodium chloride, 17) manganese sulfate, 18) ferrous sulfate heptahydrate, 19) cobalt nitrate, 20) calcium chloride, 21) zinc sulfate heptahydrate, 22) hydrated copper sulfate, 23) potassium aluminum sulfate dodecahydrate, 24) boric acid, 25) sodium molybdate, 26) sodium selenite, 27) sodium tungstate, 28) yeast extract or a culture medium containing more than half the total number of different components of yeast extract, 29) yeast extract equivalents or a culture medium containing more than half the total number of different components of yeast extract equivalents, 30) 1, 2 or 5 of the above ingredients are derived from or contained in yeast extract. proteins from a yeast extract, 31) 1, 2, or 5 nucleic acids from or contained in a yeast extract, 32) 1, 2, or 5 peptides or functional peptides from or contained in a yeast extract, 33) glutathione, 34) glucans, 35) mannans, 36) trehalose, 37) flavor nucleotides from or contained in a yeast extract, 38) B vitamins, 39) biotin, 40) 1, 2, or 5 volatile aroma compounds from or contained in a yeast extract, 41) chromium, 42) cobalt, 43) strontium, 44) nickel chloride, 45) or a culture medium containing more than half the total number of different components of a mineral elixir, 46) MnSO4, 47) NaCl, 48) FeSO4, 49) CoSO4, 50 )CaCl2, 51)ZnSO4, 52)CuSO4, 53)KAl(SO4)2, 54)H3BO3, 55)Na2MoO4, 56)NiCl2, 57)Na2SeO3, 58)peptone or a culture medium containing more than half the total number of different components of peptone, 59) a component of peptone, 60) 1, 2 or 5 proteins derived from or contained in peptone, (61) sugars derived from or contained in peptone, (62) amino acids derived from or contained in peptone, (63) ash derived from or contained in peptone, (64) fiber derived from or contained in peptone, 65) a CMR reagent, 66) boric acid, 67) an amino acid, 68) propyl amino acid, 69) arginine, 70) asparagine, 71) aspartic acid, 72) cysteine, 73) glutamine, 74) glutamic acid, 75) glycine, 76) histidine, 77) isoleucine, 78) leucine, 79) lysine, 80) methionine, 81) phenylalanine, 82) proline, 83) serine, 84) threonine, 85) tryptophan, 86) tyrosine, 87) valine, 88) a cytotoxic or toxic compound, 89) manganese sulfate, 90) copper sulfate, 91) potassium aluminum sulfate, 92) boric acid, 93) sodium tungstate, 94) a heavy metal other than iron, 95) titanium, 96) vanadium, 97) manganese, 98) nickel, 99) copper, 100) zinc, 101) gallium, 102) germanium,103) Arsenic, 104) Zirconium, 105) Niobium, 106) Molybdenum, 107) Technetium, 108) Ruthenium, 109) Rhodium, 110) Palladium, 111) Silver, 112) Cadmium, 113) Indium, 114) Tin, 115) Tellurium, 116) Lutetium, 117) Hafnium, 118) Tantalum, 119) Tungsten, 120) Rhenium, 121) Osmium, 122) Iridium, 123 ) Platinum, 125) Gold, 126) Mercury, 127) Thallium, 128) Lead, 129) Bismuth, 130) Polonium, 131) Astatine, 132) Lanthanum, 133) Cerium, 134) Praseodymium, 135) Neodymium, 136) Promethium, 137) Samarium, 138) Europium, 139) Gadolinium, 140) Terbium, 141) Dysprosium, 142) Holmium, 1 43) Erbium, 144) Thulium, 145) Ytterbium, 146) Actinium, 147) Thorium (Th), 148) Protactinium, 149) Uranium, 150) Neptunium, 151) Plutonium, 152) Americium, 153) Curium, 154) Berkelium, 155) Californium, 156) Einsteinium, 157) Fermium, 158) Nobelium, 159) Radium, 160) Lawrencium, 161) Rutherfordium, 162) Dubnium, 163) Seaborgium, 164) Bohrium, 165) Hassium, 166) Meitnerium (Mt), Darmstadtium (Ds), Roentgenium (Rg), Copernicium (Cn), Elements 113-118 (170), Helium (171), Lithium (172), Beryllium (173), Boron (174), Fluorine (175), Aluminum (176), Silicon (177), Argon (178), Scandium (179), Chromium (180), Nickel (181), Copper (182), Selenium (183), Bromine (184), Krypton (185), Rubidium (186), Yttrium (187), Sn (188), Antimony (189), Iodine (190), Xenon (191), Cesium (192), Barium (193) , 194) Lutetium, 195) Astatine, 196) Radon, 197) Francium, 198) Mendelevium, 199) Mt, 200) Ununbium, 201) Unitrium, 202) Unquadium, 203) Ununpentium, 204) Ununhexium, 205) Unus (Ununseptium), 206) Ununoctium, 207) Salts of compounds 1) to 206), and 208) derivatives thereof.

[0221] The present invention also relates to a method according to the invention, wherein the concentration of the compound that influences the growth of the nanoparticle-producing cells and / or the production of nanoparticles is, in the pre-growth, growth and / or fed-batch medium: i) greater than 1 pM, 1 μM, 1 mM, 10 -50 M, 10 -10 or 10 -5 M per liter of pre-growth, growth and / or fed-batch medium, or ii) greater than 10 -50 , 10 -10 , 10 -5 or 10 -3 Grams of compound per liter of pre-growth, growth and / or fed-batch medium.

[0222] In one embodiment of the present invention, the concentration of the compound that affects the production of nanoparticles by the nanoparticle producing cells and / or nanoparticles is such that the concentration in the pre-growth, growth and / or fed-batch medium is: i) less than 10 50 , 1, 10 -5 , 10 -6 or 10 - 9 M per liter of pre-growth, growth and / or fed-batch medium, or ii) less than 10 10 , 1, 10 -10or 10 -20 Grams of compound per liter of pre-growth, growth and / or fed-batch medium.

[0223] The present invention relates to a method according to the invention, wherein the growth medium and / or the fed-batch medium is supplemented by a fed-batch medium, and:

[0224] i) the pH of the fed-batch medium is lower than the pH of the pre-growth and / or growth medium, preferably at least 10 -5 , 0.1, 0.5, 1, 2, 3 or 5 pH units, and / or

[0225] ii) the concentration of at least one chemical element preferably selected from the group consisting of a) a phosphorus source or a phosphate source, b) a potassium source, c) a magnesium source, d) an iron source, e) a vitamin source, f) a calcium source, g) KH2PO4, h) MgSO4, i) FeCl3, j) thiamine, k) CaCl2 and l) derivatives thereof in the fed-batch medium is greater than in the pre-growth and / or growth medium, preferably at least 10 -50 , 10 -10 , 0, 1.1, 5, 10, or 10 3 times.

[0226] In one embodiment, the above conditions (i) and (ii) are verified by taking into account the pH and concentration of at least one compound:

[0227] i) for fed-batch medium, before injecting the fed-batch medium into the pre-growth and / or growth medium, and / or

[0228] ii) for pre-growth and / or growth medium, before, during or after injection of fed-batch medium into the pre-growth and / or growth medium.

[0229] The present invention also relates to a method according to the invention, wherein:

[0230] - the nanoparticle-producing cells are magnetotactic bacteria, and / or

[0231] - The nanoparticles are magnetosomes.

[0232] In another embodiment of the present invention, the nanoparticles are high purity nanoparticle producing cells.

[0233] In another embodiment of the present invention, the nanoparticles are high-purity nanoparticles, preferably nanoparticles based on high-purity iron oxide, wherein high-purity nanoparticles are preferably nanoparticles containing less than 100, 99, 90, 50, 20, 10, 1 or 0.1% by mass or number of atoms selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, copper and their derivatives. wherein high-purity iron oxide-based nanoparticles are preferably high-purity nanoparticles containing more than 1, 50, 90, 93 or 99% by mass or number of atoms of iron and / or oxygen and / or iron oxide.

[0234] The present invention also relates to a method according to the invention, wherein the pre-growth and / or growth medium comprises a calcium source, a carbon source, a nitrogen source, a phosphate or phosphorus source, a sulfur source, an iron source, a vitamin source and a calcium source, and:

[0235] - the concentration of the carbon source in the pre-growth and / or growth medium is greater, preferably at least 0, 0.5, 1.1, 2, 4, 10 or 100 times greater than the concentration of the at least one compound selected from the group consisting of phosphate or a phosphorus source, a sulfur source, a vitamin source and a calcium source in the pre-growth and / or growth medium, and / or - the concentration of the nitrogen source in the pre-growth and / or growth medium is greater, preferably at least 0, 0.5, 1.1, 2, 4, 10 or 100 times greater than the concentration of the at least one compound selected from the group consisting of phosphate or a phosphorus source, a sulfur source, a vitamin source and calcium in the pre-growth and / or growth medium.

[0236] The present invention also relates to a method according to the present invention, further comprising the step of storing, expanding, preparing or injecting a cell bank of nanoparticle-producing cells in a pre-growth and / or growth and / or fed-batch medium, wherein, preferably, the cell bank is stored, expanded or prepared in a medium containing at least 10 nanoparticles. -50 , 10 -10 , 10 -1 The incubation period is carried out in a cell bank medium containing 1% to 5% (preferably in terms of the number of compounds) the same compound as in the pre-growth and / or growth and / or fed-batch medium and preferably at least one compound, such as a vitamin, mineral, chelating agent, sucrose and / or cryoprotectant, which is different from the compound in the pre-growth and / or growth and / or fed-batch medium: i) is not in the pre-growth and / or growth and / or fed-batch medium (ii) is present in a lower concentration in the pre-growth and / or growth and / or fed-batch medium than in the cell bank medium.

[0237] In one embodiment of the present invention, the nanoparticle production cell bank is a master cell bank, a working cell bank or a research cell bank of nanoparticle production cells. In some cases, such cell banks include more than 1, 10, 10 3 , 1010 or 10 20 Nanoparticle production cells, preferably, per milliliter or per liter of pre-growth and / or growth and / or fed-batch culture medium. In some other cases, such cell banks comprise less than 10 100 , 10 50 , 10 20 , 10 10 or 10 3 Nanoparticle-producing cells.

[0238] In another embodiment of the present invention, the cell bank is a combination of at least one cell used to initiate expansion of the nanoproduction particles in pre-growth and / or growth and / or fed-batch culture.

[0239] In one embodiment of the present invention, the cell bank is prepared under the same or similar conditions as those used in the pre-growth and / or growth phases.

[0240] In another embodiment of the present invention, the cell bank is prepared by sparging or injecting gas into the cell bank culture medium with a low oxygen concentration, preferably less than 50, 10 or 1% oxygen relative to the volume of the cell bank culture medium.

[0241] The present invention relates to a method according to the invention, further comprising a purification stage for obtaining nanoparticles based on high-purity iron oxide, preferably starting from nanoparticles isolated from nanoparticle-producing cells obtained at the end of a growth stage, said purification stage preferably comprising removing at least one impurity from the nanoparticles produced during the growth stage using at least one heating stage, wherein the temperature of the nanoparticles according to the invention produced, preferably during or substantially during the growth stage, is first raised to a temperature T i , then preferably a heating time t between 1 second and 20 years hi The internal temperature is maintained at T i , where T i It is preferably 50°C to 700°C.

[0242] In one embodiment of the present invention, T i Greater than -273, -100, -50, 0, 1, 10, 20, 50, 100, 200, 500, 700, or 10 3 ℃.

[0243] In one embodiment of the present invention, T i Less than 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 0, -10 or -50℃.

[0244] In another embodiment of the present invention, T i In 10 and 10 5 Between, 50 and 10 4 Between, 100 and 10 3 between 150 and 700°C or between 200 and 500°C.

[0245] In one embodiment of the present invention, t hi Greater than 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 Second.

[0246] In another embodiment of the present invention, t hi Less than 10 100 , 10 50 , 10 10 , 10 5 , 10 3 , 10, 5, 2 or 1 second.

[0247] In another embodiment of the present invention, t hi In 10 -5 Between one second and one year, between one second and 20 years, between one second and one year, between one second and one month, between one second and one week, between one second and one day or between one second and one hour.

[0248] In one embodiment of the present invention, the temperature is preferably increased from an initial temperature, which preferably corresponds to the temperature at which the nanoparticles are injected into the device for heating the nanoparticles, to a temperature T i Time ratio t hi The time is short, preferably at least 1.1, 5, 10 or 10 3 times.

[0249] In another embodiment of the present invention, the temperature is raised to T i Time ratio t hi The time is long, preferably at least 1.1, 5, 10 or 10 3 times.

[0250] In one embodiment of the invention, iron oxide based nanoparticles are nanoparticles comprising preferably greater than 1%, 50%, 70%, 90% or 99% iron oxide by mass of iron oxide, preferably without taking into account the percentage of coating or excipient materials.

[0251] The present invention also relates to, preferably, high-purity nanoparticle producing cells obtained by the method according to the present invention, said high-purity nanoparticle producing cells comprising more than 10 -10 , 1, 5, 10, 50, 75, 80, 90, 95, 99 or 99.9% of:

[0252] i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the nanoparticles based on high-purity iron oxide, M MC is the mass of iron and metals or metalloids other than iron in the nanoparticles based on the purity of iron oxide.

[0253] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0254] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all chemical elements contained in the high purity iron oxide nanoparticles.

[0255] The present invention also relates to high-purity nanoparticle-producing cells or high-purity iron oxide-based nanoparticles obtained by expanding or growing nanoparticle-producing cells in a pure culture medium comprising less than 1, 10 -3 , 10 -6 or 10 -9 % of at least one heavy metal selected from the group consisting of cobalt, manganese, zinc, nickel, silver, aluminum, arsenic, barium, cadmium, chromium, copper, molybdenum (molybdate), lead, antimony, selenium, silicon (silicon dioxide), titanium, thallium, mercury, vanadium, gold, iridium, osmium, rhodium, ruthenium, platinum, lithium, antimony, tin, tungsten and their derivatives, wherein the percentage is based on the ratio C FeM / C MM , where C FeM is the iron concentration in pure culture, and C MM It is the concentration of iron and metals or metalloids other than iron in pure culture medium.

[0256] The present invention also relates to nanoparticles based on high-purity iron oxide, preferably obtained by the process of the present invention, said nanoparticles based on high-purity iron oxide comprising more than 10 -10 , 1, 5, 10, 50, 75, 80, 90, 93, 95, 99, or 99.9% of:

[0257] i) Iron, based on M FeN / M MN The ratio of M FeN is based on the mass of iron in the nanoparticles of high-purity iron oxide, and M MN is the mass of iron and metals or metalloids other than iron in the nanoparticles based on the purity of iron oxide.

[0258] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0259] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metals contained in the high purity iron oxide nanoparticles.

[0260] The present invention also relates to high-purity nanoparticle production cells according to the present invention and / or high-purity iron oxide-based nanoparticle production cells according to the present invention, wherein the metal or metalloid other than iron in the high-purity iron oxide nanoparticles and / or high-purity nanoparticle production cells is selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium and tungsten and their derivatives, at least 5 different metals or metalloids.

[0261] The present invention also relates to the high-purity iron oxide based nanoparticles according to the present invention, wherein the high-purity iron oxide based nanoparticles are magnetosomes.

[0262] The invention also relates to a composition comprising the high-purity iron oxide-based nanoparticles according to the invention.

[0263] The present invention also relates to high-purity nanoparticle-producing cells according to the present invention, also referred to as high-purity nanoparticle-producing cells, wherein the high-purity nanoparticle-producing cells are magnetotactic bacteria.

[0264] The present invention also relates to a composition comprising the highly pure nanoparticle-producing cells according to the present invention.

[0265] The present invention also relates to a composition comprising high-purity nanoparticle-producing cells and high-purity iron oxide nanoparticles, preferably obtained by the method according to the present invention, wherein:

[0266] High-purity nanoparticle production cells contain greater than 0, 1, 10, 50, 70, 90, 95, or 99% of:

[0267] i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the high-purity nanoparticle-producing cells, and M MC is the mass of iron and metals or metalloids other than iron in high-purity nanoparticle production cells,

[0268] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0269] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles,

[0270] as well as

[0271] Based on high purity iron oxide nanoparticles containing greater than 0, 1, 10, 50, 75, 93, 99 or 99.9% of:

[0272] i) Iron, based on M FeN / M MN The ratio of M FeN is the mass of iron in the high-purity iron oxide nanoparticles, and M MNis the mass of iron and metals or metalloids other than iron in high-purity iron oxide nanoparticles,

[0273] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0274] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles.

[0275] The present invention also relates to a composition comprising high-purity nanoparticle-producing cells and high-purity iron oxide nanoparticles, wherein:

[0276] High-purity nanoparticle production cells contain more than 0,10 -50 , 10 -10 , 10 -5 , 10 -2 , 1, 5, 10, 25, 50, 75, 90, 95, 99 or 99.9% of:

[0277] i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the high-purity nanoparticle-producing cells, and M MC is the mass of iron and metals or metalloids other than iron in high-purity nanoparticle production cells,

[0278] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0279] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles,

[0280] as well as

[0281] Based on high purity iron oxide nanoparticles containing more than 0,10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 0, 1, 5, 10, 25, 50, 75, 93, 95, 99, or 99.9% of:

[0282] i) Iron, based on M FeN / M MN The ratio of M FeN is the mass of iron in the high-purity iron oxide nanoparticles, and M MN is the mass of iron and metals or metalloids other than iron in high-purity iron oxide nanoparticles,

[0283] ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or

[0284] iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles,

[0285] The high purity nanoparticle producing cells and / or high purity iron oxide based nanoparticles are preferably obtained by culturing the nanoparticle producing cells in a growth medium that is substantially free of at least one metal or non-metal, or contains less than 10 M, or 1 M, or 10 -1 M, or 10 -3 M, or 10-6 M, or 1 nanomole of at least one metal or non-metal, wherein the at least one metal or non-metal is selected from the group consisting of: 1) cadmium, 2) lead, 3) arsenic, 4) mercury, 5) cobalt, 6) vanadium, 7) nickel, 8) lithium, 9) antimony, 10) copper, 11) V (Valadium), 12) molybdenum (molybdate), 13) selenium, 14) barium, 15) chromium, 16) strontium, 17) radioactive chemical elements, 18) beryllium, 19) rubidium, 20) ruthenium, 21) rhodium, 22) ) Palladium, 23) Promethium, 24) Ytterbium, 25) Tantalum, 26) Osmium, 27) Iridium, 28) Bismuth, 29) Polonium, 30) Francium, 31) Radium, 32) Actinium, 33) Thorium, 34) Protactinium, 35) Uranium, 36) Neptunium, 37) Plutonium, 38) Americium, 39) Curium, 40) Berkelium, 41) Californium, Einsteinium, 42) Fermium, 43) Mendelevium, 44) Nobelium, 45) Lawrencium, 46) Ruthenium, 47) Confederation, 48) Record, 49) Word, 50) Discussion, 51) 52) Darmstadtium, 53) Yuchenium, 54) Copper uranium, 55) Niobium, 56) Flerovium, 57) Molybdenum, 58) Livermorium, 59) Astatine, 60) Tennessine, 61) Oganesson, and 62) their derivatives.

[0286] In one embodiment of the present invention, high-purity nanoparticle-producing cells and / or high-purity iron oxide-based nanoparticles can be obtained by culturing and / or expanding nanoparticle-producing cells in:

[0287] a) pre-growth and / or growth medium, and / or

[0288] b) a culture medium that does not contain at least one metal or metalloid other than iron, or does not contain at least one metal or metalloid other than iron at a concentration that affects the growth of nanoparticle-producing cells, wherein the metal or metalloid other than iron is preferably selected from the group consisting of cobalt, manganese, zinc, nickel, silver, aluminum, arsenic, barium, cadmium, chromium, copper, molybdenum, lead, antimony, selenium, silicon, tantalum, thallium, mercury, vanadium, gold, iridium, osmium, rhodium, ruthenium, platinum, lithium, antimony, tin, tungsten and their derivatives.

[0289] The present invention also relates to a high-purity nanoparticle producing cell according to the present invention, and / or preferably to high-purity iron oxide-based nanoparticles obtained by a high-purity nanoparticle producing cell according to the present invention, wherein:

[0290] - High-purity nanoparticle-producing cells are magnetotactic bacteria, and / or

[0291] - High-purity iron oxide nanoparticles are magnetosomes.

[0292] The present invention also relates to a composition, a medical device, a medicament, a formulation, a suspension, a cosmetic composition, a botanical composition, a biological composition, a mineral composition and / or a nanoparticle composition comprising the high-purity nanoparticle-producing cells according to the present invention and / or the high-purity iron oxide nanoparticles according to the present invention.

[0293] The present invention also relates to, based on M FeC / M MC ratio, preferably comprising higher than 1%, 25%, 50%, 75%, 90% or 99% iron in a high purity nanoparticle production cell, wherein M FeC is the mass of iron in the high-purity nanoparticle-producing cells, M MC is the mass of iron and metals and metalloids other than iron in the high-purity nanoparticle production cells, and / or according to M FeN / M MN , preferably nanoparticles based on high purity iron oxide containing more than 1, 10, 50, 75, 93 or 99% iron, wherein M FeN is the mass of iron in high-purity iron oxide nanoparticles, M MN is the mass of iron and metals or metalloids other than iron in the high-purity iron oxide nanoparticles, wherein the high-purity nanoparticle-producing cells and / or the high-purity iron oxide-based nanoparticles are preferably obtained by culturing the nanoparticle-producing cells in a growth medium that is substantially free of metals selected from the group consisting of: cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper.

[0294] The term "substantially free" herein refers to substances that are not intentionally added to the culture medium but may be present in the culture medium as impurities.

[0295] In some cases, M FeC / M MC and / or M FeN / M MN It may be less than 100, 99, 95, 90, 50, 25, 10, 5 or 1%.

[0296] The present invention also relates to a method for preparing high-purity iron oxide nanoparticles. Preferably, the method according to the present invention uses nanoparticle production cells expanded in a pre-growth phase and a subsequent growth phase to produce high-purity iron oxide nanoparticles, wherein:

[0297] a) The pre-growth stage has at least one of the following characteristics:

[0298] a1) It comprises at least one sub-phase i, during which the nanoparticle-producing cells are grown in a volume V containing a pre-growth medium. PGSi Medium amplification,

[0299] a2)V PGSi The change between the start and end of subphase i is no greater than 1, 10, 50, 80, 90, or 99%;

[0300] a3) i is preferably comprised between 1 and 5, 1 and 10 or 1 and 1000;

[0301] a4)V PGSi+1 / V PGSi between 1.001 and 1000 or between 1.1 and 50;

[0302] a5) the duration of each subphase i is 1 second to 1 year, 1 minute to 1 month, 10 minutes to 1 week, or 60 minutes to 3 days;

[0303] a6) at least one subphase i lasts longer than 1 second or 1 hour;

[0304] a7) the temperature of the pre-growth medium of at least one sub-stage is 10-60°C, 20-50°C or 30-40°C;

[0305] a8) Concentration of oxygen or air or compressed air in the pre-growth medium:

[0306] - from: a8i) a partial pressure of oxygen, air or compressed air, preferably greater than 50 mbar, most preferably equal to 210 mbar, at the start of at least one sub-phase i, or a8ii) a percentage by volume of oxygen, air or compressed air, preferably equal to 25% to 100% relative to the maximum volume occupied by oxygen, air or compressed air in the saturated pre-growth medium, at the start of at least one sub-phase i, or a8iii) a volume of oxygen or air or compressed air, preferably between 5 and 25% relative to the volume of the pre-growth medium, at the start of at least one sub-phase i,

[0307] - decreases to: a8iv) a partial pressure of oxygen, air or compressed air, at the end of at least one sub-phase i, preferably less than 100 mbar, most preferably equal to 0 mbar, or a8v) a percentage by volume of oxygen, air or compressed air, relative to the maximum volume occupied by oxygen, air or compressed air in the saturated pre-growth medium, preferably equal to 0% to 50%, or a8vi) a volume of oxygen or air or compressed air, relative to the volume of the pre-growth medium, preferably equal to 0 to 10%, at the end of at least one sub-phase i;

[0308] a9) during at least one sub-phase i of the pre-growth phase, preferably for at least 0, 1, 5, 10 or 50% of the total duration of at least one sub-phase i, introducing a volume V PGSiThe amount of oxygen or air or compressed air is less than 100 litres, 1000 millilitres or 100 millilitres of oxygen or air or compressed air per minute;

[0309] a10) During all or part of subphase i, the speed is from 0 to 100 revolutions per minute or from 0 to 10 3 stirring the pre-growth medium at a rate of meters, wherein the rate is preferably the rate of at least one compound in the pre-growth medium, whether liquid, gaseous or solid;

[0310] a11) the pH of the pre-growth medium is not maintained at a fixed pH, preferably by not adding a fed-batch medium, preferably containing an iron source, or another medium, preferably containing an iron source in addition to said pre-growth medium, to the pre-growth medium;

[0311] a12) the pH of the pre-growth medium varies by more than 10 between the start and the end of at least one sub-phase i. -5 , 10 -1 , 0.5 or 1 pH unit, preferably from a minimum value of preferably less than 7 to a maximum value of preferably greater than 7;

[0312] a13) by adding to the pre-growth medium a mixture containing less than 10 5 , 10 3 , 10 2 , 10 or 2 μM iron or an iron source in a fed-batch medium, maintaining the pH of the pre-growth medium at 3 to 11 or 6 to 8;

[0313] a14) between the start and the end of at least one substage i, the total concentration of at least one of the iron, the iron source, the carbon, the carbon source, the nitrogen, and the nitrogen source in the pre-growth medium varies by less than 100, 50, 20, 10, 5, or 1%;

[0314] a15) The pre-growth medium contains iron or an iron source with a total concentration of less than 10 5 mM, 10 3 mM, 10 mM, 2 mM iron or iron source per liter of pre-growth medium or 10 5 , 10 3 , 10 2 , 50, 10, 5, 2, 1, 0.5 g iron or iron source per liter of pregrowth medium;

[0315] a16) The pre-growth medium contains iron or an iron source at a total concentration greater than 10 -50 M or 1 pM iron or iron source per liter of pre-growth medium or 0.4 ng iron or iron source per liter of pre-growth medium;

[0316] a17) the pre-growth medium comprises a total concentration of carbon or a carbon source of less than 2 M or 260 g of carbon or carbon source per liter of pre-growth medium;

[0317] a18) the pre-growth medium comprises a total concentration of carbon or a carbon source of greater than 0.1 nM or 0.1 ng of carbon or a carbon source per liter of pre-growth medium;

[0318] a19) the pre-growth medium comprises a total concentration of nitrogen or a nitrogen source of less than 740 mM or 40 g of nitrogen or a nitrogen source per liter of pre-growth medium;

[0319] a20) the pre-growth medium comprises nitrogen or a nitrogen source at a total concentration of greater than 0.1 nM or 0.1 ng nitrogen or a nitrogen source per liter of pre-growth medium;

[0320] a21) between the start and the end of at least one subphase i, the amount or concentration or percentage of carbon or a carbon source consumed by the nanoparticle-producing cells is greater than:

[0321] 10 -50 , 0.01, 1 or 10 g carbon or carbon source per liter of pre-growth medium, or 1 mM carbon or carbon source per liter of pre-growth medium, and / or

[0322] 10 -10 , 1, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Cf -Q Ci ) / Q Ci , where Q Cf and Q Ci are the amount of carbon contained in the pre-growth medium at the end and the beginning of at least one sub-phase i, respectively,

[0323] a22) between the start and the end of at least one subphase i, the amount or concentration or percentage of nitrogen or a nitrogen source consumed by the nanoparticle-producing cells is greater than:

[0324] 10 -50 , 0.001, 1 or 10 g nitrogen or nitrogen source per liter of pre-growth medium, or 0.1 mM nitrogen or nitrogen source per liter of pre-growth medium, and / or

[0325] 10 -10 , 1, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Nf -Q Ni ) / Q Ni , where Q Nf and Q Ni are the amount of nitrogen contained in the pregrowth medium at the end and the beginning of at least one subphase i, respectively,

[0326] a23) between the start and the end of at least one subphase i, the nanoparticle-producing cells consume an amount or concentration or percentage of iron or an iron source greater than:

[0327] 10 -10 , 0.0001, 1, 10, or 10 10 mg of iron or iron source per liter of pre-growth medium, or 0.5 μM of iron or iron source per liter of pre-growth medium, and / or

[0328] 10 -10 , 1, 20, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Fef -Q Fei ) / Q Fei , where Q Fef and Q Fei are the amount of iron contained in the pregrowth medium at the end and at the beginning of at least one subphase i, respectively;

[0329] a24) the amount of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source consumed by the nanoparticle-producing cells between the start and the end of at least one subphase i is less than the total concentration of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source in the pre-growth medium;

[0330] a25) the total concentration of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source in the pre-growth medium does not change by more than 1, 10, 20, 50, 80 or 99% between the start and the end of substage i;

[0331] a26) between the start and the end of subphase i, the concentration of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source consumed by the nanoparticle-producing cells increases by more than 1, 10, 20, 50, 80, 90 or 99%;

[0332] Wherein these conditions preferably result in nanoparticle-producing cells having at least one of the following properties:

[0333] a27) The nanoparticle-producing cells do not substantially produce nanoparticles, or the nanoparticle-producing cells produce less than 10 10 , 10 5 , 1, 10 -3 or 10 -6 mg of nanoparticles per liter of pregrowth medium, wherein this number is preferably the number of nanoparticles produced at the end of at least one subphase i, or the difference between the number of nanoparticles produced at the end of at least one subphase i and the number of nanoparticles produced at the beginning of at least one subphase i;

[0334] a28) preferably without concentration at the end of at least one partial stage i, preferably between 0 and 10 5The optical density produced by the nanoparticle producing cells is measured at nm, most preferably at 565 nm, and is characterized by at least one of the following properties:

[0335] a28i) during at least one sub-stage i of the pre-growth stage, which is 10 -20 and 10 20 or vary within an optical density range between 0.0001 and 40;

[0336] a28ii) between the start and end of at least one subphase i, it increases by a factor greater than 0, 0.5, 1, 1.1, 5, 10, 10 3 or 10 5 times, wherein the multiple is preferably the ratio between the optical density measured at the end of subphase i and the optical density measured at the beginning of subphase i;

[0337] a28iii) between the start and end of at least one sub-phase i, it increases by a factor of less than 10 10 , 10 5 , 2000, 10 3 , 10 2 , 10, 5, 2 or 1 times;

[0338] a28iv) at the end of at least one sub-phase i, it has less than 10 5 , 10 3 or a maximum value of 100;

[0339] a28v) at the beginning of at least one sub-phase i, which has a value greater than 0, 10 -50 , 10 -10 , 0.0001, 10 -3 or 10 -1 The minimum value of

[0340] a29) The nanoparticle producing cell is characterized in that, preferably during at least one subphase i of the pregrowth phase, the doubling time or the duration of the number multiplied by 2 is:

[0341] a29i) is greater than 10 -50 , 10 -5 , 1, 10, 10 2 or 10 3 minute;

[0342] a29ii) less than 10 3 , 10, 1 or 0.1 months; and / or

[0343] a29iii) 1 second to 1 month or 1 minute to 1 month;

[0344] b) a growth phase comprising expanding the nanoparticle-producing cells originating from and / or produced during the pre-growth phase in at least one growth phase, preferably in only one growth phase, most preferably the number of growth phases is smaller than the number of sub-phases i of the pre-growth phase, wherein at least one growth phase has at least one characteristic selected from the group consisting of:

[0345] b1) Nanoparticle production cells in a volume V containing growth medium GS mid-amplification;

[0346] b2)V GS is larger than the volume of at least one substage i of the pre-growth stage, preferably at least 0, 1, 1.1, 5, 10 or 10 3 times;

[0347] b3)V GS Equal to: V GS0 +V FB , where V GS0 is the volume containing the growth medium at the beginning of the growth phase, and V FB is the volume of fed-batch medium added to the growth medium during the growth phase;

[0348] b4)V GS The change between the beginning and end of at least one growth phase is greater than 10 -3 , 10 -1 , 1, 5, 10, 25, 50 or 75%, preferably by adding fed-batch medium to the growth medium during the growth phase;

[0349] b5) Volume V containing growth medium at the beginning of at least one growth phase GS0 Greater than the volume V of the fed-batch medium added to the growth medium during at least one growth phase FB , preferably at least 0, 1, 1.1, 1.5, 2, 5, 10 or 10 3 times;

[0350] b6) the duration of at least one growth phase is between 1 minute and 1 month, preferably between 40 hours and 15 days,

[0351] b7) the duration of at least one growth phase is greater than the duration of at least one sub-phase i of the pre-growth phase, preferably at least 0, 1, 1.1, 2, 5, 10 or 10 3 times,

[0352] b8) the temperature of the growth medium is 10-60°C, 20-50°C or 30-40°C;

[0353] b9) Concentration of oxygen or air or compressed air in the growth medium:

[0354] a9i) a partial pressure of oxygen, air or compressed air, preferably greater than 1 or 10 mbar, most preferably equal to 210 mbar, at the start of at least one growth phase, or a9ii) a percentage by volume of oxygen or air or compressed air, preferably equal to 10% to 100% relative to the maximum volume occupied by oxygen, air or compressed air in a saturated growth medium, at the start of at least one growth phase, or a9iii) a volume of oxygen or air or compressed air, preferably equal to 1 to 25% relative to the volume of the growth medium, at the start of at least one growth phase,

[0355] - decreases to: a9iv) a partial pressure of oxygen, air or compressed air of preferably less than 50 or 500 mbar, most preferably equal to 0 mbar, at the end of at least one growth phase, or a9v) a percentage by volume of oxygen, air or compressed air of preferably equal to 0% to 25% relative to the maximum volume occupied by oxygen, air or compressed air in a saturated growth medium at the end of at least one growth phase, or a9vi) a volume of oxygen or air or compressed air of preferably equal to 0 to 5% relative to the volume of the growth medium at the end of at least one growth phase.

[0356] b10) During the entire growth phase or a part of the growth phase, preferably for more than 1% of the total duration of the growth phase, a volume V is introduced GS The amount of oxygen, air or compressed air is greater than 1, 10 or 200 ml of oxygen, air or compressed air per minute;

[0357] b11) Introducing volume V at the end of the growth phase GS The amount of oxygen, air or compressed air is greater than at the beginning of the growth phase, preferably at least 0, 1, 1.1, 5, 10 or 10 3 times;

[0358] b12) During the entire growth phase or part of the growth phase, a volume V is introduced GS The amount of oxygen or air or compressed air increases by more than 10 per minute -10 , 10 -5 , 1, 10, or 10 5 mL oxygen or air or compressed air;

[0359] b13) during the entire growth phase or part of the growth phase, preferably at a speed (greater than that of the pre-growth medium) greater than 1, 10 or 100 revolutions per minute or 0 to 10 revolutions per minute 3agitating the growth medium at a rate of meters, wherein the rate is preferably the rate of at least one compound in the growth medium, whether liquid, gaseous or solid;

[0360] b14) the pH of the growth medium varies less than the pH of the pre-growth medium, or is maintained at a fixed pH between 1 and 14 or between 6.5 and 7.5, or is prevented from varying by more than 0.1, 0.5 or 10 pH units, preferably by adding a fed-batch medium or another medium in addition to the growth medium to the growth medium;

[0361] b15) The pH of the growth medium varies by less than 10, 0.5 or 0.1 pH units between the start and the end of the growth phase, preferably from a minimum value of less than 7.5 to a maximum value of preferably more than 6.5.

[0362] b16) the growth medium contains a total concentration of carbon, a carbon source, nitrogen, a nitrogen source, iron and / or an iron source that varies by more than 0.10% between the beginning and the end of at least one growth phase. -5 , 1 or 50%,

[0363] b17) The growth medium contains a total concentration of carbon or carbon sources greater than 10 -5 mM or 0.1mM or 10 -5 g or 0.01 g of carbon or carbon source per liter of growth medium;

[0364] b18) The growth medium contains a total concentration of carbon or carbon sources less than 10 3 M or 2M or 10 3 g or 180 g of carbon or carbon source per liter of growth medium;

[0365] b19) The growth medium contains nitrogen or nitrogen sources with a total concentration greater than 10 -50 mM or 0.01mM or 5·10 -10 g or 0.00005 g nitrogen or nitrogen source per liter of growth medium;

[0366] b20) The growth medium contains nitrogen or nitrogen sources with a total concentration less than 10 10 mM or 111mM or 10 5 g or 6 g of nitrogen or nitrogen source per liter of growth medium;

[0367] b21) The growth medium contains iron or an iron source with a total concentration greater than 10 -5 nM or 1nM or 10 -10 g or 3.10 -7 g iron or iron source per liter of growth medium;

[0368] b22) The growth medium contains iron or an iron source with a total concentration less than 10 5or 1mM or 10 5 g or 0.3 g of iron or iron source per liter of growth medium;

[0369] b23) between the beginning and the end of at least one growth phase, the nanoparticle-producing cells consume an amount, concentration, or percentage of carbon or a carbon source that is greater than:

[0370] 10 -10 , 10 -5 , 0.1, 1, 10, or 10 3 g carbon or carbon source per liter of growth medium, or 1 mM carbon or carbon or carbon source per liter of growth medium, and / or

[0371] 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Cf -Q Ci ) / Q Ci , where Q Cf and Q Ci are the amount of carbon contained in the growth medium at the end and the beginning of at least one growth phase, respectively,

[0372] b24) between the beginning and the end of at least one growth phase, the nanoparticle-producing cells consume nitrogen or a nitrogen source in an amount, concentration, or percentage greater than:

[0373] 10 -10 , 10 -5 , 0.01, 1 or 10 g nitrogen or nitrogen source per liter of growth medium, or 0.6 mM nitrogen or nitrogen source per liter of growth medium, and / or

[0374] 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Nf -Q Ni ) / Q Ni , where Q Nf and Q Ni are the amount of nitrogen contained in the growth medium at the end and the beginning, respectively, of at least one growth phase,

[0375] b25) between the beginning and the end of at least one growth phase, the nanoparticle-producing cells consume an amount, concentration, or percentage of iron or an iron source that is greater than:

[0376] 10 -10 , 10 -5, 0.01 or 1 mg iron or iron source per liter of growth medium, or 0.04 μM iron or iron source per liter of growth medium, and / or

[0377] 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 50 or 75%, wherein the percentage is preferably based on the ratio (Q Fef -Q Fei ) / Q Fei , where Q Fef and Q Fei is the amount of iron contained in the growth medium at the end and beginning of at least one growth phase, respectively;

[0378] b26) between the start and the end of at least one growth phase, the amount of iron or an iron source introduced into the growth medium, preferably via fed-batch medium, is greater than 10 -10 mg or 0.3 mg iron or iron source per liter of growth medium, or greater than 10 -3 μM or 1 μM iron or iron source per liter of growth medium;

[0379] b27) Between the start and the end of at least one growth phase, the amount of carbon or a carbon source introduced into the growth medium, preferably via fed-batch medium, is greater than 10 -50 , 10 -10 , 10 -5 or 0.07 g carbon or carbon source per liter of growth medium, or greater than 10 -10 , 10 -5 , 10 -3 , 0.8, 1, or 10 3 mM carbon or carbon source per liter of growth medium;

[0380] b28) Between the start and end of at least one growth phase, the amount of nitrogen or nitrogen source introduced into the growth medium, preferably via the fed-batch medium, is greater than 10 -10 , 0.006 or 1 g nitrogen or nitrogen source per liter of growth medium or 0.4 mM nitrogen or nitrogen source per liter of growth medium;

[0381] b29) between the start and the end of at least one growth phase, the amount of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source consumed by the nanoparticle-producing cells is less than the total concentration of carbon, carbon source, nitrogen, nitrogen source, iron and / or iron source in the growth medium;

[0382] Wherein these conditions preferably result in nanoparticle-producing cells having at least one of the following properties:

[0383] b30) Nanoparticle-producing cells produce nanoparticles, or nanoparticle-producing cells produce more than 10 nanoparticles-50 , 10 -10 , 0.01 or 1 mg per liter of growth medium, wherein the amount is preferably the amount of nanoparticles produced at the end of the growth phase, or the difference between the amount of nanoparticles produced at the end of the growth phase and the amount of nanoparticles produced at the beginning of the growth phase;

[0384] b31) preferably without concentration at the end of at least one growth phase, preferably between 0 and 10 4 The optical density produced by the nanoparticle-producing cells is measured between 100 nm and 200 nm, most preferably at 565 nm, and is characterized by at least one of the following properties:

[0385] b31i) The optical density at the end of the growth phase is greater than the optical density at the end of at least one sub-phase i of the pre-growth phase, preferably at least 0, 0.5, 1, 1.1, 1.5, 2, 5, 10 or 10 3 times;

[0386] b31ii) the optical density during the growth phase varies within the optical density range of 0.001 to 300;

[0387] b31iii) the optical density increases between the beginning and the end of at least one growth phase by a factor greater than 0, 0.5, 1, 1.1, 5, 10, or 10 3 times, wherein the multiple is preferably the ratio between the optical density measured at the end of at least one growth phase and the optical density measured at the beginning of at least one growth phase;

[0388] b31iv) the optical density increases by a factor of less than 10 between the start and end of at least one growth phase 10 , 10 4 or 10 times;

[0389] b31v) at the end of at least one growth phase, the optical density is less than 10 10 , 10 5 , 300 or a maximum of 10;

[0390] b31vi) at the beginning of at least one growth phase, the optical density is greater than 10 -50 , 10 -10 , 0.001 or a minimum value of 0.01; and / or

[0391] b32) Nanoparticle-producing cells characterized in that, preferably during the entire growth phase or a part of the entire growth phase, the doubling time or the duration of the value multiplied by 2 is:

[0392] b32i) greater than 1 minute;

[0393] b32ii) less than 1 month;

[0394] b32iii) 1 minute to 1 month; and / or

[0395] b32iv) having a doubling time lower than that of at least one substage i of the pregrowth stage, preferably by a factor of at least 1.1;

[0396] wherein preferably, the pre-growth, growth and / or fed-batch medium does not contain more than: i) 1, 2, 3 or 6 different vitamins, ii) 10 -9 mol at least one vitamin, iii) 10 -4 g of yeast extract, iv) 10 -9 mol of at least one yeast extract component, v) 1, 2, 5 or 10 yeast extract components, vi) 10 -5 g peptone, vii) 1 or 2 different CMR reagents, viii) 0.05 mg of at least one CMR reagent, ix) 1, 2 or 5 different chelating agents, x) 10 -8 mol of at least one chelating agent, xi) 1, 2 or 5 different amino acids, xii) 1 mg of at least one amino acid, xiii) 1, 2 or 5 different toxic or cytotoxic compounds, xiv) 1 mg of at least one toxic or cytotoxic compound, xv) 1, 3 or 7 different heavy metals other than iron, xvi) 1 mg of at least one heavy metal other than iron, xvii) more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 metals or chemical elements selected from cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper, xviii) 1 mg of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and / or copper, xix) 0.5 mL or 10 -8 mol Wolf vitamins, xx) 10 -9 mol of at least one ingredient of Wolff's vitamins, xxi) 1, 2, 5 or 10 different ingredients of Wolff's vitamins, xxii) 5 ml or 10 -8 mol Wolff minerals, xxiii) 10 -8 mol of at least one Wolff mineral component, xxiv) 1, 2, 5, 7, 10 or 15 different components of Wolff minerals, xxv) 10 -9 mol mineral elixir, xxvi)10 - 9 mol of at least one ingredient of a mineral elixir, and / or xxvii) 1, 2, 5, 10 or 14 different ingredients of a mineral elixir.

[0397] The present invention also relates to a method according to the invention, wherein the end of the pre-growth and / or growth phase is characterized by at least one of the following properties:

[0398] i) the optical density of the pre-growth and / or growth medium is saturated, or increases by less than 1 O.D. unit per hour during the pre-growth and / or growth phase;

[0399] ii) saturation of the pre-growth and / or growth medium with biomass, or addition of less than 1 g of nanoparticle-producing cells per hour during the pre-growth and / or growth phase;

[0400] iii) the nanoparticle-producing cells cease to produce nanoparticles, or produce less than 0.01 mg of nanoparticles per hour during the pre-growth and / or growth phases;

[0401] iv) fed-batch medium is no longer added to the pre-growth and / or growth medium;

[0402] v) oxygen is no longer sparged into the pre-growth and / or growth medium;

[0403] The present invention also relates to a method according to the invention, wherein the start of the pre-growth and / or growth phase is characterized by at least one of the following properties:

[0404] i) the optical density of the pre-growth and / or growth medium begins to increase, preferably by more than 0.1 OD units per day during the pre-growth and / or growth phase;

[0405] ii) the biomass of the pre-growth and / or growth medium begins to increase, preferably by more than 0.1 g of nanoparticle-producing cells per day during the pre-growth and / or growth phase;

[0406] iii) the nanoparticle-producing cells begin to produce nanoparticles, preferably greater than 0.01 mg of nanoparticles per day during the pre-growth and / or growth phase;

[0407] iv) fed-batch medium is initially added to the pre-growth and / or growth medium;

[0408] v) Oxygen sparging into the pre-growth and / or growth medium is initiated.

[0409] The present invention also relates to a method according to the invention, wherein the pre-growth, growth and / or fed-batch medium composition is defined hereinafter.

[0410] Preferably, the pre-growth medium comprises at least 1, 2, 3, 4, 5, 6 or 7 of the following chemical elements or derivatives, preferably the derivatives are or contain a carbon source, a nitrogen source, a phosphate (phosphorus) source, a magnesium source, a potassium source, a calcium source, a vitamin source and / or a chloride source: i) sodium lactate or a carbon source, preferably at a concentration of 10 -20or 0.0023 mol or g sodium lactate or carbon source per liter of pre-growth medium and 0.23 or 10 5 mol or g sodium lactate or carbon source per liter of pre-growth medium, the most preferred concentration is 0.023 ± 0.01 mol sodium lactate or carbon source per liter of pre-growth medium, ii) ammonium chloride or nitrogen source, preferably at a concentration of 10 -20 or 7.4·10 -4 mol or g ammonium chloride or nitrogen source per liter of pre-growth medium and 7.4·10 -2 or 10 5 mol or g ammonium chloride or nitrogen source per liter of pre-growth medium, the most preferred concentration is (7.5±1)·10 -3 mol ammonium chloride or nitrogen source per liter of pre-growth medium, iii) KH2PO4 or potassium source or phosphorus source, preferably at a concentration of 10 -20 or 1.55·10 -5 mol or g KH2PO4, potassium source or phosphorus source per liter of pre-growth medium and 1.55·10 -3 or 10 5 mol or g KH2PO4 or potassium source or phosphorus source per liter of pre-growth medium, the most preferred concentration is (1.5±1)·10 - 4 mol KH2PO4 or potassium source or phosphorus source per liter of pre-growth medium, iv) MgSO4 or magnesium source, preferably at a concentration of 10 -20 or 4.1·10 -5 mol or g MgSO4 or magnesium source per liter of pre-growth medium and 4.1·10 -3 or 10 5 mol or g MgSO4 or magnesium source per liter of pre-growth medium, the most preferred concentration is (4 ± 1) 10 -4 mol MgSO4 or magnesium source per liter of pre-growth medium, v) iron source or FeCl3, preferably at a concentration of 10 -20 or 2.10 -7 mol or g FeCl3 or iron source per liter of pre-growth medium and 10 -5 or 10 5 mol or g FeCl3 or iron source per liter of pre-growth medium, the most preferred concentration is (2±1)·10 -6 mol FeCl3 or iron source per liter of pre-growth medium, vi) thiamine or vitamins, preferably at a concentration of 10 -20 or 8.10 -9 mol or g thiamine or vitamin per liter of pre-growth medium and 8·10 -7 or 10 5 mol or g thiamine or vitamin per liter of pre-growth medium, the most preferred concentration is (8 ± 2) 10-8 mol thiamine or vitamin per liter of pre-growth medium, vii) CaCl2 or a calcium source or a chloride source, preferably at a concentration of 10 -20 or 10 -5 mol or g CaCl2 or calcium source or chloride source per liter of pre-growth medium and 10 -3 or 10 5 mol or g CaCl2 or calcium source or chloride source per liter of pre-growth medium, the optimal concentration is (1±0.8)·10 -4 mol CaCl2 or calcium source or chloride source per liter of pre-growth medium.

[0411] Preferably, the growth medium comprises at least 1, 2, 3, 4, 5, 6 or 7 of the following chemical elements or derivatives, preferably the derivatives are or contain a carbon source, a nitrogen source, a phosphorus source, a magnesium source, a potassium source, a calcium source, a vitamin source and / or a chloride source, preferably before the fed-batch medium is added to the growth medium: i) sodium lactate or a carbon source, preferably at a concentration of 10 -20 or 0.0014 mol or g sodium lactate or carbon source per liter of growth medium and 0.14 or 10 5 mol or g sodium lactate or carbon source per liter of growth medium, the most preferred concentration is (0.014 ± 0.01) mol sodium lactate or carbon source per liter of growth medium, ii) ammonium chloride or nitrogen source, preferably at a concentration of 10 -20 or 4.1·10 -4 mol or g ammonium chloride or nitrogen source per liter of growth medium and 4.1·10 -2 or 10 5 mol or g ammonium chloride or nitrogen source per liter of growth medium, the most preferred concentration is (4.1 ± 1) 10 -3 mol ammonium chloride or nitrogen source per liter of growth medium, iii) KH2PO4 or potassium source or phosphorus source, preferably at a concentration of 10 -20 or 1.55·10 -5 mol or g KH2PO4 or potassium source or phosphorus source per liter of growth medium and 1.55·10 -3 or 10 5 mol or g KH2PO4 or potassium source or phosphorus source per liter of growth medium, the most preferred concentration is (1.5±1)·10 -4 mol KH2PO4 per liter of growth medium, iv) MgSO4 or a magnesium source, preferably at a concentration of 10 -20 or 4.1·10 - 5 mol or g MgSO4 or magnesium source per liter of growth medium and 4.1·10 -3 or 10 5 MgSO4 mol or g magnesium source per liter of growth medium, the most preferred concentration is (4±1)·10-4 mol MgSO4 per liter of growth medium, v) FeCl3 or iron source, preferably at a concentration of 10 -20 or 10 -7 mol FeCl3 or iron source per liter of growth medium and 10 -5 or 10 5 mol FeCl3 or iron source per liter of growth medium, the most preferred concentration is (2±1)·10 -6 mol FeCl3 or iron source per liter of growth medium, vi) thiamine or vitamins, preferably at a concentration of 10 -20 or 8.10 -9 mol or g thiamine or vitamin per liter of growth medium and 8·10 -7 or 10 5 mol or g thiamine or vitamin per liter of growth medium, the most preferred concentration is (8 ± 2) 10 -8 mol thiamine or vitamin per liter of growth medium, vii) CaCl2 or a calcium source or a chloride source, preferably at a concentration of 10 -20 or 10 -5 mol or g CaCl2 or calcium source or chloride source per liter of growth medium and 10 -3 or 10 5 mol or g CaCl2 or calcium source or chloride source per liter of growth medium, the optimal concentration is (1±0.8)·10 -4 mol CaCl2 or calcium source or chloride source per liter of growth medium.

[0412] Preferably, the fed-batch medium comprises at least 1, 2, 3, 4, 5, 6 or 7 of the following chemical elements or derivatives, preferably the derivatives are or contain a carbon source, a nitrogen source, a phosphorus source, a magnesium source, a potassium source, a calcium source, a vitamin source and / or a chloride source, preferably before it is added to the growth medium: i) lactic acid or a carbon source, preferably at a concentration of 10 -20 or 10 -1 mol or g lactate or carbon source per liter of fed-batch medium and 10 or 10 5 mol or g lactate or carbon source per liter of fed-batch medium, the most preferred concentration is (1 ± 0.5) mol lactate or carbon source per liter of fed-batch medium, ii) ammonia or nitrogen source, preferably at a concentration of 10 -20 or 2.8·10 -2 mol or g ammonia or nitrogen source per liter of fed-batch medium and 2.8 or 10 5 mol or g ammonia or nitrogen source per liter of fed-batch culture medium, the most preferred concentration is (2.8 ± 1) 10 -1 mol ammonia or nitrogen source per liter of fed-batch culture medium, iii) KH2PO4 or potassium source or phosphorus source, preferably at a concentration of 10 -20 or 1.7·10-3 mol or g KH2PO4 or potassium source or phosphorus source per liter of fed-batch medium and 1.7·10 -1 or 10 5 mol or g KH2PO4 or potassium source or phosphorus source per liter of fed-batch culture medium, the most preferred concentration is (1.7±1)·10 - 2 mol KH2PO4 per liter of fed-batch medium, iv) MgSO4 or a magnesium source, preferably at a concentration of 10 -20 or 2.10 -4 mol or gMgSO4 or magnesium source per liter of fed-batch medium and 2·10 -2 or 10 5 mol or g MgSO4 or magnesium source per liter of fed-batch culture medium, the most preferred concentration is (2±1)·10 -3 mol MgSO4 or magnesium source per liter of fed-batch medium, v) FeCl3 or iron source, preferably at a concentration of 10 -20 or 10 -4 mol or g FeCl3 or iron source per liter of fed-batch medium and 10 -1 or 10 5 mol or g FeCl3 or iron source per liter of fed-batch culture medium, the most preferred concentration is (7±4)·10 -3 mol FeCl3 or iron source per liter of fed-batch medium, vi) thiamine or vitamins, preferably at a concentration of 10 -20 or 10 -8 mol or g thiamine or vitamin per liter of fed-batch medium and 10 -4 or 10 5 mol or g thiamine or vitamin per liter of fed-batch medium, the most preferred concentration is (2±1.5)·10 -6 mol thiamine or vitamin per liter of fed-batch medium, vii) CaCl2 or a calcium source or a chloride source, preferably at a concentration of 10 -20 or 10 -5 mol or g CaCl2 or calcium source or chloride source per liter of fed-batch medium and 10 -2 or 10 5 mol or g CaCl2 or calcium source or chloride source per liter of fed-batch culture medium, the optimal concentration is (1±0.8)·10 -3 mol CaCl2 or calcium source or chloride source per liter of fed-batch medium.

[0413] The present invention also relates to a method according to the invention, wherein the pre-growth, growth and / or fed-batch medium comprises at least one source selected from the group consisting of:

[0414] - a carbon source selected from the group consisting of: at least one compound comprising at least one carbon atom, lactic acid, sodium lactate, lactic acid, acetate, glycolate, glucose, pyruvate, succinate, carbon dioxide, glycerol, and combinations thereof, preferably at a concentration of 1 nM to 2 mol / L;

[0415] - an iron source selected from the group consisting of: at least one compound containing at least one iron atom, ferric citrate, ferric quinate, ferric chloride, ferric sulfate, FeCl3 and combinations thereof, preferably in a concentration of 1 nM to 2.10 -3 mol / L;

[0416] a nitrogen source selected from the group consisting of at least one compound containing at least one nitrogen atom, nitrates, nitrogen gas, ammonium, ammonia, ammonium salts, urea, amino acids, ammonia gas, and combinations thereof, preferably at a concentration of 1 nM to 4 mol / L;

[0417] - an oxygen source selected from the group consisting of: at least one compound containing at least one oxygen atom, oxygen, oxygen gas, air or compressed air, preferably in gaseous form, in some cases the oxygen source is bubbled or introduced into the growth medium at a gas rate preferably between 5 ml gas per minute and 50,000 ml gas per minute,

[0418] - a phosphate source consisting of at least one compound containing at least one phosphate, preferably in a concentration ranging from 1 nM to 2·10 -1 mol / L;

[0419] - a potassium source consisting of at least one compound containing at least one potassium atom, preferably in a concentration ranging from 1 nM to 2·10 -1 mol / L;

[0420] - a sulfate source or sulfur source consisting of at least one compound containing at least one sulfur atom or sulfate, preferably in a concentration ranging from 1 nM to 4·10 -1 mol / L;

[0421] - a manganese source consisting of at least one compound containing at least one manganese atom, preferably in a concentration ranging from 1 nM to 4·10 -1 mol / L;

[0422] - a vitamin source selected from the group consisting of: at least one compound containing at least one vitamin, biotin, calcium, pantothenate, folic acid, inositol, niacin, p-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine, thiamine hydrochloride and derivatives thereof and combinations thereof, preferably in a concentration of 1 nM to 10 -4 mol / L, and

[0423] - a calcium source consisting of at least one compound containing at least one calcium atom, preferably in a concentration ranging from 1 nM to 10 - 1 mol / L.

[0424] In one embodiment of the present invention, the sources of carbon, nitrogen, potassium, phosphorus, magnesium, calcium, vitamins, iron, oxygen and / or chlorine preferably include at least 1, 2, 5, 10 or 10 of these sources in their chemical formulas or molecules or components. 3 atoms of carbon, nitrogen, potassium, phosphorus, magnesium, calcium, vitamins, iron, oxygen and / or chlorine.

[0425] In another embodiment of the present invention, when compound or quantity or element or attribute P1 is higher, longer or greater than compound or quantity or element or attribute P2, this indicates that P1 = α·P2, where α is preferably a number or integer greater than 1, or P1 = α+P2, where α is preferably a number or integer greater than 0.

[0426] In another embodiment of the present invention, when compound or quantity or element or attribute P1 is lower, shorter or smaller than compound or quantity or element or attribute P2, this indicates that P1 = P2 / α, where α is preferably a number or integer greater than 1, or P1 = P2-α, where α is preferably a number or integer greater than 0.

[0427] In one embodiment of the present invention, a CMR compound is a compound that is carcinogenic, mutagenic, and / or genotoxic. In some cases, a carcinogenic compound is a compound that causes, induces, or is suspected of causing, inducing cancer, preferably in a living organism or human. In some cases, a mutagenic compound is a compound that causes, induces, or is suspected of causing, inducing, a mutation, alteration, or change in the number or size of at least one gene, DNA, RNA, DNA strand, RNA strand, and / or nucleic acid, preferably in a living organism or human. In some cases, a genotoxic compound is a compound that causes, induces, or is suspected of causing, inducing, reproductive organ, embryo, or fetal toxicity, mutation, alteration, or change, preferably in a living organism or human.

[0428] In one embodiment of the present invention, a toxic or cytotoxic compound is a compound that preferably causes or induces or is suspected of causing or inducing toxicity, death, weight loss, organ damage, behavioral changes, changes in food or water consumption, necrosis, apoptosis, cellular internalization, changes in cell number, shape and / or geometry in an individual or living organism.

[0429] In some cases, the compound is greater than 10 -6 , 10 -3 , 10 -1 , 1, 10, 10 3 or 106 At concentrations below μM, it is CMR or cytotoxic or toxic.

[0430] In some other cases, the compound is less than 10 20 , 10 6 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -6 At concentrations below μM, it is CMR or cytotoxic or toxic.

[0431] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-producing cells, comprising:

[0432] i) a pre-growth step comprising expanding the nanoparticle-producing cells in a pre-growth medium such that the nanoparticle-producing cells do not substantially produce nanoparticles, or

[0433] ii) a growth step comprising expanding the cells derived from the nanoparticle-producing step in a growth medium such that the nanoparticle-producing cells produce nanoparticles.

[0434] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-producing cells, comprising:

[0435] i) a pre-growth step comprising expanding the nanoparticle-producing cells in a pre-growth medium such that the nanoparticle-producing cells do not substantially produce nanoparticles, and / or

[0436] ii) a growth step comprising expanding the cells derived from the nanoparticle-producing step in a growth medium such that the nanoparticle-producing cells produce nanoparticles.

[0437] In one embodiment of the invention, the growth medium is supplemented by a fed-batch medium.

[0438] In one embodiment of the present invention, the growth medium supplemented by the fed-batch medium is the growth medium.

[0439] In one embodiment of the invention, the growth medium has at least one property in common with the pre-growth and / or fed-batch medium.

[0440] In one embodiment of the present invention, the growth medium and / or pre-growth medium comprises at least one source selected from the group consisting of: i) a carbon source or sodium or lactate, preferably sodium lactate, ii) an ammonium source, preferably ammonium chloride, iii) a magnesium source, preferably magnesium sulfate, iv) a potassium source, preferably potassium phosphate, v) a vitamin source, preferably thiamine, vi) a calcium source, preferably calcium chloride, and vii) an iron source, preferably ferric chloride. Each source in the growth and / or pre-growth medium is preferably present in a concentration of 10 -6 and 10 3 mM, 10 -3 and 100 mM, 0.01 and 10 mM, or 0.1 and 10 mM.

[0441] In one embodiment of the present invention, in the pre-growth medium and / or the growth medium, the concentration of the vitamin and / or calcium and / or iron source is at least 0, 0.5, 0.1, 1.1, 1.2, 1.5, 5, 10, 10 less than the concentration of the sodium source and / or ammonium source and / or magnesium source and / or potassium source. 3 or 10 5 times.

[0442] In one embodiment of the present invention, the fed-batch medium comprises at least one source selected from the group consisting of: i) a carbon source, preferably lactic acid, ii) ammonia, iii) a potassium source, preferably potassium phosphate, iv) a magnesium source, preferably magnesium sulfate, v) an iron source, preferably ferric chloride, vi) a vitamin source, preferably thiamine, vii) a calcium source, preferably calcium chloride, and vi) an iron source, preferably ferric chloride. Each source in the growth and / or pre-growth medium is preferably present at a concentration comprised between 0.001 and 100 mM, 0.01 and 10 mM, or 0.1 and 10 mM.

[0443] In one embodiment of the present invention, in the fed-batch culture medium, the concentration of the vitamin and / or calcium source is at least 0, 0.5, 1, 1, 1, 1, 2, 1, 5, 5, 10, 10 less than the concentration of the sodium source and / or ammonium source and / or magnesium source and / or potassium source and / or iron source. 3 or 10 5 times.

[0444] In one embodiment of the present invention, the nanoparticles described in the present invention are or contain more than 1, 2, 5, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 The components of the nanoparticles, nanoparticles per liter of growth medium or nanoparticles per nanoparticle-producing cell. In some cases, the iron oxide represents or is greater than 1, 10, 10 3 , 10 5, 10 10 , 10 20 , 10 50 or 10 100 Iron atoms and / or greater than 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 In some other cases, the chemical elements and / or impurities contained in the nanoparticles are or represent greater than 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 chemical elements, and / or impurities contained in the nanoparticles.

[0445] In another embodiment of the present invention, the nanoparticles according to the present invention are or contain less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 In some cases, the iron oxide may comprise less than 1, 10, 10, 5, or 2 nanoparticles per liter of growth medium or per nanoparticle-producing cell. 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 iron atoms and / or less than 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 In some other cases, the chemical element and / or impurity contained in the nanoparticle is or represents less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 5 or 2 chemical elements and / or impurities contained in the nanoparticles.

[0446] In one embodiment of the present invention, the nanoparticles contain at least one impurity.

[0447] In one embodiment of the present invention, the high purity iron oxide nanoparticles contain a small amount of impurities, for example when the method is capable of obtaining nanoparticles with a small amount of impurities. In some cases, the nanoparticles contain / do not contain at least one impurity or contain or contain or contain less than 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 5, 2, 5, 1, 10 -2 , 10 -10 , 10 -20 or 10 -50 Impurities or impurities per gram of nanoparticles, or grams of impurities per gram of nanoparticles. In some other cases, the percentage of impurities (preferably by mass) contained in or on the surface of the nanoparticles is less than 100, 90, 80, 70, 60, 50, 30, 20, 10, 5, 1, 0.1 or 0.001%. According to the present invention, in some cases, the impurity percentage can be defined as the ratio between the number of atoms, quantity, mass or volume of impurities contained in the nanoparticles divided by the total number of atoms, quantity, mass or volume of all chemical elements contained in the nanoparticles. In some cases, all chemical elements contained in the nanoparticles can be the sum of iron oxide, doping materials and impurities contained in the nanoparticles. In some other cases, the concentration of impurities contained in or on the surface of the nanoparticles is less than 10 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -50 micrograms of impurities per gram of nanoparticles.

[0448] In another embodiment of the present invention, the high purity iron oxide nanoparticles contain a significant amount of impurities, such as when the impurities are added or incorporated into the nanoparticles after the nanoparticles are prepared by the method. In some cases, the nanoparticles contain greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 1, 2, 5, 10, 10 3 , 10 5 , 10 10 , 10 20 or 10 50impurities or impurities per gram of nanoparticles or grams of impurities per gram of nanoparticles. In some cases, the nanoparticles contain a large amount of impurities. In some cases, the percentage of impurities contained in or on the surface of the nanoparticles (preferably by mass) is greater than 10 -40 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 25, 50, 75, 80 or 90%. In some other cases, the concentration of impurities contained in or on the surface of the nanoparticles is greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 or 10 10 micrograms of impurities per gram of nanoparticles.

[0449] In some cases, the impurities may be identical impurities, ie, impurities preferably comprising the same chemical element.

[0450] In some other cases, the impurities may be different impurities, ie, impurities preferably comprising at least one different chemical element.

[0451] In one embodiment of the present invention, the chemical element is selected from the group consisting of: actinides, actinium, aluminum, geranium, antimony, argon, arsenic, astatine, barium, berkelium, beryllium, bismuth, bismuth, boron, bromine, cesium, calcium, californium, carbon, cerium, chlorine, chromium, cobalt, cobalt, cadmium, copper, curium,

[0452] Darmburg, darmstadt, daptom, dysprosium, einsteinium, erbium, europium, cadmium, fluorine, francium, gadolinium, gallium, germanium, gold, hafnium, helium, tantalum, holmium, hydrogen, indium, iodine, iridium, iron, krypton, lanthanides, lanthanum, lawrencium, lead, lithium, lutetium, magnesium, manganese, deuterium, mendelevium, mercury, molybdenum, neodymium, neon, neptunium, nickel, niobium, nitrogen, niobium, osmium, oxygen, palladium, phosphorus, platinum, plutonium, polonium, potassium, praseodymium, protactinium, promethium, radium, radon, rhenium, rhodium, ergium, rubidium, ruthenium, ruthenium, samarium, selenium, silicon, silver, sodium, strontium, sulfur, scandium, selenium, tellurium, terbium, thorium, thulium, tin,

[0453] Tantalum, technetium, thallium, titanium, tungsten, Og, molybdenum, Ts (Tennessine), niobium, uranium, vanadium, xenon, ytterbium, yttrium, zinc, zirconium, and combinations of several of these chemical elements.

[0454] The invention also relates to a process according to the invention, wherein the impurity is at least one chemical element other than iron, oxygen and / or iron oxide.

[0455] The present invention also relates to a method according to the invention, wherein the impurity is preferably carbon or a carbonaceous material.

[0456] In one embodiment of the present invention, the carbonaceous material comprises at least one carbon atom, preferably but not necessarily mixed or assembled with other chemical elements other than carbon.

[0457] In yet another embodiment of the present invention, the carbon or carbon-containing material is derived from, produced by, or originates from cells that produce the nanoparticles.

[0458] The present invention also relates to a method according to the invention, wherein the nanoparticles obtained by the method comprise iron oxide, wherein the iron oxide has at least one of the following properties: i) it comprises at least one iron atom and one oxygen atom, ii) it forms a crystalline or mineral structure, iii) it may have the chemical formula FeO, FeO2, Fe3O4, Fe4O5, Fe5O6, Fe5O7, Fe 25 O 32 、Fe 13 O 19 , α-Fe2O3, β-Fe2O3, γ-Fe2O3, ε-Fe2O3, iv) it can be composed of wüstite, iron dioxide, magnetite, hematite, maghemite, v) it can be in ε phase, α phase, β phase, γ phase, vi) it can be in various oxidation levels, vii) it has the formula Fe α O β D γ , wherein α, β and / or γ are coefficients, preferably stoichiometric coefficients. In some cases, α, β and / or γ are equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 or 20. In other cases, α, β and / or γ are greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 or 20. In other cases, α, β and / or γ are less than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 or 20. In some other cases, D is the doping material of the nanoparticles. In some cases, the doping material can be selected from the group consisting of: aluminum, antimonite, barium, chromium, copper, gold, manganese, silver, tin, titanium, and zinc.

[0459] In one embodiment of the present invention, the iron oxide contained in the nanoparticles is the main chemical element of the nanoparticles. In some cases, the high purity iron oxide nanoparticles may contain a large amount of iron oxide. In some cases, the percentage of iron oxide contained in the nanoparticles (preferably by mass) is greater than 10 -40 , 10 -20 , 10-10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 25, 50, 75, 80, 90, 99 or 99.9%. According to the present invention, in some cases, the iron oxide percentage can be defined as the number of atoms, quantity, mass or volume in the iron oxide nanoparticles divided by the number of atoms, quantity, mass or volume of all chemical elements contained in the nanoparticles. In other cases, the concentration of iron oxide contained in the nanoparticles is greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 or 10 10 micrograms of iron oxide per gram of nanoparticles.

[0460] In one embodiment of the present invention, the high purity iron oxide nanoparticles contain a small amount of iron oxide, for example when the nanoparticles are processed and / or partially or completely destroyed and / or applied to an organism, or when this method cannot be achieved, a large amount of iron oxide is incorporated into the nanoparticles. In some cases, the percentage of iron oxide contained in or on the surface of the nanoparticles (preferably by mass) is less than 100%, 90%, 80%, 70%, 50%, 30%, 10%, 5%, 0.1% or 0.001%. In some other cases, the concentration of iron oxide contained in the nanoparticles can be less than 10 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -50 micrograms of iron oxide per gram of nanoparticles.

[0461] In another embodiment of the present invention, the percentage, concentration, atomic number, amount, mass or volume of iron oxide contained in the nanoparticles is greater than the percentage, concentration, atomic number, amount, mass or volume of the impurities contained in the nanoparticles, preferably 1.00001, 1.001, 1.1, 2, 5, 10, 50, 10 2 , 10 3 , 10 5 , 1010 , 10 20 or 10 50 times.

[0462] In one embodiment of the present invention, the iron oxide and / or impurities are contained or implanted: i) within the nanoparticles, ii) on the surface of the nanoparticles, iii) outside the nanoparticles, iv) in the crystalline or amorphous structure of the nanoparticles, v) in defects of the nanoparticles, and / or, vi) in vacancies of the nanoparticles.

[0463] In one embodiment of the invention, the iron oxide and / or impurities interact with the nanoparticles through, for example, electrostatic, strong, weak, nuclear, metallic, van der Waals, Debye, London or hydrogen bonding.

[0464] In one embodiment of the present invention, the iron oxide and / or impurities are located at a distance from the nanoparticles, preferably less than 10 50 , 10 20 , 10 10 , 10 5 , 10 3 In some cases, the center of a nanoparticle is an area or volume or location or assembly of chemical elements that is located in the middle of the maximum, minimum and / or average dimension of the nanoparticle, such as half the diameter of a spherical nanoparticle, or half the maximum, minimum and / or average length of a nanoparticle. In some other cases, the surface of a nanoparticle is an area or location or assembly of chemical elements that maintains a maximum distance from the center of the nanoparticle while remaining in the nanoparticle.

[0465] In another embodiment of the present invention, the iron oxide and / or impurities are located at a distance from the nanoparticles, preferably a distance greater than 0.001, 0.01, 0.1, 1, 10, 100, 10 3 , 10 5 , 10 10 , 10 20 or 10 50 nm.

[0466] In a further embodiment of the present invention, the nanoparticles according to the invention comprise a core and / or a coating, which preferably surrounds the core of the nanoparticle.

[0467] In one embodiment of the invention, the core and / or coating of the nanoparticles have at least one property in common with the nanoparticles, such as the concentration of iron oxide and / or impurities.

[0468] In one embodiment of the present invention, the nanoparticles, the core and / or the coating of the nanoparticles have at least one of the following properties:

[0469] (a) magnetic, diamagnetic, superparamagnetic, ferromagnetic, ferrimagnetic and / or paramagnetic behavior or properties, preferably greater than 10 -50 , 10 -40 , 10 -20 , 10 -10 , 10 -5 , 10 -2 or 10 -1 In the case of a magnetic field of 10 T, it is preferred to 10 , 10 5 , 10 3 , 10 2 The core and coating may have different magnetic properties. For example, the core may be ferromagnetic or superparamagnetic, while the coating may be diamagnetic or paramagnetic.

[0470] (b) a crystalline portion or structure comprising at least 1, 2, 5, 10, 50, 100, 10 3 , 10 5 , 10 7 , 10 9 , 10 20 or 10 50 The core may have a different crystal structure than the coating. For example, the core may contain more than 1, 5, 10, 10 3 or 10 5 crystal planes or crystal ordered structures, while the coating may have less than 10 5 ,10 3 ,10,5 or 2 crystal planes or crystal ordered structures.

[0471] (c) A composition of a metal or metal oxide, preferably iron oxide, most preferably a composition made of maghemite and / or magnetite. In some cases, the core comprises a different composition than the coating. For example, the core comprises greater than 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, or 99% by mass of iron oxide, while the coating comprises less than 99%, 95%, 90%, 75%, 50%, 10%, 5%, or 1% by mass of iron oxide. The percentage can be the ratio of the amount, volume, number of atoms, or mass of the iron oxide contained in the core and / or coating divided by the total amount, total volume, total number of atoms, or total mass of all chemical elements contained in the core and / or coating.

[0472] (d) Single domain, or magnetic single domain,

[0473] (e) Magnetic microstructures, characterized by the presence of magnetic field lines that can be oriented to a preferred direction, such as the easy axis of magnetization or the crystallographic direction of the nanoparticle core, for example

[111] , which can be observed under certain conditions, in particular by electron holography.

[0474] (f) Sizes between 1 nm and 10 5 Between 1 nm and 10 μm 3 Between μm, 1nm and 100μm, between 1nm and 10μm, between 1nm and 1μm, between 5nm and 1μm, between 5nm and 500nm, between 5nm and 250nm, between 5nm and 100nm, between 5nm and 80nm, between 5nm and 60nm, between 10nm and 1μm, between 10and 500nm, between 10nm and 250nm, between 10nm and 100nm, between 10nm and 80nm, between 10nm and 60nm, between 15nm and d Between 1μm, between 15nm and 500nm, between 15nm and 250nm, between 15nm and 100nm, between 15nm and 80nm, between 15nm and 60nm, between 20nm and 1μm, between 20nm and 500nm, between 20nm and 250nm, between 20nm and 100nm, between 20nm and 80nm, or between 20nm and 60nm.

[0475] (g) in some cases a size greater than 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35 or 40 nm,

[0476] (h) In certain other cases the size is less than 10 10 , 10 5 , 10 4 , 2000, 1000, 500, 400, 300, 200, 150, 120, 100, 95, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5nm,

[0477] (i) Zeta potential, charge or surface charge included in -10 10 mV and 10 10 mV, -10 5 mV and 10 5 mV, -10 4 mV and 10 4 mV, -10 3 mV, -10 2 mV and 10 2mV, -10mV and -10mV. Preferably, the pH value is between 0 and 14, between 1 and 13, between 2 and 12, between 3 and 11, between 4 and 10, between 5 and 9, or between 6 and 8.

[0478] (j) Zeta potential, electrical charge or surface charge, in some cases greater than -10 50 , -10 20 , -10 10 , -10 5 , -10 3 , -10, -5, -1, 0, 5, 10, 20, 50, or 100 mV, preferably at a pH greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0479] (k) Zeta potential, electrical charge or surface charge, greater than -10 in some other cases 50 , -10 20 , -10 10 , -10 5 , -10 3 , -10, -5, -1, 0, 5, 10, 20, 50 or 100 mV, preferably at a pH value below 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.

[0480] (l) Zeta potential, electrical charge or surface charge, less than 10 in some other cases 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10, 5, 1, 0, -5, -10, -20, -50 or 100 mV, preferably at a pH value greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0481] (m) Zeta potential, electrical charge or surface charge, less than 10 in some other cases 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10, 5, 1, 0, -5, -10, -20, -50 or -100 mV, preferably at a pH value below 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.

[0482] (n) an isoelectric point between 0 and 14, 1, 1 and 13, 2 and 12, 3 and 11, 4 and 10, 5 and 9, or between 6 and 8,

[0483] (o) in some cases, the isoelectric point is greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 in some other cases, and / or

[0484] (p) In certain other cases, the isoelectric point is less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.

[0485] In one embodiment of the invention, the core and / or coating are synthesized by the nanoparticle-producing cells.

[0486] In another embodiment of the invention, the core and / or coating are not synthesized by the nanoparticle-producing cells.

[0487] In one embodiment of the invention, the cells that generate nanoparticles (also referred to as cells or (one or more) cells that generate nanoparticles) are eukaryotic cells or prokaryotic cells. In some cases, they are cells generated by pre-growth and / or growth medium / culture medium or are contained therein or expanded therein.

[0488] In one embodiment of the invention, less than 100%, 80%, 70%, 50%, 10%, 20%, 10%, 5%, 2%, 1%, 0.1% or 10 -10 % of nanoparticle-producing cells contain or produce at least one nanoparticle. In some cases, the percentage can be the ratio between the number of cells contained in the pre-growth and / or growth medium / culture medium that contain or produce at least one nanoparticle divided by the total number of cells in the pre-growth and / or growth medium / culture medium.

[0489] In another embodiment of the invention, greater than 100, 80, 70, 50, 10, 20, 10, 5, 2, 1, 0.1 or 10-10% of the nanoparticle-producing cells contain or produce at least one nanoparticle.

[0490] In one embodiment of the invention, the cells producing the nanoparticles are whole cells.

[0491] In another embodiment of the present invention, the cell that produces the nanoparticles is a part of a cell, such as a cell membrane, vesicle, enzyme, protein, lipid, DNA, RNA, organelle, compartment, cytoplasm, virus, contained in the producing cell, or produced, originated, replicated by the producing cell.

[0492] In one embodiment of the invention, the producer cells are cells that produce nanoparticles, preferably when they are in growth and / or fed-batch medium, or when they grow or divide therein, avoiding when they are in pre-production medium, or when they divide therein, growing.

[0493] In one embodiment of the present invention, nanoparticles produced by cells are designated as cell-generated nanoparticles.

[0494] In one embodiment of the invention, the cell that produces the nanoparticles produces the nanoparticles intracellularly. The nanoparticles are preferably synthesized in the cell body when they are generated, assembled, or crystallized, partially or completely: i) by or in or near or within a part of the cell, such as an organelle, a Golgi vesicle or its apparatus, an endosome, an exosome, a ribosome, an endoplasmic reticulum, an actin filament, a nucleus, a peroxisome, a microtubule, a lysosome, a mitochondria, a filament, a centrosome, a flagellum, or a cell membrane, ii) located in an area within the cell, or iii) located less than 100 nm from a part of the cell. 5 ,10 3 ,100,10 or 1nm area.

[0495] In another embodiment of the present invention, the cell that produces the nanoparticles produces the nanoparticles extracellularly. The nanoparticles are preferably synthesized outside the cell when they are produced, assembled, or crystallized, partially or completely: i) in an area outside the cell, or ii) in a region greater than 1, 10 ... 3 or 10 5 m area.

[0496] In some cases, the cells are higher than 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 In some other cases, the cells are less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 5 or 2 cells, preferably per liter of growth medium.

[0497] In one embodiment of the present invention, the nanoparticle-producing cell is a eukaryotic cell, preferably belonging to a human, animal, plant, tree, flour, branch, mushroom, fungus, archaea, bird, fish, pigeon, trout, mammal, ant, bee or insect.

[0498] In one embodiment of the present invention, the nanoparticle-producing cells are prokaryotic cells or bacteria.

[0499] In some cases, the nanoparticle-producing cells can be mycobacteria, preferably Mycobacterium paratuberculosis, Shewanella, preferably Shewanella oneidensi, or Geothrix, preferably Geothrix fermentan. These bacteria preferably synthesize nanoparticles extracellularly.

[0500] In some other cases, the nanoparticle-producing cells can be magnetotactic bacteria, such as Magnetospirillum magneticum strain AMB-1, magnetotactic coccus strain MC-1, three facultative anaerobic Vibrio strains MV-1, MV-2 and MV-4, Magnetospirillum magnetotacticum strain MS-1, Magnetospirillum gryphiswaldense strain MSR-1, Magnetospirillum magneticum strain MGT-1 and the obligate anaerobic bacterium Desulfovibrio magneticus RS-1, which preferably produce nanoparticles intracellularly.

[0501] In one embodiment of the invention, the cells generating the nanoparticles are cultivated in a pre-growth medium or use a pre-growth medium during the pre-growth period, and / or use a growth medium during the growth period, and / or use a fed-batch medium during the growth period. In some cases, the pre-growth and / or growth medium is a medium in which the cells generating the nanoparticles expand. In some cases, a fed-batch medium is a medium that is added to the growth medium, preferably during the growth step.

[0502] In one embodiment of the present invention, the pre-growth and / or growth medium may comprise at least one chemical element, water, and a source of cells that produce nanoparticles. In some other cases, a portion of the pre-growth and / or growth medium / medium comprises at least one chemical element, a source of water, and no cells that produce nanoparticles. In other cases, the pre-growth and / or growth medium comprises only cells that produce nanoparticles.

[0503] In one embodiment of the invention, the pre-growth and / or growth and / or fed-batch medium comprises at least one source, preferably a source of a chemical element, or comprises at least one chemical element, preferably in a liquid, gaseous and / or solid state. In some cases, the pre-growth and / or growth and / or fed-batch medium is in a liquid, gaseous and / or solid state.

[0504] In one embodiment of the present invention, the concentration of a chemical element, such as iron, in the pre-growth and / or growth medium is the concentration of the chemical element in: i) the total pre-growth and / or growth medium, ii) a portion of the pre-growth and / or growth medium, or iii) the cells that produce the nanoparticles.

[0505] In one embodiment of the invention, an amount or volume of cells can be an amount or volume of growth medium containing the cells. In some other cases, an amount or volume of cells can be a number or volume of cells without water or in an anhydrous cell environment, or the aqueous environment of the cells has been removed, for example, by lyophilization.

[0506] In one embodiment of the invention, expanding the cells during the pre-growth and / or growth steps can prevent genetic variation in the cells that produce the nanoparticles. In some cases, the genetic variation in the cells that produce the nanoparticles is at least 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 50, 75, 90 or 95% of the genes, gene parts, DNA parts or nucleotides are mutated. This percentage can be the ratio between the number or amount of mutated genes, gene parts, DNA parts or nucleotides in the cell that produces the nanocellular particle and the total number or amount of mutated genes, gene parts, DNA parts or nucleotides in the cell that produces the nanocellular particle.

[0507] In another embodiment of the present invention, the pre-growth and / or growth medium comprises a majority of water, preferably purified water, deionized water or ultrapure water, preferably greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 1, 10, 50, 75, 80, 90, 99, 99.99 or 99.99999 percent or mass percentage. The percentage can be the ratio of the amount, mass, volume or number of atoms of water contained in the pre-growth and / or growth medium divided by the total amount, mass, volume or number of atoms of all chemical elements contained in the pre-growth and / or growth medium.

[0508] In one embodiment of the invention, the pre-growth and / or growth medium / medium comprises at least one chemical element or a source of a chemical element. In some cases, the concentration of a chemical element (e.g., iron) in the pre-growth and / or growth medium is the concentration of the chemical element at any time during the pre-growth and / or growth step. In some cases, this concentration can be measured by estimating the number of moles, mass, or volume occupied by the chemical element divided by the total number of moles, total mass, or total volume occupied by all chemical elements in the pre-growth and / or growth medium / medium.

[0509] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one carbon source. In some cases, the carbon source comprises a chemical element from the carbon column of the periodic table. In some cases, the carbon source can be selected from the following list: acetate, glycolate, glucose, lactate, pyruvate, succinate, carbon dioxide, glycerol, and derivatives or combinations of these compounds.

[0510] In one embodiment of the present invention, the growth and / or pre-growth medium comprises at least one nitrogen source. In some cases, the nitrogen source comprises a chemical element from the nitrogen column of the periodic table. In some cases, the nitrogen source can be selected from the group consisting of ammonium salts, nitrates, urea, amino acids, ammonium salts, ammonia, nitrogen gas, and derivatives or combinations of these compounds.

[0511] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one source of sulfur or sulfate. In some cases, the source of sulfur or sulfate comprises a chemical element that is in the same column of the periodic table as sulfur. In some cases, the source of sulfur or sulfate can be sulfate or hydrogen sulfide.

[0512] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one phosphorus or phosphate source. In some cases, the phosphorus or phosphate source comprises a chemical element that is in the phosphorus column of the periodic table. In some cases, the phosphorus or phosphate source can be a phosphate.

[0513] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one calcium source. In some cases, the calcium source comprises a chemical element that is in the same column of the periodic table as calcium. In some cases, the calcium source can be a calcium salt.

[0514] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one source of potassium. In some cases, the source of potassium comprises a chemical element from the potassium column of the periodic table. In some cases, the source of potassium is a potassium salt.

[0515] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one source of magnesium. In some cases, the source of magnesium comprises a chemical element that is a member of the magnesium column of the periodic table. In some cases, the source of magnesium is a magnesium salt.

[0516] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one oxygen source. In some cases, the oxygen source comprises a chemical element from the oxygen column of the periodic table. In some cases, the oxygen source is an organic compound, carbon dioxide, or dioxygen.

[0517] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one hydrogen source. In some cases, the hydrogen source comprises a chemical element in the periodic table of elements corresponding to hydrogen. In some cases, the hydrogen source is an organic compound or dihydrogen.

[0518] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one iron source. In some cases, the iron source comprises a chemical element from the iron column of the periodic table. In some cases, the iron source consists of or comprises iron. In some cases, the iron source is ferric citrate, ferric quinolate, ferric chloride, or ferric sulfate.

[0519] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one sulfur source. In some cases, the sulfur source comprises a chemical element in the sulfur column of the periodic table. In some cases, the sulfur source is contained in at least one vitamin.

[0520] In one embodiment of the present invention, the source of carbon, nitrogen, sulfur, sulfate phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen or iron contains more than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 25, 50, 75, 80, 90 or 95 mass percent of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen or iron. In some cases, they are in gaseous, liquid or solid state. In some other cases, they can be used to prepare pre-growth and / or growth medium. In some cases, the pre-growth and / or growth medium contains more than 2, 3, 4, 5, 10, 50, 100, 10 3 , 10 5 , 10 10 or 10 50In some other cases, the pre-growth and / or growth medium comprises less than 2, 3, 4, 5, 10, 50, 100, 10 3 , 10 5 , 10 10 or 10 50 Carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron from various sources.

[0521] In one embodiment of the invention, the source of at least one of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron in the pre-growth medium is the same as that in the growth medium.

[0522] In another embodiment of the invention, the source of at least one of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron in the pre-growth medium is different from that in the growth medium.

[0523] In another embodiment of the present invention, the pre-growth and / or growth medium contains a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron at a concentration greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 mM.

[0524] In another embodiment of the present invention, the pre-growth and / or growth medium contains a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron at a concentration of less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 , 10 -6 , 10 -9 , 10 -20 , 10 -50 or 10 -100 mM.

[0525] In another embodiment of the present invention, the pre-growth and / or growth medium is prepared using pharmaceutical grade or ultrapure chemicals or chemical elements.

[0526] In another embodiment of the present invention, culture medium impurities are impurities contained in the pre-growth and / or growth and / or fed-batch culture medium.

[0527] In one embodiment of the present invention, the pre-growth and / or growth medium contains a small amount of medium impurities. In some cases, the percentage content of medium impurities is less than 100%, 10 20 , 10 10 , 10 5 , 10 2 Preferably, the amount or concentration of medium impurities contained in the pre-growth and / or growth medium is at least 1.00001, 1.1, 1.5, 2, 5, 10, 10 less than the amount or concentration of at least one source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron. 3 , 10 10 or 10 20 times. According to the present invention, in some cases, the percentage of medium impurities can be defined as the ratio between the number of atoms, quantity, mass or volume of medium impurities contained in the pre-growth and / or growth medium divided by the total number of atoms, quantity, mass or volume of all chemical elements contained in the pre-growth and / or growth medium. In some other cases, the concentration of medium impurities contained in the pre-growth and / or growth medium is less than 10 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -50 micrograms per milliliter of pre-growth and / or growth medium.

[0528] In yet another embodiment of the present invention, the pre-growth and / or growth medium / medium comprises at least one medium impurity.

[0529] In one embodiment of the present invention, the pre-growth and / or growth medium contains a significant amount of medium impurities. In some cases, the percentage of medium impurities (preferably by mass) is greater than 10 -40 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10-1 , 1, 5, 10, 25, 50, 75, 80, or 90%. In some other cases, the concentration of medium impurities contained in the pre-growth and / or growth medium is greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 , or 10 10 micrograms per milliliter of pre-growth and / or growth medium.

[0530] In some cases, the nanoparticles produced or obtained or retrieved from the pre-growth and / or growth medium / culture medium may be magnetosomes.

[0531] In one embodiment of the present invention, the method according to the present invention comprises a pre-growth step comprising expanding the nanoparticle-producing cells in a pre-growth medium such that the nanoparticle-producing cells do not substantially produce nanoparticles.

[0532] In one embodiment of the present invention, the cells used to produce nanoparticles in the pre-growth step are cells having at least one of the following properties: i) they are cells before the pre-growth step, preferably, they are higher than 0.001, 0.1, 1, 5, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 210, 220, 230, 240, 360, 370, 380, 400, 500, 600, 700, 800, 900, 1000, 1200, 2400, 3600, 4000, 5000, 6000, 7000, 8000, 9000, 11000, 12000, 24000, 24000, 36000, 38000, 36000, 38000 3 , 10 5 or 10 10 hours, ii) they are contained in a cell bank, such as a master cell bank, a working cell bank or a research cell bank, iii) they include more than 1, 5, 10, 10 3 ,10 5 or 10 10 nanoparticles per cell, iv) they are contained in a liquid or culture medium, preferably of the same or similar composition as the pre-growth and / or growth medium, preferably containing a large amount of water, v) they are contained in a medium with an impurity concentration of less than 100, 10, 1, 10 -1 , 10 -2 , 10 -3 , 10 -5 or 10 -10 vi) they are contained in a culture medium capable of maintaining or having less than 100, 10, 1, 0.1 or 0.01 grams of impurities per gram of nanoparticles or are maintained under such conditions, v) they are included in 10 -100 and 10 100 ,10 -50and 10 50 ,10 -30 and 10 30 ,10 -20 and 10 20 ,10 -10 and 1010,10 -6 and 10 5 ,10 -6 and 10 4 ,10 -6 and 10 2 , or 10 -6 and 1 liter, vi) their volume being at least 10 times smaller than the volume of the first pregrowth step, vii) their number of cells, preferably comprising between 1 and 10 cells per liter of pregrowth and / or growth medium. 100 , 2 to 10 50 , 3 to 10 20 , or between 10 and 10 10 cells, vii), whose optical density is between 10 -50 to 10 50 , 10 -20 to 10 20 , 10 -10 to 10, 10 -5 to 10 5 , 10 -5 to 10 3 , 10 -5 to 10 2 , 10 -5 to 1, 10 -5 to 10 -1 , 10 -5 to 10 -2 , or 10 -5 to 10 -3 viii), they have a certain number of cell divisions, preferably less than 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 times per hour or per liter of pre-growth and / or growth medium, ix) they are stored or maintained at a temperature below 100, 50, 25 or 0°C, preferably 77K or -20°C.

[0533] In one embodiment of the present invention, the nanoparticle-producing cells used to initiate the pre-growth step have at least one of the following properties: i) they contain impurities in the culture medium at a concentration greater than 10 -50 , 10 -20 , 10 -10 , 10-5 , 10 -2 , 10 -1 , 1 or 10 μM in a culture medium, ii) which contains a medium capable of maintaining or having a concentration greater than 10 -40 , 10 -20 or 10 -10 gram of impurities per gram of nanoparticles in the culture medium or are maintained under such conditions, iii) they have a certain number of cell divisions, preferably greater than 1, 10, 10 per hour or per liter of pre-growth and / or growth medium. 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 iv) They contain less than 1,5,10,10 3 ,10 5 or 10 10 This can occur when / if the cells are maintained in or derived from a culture medium containing a sufficiently low iron concentration to prevent the production of nanoparticles.

[0534] In one embodiment of the invention, at least one property of the nanoparticle-producing cells used to initiate the pre-growth step is capable of preventing the death or destruction or disappearance or denaturation or inactivation of the nanoparticle-producing cells.

[0535] In one embodiment of the invention, the optical density is measured in pre-growth and / or growth medium, solution, water, preferably the growth medium is removed and the cells are redissolved in water. In some cases, the optical density of the cells is greater than 1, 2, 5, 10, 50, 100, 200, 300, 400, 450, 500, 550, 600, 900, 10 3 , 10 5 or 10 7 In some other cases, the optical density of cells is less than 10 7 , 10 5 , 10 3 , 900, 600, 550, 500, 450, 400, 300, 200, 100, 50, 10, 5, 2, or 1 nm. In some other cases, the optical density of the cells is between 1 and 10 7 nm, 50 to 10 5 nm, 100 to 10 3 nm, 200 to 900 nm, or 400 to 800 nm.

[0536] In one embodiment of the present invention, the number of cell amplifications between two time points t0 and t1 in the pre-growth and / or growth steps is equal to or proportional to: i) the ratio between the optical density measured at t1 and at t0 and / or ii) the ratio between the number of cells n(t1) at t1 and the number of cells n(t0) at t0.

[0537] In one embodiment of the present invention, the speed or rate of cell division is [n(t1) - n(t0)] / (t1 - t0).

[0538] In another embodiment, the speed or rate of cell division is: [n(t1) - n(t0)] / (t1 - t0), where V is the volume of the pre-growth and / or growth medium in which the cells are cultured or amplified.

[0539] In one embodiment of the present invention, the pre-growth step starts by thawing or heating, preferably from a temperature below 100, 50, 25, 10 or 0 °C to a temperature above 0, 10, 25, 50 or 100 °C, for the cells that generate the nanoparticles to start the pre-growth step. After that, the cells that generate the nanoparticles are filled or added to the pre-growth medium. In some cases, the initial stage of the pre-growth step occurs between 10 -50 to 10 50 、10 -50 to 10 10 、10 -30 to 10 5 、10 -20 to 10 3 、10 -10 to 10 2 、or 10 -5 to 10 hours.

[0540] In another embodiment of the present invention, the pre-growth step is divided into sub-steps 0, 1,..., i,..., j, corresponding to amplification in different, preferably increasing, volumes, volumes V0, V1,..., V i ,…,,V j 、where i is an integer representing the i-th amplification in different volumes (0 < i < j), j is an integer representing the total number of amplifications in different volumes, V0, Vi and Vj are the initial value, i th and the final amplification volume. In some cases, the cell amplification or the number of cells amplified in different volumes during the pre-growth step may be very high, for example when the pre-growth step starts with a small number of cells, preferably less than 10 100 、10 50 、10 20 、10 10 、10 5 、103 , 10 2 , 10, 5, 3 or 2 cells, preferably contained in 1 liter or 1 milliliter or 1 microliter of pre-growth medium or aqueous solution. In these cases, i and / or j can be greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 10, 10 3 , 10 5 or 10 10 In other cases, the number of cells expanded or expanded in different volumes may be low, for example when the pre-growth step starts with a large number of cells, preferably greater than 3, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 cells, preferably contained in 1 liter or 1 milliliter or 1 microliter of pre-growth medium or aqueous solution. In these cases, i and / or j are less than 10 10 , 10 5 , 10 3 , 10 2 , 10, 5, 4, 3, 2 or 1.

[0541] In one embodiment of the present invention, the ratio Vi / V i-1 Large, preferably greater than 10 -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 1.00001, 1.0001, 1.001, 1.01, 1.1, 1.2, 1.5, 2, 3, 4, 5, 7, 10, 10 2 , 10 3 , 10 5 or 10 10 In some cases, Vi / V i-1 When the number of cell divisions between sub-step i-1 of the pre-growth step and its sub-step i (preferably per hour or per hour per liter of pre-growth medium) is greater than 1, 5, 10, 10 3 , 10 10 or 10 20 When the optical density of cells increases by more than 1.00001, 1.1, 2, 5, 10, 10 3 , 10 5 or 10 7 Times.

[0542] In another embodiment of the present invention, the ratio Vi / V i-1 is low, preferably less than 10 100 、10 50 、10 10 、10 5 、10 3 、10 2 、10、5、3、2、1.01、1.001、1.000001、1、10 -5 、10 -10 or 10 -50 。In some cases, Vi / V i-1 is small when the number of cell divisions (preferably per hour or per liter of pre-growth medium per hour) is less than 10 50 、10 10 、10 3 、10 2 、10、5 or when the optical density of the cells increases by less than 1.00001、1.1、2、5、10、10 3 、10 5 or 10 7 times per hour.

[0543] In some cases, the number of pre-growth steps in different volumes can be increased by reducing Vi / Vi-1. In some other cases, the number of pre-growth steps in different volumes can be reduced by increasing Vi / Vi-1.

[0544] In one embodiment of the present invention, the pre-growth step and / or at least one of its sub-steps continue and / or occur until the optical density of the bacterial suspension in volume Vi (1 < i < j) reaches a value of: i) higher than 10 -50 、10 -30 、10 -20 、10 -10 、10 -5 、10 -3 、10 -1 、1、5、10、15、50、10 2 、10<^ 3 or 10 5 and / or ii) greater than and preferably greater than 1.01、1.1、1.5、2、5、10、10 2 、10 3 、10 5 、10 10 or 10 20 times, at the end of the pre-growth step compared to the start of the pre-growth step and / or at the end of the pre-growth step compared to at least one sub-step in the pre-growth step.

[0545] In one embodiment of the present invention, the pre-growth step and / or at least one of its sub-steps continue and / or occur until the optical density value of the cell suspension in volume Vi (1 < i < j) reaches: i) less than 10 -50 、10 -30 、10 -20 、10 -10 、10 -5 、10 -3 、10 -1 、1、5、10、15、50、10 2 、10 3 或10 5 , and / or ii) less than and preferably less than 1.000001, 1.0001, 1.01, 1.1, 1.5, 2, 5, 10, 10 2 、10 3 、10 5 、10 10 或10 20 times, at the end of the pre-growth step compared to the start of the pre-growth step and / or at the end of the pre-growth step compared to at least one sub-step during the pre-growth step.

[0546] In one embodiment of the present invention, the amplification in volume V0 starts to occur at time point t PG0b and the amplification in volume V0 ends at time point t PG0e The amplification in volume V i starts to occur at time point t PGib and the amplification in volume Vi ends at time point t PGie The amplification in volume V j starts to occur at time point t jb and / or the amplification in volume V<( j ends at time point t PGje time point.

[0547] In one embodiment of the present invention, the time length: i) separates the start of the pre-growth step, at time point t PG0b and the end of the pre-growth step, at time point t PGje equals t PGje -t PG0b and / or ii) separates the start of sub-step i, t PGib and the end of sub-step i, t PGie equals t<000(181)6> -t PGib , greater than or equal to 10 -20 、10 -10 、10 -5 、10 -3 、10 [[ID=7(9]] -2 、10 It should be noted that there seems to be a small formatting issue in the original text where the tag <( j is likely incorrect and should probably be j . Also, <000(181)6> might be a formatting error and should be PGie . The translation has been done as accurately as possible based on the provided text.-1 , 1, 2, 5, 10, 24, 100, 10 3 , 10 5 or 10 7 hours. In some cases, t PGje -t PG0 and / or t PGie -t PGib When cells are difficult or slow to divide, it is preferable to use less than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 The rate or rate of cell division is the number of cells dividing per hour or the number of cells dividing per hour per liter of pre-growth medium.

[0548] In another embodiment of the present invention, t PGje -t PG0b and / or t PGie -t PGib Less than or equal to 10 40 , 10 30 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 5, 2, 1, 10 -1 , 10 -3 , 10 -5 or 10 -10 In some cases, when cells divide easily or rapidly, t PGje -t PG0b and / or t PGie -t PGib is low, preferably greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 The rate or rate of cell divisions is the number of cell divisions per hour or the number of cell divisions per hour per liter of pre-growth medium.

[0549] In one embodiment of the present invention, the expansion of the nanoparticle-producing cells in the pre-growth step is carried out by PGib or t PG0bThe iron content of the pre-growth medium is maintained below 10% during the pre-growth step or at least one sub-step thereof. 100 , 10 20 , 10 5 , 10 3 , 10, 5, 1, 10 -1 , 10 -3 or 10 -5 μM is achieved, preferably avoiding the synthesis of nanoparticles that could prevent cell proliferation.

[0550] In another embodiment of the present invention, the nanoparticle-producing cells are expanded in the pre-growth step by PGib or t PG0b The iron content of the pre-growth medium is maintained at a concentration greater than 10% during the pre-growth step or at least one sub-step thereof. 100 , 10 20 , 10 5 , 10 3 , 10, 5, 1, 10 -1 , 10 -3 or 10 -5 μM is achieved, preferably to achieve efficient cellular metabolism.

[0551] In another embodiment of the present invention, the expansion of the nanoparticle-producing cells in the pre-growth step is carried out by PGib or t PG0b or by maintaining during the pre-growth step or in at least one sub-step thereof an iron concentration in the pre-growth medium between 10 -10 and 10 10 , 10 -5 and 10 5 , 10 -3 and 10 3 , 10 -1 and 1 μM, 10 -1 and 10 μM, or 10 -2 and 100 μM.

[0552] In another embodiment of the present invention, the nanoparticle-producing cells are expanded in the pre-growth step or at least one sub-step thereof by consuming oxygen. In some cases, the percentage of oxygen in the pre-growth growth medium is reduced from the following values: i) above 10 at tPGib or tPG0b; -50 , 10 -10 , 10 -5 , 10 -3, 1, 5, 10, 20, 50, 75, 90, 95, 99 or 99.9%, preferably starting from 21% or a value between 10% and 30%, at t PGie or t PGje , lower than 99.9, 95, 90, 80, 75, 50, 20, 5, 2, 1, 10 -3 , 10 -5 , 10 -10 or 10 -50 %, preferably 0% or a value between 10% and 30%, and / or ii) the oxygen percentage in the pre-growth medium is reduced to above 1.0001, 1.001, 1.1, 1.2, 1.5, 2, 5, 10, 50, 10 2 , 10 3 , 10 5 or 10 10 times, preferably t PG0b and t PGje between and / or t PGib and t PGie In some cases, oxygen is not added to the pre-growth medium during the pre-growth step or at least a sub-step thereof, resulting in a decrease in the oxygen percentage in the pre-growth medium due to consumption of oxygen by the bacteria. In some other cases, oxygen is added to the pre-growth medium during the pre-growth step or at least a sub-step thereof, resulting in a change in the oxygen percentage due to consumption of oxygen by the bacteria and addition of oxygen to the pre-growth medium.

[0553] In one embodiment of the present invention, the percentage of oxygen, preferably the percentage of dissolved oxygen in the pre-growth and / or growth medium, preferably oxygen in the pre-growth and / or growth medium. In some cases, a percentage of 100% may correspond to the maximum amount of dissolved oxygen in the pre-growth and / or growth medium, contained in 10 -5 and 10 20 mg of dissolved oxygen per liter of pre-growth and / or growth medium, preferably containing 1 and 10 mg of dissolved oxygen per liter.

[0554] In one embodiment of the invention, the pregrowth step comprises expanding the cells, corresponding to, or being: i) the speed or rate or number of cell divisions, preferably per unit volume, e.g. 1 liter of pregrowth medium, preferably at the beginning of the pregrowth step, at the end of one of the pregrowth step substeps, or at t PGie or tPGje Time than at t PGib or t PG0b When greater than 1.000001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 2 , 10 3, 10 5 , 10 10 , 10 20 or 10 50 10 times, ii) the speed or rate or number of cell division, preferably the number of cells per unit volume, e.g., 1 liter of pre-growth medium, or the number of cells per hour from less than or equal to 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 5 or 2 at the beginning of pregrowth or at the beginning of one of the sub-steps or at t PGib Time or t PG0b Increase to greater than or equal to 2, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 At the end of pregrowth or at the end of one of the sub-steps or t PGie or tPGje iii) optical density, preferably measured for cells contained in a fixed pre-growth volume (e.g. 1 liter), preferably 1.00001, 1.1, 1.2, 1.5, 2, 5, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 15000, 30000, 30000, 40000, 50000, 60000, 70000, 3 , 10 5 , 10 10 , 10 20 or 10 50 Multiples, or optical density from less than or equal to 10, 1, 10 -1 , 10 -2 or 10 -3 Increase to an optical density greater than or equal to 10 at the beginning of the pregrowth step or one of its substeps or at tPGib or tPG0b -10 , 10 -2 , 10 -1 , 1 or 10 at the end of the pregrowth step or one of its substeps or at t PGie or t PGje Place.

[0555] In one embodiment of the present invention, preferably during, at the beginning or at the end of the pre-growth step or at least one sub-step thereof, the cells that do not substantially produce nanoparticles have or are characterized by at least one of the following properties: i) the number of nanoparticles contained in the cells is less than 103 ; 10 2 , 50, 20, 10, 5, 2 or 1, preferably less than 10 or 5, or between 0 and 10 3 10 %, preferably between 0 and 10 or 0 and 5, ii) a percentage of cells having at least one nanoparticle is less than 100%, 99%, 90%, 80%, 50%, 20%, 10%, 1%, 0.1%, preferably less than 10% or 1%, or between 0% and 99%, 0% and 50%, 0% and 10%, preferably between 0% and 5%, wherein the percentage is preferably the ratio between the number of cells having at least one nanoparticle and the total number of cells preferably contained in the pre-growth medium, iii) an optical density greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 0.1, 0.2, 0.5, 1, 5, 10 or 100, iv) the number of cells is greater than 1, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 ,v) Their volumes are contained in more than 0.0001, 0.001, 0.1, 1, 10, 50, 10 2 , 10 3 , 10 5 or 10 10 1, vi) cell generation number includes 1 and 10 10 , 1, and 10 3 vii) the optical density OD measured at the end of the pre-growth step is between 50 and 300 PGE , and the optical density OD measured at the beginning of the pre-growth step PGB The ratio between OD PGE / OD PGB , greater than 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 , and / or viii) the optical density OD measured at the end of the pre-growth step sub-step i. PGiE and the optical density OD measured at the beginning of the pre-growth step sub-step i PGiB The ratio between OD PGiE / OD PGiB,, greater than 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 , and / or

[0556] In one embodiment of the invention, the cells that do not substantially produce nanoparticles are non-magnetic cells.

[0557] In one embodiment of the present invention, preferably at the beginning or end of the pre-growth step or at least one sub-step thereof, a percentage of non-magnetic cells is greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 50, or 75% is obtained. In some cases, the percentage of non-magnetic cells is based on the ratio n NMC / (n MC +n NMC ), where n MC and n NMC are the numbers of magnetic and non-magnetic cells, respectively.

[0558] In another embodiment of the present invention, the non-magnetic cells do not show a magnetic response, wherein the magnetic response may be an orientation of at least one cell parallel to the magnetic field or a cell movement speed proportional to the magnetic field strength, wherein the magnetic field strength may be greater than 10 -9 , 10 -3 , 10 -1 , 1, 10 3 or 10 6 mT and / or a magnetic field are preferably applied to the cells.

[0559] In one embodiment of the invention, the method includes a growing step comprising expanding the nanoparticle-producing cells derived from the pre-growth step in a growth medium such that the nanoparticle-producing cells produce nanoparticles. In some cases, the growing step is performed in a fermentor or apparatus in which the temperature, pH, iron concentration, and / or oxygen concentration of the growth medium can be controlled.

[0560] In another embodiment of the invention, the growing step begins by placing the cells obtained from the pre-growing step into a growth medium. In some cases, the growing step or at least one sub-step thereof is performed within 10 -50 and 10 50 , 10 -50 and 10 10 , 10 -30 and 105 and 10 -20 and 10 3 and 10 -10 and 10 2 or during a time period between 10 -5 and 24 hours. In some other cases, the growth step or at least one of its sub-steps occurs at a temperature below 10 20 and 10 10 and 10 5 and 10 3 and 10 2 and 10, 5, 2, 1, 10 -1 and 10 -2 and 10 -3 and 10 -5 and 10 -10 or 10 -20 of a time period. In some other cases, at least one of the growth step or its sub-steps occurs at a temperature greater than 10[[ID=***]] -20 and 10 -10 and 10 -5 and 10 -3 and 10 -2 and 10 -1 and 1, 2, 5, 10, 10 2 and 10 3 and 10 5 and 10 10 or 10 20 of a time period.

[0561] In one embodiment of the present invention, the growth step includes amplifying cells during successive sub-steps GS O …GS i …GS j where t GSOb , t GSib , t GSjb are the start times of steps 0, i and j, and t GSOe , t GSie , t GSje are the end times of steps 0, i and j, where 0 < i < j. In some cases, i and / or j are greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 10, 10 3 and 10 5 or 10 10 . In some other cases, i and / or j are less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 10, 10 3 and 10 5 or 10 10 .

[0562] In one embodiment of the invention, each subset i comprises sparging the growth medium or introducing different amounts of oxygen into the growth medium and / or introducing different amounts of iron into the growth medium / preferably with the aid of fed-batch medium.

[0563] In one embodiment of the invention, the growing step comprises at least one of the following sub-steps, during which a gas, such as compressed air or a gas containing more than 1% O2, is introduced into the growth medium, preferably under stirring conditions of 1 to 10 10 ,5 to 10 5 ,10 to 10 4 ,100 to 10 3 or less than 100 to 300 rpm, and wherein:

[0564] - Lasts for 10 -3 to 10 3 During the first sub-step, which lasts from 2 to 16 hours or preferably from 2 to 16 hours, the flow rate of the gas is between 0 and 10 10 between 0.001 and 40 ml per minute per liter of growth medium, so that the optical density of the cells increases from 10 at the beginning of the first substep. -10 and 10 3 between 0.08 and 0.12, and the value at the end of the first sub-step is 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 greater than the value at the beginning of the first sub-step. 3 times or greater than 10 -9 and 10 4 A value between , preferably between 0.2 and 1.

[0565] - Lasts for 10 -3 to 10 3 During the second sub-step lasting from 2 to 120 hours or preferably from 2 to 120 hours, the flow rate of the gas is increased by 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 more than that in the first sub-step. 3 times or between 0 and 10 10 between 1 and 50 ml per minute per liter of growth medium, so that the optical density of the cells increases from the value at the beginning of the second sub-step, that is, equal to the value at the end of the first sub-step or included in 10 -9 and 10 4 between 0.2 and 1, and the value at the end of the second sub-step is 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 greater than the value at the beginning of the second sub-step. 3 times or 10 -9 and 10 4 A value between , preferably between 0.5 and 4 inclusive.

[0566] - Lasts for 10 -3 to 10 3 During the third sub-step lasting from 2 to 120 hours or preferably from 2 to 120 hours, the flow rate of the gas is increased by 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 more than that in the second sub-step. 3 times or between 0 and 10 10 between 50 and 120 ml / min per liter of growth medium, so that the optical density of the cells increases from the beginning of the third sub-step, that is, equal to the end of the second sub-step, or is included in 10 -9 and 10 4 between 0.5 and 4, preferably between 0.5 and 4, and the value at the end of the third sub-step is 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 greater than the value at the beginning of the third sub-step. 3 times or 10 -9 and 10 4 , preferably between 1 and 8 included at the end of the third sub-step.

[0567] - Lasts for 10 -3 to 10 3 During the fourth sub-step lasting from 2 to 120 hours or preferably from 2 to 120 hours, the flow rate of the gas is increased by 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 more than that in the third sub-step. 3 times or between 0 and 10 10 between 200 and 300 milliliters per minute per liter of growth medium, so that the optical density of the cells increases from the beginning of the fourth sub-step, that is, equal to the end of the third sub-step, or is included in 10 -9 and 10 4 between 1 and 8, preferably between 1 and 8, the value at the end of the fourth sub-step being 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 greater than the value at the beginning of the fourth sub-step 3 times or 10 -9 and 10 4 , preferably between 2 and 16 included at the end of the fourth sub-step.

[0568] - Lasts for 10 -3 to 10 3 During the fifth sub-step lasting from 2 to 120 hours or preferably from 2 to 120 hours, the flow rate of the gas is increased by 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 more than that in the fourth sub-step. 3 times or between 0 and 10 10 between 300 and 500 milliliters per minute per liter of growth medium, so that the optical density of the cells increases from the beginning of the fifth sub-step, that is, equal to the end of the fourth sub-step, to or including 10-9 and 10 4 between 1 and 8, preferably between 1 and 8, the value at the end of the fourth sub-step being 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 greater than the value at the beginning of the fourth sub-step 3 times or 10 -9 and 10 4 , preferably between 4 and 32 included at the end of the fifth sub-step.

[0569] In one embodiment of the present invention, during sub-step i, preferably sub-steps 2 to 5: i) the percentage of oxygen is maintained above 0.01% or 0.1 mBar by air flow, and below 0.9% or 9 mBar due to oxygen consumption by cells, ii) the gas flow is between 0 and 10 10 , 1, and 10 5 , 5 and 10 4 , 10 and 10 3 ml per minute per liter of growth medium, iii) growth medium at 1 to 10 5 ,10 and 10 4 , 50 and 10 3 , or is stirred at a speed of 100 to 500 revolutions per minute, iv) the flow rate of the gas can be reduced by increasing the stirring rate of the culture medium v) the flow rate of the gas can be reduced by decreasing the stirring rate of the culture medium, and / or vi) the optical density of the cells is increased from the value preferably contained in 10 at the beginning of sub-step i. -50 and 10 3 The value between increases to preferably comprise 10 at the end of sub-step i -20 and 10 5 Value between.

[0570] In one embodiment of the invention, the growing step comprises at least one sub-step, in which: i) the pH of the growth medium is maintained at a fixed or determined or selected pH comprised between 0 and 14, 2 and 13, 4 and 11, 5 and 10, preferably comprised between 5 and 8, most preferably equal to 6.9, preferably by adding an acidic iron source comprised in the fed-batch medium, preferably between 1 and 10 10 In some cases, the growth medium comprises an iron concentration at the beginning of the growth step or one of its substeps, i.e., i) less than 10 10 , 10 5 or 10 2 μM, preferably less than 10 or 2 μM, and / or ii), contained in 10 -10 and 10 10 , 10 -5 and 10 5 , 10-3 and 10 3 In some other cases, during the growth step or one of its sub-steps, the iron concentration of the growth medium is increased, preferably by adding fed-batch medium to the growth medium, to reach a value at the end of the growth medium or one of its sub-steps of:

[0571] i) greater than 10 -10 ,10 -5 ,10 -1 or 1 μM, preferably greater than 2 μM and / or ii) contained in 10 -10 and 10 10 Between μM, preferably between 2 μM and 5 mM or between 2 μM and 0.5 mM.

[0572] - In one embodiment of the invention, the growing step comprises at least one of the following sub-steps, wherein: in the first sub-step (lasting 10 -3 to 10 3 , preferably 2 to 16 hours), the fed-batch medium is added to the growth medium to bring the iron concentration in the growth medium to 10 -10 μM and 10 10 between 2 and 20 μM. In some cases, the consumption of iron by bacteria is not taken into account, and in some other cases, the consumption of iron by bacteria is taken into account. This preferably results in an increase in the production of nanoparticles from the beginning of the first substep to the end of the first substep of more than 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 3 times or from 10 -10 to 10 10 Preferably, at the beginning of the first sub-step, 0.001 to 0.1 mg of nanoparticles per liter of growth medium is between 10 -10 to 10 10 A value between 1 and 2, preferably between 1 and 10 mg of nanoparticles per liter of growth medium at the end of the first sub-step.

[0573] - In the second sub-step (lasting 10 -3 to 10 3 , preferably 2 to 120 hours), the fed-batch medium is injected into the growth medium to maintain the iron concentration in the growth medium between 10-10 and 10 10 The fed-batch medium was added to the growth medium to keep the iron concentration in the growth medium between 10 -10 μM and 10 10between 20 and 40 μM. In some cases, the consumption of iron by bacteria is not taken into account, and in some other cases, the consumption of iron by bacteria is taken into account. This preferably results in an increase in the production of nanoparticles from the beginning of the second sub-step to the end of the second sub-step of more than 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 3 times or from 10 -10 to 10 10 Preferably, at the beginning of the second sub-step, 1 to 10 mg nanoparticles per liter of growth medium is between 1 and 10 -10 to 10 10 A value between 1 and 20 mg nanoparticles per liter of growth medium is preferably present at the end of the second sub-step.

[0574] - In the third sub-step (lasting 10 -3 to 10 3 , preferably 2 to 120 hours), the fed-batch medium is injected into the growth medium to maintain the iron concentration in the growth medium between 10-10 and 10 10 The fed-batch medium was added to the growth medium to keep the iron concentration in the growth medium between 10 -10 μM and 10 10 between 40 and 150 μM. In some cases, the consumption of iron by bacteria is not taken into account, and in some other cases, the consumption of iron by bacteria is taken into account. This preferably results in an increase in the production of nanoparticles from the beginning of the third substep to the end of the third substep of more than 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 3 times or from 10 -10 to 10 10 Preferably, at the beginning of the third sub-step, the nanoparticles are present in a range of 2 to 20 mg per liter of growth medium to 10 -10 to 10 10 A value between 4 and 40 mg of nanoparticles per liter of growth medium is preferably achieved at the end of the third sub-step.

[0575] - In the fourth sub-step (lasting 10 -3 to 10 3 , preferably 2 to 120 hours), the fed-batch medium is injected into the growth medium to maintain the iron concentration in the growth medium between 10-10 and 10 10 The fed-batch medium was added to the growth medium to keep the iron concentration in the growth medium between 10 -10 μM and 10 10Preferably, the concentration of the nanoparticles is between 150 and 500 μM, in some cases not taking into account the consumption of iron by the bacteria, in some other cases taking into account the consumption of iron by the bacteria. This preferably results in an increase in the production of nanoparticles by more than 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 from the beginning of the fourth sub-step to the end of the fourth sub-step. 3 times or from 10 -10 to 10 10 Preferably, at the beginning of the fourth sub-step, the nanoparticles are present in a range of 4 to 40 mg per liter of growth medium to 10 -10 to 10 10 A value between 1 and 2, preferably between 8 and 80 mg of nanoparticles per liter of growth medium at the end of the third sub-step.

[0576] - In the fifth sub-step (lasting 10 -3 to 10 3 , preferably 2 to 120 hours), the fed-batch medium is injected into the growth medium to maintain the iron concentration in the growth medium between 10-10 and 10 10 The fed-batch medium was added to the growth medium to keep the iron concentration in the growth medium between 10 -10 μM and 10 10 Preferably, the concentration of the nanoparticles is between 500 and 1000 μM, in some cases not taking into account the consumption of iron by the bacteria, and in some other cases taking into account the consumption of iron by the bacteria. This preferably results in an increase in the production of nanoparticles by more than 1.0000001, 1.1, 1.5, 2, 5, 10 or 10 from the beginning of the fifth substep to the end of the fifth substep. 3 times or from 10 -10 to 10 10 Preferably, at the beginning of the fourth sub-step, the nanoparticles are present in an amount of 8 to 80 mg per liter of growth medium to 10 -10 to 10 10 A value between 16 and 160 mg of nanoparticles per liter of growth medium is preferably achieved at the end of the third sub-step.

[0577] In one embodiment of the invention, during the growing step or sub-step i of the growing step, preferably sub-steps 1 to 5: i) the iron concentration of the growth medium is increased to 10 -10 μM or more, preferably 2 μM, preferably by adding a fed-batch medium containing iron to the growth medium and less than 10 10 mM, preferably 5 mM, since cells consume iron, ii) the total amount of iron added to the growth medium per liter of growth medium is included in 10 -6 Between 1 and 15, preferably 2.10 -4and 1.5 g iron per liter of growth medium, and / or iii) the amount of nanoparticles is increased from a value between 0 and 500 mg, preferably between 0 and 80 mg at the beginning of the growth step or one of its substeps to between 1 and 10 mg at the end of the growth step or one of its substeps 5 The amount of nanoparticles present in the nanoparticles may range from 10 to 200 mg per liter of growth medium.

[0578] In one embodiment of the invention, the growth step comprises cell expansion, which is related to or corresponds to the speed or rate or number or optical density of cell division, and is preferably greater than 1.000001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 2 , 10 3 , 10 5 , 10 10 , 10 20或 10 50 times.

[0579] In one embodiment of the present invention, preferably during the growing step, during its beginning or at its end or at least one sub-step thereof, the cells producing the nanoparticles have at least one of the following properties: i) a number of nanoparticles contained in the cells is greater than 1, 2, 5, 10, 50, 10 2 or 10 3 , preferably greater than 0, 1 or 2, or from 0 to 10 3 between 0 and 100, preferably between 0 and 10, ii) a certain percentage of cells have at least one nanoparticle greater than 10 -4 ,10 -2 ,10 -1 , 1, 5, 10, 50, 75 or 95, preferably greater than 10 or 50% or comprised between 0 and 99%, 10% and 75%, 5% and 90%, preferably between 20% and 100%, iii) an optical density greater than 10 -50 ,10 -20 ,10 -10 ,10 -5 ,10 -3 ,10 -2 ,0.1,0.2,0.5,1,5,10 or 100,iv) a certain number of cells greater than 1,5,10,10 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100v) Cells contained in greater than 0.0001, 0.001, 0.1, 1, 10, 50, 10 2 ,10 3 ,10 5 or 10 10 vi) The number of cells produced in a volume of 1 to 10 10 ,1 and 10 3 between 50 and 300, preferably between 50 and 300, vii) the optical density, OD, measured at the end of the growth step GE and the optical density OD measured at the beginning of the growth step GB The ratio between GE / OD GB , greater than 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 , or viii) the optical density OD measured at the end of sub-step i of the growth step GiE The ratio ODGiE / ODGiB of the optical density ODGiB measured at the beginning of sub-step I is greater than 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 .

[0580] In one embodiment of the invention, the cells that do not substantially produce nanoparticles are non-magnetic cells.

[0581] In one embodiment of the invention, during the growth step, at the beginning or at the end thereof, or at least one sub-step thereof, the percentage of magnetic cells obtained is greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 50 or 75%. In some cases, the percentage of magnetic cells is equal to n MC / (n MC +n NMC ), where n MC and n NMC are the numbers of magnetic cells and non-magnetic cells, respectively. In another embodiment of the present invention, the magnetic cells are cells that show a magnetic response.

[0582] In some cases, the pre-growth step, the growth step, or at least one sub-step thereof, is performed at a temperature greater than -250, -200, -150, -100, -50, -20, -10, -5, -2, -1, 0, 1, 2, 5, 10, 20, 50, 75, 100, 10 3 , 10 5 or 10 7 ℃, or its temperature changes by more than 10 -5 , 10 -3 , 10 -2 , 0.1, 1, 5, 10, 50, 100 or 150° C. In some other cases, the pre-growth step, the growth step or at least one sub-step thereof is performed at a temperature below 0 7 , 10 5 , 10 3 , 100, 75, 50, 40, 30, 20, 10, 5, 2, 1, 0, -1, -2, -5, -10, -20, -50, -100, -150, -200 or -250°C, or a temperature variation of less than 10 5 , 10 3 , 10 2 , 50, 20, 10, 5, 2, 1 or 0.1℃.

[0583] In some cases, the pre-growth step, the growth step, or at least one sub-step thereof is performed at a pH greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or a pH change of greater than 10 -10 , 10 -7 , 10 -5 , 10 -4 , 10 -3 , 10 -1 In some other cases, the pre-growth step, the growth step, or at least one sub-step thereof is performed at a pH lower than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pH units or a pH change of less than 10. -10 , 10 -7 , 10 -5 , 10 -4 , 10 -3 , 10 -1 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 pH units.

[0584] In some cases, the temperature, temperature change, pH, or pH change is large enough to allow at least 1, 5, 10, 10 3 , 105 , 10 10 or 10 50 cell divisions or cell divisions per hour.

[0585] In some other cases, the temperature, temperature change, pH or pH change is low enough to prevent the above 1, 5, 10, 10 3 , 10 5 , 10 10 or 10 50 The destruction, disappearance or degeneration of cells or cells per hour.

[0586] The present invention also relates to a method according to the invention, wherein the pre-growth medium does not comprise iron or at least one iron source.

[0587] The present invention relates to a method according to the invention, wherein the pre-growth medium comprises iron or at least one iron source, wherein the nature and / or amount of the iron or iron source preferably does not allow simultaneous nanoparticle production and cell growth by the cells.

[0588] In one embodiment of the present invention, the property of the iron source is the composition, chemical formula, type of iron source, or the iron source itself. In some cases, the iron source is a source of iron (III) or iron (II). In some cases, it can be or contain or be made of or have the chemical formula Cl3Fe, C 10 H 12 FeN2NaO8, Fe2O 12 S3, C6H8FeNO7, C6H5FeO7, FeH 18 N3O 18 、C 30 H 21 FeN3O 15 -3 、FeO4P、C6H7FeO8、Fe2H2O 13 S3、Fe2H 12 O 18 S3, C 10 H 12 FeN2NaO8、C 10 H 13 FeN2O8、FeH 28 NO 20 S2, C 10 H 15 FeN2NaO8、C 10 H 14 FeN2NaO8 +4 、C 14 H 21 FeN3O 10 、C 18 Fe7N 18、Fe4H2O 22 S5、Fe4O 21 P6、F3Fe、C6H 11 FeNO7 +3 、C6H 11 FeNO7、C 18 H 15 FeO9、C 12 H 29 Fe5Na2O 23 、C 12 H 22 Fe2O 14 、C 15 H 21 FeO6、C 15 H 24 FeO6、C6H5FeO7、C 10 H 16 FeN3O8、C4H 10 FeO5、C 54 H 105 FeO6、AsFeH 13 O9 + 、AsFeO4、Fe +3 、C6H 12 FeN3O 12 、C6H 18 As3FeO6、FeH2O5P、C 21 H 21 FeO9S3、C6H 11 FeNaO7 +3 、C 14 H 22 FeN3NaO 10 、FeNaO7P2、C3H9As3Fe3O9、C 18 H 24 Fe4O 42 P6、C6H 11 FeO 10 、C9H 18 FeN3S6、Cl3FeO 12 、C6H9FeNO7 + 、Cr3Fe2O 12 、C6H 10 FeNO8、FeH3O3、C 15 H 30 FeN3S6、C 30 H 27 FeN3O 15 、C3FeN3S3、C6H 12 FeKO6 +4、FeH3O3、FeN3O9、C3H3FeO6、C6H8FeO7、C 24 H 45 FeO6、FeO6P3、Fe2H 14 O 19 S3、C 18 H 33 FeO 21 、C6H9FeO9、C 18 H 27 FeO 24 、C6FeN6 -3 、C 10 H 12 FeN2O8 - 、C 22 H 36 N4O 13 、C3FeN3、C6H 12 FeN3O 12 、C6H9FeO6、C 15 H 27 FeO6、FeH4O6P、C 21 H 15 FeO9、FeH8O8P、C6H6FeNO6、C4FeKO8、C 12 H 12 Fe2O 18 、C 33 H 35 FeN4O4、Cl3FeH4O2、C 24 H 45 FeO6、C 10 H 15 FeN2O7、FeH4NO8S2、C 32 H 31 FeN4O5、Fe2H6O3、AlF6Fe、C4H4FeNO8、C 81 H 84 FeN3O 33 、Fe2S3、Cl3FeH 14 O7、C 18 H6FeN9O 21 、Cl3FeO9、FeI3、C6H 14 FeO 10 、C6H 10 FeO8、C 55 H 80 FeN 17 O 21 S3、C 10 H 16 FeN5O 13 P3、C 18 H34 FeO 16 +3 、C 12 H 12 Fe2O 15 、C6FeNa3O 12 、C 10 H 12 FeKN2O8、C 21 H 24 FeN3O9、C6H6Fe2O 12 、C6Fe2O 12 、AsFe、C 35 H 33 FeN5O 11 -3 、Cl3FeH2O、C 18 H 30 Fe2N6O 12 、FeI3O9、C 10 H 18 FeN2NaO 11 、Cl3FeH 18 O9、Cr2FeH4NO8、C9H 21 Fe2O 18 P3、C 18 H 34 FeO2、C 30 H 27 FeO6、C 30 H 24 FeN3O 15 、C 54 H 102 FeO6、Fe4H 18 O 30 P6、Fe2Se3、C 54 H 99 FeO6、C 15 H 21 FeO6、C 10 H 18 FeN2O7 +2 、C 10 H 18 FeN2O7 +2 、C 10 H 19 FeN3O8、C 22 H 14 FeO4、C 39 H 63 FeN6O 15 +3 、C 10 H 19 FeN3O8、C4FeNaO8、FeO4V、C6H15 FeN3O 12 、C6Fe2O 12 、C 18 H 24 Fe2O 24 -6 、C 18 H 19 FeN2NaO6、C 18 H 19 FeN2NaO6、C 12 H 18 Fe2O 12 、C6FeK3N6、C 24 H 47 FeO 25 - 、C 18 H 38 FeO 19 、C 15 H 21 FeO6、C 18 H 39 FeO 24 、C6H 11 FeNO7、C6H 12 FeO6、C 12 H 28 FeO 14 、FeHO2、C 45 H 36 FeN3O6、Fe3H2O4、Fe2O3、C 36 H 72 FeO6、C 12 H 18 Fe2O 15 、C9H 18 FeO9、FeH6O3、C 54 H 102 FeO6、C 42 H 84 FeO6、C 16 H 31 FeO2 +2 、C 36 H 69 FeO6、Fe3H8O4、C8H 15 Fe2O2 +4 、C 12 H 48 Fe2N 12 Oh 12 S3、C 48 H 96 FeO6、C9H 15 FeO9、C 35 H 39<h2 style=";text-align:left;direction:ltr">N5O<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 42 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 81 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 48 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 93 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 24 <h2 style=";text-align:left;direction:ltr"> O2、Fe2H<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> S3、FeH<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> N3O<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 24 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 23 <h2 style=";text-align:left;direction:ltr"> FeN<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> O6S2、C<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> Cl3FeN<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> S2、C<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> FeO6、Fe2H<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> S3、C<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> FeN3O8、C<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> FeN2NaO6、C3F9FeO9S3、C5H<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> FeO4、C6H<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> FeNO<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> FeN2NaO6、C<h2 style=";text-align:left;direction:ltr"> 32 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> N4O9、C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 24 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> F9FeO6、C<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> Cl3FeIS、C6H<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> Fe2O<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> 、C6H<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 、C6H<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 、C6H<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> Fe2O<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> 、C6H8FeO7、C6H<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 、C6H4Fe2N7、FeH2O4S、C<h2 style=";text-align:left;direction:ltr"> 42 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 60 <h2 style=";text-align:left;direction:ltr"> N<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> 、C6Fe2N6、C3Fe2O9、C<h2 style=";text-align:left;direction:ltr"> 162 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 297 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 27-6 、C 21 H 27 Cl4FeN2O、C6H4FeNaO7、C 27 H 50 FeN6O 10 、C 25 H 48 FeN6O8、C 27 H 48 FeN6O9、C6H7FeO6 +2 、Fe2H2O4、C 14 H 26 FeN5O 10 、Cl4FeH4N、Cl3FeH 12 O 18 、C6H 17 FeN2NaO7 +3 、C 10 H 11 FeNO6、C 15 H 15 F9FeO6、C6Fe2N6Na、C9H 21 Fe2O 18 P3、C 21 H 27 ClN2O、C2H3FeO、C 10 H 12 Fe2N2O8、FeH3O3P、C7H5FeO2、C7H5FeO2、FeI3O 12 、C3H4FeNO2S、C2H2FeNO2、C 12 H 12 Fe2O 12 、C8H7FeNO3、C2HFe、C6H7FeO2S4、C6H 11 FeO6、C 14 H 19 FeO 12 、BFeH3O3、C 21 H 18 FeO 15 、C 35 H 56 FeN6O 13 、C 12 H 30 FeO3、CHFe、C 47 H 48 FeNO 14 、Fe2H6O3、Fe2O9Sn3、C 18 H 18FeO3、Fe2O9Se3、Fe2O9Si3、Fe2O9S3、Br3FeO9、FeN3O6、C 24 H 54 FeO3、C 66 H 129 FeO6、FeP、C6H 18 FeO 24 P6 +3 、C 33 H 72 FeO3、C 40 H 75 FeO4、C2H3FeS、C3FeN3、C 21 H 39 FeO6、FeSi、C 30 H 29 FeN3O 16 、C 22 H 36 FeN4O 13 、C 30 H 57 FeO6、C 60 H 117 FeO6、C 18 H 12 FeN3O6、C 18 H 31 FeO2 +2 、FeS2、C6H 11 FeN4O2、C6H5FeO7、C6H5FeS、C 10 H 13 FeN2O 10 - 、C8H 13 FeOS2、C 27 H 51 FeO6、C 24 H 44 FeO 25 - ,C6H 15 FeN3O6、C6H 12 FeO9、Cl3FeO9S3、CFeNS、Fe4H 12 O 12 Si3、C3H6FeO 12 、C4H3FeO4S2、C4H4FeO6、C6H3FeN3O6、C5H5FeO2、C 10 H 24 FeN4O9、C 14 H 19 FeN3NaO 10 、C 10 H14 FeN2Na2O8、C 36 H 44 FeN4、C6FeNa3O 12 、Fe2H3OS3、C 16 H 27 FeO4、C6H8Fe2O 13 、C6H7FeO3、C4H4FeO6S2、C2H5FeN2、C5H7FeOS2、C 18 H 18 FeNa6O 21 、C3H9FeO9S3、C 24 H 54 FeO 12 P3、C 36 H 55 FeN6O 11 、Fe2H2O 10 Si3、C2H4FeNO2、C4H 11 FeN2O4、AsFeH2O5、C 12 H 13 FeO 13 、C 36 H 67 FeO6、C 12 H 13 FeO 13 、C3H6FeN3O6、C 18 H 15 FeO9S3、C 36 H 75 FeO 12 S3、Fe2H4O5、C 28 H 24 FeN4 +3 、F3Fe、C 30 H 30 FeO6、BFe、C2H8N2O4、C8H5FeN2O5、Fe2H4O 11 Se3、C6H7FeO6S4、C4H 10 FeN3、C6H 12 Fe2O 15 、C 15 H 23 FeO5、C8H 12 FeNO 12 、C 49 H 56 ClFeN4O6、FeH4NO8S2、C 36 H 75 FeO9S3、B3F 12 Fe、FeP、Fe2H20 O 22 S3、Cl3FeH 12 O 15 、C 18 H9FeN6、Fe2H 12 O 15 Se3、C 56 H 51 FeN4、Fe2H8O 13 Se3、C 44 H 27 FeN4、C 33 H 30 FeN4O6 -2 、CrFeO3、C 18 H 12 FeN3O 15 S3、Cl3FeH 18 O 21 、C6H5FeNa3O 13 、C 18 H 14 FeN 13 O9S2、C 15 H 24 FeO6、C 24 H 27 FeO9S3、C 27 H 54 FeN3S6、Cl3FeH 12 O6、C 16 H 36 Pb、C8H 18 Fe2O 12 P2、Cl3FeH 24 O 12 、C 24 H 30 FeO9S3、C 21 H 24 FeO9S3、C 18 H 15 FeO 12 S3、Cl3FeH 20 O 10 、C 28 H 24 FeN6O6 + ,C 66 H 121 Fe2NaO 65 、Cr3FeH3O 12 、C 12 H 28 Fe2O 14 、C3H8FeNS2Zn - ,F3FeH6O3、C<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 51 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 48 <h2 style=";text-align:left;direction:ltr"> Fe4N6O<h2 style=";text-align:left;direction:ltr"> 24 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> FeN3O6、C<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> FeO4、C6H6FeK3O<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H6F<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> FeN2O8、C6FeN6、C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H3F<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> FeN3O3S3、C<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 23 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> S3、FeH2O<h2 style=";text-align:left;direction:ltr"> +3 <h2 style=";text-align:left;direction:ltr"> C<h2 style=";text-align:left;direction:ltr"> 24 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> FeNaO<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> 、Cr3FeO6、Fe2H2O<h2 style=";text-align:left;direction:ltr"> +6 <h2 style=";text-align:left;direction:ltr"> 、C6H<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> FeN9、FeH5NO4S、C2K2O4、C<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> FeN6、C<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 27 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 34 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 38 <h2 style=";text-align:left;direction:ltr"> N4O4、Cl3FeH<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> 、C6H<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> FeO6P3S6、C6H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> ClFeNO<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> S2、C5H4F3FeO2<h2 style=";text-align:left;direction:ltr"> +2 <h2 style=";text-align:left;direction:ltr"> 、C6H6Cr2O<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 、C4H3CrKO8、C2MgO4、C<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 25 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> 、C2H2MgO4<h2 style=";text-align:left;direction:ltr"> +2 <h2 style=";text-align:left;direction:ltr"> 、C2CrO4<h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> ,C2HNaO4、C2HKO4、C6Cr2O<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 、C2H2FeO4、C2H4MgO6、C6AlO<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> -3 <h2 style=";text-align:left;direction:ltr"> 、C6Al2O<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 、C2Li2O4、C2MgO4、C<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> N4O12 S4、C 10 H 19 FeN2NaO 10 、C5H4CuFeN6O3、C 10 H 14 FeN2NaO9、C 30 H 15 FeN3Na3O 15 S3、C 27 H 15 FeN 12 O6、C9H 18 FeN3S6、C 30 H 30 FeN3O 15 +3 、C9H 18 FeN3S6、C6FeN6、C 18 Fe7N 18 、C 18 H 18 FeN2NaO6、C 30 H 21 FeN 12 O6、C 44 H 30 FeN4 +3 、C 14 H 18 FeK2N3O 10 、C 10 H 16 FeN2NaO8、C 33 H 29 FeNO 11 + 、C 25 H 18 FeN4O6S + ,C 35 H 24 FeN6O2S + ,C 32 H 32 ClFeN4O6、C 30 H 12 F9FeN 12 O6、C 30 H 18 Cl3FeN 12 O9、C 60 H 72 FeN9O9 +3 、C 60 H 66 FeN9O9 +3 、C 15 H 24 FeO6、C22 H 25 Cl2FeN3O9 + ,C 18 H 23 Cl3FeN3O 12 、C 11 H 24 FeNO 11 、C 49 H 54 FeN4O9 + ,C 42 H 54 Cl8Fe2N4O2、C 44 H 26 Cl4FeN4 +3 、C 34 H 32 FeN4O4 + ,C 44 H 38 FeN8 +7 、C9H 11 Cl2FeN4O2S、C 18 H 32 FeN4O8 +3 、C 34 H 32 ClFeN4O6, or C 19 H 25 In some other cases, the source of iron can be or contain or consist of or have the chemical formula: Fe +2 、FeH 14 O 11 S, FeH8N2O8S2, FeO4S, Cl2Fe, FeS, C4H2FeO4, C 12 H 26 FeO 16 、C4H5FeNO4、C 12 H 10 Fe3O 14 、C 16 H 30 FeO4, FeH2O5S, C 10 H 12 FeN2Na2O8, As2Fe3O8, CFeO3, C6H 12 FeO6、FeH 12 N2O 12 、C 12 H 10 Fe3O 14 、C6H5FeNaO7、C 34 H 32 FeN4O4、C 12 H22 FeO 14 、C 12 H 14 FeO 12 、C6H 10 FeO6、C4H8FeN2O4、C 12 H 28 FeO 16 、FeI2、FeH4N2O6S2、C 34 H 32 FeN4O4 -2 、C 34 H 32 FeN4O4、F2Fe、C6H 18 FeO9、C6H5FeO7 - 、C2FeO4、C4H4FeO4、Cl2FeO8、Fe3O8P2、FeO、B2F8Fe、FeH8O8S、C4H6FeO4、C4H4FeO4、C 12 H 10 FeNa4O 14 、C 22 H 14 FeO4、C2H4FeO6、C 12 H 24 FeO 14 、C 14 H 20 FeN3O 10 - 、Cl2FeH8O4、C 12 H8FeN2O4、C4H8FeO4、C5H7FeNO4、C8H 12 FeN2O8、C 12 H 10 Fe3O 14 、C6H 16 FeO9、C 19 H 19 FeN7O 10 S、C 10 H 16 FeN2O8、C 12 H 10 Ca2FeO 14 、C2H6FeO6、C 36 H 70 FeO4、C6H6FeO7、C4H2FeO4、C 36 H 21 Cl2FeN9O 14 、C 32 H 62FeO4、FeH2O2、C4H6FeO6、C6H8CaFeO7 +4 、C4H 10 Cl2FeN2O4、C 36 H 24 Cl2FeN6O8、C6H 14 FeO7、C 12 H 16 FeO 12 、BFe、C 32 H 16 FeN8、C 12 H 26 FeO 15 、C 12 H 10 Fe3O 14 、FeH8I2O4、C4H 10 FeN2O8S、C 30 H 24 Cl2FeN6O8、C 39 H 30 Cl2FeN6O8、C 12 H 14 FeO 12 、C 30 H 24 FeN6 +2 、C4H2FeO4 -2 、C4H4FeO4、C 10 H 16 FeO4、C 36 H 24 FeN6O4S、C2H4FeO6、C2H2FeO6、C8H 15 Fe2O2 +4 、C 32 H 16 FeN8、C 12 H 16 Fe3O 14 、C 12 H 24 FeO 14 、C2FeN2S2、C 12 H 16 FeN6O4、C 14 H 20 FeN3O 10 、C 12 H7FeN3O6S、C 20 H 12 FeN4、C 12 H 16 Ca2FeO 14 、C 46 H54 FeO9、C6H5FeO7、FeH4O6S、C 10 H 15 FeN2NaO7、C 10 H6FeN4O8、Fe2P、C4H4FeO6、C 14 H 26 FeO 16 、Cl2FeH 12 O 14 、C4H8Cl2FeN2O4、C6Fe3N6、C4H 12 As2FeO8、C 10 H 16 FeO4、FeH 20 N2O 14 S2、C 16 H 30 FeO4、C 40 H 40 FeN8O4 + 、Fe2Na8O 21 P6、C 14 H8FeO 10 、C 14 H8FeO4、C 12 H 20 FeO4、C8H8FeS、C5H4FeO、C2H3FeNO2、C 10 H 14 FeN2O8、C6H2FeN3O7 + 、C2H2Fe、C 10 H6FeN2、C6H 15 FeN3O7、C 72 H 124 FeO8 -2 、FeH 22 N2O 15 S2、C 40 H 78 FeO4、FeH2N2O6 +2 、C 44 H 86 FeO4、C 10 H 20 FeN2O8S2、C 20 H 38 FeO4、C 36 H 66 FeO4、C 24 H 46 FeO4、C 29 H 26 FeP + 、C 36 H<h2 style=";text-align:left;direction:ltr"> 64 <h2 style=";text-align:left;direction:ltr"> FeO6、C<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 26 <h2 style=";text-align:left;direction:ltr"> FeO4、C<h2 style=";text-align:left;direction:ltr"> 26 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> FeNP、C<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 54 <h2 style=";text-align:left;direction:ltr"> FeO4、C<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 32 <h2 style=";text-align:left;direction:ltr"> FeN4O4、C<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> FeN4O8、C6H9FeNO7<h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> 、C5H6FeO2、C4H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> BFeO4、C8H<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> BFeO4、C4H4FeO4S2、C6H6FeO7、C<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 34 <h2 style=";text-align:left;direction:ltr"> FeO4、C<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> FeO<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> 、C4H4FeO6、C5H7FeNO3、Fe3H8O4、C2FeN2S2、FeH2O2、Fe3H2O4、C<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> FeN4、C2H6FeO5、Fe2H6O<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> S2、C3H4FeN2O3、Fe3H2O9P2、C6H<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> Fe3N3O7<h2 style=";text-align:left;direction:ltr"> -3 <h2 style=";text-align:left;direction:ltr"> C4H<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> FeN2O6、Cl2FeH2O、FeO4W、C6H5FeO3P、C6H8FeO7、FeTe、C4H2FeO4、C<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> Cl2FeN8、C<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> FeO6、C3H3FeO7P、C4H7FeNO4、FeO3Si、Cl2FeH<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> O6、Cl2FeH2O9、FeH<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> O9S、FeH<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> S、C8H<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> FeO3P、C4H<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> FeO8、Fe3H<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> O<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> P2、F6FeSi、C<h2 style=";text-align:left;direction:ltr"> 72 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 42 <h2 style=";text-align:left;direction:ltr"> FeN6Na6O<h2 style=";text-align:left;direction:ltr"> 22 <h2 style=";text-align:left;direction:ltr"> S7、FeH4O5S、C<h2 style=";text-align:left;direction:ltr"> 39 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> FeN6O4S、C<h2 style=";text-align:left;direction:ltr"> 40 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> O4、C4H10 FeN2O4、C2H4FeN2O4S、Br2FeH2O、C 98 H 200 FeN 10 、C 36 H 21 FeN9O 10 S、C 10 H 10 Fe、C2H6FeN2、F6FeH 12 O6Si、C 48 H 48 FeN6O4S、FeO4S、C2H 10 FeN2O8S2、C 44 H 27 FeN5O、C 30 H 24 FeN6O4S、C6H8O6、C6H7NaO6、FeH4O2 +2 、FeH2O +2 、C3H7FeNO7S、C 30 H 18 FeN3NaO6、C2H 18 FeN2O 12 S2、C4H4FeO4、C7H7FeN4O + 、Br2Fe、C 18 H 22 Cl2FeN2、C 32 H 28 FeN6O6S2、C 12 H 14 MgO 12 、C2H5FeNO6S、C 45 H 60 FeN2O8、C 30 H 22 Cl2FeN2、C 38 H 26 FeN8O2S2、C 30 H 28 FeN2O6、C 14 H 12 Cl6FeO4、C 12 H 14 Fe、C 36 H 36 Cl2FeN6O8、C 17 H 14 FeN4O4S、C 24 H 30 FeN4O4、C 34 H 32 ClFeN4O6、C12 H 12 Fe、Fe3H 14 O 12 P2 +6 、C 32 H 16 FeN8、FeS2、C 16 H 15 FeNO2 +2 、C 29 H 20 FeO6、C 23 H 28 FeO2、C 11 H 10 FeO2、C 13 H 14 FeO2、C 12 H 12 FeO2、C 46 H 48 FeN4O6 +2 、C 47 H 59 FeN 13 O8 +2 、C 46 H 59 FeN 13 O8 +2 、C 48 H 62 FeN 12 O8S +2 、C 50 H 65 FeN 13 O8 +2 、C 48 H 63 FeN 13 O8 +2 、C 48 H 62 FeN 12 O8S +2 、C 55 H 76 FeN 14 O9 +2 、C 25 H 19 FeN3、C 15 H 17 FeN3OS +2 、C 22 H 23 FeN3OS +2 、C 26 H 28 ClFeN3、C 28 H 33 ClFeN4、C27 H 31 ClFeN4、C 29 H 35 ClFeN4、C 30 H 37 ClFeN4、C 28 H 33 ClFeN4、C 27 H 30 ClFeN3、C 26 H 28 ClFeN3、C 29 H 35 ClFeN4、C 27 H 30 ClFeN5O +2 、C 41 H 38 ClFeN5O3 +2 、C 42 H 41 FeN5O3 +2 、C 41 H 38 FFeN5O3 +2 、C 42 H 47 FeN5O3 +2 、C 43 H 49 FeN5O3 +2 、C 42 H 41 FeN5O3 +2 、C 42 H 40 ClFeN5O3 +2 、C 42 H 40 ClFeN5O3 +2 、C 42 H 40 FFeN5O3 +2 、C 41 H 45 FeN5O3 +2 、C 42 H 47 FeN5O3 +2 、C 41 H 39 FeN5O3 +2 、C 22 H 25 FeN5O5 +2 、C 24 H 23 ClFeN4O2 +2 、C 24 H 23FFeN4O2 +2 、C 24 H 24 FeN4O2 +2 、C 15 H 21 FeN3S +2 、C 29 H 34 FeN4O2 +2 、C 28 H 31 ClFeN4O2 +2 、C 28 H 31 FFeN4O2 +2 、C 30 H 35 ClFeN4O3 +2 、C 30 H 35 FFeN4O3 +2 、C 28 H 32 FeN4O2 +2 、C 27 H 30 FeN4O2 +2 、C 26 H 27 ClFeN4O2 +2 、C 30 H 36 FeN4O3 +2 、C 28 H 31 ClFeN4O3 +2 、C 28 H 31 FFeN4O3 +2 、C 28 H 32 FeN4O3 +2 、C 27 H 29 ClFeN4O3 +2 、C 26 H 27 FFeN4O2 +2 、C 26 H 28 FeN4O2 +2 、C 26 H 28 FeN4O2 +2 、C 27 H 29 FFeN4O3 +2 、C 27 H 30 FeN4O3 +2 、C26 H 27 ClFeN4O3 +2 、C 26 H 27 FFeN4O3 +2 、C 26 H 28 FeN4O3 +2 、C 25 H 25 ClFeN4O3 +2 、C 25 H 25 FFeN4O3 +2 、C 25 H 26 FeN4O3 +2 、C 24 H 23 ClFeN4O3 +2 、C 24 H 23 FFeN4O3 +2 、C 24 H 24 FeN4O3 +2 、C 25 H 25 ClFeN4O2 +2 、C 25 H 25 FFeN4O2 +2 、C 25 H 26 FeN4O2 +2 、C 25 H 26 FeN4O2 +2 、C 29 H 32 ClFeN7 +2 、C 33 H 32 ClFeN7 +2 、C 22 H 27 ClFeN3RuS + 、C 18 H 19 ClFeN3RuS + 、C 19 H 19 BFeO3 +2 、C 28 H 25 ClFeN4O +2 、C 31 H 38 FeN4O3、C 29 H 34 FeN4O3、C31 H 41 FeN3O、C 28 H 32 FeN4O3、C 26 H 29 FeN3O2、C 26 H 30 FeN2O、C 31 H 36 FeN4O3、C 30 H 35 FeN5O4、C 29 H 35 FeN5O3、C 32 H 41 FeN5O3、C 35 H 38 FeN4O3、C 32 H 40 FeN4O3、C 19 H 13 BBr2F2FeO2、C 19 H 14 BClF2FeO2、C 19 H 14 BBrF2FeO2、C 19 H 15 BF2FeO2、C 21 H 20 FeO4、C 20 H 18 FeO3、C 20 H 18 FeO3、C 20 H 18 FeO3、C 19 H 14 F2FeO2、C 19 H 14 Br2FeO2、C 19 H 15 BrFeO2、C 14 H 12 FeO3、C 21 H 19 BF2FeO4、C 20 H 17 BF2FeO3、C 20 H 17 BF2FeO3、C 20 H 17 BF2FeO3、C 19 H 13 BF4FeO2、C 19 H 13 BCl2F2FeO2、C 21 H29 AuCl2FeN4S + 、C 30 H 24 Cl2FeN6 +2 、C 22 H 21 Cl2FeN3 +2 、C 23 H 22 FeN6 +2 、C 21 H 19 FeN7 +2 、C 23 H 24 FeN6O +2 、C 47 H 64 FeN 14 O9、C 46 H 60 FeN 12 Oh 10 、C 41 H 53 FeN 11 O7、C 47 H 65 FeN 15 O8、C 45 H 59 FeN 13 O9、C 42 H 54 FeN 12 O7、C 43 H 67 FeN 15 O8、C 48 H 65 FeN 13 O8、C 47 H 64 FeN 12 O8、C 54 H 77 FeN 17 O9、C 51 H 71 FeN 15 Oh 10 、C 19 H 16 FeO2、C 44 H 48 FeN9O 17 P3、C 13 H9Cl2FeN3O6S, C 19 H 15 FeNO3、C 20 H 18 FeO2、C 20H 18 FeO3, C 21 H 20 FeO3, C 17 H 20 FeN2O2、C 18 H 15 FeNO, C 17 H 14 FeOS, C 17 H 14 FeOS, C 17 H 14 FeO2, C 22 H 22 FeO4, C 20 H 18 FeO2, C 20 H 18 FeO2, C 19 H 14 Cl2FeO、C 21 H 20 FeO3, C 48 H 28 FeN4O8、C 17 H 15 FeNS, C 34 H 30 FeN4O4 -2 、C 30 H 26 Br2FeN4O4、C 10 H 18 FeN2O7 +2 、C 14 H 12 FeO4, C 44 H 20 Cl8FeN4、C 64 H 64 FeN8O 12 S4, C 56 H 56 FeN8O8S4、C 56 H 44 Br8FeN4、C 56 H 52 FeN4、C 52 H 40 FeN8O 12 S4, C 44 H 32 FeN8O8S4, or C 44 H 28 FeN4. In some other cases, the source of iron has the formula C a H b Fe c Od N e S f Br g Cl h P i Na j As k K l Al m C rn V o I p B q F r Te s W twherein the coefficients a, b, c, d, e, f, g, h, I, j, kl, m, n, o, p, q, r, s, t are any integers between 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any integer between 21 and 1000000000000, wherein the C, H, Fe, O, N, S, Br, Cl, P, Na, As, K, Al, Cr, V, I, B, F, Te and W atoms preferably occupy the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth and twentieth positions, respectively. In some cases, at least one atom of the formula may occupy any of the 20 positions of the formula.In some other cases, the source of iron may include a chemical functional group selected from the group consisting of alkanes (R(CH2)nH), alkenes (R2C=CR2), alkynes (RC≡CR'), benzene derivatives (RC6H5); groups containing halogens, alkyl halides (RX), oxygen-containing, alcohol groups (ROH), carbonyls (RCOR'), aldehydes (RCHO), acyl halides (RCOX), carbonates (ROCOOR'), carboxylates (RCOO-), carboxylic acids (RCOOH), esters (RCOOR'), methoxy groups (ROCH3), hydroperoxides (ROOH), peroxides (ROOR'), ethers (ROR'), hemiacetals (RCH(OR')(OH)), hemiketal ((RC(OR") (OH)R'), acetal (RCH(OR')(OR")), ketal ((RC(OR") (OR")')R'), orthoester (RC(OR')(OR") (OR'")), heterocycle (PhOCOPh), orthocarbonate (C(OR)(OR')(OR") (OR")), containing nitrogen, amide (RCONR2), amine (RNH2, R2NH, R3N, R4N+), imine ((RC(=NH)R', RC(=NR")R', RC(=NH)H, RC(=NR')H, acylide Amines ((RCO)2NR'), azides (RN3), azo compounds (RN2R'), cyanates (ROCN, RNCO), nitrates (RONO2), nitriles (RCN, RNC), nitrites (RONO), nitro compounds (RNO2), nitroso compounds (RNO), oximes (RCH=NOH), pyridine derivatives (RC5H4N), sulfur-containing groups, thiols (RSH), sulfides (RSR'), disulfides (RSSR'), sulfoxides (RSOR')), sulfones (RSO2R'), sulfinic acids (RSO2H), sulfonic acids (RSO3H), thiocyanates (RSCN, R In some cases, the iron source can be an iron chelator. In some cases, the amount of the iron source is an iron source or an amount or concentration of iron, preferably derived from an iron source, preferably derived from a pre-growth and / or growth medium.

[0589] The present invention also relates to a method according to the invention, wherein the iron or iron source in the pre-growth medium consists of Fe 2+ and / or Fe 3+ Composed of or containing Fe 2+ and / or Fe3+ .

[0590] In some cases, when the iron source includes Fe2 in its chemical formula, the iron source includes Fe 2+ In some other cases, when the iron source includes Fe 3 in its chemical formula, the iron source includes Fe 3+ .

[0591] The present invention relates to a method according to the invention, wherein the concentration of iron or an iron source in the pre-growth medium is less than 20 μM. In some cases, the concentration of iron or an iron source in the pre-growth medium is less than 10 μM. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 In some other cases, the concentration of iron or iron source in the pre-growth medium is greater than 0, 10 -50 , 10 -20 , 10 -5 , 10 -1 , 1, 5, 10 or 20 μM. In other cases, the concentration of iron or iron source in the pre-growth medium is 10 -50 and 10 50 , 10 -10 and 10 10 , 10 -10 and 10 5 , 10 -10 and 10 3 Between or within 10 -10 and 1 μM.

[0592] The present invention relates to a method according to the invention, wherein the growth medium comprises iron or at least one iron source, wherein the nature and / or amount of the iron or iron source allows nanoparticle production by the nanoparticle-producing cells and cell growth.

[0593] The present invention relates to a method according to the invention, wherein the iron source of the growth medium is the same as the iron source of the pre-growth medium.

[0594] The present invention relates to a method according to the invention, wherein the concentration of the iron or the iron source in the growth medium is greater than or equal to the concentration of the iron or the iron source in the pre-growth medium.

[0595] In some cases, the concentration of iron or iron source in the growth medium is less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 102 In some other cases, the concentration of iron or iron source in the growth medium is greater than 0, 10 -50 , 10 -20 , 10 -5 , 10 -1 , 1, 5, 10 or 20 μM. In other cases, the concentration of iron or iron source in the growth medium is 10 -50 and 10 50 ,10 -10 and 10 10 ,10 -10 and 10 5 ,10 -10 and 10 3 Between, or 10 -10 and 1 μM.

[0596] The present invention relates to a method according to the invention, wherein the growth medium is supplemented by a fed-batch medium.

[0597] In one embodiment of the invention, the fed-batch medium comprises at least one source in common with the pre-growth and / or growth medium. In some cases, the concentration of the source in the fed-batch medium is equal to or greater than that in the pre-growth and / or growth medium / medium by at least 1.00001, 1.1, 2, 5, 10, 10 3 or 10 5 In some other cases, the concentration of the source in the fed-batch medium is at least 10% lower than in the pre-growth and / or growth medium / medium. 5 ,10 3 ,10,1,1.1 or 1.00000001.

[0598] The present invention relates to a method according to the invention, wherein the fed-batch culture medium comprises a concentration of greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 20, 50, 100, 10 3 or 10 5 In some other cases, the fed-batch medium contains a concentration of less than 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10 -2 , 10 -10 or 10 -20 In some other cases, the fed-batch medium contains iron or an iron source at a concentration of 10 -50and 10 50 ,10 -15 and 10 15 ,10 -10 and 10 5 ,10 -5 and 10 5 , 10 -3 and 10 3 The concentration of the feed medium is preferably between 0.5 μM and 50 μM, or between 0.5 nM and 50 μM, preferably before the fed-batch medium is added to the growth medium.

[0599] In one embodiment of the present invention, the fed-batch medium is acidic or has a pH value below 7, 6, 5, 4 or 3, preferably below 2. In some cases, the fed-batch medium has a pH greater than 0 or 1.

[0600] The present invention relates to a method according to the invention, wherein the fed-batch culture medium is fed at 10 -15 Liters per hour to 10 15 Liters per hour or 10 -15 micromoles iron per hour to 10 15 In some cases, the fed-batch medium is added to the growth medium at a low rate, preferably less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 5, 1, 10 -2 , 10 -3 , 10 -5 or 10 -10 The fed-batch medium is added to the growth medium at a rate of 10 μmol iron per minute or micromoles iron per minute, preferably when the number of cell divisions in the growth medium is low, preferably less than 10 μmol iron per minute. 20 , 10 10 , 10 5 , 10 3 , 10, cell divisions per second or per hour or per day or per month. In some other cases, the fed-batch medium is added to the growth medium at a high rate, preferably at a rate greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 1, 5, 10, 10 2 , 10 3 , 10 5 or 10 10Feeding of the fed-batch medium per minute or micromoles of iron per minute, preferably when the number of cell divisions in the growth medium is large, preferably greater than 1, 2, 5, 10, 10 3 , 10 5 , 10 10 or 10 20 , the cells divide every second or every hour or every day or every month. In some cases, between two sub-steps of the growth step, the flow rate of the fed-batch medium is reduced, preferably by more than a factor of 1.0000001, 1.00001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 , 10 5 , 10 7 or 10 9 In some other cases, between two sub-steps of the growth step, the flow rate of the fed-batch medium is increased, preferably by a factor of more than 0.000001, 1.00001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 , 10 5 , 10 7 or 10 9 .

[0601] In one embodiment of the invention, the iron concentration of the fed-batch medium prior to addition to the growth medium is at least 1.000001, 1.001, 1, 1.5, 2, 5, 10, 10 or more greater than the iron concentration of the growth medium. 2 , 10 3 or 10 5 times.

[0602] In another embodiment of the present invention, the fed-batch medium becomes part of the growth medium, preferably after addition of the growth medium.

[0603] The present invention relates to a method according to the invention, wherein the pre-growth and / or growth medium according to the invention comprises only one vitamin selected from the group consisting of calcium pantothenate, folic acid, inositol, niacin, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride and any derivatives of these vitamins.

[0604] In some cases, the pre-growth and / or growth medium may contain less than 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 100, 75, 50, 25, 10, 5, 3, 2 or 1 vitamin or different vitamins. In some cases, different vitamins can be a vitamin comprising at least 1, 2, 5, 10, 10 2 , 103 , 10 5 , 10 10 , 10 20 or 10 50 In some other cases, the pre-growth and / or growth medium contains more than 1, 2, 5, 10, 10 2 , 10 3 , 10 5 or 10 10 vitamins or different vitamins.

[0605] In some cases, the vitamin may be a water-soluble vitamin. In other cases, the vitamin may be a fat-soluble vitamin. In other cases, the vitamin may be vitamin A, D, E, K, B1, B2, B3, B5, B6, B7, B9, B12, or vitamin C. In other cases, the vitamin is not produced by the cells that produce the nanoparticles. In other cases, the vitamin may be a vitamin used to treat a disease caused by or associated with vitamin deficiency.

[0606] In another embodiment of the present invention, the vitamin is selected from the group consisting of adenosylcobalamin, aminobenzoic acid, ascorbic acid, biotin, calcium D-(+)-pantothenate, carotene thiamine, carotenoid beta, cholecalciferol (D3), cyanocobalamin, cyanocobalamin, ergocalciferol (D2), folic acid, folic acid, folinic acid, hydroxocobalamin, inositol, menaquinone (K2), methylcobalamin, niacin, nicotinamide, nicotinamide, nicotinamide riboside, niacin, pantothenic acid, phylloquinone (K1), pyridoxal, pyridoxamine, pyridoxine, para-lipoic acid, pyridoxal, pyridoxamine, pyridoxine, pyridoxine hydrochloride, retinal, retinoic acid, retinol, riboflavin, thiamine, tocopherol or tocotrienol, and derivatives or combinations of one or more of these vitamins.

[0607] In another embodiment of the present invention, the at least one vitamin included in the growth medium is biotin, folic acid, riboflavin, niacin or thiamine.

[0608] In some cases, the vitamins included in the pre-growth medium are the same as the vitamins included in the growth medium. In some other cases, the vitamins included in the pre-growth medium are different from the vitamins included in the growth medium.

[0609] The present invention also relates to a method according to the invention, wherein the concentration of at least one vitamin contained in the pre-growth and / or growth medium / medium is less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 103 , 10, 1, 10 -1 , 10 -3 , 10 -4 , 10 -6 , 10 -9 , 10 -20 , 10 -50 or 10 -100 M, or preferably less than 0.002 mol / L.

[0610] In another embodiment of the present invention, the pre-growth and / or growth medium / medium comprises at least one vitamin or one chemical element in which the concentration of at least one vitamin is greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -4 , 10 -3 , or 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 M.

[0611] The present invention also relates to a method according to the invention, wherein the pre-growth and / or growth medium comprises at least one vitamin in a lower concentration than a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron, preferably at a concentration of less than, preferably greater than 1.0001, 1.2, 1.5, 2, 5, 10, 10 3 or 10 5 In some cases, cells do not require large amounts of vitamins to grow, divide, and / or synthesize nanoparticles.

[0612] The present invention also relates to a method according to the invention, wherein the pre-growth and / or growth medium / culture medium comprises, per gram or per milliliter of growth or pre-growth medium, less than: i) 1 mg yeast extract, ii) 1 mg of at least one component of yeast extract, iii) 1 mg peptone, iv) 1 mg of at least one peptone fraction, v) 1 mg CMR reagent, vi) 1 mg of at least one chelating agent, vii) 1 mg of at least one amino acid, viii) 1 mg of a toxic or cytotoxic compound, and / or ix) 1 mg of at least one heavy metal.

[0613] In one embodiment of the invention, the pre-growth and / or growth medium comprises yeast extract, peptone, CMR reagent, chelating agent, amino acid, toxic or cytotoxic compound and / or heavy metal in a concentration less than 10 100 , 10 50 , 1020 , 10 10 , 10 5 , 10 2 , 10, 1, 10 -3 or 0 -5 μg yeast extract, peptone, CMR agents, chelating agents, amino acids, and / or heavy metals per liter or milliliter of pre-growth and / or growth medium / media. In some cases, this occurs when yeast extract, peptone, CMR agents, chelating agents, amino acids, toxic or cytotoxic compounds, and / or heavy metals have been removed from the pre-growth and / or growth medium or are not included in the pre-growth.

[0614] In one embodiment of the present invention, the first and / or second culture medium / medium comprises yeast extract, peptone, amino acids and / or heavy metals in a concentration greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 10, 10 3 or 10 5 μg yeast extract, peptone, amino acids and / or heavy metals per liter or milliliter of pre-growth and / or growth medium / culture medium. In some cases, this may occur when yeast extract, peptone, CMR agent, chelating agent, amino acids and / or heavy metals have been (preferably not intentionally) added to the pre-growth and / or growth medium / culture medium.

[0615] In one embodiment of the present invention, the yeast extract is or comprises a peptide, an amino acid, a purine base, a pyrimidine base and / or a water-soluble vitamin of Group B. In one embodiment of the present invention, the amino acid is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and / or valine.

[0616] In one embodiment of the present invention, the heavy metal is arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), selenium (Se) and / or zinc (Zn).

[0617] In one embodiment of the present invention, the agent that is carcinogenic, mutagenic or toxic to reproduction (also referred to as a CMR agent) is preferably nitriloacetic acid, trisodium salt and / or boric acid.

[0618] In one embodiment of the present invention, a toxic or cytotoxic compound is a compound that causes cell death, preferably of cells producing nanoparticles, when introduced into a pre-growth and / or growth medium, preferably at a concentration greater than 10 -10 , 10 -5 , 10 -2 , 1, 5, 10, 10 3 or 10 μM, preferably 10 -10 and 10 10 Between μM.

[0619] In one embodiment of the present invention, the pre-growth and / or growth medium / medium contains / does not contain a mineral selected from the group consisting of: C6H6NO6Na3, nitriloacetic acid trisodium salt, MnO4SH2O, manganese (II) sulfate monohydrate, NaCl, sodium chloride, CoN2O6 6H 2O, cobalt (II) nitrate hexahydrate, O 4SZn 7H 2O, zinc sulfate heptahydrate, CuO 4S 5H2O, copper (II) sulfate pentahydrate, AlKO 8S2 12H 2O, potassium aluminum sulfate dodecahydrate, H3BO3, boric acid, Na2MoO4·2H2O, sodium molybdate dihydrate, Cl2Ni6H2O, nickel (II) chloride hexahydrate, Na2SeO3, sodium selenite, and derivatives or combinations of one or more of these compounds.

[0620] In one embodiment of the present invention, the pre-growth and / or growth medium comprises a concentration of minerals which is less than 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 1, 10 -1 , 10 -3 or 10 -5 μg mineral per liter or milliliter of growth medium. In some cases, this may occur when minerals are removed from pre-growth and / or growth medium.

[0621] In one embodiment of the present invention, the first (pre-growth) and / or second growth medium comprises a concentration of minerals greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 10, 10 3 or 10 5μg mineral per litre or millilitre of growth medium. In some cases this may occur when minerals have been (preferably unintentionally) removed from the growth and / or growth medium / culture medium.

[0622] The present invention also relates to a method according to the invention comprising the additional step of purifying the high-purity iron oxide nanoparticles by removing at least one impurity from the nanoparticles.

[0623] In one embodiment of the present invention, an additional step of purifying the high purity iron oxide nanoparticles consists in removing impurities from the nanoparticles and / or denaturing and / or destroying impurities contained in the nanoparticles.

[0624] In one embodiment of the invention, the additional step of purifying the nanoparticles is preceded by the aforementioned step of isolating or extracting the nanoparticles from the cells. In some cases, the aforementioned step is a step of recovering the nanoparticles. In some cases, the aforementioned step is performed by: i) mixing the cells, preferably obtained from the growing step, with a detergent such as KOH or NaOH, ii) purifying the nanoparticles at a temperature greater than -270, -250, -200, -150, -100, -50, -30, -10, -5, 0, 5, 10, 20, 30, 50, 75, 100, 150, 200, 500, 10 3 , 10 5 or 10 10 ℃, or between -270 and 10 10 Between - 100 and 10 5 or heating the cells between 0 and 100°C, iii) with a temperature gradient greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 2, 5, 10, 10 3 , 10 5 or 10 10 °C / hour, minute or second, or between 10 -50 and 0 10 ℃ per hour, minute or second, iii) applying pressure to the cells, preferably greater than 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 atmospheres, or between 1 and 10 9 atmospheres, for example using French pressure, and / or, iv) preferably at a pressure greater than 10 -50, 10 -20 , 10 -5 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 The cells were sonicated at a power of 10 W.

[0625] In another embodiment of the present invention, an additional step of purifying the nanoparticles can remove: i) more than 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 25, 50, 75, 80 or 90% by mass of impurities ii) greater than 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 , 10 5 or 10 10 μg impurities per gram of nanoparticles.

[0626] In one embodiment of the invention, the impurities removed by the additional step of purifying the nanoparticles are preferably carbon or carbonaceous materials and / or are not iron oxides. Preferably, such / these impurities are located in the coating of the nanoparticles.

[0627] In one embodiment of the present invention, impurities are removed from the core and / or coating of the nanoparticles, preferably from the coating of the nanoparticles. Preferably, the impurities removed are shallow impurities. In some other cases, the impurities removed are deep impurities.

[0628] The present invention also relates to an additional step of a method for purifying high purity iron oxide nanoparticles by removing at least one impurity from the nanoparticles, comprising at least one heating step, wherein the temperature of the nanoparticles is increased to a temperature T0 and then maintained at T0 for a heating time, the heating time preferably being comprised between 1 second and 1 minute, 1 second and 1 hour, 1 second and 12 hours, 1 second and 1 day, 1 second and 1 week, 1 second and 1 month, or 1 second to 1 year, wherein T0 is preferably comprised between -200 and 10 5 , -100 and 10 5 , -50 and 10 5 , -10 and 10 5 ,0 and 10 5 °C, 10 and 10 5 , 20 and 10 5 , 30 and 10 5 ,100 and 105 , 200 and 10 5 , 100 and 10 4 ,100 and 10 3 , or between 100 and 500°C.

[0629] The present invention also relates to an additional step of a method for purifying high purity iron oxide nanoparticles by removing at least one impurity from the nanoparticles, comprising at least two heating steps, wherein:

[0630] - During step 1, the temperature of the nanoparticles is raised to a temperature T1 and then maintained at T1 for a heating time ranging from 1 second to 20 years, wherein T1 is between 150°C and 250°C.

[0631] - During step 2, the temperature of the nanoparticles is raised to temperature T2 and then maintained at T2 for a heating time ranging from 1 second to 20 years, wherein T2 is between 350°C and 450°C.

[0632] In some cases, the method of purifying high-purity iron oxide nanoparticles or the additional step of the heating step may be referred to as a purification method.

[0633] The present invention relates to a method for removing at least one impurity from high-purity iron oxide nanoparticles, comprising an additional step between steps 1 and 2, wherein the temperature of the nanoparticles is increased to a temperature T3 and then maintained at T3 for a heating time ranging from 1 second to 20 years, wherein T3 is 250° C. to 350° C.

[0634] In some cases, the temperature of the nanoparticles is maintained at temperature T1, T2 and / or T3 during a heating time of less than 100 years, 50 years, 20 years, 10 years, 5 years, 2 years, 1 year, 11 months, 6 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 3 days, 1 day, 23 hours, 12 hours, 6 hours, 1 hour, 50 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 2 minutes, 1 minute, 50 seconds, 30 seconds, 10 seconds, 1 second, 1 millisecond or 1 microsecond. In some other cases, the temperature of the nanoparticles is maintained at temperature T1, T2, and / or T3 for a heating period of greater than 1 microsecond, 1 millisecond, 1 second, 10 seconds, 30 seconds, 50 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 1 hour, 6 hours, 12 hours, 23 hours, 1 day, 3 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 11 months, 1 year, 2 years, 5 years, 10 years, 20 years, 50 years, or 100 years. In some other cases, the temperature of the nanoparticles is maintained at temperature T1, T2, and / or T3 for a heating period of between 1 microsecond and 100 years, 1 second and 20 years, 1 second and 1 year, 1 second and 1 month, 1 second and 1 day, 1 minute and 1 day, 5 minutes and 1 day, 10 minutes and 12 hours, 30 minutes and 6 hours, or 30 minutes and 3 hours.

[0635] In one embodiment of the present invention, the heating time is greater than the duration of the temperature increase to T1, T2 and / or T3, preferably greater than 1.001, 1.1, 1.5, 2, 5, 10, 10 3 ,10 5 or 10 10 times.

[0636] In some cases, T1 is between -273°C and 250°C, -200°C and 250°C, -100°C and 250°C, 0 and 250°C, 50°C and 250°C, 150°C and 250°C, or between 180°C and 220°C. In some other cases, T2 is between 200 and 10 5 , 250 and 10 5 , 300 and 10 5 , 350 and 10 5 , 350 and 10 3 , between 350 and 500, 350 and 450, or between 360 and 400°C. In some cases, T3 is included between -273 and 10 5 , -200 and 10 3 , between -100 and 500, -50 and 200, 0 and 500, 100 and 500, 200 and 500, 200 and 400, or between 250 and 350°C.

[0637] In another embodiment of the present invention, T3 is included between T1 and T2. In some cases, T3 is lower than T2, preferably higher than 1.0001, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 or 10 5 In some other cases, T3 is greater than T1, preferably greater than 1.0001, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 or 10 5 times.

[0638] In one embodiment of the present invention, the temperature of the nanoparticles is the temperature of a heating device or furnace used to heat the nanoparticles and / or containing the nanoparticles, preferably before, during or after treatment of the nanoparticles by a purification method.

[0639] In one embodiment of the invention, the temperature interval separating T1 and T2, designated as [T1, T2], is such that: i) the nanoparticles exhibit a maximum change or loss in weight or mass as a function of temperature, and / or ii) the derivative of the function of the change or loss in weight or mass of the nanoparticles as a function of time is maximum.

[0640] In one embodiment of the present invention, the ratio [% W (T2) - % W (T1)] / (T2 - T1), wherein % W (T2) and % W (T1) are the weight or mass percentages of the nanoparticles at T2 and T1, respectively, -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.5, 1, 10, or 10 5 In some cases, when the mass percentage of carbon in the nanoparticles (preferably before the nanoparticles are treated by a purification method) is large, the ratio is large, preferably greater than 10. -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 20, 50, 75, 85, 95 or 100% / ℃.

[0641] In another embodiment of the present invention, the ratio [% W (T2) - % W (T1)] / (T2 - T1) is less than 10 50 , 10 30 , 10 20 , 10 10 , 10 5, 10, 5, 2, 1, 0.5, 0.05, 10 -3 , 10 -5 , 10 -10 or 10 -20 % / °C. In some cases, when the mass percentage of carbon in the nanoparticles (preferably before the nanoparticles are treated by or through a purification method) is low, the ratio is low, preferably less than 100, 95, 80, 70, 50, 30, 20, 10, 5, 2, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -20 % / ℃.

[0642] In one embodiment of the present invention, the temperature of the nanoparticles is maintained at T1, T2 and / or T3, and when T1, T2 and / or T3 change by less than 10 5 , 10 3 , 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 3, 2, 1, 10 -5 , 10 -10 or 10 -20 %. In some cases, for each temperature T1, T2, and / or T3, the percentage is equal to T maxi -T mini / T avi , where T maxi 、T mini , and T avi (i=1, 2, 3) is the maximum, minimum, and average temperature reached during the heating time or during the heating step, preferably after the temperature is maintained at temperature T1, T2, and / or T3 or when the temperature is maintained at temperature T1, T2, and / or T3. In some cases, this percentage is low when the furnace or heating device is able to maintain temperature stability without large fluctuations and / or when the nanoparticles are not prone to endothermic and / or exothermic reactions. In some cases, an endothermic reaction is a reaction in which heat or energy is transferred from the medium surrounding the nanoparticle to the nanoparticle. In some other cases, an exothermic reaction is a reaction in which heat or energy is transferred from the nanoparticle to the medium surrounding the nanoparticle.

[0643] In one embodiment of the present invention, when the temperature of the nanoparticles changes by more than 10 5 , 10 3 , 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 3, 2, 1, 10 -5 , 10 -10 or 10 -20%, the temperature of the nanoparticles is not maintained at T1, T2 and / or T3. In some cases, this percentage is large when the furnace or heating device cannot maintain a stable temperature without large fluctuations and / or when the nanoparticles are prone to endothermic and / or exothermic reactions.

[0644] In one embodiment of the present invention, the temperature T1 and / or T2 is determined by the following formula:

[0645] i) Measure the change of weight or mass percentage of nanoparticles with temperature when the nanoparticles are at two temperatures T T<T1 and T T>T2 When heated, T T<T1 Lower than T1 and T T> T2 is greater than T2,

[0646] ii) measuring or representing or considering or examining or using at least one peak value of the derivative of the percentage change as a function of temperature,

[0647] iii) estimating or inferring the change in weight or mass percentage of the nanoparticles as a function of temperature, the temperature interval in which the change is greatest, wherein the minimum and maximum temperatures of the temperature interval are T1 and T2, respectively;

[0648] iv) estimating or deriving two temperatures, T1 and T2, from the position of at least one peak in a derivative curve of the weight or mass percentage change of the nanoparticles, where the minimum values ​​of the derivative curve at the beginning and end of the peaks are obtained, respectively, and T3 is obtained from the maximum value between the peaks of the derivative curve, and

[0649] v) estimating or inferring the temperature T1, T2, T3 from the position of at least one peak in the curve of the heat flux of the nanoparticles as a function of temperature, preferably at the beginning of the at least one peak.

[0650] In some cases, the temperature at which the derivative of the change in the weight or mass percentage of the nanoparticles decreases with temperature is at the beginning of the peak. In some other cases, the temperature at which the derivative of the change in the weight or mass percentage of the nanoparticles increases with temperature is at the end of the peak.

[0651] In another embodiment of the present invention, the heat flux of the nanoparticles is the heat flux generated by or released from or derived from the nanoparticles, preferably when the nanoparticles are heated with a heating device such as a furnace. Preferably, the heat flux can be measured using an apparatus or using a thermal analysis method, or using differential thermal analysis (DTA) or using differential scanning calorimetry (DSC).

[0652] In one embodiment of the present invention, the heating step i of the purification method according to the present invention, wherein i is preferably an integer greater than or equal to 0, comprises at least one of the following stages, wherein:

[0653] - During the first phase, at t i1P During this time period, the temperature of the nanoparticles changes from temperature T i Raised to temperature T iav ,

[0654] - During the second phase, at t i2P During this time period, the temperature of the nanoparticles is maintained at temperature T iav ,as well as

[0655] - During the third phase, at t i3P During this time period, the temperature of the nanoparticles changes from T iav Reduce to T f .

[0656] The present invention also relates to a purification process according to the invention comprising at least one heating step i, wherein the heating step comprises at least one of the following first, second and / or third stages, wherein:

[0657] - During the first phase, at t i1P During this time period, the temperature of the nanoparticles changes from temperature T i Raised to temperature T iav ,

[0658] - During the second phase, at t i2P During this time period, the temperature of the nanoparticles is maintained at temperature T iav ,as well as

[0659] - During the third phase, at t i3P During this time period, the temperature of the nanoparticles changes from T iav Reduce to T f .

[0660] In some cases, T i and / or t i1P T iav and / or t i2P At least 1.0001, 1.1, 1.5, 2, 5, 10, or 100 times lower. In some cases, T iav Equal to T1, T2 or T3, and / or t i1P Equal to the temperature increase to T i In some other cases, T f and / or t i3P With T i and / or t i1PThe difference is no more than 1.0001, 1.1, 1.5, 2, 5, 10, 10 2 or 10 5 times.

[0661] The present invention also relates to a method according to the invention, preferably a purification method, wherein more than 10% by mass of carbon or carbon-containing material is removed from the nanoparticles, wherein this percentage is based on the ratio (% C AT -%C BT ) / %C BT , where %C AT and %C BT are the percentages of carbon or carbonaceous material before and after the nanoparticles are treated with the method, respectively.

[0662] In some cases, (%C AT -%C BT ) / %C BT Greater than 10 -50 , 10 -20 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 50, 75, 90, 95 or 99%. This may be the case when the purification method is effective or when the amount of carbon or carbonaceous material contained in the nanoparticles before the nanoparticles are treated with the purification method is below a certain threshold, preferably below 9, 90, 70, 60, 50, 40, 30, 20, 10 or 1%.

[0663] In some other cases, (%C AT -%C BT ) / %C BT Less than 99%, 90%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 1%. This may be the case when the purification process is not efficient or when the amount of carbon or carbonaceous material contained in the nanoparticles before the nanoparticles are subjected to the purification process is greater than a certain threshold, preferably greater than 99, 90, 70, 60, 50, 40, 30, 20, 10 or 1%.

[0664] In some other cases, (%C AT -%C BT ) / %C BT Between 0.1 and 100, 1 and 99, 10 and 99, 50 and 99, or between 80 and 99%.

[0665] The present invention relates to high-purity nanoparticles based on iron oxide obtained by the process according to the invention.

[0666] The present invention also relates to high-purity nanoparticles or high-purity nanoparticles not obtainable by this method.

[0667] In one embodiment of the present invention, the high purity nanoparticles, preferably the coating of these nanoparticles, contain: i) 0.8 to 0.999999999 g of iron oxide per gram of nanoparticles, and / or ii) 10 -40 to 10 5 Impurities per gram of nanoparticles range between μg.

[0668] In one embodiment of the invention, the high purity nanoparticles comprise less than 90%, 10%, 5%, 2%, preferably 1%, 0.5%, 0.4% or 0.3% by weight of carbon or carbonaceous material. In some cases, such a low carbon mass percentage enables the nanoparticles to be coated with a coating that is not derived from the cells that produced the nanoparticles.

[0669] In one embodiment of the present invention, the SAR (Specific Absorption Rate) of the high purity iron oxide nanoparticles is greater than 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 Watts per gram of nanoparticles. In some cases, the SAR of the nanoparticles is greatest when the amount of impurities in the nanoparticles is lowest. In some cases, the SAR of high-purity iron oxide nanoparticles is 10 -100 and 10 100 , 10 -1 and 10 5 , or 0.1 and 10 3 Watts per gram of nanoparticles. In some cases, the SAR of a nanoparticle is proportional to the slope (preferably the initial slope) of the change in temperature of the nanoparticle over time (ΔT / δt), preferably surrounded by, for example, water, biological material, a host site, or tissue, wherein (ΔT / δt) is preferably estimated in °C / sec, where SAR = α(ΔT / δt). In some cases, α = C v / C nano , where C v is the specific heat capacity, preferably of water, biomaterial, body part or tissue containing the nanoparticles, and C nano is the concentration of nanoparticles or the number or total amount of nanoparticles, preferably contained in water, biological material, body part or tissue. In some cases, SAR is measured by exposing high-purity iron oxide nanoparticles to radiation, preferably radiation that generates heat, preferably laser light, a magnetic field, an alternating magnetic field, sound waves, ultrasound, or radio frequency.

[0670] In one embodiment of the present invention, the size distribution of high purity iron oxide nanoparticles is less than 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 1, 10 -1 , 10 -2 or 10 -5 In some cases, the nanoparticle size distribution is low when the methods according to the present invention are capable of producing nanoparticles having a low size distribution.

[0671] In another embodiment of the present invention, high purity iron oxide nanoparticles, preferably at a concentration greater than 0 -6 , 10 -3 , 10 -1 , 1 or 10 mg nanoparticles per milliliter or per millicubic meter or per cell, destroying more than 1, 10, 10 3 , 10 6 or 10 9 cells.

[0672] The present invention also relates to high purity iron oxide nanoparticles with a concentration of more than 10 -50 , 10 -30 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 50, 10 2 , 10 3 or 10 5 Milligrams of nanoparticles or nanoparticles containing milligrams of iron are generated, preferably per cell, preferably per liter of pre-growth and / or growth medium / media.

[0673] The present invention also relates to nanoparticles obtained by the process according to the invention, wherein the yield of the nanoparticles is less than 10 50 , 10 30 , 10 10 , 10 5 , 10 2 , 10, 5, 1, 10 -1 , 10 -2 , 10 -3 or 10 -5 mg of nanoparticles or mg of iron contained in the nanoparticles, preferably per cell, preferably per liter of pre-growth and / or growth medium.

[0674] The present invention also relates to nanoparticles based on high-purity iron oxide according to the invention, wherein the high-purity iron oxide nanoparticles are magnetosomes.

[0675] In one embodiment of the present invention, the magnetosomes are nanoparticles produced by magnetotactic bacteria, which are preferably processed after at least one of the following steps: i) the nanoparticles are extracted and / or separated from the bacteria, preferably to obtain magnetosomes comprising crystalline minerals covered by a biofilm; ii) the biofilm is preferably removed using a purification step; iii) the magnetosomes are coated with a coating that does not originate from the nanoparticle-producing cells to stabilize them, preferably to prevent aggregation and / or deposition of the magnetosomes. The present invention also relates to a composition, medicament, medical device, diagnostic composition, therapeutic composition, or cosmetic composition comprising the high-purity iron oxide nanoparticles according to the present invention.

[0676] In another embodiment of the present invention, the high purity iron oxide nanoparticles produce: i) medical or therapeutic activity, such as by destroying pathological cells, viruses, bacteria, cancer cells, or by being less toxic to healthy cells than pathological cells, viruses, bacteria, cancer cells, ii) diagnostic activity, such as by detecting pathological cells, viruses, bacteria, cancer cells, or by being less toxic to healthy tissue, and / or iii) cosmetic activity, such as by improving a person's appearance.

[0677] In another embodiment of the present invention, the high purity iron oxide nanoparticles are non-immunogenic or non-pyrogenic. In this case, they preferably: i) attract or cause the appearance of a small number of immune cells, preferably less than 1, 5, 10, 10 3 , 10 10 , 10 50 or 10 100 immune cells and / or ii) produce an increase in the temperature of the organism below 10 5 , 10 3 , 10 2 , 50, 20, 10, 5, 2, 1 or 0.1C.

[0678] The present invention also relates to nanoparticles according to the invention, preferably nanoparticles based on high-purity iron oxide, for use in treating a disease, which is preferably selected from the group consisting of: i) diseases associated with cell proliferation which differs from cell proliferation in a healthy individual, ii) diseases associated with the presence of pathological cells, such as tumors or cancer cells in a body part or an individual, iii) diseases associated with the presence of a pathological site, i.e. a site in an individual or a body part containing pathological cells iv) a disease or condition or dysfunction of a body site, v) a disease associated with the presence of cells which are resistant to radiation, sound waves, laser light or magnetic fields, vi) infectious diseases, vii) autoimmune diseases, viii) neuropathologies, ix) cancer, x) tumors, xi) diseases or tumor cells which contain or are caused by at least one cancer, xii) skin diseases, xiii) endocrine diseases, xiv) eye diseases or disorders, xv) intestinal diseases, xvi) communication disorders, xvii) genetic diseases, xviii) nervous system diseases, xix) speech disorders, xx) vulvovaginal diseases, xxi) liver diseases, xxii) heart diseases, xxiii) heating disorders, xxiv) mood disorders, xxv) anemia, preferably iron deficiency, xxvi) personality disorders, xxvii) AIDS, especially neurological disorders, xxviii) Parkinson's disease, xxix) Alzheimer's disease, xxx) bacterial and / or fungal infections or contamination, xxxi) blood diseases, for example due to the absence or lack of effective coagulation, and xxxii) diseases due to immune deficiencies or immune diseases.

[0679] In one embodiment of the present invention, the cancer or tumor is selected from the group consisting of organ cancer, blood cancer, biological system cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, heart cancer, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma cancer, ovarian cancer, pancreatic cancer, pancreatic cancer, Penile cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma cancer, vaginal cancer, vulvar cancer, Waldenstrom disease, Castillo disease, Ewing family of tumors, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, myelodysplastic syndrome pituitary tumors, as well as gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, malignant mesothelioma and multiple myeloma.

[0680] In another embodiment of the present invention, the use of the nanoparticles according to the present invention for disease treatment occurs or is activated when the nanoparticles are exposed to radiation, and preferably is not initiated or activated when the nanoparticles are not exposed to radiation.

[0681] In another embodiment, the radiation is laser light, sound waves, such as ultrasound, X-rays, gamma rays and / or a magnetic field, preferably an alternating magnetic field.

[0682] In another embodiment, the power or intensity of the radiation is between 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 ,0,1mT,T,W,W / cm,W / cm 2 or W / cm 3 and 1, 5, 10, 10 3 , 10 5 , 10 10 , 10 20 or 10 50 mT, T, W, W / cm, W / cm 2 or W / cm 3 between.

[0683] In another embodiment of the present invention, the properties or characteristics, preferably of the nanoparticle or method, described in each individual embodiment or part or sentence of the present patent application can be combined to produce properties or characteristics, preferably of the nanoparticle or method.

[0684] In another embodiment of the present invention, when a compound such as a nanoparticle or a chemical element has a property in condition 1 (P1) that is higher, longer or greater by a multiple α than in condition 2 (P2), this means P1 = αP2 or P1 = α+P2, where α is preferably a number or integer greater than 1 or 0.

[0685] In another embodiment of the present invention, when a compound such as a nanoparticle or a chemical element has a smaller or shorter factor α in condition 1 (P1) than in condition 2 (P2), this means P1 = P2 / α or P1 = P2-α, where α is preferably a number or integer greater than 1 or 0.

[0686] The present invention is further disclosed by the following non-limiting examples.

[0687] example

[0688] Materials and Methods

[0689] Optical density measurements of all magnetotactic bacterial suspensions used to evaluate bacterial growth: The optical density of different suspensions of magnetotactic bacteria was measured at 565 nm and designated as OD 565nm , using Secomam UviLine9400 spectrophotometer. OD 565nm The value is proportional to the concentration of bacteria in the suspension.

[0690] Live magnetotactic bacteria were observed and their magnetic response was measured using an optical microscope under an applied magnetic field: 1 mL of the MSR-1 magnetotactic bacterial suspension was centrifuged at 14500 rpm for 10 min. The growth medium was removed and replaced with a volume of PBS 0.1X to reach an OD of 565nm 1 μL of this MSR-1 magnetotactic bacterial suspension was placed on parallel microscope slides (Menzel- 24 mm × 60 mm, 0.13-16 mm thickness) and microscopic observations were performed using a Zeiss Primo Vert optical microscope with 40x magnification. Four small cubic neodymium magnets with a strength of 1.3 T (Supermagnet, N42W-10-N 10x10x10 mm) were placed on the microscope stage and at a distance of 2 cm from the bacterial suspension so that the magnetic field they generated was parallel to the position of the observer or parallel to the line between the two binoculars (position 1) or perpendicular to it (position 2). After positioning the magnet in position 1 or 2 for 20 seconds, the percentage of bacteria aligned in the direction of the magnetic field was estimated by considering 200 magnetotactic bacteria. Bacteria that were not aligned in the same direction as the magnetic field generated by the magnet were considered non-magnetic. Their number was designated n BNM Bacteria that align in the direction of the magnetic field produced by the magnet are said to be magnetic. Their number is designated as n BM Then through n BM / (n BM +n BNM ) gives the percentage of magnetic bacteria. The positive magnetic response of magnetotactic bacteria corresponds to n BM / (n BM +n BNM )>0.5. The negative magnetic response of magnetotactic bacteria corresponds to n BM / (n BM +n BNM ) < 0.5. In some cases, the percentage of magnetic bacteria can be measured by optical observation under a microscope in the presence of a magnetic field.

[0691] Measurement of intracellular iron concentration: The iron concentration in magnetotactic bacteria was determined by a destructive iron assay. To this end, 2 mL of MSR-1 magnetotactic bacteria were centrifuged at 14500 g for 10 minutes. The bacterial pellet was then washed twice with 1X PBS and MilliQ water. After the second wash, the bacterial pellet was collected and 1 mL of 12N hydrochloric acid (HCl) was added to the pellet under a chemical hood. The sample was heated at 75°C for 2 hours with stirring at 300 rpm to convert the intracellular iron into Fe 3+ and Fe 2+ ions. Then, Fe 2+Ions are oxidized to Fe 3+ The addition of potassium thiocyanate (KCN, 2 mol / L) to an acidic medium showed that Fe 3+ ions, which results in the formation of a reddish-orange solution, the color of which depends on the Fe 3+ Once KCN is added, the absorbance of the solution is measured at 476 nm. The iron concentration in the sample is then estimated using the established relationship between the absorbance value measured at 476 nm and the concentration in iron (III) chloride. This method can estimate the total intracellular iron concentration.

[0692] The elemental chemical composition of the magnetosomes was analyzed by ICP-AES. After fermentation, MSR-1 magnetotactic bacteria were concentrated in a 5-liter volume by cross-sectional flow filtration to an optical density of 25-30. The bacteria were then dissolved in a 1 M KOH solution over one hour at 80°C and stirred at 150 rpm. The bacterial lysate containing the magnetosomes was placed on a neodymium magnet for 12 hours. The magnetosomes were then separated from the bacterial lysate and resuspended in 10X PBS. This washing procedure was repeated twice with 10X PBS and three times in MilliQ water. The magnetosomes were then lyophilized and heated in a muffle furnace under the following conditions to obtain a magnetosome powder consisting of high-purity iron oxide crystals with a low carbon content. To analyze the elemental chemical composition, a solution of 500 μg of this powder was mixed with 200 μl of 12N HCl and 10 ml of 2% filtered HNO₃. ICP-AES measurement of the powder gives the amount of chemical elements contained in the magnetosomes in μg of these chemical elements per gram of iron contained in the magnetosomes (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Si, Sn, Ti, Tl, W and Zn).

[0693] Chemical products used for the preparation of growth medium: potassium aluminum sulfate dodecahydrate (AlK(SO4)212H2O, ref. NFGA6435, Merck); ammonium hydroxide (NH4OH, ref. NFG 1336-21-6, Acros Organics; ref. FG 105422, Merck); ammonium chloride (NH4Cl, ref. FNG A9434, Merck; ref. FG 1011420001, Merck); ammonium sulfate (NH4)2SO4, ref. NFG A4418); biotin ((C 10 H 16N2O3S, cf. NFG B4639, Merck; cf. FG B301, Merck); boric acid (H3BO3, cf. NFG B6768, Merck); calcium chloride (CaCl2, cf. FNG 223506, Merck; cf. FG1.42002, Merck); calcium pantothenate (HOCH2C(CH3)2CH(OH)CONHCH2CH2CO2·1 / 2Ca, cf. FG C0400000, Merck); cobalt(II) nitrate hexahydrate (cobalt(II) hexahydrate, cf. FG 239267, Merck); copper(II) sulfate pentahydrate (CuO4S·5H2O) (cf. NFG C8027, Merck), DL-methionine (CH3SCH2CH2CH(NH2)COOH, cf. NFG M2768, Merck), DL-tryptophan (C11H12N2O2, reference NFG T3300, Merck); EDTA ((HO2CCH2)2NCH2CH2N(CH2CO2H)2, reference NFG E6758, Merck); ferric citrate (C6H5FeO7, reference NFG F3388, Merck; reference FG B301, Merck); folic acid (C 19 H 19 N7O6, reference NFG F7876, Merck; reference FG F0300000, Merck); inositol (C6H 12O6, reference FG PHR1351, Merck); iron (II) sulfate heptahydrate (FeO4S.7H2O, reference NFG F8633, Merck; reference FG 1.03963, Merck); iron (III) oxalate hexahydrate (Fe2(C2O4)3.6H2O, reference NFG381446, Merck); L-histidine (C6H9N3O2, reference FG PHR1108, Merck); magnesium sulfate heptahydrate (MgSO4.7H2O, reference NFG 63138, Merck; reference FG 105882, Merck); manganese (II) sulfate monohydrate (MnO4S.H2O, reference NFG M7899, Merck); nickel (II) chloride hexahydrate (Cl2Ni.6H2O, reference NFG N6136, Merck); nicotinic acid (C6H5NO2, reference NFG N4126, Merck); trisodium nitrilotriacetic acid ((C6H6NO6Na3, reference NFGN0253, Merck); p-aminobenzoic acid (H2NC6H4CO2H, reference NFG A9878, Merck); dipotassium hydrogen phosphate (K2HPO4, reference NFG P8281, Merck; reference FG 105101, Merck); potassium dihydrogen phosphate (KH2PO4, reference NFGP9791, Merck); protoporphyrin IX (C 34 H 34 N4O4, ref. NFG P8293, Merck); pyridoxine hydrochloride (C 12 H 17 ClN4OS.HCl, ref. FNG P9755, Merck); riboflavin (C 17 H 20 N4O6, ref. NFG R9504, Merck; ref. FG PHR1054, Merck); sodium chloride (NaCl, ref. FNG S7653, Merck); sodium lactate (C3H5NaO3, ref. NFGL1375, Merck; ref. FG 106522, Merck); sodium molybdate dihydrate (Na2Mo4.2H2O, ref. NFG M1003, Merck); sodium selenite pentahydrate (Na2SeO3.5H2O, ref. FG 89771, Merck); thiamine HCL (C 12 H 17ClN₄OS.HCl, ref. FNG47858, Merck, ref. FG PHR1037, Merck); yeast extract (ref. NFGY1625, Merck); zinc sulfate heptahydrate (O₄SZn.7H₂O, ref. NFG Z0251, Merck). NFG designates non-pharmaceutical-grade chemicals used to prepare growth medium; FG designates pharmaceutical-grade chemicals used to prepare growth medium. We also used deionized water (H₂O) with a resistivity of 15 MΩ.

[0694] Composition of different mineral elixirs: The composition of different mineral elixirs (V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12, CB13) is shown in Table 6, where the quantities (in grams) represent the different chemicals used to prepare 1 liter of these elixirs.

[0695] Composition of Different Yeast Extracts: The compositions of different yeast extracts (YE, YNBWAA, YNBWoAA, YNBWoAA.AS) are given in Table 7, where the amounts of different chemicals used to prepare 1 liter of these yeast extracts are given in grams. YNBWAA, YNBWoAA, and YNBWoAA.AS represent reduced yeast extracts, while YE represents non-reduced yeast extract (reference: Y0875, Sigma). YE contains nitrogenous compounds, carbon, sulfur, micronutrients, vitamin B complex, and other important growth factors.

[0696] Composition of different vitamin cocktails: The composition of different vitamin cocktails (Vit1X, Vit5X, Vit10X, Vit0.5X, Vit0.1X) is given in Table 8, where the amounts of different chemicals (in grams) used to prepare 1 liter of these vitamin cocktails are given.

[0697] The composition of the pregrowth medium for condition 1 (Table 1): 1 liter of pregrowth medium contained 1 liter of deionized water, 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g potassium phosphate dibasic, 0.1 g yeast extract YE (Table 7) and 0.5 ml of any one of mineral elixir V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12 or CB13 (Table 6).

[0698] Composition of the growth medium for Condition 1 (Table 1): 1 L of growth medium contained 1 L of deionized water, 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g potassium phosphate dibasic, 0.1 g yeast extract YE (Table 7), 0.5 mL of any one of mineral elixir V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12 or CB13 (Table 6) and 10 mL of ferric citrate (20 mM initial concentration).

[0699] Composition of the pre-growth medium for condition 2 (Table 2): 1 liter of pre-growth medium contained 1 liter of deionized water: 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g dipotassium phosphate, 0.1 g yeast extract any one of YE, YNBWAA, YNBWoAA, YNBWoAA.AS (Table 7) and 0.5 mL mineral elixir CB3 (Table 6).

[0700] Composition of the growth medium for Condition 2 (Table 2): 1 L of growth medium contained 1 L of deionized water, 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of potassium phosphate dibasic, 0.1 g of yeast extract YE, YNBWAA, YNBWoAA or YNBWoAA.AS (Table 7), 0.5 mL of mineral elixir CB3 (Table 6) and 10 mL of ferric citrate (20 mM initial concentration).

[0701] The composition of the pre-growth medium for condition 3 (Table 3): 1 liter of pre-growth medium contained 1 liter of deionized water, 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of potassium phosphate dibasic, 0.1 mM of any one of vitamins Vit1X, Vit5X, Vit10X, Vit5X, Vit0.5X or Vit0.1X (Table 8) and 0.5 mL of mineral elixir CB3 (Table 6).

[0702] The composition of the growth medium for condition 3 (Table 3): 1 liter of growth medium contained 1 liter of deionized water, 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g dipotassium phosphate, 0.1 mL of vitamin cocktail Vit1X, Vit5X, Vit10X, Vit5X, Vit0.5X or Vit0.1X (Table 8), 0.5 mL of mineral elixir CB3 (Table 6) and 10 mL of ferric citrate (20 mM initial concentration).

[0703] The composition of the pre-growth medium for condition 4 (Table 4): 1 liter of pre-growth medium contained 1 liter of deionized water, 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g dipotassium phosphate, 0.1 mL of any one of vitamin Bt, CP, FA, I, NA, AA, P, R or T (Table 9), and 0.5 mL of mineral elixir CB3 (Table 6).

[0704] Composition of growth medium for condition 4 (Table 4): 1 L of growth medium contained 1 L of deionized water, 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g potassium phosphate dibasic, 0.1 mL of individual vitamins Bt, CP, FA, I, NA, AA, P, R or T (Table 9), 0.5 mL of mineral elixir CB3 (Table 6) and 10 mL of ferric citrate (20 mM initial concentration).

[0705] Composition of the pre-growth medium for condition 5 (Table 5): Table 5 gives the compositions of different pre-growth media with different concentrations of the main components of the pre-growth medium, namely sodium lactate, ammonium chloride, magnesium sulfate heptahydrate, dibasic potassium phosphate (N, SL0, SL0.5X, SL0.2X, SL0.1X, ACO, AC0.5X, AC0.2X, AC0.1X, MG0, MG0.5X, MG0, MG0.5X, MG0.2X, MG0.1X, P, P0.5X, P0.2X, P0.1X), where the amounts of different chemicals used to prepare 1 liter of these pre-growth media are shown (in grams).

[0706] The composition of the pre-growth medium, growth medium, and fed-batch medium for Condition 6 prepared using non-pharmaceutical grade chemicals (Table 14(a)): Pre-growth medium B1 and B4 contained 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g potassium phosphate dibasic, 0.1 mL vitamin mix Vit 0.1X (Table 8), and 0.5 mL mineral elixir CB3 (Table 6) in 1 liter of deionized water. Growth medium B1 and B4 contained 104 g sodium lactate, 16 g ammonium chloride, 1.2 g magnesium sulfate heptahydrate, 2.8 g potassium phosphate dibasic, 3.2 mL vitamin mix Vit 0.1X (Table 8), and 2.8 mL mineral elixir CB3 (Table 6) in 1 liter of deionized water. Fed-batch media B1 and B4 contained in 1 liter water, 100 g lactic acid, 4.8 g ammonia, 6 g potassium phosphate dibasic, 2.4 g magnesium sulfate heptahydrate, 1 mL vitamin mixture Vit 0.1X (Table 8), 7 mL mineral elixir CB3 (Table 6), and 1.8 g ferric citrate (B1) or 2 g iron III chloride (B4).

[0707] The compositions of the pre-growth medium, growth medium, and fed-batch medium for Condition 6 were prepared using pharmaceutical grade chemicals (Table 14(b)): Pre-growth medium B2 and B3 contained 2.6 g sodium lactate, 0.4 g ammonium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g potassium phosphate dibasic, 0.1 mL vitamin mix Vit 0.1X (Table 8), and 0.5 mL mineral elixir CB3 (Table 6) in 1 liter of deionized water. Growth medium B1 and B4 contained 104 g sodium lactate, 16 g ammonium chloride, 1.2 g magnesium sulfate heptahydrate, 2.8 g potassium phosphate dibasic, 3.2 mL vitamin mix Vit 0.1X (Table 8), and 2.8 mL mineral elixir CB3 (Table 6) in 1 liter of deionized water. Fed-batch media B1 and B4 contained 100 g lactic acid, 4.8 g ammonia, 6 g potassium phosphate dibasic, 2.4 g magnesium sulfate heptahydrate, 1 mL vitamin mixture Vit 0.1X (Table 8), 7 mL mineral elixir CB3 (Table 6), and 1.8 g ferric citrate (B2) or 2 g ferric chloride (B3) in 1 liter of water.

[0708] Stocks of MSR-1 magnetotactic bacteria for different cultures: MSR-1 magnetotactic bacteria are commercialized by DSMZ under the identifier DSM 6361. Upon receipt, MSR-1 bacterial suspensions were stored at an OD of 0.01. 565nm (optical density measured at 565 nm), equivalent to a bacterial concentration of 5.10 in a 15 mL test tube in a -80 °C refrigerator. 7 For each milliliter of culture medium (medium DSMZ 380 for culturing the MSR1 strain DSMZ 6361), add (5 mL of bacterial suspension per tube) or a 1.5 mL Eppendorf tube (600 μL of bacterial suspension per tube). The suspension of MSR-1 bacteria was stored in a -80°C freezer to form a cell stock. In some cases, the culture medium and / or pre-growth medium can be the same as the growth and / or pre-growth medium.

[0709] Note: The number X after the D in DX indicates the number of days after the start of the pregrowth step, preferably the day when magnetotactic bacteria are first added to the pregrowth medium or the first sub-step of the pregrowth step.

[0710] Example 1: Determination of the minimum mineral elixir capable of bacterial growth and magnetosome synthesis: This example describes an experimental protocol for minimizing the composition of the mineral elixir while allowing the growth of MSR-1 magnetotactic bacteria and the synthesis of magnetosomes by these cells. In this example, non-pharmaceutical grade chemicals were used to prepare the growth medium. The composition of 1 liter of pre-growth and growth medium used in this example (Condition 1) is shown in Table 1. On the first day of the experiment (D1), the first step involved removing a 15 mL test tube containing a 5 mL MSR-1 cell stock tube from the freezer at -80°C and placing it at room temperature for 10 minutes to thaw. In a fume hood, we collected 100 μl of 5.10 MSR-1 cell stock tubes from these tubes. 6 MSR-1 magnetotactic bacteria were placed in a 50 mL tube containing 8 mL of filtered pre-growth medium. Overall, the 13 different culture conditions tested corresponded to the 13 different mineral elixirs tested. The 50 mL tubes were placed in an incubator at 29.5°C and incubated with shaking at 150 rpm for 6 days between D1 and D6. The second step involved adding an iron source to the growth medium to enable the MSR-1 bacteria to synthesize magnetosomes. After 6 days of pre-growth on D6, the 50 mL tubes were placed in a fume hood. 30 mL of filtered medium was then added to the 50 mL tubes, and the bacteria were grown between D6 and D13. A positive magnetic response at D13 and a ratio of optical density at D13 to optical density at D6 greater than 1 were observed for V0, V2, CB2, CB3, CB4, CB5, CB7, CB10, CB11, CB12, and CB13 (Condition 1). In contrast, there was no magnetic response under conditions CB1 and CB9, where the concentrations of chemical elements constituting the mineral elixir were less than 10 -5 g / L. In short, it can greatly increase the optical density (OD 565nmD13 / OD 565nmD6 The minimum mineral elixir for the growth of MSR-1 bacteria that exhibited a pH greater than 4.8 and magnetosome synthesis (positive magnetic response) was CB13, consisting only of ferric sulfate heptahydrate at a concentration of 1 g / L and calcium chloride at a concentration of 20 g / L.

[0711] Example 2: Determination of growth medium without yeast extract to allow the growth of magnetotactic bacteria and the synthesis of magnetosomes: This example describes an experimental protocol for determining the ability of reducing medium to replace yeast extract to allow the growth of MSR-1 magnetotactic bacteria and the synthesis of magnetosomes with these bacteria. In this example, we used non-pharmaceutical grade chemicals to prepare the growth medium. The composition of the pre-growth and growth medium in 1 liter of deionized water is shown in Table 2 (Condition 2), Table 3 (Condition 3) and Table 4 (Condition 4). On the first day of the experiment (D1), the first step was to collect 15 ml test tubes containing 5 mL MSR-1 cell stock tubes from the refrigerator at -80°C and thaw the test tubes by placing them at room temperature for 10 minutes. In the fume hood, we collected 100 μl of 5.10 6 MSR-1 magnetotactic bacteria were placed in a 50 mL tube filled with 8 mL of filtered pre-growth medium (Condition 2 (Table 2), Condition 3 (Table 3)), or Condition 4 (Table 4)). The 50 mL tubes were placed in an incubator at 29.5°C and cultured for 6 days between D1 and D6 with shaking at 150 rpm. The second step involved adding an iron source to the growth medium to enable the MSR-1 bacteria to synthesize magnetosomes. After 6 days of pre-growth on D6, the 50 mL tubes were placed in a fume hood and 30 mL of filtered growth medium was added to the 50 mL tubes (Condition 2, Table 2, Condition 3, Table 3, Condition 4, Table 4), and the bacteria were grown between D6 and D13. Tables 11 and 12 show that for yeast extracts YE, YNBWAA, YNBWAA, YNBWoAA, YNBWoAA, YNBWoAA.AS (Condition 2), for Vit1X, Vit0.5X, Vit0.1X, (Condition 3), biotin (Bt), folic acid (FA), niacin (NA), riboflavin (R), thiamine (T) (Condition 4), the magnetic response at D13 was greater than 90% and the ratio between the optical density measured at D13 and the optical density measured at D6 was greater than 1. In contrast, for the conditions Vit5X, Vit10X (Condition 3), the magnetic response was 0, and for the conditions CP, I, AA, P (Condition 4), the magnetic response was very low. In summary, yeast extract can be replaced with a single vitamin: biotin, folic acid, riboflavin, niacin, or thiamine. The OD values ​​generated by these vitamins were significantly higher than those of the control group. 565nmD13 / OD 565nmD6 The values ​​were 9.8 (biotin), 2.9 (folic acid), 4.8 (riboflavin), 2.4 (niacin), 5.8 (thiamine) and a magnetic response of 90% (Table 11).

[0712] Example 3: Determination of Minimum Concentrations of the Main Components of the Growth Medium (Sodium Lactate, Ammonium Chloride, Magnesium Sulfate, Potassium Phosphate) for Growth of Magnetotactic Bacteria and Synthesis of Magn...

Claims

1. A method for preparing high-purity iron oxide nanoparticles using nanoparticle production cells, comprising: a) a pre-growth phase comprising expanding said nanoparticle producing cells in pre-growth and / or fed-batch culture medium, as well as b) a growth phase comprising expanding said nanoparticle-producing cells derived from said pre-growth phase in a growth and / or fed-batch medium, Wherein, the pre-growth and / or fed-batch medium and / or the growth and / or fed-batch medium comprises per kilogram or per liter of the pre-growth and / or fed-batch medium and / or the growth and / or fed-batch medium: i) not more than 0.005 g of yeast extract, as well as ii) not more than 0.001 g of a CMR agent selected from the group consisting of boric acid and nitrilotriacetic acid, wherein, when a fed-batch medium is present, the fed-batch medium is a medium supplementing the pre-growth and / or growth medium, and The nanoparticles generated in the growth stage are more than those generated in the pre-growth stage.

2. The method of claim 1 , wherein the growth stage is distinguished from the pre-growth stage by at least one characteristic selected from the group consisting of: i)C FeGS / C FePGS The ratio is greater than 1, where C FeGS and C FePGS are the concentrations of iron or iron sources in the growth medium and pre-growth medium, respectively, ii)C CGS / C CPGS The ratio is greater than 1, where C CGS and C CPGS are the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively, iii)C NGS / C NPGS The ratio is greater than 1, where C NGS and C NPGS are the concentrations of nitrogen or nitrogen sources in the growth medium and pre-growth medium, respectively, iv)ΔpH GS / ΔpH PGS The ratio is less than 1, where ΔpH GS and / ΔpH PGS are the pH changes in the growth medium and pre-growth medium, v)Q GGS / Q GPGS The ratio is greater than 1, where Q GGS and Q GPGS is the amount of gas, oxygen or air introduced or bubbled into the growth medium and pre-growth medium, respectively, vi)N SSGS / N SSPGS The ratio is less than 1, where N SSGS and N SSPGS are the number of sub-stages of the growth stage and the number of sub-stages of the pre-growth stage, respectively, wherein the two sub-stages are separated from each other by transferring the nanoparticle-producing cells from the first sub-stage to the second sub-stage, and vii) The growth medium is supplemented by the fed-batch medium, whereas the pre-growth medium is not supplemented by the fed-batch medium.

3. The method according to claim 1, wherein The pre-growth and / or fed-batch medium and / or the growth and / or fed-batch medium comprises per kilogram or per liter of pre-growth and / or fed-batch medium and / or growth and / or fed-batch medium less than: i) By mass or volume 5.10 -3 % or 0.5g or 0.5mL or 10 -8 mol or 10 -9 mol of a vitamin or chemical component selected from the group consisting of folic acid, folate, pyridoxine, pyridoxine hydrochloride, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine hydrochloride, niacin, pantothenic acid, calcium pantothenate, inositol, p-aminobenzoic acid, aminobenzoic acid, lipoic acid, all-trans retinol, retinal, alternative provitamin A-functionalized carotenoids including all-trans beta-carotene, niacin, niacinamide, niacinamide, nucleosides, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherols, tocotrienols, theoquinone, menadione, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 , Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i and their derivatives, where V can be any letter from A to Z, and i can be any integer from 1 to 100, ii) 6 different vitamins or chemical components selected from the group consisting of folic acid, folate, pyridoxine, pyridoxine hydrochloride, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine hydrochloride, niacin, pantothenic acid, calcium pantothenate, inositol, para-aminobenzoic acid, aminobenzoic acid, lipoic acid, all-trans retinol, retinal, alternative provitamin A-functionalized carotenoids including all-trans beta-carotene, niacin, niacinamide, niacinamide, nucleosides, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherols, tocotrienols, theoquinone, menadione, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 , Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i and their derivatives, where V can be any letter from A to Z, and i can be any integer from 1 to 100, iii) by mass or volume 10 -2 % or 1g or 1mL or 10 -7 mol or 10 -8 mol of a mineral or chemical component selected from the group consisting of nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, hydrated copper sulfate, potassium aluminum sulfate, potassium aluminum sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and derivatives thereof, iv) 7 minerals or chemical components selected from the group consisting of nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, hydrated copper sulfate, potassium aluminum sulfate, potassium aluminum sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and derivatives thereof, v) 0.005g or 10 -8 at least one component of a yeast extract of M or at least one compound derived from a yeast extract, said component or compound being selected from the group consisting of: at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, glucan, mannan, trehalose, flavor nucleotides, B vitamins, biotin, at least one volatile aroma compound, calcium, phosphorus, zinc, iron, chromium, potassium, cobalt, manganese, strontium, magnesium, and derivatives thereof, vi) 5 different components of yeast extract or different compounds derived from yeast extract, said components or compounds being selected from the group consisting of at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, glucan, mannan, trehalose, flavor nucleotides, B vitamins, biotin, at least one volatile aroma compound, calcium, phosphorus, zinc, iron, chromium, potassium, cobalt, manganese, strontium, magnesium and derivatives thereof, vii) 0.01 g or 10 -8 at least one component of peptone or at least one compound derived from peptone of M, said component or compound being selected from the group consisting of ash, protein, sucrose, stachyose, raffinose, neutral detergent fiber, ether extracts and derivatives thereof, viii) 5 different components of peptone or different compounds derived from peptone, said components or compounds being selected from the group consisting of ash, protein, sucrose, stachyose, raffinose, neutral detergent fiber, ether extracts and derivatives thereof, ix) 0.001 g of EDTA, x) 0.001 g of at least one amino acid, xi) 5 different amino acids, xii) 12 different CMR, toxic or cytotoxic compounds selected from the group consisting of nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride and their derivatives, xiii) 5 chemical elements or heavy metals selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper and their derivatives, xiv)10 -5 g of a chemical element or heavy metal selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony and copper and their derivatives, xv)10 -5 g at ​​least one CMR, toxic or cytotoxic compound selected from the group consisting of nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride and derivatives thereof, and / or xvi) 0.01 g of peptone.

4. The method according to claim 1, wherein the concentration of at least one compound in the pre-growth and / or growth medium is C2 or C 总量 =C1+C2, where: C1 is the concentration of the at least one compound in the pre-growth medium and / or growth medium that has not been consumed by the nanoparticle-producing cells, C2 is the concentration of the at least one compound in the pre-growth medium and / or the growth medium consumed by the nanoparticle-producing cells, as well as C1 and C2 are measured or taken into account at the beginning, during or at the end of the pre-growth phase and / or the growth phase.

5. A high-purity nanoparticle-producing cell obtained by the method according to claim 1, wherein the high-purity nanoparticle-producing cell comprises greater than 50% of: i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the high-purity nanoparticle-producing cells, M MC is the mass of iron and metals or metalloids other than iron in the high-purity nanoparticle production cell, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all chemical elements contained in the high purity iron oxide nanoparticles. A composition comprising the high-purity nanoparticle-producing cell according to claim 5 .

7. Nanoparticles based on high-purity iron oxide obtained by the method according to claim 1, said nanoparticles based on high-purity iron oxide comprising more than 93% of: i) Iron, based on M FeN / M MN The ratio of M FeN is the mass of iron in the high-purity iron oxide nanoparticles, and M MN is the mass of iron and metals or metalloids other than iron in high-purity iron oxide nanoparticles, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles.

8. A composition comprising the high-purity iron oxide-based nanoparticles according to claim 7.

9. A composition comprising high-purity nanoparticle-producing cells obtained by the method according to claim 1 and high-purity iron oxide nanoparticles, wherein: The high-purity nanoparticle production cells contain greater than 50% of: i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the high-purity nanoparticle-producing cells, and M MC is the mass of iron and metals or metalloids other than iron in the high-purity nanoparticle-producing cell, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles, as well as The high-purity iron oxide-based nanoparticles contain greater than 93% of: i) Iron, based on M FeN / M MN The ratio of M FeN is the mass of iron in the high-purity iron oxide nanoparticles, and M MN is the mass of iron and metals or metalloids other than iron in high-purity iron oxide nanoparticles, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles.

10. A composition comprising high-purity nanoparticle-producing cells and / or high-purity iron oxide nanoparticles, wherein: The high-purity nanoparticle production cells contain greater than 50% of: i) Iron, based on M FeC / M MC The ratio of M FeC is the mass of iron in the high-purity nanoparticle-producing cells, and M MC is the mass of iron and metals or metalloids other than iron in the high-purity nanoparticle-producing cell, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles, as well as The high-purity iron oxide-based nanoparticles contain greater than 93% of: i) Iron, based on M FeN / M MN The ratio of M FeN is the mass of iron in the high-purity iron oxide nanoparticles, and M MN is the mass of iron and metals or metalloids other than iron in high-purity iron oxide nanoparticles, ii) iron and at least one other metal other than iron selected from the group consisting of sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungsten, based on the ratio M1 / M2, where M1 is the mass of the iron and the at least one other metal selected from the above group in the high purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high purity iron oxide nanoparticles, and / or iii) iron and at least one other non-metal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon and oxygen, based on the ratio M3 / M4, wherein M3 is the mass of the iron and at least one other non-metal selected from the above group in the high purity iron oxide nanoparticles, and M4 is the mass of all non-metal elements contained in the high purity iron oxide nanoparticles, wherein the high purity nanoparticle producing cells and / or the high purity iron oxide based nanoparticles are obtained by culturing nanoparticle producing cells in a growth medium that is substantially free of at least one metal or metalloid, or contains less than 1 nanomole of at least one metal or metalloid, wherein the at least one metal or metalloid is selected from the group consisting of: 1) cadmium, 2) lead, 3) arsenic, 4) mercury, 5) cobalt, 6) vanadium, 7) nickel, 8) lithium, 9) antimony, 10) copper, 11) vanadium, 12) molybdenum, 13) Selenium, 14) Barium, 15) Chromium, 16) Strontium, 17) Radioactive Chemical Elements, 18) Beryllium, 19) Rubidium, 20) Ruthenium, 21) Rhodium, 22) Palladium, 23) Promethium, 24) Ytterbium, 25) Tantalum, 26) Osmium, 27) Iridium, 28) Bismuth, 29) Polonium, 30) Francium, 31) Radium, 32) Actinium, 33) Thorium, 34) Protactinium, 35) Uranium, 36) Neptunium, 37) Plutonium, 38) Americium, 39) Curium, 40) Berkelium, 41) Californium, Einsteinium, 42) Fermium, 43) Mendelevium, 44) Nobelium, 45) Lawrencium, 46) Ruthenium, 47) 48) 49) 50) 51) 52) dammium, 53) terahertz, 54) cooperitonium, 55) niobium, 56) ferrum, 57) molybdenum, 58) lead, 59) astatine, 60) thorium, 61) argon, and 62) their derivatives.

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