Nanoparticles and preparation process

Abstract

Nanoparticles and preparation method The present invention relates to nanoparticles of metal chosen from platinum, gold or bismuth, functionalized at their surface by functionalized polyethylene glycol in particular comprising at least one OH, COOH, NH2 or SH function, and their preparation method comprising the following steps: a) Mixing a nanoparticle precursor with a functionalized polyethylene glycol in particular comprising at least one OH, COOH, NH2 or SH function in water; b) Exposing the mixture to ionizing radiation. Figure for abstract: none

Description

Title of the invention: Nanoparticles and preparation process

[0001] The present invention relates to platinum, gold and bismuth nanoparticles, usable in particular in the field of diagnosis and therapy and a process for preparing these nanoparticles.

[0002] Nanoparticles are generally synthesized chemically in several steps. Consequently, these syntheses have yields of less than 100% and are often difficult to reproduce. Furthermore, most of these methods require the use of more or less toxic chemical solvents and reducers. It is therefore necessary to provide rinsing and sterilization steps to make these particles biocompatible and non-toxic, thus allowing them to be injected into humans. These additional rinsing and sterilization steps lengthen the preparation time of the nanoparticles and reduce the yields.

[0003] There is therefore an interest in providing a process for preparing nanoparticles with a high yield making it possible to obtain non-toxic and biocompatible particles in a single step.

[0004] An objective of the present invention is to provide a method for preparing nanoparticles with high yield and with the possibility of scale-up.

[0005] Another objective of the present invention is to provide such a method making it possible to obtain non-toxic and biocompatible particles in a single step.

[0006] Yet another objective of the present invention is to provide non-toxic, biocompatible and sterile nanoparticles which can in particular be used in diagnosis and therapy by ionizing radiation (radiotherapy, hadrontherapy, protontherapy, Curie therapy).

[0007] Still other objectives will appear on reading the description of the invention which follows.

[0008] The present invention relates to nanoparticles of metal chosen from platinum, gold or bismuth, functionalized at their surface by functionalized polyethylene glycol.

[0009] In the context of the present invention, functionalized polyethylene glycol is understood to mean a polyethylene glycol comprising at least one function allowing interaction or ligandation with the surface of platinum, gold or bismuth, for example these functions are OH, NH2, SH, COOH functions.

[0010] Preferably, the nanoparticles have an average diameter of between 1 nm and 100 nm, preferably between 1 nm and 10 nm. The average diameter of the nanoparticles is obtained by transmission electron microscopy (TEM).

[0011] Preferably, the nanoparticles have an average hydrodynamic diameter of between 5 and 100 nm, preferably between 10 and 20 nm. In the context of the present invention, the hydrodynamic diameter is understood to mean that which is measured by DLS.

[0012] Preferably, the nanoparticles of the invention have an average zeta potential of between -5 and -30 mV, preferably between -10 and -20 mV. The average zeta potential is preferably measured by zetametry.

[0013] In a particularly advantageous and preferred manner, the nanoparticles of the invention do not present any trace of solvent other than water.

[0014] The present invention also relates to an aqueous solution comprising the nanoparticles according to the invention described above.

[0015] The present invention also relates to a process for preparing nanoparticles of metal chosen from platinum, gold or bismuth, in particular the nanoparticles of the invention described above, comprising the following steps: a) Mixing a nanoparticle precursor with a functionalized polyethylene glycol in water; b) Expose the mixture to ionizing radiation: gamma radiation, X-rays, accelerated electrons or accelerated ions.

[0016] Preferably the metal is platinum.

[0017] In the context of the present invention, the term “nanoparticle precursor” means any compound known to those skilled in the art that allows the preparation of nanoparticles. In particular, the precursors are chosen from platinum salts (in particular tetraamine platinum (II) chloride (Pt(NH3)4Cl2, H2O), chloroplatinic acid (H2PtCl6), potassium tetrachloroplatinate (K2PtCl4)), gold salts (in particular chloroauric acid (HAuC14)), bismuth salts (in particular bismuth chloride (BiCl3), bismuth nitrate (Bi(NO3)3), and oxides (in particular bismuth oxide (Bi2O3)).

[0018] After step b), the particles can be lyophilized in particular for storage, the particles then being able to be resuspended, for example in water, before use.

[0019] Preferably, the mixture is degassed under inert gas, for example nitrogen or argon, before exposure to ionizing radiation.

[0020] Preferably, the solution is irradiated with ionizing radiation, preferably with energies between 5 keV and 5 MeV, such as gamma radiation from a Cobalt 60 source, X-rays, accelerated electron beams or accelerated ions, in particular medical radiation from carbon ions as used in hadrontherapy.

[0021] Particularly advantageously, the doses used for the reduction of platinum, gold or bismuth depend on the precursor, the initial precursor concentration and the irradiation source: they vary from 1 to 20 kGy for metal concentrations of 0.5 to 20 mM, for example it is typically 10 kGy to completely reduce 103 mol / L of metal complex containing a salt comprising Pt2+. The gamma radiation exposure step thus lasts between 1 and 20 hours, at a dose rate of 95.5 Gy.min *.

[0022] The functionalized polyethylenes used in the process of the invention are, for example, PEG-OH, HO-PEG-COOH, NH2-PEG-SH, PEG-2NH2 and PEG-SH. Advantageously, the use of functionalized polyethylene glycol makes it possible to provide stability and biocompatibility to the nanoparticles obtained. Preferably, the polyethylene glycols used according to the invention have a molar mass MW of between 600 and 3000, preferably between 1000 and 2000 g / mol. In a particularly preferred manner, the polyethylene glycols used according to the invention are PEG-OH and PEG-2NH2, preferably having a MW of between 1000 and 2000 g / mol.

[0023] In the process according to the invention, the nanoparticle precursor is preferably used in a proportion of 10 4 mol / L to 10 2 mol / L, preferably 3x10 3 mol / L to 5x10 4 mol / L, in molar concentration in the mixture of step a).

[0024] In the process according to the invention, the functionalized polyethylene glycol is preferably used at a rate of 103 to 5x10 1 mol / L and preferably 102 to 10 1 mol / L in molar concentration in the mixture of step a).

[0025] Preferably, in the process of the invention, the molar ratio between the nanoparticle precursor and the functionalized polyethylene glycol is between 10 and 1000, preferably between 25 and 100.

[0026] In a particularly advantageous and preferred manner, the method of the invention is carried out with water as the sole solvent. Thus, in a particularly advantageous manner, the method of the present invention does not use any solvent, in particular any organic solvent, any toxic solvent. This advantageously makes it possible to obtain nanoparticles or a suspension of nanoparticles in water that is biocompatible and can be used directly without an intermediate washing step.

[0027] The process of the present invention advantageously makes it possible to obtain ready-to-use nanoparticles in a sterile solution in a single step. The reducing species are derived from the radiolysis of water (electrons and H radicals); no chemical reducing agent is added to the solution. It is therefore a “clean” reduction process.

[0028] The implementation of radiolysis (gamma radiation, accelerated electrons or ions, X-rays), while allowing the synthesis of nanoparticles, kills any living organism that would be present and therefore sterilizes the solution obtained.

[0029] Furthermore, and particularly advantageously, the process of the present invention allows 100% conversion of the metal precursor. Since the process only uses water, it is not necessary to carry out tedious and costly steps in terms of product and time, such as washing the nanoparticles, which avoids product losses (as generally observed when a filtration step is necessary, which is not the case here).

[0030] Thus, the method of the present invention makes it possible to obtain nanoparticles of homogeneous size and to obtain a sterile colloidal solution composed exclusively of nanoparticles of metal chosen from platinum, gold or bismuth.

[0031] Furthermore, the method of the invention allows stable nanoparticles to be obtained. By stable nanoparticles is meant nanoparticles which do not change in size (in particular no modification of the metallic body of the nanoparticles), nor in oxidation degrees over a period of several weeks.

[0032] The present invention also relates to an aqueous solution of nanoparticles capable of being obtained by the method described above.

[0033] The present invention also relates to nanoparticles capable of being obtained by the method of the invention.

[0034] Particularly advantageously, the presence of the biocompatible polymer, functionalized PEG, on the surface of the nanoparticles as well as their small size promotes the accumulation of said nanoparticles for example in tumors, in particular by EPR effect (enhanced permeability and retention). The nanoparticles of the invention, or capable of being obtained by the method of the invention, have the property of amplifying the effects of ionizing radiation (photonic or hadronic) such as radio-induced cell death. Thus, the present invention also relates to nanoparticles according to the invention or capable of being obtained by the method of the invention for their use in the treatment of cancers and tumors, in particular to amplify the effects of medical radiation used for the treatment of these cancers or tumors.

[0035] The present invention also relates to a method for amplifying the effects of ionizing radiation used for the treatment of cancer or tumor of a patient comprising the injection into said patient of an effective quantity of nanoparticles according to the invention or capable of being obtained by the method of the invention.

[0036] Particularly advantageously, the nanoparticles of the invention, or capable of being obtained by the method of the invention, are composed of metals with a high atomic number and electron density, which allows them to be imaged, in particular by CT (Computed Tomography in English, or tomodensitometry). Thus, the present invention also relates to nanoparticles according to the invention or capable of being obtained by the method of the invention for their use in medical imaging for diagnosis.

[0037] The present invention will now be described with the aid of non-limiting examples.

[0038] [Fig-1] [Fig.l] is an image of platinum nanoparticles functionalized by PEG-OH visualized by MET-HR.

[0039] [Fig.2] The [Fig.2] an image of the platinum nanoparticles functionalized by PEG-2NH2 visualized by MET-HR.

[0040] [Fig.3] [Fig.3] shows the internalizations of particles by HeLa cells.

[0041] [Fig.4] [Fig.4] shows the mitotic survival of HeLa cells in the presence or absence of PtNPs (6h incubation at 0.5 mM and 1 mM).

[0042] [Fig.5] [Fig.5] shows the survival of HeLa cells treated with ion radiation C6+ or Cs 137 gamma photons, with and without platinum nanoparticles Example 1: Preparation of nanoparticles

[0043] Platinum nanoparticles (PtNPs) are synthesized using a platinum precursor, tetraamineplatinum(II) chloride (Pt(NH3)4Cl2, 2H2O). This precursor is diluted in water at a rate of 50 mg in 10 mL. A sample of this solution (6.67 mL) is mixed with 2 mL of polyethylene glycol 1000 (PEG-OH) (5 M) or polyethylene diamine 2000 (PEG-2NH2), the mixture is diluted in water (1.33 mL). This solution is degassed under nitrogen. It is then exposed for approximately 17.5 hours to radiation from a cobalt 60 gamma source (dose rate: 95.5 Gy / min). The result is a sterile, black colloidal solution composed exclusively of homogeneous platinum nanoparticles functionalized with PEG (-OH or -2NH2).

[0044] In the case of PEG-OH, the platinum core is spherical in shape and has a diameter of 3.2 nm ([Fig.l]). In the case of PEG-2NH2, flower-shaped aggregates are obtained, their size is 14.6 nm and they contain NPs (nanoparticles) of 3.2 nm in diameter ([Fig.2]).

[0045] The hydrodynamic diameter of the nanoparticles is obtained using DLS (Dynamic Light Scattering) measurements. It is 8.8 nm in the case of PEG-OH and 16.1 nm in the case of PEG-2NH2. The platinum nanoparticles functionalized with PEG-OH have an average zeta potential of -17 mV. Additional measurements by XPS (X-ray Photoelectron Spectrometry) confirmed the absence of platinum precursor in the PtNPs (platinum nanoparticles) solution and shows that all the platinum is reduced (zero oxidation number), which proves the 100% yield of this synthesis method. Example 2: Implementation of nanoparticles

[0046] After incubation for 6 hours of cells (HeLa) with a solution of PtNPs functionalized with PEG-OH containing 0.5 mM platinum (obtained in the example 1), these are internalized by HeLa cells ([Fig.3]) at a rate of 1.6 pg per cell, which corresponds to 49xl05 PtNPs per cell (quantification carried out by ICP-MS, Inductively Coupled Plasma Mass Spectrometry).

[0047] PtNPs functionalized with PEG-OH or PEG-2NH2 have low cytotoxicity on human cell lines. Indeed, HeLa cells incubated for 6 hours with a solution of PtNPs (platinum nanoparticles) containing respectively 0.5 mM or ImM of platinum, show low mitotic death (<10% + / - 5%) ([Fig.4]).

[0048] When nanoparticles are activated by ionizing radiation (gamma radiation from Cobalt 60 or Cesium 137, X-rays, or medical radiation from carbon ions as used in hadrontherapy), they have the property of amplifying radiation-induced molecular damage and cell death. For example, preliminary studies on nano-bioprobes show that the presence of platinum nanoparticles functionalized with PEG-OH amplifies by a factor of 2 the number of nanometric-sized molecular damages induced by ionic radiation.

[0049] Studies on human tumor cells (HeLa) show that the presence of platinum nanoparticles functionalized with PEG-OH amplifies the radiotoxicity (cell death) of a medical beam such as carbon ions or gamma photons. Indeed, when HeLa cells are incubated for 6 hours with a PtNPs solution containing 0.5 mM platinum, they exhibit cell death (as measured by clonogenic survival) induced by irradiation (by accelerated carbon ions or Cs 137 gamma photons), greater than the cell death of control cells (not containing NPs) ([Fig.5]).

Claims

Claims

1. Nanoparticles of metal chosen from platinum or bismuth, functionalized at their surface by functionalized polyethylene glycol comprising at least one OH, COOH, NH2 or SH function.

2. Process for preparing a sterile and biocompatible colloidal solution of nanoparticles of metal chosen from platinum or bismuth, comprising the following steps: a) Mixing a nanoparticle precursor with a functionalized polyethylene glycol comprising at least one OH, COOH, NH2 or SH function in water; b) Exposing the mixture to ionizing radiation.

3. Method according to claim 2, in which the ionizing radiation is chosen from gamma radiation from a source of Cobalt 60 or Cesium 137, electrons or accelerated ions.

4. Method according to claims 2 and 3, wherein the step of exposing to gamma radiation lasts between 1 and 20 hours, preferably between 1 and 2 hours for a dose of 10 kGy applied with a Co60 source gamma radiation source with a dose rate of about 95.5 Gy / min to completely reduce 103 mol / L 1 of platinum complex.

5. Process according to any one of claims 2 to 4 in which the functionalized polyethylenes are chosen from PEG-OH, PEG-2NH2, OH-PEG-COOH and PEG-SH.

6. Process according to any one of claims 2 to 5, in which the precursor is used at a rate of 10 4 to 10 2 mol / L, in molar concentration of the total mixture of step a) and / or the functionalized polyethylene glycol is used at a rate of 10 3 to 10 1 mol / L in molar concentration of the total mixture of step a).

7. A method according to any one of claims 2 to 6, wherein the molar ratio between the precursor and the functionalized polyethylene glycol is between 10 and 100.

8. A method according to any one of claims 2 to 7 carried out in the absence of a solvent other than water.

9. Nanoparticles according to claim 1, in which the functionalized polyethylenes are chosen from PEG-OH, PEG-2NH2, OH-PEG-COOH and PEG-SH.

10. Nanoparticle according to claim 1, for its use in the treatment of cancers and tumors, in particular to amplify the effects of medical radiation used for the treatment of these cancers or tumors.

11. Nanoparticle according to claim 1, for its use in medical imaging for diagnosis, for example by computed tomography (CT).

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