METHOD FOR THE PREPARATION OF CONTROLLED PEPTIDE-BASED POLYMERS AND COPOLYMERS IN AQUEOUS SOLUTION

Description

[0001] The present invention relates to a process for preparing controlled polymers and copolymers based on peptides in aqueous solution, as well as the products as obtained.

[0002] The self-assembly of amphiphilic copolymers is a promising strategy for designing advanced nanomaterials with unique functionalities. Among amphiphilic copolymers, polypeptides constitute an emerging class of biomaterials used as carriers for active ingredients in pharmaceutical and cosmetic applications. To date, the most economical and efficient method for preparing amphiphilic polypeptide-based nanomaterials is a multi-step process, notably involving ring-opening polymerization of N-carboxyanhydride monomers (ROP or ROPISA).

[0003] This controlled polymerization uses the simplest reagents but still suffers from significant limitations, including: 1) tedious purification steps for the N-carboxyanhydride monomer; 2) significant sensitivity to water and humidity; and 3) implementation in toxic organic solvents such as DMF, which must then be disposed of. Designing nanoparticles from amphiphilic polypeptides involves at least one second formulation step, nanoprecipitation, which consists of adding a non-solvent for the hydrophobic segment to a copolymer solution in a solvent common to both blocks. The nanoprecipitation step is typically performed using toxic or volatile organic solvents, under relatively dilute conditions (<1 wt%) and is susceptible to scaling issues, which generally hinders regulatory approval of the process.

[0004] Polymerization-induced self-assembly (PISA) is a simple and robust approach to accessing amphiphilic polymers with the advantage of simultaneously obtaining nanoparticles of these same polymers. The PISA method involves the growth in situ of a living amphiphilic polymer chain that spontaneously self-assembles into nanostructures. To date, the PISA method has been implemented using radical polymerization processes (RAFT, ATRP, NMP, CMP, TERP), either in dispersion or emulsion.

[0005] The present invention aims to provide functionalized nano-objects in a single preparation step.

[0006] Another objective of the present invention is to provide a simple and rapid one-step "one pot" process in aqueous medium to obtain amphiphilic peptide copolymers, without the need for further purification.

[0007] The present invention also aims to provide a polymerization process with very rapid polymerization kinetics, enabling the production of functional, bioassimilable, biocompatible and biodegradable polymers.

[0008] Thus, the present invention relates to a "one-pot" method for preparing an aqueous solution of amphiphilic block copolymer nanoparticles comprising polypeptide motifs, said method comprising at least one step (E1), in an aqueous solvent free of organic solvent, consisting of bringing together: at least one hydrophilic polymer (P1) comprising at least one amine function, and at least one hydrophobic α-amino acid (NCA) N-carboxyanhydride monomer in which the temperature of step (E1) is from -10°C to 80°C, said process being a ring-opening polymerization-induced self-assembly process of N-carboxyanhydride.

[0009] The process of the invention therefore makes it possible to obtain an aqueous solution of amphiphilic block copolymer nanoparticles based on polypeptides, and this in an aqueous medium.

[0010] It is based on a polymerization-induced self-assembly process (PISA). Thus, by adapting the PISA process to NCA monomers, the inventors fortuitously discovered that the spontaneous self-assembly of a PISA process made it possible to protect them from hydrolysis.

[0011] The present invention therefore relates to the preparation of amphiphilic polypeptides in aqueous solutions by ring-opening polymerization.

[0012] The process of the invention is a process that does not require the use of an organic solvent. According to the invention, the process is carried out in the absence of any organic solvent.

[0013] According to the invention, the aqueous solvent does not include any organic solvent.

[0014] The present invention therefore relates to a process for preparing controlled peptide-based polymers and copolymers in aqueous solution and their spontaneous self-formulation during this same process, thus enabling the formation of stable and functional nanoparticles in a single preparation step, which may be used, for example and non-exclusively, in the preparation of pharmaceutical or cosmetic compositions.

[0015] The process of the invention consists of preparing amphiphilic polypeptide copolymers without organic solvents, in a rapid and controlled manner, which also allows for the concomitant formation of nanoparticles, all at dry extract levels exceeding 10% by mass, thus reducing the complexity of existing processes. In particular, the present invention relates to a faster process for preparing amphiphilic polypeptide copolymers and for spontaneous self-formulation by carrying out these two steps simultaneously and without purification, in an aqueous medium.

[0016] As mentioned above, the process of the invention includes the implementation of at least one hydrophobic NCA monomer.

[0017] Any hydrophobic NCA monomer can be used. Depending on the nature of the NCA monomer used, it is possible to modify it, notably via a hydrophobic protecting group, to make it hydrophobic.

[0018] According to one embodiment, the hydrophobic α-amino acid N-carboxyanhydride monomer corresponds to the following formula (I): in which R represents the side chain of a natural or modified, hydrophobic, possibly protected α-amino acid.

[0019] When the NCA monomer used is a hydrophilic α-amino acid NCA (including OH, COOH or NH2 functions in particular), then the aforementioned R group includes a hydrophobic protecting group, in order to make said monomer hydrophobic.

[0020] This protection is not necessary when the NCA used is an NCA of an α-amino acid that is hydrophobic by nature.

[0021] The following compounds are used in particular as N-carboxyanhydride monomers of hydrophobic α-amino acids:

[0022] Preferably, we can cite the NCA monomers of γ-benzyl- L -glutamate, from ε-Boc- L-Lysine, L-leucine or L-Phenylalanine.

[0023] As mentioned above, the process of the invention includes the implementation of at least one hydrophilic polymer (P1) comprising at least one amine function. This hydrophilic polymer serves as a macro-initiator.

[0024] According to one embodiment, the polymer (P1) is chosen from the group consisting of polyethers, polyesters, poly(met)arylates, polysaccharides, polypeptides, polypeptoids, DNA derivatives and proteins, in particular elastin-like polypeptides (ELP) comprising at least one amine function, and is preferably chosen from poly(ethylene oxide)s comprising at least one amine function.

[0025] Preferably, the polymer (P1) is a PEG.

[0026] Preferably, the hydrophilic polymer (P1) has a molecular weight greater than 500 g / mol and preferably has a molecular weight between 2000 g / mol and 10000 g / mol.

[0027] Preferably, the polymer (P1) conforms to the following formula: in which x is between 16 and 500.

[0028] According to the process of the invention, the starting product can be considered as a suspension, namely an opaque heterogeneous medium, and the final product obtained is preferably in the form of a homogeneous transparent solution.

[0029] The process of the invention therefore advantageously allows the transformation of a very heterogeneous medium, in particular a solid dispersed in water, into a solution of well-defined nanoparticles based on amphiphilic copolymers of controlled macromolecular architecture.

[0030] The process of the invention is a "one pot" process, that is to say a process in which the reactants undergo one or more successive or simultaneous reactions, but in a single reaction mixture.

[0031] According to one embodiment, in the process of the invention, the aqueous solvent is water or a buffer.

[0032] For example, an aqueous solvent is water with a buffer solution added.

[0033] According to one embodiment, the aqueous solvent further comprises a buffer solution comprising a salt at concentrations ranging from 0.01 M to 1 M, in particular selected from the group consisting of sodium hydrogen carbonate solutions and phosphate buffer solutions.

[0034] Preferably, the process of the invention uses an aqueous solution of NaHCO3.

[0035] Preferably, the pH of the aqueous solvent is between 2 and 12, and in particular between 7 and 10.

[0036] According to the invention, the temperature of the step (E1) is from -10°C to 80°C, and preferably from 0°C to 4°C.

[0037] According to one embodiment, step (E1) is carried out under stirring from a dispersion of the hydrophobic α-amino acid N-carboxyanhydride monomer.

[0038] The process of the invention can also be applied to obtain multiblock copolymers.

[0039] Thus, according to one embodiment, the aqueous solution of amphiphilic block copolymer nanoparticles obtained at the end of step (E1) is subsequently contacted with a second hydrophobic α-amino acid N-carboxyanhydride monomer, identical or different from that of step (E1), which makes it possible to obtain an aqueous solution of modified amphiphilic block copolymer nanoparticles.

[0040] Thus, according to this embodiment, it is possible to obtain multiblock, gradient or statistical copolymers when at least one second N-carboxyanhydride monomer of a hydrophobic α-amino acid different from that of step (E1) is added.

[0041] The process of the invention can be applied to obtain core-crown nanoparticles, preferably elongated, preferably rigid, and preferably with a size ranging from 2 nm to 1 µm. Thus, according to this embodiment, it is possible to obtain anisotropic nanoparticles having optical properties.

[0042] Amphibious block copolymer nanoparticles comprising polypeptide motifs are obtained according to the process as defined above.

[0043] Amphibious block copolymer nanoparticles comprising polypeptide motifs can also be obtained according to the process as defined above, said nanoparticles having a core-shell structure and a particle size of 2 nm to 1 µm.

[0044] An aqueous composition comprising nanoparticles as defined above may also be obtained, the weight content of said nanoparticles being at least 2% relative to the weight of said aqueous composition, and preferably between 2% by weight and 15% by weight relative to the weight of said aqueous composition.

[0045] This solids content is measured after polymerization and after purification of the salts by dialysis by performing freeze-drying (comparison of the mass of residues with the mass before freeze-drying). DESCRIPTION DES FIGURES

[0046] There Figure 1 This concerns size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG copolymer from Example 1. Left: IR detection, Right: absorbance detection. Figure 2 represents the 1<H NMR spectrum obtained in the CDCl3 + 15% TFA of the PEG 5k-b-PBLG copolymer from Example 1. Figure 3 represents the diffuse intensity distribution of the hydrodynamic diameter (Dh) of the PEG 5k-b-PBLG nanoparticles from Example 1 (in ultrapure water). Figure 4 represents Transmission Electron Microscopy (Cryo-TEM) images of cryogenically frozen nanoparticles: nanoparticles of the PEG 5k-b-PBLG copolymer from Example 1. Figure 5 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG copolymer from Example 2. Left: IR detection, Right: absorbance detection. Figure 6 represents the 1<H NMR spectrum obtained in the CDCl3 + 15% TFA of the PEG 5k-b-PBLG copolymer from Example 2. Figure 7 represents a Cryo-TEM image of the nanoparticles of the PEG 5k-b-PBLG copolymer from Example 2. Figure 8 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG copolymer from Example 3. Left: IR detection, Right: absorbance detection. Figure 9 represents the 1<H NMR spectrum obtained in the CDCl3 + 15% TFA of the PEG 5k-b-PBLG copolymer from Example 3. Figure 10 represents the distribution (in scattered intensity) of the hydrodynamic diameter (Dh) of the PEG 5k-b-PBLG nanoparticles from example 3 (in ultrapure water). Figure 11 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG copolymer from Example 4. Left: IR detection, Right: absorbance detection. Figure 12 represents the 1<H NMR spectrum obtained in the CDCl3 + 15% TFA of the PEG 5k-b-PBLG copolymer from example 4. Figure 13 represents the distribution (in scattered intensity) of the hydrodynamic diameter (Dh) of the PEG 5k-b-PBLG nanoparticles from example 4 (in ultrapure water). Figure 14 represents a cryo-TEM image of the nanoparticles of the PEG 5k-b-PBLG copolymer from example 4. Figure 15 This concerns size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 2k-b-PBLG copolymer from Example 5. RI detection is shown in black and gray, and absorbance detection is shown as a dashed gray line. Figure 16 represents the distribution (in scattered intensity) of the hydrodynamic diameter (Dh) of the PEG 2k-b-PBLG nanoparticles from example 5 (in ultrapure water). Figure 17 This represents the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 10k-b-PBLG copolymer from Example 6. Left: IR detection, Right: absorbance detection. Figure 18 represents the 1<H NMR spectrum obtained in the CDCl3 + 15% TFA of the PEG 10k-b-PBLG copolymer from Example 6. Figure 19 represents a cryo-TEM image of the nanoparticles of the PEG 10k-b-PBLG copolymer from Example 6. Figure 20 represents A) the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the ELPM40-b-PBLG copolymer from Example 7 (RI detection); B) a TEM (uranyl acetate staining) image of the nanoparticles of the ELPM40-b-PBLG copolymer from Example 7. Figure 21 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PLys copolymer from Example 8. RI detection. The Figure 22 represents the 1<H NMR spectrum obtained in the DMF-d6 of the PEG 5k -b-PLys copolymer from Example 8. The Figure 23 represents the distribution (in scattered intensity) of the hydrodynamic diameter (Dh) of the PEG 5k-b-PLys nanoparticles from example 8 (in ultrapure water). Figure 24 represents a cryo-TEM image of the nanoparticles of the PEG 5k-b-PLys copolymer from example 8. Figure 25 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PLys-b-PBLG copolymer from Example 9. RI detection. The Figure 26 represents A) the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PLeu-b-PBLG copolymer from Example 10 (RI detection); B) a TEM image (uranyl acetate staining) of the nanoparticles of the PLeu-b-PBLG copolymer from Example 10. Figure 27 represents A) the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PPhe-b-PBLG copolymer from Example 11 (RI detection); B) a TEM image (uranyl acetate staining) of the nanoparticles of the PPhe-b-PBLG copolymer from Example 11. Figure 28 represents A) the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG-b-PEG 5k copolymer from Example 12 (RI Detection); B) a TEM (uranyl acetate staining) image of the nanoparticles of the PEG 5k-b-PBLG-b-PEG 5k copolymer from Example 12. Figure 29 represents A) the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG-4arm-b-(PBLG) 4 copolymer from Example 13 (RI Detection); B) a TEM (uranyl acetate stain) image of the nanoparticles of the PEG-4arm-b-(PBLG) 4 copolymer from Example 13. Figure 30 represents A) the size-exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PSar-b-PBLG copolymer from Example 14 (RI detection); B) a TEM (ureanyl acetate staining) image of the nanoparticles of the PSar-b-PBLG copolymer from Example 14. Figure 31 represents A) size exclusion chromatography performed in H2O (Juanito buffer) of the PGA-b-PBLG copolymer from Example 15 after deprotection (RI detection); B) a TEM (urea acetate staining) image of the nanoparticles of the PGA-b-PBLG copolymer from Example 15. Figure 32 represents A) an infrared analysis of example 16 (powder); B) a TEM (urea acetate staining) image of the copolymer nanoparticles of example 16. Figure 33 represents the size exclusion chromatography performed in DMF (+1 mg / mL LiBr) of the PEG 5k-b-PBLG copolymer from Example 17 (RI Detection); The Figure 34 represents A) a TEM (acetate uranyl staining) image of the copolymer nanoparticles of Example 18; B) the distribution (in scattered intensity) of the hydrodynamic diameter (Dh) of the nanoparticles of Example 18 (in ultrapure water); C) an electrophoresis gel showing the formation of the copolymer of Example 18. EXEMPLES Exemple 1 : Concomitant synthesis of an amphiphilic poly(ethylene glycol) 5k peptidic diblock copolymer - block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0047] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0048] The NCA monomer of γ-benzyl- L-glutamate (300 mg, 1.14 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 85 ± 3% (. Figures 1 à 4 ). Exemple 2 : Concomitant synthesis and in the absence of salts of an amphiphilic poly(ethylene glycol) 5k peptidic diblock copolymer - block -poly(γ-benzyl- L- glutamate) and their corresponding nanoparticles

[0049] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0050] The NCA monomer of γ-benzyl- L-glutamate (300 mg, 1.14 mmol) is placed in an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, the macroinitiator PEG 5k-NH₂ (8 mL, 300 mg, 0.06 mmol) is diluted in ultrapure water and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution, which is then transferred to a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained ( Figures 5 à 7 ). Exemple 3 : Concomitant and high solids-content synthesis of an amphiphilic poly(ethylene glycol) 5k peptidic diblock copolymer - block -poly(γ-benzyl- L- glutamate) and their corresponding nanoparticles

[0051] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0052] The NCA monomer of γ-benzyl- L-glutamate (600 mg, 2.28 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 600 mg, 0.12 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 77% (. Figures 8 à 10 ). Example 4: Chain extension procedure of an amphiphilic poly(ethylene glycol) 5k peptidic diblock copolymer - block -poly(γ-benzyl-L-glutamate)

[0053] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0054] The NCA monomer of γ-benzyl- L-glutamate (300 mg, 1.14 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, an aqueous solution of 0.05 M NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While remaining in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. After 90 min, a second addition of γ-benzyl- NCA L-glutamate (300 mg, 1.14 mmol) is carried out. The reaction is left under stirring 1) first in an ice water bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred to a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 85% ( Figures 11 à 14 ). Example 5: Concomitant synthesis of an amphiphilic poly(ethylene glycol) 2k- peptide diblock copolymer block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0055] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 2k-NH2 (Mp = 2022 Da, Δ = 1.04) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0056] The NCA monomer of γ-benzyl- L-glutamate (30 mg, 0.11 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.2 M aqueous solution of NaHCO3 containing the macroinitiator PEG 2k-NH2 (800 µL, 30 mg, 0.015 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into a colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained (. Figures 15 And 16 ). Example 6: Concomitant synthesis of an amphiphilic poly(ethylene glycol) 10k- peptide diblock copolymer block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0057] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 10k -NH₂ (Mp = 11153 Da, Δ = 1.05) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0058] The NCA monomer of γ-benzyl- L-glutamate (300 mg, 1.14 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 10k -NH2 (8 mL, 300 mg, 0.03 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion transforms into a gel which is then transferred to a dialysis pack (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained ( Figures 17 à 19 ). Example 7: Concomitant synthesis of an amphiphilic peptide diblock copolymer (Elastin-like polypeptide)- block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0059] The γ-Benzyl- monomer L -glutamate N BLG-NCA is a commercial chemical reagent distributed by ISOCHEM. Elastin-like polypeptide (ELP) is a recombinant protein produced in bacteria. E. Coli at the Laboratory of Organic Polymer Chemistry in Bordeaux, France. The ELP used has a primary amine at its N-terminus. It has the structure MW (VPGVP VPGMG VPGVG VPGVG) 10 and a molar mass of 17,035 Da. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0060] The NCA monomer of γ-benzyl- L-glutamate (10 mg, 0.04 mmol) is placed in an inert atmosphere in a test tube containing a magnetic stir bar. The tube is cooled in an ice bath for at least 10 minutes. Concurrently, an aqueous solution of 0.05 M NaHCO3 containing the macroinitiator ELP (2.7 mL, 10 mg, 0.001 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While remaining in the ice bath, the aqueous solution is added to the NCA powder under vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left under magnetic stirring at 4°C for 20 h. The milky dispersion evolves into a turbid dispersion at room temperature, which is then transferred to a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days at 4°C. After freeze-drying, a white powder is obtained ( Figure 20 ). Example 8: Concomitant synthesis of an amphiphilic poly(ethylene glycol)- peptide diblock copolymer block - poly(ε-Boc- L -Lysine) and their corresponding nanoparticles

[0061] The ε-tert-butyloxycarbonyl- monomer L -lysine N LysBOC-NCA is a commercial product distributed by ISOCHEM. PEG 5k-NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0062] The NCA monomer of ε-Boc- LLysine (310 mg, 1.14 mmol) is placed in an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kD dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 79% (. Figures 21 à 24 ). Example 9: Concomitant synthesis of an amphiphilic poly(ethylene glycol)- peptide triblock copolymer block -poly(ε - Boc- L -Lysine)- block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0063] γ-Benzyl- monomers L -glutamate N -carboxyanhydride (BLG-NCA) and ε-tert-butyloxycarbonyl- L -lysine N -carboxyanhydride (LysBOC-NCA) are commercial products distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0064] The NCA monomer of ε-Boc- LLysine (300 mg, 1.10 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.2 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While remaining in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left to stir in an ice-water bath for approximately 15 minutes. Concurrently, the NCA monomer of γ-benzyl- L-glutamate (BLG-NCA, 300 mg, 1.14 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. While remaining in this ice bath, the opalescent colloidal aqueous solution is mixed with the BLG-NCA powder under vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is allowed to proceed under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution, which is then transferred to a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 75 ± 3% ( Figure 25 ). Example 10: Concomitant synthesis of an amphiphilic poly(ethylene glycol) 5k- peptide diblock copolymer block- poly( L -leucine) and their corresponding nanoparticles

[0065] The monomer L -leucine N-carboxyanhydride (LEU-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0066] The leucine monomer NCA (300 mg, 1.9 mmol) is placed in an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, an aqueous solution of 0.05 M NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 77% (. Figure 26 ). Example 11: Concomitant synthesis of an amphiphilic poly(ethylene glycol) 5k- peptide diblock copolymer block -poly( L -phenylalanine) and their corresponding nanoparticles

[0067] The monomer L -phenylalanine N PEG-carboxyanhydride (PHE-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG-5k-NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0068] Phenylalanine monomer NCA (150 mg, 0.8 mmol) is placed in an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk tube is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 72% (. Figure 27 ). Example 12: Concomitant synthesis of an amphiphilic poly(γ-benzyl- peptide triblock copolymer L -glutamate)- block -poly(ethylene glycol) 5k - block- poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0069] The γ-Benzyl- monomer L -glutamate N NH₂-carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. NH₂-PEG 5k-NH₂ (PEG-2arm) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0070] The NCA monomer of γ-Benzyl- L -glutamate NNCA-carboxyanhydride (BLG-NCA) (29 mg, 0.11 mmol) is placed in an inert atmosphere in a test tube containing a magnetic stir bar. The tube is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator NH2-PEG 5k-NH2 (0.85 mL, 35 mg) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained (. Figure 28 ). Exemple 13 : Concomitant synthesis of an amphiphilic poly(γ-benzyl- peptide star copolymer L -glutamate) 4 - block -poly(ethylene glycol) 5k and their corresponding nanoparticles

[0071] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG-4arm is distributed by RAPP Polymer (star: 4-terminal NH₂). The other reagents are distributed by Sigma-Aldrich.

[0072] The NCA monomer of γ-Benzyl -L -glutamate N18 mg of NCA (BLG-NCA) (0.07 mmol) is placed in an inert atmosphere in a test tube containing a magnetic stir bar. The tube is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-4arm (0.70 mL, 35 mg) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, and 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained (. Figure 29 ). Example 14: Concomitant synthesis of an amphiphilic poly(sarcosine)- peptide diblock copolymer block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0073] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. Poly(sarcosine) PSar (Mp = 2100 Da, Δ = 1.03) is synthesized by conventional ring-opening polymerization in the laboratory. The other reagents are distributed by Sigma-Aldrich.

[0074] The NCA monomer of γ-benzyl- L-glutamate (120 mg, 0.46 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator (4 mL, 50 mg, 0.02 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained with a yield of 55% (. Figures 30 ). Example 15: Concomitant synthesis of an amphiphilic poly(peptide diblock copolymer L -glutamic)- block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0075] The γ-Benzyl- monomer L -glutamate N L-carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. Poly(L-glutamic acid) PGA (Mw = 6600 g / mol) is synthesized by conventional ring-opening polymerization in the laboratory. The other reagents are distributed by Sigma-Aldrich.

[0076] The NCA monomer of γ-benzyl- L-glutamate (300 mg, 1.14 mmol) is placed under an inert atmosphere in a Schlenk tube containing a magnetic stir bar. The Schlenk is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.20 M aqueous solution of NaHCO3 containing the macroinitiator (8 mL, 300 mg, 0.45 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days (TEM image, figure 31 After lyophilization, in order to analyze the copolymer, the PBLG block is deprotected under mild acidic conditions (MSA, TFA) to obtain a white powder that can be analyzed in aqueous SEC (Juanito buffer) ( Figure 31 ). Example 16: Concomitant synthesis of an amphiphilic peptide diblock polysaccharide copolymer block- poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles

[0077] The γ-Benzyl- monomer L -glutamate N β-carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. The polyaminosaccharide (Polysacc., Mw = 7750 g / mol) is synthesized by anionic polymerization of β-lactam monomers in the laboratory. The other reagents are distributed by Sigma-Aldrich.

[0078] The NCA monomer of γ-benzyl- L-glutamate (13 mg, 0.05 mmol) is placed in an inert atmosphere in a test tube containing a magnetic stir bar. The tube is cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator (2 mL, 20 mg, 0.003 mmol) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder with vigorous stirring. A milky dispersion results from the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first in an ice bath for approximately 2 hours, 2) then at room temperature for 16 hours. The milky dispersion evolves into a whitish colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained (. Figure 32 ). Example 17: Concomitant synthesis of an amphiphilic poly(ethylene glycol) 5k- peptide diblock copolymer block -poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles at very small scale

[0079] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG 5k -NH₂ (Mp = 5516 Da, Δ = 1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0080] The NCA monomer of γ-benzyl- L-glutamate (3 mg, 0.01 mmol) is placed under an inert atmosphere in an Eppendorf tube containing a small magnetic stir bar, which is then cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator PEG 5k-NH2 (80 µL, 3 mg) is prepared and then cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder under strong magnetic stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is then carried out under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution which is then transferred into a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days.After freeze-drying, a white powder is obtained (. Figure 33 ). Example 18: Concomitant synthesis of an amphiphilic peptide diblock copolymer with deoxyribonucleic acid block- poly(γ-benzyl- L -glutamate) and their corresponding nanoparticles at very small scale

[0081] The γ-Benzyl- monomer L -glutamate N -carboxyanhydride (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. DNA deoxyribonucleic acid (TTT) 15 (Mw=4680 g / mol) is distributed by IDT Technologies. The other reagents are distributed by Sigma-Aldrich.

[0082] The NCA monomer of γ-benzyl- L-glutamate (5 mg) is placed under an inert atmosphere in an Eppendorf tube, which is then cooled in an ice bath for at least 10 minutes. Concurrently, a 0.05 M aqueous NaHCO3 solution containing the DNA macroinitiator (1 mL, 5 mg) is prepared and cooled in an ice bath for at least 10 minutes. While still in the ice bath, the aqueous solution is added to the NCA powder under strong magnetic stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is allowed to proceed under stirring 1) first in an ice bath for approximately 2 hours, 2) and then at room temperature for 16 hours. The milky dispersion evolves into an opalescent colloidal aqueous solution, which is then transferred to a dialysis drum (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After freeze-drying, a white powder is obtained ( Figure 34 ).

[0083] Tables 1 and 2 below provide the molecular and physicochemical characteristics of the copolymers synthesized by ROPISA and of their nanoparticles according to the examples above. [Table 1] Copolymère Tampon Initiateur M / I g / mol T s (%) M / I M n g / mol M n g / mol Ð Th. 1< H NMR SEC* PEG 5k -NH 2 - - - - - - 6996 1.02 Ex.1 NaHCO 3 50mM PEG 5k -NH 2 19 7 20 9380 11940 1.05 Ex.2 MQ PEG 5k -NH 2 19 7 20 9380 13470 1.14 Ex.3 NaHCO 3 50mM PEG 5k -NH 2 19 13 21 9600 12250 1.06 Ex.4 NaHCO 3 50mM PEG 5k -NH 2 38 10 35 12670 14370 1.09 Ex.5 NaHCO 3 200mM PEG 2k -NH 2 8 13 - - 4067 1.20 Ex.6 NaHCO 3 50mM PEG 10k -NH 2 38 7 37 18100 24660 1.12 Ex.7 NaHCO 3 50mM ELPM40 65 0.7 30 23600 27000 1.03 Ex.8 NaHCO 3 50mM PEG 5k -NH 2 19 7 21 9790 11750 1.09 Ex.9 NaHCO 3 200mM PEG 5k -NH 2 38 13 - - 12220 1.05 Ex.10 NaHCO₃ 50mM PEG 5k -NH 2 32 7 - - 10600 1.04 Ex.11 NaHCO₃ 50mM PEG 5k -NH 2 2arms- 13 5 - - - - Ex.12 NaHCO₃ 50mM PEG 5k -NH 2 4arms- 19 8 18 9900 14700 1.10 Ex.13 NaHCO₃ 50mM PEG 5k -NH 2 10 8 16 8500 11700 1.32 Ex.14 NaHCO₃ 50mM PSar 19 7 - - 7100 1.34 Ex.15 NaHCO₃ 50mM PGA Ex.16 NaHCO₃ 50mM Polysaccharides. 19 1.6 - - - - Ex.17 NaHCO₃ 50mM PEG 5k -NH 2 19 7 20 9380 11900 1.05 Ex.18 NaHCO₃ 50mM DNA 19 5 - - - - * Number-average molecular mass ( M n ) and dispersity ( Ð ) determined by SEC in DMF (+LiBr) using a polystyrene calibration curve. [Table 2] Copolymere Tampon Initial D h (s) nm Rendering % DLS PEG 5k -NH 2 - - - - Ex.1 NaHCO₃ 50mM PEG 5k -NH 2 79 (0.08) 87 Ex.2 MQ PEG 5k -NH 2 - - Ex.3 NaHCO₃ 50mM PEG 5k -NH 2 88 (0.17) 77 Ex.4 NaHCO₃ 50mM PEG 5k -NH 2 99 (0.12) 85 Ex.5 NaHCO₃ 200mM PEG 2k -NH 2 polydisperse - Ex.6 NaHCO₃ 50mM PEG 10k-NH2 gel 83 Ex.7 NaHCO₃ 200mM ELPM40 Aggrégé à TA - Ex.8 NaHCO₃ 50mM PEG 5k -NH 2 131 (0.19) 79 Ex.9 NaHCO₃ 200mM PEG 5k -NH 2 - 75 Ex.10 NaHCO₃ 50mM PEG 5k -NH 2 92 (0.22) 77 Ex.11 NaHCO₃ 50mM PEG 5k -NH 2 125 (0.33) 72 Ex.12 NaHCO₃ 50mM 2arms-PEG 5k -NH 2 - - Ex.13 NaHCO₃ 50mM 4arms-PEG 5k -NH 2 - - Ex.14 NaHCO₃ 50mM PSar - 55 Ex.15 NaHCO₃ 50mM PGA 99 (0.7) - Ex.16 NaHCO₃ 50mM Polysaccharides. 414 (0.19) - Ex.17 NaHCO₃ 50mM PEG 5k -NH 2 - - Ex.18 NaHCO₃ 50mM DNA 460 (0.26) -

Claims

1. A "one pot" method for preparing an aqueous solution of nanoparticles of amphiphilic block copolymers and comprising polypeptide units, said method comprising at least one step (E1) in an aqueous solvent free of organic solvent, consisting of bringing together: - at least one hydrophilic polymer (P1) comprising at least one amine function, and - at least one hydrophobic α-amino acid N-carboxyanhydride monomer wherein the temperature of the step (E1) is from -10°C to 80°C, the said method being a self-assembly method induced by ring-opening polymerisation of N-carboxyanhydride.

2. The method according to claim 1 wherein the hydrophobic α-amino acid N-carboxyanhydride monomer has the following formula (I): where R is the side chain of an optionally protected, natural or modified, hydrophobic α-amino acid.

3. The method according to claim 1 or 2, wherein the polymer (P1) is selected from the group consisting of: polyethers, polyesters, poly(meth)acrylates, polysaccharides, polypeptides, polypeptoids, DNA and protein derivatives, in particular elastin-like polypeptides (ELPs) comprising at least one amine function, and it is preferably selected from among poly(ethylene oxides) having at least one amine function.

4. The method according to any of claims 1 to 3, wherein the polymer (P1) has the following formula: where x is from 16 to 500.

5. The method according to any of claims 1 to 4, wherein the aqueous solvent is water or a buffer.

6. The method according to any of claims 1 to 5, wherein the aqueous solvent also comprises a buffer solution comprising an inorganic salt at concentrations ranging from 0.01 M to 1 M, selected in particular from the group consisting of sodium hydrogen carbonate solutions and phosphate buffer solutions.

7. The method according to any of claims 1 to 6, wherein the pH of the aqueous solvent is from 2 to 12.

8. The method according to any of claims 1 to 7, wherein the temperature at step (E1) is from 0°C to 4°C.

9. The method according to any of claims 1 to 8, wherein step (E1) is conducted under agitation from a dispersion of the hydrophobic α-amino acid N-carboxyanhydride monomer.

10. The method according to any of claims 1 to 9, wherein the aqueous solution of nanoparticles of amphiphilic block copolymers obtained after step (E1) is subsequently contacted with a second hydrophobic α-amino acid N-carboxyanhydride monomer, the same or differing from the one at step (E1), which allows an aqueous solution to be obtained of modified nanoparticles of modified amphiphilic block copolymers