Methods for preparing controlled peptide-based polymers and copolymers in aqueous solutions - Patents.com

Abstract

The present invention relates to a "one-pot" method for preparing an aqueous solution of nanoparticles comprising amphiphilic block copolymers and comprising polypeptide units, comprising at least one step (E1) consisting of combining, in an aqueous solvent, at least one hydrophilic polymer (P1) comprising at least one amine functional group and at least one hydrophobic α-amino acid N-carboxyanhydride monomer.

Description

[Technical field]

[0001] The subject of the present invention is a method for the preparation of controlled peptide-based polymers and copolymers in aqueous solution, as well as the products obtained thereby. [Background technology]

[0002] Self-assembly of amphiphilic copolymers is a promising strategy for designing advanced nanomaterials with unique properties. Among amphiphilic copolymers, polypeptides form a new class of biomaterials that are used as active vectors for pharmaceutical and cosmetic applications. To date, the most economical and most efficient method for preparing nanomaterials based on amphiphilic polypeptides is the method that requires several steps, including in particular the ring-opening polymerization of N-carboxyanhydride monomers (ROP or ROPISA).

[0003] Although this controlled polymerization relies on the simplest reagents, it still suffers from major limitations, in particular: 1) the laborious purification of the N-carboxyanhydride monomers, 2) its extreme sensitivity to water and humidity, and 3) its implementation in toxic organic solvents (such as DMF) that must then be removed. The preparation of nanoparticles from amphiphilic polypeptides requires at least one second formation step, nanoprecipitation, consisting of adding a non-solvent for the hydrophobic segments to a solution of the copolymer in a solvent common to both blocks. This nanoprecipitation step is generally carried out under relatively dilute conditions (<1% by weight) and using toxic or volatile organic solvents, making it sensitive to problems related to scaling, which is generally detrimental to the regulatory approval of this method.

[0004] Polymerization-induced self-assembly (PISA) is a simple and robust approach to access amphiphilic polymers, with the advantage of simultaneously obtaining nanoparticles of the same amphiphilic polymers. The PISA method refers to the in situ growth of living amphiphilic polymer chains that spontaneously self-assemble within nanostructures. To date, the PISA method has been realized using radical polymerization processes (RAFT, ATRP, NMP, CMP, TERP) in dispersions or emulsions. Summary of the Invention [Problem to be solved by the invention]

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

[0006] A further object of the present invention is to provide a simple and rapid "one-pot" method for obtaining amphiphilic peptide copolymers in a single step in aqueous medium without the need for subsequent purification.

[0007] A further object of the present invention is to provide a polymerization process with very fast polymerization kinetics, making it possible to obtain functional, bioassimilable, biocompatible and biodegradable polymers. [Means for solving the problem]

[0008] The present invention therefore relates to a "one-pot" method for preparing an aqueous solution of nanoparticles of amphiphilic block copolymers comprising polypeptide units, comprising the steps of: at least one hydrophilic polymer (P1) containing at least one amine functional group; and - at least one hydrophobic α-amino acid N-carboxyanhydride (NCA) monomer; in an aqueous solvent free of organic solvent.

[0009] Thus, using the method of the present invention, aqueous solutions of nanoparticles of polypeptide-based amphiphilic block copolymers can be obtained in an aqueous medium.

[0010] It is based on a polymerization-induced self-assembly (PISA) method. By adapting the PISA method to NCA monomers, the inventors serendipitously found that the spontaneous self-assembly of the PISA method provides protection against hydrolysis to the NCA monomers.

[0011] Thus, the present invention relates to the preparation of amphiphilic polypeptides in aqueous solution via ring-opening polymerization.

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

[0013] In the present invention, the aqueous solvent does not include an organic solvent.

[0014] The present invention thus relates to a method for the preparation of controlled peptide-based polymers and copolymers in aqueous solution, and the spontaneous self-formulation during the implementation of the same, which allows the formation of stable and functionalized nanoparticles in a single preparation step, such as, but not limited to, the preparation of pharmaceutical or cosmetic products.

[0015] The method of the present invention consists of a step of preparing amphiphilic polypeptide copolymers without organic solvents, which is rapid, controlled and allows a simultaneous step of forming nanoparticles with a dry extract rate higher than 10% by weight, thereby reducing the complexity of already known methods.In particular, the present invention relates to a method of preparing amphiphilic polypeptide copolymers that exhibit faster spontaneous self-assembly by carrying out these two steps in an aqueous medium simultaneously and without purification.

[0016] As mentioned above, the methods of the present invention involve the use of at least one hydrophobic NCA monomer.

[0017] Any hydrophobic NCA monomer may be used. Depending on the type of NCA monomer used, the NCA monomer may be modified to render it hydrophobic, particularly via a hydrophobic protecting group.

[0018] In one embodiment, the hydrophobic α-amino acid N-carboxyanhydride monomer has the following formula (I):

[0019] [ka]

[0020] where R is the optionally protected side chain of a hydrophobic α-amino acid, natural or modified. has.

[0021] When the NCA monomer used is a hydrophilic α-amino acid NCA (an NCA that in particular contains an OH, COOH or NH2 functional group), the aforementioned R group contains a hydrophobic protecting group in order to render the monomer hydrophobic.

[0022] This protection is not necessary if the NCA used is an α-amino acid NCA, which is hydrophobic in nature.

[0023] As hydrophobic α-amino acid N-carboxyanhydride monomers the following compounds are in particular used:

[0024] [ka]

[0025] Preferred examples include NCA monomers such as γ-benzyl-L-glutamate, ε-Boc-L-lysine, L-leucine, or L-phenylalanine.

[0026] As mentioned above, the method of the present invention involves the use of at least one hydrophilic polymer (P1) containing at least one amine functional group, which is used as a macroinitiator.

[0027] In one embodiment, 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) containing at least one amine function, preferably the polymer (P1) is selected among poly(ethylene oxide)s having at least one amine function.

[0028] Preferably, the polymer (P1) is PEG.

[0029] Preferably, the hydrophilic polymer (P1) has a molecular weight higher than 500 g / mol, more preferably between 2000 g / mol and 10 000 g / mol.

[0030] Preferably, the polymer (P1) has the following formula:

[0031] [ka]

[0032] [In the formula, x is 16 to 500] has.

[0033] In the process of the invention, the starting product can be compared to a suspension, ie an opaque, heterogeneous medium, and the final product obtained is preferably in the form of a homogeneous, clear solution.

[0034] Thus, the method of the present invention can advantageously be used to convert solids dispersed in highly heterogeneous media, especially water, into solutions of well-defined nanoparticles based on amphiphilic copolymers of controlled macromolecular architecture.

[0035] The methods of the present invention are "one-pot" methods, ie, methods in which reagents undergo one or more sequential or simultaneous reactions in a single reaction mixture.

[0036] In one embodiment, in the methods of the invention, the aqueous solvent is water or a buffer.

[0037] For example, the aqueous solvent is water to which a buffer solution has been added.

[0038] In one embodiment, the aqueous solvent also comprises a buffer solution containing a salt at a concentration ranging from 0.01 M to 1 M, in particular a buffer solution selected from the group consisting of sodium bicarbonate solution and phosphate buffer solution.

[0039] Preferably, the method of the present invention uses an aqueous solution of NaHCO3.

[0040] Preferably, the pH of the aqueous medium is between 2 and 12, in particular between 7 and 10.

[0041] In one embodiment, the temperature in step (E1) is from -10°C to 80°C, preferably from 0°C to 4°C.

[0042] In one embodiment, step (E1) is carried out from a dispersion of the hydrophobic α-amino acid N-carboxyanhydride monomer under stirring.

[0043] The method of the present invention can also be applied to obtain multi-block copolymers.

[0044] For this reason, in one embodiment, the aqueous solution of nanoparticles of the amphiphilic block copolymer obtained after step (E1) is subsequently contacted with a second hydrophobic α-amino acid N-carboxyanhydride monomer, which is the same or different from that in step (E1), thereby obtaining an aqueous solution of modified nanoparticles of the modified amphiphilic block copolymer.

[0045] Thus, in this embodiment, the addition of at least one second hydrophobic α-amino acid N-carboxyanhydride monomer different from that used in step (E1) allows gradient or statistical multiblock copolymers to be obtained.

[0046] The method of the present invention can be applied to obtain nanoparticles, preferably core-corona, preferably elongated, preferably rigid, preferably with dimensions in the range of 2 nm to 1 μm, thus in this embodiment anisotropic nanoparticles with optical properties can be obtained.

[0047] The present invention also relates to nanoparticles of amphiphilic block copolymers comprising polypeptide units, obtainable using the process defined above.

[0048] The present invention therefore also relates to nanoparticles of amphiphilic block copolymers comprising polypeptide units, obtained using the method defined above, having a core-shell structure and a particle size between 2 nm and 1 μm.

[0049] The present invention therefore relates to an aqueous composition comprising nanoparticles as defined above, wherein the mass content of said nanoparticles is at least 2% relative to the mass of said aqueous composition, preferably between 2% and 15% by mass relative to the mass of said aqueous composition.

[0050] This solids content is determined after polymerization and after purification of the salt by dialysis by carrying out lyophilization (by comparing the residue mass with the mass before lyophilization). [Brief description of the drawings]

[0051] [Figure 1] Size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 1 performed in DMF (+1 mg / mL LiBr). Left: RI detection. Right: absorbance detection. [Diagram 2]FIG. 1 shows the 1H NMR spectrum of the PEG5k-b-PBLG copolymer in Example 1 obtained in CDCl3+15% TFA. [Diagram 3] FIG. 1 shows the distribution of hydrodynamic diameter (Dh) at the diffusion intensity of PEG5k-b-PBLG nanoparticles (in ultrapure water) in Example 1. [Figure 4] FIG. 1 shows images of cryogenized nanoparticles: PEG5k-b-PBLG copolymer nanoparticles in Example 1 taken under a transmission electron microscope (Cryo-TEM). [Diagram 5] Size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 2 carried out in DMF (+1 mg / mL LiBr). Left: RI detection. Right: absorbance detection. [Figure 6] FIG. 1 shows the 1H NMR spectrum of PEG5k-b-PBLG copolymer in Example 2 carried out in CDCl3+15% TFA. [Figure 7] FIG. 1 shows a Cryo-TEM image of PEG5k-b-PBLG copolymer nanoparticles in Example 2. [Figure 8] Size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 3 performed in DMF (+1 mg / mL LiBr). Left: RI detection. Right: absorbance detection. [Figure 9] FIG. 1 shows the 1H NMR spectrum of PEG5k-b-PBLG copolymer in Example 3 carried out in CDCl3+15% TFA. [Figure 10] FIG. 13 is a diagram showing the distribution of hydrodynamic diameter (Dh) (diffusion intensity) of PEG5k-b-PBLG nanoparticles (in ultrapure water) in Example 3. [Figure 11] Size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 4 performed in DMF (+1 mg / mL LiBr). Left: RI detection. Right: absorbance detection. [Figure 12]FIG. 1 shows the 1H NMR spectrum of PEG5k-b-PBLG copolymer in Example 4 carried out in CDCl3+15% TFA. [Figure 13] FIG. 13 is a diagram showing the distribution of hydrodynamic diameter (Dh) (diffusion intensity) of PEG5k-b-PBLG nanoparticles (in ultrapure water) in Example 4. [Figure 14] FIG. 1 shows a Cryo-TEM image of PEG5k-b-PBLG copolymer nanoparticles in Example 4. [Figure 15] Figure 1 shows size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 5, performed in DMF (+1 mg / mL LiBr), RI detection in black and grey, absorbance detection in grey dotted line. [Figure 16] FIG. 13 shows the distribution of hydrodynamic diameter (Dh) (diffusion intensity) of PEG5k-b-PBLG nanoparticles (in ultrapure water) in Example 5. [Figure 17] Size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 6, performed in DMF (+1 mg / mL LiBr). Left: RI detection. Right: absorbance detection. [Figure 18] FIG. 1 shows the 1H NMR spectrum of PEG5k-b-PBLG copolymer in Example 6 carried out in CDCl3+15% TFA. [Figure 19] FIG. 13 shows a Cryo-TEM image of PEG5k-b-PBLG copolymer nanoparticles in Example 6. [Figure 20] A) shows size exclusion chromatography (RI detection) of the ELPM40-b-PBLG copolymer in Example 7 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the ELPN40-b-PBLG copolymer in Example 7. [Figure 21] Size exclusion chromatography of PEG5k-b-PLys copolymer in Example 8, performed in DMF (+1 mg / mL LiBr). RI detection. [Figure 22] FIG. 1 shows the 1H NMR spectrum of the PEG5k-b-PLys copolymer in Example 8 carried out in DMF-d6. [Figure 23] FIG. 13 shows the distribution of hydrodynamic diameter (Dh) (diffusion intensity) of PEG5k-b-PLys nanoparticles (in ultrapure water) in Example 8. [Figure 24] FIG. 13 shows a Cryo-TEM image of nanoparticles of PEG5k-b-PLys copolymer in Example 8. [Diagram 25] Figure 1 shows size exclusion chromatography of PEG5k-b-PLys-b-PBLG copolymer in Example 9 in DMF (+1 mg / mL LiBr). RI detection. [Figure 26] A) shows size exclusion chromatography (RI detection) of the PLeu-b-PBLG copolymer in Example 10 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PLeu-b-PBLG copolymer in Example 10. [Figure 27] A) shows size exclusion chromatography (RI detection) of the copolymer PPhe-b-PBLG in Example 11 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PPhe-b-PBLG copolymer in Example 11. [Figure 28] A) shows size exclusion chromatography (RI detection) of the PEG5k-b-PBLG-b-PEG5k copolymer in Example 12 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PEG5k-b-PBLG-b-PEG5k copolymer in Example 12. [Figure 29]A) shows size exclusion chromatography (RI detection) of the PEG-4arm-b-(PBLG)4 copolymer in Example 13 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PEG-4arm-b(PBLG)4 copolymer in Example 13. [Diagram 30] A) shows size exclusion chromatography (RI detection) of the PSar-b-PBLG copolymer in Example 14 carried out in DMF (+1 mg / mL LiBr). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PSar-b-PBLG copolymer in Example 14. [Diagram 31] A) shows size exclusion chromatography (RI detection) after deprotection of the PGA-b-PBLG copolymer in Example 15, performed in HO (Juanito buffer). B) shows a TEM image (uranyl acetate staining) of nanoparticles of the PGA-b-PBLG copolymer in Example 15. [Diagram 32] A) Infrared analysis (powder) of Example 16. B) TEM image (uranyl acetate stain) of the copolymer nanoparticles in Example 16. [Diagram 33] FIG. 13 shows size exclusion chromatography of PEG5k-b-PBLG copolymer in Example 17 carried out in DMF (+1 mg / mL LiBr) (RI detection). [Figure 34A] FIG. 13 shows a TEM image (stained with uranyl acetate) of the copolymer nanoparticles in Example 18. [Figure 34B] FIG. 12 is an electrophoretic gel showing copolymer formation in Example 18. [Figure 34C] FIG. 13 shows the distribution of hydrodynamic diameter (Dh) (diffusion intensity) of nanoparticles (in ultrapure water) in Example 18. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] [Example] Example 1 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k -synthesis of -block-poly(γ-benzyl-L-glutamate)s and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0053] The NCA monomer of γ-benzyl-L-glutamate (300 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 5k Prepare a 0.05 M aqueous solution of NaHCO3 containing -NH2 (8 mL, 300 mg, 0.06 mmol) and leave to cool in an ice bath for at least 10 min. While still in the ice bath, add the aqueous solution to the 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 left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tubing (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 ± 3% (Figures 1-4).

[0054] Example 2 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Synthesis of -block-poly(γ-benzyl-L-glutamate) and their corresponding nanoparticles in the absence of salts γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k-NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0055] The NCA monomer of γ-benzyl-L-glutamate (300 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 5k -NH2 (8 mL, 300 mg, 0.06 mmol) is diluted in ultrapure water and left to cool in an ice bath for at least 10 min. While still in the ice bath, the aqueous solution is added to the 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 left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tubing (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figures 5-7).

[0056] Example 3 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Simultaneous synthesis of -block-poly(γ-benzyl-L-glutamate) and their corresponding nanoparticles at high solids content γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0057] The NCA monomer of γ-benzyl-L-glutamate (600 mg, 2.28 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 5kPrepare a 0.05 M aqueous solution of NaHCO3 containing -NH2 (8 mL, 600 mg, 0.12 mmol) and leave to cool in an ice bath for at least 10 min. While still in the ice bath, add the aqueous solution to the 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 left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tubing (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 77% (Figures 8-10).

[0058] Example 4 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Procedure for chain extension of -block-poly(γ-benzyl-L-glutamate) γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0059] The NCA monomer of γ-benzyl-L-glutamate (300 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 5kA 0.05 M aqueous solution of NaHCO3 containing -NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool in an ice bath for at least 10 min. While still in the ice bath, the aqueous solution is added to the NCA powder under 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 NCA γ-benzyl-L-glutamate is made (300 mg, 1.14 mmol). The reaction is left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (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 11-14).

[0060] Example 5 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 2k -synthesis of -block-poly(γ-benzyl-L-glutamate)s and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 2k -NH2 (Mp=2022 Da, D=1.04) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0061] The NCA monomer of γ-benzyl-L-glutamate (30 mg, 0.11 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 2kA 0.2 M aqueous solution of NaHCO3 containing -NH2 (800 μL, 30 mg, 0.015 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figures 15 and 16).

[0062] Example 6 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 10k -synthesis of -block-poly(γ-benzyl-L-glutamate)s and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 10k -NH2 (Mp=11153 Da, D=1.04) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0063] The NCA monomer of γ-benzyl-L-glutamate (300 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG is added to the flask. 10kA 0.05 M aqueous solution of NaHCO3 containing -NH2 (8 mL, 300 mg, 0.03 mmol) is prepared and left to cool in an ice bath for at least 10 min. While still in the ice bath, the aqueous solution is added to the 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 left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into a gel, which is transferred to a dialysis tubing (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figures 17-19).

[0064] Example 7 Simultaneous synthesis of amphiphilic peptide diblock copolymers (elastin-like polypeptide)-block-poly(.GAMMA.-benzyl-L-glutamate) and their corresponding nanoparticles Gamma-benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. Elastin-like polypeptide (ELP) is a recombinant protein produced in pathogenic E. coli at the Laboratoire de Chimie des Polymeres Organiques de Bordeaux, France. The ELP used has a primary amine at its N-terminus. It has the structure MW (VPGVP VPGMG VPGVG VPGVG) 10 and molecular weight of 17035 Da. Other unconventional reagents are supplied by Sigma-Aldrich.

[0065] The NCA monomer of γ-benzyl-L-glutamate (10 mg, 0.04 mmol) is placed in a test tube containing a magnetic stir bar under an inert atmosphere. The tube is cooled in an ice bath for at least 10 min. In parallel, a 0.05 M aqueous solution of NaHCO3 containing the ELP macroinitiator (2.7 mL, 10 mg, 0.001 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and under vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left for 20 h at 4 °C under magnetic stirring. The milky dispersion turns into a turbid dispersion at ambient temperature, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water at 4 °C for 2 days. After lyophilization, a white powder is obtained (Figure 20).

[0066] Example 8 Simultaneous synthesis of amphiphilic peptide diblock copolymers poly(ethylene glycol)-block-poly(ε-Boc-l-lysine) and their corresponding nanoparticles The monomer ε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride (LysBOC-NCA) is a commercial product supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other unconventional reagents were supplied by Sigma-Aldrich.

[0067] The NCA monomer of ε-Boc-L-lysine (310 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG 5kA 0.05 M aqueous solution of NaHCO3 containing -NH2 (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool in an ice bath for at least 10 min. While still in the ice bath, the aqueous solution is added to the 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 left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tubing (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained in 79% yield (Figures 21-24).

[0068] Example 9 Simultaneous synthesis of amphiphilic peptide triblock copolymers poly(ethylene glycol)-block-poly(ε-Boc-L-lysine)-block-poly(γ-benzyl-L-glutamate) and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) and ε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride monomer (LysBOC-NCA) are commercial products supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other unconventional reagents were supplied by Sigma-Aldrich.

[0069] The NCA monomer of ε-Boc-L-lysine (300 mg, 1.10 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk flask is cooled in an ice bath for at least 10 min. In parallel, PEG 5kA 0.2 M aqueous solution of NaHCO3 containing -NH2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and allowed to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and with vigorous stirring. This gives a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring for about 15 min in an ice-water bath. In parallel, the NCA monomer of γ-benzyl-L-glutamate (BLG-NCA, 300 mg, 1.14 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is allowed to cool in an ice bath for at least 10 min. The opalescent aqueous colloidal solution is mixed with the BLG-NCA powder while remaining in the ice bath and with vigorous stirring. This gives a milky dispersion due to the immiscibility of the monomer in the aqueous phase. The reaction is left under stirring 1) first for about 2 hours in an ice-water bath, and 2) then for 16 hours at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred into a dialysis tube (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).

[0070] Example 10 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Simultaneous synthesis of -block-poly(L-leucine)s and their corresponding nanoparticles L-Leucine N-carboxyanhydride monomer (LEU-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0071] The NCA monomer of leucine (300 mg, 1.9 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, PEG 5kA 0.05 M aqueous solution of NaHCO3 containing -NH2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 77% (Figure 26).

[0072] Example 11 Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Simultaneous synthesis of -block-poly(L-phenylalanine)s and their corresponding nanoparticles L-Phenylalanine N-carboxyanhydride monomer (PHE-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0073] The NCA monomer of phenylalanine (150 mg, 0.8 mmol) is placed in a Schlenk flask containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, PEG 5kA 0.05 M aqueous solution of NaHCO3 containing -NH2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 72% (Figure 27).

[0074] Example 12 Amphiphilic peptide triblock copolymer, poly(γ-benzyl-L-glutamate)-block-poly(ethylene glycol) 5k -synthesis of -block-poly(γ-benzyl-L-glutamate)s and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (PEG-2arm) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0075] The NCA monomer of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) (29 mg, 0.11 mmol) is placed in a test tube containing a magnetic stir bar under an inert atmosphere. The tube is cooled in an ice bath for at least 10 min. In parallel, NH2-PEG 5kA 0.05 M aqueous solution of NaHCO3 containing -NH2 macroinitiator (0.85 mL, 35 mg) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under vigorous stirring. This results in a milky dispersion due to the immiscibility of the monomers in the aqueous phase. The reaction is left under stirring 1) first for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figure 28).

[0076] (Example 13) Amphiphilic peptide star copolymer poly(γ-benzyl-L-glutamate) 4-block-poly(ethylene glycol) 5k and simultaneous synthesis of their corresponding nanoparticles. γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. PEG-4arm is a commercially available chemical reagent supplied by RAPP Polymer (star: 4N H2 The reagents were supplied by Sigma-Aldrich.

[0077] The NCA monomer of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) (18 mg, 0.07 mmol) is placed in a test tube containing a magnetic stir bar under an inert atmosphere. The tube is cooled in an ice bath for at least 10 min. In parallel, PEG 5kA 0.05 M aqueous solution of NaHCO3 containing the -4arm macroinitiator (0.70 mL, 35 mg) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figure 29).

[0078] Example 14 Simultaneous synthesis of amphiphilic peptide diblock copolymers poly(sarcosine)-block-poly(.GAMMA.-benzyl-L-glutamate) and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent supplied by ISOCHEM. Poly(sarcosine) PSar (Mp=2100 Da, D=1.03) is synthesized in the laboratory by conventional ring-opening polymerization. Other reagents are supplied by Sigma-Aldrich.

[0079] The NCA monomer of γ-benzyl-L-glutamate (120 mg, 0.46 mmol) is placed in a Schlenk tube containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator (4 mL, 50 mg, 0.02 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 55% (Figure 30).

[0080] Example 15 Simultaneous synthesis of amphiphilic peptide diblock copolymers poly(L-glutamic acid)-block-poly(.GAMMA.-benzyl-L-glutamate) and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent supplied by ISOCHEM. Poly(L-glutamic acid) PGA (Mw=6600 g / mol) is synthesized in the laboratory by conventional ring-opening polymerization. Other reagents are supplied by Sigma-Aldrich.

[0081] The NCA monomer of γ-benzyl-L-glutamate (300 mg, 1.14 mmol) is placed in a Schlenk tube containing a magnetic stir bar under an inert atmosphere. The Schlenk is cooled in an ice bath for at least 10 min. In parallel, a 0.20 M aqueous solution of NaHCO3 containing the macroinitiator (8 mL, 300 mg, 0.45 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days (TEM image, Figure 31). After lyophilization, for analysis of the copolymers, the PBLG blocks are deprotected under mild acid conditions (MSA, TFA) to give a white powder that can be analyzed in aqueous SEC (Juanito buffer) (Figure 31).

[0082] (Example 16) Simultaneous synthesis of amphiphilic peptide diblock copolymers, polysaccharide-block-poly(.GAMMA.-benzyl-L-glutamate) and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent supplied by ISOCHEM. Polyaminosugar (polysaccharide, Mw=7750 g / mol) is synthesized in the laboratory by anionic polymerization of β-lactam monomers. Other reagents are supplied by Sigma-Aldrich.

[0083] The NCA monomer of γ-benzyl-L-glutamate (13 mg, 0.05 mmol) is placed in a test tube containing a magnetic stirring bar under an inert atmosphere. The tube is cooled in an ice bath for at least 10 min. In parallel, a 0.05 M aqueous solution of NaHCO3 containing the macroinitiator (2 mL, 20 mg, 0.003 mmol) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into a whitish aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figure 32).

[0084] (Example 17) Poly(ethylene glycol), an amphiphilic peptide diblock copolymer 5k Simultaneous synthesis of -block-poly(γ-benzyl-L-glutamate) and their corresponding nanoparticles on a very small scale γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercially available chemical reagent supplied by ISOCHEM. 5k -NH2 (Mp=5516 Da, D=1.02) was supplied by RAPP Polymer, Inc. Other reagents were supplied by Sigma-Aldrich.

[0085] The NCA monomer of γ-benzyl-L-glutamate (3 mg, 0.01 mmol) is placed in an Eppendorf containing a small magnetic stir bar under an inert atmosphere and cooled in an ice bath for at least 10 min. In parallel, the macroinitiator PEG 5k A 0.05 M aqueous solution of NaHCO3 containing -NH2 (80 μL, 3 mg) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while still in the ice bath and under 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figure 33).

[0086] (Example 18) Very small-scale simultaneous synthesis of amphiphilic peptide diblock copolymers, deoxyribonucleic acid-block-poly(γ-benzyl-L-glutamate) and their corresponding nanoparticles γ-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent supplied by ISOCHEM. Deoxyribonucleic acid DNA (TTT)15 (Mw=4680 g / mol) is supplied by IDT Technologies. Other reagents are supplied by Sigma-Aldrich.

[0087] The NCA monomer of γ-benzyl-L-glutamate (5 mg) is placed in an Eppendorf under inert atmosphere and cooled in an ice bath for at least 10 min. In parallel, a 0.05 M aqueous solution of NaHCO3 containing the DNA macroinitiator (1 mL, 5 mg) is prepared and left to cool in an ice bath for at least 10 min. The aqueous solution is added to the NCA powder while remaining in the ice bath and under vigorous magnetic 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 for about 2 h in an ice-water bath and 2) then for 16 h at ambient temperature. The milky dispersion turns into an opalescent aqueous colloidal solution, which is transferred to a dialysis tube (3.5 kDa dialysis membrane) and dialyzed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (Figure 34).

[0088] Tables 1 and 2 below show the molecular and physicochemical properties of the copolymers synthesized by ROPISA and their nanoparticles according to the above examples.

[0089] [Table 1]

[0090] [Table 2]

Claims

1. A "one-pot" method for preparing an aqueous solution of nanoparticles of amphiphilic block copolymers comprising polypeptide units, comprising: at least one hydrophilic polymer (P1) containing at least one amine functional group, and at least one hydrophobic α-amino acid N-carboxyanhydride monomer; in an aqueous solvent free of organic solvent.

2. The hydrophobic α-amino acid N-carboxyanhydride monomer has the following formula (I): 【Chemistry 1】 where R is the optionally protected side chain of a hydrophobic α-amino acid, natural or modified.

2. The method of claim 1, comprising:

3. 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.

4. The polymer (P1) has the following formula: 【Chemistry 2】 [In the formula, x is 16 to 500] 4. The method according to claim 1, further comprising:

5. 5. The method according to claim 1, wherein the aqueous solvent is water or a buffer solution.

6. The method according to any one of claims 1 to 5, wherein the aqueous solvent also comprises a buffer solution comprising an inorganic salt at a concentration in the range of 0.01M to 1M.

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

8. The process according to any one of claims 1 to 7, wherein the temperature in step (E1) is from -10°C to 80°C.

9. 9. The method according to claim 1, wherein step (E1) is carried out from a dispersion of the hydrophobic α-amino acid N-carboxyanhydride monomer under stirring.

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

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