METHOD FOR PREPARING CONTROLLED PEPTIDE-BASED POLYMERS AND COPOLYMERS IN AN AQUEOUS SOLUTION

Abstract

The present invention relates to a “one-pot” method for preparing an aqueous solution of nanoparticles with amphiphilic block copolymers and comprising polypeptide units, the method comprising at least one step (E1), in an aqueous 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.

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.

2. The method according to claim 1 wherein the hydrophobic α-amino acid N-carboxyanhydride monomer has the following formula (I): ##STR00004## where R is the side chain of an optionally protected, natural or modified, hydrophobic α-amino acid.

3. The method according to claim 1, 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 method according to claim 1, wherein the polymer (P1) has the following formula: ##STR00005## where x is from 16 to 500.

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

6. The method according to claim 1, wherein the aqueous solvent also comprises a buffer solution comprising an inorganic salt at concentrations ranging from 0.01 M to 1 M.

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

8. The method according to claim 1, wherein the temperature at step (E1) is from −10° C. to 80° C.

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

10. The method according to claim 1, 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

11. An aqueous composition of nanoparticles of amphiphilic block copolymers comprising polypeptide units obtained with the method according to claim 1, said nanoparticles having a core-shell structure and particle size of 2 nm to 1 μm, the weight content of said nanoparticles being at least 2% by weight relative to the weight of said aqueous composition.

12. The method of claim 1, wherein the polymer (P1) is selected from the elastin-like polypeptides (ELPs) comprising at least one amine function.

13. The method of claim 6, wherein the aqueous solvent also comprises a buffer solution is selected from the group consisting of sodium hydrogen carbonate solutions and phosphate buffer solutions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 concerns size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 1. Left: RI detection. Right: absorbance detection.

[0052] FIG. 2 illustrates the .sup.1H NMR spectrum obtained in CDCl.sub.3+15% TFA of the PEG.sub.5k-b-PBLG copolymer in Example 1.

[0053] FIG. 3 illustrates the distribution of hydrodynamic diameter (Dh) in diffusion intensity of the nanoparticles of PEG.sub.5k-b-PBLG in Example 1 (in ultrapure water).

[0054] FIG. 4 gives images taken under Transmission Electron Microscopy of cryogenized nanoparticles (Cryo-TEM): nanoparticles of the PEG.sub.5k-b-PBLG copolymer in Example 1.

[0055] FIG. 5 illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 2. Left: RI detection. Right: absorbance detection.

[0056] FIG. 6 illustrates the .sup.1H NMR spectrum performed in CDCl.sub.3+15% TFA of the PEG.sub.5k-b-PBLG copolymer in Example 2.

[0057] FIG. 7 gives a cryo-TEM image of the nanoparticles of the PEG.sub.5K-b-PBLG copolymer in Example 2.

[0058] FIG. 8 illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 3. Left: RI detection. Right: absorbance detection.

[0059] FIG. 9 illustrates the .sup.1H NMR spectrum performed in CDCl.sub.3+15% TFA of the PEG.sub.5k-b-PBLG copolymer in Example 3.

[0060] FIG. 10 gives the distribution (in diffusion intensity) of the hydrodynamic diameter (Dh) of the nanoparticles of PEG.sub.5k-b-PBLG in Example 3 (in ultrapure water).

[0061] FIG. 11 illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 4. Left: RI detection. Right: absorbance detection.

[0062] FIG. 12 gives the .sup.1H NMR spectrum performed in CDCl.sub.3+15% TFA of the PEG.sub.5k-b-PBLG copolymer in Example 4.

[0063] FIG. 13 gives the distribution (in diffusion intensity) of the hydrodynamic diameter (Dh) of the nanoparticles of PEG.sub.5k-b-PBLG in Example 4 (in ultrapure water).

[0064] FIG. 14 gives a Cryo-TEM image of the nanoparticles of the PEG.sub.5k-b-PBLG copolymer in Example 4.

[0065] FIG. 15 shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 5. RI detection in black and grey and absorbance detection in dotted grey line.

[0066] FIG. 16 gives the distribution (in diffusion intensity) of the hydrodynamic diameter (Dh) of the nanoparticles of PEG.sub.5k-b-PBLG in Example 5 (in ultrapure water).

[0067] FIG. 17 illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 6. Left: RI detection. Right: absorbance detection.

[0068] FIG. 18 gives the .sup.1H NMR spectrum performed in CDCl.sub.3+15% TFA of the PEG.sub.5k-b-PBLG copolymer in Example 6.

[0069] FIG. 19 gives a Cryo-TEM image of the nanoparticles of the PEG.sub.5k-b-PBLG copolymer in Example 6.

[0070] FIG. 20A illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the ELPM40-b-PBLG copolymer in Example 7 (RI detection); FIG. 20B illustrates a TEM image (uranyl acetate staining) of the nanoparticles of the ELPN40-b-PBLG copolymer in Example 7.

[0071] FIG. 21 illustrates size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PLys copolymer in Example 8. RI detection.

[0072] FIG. 22 gives the .sup.1H NMR spectrum performed in DMF-d6 of the PEG.sub.5k-b-PLys copolymer in Example 8.

[0073] FIG. 23 gives the distribution (in diffusion intensity) of the hydrodynamic diameter (Dh) of the nanoparticles of PEG.sub.5k-b-PLys in Example 8 (in ultrapure water).

[0074] FIG. 24 gives a Cryo-TEM image of the nanoparticles of the PEG.sub.5k-b-PLys copolymer in Example 8.

[0075] FIG. 25 illustrates size exclusion chromatography in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PLys-b-PBLG copolymer in Example 9. RI detection.

[0076] FIG. 26A shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PLeu-b-PBLG copolymer in Example 10 (RI detection); FIG. 26B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PLeu-b-PBLG copolymer in Example 10.

[0077] FIG. 27A shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the copolymer PPhe-b-PBLG in Example 11 (RI detection); FIG. 27B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PPhe-b-PBLG copolymer in Example 11.

[0078] FIG. 28A shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG-b-PEG.sub.5k copolymer in Example 12 (RI detection); FIG. 28B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PEG.sub.5k-b-PBLG-b-PEG.sub.5k copolymer in Example 12.

[0079] FIG. 29A shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG-4arm-b-(PBLG).sub.4 copolymer in Example 13 (RI detection); FIG. 29B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PEG-4arm-b-(PBLG).sub.4 copolymer in Example 13.

[0080] FIG. 30A shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PSar-b-PBLG copolymer in Example 14 (RI detection); FIG. 30B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PSar-b-PBLG copolymer in Example 14.

[0081] FIG. 31A shows size exclusion chromatography performed in H.sub.2O (Juanito buffer) of the PGA-b-PBLG copolymer in Example 15 after deprotection (RI detection); FIG. 31B shows a TEM image (uranyl acetate staining) of the nanoparticles of the PGA-b-PBLG copolymer in Example 15.

[0082] FIG. 32A shows infrared analysis of Example 16 (powder); FIG. 32B shows a TEM image (uranyl acetate staining) of the nanoparticles of the copolymer in Example 16.

[0083] FIG. 33 shows size exclusion chromatography performed in DMF (+1 mg/mL LiBr) of the PEG.sub.5k-b-PBLG copolymer in Example 17 (RI detection).

[0084] FIG. 34A shows a TEM image (uranyl acetate staining) of the nanoparticles of the copolymer in Example 18; FIG. 34B shows the distribution (in diffusion intensity) of the hydrodynamic diameter (Dh) of the nanoparticles in Example 18 (in ultrapure water); FIG. 34C shows electrophoresis gel showing the formation of the copolymer in Example 18.

DETAILED DESCRIPTION

Examples

Example 1: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(y-benzyl-L-glutamate) and of Their Corresponding Nanoparticles

[0085] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0086] The NCA monomer of y-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.5k-NH.sub.2 (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 85±3% (FIGS. 1 to 4).

Example 2: Concomitant Synthesis, in the Absence of Salts, of an Amphiphilic Peptide Diblock Copolymer Poly(Ethylene Glycol).SUB.5k.-Block-Poly(y-Benzyl-L-Glutamate) and of their Corresponding Nanoparticles

[0087] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0088] The NCA monomer of y-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. In parallel, the macroinitiator PEG.sub.5k-NH.sub.2 (8 mL, 300 mg, 0.06 mmol) is diluted in ultrapure water and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 5 to 7).

Example 3: Concomitant Synthesis with High Solid Content of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0089] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0090] The NCA monomer of y-benzyl-L-glutamate (600 mg, 2.28 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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.5k-NH.sub.2 (8 mL, 600 mg, 0.12 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 77% (FIGS. 8 to 10).

Example 4: Procedure for Extending the Chain of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(y-benzyl-L-glutamate)

[0091] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0092] The NCA monomer of y-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.5k-NH.sub.2 (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. After 90 minutes, a second addition of NCA y-benzyl-L-glutamate is made (300 mg, 1.14 mmol). The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 85% (FIGS. 11 to 14).

Example 5: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.2k.-block-poly(y-benzyl-L-glutamate) and of their corresponding nanoparticles

[0093] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.2k-NH.sub.2 (Mp=2022 Da, Ð=1.04) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0094] The NCA monomer of y-benzyl-L-glutamate (30 mg, 0.11 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. In parallel, an aqueous 0.2 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.2k-NH.sub.2 (800 μL, 30 mg, 0.015 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 15 and 16).

Example 6: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.10k.-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0095] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.10k-NH.sub.2 (Mp=11153 Da, Ð=1.04) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0096] The NCA monomer of y-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.10k-NH.sub.2 (8 mL, 300 mg, 0.03 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to a gel which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 17 to 19).

Example 7: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer (Elastin-like polypeptide)-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0097] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. The elastin-like polypeptide (ELP) is a recombinant protein produced in the bacterium E. coli at the laboratory: Laboratoire de Chimie des Polymères Organiques de Bordeaux, France. The ELP use has a primary amine at its N-terminal end. It has the structure MW (VPGVP VPGMG VPGVG VPGVG).sub.10 and a molecular weight of 17 035 Da. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0098] The NCA monomer of y-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 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 minutes. While remaining in the ice bath, the aqueous solution is added to the NCA powder under vigorous stirring. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under magnetic agitation at 4° C. for 20 h. The milky dispersion changes to a turbid dispersion at ambient temperature and it is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days at 4° C. After lyophilization, a white powder is obtained (FIGS. 20A-20B).

Example 8: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol)-block-poly(ε-Boc-L-Lysine) and of their Corresponding Nanoparticles

[0099] The monomer of ε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride (LysBOC-NCA) is a commercial product distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0100] The NCA monomer of ε-Boc-L-Lysine (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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.5k-NH.sub.2 (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 79% (FIGS. 21 to 24).

Example 9: Concomitant Synthesis of an Amphiphilic Peptide Triblock Copolymer poly(ethylene glycol)-block-poly(ε-Boc-L-Lysine)-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0101] The y-Benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) and ε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride monomers (LysBOC-NCA) are commercial products distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other non-conventional reagents are distributed by Sigma-Aldrich.

[0102] The NCA monomer of ε-Boc-L-Lysine (300 mg, 1.10 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. In parallel, an aqueous 0.2 M solution of NaHCO.sub.3 containing the PEG.sub.5k-NH.sub.2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation in an iced water bath for about 15 minutes. In parallel, the NCA monomer of y-benzyl-L-glutamate (BLG-NCA, 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. While remaining in the ice bath, the aqueous opalescent colloidal solution is mixed with the BLG-NCA powder under vigorous stirring. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 75±3% (FIG. 25).

Example 10: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(L-leucine) and of their Corresponding Nanoparticles

[0103] The L-leucine N-carboxyanhydride monomer (LEU-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0104] The NCA monomer of leucine (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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the PEG.sub.5k-NH.sub.2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 77% (FIGS. 26A-26B).

Example 11: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(L-phenylalanine) and of their Corresponding Nanoparticles

[0105] The L-phenylalanine N-carboxyanhydride monomer (PHE-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0106] The NCA monomer of phenylalanine (150 mg, 0.8 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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the PEG.sub.5k-NH.sub.2 macroinitiator (8 mL, 300 mg, 0.06 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 h. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 72% (FIGS. 27A-27B).

Example 12: Concomitant Synthesis of an Amphiphilic Peptide Triblock Copolymer poly(y-benzyl-L-glutamate-block-poly(ethylene glycol).SUB.5k.-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0107] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. NH.sub.2-PEG.sub.5k-NH.sub.2 (PEG-2arm) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0108] The NCA monomer of y-Benzyl-L-glutamate N-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the NH.sub.2-PEG.sub.5k-NH.sub.2 macroinitiator (0.85 mL, 35 mg) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 28A-28B).

Example 13: Concomitant Synthesis of an Amphiphilic Peptide Star Copolymer poly(y-benzyl-L-glutamate).SUB.4.-block-poly(ethylene glycol).SUB.5k .and of their Corresponding Nanoparticles

[0109] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG-4arm is distributed by RAPP Polymer (star: 4 NH.sub.2 ends). The other reagents are distributed by Sigma-Aldrich.

[0110] The NCA monomer of y-Benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) (18 mg, 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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the PEG.sub.5k-4arm macroinitiator (0.70 mL, 35 mg) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 29A-29B).

Example 14: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(sarcosine)-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0111] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. Poly(sarcosine) PSar (Mp=2100 Da, Ð=1.03) is synthesized in the laboratory by conventional ring opening polymerization. The other reagents are distributed by Sigma-Aldrich.

[0112] The NCA monomer of y-benzyl-L-glutamate (120 mg, 0.46 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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator (4 mL, 50 mg, 0.02 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained with a yield of 55% (FIGS. 30A-30B).

Example 15: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer of poly(L-glutamic acid)-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0113] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (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.

[0114] The NCA monomer of y-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. In parallel, an aqueous 0.20 M solution of NaHCO.sub.3 containing the macroinitiator (8 mL, 300 mg, 0.45 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days (TEM image, FIGS. 31A-31B). After lyophilization, for analysis of the copolymer, the PBLG block is deprotected under mild acid conditions (MSA, TFA) to obtain a white powder that can be analysed in aqueous SEC (Juanito buffer) (FIGS. 31A-31B).

Example 16: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer polysaccharide-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles

[0115] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (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.

[0116] The NCA monomer of y-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. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator (2 mL, 20 mg, 0.003 mmol) is prepared and left to cool 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. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous whitish colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 32A-32

Example 17: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer poly(ethylene glycol).SUB.5k.-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles on Very Small Scale

[0117] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. PEG.sub.5k-NH.sub.2 (Mp=5516 Da, Ð=1.02) is distributed by RAPP Polymer. The other reagents are distributed by Sigma-Aldrich.

[0118] The NCA monomer of y-benzyl-L-glutamate (3 mg, 0.01 mmol) is placed in an inert atmosphere in a Eppendorf containing a small magnetic stir bar which is cooled in an ice bath for at least 10 minutes. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the macroinitiator PEG.sub.5k-NH.sub.2 (804, 3 mg) is prepared and left to cool 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 magnetic agitation. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIG. 33).

Example 18: Concomitant Synthesis of an Amphiphilic Peptide Diblock Copolymer deoxyribonucleic acid-block-poly(y-benzyl-L-glutamate) and of their Corresponding Nanoparticles on Very Small Scale

[0119] The y-Benzyl-L-glutamate N-carboxyanhydride monomer (BLG-NCA) is a commercial chemical reagent distributed by ISOCHEM. Deoxyribonucleic acid DNA (TTT)15 (Mw=4680 g/mol) is distributed by IDT Technologies. The other reagents are distributed by Sigma-Aldrich.

[0120] The NCA monomer of y-benzyl-L-glutamate (5 mg) is placed in an inert atmosphere in an Eppendorf which is cooled in an ice bath for at least 10 minutes. In parallel, an aqueous 0.05 M solution of NaHCO.sub.3 containing the DNA macroinitiator (1 mL, 5 mg) is prepared and left to cool 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 magnetic agitation. This gives a milky dispersion resulting from non-miscibility of the monomer in the aqueous phase. The reaction is left under agitation 1) first in an iced water bath for about 2 hours, 2) then at ambient temperature for 16 hours. The milky dispersion changes to an aqueous opalescent colloidal solution which is transferred to a dialysis tube (dialysis membrane of 3.5 kDa) and dialysed against ultrapure water for 2 days. After lyophilization, a white powder is obtained (FIGS. 34A-34C).

[0121] Tables 1 and 2 below give the molecular and physicochemical characteristics of the copolymers synthesized by ROPISA, and of their nanoparticles, according to the above examples.

TABLE-US-00001 TABLE 1 Th. .sup.1H NMR SEC* Copolymer Buffer Initiator M/I g/mol Ts (%) M/I Mn g/mol Mn g/mol Ð PEG.sub.5k-NH.sub.2 — — — — — — 6996 1.02 Ex. 1 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 19 7 20 9380 11940 1.05 Ex. 2 MQ PEG.sub.5k-NH.sub.2 19 7 20 9380 13470 1.14 Ex. 3 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 19 13 21 9600 12250 1.06 Ex. 4 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 38 10 35 12670 14370 1.09 Ex. 5 NaHCO.sub.3 200 mM PEG.sub.2k-NH.sub.2 8 13 — — 4067 1.20 Ex. 6 NaHCO.sub.3 50 mM PEG.sub.10k-NH.sub.2 38 7 37 18100 24660 1.12 Ex. 7 NaHCO.sub.3 50 mM ELPM40 65 0.7 30 23600 27000 1.03 Ex. 8 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 19 7 21 9790 11750 1.09 Ex. 9 NaHCO.sub.3 200 mM PEG.sub.5k-NH.sub.2 38 13 — — 12220 1.05 Ex. 10 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 32 7 — — 10600 1.04 Ex. 11 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 2arms- 13 5 — — — — Ex. 12 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 4arms- 19 8 18 9900 14700 1.10 Ex. 13 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 10 8 16 8500 11700 1.32 Ex. 14 NaHCO.sub.3 50 mM PSar 19 7 — — 7100 1.34 Ex. 15 NaHCO.sub.3 50 mM PGA Ex. 16 NaHCO.sub.3 50 mM Polysacc. 19 1.6 — — — — Ex. 17 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 9 7 20 9380 11900 1.05 Ex. 18 PEG.sub.5k-NH.sub.2 DNA 19 5 — — — — *Number average molecular weight (M.sub.n) and dispersity (Ð) determined by SEC in DMF (+LiBr) using a polystyrene calibration curve.

TABLE-US-00002 TABLE 2 D.sub.h (σ) nm Copolymer Buffer Initiator DLS Yield % PEG.sub.5k-NH.sub.2 — — — — Ex. 1 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2  79 (0.08) 87 Ex. 2 MQ PEG.sub.5k-NH.sub.2 — — Ex. 3 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2  88 (0.17) 77 Ex. 4 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2  99 (0.12) 85 Ex. 5 NaHCO.sub.3 200 mM PEG.sub.2k-NH.sub.2 polydisperse — Ex. 6 NaHCO.sub.3 50 mM PEG.sub.10k-NH.sub.2 gel 83 Ex. 7 NaHCO.sub.3 200 mM ELPM40 Aggregated at T.sub.a — Ex. 8 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 131 (0.19) 79 Ex. 9 NaHCO.sub.3 200 mM PEG.sub.5k-NH.sub.2 — 75 Ex. 10 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2  92 (0.22) 77 Ex. 11 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 2arms- 125 (0.33) 72 Ex. 12 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 4arms- — — Ex. 13 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 — — Ex. 14 NaHCO.sub.3 50 mM PSar — 55 Ex. 15 NaHCO.sub.3 50 mM PGA 99 (0.7) — Ex. 16 NaHCO.sub.3 50 mM Polysacc. 414 (0.19) — Ex. 17 NaHCO.sub.3 50 mM PEG.sub.5k-NH.sub.2 — — Ex. 18 NaHCO.sub.3 50 mM DNA 460 (0.26) —