Process for the preparation of dispersible colloidal systems from a protein in nanoparticle form

Agent (s): CABINET LAVOIX

The present invention relates to a new process for the preparation of colloidal dispersible systems of a protein in the form of spherical particles of matrix type and of size less than

500 nm (nanoparticles).

The main patent N * 86 18446 relates to a process for the preparation of dispersible colloidal systems of a substance, in the form of spherical particles of matrix type and of size less than

500 nm. (nanoparticles), characterized in that:

(1) preparing a liquid phase consisting essentially of a solution of the substance in a solvent or in a mixture of solvents, and which may be added with one or more surfactants, (2) preparing a second liquid phase consisting essentially of a non-solvent or a mixture of non-solvents for the substance and which may be added with one or more surfactants, the non-solvent or the mixture of non-solvents of the substance being miscible in all proportions with the solvent or mixture of solvents of the substance, (3) one of the liquid phases obtained under (1) or (2) to the other, so as to obtain almost instantaneously a colloidal suspension of nanoparticles of the substance, and (4) if desired, all or part of the solvent or mixture of solvents is removed from the substance and the non-solvent or mixture of non-solvents from the substance. so as to obtain a colloidal suspension of desired concentration of nanoparticles or to obtain a powder of nanoparticles.

According to the present invention, said substance is a protein and optionally a biologically active substance, said solvent is water or an aqueous mixture at a temperature below the coagulation temperature of the protein, said non-solvent of the substance is l 'water at a temperature above the coagulation temperature of the protein and may optionally contain a biologically active substance, and said two liquid phases (1) and (2) are brought together under conditions of.pH away from the isoelectric point of the protein.

Thus, the present method is characterized in that:

(1) a liquid phase is prepared consisting essentially of a solution of the protein and optionally of a biologically-active substance in water or in an aqueous mixture at a temperature below the coagulation temperature of the protein, and which can be with one or more surfactants added, (2) a second liquid phase is prepared consisting essentially of water or of an aqueous mixture at a temperature above the coagulation temperature of the protein, which may contain a biologically active substance and which may be added with one or more several surfactants, (3) is added with moderate stirring, one of the liquid phases obtained under (1) or (2) to the other in. pH conditions far from the isoelectric point of the protein, so as to obtain practically instantaneously a colloidal suspension of nanoparticles of the protein and possibly of the biologically active substance, and (4) if desired, all or part of the water or an aqueous mixture, so as to obtain a colloidal suspension of desired concentration of nanoparticles or to obtain a powder of nanoparticles.

The protein is in particular a natural protein such as a serum albumin (for example human or bovine serum albumin) or an elastin (bovine, etc.). The biologically active substance can be a medicinal active principle or a medicinal precursor, a biological reagent or a cosmetic principle. The invention makes it possible to obtain nanoparticles of protein alone (which can be used as such) or with the biologically active substance. It is also possible and even desirable in the event of thermal instability, to bind the biologically active substance to the protein nanoparticles already formed.

The solvent of phase (1) which is water or an aqueous mixture (for example acidified or basified water) is in particular at a temperature ranging from 0 ° to 50 ° C., for example approximately at room temperature. .

The non-solvent for the protein in phase (2), which is water or an aqueous mixture (for example, acidified or basified water) is found in particular at a temperature ranging from 00 'to 100 ° C. (at atmospheric pressure), for example at about the boiling temperature.

The pH of the mixture of phases (1) and (2) must be far from the electrical isolating point of the protein in order to avoid its flocculation. This desirable pH difference is on the order of 2 to 3. Since natural proteins often have a pH in the range of 5 to 6, it is desirable that the final solution have a pH of about 3 or about. 9. For this purpose, the acid or the base can be added (e) indifferently to phase (1) or (2).

The protein concentration in phase (1, can vary from 0.1 to 10Z, preferably from

0.5 to 4Z.

263^397

The phase (1) / phase (2) volume ratio can vary from 0.1 to 1, preferably from 0.2 to 0.6.

Finally, the colloidal solution of nanoparticles can be concentrated, sterilized, buffered (for example at physiological pH), lyophilized or crosslinked at will.

The invention makes it possible to obtain protein nanoparticles, in particular from 150 to 300 nm.

The following examples illustrate the invention:

Example

Preparation of human serum bumine (HSA) nanoparticles.

Phase 1

SAH 1.0g

Hydrochloric acid N 0.3g

Demineralized or distilled water 100.0 g at room temperature

Phase 2

Demineralized or distilled water brought to the boil 180.0 g

Phase 1 is added with magnetic stirring to phase 2. The medium immediately becomes opalescent by formation of HSA nanoparticles. The mean diffractometer size of the nanoparticles, measured in a laser beam R (Nanosizer a laser beam (Nanosizer from Coultronics) is 190 nm with an average dispersion index of 0.5.

The suspension can be -concentrated under reduced pressure to the desired volume, for example 100 3 cm

Example 2

Preparation of sterile human serum albumin nanoparticles.

The procedure is as in Example 1, then the

2 ^ 34397 suspension is sterilized by autoclaving at 134 ° C for 15 minutes. The average particle size remains virtually unchanged after sterilization. Example 3: Preparation of lyophilized human serumal5 bumine nanoparticles.

The procedure is as in Example 2, then the sterile suspension is lyophilized.

The addition of a cryoprotectant (maltose, trehalose, etc.) is not essential, but favors the resuspension of the lyophilisate. The average particle size remains unchanged after lyophilization.

Example 4 Preparation of crosslinked human serum albumin nanoparticles.

The procedure is as in Example 1, but adding 0.06 g of an aqueous 257 (w / v) glutaraldehyde solution to phase 1. The average particle size remains unchanged after crosslinking. Example 5: Preparation of bovine serumal bumine (BSA) nanoparticles.

The procedure is as in Example 1, replacing HSA with BSA and replacing normal hydrochloric acid with the same amount of 0.01N sodium hydroxide. The average size of the nanoparticles is 150 nm with an average dispersion index of 0.5.

BSA nanoparticles can be crosslinked, autoclaved and lyophilized like HSA nanoparticles.

Example 6: Preparation of elastin nanoparticles.

³⁰ The procedure is as in Example 1 but replacing the HSA with elastin. The average size of the nanoparticles is 280 nm with an average dispersion index of 1.

Elastin nanoparticles can be

26 ^ 397 crosslinked, autoclaved and lyophilized like those of SAH.

Example 7: Adsorption of an active principle on protein nanoparticles.

To the nanoparticles prepared according to Example (HSA) or according to Example 5 (SAB), increasing amounts (from 0.25 g to 2.50 g) of sodium salicylate are added. The rate of binding of sodium salicylate to the nanoparticles, measured after ultracentrifugation, is 60% of the amount used, regardless of the amount of active principle added.

Example 8: Preparation of nanoparticles in the presence of an active principle.

The procedure is carried out according to Example 1 (1g of HSA) or according to Example 5 (1g of BSA), but in the presence of

0.50 g of sodium salicylate dissolved in phase 1. The average size of the nanoparticles is 200 nm with an average dispersion index of 0.5. The rate of binding of sodium salicylate to the nanoparticles, measured after ultracentrifugation, is 60% of the amount used.

Example 9: Variant of example 8.

The procedure is according to Example 8, but the sodium salicylate is dissolved in phase 2. The nanoparticles obtained have the same characteristics as those of Example 6.

Example 10: Preparation of protein nanoparticles of doxorubicin.

With nanoparticles prepared according to the example

1 (HSA) or according to Example 5 (BSA), 50 mg of doxorubicin are added. The rate of binding of on the nanoparticles, measured after hydrochloride of doxorubicin ultracentrifugation, is 90% of the amount used.