Process for preparing colloidal dispersive protein systems in the shape of nanoparticles.

The present invention relates to a new system process for preparing colloidal dispersible protein in the form of spherical particles of matrix type and size of less than 500 nm (nanoparticles).

EP-A-0275796 relates to a process for preparing dispersible colloidal systems of a substance in the form of matrix type spherical particles and of a size less than 500 nm. (Nanoparticles), characterized in that:<ul><li>(1) a liquid phase is prepared constituted essentially by a solution of the substance in a solvent or in a mixture of solvents, and can be supplemented with one or more surfactants,</li><li>(2) a second liquid phase is prepared constituted essentially by a non-solvent or a mixture of non-solvents of the substance and may be supplemented with one or more surfactants, the non-solvent or mixture of non-solvents the substance being miscible in all proportions with the solvent or mixture of solvents of the substance,</li><li>(3) is added with moderate stirring, 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</li><li>(4) if desired, are removed all or part of the solvent or solvent mixture of the substance and the non-solvent or non-solvents mixture of the substance, so as to obtain a colloidal suspension of the desired concentration nanoparticulate or to obtain a powder of nanoparticles.</li></ul>

The present invention relates to a variant of the above method wherein said substance is a protein and optionally a biologically active substance, said solvent is water or an aqueous mixture to a temperature below the protein coagulation temperature, said non-solvent for the substance is water at a temperature above the coagulation temperature of protein and may optionally contain a biologically active substance, and said two liquid phases (1) and (2) are combined in the pH conditions the distance to the isoelectric point of the protein.

Thus, the present process is characterized in that:<ul><li>(1) a liquid phase is prepared constituted essentially by a solution of the protein and optionally a biologically active substance in water or in an aqueous mixture at a temperature below the protein coagulation temperature, and can be added one or more surfactants,</li><li>(2) a second liquid phase is prepared constituted essentially by water or an aqueous mixture to a temperature above the protein coagulation temperature, which can contain a biologically active substance and can be supplemented with one or more surfactants,</li><li>(3) is added with moderate stirring, one of the liquid phases obtained under (1) or (2) to one another in conditions of pH away from the isoelectric point of the protein, so as to obtain almost instantaneously a colloidal suspension nanoparticles of the protein and optionally of the biologically active substance, and</li><li>(4) if desired, are removed all or part of the water or aqueous mixture, so as to obtain a colloidal suspen sion desired concentration of nanoparticles or to obtain a powder of nanoparticles.</li></ul>

The protein is in particular a natural protein such as serum albumin (for example human or bovine serum albumin) or elastin (bovine, etc.). The biologically active substance may be a medicinal active principle or a medicinal precursor, a biological reagent or a cosmetic principle. The invention allows to obtain single protein nanoparticles (used as such) or with the biologically active substance. it is also possible and desirable in the event of thermal instability, to fix the biologically active substance on the protein nanoparticles already formed.

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

The non-solvent of the protein in step (2), which is water or an aqueous mixture (e.g. water acidified or basified) is in particular at a temperature ranging from 80 ° to 100 ° C ( under atmospheric pressure), for example approximately at boiling temperature.

The pH of the mixture of phases (1) and (2) is to be remote from the isoelectric point of the protein to avoid flocculation. This desirable pH difference is of the order of 2 to 3. The natural proteins often having a pH of about 5 to 6, it is desirable that the final solution has a pH of about 3 or about 9 . for this purpose, the acid or base can be added (e) either to the phase (1) or (2).

The expression "moderate stirring" mild agitation of 10 to 500 rpm, p. ex. about 100 rpm, including magnetic stirrer.

The protein concentration in step (1) may vary from 0.1 to 10%, preferably from 0.5 to 4%.

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

Finally, the colloidal nanoparticle solution may be concentrated, sterilized, buffered (for example to physiological pH), freeze-dried or crosslinked.

The invention allows to obtain protein nanoparticles in particular from 150 to 300 nm.

The following examples illustrate the invention:

example 1

Preparation of human serum albumin nanoparticles (SAH).

Phase 1

SAH 1.0 g N hydrochloric acid 0.3 g demineralized or distilled water at room temperature 100.0 g

Phase 2

demineralized or distilled water brought to boiling 180.0 g

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

The suspension may be concentrated under reduced pressure to the desired volume, for example 100 cc.

example 2

: Preparation of sterile human serum albumin nanoparticles.

The procedure is as in Example 1, and the suspension is sterilized by autoclaving at 134 ° C for 15 minutes. The average particle size remains virtually unchanged after sterilization.

example 3

Preparation of nanoparticles of lyophilized human serum albumin.

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

The addition of a cryoprotectant (maltose, trehalose etc ...) is not essential, but favors the resuspension of the freeze-dried. 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 solution of glutaraldehyde at 25% (w / v) in Phase 1. The average particle size remained unchanged after crosslinking.

example 5

Preparation of bovine serum albumin nanoparticles (SAB).

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

Nanoparticles BSA can be crosslinked, autoclaved and lyophilized as those of HSA.

example 6

Preparation of elastin nanoparticles.

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

Elastin nanoparticles may be cross-linked, sterilized by autoclaving and freeze-dried such as HSA.

example 7

Adsorption of an active ingredient on protein nanoparticles.

The nanoparticles prepared according to Example 1 (SAH) or according to Example 5 (BSA) are added increasing amounts (0.25 g to 2.50 g) of sodium salicylate. Sodium salicylate fixation rates on nanoparticles, measured after ultracentrifugation, is 60% of the amount used, regardless of the amount of active ingredient added.

example 8

Preparation of nanoparticles in the presence of an active ingredient.

The procedure of Example 1 (1g HSA) or according to Example 5 (1g 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. Sodium salicylate fixation rates on nanoparticles, measured after ultracentrifugation, is 60% of the amount used.

example 9

: Variant of Example 8.

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

example 10

Preparation of protein doxorubicin nanoparticles.

The nanoparticles prepared according to Example 1 (SAH) or according to Example 5 (BSA) was added 50 mg of doxorubicin hydrochloride. The fixation rate of doxorubicin to the nanoparticles, measured after ultracentrifugation, is 90% of the amount employed.