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

0001The present invention relates to a new process for the preparation of dispersible colloidal systems of a protein in the form of spherical particles of matrix type and of size less than 500 nm (nanoparticles).

0002DE-A-1542261 describes a process for the preparation of microcapsules whose walls consist of a polymer, by dispersing the material to be encapsulated in a solution of a coagulable polymer for the effect of heat.

0003EP-A-0 275 796 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:<ul id="ul0001" list-style="none"><li>(1) a liquid phase is prepared consisting essentially of a solution of the substance in a solvent or in a mixture of solvents, and which can be added with one or more surfactants,</li><li>(2) a second liquid phase is prepared consisting essentially of a non-solvent or a mixture of non-solvents of 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,</li><li>(3) one of the liquid phases obtained under (1) or (2) is added with moderate stirring to the other, so as to obtain almost instantaneously a colloidal suspension of nanoparticles of the substance, and</li><li>(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 into nanoparticles or to obtain a powder of nanoparticles.</li></ul>

0004The present invention relates to a variant of the above process in which, 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 for the substance is water at a temperature higher than the coagulation temperature of the protein and may optionally contain a biologically active substance, and said two liquid phases (1) and (2) are combined under pH conditions far from the isoelectric point of the protein.

0005Thus, the present process is characterized in that:<ul id="ul0002" list-style="none"><li>(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 solution at a temperature below the coagulation temperature of the protein, and which can be added one or more surfactants,</li><li>(2) a second liquid phase is prepared, consisting essentially of water or an aqueous solution at a temperature higher than the coagulation temperature of the protein, which may contain a biologically active substance and which may be supplemented with one or more several surfactants,</li><li>(3) one of the liquid phases obtained under (1) or (2) is added with moderate stirring to the other under pH conditions far from the isoelectric point of the protein, so as to obtain almost instantaneously a colloidal suspension nanoparticles of the protein and possibly of the biologically active substance, and</li><li>(4) if desired, all or part of the water or aqueous mixture is eliminated, so as to obtain a colloidal suspension of desired concentration of nanoparticles or to obtain a powder of nanoparticles.</li></ul>

0006The 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 protein nanoparticles alone (usable as such) or with the biologically active substance. It is also possible and even desirable in the event of thermal instability, to fix the biologically active substance on the protein nanoparticles already formed.

0007The solvent of phase (1) which is water or an aqueous solution (for example acidified or basified water) is found in particular at a temperature ranging from 0 ° to 50 ° C., for example approximately at room temperature .

0008The non-solvent for the protein in phase (2), which is water or an aqueous solution (for example acidified or basified water) is found in particular at a temperature ranging from 80 ° to 100 ° C ( under atmospheric pressure), for example approximately at the boiling point.

0009The pH of the mixture of phases (1) and (2) must be removed from the isolelectric point of the protein in order to avoid its flocculation. This desirable pH difference is on the order of 2 to 3. Natural proteins often having a pH on the order of 5 to 6, it is desirable that the final solution has a pH of about 3 or about 9 To this end, the acid or the base can be added indifferently to phase (1) or (2).

0010By "moderate agitation" is meant slight agitation of 10 to 500 rpm, p. ex. around 100 rpm, in particular by magnetic stirrer.

0011The protein concentration in phase (1) can vary from 0.1 to 10%, preferably from 0.5 to 4%.

0012The phase (1) / phase (2) volume ratio can vary from 0.1 to 1, preferably from 0.2 to 0.6.

0013Finally, the colloidal solution of nanoparticles can be concentrated, sterilized, buffered (for example at physiological pH), lyophilized or crosslinked at will.

0014The invention makes it possible to obtain protein nanoparticles, in particular from 150 to 300 nm.

0015The following examples illustrate the invention:

Example 1

: Preparation of nanoparticles of human serum albumin (SAH).

0016<tables id="tabl0001" num="0001"><img file="EP0349428B1_D0001.tif" /></tables>

0017Phase 1 is added with magnetic stirring to phase 2. The medium immediately becomes opalescent by the formation of nanoparticles of SAH. The average size of nanoparticles, measured in a laser beam diffractometer (Nanosizer^R from Coultronics) is 190 nm with an average dispersion index of 0.5.

0018The suspension can be concentrated under reduced pressure to the desired volume, for example 100 cm³.

Example 2

: Preparation of sterile human serum albumin nanoparticles.

0019The procedure is as in Example 1, then the suspension is sterilized in an autoclave at 134 ° C for 15 minutes. The average particle size remains practically unchanged after sterilization.

Example 3

: Preparation of lyophilized human serum albumin nanoparticles.

0020The procedure is as in Example 2, then the sterile suspension is lyophilized.

0021The addition of a cryoprotective (maltose, trehalose ... etc) is not essential, but promotes the resuspension of the lyophilisate. The average particle size remains unchanged after lyophilization.

Example 4

: Preparation of crosslinked human serum albumin nanoparticles.

0022The procedure is as in Example 1, but adding 0.06 g of an aqueous glutaraldehyde solution at 25% (w / v) to phase 1. The average particle size remains unchanged after crosslinking.

Example 5

: Preparation of bovine serum albumin (SAB) nanoparticles.

0023The procedure is as in Example 1, replacing SAH with SAB 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.

0024SAB nanoparticles can be crosslinked, autoclaved and lyophilized like those of SAH.

Example 6

: Preparation of elastin nanoparticles.

0025The procedure is as in Example 1 but replacing the SAH with elastin. The average size of the nanoparticles is 280 nm with an average dispersion index of 1.

0026The elastin nanoparticles can be crosslinked, sterilized in an autoclave and lyophilized like those of SAH.

Example 7

: Adsorption of an active principle on protein nanoparticles.

0027To the nanoparticles prepared according to Example 1 (SAH) or according to Example 5 (SAB) are added increasing amounts (from 0.25 g to 2.50 g) of sodium salicylate. The rate of fixation of sodium salicylate on the nanoparticles, measured after ultracentrifugation, is 60% of the amount used, whatever the amount of active ingredient added.

Example 8

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

0028The procedure is according to Example 1 (1g of SAH) or according to Example 5 (1g of SAB), but in the presence of 0.50g 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 fixation of sodium salicylate on the nanoparticles, measured after ultracentrifugation, is 60% of the amount used.

Example 9

: Variant of Example 8.

0029The procedure is as in Example 8, but the sodium salicylate is dissolved in phase 2. The nanoparticles obtained have the same characteristics as those of Example 8.

Example 10

: Preparation of doxorubicin protein nanoparticles.

0031To the nanoparticles prepared according to Example 1 (SAH) or according to Example 5 (SAB) are added 50 mg of doxorubicin hydrochloride. The rate of fixation of doxorubicin on the nanoparticles, measured after ultracentrifugation, is 90% of the amount used.