RU2424516C1 - Method of mixture recovery for preparing water dispersions of spherical nanoparticles - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of mixture recovery for preparing water dispersions of spherical nanoparticles of mixed poorly water-soluble birch bark triterpenoids including injection of excess water in the birch bark triterpenoid solution in a water-mixed organic solvent to form a dispersion containing spherical nanoparticles and crystals of birch bark triterpenoids differing by the fact the produced dispersion is filtered or centrifuged with separating a fraction of the spherical nanoparticles from crystals; the separated nanoparticles are evaporated to produce a solid mixture of triterpenoids for morphologically homogeneous spherical nanoparticles by multiple injection.

EFFECT: high morphological uniformity of the spherical nanoparticles.

2 cl, 3 ex, 2 dwg

Description

The invention relates to the field of biotechnology, mainly associated with the preparation of dispersions of nanoparticles for use in medicine. These dispersions exhibit biological activity on their own when systemically introduced into the body, and can also be used as systems for the delivery of biologically active substances into living organisms.

Birch bark triterpenoids have multiple activity, however, low solubility in water dramatically reduces the bioavailability and, as a consequence, the effectiveness of these substances. Known methods for increasing the bioavailability of poorly soluble in water triterpenoids by obtaining nanodispersions of these substances. The essence of the methods for producing dispersions is the injection of excess water (up to twenty-five) into a solution of triterpenoids of birch bark (up to 5 mg / ml) in water-miscible organic solvents. The closest technical solution to the proposed invention is the method described in the patent of the Russian Federation No. 2322091 "Composition of biologically active substances and a method for producing its nanodispersions"; A23L 1/30; A61K 36/00; published on April 20, 2008. This method uses directly dry birch bark extract without determining the optimal composition of the mixture and without changing the content of the components, which is associated with the following disadvantages. Dispersions obtained from such extracts are metastable. Heating the resulting dispersions, for example, for thermal sterilization, causes crystallization of betulin and a decrease in the diameter of the nanoparticles, apparently due to a decrease in the viscosity of the particle material. In addition to spherical nanoparticles that are optimal for use, such dispersions can also contain crystals, including and going beyond the dimensions acceptable for dispersions, the use of which involves intravascular administration. In addition, according to preliminary data, crystalline nanoparticles of birch bark triterpenoids can be toxic to eukaryotic cells.

Since spherical nanoparticles from the composition of triterpenoids of birch bark, indicated in RF patent No. 2322091, are metastable, their preparation is possible only under conditions of kinetic control.

The technical result of the invention is the isolation of a mixture for the preparation of stable aqueous dispersions of spherical nanoparticles with high morphological homogeneity (i.e., containing exclusively spherical particles) from triterpenoids of the birch bark without sonication and at room temperature.

To achieve the specified technical result, one should obtain nanoparticles in thermodynamically controlled conditions. In this case, as a result of injection, in addition to amorphous spherical nanoparticles of optimal composition, crystal fractions are formed that can be separated by filtration or centrifugation. A mixture of triterpenoids obtained after evaporation of spherical amorphous nanoparticles to dryness can be analyzed to determine the ratio of triterpenoids of the birch bark, optimal for the formation of spherical amorphous nanoparticles, or used without determining the quantitative ratio of components to obtain an aqueous dispersion of spherical amorphous nanoparticles by re-injection.

Example 1

50 mg of the extract (extractant: toluene, or acetone, or isopropyl alcohol) of birch bark was dissolved in 10 ml of tetrahydrofuran (5 mg / ml). 250 ml of water was injected into this solution, as a result of which a dispersion was formed containing both submicron particles, as evidenced by the opalescence of the solution, and crystals. Crystalline impurities were removed by filtration, the remaining particles were evaporated to dryness on a rotary evaporator.

The isolated solid mixture of triterpenoids was analyzed by TLC followed by densitometric treatment and includes,% wt.:

lupeol ~ 50 3-O-betulin coffee ~ 20 betulin ~ 30

This mixture was dissolved in tetrahydrofuran to a concentration of 5 mg / ml and injected into the resulting solution, as in the first case, a twenty-five-fold excess of water. In the dispersion formed during this, coarse suspensions were not observed, the resulting nanoparticles had an exclusively spherical shape and size up to 150 nm (Fig. 1). The dispersion is stable for 6 months.

Example 2

Analogously to example 1, except for the use of centrifugation to separate crystalline impurities.

Example 3

The proposed method for isolating a mixture of optimal composition to obtain spherical nanoparticles of their birch bark triterpenoids was confirmed by the following experiment.

Individually isolated birch bark triterpenoids were mixed in the ratio determined in Example 1: lupeol (10 mg), betulin 3-O-caffeate (4 mg) and betulin (6 mg) and dissolved in 4 ml of tetrahydrofuran. A twenty-five-fold excess of water was injected into the solution. The resulting dispersion (figure 2) contains only spherical nanoparticles with a diameter of about 180 nm and is stable for 6 months.

Particle size and distribution were determined using a NICOMP-380 spectrometer (Particle Sizing Systems, USA). The morphology of nanoparticles was determined by electron micrographs.

Using the proposed method for isolating a mixture of optimal mixture composition to obtain dispersions of nanoparticles from a mixture of triterpenoids provides the following advantages:

a) the use of a mixture of optimal composition increases the uniformity of the morphology of the obtained nanoparticles, which allows to obtain nanoparticles of exclusively spherical shape, without impurities of crystals, as well as other non-spherical nanoparticles;

b) the use of a mixture of optimal composition allows one to obtain dispersions of nanoparticles under thermodynamically controlled conditions and does not require cooling of solutions and / or sonication upon receipt of nanoparticles.

Claims (2)

1. The method of separation of the mixture to obtain aqueous dispersions of spherical nanoparticles from a mixture of poorly water-soluble birch bark triterpenoids, including the injection of excess water into a solution of birch bark triterpenoids in a water-miscible organic solvent with the formation of a dispersion containing spherical nanoparticles and crystals of birch bark triterpenoids, characterized the fact that the resulting dispersion is filtered or centrifuged, separating the fraction of spherical nanoparticles from the crystals, I evaporate the separated nanoparticles to obtain a solid mixture of triterpenoids for the formation of morphologically homogeneous spherical nanoparticles by reinjection. 2. The method according to claim 1, characterized in that tetrahydrofuran is used as an organic solvent for injection.

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