Progesterone sustained-release nanoparticles and preparation method thereof

Abstract

The invention relates to the technical field of progesterone sustained-release nanoparticle production, in particular to a progesterone sustained-release nanoparticle which comprises progesterone and further comprises solid lipid and liquid lipid according to the formula ratio of 1: 1.4: 1.2: 0.8. A preparation method of the progesterone sustained-release nanoparticle comprises raw material preparation of progesterone, solid lipid and liquid lipid and melt emulsification. The prepared progesterone lipid nanoparticles have high drug entrapment efficiency which can reach 77.48% at most, and have obvious drug sustained and controlled release performance, although the particle size of the nanoparticles is increased along with the increase of the content of liquid lipid-oleic acid in the lipid nanoparticles, the drug entrapment efficiency is improved, the in-vitro release of the drug is accelerated, and the progesterone lipid nanoparticles have good application prospects. According to the progesterone nanostructure lipid carrier, the drug content in the preparation process of the nanoparticles is improved, the drug encapsulation efficiency of the prepared progesterone nanostructure lipid carrier is reduced, but the drug loading capacity of the nanoparticles can be improved, and when the dosage reaches 20%, the drug loading capacity of the nanoparticles can reach 11.57%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of progesterone sustained-release nanoparticles, in particular to a kind of progesterone sustained-release nanoparticles and preparation method thereof. BACKGROUND

[0002] Progesterone is an important component for regulating female reproductive system, mainly acting on uterus, ovary, breast and central nervous system, and the progesterone blood concentration in female body is very high during luteal phase of menstrual cycle and pregnancy, progesterone is produced in corpus luteum in non-pregnant period and early pregnancy, and in placenta in late pregnancy, endogenous progesterone can make endometrium transform from proliferative phase to secretory phase, and maintain normal physiological changes.

[0003] Solid lipid nanoparticles (SLN) is a new type of microparticle drug delivery system developed in recent years after emulsion, liposome, microparticle and polymer nanoparticle, SLN has the advantages of high physical stability and slow drug leakage of polymer nanoparticles, and also has the advantages of low toxicity and large-scale production of emulsion and liposome, and is a new type of drug delivery system carrier with great development prospect due to good physiological compatibility, controllable drug release and good targeting, but SLN also has some shortcomings, such as limited drug loading capacity, drug displacement caused by crystal transformation of lipid material during storage, and high water content of dispersion liquid. Due to the adhesion of drug-loaded nanoparticles on the surface and small particle size, it is beneficial to increase the retention of local drug use, increase the contact time and contact area of drug and intestinal wall, and improve the bioavailability of oral drug absorption; drug-loaded nanoparticles can change the membrane transport mechanism, increase the permeability of drug to biological membrane, and be beneficial to transdermal absorption of drug and exertion of intracellular drug efficacy, so drug-loaded nanoparticles are mainly used in the direction of targeting, sustained release and improving the bioavailability of oral, eye and transdermal drug delivery.

[0004] Traditional progesterone nanoparticles are prepared by aqueous solvent diffusion method or release medium redispersion method, the drug encapsulation efficiency of progesterone lipid nanoparticles prepared by aqueous solvent diffusion method is low, only 45.9%, and the in vitro drug release has obvious burst release characteristics, which shows that the drug is mainly distributed in the superficial layer of nanoparticles, and the release medium redispersion method has the problem of incomplete drug release: the diffusion rate of drug itself through the diffusion cell seriously affects the in vitro release behavior of progesterone from lipid nanoparticles; drug can quickly diffuse through the dialysis bag, which has little effect on the in vitro release of progesterone from nanoparticles. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art, the present application provides a kind of progesterone sustained-release nanoparticles and preparation method thereof, which can effectively solve the problems proposed in the background art.

[0006] To achieve the above object, the present application is realized by the following technical solutions: The present application provides a progesterone sustained-release nanoparticle, comprising progesterone, characterized in further comprising: solid lipid and liquid lipid; The solid lipid comprises stearic acid and glycerol monostearate; The liquid lipid comprises oleic acid; The formula ratio of progesterone, stearic acid, glycerol monostearate and oleic acid is 1:1.4:1.2:0.8.

[0007] Further, the particle size of the solid gel prepared from progesterone, stearic acid, glycerol monostearate and oleic acid is between 50-1000 nanometers.

[0008] A preparation method of the progesterone sustained-release nanoparticle, comprising the following steps: S1: Progesterone preparation; dry toluene is added into a dry reaction pot, cyclohexanone and pregnenolone are added, stirring and dissolving, toluene is removed by steam dehydration, aluminum isopropoxide is quickly added, oxidation reaction is carried out at 115℃ for 2 hours, cooling to about 80℃, 5% dilute sulfuric acid is added under stirring, standing and separating, the water layer is removed, the toluene layer is washed with water until neutral, then water vapor distillation is carried out, toluene and cyclohexanone are distilled out, cooling, filtering, the filter residue is stirred into a slurry with petroleum ether, filtering, washing with petroleum ether, drying the crude progesterone, the crude product is dissolved in ethanol, decolorizing with activated carbon, recrystallizing the finished product, the yield is 80%, then the product is crushed into 1 micron fine powder particles by a superfine grinder, and the product is sealed for use; S2: Solid lipid preparation; comprising preparation of stearic acid and preparation of glycerol monostearate; S2.1: The preparation of stearic acid is prepared by alkali catalysis and high temperature method with palm oil as raw material; S2.2: The preparation of glycerol monostearate is prepared by esterification reaction of stearic acid and glycerol, comprising the following steps: S2.2.1: Raw material preparation: stearic acid is extracted from animal and plant fats, and glycerol is obtained from the hydrolysis of plant or animal fats; S2.2.2: Esterification reaction: the prepared stearic acid and glycerol are added into a reaction kettle according to a certain molar ratio, and then esterification reaction is carried out under appropriate temperature and pressure; S2.2.3: Neutralization and water washing: after the esterification reaction is completed, the reaction mixture is cooled to room temperature, and then an appropriate amount of alkali solution is added for neutralization reaction; S2.2.4: Refining and decolorizing: the neutralized mixture is added into a precipitation separation device to separate the water phase and the organic phase, and the organic phase is further subjected to decolorizing treatment; S2.2.5: Desolventization: using the method of distillation or evaporation concentration to remove the solvent to obtain pure glycerol monostearate.

[0009] S3: liquid lipid preparation; including the preparation of oleic acid, by cooling to make the high melting point fatty acid hexadecanoic acid, stearic acid first form crystals, then through the membrane filter separation, the saturated fatty acid and low melting point unsaturated fatty acid are effectively separated, after continuous stirring and heating, the crystallization slurry is sent into the membrane filter press for filtration, and finally the oleic acid is obtained; S4: material melting emulsification; the obtained progesterone, stearic acid, glycerol monostearate and oleic acid are respectively put into a reactor according to equal proportion, the raw materials are heated by a heating unit in the reactor; under the condition of 400 revolutions per minute, water solution is slowly added into the oil phase under constant stirring, and the emulsion matrix is prepared after stirring and condensation, and progesterone nanoparticles are obtained by freeze-drying after solidification and concentration.

[0010] Compared with the known prior art, the technical scheme provided by the present application has the following beneficial effects: The progesterone lipid nanoparticles prepared by the melting emulsification method have a high drug encapsulation rate, which can reach 77.48% at most, and have obvious drug sustained and controlled release performance. The research results of the progesterone nanostructured lipid carriers prepared by the melting emulsification method show that although the particle size of the nanoparticles increases with the increase of the content of liquid lipid-oleic acid in the nanoparticles, the drug encapsulation rate increases, the in vitro release of the drug is accelerated, the drug content in the preparation process of the nanoparticles is increased, the drug encapsulation rate of the prepared progesterone nanostructured lipid carriers decreases, but the drug loading capacity of the nanoparticles can be increased, and when the drug dosage reaches 20%, the drug loading capacity of the nanoparticles can be as high as 11.57%. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical scheme in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creating any creative labor.

[0012] Figure 1 It is a process flow diagram of the present application. DETAILED DESCRIPTION

[0013] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments but not all of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0014] The present application will be further described below with reference to the embodiments.

[0015] Embodiment 1 Reference Figure 1 For the first embodiment of the present application, a progesterone sustained-release nanoparticle comprises progesterone, characterized in that it further comprises solid lipid and liquid lipid, wherein the solid lipid comprises stearic acid and glycerin monostearate, and wherein the liquid lipid comprises oleic acid. The formula ratio of progesterone, stearic acid, glycerin monostearate and oleic acid is 1:1.4:1.2:0.8, the particle size of the solid colloidal particles prepared from progesterone, stearic acid, glycerin monostearate and oleic acid is between 50-1000 nanometers, the particle size of the nanoparticle is increased, the drug encapsulation efficiency is improved, the in-vitro release of the drug is accelerated, and the drug content in the preparation process of the nanoparticle is increased.

[0016] Embodiment 2 Reference Figure 1 For the second embodiment of the present application, the difference between this embodiment and the first embodiment is that the embodiment is a preparation method of a progesterone sustained-release nanoparticle, comprising the following steps: S1: Progesterone preparation; dry toluene is added into a dry reaction pot, cyclohexanone and pregnenolone are added, stirring and dissolving, toluene is evaporated to dryness, aluminum isopropoxide is quickly added, oxidation reaction is carried out at 115℃ for 2 hours, cooling to about 80℃, 5% dilute sulfuric acid is added under stirring, standing and separating, the water layer is removed, the toluene layer is washed with water until neutral, then water vapor distillation is carried out, toluene and cyclohexanone are evaporated, cooling, filtration, the filter residue is stirred into a slurry with petroleum ether, filtration, washing with petroleum ether, drying of the crude progesterone, the crude product is dissolved in ethanol, decolorizing with activated carbon, recrystallizing the finished product, the yield is 80%, then the product is crushed into 1 micron fine powder particles by a superfine grinder, and sealed for use; S2: Solid lipid preparation; including preparation of stearic acid and preparation of glycerin monostearate; S2.1: Preparation of stearic acid is to prepare stearic acid by alkali catalysis and high temperature method with palm oil as raw material, a composite alkali is used as catalyst, the composite alkali and the palm oil raw material molecules react by accepting protons or giving electrons to form a reactive negative carbon ion intermediate compound, and then the stearic acid is decomposed and sealed for use; S2.2: Preparation of glyceryl monostearate is made by esterification reaction of stearic acid and glycerol, including the following steps: S2.2.1: Raw material preparation: stearic acid is extracted from animal and plant fats, while glycerol is obtained from the hydrolysis of plant or animal fats; S2.2.2: Esterification reaction: the prepared stearic acid and glycerol are added to the reaction kettle according to a certain molar ratio. Then, the esterification reaction is carried out at a suitable temperature and pressure, usually the reaction temperature is 150-180°C, and the reaction time is 2-4 hours; S2.2.3: Neutralization and water washing: after the esterification reaction is completed, the reaction mixture is cooled to room temperature and then an appropriate amount of alkali solution is added for neutralization reaction. Neutralization reaction can help remove unreacted acid and acid catalyst. Next, use an appropriate amount of hot water for water washing to remove residual alkali and impurities; S2.2.4: Refining and decolorization: the neutralized mixture is added to a sedimentation separation device to separate the water phase and the organic phase, and the organic phase is further treated with decolorization, usually using activated carbon adsorbent to remove pigments and impurities; S2.2.5: Desolventization treatment: a small amount of solvent may still remain in the decolorized organic phase, which needs to be treated with desolventization. Usually, distillation or evaporation concentration methods are used to remove the solvent to obtain pure glyceryl monostearate.

[0017] S3: Liquid lipid preparation; including the preparation of oleic acid, specifically by cooling to make fatty acid high melting point hexadecanoic acid, stearic acid first form crystals, then through the membrane filter separation, the saturated acid fatty acid and low melting point of unsaturated fatty acid are effectively separated, and then the separated saturated material is sent to a crystallization tank equipped with a coil or a jacket with stirring, at a speed of 200 revolutions per minute, with a cooling frequency of 100 degrees Celsius per 15 minutes, and finally the crystallization slurry is sent to a membrane filter for filtration, and finally oleic acid is obtained; S4: Material melting emulsification; the obtained progesterone, stearic acid, glyceryl monostearate and oleic acid are placed in the reactor according to equal proportion, the raw materials are heated by the heating unit in the reactor, the heating temperature is 80-120 degrees Celsius, after the raw materials are completely melted, the water-soluble ingredients are dissolved in water and heated to slightly higher than the oil phase temperature to prevent the components in the oil phase from precipitating or coagulating too early when the two phases are mixed; slowly add the aqueous solution to the oil phase under constant stirring at a speed of 400 revolutions per minute, and stir until condensation to prepare an emulsion base, then after solidification and concentration, the progesterone nanoparticles are obtained by freeze-drying machine; S5: Particle size measurement; a small amount of progesterone nanoparticles were taken and randomly arranged into multiple groups, and each group of progesterone nanoparticles was placed into a particle size measuring instrument to measure the size of the progesterone nanoparticles, and the average value of the measurement results was obtained; S6: Potential measurement; potential measurement was performed by a Zeta potential measuring instrument, the Zeta potential measurement mode was switched in the control software, the electrode was inserted into the sample cell according to the guide operation, the electric field intensity was adjusted as needed, and the measurement was started, after the measurement was completed, the data was recorded and analyzed.

[0018] S7: Drug content determination; the drug content was determined by high performance liquid chromatography, specifically, the mobile phase in the solvent reservoir was pumped in, mixed by a gradient controller according to a certain gradient and then output, the pressure and flow rate were measured, the sample valve was introduced, and the sample was separated by a protection column and a separation column and then detected by a detector, the data was processed by a data processing device or a chromatogram was recorded by a recorder, and the fraction collector collected the fraction as waste liquid.

[0019] S8: Packaging and storage; the progesterone nanoparticles that passed the detection were sent into a packaging machine in a low-temperature environment, an equal amount of progesterone nanoparticles was supplemented and uniformly fed, a packaging bottle with consistent specifications and batch size was formed, and the progesterone nanoparticles were stored in a refrigerated cabinet in a low-temperature refrigerated manner, and the environmental temperature was 4 degrees Celsius.

[0020] The remaining structure is the same as that of Example 1.

[0021] The oral progesterone nanoparticle biological experiment shows that the blood drug-time curve appears a double-peak phenomenon, the first peak appears about 2 hours after administration, and the second peak appears at a time that is obviously different from that of the control group; after administration of NLC, the peak appears relatively late because of the lymphatic transport pathway, and the peak value is also large; the second peak value of the PEG modified nanoparticle after administration is higher, and the duration time is longer, which reflects the long circulation effect; compared with the AUC of the oil solution administration system, the AUC of the NLC administration system is improved, the bioavailability is 1.93%, and after modification of PEG, a larger AUC is shown, the bioavailability is improved to 2.53%, and compared with NLC administration, it is also improved by 1.31%.

[0022] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the protection scope of the technical solutions of the embodiments of the present application.

Claims

1. A progesterone sustained-release nanoparticle, comprising progesterone, characterized in that, Also includes: Solid lipids and liquid lipids; The solid lipids include stearic acid and glyceryl monostearate; The liquid lipids include oleic acid; The formulation ratio of progesterone, stearic acid, glyceryl monostearate, and oleic acid is 1:1.4:1.2:0.

8.

2. The progesterone sustained-release nanoparticle according to claim 1, characterized in that, The solid particles made from progesterone, stearic acid, glyceryl monostearate, and oleic acid have a particle size between 50 and 1000 nanometers.

3. A method for preparing progesterone sustained-release nanoparticles, comprising using a progesterone sustained-release nanoparticle as described in claim 2, characterized in that, Includes the following steps: S1: Preparation of progesterone; Add dried toluene to a dry reaction vessel, add cyclohexanone and pregnenolone, stir to dissolve, evaporate toluene to remove all water, quickly add aluminum isopropoxide, oxidize at 115℃ for 2 hours, cool to about 80℃, add 5% dilute sulfuric acid under stirring, let stand to separate the layers, separate the water layer, wash the toluene layer with water until neutral, then perform steam distillation to evaporate toluene and cyclohexanone, cool, filter, stir the filter residue into a slurry with petroleum ether, filter, wash with petroleum ether, dry crude progesterone product, dissolve the crude product in ethanol, decolorize with activated carbon, recrystallize the product, yield 80%, then grind into 1-micron fine powder particles through an ultrafine pulverizer, seal for later use. S2: Preparation of solid lipids; including the preparation of stearic acid and glyceryl monostearate; S2.1: Stearic acid is prepared by using palm oil as raw material and obtaining stearic acid through alkaline catalysis and high temperature. S2.2: Glyceryl monostearate is prepared by the esterification reaction of stearic acid and glycerol, including the following steps: S2.2.1： Raw material preparation: Stearic acid is extracted from animal and plant fats, while glycerol is obtained from the hydrolysis of plant or animal fats; S2.2.2： Esterification reaction: Stearic acid and glycerol are added to a reaction vessel in a specific molar ratio. Then, the esterification reaction is carried out under appropriate temperature and pressure. S2.2.3： Neutralization and washing: After the esterification reaction is completed, the reaction mixture is cooled to room temperature and then an appropriate amount of alkaline solution is added for neutralization. S2.2.4： Refining and decolorization: The neutralized mixture is added to a precipitation separation device to separate the aqueous phase and the organic phase, and the organic phase is further decolorized. S2.2.5： Solvent removal: The solvent is removed by distillation or evaporation to obtain pure glyceryl monostearate. 4.S3: Liquid lipid preparation; including the preparation of oleic acid, which involves first precipitating high-melting-point fatty acids such as hexadecanoic acid and stearic acid by cooling, and then separating them by membrane pressure filtration to effectively separate saturated fatty acids and low-melting-point unsaturated fatty acids. After continuous stirring and heating, the crystallized slurry is sent to a membrane filter press for filtration to finally obtain oleic acid. S4: Material melt emulsification; The obtained progesterone, stearic acid, glyceryl monostearate and oleic acid are put into the reactor in equal proportions, and the raw materials are heated by the heating unit in the reactor; The aqueous solution is slowly added to the oil phase at a speed of 400 rpm and stirred until condensation is achieved to form an emulsion matrix. After solidification and concentration, it is freeze-dried by a freeze dryer to obtain progesterone nanoparticles.

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