A retinoic acid-lactoferrin nanocrystal preparation and its preparation method and application

Abstract

The present application provides a retinoic acid-lactoferrin nanocrystal preparation, which is prepared by an ultrasonic-solvent volatilization method; its particle size is 215nm and its potential is 36.47mV. Compared with the retinoic acid original drug, the nanocrystal of the present application has better stability, improved oral bioavailability, and plays a good therapeutic role in preventing / treating ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy. Specifically, the present application provides a retinoic acid-lactoferrin nanocrystal preparation and a preparation method and application thereof. Background Art

[0002] Retinoic acid is an active metabolite of vitamin A and plays a vital role in a variety of biological processes, including embryonic morphogenesis and organogenesis, cell proliferation, differentiation and apoptosis, and regulation of immune balance in the body. Among them, retinoic acid not only participates in the normal physiological activities of the central nervous system, but also plays a good protective role in various pathological states of the central nervous system. Studies have confirmed that retinoic acid can protect brain diseases through various mechanisms such as anti-inflammatory, anti-oxidation, promoting nerve regeneration, and regulating immune cells, such as reducing cerebral ischemia-reperfusion injury and reducing brain damage to the blood-brain barrier. These characteristics make retinoic acid very attractive in the field of stroke prevention and treatment research.

[0003] However, due to the limitations of some physical and chemical properties of retinoic acid, its clinical application is not ideal. First, retinoic acid is poorly soluble in water. At room temperature, its solubility in an aqueous solution of pH 7.3 is only 0.21µm. At the same time, retinoic acid is sensitive to light and has poor stability, which makes it difficult to administer retinoic acid. Second, retinoic acid can cause irritation when applied to the skin, and its degradation is rapid when administered intravenously, with a short half-life in the body (t 1 / 2 =45min), thus reducing its therapeutic effect; in addition, retinoic acid is involved in many biological processes, so systemic administration of large doses of retinoic acid can cause side effects. All these limitations have prompted researchers to look for new and better-tolerated drug delivery carriers and develop retinoic acid delivery preparations based on gels, liposomes, microparticles, nanoparticles, and micro / nanofibers. The development of these drug delivery systems provides several advantages for retinoic acid-based therapies, including improved dissolution, prolonged circulation, reduced toxicity, sustained drug release, and improved efficacy. However, these new drug delivery systems often have complex preparation processes, more prescription excipients, high production costs, and difficult clinical transformation. Therefore, it is particularly important to develop a simpler and more accessible drug delivery carrier for retinoic acid.

[0004] Since retinoic acid is insoluble and highly unstable in aqueous media, retinoic acid in the human body increases its solubility and stability by binding to proteins and achieving fluid transport. Physiological studies have found that serum albumin, transthyretin, epididymal retinoic acid-binding protein, retinol-binding protein, and cellular retinoic acid-binding protein are all transporters of retinoic acid in the body. In vitro studies have found that retinoic acid easily forms ionic bonds with the amino groups on the transport protein using the carboxyl groups in the structure to form co-crystals. Using these characteristics, retinoic acid and suitable proteins can be prepared into nanocrystals through nanotechnology.

[0005] Nanocrystals are an important strategy to solve the challenges of low solubility, low bioavailability and formulation of poorly soluble small molecule drugs. The drug is encapsulated by a stabilizer to form nanoparticles. Nanocrystals do not require a large amount of surfactants or carrier materials and have a high drug content. The saturated solubility of nanocrystals is increased due to the reduction in particle size. At the same time, nanocrystals increase the bioadhesion to the gastrointestinal mucosa through electrostatic adsorption and van der Waals forces, which can improve the oral bioavailability of drugs and reduce the toxic and side effects of drugs by reducing the dosage.

[0006] Lactoferrin is a whey protein with considerable potential as a functional ingredient in food, cosmetic and pharmaceutical applications. Lactoferrin has multiple biological functions such as antibacterial, antiviral, antitumor, antifungal, anti-inflammatory, immunomodulatory, analgesic, antioxidant, and lipid metabolism enhancement. Recent studies have shown that lactoferrin-based monotherapy has a unique and powerful effect in reducing brain damage after intracerebral hemorrhage. Lactoferrin can alleviate neurological damage caused by intracerebral hemorrhage in spontaneously hypertensive rats, protect neurons from the toxic effects of erythrocytes, and promote microglial phagocytosis of erythrocytes and dead neurons, indicating that lactoferrin has great potential for the treatment of stroke. In addition, lactoferrin is low-cost and has no toxic side effects. The main challenge hindering its widespread application is its poor in vivo stability due to rapid degradation by proteolytic enzymes. When injected intravenously, the plasma half-life of lactoferrin is a few minutes, and frequent administration is required to achieve therapeutic effects. Oral administration is the main way to supplement the body with lactoferrin because the important receptors of lactoferrin in the body are distributed on the intestinal mucosa and gut-associated lymphoid tissue-related cells. Therefore, there is a need to prepare a new formulation suitable for oral delivery of lactoferrin.

[0007] Although studies have confirmed that retinoic acid and lactoferrin play a certain role in reducing neuronal damage and blood-brain barrier damage caused by brain diseases, promoting neural repair after brain injury, and increasing the survival rate of newborn neurons, there are currently no reports on the combined use of retinoic acid and lactoferrin for the prevention / treatment of brain diseases. Summary of the invention

[0008] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a retinoic acid-lactoferrin nanocrystal preparation and a preparation method and application thereof, which are used to solve the problems of complex preparation process, multiple prescription excipients and drug bioavailability of retinoic acid and lactoferrin preparations in the prior art. At the same time, the present invention combines the pharmacological effects of retinoic acid and lactoferrin in preventing and treating brain injury diseases, studies the protective effect of retinoic acid-lactoferrin nanocrystal preparations on ischemic stroke reperfusion injury, investigates the synergistic effect of the combined use of the two, and provides a basis for new preparations and new treatment plans of retinoic acid and lactoferrin.

[0009] In view of the shortcomings of the prior art, the first aspect of the present invention provides a retinoic acid-lactoferrin nanocrystal preparation, one of the purposes of which is to overcome the shortcomings of poor stability and low water solubility of retinoic acid by preparing retinoic acid into a nanoscale drug, while improving the problem of low oral bioavailability of retinoic acid and lactoferrin, reducing the dosage of the drug and thus reducing drug toxicity and adverse reactions.

[0010] In one aspect, the present application provides a retinoic acid-lactoferrin nanocrystal preparation, which is prepared by an ultrasound-solvent evaporation method.

[0011] Furthermore, the lactoferrin is human lactoferrin or bovine lactoferrin, preferably bovine lactoferrin.

[0012] Furthermore, the mass ratio of retinoic acid to lactoferrin in the retinoic acid-lactoferrin nanocrystal preparation is 1:5-20, preferably 1:5.

[0013] Furthermore, the preparation method of the retinoic acid-lactoferrin nanocrystal preparation comprises:

[0014] (1) Completely dissolving retinoic acid in an organic solvent; completely dissolving lactoferrin in an aqueous solution;

[0015] (2) mixing the two solutions in step (1) and performing ultrasound treatment;

[0016] (3) The mixed solution obtained in step (2) is subjected to rotary evaporation to remove the organic solvent, thereby obtaining the retinoic acid-lactoferrin nanocrystal preparation.

[0017] Furthermore, the organic solvent in step (1) is selected from any one or more combinations of methanol, ethanol, acetone, acetonitrile, dichloromethane, dimethyl sulfoxide, and carbon tetrachloride; preferably methanol, ethanol or dichloromethane; more preferably dichloromethane.

[0018] In step (1), the mass / volume ratio of retinoic acid to dichloromethane is 4:1 to 20:1, preferably 10:1; and the mass / volume ratio of lactoferrin to water is 4:1 to 10:1, preferably 6.25:1.

[0019] Furthermore, in step (1), the ratio of retinoic acid to dichloromethane is 20-5 mg: 0.5-5 mL; preferably, the ratio of retinoic acid to dichloromethane is 10 mg: 1 mL.

[0020] Furthermore, in step (1), the ratio of lactoferrin to water is 20-100 mg: 5-10 mL; preferably, the ratio of lactoferrin to water is 50 mg: 8 mL.

[0021] Furthermore, in step (2), the ultrasonic power is 150-400 W, preferably 200-300 W, and most preferably 260 W; the ultrasonic power time is 1-30 min, preferably 2.5-10 min, and most preferably 5 min.

[0022] On the other hand, the present application provides the use of the above-mentioned retinoic acid-lactoferrin nanocrystal preparation in the preparation of a drug for treating stroke, brain injury, depression, liver injury, inflammation or tumor; preferably, the use of the above-mentioned retinoic acid-lactoferrin nanocrystal preparation in the preparation of a drug for treating cerebral ischemia / reperfusion injury.

[0023] Furthermore, the drug has any one or more of the following effects:

[0024] (1) Reduce the body's inflammation level; (2) Fight oxidative stress damage; (3) Improve neurological deficits; (4) Reduce cerebral infarction volume; (5) Regulate immune cell function.

[0025] Retinoic acid and lactoferrin can be selected from a variety of commercial products or prepared by yourself, and different grades and purities of products can be used according to different regulations. The chemical formula of retinoic acid is as follows:

[0026]

[0027] Beneficial effects:

[0028] The preparation of retinoic acid-lactoferrin nanocrystal preparation according to the present invention can greatly improve the solubility and stability of retinoic acid.

[0029] The retinoic acid-lactoferrin nanocrystals prepared according to the present invention can greatly improve the oral bioavailability of the drug.

[0030] In the retinoic acid-lactoferrin nanocrystals prepared according to the present invention, lactoferrin serves as a pharmacologically active ingredient to synergistically enhance the pharmacological effect of retinoic acid against cerebral ischemia / reperfusion injury. At the same time, lactoferrin also serves as a carrier protein for retinoic acid and plays a role as a stabilizer, greatly improving the delivery efficiency of retinoic acid and achieving a multifunctional effect of one ingredient.

[0031] The retinoic acid-lactoferrin prescription provided by the invention does not involve other pharmaceutical excipients and has a simple prescription. The preparation process of the nanocrystal is simple and rapid, the conditions are mild, and the yield is high; it is conducive to large-scale production and has broad application and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1 is a morphological diagram of the retinoic acid-lactoferrin nanocrystal preparation and the freeze-dried retinoic acid-lactoferrin nanocrystal preparation in Example 1.

[0033] Figure 2 This is the particle size distribution diagram of the retinoic acid-lactoferrin nanocrystal preparation in Example 1.

[0034] Figure 3 This is a transmission electron microscopy image of the retinoic acid-lactoferrin nanocrystal preparation in Example 1.

[0035] Figure 4 The scanning electron microscope images of the retinoic acid raw material and the retinoic acid-lactoferrin nanocrystal preparation in Example 1 are shown.

[0036] Figure 5 This is the stability result of the retinoic acid nanocrystal-lactoferrin preparation in Example 1.

[0037] Figure 6 The in vitro drug release curves of the retinoic acid bulk drug and the retinoic acid-lactoferrin nanocrystal preparation in Example 1 in phosphate buffer (PBS, pH 7.4), artificial intestinal fluid (SIF, pH 6.8) and artificial gastric fluid (SGF, pH 1.2).

[0038] Figure 7 1 is the drug-time curve of SD rats after oral administration of the retinoic acid raw material and the retinoic acid-lactoferrin nanocrystal preparation in Example 1.

[0039] Figure 8 The drug tissue distribution in SD rats after oral administration of the retinoic acid raw material and the retinoic acid-lactoferrin nanocrystal preparation in Example 1.

[0040] Fig. 9The blood flow change diagram (a) and blood flow quantification diagram (b) of the rat brain before and after the middle cerebral artery occlusion (MCAO) model prepared by the suture occlusion method and after blood reperfusion in Example 1. Before occlusion: cerebral blood flow diagram of rats before suture occlusion; occlusion: cerebral blood flow diagram of rats after suture occlusion; Reperfusion: cerebral blood flow diagram of rats after reperfusion.

[0041] Fig.10 The neurological function scores of rats with cerebral ischemia / reperfusion injury model after preventive administration of retinoic acid-lactoferrin nanocrystal preparation in Example 1. RA: intragastric administration of free retinoic acid solution; LF: intragastric administration of lactoferrin solution; LF-RA: intragastric administration of retinoic acid-lactoferrin nanocrystal preparation; RA-IP: intraperitoneal injection of retinoic acid solution.

[0042] Fig.11 The results of brain tissue infarction staining in rats with cerebral ischemia / reperfusion injury model after preventive administration of retinoic acid-lactoferrin nanocrystal preparation in Example 1. The captions are as above.

[0043] Fig.12 This is the brain tissue infarction volume of rats with cerebral ischemia / reperfusion injury model after preventive administration of retinoic acid-lactoferrin nanocrystal preparation in Example 1. The caption is as above.

[0044] Fig.13 The levels of inflammatory factors in the serum of rats with cerebral ischemia / reperfusion injury model after preventive administration of retinoic acid-lactoferrin nanocrystal preparation in Example 1. The captions are as above.

[0045] Fig.14 The results of routine blood test after intragastric administration of the retinoic acid-lactoferrin nanocrystal preparation in Example 1. RA: intragastric administration of free retinoic acid solution; LF: intragastric administration of lactoferrin solution; LF-RA Low: intragastric administration of low-concentration retinoic acid-lactoferrin nanocrystal preparation; LF-RA High: intragastric administration of high-concentration retinoic acid-lactoferrin nanocrystal preparation. DETAILED DESCRIPTION

[0046] Example 1

[0047] Provided is a retinoic acid-lactoferrin nanocrystal preparation, which comprises active drugs retinoic acid and lactoferrin.

[0048] The retinoic acid is a hydrophobic drug and a derivative of vitamin A. It was purchased from Sigma-Aldrich (R2625, Merck, Germany) with a purity ≥98% (HPLC); the lactoferrin is derived from bovine milk and was purchased from Nanjing Kangmanlin Chemical Industry Co., Ltd. (production batch number: 20200126, Jiangsu, China), with a protein content of 98.3% and an iron saturation of 15%.

[0049] As an embodiment of the present invention, the retinoic acid-lactoferrin nanocrystal preparation comprises, by mass ratio:

[0050] Retinoic acid: 10 mg

[0051] Lactoferrin: 50mg

[0052] The preparation method of the retinoic acid-lactoferrin nanocrystal preparation is as follows:

[0053] Step 1: Dissolve retinoic acid completely in an organic solvent; dissolve lactoferrin completely in water: weigh 10 mg of retinoic acid and add it to 1 mL of dichloromethane to fully dissolve; weigh 50 mg of lactoferrin and add it to 8 mL of ultrapure water to fully dissolve;

[0054] Step 2: slowly add the lactoferrin solution obtained in step 1 to the retinoic acid solution, vortex mix while adding, and then ultrasonicate the mixture at an ultrasonic power of 260 W for 5 min;

[0055] Step 3: The mixed solution after ultrasonication in step 2 is subjected to rotary evaporation to remove the organic solvent. Since retinoic acid is sensitive to heat and light, the mixture is protected from light and rotary evaporation is performed at room temperature to obtain a retinoic acid-lactoferrin nanocrystal preparation.

[0056] Property observation: Figure 1 Part (a) is the appearance morphology of the retinoic acid-lactoferrin nanocrystal preparation, which is a light yellow clear solution without stratification or precipitation. Figure 1 Part (b) is the appearance morphology of the freeze-dried retinoic acid-lactoferrin nanocrystal preparation. After freeze-drying, it presents an elegant cake-like morphology without collapse. After being dissolved in water, the nanocrystal particles are retained and the particle size remains unchanged. The surface potential decreases slightly due to the influence of the freeze-drying protective agent microcrystalline cellulose, indicating that freeze-drying has no effect on retinoic acid-lactoferrin nanocrystals, and it can be prepared into a freeze-dried product for storage.

[0057] Particle size and surface potential determination: Zetasizer Nano ZS90 laser particle size analyzer (Malvern, UK) was used to determine the particle size, particle size distribution, and potential of nanocrystals. 50 µL of retinoic acid-lactoferrin nanocrystals was diluted to 3 mL with ultrapure water, and the above sample was added to the sample cell. After removing the bubbles, the test was performed. The measurement temperature was set at 25 °C. Figure 2 This is a statistical diagram of the particle size distribution of the retinoic acid-lactoferrin nanocrystal preparation. The average particle size of the retinoic acid-lactoferrin nanocrystal preparation is 215 nm; the polydispersity index (PDI) is 0.202, and the particle size distribution is uniform; the surface potential is 36.47 mV.

[0058] Morphological observation: Take retinoic acid-lactoferrin nanocrystal preparation, drop it on the copper mesh, let it stand for 3 minutes, then stain it with 2% phosphotungstic acid for 2 minutes, and use filter paper to absorb the excess dye on the copper mesh. Dry the sample at room temperature, place it under a transmission electron microscope, observe the sample at 200kV and take pictures, such as Figure 3 As shown, the retinoic acid-lactoferrin nanocrystal particles are polygonal spindle-shaped crystals, uniformly dispersed, and have a particle size of about 200 nm, which is consistent with the results of the laser particle size analyzer.

[0059] Retinoic acid API and tretinoin-lactoferrin nanocrystal preparation were dispersed on the conductive glue, and then sprayed, and placed under a scanning electron microscope for observation and image acquisition. Figure 4 As shown in the figure, the crystals of retinoic acid raw material are irregular long strip crystals with wide particle size distribution, and the particle size ranges from 20 to 100 μm; while the retinoic acid-lactoferrin nanocrystal preparation has polygonal spindle-shaped particles, the surface of which is covered with a stabilizing layer, and the particle size distribution is uniform, and the particle size ranges from 200 to 250 nm.

[0060] Crystal stability study: The retinoic acid-lactoferrin nanocrystal preparation was stored in a dark environment at 4°C, and taken out at 1, 2, 3, 7, 20, and 30 days, and the particle size and PDI distribution of the nanoparticles were measured using a Zetasizer Nano ZS90 laser particle size analyzer to investigate the stability of the retinoic acid-lactoferrin nanocrystals. Figure 5 As shown, it can be observed that the particle size and PDI of the retinoic acid-lactoferrin nanocrystal preparation did not change significantly within 30 days, and the PDI was less than 0.3, indicating that the retinoic acid-lactoferrin nanocrystal preparation was relatively stable when refrigerated in the dark.

[0061] Release behavior study: Take the retinoic acid-lactoferrin nanocrystal preparation solution and retinoic acid solution, place them in dialysis bags (Mw: 8000-14000), fix and seal them, and place them in phosphate buffer solution (pH7.4) containing 1% Tween 80 and 20% ethanol, artificial intestinal fluid (pH 6.8) containing 1% Tween 80 and 20% ethanol, and artificial gastric fluid (pH1.2) containing 2% Tween 80 and 40% ethanol. Transfer the dialysis device to a 37°C constant temperature oscillator, set the speed to 120r / min, take 1mL of samples at 2, 5, 8, 10, 12, 24, 36, 48, 60, 72, 84, and 96h, replace 1mL of the corresponding fresh medium, filter the samples with a 0.22μm microporous membrane, collect the filtrate for high performance liquid chromatography-ultraviolet chromatography (HPLC-UV) analysis, and determine the drug concentration in the dialysis medium at different time points. The monitoring conditions were as follows: the chromatographic column was Kromasil 100-5-C18 (4.6×250 nm); the mobile phase was acetonitrile and 2% glacial acetic acid solution in a ratio of 96:4, the detection wavelength was 350 nm, the column temperature was 30°C, the injection volume was 10 μL, and the flow rate was 1 mL / min.

[0062] Figure 6 The drug release curves of retinoic acid API and retinoic acid-lactoferrin nanocrystal preparation in phosphate buffer (pH 7.4), artificial intestinal fluid (pH 6.8) and artificial gastric fluid (pH 1.2) are shown in the figure. Figure 6 A), the free drug release reached 90% in 10 hours and was almost complete in 24 hours; while the nanocrystal preparation released about 60% in 72 hours and entered a plateau phase. Figure 6 B), the cumulative release of free drug reached 80% in 8 hours, while the cumulative release of nanocrystal preparation was 38% in 72 hours, entering the plateau phase; however, compared with PBS, in gastric juice ( Figure 6 C), the free drug release reached 40% in 8 hours and entered the plateau phase, and the nanocrystal preparation released 20% in 72 hours and entered the plateau phase. In general, the nanocrystal preparation showed sustained release behavior in PBS, gastric juice, and intestinal juice, and the release of the nanocrystal preparation was slower in gastric juice and intestinal juice. Since retinoic acid is sensitive to both acid and heat, retinoic acid was degraded in the medium 24 hours after release, and the degradation was more serious in low-acid gastric juice.

[0063] Oral bioavailability study: 10 SPF SD male rats weighing 200-250g were selected and divided into groups by simple random method: free retinoic acid (RA) group and retinoic acid-lactoferrin nanocrystal group (LF-RA). The free group was gavaged with retinoic acid solution (10 mg / kg), and the preparation group was gavaged with retinoic acid-lactoferrin nanocrystal (retinoic acid 10 mg / kg, lactoferrin 50 mg / kg). After gavage, the rats were blooded from the orbital vein at 0min, 15min, 30min, 45min, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 6h, 8h, 12h, and 24h, and serum was collected. 55μL serum was added with internal standard 10μL ibuprofen solution, 50μL acetonitrile and 150μL methanol, vortexed for 2min, centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken for HPLC-UV detection. Figure 7 Table 2 shows the pharmacokinetic parameters. The results show that the prepared LF-RA significantly increases the area under the drug-time curve of RA, and that LF-RA significantly improves the oral bioavailability of RA compared with free RA.

[0064] Table 1. Pharmacokinetic parameters of RA and LF-RA in rats after oral administration

[0065]

[0066] Tissue distribution study: 20 SPF SD male rats weighing 200-250g were selected and divided into groups by simple random method: free retinoic acid (RA) and retinoic acid-lactoferrin nanocrystal group (LF-RA). The free group was gavaged with retinoic acid solution (10 mg / kg), and the preparation group was gavaged with retinoic acid-lactoferrin nanocrystal (retinoic acid 10 mg / kg, lactoferrin 50 mg / kg). The rats were killed 2 hours after gavage, and the heart, liver, spleen, lung, kidney, and brain were immediately removed. The surface blood was washed with PBS buffer solution, and the floating blood was dried with filter paper. 0.2g of tissue was weighed and added to 0.6 mL of physiological saline to prepare tissue homogenate. 55uL of tissue homogenate was added to 200μL of methanol solution, vortexed for 2min, centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken for HPLC-UV detection. Figure 8 The tissue distribution of RA and LF-RA 2 hours after oral administration. LF-RA significantly increased the drug content in various tissues and successfully entered the brain tissue.

[0067] Evaluation of preventive drug administration effect: 40 SPF SD male rats weighing 200-250g were selected and divided into groups by simple random method: sham operation group, model group, free retinoic acid group, lactoferrin group, preparation group, and positive control group. The rat cerebral ischemia model (middle cerebral artery occlusion, MCAO) was prepared by thread embolization. Before modeling, the free drug group was given retinoic acid solution (5 mg / kg) by gavage, the lactoferrin group was given lactoferrin aqueous solution (25 mg / kg) by oral gavage, and the preparation group was given retinoic acid-lactoferrin nanocrystal solution (retinoic acid 5 mg / kg, lactoferrin 25 mg / kg). The sham operation group and the model group were given equal volumes of normal saline, and the positive group was intraperitoneally injected with dimethyl sulfoxide solution containing retinoic acid (5 mg / kg). Once a day, the drug was administered for 4 consecutive days. On the 5th day, the rat focal cerebral ischemia reperfusion injury model (CIRI) was established by thread embolization method to induce rat cerebral ischemia. During the operation, the cerebral blood flow of SD rats was detected by laser Doppler blood flow monitor to confirm that the cerebral ischemia-reperfusion injury model was successfully established ( Fig. 9 ).

[0068] After 24 hours of feeding after modeling, the Longa 5-point scoring standard (as shown in Table 1) was used to score neurological function. After the scoring was completed, SD rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution. After anesthesia, the brain tissue of SD rats was quickly peeled off on ice, fixed in a -20℃ refrigerator for 20 minutes, sliced, and placed in 2,3,5-triphenyltetrazolium chloride (TTC) dye solution, reacted at 37℃ for 60 minutes. After ensuring that the staining was sufficient, the brain tissue slices were placed in 4% paraformaldehyde solution, fixed in a 4℃ refrigerator for 48 hours, and then photographed and the infarcted part of the brain tissue was removed. The weight of the entire brain tissue and the infarcted part was weighed respectively, and the percentage of cerebral infarction volume was calculated. The calculation formula is as follows;

[0069] Percentage of cerebral infarction volume = (W 脑梗死 / W 脑组织 )× 100 %.

[0070] W 脑梗死 W is the weight of brain tissue in cerebral ischemia infarction; 脑组织 is the weight of the entire brain tissue.

[0071] Table 2. Longa 5-point scoring criteria

[0072]

[0073] Fig.10The figure shows the neurological function score of model rats established after preventive administration. The average neurological function score of CIRI model rats pretreated with nanocrystals was 1.3, which was significantly lower than that of the model group (average: 3.5), free retinoic acid group (average: 3.5), and lactoferrin group (average: 2.67), and the neurological function was significantly improved. Fig.11 and Fig.12 The representative TTC staining diagram of cerebral infarction in rats with model after preventive administration is the statistical diagram of cerebral infarction volume percentage. Fig.11 As shown, the retinoic acid-lactoferrin nanocrystal preparation group significantly reduced the cerebral infarction volume of CIRI model rats after pretreatment compared with other experimental groups, and the preventive effect was better than that of retinoic acid free drug and intraperitoneal injection positive group.

[0074] Investigation of inflammatory factor levels: 40 SPF SD male rats weighing 200-250g were randomly divided into groups: sham operation group, model group, free retinoic acid group, lactoferrin group, preparation group, and positive control group. The rats in each experimental group were given preventive medication for 4 consecutive days. On the 5th day, a rat focal cerebral ischemia-reperfusion injury model (CIRI) was established, and the success of the model was determined by a laser Doppler blood flow monitor. On the 6th day, blood was collected from the abdominal aorta of the rats, and after standing for 3 hours, it was centrifuged at 3000 rpm / min for 20 min, and the upper serum was taken. The inflammatory factors and oxidative stress indicators in the serum were determined by enzyme-linked immunosorbent assay. First, take the protein-coated ELISA plate, set up standard wells, sample wells, and blank wells (blank control wells do not add samples and ELISA reagents), and add 50μL of standards of different concentrations to each standard well; first add 40μL of sample diluent to the sample to be tested on the ELISA coated plate, and then add 10μL of the sample to be tested (the final sample dilution is 5 times), add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix. Add 100μL of ELISA reagent to each well, except for the blank well. Seal the plate with a sealing film and incubate at 37℃ for 60min. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30s and discard it, repeat this 5 times, and pat dry. Then add the color developer to each well, gently shake and mix, and color at 37℃ in the dark for 15min. Add 50μL of stop solution to each well to stop the reaction, adjust the blank well to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm.

[0075] Fig.13 The levels of inflammatory factors and oxidative stress in the model rats after preventive drug administration were studied. The results showed that after pretreatment with retinoic acid-lactoferrin nanocrystals, the inflammatory factors IL-6 and TNF-𝛂 in CIRI model rats were significantly reduced, which can effectively prevent reperfusion injury after cerebral ischemia.

[0076] Preliminary in vivo safety evaluation: 12 SPF SD male rats weighing 200-250g were randomly divided into normal group, free retinoic acid group, lactoferrin group, low-dose preparation group, and high-dose preparation group. The free retinoic acid group was gavaged with retinoic acid solution (5 mg / kg), the lactoferrin group was gavaged with lactoferrin solution (25 mg / kg), the low-dose preparation group was gavaged with retinoic acid-lactoferrin nanocrystals (retinoic acid 5 mg / kg, lactoferrin 25 mg / kg), and the high-dose preparation group was gavaged with retinoic acid-lactoferrin nanocrystals (retinoic acid 20 mg / kg, lactoferrin 100 mg / kg). The normal group was given an equal volume of normal saline once a day for 4 days. After the end of the administration, they were kept normally for 3 days. On the 8th day, whole blood was collected from the abdominal aorta in EDTA anticoagulant tubes for blood cell detection.

[0077] Fig.14 The results of the preliminary in vivo safety evaluation showed that there was no significant difference in the number of white blood cells (14-A), lymphocytes (14-B), monocytes (14-C), red blood cells (14-D) and platelets (14-E) in the low-dose and high-dose groups compared with the normal group, indicating that oral administration of retinoic acid-lactoferrin nanocrystals had little effect on blood cells. Since lactoferrin can inhibit antibody-dependent cell-mediated reactions and enhance monocyte activity, the number of monocytes in the lactoferrin group increased compared with the normal group.

Claims

1. A retinoic acid-lactoferrin nanocrystal preparation, characterized in that: The preparation method of the retinoic acid-lactoferrin nanocrystal preparation comprises: (1) Dissolve retinoic acid completely in an organic solvent and dissolve bovine lactoferrin completely in water: weigh 10 mg of retinoic acid and add it to 1 mL of dichloromethane to fully dissolve it to obtain a retinoic acid solution; weigh 50 mg of bovine lactoferrin and add it to 8 mL of ultrapure water to fully dissolve it to obtain a lactoferrin solution; (2) slowly adding the lactoferrin solution obtained in step (1) to the retinoic acid solution, vortexing and mixing; then subjecting the obtained mixed solution to ultrasound to obtain an ultrasonically treated mixed solution, with an ultrasound power of 260 W and an ultrasound time of 5 min; (3) The mixed solution subjected to ultrasonic treatment obtained in step (2) is subjected to rotary evaporation to remove dichloromethane, thereby obtaining the retinoic acid-lactoferrin nanocrystal preparation.

2. The method for preparing the retinoic acid-lactoferrin nanocrystal preparation according to claim 1, characterized in that: The method comprises: (1) Dissolve retinoic acid completely in an organic solvent and dissolve bovine lactoferrin completely in water: weigh 10 mg of retinoic acid and add it to 1 mL of dichloromethane to fully dissolve it to obtain a retinoic acid solution; weigh 50 mg of bovine lactoferrin and add it to 8 mL of ultrapure water to fully dissolve it to obtain a lactoferrin solution; (2) slowly adding the lactoferrin solution obtained in step (1) to the retinoic acid solution, vortexing and mixing; then subjecting the obtained mixed solution to ultrasound to obtain an ultrasonically treated mixed solution, with an ultrasound power of 260 W and an ultrasound time of 5 min; (3) The mixed solution subjected to ultrasonic treatment obtained in step (2) is subjected to rotary evaporation to remove dichloromethane, thereby obtaining the retinoic acid-lactoferrin nanocrystal preparation.

3. Use of the retinoic acid-lactoferrin nanocrystal preparation according to claim 1 in the preparation of a medicament for treating cerebral ischemia / reperfusion injury.

4. The use according to claim 3, characterized in that: The drug has any one or more of the following effects: (1) Reduce the body's inflammation level; (2) Fight oxidative stress damage; (3) Improve neurological deficits; (4) Reduce cerebral infarction volume; (5) Regulate immune cell function.

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