Rapamycin-loaded microemulsion gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses rapamycin-loaded microemulsion gel as well as a preparation method and application thereof. The preparation method of the microemulsion gel comprises the following steps: dissolving N, N-dimethyl octadecyl carboxymethyl chitosan in a water phase, then adding an oil phase, a cosurfactant and rapamycin, and stirring to obtain rapamycin-loaded microemulsion; the preparation method comprises the following steps: dissolving a high polymer material in a PBS solution, heating until the high polymer material is completely dissolved, then adding dopamine, stirring for reaction, and finally dialyzing and freeze-drying to obtain the adhesive. And adding a gel matrix containing a thickening agent and an adhesive into the microemulsion, and stirring to obtain the rapamycin-loaded microemulsion gel. According to the prepared microemulsion gel, transdermal drug delivery of hydrophobic drugs is achieved, testis are coated with the microemulsion gel loaded with rapamycin, the blocking effect of damp-heat stress on differentiation and development of testis interstitial cells can be relieved, and the functionality of a male reproduction endocrine system is recovered.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a microemulsion gel loaded with rapamycin, its preparation method, and its application. Background Technology

[0002] The impact of heat and humidity stress on the male reproductive system should not be underestimated, as it exerts a broad inhibitory effect on the secretion of reproductive hormones through external environmental factors. Hormonal imbalances not only lead to decreased libido but also result in a significant reduction in sperm count, decreased sperm motility, and impaired fertilization capacity. Simultaneously, heat and humidity stress increases the risk of testicular diseases, severely affecting male reproductive function; therefore, timely intervention is necessary to prevent complications. Studies have shown that heat and humidity stress can lead to excessive activation of the mTOR signaling pathway in testicular interstitial cells, thereby inhibiting autophagy levels, interfering with the normal differentiation and development of interstitial cells, and causing functional arrest. These mechanisms provide an important pathophysiological basis for understanding male reproductive dysfunction caused by environmental factors and also offer potential targets for the development of relevant intervention strategies.

[0003] While rapamycin plays an effective role in alleviating excessive activation of mTOR in interstitial cells, long-term oral or injectable administration of rapamycin may impair receptor function and induce nephrotoxicity. Furthermore, rapamycin's poor solubility and low absorption rate severely affect therapeutic efficacy. Therefore, it is necessary to develop a dosing method with high bioavailability, good patient adaptability, good administration safety, reduced need for frequent dosing, and improved patient compliance.

[0004] Modern pharmacotherapeutic science not only requires drugs to have controlled release characteristics, but also emphasizes targeted delivery, enabling precise accumulation of drugs at the lesion site to improve bioavailability and therapeutic efficacy while minimizing toxic side effects. Therefore, constructing a delivery system capable of achieving drug accumulation has become a key scientific problem and technical challenge in the current field of drug delivery and clinical translational research. Microemulsion gels, as multifunctional drug carriers, can efficiently encapsulate drugs and, combined with transdermal administration, achieve the accumulation of active ingredients in diseased tissues, thereby optimizing therapeutic effects and reducing systemic adverse reactions, demonstrating excellent drug delivery capabilities. Therefore, utilizing microemulsion gels to encapsulate rapamycin and administering it transdermally to treat the effects of heat and dampness stress on male reproductive function has enormous application potential. Summary of the Invention

[0005] The purpose of this invention is to provide a microemulsion gel loaded with rapamycin, its preparation method, and its application, in order to solve the above-mentioned technical problems.

[0006] To achieve the above objectives, the present invention provides a method for preparing a microemulsion gel loaded with rapamycin, comprising the following steps: Step 1: Dissolve N,N-dimethyloctadecylcarboxymethyl chitosan in the aqueous phase, then add the oil phase, co-surfactant and rapamycin, and stir to obtain a microemulsion loaded with rapamycin; Step 2: Dissolve the polymer material in PBS solution, heat until completely dissolved, then add dopamine and stir to react. Finally, dialyze and freeze dry to obtain the adhesive. Step 3: Add the gel matrix containing thickener and binder to the microemulsion obtained in Step 1 and stir to obtain a microemulsion gel loaded with rapamycin.

[0007] Further, in step 1, the aqueous phase is deionized water; the oil phase is at least one of oleic acid, isopropyl palmitate, caprylic triglyceride, soybean oil, olive oil, castor oil, and mineral oil.

[0008] Furthermore, in step 1, the co-surfactant is at least one of ethanol, n-propanol, and isopropanol.

[0009] Furthermore, in the microemulsion of step 1, the mass-to-volume ratio of N,N-dimethyloctadecylcarboxymethyl chitosan to the aqueous phase is 1:150; the mass-to-volume ratio of the oil phase, co-surfactant, and rapamycin is 150:150:1.

[0010] Furthermore, in the microemulsion of step 1, the concentration of rapamycin is 0.1 mg / ml-10 mg / ml.

[0011] Furthermore, in step 2, the polymer material is at least one of gelatin, hyaluronic acid, sodium alginate, carboxymethyl cellulose, carboxymethyl chitosan, and polyacrylic acid.

[0012] Furthermore, in step 2, the mass ratio of the polymer material to dopamine is 2:1.

[0013] Further, in step 3, the thickener is at least one of aloe polysaccharide, carboxymethyl cellulose, agar, guar gum, carrageenan, xanthan gum, carbomer, sodium alginate, and polyvinyl alcohol; the mass fraction of the thickener in the gel matrix is ​​10%-50%, the mass fraction of the binder is 1%-10%, and the remainder is water.

[0014] Furthermore, in step 3, the mass-to-volume ratio of the gel matrix to the microemulsion is 2:1.

[0015] Furthermore, the present invention also provides a microemulsion gel loaded with rapamycin, prepared by the above-described preparation method.

[0016] Furthermore, the present invention also provides the application of the above-mentioned microemulsion gel in the preparation of a drug for preventing and / or treating male reproductive endocrine system disorders caused by damp heat stress, wherein the male reproductive endocrine system disorders caused by damp heat stress are characterized by the obstruction of the differentiation and development process of testicular interstitial cells due to damp heat stress.

[0017] Furthermore, the present invention also provides a medicament for preventing and / or treating male reproductive endocrine system disorders caused by hot and humid stress, wherein the active ingredient of the medicament includes the aforementioned microemulsion gel.

[0018] The drug can be applied to the patient's testes to achieve transdermal delivery of rapamycin. As an autophagy agonist, rapamycin inhibits the mTOR pathway, alleviating the obstruction of interstitial cell differentiation and development induced by heat and humidity stress, thereby restoring the function of the male reproductive endocrine system.

[0019] The advantages and positive effects of the microemulsion gel loaded with rapamycin described in this invention, its preparation method, and its application are as follows: 1. The microemulsion gel in this invention has excellent loading capacity for the hydrophobic drug rapamycin, and the nanoscale size of the microemulsion is conducive to penetration into the skin to achieve the purpose of local drug delivery.

[0020] 2. The microemulsion gel in this invention increases the residence time of the microemulsion gel on the skin surface by adding thickeners and adhesives, thereby promoting drug absorption and enhancing the therapeutic effect of the drug.

[0021] 3. The microemulsion gel in this invention achieves transdermal drug delivery, avoiding the first-pass effect of the gastrointestinal tract and liver, and has advantages such as high drug utilization, strong patient adaptability, and good drug delivery safety.

[0022] 4. The microemulsion gel in this invention has a wide range of material options and is inexpensive. The preparation process does not require complex equipment or instruments, making it easy to produce on a large scale.

[0023] 5. Chitosan has excellent antibacterial effects, which allows the prepared microemulsion gel loaded with rapamycin to be preserved for a longer period of time without the need for preservatives.

[0024] 6. The microemulsion gel loaded with rapamycin of the present invention is easy to use and non-irritating to the skin. It can be used as a highly efficient drug delivery system to treat the effects of hot and humid stress on male reproductive function and has good application prospects.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1This is a schematic diagram illustrating the preparation and application of microemulsion gel loaded with rapamycin in an embodiment of the present invention; Figure 2 The microemulsion exhibits performance characteristics in this embodiment of the invention, wherein A is a TEM image of the microemulsion; B is a particle size distribution diagram of the microemulsion; and C is an experimental diagram of the drug loading capacity of the microemulsion. Figure 3 The figures illustrate the performance of the microemulsion gel in this embodiment of the invention, where A is an experimental diagram showing the thickening effect of the gel matrix; and B is an experimental diagram showing the adhesion effect of the gel matrix. Figure 4 To illustrate the application and biosafety of the microemulsion gel in this invention, A is a photograph of the microemulsion gel loaded with rapamycin applied to the rat testis; B is an H&E staining image of the rat scrotum skin. Figure 5 This invention illustrates the therapeutic effect of rapamycin-loaded microemulsion gel on damp heat stress. Figure A shows the experimental results of testosterone concentration in rat plasma; Figure B shows the experimental results of PDGFRα and 3β-HSD expression levels in rat testicular interstitial cells; and Figure C shows the experimental results of PDGFRα and 3β-HSD immunofluorescence staining in rat testicular interstitial cells. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0029] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0030] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0031] Example 1 (1) Weigh 40 mg of N,N-dimethyloctadecyl carboxymethyl chitosan and dissolve it in 6 mL of deionized water. Then add 12 mg of rapamycin and 6 mL of oleic acid and vortex the solution to mix the oil phase and the water phase thoroughly. Then slowly add ethanol dropwise while stirring continuously. When the solution changes from turbid to clear, stop adding ethanol to obtain a microemulsion solution loaded with rapamycin.

[0032] (2) 2.0 g of gelatin was dissolved in 100 mL of phosphate-buffered saline (PBS, pH 7.4) at 60 °C. Then, 0.5 g of ethyldimethylaminopropylcarbodiimide and 0.3 g of N-hydroxysuccinimide were added to the solution sequentially, and the pH of the reaction solution was adjusted to 4-5 using 1 M hydrochloric acid. After stirring for 30 minutes, 1.0 g of dopamine hydrochloride was added to the solution, and the solution was stirred for 24 hours. Finally, the reaction solution was dialyzed against deionized water for three days, and then lyophilized to obtain gelatin grafted with dopamine groups.

[0033] (3) 5.0 g of aloe polysaccharide and 1.0 g of dopamine-grafted gelatin were dissolved in 5 ml of microemulsion solution loaded with rapamycin to prepare microemulsion gel loaded with rapamycin.

[0034] The preparation process and application method of the rapamycin-loaded microemulsion gel obtained in this embodiment are as follows: Figure 1 As shown, an oil-in-water microemulsion was prepared by vortexing using amphiphilic N,N-dimethyloctadecyl carboxymethyl chitosan as the surfactant, oleic acid as the oil phase, ethanol as the co-surfactant, and deionized water as the aqueous phase. Aloe polysaccharide, a natural plant extract, was selected as the thickener, and dopamine-grafted gelatin was used as the binder to disperse the rapamycin-loaded microemulsion into a gel matrix, thus preparing a microemulsion gel for convenient daily medication administration.

[0035] The microemulsion morphology in this embodiment is as follows: Figure 2 As shown in Figure A, the prepared microemulsion is bright inside and dark outside, indicating that it is an oil-in-water microemulsion. The microemulsion size in this embodiment is as follows: Figure 2 As shown in Figure B, the average particle size of the unloaded rapamycin microemulsion was approximately 22.53 ± 0.45 nm, while the particle size of the microemulsion loaded with rapamycin was 22.27 ± 0.98 nm. There was no significant change in the size of the microemulsion before and after drug loading. Simultaneously, the nanoscale size facilitates the microemulsion's ability to penetrate the skin barrier and enter the testis, achieving more efficient and safer rapamycin delivery. The drug loading performance of the microemulsion in this embodiment is as follows: Figure 2 As shown in Figure C, the maximum loading capacity of the microemulsion for rapamycin is 2.82 ± 0.13 mg / mL, which is much higher than the solubility of rapamycin in aqueous solution, indicating that the microemulsion has a good loading capacity for the hydrophobic drug rapamycin.

[0036] The thickening effect of aloe polysaccharides in this embodiment is as follows: Figure 3 As shown in Figure A, the microemulsion solution rapidly flows to the bottom of the inverted vial, while the microemulsion gel, after the addition of aloe polysaccharide, flows slowly along the vial wall, demonstrating the thickening effect of aloe polysaccharide on the nanoemulsion. The tissue adhesion effect of the dopamine-grafted gelatin as a binder in this embodiment is as follows: Figure 3 As shown in Figure B, when equal volumes of microemulsion solution (Rapa / DCMC), microemulsion gel with added aloe polysaccharide (Rapa / DCMC / AP), and microemulsion gel with gelatin grafted with aloe polysaccharide and dopamine groups (Rapa / DCMC / GelDA / AP) were applied to the skin, it was found that the microemulsion solution was rapidly lost. Although the microemulsion gel with added aloe polysaccharide had a higher viscosity, it still could not be fixed on the skin surface. However, with the addition of gelatin grafted with dopamine groups, the microemulsion gel adhered firmly to the skin surface, significantly prolonging the drug's contact time with the skin and thus enhancing the drug's absorption efficiency.

[0037] Example 2 (1) Weigh 40 mg of N,N-dimethyloctadecyl carboxymethyl chitosan and dissolve it in 6 mL of deionized water. Then add 12 mg of rapamycin and 6 mL of oleic acid and vortex the solution to mix the oil phase and the water phase thoroughly. Then slowly add ethanol dropwise while stirring continuously. When the solution changes from turbid to clear, stop adding ethanol to obtain a microemulsion solution loaded with rapamycin.

[0038] (2) 2.0 g of gelatin was dissolved in 100 mL of phosphate-buffered saline (PBS, pH 7.4) at 60 °C. Then, 0.5 g of ethyldimethylaminopropylcarbodiimide and 0.3 g of N-hydroxysuccinimide were added to the solution sequentially, and the pH of the reaction solution was adjusted to 4-5 using 1 M hydrochloric acid. After stirring for 30 minutes, 1.0 g of dopamine hydrochloride was added to the solution, and the solution was stirred for 24 hours. Finally, the reaction solution was dialyzed against deionized water for three days, and then lyophilized to obtain gelatin grafted with dopamine groups.

[0039] (3) 5.0 g of aloe polysaccharide and 1.0 g of dopamine-grafted gelatin were dissolved in 5 ml of microemulsion solution loaded with rapamycin to prepare microemulsion gel loaded with rapamycin.

[0040] Establishment of a rat model of heat and damp stress: Adult male SD rats, 8 weeks old, weighing 200±20g. Temperature: 24±0.5℃, humidity: 50±10%, 12-hour light / dark cycle. Rats were placed under the above conditions to establish a heat and damp stress model. Rats raised at room temperature served as the control group, with no fewer than 5 rats in each group.

[0041] The rapamycin-loaded microemulsion gel obtained in Example 2 was applied to the testes of rats in a wet heat stress model at a frequency of 2 ml / time, twice a day. Two weeks after application, rat scrotal skin was collected for H&E staining to test the concentration of testosterone in rat plasma and the expression levels of PDGFRα and 3β-HSD in rat testicular interstitial cells.

[0042] The procedure for using this embodiment on the testes of a rat model of damp heat stress is as follows: Figure 4 As shown in Figure A; two weeks after microemulsion gel administration, H&E staining of rat scrotal skin is as follows. Figure 4 As shown in Figure B, the epidermal structure of the rat scrotum remained largely intact, similar to that of untreated rats. No significant increase in inflammatory cells was observed, indicating that the microemulsion gel is biosafe when used as a transdermal drug delivery formulation.

[0043] After two weeks of use in the testes of a rat model of damp heat stress, the serum testosterone concentration in this embodiment was as follows: Figure 5 As shown in Figure A, compared with the microemulsion gel control group, the microemulsion gel loaded with rapamycin (Rapa / DCMC / GelDA / AP) significantly increased testosterone levels in a rat model under heat and humidity stress; protein expression in rat testicular interstitial cells was as follows. Figure 5 China B and Figure 5 As shown in Figure C, the protein expression of PDGFRα and 3β-HSD was significantly increased, indicating a reversal of impaired mesenchymal cell development.

[0044] Comparative Example 1 Unlike Example 1, the surfactant used to prepare the microemulsion gel loaded with rapamycin was carboxymethyl chitosan. The results showed that rapamycin could not dissolve sufficiently and deposited in the prepared microemulsion solution.

[0045] Comparative Example 2 Unlike Example 1, glucose was used as the thickener in the preparation of the rapamycin-loaded microemulsion gel. The results showed that the obtained rapamycin-loaded microemulsion gel had low viscosity and high flowability.

[0046] Comparative Example 3 Unlike Example 1, gelatin was used as the binder to prepare the rapamycin-loaded microemulsion gel. The results showed that the obtained rapamycin-loaded microemulsion gel had poor adhesion and could not be effectively fixed to the skin surface.

[0047] This invention provides a microemulsion gel loaded with rapamycin and its preparation method. The microemulsion prepared by this method exhibits excellent drug-loading performance for the hydrophobic drug rapamycin, and its small nanoscale size facilitates skin penetration for transdermal drug delivery. Furthermore, the gel matrix possesses excellent tissue adhesion and thickening effects, which can effectively prolong the residence time of the microemulsion gel on the skin surface and enhance drug absorption efficiency. The microemulsion gel preparation method of this invention is simple, uses inexpensive and readily available raw materials, is convenient and safe to use, and can effectively load rapamycin for efficient delivery. It can serve as a high-performance pharmaceutical formulation for the precise treatment of the effects of heat and humidity stress on male reproductive function.

[0048] Therefore, the microemulsion gel prepared in this invention enables transdermal drug delivery of hydrophobic drugs. Coating the testis with the microemulsion gel loaded with rapamycin can alleviate the inhibitory effect of heat and humidity stress on the differentiation and development of testicular interstitial cells and restore the function of the male reproductive endocrine system.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A process for the preparation of a rapamycin-loaded microemulsion gel, characterized in that, The method comprises the following steps: Step 1, dissolving N,N-dimethyl octadecyl carboxymethyl chitosan in the aqueous phase, then adding the oil phase, the co-surfactant and rapamycin, and stirring to obtain the rapamycin-loaded microemulsion; Step 2, dissolving the polymer material in the PBS solution, heating to complete dissolution, then adding dopamine and stirring to react, and finally dialyzing and freeze-drying to obtain the adhesive; Step 3, adding the gel matrix containing the thickening agent and the adhesive to the microemulsion obtained in Step 1, and stirring to obtain the rapamycin-loaded microemulsion gel.

2. A process for the preparation of a rapamycin-loaded microemulsion gel according to claim 1, characterized in that: In Step 1, the aqueous phase is deionized water; the oil phase is at least one of oleic acid, isopropyl palmitate, caprylic triglyceride, soybean oil, olive oil, castor oil and mineral oil; and in Step 1, the co-surfactant is at least one of ethanol, n-propanol and isopropanol.

3. A process for the preparation of a rapamycin-loaded microemulsion gel according to claim 1, characterized in that: In the microemulsion of Step 1, the mass ratio of N,N-dimethyl octadecyl carboxymethyl chitosan to the aqueous phase is 1:150; and the mass ratio of the oil phase, the co-surfactant and rapamycin is 150:150:

1.

4. A process for the preparation of a rapamycin-loaded microemulsion gel according to claim 1, characterized by: In Step 2, the polymer material is at least one of gelatin, hyaluronic acid, sodium alginate, carboxymethyl cellulose, carboxymethyl chitosan and polyacrylic acid.

5. A process for preparing a rapamycin-loaded microemulsion gel according to claim 1, characterized by: In Step 2, the mass ratio of the polymer material to dopamine is 2:

1.

6. A process for preparing a rapamycin-loaded microemulsion gel according to claim 1, characterized by: In Step 3, the thickening agent is at least one of aloe polysaccharide, carboxymethyl cellulose, agar, guar gum, carrageenan, xanthan gum, carbomer, sodium alginate and polyvinyl alcohol; the mass fraction of the thickening agent in the gel matrix is 10%-50%, the mass fraction of the adhesive is 1%-10%, and the rest is water.

7. A process for preparing a rapamycin-loaded microemulsion gel according to claim 1, characterized by: In Step 3, the mass-volume ratio of the gel matrix to the microemulsion is 2:

1.

8. A rapamycin-loaded microemulsion gel characterized in that: Prepared by the preparation method of any one of claims 1-7.

9. The use of the microemulsion gel of claim 8 in the preparation of a drug for preventing and / or treating the disorder of the male reproductive endocrine system caused by damp-heat stress, characterized in that: The disorder of the male reproductive endocrine system caused by heat stress is that the differentiation and development process of testicular interstitial cells is hindered by heat stress.

10. A medicament for preventing and / or treating male reproductive endocrine system disorder caused by damp-heat stress, characterized in that: The effective component of the drug comprises the microemulsion gel prepared by the preparation method of any one of claims 1-8 or the microemulsion gel of claim 8.

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