A nanocarrier for targeted and sustained release of plumbagin and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to a targeted sustained-release nanometer drug delivery system of geniposide and a preparation method and application thereof. A polylactic acid-polyglycolic acid copolymer is used as a matrix, polyvinyl alcohol is used as a crosslinking agent, and the matrix and the crosslinking agent are crosslinked to form coated drug-loaded microspheres; then chitosan is used as a protective agent, and the chitosan and the coated drug-loaded microspheres are crosslinked to form double-wrapped composite drug-loaded microspheres, wherein the chitosan in the composite drug-loaded microspheres is a shell, and the coated drug-loaded microspheres are a core, so that the targeted sustained-release nanometer drug delivery system of geniposide is obtained. The application enhances the targeting of the cornea and the lacrimal gland through double-target design, improves the treatment efficiency of the drug, realizes stable release and sustained release of the geniposide, and overcomes the problem of unstable drug release; and the targeted sustained-release nanometer drug delivery system reduces the side effects on the eyes by combining the anti-inflammatory activity and the mitochondrial protection function of the geniposide, and improves the safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and specifically relates to a Linarin targeted sustained-release nanocarrier system, a preparation method and application thereof. BACKGROUND

[0002] Dry Eye Disease (DED) is a common eye disease characterized by insufficient tear secretion or excessive tear evaporation, leading to symptoms such as dryness, burning, foreign body sensation, etc. Dry eye disease is closely related to eye inflammation, corneal epithelial damage, and eye discomfort. Current treatment methods include the use of artificial tears, anti-inflammatory drugs, and immunosuppressive agents. However, these methods can only alleviate symptoms and cannot fundamentally solve the problem, and long-term use may cause side effects.

[0003] Linarin (LA) has anti-inflammatory, antioxidant and other biological activities, and has potential therapeutic effects on mitochondrial damage and inflammatory response. However, the low solubility and poor bioavailability of Linarin limit its clinical application.

[0004] Nanoparticle Drug Delivery Systems (NDDS) show great potential in the field of drug delivery. These systems can improve drug bioavailability, reduce drug side effects, and achieve targeted therapy. Common nanoparticle carriers include polylactic acid-polyglycolic acid (PLGA) nanoparticles and other biodegradable polymers. However, these carrier systems still have problems such as insufficient targeting, difficult drug release control, and side effects in the treatment of specific diseases. SUMMARY

[0005] To solve the above problems, the present application provides a Linarin targeted sustained-release nanocarrier system, a preparation method and application thereof. The existing Nanoparticle Drug Delivery Systems still have problems such as insufficient targeting, difficult drug release control, and side effects. The development of a dual-targeting, multifunctional Linarin targeted sustained-release nanocarrier system can improve targeting, enhance drug stability and control release, and more effectively regulate mitochondrial damage and macrophage polarization, thereby reducing the inflammatory response of dry eye disease. This new nanoparticle system is expected to overcome the shortcomings of existing technology and improve treatment efficacy.

[0006] The present application solves the above technical problems through the following technical solutions.

[0007] The first object of the present application is to provide a targeted sustained-release nanocarrier system of plumbagin, which is formed by coating drug-loaded microspheres through cross-linking grafting of a substrate and a cross-linking agent, and then forming double-wrapped composite drug-loaded microspheres by cross-linking grafting of chitosan and the coated drug-loaded microspheres, wherein the chitosan in the composite drug-loaded microspheres is a shell, and the coated drug-loaded microspheres are a core, i.e., the targeted sustained-release nanocarrier system of plumbagin is obtained, and the substrate is loaded with plumbagin.

[0008] The second object of the present application is to provide a preparation method of the targeted sustained-release nanocarrier system of plumbagin, which comprises the following steps:

[0009] S1, mixing plumbagin and polylactic acid-polyglycolic acid copolymer in a solvent to obtain a mixed solution, adding a polyvinyl alcohol solution to the mixed solution, and stirring to cross-link and graft to perform nanometer precipitation, and then dialyzing to prepare a PLGA@LA nanoparticle dispersion solution.

[0010] S2, adding chitosan solution dropwise to the PLGA@LA nanoparticle dispersion solution, stirring at room temperature to uniformly disperse the chitosan on the surface of the PLGA@LA nanoparticles, cross-linking and grafting to form a coating, forming a coated composite drug-loaded microsphere, and obtaining the targeted sustained-release nanocarrier system of plumbagin.

[0011] Further, the mass-volume ratio of plumbagin, polylactic acid-polyglycolic acid copolymer and solvent is 1mg-2mg:4mg-5mg:500μL.

[0012] Further, the mass-volume ratio of polylactic acid-polyglycolic acid copolymer and polyvinyl alcohol solution is 1mg:0.9mL-1.25mL, and the concentration of the polyvinyl alcohol solution is 10mg / mL-15mg / mL.

[0013] Further, the specific method of cross-linking grafting and nanometer precipitation is as follows: the mixed solution is added dropwise to the polyvinyl alcohol solution under the condition of stirring at a speed of 400rpm-500rpm, and after the dropwise addition is completed, the stirring is continued for 2min-3min, and then dialysis is performed overnight at 5KDa-15KDa to obtain the PLGA@LA nanoparticle dispersion solution.

[0014] Further, the volume ratio of the PLGA@LA nanoparticle dispersion solution and the chitosan solution is 100-300:1, and the concentration of the chitosan solution is 5mg / mL-10mg / mL.

[0015] Further, the specific way of cross-linking grafting to form the coating is: under the condition of stirring at a speed of 200 rpm to 400 rpm at room temperature, the chitosan solution is added dropwise into the PLGA@LANPs nanoparticle dispersion solution, and after the dropwise addition is completed, the stirring is continued for 2 h to 3 h to form the coated composite drug-loaded microspheres.

[0016] A third object of the present application is to provide the use of the above-mentioned targeted sustained-release nanodrug delivery system of plumbagin in the preparation of a dry eye disease drug.

[0017] Further, the dry eye disease drug is eye drops.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] (1) After the plumbagin is loaded with polylactic acid-glycolic acid copolymer, the coated drug-loaded microspheres are formed by cross-linking grafting of polyvinyl alcohol, and then coated with chitosan to form double-wrapped composite drug-loaded microspheres, which can effectively protect the plumbagin from degradation or inactivation in the body environment; the core layer of PLGA can provide a stable drug carrier, and the outer layer of chitosan further enhances this protective effect, ensuring that the drug remains stable before release, reducing the irritation of the drug to the ocular tissue, increasing the safety of the nanoparticles, reducing side effects, and promoting local absorption of the drug, enhancing adhesion, sustained release, targeting and bioavailability. Through double-target design, the targeting of the cornea and lacrimal gland is enhanced, the therapeutic efficiency of the drug is improved, the stable and sustained release of plumbagin is achieved, and the problem of unstable drug release is overcome; and the targeted sustained-release nanodrug delivery system reduces the side effects on the eye by combining the anti-inflammatory activity and mitochondrial protection function of plumbagin, and improves the safety.

[0020] (2) The plumbagin targeted sustained-release nanodrug delivery system prepared by the present application has the following advantages: first, the size of the nanodrug delivery system particles is less than 200 nm, and this small size is beneficial for them to pass through biological barriers such as cell membranes, thereby effectively entering the lacrimal gland. Not only can it act on the cornea, but also can enter the lacrimal gland, thereby realizing double targeting; secondly, the combination of chitosan and PLGA can achieve controlled release of the drug, i.e. the drug can be slowly released near the lacrimal gland, prolonging its action time and forming a controlled release. This sustained release mechanism can improve the bioavailability of the drug, so that the concentration of the drug in the target area remains within the effective range; finally, due to the physiological characteristics of the lacrimal gland, these nanoparticles can be transported to the lacrimal gland in the flow of tears, promoting the distribution of the drug. This makes it easier for the wrapped drug to reach the target area, and chitosan has cationic properties that can interact with the anionic sites on the lacrimal gland cell membrane through electrostatic interaction, thereby enhancing cell uptake. This interaction helps the nanoparticles to penetrate the cell membrane more effectively and enter the lacrimal gland.

[0021] (3) The preparation method of the plumbagin targeted sustained-release nano drug delivery system of the present application has simple preparation process, and can realize large-scale industrial production conveniently. Meanwhile, the prepared plumbagin targeted sustained-release nano drug delivery system has targeting property, good long-term stability, long sustained-release time, excellent biocompatibility and biodegradability, and can significantly improve the survival rate of corneal epithelial cells. The operation is simple, the cost is low, and the repeatability is good.

[0022] (4) As a dry eye disease drug, the plumbagin targeted sustained-release nano drug delivery system of the present application can maintain the drug concentration at a therapeutic level and prolong the efficacy by slow release of the drug through diffusion and degradation due to the structure of the polymer. The chitosan has good adhesion and can effectively adhere to the ocular surface to prolong the drug residence time and reduce the need for frequent drug administration. The positively charged nanoparticles have strong adsorption capacity on the ocular surface because the ocular tissue surface is usually negatively charged. The positively charged nanoparticles are combined with the cell membrane, glycosaminoglycans and other components of the ocular surface through electrostatic interaction, thereby prolonging the residence time of the drug on the ocular surface and reducing the loss of the drug. This electrostatic adsorption effect helps to slowly release the drug, so that the local concentration of the drug in the eye can be maintained for a long time.

[0023] (5) As a dry eye disease drug, the plumbagin targeted sustained-release nano drug delivery system of the present application has mitochondrial protection function. According to the results of mouse cornea, it can be observed that after drug treatment, the morphology of mitochondria in corneal epithelial cells is obviously improved, the structure of mitochondria tends to be normal, the membrane integrity is restored, and the swelling and lysis phenomenon is reduced. This indicates that the drug can reduce the excessive generation of reactive oxygen species (ROS) by reducing mitochondrial damage, thereby reducing the oxidative stress caused by mitochondrial damage. The recovery of mitochondrial function helps to reduce the activation of inflammasomes and the excessive release of cytokines (such as IL-1β, TNF-α, etc.), thereby effectively inhibiting local inflammatory reactions. Therefore, the drug can effectively control the inflammatory response and promote the repair and recovery of the cornea by improving the health status of mitochondria. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a transmission electron microscopy (TEM) image of the PLGANPs and CTS@PLGA@LANPs of Example 1 of the present application, wherein, Figure 1 A is a TEM image of the PLGANPs, and the small image in the upper left corner of A is an enlarged image of the nanoparticles in A; B is a TEM image of the CTS@PLGA@LANPs, and the small image in the upper left corner of B is an enlarged image of the nanoparticles in B.

[0025] Figure 2 Figure 2 is a graph of the encapsulation efficiency of the CTS@PLGA@LANPs of Example 1 of the present application.

[0026] Figure 3Fig. 1 is a particle size data graph of the PLGA NPs and CTS@PLGA@LANPs of Example 1 of the present application.

[0027] Figure 4 Fig. 2 is a potential data graph of the PLGA NPs and CTS@PLGA@LANPs of Example 1 of the present application.

[0028] Figure 5 Fig. 3 is a release graph of the PLGA@LANPs and CTS@PLGA@LANPs of Example 1 of the present application under different environments.

[0029] Figure 6 Fig. 4 is a UV-visible absorption spectrum graph of the CTS@PLGA@LANPs of Example 1 of the present application.

[0030] Figure 7 Fig. 5 is a graph of the influence of the CTS@PLGA@LANPs of Example 1 of the present application on the inflammatory factors of macrophage THP-1 cells, wherein, Figure 7 A is a graph of the level change of the inflammatory factor TNF-α, and B is a graph of the level change of the inflammatory factor IL-1β.

[0031] Figure 8 Fig. 6 is a distribution graph of the CTS@PLGA@LANPs of Example 1 of the present application in the cornea and lacrimal gland.

[0032] Figure 9 Fig. 7 is an H&E staining graph of the corneal tissue of the present application.

[0033] Figure 10 Fig. 8 is an H&E staining graph of the lacrimal gland tissue of the present application.

[0034] Figure 11 Fig. 9 is a graph of the change of mitochondria in the corneal tissue of the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] It should be noted that the professional terms used in the present application are only for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present application can be purchased from the market or prepared by the existing method.

[0037] Linarin (LA) has anti-inflammatory, antioxidant and other biological activities, and has potential therapeutic effects on mitochondrial injury and inflammatory response. However, the low solubility and poor bioavailability of Linarin limit its clinical application.

[0038] Common nanoparticle carriers include polylactic acid-polyglycolic acid (PLGA) nanoparticles and other biodegradable polymers. However, these drug delivery systems also have some problems in treating certain diseases:

[0039] Insufficient targeting: existing nanoparticle drug delivery systems such as PLGA nanoparticles have limited targeting in the treatment of eye diseases, making it difficult to accurately reach the corneal lacrimal lesion site. Drug stability and release control: the release rate and stability of drugs in the body are difficult to accurately control, affecting the persistence of treatment effect. Side effect problem: long-term use of anti-inflammatory drugs and immunosuppressive agents may cause side effects such as eye infection, exacerbation of dry eye, etc.

[0040] Based on the above problems, the present application provides a Linarin targeted sustained-release nanoparticle drug delivery system, which uses polylactic acid-polyglycolic acid copolymer as the matrix, polyvinyl alcohol as the crosslinking agent, and forms a coated drug-loaded microsphere by crosslinking grafting of the matrix and the crosslinking agent; then chitosan is used as a protective agent to crosslink graft chitosan and coated drug-loaded microspheres to form a double-wrapped composite drug-loaded microsphere, wherein the chitosan in the composite drug-loaded microsphere is the shell, and the coated drug-loaded microsphere is the core, i.e. the Linarin targeted sustained-release nanoparticle drug delivery system, wherein the matrix is loaded with Linarin.

[0041] After loading Linarin LA with PLGA, the present application forms a coated drug-loaded microsphere by crosslinking grafting with polyvinyl alcohol (PVA), and then coats it with chitosan (CTS) to form a double-wrapped composite drug-loaded microsphere, which can effectively protect Linarin from degradation or inactivation in the body environment; wherein the core layer of PLGA can provide a stable drug carrier, and the outer layer of chitosan further enhances this protective effect, ensuring that the drug remains stable before release, reducing the irritation of the drug to the eye tissue, increasing the safety of the nanoparticles, reducing side effects, and at the same time promoting the local absorption of the drug, enhancing adhesion, sustained release, targeting and bioavailability.

[0042] The present application enhances the targeting of the cornea and lacrimal gland through double-target design, improves the therapeutic efficiency of the drug, realizes the stable release and sustained release of Linarin, and overcomes the problem of unstable drug release; and the targeted sustained-release nanoparticle drug delivery system reduces the side effects on the eye by combining the anti-inflammatory activity and mitochondrial protection function of Linarin, and improves the safety.

[0043] The preparation method of the above-mentioned Linarin targeted sustained-release nanoparticle drug delivery system comprises the following steps:

[0044] S1, mix the glycosides of biotperin and polylactic acid-polyglycolic acid copolymer in a solvent to obtain a mixed solution, add a polyvinyl alcohol solution to the mixed solution, stir and crosslink graft to perform nano precipitation, and prepare a PLGA@LA nanoparticle dispersion solution after dialysis.

[0045] S2, drop the chitosan solution into the PLGA@LA nanoparticle dispersion solution, stir at room temperature to uniformly disperse the chitosan on the surface of the PLGA@LA nanoparticles, crosslink graft to form a coating, form a coated composite drug-loaded microsphere, and obtain a glycosides of biotperin targeted sustained-release nano drug delivery system.

[0046] The preparation method of the glycosides of biotperin targeted sustained-release nano drug delivery system of the application has a simple preparation process, is convenient for large-scale industrial production, and at the same time, the prepared glycosides of biotperin targeted sustained-release nano drug delivery system has targeting, good long-term stability, long sustained release time, and excellent biocompatibility and biodegradability, significantly improves the survival rate of corneal epithelial cells, is simple to operate, low in cost, and good in repeatability.

[0047] The prepared glycosides of biotperin targeted sustained-release nano drug delivery system has the following advantages: firstly, the size of the nano drug delivery system particles is less than 200 nm, and such a small size is conducive to their passing through biological barriers such as cell membranes, thereby effectively entering the lacrimal gland. Not only can it act on the cornea, but also can enter the lacrimal gland, thereby realizing double targeting; secondly, the combination of chitosan and PLGA can realize controlled release of the drug, that is, the drug can be slowly released near the lacrimal gland, thereby prolonging the action time and forming controlled release. This slow-release mechanism can improve the bioavailability of the drug and keep the concentration of the drug in the target area within an effective range; finally, due to the physiological characteristics of the lacrimal gland, these nanoparticles can be transported to the lacrimal gland in the flow of tears, thereby promoting the distribution of the drug. This makes the wrapped drug more easily reach the target area, and chitosan has cationic properties, which can interact with the anionic sites on the cell membrane of the lacrimal gland, thereby enhancing cell uptake. This interaction helps the nanoparticles penetrate the cell membrane more effectively and enter the lacrimal gland.

[0048] In some specific embodiments, the mass-volume ratio of the glycosides of biotperin, polylactic acid-polyglycolic acid copolymer and solvent is 1 mg-2 mg:4 mg-5 mg:500 μL. Preferably, the mass-volume ratio of the glycosides of biotperin, polylactic acid-polyglycolic acid copolymer and solvent is 1 mg:5 mg:500 μL.

[0049] In the application, PLGA is a material commonly used in drug sustained-release systems, and its biodegradability and controlled release characteristics enable it to gradually release drugs over a long period of time. This slow-release mechanism is particularly important for treating dry eye symptoms, as it can achieve long-term drug efficacy and reduce the frequent use of drugs, thereby improving patient compliance.

[0050] Chitosan has excellent biocompatibility and biodegradability, which makes it an ideal material in drug delivery systems. It can reduce the irritation of drugs to ocular tissues, increase the safety of nanoparticles, reduce side effects, and promote the local absorption of drugs.

[0051] The solvent used only needs to ensure the complete dissolution of geniposide and polylactic acid-polyglycolic acid copolymer. Preferably, the solvent is dimethyl sulfoxide (DMSO), and the specific mixing method is ultrasonic water bath at 37°C until complete dissolution.

[0052] In some specific embodiments, the mass-volume ratio of the polylactic acid-polyglycolic acid copolymer and the polyvinyl alcohol solution is 1 mg: 0.9 mL ~ 1.25 mL, and the concentration of the polyvinyl alcohol solution is 10 mg / mL ~ 15 mg / mL. Preferably, the mass-volume ratio of the polylactic acid-polyglycolic acid copolymer and the polyvinyl alcohol solution is 5 mg: 4.5 mL, and the concentration of the polyvinyl alcohol solution is 10 mg / mL.

[0053] In some specific embodiments, the specific method of cross-linking grafting and nano-precipitation is to add the mixed solution to the polyvinyl alcohol solution under stirring at a speed of 400 rpm ~ 500 rpm, continue stirring for 2 min ~ 3 min after the addition is completed, and then dialyze overnight at 5 KDa ~ 15 KDa to obtain a PLGA@LA nanoparticle dispersion solution.

[0054] The dropwise addition method is used to ensure the uniformity of the reaction between polylactic acid-polyglycolic acid copolymer and polyvinyl alcohol, so as to prevent local supersaturation. During the dropwise addition process, the mixing state needs to be observed to ensure that there is no precipitation or stratification. After the dropwise addition is completed, the mixture is continued to be stirred at a speed of 400 rpm ~ 500 rpm for 2 min ~ 3 min. This step helps to form preliminary nanoparticles and ensures uniformity.

[0055] Preferably, the molecular weight used for overnight dialysis is 5 KDa, 10 KDa or 15 KDa. When dialysis is performed overnight, the dialysis bag needs to be pre-washed with appropriate ultrapure water to remove possible impurities.

[0056] In some specific embodiments, the volume ratio of the PLGA@LA nanoparticle dispersion solution and the chitosan solution is 100 ~ 300: 1, and the concentration of the chitosan solution is 5 mg / mL ~ 10 mg / mL. Preferably, the volume ratio of the PLGA@LA nanoparticle dispersion solution and the chitosan solution is 100: 1, and the concentration of the chitosan solution is 10 mg / mL.

[0057] In some specific embodiments, the specific way of forming the coating by crosslinking grafting is: the chitosan solution is added dropwise into the PLGA@LANP nanoparticle dispersion solution under stirring at a speed of 200 rpm to 400 rpm at room temperature, and stirring is continued for 2 h to 3 h after the dropwise addition is completed, to form the coated composite drug-loaded microspheres.

[0058] Before the PLGA@LA nanoparticle dispersion solution is added into the chitosan solution, the PLGA@LA nanoparticle dispersion solution is fully stirred to obtain a uniform dispersion. To avoid violent reaction, a dropwise addition method is adopted, and gentle stirring is performed to promote uniform mixing. After the dropwise addition is completed, stirring is continued for 2 h. During this period, the state of the mixture can be observed regularly to ensure that no precipitation or stratification occurs.

[0059] In addition, the application provides a use of the above-mentioned oleracein targeted sustained-release nano drug delivery system in the preparation of a dry eye disease drug. In a specific embodiment, the dry eye disease drug is an eye drop.

[0060] The oleracein targeted sustained-release nano drug delivery system of the application, as a dry eye disease drug, slowly releases the drug through diffusion and degradation due to the structure of the polymer, thereby maintaining the drug concentration at a therapeutic level and prolonging the efficacy. Chitosan has good adhesion and can effectively adhere to the ocular surface, thereby prolonging the drug residence time and reducing the need for frequent drug administration. The positively charged nanoparticles have strong adsorption capacity on the ocular surface because the ocular tissue surface is usually negatively charged. The positively charged nanoparticles are combined with the cell membrane, glycosaminoglycans and other components of the ocular surface through electrostatic interaction, thereby increasing the residence time of the drug on the ocular surface and reducing the loss of the drug. This electrostatic adsorption helps to slowly release the drug, so that the local concentration of the drug in the eye can be maintained for a long time.

[0061] The oleracein targeted sustained-release nano drug delivery system of the application, as a dry eye disease drug, has a mitochondrion protection function. According to the results of the mouse cornea, it can be observed that after drug treatment, the morphology of the mitochondria in the corneal epithelial cells is obviously improved, the structure of the mitochondria tends to be normal, the membrane integrity is restored, and the swelling and lysis phenomenon is reduced. This indicates that the drug can reduce the excessive generation of reactive oxygen species (ROS) by reducing mitochondrial damage, thereby reducing the oxidative stress triggered by mitochondrial damage. The recovery of mitochondrial function helps to reduce the activation of inflammasomes and the excessive release of cytokines (such as IL-1β, TNF-α, etc.), thereby effectively inhibiting the local inflammatory response. Therefore, the drug can effectively control the inflammatory response and promote the repair and recovery of the cornea by improving the health status of the mitochondria.

[0062] The following is further illustrated by specific embodiments.

[0063] Example 1

[0064] A preparation method of a glycoside targeting sustained-release nano drug delivery system, comprising the following steps:

[0065] S1, 1 mg of LA and 5 mg of PLGA were dissolved in 500 μL of DMSO, and ultrasonic treatment was carried out at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0066] S2, 0.045 g of PVA powder was weighed and added to 4.5 mL of distilled water, and stirred until completely dissolved to obtain a clear and particle-free PVA solution.

[0067] S3, the stirring speed was set to 400 rpm, the LA / PLGA mixed solution was slowly added to the PVA solution, after the addition was completed, the mixture was continuously stirred at a speed of 400 rpm for 2 min, and then the mixture was transferred to a 10KDa dialysis bag, the dialysis bag was placed in a container filled with ultrapure water, and the dialysis bag was completely immersed in the buffer solution, and dialysis was carried out overnight to obtain a PLGA@LANPs dispersion solution.

[0068] S4, 1 mL of PLGA@LANPs dispersion solution was taken, and 0.01 mL of CTS solution with a concentration of 10 mg / mL was slowly added to 1 mL of PLGA@LANPs solution, and stirring was carried out at a speed of 200 rpm at room temperature to ensure uniform distribution of CTS on the surface of PLGA@LA NPs, and the stirring was continued for 2 h, and the reaction was completed to form coated composite drug-loaded microspheres, i.e. glycoside targeting sustained-release nano drug delivery system, named CTS@PLGA@LANPs.

[0069] Example 2

[0070] A preparation method of a glycoside targeting sustained-release nano drug delivery system, comprising the following steps:

[0071] S1, 1 mg of LA and 5 mg of PLGA were dissolved in 500 μL of DMSO, and ultrasonic treatment was carried out at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0072] S2, 0.045 g of PVA powder was weighed and added to 4.5 mL of distilled water, and stirred until completely dissolved to obtain a clear and particle-free PVA solution.

[0073] S3, set the stirring speed to 500 rpm, slowly drop the LA / PLGA mixed solution into the PVA solution, after the drop is completed, continue to stir the mixture at a speed of 500 rpm for 2 min, after the stirring is completed, transfer the mixture to a dialysis bag with a molecular weight of 15 KDa, place the dialysis bag in a container filled with ultrapure water, ensure that the dialysis bag is completely immersed in the buffer, dialyze overnight to obtain a PLGA@LANPs dispersion solution.

[0074] S4, take 3 mL of the PLGA@LANPs dispersion solution, stir thoroughly to obtain a uniform dispersion, slowly drop 0.02 mL of a CTS solution with a concentration of 10 mg / mL into 3 mL of the PLGA@LANPs solution, stir at a speed of 400 rpm at room temperature, ensure that the CTS is uniformly distributed on the surface of the PLGA@LANPs, continue to stir for 3 h, after the reaction is completed, form coated composite drug-loaded microspheres, i.e. obtain a plumbagin targeted slow-release nanodrug system, named CTS@PLGA@LANPs.

[0075] Example 3

[0076] A preparation method of a plumbagin targeted slow-release nanodrug system, comprising the following steps:

[0077] S1, dissolve 1 mg of LA and 4 mg of PLGA in 500 μL of DMSO, ultrasonicate in a water bath at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0078] S2, weigh 0.054 g of PVA powder, add 4.5 mL of distilled water, stir until completely dissolved to obtain a clear and particle-free PVA solution.

[0079] S3, set the stirring speed to 300 rpm, slowly drop the LA / PLGA mixed solution into the PVA solution, after the drop is completed, continue to stir the mixture at a speed of 300 rpm for 3 min, after the stirring is completed, transfer the mixture to a dialysis bag with a molecular weight of 5 KDa, place the dialysis bag in a container filled with ultrapure water, ensure that the dialysis bag is completely immersed in the buffer, dialyze overnight to obtain a PLGA@LANPs dispersion solution.

[0080] S4, take 2 mL of the PLGA@LANPs dispersion solution, stir thoroughly to obtain a uniform dispersion, slowly drop 0.02 mL of a CTS solution with a concentration of 8 mg / mL into 1 mL of the PLGA@LANPs solution, stir at a speed of 350 rpm at room temperature, ensure that the CTS is uniformly distributed on the surface of the PLGA@LANPs, continue to stir for 3 h, after the reaction is completed, form coated composite drug-loaded microspheres, i.e. obtain a plumbagin targeted slow-release nanodrug system, named CTS@PLGA@LANPs.

[0081] Comparative Example 1

[0082] A preparation method of a plumbagin targeted sustained-release nano drug delivery system, comprising the following steps:

[0083] S1, 1 mg of LA and 5 mg of PLGA were dissolved in 500 μL of DMSO, and ultrasonic treatment was carried out at 37℃ for 5 min to obtain a uniform LA / PLGA mixed solution.

[0084] S2, 0.045 g of PVA powder was weighed and added to 4.5 mL of distilled water, and stirred until completely dissolved to obtain a clear and particle-free PVA solution.

[0085] S3, the stirring speed was set to 400 rpm, and the LA / PLGA mixed solution was slowly added to the PVA solution, after the addition was completed, the mixture was continuously stirred at a speed of 400 rpm for 2 min, and then the mixture was transferred to a 10 KDa dialysis bag, the dialysis bag was placed in a container filled with ultrapure water, and the dialysis bag was completely immersed in the buffer solution, and dialysis was carried out overnight to obtain a PLGA@LANPs dispersion solution.

[0086] The CTS@PLGA@LANPs prepared in Examples 1-3 have similar structures and effects, and the CTS@PLGA@LANPs prepared in Example 1 are further described.

[0087] The morphology and structure of the CTS@PLGA@LANPs prepared in Example 1 and the PLGA@LANPs dispersion solution of Comparative Example 1 were tested, and the results are as follows:

[0088] Figure 1 The transmission morphology of the PLGANPs and CTS@PLGA@LANPs of Example 1 is shown in the following figure: Figure 1 A is the transmission morphology of the PLGANPs, and the small graph in the upper left corner of A is an enlarged view of the nanoparticles in A, B is the transmission morphology of the CTS@PLGA@LANPs, and the small graph in the upper left corner of B is an enlarged view of the nanoparticles in B. Figure 1 As shown in the figure, the PLGANPs provided by Example 1 have good particle size uniformity, and the particle size distribution is 150 nm-250 nm. The CTS@PLGA@LANPs are further optimized to be 80 nm-120 nm, and have good uniformity.

[0089] The standard curve was obtained by determining the absorbance of CTS@PLGA@LANPs standard solution with different concentrations (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL), plotting the scatter plot of concentration and response value, and performing linear regression analysis. The entrapment efficiency (EE) was calculated according to the following formula: Entrapment efficiency (EE, %) = (amount of encapsulated drug / total amount of drug) x 100%.

[0090] Figure 2 The encapsulation efficiency curve of CTS@PLGA@LANPs in Example 1 of the present application is shown in FIG. 1. As shown in FIG. 1, the encapsulation efficiency of CTS@PLGA@LANPs was calculated to be 68.22%. Figure 2

[0091] The PLGANPs and CTS@PLGA@LANPs samples were dispersed in deionized water, and the concentration was adjusted to 30 μg / mL-50 μg / mL for particle size analysis. The average particle size and its distribution range were calculated. All measurements were performed at 25°C, and repeated three times to ensure the accuracy and repeatability of the data. Figure 3 The particle size data of PLGANPs and CTS@PLGA@LANPs in Example 1 of the present application are shown in FIG. 2. As shown in FIG. 2, the particle size of PLGANPs was 123.7 ± 11.40 nm, and the particle size of CTS@PLGA@LANPs was 140.53 ± 3.11 nm. As can be seen, the particle size of CTS@PLGA@LANPs was larger than that of PLGANPs, indicating that the surface modification of CTS on the basis of PLGANPs resulted in an increase in particle size. This increase may be related to the distribution of the chitosan layer on the surface of the nanoparticles. The change in particle size can affect the drug loading, release characteristics and distribution of the nanoparticles in the body, indicating that CTS@PLGA@LANPs have potential improvement effects for improving the stability and biocompatibility of the drug delivery system. Figure 3 The PLGANPs and CTS@PLGA@LANPs samples were dispersed in appropriate solvents, respectively, to ensure that the concentration of the solution met the measurement requirements. Then, the dispersion was placed in the sample cell of the potentiometer, and the potential value of the sample was measured. During the test, the instrument calculated the Zeta potential of the sample by electrophoretic mobility speed. All measurements were performed at room temperature, and each sample was measured at least three times to ensure the accuracy and consistency of the results.

[0092] The potential data of PLGANPs and CTS@PLGA@LANPs provided in Example 1 of the present application are shown in FIG. 3. As shown in FIG. 3, the Zeta potential of PLGANPs was - 32.6 ± 0.6 mV, and the Zeta potential of CTS@PLGA@LANPs was - 22.6 ± 0.6 mV. As can be seen, the Zeta potential of CTS@PLGA@LANPs was smaller than that of PLGANPs, indicating that the surface modification of CTS on the basis of PLGANPs resulted in a decrease in Zeta potential. This decrease may be related to the distribution of the chitosan layer on the surface of the nanoparticles. The change in Zeta potential can affect the drug loading, release characteristics and distribution of the nanoparticles in the body, indicating that CTS@PLGA@LANPs have potential improvement effects for improving the stability and biocompatibility of the drug delivery system. Figure 4 Figure 4 ​​As shown, the Zeta potential of PLGA NPs was -29.8 ± 0.44 mV, while that of CTS@PLGA@LANPs was 19.47 ± 1.15 mV. The results indicate that the potential of CTS@PLGA@LANPs is positive, suggesting that the surface charge changed from negative to positive after CTS modification, possibly due to the positive charge of the amino groups in chitosan. This potential change may improve the biocompatibility of CTS@PLGA@LANPs, making it more conducive to drug adhesion on the ocular surface.

[0093] The release behavior of PLGA@LANPs and CTS@PLGA@LANPs was tested in vitro under simulated pH conditions (pH=5.4 and pH=7.4). First, equal amounts of PLGA@LANPs and CTS@PLGA@LANPs were added to centrifuge tubes containing buffer solutions of different pH values ​​(pH=5.4 and pH=7.4), and incubated in a 37°C constant-temperature shaker. The LA concentration in the supernatant was measured at set time points (e.g., 12h, 24h, 36h, 48h, 60h, 72h).

[0094] Figure 5 This is a release diagram of PLGA@LANPs and CTS@PLGA@LANPs under different environments in Embodiment 1 of the present invention. Figure 5 As shown, the release data of PLGA@LANPs and CTS@PLGA@LANPs under different environments indicate that there are significant differences in the drug release characteristics of PLGA@LANPs and CTS@PLGA@LANPs under different environmental conditions. CTS@PLGA@LANPs exhibits a more stable release pattern compared to PLGA@LANPs, which may be due to the sustained-release effect of the dense layer formed by CTS outside the PLGA matrix, thus prolonging the drug release time. Furthermore, the positive charge of CTS may affect the drug release rate. Overall, the modification of CTS@PLGA@LANPs helps to achieve more sustained and controllable drug release under specific environmental conditions, providing new insights for the optimization of drug delivery systems.

[0095] Disperse CTS@PLGA@LANPs in deionized water and adjust the concentration to 30 μg / mL–50 μg / mL. Transfer the solution to a cuvette in a UV-Vis spectrophotometer, ensuring the cuvette is clean and free of air bubbles. Perform baseline correction to ensure a stable baseline. Set the wavelength scan range (200 nm to 800 nm) and begin measurement, recording the absorbance values ​​of the CTS@PLGA@LANPs solution across the entire wavelength range. Figure 6 This is the UV-Vis absorption spectrum of CTS@PLGA@LANPs in Example 1 of the present invention.Figure 6 As shown, there is a characteristic peak of LA (323 nm) in CTS@PLGA@LANPs.

[0096] Example 1 CTS@PLGA@LANPs were used to investigate the effect on inflammatory cytokines of macrophage THP-1 cells. First, THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to the logarithmic growth phase, and treated with 100 ng / ml PMA for 24 h to induce cell differentiation into macrophage-like morphology. Then, IFN-γ (20 ng / mL) and LPS (100 ng / mL) were used to induce inflammation, and LA, PLGA@LANPs, and CTS@PLGA@LANPs were used for treatment for 24 h, respectively. After treatment, the levels of inflammatory cytokines (TNF-α and IL-1β) in the supernatant were detected by ELISA to analyze the effect of CTS@PLGA@LA nanoparticles on inflammatory cytokines of macrophages. All experiments were repeated three times, and statistical analysis was performed to ensure the reliability of the results. Figure 7 Figure showing the effect of Example 1 CTS@PLGA@LANPs on inflammatory cytokines of macrophage THP-1 cells, wherein, Figure 7 A is a graph showing the change in the level of inflammatory cytokine TNF-α, and B is a graph showing the change in the level of inflammatory cytokine IL-1β. As Figure 7 shown, the levels of inflammatory cytokines in THP-1 macrophages treated with CTS@PLGA@LA NPs changed significantly. Compared with the control group, the secretion of pro-inflammatory cytokines (TNF-α and IL-1β) in the CTS@PLGA@LANPs treatment group was significantly reduced, indicating that CTS@PLGA@LANPs have an inhibitory effect on inflammatory cytokines, and may exert its potential anti-inflammatory effect by regulating the immune function of macrophages.

[0097] The distribution of Ce6, PLGA@Ce6, and CTS@PLGA@Ce6 in the cornea and lacrimal gland of mice was studied by local administration. First, each group of liquid was precisely dropped into the eyes (cornea) of mice by microsyringe, and it was ensured that the particles could fully contact the ocular surface. Then, the cornea and lacrimal gland tissues of the animals were sampled at 12 h, and the fluorescence images of the main organs were captured using system. Figure 8 Figure showing the distribution of Example 1 CTS@PLGA@LANPs in the cornea and lacrimal gland. As Figure 8As shown, the distribution of the fluorescence-labeled CTS@PLGA@LANPs in the cornea and lacrimal gland showed that the distribution of CTS@PLGA@LANPs in the local tissue had obvious targeting. The experimental results showed that CTS@PLGA@LANPs significantly accumulated in the corneal and lacrimal gland tissues and could maintain a long-term local presence. Compared with the control group, the permeability and distribution density of nanoparticles in the corneal epithelial cells and lacrimal glands were significantly increased, indicating that CTS@PLGA@LANPs could effectively penetrate the corneal barrier and accumulate in the lacrimal gland, and had excellent biodistribution characteristics. These results showed that CTS@PLGA@LANPs had a targeting delivery potential in ocular tissues, and were expected to be used for precise treatment of ocular drugs.

[0098] The animal model was established and analyzed according to the ethical requirements of animal experiments. 50 mice were 6-8 week old female C57BL / 6J mice, which were randomly divided into 8 groups. 0.2% benzalkonium chloride solution was used to establish a dry eye mouse model, and treatment was given by ocular surface drug administration for 14 days (Control group; Model group; LA group; PLGA@LA NPs group; CTS@PLGA@LANPs group).

[0099] The histological analysis of corneal tissue was performed by H&E staining method to observe the morphological changes. First, the corneal tissue was taken out after the experimental animals were sacrificed, and was quickly fixed using 10% formalin solution for 24 h. The fixed tissue samples were processed by conventional dehydration, transparency, and paraffin immersion program, and were cut into 5 μm thick tissue sections after embedding in paraffin. The sections were treated by conventional Hematoxylin and Eosin (H&E) staining, first stained with Hematoxylin for 15 min, then stained with Eosin for 5 min, washed and dehydrated, and finally mounted. The stained sections were observed and photographed under a microscope, and the morphological characteristics of the corneal tissue were evaluated.

[0100] Figure 9 The H&E staining diagram of the corneal tissue of the application is shown in FIG. 1. As shown in FIG. 1, CTS@PLGA@LANPs can reduce the loss of corneal epithelial layers, the exfoliation of epidermal upper layer cells, and the disordered arrangement of stromal cells. Figure 9

[0101] The H&E staining diagram of the lacrimal gland tissue of the application is shown in FIG. 2. As shown in FIG. 2, CTS@PLGA@LANPs can promote the enlargement or restore the normal morphology of the lacrimal gland, reduce the inflammatory response, and improve the function of the lacrimal gland. Figure 10 Figure 10 The H&E staining diagram of the lacrimal gland tissue of the application is shown in FIG. 2. As shown in FIG. 2, CTS@PLGA@LANPs can promote the enlargement or restore the normal morphology of the lacrimal gland, reduce the inflammatory response, and improve the function of the lacrimal gland.

[0102] ​The changes of mitochondria in mouse corneal tissue were observed by transmission electron microscopy (TEM). The experimental mice were first sacrificed and the corneal tissue was quickly removed, and the blood and impurities were removed by washing with 0.1M phosphate buffer (PBS). Then, the corneal tissue was placed in a special fixing liquid for electron microscopy, and fixed at 4°C for 24h. The fixed sample was dehydrated by gradient ethanol, then further dehydrated in acetone, and finally embedded in epoxy resin. The tissue sections with a thickness of about 70nm were cut using an ultramicrotome, and the sections were contrasted stained in uranyl lead solution. The stained sections were observed under a transmission electron microscope to analyze the morphological characteristics of mitochondria in corneal tissue. Figure 11 The figure of the changes of mitochondria in mouse corneal tissue of the present application is shown as follows. Figure 11 As shown, after CTS@PLGA@LANPs treatment, the morphology of mitochondria in corneal epithelial cells was obviously improved, the structure of mitochondria tended to be normal, the membrane integrity was restored, and the swelling and lysis phenomenon was reduced. The recovery of mitochondrial function helps to reduce the activation of inflammasomes and the excessive release of cytokines to inhibit local inflammatory response. Therefore, the drug can effectively control the inflammatory response by improving the health status of mitochondria, and promote the repair and recovery of cornea.

[0103] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments of the present application are described in order to prevent redundancy. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0104] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A preparation method of a targeted sustained-release nanocarrier system of oleanolic acid for preparing a dry eye disease drug, characterized in that, The method comprises the following steps: Mixing and dissolving the platinin and polylactic acid-polyglycolic acid copolymer in dimethyl sulfoxide to obtain a mixed solution, adding the mixed solution into a polyvinyl alcohol solution at a stirring speed of 400 rpm to 500 rpm, continuing to stir for 2 min to 3 min after the addition is completed, and then dialyzing overnight at 5 KDa to 15 KDa to obtain a PLGA@LA nanoparticle dispersion solution; At room temperature, adding the chitosan solution into the PLGA@LA nanoparticle dispersion solution at a stirring speed of 200 rpm to 400 rpm to uniformly disperse the chitosan on the surface of the PLGA@LA nanoparticles, continuing to stir for 2 h to 3 h after the addition is completed, forming a coated composite drug-loaded microsphere, and obtaining a platinin targeted sustained-release nano drug delivery system; The mass-volume ratio of the platinin, the polylactic acid-polyglycolic acid copolymer and the dimethyl sulfoxide is 1 mg to 2 mg: 4 mg to 5 mg: 500 μL; The mass-volume ratio of the polylactic acid-polyglycolic acid copolymer and the polyvinyl alcohol solution is 1 mg: 0.9 mL to 1.25 mL, and the concentration of the polyvinyl alcohol solution is 10 mg / mL to 15 mg / mL; The volume ratio of the PLGA@LA nanoparticle dispersion solution and the chitosan solution is 100 to 300: 1, and the concentration of the chitosan solution is 5 mg / mL to 10 mg / mL.

2. The platinin targeted sustained-release nano drug delivery system for preparing a dry eye disease medicine prepared by the preparation method of claim 1.

3. The use of the platinin targeted sustained-release nano drug delivery system of claim 2 in the preparation of a dry eye disease medicine.

4. Use according to claim 3, characterized in that, The dry eye disease medicine is an eye drop.

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