Blinarin targeted slow-release nano drug-loading system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a linarin targeted slow-release nano drug-loading system as well as a preparation method and application thereof. A polylactic acid-polyglycolic acid copolymer is used as a matrix, polyvinyl alcohol is used as a cross-linking agent, and coated drug-loaded microspheres are formed through cross-linking and grafting of the matrix and the cross-linking agent; and then chitosan is used as a protective agent, chitosan and the coated drug-loaded microspheres are crosslinked and grafted to form double-coated composite drug-loaded microspheres, the chitosan in the composite drug-loaded microspheres is used as a shell, the coated drug-loaded microspheres are used as a core, and the linarin targeted slow-release nano drug-loaded system is obtained. Through the double-target design, the targeting property to cornea and lacrimal gland is enhanced, the treatment efficiency of the medicine is improved, stable release and continuous release of linarin are realized, and the problem of unstable release of the medicine is solved; in addition, the targeted slow-release nano drug-loading system is combined with the anti-inflammatory activity and the mitochondrial protection function of linarin, so that the side effects on eyes are reduced, and the safety is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a linalool targeted sustained-release nano drug delivery system and a preparation method and application thereof. Background Art

[0002] Dry Eye Disease (DED) is a common eye disease characterized by insufficient tear secretion or rapid tear evaporation, leading to symptoms such as dry eyes, burning, and foreign body sensation. The occurrence of dry eye is closely related to eye inflammation, corneal epithelial damage, and eye discomfort. Current treatments include the use of artificial tears, anti-inflammatory drugs, and immunosuppressants. However, these methods usually only relieve symptoms but cannot fundamentally solve the cause, and long-term use may cause side effects.

[0003] Linarin (LA) has multiple biological activities such as anti-inflammatory and antioxidant, 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 have shown great potential in the field of drug delivery. These systems can improve the bioavailability of drugs, reduce the side effects of drugs, 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, difficulty in controlling drug release, and side effects in treating specific diseases. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a targeted sustained-release nano drug delivery system of lignocerin and its preparation method and application. The existing nano drug delivery system still has the problems of insufficient targeting, difficult drug release control and side effects, and develops a dual-target, multifunctional lignocerin targeted sustained-release nano drug delivery system to more effectively regulate mitochondrial damage and macrophage polarization by improving targeting, enhancing drug stability and controlling release, thereby reducing the ocular inflammatory response of dry eye disease. This new nanoparticle system is expected to overcome the shortcomings of the existing technology and improve the treatment effect.

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

[0007] The first purpose of the present invention is to provide a targeted sustained-release nano drug delivery system for limonene glycoside, which uses polylactic acid-polyglycolic acid copolymer as a matrix and polyvinyl alcohol as a cross-linking agent, and forms coated drug-loaded microspheres through cross-linking and grafting of the matrix and the cross-linking agent; then uses chitosan as a protective agent, cross-links and grafts chitosan and the coated drug-loaded microspheres to form double-wrapped composite drug-loaded microspheres, wherein the chitosan in the composite drug-loaded microspheres is the shell and the coated drug-loaded microspheres is the core, so as to obtain a targeted sustained-release nano drug delivery system for limonene glycoside, wherein the matrix is ​​loaded with limonene glycoside.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned linaloside targeted sustained-release nano drug delivery system, comprising the following steps:

[0009] S1. Dissolve linalool and polylactic acid-polyglycolic acid copolymer in a solvent and mix them to obtain a mixed solution. Add polyvinyl alcohol solution to the mixed solution and stir to crosslink and graft for nanoprecipitation. After dialysis, prepare a PLGA@LA nanoparticle dispersion solution.

[0010] S2. Add the chitosan solution dropwise into the PLGA@LA nanoparticle dispersion solution, stir at room temperature to evenly disperse the chitosan on the surface of the PLGA@LA nanoparticles, perform cross-linking and grafting to form a coating, and form coated composite drug-loaded microspheres, thereby obtaining a targeted sustained-release nano drug-loading system of linalool.

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

[0012] Furthermore, 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.

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

[0014] Furthermore, 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.

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

[0016] The third object of the present invention is to provide the use of the above-mentioned linalool targeted sustained-release nano drug delivery system in the preparation of dry eye disease drugs.

[0017] Furthermore, the dry eye disease medicine is eye drops.

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

[0019] (1) After the polylactic acid-glycolic acid copolymer is loaded on mongoside, the coated drug-loaded microspheres are formed by crosslinking and grafting with polyvinyl alcohol, and then coated with chitosan to form double-wrapped composite drug-loaded microspheres, which can effectively protect mongoside and prevent it 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, which can reduce the stimulation of the drug to the eye tissue, increase the safety of nanoparticles, reduce side effects, and promote local absorption of the drug, enhance adhesion, sustained release, targeting and bioavailability. Through the dual-target design, the targeting to the cornea and lacrimal gland is enhanced, the therapeutic efficiency of the drug is improved, the stable release and sustained release of mongoside is achieved, and the problem of unstable drug release is overcome; and the targeted sustained-release nano drug delivery system reduces the side effects to the eye and improves safety by combining the anti-inflammatory activity and mitochondrial protection function of mongoside.

[0020] (2) The targeted sustained-release nano drug delivery system of lignocerin prepared by the present invention, firstly, the size of the nano drug delivery system particles is less than 200nm, and this tiny size is conducive to their passing through biological barriers, such as cell membranes, and thus effectively entering the lacrimal glands. It can not only act on the cornea, but also enter the lacrimal glands, thereby achieving dual targeting; secondly, the combination of chitosan and PLGA can achieve controlled release of drugs, that is, the drugs can be slowly released near the lacrimal glands, prolonging their action time, forming controlled release, and this sustained-release mechanism can improve the bioavailability of drugs, so that the concentration of drugs in the targeted area is maintained within an effective range; finally, due to the physiological characteristics of the lacrimal glands, these nanoparticles can be transported to the lacrimal glands in the flow of tears, promoting the distribution of drugs. This makes it easier for the encapsulated drugs to reach the target area, and chitosan has cationic properties, which can electrostatically interact with the anionic sites on the lacrimal gland cell membranes, thereby enhancing cell uptake. This interaction helps the nanoparticles to more effectively penetrate the cell membrane and enter the lacrimal glands.

[0021] (3) The preparation method of the montmorillonine targeted sustained-release nano-drug delivery system of the present invention has a simple preparation process and is convenient for large-scale industrial production. At the same time, the montmorillonine targeted sustained-release nano-drug delivery system with targeted properties, good long-term stability and a long sustained release time is prepared, and has excellent biocompatibility and biodegradability, significantly improving the survival rate of corneal epithelial cells. The operation is simple, the cost is low and the repeatability is good.

[0022] (4) The targeted sustained-release nano drug delivery system of the present invention is used as a drug for dry eye disease. Through the structure of the polymer, the drug is slowly released through diffusion and degradation, thereby maintaining the drug concentration at the therapeutic level and prolonging the therapeutic effect; chitosan has good adhesion and can effectively adhere to the surface of the eye, prolonging the drug residence time and reducing the need for frequent administration. The positively charged nanoparticles have a strong adsorption capacity on the ocular surface because the surface of the ocular tissue is usually negatively charged. The positively charged nanoparticles bind to the cell membrane, glycosaminoglycans and other components of the ocular surface through electrostatic action, thereby increasing 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 and maintain the local concentration of the drug in the eye for a longer time.

[0023] (5) The targeted sustained-release nano drug delivery system of the present invention is a drug for dry eye disease, which has mitochondrial protection function. According to the results of mouse cornea, it can be observed that after drug treatment, the mitochondrial morphology in corneal epithelial cells is significantly improved, the mitochondrial structure tends to be normal, the membrane integrity is restored, and the swelling and lysis phenomena are reduced. This shows that the drug may reduce the oxidative stress caused by mitochondrial damage by reducing mitochondrial damage and excessive generation of reactive oxygen species (ROS). The restoration 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 of mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a transmission morphology image of PLGANPs and CTS@PLGA@LANPs in Example 1 of the present invention, wherein: Figure 1 A in the figure is a transmission morphology image of PLGANPs, and the small image in the upper left corner of A is an enlarged image of the nanoparticles in Figure A. B is a transmission morphology image of CTS@PLGA@LANPs, and the small image in the upper left corner of B is an enlarged image of the nanoparticles in Figure B.

[0025] Figure 2 This is the encapsulation efficiency curve of CTS@PLGA@LANPs in Example 1 of the present invention.

[0026] Figure 3This is a graph showing the particle size data of PLGANPs and CTS@PLGA@LANPs in Example 1 of the present invention.

[0027] Figure 4 This is a potential data diagram of PLGANPs and CTS@PLGA@LANPs provided in Example 1 of the present invention.

[0028] Figure 5 This is the release diagram of PLGA@LANPs and CTS@PLGA@LANPs under different environments in Example 1 of the present invention.

[0029] Figure 6 This is the UV-visible absorption spectrum of CTS@PLGA@LANPs in Example 1 of the present invention.

[0030] Figure 7 This is a graph showing the effect of CTS@PLGA@LANPs on inflammatory factors in macrophage THP-1 cells in Example 1 of the present invention, wherein: Figure 7 A in the figure is a graph showing changes in the levels of the inflammatory factor TNF-α, and B is a graph showing changes in the levels of the inflammatory factor IL-1β.

[0031] Figure 8 This is the distribution diagram of CTS@PLGA@LANPs in the cornea and lacrimal gland of Example 1 of the present invention.

[0032] Fig. 9 This is the H&E staining picture of the corneal tissue of the present invention.

[0033] Fig.10 This is the H&E staining picture of the lacrimal gland tissue of the present invention.

[0034] Fig.11 This is a diagram showing the mitochondrial changes in the mouse corneal tissue of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0037] Linarin (LA) has multiple biological activities such as anti-inflammatory and antioxidant, and has potential therapeutic effects on mitochondrial damage 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 when treating specific diseases:

[0039] Insufficient targeting: Existing nano-drug delivery systems such as PLGA nanoparticles have limited targeting in the treatment of eye diseases and are difficult to accurately reach the site of corneal tear gland lesions. Drug stability and release control: The release rate and stability of drugs in the body are difficult to accurately control, which affects the sustainability of the treatment effect. Side effect issues: Long-term use of anti-inflammatory drugs and immunosuppressants may cause side effects, such as eye infections and aggravation of dry eye.

[0040] Based on the above problems, the present invention provides a leucoside targeted sustained-release nano drug delivery system, which uses polylactic acid-polyglycolic acid copolymer as a matrix and polyvinyl alcohol as a cross-linking agent, and forms coated drug-loaded microspheres by cross-linking and grafting the matrix and the cross-linking agent; then, using chitosan as a protective agent, the chitosan and the coated drug-loaded microspheres are cross-linked and grafted to form double-wrapped composite drug-loaded microspheres, wherein the chitosan in the composite drug-loaded microspheres is the shell and the coated drug-loaded microspheres is the core, so as to obtain a leucoside targeted sustained-release nano drug delivery system, wherein the matrix is ​​loaded with leucoside.

[0041] After LA is loaded with PLGA, polyvinyl alcohol (PVA) is cross-linked and grafted to form coated drug-loaded microspheres, which are then coated with chitosan (CTS) to form double-wrapped composite drug-loaded microspheres, which can effectively protect LA and prevent it from being degraded or inactivated in the body environment; wherein the core layer of PLGA can provide a stable drug carrier, and the outer layer of chitosan further enhances the protective effect, ensuring that the drug remains stable before release, which can reduce the stimulation of the drug to eye tissues, increase the safety of nanoparticles, reduce side effects, and promote local absorption of the drug, and enhance adhesion, sustained release, targeting and bioavailability.

[0042] The present invention enhances the targeting to the cornea and lacrimal glands through a dual-target design, improves the therapeutic efficiency of the drug, achieves stable and sustained release of lignoceramide, and overcomes the problem of unstable drug release; and the targeted sustained-release nano-drug delivery system reduces side effects on the eyes and improves safety by combining the anti-inflammatory activity and mitochondrial protection function of lignoceramide.

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

[0044] S1. Dissolve linalool and polylactic acid-polyglycolic acid copolymer in a solvent and mix them to obtain a mixed solution. Add polyvinyl alcohol solution to the mixed solution and stir to crosslink and graft for nanoprecipitation. After dialysis, prepare a PLGA@LA nanoparticle dispersion solution.

[0045] S2. Add the chitosan solution dropwise into the PLGA@LA nanoparticle dispersion solution, stir at room temperature to evenly disperse the chitosan on the surface of the PLGA@LA nanoparticles, perform cross-linking and grafting to form a coating, and form coated composite drug-loaded microspheres, thereby obtaining a targeted sustained-release nano drug-loading system of linalool.

[0046] The preparation method of the limonene glycoside targeted sustained-release nano drug delivery system of the present invention has a simple preparation process and is convenient for large-scale industrial production. At the same time, the limonene glycoside targeted sustained-release nano drug delivery system with targeting, good long-term stability and long sustained release time is prepared, and has 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 targeted sustained-release nano drug delivery system of mongoside has the following characteristics: first, the size of the nano drug delivery system particles is less than 200nm, and this tiny size is conducive to their passing through biological barriers, such as cell membranes, and thus effectively entering the lacrimal glands. It can not only act on the cornea, but also enter the lacrimal glands, thereby achieving dual targeting; secondly, the combination of chitosan and PLGA can achieve controlled release of drugs, that is, the drugs can be slowly released near the lacrimal glands, prolonging their action time and forming controlled release. This sustained-release mechanism can improve the bioavailability of drugs and keep the concentration of drugs in the targeted area within an effective range; finally, due to the physiological characteristics of the lacrimal glands, these nanoparticles can be transported to the lacrimal glands in the flow of tears, promoting the distribution of drugs. This makes it easier for the encapsulated drugs to reach the target area, and chitosan has cationic properties, which can electrostatically interact with the anionic sites on the lacrimal gland cell membranes, thereby enhancing cell uptake. This interaction helps the nanoparticles to penetrate the cell membrane more effectively and enter the lacrimal glands.

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

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

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

[0051] The solvent used only needs to ensure that the lignocerin and the polylactic acid-polyglycolic acid copolymer are completely dissolved. Preferably, the solvent is dimethyl sulfoxide (DMSO), and the specific mixing method is to perform water bath ultrasound at 37°C until they are completely dissolved.

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

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

[0054] The dripping method is used to ensure the uniformity of the reaction of polylactic acid-polyglycolic acid copolymer and polyvinyl alcohol to prevent local oversaturation. During the dripping process, it is necessary to observe the mixing state to ensure that there is no precipitation or stratification. After the dripping is completed, the mixed liquid is stirred at a speed of 400rpm to 500rpm for 2min to 3min. This step helps to form preliminary nanoparticles and ensure uniformity.

[0055] Preferably, the molecular weight used for overnight dialysis is 5 KDa, 10 KDa or 15 KDa. During overnight dialysis, 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 to 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 to the chitosan solution is 100:1, and the concentration of the chitosan solution is 10 mg / mL.

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

[0058] Before adding the PLGA@LA nanoparticle dispersion solution to the chitosan solution, the PLGA@LA nanoparticle dispersion solution was fully stirred to obtain a uniform dispersion. In order to avoid violent reactions, the dropwise addition method was adopted, and gentle stirring was performed to promote uniform mixing. After the dropwise addition, stirring was continued for 2 h. During this period, the state of the mixture could be regularly observed to ensure that no precipitation or stratification occurred.

[0059] In addition, the present invention provides the use of the above-mentioned linalool targeted sustained-release nano drug delivery system in the preparation of dry eye disease drugs. In a specific embodiment, the dry eye disease drug is eye drops.

[0060] The targeted sustained-release nano drug delivery system of the present invention is used as a drug for dry eye disease. The drug is slowly released through diffusion and degradation through the structure of the polymer, thereby maintaining the drug concentration at the therapeutic level and prolonging the therapeutic effect; chitosan has good adhesion and can effectively adhere to the surface of the eye, prolonging the drug residence time and reducing the need for frequent administration. The positively charged nanoparticles have a strong adsorption capacity on the ocular surface because the surface of the ocular tissue is usually negatively charged. The positively charged nanoparticles are combined with the cell membrane, glycosaminoglycan and other components of the ocular surface through electrostatic action, thereby increasing 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 and maintain the local concentration of the drug in the eye for a long time.

[0061] The targeted sustained-release nano drug delivery system of the present invention is a drug for dry eye disease, which has mitochondrial protection function. According to the results of mouse cornea, it can be observed that after drug treatment, the mitochondrial morphology in corneal epithelial cells is significantly improved, the structure of mitochondria tends to be normal, the membrane integrity is restored, and the swelling and lysis phenomena are reduced. This shows that the drug may reduce the oxidative stress caused by mitochondrial damage by reducing the excessive generation of reactive oxygen species (ROS). The restoration 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 of mitochondria.

[0062] The invention is further described below through specific examples.

[0063] Example 1

[0064] A method for preparing a targeted sustained-release nano drug delivery system of linalool comprises the following steps:

[0065] S1. Dissolve 1 mg of LA and 5 mg of PLGA in 500 μL of DMSO and ultrasonicate in a water bath at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0066] S2. Weigh 0.045 g of PVA powder, add 4.5 mL of distilled water, and stir until completely dissolved to obtain a clear and particle-free PVA solution.

[0067] S3. Set the stirring speed to 400 rpm, and slowly drop the LA / PLGA mixed solution into the PVA solution. After the addition is completed, continue to stir the mixed solution at 400 rpm for 2 minutes. After the stirring is completed, transfer the mixed solution to a 10 KDa dialysis bag, and place the dialysis bag in a container filled with ultrapure water to ensure that the dialysis bag is completely immersed in the buffer solution. Perform dialysis overnight to obtain a PLGA@LANPs dispersed solution.

[0068] S4. Take 1 mL of PLGA@LANPs dispersion solution and stir it thoroughly to obtain a uniform dispersion. Slowly add 0.01 mL of 10 mg / mL CTS solution into 1 mL of PLGA@LANPs solution. Stir at 200 rpm at room temperature to ensure that CTS is evenly distributed on the surface of PLGA@LA NPs. Continue stirring for 2 h. After the reaction is completed, coated composite drug-loaded microspheres are formed, and the targeted sustained-release nano-drug delivery system of glycosides is obtained, which is named CTS@PLGA@LANPs.

[0069] Example 2

[0070] A method for preparing a targeted sustained-release nano drug delivery system of linalool comprises the following steps:

[0071] S1. Dissolve 2 mg of LA and 5 mg of PLGA in 500 μL of DMSO and ultrasonicate in a water bath at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0072] S2. Weigh 0.045 g of PVA powder, add 3 mL of distilled water, and stir until completely dissolved to obtain a clear and particle-free PVA solution.

[0073] S3. Set the stirring speed to 500 rpm, and slowly add the LA / PLGA mixed solution to the PVA solution. After the addition is completed, continue to stir the mixed solution at a speed of 500 rpm for 2 minutes. After the stirring is completed, transfer the mixed solution to a 15 KDa dialysis bag, and place the dialysis bag in a container filled with ultrapure water to ensure that the dialysis bag is completely immersed in the buffer solution. Perform dialysis overnight to obtain a PLGA@LANPs dispersed solution.

[0074] S4. Take 3 mL of PLGA@LANPs dispersion solution and stir it thoroughly to obtain a uniform dispersion. Slowly add 0.02 mL of 10 mg / mL CTS solution into 3 mL of PLGA@LANPs solution. Stir at 400 rpm at room temperature to ensure that CTS is evenly distributed on the surface of PLGA@LA NPs. Continue stirring for 3 h. After the reaction is completed, coated composite drug-loaded microspheres are formed, and the targeted sustained-release nano-drug delivery system of glycosides is obtained, which is named CTS@PLGA@LANPs.

[0075] Example 3

[0076] A method for preparing a targeted sustained-release nano drug delivery system of linalool comprises the following steps:

[0077] S1. Dissolve 1 mg of LA and 4 mg of PLGA in 500 μL of DMSO and 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, and stir until completely dissolved to obtain a clear and particle-free PVA solution.

[0079] S3. Set the stirring speed to 300 rpm, and slowly drop the LA / PLGA mixed solution into the PVA solution. After the addition is completed, continue to stir the mixed solution at 300 rpm for 3 minutes. After the stirring is completed, transfer the mixed solution to a 5 KDa dialysis bag, and place the dialysis bag in a container filled with ultrapure water to ensure that the dialysis bag is completely immersed in the buffer solution. Perform dialysis overnight to obtain a PLGA@LANPs dispersed solution.

[0080] S4. Take 2 mL of PLGA@LANPs dispersion solution and stir it thoroughly to obtain a uniform dispersion. Slowly add 0.02 mL of 8 mg / mL CTS solution into 1 mL of PLGA@LANPs solution. Stir at 350 rpm at room temperature to ensure that CTS is evenly distributed on the surface of PLGA@LA NPs. Continue stirring for 3 h. After the reaction is completed, coated composite drug-loaded microspheres are formed, and the targeted sustained-release nano-drug delivery system of glycosides is obtained, which is named CTS@PLGA@LANPs.

[0081] Comparative Example 1

[0082] A method for preparing a targeted sustained-release nano drug delivery system of linalool comprises the following steps:

[0083] S1. Dissolve 1 mg of LA and 5 mg of PLGA in 500 μL of DMSO and ultrasonicate in a water bath at 37°C for 5 min to obtain a uniform LA / PLGA mixed solution.

[0084] S2. Weigh 0.045 g of PVA powder, add 4.5 mL of distilled water, and stir until completely dissolved to obtain a clear and particle-free PVA solution.

[0085] S3. Set the stirring speed to 400 rpm, and slowly drop the LA / PLGA mixed solution into the PVA solution. After the addition is completed, continue to stir the mixed solution at 400 rpm for 2 minutes. After the stirring is completed, transfer the mixed solution to a 10 KDa dialysis bag, and place the dialysis bag in a container filled with ultrapure water to ensure that the dialysis bag is completely immersed in the buffer solution. Perform dialysis overnight to obtain a PLGA@LANPs dispersed solution.

[0086] The CTS@PLGA@LANPs prepared in Examples 1 to 3 have similar structures and similar effects, and the CTS@PLGA@LANPs prepared in Example 1 is used for further explanation.

[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 This is a transmission morphology image of PLGANPs and CTS@PLGA@LANPs in Example 1 of the present invention, wherein: Figure 1 A in the figure is a transmission morphology image of PLGANPs, and the small image in the upper left corner of A is an enlarged image of the nanoparticles in Figure A. B is a transmission morphology image of CTS@PLGA@LANPs, and the small image in the upper left corner of B is an enlarged image of the nanoparticles in Figure B. Figure 1 As shown, the particle size uniformity of PLGANPs provided in Example 1 is good, and the particle size distribution is 150nm to 250nm. CTS@PLGA@LA NPs are more preferably 80nm to 120nm, and the uniformity is relatively good.

[0089] By measuring the absorbance of standard solutions of different concentrations of CTS@PLGA@LANPs (5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, 25μg / mL, 30μg / mL), a scatter plot of concentration and response value was drawn, and a linear regression analysis was performed to obtain a standard curve. The encapsulation efficiency (EE, %) was calculated according to the following formula: Entrapment efficiency (EE, %) = (encapsulated drug amount / total drug amount) × 100%.

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

[0091] PLGANPs and CTS@PLGA@LANPs samples were dispersed in deionized water and the concentration was adjusted to 30μg / mL~50μg / 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 Figure 1 is a particle size data diagram of PLGANPs and CTS@PLGA@LANPs in Example 1 of the present invention. Figure 3 As shown in the figure, the particle size of PLGANPs is 123.7±11.40nm, while the particle size of CTS@PLGA@LANPs is 140.53±3.11nm. It can be seen that the particle size of CTS@PLGA@LANPs is larger than that of PLGANPs, indicating that the surface modification of PLGANPs by CTS has led to an increase in particle size. This increase may be related to the distribution of the chitosan layer attached to the surface of the nanoparticles. The change in particle size may affect the drug loading, release characteristics and distribution of the nanoparticles in the body, indicating that CTS@PLGA@LANPs has a potential improvement effect for improving the stability and biocompatibility of drug delivery systems.

[0092] The PLGANPs and CTS@PLGA@LANPs samples were dispersed in appropriate solvents to ensure that the concentration of the solution met the measurement requirements. Then, the dispersion was placed in the sample cell of the potential meter to measure the potential value of the sample. During the test, the instrument calculated the Zeta potential of the sample by the electrophoretic migration 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. Figure 4 This is a potential data diagram of PLGANPs and CTS@PLGA@LANPs provided in Example 1 of the present invention. Figure 4As shown in the figure, the Zeta potential of PLGA NPs is -29.8±0.44mV, while that of CTS@PLGA@LANPs is 19.47±1.15mV. The results show that the potential of CTS@PLGA@LANPs is positive, indicating that the surface charge changes from negative to positive after the surface is modified by CTS, which may be due to the positive charge of the amino group of chitosan. This change in potential may improve the biocompatibility of CTS@PLGA@LANPs, which is more conducive to the adhesion of drugs on the ocular surface.

[0093] The release behavior of PLGA@LANPs and CTS@PLGA@LANPs was tested by simulating different pH environments (pH=5.4 and pH=7.4) in vitro. 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), respectively, and incubated in a 37°C constant temperature oscillator. At set time points (such as 12h, 24h, 36h, 48h, 60h, 72h, etc.), the LA concentration in the supernatant was measured.

[0094] Figure 5 The release diagrams of PLGA@LANPs and CTS@PLGA@LANPs under different environments in Example 1 of the present invention are shown in FIG. Figure 5 As shown in the figure, 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 showed a more stable release pattern compared with PLGA@LANPs, which may be due to the fact that the dense layer formed by CTS outside the PLGA matrix has a sustained release effect on drug release, thereby prolonging the drug release time. In addition, the positive charge of CTS may affect the release rate of the drug. Overall, the modification of CTS@PLGA@LANPs helps to achieve more sustained and controllable drug release under specific environmental conditions, providing new ideas for the optimization of drug delivery systems.

[0095] Disperse CTS@PLGA@LA NPs in deionized water and adjust the concentration to 30 μg / mL to 50 μg / mL. Transfer the solution to the cuvette of the UV-Vis spectrophotometer, making sure that the cuvette is clean and free of bubbles. Perform baseline calibration to ensure that the baseline is stable. Set the wavelength scan range (200 nm to 800 nm) and start measuring, recording the absorbance value of the CTS@PLGA@LANPs solution over the entire wavelength range. Figure 6 This is the UV-visible 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 was used for macrophage THP-1 cell inflammatory factors. 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 100ng / ml PMA was used to treat for 24h to induce cell differentiation into a macrophage-like morphology. Then, IFN-γ (20ng / mL) and LPS (100ng / mL) were induced as an inflammatory model, and LA, PLGA@LANPs, and CTS@PLGA@LANPs were treated for 24h, respectively. After treatment, the levels of inflammatory factors (TNF-α and IL-1β) in the supernatant were detected by ELISA, and the effects of CTS@PLGA@LA nanoparticles on macrophage inflammatory factors were analyzed. All experiments were repeated three times and statistically analyzed to ensure the reliability of the results. Figure 7 This is a graph showing the effect of CTS@PLGA@LANPs on inflammatory factors in macrophage THP-1 cells in Example 1 of the present invention, wherein: Figure 7 A in the figure is the level change diagram of the inflammatory factor TNF-α, and B is the level change diagram of the inflammatory factor IL-1β. Figure 7 As shown in the figure, the levels of inflammatory factors in THP-1 macrophages treated with CTS@PLGA@LA NPs changed significantly. Compared with the control group, the secretion of pro-inflammatory factors (TNF-α and IL-1β) in the CTS@PLGA@LANPs-treated group was significantly reduced, indicating that CTS@PLGA@LANPs has the effect of inhibiting inflammatory factors and may exert its potential anti-inflammatory effect by regulating the immune function of macrophages.

[0097] Ce6, PLGA@Ce6, and CTS@PLGA@Ce6 were topically applied to mice to study their distribution in the cornea and lacrimal glands. First, each group of liquid was precisely dripped into the mouse eye (cornea) using a microsyringe, and the particles were ensured to fully contact the ocular surface. Then, the cornea and lacrimal gland tissues of the animals were sampled 12 hours later. The system captures fluorescent images of major organs. Figure 8 The distribution diagram of CTS@PLGA@LANPs in cornea and lacrimal gland of Example 1 of the present invention is shown in FIG. Figure 8As shown in the figure, the distribution study of fluorescently labeled CTS@PLGA@LANPs in the cornea and lacrimal glands showed that the distribution of CTS@PLGA@LA NPs in local tissues has obvious targeting. The experimental results showed that CTS@PLGA@LANPs accumulated significantly in the cornea and lacrimal gland tissues and were able to maintain local presence for a long time. Compared with the control group, the permeability and distribution density of nanoparticles in corneal epithelial cells and lacrimal glands increased significantly, indicating that CTS@PLGA@LANPs can effectively penetrate the corneal barrier and accumulate in the lacrimal glands, with excellent biodistribution characteristics. These results show the targeted delivery potential of CTS@PLGA@LANPs in ocular tissues, which is expected to be used for the precision treatment of ocular drugs.

[0098] Animal models were established and analyzed according to the ethical requirements of animal experiments. Fifty female C57BL / 6J mice aged 6-8 weeks were randomly divided into 8 groups, and 0.2% benzalkonium chloride solution was dripped into the eyes to establish a dry eye mouse model, and the treatment was given by ocular surface drops for 14 days (Control group; Model group; LA group; PLGA@LA NPs group; CTS@PLGA@LANPs group).

[0099] The corneal tissue was histologically analyzed by H&E staining to observe its morphological changes. First, the corneal tissue was removed after the experimental animals were killed, and it was quickly fixed and fixed with 10% formalin solution for 24 hours. The fixed tissue samples were processed by conventional dehydration, transparency, and wax immersion procedures, and then embedded in paraffin and cut into 5μm thick tissue sections. The sections were processed by conventional Hematoxylin and Eosin (H&E) staining, first stained with hematoxylin for 15 minutes, then stained with eosin for 5 minutes, washed and dehydrated, and finally sealed. The stained sections were observed and photographed under a microscope to evaluate the morphological characteristics of the corneal tissue.

[0100] Fig. 9 This is the H&E staining picture of the corneal tissue of the present invention. Fig. 9 As shown, CTS@PLGA@LANPs can alleviate the loss of corneal epithelial layers, shedding of upper epidermal cells, disordered arrangement of stromal cells, and edema.

[0101] Fig.10 This is the H&E staining picture of the lacrimal gland tissue of the present invention. Fig.10 As shown in the results, CTS@PLGA@LA NPs may promote the enlargement of the lacrimal gland or restore its normal morphology, reduce the inflammatory response, and improve the function of the lacrimal gland.

[0102] Transmission electron microscopy (TEM) was used to observe the changes of mitochondria in mouse corneal tissue. The experimental mice were first killed and the corneal tissue was quickly removed, and 0.1M phosphate buffered saline (PBS) was used to wash and remove blood and impurities. Then, the corneal tissue was placed in a special fixative for electron microscopy and fixed at 4°C for 24 hours. The fixed samples were dehydrated with gradient ethanol, then further dehydrated in acetone, and finally embedded in epoxy resin. An ultrathin slicer was used to cut tissue sections of about 70nm thickness, and the sections were contrast stained in uranium-lead solution. The stained sections were observed under a transmission electron microscope to analyze the morphological characteristics of mitochondria in corneal tissue. Fig.11 Figure 2 is a graph showing changes in mitochondria in the corneal tissue of mice of the present invention. Fig.11 As shown in the figure, after CTS@PLGA@LANPs treatment, the mitochondrial morphology in corneal epithelial cells was significantly improved, the mitochondrial structure tended to be normal, the membrane integrity was restored, and the swelling and lysis phenomena were reduced. The restoration of mitochondrial function helps to reduce the activation of inflammasomes and the excessive release of cytokines, 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 of mitochondria.

[0103] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0104] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A targeted sustained-release nano drug delivery system of linalool, characterized in that: Polylactic acid-polyglycolic acid copolymer is used as a matrix and polyvinyl alcohol is used as a cross-linking agent. The matrix and the cross-linking agent are cross-linked and grafted to form coated drug-loaded microspheres. Then, chitosan is used as a protective agent, and chitosan and the coated drug-loaded microspheres are cross-linked and grafted to form double-wrapped composite drug-loaded microspheres, wherein the chitosan in the composite drug-loaded microspheres is the shell and the coated drug-loaded microspheres is the core, so that a targeted sustained-release nano drug-loading system of lignoside is obtained, and the matrix is ​​loaded with lignoside.

2. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 1, characterized in that: The following steps are involved: The linalool and polylactic acid-polyglycolic acid copolymer are dissolved in a solvent and mixed to obtain a mixed solution, a polyvinyl alcohol solution is added to the mixed solution, and then the mixture is stirred for cross-linking and grafting to perform nanoprecipitation, and a PLGA@LA nanoparticle dispersion solution is prepared after dialysis; The chitosan solution was added dropwise to the PLGA@LA nanoparticle dispersion solution, and the chitosan was evenly dispersed on the surface of the PLGA@LA nanoparticles by stirring at room temperature. Cross-linking and grafting were performed to form a coating to form coated composite drug-loaded microspheres, thereby obtaining a targeted sustained-release nano drug-loading system of linalool.

3. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 2, characterized in that: The mass volume ratio of linalool, polylactic acid-polyglycolic acid copolymer and solvent is 1mg-2mg:4mg-5mg:500μL.

4. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 2, characterized in that: 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.

5. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 2, characterized in that: The specific method of cross-linking and grafting for nanoprecipitation is: under stirring at a speed of 400 rpm to 500 rpm, the mixed solution is added dropwise to the polyvinyl alcohol solution, stirring is continued for 2 min to 3 min after the addition is completed, and then dialyzed at 5 KDa to 15 KDa overnight to obtain a PLGA@LA nanoparticle dispersion solution.

6. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 1, characterized in that: The volume ratio of the PLGA@LA nanoparticle dispersion solution to the chitosan solution is 100-300:1, and the concentration of the chitosan solution is 5 mg / mL-10 mg / mL.

7. The method for preparing the targeted sustained-release nano drug delivery system of linalool according to claim 2, characterized in that: The specific method of cross-linking grafting to form coating is: at room temperature, under stirring at 200rpm to 400rpm, the chitosan solution is added dropwise to the PLGA@LANPs nanoparticle dispersion solution, and stirring is continued for 2h to 3h after the addition is completed to form coated composite drug-loaded microspheres.

8. Use of the targeted sustained-release nano drug delivery system of linalool according to claim 1 in the preparation of drugs for dry eye disease.

9. The use according to claim 8, characterized in that: Dry eye medications come as eye drops.

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