Multi-layer drug-loaded microsphere for intraocular sustained-release treatment and preparation process of multi-layer drug-loaded microsphere

Abstract

The invention relates to the technical field of drug delivery, and discloses a multilayer drug-loaded microsphere for intraocular sustained-release therapy and a preparation process thereof.The multilayer drug-loaded microsphere comprises a core layer, a middle layer and a shell layer, the core layer is composed of a polylactic acid-glycolic acid copolymer (PLGA) and a first hydrophobic drug, the middle layer is formed by wrapping the core layer with carboxymethyl chitosan, and the shell layer is composed of a first hydrophobic drug and a second hydrophobic drug. The first hydrophobic drug comprises a first therapeutic drug, the middle layer comprises a second therapeutic drug, the shell layer is composed of a polylactic acid-caprolactone copolymer PLCL and a third hydrophilic drug, and the first hydrophobic drug, the second therapeutic drug and the third hydrophilic drug are different from one another. Through the layered design of the core layer, the middle layer and the shell layer and in combination with differential degradation rates of PLGA, carboxymethyl chitosan and PLCL materials, gradient release of hydrophobic and hydrophilic drugs is achieved, a drug release curve is matched with the pathological stage of intraocular diseases, macular degeneration rapidly inhibits vascular leakage in the early stage and continuously resists inflammation in the later stage, and the effect of treating macular degeneration is achieved. And the layered slow-release synergistic treatment effect is verified.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, in particular to multilayer drug-loaded microspheres for intraocular sustained-release therapy and a preparation process thereof. Background Art

[0002] In the field of ophthalmology, intraocular sustained-release therapy plays a vital role in many intraocular diseases. Intraocular diseases such as glaucoma, cataracts, macular degeneration and uveitis often require continuous release of drugs in the eye to achieve the ideal therapeutic effect, control the progression of the disease and relieve the patient's pain. However, frequent drug administration may irritate the eye tissue, causing discomfort or other complications. In order to improve biocompatibility and reduce irritation to the eye tissue and potential adverse reactions, a multilayer drug-loaded microsphere for intraocular sustained-release therapy is needed.

[0003] Traditional intraocular drug treatments often have the problem of short drug action time and frequent drug administration. This not only brings many inconveniences to patients, such as frequent eye drops can easily cause patients to forget to take medicine or take medicine late, but may also cause unstable drug concentration in the eye, affecting the treatment effect. At the same time, although previous intraocular sustained-release preparations can prolong the drug action time to a certain extent, they still have many defects. This type of microspheres is prone to drug burst release, that is, the drug is released rapidly in large quantities in the initial stage, which may lead to excessively high drug concentration in the eye, thereby irritating the eye tissue and affecting the normal physiological environment in the eye. In the subsequent stage, the drug release rate is too slow to maintain an effective therapeutic concentration, resulting in poor treatment effect. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a multilayer drug-loaded microsphere for intraocular sustained-release therapy and its preparation process, which solves the problems of traditional intraocular drug treatment methods, which often have short drug action time and require frequent administration.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A multilayer drug-loaded microsphere for intraocular sustained-release therapy, comprising:

[0007] Core layer: composed of polylactic acid-glycolic acid copolymer PLGA and a first hydrophobic drug, wherein the first hydrophobic drug is a glucocorticoid, the PLGA molecular weight is 10,000-50,000 g / mol, and the mass proportion of the first hydrophobic drug is 10%-20%;

[0008] The middle layer is formed by wrapping the core layer with carboxymethyl chitosan, the carboxymethyl chitosan has a degree of substitution of 0.6-0.9 and a molecular weight of 30,000-100,000 g / mol, and contains a second therapeutic drug, which is an anti-vascular endothelial growth factor drug, and its drug loading is 20%-30% of the total weight of the microspheres;

[0009] The outer shell layer is composed of polylactic acid-caprolactone copolymer PLCL and a third hydrophilic drug. The third hydrophilic drug is an antibody drug. The molecular weight of PLCL is 20,000-50,000 g / mol, and the weight proportion of the third hydrophilic drug is 15%-25%. The surface is modified with hyaluronic acid.

[0010] The first hydrophobic drug, the second therapeutic drug, and the third hydrophilic drug are different, and the diameter of the microspheres is 50-300 μm.

[0011] This technical solution utilizes a layered design of core, intermediate, and outer shell layers, combined with the differentiated degradation rates of PLGA, carboxymethyl chitosan, and PLCL materials, to achieve a gradient release of hydrophobic and hydrophilic drugs. The core layer exhibits a sustained release rate of 0.5%-3% / day for dexamethasone, while the outer shell layer exhibits a rate of 1%-5% / day for ranibizumab. The in vitro release period lasts for 14-90 days, with the outer shell layer degrading 20%-50% faster than the core layer. This structure aligns the drug release profile with the pathological stage of the intraocular disease, demonstrating rapid inhibition of vascular leakage in the early stages of macular degeneration and sustained anti-inflammatory effects in the later stages, demonstrating the synergistic therapeutic effect of layered sustained release.

[0012] Preferably, the first hydrophobic drug is selected from one of dexamethasone, triamcinolone acetonide or non-steroidal anti-inflammatory drugs, the second therapeutic drug is selected from anti-vascular endothelial growth factor drugs or antibiotics, the third hydrophilic drug is selected from ranibizumab or glucocorticoids, and the mass ratio of hydrophobic to hydrophilic drugs is 1:0.5 to 1:2.

[0013] Preferably, the thickness of the core layer is 10%-15% of the diameter of the microsphere, the thickness of the middle layer is 20%-25%, and the thickness of the outer shell layer is 25%-30%; the cross-linked porosity of the middle layer is 10%-30%, which is achieved by a genipin cross-linking agent concentration of 0.5%-2%; and the hyaluronic acid coverage of the outer shell layer is 60%-90%.

[0014] Preferably, the in vitro release time of the microspheres is achieved by a PLGA molecular weight of 10,000-50,000 g / mol, a PLCL molecular weight of 20,000-50,000 g / mol, and a gradient drying process, with a core layer drug release rate of 0.5%-3% / day and a shell layer drug release rate of 1%-5% / day;

[0015] The degradation time is achieved by carboxymethyl chitosan substitution degree of 0.6-0.9 and hyaluronic acid modification of the outer shell. The degradation rate of the outer shell is 20%-50% faster than that of the core layer.

[0016] Preferably, a transition layer is provided between the core layer and the intermediate layer, which is composed of a blend of PLGA and PEG, the molecular weight of PEG is 2000-10000 g / mol, the thickness of the transition layer is 0.5-3 μm, and the mass ratio of PLGA to PEG in the transition layer is 3:1 to 8:1.

[0017] Preferably, a process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy, used for said multilayer drug-loaded microspheres for intraocular sustained-release therapy, comprises the following steps:

[0018] S1. Preparation of the core layer: PLGA and the first hydrophobic drug are dissolved in dichloromethane, chloroform or ethyl acetate at a weight ratio of 5:1 to 10:1, with a PLGA concentration of 8%-15% (w / v). The coaxial electrostatic spray method or the liquid paraffin emulsification method is used. The coaxial spray voltage is 10-20 kV and the liquid paraffin stirring speed is 500-800 r / min. After collecting the microspheres, vacuum drying is performed for 6-12 hours at a vacuum degree of -0.09 to -0.08 MPa.

[0019] S2. Intermediate layer coating: dissolving carboxymethyl chitosan in acetate buffer at pH 5.0-6.5, adding genipin crosslinker and second therapeutic drug, genipin concentration is 0.5%-2% (w / w), crosslinking time is 1-4 hours, dispersing core layer microspheres in the solution, stirring speed is 400-600 rpm, centrifuging and freeze drying, freezing temperature is -40 to -20°C, and drying time is 24-48 hours;

[0020] S3. Shell layer coating: PLCL and a third hydrophilic drug are dissolved in hexafluoroisopropanol or ethyl acetate at a weight ratio of 6:1 to 10:1, and hyaluronic acid is added, with a mass ratio of PLCL to hyaluronic acid of 5:1 to 10:1; an emulsion cross-linking method or a solvent evaporation method is used, and Span80 emulsifier is added at a concentration of 0.1%-0.5% (w / v), with a stirring speed of 500-1000 r / min, and gradient drying is performed with a first stage of drying at 30-40°C and a humidity of 40%-60% for 2-4 hours, and a second stage of drying at 40-50°C and a humidity of 20%-30% for 4-8 hours;

[0021] S4. Plasma treatment: Place the gradient dried microspheres in a plasma reactor, introduce a mixed gas of argon and oxygen at a volume ratio of 4:1 to 8:1, a power of 50-100 W, and a treatment time of 5-15 minutes.

[0022] Preferably, the organic solvent in S1 is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 3:1 to 6:1; the organic solvent in S2 is acetone or methanol; and the organic solvent in S3 is ethyl acetate or tetrahydrofuran.

[0023] Preferably, the ambient temperature during the stirring process of S1-S3 is controlled at 20-25°C, and the heating rate of the gradient drying is 1-3°C / min, and the cooling rate is 0.5-2°C / min.

[0024] Preferably, during the vacuum drying process of S1-S3, the vacuum degree is maintained at -0.09 to -0.08 MPa, and the surface porosity of the freeze-dried microspheres is 50-200 nm.

[0025] Preferably, the contact angle of the microsphere surface after the plasma treatment is reduced to 20°-40°, and the centrifugal shedding rate of hyaluronic acid is ≤5%.

[0026] The present invention provides a multilayer drug-loaded microsphere for intraocular sustained-release therapy and its preparation process. It has the following beneficial effects:

[0027] 1. This invention utilizes a layered design of core, intermediate, and outer layers, combined with the differentiated degradation rates of PLGA, carboxymethyl chitosan, and PLCL materials, to achieve gradient release of hydrophobic and hydrophilic drugs. The core layer exhibits a sustained release rate of 0.5%-3% / day for dexamethasone, while the outer layer exhibits a rate of 1%-5% / day for ranibizumab. The in vitro release period lasts for 14-90 days, and the outer layer degrades 20%-50% faster than the core layer. This structure aligns the drug release profile with the pathological stage of the intraocular disease, demonstrating rapid inhibition of vascular leakage in the early stages of macular degeneration and sustained anti-inflammatory effects in the later stages, demonstrating the synergistic therapeutic effect of layered sustained release.

[0028] 2. The present invention adopts a switching process of coaxial electrostatic spraying and liquid paraffin emulsification, combined with gradient drying (humidity controlled in stages at 30-50°C) and plasma treatment (argon-oxygen mixed gas, power 50-100W), to increase the drug loading of microspheres to 65%, the surface hydrophilicity by 20%-40% (the contact angle is reduced from 75° to 28°), and the hyaluronic acid binding strength by 15%-30% (centrifugal shedding rate ≤5%). After optimization, the burst release effect of the microspheres is reduced by 58.9%, the interlayer bonding force is improved, and the ultrasonic treatment delamination rate is <5%, which proves that the process significantly improves drug stability and intraocular adhesion.

[0029] 3、The application solves the problems of low drug loading (≤40%) and high burst release rate (>12%) of traditional single-layer microspheres by designing a PLGA / PEG transition layer (mass ratio 3:1-8:1) and cross-linking porosity regulation (10%-30%) of carboxymethyl chitosan. In Example 2, hydrophobic drugs (20%), antibiotics (30%), and hydrophilic drugs (15%) are loaded in three layers, with a total drug loading of 65%, and the IL-6 inflammatory factor inhibition rate is increased by 22% compared with the single-drug group, verifying the synergistic treatment potential under high drug loading, which is suitable for complex conditions such as severe uveitis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A process flow chart for preparing the multi-layer drug-loaded microspheres for intraocular sustained-release treatment of the application. DETAILED DESCRIPTION

[0031] The technical solutions of the application will be described below in conjunction with the drawings of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0032] The embodiment of the application provides a multi-layer drug-loaded microsphere for intraocular sustained-release treatment, comprising:

[0033] The core layer is composed of polylactic acid-glycolic acid copolymer PLGA and a first hydrophobic drug, the first hydrophobic drug is a glucocorticoid, the molecular weight of PLGA is 10000-50000 g / mol, and the mass fraction of the first hydrophobic drug is 10%-20%;

[0034] The intermediate layer is formed by wrapping the core layer with carboxymethyl chitosan, the degree of substitution of carboxymethyl chitosan is 0.6-0.9, the molecular weight is 30000-100000 g / mol, the intermediate layer contains a second therapeutic drug, the second therapeutic drug is an anti-vascular endothelial growth factor drug, and the drug loading is 20%-30% of the total weight of the microsphere;

[0035] The outer shell layer is composed of polylactic acid-caprolactone copolymer PLCL and a third hydrophilic drug, the third hydrophilic drug is an antibody drug, the molecular weight of PLCL is 20000-50000 g / mol, the mass fraction of the third hydrophilic drug is 15%-25%, and the surface is modified with hyaluronic acid;

[0036] The first hydrophobic drug, the second therapeutic drug, and the third hydrophilic drug are all different, and the diameter of the microsphere is 50-300 μm.

[0037] Specifically, the core layer is the inner core support of the microsphere, carrying the first hydrophobic drug, which accounts for 10%-20% of the mass, which can not only ensure the effective drug dosage, but also avoid the initial burst release caused by excessive drug loading, playing a role in long-term stable sustained release of the first hydrophobic drug, laying the foundation for the long-term treatment of intraocular diseases; the middle layer is composed of carboxymethyl chitosan wrapped around the core layer, which plays the role of transition and bonding the core layer and the outer shell layer, and at the same time loads the second therapeutic drug, which can effectively enhance the interlayer bonding force of the microspheres, reduce the risk of stratification, and overcome the problem of limited drug loading of traditional single polymer microspheres.

[0038] The first hydrophobic drug is selected from one of dexamethasone, triamcinolone acetonide or non-steroidal anti-inflammatory drugs, the second therapeutic drug is selected from anti-vascular endothelial growth factor drugs or antibiotics, and the third hydrophilic drug is selected from ranibizumab or glucocorticoids, and the mass ratio of hydrophobic to hydrophilic drugs is 1:0.5 to 1:2.

[0039] Specifically, dexamethasone, triamcinolone acetonide, and nonsteroidal anti-inflammatory drugs are all common ophthalmic treatments. Dexamethasone and triamcinolone acetonide belong to the glucocorticoid class of drugs, which have powerful anti-inflammatory, anti-allergic, and immunosuppressive effects and can effectively reduce intraocular inflammatory reactions. Nonsteroidal anti-inflammatory drugs exert anti-inflammatory, analgesic, and antipyretic effects by inhibiting cyclooxygenase activity and reducing prostaglandin synthesis, and can be used to relieve intraocular inflammation and pain symptoms. Anti-vascular endothelial growth factor (VEGF) drugs are mainly used to inhibit the formation of new blood vessels in the eye. For example, in diseases such as macular degeneration and diabetic retinopathy, they can effectively reduce vascular leakage and new blood vessel proliferation, preventing further vision loss. Antibiotics are used to prevent and treat intraocular infections and play a key therapeutic role in infectious eye diseases such as bacterial endophthalmitis.

[0040] The thickness of the core layer is 10%-15% of the microsphere diameter, the thickness of the middle layer is 20%-25%, and the thickness of the outer shell layer is 25%-30%; the cross-linked porosity of the middle layer is 10%-30%, which is achieved by a genipin cross-linking agent concentration of 0.5%-2%; the hyaluronic acid coverage of the outer shell layer is 60%-90%.

[0041] Specifically, the thickness of the core layer is 10%-15% of the diameter of the microsphere. Such a thickness design can ensure that the core layer has sufficient structural stability to carry the first hydrophobic drug and maintain its slow release, while not being too thick so that the drug release is too slow or the overall degradation time of the microsphere is too long; the thickness of the middle layer accounts for 20%-25% of the diameter of the microsphere, which can enable the middle layer to play a good transition and bonding role in the microsphere structure, while providing a suitable drug-loading space for the second therapeutic drug; the thickness of the outer shell layer is 25%-30% of the diameter of the microsphere. This thickness enables the outer shell layer to quickly respond to the initial treatment needs of intraocular diseases, realize the rapid release of the third hydrophilic drug, and timely relieve the symptoms of the disease.

[0042] The in vitro release time of the microspheres is achieved by a PLGA molecular weight of 10,000-50,000 g / mol, a PLCL molecular weight of 20,000-50,000 g / mol, and a gradient drying process, with a drug release rate of 0.5%-3% / day in the core layer and 1%-5% / day in the shell layer;

[0043] The degradation time is achieved by carboxymethyl chitosan substitution degree of 0.6-0.9 and hyaluronic acid modification of the outer shell. The degradation rate of the outer shell is 20%-50% faster than that of the core layer.

[0044] Specifically, the lower drug release rate of the core layer helps to achieve long-term and stable drug release in the eye, especially for chronic intraocular diseases. It can continuously provide drug efficacy for a longer period of time, maintain the effective drug concentration in the intraocular tissue, and reduce the recurrence of the disease caused by fluctuations in drug concentration.

[0045] A transition layer is provided between the core layer and the middle layer, which is composed of a blend of PLGA and PEG, the molecular weight of PEG is 2000-10000 g / mol, the thickness of the transition layer is 0.5-3 μm, and the mass ratio of PLGA to PEG in the transition layer is 3:1 to 8:1.

[0046] Specifically, the transition layer is located between the core layer and the intermediate layer, playing a key connecting role. It can effectively improve the compatibility between the core layer and the intermediate layer, reduce interface defects caused by material differences, enhance interlayer bonding, and prevent stratification of microspheres during drug release.

[0047] Please see the attached Figure 1 A process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy, which is used for the above-mentioned multilayer drug-loaded microspheres for intraocular sustained-release therapy, comprises the following steps:

[0048] S1. Preparation of the core layer: PLGA and the first hydrophobic drug are dissolved in dichloromethane, chloroform or ethyl acetate at a weight ratio of 5:1 to 10:1, with a PLGA concentration of 8%-15% (w / v). The coaxial electrostatic spray method or the liquid paraffin emulsification method is used. The coaxial spray voltage is 10-20 kV and the liquid paraffin stirring speed is 500-800 r / min. After collecting the microspheres, vacuum drying is performed for 6-12 hours at a vacuum degree of -0.09 to -0.08 MPa.

[0049] S2. Intermediate layer coating: dissolving carboxymethyl chitosan in acetate buffer at pH 5.0-6.5, adding genipin crosslinker and second therapeutic drug, genipin concentration is 0.5%-2% (w / w), crosslinking time is 1-4 hours, dispersing core layer microspheres in the solution, stirring speed is 400-600 rpm, centrifuging and freeze drying, freezing temperature is -40 to -20°C, and drying time is 24-48 hours;

[0050] S3. Shell layer coating: PLCL and a third hydrophilic drug are dissolved in hexafluoroisopropanol or ethyl acetate at a weight ratio of 6:1 to 10:1, and hyaluronic acid is added, with a mass ratio of PLCL to hyaluronic acid of 5:1 to 10:1; an emulsion cross-linking method or a solvent evaporation method is used, and Span80 emulsifier is added at a concentration of 0.1%-0.5% (w / v), with a stirring speed of 500-1000 r / min, and gradient drying is performed with a first stage of drying at 30-40°C and a humidity of 40%-60% for 2-4 hours, and a second stage of drying at 40-50°C and a humidity of 20%-30% for 4-8 hours;

[0051] S4. Plasma treatment: Place the gradient dried microspheres in a plasma reactor, introduce a mixed gas of argon and oxygen at a volume ratio of 4:1 to 8:1, a power of 50-100 W, and a treatment time of 5-15 minutes.

[0052] Please see the attached Figure 1 The organic solvent in S1 is a mixed solvent of dichloromethane and ethyl acetate with a volume ratio of 3:1 to 6:1; the organic solvent in S2 is acetone or methanol; and the organic solvent in S3 is ethyl acetate or tetrahydrofuran.

[0053] Specifically, dichloromethane has good solubility and can effectively dissolve PLGA and hydrophobic drugs, allowing the materials to be evenly mixed to form a stable solution; ethyl acetate can adjust the volatilization rate and viscosity of the solution, and a lower volume ratio can provide suitable solution rheological properties during the spraying process, which is conducive to the formation of microspheres; acetone or methanol, as both solvents, have strong polarity and solubility, and can well dissolve carboxymethyl chitosan and the second therapeutic drug, so that the intermediate layer solution has good fluidity, which is convenient for wrapping the core layer; ethyl acetate has suitable solubility and volatilization rate, and can effectively dissolve PLCL and hydrophilic drugs; tetrahydrofuran has good chemical stability, which can ensure the stability of components such as PLCL and hyaluronic acid in the solution and prevent them from degradation or denaturation.

[0054] Please see the attached Figure 1 During the stirring process of S1-S3, the ambient temperature is controlled at 20-25°C, and the heating rate of the gradient drying is 1-3°C / min, and the cooling rate is 0.5-2°C / min.

[0055] Specifically, the temperature range of 20-25°C is a relatively stable temperature range for most organic solvents and polymer systems. It can ensure a moderate volatilization rate of the organic solvent, avoid rapid volatilization of the solvent due to excessively high temperature, and cause a sharp change in the viscosity of the solution, affecting the stirring effect and mixing uniformity; at the same time, it prevents the temperature from being too low, resulting in a decrease in the solubility of the drug or polymer, and the occurrence of crystallization or precipitation; through slow and controllable temperature changes, the microspheres gradually lose the solvent during the drying process, reducing the internal stress concentration of the microspheres caused by sudden temperature changes.

[0056] Please see the attached Figure 1 ,During the vacuum drying process of S1-S3, the vacuum degree was maintained at -0.09 to -0.08 MPa, and the surface porosity of the freeze-dried microspheres was 50-200 nm.

[0057] Specifically, the vacuum range of -0.09 to -0.08 MPa can effectively lower the boiling point of the solvent, allowing the solvent to evaporate and escape quickly at a lower temperature, while avoiding excessive vacuum causing a sudden drop in pressure inside the microsphere structure, causing the microspheres to collapse or deform, thereby ensuring the physical integrity of the microspheres during the drying process; the surface porosity of the microspheres after drying is 50-200 nm. This moderate porosity is conducive to the diffusion and release of drugs inside the microspheres, allowing the drugs to be delivered from the inside of the microspheres to the intraocular tissue at a designed rate.

[0058] Please see the attached Figure 1 The contact angle of the microsphere surface after plasma treatment was reduced to 20°-40°, and the hyaluronic acid centrifugal shedding rate was ≤5%.

[0059] Specifically, the improvement in hydrophilicity helps to enhance the interaction between the microspheres and the intraocular tissue fluid, making the microspheres more easily wetted by physiological fluids in the intraocular environment, and promoting the diffusion and release of drugs from the surface of the microspheres to the surrounding tissues; the hyaluronic acid binding strength is increased by 15%-30%, making the hyaluronic acid coverage on the microsphere surface more firm and stable, preventing the hyaluronic acid from falling off or degrading prematurely in the physiological environment of the eye.

[0060] Example 1: Basic PLGA core layer / carboxymethyl chitosan intermediate layer / PLCL shell layer microspheres

[0061] 1. Technical Solution

[0062] 1. Core layer preparation: PLGA with a molecular weight of 30,000 g / mol was selected as the substrate, and a mixture of dichloromethane and ethyl acetate in a volume ratio of 4:1 was used as the solvent. The drug loading of the hydrophobic drug dexamethasone was controlled at 15%. Coaxial electrostatic spray technology was used, and the voltage was set to 15 kV for spraying. Subsequently, the microspheres were placed in a vacuum drying oven with a vacuum degree maintained at -0.09 MPa for 10 hours to obtain the PLGA core layer microspheres. This layer of microspheres is mainly responsible for the long-term sustained release of dexamethasone, exerting a long-term anti-inflammatory effect.

[0063] 2. Construction of the middle layer: Carboxymethyl chitosan with a degree of substitution of 0.7 and a molecular weight of 50,000 g / mol was used as the middle layer material. Genipin at a concentration of 1.2% was added as a cross-linker to load the anti-VEGF drug bevacizumab. After a 2-hour cross-linking reaction, the freeze-drying method was used, with a set temperature of -30°C and a drying time of 36 hours, so that the middle layer tightly covered the core layer. This layer mainly played a transitional role, taking into account both drug loading and interlayer bonding. The biocompatibility of carboxymethyl chitosan and the stability after cross-linking were utilized to ensure the stable release of the drug in the medium term.

[0064] 3. Shell Formation: PLCL with a molecular weight of 35,000 g / mol was selected to prepare the shell layer. The drug loading of the hydrophilic drug ranibizumab was set to 20%. Hyaluronic acid and PLCL were mixed in a mass ratio of 7:1. Emulsion cross-linking was used, and the Span80 concentration was controlled at 0.3%. Finally, a gradient drying process was used, first at 35°C for 3 hours and then at 45°C for 6 hours, to uniformly wrap the shell layer around the middle layer. This layer is used for the rapid early release of ranibizumab and timely inhibition of vascular leakage.

[0065] 4. Surface modification: The prepared multilayer microspheres are subjected to plasma treatment. A mixed gas of argon and oxygen is introduced at a volume ratio of 6:1. The power is set to 75W and the treatment time is 10 minutes. With the help of the plasma bombardment, the hydrophilicity of the microsphere surface is improved, and its adaptability and stability in the intraocular environment are enhanced.

[0066] 2. Technical Effect Verification

[0067] 1. In vitro release test: Based on the ISO10993-12 standard, using HPLC testing, the release rate of dexamethasone in the core layer was stable at 1.8% / day, with a cumulative release of 25.2% over 14 days. The release rate of ranibizumab in the outer shell layer was 3.2% / day, with a cumulative release of 22.4% over 7 days. These results precisely match the treatment rhythm of macular degeneration, which requires rapid inhibition of vascular leakage in the early stages and relies on continuous anti-inflammatory treatment in the later stages.

[0068] 2. Degradation experiment: The microsphere samples were immersed in PBS solution and their degradation was observed using SEM. The results showed that the outer shell degraded by 50% in 60 days and the core layer degraded by 50% in 90 days, indicating that the degradation rate of the outer shell was 33% faster than that of the core layer, realizing the ingenious design of sequential release of drugs in different layers.

[0069] 3. Adhesion test: According to the GB / T16886-2011 standard, a centrifugal shedding rate experiment was conducted. It was found that after plasma treatment, the shedding rate of hyaluronic acid dropped to 4.2%. Compared with the untreated control group (shedding rate 18.5%), the surface hydrophilicity increased by 30%, and the contact angle decreased significantly from 75° to 28°, greatly enhancing the adhesion of the microspheres in the eye.

[0070] 3. Comparative Experiment

[0071] Compared with the comparative example without adding the transition layer (other conditions are the same):

[0072] 1. Burst release effect: The burst release rate of dexamethasone in Example 1 was only 5.2% on the first day, while that in the control group was as high as 12.7%. The burst release effect was reduced by 58.9%, effectively avoiding the risk of excessive initial drug release.

[0073] 2. Interlayer bonding strength: When the microspheres were placed in an ultrasonic treatment environment (40 kHz frequency, 30 minutes), the delamination rate of the microspheres in Example 1 was less than 5%, while that in the comparative example was 22%, highlighting the significant effect of the transition layer design on enhancing interlayer bonding strength.

[0074] In summary, the introduction of the transition layer cleverly resolves the problems of weak interlayer bonding and obvious burst release effect, fully unleashes the therapeutic potential of the synergistic sustained release of the multi-layer structure, and provides a better microsphere carrier for intraocular sustained release treatment.

[0075] Example 2: High-loaded hydrophobic-hydrophilic drug combination microspheres with high drug loading

[0076] 1. Technical Solution

[0077] 1. Core layer construction: PLGA with a molecular weight of 50,000 g / mol was prepared using the liquid paraffin emulsification method. The stirring speed was controlled at 700 r / min, and the drug loading of the hydrophobic drug triamcinolone acetonide was set to 20%. Subsequently, vacuum drying was performed at a vacuum degree of -0.08 MPa for 8 hours to obtain core layer microspheres with a high drug loading, aiming to lay the foundation for long-term anti-inflammatory treatment.

[0078] 2. Construction of the intermediate layer: Carboxymethyl chitosan with a degree of substitution of 0.9 and a molecular weight of 100,000 g / mol was selected, combined with a 2% concentration of genipin crosslinker, and loaded with the antibiotic tobramycin. After a 4-hour crosslinking reaction, the mixture was freeze-dried at -20°C for 48 hours to form an intermediate layer. This enhanced the drug's antibacterial efficacy in the medium term and utilized a high degree of crosslinking to increase drug loading and stability.

[0079] 3. Shell Formation: The shell layer was prepared using PLCL with a molecular weight of 50,000 g / mol. The drug loading of the hydrophilic glucocorticoid dexamethasone sodium phosphate was set to 25%. Hyaluronic acid and PLCL were mixed in a mass ratio of 10:1. Using a solvent evaporation method with ethyl acetate as the solvent, a gradient drying process was used. The shell layer was first dried at 40°C for 2 hours and then dried at 50°C for 8 hours. This ensured that the shell layer firmly wrapped around the middle layer, achieving rapid early anti-inflammatory effects while also taking into account the synergistic release of drugs in the core and middle layers.

[0080] 4. Surface modification: Plasma treatment was performed by introducing a mixture of argon and oxygen (volume ratio 4:1) at a power of 100 W for 15 minutes to optimize the surface properties of the microspheres and improve their biocompatibility and compatibility within the eye.

[0081] 2. Technical Effect Verification

[0082] 1. Antibacterial performance evaluation: According to the ISO20776-1 drug sensitivity test standard, the test found that after 14 days of sustained-release of tobramycin, the diameter of the inhibition zone formed against Staphylococcus aureus can still reach 12mm (initial 18mm), and the effective antibacterial concentration is continuously and stably maintained to ensure the prevention and treatment of intraocular infection.

[0083] 2. Anti-inflammatory synergy detection: The ELISA method was used to detect the expression of IL-6 in corneal epithelial cells. The results showed that the combined release of dexamethasone sodium phosphate and triamcinolone acetonide reduced IL-6 expression by 72%, compared with the single drug group (reduction of 45-50%), demonstrating the significant synergistic anti-inflammatory advantage of the drug combination.

[0084] 3. Porosity control analysis: Using the BET method, the porosity of the cross-linked intermediate layer is 25%, and the pore diameter is 150 nm. Under the condition of 2% genipin concentration, compared with Example 1 (porosity 18%), it is more conducive to drug diffusion, accelerates the drug release rate, and improves the speed of treatment.

[0085] 3. Comparative Experiment

[0086] Compared with single drug-loaded microspheres (only the outer shell contains dexamethasone sodium phosphate):

[0087] 1. Duration of inflammation suppression: The microspheres in Example 2 can maintain the IL-6 inhibition effect for more than 28 days, while the single-drug group can only maintain the IL-6 inhibition effect for 14 days, nearly doubling the inflammation control period.

[0088] 2. Drug loading limit: The total drug loading in Example 2 can reach 65% (20% hydrophobic, 30% antibiotic, 15% hydrophilic), far exceeding the drug loading limit of single-layer microspheres (≤40%), breaking the bottleneck of traditional drug loading and meeting the urgent need for high-dose drug combination therapy for complex eye diseases such as severe uveitis.

[0089] In summary, the multi-layer loading strategy cleverly combines high-drug-loading hydrophobic and hydrophilic drugs, deeply explores the drug-loading potential of microspheres, promotes the treatment of intractable eye diseases such as severe uveitis to a new level, achieves synergistic enhancement between drugs, and provides a powerful weapon for clinical critical care.

[0090] Example 3: Acute Therapy-Adapted Rapid-Release Microspheres

[0091] 1. Technical Solution

[0092] 1. Core layer preparation: PLGA with a molecular weight of 10,000 g / mol was selected. Coaxial electrostatic spray technology was used, and the voltage was set to 20 kV. The drug loading of the non-steroidal anti-inflammatory drug diclofenac was controlled at 10%. The microspheres were then placed in a vacuum drying oven with a vacuum degree of -0.09 MPa and dried for 12 hours to form core layer microspheres, preparing for rapid anti-inflammatory in the acute phase.

[0093] 2. Construction of the intermediate layer: Use carboxymethyl chitosan with a degree of substitution of 0.6 and a molecular weight of 30,000 g / mol, add 0.5% concentration of genipin crosslinker, undergo a 1-hour crosslinking reaction, and freeze-dry at -40°C for 24 hours to create the intermediate layer, which plays the role of transition and initial drug release, and ensures the stability of the microsphere structure transition.

[0094] 3. Shell Formation: The shell layer was prepared using PLCL with a molecular weight of 20,000 g / mol. The drug loading of the anti-VEGF drug aflibercept was set to 15%. Hyaluronic acid and PLCL were mixed in a mass ratio of 5:1. The emulsion cross-linking method was used, and the Span80 concentration was 0.5%. A gradient drying process was used, first drying at 30°C for 4 hours, then heating to 40°C and drying for 4 hours. This enabled the shell layer to respond quickly and accelerate the release of aflibercept, meeting the urgent need for rapid blood pressure reduction in the acute phase of glaucoma treatment.

[0095] 4. Surface modification: Plasma treatment was performed by introducing a mixture of argon and oxygen (8:1 by volume) at a power of 50 W for 5 minutes to optimize the surface properties of the microspheres, making them better suited for acute treatment scenarios and ensuring rapid drug release while maintaining good biocompatibility.

[0096] 2. Technical Effect Verification

[0097] 1. Acute release rate detection: Using HPLC technology, the release rate of aflibercept in the outer shell was measured to be as high as 8.5% on the first day (traditional microspheres ≤3%), and the cumulative release after three days reached 35%, providing strong support for rapidly reducing intraocular pressure and controlling disease progression in the acute phase of glaucoma.

[0098] 2. Mechanical Strength Test: For PLCL microspheres with a molecular weight of 20kDa, the breakage rate was less than 2% under a simulated intraocular fluid shear force environment. Compared with the PLCL 50kDa group (breakage rate of 8%), while ensuring rapid release, they maintained good mechanical stability, ensuring the structural integrity of the microspheres during intraocular treatment.

[0099] 3. Biocompatibility assessment: Based on the ISO10993-5 standard, the CCK-8 method was used to test the effect of microsphere extracts on human corneal cell culture. The results showed that the cell survival rate exceeded 95% after 72 hours, verifying the good biocompatibility of the microspheres and ensuring the safety of intraocular application.

[0100] 3. Comparative Experiment

[0101] Compared with the comparative example without optimizing the molecular weight of PLCL (PLCL50kDa, other conditions are the same):

[0102] 1. Drug release rate: The release rate of the microspheres in Example 3 on the third day was as high as 35%, while that of the control group was only 18%, achieving a faster drug release response, which meets the urgent needs of acute treatment.

[0103] 2. In terms of intraocular retention time: The residual amount of the microspheres in Example 3 in the rabbit vitreous body after 14 days was 80%, while that in the comparative example was 92%. While ensuring rapid release, it also takes into account the long-term sustained-release effect, prolonging the duration of drug action in the eye and improving the overall effectiveness of treatment.

[0104] In general, Example 3 accelerates drug release by selecting a low-molecular-weight PLCL shell layer and optimizes surface properties through plasma treatment, precisely adapting to the combined treatment scenario of acute glaucoma, achieving an organic combination of rapid blood pressure reduction and long-term control, and providing a practical microsphere treatment solution for the emergency treatment of acute eye diseases.

[0105] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A multilayer drug-loaded microsphere for intraocular sustained-release therapy, characterized in that: include: Core layer: composed of polylactic acid-glycolic acid copolymer PLGA and a first hydrophobic drug, wherein the first hydrophobic drug is a glucocorticoid, the PLGA molecular weight is 10,000-50,000 g / mol, and the mass proportion of the first hydrophobic drug is 10%-20%; The middle layer is formed by wrapping the core layer with carboxymethyl chitosan, the carboxymethyl chitosan has a degree of substitution of 0.6-0.9 and a molecular weight of 30,000-100,000 g / mol, and contains a second therapeutic drug, which is an anti-vascular endothelial growth factor drug, and its drug loading is 20%-30% of the total weight of the microspheres; The outer shell layer is composed of polylactic acid-caprolactone copolymer PLCL and a third hydrophilic drug. The third hydrophilic drug is an antibody drug. The molecular weight of PLCL is 20,000-50,000 g / mol, and the weight proportion of the third hydrophilic drug is 15%-25%. The surface is modified with hyaluronic acid. The first hydrophobic drug, the second therapeutic drug, and the third hydrophilic drug are different, and the diameter of the microspheres is 50-300 μm.

2. The multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 1, characterized in that: The first hydrophobic drug is selected from one of dexamethasone, triamcinolone acetonide or non-steroidal anti-inflammatory drugs, the second therapeutic drug is selected from anti-vascular endothelial growth factor drugs or antibiotics, the third hydrophilic drug is selected from ranibizumab or glucocorticoids, and the mass ratio of hydrophobic to hydrophilic drugs is 1:0.5 to 1:

2.

3. The multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 1, characterized in that: The thickness of the core layer is 10%-15% of the diameter of the microsphere, the thickness of the middle layer is 20%-25%, and the thickness of the outer shell layer is 25%-30%; the cross-linking porosity of the middle layer is 10%-30%, which is achieved by a genipin cross-linking agent concentration of 0.5%-2%; the hyaluronic acid coverage of the outer shell layer is 60%-90%.

4. The multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 1, characterized in that: The in vitro release time of the microspheres is achieved by a PLGA molecular weight of 10,000-50,000 g / mol, a PLCL molecular weight of 20,000-50,000 g / mol, and a gradient drying process, with a drug release rate of 0.5%-3% / day in the core layer and 1%-5% / day in the shell layer; The degradation time is achieved by carboxymethyl chitosan substitution degree of 0.6-0.9 and hyaluronic acid modification of the outer shell. The degradation rate of the outer shell is 20%-50% faster than that of the core layer.

5. The multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 1, characterized in that: A transition layer is provided between the core layer and the intermediate layer, which is composed of a blend of PLGA and PEG, wherein the molecular weight of PEG is 2000-10000 g / mol, the thickness of the transition layer is 0.5-3 μm, and the mass ratio of PLGA to PEG in the transition layer is 3:1 to 8:

1.

6. A process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy, characterized in that: The multilayer drug-loaded microspheres for intraocular sustained-release therapy according to any one of claims 1 to 5 comprise the following steps: S1. Preparation of the core layer: PLGA and the first hydrophobic drug are dissolved in dichloromethane, chloroform, or ethyl acetate at a weight ratio of 5:1 to 10:1, with a PLGA concentration of 8%-15% (w / v). The microspheres are collected and vacuum dried for 6-12 hours at a vacuum degree of -0.09 to -0.08 MPa using a coaxial electrostatic spray method or a liquid paraffin emulsification method. The coaxial spray voltage is 10-20 kV and the liquid paraffin stirring speed is 500-800 r / min. S2. Intermediate layer coating: dissolving carboxymethyl chitosan in acetate buffer at pH 5.0-6.5, adding genipin crosslinker and second therapeutic drug, genipin concentration is 0.5%-2% (w / w), crosslinking time is 1-4 hours, dispersing core layer microspheres in the solution, stirring speed is 400-600 rpm, centrifuging and freeze drying, freezing temperature is -40 to -20°C, and drying time is 24-48 hours; S3. Shell layer coating: PLCL and a third hydrophilic drug are dissolved in hexafluoroisopropanol or ethyl acetate at a weight ratio of 6:1 to 10:1, and hyaluronic acid is added, with a mass ratio of PLCL to hyaluronic acid of 5:1 to 10:1; an emulsion crosslinking method or a solvent evaporation method is used, and Span80 emulsifier is added at a concentration of 0.1%-0.5% (w / v), with a stirring speed of 500-1000 r / min, and gradient drying is performed with a first stage of drying at 30-40°C and a humidity of 40%-60% for 2-4 hours, and a second stage of drying at 40-50°C and a humidity of 20%-30% for 4-8 hours; S4. Plasma treatment: Place the gradient dried microspheres in a plasma reactor, introduce a mixed gas of argon and oxygen at a volume ratio of 4:1 to 8:1, a power of 50-100 W, and a treatment time of 5-15 minutes.

7. The process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 6, characterized in that: The organic solvent in S1 is a mixed solvent of dichloromethane and ethyl acetate with a volume ratio of 3:1 to 6:1; the organic solvent in S2 is acetone or methanol; and the organic solvent in S3 is ethyl acetate or tetrahydrofuran.

8. The process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 7, characterized in that: During the stirring process of S1-S3, the ambient temperature is controlled at 20-25°C, and the heating rate of the gradient drying is 1-3°C / min, and the cooling rate is 0.5-2°C / min.

9. The process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 8, characterized in that: During the vacuum drying process of S1-S3, the vacuum degree was maintained at -0.09 to -0.08 MPa, and the surface porosity of the freeze-dried microspheres was 50-200 nm.

10. The process for preparing multilayer drug-loaded microspheres for intraocular sustained-release therapy according to claim 9, characterized in that: The contact angle of the microsphere surface after the plasma treatment is reduced to 20°-40°, and the centrifugal shedding rate of hyaluronic acid is less than or equal to 5%.

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