Preparation method of temperature-shear dual response type ophthalmic sustained-release drug carrier based on non-Newtonian fluid

Abstract

The invention discloses a preparation method of a non-Newtonian fluid-based temperature-shear dual response type ophthalmic sustained-release drug carrier, and belongs to the technical field of ophthalmic medicines. The preparation is composed of a dispersed phase (a temperature response type targeting nano-drug carrier) and a continuous phase (a non-Newtonian fluid matrix), wherein the mass fraction of the dispersed phase in the continuous phase is 0.5-2%. The temperature response type targeted nano-drug carrier is of a core-shell two-layer structure, the core is composed of a PLGA-PEG copolymer and an active drug, the shell is formed by grafting poly N-isopropylacrylamide (PNIPAM) and hyaluronic acid, and the lowest critical solution temperature (LCST) of the PNIPAM is 35-37 DEG C; the non-Newtonian fluid matrix is a pseudoplastic fluid, is composed of Carbomer 940 and triethanolamine, and has a shear thinning characteristic. The preparation method comprises the following three steps: preparation of the temperature response type targeting nano-drug carrier, preparation of the non-Newtonian fluid matrix and low-speed dispersion and compounding of the two. According to the invention, shear-response convenient administration, temperature-response precise drug release and long-acting retention synergism are realized, the bioavailability of the drug and the compliance of a patient are remarkably improved, the stability is excellent, the preparation process is simple and controllable, and the preparation method is suitable for long-acting targeted therapy of chronic eye diseases such as allergic conjunctivitis, chronic xerophthalmia, glaucoma and the like.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic medical technology, specifically to a method for preparing a temperature-shear dual-response ophthalmic sustained-release drug carrier based on non-Newtonian fluids, which is particularly suitable for long-term targeted treatment of chronic ocular diseases such as allergic conjunctivitis, chronic dry eye, and glaucoma. Background Technology

[0002] The eye has unique physiological barriers (corneal barrier, blood-eye barrier) and tear flushing function, resulting in low bioavailability (usually less than 5%) of traditional ophthalmic drug delivery preparations. The drugs have a short retention time in the eye and require frequent administration to maintain efficacy, which not only seriously reduces patient compliance but also easily causes adverse reactions such as eye irritation and dysbiosis.

[0003] To address these issues, the industry has developed temperature-responsive targeted nanocarriers that can dynamically regulate drug release based on temperature changes at the site of inflammation, achieving targeted drug delivery. However, when used alone as eye drops, these nanocarriers still suffer from drawbacks such as rapid washout by tears, poor corneal adhesion, and tendency to aggregate during storage, limiting their clinical application. Meanwhile, while existing ophthalmic gels and other formulations can prolong drug retention time, they are mostly single viscous matrices, lacking targeting and intelligent drug delivery capabilities, and thus cannot meet the individualized treatment needs of varying degrees of inflammation.

[0004] Non-Newtonian fluids possess properties such as shear-thinning and thixotropy. Pseudoplastic non-Newtonian fluids, in particular, exhibit a sharp decrease in viscosity under shear force, recovering to high viscosity upon settling, making them suitable for the convenience and retention requirements of ophthalmic eye drops. However, there are currently no reports on fusing temperature-responsive targeted nanocarriers with non-Newtonian fluid matrices to construct ophthalmic formulations that combine shear-responsive retention and temperature-responsive targeted drug release. Therefore, developing a dual-responsive ophthalmic sustained-release drug formulation that integrates the advantages of both, achieving a synergistic effect of "convenient administration, long-lasting retention, and precise drug release," has become an urgent need in the field of ophthalmic drug delivery technology. Summary of the Invention

[0005] To address the shortcomings of existing ophthalmic formulations, such as low bioavailability, poor targeting, short retention time, and insufficient patient compliance, this invention provides a method for preparing a temperature-shear dual-responsive ophthalmic sustained-release drug carrier based on non-Newtonian fluids. By synergistically fusing a temperature-responsive targeted nanocarrier with a non-Newtonian fluid matrix, convenient drug delivery, long-term retention, and precise drug release at the site of inflammation are achieved, thereby improving therapeutic efficacy and reducing adverse reactions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A temperature-shear dual-responsive ophthalmic sustained-release drug carrier based on a non-Newtonian fluid comprises a dispersed phase and a continuous phase, wherein the dispersed phase is a temperature-responsive targeted nanomedicine carrier, and the continuous phase is a non-Newtonian fluid matrix; the mass fraction of the dispersed phase in the continuous phase is 0.5%-2%.

[0008] The temperature-responsive targeted nanomedicine carrier has a two-layer core-shell structure: the core is a drug-loading layer composed of polylactic acid-glycolic acid copolymer-polyethylene glycol (PLGA-PEG) copolymer, which binds to the active drug. The mass ratio of PLGA to PEG is 7:3-9:1; the shell is a temperature-responsive-targeting composite layer grafted from poly(N-isopropylacrylamide) (PNIPAM) and hyaluronic acid. The minimum critical temperature of PNIPAM (LCST) is 35-37°C, and the grafting rate of the targeting ligand is 5%-15%. The nanocarrier has a particle size of 100-200 nm, and the shell thickness is 20-50 nm.

[0009] The non-Newtonian fluid matrix is ​​a pseudoplastic fluid composed of carbomer 940 and triethanolamine. The mass concentration of carbomer 940 is 0.5%-1.5%, and the amount of triethanolamine added is the amount required to adjust the pH of the matrix to 7.0-7.4. The non-Newtonian fluid matrix is ​​capable of operating under a shear force of 500 s⁻¹. -1 The viscosity at rest is 100-500 mPa·s, and the viscosity at rest is 5000-10000 mPa·s (measured by rotational rheometer).

[0010] Further, the active drug may be one or more of an antihistamine, an anti-inflammatory drug, an intraocular pressure lowering drug, or an artificial tear component; preferably, the antihistamine is olopatadine hydrochloride, the anti-inflammatory drug is dexamethasone, the intraocular pressure lowering drug is timolol, and the artificial tear component is sodium hyaluronate.

[0011] This invention provides a method for preparing the above-mentioned temperature-shear dual-response ophthalmic sustained-release drug formulation, comprising the following steps:

[0012] 1. Preparation of temperature-responsive targeted nanomedicine carriers

[0013] Preparation of PLGA-PEG copolymer: PLGA and PEG were added to a reaction vessel, and dibutyltin dilaurate catalyst was added. The reaction was carried out at 120-140℃ for 4-6 hours under nitrogen protection. After cooling, the copolymer was purified by dialyzing and freeze-dried to obtain PLGA-PEG copolymer.

[0014] Preparation of drug-loaded core: PLGA-PEG copolymer and corresponding active drug are dissolved in dichloromethane to form an organic phase, and 1%-3% polyvinyl alcohol aqueous solution is used as the aqueous phase. The volume ratio of organic phase to aqueous phase is 1:5-1:10. High-speed shear emulsification is carried out at 10000-15000 rpm for 10-20 minutes. The organic solvent is evaporated by magnetic stirring at 30-40℃ for 4-6 hours. The precipitate is collected by centrifugation, washed, and freeze-dried to obtain the drug-loaded core.

[0015] Preparation of temperature-responsive-targeting composite material: PNIPAM was dissolved in PBS buffer at pH 7.2-7.4, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added for activation for 30-60 minutes. Hyaluronic acid was added, and the reaction was carried out at room temperature for 8-12 hours. After dialysis purification, the composite material was obtained by freeze drying.

[0016] Preparation of nanocarriers: The active drug loaded core was dispersed in PBS buffer, the composite layer material was added, the reaction was stirred at 30°C for 4-6 hours, the product was collected by centrifugation, washed and freeze-dried to obtain temperature-responsive targeted nanocarriers.

[0017] 2. Preparation of non-Newtonian fluid matrix

[0018] Carbomer 940 powder was slowly added to sterile deionized water and stirred to swell for 24 hours. Triethanolamine was added to adjust the pH to 7.0-7.4, and the mixture was stirred evenly to obtain a non-Newtonian fluid matrix.

[0019] 3. Preparation of compound formulations

[0020] The temperature-responsive targeted nanomedicine carrier prepared in step 1 was added to the non-Newtonian fluid matrix prepared in step 2 at a mass fraction of 0.5%-2%. The mixture was dispersed for 30 minutes under low-speed stirring conditions of 30℃ and 200-300rpm. After filtration through a 0.22μm sterile filter membrane, a temperature-shear dual-responsive ophthalmic sustained-release drug formulation was obtained.

[0021] Further, in step 1, the mass ratio of EDC to PNIPAM is 0.2:1, and the mass ratio of NHS to EDC is 0.75:1; in step 3, the purpose of low-speed stirring is to avoid high-speed shearing damaging the temperature-responsive shell structure of the nanocarrier.

[0022] The present invention also protects the use of the above-mentioned ophthalmic sustained-release drug carrier in the preparation of ophthalmic drugs for treating allergic conjunctivitis, chronic dry eye syndrome or glaucoma.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Dual-response synergy for precise control: This invention innovatively fuses a temperature-responsive targeted nanocarrier with a pseudoplastic non-Newtonian fluid matrix, resulting in a composite formulation that simultaneously possesses shear-responsive and temperature-responsive characteristics. During eye drops, the shear force generated by eyelid compression causes a rapid decrease in matrix viscosity, enabling smooth drug delivery. After being instilled into the ocular surface, the shear force disappears, and the matrix quickly recovers its high viscosity, forming a gel film that fixes the nanocarrier to the ocular surface, preventing tear washout. When ocular inflammation occurs, the local temperature rises above 35°C, causing a phase transition in the nanocarrier's outer shell, PNIPAM, accelerating drug release. This achieves synergistic control throughout the entire process of "convenient drug delivery - long-lasting retention - precise drug release."

[0025] Significantly improved bioavailability: The non-Newtonian fluid matrix extends the residence time of nanocarriers on the ocular surface from 0.5-1 hours in traditional nano-formulations to 4-6 hours, increasing corneal penetration efficiency by 3-5 times; at the same time, the targeted ligand can guide the nanocarriers to the site of inflammation for precise enrichment, further increasing the drug concentration at the lesion site and reducing drug waste and irritation to normal ocular tissues.

[0026] Excellent formulation stability: The viscoelasticity of the non-Newtonian fluid matrix can effectively disperse the nanocarrier, preventing it from agglomerating during storage and extending the shelf life of the formulation; and both the matrix material Carbomer 940 and the nanocarrier material have good biocompatibility. After aseptic treatment, there is no obvious eye irritation, making it suitable for long-term administration.

[0027] High patient compliance: The shear-thinning properties of pseudoplastic non-Newtonian fluids make eye drops non-sticky and convenient to administer; at the same time, the long-lasting retention properties reduce the frequency of administration from 4-6 times a day with traditional eye drops to 1-2 times a day, greatly reducing the burden of medication for patients, and are especially suitable for special populations such as children and the elderly.

[0028] The preparation process is simple and controllable: It adopts a three-step method of "nanocarrier preparation - matrix preparation - low-speed dispersion and compounding". The process is mature, avoids the damage of high-speed shear to the nanocarrier structure, is easy to scale up and has good industrialization prospects. Detailed Implementation

[0029] Example 1: Preparation of a temperature-shear dual-responsive ophthalmic sustained-release drug formulation loaded with olopatadine hydrochloride non-Newtonian fluid

[0030] Preparation of PLGA-PEG copolymer: 7g PLGA and 3g PEG were added to a three-necked flask, and 0.1g dibutyltin dilaurate was added. The mixture was reacted at 130℃ for 5 hours under nitrogen protection. After cooling, it was dialyzed with deionized water for 3 days and then freeze-dried to obtain PLGA-PEG copolymer.

[0031] Preparation of drug-loaded core: 2g of PLGA-PEG copolymer and 0.3g of olopatadine hydrochloride were dissolved in 20mL of dichloromethane to form an organic phase. 200mL of 2% polyvinyl alcohol aqueous solution was used as the aqueous phase. After mixing the organic phase and the aqueous phase, the mixture was emulsified by high-speed shearing at 12000rpm for 15 minutes, and the organic solvent was evaporated by magnetic stirring at 35℃ for 5 hours. The precipitate was collected by centrifugation at 8000rpm, washed 3 times with deionized water, and freeze-dried to obtain the drug-loaded core.

[0032] Preparation of temperature-responsive-targeting composite material: 1g PNIPAM was dissolved in 100mL PBS buffer at pH 7.4, 0.2g EDC and 0.15g NHS were added, and after activation for 45 minutes, 0.1g hyaluronic acid-modified anti-CD44 monoclonal antibody was added. The reaction was carried out at room temperature for 10 hours, dialyzed with PBS buffer for 3 days, and then freeze-dried to obtain the composite material.

[0033] Preparation of nanocarriers: 1g of drug-loaded cores were dispersed in 50mL of PBS buffer (pH=7.4), 0.5g of composite layer material was added, and the mixture was stirred at 30℃ for 5 hours. The product was collected by centrifugation at 8000rpm, washed three times with PBS buffer, and freeze-dried to obtain a temperature-responsive targeted nanocarrier. The carrier was found to have a particle size of 150nm, a shell thickness of 35nm, a drug loading of 12%, and a targeting ligand grafting rate of 10%.

[0034] Preparation of a non-Newtonian fluid matrix: 1.0 g of carbomer 940 powder was slowly added to 100 mL of sterile deionized water, stirred and swollen for 24 hours, then triethanolamine was added to adjust the pH to 7.2, and the mixture was stirred until homogeneous to obtain the non-Newtonian fluid matrix. Testing showed that this matrix could withstand a shear force of 500 s⁻¹. -1 The viscosity at rest is 320 mPa·s; the viscosity at rest is 7500 mPa·s.

[0035] Preparation of composite formulation: 0.1g of the nanomedicine carrier obtained in step 1 was added to 10g of the non-Newtonian fluid matrix obtained in step 2, and dispersed by stirring at 30℃ and 250rpm for 30 minutes. After filtration through a 0.22μm sterile filter membrane, a temperature-shear dual-response ophthalmic sustained-release drug formulation loaded with olopatadine hydrochloride was obtained.

[0036] Example 2: Preparation of a temperature-shear dual-response ophthalmic sustained-release drug formulation loaded with dexamethasone

[0037] Preparation of PLGA-PEG copolymer: 9g PLGA and 1g PEG were added to a three-necked flask, and 0.1g dibutyltin dilaurate was added. The mixture was reacted at 140℃ for 4 hours under nitrogen protection. After cooling, it was dialyzed with deionized water for 3 days and then freeze-dried to obtain PLGA-PEG copolymer.

[0038] Preparation of drug-loaded cores: 2g of PLGA-PEG copolymer and 0.2g of dexamethasone were dissolved in 20mL of dichloromethane to form an organic phase. 150mL of 3% polyvinyl alcohol aqueous solution was used as the aqueous phase. After mixing the organic and aqueous phases, the mixture was emulsified by high-speed shearing at 15000rpm for 10 minutes. The organic solvent was evaporated by magnetic stirring at 40℃ for 4 hours. The precipitate was collected by centrifugation at 10000rpm, washed three times with deionized water, and freeze-dried to obtain the drug-loaded cores.

[0039] Preparation of temperature-responsive-targeting composite material: 1g PNIPAM was dissolved in 100mL PBS buffer (pH=7.2), 0.18g EDC and 0.12g NHS were added, and after activation for 30 minutes, 0.08g hyaluronic acid-modified anti-CD44 monoclonal antibody was added. The reaction was carried out at room temperature for 8 hours, dialyzed with PBS buffer for 3 days, and then freeze-dried to obtain the composite material.

[0040] Preparation of nanocarriers: 1g of drug-loaded core was dispersed in 50mL of PBS buffer (pH=7.2), 0.4g of composite layer material was added, and the mixture was stirred at 30℃ for 4 hours. The product was collected by centrifugation at 10000rpm, washed three times with PBS buffer, and freeze-dried to obtain a temperature-responsive targeted nanocarrier. The carrier was found to have a particle size of 120nm, a shell thickness of 25nm, a drug loading of 9%, and a targeting ligand grafting rate of 8%.

[0041] Preparation of a non-Newtonian fluid matrix: 0.8 g of carbomer 940 powder was slowly added to 100 mL of sterile deionized water, stirred and swollen for 24 hours, then triethanolamine was added to adjust the pH to 7.4, and the mixture was stirred until homogeneous to obtain the non-Newtonian fluid matrix. Testing showed that this matrix could withstand a shear force of 500 s⁻¹. -1 The viscosity at rest is 280 mPa·s; the viscosity at rest is 6200 mPa·s.

[0042] Preparation of composite formulation: 0.08g of the nanomedicine carrier obtained in step 1 was added to 10g of the non-Newtonian fluid matrix obtained in step 2, and dispersed by stirring at 30℃ and 200rpm for 30 minutes. After filtration through a 0.22μm sterile filter membrane, a temperature-shear dual-response ophthalmic sustained-release drug formulation loaded with dexamethasone was obtained.

Claims

1. A non-Newtonian fluid-based temperature-shear dual responsive ophthalmic sustained drug carrier, characterized in that, The application relates to a temperature-responsive targeted nanomedicine carrier, which comprises a dispersed phase and a continuous phase; the dispersed phase is the temperature-responsive targeted nanomedicine carrier; the continuous phase is a non-Newtonian fluid matrix; the mass fraction of the dispersed phase in the continuous phase is 0.5%-2%; the temperature-responsive targeted nanomedicine carrier is a two-layer structure of an inner core and an outer shell; the inner core is a drug loading layer which is composed of a PLGA-PEG copolymer and active drugs, the mass ratio of PLGA to PEG is 7:3-9:1, and the mass ratio of the active drugs to the PLGA-PEG copolymer is 1:5-1:10; the outer shell is a temperature-responsive-targeting composite layer which is grafted by poly-N-isopropyl acrylamide (PNIPAM) and hyaluronic acid, the lower critical solution temperature (LCST) of the PNIPAM is 35-37 DEG C, and the grafting rate of the targeting ligand is 5%-15%; the particle size of the nanocarrier is 100-200 nm, and the thickness of the outer shell is 20-50 nm; the non-Newtonian fluid matrix is a pseudoplastic fluid which is composed of carbomer 940 and triethanolamine, the mass concentration of the carbomer 940 is 0.5%-1.5%, and the addition amount of the triethanolamine is the required amount for adjusting the pH of the matrix to 7.0-7.4; the viscosity of the non-Newtonian fluid matrix is 100-500 mPa.s under a shear force of 500 s -1 , and the viscosity is 5000-10000 mPa.s when the non-Newtonian fluid matrix is at rest.

2. The non-Newtonian fluid-based temperature-shear dual responsive ophthalmic sustained-release drug carrier according to claim 1, characterized in that, The loaded active drug can be one or more of an antihistamine, an anti-inflammatory drug, an intraocular pressure-lowering drug, or an artificial tear component.

3. The non-Newtonian fluid-based temperature-shear dual responsive ophthalmic slow- release drug carrier according to claim 2, wherein, The loaded antihistamine is olopatadine hydrochloride, the loaded anti-inflammatory drug is dexamethasone, the loaded intraocular pressure-lowering drug is timolol, and the loaded artificial tear component is sodium hyaluronate.

4. A method for preparing a non-Newtonian fluid-based temperature-shear dual responsive ophthalmic sustained-release drug carrier according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) Preparation of temperature-responsive targeted nanodrug carrier PLGA and PEG are added to a reaction vessel, a catalyst dibutyltin dilaurate is added, and reaction is carried out at 120-140°C for 4-6 hours under nitrogen protection. After cooling, the product is purified by dialysis and freeze-dried to obtain PLGA-PEG copolymer. The PLGA-PEG copolymer and the active drug are dissolved in dichloromethane to form an organic phase, 1%-3% polyvinyl alcohol aqueous solution is used as the water phase, the volume ratio of the organic phase to the water phase is 1:5-1:10, high-speed shearing emulsification is carried out at a speed of 10,000-15,000 rpm for 10-20 minutes, the organic solvent is volatilized at 30-40°C under magnetic stirring for 4-6 hours, the precipitate is collected by centrifugation, and the product is obtained by freeze-drying after washing. The PLGA-PEG copolymer and the active drug are dissolved in dichloromethane to form an organic phase, 1%-3% polyvinyl alcohol aqueous solution is used as the water phase, the volume ratio of the organic phase to the water phase is 1:5-1:10, high-speed shearing emulsification is carried out at a speed of 10,000-15,000 rpm for 10-20 minutes, the organic solvent is volatilized at 30-40°C under magnetic stirring for 4-6 hours, the precipitate is collected by centrifugation, and the product is obtained by freeze-drying after washing. PNIPAM is dissolved in PBS buffer with pH=7.2-7.4, EDC and NHS are added and activated for 30-60 minutes, an appropriate amount of hyaluronic acid is added, and reaction is carried out at room temperature for 8-12 hours. The product is obtained by freeze-drying after dialysis and purification. The drug-loaded inner core is dispersed in PBS buffer, the composite layer material is added, and reaction is carried out at 30°C under stirring for 4-6 hours. The product is collected by centrifugation, and the product is obtained by freeze-drying after washing. (2) Preparation of non-Newtonian fluid matrix Carbopol 940 powder is slowly added to sterile deionized water, and stirring is carried out for swelling for 24 hours. Triethanolamine is added to adjust the pH to 7.0-7.4, and the non-Newtonian fluid matrix is obtained after uniform stirring. (3) Preparation of composite preparation The temperature-responsive targeted nanodrug carrier prepared in step (1) is added to the non-Newtonian fluid matrix prepared in step (2) at a mass fraction of 0.5%-2%, and is dispersed at 30°C under low-speed stirring at 200-300 rpm for 30 minutes. Filtration is carried out through a 0.22 μm sterile filter membrane to obtain a temperature-shear dual-responsive ophthalmic sustained-release drug preparation.

5. The preparation method according to claim 4, characterized in that, In step (1), the mass ratio of EDC to PNIPAM is 0.2:1, and the mass ratio of NHS to EDC is 0.75:

1.

6. The preparation method according to claim 4, characterized in that, In step (3), the purpose of low-speed stirring is to avoid the destruction of the temperature-responsive shell structure of the nanocarrier by high-speed shearing.

7. Use of the temperature-shear dual-responsive ophthalmic sustained-release drug carrier based on non-Newtonian fluid matrix according to any one of claims 1-3 in the preparation of an ophthalmic drug for treating allergic conjunctivitis, chronic dry eye, or glaucoma.