Drug-loaded polyacrylic resin nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drugs, and discloses drug-loaded polyacrylic resin nanoparticles as well as a preparation method and application thereof. Compared with a pharmaceutical preparation, the drug-loaded polyacrylic resin nanoparticle has the advantages of good slow release effect, no stimulation, long residence time, better treatment effect and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to drug-loaded polyacrylic acid resin nanoparticles and a preparation method and application thereof. Background Art

[0002] Glaucoma is a serious eye disease characterized by increased intraocular pressure, which is clinically manifested as pain, decreased vision, visual impairment, and in severe cases, can lead to complete blindness. Drugs for the treatment of glaucoma often include prostate analogs, cholinergic agonists, carbonic acid glycosides inhibitors, beta-blockers and other categories. Among them, timolol maleate is a non-selective beta-blocker. Because of its low cost and good efficacy, it has been used in the clinic for the treatment of glaucoma earlier. Currently, timolol eye drops used to treat glaucoma on the domestic and foreign markets have an intraocular pressure-lowering effect on both normal and high intraocular pressure patients. Timolol maleate lowers intraocular pressure quickly and reaches the maximum effect in 1 to 2 hours. Due to various physiological barriers and blinking reflexes in the eye, after administration, the drug stays in front of the cornea for a short time, and a large amount of drug is lost. In order to maintain the efficacy, this requires patients to increase the number of administrations. Part of the lost drug is absorbed into the blood and acts on the beta receptors of cells or tissues other than the eyes through the human circulatory system, causing adverse reactions such as bradycardia, arrhythmia, hypotension, headache, aggravated myasthenia gravis, and respiratory failure.

[0003] ABBVIE, a US company, launched a It is an eye drop for lowering intraocular pressure containing brimonidine tartrate and timolol maleate. The concentration of timolol maleate (0.5%) is not only high, but also needs to be administered twice a day; COSOPT, an eye drop for lowering intraocular pressure announced by THEA PHARMA, contains dorzolamide hydrochloride and timolol maleate. The concentration of timolol maleate is still very high (0.5%), and this eye drop is also administered twice a day on average; Although it can achieve an average of one dose per day, the high concentration of timolol maleate (0.5%) will also cause many adverse reactions. Duo, a drug launched by Alcon in the UK Although it is also administered once a day, it contains a high concentration of timolol (0.5%); The concentration of timolol maleate contained in the eye drops is high (0.5%) and also needs to be administered twice a day. Most of the timolol maleate eye drops available in China also have a concentration of 0.5% timolol maleate, and the 0.25% specification also needs to be administered twice a day.

[0004] Based on the above situation, theoretically speaking, a good glaucoma drug sustained-release system can not only improve bioavailability and have sustained-release ability, but also reduce the toxic and side effects of the drug (with higher safety). Summary of the invention

[0005] The first aspect of the present invention aims to provide drug-loaded polyacrylic acid resin nanoparticles.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention.

[0007] The third aspect of the present invention aims to provide an in-situ gel of drug-loaded polyacrylic acid resin nanoparticles.

[0008] The purpose of the fourth aspect of the present invention is to provide a method for preparing the in-situ gel of drug-loaded polyacrylic acid resin nanoparticles according to the third aspect of the present invention.

[0009] The purpose of the fifth aspect of the present invention is to provide the use of the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention or the drug-loaded polyacrylic acid resin nanoparticles in situ gel of the third aspect of the present invention in the preparation of eye drops.

[0010] The sixth aspect of the present invention aims to provide an eye drop.

[0011] The purpose of the seventh aspect of the present invention is to provide a method for preparing the eye drops of the sixth aspect of the present invention.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides drug-loaded polyacrylic acid resin nanoparticles, wherein the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticles include: drugs, polyacrylic acid resin, basic polysaccharides, and acidic mucopolysaccharides.

[0014] Preferably, the drug comprises at least one of timolol maleate, betaxolol hydrochloride, brimonidine tartrate, dexamethasone acetate, carteolol hydrochloride, levobunolol hydrochloride, and metipranolol hydrochloride; further comprises timolol maleate, betaxolol hydrochloride, and brimonidine tartrate; and further comprises timolol maleate.

[0015] Preferably, the polyacrylic acid resin comprises at least one of EDURAGIT RS PO, EDURAGIT RL PO, EDURAGIT EPO, EDURAGIT L 100-55, EDURAGIT 30D-55, EDURAGIT L 100, EDURAGIT RL 30D, EDURAGITRS 30D, EDURAGIT NE 30D, EDURAGIT RD 100, EDURAGIT E 100, EDURAGIT RL 100, EDURAGIT RS100, EDURAGIT R 100, EDURAGIT S100, polyacrylic acid resin II, polyacrylic acid resin III, polyacrylic acid resin IV, and carbomer cross-linked polyacrylic acid resin; and further comprises at least one of EDURAGIT RSPO, EDURAGIT RL PO, EDURAGIT E PO, EDURAGIT RL 100, and EDURAGIT RS100, and further comprises EDURAGIT RS PO.

[0016] Preferably, the alkaline polysaccharide comprises at least one of chitosan oligosaccharide, chitosan and chitin; further comprises chitosan.

[0017] Preferably, the acidic mucopolysaccharide comprises at least one of chondroitin sulfate, hyaluronic acid, dermatan sulfate, heparin and heparan sulfate; and further comprises hyaluronic acid.

[0018] Preferably, the mass ratio of the drug, polyacrylic acid resin, alkaline polysaccharide, and acidic mucopolysaccharide is 2040:(40-100):(50-125):(40-140); further 2040:(50-80):(75-100):(50-125); further 2040:(60-70):(90-100):(50-60).

[0019] Preferably, the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticles further include: an emulsifier.

[0020] Preferably, the emulsifier comprises at least one of poloxamer 188, poloxamer 407, Tween, polyvinyl alcohol, sodium cholate, and sodium deoxycholate; further comprises at least one of poloxamer 188 and Tween; and further comprises poloxamer 188.

[0021] Preferably, the mass ratio of the drug to the emulsifier is 2040:(100-400); further 2040:(112.5-375); further 2040:(325-375).

[0022] Preferably, the alkaline polysaccharide is an alkaline polysaccharide solution.

[0023] Preferably, the pH of the alkaline polysaccharide solution is 4-6; further 4.5-5.5.

[0024] Preferably, the solvent of the alkaline polysaccharide solution comprises at least one of water and phosphate buffer; further comprises water.

[0025] Preferably, the concentration of the alkaline polysaccharide solution is 0.0007-0.0011 g / mL; further 0.00075-0.001 g / mL; further 0.0009-0.001 g / mL.

[0026] Preferably, the alkaline polysaccharide solution is prepared by mixing the alkaline polysaccharide with a solvent, swelling (preferably swelling overnight), and adjusting the pH.

[0027] Preferably, the method for preparing the alkaline polysaccharide solution further comprises: filtering (preferably through a 0.45 μm microporous filter membrane).

[0028] Preferably, the acidic mucopolysaccharide is an acidic mucopolysaccharide solution.

[0029] Preferably, the solvent of the acidic mucopolysaccharide solution comprises at least one of water and phosphate buffer; further comprises water.

[0030] Preferably, the concentration of the permeabilized mucopolysaccharide solution is 0.0004-0.0013 g / mL; further 0.0005-0.00125 g / mL; further 0.0005-0.0006 g / mL.

[0031] Preferably, the preparation method of the acidic mucopolysaccharide solution is as follows: the acidic mucopolysaccharide is mixed with a solvent and swelled (preferably overnight swelling).

[0032] Preferably, the method for preparing the acidic mucopolysaccharide solution further comprises: filtering (preferably through a 0.45 μm microporous filter membrane).

[0033] Preferably, the polyacrylic acid resin is a polyacrylic acid resin solution.

[0034] Preferably, the solvent of the polyacrylic acid resin solution is an organic solvent.

[0035] Preferably, the organic solvent comprises at least one of ethanol, dichloromethane, chloroform, vinyl chloride, dimethyl sulfoxide, acetone, methyl acetate, and ether; further comprises ethanol; and further comprises anhydrous ethanol.

[0036] Preferably, the concentration of the polyacrylic acid resin solution is 0.005-0.020 g / mL; further 0.01-0.015 g / mL; further 0.013-0.014 g / mL.

[0037] Preferably, the polyacrylic acid resin solution is prepared by mixing and dissolving the polyacrylic acid resin with an organic solvent.

[0038] Preferably, the dissolution condition is ultrasonic treatment for 5-15 min.

[0039] Preferably, the average particle size of the drug-loaded polyacrylic acid resin nanoparticles is 240-1330 nm; further 307-335 nm; further 321±13.75 nm.

[0040] Preferably, the PDI of the drug-loaded polyacrylic acid resin nanoparticles is 0.17-0.48; further 0.34-0.36; further 0.350±0.004.

[0041] Preferably, the Zeta potential of the drug-loaded polyacrylic acid resin nanoparticles is +(5-22.7) mV; further +(17.4-21) mV; further +(19.26±1.77) mV.

[0042] The second aspect of the present invention provides a method for preparing the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention, wherein the method is a1) or a2):

[0043] a1):

[0044] mixing the drug with the alkaline polysaccharide to obtain a mixture A;

[0045] Mixing mixture A with acidic mucopolysaccharide to obtain mixture B;

[0046] Mixing the mixture B with polyacrylic acid resin to obtain drug-loaded polyacrylic acid resin nanoparticles;

[0047] a2):

[0048] Mixing the drug, the emulsifier and the alkaline polysaccharide to obtain a mixture A;

[0049] Mixing mixture A with acidic mucopolysaccharide to obtain mixture B;

[0050] The mixture B is mixed with polyacrylic acid resin to obtain drug-loaded polyacrylic acid resin nanoparticles (when the preparation raw materials include an emulsifier).

[0051] Preferably, in a1) and a2), the mixture A is mixed with the acidic mucopolysaccharide by adding the acidic mucopolysaccharide into the mixture A while stirring.

[0052] Preferably, the stirring speed is 500-1200 rpm; further 700-1000 rpm.

[0053] Preferably, the stirring time is 20-50 min, further 27-36 min.

[0054] Preferably, the stirring temperature is 16-33°C; further 20-30°C.

[0055] Preferably, the adding is dropwise addition; further, the adding is dropwise addition at a speed of 0.5-1.5 mL / min.

[0056] Preferably, in a1) and a2), the mixture B and the polyacrylic acid resin are mixed by ultrasonic mixing.

[0057] Preferably, the power of the ultrasound is 200-500W; further 300-450W; further 340-400W.

[0058] Preferably, the ultrasonication time is 5-20 min, further 10-20 min.

[0059] Preferably, in a1) and a2), the mixing of the mixture B and the polyacrylic acid resin further comprises a step of volatilizing the organic solvent.

[0060] Preferably, the method of volatilizing the organic solvent is stirring.

[0061] Preferably, the stirring rotation speed is 100-500 rpm, further 150-350 rpm; further 170-250 rpm.

[0062] Preferably, the stirring time is 1-3.5 h; further 1.5-3 h.

[0063] The third aspect of the present invention provides an in-situ gel of drug-loaded polyacrylic acid resin nanoparticles, wherein the raw materials for preparing the in-situ gel of drug-loaded polyacrylic acid resin nanoparticles include: the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention.

[0064] Preferably, the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel further include: an in-situ gel matrix and a thickener.

[0065] Preferably, the in situ gel matrix comprises at least one of gellan gum, methylcellulose, sodium alginate, poloxamer, carbomer, and chitosan; further comprises gellan gum.

[0066] Preferably, the thickener comprises at least one of xanthan gum, carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, propylene glycol alginate, sodium starch phosphate, sodium alginate, casein, sodium polyacrylate, polyoxyethylene, and polyvinyl pyrrolidone; and further comprises xanthan gum.

[0067] Preferably, the mass ratio of the in situ gel matrix, the thickener and the drug in the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention is (280-360):(280-360):204; further (300-340):(300-340):204; further (310-330):(310-330):204.

[0068] Preferably, the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel further include: an inorganic solvent.

[0069] Preferably, the inorganic solvent comprises at least one of water and phosphate buffer; further comprises water.

[0070] Preferably, the mass volume ratio (g:mL) of the mixture of the in-situ gel matrix and the thickener to the inorganic solvent is (0.004-0.012):1; further (0.006-0.01):1.

[0071] Preferably, the preparation raw materials further include: drugs, and the drugs are the drugs in the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention.

[0072] Preferably, the mass ratio of the drug to the drug in the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention is (110-150):204; further (120-140):204.

[0073] Preferably, the average particle size of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is 345-390 nm; further 367±21.94 nm.

[0074] Preferably, the PDI value of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is 0.38-0.40; further 0.390±0.053.

[0075] Preferably, the Zeta potential of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is +(18.5-22) mV; further, +(20.15±1.62) mV.

[0076] The fourth aspect of the present invention provides a method for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel of the third aspect of the present invention, which is b1) or b2):

[0077] b1):

[0078] The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A;

[0079] Mixing the mixture A with the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention to obtain an in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles;

[0080] b2):

[0081] The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A;

[0082] The mixture A, the drug and the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention are mixed to obtain an in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles (when the preparation raw materials include the drug).

[0083] Preferably, the in-situ gel matrix, thickener and inorganic solvent are mixed under the conditions of heating and stirring until dissolved.

[0084] Preferably, in b1) and b2), after the in-situ gel matrix, thickener and inorganic solvent are mixed, a filtering step is further included (preferably using a 0.8 μm microporous filter membrane for filtration).

[0085] The fifth aspect of the present invention provides the use of the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention or the drug-loaded polyacrylic acid resin nanoparticles in situ gel of the third aspect of the present invention in the preparation of eye drops.

[0086] The sixth aspect of the present invention provides an eye drop comprising: the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention, or the drug-loaded polyacrylic acid resin nanoparticles in situ gel of the third aspect of the present invention.

[0087] Preferably, the eye drops further comprise: at least one of an osmotic pressure regulator, a pH regulator, and an antibacterial agent; and further comprise an osmotic pressure regulator, a pH regulator, and an antibacterial agent.

[0088] Preferably, the raw materials for preparing the eye drops include: the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention, an osmotic pressure regulator, a pH regulator, an antibacterial agent, the in situ gel matrix, a thickener, a drug, and an inorganic solvent of the third aspect of the present invention.

[0089] Preferably, the osmotic pressure regulator comprises at least one of mannitol, lactose, sucrose, sodium chloride and glycerol; further comprises mannitol.

[0090] Preferably, the pH adjuster comprises at least one of tris(hydroxymethyl)aminomethane, sodium hydroxide, triethylamine, boric acid, hydrochloric acid, and phosphoric acid; further comprises tris(hydroxymethyl)aminomethane.

[0091] Preferably, the antibacterial agent comprises at least one of benzalkonium chloride, benzalkonium bromide, chlorobutanol and potassium sorbate; further comprising benzalkonium chloride.

[0092] Preferably, the concentration of the antibacterial agent in the eye drops is 0.1-0.67 mg / mL.

[0093] Preferably, the osmotic pressure of the eye drops is 270-320 mOsmol / kg; further 280-310 mOsmol / kg; further 301-305 mOsmol / kg.

[0094] Preferably, the pH of the eye drops is 6.0-8.0; further 6.5-7.5; further 7.0-7.5.

[0095] The seventh aspect of the present invention provides a method for preparing the eye drops of the sixth aspect of the present invention, which is c1), c2), or c3):

[0096] c1): mixing an osmotic pressure regulator, a pH regulator, and an antibacterial agent with the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention to obtain eye drops;

[0097] c2): mixing the osmotic pressure regulator, pH regulator, antibacterial agent with the in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles of the third aspect of the present invention to obtain eye drops;

[0098] c3):

[0099] The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A;

[0100] Mixing mixture A, the drug and antibacterial agent in the third aspect of the present invention and the drug-loaded polyacrylic acid resin nanoparticles in the first aspect of the present invention to obtain mixture B;

[0101] Mixture B is mixed with an osmotic pressure regulator and a pH regulator to obtain eye drops (the raw materials for preparing the eye drops include: the drug-loaded polyacrylic acid resin nanoparticles of the first aspect of the present invention, an osmotic pressure regulator, a pH regulator, an antibacterial agent, and the in-situ gel matrix, thickener, drug, and inorganic solvent of the third aspect of the present invention).

[0102] Preferably, the in-situ gel matrix, thickener and inorganic solvent are mixed under the conditions of heating and stirring until dissolved.

[0103] Preferably, in c3), after the in-situ gel matrix, thickener and inorganic solvent are mixed, a filtering step is further included (preferably using a 0.8 μm microporous filter membrane for filtration).

[0104] The beneficial effects of the present invention are:

[0105] The invention provides a drug-loaded polyacrylic acid resin nanoparticle. Compared with drug preparations, the drug-loaded polyacrylic acid resin nanoparticle has the advantages of good sustained-release effect, no stimulation, long retention time, better therapeutic effect, etc.

[0106] The present invention provides a drug-loaded polyacrylic acid resin nanoparticle in-situ gel, which has the advantages of good sustained-release effect, better therapeutic effect, etc. compared with drug-loaded polyacrylic acid resin nanoparticles, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 This is a physical picture of the diluted TM-HA / CS@ED NPs eye drops obtained in step (7) of Example 1 and a particle size distribution diagram of the TM-HA / CS@ED NPs therein.

[0108] Figure 2 It is an in vitro release curve diagram of timolol maleate aqueous solution (TM solution), TM-HA / CS@EDNPs eye drops prepared in Example 1, and timolol maleate polyacrylic acid resin nanoparticle in situ gel (TM-HA / CS@ED NPs ISG) eye drops prepared in Example 7, wherein n=3.

[0109] Figure 3 3 is a graph showing the mucin adhesion amount of the TM-HA / CS@ED NPs eye drops prepared in Example 1, wherein n=3.

[0110] Figure 4 It is a graph showing the results of hemolytic tests of the normal saline group (Negative group), deionized water group (Positive group), TMsolution group, TM-HA / CS@ED NPs eye drops group prepared in Example 1, and TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7.

[0111] Figure 5 It is a graph of the chicken embryo chorioallantoic membrane test results of the normal saline group (Negative group), sodium hydroxide group (Positive group), TMsolution group, TM-HA / CS@ED NPs eye drops group prepared in Example 1, and TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7.

[0112] Figure 6It is a graph of the trypan blue adsorption amount of the normal saline group (Negative group), the sodium hydroxide group (Positive group), the TMsolution group, the TM-HA / CS@ED NPs eye drops group prepared in Example 1, and the TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7, wherein n=3.

[0113] Figure 7 It is a graph showing the results of precorneal fluorescence retention of the TM solution group, the TM-HA / CS@ED NPs eye drops group prepared in Example 1, and the TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7.

[0114] Figure 8 The diagrams are the ocular hypotensive efficacy results of the normal saline group, the TM solution group, the TM-HA / CS@ED NPs eye drops group prepared in Example 1, and the TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7: wherein, (A) is a diagram of the changes in intraocular pressure during the construction of the chronic ocular hypertension model; (B) is a diagram of the ocular hypotensive efficacy results of the normal saline group, the TM solution group, the TM-HA / CS@ED NPs eye drops group prepared in Example 1, and the TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7, wherein n=3. DETAILED DESCRIPTION

[0115] The present invention is further described in detail below through specific examples.

[0116] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0117] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0118] In the present invention, "room temperature" is 25±5°C.

[0119] The reagents and their manufacturers used in the following examples are shown in Table 1.

[0120] Table 1

[0121]

[0122]

[0123] Note: The molecular weight of hyaluronic acid is 10-20k; the viscosity of chitosan is less than 200mpa.s.

[0124] Example 1 A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0125] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0126] (1) Accurately weigh 0.01 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5 and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0127] (2) Accurately weigh 0.005 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0128] (3) Accurately weigh 0.0375 g of poloxamer 188 and 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0129] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0130] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0131] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0132] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL, and the subsequent addition of mannitol, tris(hydroxymethyl)aminomethane, and antibacterial agent is very small, which basically does not affect the drug concentration, and the addition of antibacterial agent also basically does not affect the osmotic pressure and pH of the eye drops, and other embodiments are similar), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure the osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0133] Example 2 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0134] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0135] (1) Accurately weigh 0.01 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5 and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0136] (2) Accurately weigh 0.0125 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0137] (3) Accurately weigh 0.0375 g of poloxamer 188 and 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0138] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0139] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0140] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0141] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute it (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure its osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0142] Example 3 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0143] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0144] (1) Accurately weigh 0.0075 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5 and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0145] (2) Accurately weigh 0.005 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0146] (3) Accurately weigh 0.0375 g of poloxamer 188 and 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0147] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0148] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0149] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0150] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute it (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure its osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0151] Example 4 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0152] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0153] (1) Accurately weigh 0.01 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5, and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0154] (2) Accurately weigh 0.005 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0155] (3) Accurately weigh 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0156] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0157] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0158] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0159] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute it (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure its osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0160] Example 5 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0161] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0162] (1) Accurately weigh 0.01 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5, and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0163] (2) Accurately weigh 0.005 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0164] (3) Accurately weigh 0.01125 g of poloxamer 188 and 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0165] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0166] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0167] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0168] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute it (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure its osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0169] Example 6 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs)

[0170] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs), comprising the following steps:

[0171] (1) Accurately weigh 0.01 g of chitosan, add 10 mL of deionized water, seal, and swell overnight at 4°C, then adjust the pH to 5 and filter through a 0.45 μm microporous filter to obtain a chitosan solution.

[0172] (2) Accurately weigh 0.005 g of hyaluronic acid, add 10 mL of deionized water, swell overnight at 4°C, and filter through a 0.45 μm microporous filter to obtain a hyaluronic acid solution.

[0173] (3) Accurately weigh 0.01125 g of Tween 80 and 0.204 g of timolol maleate into the chitosan solution to obtain solution 1.

[0174] (4) At a rotation speed of 900 rpm and room temperature, the hyaluronic acid solution was dripped into solution 1 at a rate of 1 mL / min and stirred for 30 min to obtain solution 2.

[0175] (5) Accurately weigh 0.0066 g of EDURAGIT RS PO, add 0.5 mL of anhydrous ethanol, and sonicate for 10 min to dissolve the mixture to obtain Solution 3.

[0176] (6) Solution 3 was slowly added to solution 2, and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) (345 W, 10 min), and stirred at 200 rpm at room temperature for 2 h to evaporate the anhydrous ethanol to obtain timolol maleate polyacrylic acid resin nanoparticles (TM-HA / CS@ED NPs).

[0177] (7) Take 1 mL of the TM-HA / CS@ED NPs obtained above (the drug concentration of timolol maleate is 10.2 mg / mL), add 2 mL of deionized water to dilute it (the drug concentration of timolol maleate in the diluted TM-HA / CS@ED NPs is 3.4 mg / mL), and then add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure its osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg, and the pH value is 7.00. Finally, 0.002 g of benzalkonium chloride is added as an antibacterial agent to obtain TM-HA / CS@ED NPs eye drops.

[0178] Example 7 Preparation method of eye drops containing timolol maleate polyacrylic acid resin nanoparticles in situ gel (TM-HA / CS@EDNPs)

[0179] A method for preparing eye drops containing timolol maleate polyacrylic acid resin nanoparticles in situ gel (TM-HA / CS@ED NPs), comprising the following steps:

[0180] (1) Accurately weigh 0.32 g of gellan gum and 0.32 g of xanthan gum, add 80 mL of deionized water, heat and stir at 900 rpm to dissolve, and then filter through a 0.8 μm microporous membrane while hot.

[0181] (2) 0.136 g of timolol maleate, 0.01 g of benzalkonium chloride and TM-HA / CS@ED NPs (20 mL, 10.2 mg / mL based on the drug concentration of timolol maleate) prepared in step (6) of Example 1 were added and stirred evenly to obtain a mixture with a drug concentration of 3.4 mg / mL based on the drug concentration of timolol maleate.

[0182] (3) Then, mannitol and tris(hydroxymethyl)aminomethane were added in small amounts and repeatedly, stirred evenly, and the osmotic pressure and pH value were continuously measured. The final osmotic pressure value was 301 mOsmol / kg, and the pH value was 7, thus obtaining timolol maleate polyacrylic acid resin nanoparticles in situ gel (TM-HA / CS@ED NPs ISG) eye drops.

[0183] Comparative Example 1 A method for preparing eye drops containing timolol maleate nanoparticles (TM-HA / CS NPs)

[0184] A method for preparing eye drops containing timolol maleate nanoparticles is the same as Example 1, except that EDURAGIT RS PO is not included to obtain TM-HA / CS NPs eye drops.

[0185] Effect Example

[0186] 1. The diluted TM-HA / CS@ED NPs eye drops obtained in step (7) of Example 1-6 and the TM-HA / CS@ED NPs ISG eye drops obtained in step (3) of Example 7 were placed in sample pools, and the particle size, polydispersity index (PDI) and Zeta potential of the nanoparticles were measured using a nano laser particle size analyzer (Winner802, Jinan Micro-Nano Particle Instrument Co., Ltd.) and a ZETA potential meter (JS94J, Shanghai Zhongchen Digital Technology Equipment Co., Ltd.); the average particle size of the nanoparticles (TM-HA / CS@ED NPs) in the TM-HA / CS@ED NPs eye drops prepared in Examples 1-6 was 321±13.75 nm ( Figure 1 )、929.83±18.04nm, 261.02±23.23nm, 514.34±62.36nm, 642.61±18.46nm, 1244.19±84.64nm; the PDI values ​​were 0.350±0.004, 0.400±0.071, 0.355±0.026, 0.340±0.0 The average particle size of the nanoparticles (TM-HA / CS@ED NPs ISG) in the TM-HA / CS@ED NPs ISG eye drops prepared in Example 7 was 367±21.94nm, the PDI value was 0.390±0.053, and the Zeta potential was +(20.15±1.62)mV.

[0187] 2. TM solution is 100 mL of 3.4 mg / mL timolol maleate aqueous solution, add 0.01 g of benzalkonium chloride, add mannitol and tris(hydroxymethyl)aminomethane in small amounts and repeatedly, and continuously measure the osmotic pressure and pH value. The final osmotic pressure value is 301 mOsmol / kg and the pH value is 7.

[0188] 3. Preparation of artificial tears: Accurately weigh 0.678g NaCl, 0.138g KCl, 0.218g NaHCO 3 and 0.008 g CaCl2 ·2H 2 O in a beaker, add 100 mL of deionized water, stir until completely dissolved, and then adjust the pH value to 7.4 with hydrochloric acid.

[0189] The in vitro release of tacrolimus lipid microspheres was investigated by dynamic dialysis method, as follows:

[0190] (1) 4 mL of TM solution, TM-HA / CS@ED NPs eye drops prepared in Example 1, and TM-HA / CS@ED NPs ISG eye drops prepared in Example 7 were added to 8 cm dialysis bags (MD25-8000-14000, molecular weight cutoff 8000-14000D, purchased from Hunan YiBo Biotechnology Co., Ltd.), and both ends of the dialysis bag were tightened with dialysis clamps to ensure no leakage.

[0191] (2) Immerse the dialysis bag containing the sample in a beaker containing 100 mL of release medium (artificial tears). Start timing in a constant temperature oscillator set at 34°C and an oscillation rate of 120 rpm. Take 5 mL of drug-containing release medium at a specific time point and supplement with an equal amount of blank artificial tears release medium.

[0192] (3) The drug-containing release medium was filtered through a 0.22 μm microporous filter membrane, and the absorbance of the filtrate was measured in an ultraviolet spectrophotometer at a wavelength of 294 nm to obtain the in vitro release rate (cumulative release amount) of TM solution, TM-HA / CS@ED NPs eye drops prepared in Example 1, and TM-HA / CS@ED NPs ISG eye drops prepared in Example 7. The calculation formula is:

[0193]

[0194] Where Q n is the in vitro release rate, W is the drug content in 4 mL sample; C n is the drug concentration of the sample at the nth sampling; V1 is the sample volume of each sampling, and V is the volume of the release medium.

[0195] The results are as follows Figure 2 As shown in the figure, according to the requirements of the 2020 edition of the Chinese Pharmacopoeia for sustained-release preparations, in the in vitro release experiment, the cumulative release of sustained-release preparations within 0.5h must be less than 40%. The cumulative release rate of timolol maleate aqueous solution (TM aqueous solution, TM solution) in 0.5h reached 40.16%, the cumulative release rate of TM-HA / CS@ED NPs eye drops in 0.5h was 29.76%, and the cumulative release rate of TM-HA / CS@ED NPs ISG eye drops in 0.5h was 18.93%.

[0196] 4. Take different volumes (1-8 mL) of TM-HA / CS NPs eye drops prepared in Comparative Example 1 and TM-HA / CS@ED NPs eye drops prepared in Example 1, respectively, at 0.03 mg.mL -1 Two volumes of Bengal Rose Red (RB) solution were incubated together in an air bath constant temperature oscillator at room temperature in the dark for 2 hours. The samples were centrifuged for 30 minutes. The supernatant was filtered through a microporous filter membrane, and the absorbance was measured at the maximum absorption wavelength of RB. The corresponding concentration was calculated and the K value was calculated according to the Scatchard formula:

[0197]

[0198] where a is the RB concentration at equilibrium, r is the RB concentration bound to the eye drops, N is the maximum RB binding amount, and K is the hydrophobic binding constant.

[0199] The results are shown in Table 2: The K value of TM-HA / CS@ED NPs eye drops is much greater than that of TM-HA / CS NPs eye drops, indicating that TM-HA / CS@ED NPs encapsulated by EDURAGIT RS PO are highly hydrophobic and have affinity with the hydrophobic cornea, providing conditions for prolonging the retention time of the drug in front of the cornea.

[0200] Table 2 Hydrophobic binding constants of TM-HA / CS NPs eye drops and TM-HA / CS@ED NPs eye drops

[0201]

[0202] 5. Dissolve a certain amount of porcine gastric mucin powder in PBS solution (pH 7.4), shake well in an air bath at 34°C and 120 rpm for a period of time, and prepare 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL porcine gastric mucin solutions. The TM-HA / CS@ED NPs eye drops prepared in Example 1 and mucin solutions of different concentrations were placed in a centrifuge tube at a volume ratio of 1:1. The results are shown in Figure 3 As shown in the figure, with the increase of mucin concentration, the content of mucin adsorbed by the nanoparticles also increased further, and the two were highly positively correlated, indicating that TM-HA / CS@ED NPs eye drops had a strong interaction with mucin in the tear film, which was beneficial to prolong the retention time of nanoparticles in front of the cornea.

[0203] 6. Take fresh blood from New Zealand white rabbits in a centrifuge tube and add 0.1% (w / v) sodium heparin for anticoagulation. Add physiological saline to the blood for washing and centrifugation several times until the supernatant is clear to separate the red blood cells. Physiological saline was added to the red blood cell suspension as a negative control (Negative), and deionized water was added to the red blood cell suspension as a positive control (Positive). Each preparation (TM solution, TM-HA / CS@ED NPs eye drops prepared in Example 1, and TM-HA / CS@ED NPs ISG eye drops prepared in Example 7) was mixed with physiological saline (NS) in different volume ratios and added to the red blood cell suspension as the experimental group. All groups of suspensions were incubated in a constant temperature air bath oscillator (120rpm, 34°C) for 2h, and finally centrifuged to obtain the supernatant to observe the red blood cell hemolysis. Figure 4 It can be seen that the supernatant of the negative control group is clear and there is no erythrocyte hemolysis, the supernatant of the positive control group is obviously blood red and there is severe erythrocyte hemolysis, and the supernatant of the TM HA / CS@ED NPs eye drops group prepared in Example 1 and the TM HA / CS@ED NPs ISG eye drops group prepared in Example 7 are lighter in color than the timolol maleate aqueous solution (TM solution), indicating that TM HA / CS@ED NPs eye drops and TM HA / CS@ED NPs ISG eye drops are less irritating to erythrocytes.

[0204] 7. After fertilized eggs were wiped dry with 75% alcohol disinfection, they were placed in an incubator, the temperature was set to 37.8°C, appropriate amount of water was added to the water tank to maintain relative humidity between 65%-75%, and eggs were manually turned over, five times a day, and water was replenished in time when the hatching relative humidity decreased. When hatching on the 10th day, the chick embryo chorioallantoic membrane was basically formed, and eggs were taken out, and the air chamber position was observed and drawn according to the eggs. The eggshell was removed gently with tweezers along the line position, a little warm physiological saline was poured on the eggshell membrane surface to make it fully moist, and the eggshell membrane was carefully peeled off to expose the chick embryo chorioallantoic membrane (CAM), and the blood vessels of the CAM were observed to be broken to assess whether it could carry out subsequent experiments. Physiological saline was used as the negative control group (Negative), 0.1% sodium hydroxide solution was used as the positive control group (Positive), and timolol maleate aqueous solution (TM solution), TM HA / CS@ED NPs eye drops prepared in Example 1, and TM HA / CS@ED NPs ISG eye drops prepared in Example 7 were used as the experimental groups. 0.3 mL of each of the above solutions was dripped into the CAM and allowed to fully diffuse. After contact for 5 minutes, the surface of the CAM was gently rinsed with physiological saline within 30 seconds, and the rupture of the CAM blood vessels was observed immediately after the liquid was poured out. The irritation score (IS, the scoring criteria are shown in Table 3) of each group was calculated according to the formula. The results are shown in Table 4, Figure 5 After the observation, 0.5 mL of 0.1% (w / v) trypan blue PBS solution (pH 7.4) was immediately added to the CAM surface. After 1 min of contact, the CAM in the marked area was cut and rinsed with physiological saline several times and dry-cleaned. The excess water was absorbed by filter paper and weighed. 5 mL of formamide was added for overnight extraction. The supernatant was centrifuged and the absorbance was measured at 587 nm to obtain the trypan blue absorption, thereby calculating the trypan blue absorption per unit weight of CAM ( Figure 6 ). This indicates that both TM HA / CS@ED NPs eye drops and TM HA / CS@ED NPs ISG eye drops meet the safety requirements for ophthalmic preparations.

[0205] The calculation formula of IS is:

[0206]

[0207] Wherein, sec H is the initial time of vascular bleeding (s), sec L is the initial time of vascular lysis (s), and sec C is the initial time of vascular coagulation (s).

[0208] Table 3 Chicken embryo chorioallantoic membrane irritation scoring standard

[0209]

[0210] Table 4 Chicken embryo chorioallantoic membrane irritation scoring results

[0211]

[0212] 8. Nine healthy New Zealand white rabbits without eye diseases were selected and randomly divided into three groups, namely, the timolol maleate aqueous solution (TM solution) group, the TM HA / CS@ED NPs eye drops group prepared in Example 1, and the TM HA / CS@ED NPs ISG eye drops group prepared in Example 7. 0.008 g of sodium fluorescein was mixed with 4 mL of TM solution, TM HA / CS@ED NPs eye drops, and TM HA / CS@ED NPs ISG eye drops prepared in Example 7, respectively, and incubated in the dark at room temperature for 4 hours. Before the experiment, the rabbit eye condition was examined under normal illumination of a slit lamp, and then 100 μL of TM solution, TM HA / CS@ED NPs eye drops, and TM HA / CS@ED NPs ISG eye drops incubated with sodium fluorescein were dripped into the conjunctival sac of one side of the New Zealand rabbit (purchased from the Animal Center of Southern Medical University). The eyes were gently closed manually for 10 seconds, and the nasolacrimal duct was pressed at the same time to reduce sample loss. At specific time points, the fluorescence intensity was observed using a slit lamp and photographed under cobalt blue light. Figure 7As shown in the data, the in vivo corneal fluorescence retention time of the TMsolution group was 15.70±2.10min, that of the TM HA / CS@ED NPs eye drops group was 31.15±0.64min, and that of the TM HA / CS@ED NPs ISG eye drops group was 31.67±3.51min, indicating that TM HA / CS@ED NPs eye drops and TM HA / CS@ED NPs ISG eye drops can quickly gel in the conjunctival sac after contact with tears.

[0213] 9. A chronic ocular hypertension model was established by injecting compound carbomer suspension into the intracameral chamber of healthy New Zealand rabbits without eye disease (refer to the following literature for the method: LIU S, HAN X, LIU H, et al. Incorporation of ion exchangefunctionalized-mon tmorillonite into solid lipid nanoparticles with lowirritation enhances drug bioavailability for glaucoma treatment[J]. 2020, 27(1): 652-61.). The rabbits with successful modeling were randomly divided into 4 groups, with 3 rabbits in each group, namely, a normal saline group, a timolol maleate aqueous solution (TM solution) group, a TM HA / CS@ED NPs eye drops group prepared in Example 1, and a TM-HA / CS@ED NPs ISG eye drops group prepared in Example 7. 50 μL of saline, TM solution, TM HA / CS@ED NPs eye drops, and TM-HA / CS@ED NPs ISG eye drops were instilled into the conjunctival sac of one side of each rabbit, and the eyes were closed manually and the nasolacrimal duct was pressed for 10 seconds. Each preparation was repeated in 3 groups (n=3). IOP was measured at specific time points after administration. Figure 8 As shown in the figure: the IOP-lowering effect of TM-HA / CS@ED NPs ISG eye drops and TM HA / CS@ED NPs eye drops is longer than 12 hours, and the effective duration of IOP-lowering is significantly longer than the 6 hours of timolol maleate aqueous solution (TM solution), but the IOP-lowering amplitude of TM-HA / CS@ED NPs ISG eye drops is greater; where ΔIOP(%)=((IOP 0 -IOP t ) / IOP 0 )*100;IOP 0 is the initial intraocular pressure value, IO P t The intraocular pressure value measured at a certain point in time.

[0214] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A drug-loaded polyacrylic acid resin nanoparticle, wherein the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticle include: a drug, a polyacrylic acid resin, a basic polysaccharide, and an acidic mucopolysaccharide.

2. The drug-loaded polyacrylic acid resin nanoparticles according to claim 1, characterized in that: The drug comprises at least one of timolol maleate, betaxolol hydrochloride, brimonidine tartrate, dexamethasone acetate, carteolol hydrochloride, levobunolol hydrochloride, and metipranolol hydrochloride; further comprises timolol maleate, betaxolol hydrochloride, and brimonidine tartrate; further comprises timolol maleate; Preferably, the polyacrylic acid resin comprises at least one of EDURAGIT RS PO, EDURAGIT RL PO, EDURAGIT E PO, EDURAGIT L 100-55, EDURAGIT 30D-55, EDURAGIT L 100, EDURAGIT RL 30D, EDURAGIT RS30D, EDURAGIT NE 30D, EDURAGIT RD 100, EDURAGIT E 100, EDURAGIT RL 100, EDURAGIT RS100, EDURAGIT R 100, EDURAGIT S100, polyacrylic acid resin II, polyacrylic acid resin III, polyacrylic acid resin IV, and carbomer cross-linked polyacrylic acid resin; and further comprises at least one of EDURAGIT RS PO, EDURAGIT RL PO, EDURAGIT E PO, EDURAGIT RL 100, and EDURAGIT RS100; Preferably, the alkaline polysaccharide comprises at least one of chitosan oligosaccharide, chitosan and chitin; Preferably, the acidic mucopolysaccharide comprises at least one of chondroitin sulfate, hyaluronic acid, dermatan sulfate, heparin and heparan sulfate; Preferably, the mass ratio of the drug, polyacrylic acid resin, alkaline polysaccharide, and acidic mucopolysaccharide is 2040:(40-100):(50-125):(40-140).

3. The drug-loaded polyacrylic acid resin nanoparticles according to claim 1 or 2, characterized in that: The raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticles further include: an emulsifier; Preferably, the emulsifier comprises at least one of poloxamer 188, poloxamer 407, Tween, polyvinyl alcohol, sodium cholate, and sodium deoxycholate; and further comprises at least one of poloxamer 188 and Tween; Preferably, the mass ratio of the drug to the emulsifier is 2040:(100-400); Preferably, the average particle size of the drug-loaded polyacrylic acid resin nanoparticles is 240-13300 nm; Preferably, the PDI of the drug-loaded polyacrylic acid resin nanoparticles is 0.17-0.48; Preferably, the Zeta potential of the drug-loaded polyacrylic acid resin nanoparticles is +(5-22.7) mV.

4. Any of a1)-a2) methods: a1) A method for preparing drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 2, comprising the following steps: mixing the drug with the alkaline polysaccharide to obtain a mixture A; Mixing mixture A with acidic mucopolysaccharide to obtain mixture B; Mixing the mixture B with polyacrylic acid resin to obtain drug-loaded polyacrylic acid resin nanoparticles; a2) The method for preparing the drug-loaded polyacrylic acid resin nanoparticles according to claim 3, comprising the following steps: Mixing the drug, the emulsifier and the alkaline polysaccharide to obtain a mixture A; Mixing mixture A with acidic mucopolysaccharide to obtain mixture B; The mixture B is mixed with polyacrylic acid resin to obtain drug-loaded polyacrylic acid resin nanoparticles.

5. An in-situ gel of drug-loaded polyacrylic acid resin nanoparticles, wherein the raw materials for preparing the in-situ gel of drug-loaded polyacrylic acid resin nanoparticles comprise: the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3.

6. The drug-loaded polyacrylic acid resin nanoparticle in-situ gel according to claim 5, characterized in that: The raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel further include: an in-situ gel matrix and a thickener; Preferably, the in situ gel matrix comprises at least one of gellan gum, methylcellulose, sodium alginate, poloxamer, carbomer, and chitosan; Preferably, the thickener comprises at least one of xanthan gum, carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, propylene glycol alginate, sodium starch phosphate, sodium alginate, casein, sodium polyacrylate, polyoxyethylene, and polyvinyl pyrrolidone; Preferably, the mass ratio of the in situ gel matrix, the thickener and the drug in the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3 is (280-360): (280-360): 204; Preferably, the raw materials for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel further include: an inorganic solvent; Preferably, the inorganic solvent comprises at least one of water and phosphate buffer; Preferably, the preparation raw materials further comprise: the drug in the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3; Preferably, the average particle size of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is 345-390 nm; Preferably, the PDI value of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is 0.38-0.40; Preferably, the Zeta potential of the drug-loaded polyacrylic acid resin nanoparticle in-situ gel is +(18.5-22) mV.

7. The method for preparing the drug-loaded polyacrylic acid resin nanoparticle in-situ gel according to claim 6, which is b1) or b2): b1)： The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A; Mixing the mixture A with the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3 to obtain an in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles; b2)： The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A; The mixture A, the drug described in claim 6 and the drug-loaded polyacrylic acid resin nanoparticles described in any one of claims 1 to 3 are mixed to obtain an in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles.

8. An eye drop comprising: the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3, or the drug-loaded polyacrylic acid resin nanoparticle in-situ gel according to any one of claims 5 to 6.

9. The eye drops according to claim 8, characterized in that: The eye drops further comprise: at least one of an osmotic pressure regulator, a pH regulator, and an antibacterial agent; further comprise an osmotic pressure regulator, a pH regulator, and an antibacterial agent; Preferably, the raw materials for preparing the eye drops include: the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3, an osmotic pressure regulator, a pH regulator, an antibacterial agent, the in-situ gel matrix according to claim 6, a thickener, a drug, and an inorganic solvent; Preferably, the osmotic pressure regulator comprises at least one of mannitol, lactose, sucrose, sodium chloride, and glycerol; Preferably, the pH adjuster comprises at least one of tris(hydroxymethyl)aminomethane, sodium hydroxide, triethylamine, boric acid, hydrochloric acid, and phosphoric acid; Preferably, the antibacterial agent comprises at least one of benzalkonium chloride, benzalkonium bromide, chlorobutanol, and potassium sorbate; Preferably, the osmotic pressure of the eye drops is 270-320 mOsmol / kg; Preferably, the pH of the eye drops is 6.0-8.

0.

10. The method for preparing the eye drops according to claim 9, which is c1), c2), or c3): c1): mixing an osmotic pressure regulator, a pH regulator, an antibacterial agent with the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3 to obtain eye drops; c2): mixing the osmotic pressure regulator, pH regulator, antibacterial agent with the in-situ gel of the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 5 to 6 to obtain eye drops; c3): The in-situ gel matrix, the thickener and the inorganic solvent are mixed to obtain a mixture A; Mixing the mixture A, the drug, the antibacterial agent and the drug-loaded polyacrylic acid resin nanoparticles according to any one of claims 1 to 3, A mixture B is obtained; The mixture B is mixed with an osmotic pressure regulator and a pH regulator to obtain eye drops.

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