Ophthalmic polyzwitterionic nanopharmaceutical carrier, nanofomulation and use

Abstract

The application belongs to the field of ophthalmic nano-preparations, and particularly relates to an ophthalmic poly-nitrogen-oxygen amphoteric nano-drug carrier, an ophthalmic nano-preparation and a use. The nano-drug carrier is prepared by using a random copolymer of 2-(N-oxide-N,N-diethylamino)ethyl methacrylate and butyl methacrylate, has an average particle size of 7-12 nm, and can be used for loading ophthalmic drugs such as cyclosporine A. Research shows that the nano-drug carrier has good corneal retention characteristics and corneal permeability, and after loading cyclosporine A, has a good relieving effect on corneal damage and local inflammation of dry eye model animals, and the safety of the nano-drug carrier is good, and the nano-drug carrier has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of ophthalmic nanoparticle technology, specifically relating to an ophthalmic polynitrogen-oxygen zwitterionic nanoparticle drug carrier, nanoparticle formulation, and its uses. Background Technology

[0002] Treatment for eye diseases such as glaucoma, cataracts, and dry eye usually requires oral medications, injectable medications, or topical ophthalmic preparations, such as eye drops. Among these, topical ophthalmic preparations such as eye drops can overcome the loss caused by the first-pass effect of oral medications and the limited distribution of oral medications in the eye, as well as the inconvenience of injectable medications.

[0003] Treatment of ocular diseases requires medications to remain on the ocular surface for extended periods and penetrate deep into the cornea. However, multiple physiological mechanisms within the eye, including rapid tear clearance, nasolacrimal duct drainage, the mucus layer, the corneal barrier, and the blood-ocular barrier, significantly limit drug retention time and penetration efficiency. Therefore, precisely delivering medications to specific sites in the anterior or posterior segment of the eye has become a major challenge in the field of ocular drug delivery.

[0004] Wei et al. prepared zwitterionic micelles composed of amphiphilic molecules such as poly(N-oxide-N,N-diethylamino)ethyl methacrylate-block-polycaprolactone (OPDEA-PCL) as corneal drug delivery carriers, achieving non-invasive intraocular delivery of brinzolamide (Wei Q, Zhu C, Yuan G, et al. Active trans-corneal drug delivery with ocular adhesive micelles for efficient glaucomatherapy[J]. Journal of Controlled Release, 2025, 377:578-590.). However, its ocular retention effect and drug delivery characteristics still need to be improved. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide an ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier, nanoformulation, and its uses. This invention includes the following technical solutions:

[0006] An ophthalmic polyoxo-zwitterionic nanomedicine carrier is disclosed, wherein the ophthalmic polyoxo-zwitterionic nanomedicine carrier is prepared from a random copolymer of 2-(N-oxide-N,N-diethylamino)ethyl methacrylate (ODEA) and butyl methacrylate (BMA). This nanomedicine carrier is named poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-random-butyl methacrylate], abbreviated as P(ODEA-co-BMA].

[0007] Preferably, the average particle size of the ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier is 7-12 nm.

[0008] Preferably, the preparation method of the ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier includes the following steps:

[0009] Synthesis of S1-oxidized 2-(N,N-diethylamino)ethyl methacrylate: 2-(N,N-diethylamino)ethyl methacrylate and 3-chloroperoxybenzoic acid were weighed and dissolved separately in dichloromethane. Under ice bath conditions for 30 min, the dichloromethane solution of 2-(N,N-diethylamino)ethyl methacrylate was added dropwise to the dichloromethane solution of 3-chloroperoxybenzoic acid. The reaction was stirred overnight at room temperature. After the reaction was completed, the oxidized 2-(N,N-diethylamino)ethyl methacrylate was obtained by purification, namely 2-(N-oxide-N,N-diethylamino)ethyl methacrylate.

[0010] Preparation of S2 polynitrogen-oxygen zwitterionic nanoparticle drug carrier: 2-bromoisobutyrate ethyl ester, 2-(N-oxide-N,N-diethylamino) ethyl methacrylate, n-butyl methacrylate, and pentamethyldiethylenetriamine were placed in a sealed tube. Anhydrous N,N-dimethylformamide was added to dissolve the mixture. Before sealing the tube, a magnetic pole was added, and the solution was frozen through liquid nitrogen. After evacuation and thawing, the freeze-thaw cycle was repeated. CuBr was added, and the tube opening was sealed under vacuum. The reaction was carried out in an oil bath at 30℃~60℃ for 60h~96h. After the reaction, the product was purified. The filtrate was rotary evaporated and precipitated with petroleum ether. The precipitate was dissolved, transferred to a dialysis bag, and dialyzed with pure water. The dialysis product was freeze-dried to obtain poly[2-(N-oxide-N,N-diethylamino) ethyl methacrylate-random-butyl methacrylate].

[0011] Preferably, in step S1, the molar ratio of 2-(N,N-diethylamino) methacrylate to 3-chloroperoxybenzoic acid is (1~3):(2~4); in step S2, the molar ratio of 2-(N-oxide-N,N-diethylamino) methacrylate to n-butyl methacrylate is (198~200):(197~200), the molar ratio of ethyl 2-bromoisobutyrate to pentamethyldiethylenetriamine is (495~499):(498~502), and the molar ratio of ethyl 2-bromoisobutyrate to 2-(N-oxide-N,N-diethylamino) methacrylate is (4.95~4.99):(198~200).

[0012] An ophthalmic nano-formulation, wherein the drug carrier of the ophthalmic nano-formulation is the aforementioned ophthalmic polynitrogen-oxygen zwitterionic nano-drug carrier. Preferably, the active ingredient of the ophthalmic nano-formulation is cyclosporine A.

[0013] The aforementioned ophthalmic polynitrogen amphoteric nanoparticle drug carrier is used in the preparation of ophthalmic topical drug delivery formulations for dry eye syndrome.

[0014] Beneficial effects: Compared with existing ophthalmic nanoparticles and carriers, the ophthalmic nanoparticle carrier of the present invention exhibits significantly improved corneal retention and penetration, and demonstrates good safety. The cyclosporine A ophthalmic nanoparticle prepared therefrom has a mitigating effect on corneal damage in a mouse model of dry eye, with a rapid improvement rate, and significantly enhances the reduction of ocular inflammatory factor levels in the mouse model of dry eye. Attached Figure Description

[0015] Figure 1 The 1H NMR spectrum of PEG-PCL in Example 1;

[0016] Figure 2 The 1H NMR spectrum of cRGD-PEG-PCL in Example 1;

[0017] Figure 3 The 1H NMR spectra of ODEA (a), PCL-OH / PCL-Br (b) and PODEA-PCL (c) in Example 1;

[0018] Figure 4 The 1H NMR spectrum of P(OEGMA-co-BMA) in Example 1;

[0019] Figure 5 The following are examples from Example 1: (a) the 1H NMR spectra of different P(ODEA-co-BMA) and (b) the calculated actual molar ratio of ODEA repeating units;

[0020] Figure 6 Particle size distribution diagrams of different formulations in Example 1;

[0021] Figure 7 Zeta potential diagrams for different formulations in Example 1;

[0022] Figure 8 The in vitro release curves of different formulations in Example 1 are shown.

[0023] Figure 9 The results of the concentration detection experiments of different formulations in an in vitro (a) monolayer corneal epithelial cell model and (b) monolayer corneal epithelial cell and mucus layer model in Example 2 are shown.

[0024] Figure 10 Example 2 shows the ocular surface retention experiment of different formulations in mice with dry eye disease: (a) in vivo imaging and (b) results of fluorescence quantitative analysis of the in vivo imaging.

[0025] Figure 11 Example 2 shows the rabbit corneal permeability experiment with different formulations: (a) fluorescence images of the corneal epithelium and endothelium, (b) quantitative fluorescence analysis results of the corneal epithelium, and (c) quantitative fluorescence analysis results of the corneal endothelium.

[0026] Figure 12 The results of the experiment on the regulation of inflammatory factors (a) TNF-α, (b) IL-1β and (c) IL-6 by different formulations in Example 3 are shown. Detailed Implementation

[0027] The technical solutions and effects of the present invention will be shown and described below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection.

[0028] The source information of the chemical materials used in the following examples is shown in Table 1. Unless otherwise specified, the chemical raw materials and reagents used in the research of the examples are all commercially available or prepared using existing technology methods.

[0029] Table 1. Sources of chemical materials and reagents used in the examples

[0030]

[0031]

[0032] Example 1: Preparation of different ophthalmic nanoformulations and study on loading efficiency and in vitro release

[0033] This embodiment provides the preparation methods and in vitro release test results of the ophthalmic nanoformulations and control formulations involved in this study. Using cyclosporine A (CsA) as a model drug, polyethylene glycol-polycaprolactone nanoformulations were constructed, namely PEG-PCL-CsA, cRGD peptide-modified polyethylene glycol-polycaprolactone nanoformulations, namely cRGD-PEG-PCL-CsA, poly(2-(N-oxide-N,N-diethylamino)ethyl methacrylate-polycaprolactone nanoformulations, namely PODEA-PCL-CsA, and poly[(oligoethylene glycol) methyl ether methacrylate-random-tert-butyl methacrylate] nanoformulations, namely P(… OEGMA-co-BMA)-CsA; poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-random-n-butyl methacrylate] nanoformulation, namely P(ODEA-co-BMA)-CsA; the synthesized carrier was characterized by proton nuclear magnetic resonance spectroscopy, the particle size and potential of the nanoformulation were characterized by dynamic light scattering (DLS), and finally the drug loading efficiency and in vitro release characteristics of the nanoformulation were studied by ultra-high performance liquid chromatography (UPLC).

[0034] 1 Synthesis of Polymers for Nanoparticle Carriers

[0035] 1.1 Synthesis of PEG-PCL

[0036] Accurately weigh monohydroxy polyethylene glycol (PEG) with a molecular weight of approximately 2000. 45 -OH (4 g, 0.002 mol) and ε-caprolactone (9.12 g, 0.08 mol) were added to a dried 250 mL reaction flask. The reaction was carried out under vacuum, with 20 mL of dehydrated toluene added and the oil bath temperature maintained at 45°C. Dehydration was achieved through azeotropic distillation with toluene until no bubbles were generated in the large reaction flask within 3 minutes. This azeotropic distillation process was repeated twice more. After the final azeotropic distillation, the toluene was evaporated completely. Stannous isooctanoate (81 mg, 0.0002 mol) was accurately weighed into a 50 mL reaction flask, and approximately 10 mL of dehydrated toluene was added. The reaction was then carried out under vacuum, with azeotropic distillation of toluene to remove any remaining water.

[0037] After the toluene in both the large and small reaction flasks had completely evaporated, nitrogen gas was introduced into the small reaction flask. 40 mL of dehydrated toluene was added to the small reaction flask in portions. Stannous isooctanoate was then added to the large reaction flask using a solvent transfer needle. After complete transfer, the large reaction flask was filled with nitrogen gas and reacted at 105°C for 6 h. After the reaction was complete and cooled to room temperature, the product was dissolved in tetrahydrofuran and precipitated twice with petroleum ether. The supernatant was removed, and the lower layer was collected and dried under vacuum to obtain the polyethylene glycol-polycaprolactone block copolymer PEG-PCL. An appropriate amount of the product was subjected to NMR analysis. The 1H NMR spectrum of the PEG-PCL product is shown below. Figure 1As shown in the image, the peak at position b at 3.64 ppm corresponds to the methylene proton of the –O–CH2–CH2– segment in the polyethylene glycol chain, and is the strongest peak in the spectrum. The triplet at 4.06 ppm and the peak at position g correspond to the methylene proton (–O–CH2–) at the end of the polycaprolactone segment bonded to an oxygen atom, indicating the successful preparation of PEG-PCL. The degree of polymerization was calculated: PEG has a degree of polymerization of 45, and PCL has a degree of polymerization of 40 (hereinafter referred to as PEG-PCL).

[0038] 1.2 Synthesis of cRGD-PEG-PCL

[0039] Accurately weigh PEG-PCL (120 mg, 11 µmol) and add it to 4.5 mL of borate buffer (0.01 M, pH=9.1) containing the penetrating peptide (also known as the membrane-penetrating peptide) cRGD (13.6 mg, 22 µmol). Then add 1.5 mL of dimethyl sulfoxide solution and stir magnetically at room temperature for 16 h. After the reaction is complete, dialyze the product in pure water (molecular weight cutoff 3500 Da) for 48 h. After freeze-drying, the cRGD-modified polyethylene glycol-polycaprolactone amphiphilic block copolymer, cRGD-PEG-PCL, is obtained. Take an appropriate amount of the product for NMR analysis. The 1H NMR spectrum of cRGD-PEG-PCL is shown below. Figure 2 As shown. Analysis using DMSO-d6 as the solvent revealed a solvent peak at 2.51 ppm and a water peak at 3.37 ppm. The absorption peak at 3.51 ppm corresponds to hydrogen atoms in the repeating unit of polyethylene glycol. The absorption peaks at 1.30 ppm, 1.54 ppm, 2.30 ppm, and 3.99 ppm, at positions a, b, c, and d, correspond to polycaprolactone hydrogen atoms in the structure. The peaks at 7.22 ppm, at positions g and f, correspond to cRGD in the structure. This indicates that cRGD was successfully grafted onto PEG-PCL.

[0040] 1.3 Synthesis of PODEA-PCL diblock copolymer support

[0041] 1.3.1 Synthesis of Monomer ODEA

[0042] Accurately weigh 3.7 g (0.02 mol) of 2-(N,N-diethylamino)methacrylate (DEA) and 5.18 g (0.03 mol) of 3-chloroperoxybenzoic acid (m-CPBA), respectively, and dissolve them in 30 mL and 50 mL of dichloromethane, respectively. Under ice bath conditions for 30 min, the dichloromethane solution of DEA was added dropwise to the dichloromethane solution of m-CPBA, and the reaction was carried out overnight at room temperature with magnetic stirring. After the reaction was complete, the crude product was first concentrated by rotary evaporation, then passed through a neutral alumina column using methanol:ethyl acetate = 1:2 as the eluent. The corresponding sample solution was collected, and finally the solvent was removed by rotary evaporation to obtain a pale yellow clear liquid (2.97 g, yield: 80.27%), namely 2-(N-oxide-N,N-diethylamino)methacrylate (ODEA). An appropriate amount of the product was subjected to NMR analysis. The 1H NMR spectrum of ODEA is shown below. Figure 3 As shown in Figure a, the absorption peaks at 3.28 ppm and 3.52 ppm represent two hydrogen atoms in different environments surrounding nitrogen and oxygen, thus confirming the synthesis of the product ODEA.

[0043] 1.3.2 Hydrophobic Block PCL 38 Synthesis of -OH

[0044] Accurately weigh dodecanol (0.373 g, 0.002 mol) and ε-caprolactone (9.12 g, 0.08 mol) into a dried 250 mL reaction flask. Under vacuum, add 20 mL of dehydrated toluene and maintain an oil bath temperature of 60 °C. Remove water by azeotropic distillation with toluene until no bubbles are generated in the large reaction flask within 3 minutes. Repeat the azeotropic distillation process twice more, adding 20 mL of dehydrated toluene. After the final azeotropic distillation, evaporate until the toluene is completely evaporated. Accurately weigh stannous isooctanoate (81 mg, 0.0002 mol) into a 50 mL reaction flask, add approximately 2 mL of dehydrated toluene, and maintain vacuum. Remove any remaining water from the stannous isooctanoate by azeotropic distillation with toluene.

[0045] After the toluene in both the large and small reaction flasks had completely evaporated, nitrogen gas was introduced into the small reaction flask. 40 mL of dehydrated toluene was added to the small reaction flask in portions. Stannous isooctanoate was then added to the large reaction flask using a solvent transfer needle. After complete transfer, the large reaction flask was filled with nitrogen gas and reacted at 105°C for 7 hours. The product was dissolved in tetrahydrofuran and then precipitated twice with petroleum ether at a 1:5 (volume ratio). The supernatant was removed, and the lower layer was collected and dried under vacuum. An appropriate amount of the product was analyzed by NMR. The product PCL... 38 The 1H NMR spectrum of -OH is as follows: Figure 3 As shown in b, the absorption peak at 2.32 ppm represents the hydrogen atoms in the 2n repeating units of polycaprolactone under the same conditions, with a peak area of ​​76.47 / 2 = 38.23. The NMR integral results confirm that the degree of polymerization is 38.

[0046] 1.3.3 Initiator PCL 38 Synthesis of -Br

[0047] Accurately weigh polycaprolactone (PCL) 38 -OH (2.72 g, 0.0006 mol) and triethylamine (0.61 g, 0.006 mol) were placed in a 250 mL reaction flask and dissolved in 20 mL of dichloromethane. Then, 1.38 g, 0.006 mol of 2-bromoisobutyryl bromide diluted in dichloromethane was added dropwise under ice bath conditions. After the addition was complete, the mixture was stirred at room temperature for two days. After the reaction was complete, the solution was concentrated to approximately 10 mL using a rotary evaporator. Precipitation was then carried out with petroleum ether, and the petroleum ether was removed by filtration. The precipitate was dissolved in tetrahydrofuran, and petroleum ether was added dropwise to precipitate the precipitate. The precipitate was then filtered and dried under vacuum to obtain a white solid (2.25 g, yield: 80.4%), namely polycaprolactone-bromine (PCL). 38 -Br). Take an appropriate amount of the product for NMR analysis. PCL 38 -Br 1H NMR spectrum as follows Figure 3 As shown in b, an absorption peak appears at 1.9 ppm, which is PCL. 38 -Br contains hydrogen atoms on two methyl groups in the same environment.

[0048] 1.3.4 Synthesis of PODEA-PCL

[0049] Accurately weigh PCL 38 -Br(199 mg, 4.26 × 10⁻⁶) -5 2-(N-oxide-N,N-diethylamino)ethyl methacrylate (ODEA) (400 mg, 1.99 × 10⁻⁶ mol) -3 mol), pentamethyldiethylenetriamine, i.e., PMDETA (8.65 mg, 5.00 × 10⁻⁶ mol), -5 Add 1.8 mL of dehydrated N,N-dimethylformamide to a 5 mL sealed tube. Before sealing, add a magnetic stir bar, pass the solution through liquid nitrogen to freeze, evacuate for 5 minutes, and then thaw. Repeat the freeze-thaw operation three times. After the fourth freezing, add CuBr (7.2 mg, 5.00 × 10⁻⁶ mol) to the solution. -5The tube was heated to 1 mol under vacuum for 15 min, and the tube opening was sealed with a blowtorch under vacuum. The tube was then placed in an oil bath at 40°C for 72 h. After the reaction, the crude product was passed through a neutral alumina column using a tetrahydrofuran:methanol = 1:1 eluent. The solution was concentrated to approximately 10 mL and transferred to a dialysis bag (molecular weight cutoff 3500 Da). Dialysis was performed with pure water for 24 h. After freeze-drying, a pale yellow powder (413 mg, yield: 68.95%) was obtained, namely poly(N-oxide-N,N-diethylamino)ethyl methacrylate-polycaprolactone (PODEA-PCL). An appropriate amount of the product was analyzed by NMR. The 1H NMR spectrum of PODEA-PCL is shown below. Figure 3 As shown in c. The absorption peak at 2.32 ppm corresponds to the hydrogen atoms in the 2n repeating units of polycaprolactone under the same conditions, and the absorption peak at around 3.28 ppm corresponds to the hydrogen atoms in the 4n repeating units of 2-(N-oxide-N,N-diethylamino)ethyl methacrylate under the same conditions. The degree of polymerization is calculated to be (36.09 + 86.4) / 6 = 20.41, and the degree of polymerization of PODEA is 20. The NMR integral results are consistent with the target product PODEA. 20 -PCL 38 (Hereinafter referred to as PODEA-PCL).

[0050] 1.4 Synthesis of P(OEGMA-co-BMA)

[0051] Accurately weigh 20 mg of ethyl 2-bromoisobutyrate (1.03 × 10⁻⁶). -4 mol), poly(oligomeric glycol) methyl ether methacrylate (OEGMA) (4.8721 g, 0.009744 mol), n-butyl methacrylate (BMA) (2.7837 g, 0.0196 mol), pentamethyldiethylenetriamine (PMDETA) (33.74 mg, 1.95 × 10⁻⁶ mol), poly(oligomeric glycol) methyl ether methacrylate (OEGMA) (4.8721 g, 0.009744 mol), poly(oligomeric glycol) methacrylate (BMA) (2.7837 g, 0.0196 mol), poly(oligomeric glycol) methacrylate (PMDETA) (33.74 mg, 1.95 × 10⁻⁶ mol), poly(oligomeric glycol) methacrylate (OEGMA) (4.8721 g, 0.0097 -4 Add 7.2 mg of CuBr (5.00 × 10⁻⁶ mol) to a 50 mL sealed tube, then add 20 mL of dehydrated tetrahydrofuran to dissolve it. After sealing the tube with a magnetic buoy, pass it through liquid nitrogen to freeze the solution. After evacuating for 5 minutes, thaw the solution. Repeat the freeze-thaw cycle three times. After the fourth freezing, add CuBr (7.2 mg, 5.00 × 10⁻⁶ mol). -5The product was evacuated to a vacuum for 15 minutes, and the tube opening was sealed with a blowtorch under vacuum. The mixture was then placed in an oil bath at 40°C for 24 hours. After the reaction, the crude product was passed through a neutral alumina column using tetrahydrofuran as the eluent, followed by rotary evaporation for concentration. The product was precipitated with petroleum ether; the precipitate was viscous. The viscous liquid was dissolved and transferred to a dialysis bag (molecular weight cutoff 1000 Da), and dialyzed with pure water for two days. After freeze-drying, a pale yellow viscous solid (3.904 g, yield: 51.2%) poly[(oligoethylene glycol) methyl ether methacrylate-random-tert-butyl methacrylate], i.e., P(OEGMA-co-BMA), was obtained. An appropriate amount of the product was analyzed by NMR. The 1H NMR spectrum of P(OEGMA-co-BMA) is shown below. Figure 4 As shown. In the range of 3.5–4.5 ppm, the peaks at positions f and j correspond to methylene hydrogen atoms directly bonded to oxygen atoms in the structure. In the range of 0.5–2.0 ppm, the peaks at positions a, c, d, e, h, i, k, l, and m correspond to alkyl hydrogen atoms in the structure, with the characteristic peak at position g also located near the shift range of alkyl methylene hydrogen atoms bonded to oxygen atoms within this range.

[0052] 1.5 Synthesis of P(ODEA-co-BMA) and Screening of ODEA / BMA Feed Ratio

[0053] Accurately weigh ethyl 2-bromoisobutyrate (9.69 mg, 4.97 × 10⁻⁶). -5 2-(N-oxide-N,N-diethylamino)ethyl methacrylate, i.e., ODEA (400 mg, 1.99 × 10⁻⁶ mol), is a methyl methacrylate (Methyl methacrylate). -3 mol), n-butyl methacrylate, i.e., BMA (282.5 mg, 1.98 × 10⁻⁶ mol), -3 mol), pentamethyldiethylenetriamine, i.e., PMDETA (8.65 mg, 5.00 × 10⁻⁶ mol), -5 Add 1 mol of ODEA to a 5 mL sealed tube, then add 2 mL of dehydrated N,N-dimethylformamide to dissolve it (where the molar ratio of ODEA to BMA is 199:198). Before sealing the tube, add a magnetic stir bar, pass the solution through liquid nitrogen to freeze, evacuate for 5 minutes, and then thaw. Repeat the above freeze-thaw operation three times. After the fourth freezing, add CuBr (7.2 mg, 5.00 × 10⁻⁶ mol). -5The product was evacuated to a vacuum for 15 min, and the tube opening was sealed with a blowtorch under vacuum. The mixture was then placed in an oil bath at 40°C for 72 h. After the reaction, the crude product was passed through a neutral alumina column using a tetrahydrofuran:methanol = 1:1 eluent. The filtrate was concentrated by rotary evaporation and precipitated with petroleum ether. The precipitate was viscous; the viscous liquid was dissolved and transferred to a dialysis bag (molecular weight cutoff 1000 Da). Dialysis was performed with pure water for two days. After freeze-drying, a pale yellow viscous solid (276.3 mg, yield: 40.48%) poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-random-n-butyl methacrylate], i.e., P(ODEA-co-BMA), was obtained. An appropriate amount of the product was analyzed by NMR. The 1H NMR spectrum of P(ODEA-co-BMA) is shown below. Figure 5 As shown in Figure a, the solvent peak for deuterated chloroform (CDCl3) is at 7.26 ppm, while the solvent peak for deuterated methanol (CD3OD) is at 3.3 ppm, and the water peak is at 4.7 ppm. The absorption peak around 3.28 ppm corresponds to the 4n hydrogen atoms in the repeating unit of 2-(N-oxide-N,N-diethylamino)ethyl methacrylate under the same conditions. In the range of 0.5–2.0 ppm, peaks a, c, d, e, j, k, g, h, and i correspond to alkyl hydrogen atoms in the structure. Peaks l, m, n, and o correspond to hydrogen atoms on zwitterionic groups in the structure. The NMR integral results are consistent with the target product P(ODEA-co-BMA).

[0054] To investigate the effect of different monomer feed ratios on the copolymer composition, three P(ODEA-co-BMA) random copolymers with different compositions were prepared using the same synthesis method as described above, by adjusting the molar ratio of ODEA to BMA. The molar ratios of ODEA to BMA were 199:66.3, 199:198, and 199:597, respectively. A suitable amount of the product was taken, and the actual composition of the copolymers was characterized by 1H NMR spectroscopy. Figure 5 As shown in Figure a, the actual molar fraction of ODEA repeating units in the copolymer (defined as x) was calculated based on the integral area ratio of the characteristic proton peaks of ODEA and BMA in the spectra. Correspondingly, the molar fraction of BMA repeating units was 1-x. The results show that when the feed ratio is 199:66.3, x is 0.81; when the feed ratio is 199:198, x is 0.45; and when the feed ratio is 199:597, x is 0.18. The results indicate that as the proportion of ODEA feed increases, the actual molar fraction of ODEA structural units in the copolymer increases accordingly.

[0055] Three different ODEA molar ratios of P(ODEA-co-BMA) copolymers were dissolved in 200 μL of ethyl acetate using ultrasonication at 5 mg each. Then, 1 mL of PBS (pH 7.4) was added, followed by ultrasonic emulsification and rotary evaporation to remove the ethyl acetate, resulting in the self-assembly of blank nanoparticles. The hydrated particle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS). The results are as follows: Figure 5 As shown in b and Table 2, when x is 0.45 (ODEA to BMA molar ratio of 199:198), the resulting nanoparticles have the smallest size and narrowest particle size distribution; while when x is 0.18 or 0.81, the particle size increases significantly. Simultaneously, when x is 0.45, the PDI is around 0.17, indicating good distribution and relatively uniform particle size in PBS solution; while when x is 0.18 or 0.81, the PDI is 0.477 and 0.949 respectively, showing instability in PBS solution. Therefore, the P(ODEA-co-BMA) copolymer with x of 0.45 (ODEA to BMA molar ratio of 199:198) was selected for the subsequent preparation of drug-loaded nanoformulations.

[0056] Table 2. DLS measurement results of different P(ODEA-co-BMA) blank nanoparticles

[0057]

[0058] 2. Preparation of CsA ophthalmic nano-formulations

[0059] 2.1 Preparation of CsA ophthalmic nanoformulation

[0060] Weigh 5 mg of P(ODEA-co-BMA) and 1 mg of CsA into a 1.5 mL centrifuge tube, add 200 μL of ethyl acetate and sonicate to dissolve. Transfer to a 15 mL centrifuge tube and add 1 mL of PBS (pH=7.4). Continue sonication for about 1 min, then sonicate for 2 min using an ultrasonic cell disruptor under ice bath conditions. Remove ethyl acetate by rotary evaporation to obtain the CsA nano-ophthalmic formulation: P(ODEA-co-BMA)-CsA.

[0061] By replacing the polymer material of P(ODEA-co-BMA)-CsA with PEG-PCL, cRGD-PEG-PCL, PODEA-PCL, and P(OEGMA-co-BMA), respectively, and using the same assembly method and dosage ratio (mass ratio of polymer carrier to CsA), PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, and P(OEGMA-co-BMA)-CsA were prepared, resulting in a total of five CsA ophthalmic nano-formulations.

[0062] 2.2 Preparation of Free CsA

[0063] Weigh 0.4 mg CsA, add 200 μL of anhydrous ethanol, sonicate for 10 min, and then add 600 μL of PEG. 200 (i.e., polyethylene glycol 200), vortex for 30 s, then sonicate for 15 min. Add 1200 μL of physiological saline while sonicating, and sonicate for 10 min to obtain free CsA.

[0064] Different prepared CsA ophthalmic nano-formulations were taken and diluted to 0.2 mg / mL with PBS. Free CsA of the same concentration and an equal volume of PBS were used as controls. The nano-formulations were placed in front of a blank background, observed, and photographed.

[0065] 3. Establishment of in vitro analytical method for CsA ophthalmic nano-formulation

[0066] 3.1 Chromatographic conditions

[0067] Column: Waters ACQUITYUPLC CSH TM C18(100mm×2.1 mm, 1.7 μm) Column;

[0068] Mobile phase: methanol-water (90:10); flow rate: 0.2 mL / min; detection wavelength: 210 nm; column temperature: 30 ℃; injection volume: 5 µL.

[0069] 3.2 Solution Preparation

[0070] Preparation of reference solution: Accurately weigh 20 mg of CsA into a 10 mL volumetric flask, dissolve in methanol and dilute to 10 mL, shake well to obtain a 2 mg / mL reference solution for later use.

[0071] Preparation of test solution: Prepare nano-formulations of CsA-loaded PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA according to 2.1, and then dilute to 1 mg / mL for later use.

[0072] 3.3 Establishment of the CsA standard curve

[0073] Take the reference solution from step "3.2" and dilute it sequentially to 1000, 500, 250, 100, and 50 μg / mL.

[0074] The peak area was obtained according to the chromatographic conditions in 3.1, and a standard curve was plotted. The horizontal axis corresponds to the CsA concentration, and the vertical axis is the measured peak area.

[0075] 3.4 Determination of drug loading rate and encapsulation efficiency

[0076] The loading rate of CsA on the five nanocarriers was determined by UPLC under the chromatographic conditions described in 3.1. The P(ODEA-co-BMA)-CsA prepared in “2.1” was centrifuged in a 1.5 mL centrifuge tube at 12000 rpm for 15 min. The supernatant was separated, and the concentration of CsA in the supernatant was determined by UPLC. The amount of free CsA (W1) was then calculated. Different nanoformulations (PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA) were replaced with the same treatment method, and then the drug loading (DL) and encapsulation efficiency (EE) were calculated, where W0 is the weight of CsA and W is the weight of the carrier. The formulas for calculating drug loading and encapsulation efficiency are as follows: DL(%)=(W0-W1) / (W+W0-W1)×100%; EE(%)=(W0-W1) / W0×100%.

[0077] 3.5 In vitro release study of CsA ophthalmic nanoformulation

[0078] One mL each of CsA, PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA (all with a CsA concentration of 0.2 mg / mL) was placed into a pre-treated dialysis bag (molecular weight cutoff of 8000 Da). The dialysis bags were tied tightly at both ends with cotton thread and placed in an Erlenmeyer flask. 35 mL of release medium preheated to 37°C (containing 0.1% sodium dodecyl sulfate (SDS) and PBS at pH 7.4) was added. The Erlenmeyer flask was placed in a constant-temperature shaking incubator with the temperature and shaking speed set to 37°C and 100 rpm, respectively, to allow the release medium to operate dynamically and better simulate the in vivo environment. Samples were taken at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours. mL, and replenish with blank release medium of the same temperature and volume. Dilute the sample taken out several times, centrifuge, filter through a membrane, and analyze by UPLC under the chromatographic conditions in 3.1. Repeat 3 times, plot the CsA release curve, and calculate the following formula: M n = C n V0+∑C i V i Q n (%) = M n / M×100%. Where: M n This represents the cumulative release of CsA at the nth time point, where M is the total weight of CsA in the nano-formulation, and C...n C is the concentration of the drug at the nth time point when the sample is taken. i V is the concentration of the drug at the i-th time point (where i is an integer not greater than n-1). i Q is the volume of each sample taken. n V0 is the cumulative percentage of drug release at a single time point, and V0 is the total volume of the release medium.

[0079] 4. Experimental Results

[0080] In Example 1, photos were taken and compared after different preparations were left for different times. The results showed that all of them had good solubility and stability.

[0081] 4.1 Particle size and particle size distribution of CsA ophthalmic nanoformulations

[0082] The particle size and particle size distribution of CsA ophthalmic nanoparticles were determined using dynamic light scattering (DLS). The results are shown in Table 3. Figure 6 As shown.

[0083] Table 3. Particle size, PDI, drug loading (DL), and encapsulation efficiency (EE) data for CsA ophthalmic nanoformulations.

[0084]

[0085] Among the five CsA ophthalmic nanoformulations, P(ODEA-co-BMA)-CsA and P(OEGMA-co-BMA)-CsA both have a particle size of approximately 10 nm. This size allows them to penetrate corneal epithelial cells via passive diffusion, exhibiting good corneal permeability. However, P(OEGMA-co-BMA)-CsA, lacking zwitterionic groups in its structure, can only achieve drug delivery through passive diffusion and cannot further enhance corneal permeability through endocytosis. In contrast, P(ODEA-co-BMA)-CsA, containing zwitterionic groups, can further improve corneal permeability efficiency through endocytosis in addition to passive diffusion. The other CsA ophthalmic nanoformulations have a particle size of approximately 100 nm, maintaining a certain level of corneal permeability and theoretically facilitating a certain retention time on the corneal surface. Furthermore, the PDI (particulate index) of all five CsA ophthalmic nanoformulations is approximately 0.2, indicating good distribution and relatively uniform particle size in PBS solution.

[0086] Further measurements of the Zeta potential (also known as zeta electric potential or surface potential) of five CsA ophthalmic nanoformulations showed that all of them carried a negative surface charge, as shown in the following results. Figure 7As shown, their Zeta potentials are approximately -10 mV and -4 mV, respectively. The surface of ocular tissues (such as the cornea and conjunctiva) typically carries a negative charge, primarily originating from glycoproteins on cell surfaces, the mucus layer, and charged components in tears. Since both nanoparticles and ocular surface tissues carry a negative charge, an electrostatic repulsion occurs between them. This electrostatic repulsion reduces direct contact between the nanoparticles and ocular surface tissues, thereby preventing rapid clearance of the nanoparticles and improving drug bioavailability.

[0087] Further determination of the CsA drug loading and encapsulation effect of five CsA ophthalmic nanoformulations is shown in Table 3. The drug loading and encapsulation rate of P(OEGMA-co-BMA)-CsA were too low, while the CsA drug loading and encapsulation effect of the other four CsA ophthalmic nanoformulations were similar.

[0088] In vitro, five CsA ophthalmic nano-formulations released approximately 30% of CsA within 72 hours in phosphate-buffered saline (PBS), demonstrating good stability and sustained-release effect. The 72-hour release amount was significantly lower than that of CsA loaded with non-nano-formulations. Results are as follows... Figure 8 As shown in the figure, P(ODEA-co-BMA)-CsA has a similar sustained-release effect to P(OEGMA-co-BMA)-CsA and PEG-PCL-CsA, and is superior to the other two CsA ophthalmic nano-formulations.

[0089] Example 2: In vitro safety testing and corneal retention and permeation experiments of different ophthalmic nano-formulations

[0090] This embodiment is an in vitro safety test and corneal retention and permeation experiment conducted on different ophthalmic nanoparticle carriers in Example 1.

[0091] 1 Experimental Methods

[0092] 1.1 Cytotoxicity test of CsA ophthalmic nanoformulation

[0093] To test the toxicity of different concentrations of CsA ophthalmic nano-formulations to human corneal epithelial cells (HCECs Cells) and human retinal cells (ARPE-19 Cells), the MTT assay was used to study the in vitro cytotoxicity of each group of nano-formulations.

[0094] 1) HCECs Cells and ARPE-19 Cells: Purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; HCECs Cells' dedicated culture medium (Pronosai) consisted of: DMEM / F12 + 15% FBS + 5 μg / mL Insulin + 10 ng / mL human EGF + 1% P / S. ARPE-19 Cells' complete culture medium consisted of: DMEM / F12 + 10% FBS + 1% P / S.

[0095] 2) Culture of HCECs Cells and ARPE-19 Cells: The cell culture incubator temperature was 37℃ and the CO2 content was 5%. HCECs Cells were passaged once a day and ARPE-19 Cells were passaged once every three days, with a passage ratio of 1:2 or 1:3.

[0096] 3) MTT treatment steps: After digestion and centrifugation, HCECs and ARPE-19 cells were cultured in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells per well, replicated 6 times. The culture medium was discarded the following day, and 100 μL of nano-formulation (loaded with CsA) was added to each well. HCECs cells contained nano-formulation at concentrations of 250, 100, 50, 25, 10, 5, and 0 μg / mL, while ARPE-19 cells contained nano-formulation at concentrations of 100, 50, 25, 10, 5, and 0 μg / mL, for a total of 9 columns. Cells were incubated for 48 h. 10 μL of MTT solution was added to each well, followed by incubation in the dark for another 4 h. The culture medium was then aspirated, and 150 μL of dimethyl sulfoxide was added to each well to dissolve formazan for 10 min. The absorbance was measured using a microplate reader at a wavelength of 570 nm. Finally, the cytotoxicity of the nano-formulation was assessed based on cell viability. Untreated cells served as a negative control, and benzalkonium chloride (BAC) treated cells served as a positive control.

[0097] 1.2 Erythrocyte hemolysis test of CsA ophthalmic nano-formulation

[0098] 1) Sample pretreatment: After anticoagulating New Zealand rabbit blood, mix it thoroughly with PBS in a centrifuge tube, centrifuge for 5 minutes at 2000 rpm, remove the supernatant after centrifugation, and repeat the above operation five times to obtain pure red blood cells, which are used as solution A.

[0099] Positive group: 0.98 mL deionized water + 0.02 mL solution A;

[0100] Negative group: 0.98 mL PBS + 0.02 mL solution A.

[0101] 2) Take P(ODEA-co-BMA)-CsA, PODEA-PCL-CsA and free CsA prepared according to Example 1, and dilute them with PBS to 1000, 500, 250, 100, 50, 25 and 10 μg / mL respectively, as solutions B (B1 to B7).

[0102] Sample group: 0.02 mL solution A + 0.98 mL solution B: Take 0.98 mL of solution B (B1~B7) into a 1.5 mL centrifuge tube, add 0.02 mL of solution A, and incubate on a shaker for 1 h at 37℃. Then centrifuge for 5 min at 3000 rpm, observe the hemolysis and take pictures.

[0103] The hemolysis rate was measured using the supernatant at a wavelength of 540 nm. The hemolysis rate was calculated as (sample absorbance - negative absorbance) / (positive absorbance - negative absorbance). After calculation, a curve was plotted showing the concentration value (x-axis) versus the hemolysis rate (y-axis).

[0104] 1.3 In vitro corneal permeation experiment of polynitrogen-oxygen zwitterionic carriers

[0105] 1.3.1 Preparation of pyrene-labeled nanoformulations

[0106] Preparation and characterization of pyrene-loaded nano-formulations: Pyrene-loaded nano-formulations were prepared by ultrasonic emulsification. 1 mg of pyrene and 5 mg of polymer carriers (PODEA-PCL, P(ODEA-co-BMA), PEG-PCL, P(OEGMA-co-BMA), and cRGD-PEG-PCL) were accurately weighed into 15 mL centrifuge tubes. 200 μL of ethyl acetate was added to each tube, and after ultrasonic dissolution, 1 mL of PBS was added. The tubes were then ultrasonicated for 30 s, followed by ultrasonic dispersion in an ice bath for about 2 min. Finally, the organic phase was removed by rotary evaporation to obtain the pyrene-loaded nano-formulations (PODEA-PCL-Pyrene, P(ODEA-co-BMA)-Pyrene, PEG-PCL-Pyrene, P(OEGMA-co-BMA)-Pyrene, and cRGD-PEG-PCL-Pyrene). After dilution with PBS, the particle size and particle size distribution of the nanoformulation were detected by DLS, and the pyrene loading and encapsulation efficiency were detected by UPLC.

[0107] 1.3.2 Establishment of the Pyrene Standard Curve

[0108] 1) Chromatographic conditions for pyrene: Thermo Scientific C18 column (250 mm × 4.6 mm, 5 μm), acetonitrile-pure water (70:30, v / v) as mobile phase, flow rate 0.200 mL / min, column temperature 30 ℃, detection wavelength 245 nm, injection volume 5 μL.

[0109] 2) Preparation of pyrene reference standard and test solution: Accurately weigh 6.4 mg of pyrene, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to volume to obtain a reference solution with a concentration of 640 μg / mL. Five pyrene-loaded nanoformulations prepared according to the method in “1.3.1” of this example were used as test solutions. Nanomicelle test solutions were prepared from the five pyrene-loaded nanomicelles respectively.

[0110] 3) Specificity assessment: Take the reference solution and the test solution respectively, analyze them under the above chromatographic conditions, record the chromatograms, and compare the specificity.

[0111] 4) Linearity Assessment: Accurately pipette appropriate amounts of pyrene reference solution, add methanol to dilute to volume, and shake well to prepare a series of reference solutions with concentrations of 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 μg / mL. Take 100 μL of each solution and add it to 900 μL of blank culture medium. After mixing, filter the organic phase through a 0.22 μm organic membrane and determine the pyrene concentration under the chromatographic conditions, recording the peak area. Plot a standard curve with the concentration of the analyte (X, μg / mL) on the x-axis and the corresponding peak area (Y) on the y-axis.

[0112] 1.3.3 In vitro corneal permeation experiment in a Transwell chamber

[0113] Corneal cell permeation assay: HCECs cells were per well at a density of 5 × 10⁶ cells / well. 4 Cells were seeded at a density of 100 μL in Transwell chambers. After cell monolayer formation, 200 μL of nanoparticles with different pyrene loadings at a concentration of 200 μg / mL were added to the cell monolayer, followed by 700 μL of serum-free culture medium (DMEM) in the recipient chamber. Transwell plates were incubated in a cell culture incubator at 37°C in the dark for 6 h, and then 100 μL was removed from the recipient chamber to quantify the pyrene penetration rate using UPLC.

[0114] 1.4 Mucus Layer Permeability Experiment of Polynitrogen-Oxygen Zwitterions

[0115] 20 μL of a 200 μg / mL mucin solution was added to the HCECs Cell monolayer to simulate the ocular surface mucus layer; the mixture was then co-incubated with five pyrene-loaded nanoparticles for 6 h, and the pyrene content in the receiving chamber was measured and the pyrene penetration rate was calculated.

[0116] 1.4.1 Preparation of mucin solution

[0117] Preparation of mucin solution: Accurately weigh 1 mg of mucin powder into a 5 mL centrifuge tube, add 5 mL of PBS and sonicate to dissolve, so as to prepare a mucin solution with a concentration of 200 μg / mL to simulate the mucus layer on the surface of the eye, and store it in a refrigerator at 4°C for later use.

[0118] 1.4.2 Mucus Layer Permeability Experiment of Polynitrogen-Oxygen Zwitterions

[0119] Experimental method for polynitrogen-oxygen zwitterionic carriers to penetrate the mucus layer: HCEC cells were placed at 5 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 100 μL in Transwell chambers. After cell monolayer formation, 20 μL of a 200 μg / mL mucin solution was added to the cell monolayer to simulate the ocular surface mucus layer. 700 μL of serum-free culture medium (DMEM) was added to the recipient chamber. Then, 200 μL of five nanoparticles loaded with pyrene at a concentration of 200 μg / mL were added to the recipient chamber. The Transwell plates were incubated at 37°C on a shaker (150 rpm) for 6 h. 100 μL of the liquid was then removed from the recipient chamber, and the pyrene content in the receiving chamber was determined by UPLC.

[0120] 1.5 Retention of polynitrogen-oxygen zwitterions on the ocular surface of mice with dry eye syndrome

[0121] We used small animal in vivo imaging technology to evaluate the corneal retention capacity of five nano-formulations (loaded with Nile Red) in the eyes of mice with dry eye syndrome, i.e., the residence time of the nano-formulations on the corneal surface.

[0122] 1) Preparation of Nile Red-labeled nano-formulations

[0123] Nile Red (NR) is used to label ophthalmic nanoformulations.

[0124] Preparation of Nile Red stock solution: Accurately weigh 5 mg of Nile Red, add ethyl acetate and dissolve by sonication, then bring the volume to 10 mL to obtain a Nile Red standard stock solution with a concentration of 500 μg / mL. Store at 4°C in the dark.

[0125] Preparation of free Nile Red group: Accurately weigh 1 mg of Nile Red, add olive oil and dissolve by sonication, and make up to 10 mL to obtain a free control group with a concentration of 100 μg / mL. Store at 4℃ protected from light.

[0126] Preparation of Nile Red-labeled nano-formulations (Nile Red-labeled ophthalmic nano-formulations): Accurately weigh 5 mg of P(ODEA-co-BMA) carrier, add 200 μL of Nile Red standard stock solution to obtain Nile Red with a concentration of 100 μg / mL. Dissolve by sonication, transfer to a 15 ml centrifuge tube, add 1 mL of pH 7.4 PBS, and continue sonication for approximately 30 s. Disperse by sonication for 2 min using an ultrasonic cell disruptor under ice bath conditions. Remove ethyl acetate by rotary evaporation to obtain NR-labeled P(ODEA-co-BMA) nano-formulations with a concentration of 100 μg / mL. Replace the polymer carriers (PEG-PCL, cRGD-PEG-PCL, PODEA-PCL, P(OEGMA-co-BMA)) with the same assembly method and proportions to prepare NR-labeled nano-formulations (PODEA-PCL-NR, cRGD-PEG-PCL-NR, P(OEGMA-co-BMA)-NR, PEG-PCL-NR), all with a concentration of 100 μg / mL. μg / mL.

[0127] 2) Turn on the IVIS Lumina imaging system (PerkinElmer) and set the appropriate filter (excitation / emission wavelength 535 / 600nm). Adjust the imaging system parameters to ensure that the fluorescence signal can be clearly captured.

[0128] 3) A mouse model of dry eye (male C57BL / 6J mice, Lin Z, Liu X, Zhou T, Wang Y, Bai L, He H, Liu Z. A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol Vis. 2011 Jan 25;17:257-64.) was established according to the literature method. The mice were anesthetized with isoflurane. 5 μL of NR-loaded nanoparticle suspension was dropped onto the corneal surface of the dry eye mouse model. A control group was also established, with an equal volume (5 μL) of free nylon red solution dropped onto the corneal surface of the control group. The mice were placed in the dark chamber of the imaging system and fixed on the imaging stage, ensuring head stability. Fluorescence images were captured at intervals from the start of solution application for 30 min.

[0129] 1.6 Rabbit corneal permeability experiment of polynitrogen-oxygen zwitterionic carriers

[0130] 1.6.1 Feeding and Grouping of New Zealand White Rabbits

[0131] Six healthy New Zealand White rabbits (provided by Nanjing Pukou District Laifu Rabbit Farm, Laboratory Animal Use Permit No. SYXK 2024-011), of any sex and weighing 2–2.5 kg, were used for an acclimatization period of approximately one week at the animal facility of the Research and Technology Center of Anhui University of Traditional Chinese Medicine. All animal protocols were approved by the Animal Ethics Committee of Anhui University of Traditional Chinese Medicine (AHUCM-rabbit-20230050).

[0132] Rabbits were randomly divided into 6 groups (5 Nile Red-labeled ocular nano-formulation groups and free Nile Red group), with 2 eyes in each group for the experiment.

[0133] 1.6.2 Preparation and Administration of Nile Red-Labeled Nanoparticles

[0134] Nile red-labeled ophthalmic nanoparticles were prepared according to Part 1.5 of this Example, and the penetration behavior of Nile red in rabbit cornea was observed.

[0135] Six groups of Nile red-labeled ophthalmic nanoparticles and free Nile red were administered at a dose of 30 μL per eye, once every 5 minutes, for a total of four instillations. The instillations were placed onto the corneal surface of New Zealand white rabbits. Timing began at the end of the fourth instillation. After 5 hours, the ocular surface was rinsed with physiological saline, and the rabbits were euthanized by air embolism. The eyes were then enucleated, and the adjoint tissues connecting the sclera and conjunctiva were removed. The cornea, with a 2 mm scleral ring, was separated. The cornea was rinsed with physiological saline and cryopreserved for later use. In vitro Franz diffusion cell experiments were initiated within 20 minutes.

[0136] 1.6.3 Rabbit corneal permeation experiment of polynitrogen-oxygen zwitterionic carriers

[0137] The treated rabbit corneas were fixed between the supply and receiving cells of the Franz diffusion cell, with the corneal epithelium facing the supply cell, and secured with screws. 5 mL of PBS solution was added to the receiving cell as the receiving fluid, ensuring complete contact between the fluid and the cornea without air bubbles. To simulate a realistic drug penetration environment, the Franz diffusion cell was maintained at 37°C throughout the experiment. Magnetic stirring at 100 rpm was used during the experiment. To minimize errors caused by evaporation, the openings of the supply and receiving cells were sealed with sealing strips. Five types of Nile Red-labeled ophthalmic nanoparticles and free Nile Red were used to track drug release, diffusion, and penetration in isolated corneas. 200 μL of each group of nanoparticles at a concentration of 100 μg / mL was added to the supply cell.

[0138] Three hours later, the cornea was immediately removed and cryopreserved in liquid nitrogen in the dark. The quick-frozen corneal tissue was fixed on a cryostat and cut into sections of appropriate thickness. The frozen sections were removed and thawed at room temperature for 3 minutes. Fixative solution (4% paraformaldehyde) was added to the sections and fixed at room temperature for 15-20 minutes. The sections were washed three times with PBS and covered with coverslips to prepare temporary sections. The sections were observed under a fluorescence microscope (λex=543nm, λem=598nm) to assess the drug permeability in different layers of the corneal tissue.

[0139] 2. Experimental Results

[0140] 2.1 CsA ophthalmic nano-formulation cytotoxicity test

[0141] Using benzalkonium chloride (BAC), a commonly used preservative in eye drops, as a positive control, the MTT assay was used to investigate the cytotoxicity of PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA nanoparticles on human corneal epithelial cells (HCECs cells). The cytotoxicity of P(ODEA-co-BMA)-CsA and PODEA-PCL-CsA nanoparticles on human retinal cells (ARPE-19 cells) was also investigated.

[0142] The results showed that BAC exhibited significant toxicity to human corneal epithelial cells (HCECs), and this toxicity increased with increasing BAC concentration. Experimental results indicated that after 48 h of incubation, when the concentration of the CsA ophthalmic nano-formulation was below 250 μg / mL, PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA all showed good cell tolerance and high cell viability. No significant cytotoxicity was observed in human retinal cells (ARPE-19 cells) with P(ODEA-co-BMA)-CsA and PODEA-PCL-CsA. The experimental results demonstrate that all groups of ophthalmic nano-formulations exhibited good biocompatibility with the eye.

[0143] 2.2 Erythrocyte hemolysis test of CsA ophthalmic nanoformulation

[0144] CsA is an immunosuppressant whose primary mechanism of action is immunosuppression through the inhibition of intracellular signaling pathways, rather than through cell membrane disruption. Therefore, even at high concentrations, free CsA exhibits minimal cell membrane disruption. P(ODEA-co-BMA)-CsA and PODEA-PCL-CsA show almost no hemolysis at concentrations up to 250 μg / mL, indicating extremely low cell membrane disruptive activity. This characteristic makes P(ODEA-co-BMA)-CsA and PODEA-PCL-CsA ideally suited for ocular delivery systems, maintaining stability in the ocular environment, reducing potential damage to ocular tissues, and thus improving therapeutic efficacy and safety.

[0145] 2.3 In vitro corneal permeability test and mucus layer permeability

[0146] A standard curve for pyrene was established using the chromatographic method of this embodiment. Regression analysis was performed with the concentration of pyrene as the abscissa (X) and the peak area as the ordinate (Y). The regression equation obtained showed that the detection method had a good linear relationship in the concentration range of 2.5 ~ 640 μg / mL.

[0147] In vitro Transwell cell experiments were conducted by adding a mucus solution simulating the ocular surface mucus layer to a single layer of human corneal epithelial cells and above it. Results are shown below. Figure 9 . Figure 9 a and Figure 9 In section b, the significance signs at the top of the bars indicate statistically significant differences between each experimental group and the PEG-PCL-Pyrene group: *** represents P < 0.001, ** represents P < 0.01, * represents P < 0.05, and # represents no significant difference. *** on the inter-group line indicates a highly significant difference (p < 0.001) between each group and the P(ODEA-co-BMA)-Pyrene group. Figure 9 It is evident that, regardless of the presence or absence of mucin, the permeation concentration of P(OEGMA-co-BMA)-Pyrene was significantly lower than that of the other groups, while the permeation concentration of P(ODEA-co-BMA)-Pyrene was significantly higher than that of the other groups, including PODEA-PCL-Pyrene. Although the particle sizes of the P(OEGMA-co-BMA)-Pyrene and P(ODEA-co-BMA)-Pyrene carriers were similar, the final permeation effects differed greatly.

[0148] 2.4 Retention test on ocular surface in mice with dry eye syndrome

[0149] Prolonging drug retention time on the ocular surface is crucial for effective ocular drug delivery, as rapid clearance by blinking and tear drainage significantly limits drug bioavailability and therapeutic efficacy. To evaluate the performance of different drug carriers, the ocular absorption and retention of nanoformulations such as P(ODEA-co-BMA)-NR and PODEA-PCL-NR were investigated in a mouse model of dry eye using a small animal in vivo imaging system. Figure 10 As shown, after local administration, compared with the rapidly cleared control groups such as PEG-PCL-NR and P(OEGMA-co-BMA)-NR, the retention time of P(ODEA-co-BMA)-NR and PODEA-PCL-NR on the ocular surface was significantly prolonged. PEG-PCL-NR retained only 32.44% of the initial dose at 30 min after administration, while PODEA-PCL-NR and cRGD-PEG-PCL-NR retained more than 50% of the initial dose at 30 min, and P(ODEA-co-BMA)-NR retained 75% of the initial dose. The retained dose was significantly higher than other nano-formulations, indicating that this ocular nano-formulation carrier has an unexpected ocular surface retention effect.

[0150] Rabbit corneal permeability experiment of 2.5 nanoparticle formulation

[0151] Rabbit corneas were treated with five nano-formulations loaded with Nile Red and free Nile Red. The corneas were then removed and frozen sections were prepared 5 hours after instillation into the rabbit eyes to investigate the permeability of the rabbit corneas with different nano-formulation carriers.

[0152] See results Figure 11 . Figure 11 b and Figure 11 In column c, the significance signs at the top of the bars indicate statistical differences between each experimental group and the free-NR group: *** represents P < 0.001, ** represents P < 0.01, * represents P < 0.05, # represents no significant difference, and ns represents no statistical difference. The *** on the inter-group line indicates a highly significant difference (p < 0.001) between the PEG-PCL-NR group, the cRGD-PEG-PCL-NR group, the P(OEGMA-co-BMA)-NR group, and the P(ODEA-co-BMA)-NR group; the * on the inter-group line indicates a significant difference (p < 0.05) between the PODEA-PCL-NR group and the P(ODEA-co-BMA)-NR group.

[0153] Free Nile red primarily resides on the surface of the corneal epithelium, indicating that free Nile red molecules have difficulty penetrating the multilayered structure of the cornea. However, when a nano-formulation containing Nile red is instilled into the cornea, Nile red is observed not only in the corneal epithelium but also diffuses into the corneal stroma and endothelium. This demonstrates that the nano-formulation carrier can effectively promote the corneal penetration of Nile red.

[0154] Among them, PEG-PCL-NR has difficulty effectively penetrating the corneal barrier. The physiological structural characteristics of the cornea (tight junctions and the density of the stroma) limit the penetration of macromolecules and nanoparticles. PEG-PCL-NR, with its relatively large particle size, struggles to pass through intercellular spaces and cell membranes, mostly remaining on the surface of the corneal epithelium and rarely reaching the stroma. cRGD-PEG-PCL-NR exhibits strong adsorption to the cornea, with noticeable fluorescence signals observed in the corneal epithelium, but rarely reaches the stroma. While P(OEGMA-co-BMA)-NR has a smaller particle size, it lacks targeting specific corneal cells, and the clearing action of tears rapidly removes nanoparticles from the ocular surface, resulting in most remaining on the corneal epithelium and rarely reaching the stroma.

[0155] In contrast, PODEA-PCL-NR significantly improves corneal permeability. Its zwitterionic properties give it a unique affinity for cell membranes, but it does not adhere to plasma proteins. Zwitterions can cross corneal epithelial cells via transcytosis, a property that allows the PODEA-PCL carrier to interact with corneal cells, thereby significantly improving the transcorneal transport efficiency and intraocular accumulation of Nile Red.

[0156] P(ODEA-co-BMA)-NR also contains polynitrogen-oxygen zwitterions, and therefore can also cross corneal epithelial cells via transcytosis. However, it exists in the corneal epithelium, stroma, and endothelium, and its content is significantly higher than other ophthalmic nanoparticle carriers, including PODEA-PCL.

[0157] Example 3: The ameliorative effect of different CsA ophthalmic nanoformulations on a dry eye animal model

[0158] This embodiment is used to test the in vivo efficacy of the CsA ophthalmic nanoformulation prepared in Example 1 and to examine its in vivo safety.

[0159] 1 Experimental Methods

[0160] 1.1 Mice used for modeling and their feeding

[0161] Male C57BL / 6J mice (20±2.0g, purchased from Anhui Medical University) were acclimatized for about one week. Before establishing the mouse dry eye model, mice with eye diseases were removed by slit-lamp examination.

[0162] 1.2 Establishment and grouping of a mouse model of dry eye

[0163] Male C57BL / 6J mice were used in the experiment. A benzalkonium chloride (BAC)-induced ocular surface injury model was established according to the method reported in the literature (Lin Z, Liu X, Zhou T, Wang Y, Bai L, He H, Liu Z. A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol Vis. 2011 Jan 25;17:257-64.). During the experiment, mice were housed in the following standard environment: temperature (25±1.0)℃, relative humidity (60±10)%, simulating diurnal variation. 5 μL of 0.2% BAC was administered topically to the right eye of each mouse twice daily for 14 days to induce ocular inflammation. On day 14, mice with more than 90% of their corneal surface showing fluorescent staining (indicating successful establishment of the dry eye model) were selected for subsequent experiments.

[0164] 1.3 Pharmacodynamic experiments and pathological examinations in mice with dry eye syndrome

[0165] Dry eye model mice were divided into 8 groups, with 8 mice in each group. The experimental groups were treated with the following test substances and labeled accordingly: normal group (no modeling, no drug administration), PBS control group (negative treatment control; modeling, no drug administration), commercially available CsA eye drops group (positive treatment control), PEG-PCL-CsA group, cRGD-PEG-PCL-CsA group, PODEA-PCL-CsA group, P(OEGMA-co-BMA)-CsA group, and P(ODEA-co-BMA)-CsA group. The concentration of each nano-formulation group was adjusted to 0.5 mg / mL according to the CsA content, and 5 μL was instilled into the right eye each time (positive and negative treatment controls were administered the same volume and dose), once daily. During the treatment period (days 14-21), all treated mice were given 5 μL of 0.2% BAC in their right eye to continuously induce ocular inflammation. During the experiment, the left eye of all mice was not treated and served as a self-control.

[0166] (1) On days 14, 15, 17, 19 and 21, 5 μL of 0.25% sodium fluorescein solution (Tianjin Jingming, China) was instilled into the right eye of mice. After 90 seconds of instillation, the corneal epithelium was stained with sodium fluorescein under cobalt blue light under a slit-lamp microscope to assess corneal integrity.

[0167] The cornea was divided into four quadrants: superior nasal, inferior nasal, superior temporal, and inferior temporal. Each quadrant was scored separately, and all scores were summed to obtain the final score. The staining scoring criteria were as follows: fluorescein-positive patchy staining = 4 points; obvious diffuse staining without patchy staining = 3 points; moderate punctate fluorescence diffuse staining = 2 points; mild fluorescence resembling sparse punctate staining = 1 point; no fluorescence = 0 points. The scores for each eye were summed and analyzed.

[0168] (2) After administration on day 21 of the experiment, mice were sacrificed, and fresh orbital tissue containing the eyeball was fixed in 4% paraformaldehyde for 48 hours. The tissue was then dehydrated and embedded. The embedded tissue was sectioned. The area connecting the skin behind the mouse's ear to the mandible was cut open with a scalpel to expose the muscle tissue, locate the lacrimal gland, find the lacrimal duct connecting to the orbit, and remove the entire lacrimal gland tissue. The obtained tissue was subjected to routine HE staining and pathological examination.

[0169] (3) After the administration of the drug on day 21 of the experiment, three corneal samples were taken from each group. The corneas were cut into small pieces, and then 300 μL of ice-cold PBS was added to homogenize the tissue. The homogenized corneal samples were centrifuged and the supernatant was collected. The levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the supernatant were detected by ELISA.

[0170] 1.4 Ocular irritation test in healthy mice in vivo

[0171] The ocular irritation of different CsA ophthalmic nanoformulations was evaluated using a modified Draize test. BALB / c mice (Hangzhou Resource Experimental Animal Technology Co., Ltd.) were randomly divided into 8 groups of 3 mice (6 eyes) each, and administered PBS (negative control), 0.5% SDS (sodium dodecyl sulfate, positive control), 0.1% BAC, PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA. 5 μL was instilled into the eyes twice daily (9:00 AM and 9:00 PM). On day 7 of administration, slit-lamp examination was used to observe the irritation and toxicity of the cornea and conjunctiva in the mice.

[0172] 2. Experimental Results

[0173] 2.1 Results of the dry eye experiment in mice

[0174] Corneal epithelial fluorescein staining under cobalt blue light using a slit-lamp microscope revealed that the PBS group failed to alleviate corneal damage caused by bacillary sclerosis (BAC). All groups containing CsA nanoparticles showed varying degrees of repair for BAC-induced corneal damage. The P(ODEA-co-BMA)-CsA group exhibited the fastest repair speed and the lowest staining score, followed by the PODEA-PCL-CsA group.

[0175] Further inflammatory factor detection was performed, and the results are shown below. Figure 12 . Figure 12 a, Figure 12 b and Figure 12 In c, the significance signs at the top of the bars indicate the statistical differences between each experimental group and the Mock group: *** represents P < 0.001, ** represents P < 0.01, * represents P < 0.05, and # represents no significant difference.

[0176] Figure 12 a and Figure 12 c: The *** on the intergroup line indicates a highly significant difference (p<0.001) between the PBS group, PEG-PCL-CsA group, cRGD-PEG-PCL-CsA group, P(OEGMA-co-BMA)-CsA group, and P(ODEA-co-BMA)-CsA group; the # on the intergroup line indicates no significant difference between the PODEA-PCL-CsA group, the Market group, and the P(ODEA-co-BMA)-CsA group.

[0177] Figure 12 b: *** on the inter-group line indicates a highly significant difference (p<0.001) between the PBS group, PEG-PCL-CsA group, P(OEGMA-co-BMA)-CsA group, and P(ODEA-co-BMA)-CsA group; * on the inter-group line indicates a significant difference (p<0.05) between the cRGD-PEG-PCL-CsA group and P(ODEA-co-BMA)-CsA group; # on the inter-group line indicates no significant difference between the PODEA-PCL-CsA, Market group, and P(ODEA-co-BMA)-CsA group.

[0178] The expression levels of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α in the cornea of ​​the PBS group were higher than those of the normal control group (Mock group). The PEG-PCL-CsA group, cRGD-PEG-PCL-CsA group, and P(OEGMA-co-BMA)-CsA group could slightly reduce inflammation, but the therapeutic effect was limited; the commercially available formulation group, PODEA-PCL-CsA group, and P(ODEA-co-BMA)-CsA group could significantly reduce inflammation and reverse the increase of the above-mentioned inflammatory factors. Among them, the P(ODEA-co-BMA)-CsA group showed the best anti-inflammatory effect: the level of the core inflammatory factor TNF-α (which plays a key role in inflammatory response and immune regulation) was significantly lower than that of the PEG-PCL-CsA group, the cRGD-PEG-PCL-CsA group, and the P(OEGMA-co-BMA)-CsA group (P<0.001), and slightly lower than that of the commercially available formulation group; the level of IL-1β (which acts as a key amplification mediator and stimulates cells to secrete more inflammatory factors) was significantly lower than that of the PEG-PCL-CsA group, the cRGD-PEG-PCL-CsA group, and the P(OEGMA-co-BMA)-CsA group (P<0.001); the level of IL-6 (which inhibits corneal epithelial repair and amplifies ocular surface immune damage) was significantly lower than that of the PEG-PCL-CsA group and the P(OEGMA-co-BMA)-CsA group (P<0.001). The above results indicate that, under in vivo experimental conditions, the P(ODEA-co-BMA)-CsA group significantly improved corneal damage and abnormally elevated inflammatory factors in a dry eye model, with its anti-inflammatory effect superior to most experimental groups and slightly better than the commercially available formulation. Pathological observation of the sampled tissues showed no drug-related tissue damage in the P(ODEA-co-BMA)-CsA group.

[0179] 2.2 Results of ocular irritation experiments in healthy mice in vivo

[0180] Slit-lamp observation stimulation tests showed that PEG-PCL-CsA, cRGD-PEG-PCL-CsA, PODEA-PCL-CsA, P(OEGMA-co-BMA)-CsA, and P(ODEA-co-BMA)-CsA nanoformulations all exhibited good ocular tolerability. After administration of each group of nanoformulations to healthy mice, no ocular irritation symptoms were observed, including conjunctival redness or swelling.

[0181] Both the SDS and BAC groups showed significant eye irritation. Mice in the SDS group exhibited severe conjunctival hyperemia, edema, and mucopurulent discharge, with obvious fluorescein staining.

[0182] The above in vitro and in vivo experiments investigated the particle size distribution, in vitro release, corneal permeability and retention characteristics, in vivo efficacy and safety of P(ODEA-co-BMA) ophthalmic nanoparticle carrier, P(ODEA-co-BMA)-CsA ophthalmic nanoparticle formulation, and four other comparative ophthalmic nanoparticle carriers and CsA ophthalmic nanoparticle formulations. The results demonstrated that the P(ODEA-co-BMA) ophthalmic nanoparticle carrier and P(ODEA-co-BMA)-CsA ophthalmic nanoparticle formulation showed good safety. This nanoparticle carrier significantly improved corneal retention and demonstrated good corneal permeability, and significantly reduced corneal damage and inflammatory factor levels in dry eye mice, indicating promising application prospects.

Claims

1. An ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier, characterized in that, The ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier is prepared by a random copolymer of 2-(N-oxide-N,N-diethylamino) ethyl methacrylate and n-butyl methacrylate; The average particle size of the ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier is 7-12 nm. The preparation method of the ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier includes the following steps: Synthesis of S1-oxidized N,N-diethylaminoethyl methacrylate: 2-(N,N-diethylamino)ethyl methacrylate and 3-chloroperoxybenzoic acid were weighed and dissolved in dichloromethane. Under ice bath conditions for 30 min, the dichloromethane solution of 2-(N,N-diethylamino)ethyl methacrylate was added dropwise to the dichloromethane solution of 3-chloroperoxybenzoic acid. The mixture was stirred overnight at room temperature. After the reaction was completed, 2-(N-oxide-N,N-diethylamino)ethyl methacrylate was obtained by purification. Preparation of S2 polynitrogen-oxygen zwitterionic nanoparticle drug carrier: 2-bromoisobutyrate, 2-(N-oxide-N,N-diethylamino)ethyl methacrylate, n-butyl methacrylate, and pentamethyldiethylenetriamine were placed in a sealed tube. Anhydrous N,N-dimethylformamide was added to dissolve the mixture. Before sealing, a magnetic wave was added, and the solution was frozen through liquid nitrogen. After evacuation and thawing, the freeze-thaw cycle was repeated. CuBr was then added, and the tube was evacuated and sealed under vacuum. The reaction was carried out in an oil bath at 30℃~60℃ for 60h~96h. After the reaction, the product was purified. The filtrate was rotary evaporated, precipitated with petroleum ether, and the precipitate was dissolved and transferred to a dialysis bag. Dialysis was performed using pure water, and the dialysis product was freeze-dried to obtain poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-random-n-butyl methacrylate]. In step S2, the molar ratio of 2-(N-oxide-N,N-diethylamino)ethyl methacrylate to n-butyl methacrylate is (198~200):(197~200).

2. The ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier according to claim 1, characterized in that, In step S1, the molar ratio of 2-(N,N-diethylamino) methacrylate to 3-chloroperoxybenzoic acid is (1~3):(2~4).

3. The ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier according to claim 1, characterized in that, In step S2, the molar ratio of ethyl 2-bromoisobutyrate to pentamethyldiethylenetriamine is (495~499):(498~502).

4. The ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier according to claim 1, characterized in that, In step S2, the molar ratio of ethyl 2-bromoisobutyrate to ethyl 2-(N-oxide-N,N-diethylamino)methacrylate is (4.95~4.99):(198~200).

5. An ophthalmic nano-formulation, characterized in that, The drug carrier for the ophthalmic nano-formulation is the ophthalmic polynitrogen amphoteric nano-drug carrier as described in any one of claims 1 to 4.

6. The ophthalmic nano-formulation according to claim 5, characterized in that, The active ingredient in the ophthalmic nano-formulation is cyclosporine A.

7. Use of the ophthalmic polynitrogen-oxygen zwitterionic nanomedicine carrier according to any one of claims 1 to 4 in the preparation of ophthalmic topical drug delivery formulations for dry eye syndrome.

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