Liposome drug carrier with mucosal adhesion and its preparation method and application

Abstract

The application discloses a liposome drug carrier with mucosal adhesion, a preparation method and application thereof, and specifically, the drug carrier is DSPE-PEG-PBA, which can be used for loading cyclosporine A and crocin I, and the carrier of the application is used for loading cyclosporine A and crocin I. The liposome loaded with the two drugs has good stability and adhesion, can inhibit the side effect of cyclosporine A on the increase of ROS level, and has a wide application prospect in the treatment of inflammation and epithelial tissue damage.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a liposome drug carrier with mucosal adhesion, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome, also known as keratoconjunctivitis sicca, is an eye disease characterized by impaired tear secretion, with dryness of the eyes as the primary symptom. It is often accompanied by itching, foreign body sensation, burning sensation in both eyes, or photophobia, blurred vision, and fluctuating visual acuity. Ocular inflammation is one of the key causes of dry eye syndrome, and cyclosporine A (CsA) is a commonly used medication for its treatment. CsA effectively treats dry eye syndrome by increasing tear secretion through inhibiting the infiltration of lymphocytes in the lacrimal glands, reducing inflammation, and suppressing scarring of the lacrimal gland tissue.

[0003] However, systemic administration of CsA can cause hepatotoxicity and nephrotoxicity. Studies have shown that this toxicity is induced by increasing the levels of ROS (an upstream substance in a major inflammatory signaling pathway) in hepatic and renal cells, thus significantly limiting the practical application of CsA. In the treatment of dry eye, the excessive production of ROS is both a characteristic of dry eye and a factor contributing to its further deterioration. Current research indicates that topical administration of CsA to the ocular surface can cause irritation, primarily manifesting as ocular pain and conjunctival hyperemia. Based on this, researchers conducted experiments using human corneal epithelial cells, demonstrating that CsA can indeed increase the ROS levels of normal corneal epithelial cells, and thus hypothesizing that the irritant effect of CsA on the ocular surface is due to its stimulation of increased ROS levels in normal ocular surface cells.

[0004] With a deeper understanding of the structure and composition of ocular tissues, it has been discovered that the surface of the eye is covered by a complex tear film, consisting of a lipid layer, an aqueous layer, and a mucus layer from the outside in. The mucus layer contains negatively charged mucins. The ocular surface mucus layer, composed of mucins and tears, has a dual effect on the ocular bioavailability of drugs. For local drug delivery, the ocular mucus layer acts as a barrier (secretory proteins in the tears act as a barrier to drug entry into the cornea, significantly affecting the drug's residence time in the cornea and thus its ocular bioavailability, especially for lipophilic compounds, large biomolecules, or drugs formulated in micro and nanoparticle form). It also increases the retention of cationic drug delivery systems on the ocular surface. The degree of drug bioavailability in the eye depends on the strength of the barrier and adhesion effects of the ocular mucus layer. CsA has poor water solubility. Although it is well soluble in organic solvents such as ethanol and acetone, its solubility in water is 6.6~106 μg / mL and has a significant temperature dependence. Therefore, CsA drug particles have a short residence time on the ocular surface and low bioavailability when used topically in the eye.

[0005] Therefore, there is an urgent need in the field of dry eye treatment for a drug that has mucin adhesion properties and can inhibit ROS levels. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a liposome drug carrier with mucosal adhesion, its preparation method and application, so as to obtain at least one dry eye treatment drug with strong mucosal adhesion and no eye irritation.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A drug carrier, characterized in that: the carrier is DSPE-PEG-PBA, with the following structural formula:

[0009] .

[0010] Furthermore, the method for preparing the drug carrier includes the following steps:

[0011] Benzylboronic acid, N-hydroxythiosuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) with carboxyl or amino groups substituted on the benzene ring are dissolved in a mixed solvent of dichloromethane and methanol (v / v=2:1). Then, DSPE-PEG is added to carry out a cross-linking reaction. The reaction solution is then subjected to dialysis and lyophilization to obtain the drug carrier.

[0012] Furthermore, the phenylboronic acid with a carboxyl-substituted group on the benzene ring is preferably 4-carboxy-3-fluoro-phenylboronic acid (hereinafter abbreviated as PBA).

[0013] When the substituted phenylboronic acid is a phenylboronic acid with a carboxyl group substituted on the benzene ring, the DSPE-PEG is selected as DSPE-PEG-NH2; when the substituted phenylboronic acid is a phenylboronic acid with an amino group substituted on the benzene ring, the DSPE-PEG is selected as DSPE-PEG-COOH.

[0014] The molar ratio of 4-carboxy-3-fluoro-phenylboronic acid, thio-NHS, EDC and DSPE-PEG-NH2 is 1:3:2:0.6-0.8.

[0015] Furthermore, the crosslinking reaction is carried out at a temperature of 35-40°C for a time of 36-72 hours.

[0016] Preferably, the crosslinking reaction is carried out at a temperature of 37°C and a reaction time of 48 hours.

[0017] Furthermore, after adding the 4-carboxy-3-fluoro-phenylboronic acid, thioNHS, EDC and MW 2000 to the mixed solvent, it is necessary to stir and activate them at 35-40°C for 30-60 minutes.

[0018] Furthermore, the drug carrier is used to encapsulate anti-inflammatory drugs.

[0019] Furthermore, the anti-inflammatory drugs include nonsteroidal anti-inflammatory drugs and corticosteroids or immunosuppressants.

[0020] The immunosuppressants include cyclosporine A.

[0021] Furthermore, the method for encapsulating the anti-inflammatory drug is either thin-film dispersion or reverse evaporation.

[0022] Specifically, when the anti-inflammatory drug is a lipid-soluble drug, a thin-film dispersion method is used; when the anti-inflammatory drug is a water-soluble drug, a reverse evaporation method is used.

[0023] The method for encapsulating cyclosporine A using the thin-film dispersion method is as follows:

[0024] The DSPE-PEG-PBA, soybean lecithin (SPC), cholesterol (CHO), and cyclosporine A (CsA) were added to a mixed solvent of dichloromethane and methanol (v / v=2:1) ​​and rotary evaporated to form a membrane. Then, crocin I (Cro) was added, and the mixture was hydrated and sonicated to obtain a liposome solution. The free cyclosporine A and crocin I were removed from the liposome solution by centrifugation and dextran gel chromatography, respectively, to obtain CsA / Cro PBA Lip loaded with cyclosporine A and crocin I.

[0025] Furthermore, the CsA / Cro PBA Lip loaded with cyclosporine A and crocin I is used to treat inflammation and promote epithelial tissue healing.

[0026] Furthermore, the molar ratio of DSPE-PEG-PBA, soybean lecithin, cholesterol, cyclosporine A, and crocin I is 0.05-0.10:2:1:0.05-0.10:0.05.

[0027] Furthermore, the conditions for rotary evaporation to form the film are 25-37°C and 60-80 rpm.

[0028] Furthermore, the hydration conditions are 25-37℃, 60-100rpm, and hydration time is 15-30min.

[0029] Furthermore, the ultrasound conditions are 80W and the ultrasound time is 3-5 minutes.

[0030] The beneficial effects of this invention are:

[0031] 1. The carrier of the present invention is used to encapsulate cyclosporine A and crocin I. The carrier of the present invention has good stability and adhesion.

[0032] 2. The liposome drug obtained by encapsulating cyclosporine A and crocin I in the carrier of the present invention can inhibit the side effect of increased ROS levels caused by cyclosporine A.

[0033] 3. The carrier of the present invention, after encapsulating cyclosporine A and crocin I, has broad application prospects in the treatment of inflammation and epithelial tissue damage. Attached Figure Description

[0034] Figure 1 The 1H NMR spectra of DSPE-PEG-NH2, 4-carboxy-3-fluoro-phenylboronic acid (PBA), and DSPE-PEG-PBA are shown.

[0035] Figure 2 Infrared spectra of DSPE-PEG-NH2, 4-carboxy-3-fluoro-phenylboronic acid and DSPE-PEG-PBA;

[0036] Figure 3 This is the effect of different concentrations of CsA on increasing ROS levels in HCEC cells in Experiment Example 1 (n=3, mean±SD);

[0037] Figure 4 The effect of different concentrations of Cro on the increase of ROS level in HCEC cells stimulated by 5 μM CsA in Experiment 1 (n=3, mean±SD);

[0038] Figure 5 The inhibitory effects of different formulations on the increase of ROS levels in HCEC cells stimulated by 200 μM H2O2 in Experiment Example 2 (n=3, mean±SD);

[0039] Figure 6 The images show fluorescence images of the inhibitory effects of different formulations on the increased ROS levels in HCEC cells caused by 200 μM H2O2 in Experiment Example 2.

[0040] Figure 7 For example 3, the wound healing status 12 hours after treatment (n=3, mean±SD);

[0041] Figure 8 For Experiment 4, different formulations inhibited the expression of LPS-induced inflammatory factor proteins in HCEC cells and were semi-quantitatively analyzed (n=3, mean±SD).

[0042] Figure 9For example 5, the same formulation inhibited the expression of LPS-induced NF-κb pathway-related proteins and performed a semi-quantitative analysis (n=3, mean±SD);

[0043] Figure 10 For example 6, a) flowchart of mouse modeling and drug administration and b) H&E staining images of corneal sections from normal mice and model mice;

[0044] Figure 11 For example 6, the following are the corneal fluorescein sodium staining images and corneal fluorescein staining scores (n=5, mean±SD) of mice in each group from day 0 to day 3.

[0045] Figure 12 Corneal sections of mice with dry eye disease after 7 days of treatment: a) H&E staining and b) TUNEL staining (H&E scale bar: 100 μm, TUNEL scale bar: 50 μm).

[0046] Figure 13 For example 6, corneal immunohistochemical staining sections of mice with dry eye syndrome 7 days after treatment (scale bar: 50 μm).

[0047] Figure 14 In Experiment 6, the cornea of ​​normal mice was stained with H&E after 7 days of drug administration;

[0048] Figure 15 The attached diagram shows the mucins of Lip and PBA Lip in Experiment Example 7.

[0049] Figure 16 The results of flow cytometry analysis of HCEC cell uptake of Lip and PBA Lip are shown in Experiment Example 8.

[0050] Figure 17 This is a fluorescence confocal image of HCEC cells taking up PBA-modified liposomes in Experiment Example 8 (scale bar: 20 μm).

[0051] Figure 18 This is a statistical graph of in vivo adhesion in mice, measured by an in vivo fluorescence imaging system, in Experiment Example 9.

[0052] Figure 19 The graph shows the particle size stability of liposomes over 7 days in Experiment Example 10. Detailed Implementation

[0053] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0054] Example 1

[0055] The specific method for preparing a drug carrier is as follows:

[0056] 7.4 mg of 4-carboxy-3-fluoro-phenylboronic acid, 9.2 mg of N-hydroxythiosuccinimide, and 23 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in a mixed solvent of dichloromethane and methanol (v / v = 2:1). The mixture was stirred and activated at 37 °C for 30 min. Then, 34 mg of DSPE-PEG-NH2 was added, and the reaction was carried out at 37 °C for 48 h. The reaction solution was transferred to a dialysis bag (MWCO1000) and dialyzed in ultrapure water for 72 h. After lyophilization, DSPE-PEG-PBA was obtained.

[0057] Using deuterated DMSO as a solvent, 1H NMR spectroscopy was performed to identify DSPE-PEG-PBA. Figure 1 As shown, the characteristic peak of the carboxyl hydrogen of 4-carboxy-3-fluoro-phenylboronic acid at 9.5 ppm disappeared in the 1H NMR spectrum of DSPE-PEG-PBA, indicating the successful synthesis of DSPE-PEG-PBA.

[0058] Take a small amount of DSPE-PEG-PBA and perform infrared spectroscopy identification on it using methanol as a solvent. Figure 2 As shown, the infrared spectrum of PBA shows a characteristic OH peak of the carboxyl group around 3300, the infrared spectrum of DSPE-PEG-NH2 shows a characteristic NH peak around 3500, while the infrared spectrum of DSPE-PEG-PBA shows the disappearance of the NH peak around 3500, and the presence of CH peaks around 3000 and CO peaks between 1000-1250 for DSPE-PEG-NH2, indicating the successful synthesis of DSPE-PEG-PBA.

[0059] Example 2

[0060] Preparation of CsA / Cro PBA Lip:

[0061] 5.6 mg soybean lecithin (SPC), 1.4 mg cholesterol (CHO), 2.6 mg DSPE-PEG-PBA, and 0.48 mg cyclosporine A (CsA) were added to a mixed solvent of dichloromethane and methanol (v / v = 2:1). The mixture was rotary evaporated at 37°C and 80 rpm to form a membrane. 1 mL of 1 mg / mL crocin I (Cro) was added, and the mixture was hydrated at 37°C and 80 rpm for 20 min. The membrane was then sonicated at 80 W for 3 min to homogenize the particle size, resulting in a slightly opalescent liposome solution. The sonicated liposome solution was centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was then subjected to dextran gel chromatography to obtain CsA / Cro PBA Lip, free of cyclosporine A and crocin I.

[0062] Comparative Example

[0063] The CsA PBA Lip was prepared using the same method as in Example 2, except that crocin I was not added.

[0064] The preparation of Cro PBA Lip was the same as in Example 2, except that cyclosporine A was not added.

[0065] The CsA / Cro Lip was prepared using the same method as in Example 2, except that DSPE-PEG-PBA was replaced with DSPE-PEG.

[0066] Experimental Example 1

[0067] Evaluation of the side effects of Cro anti-CsA increasing cellular ROS levels:

[0068] HCEC cells were loaded at 5 × 10 5 Cells were evenly seeded in six-well plates at a density of 1 cell / well and cultured until the cells reached 80-90% coverage. After co-incubating the cells with CsA at concentrations of 2, 5, 10, 20, and 40 μM for 4 h, the cells were washed once with PBS, stained with DCFDA for 30 min, washed three times with PBS, digested with 0.25% trypsin containing 0.02% EDTA, resuspended in PBS, washed once, and then resuspended in PBS again before being analyzed by flow cytometry.

[0069] like Figure 3 As shown, cyclosporine A can significantly upregulate the ROS level of HCEC cells in the concentration range of 2 μM to 80 μM, stimulate human corneal epithelial cells, and the stimulatory effect does not increase with increasing concentration, reaching a maximum at 5 μM.

[0070] HCEC cells were loaded at 5 × 10 5 Cells were evenly seeded in six-well plates at a density of 1 cell / well and cultured until the cells reached 80-90% coverage. The CsA concentration was controlled at 5 μM, and the CsA to Cro concentration ratios were 1:0, 1:1, 1:2, and 1:4. Cells were co-incubated with culture media containing different CsA and Cro concentrations for 4 h. Cells were then washed once with PBS, stained with DCFDA for 30 min, washed three times with PBS, digested with 0.25% trypsin containing 0.02% EDTA, resuspended in PBS, washed once, and resuspended in PBS again before being analyzed by flow cytometry.

[0071] like Figure 4As shown, the ROS level of HCEC cells was significantly increased under the stimulation of 5 μM cyclosporine A, while the ROS level of HCEC cells was significantly decreased after administration of crocin, indicating that crocin I can effectively inhibit the ROS production caused by cyclosporine A and has the effect of alleviating the side effects of cyclosporine A.

[0072] Experiment Example 2

[0073] Quantitative and qualitative evaluation of the anti-ROS ability of CsA / Cro PBA Lip:

[0074] ① HCEC cells were loaded at a rate of 5 × 10⁻⁶ 5 Cells were evenly seeded in six-well plates at a density of 80-90% coverage. Cells were then co-incubated with 200 μM H₂O₂, 200 μM H₂O₂ + Free-CsA / Cro, 200 μM H₂O₂ + CsA PBA Lip, 200 μM H₂O₂ + Cro PBA Lip, and 200 μM H₂O₂ + CsA / Cro PBA Lip (with final concentrations of 5 μM for both CsA and Cro) for 4 h. Cells were washed once with PBS, and each well was stimulated with 200 μM H₂O₂ for 1 h. Cells were washed once with PBS, stained with DCFDA for 30 min, washed three times with PBS, digested with 0.25% trypsin containing 0.02% EDTA, resuspended in PBS, washed once, and resuspended in PBS again before analysis by flow cytometry.

[0075] like Figure 5 As shown, H2O2 stimulation significantly upregulated ROS levels in HCEC cells, while administration of Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBA Lip significantly downregulated ROS levels, demonstrating good antioxidant capacity. Among these, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBA Lip exhibited stronger inhibitory effects on ROS production compared to Free-CsA / Cro, indicating that encapsulating drugs in liposomes enhances cellular absorption and utilization, leading to better therapeutic effects.

[0076] ② HCEC cells were loaded at a rate of 5 × 10⁻⁶ 5Cells were evenly seeded in six-well plates at a density of cells / well and cultured until the cell coverage reached 80-90%. Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, CsA / Cro PBA Lip, and CsA and Cro PBA Lip, with a final concentration of 5 μM for both CsA and Cro, were co-incubated with the cells for 4 h. Then, 200 μM H2O2 was added and the cells were incubated for 1 h. The cells were washed once with PBS, stained with DCFDA for 30 min, washed three times with PBS, and observed and photographed using an inverted fluorescence microscope.

[0077] like Figure 6 As shown, the FITC fluorescence of the group stimulated with 200 μM H2O2 alone was significantly enhanced compared with the PBS group, while the FITC fluorescence of the groups pre-incubated with Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBA Lip was significantly weakened compared with the group stimulated with 200 μM H2O2 alone, which is consistent with the flow cytometry results.

[0078] Experimental Example 3

[0079] Evaluation of corneal epithelial repair capacity of CsA / Cro PBA Lip:

[0080] The corneal epithelial repair properties of the formulation were evaluated using a scratch assay. HCEC cells were evenly seeded in six-well plates at a density of 5 × 10⁵ cells / well and cultured until 100% cell coverage was achieved. Scratches were created in the six-well plates using a 200 μL pipette tip. The cells were then co-incubated with PBS, Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBA Lip, with CsA and Cro at a final concentration of 5 μM, for 12 h, followed by photographic recording.

[0081] like Figure 7 As shown, 12 h after cell line marking and drug administration, all groups, compared with the PBS group, promoted corneal epithelial healing. The healing rate of the PBS group was 6.45±0.80%, the healing rate of Free-CsA / Cro was 9.16±2.47%, the healing rate of CsAPBA Lip was 22.14±0.25%, the healing rate of Cro PBA Lip was 17.40±0.62%, and the CsA / Cro PBALip had the strongest effect on promoting scratch healing, with a healing rate of 62.74±1.65%.

[0082] Experiment Example 4

[0083] Evaluation of the anti-inflammatory effect of CsA / Cro PBA Lip:

[0084] HCEC cells were seeded uniformly in six-well plates at a density of 5 × 10⁵ cells / well and cultured until tumor cells reached 80–90% coverage. The medium was then replaced with serum-free medium, and LPS was added at a concentration of 1 μg / mL to the cells for 1 h. The serum-free medium containing LPS was discarded, and medium containing Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, or CsA / Cro PBA Lip was added. The final concentrations of CsA and Cro were both 5 μM. After incubation for 5 h, the cells were digested and centrifuged to collect them. The cell lysis buffer was mixed with 100 mmol / L benzyl sulfonyl fluoride (PMSF) in isopropanol at a volume ratio of 100:1 to obtain the experimental cell lysis buffer. Cells were lysed using this lysis buffer at 4 °C and 12,000 rpm, and total protein was extracted. The protein was mixed with protein loading buffer at a ratio of 4:1 and boiled for 10 min to obtain the Western blot protein sample.

[0085] The specific procedures for Western blotting are as follows: Use a rapid gel preparation kit and select a separating gel of appropriate concentration based on the molecular weight of the target protein. After correctly assembling the apparatus, add the separating gel 1.5-2 cm from the top of the glass plate, quickly pour in the upper stacking gel, and insert the comb. After the stacking gel solidifies, remove the comb, pour in UP water, transfer the plate to the electrophoresis tank, and add electrophoresis buffer to submerge the plate. Add the sample and protein molecular weight standards, and perform electrophoresis at a constant voltage of 90 V to compress the protein sample in the stacking gel. Once the sample enters the separating gel, switch to 150 V for electrophoresis. Stop the electrophoresis once the protein sample is fully separated. Under a constant current of 300 mA and an ice bath, transfer the protein to a PVDF membrane and block it with TBST solution containing 5% skim milk powder at 37°C for 2 h. After washing with TBST, the sample was incubated in 3% skim milk with rabbit anti-TNF-α primary antibody, rabbit anti-IL-1β primary antibody, rabbit anti-IL-6 primary antibody, or rabbit anti-GAPDH primary antibody at 37°C for 1 hour in a shaker, followed by overnight incubation at 4°C. After washing 5 times with TBST, the sample was incubated in 3% skim milk with HRP-labeled goat anti-rabbit secondary antibody at 37°C for 1 hour, washed 5 times, and then developed with chemiluminescent developing solution. Finally, the sample was photographed using a gel imaging system.

[0086] like Figure 8As shown, LPS significantly induced an inflammatory response in HCEC cells, leading to increased levels of IL-6, IL-1β, and TNF-α. However, after incubation with Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBALip, the production of IL-6, IL-1β, and TNF-α in HCEC cells was significantly reduced. Among these, CsA / Cro PBALip showed the best anti-inflammatory factor production, with significant differences compared to the other groups. This indicates that encapsulating cyclosporine A and crocin I in liposomes can enhance cellular absorption and utilization of the drugs, achieving synergistic drug action and resulting in better anti-inflammatory effects.

[0087] Experimental Example 5

[0088] Research on the anti-inflammatory mechanism of CsA / Cro PBA Lip:

[0089] HCEC cells were loaded at 5 × 10 5 Tumor cells were evenly seeded in six-well plates at a density of 1 cell / well and cultured until the coverage reached 80-90%. The medium was then replaced with serum-free medium, and LPS was added at a concentration of 1 μg / mL for 1 h. The serum-free medium containing LPS was discarded, and medium containing Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, or CsA / Cro PBA Lip was added. The final concentrations of CsA and Cro were both 5 μM. After incubation for 5 h, the cells were digested and centrifuged to collect them. The cell lysis buffer was mixed with 100 mmol / L isopropanol solution of benzyl sulfonyl fluoride (PMSF) and a phosphatase inhibitor at a volume ratio of 100:1:1 to prepare the experimental cell lysis buffer. Cells were lysed using this lysis buffer at 4°C and 12000 rpm, and total protein was extracted. The protein was mixed with protein loading buffer at a ratio of 4:1 and boiled for 10 min to obtain Western blot protein samples.

[0090] like Figure 9 As shown, after LPS stimulation, the levels of P65 and P-P65 proteins in HCEC cells were significantly upregulated, indicating the activation of the NF-κB pathway. However, after co-incubation with Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, and CsA / Cro PBA Lip, the levels of P65 and P-P65 proteins were significantly downregulated compared with the LPS group, indicating that CsA / Cro PBA Lip can inhibit the expression of inflammatory factors by inhibiting the NF-κB pathway, thereby exerting an anti-inflammatory effect.

[0091] Experimental Example 6

[0092] Animal experiments and in vivo pharmacodynamic evaluation of CsA / Cro PBA Lip:

[0093] ① Establishment of animal models and drug administration regimens:

[0094] A mouse model of dry eye was established by instilling 5 μL of 0.2% benzalkonium chloride solution into the right eye twice daily for 14 days in 8-week-old female C57 mice weighing 18–20 g. The mice were then randomly divided into 6 groups of 6 mice each: PBS, Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, CsA / Cro Lip, and CsA / Cro PBA Lip. CsA and Cro were administered topically to the right eye of the modeling mice at final concentrations of 0.5 mg / mL and 0.4 mg / mL, respectively, 5 μL per mouse, twice daily for 7 days.

[0095] like Figure 10 As shown, the cornea of ​​a normal mouse is clearly defined and intact, with a distinct three-layer structure consisting of the epithelium, stroma, and endothelium. The corneal epithelial cells are arranged in multiple layers and are tightly packed, exhibiting a certain degree of polarity. In the modeling mouse, the corneal epithelium is damaged, resulting in obvious corneal epithelial defects and inflammatory cell infiltration in the cornea, indicating that the mouse dry eye model has been successfully established.

[0096] ② Fluorescein sodium staining test:

[0097] Fluorescein staining was performed on days 0, 1, 2, and 3. Mice were anesthetized, and a 1 mg / mL solution of fluorescein was instilled into the right eye of each mouse.

[0098] 5 μL of sodium fluorescein was applied. After manually closing the mouse's eyelids three times, waited 60 seconds, and gently wiped away excess sodium fluorescein with paper. The mouse was then placed on its side with its right eye under a slit lamp cobalt blue light for observation and photographing. Corneal staining was scored according to the following rules: the eye was divided into 5 parts (central, nasal, temporal, superior, and inferior). Evaluation criteria: 0 = no staining, 1 = slight punctate staining, 2 = obvious punctate staining or slight confluent staining, 3 = obvious confluent staining or slight patchy staining, 4 = obvious patchy staining. The scores for each group were recorded and statistically analyzed.

[0099] like Figure 11As shown, on day 0, large areas of patchy staining were observed in the corneal fluorescein staining of all groups of mice, indicating corneal defects and easy penetration and retention of fluorescein dye. After one day of treatment with each formulation, the PBS group still showed large areas of obvious patchy staining in the central, nasal, temporal, and upper regions; the Free-CsA / Cro, CsALip, and CsA PBA Lip groups still showed obvious patchy staining in the central region; the Cro PBA Lip and CsA / Cro Lip groups showed slight patchy staining in the central region; and the CsA / Cro PBA Lip group showed obvious fused staining in the lower region. On day 2, the PBS group still showed patchy staining in the central region; the Free-CsA / Cro, Cro PBA Lip, and CsA PBA Lip groups showed obvious fused staining in the central region; and the CsA / Cro PBA Lip group showed only a few punctate staining. On the third day of treatment, the PBS group showed obvious fused staining in the central, nasal, temporal, upper, and lower regions. The Free-CsA / Cro, CsA PBA Lip, and CroPBA Lip groups showed obvious punctate staining. The CsA / Cro Lip group showed only slight punctate staining, while the CsA / Cro PBA Lip group showed almost no staining. This indicates that the CsA / Cro PBA Lip group had the best therapeutic effect on dry eye in mice. The corneal staining scores of each group also confirmed this result.

[0100] ③ Pathological observation of the cornea:

[0101] After the mice were euthanized, their right eyeballs were fixed with 4% paraformaldehyde, stained with hematoxylin and eosin (HE) and TUNEL, and observed under a microscope.

[0102] like Figure 12 As shown, after seven days of treatment, the corneal structure in the PBS group was unclear, the corneal epithelium was thin with only a single layer of cells and obvious defects, and there was significant inflammatory cell infiltration in the cornea, with corneal edema. The corneal epithelial cell layer in the Free-CsA / Cro and CsALip groups was also thin, with only a single layer of cells, and the cells were irregularly arranged without polarity, with mild corneal edema. The corneal structure in the CsA / Cro Lip and CsA / Cro PBA Lip groups was clear, divided into three distinct layers: epithelium, stroma, and endothelium. The corneal epithelium was thicker, with multiple layers of cells arranged tightly and showing obvious polarity, and there was virtually no immune cell infiltration in the cornea, indicating that their dry eye symptoms had largely resolved.

[0103] like Figure 12As shown, a large number of apoptotic cell nuclei stained dark brown were observed in the PBS group and the Free-CsA / Cro group, while a small number of apoptotic cells were still observed in the CsA Lip group. However, no obvious corneal epithelial cell apoptosis was observed in the CsA PBA Lip, Cro PBA Lip, CsA / Cro Lip, and CsA / Cro PBALip groups. This indicates that encapsulating the drug in liposomes for drug delivery can effectively increase the absorption and utilization of the drug by corneal epithelial cells and reduce corneal epithelial cell apoptosis caused by dry eye syndrome.

[0104] ④ Immunohistochemical analysis:

[0105] After the obtained eyeball tissue was fixed, immunohistochemical staining was performed, and microscopic images were taken to analyze the expression of TNF-α, IL-1β, and IL-6 proteins in the tumor tissue.

[0106] Immunohistochemistry: After preparing paraffin sections using the aforementioned method, incubate with 3% H2O2 at room temperature for 5-10 min.

[0107] Rinse with PBS and soak for 5 min. Block with 5-10% normal goat serum (diluted in PBS) and incubate at room temperature for 10 min. Discard the serum, add an appropriate amount of diluted primary antibody or working solution, incubate at 37°C for 1-2 hours or at 4°C overnight, wash 3 times with PBS for 5 min each time, add an appropriate amount of diluted biotin-labeled secondary antibody (1% BSA-PBS), incubate at 37°C for 10-30 min, and then wash 3 times with PBS for 5 min each time.

[0108] Add horseradish enzyme-labeled streptavidin (PBS diluted appropriately), incubate at 37°C for 10-30 min, wash three times with PBS for 5 min each time, then develop with DAB. Rinse thoroughly with tap water, counterstain, mount, and then observe under a microscope.

[0109] like Figure 13 As shown, semi-quantitative analysis revealed higher levels of inflammatory factors in the PBS group. After seven days of treatment, except for the Free-CsA / Cro group which resulted in an increase in IL-1β levels, all other groups showed a decrease in TNF-α, IL-1β, and IL-6 levels. The CsA / Cro PBA Lip group exhibited the lowest levels of TNF-α, IL-1β, and IL-6, demonstrating the best inhibitory effect on inflammation. This result is largely consistent with the Western blot (WB) results from in vitro cell experiments.

[0110] ⑤ Safety assessment:

[0111] Healthy 8-week-old female C57 mice, weighing 18-20g, were randomly divided into 6 groups of 5 mice each: PBS, Free-CsA / Cro, CsA PBA Lip, Cro PBA Lip, CsA / Cro Lip, and CsA / Cro PBA Lip. The formulations were administered topically to the right eye of each mouse via intraocular drops. The final concentrations of CsA and Cro were 0.5 mg / mL and 0.4 mg / mL, respectively. 5 μL was administered to each mouse twice daily for 7 days. After 7 days, the mice were euthanized by cervical dislocation. The right eye was collected, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (HE) to assess the safety of the formulations.

[0112] like Figure 14 As shown, the corneal structures in each group were clear and intact, with no obvious defects in the corneal epithelium, indicating that the preparations in each group were safe and had no obvious toxicity to the cornea.

[0113] Experimental Example 7

[0114] Evaluation of the in vitro adhesion of PBA Lip:

[0115] Unmodified PBA liposomes and PBA liposomes were separately suspended in a 1 mg / mL mucin solution and incubated at 37°C for 1 h. The suspensions were then centrifuged at 10,000 rpm for 10 min, and the free mucin in the supernatant was determined by periodic acid-Schiff (PAS) staining. The supernatant was mixed with 200 μL of periodic acid reagent and incubated at 37°C for 2 h, followed by the addition of 200 μL of Schiff reagent and incubation at room temperature for 30 min. The absorbance was measured at 555 nm. The amount of mucin adsorbed on the liposomes was determined by subtracting the amount of free mucin from the initial protein amount. Figure 15 As shown, in vitro experiments have demonstrated that PBA-modified liposomes can adhere to more mucoproteins than ordinary liposomes, thus achieving mucosal adhesion.

[0116] Experimental Example 8

[0117] Qualitative and quantitative evaluation of PBA Lip intake:

[0118] HCEC cells were loaded at 5 × 10 5 Cells were evenly seeded at a density of [number] cells / well in six-well plates and cultured until 80-90% cell coverage was achieved. Preparations of ordinary liposomes and PBA liposomes loaded with DID (diffuse membrane far-infrared probe) were added to the wells to a final DID concentration of 0.5 μg / mL, and incubated for 0.5 h, 1 h, and 2 h, respectively. Cells were washed three times with PBS, digested with 0.25% trypsin containing 0.02% EDTA, resuspended in PBS, washed once, and then resuspended in PBS again before flow cytometry analysis. Figure 16As shown, the uptake of PBA Lip and Lip increased significantly with time. At 0.5 h, 1 h and 2 h, the fluorescence intensity of PBA-modified liposomes was significantly enhanced compared with that of ordinary liposomes, indicating that the cells uptake PBA Lip significantly stronger than Lip.

[0119] HCEC cells were loaded at 5 × 10 5 Cells were evenly seeded onto glass slides at a density of 1 cell / well and cultured until 80-90% cell coverage was achieved. Different formulations of DID were added to the wells to achieve a final DID concentration of 0.5 μg / mL, and incubated for 0.5 h, 1 h, and 2 h, respectively. After washing three times with PBS, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, stained with 1 μg / mL DAPI solution for 5 min, washed three times with PBS, and then fixed with an anti-fluorescence quencher. The slides were then mounted and observed and photographed under a laser confocal microscope. Figure 17 As shown, the uptake of PBA Lip and Lip increased significantly with time. The PBA-modified liposomes showed more pronounced red fluorescence at 0.5 h, 1 h, and 2 h compared to ordinary liposomes, which is consistent with the flow cytometry results.

[0120] Experimental Example 9

[0121] Evaluation of PBA Lip's in vivo adhesion:

[0122] Free-DID (liposomes without DID), DID-Lip (a formulation containing DID and ordinary liposomes), and DID-PBA Lip (a liposome formulation containing DID and modified with phenylboronic acid) were administered as ocular drops to the right eye of each mouse, at a concentration of 0.2 μg / mouse. Mice were anesthetized and photographed in vivo using a live imaging system before and at 1 min, 5 min, 10 min, 20 min, and 30 min after administration. Figure 18 As shown, free DID showed no obvious fluorescence at any time point, while the fluorescence intensity of the PBA Lip group was stronger than that of the Lip group at all time points. This indicates that the liposomes modified with phenylboronic acid can effectively enhance mucosal adhesion, enhance drug retention on the ocular surface, and prolong the duration of drug action.

[0123] Experimental Example 10

[0124] Stability assessment of CsA / Cro PBA Lip:

[0125] The liposomes prepared in Example 2 and the comparative group were diluted with ultrapure water to a suitable concentration and stored at 4°C for 7 days. The particle size was measured daily using a laser particle size analyzer to evaluate their stability.

[0126] Depend on Figure 19 As shown, the liposomes were stored at 4℃ for 7 days, and their particle size did not change significantly, indicating that the liposome solution has good stability under low temperature conditions.

[0127] Experimental Example 11

[0128] Determination of PBA content in CsA / Cro PBA Lip:

[0129] DSPE-PEG-FITC (prepared in the same way as in Example 2, except that FITC is used instead of PBA) was used instead of DSPE-PEG-PBA, and the content of PBA on the liposomes was determined by fluorescence method. The prepared FITC Lip was placed in a dialysis bag at 8000-14000 and dialyzed for 4 hours, with the water changed every 2 hours to remove unloaded DSPE-PEG-FITC. The dialyzed liposomes and the undialyzed liposomes were mixed with an equal volume of methanol solution and vortexed to break the emulsion. The fluorescence intensity was measured by fluorescence spectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The fluorescence intensity of the treated liposomes was divided by the fluorescence intensity of the liposomes before treatment to obtain the PBA content on the liposomes as 6.28 ± 0.03%.

[0130] Experimental Example 12

[0131] Determination of CsA and Cro encapsulation efficiency and drug loading in CsA / Cro PBA Lip:

[0132] The CsA / Cro PBA Lip prepared in Example 2 was divided into three equal parts. The first part was subjected to dextran gel chromatography to remove unencapsulated Cro, concentrated by ultrafiltration, and then mixed with methanol at a volume ratio of 1:1. The second part was centrifuged at 10,000 rpm for 10 min to remove water-insoluble free CsA, and the supernatant was mixed with methanol at a volume ratio of 1:1. The third part was directly mixed with methanol at a volume ratio of 1:1, which was taken as the total feed amount. The above samples mixed with methanol were vortexed for 5 min to break the emulsion, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The encapsulation efficiency was obtained by calculating the peak area ratio by HPLC. The results are shown in Table 1.

[0133] Table 1

[0134]

[0135] It can be seen that the liposome carrier of the present invention achieves encapsulation rates of over 96% for CsA and over 52% for Cro.

[0136] Experimental Example 13

[0137] Experimental study on the encapsulation effect of DSPE-PEG-PBA on nonsteroidal anti-inflammatory drugs and corticosteroids:

[0138] For corticosteroids, dexamethasone (DEX) was selected as the model drug, and for nonsteroidal anti-inflammatory drugs, pranofen (Pra) was selected as the model drug.

[0139] DEX PBA Lip and Pra PBALip were prepared using the same method as in Example 2, except that CsA was replaced with DEX and Pra respectively, and Cro was not added.

[0140] The encapsulation efficiency and drug loading of DEX and Pra in DEX PBA Lip and Pra PBALip were determined using the same method as in Experiment 2. The results are shown in Table 2.

[0141] Table 2

[0142]

[0143] It can be seen that the liposome carrier of the present invention has an encapsulation rate of over 80% for both nonsteroidal anti-inflammatory drugs and corticosteroid hormones, which meets the quality standards for liposome carriers specified in the pharmacopoeia.

[0144] The CsA / Cro PBA Lip from Example 2, as well as the CsA PBA Lip, Cro PBA Lip, and CsA / Cro Lip from the comparative examples, are used as references.

[0145] The liposomes prepared above, as well as the CsA / Cro PBA Lip from Example 2 and the CsA PBALip, Cro PBA Lip, and CsA / Cro Lip from the comparative examples, were diluted with ultrapure water to a suitable concentration, and their particle size and potential were measured using a laser particle size analyzer. As shown in Table 3, the particle size of each group of liposomes was around 100 nm, and the polydispersity index (PDI) was below 0.3. The potential of the unmodified PBA liposomes was around -6 mV, while the potential of the PBA-modified liposomes was around 0 mV, indicating that PBA modification caused the electrical properties of the liposomes to change towards neutral or even positive charge.

[0146] Table 3

[0147]

[0148] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. The CsA / Cro PBA Lip is used in the preparation of a drug for treating dry eye syndrome, characterized in that: Preparation methods include: DSPE-PEG-PBA, soybean lecithin, cholesterol, and cyclosporine A were added to dichloromethane solvent and rotary evaporated in a mixed solvent of methanol and water to form a film. Then, crocin I was added, and the mixture was hydrated and sonicated to obtain a liposome solution. The free cyclosporine A and crocin I were removed from the liposome solution by centrifugation and dextran gel chromatography, respectively, to obtain CsA / Cro PBA Lip loaded with cyclosporine A and crocin I.

2. The drug application according to claim 1, characterized in that: The molar ratio of DSPE-PEG-PBA, soybean lecithin, cholesterol, cyclosporine A and crocin I is 0.05-0.10:2:1:0.05-0.10:0.

05.

3. The drug application according to claim 1, characterized in that: The treatment for dry eye includes promoting epithelial tissue healing.

4. The drug application according to claim 1, characterized in that: The structural formula of the DSPE-PEG-PBA is as follows: ； The DSPE-PEG-PBA is prepared by cross-linking phenylboronic acid with carboxyl-substituted groups on the benzene ring with DSPE-PEG-NH2, and has the ability to adhere to mucous membranes.

5. The drug application according to claim 4, characterized in that: The preparation method of the DSPE-PEG-PBA includes the following steps: Benzeneboronic acid with carboxyl substitution on the benzene ring, N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in a mixed solvent of dichloromethane and methanol, and then DSPE-PEG was added to carry out an acylation reaction. The reaction solution was then dialyzed and lyophilized to obtain the DSPE-PEG-PBA. Wherein, the phenylboronic acid with a carboxyl-substituted group on the benzene ring is 4-carboxy-3-fluoro-phenylboronic acid; The DSPE-PEG is DSPE-PEG-NH2; The molar ratio of 4-carboxy-3-fluoro-phenylboronic acid, N-hydroxythiosuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and DSPE-PEG-NH2 is 1:3:2:0.6-0.

8.

6. The drug application according to claim 5, characterized in that: The acylation reaction is carried out at a temperature of 35-40℃ for 36-72 hours.

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