Nanoparticle penetrating cornea as well as preparation method and application thereof

Abstract

The invention provides a nanoparticle penetrating cornea as well as a preparation method and application thereof. The nanoparticle comprises a cationic liposome G2-C14, an inner core formed by aliphatic polyester and an amphiphilic polymer, and a shell modified by polysaccharide, the hydrophobic drug and the siRNA are loaded in the inner core. According to the nano-particles provided by the invention, through polysaccharide surface modification, ocular surface adhesion and trans-epithelium transport capability are enhanced, multi-barrier penetration of the eye is realized, the nano-particles are effectively delivered to a focus area of posterior segment of the eye, the delivered siRNA targets to silence the expression of age-related macular degeneration pathogenic genes, and the hydrophobic drug removes active oxygen and inhibits release of inflammatory factors, so that the anti-aging effect of the nano-particles is improved. The focus thickness is synergistically reduced.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and nanotechnology, specifically relating to a corneal-penetrating nanoparticle, its preparation method, and its application. Background Technology

[0002] Choroidal neovascularization (CNV) is the most significant pathological mechanism leading to irreversible vision loss in wet age-related macular degeneration (wAMD), high myopia-related CNV, and other retinal vascular diseases. Its core pathological processes include retinal pigment epithelial (RPE) cell dysfunction, chronic oxidative stress, local inflammatory responses, and abnormally high expression of vascular endothelial growth factor (VEGF). VEGF, currently recognized as the most crucial pro-angiogenic factor, not only drives the growth, leakage, and hemorrhage of abnormal choroidal vessels into the subretinal space through excessive secretion, but also amplifies the inflammatory cascade by activating the downstream NF-κB pathway, creating a vicious cycle that ultimately leads to retinal structural damage, macular edema, and permanent central vision loss.

[0003] Small interfering RNA (siRNA) technology can specifically silence the expression of pathogenic genes, providing a new strategy for targeted therapy of AMD. For example, siRNAs targeting genes such as vascular endothelial growth factor (VEGF) and complement factors (e.g., CFB, C3) hold promise for inhibiting angiogenesis in AMD at its source or regulating the inflammatory response in dry AMD. However, siRNAs themselves have difficulty penetrating the multiple physiological barriers of the eye (especially the cornea and blood-retinal barrier), and systemic administration can lead to off-target effects, while intravitreal injection also presents invasiveness issues.

[0004] Lutein is a natural carotenoid that accumulates in the macular region of the retina. It possesses powerful antioxidant and blue light filtering properties and is a key nutrient for maintaining the health of photoreceptor cells. Clinical studies have confirmed that lutein supplementation can slow the progression of AMD. However, lutein has extremely poor water solubility, low oral bioavailability, and difficulty penetrating the retina when used topically as eye drops. Furthermore, there are few reports on the application of the synergistic effect of lutein's anti-inflammatory properties and siRNA gene silencing in the treatment of fundus diseases.

[0005] In conclusion, developing a drug delivery system that can non-invasively and efficiently deliver gene therapy drugs (siRNA) and retinal nutritional protectants (such as lutein) to the posterior segment of the eye to achieve multi-target synergistic treatment of AMD has significant clinical needs and practical implications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a corneal-penetrating nanoparticle, its preparation method, and its application. The nanoparticles provided by the present invention can penetrate multiple eye barriers, effectively deliver to the posterior segment lesion area, target and silence the expression of age-related macular degeneration pathogenic genes, scavenge reactive oxygen species and inhibit the release of inflammatory factors, and synergistically reduce lesion thickness.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a corneal-penetrating nanoparticle, the nanoparticle comprising a core formed of cationic liposome G2-C14, an aliphatic polyester and an amphiphilic polymer, and a polysaccharide-modified shell; a hydrophobic drug and siRNA are loaded in the core.

[0008] The nanoparticles provided by this invention enhance ocular surface adhesion and transepithelial transport capabilities through polysaccharide surface modification, enabling them to penetrate multiple ocular barriers and effectively deliver to the posterior segment lesion area. The delivered siRNA targets and silences the expression of age-related macular degeneration pathogenic genes, while the hydrophobic drug scavenges reactive oxygen species and inhibits the release of inflammatory factors, synergistically reducing lesion thickness.

[0009] Compared to conventional cationic liposomes G0-C14, cationic liposomes G2-C14 have a higher effective cation density and multivalent interaction capability, resulting in better performance in siRNA complex stability, cellular uptake and endosome escape, while maintaining acceptable biocompatibility under appropriate formulation.

[0010] Preferably, the mass ratio of the cationic liposome G2-C14, the aliphatic polyester, and the amphiphilic polymer is 1:(3~7):(90~110).

[0011] The specific point values ​​in (3~7) can be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7, etc.

[0012] The specific point values ​​in (90~110) can be 90, 92, 95, 97, 100, 103, 105, 108 or 110, etc.

[0013] Preferably, the mass ratio of the cationic liposome G2-C14 to the polysaccharide is 1:(0.1~5).

[0014] The specific point values ​​in (0.1~5) can be 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0015] Preferably, the mass ratio of the cationic liposome G2-C14 to the polysaccharide is 1:(0.5~2).

[0016] The specific point values ​​in (0.5~2) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.8 or 2, etc.

[0017] Preferably, the mass ratio of the cationic liposome G2-C14 to the hydrophobic drug is 1:(0.5~10).

[0018] The specific point values ​​in (0.5~10) can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0019] Preferably, the ratio of cationic liposome G2-C14 to siRNA is 1 μg:(0.001~0.01) nmol.

[0020] The specific point values ​​in (0.001~0.01) can be 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.01, etc.

[0021] Preferably, the aliphatic polyester comprises any one or a combination of at least two of polylactic acid-glycolic acid copolymer, polylactic acid, or polyacetin.

[0022] Preferably, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (50~75):(50~25).

[0023] The specific point values ​​in (50~75) can be 50, 52, 55, 57, 60, 63, 65, 68, 70 or 75, etc.

[0024] The specific point values ​​in (50~25) can be 50, 48, 45, 43, 40, 37, 35, 32, 30 or 25, etc.

[0025] Preferably, the amphiphilic polymer comprises any one or a combination of at least two of the following: distearylphosphatidylethanolamine-polyethylene glycol, dimyristoylglycerol-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, or dioleoylphosphatidylethanolamine-polyethylene glycol.

[0026] Preferably, the number average molecular weight of the polyethylene glycol in the distearylphosphatidylethanolamine-polyethylene glycol, dimyristoylglycerol-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, or dioleoylphosphatidylethanolamine-polyethylene glycol is 1000~8000 Da, for example, it can be 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, or 8000 Da.

[0027] Preferably, the polysaccharide comprises chitosan and / or chitosan quaternary ammonium salt.

[0028] Preferably, the hydrophobic drug includes any one or a combination of at least two of lutein, astaxanthin, or curcumin.

[0029] Preferably, the siRNA includes siRNA that targets genes causing age-related macular degeneration.

[0030] Preferably, the age-related macular degeneration pathogenic gene includes any one or a combination of at least two of VEGF, complement factor H, complement factor B, ARMS2, or HTRA1.

[0031] Preferably, the siRNA is a siRNA that targets VEGF.

[0032] Preferably, the average particle size of the nanoparticles is 50~200 nm, for example, it can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm, 180 nm or 200 nm, etc.

[0033] Preferably, the encapsulation rates of the hydrophobic drug and siRNA in the nanoparticles are each independently not less than 40%, for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

[0034] In a second aspect, the present invention provides a method for preparing nanoparticles as described in the first aspect, the method comprising: An organic solvent containing cationic liposomes G2-C14 was mixed with an aqueous solution of siRNA, then mixed with an aliphatic polyester and a hydrophobic drug, and finally mixed with an aqueous solution of an amphiphilic polymer. The mixture was centrifuged, the precipitate was collected, soaked in a polysaccharide solution, centrifuged again, and the precipitate was collected to obtain the nanoparticles.

[0035] In this invention, cationic liposomes G2-C14 and siRNA are fully bound through electrostatic interactions. Then, hydrophobic drugs are loaded onto aliphatic polyesters, and amphiphilic polymers stabilize the particle structure and prolong the in vivo circulation time. Finally, the outer layer is modified with polysaccharides to enhance transcorneal transport capacity.

[0036] Preferably, the organic solvent includes acetone and / or N,N-dimethylformamide.

[0037] Preferably, the centrifugation speed is 800~1200 rpm, for example, 800 rpm, 850 rpm, 900 rpm, 950 rpm, 1000 rpm, 1050 rpm, 1100 rpm, 1150 rpm or 1200 rpm; the time is 15~30 min, for example, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 22 min, 25 min, 28 min or 30 min.

[0038] Preferably, the process of collecting the sediment further includes a water washing step.

[0039] Preferably, the soaking time is 8 to 12 hours, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours.

[0040] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0041] Thirdly, the present invention provides the use of the nanoparticles as described in the first aspect in the preparation of medicaments for treating age-related macular degeneration.

[0042] Fourthly, the present invention provides an ophthalmic formulation for treating age-related macular degeneration, the ophthalmic formulation comprising nanoparticles as described in the first aspect and a pharmaceutically acceptable carrier.

[0043] Preferably, the dosage form of the ophthalmic preparation includes any one of eye drops, ophthalmic gel, or ophthalmic ointment.

[0044] Preferably, the ophthalmic preparation is administered via ocular surface application.

[0045] Compared with the prior art, the present invention has the following beneficial effects: The nanoparticles provided by this invention, through polysaccharide surface modification, enhance ocular surface adhesion and transepithelial transport capabilities, enabling penetration through multiple ocular barriers and effective delivery to the posterior segment lesion area. The delivered siRNA targets and silences the expression of age-related macular degeneration pathogenic genes, while the hydrophobic drug scavenges reactive oxygen species and inhibits the release of inflammatory factors, synergistically reducing lesion thickness. The local drug delivery system minimizes systemic exposure and side effects, and the carrier material exhibits good biocompatibility. This non-invasive approach transforms siRNA therapy, which previously required intraocular injection, into a self-administered eye drop, significantly improving treatment convenience and patient compliance while reducing the risk of infection. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the preparation process of Chi@Lu / si NPs nanoparticles in Example 1; Figure 2 The average hydrated particle size distribution (A), transmission electron microscope image (B), and zeta potential distribution (C) of the Si NPs nanoparticles in Comparative Example 1 are shown. Figure 3 The UV-Vis absorption spectrum of Lu / si NPs, the nanoparticle intermediate in Example 1, is shown below. Figure 4 The encapsulation efficiency (A) and average hydrated particle size (B) of the Chi@Lu / si NPs prepared in Example 1 when the mass ratio of cationic liposome G2-C14 to lutein was 1:0.5, 1:1, 1:2, 1:4, 1:8, and 1:10. Figure 5 Fluorescence emission spectrum of Example 1 nanoparticles (Chi@Lu / si NPs) labeled with fluorescent groups; Figure 6 The images show the average hydrated particle size distribution (A), zeta potential distribution (B), and transmission electron microscope image (C) of the Chi@Lu / si NPs prepared in Example 1 when the concentrations of the chitosan solution were 0.01%w / v, 0.02%w / v, 0.03%w / v, and 0.06%w / v. Figure 7 The in vitro release curve of lutein from Chi@Lu / si NPs nanoparticles in Example 1 is shown. Figure 8 The images shown are laser confocal microscopy (A) and fluorescence quantitative PCR (B) images of HCE cells after siRNA in Chi@Lu / si NPs nanoparticles labeled with FAM in Example 1 were incubated with HCE cells for 4 h and 24 h. Figure 9The cell viability graphs are shown for Chi@Lu / si NPs nanoparticles from Example 1 after co-incubation with 293T cells (A), HCE cells (B), and ARPE-19 cells (C) for 24 h. Figure 10 Bioluminescence imaging (A), average luminescence intensity quantification (B), and relative luminescence intensity quantification (C) of 293T cells expressing luciferase after incubation with 0 nM, 12.5 nM, 25 nM, and 50 nM siRNA-containing nanoparticles Chi@Lu / si NPs and si NPs containing 50 nM siRNA-containing luciferase-targeting nanoparticles Chi@Lu / si NPs and si NPs for 24 h. Figure 11 The images show the DCFH-DA fluorescence staining pattern (B) and the DCF relative fluorescence intensity quantification pattern (A) of ARPE-19 cells stimulated with hydrogen peroxide after incubation of Chi@Lu / si NPs nanoparticles for 24 h in Example 1. Figure 12 The DCF mean fluorescence intensity was measured by flow cytometry after incubating the nanoparticles Chi@Lu / si NPs of Example 1 with hydrogen peroxide-stimulated ARPE-19 cells for 24 h. Figure 13 The images show the fluorescence staining of JC-1 monomers and JC-1 aggregates and the quantitative fluorescence intensity ratio of the nanoparticles Chi@Lu / si NPs in ARPE-19 cells stimulated by hydrogen peroxide for 24 h after incubation. (A) Figure 14 This is a quantitative graph showing the expression of TNF-α (A), IL-6 (B), and IL-12 p70 (C) in RAW264.7 cells stimulated with LPS after incubation of Chi@Lu / si NPs nanoparticles with LPS for 24 h in Example 1. Figure 15 The images show the relative mRNA expression levels of vascular endothelial growth factor (VEGF) after Chi@Lu / si NPs nanoparticles were incubated with HCE cells and HUVEC cells for 24 h (A-B) and the protein expression levels were detected by Western blot (C-D). Figure 16 The images show representative migration diagrams (A) and quantitative diagrams (B) of 24-hour wound healing results for Chi@Lu / si NPs nanoparticles in the scratch test model of Example 1. Figure 17The images show representative migration patterns (A) and quantitative 24-hour migration cell area (B) of Chi@Lu / si NPs nanoparticles in the Transwell migration model of Example 1. Figure 18 The figures (A), (B), and (C) show the 6-hour in vitro lumen formation experiment of Chi@Lu / si NPs nanoparticles in Example 1, respectively.

[0047] Figure 19 This is a confocal staining image of the cytoskeleton of HUVEC cells after incubation of Chi@Lu / si NPs nanoparticles for 24 h, as described in Example 1. Figure 20 This is an immunofluorescence staining image of ZO-1 after incubating Chi@Lu / si NPs nanoparticles with HCE cells for 24 h, as shown in Example 1. Figure 21 The diagram shows the structure of the in vitro simulated corneal epithelial barrier Transwell model treated with Chi@Lu / si NPs in Example 1 (A), the quantitative transepithelial resistance (B), the quantitative relative fluorescence intensity of DiO in the lower chamber (C), the laser confocal imaging of ARPE-19 cells (D), and the quantitative mean fluorescence intensity of ARPE-19 cells by flow cytometry (E). Figure 22 Confocal microscopy images of ocular tissue sections after 6 h of Chi@Lu / si NPs (Example 1) being added to C57BL / 6J mice (A) and a schematic diagram of the mechanism by which Chi@Lu / si NPs enhance transmucosal transport (B). Figure 23 Representative fundus color photographs (A), representative optical coherence tomography (B), and HE staining (C) of C57BL / 6J mice after the addition of Chi@Lu / si NPs nanoparticles from Example 1 to the eyes; Figure 24 On day 14 after adding Chi@Lu / si NPs (Example 1) to CNV mice, fundus fluorescein angiography (A), quantitative relative fluorescence intensity (B), and quantitative relative leakage area of ​​neovascularization (C) were performed on individual lesions. Figure 25 Representative optical coherence tomography (A) and quantitative thickness map of CNV lesions on day 14 after adding Chi@Lu / si NPs (Example 1) to CNV mice; Figure 26 HE staining image (A) and relative CNV thickness quantification image (B) of individual lesions on day 14 after adding Chi@Lu / si NPs of Example 1 to CNV mice; Figure 27 A schematic diagram illustrating the mechanism of action of Chi@Lu / si NPs nanoparticles in treating age-related macular degeneration. Detailed Implementation

[0048] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0050] siRNAs targeting VEGF: SEQ ID NO.1 (si-VEGF justice chain): GAAGUUCAUGGAUGUCUAUTT; SEQ ID NO.2 (si-VEGF antisense chain): AUAGACAUCCAUGAACUUCTT; siRNA targeting luciferase: SEQ ID NO.3 (si-Luci Justice Chain): GGGAGAACCGUAUAUAAGUTT; SEQ ID NO.4 (si-Luci antisense chain): ACUUAUAUACGGUUCUCCCTT; The structural formula of cationic liposome G2-C14 is as follows: .

[0051] Example 1 This embodiment provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of N,N-dimethylformamide to prepare solution 1; 0.4 nmol of VEGF-targeting siRNA (its sequence is shown in SEQ ID NO.1~SEQ ID NO.2, abbreviated as si-VEGF) was dissolved in 6 μL of nuclease-free water to prepare solution 2; 50 μg of lutein was dissolved in 10 μL of N,N-dimethylformamide to prepare solution 3; 250 μg of polylactic acid-glycolic acid copolymer (PLGA) (lactic acid and glycolic acid molar ratio 50:50) was dissolved in 100 μL of N,N-dimethylformamide to prepare solution 4; 5 mg of distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) was dissolved in 5 mL of water to prepare solution 5; chitosan was dissolved in 0.1 mL of water. Prepare a 0.03% (w / v) polysaccharide solution in an aqueous solution of M acetic acid.

[0052] Solutions 1 and 2 were mixed and shaken for 10 s to form a complex (abbreviated as siRNA / G2-C14) through electrostatic interaction. Solutions 3 and 4 were then added, gently mixed, and added dropwise to solution 5. The mixture was stirred continuously at 1000 rpm for 30 min, followed by centrifugation at 800–1200 rpm for 15–30 min. The precipitate (abbreviated as Lu / siNPs) was collected, washed three times with water, and resuspended in PBS. This precipitate was then added dropwise to the polysaccharide solution, stirred continuously at 400 rpm for 60 min, and soaked at 4°C for 8–12 h. The mixture was then centrifuged at 800–1200 rpm for 15–30 min, and the precipitate was collected. The precipitate was washed three times with water and resuspended in PBS to obtain the nanoparticles (abbreviated as Chi@Lu / siNPs). A schematic diagram of the nanoparticle preparation process is shown below. Figure 1 As shown.

[0053] Example 2 This embodiment provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of N,N-dimethylformamide to prepare solution 1; 0.5 nmol of VEGF-targeting siRNA was dissolved in 6 μL of nuclease-free water to prepare solution 2; 250 μg of lutein was dissolved in 10 μL of N,N-dimethylformamide to prepare solution 3; 150 μg of polylactic acid was dissolved in 100 μL of N,N-dimethylformamide to prepare solution 4; 4.5 mg of dimyristoylglycerol-polyethylene glycol 2000 was dissolved in 5 mL of water to prepare solution 5; chitosan was dissolved in 0.1M acetic acid aqueous solution to prepare a 0.02% (w / v) polysaccharide solution.

[0054] Solution 1 and Solution 2 were mixed and shaken for 10 s. Then Solution 3 and Solution 4 were added and gently mixed. The mixture was then added dropwise to Solution 5. The mixture was stirred continuously at 1000 rpm for 30 min. After that, it was centrifuged at 800-1200 rpm for 15-30 min, and the precipitate was collected. The precipitate was washed three times with water and resuspended in PBS. Then, it was added dropwise to the polysaccharide solution and stirred continuously at 400 rpm for 60 min. The mixture was soaked at 4℃ for 8-12 h. After that, it was centrifuged at 800-1200 rpm for 15-30 min, and the precipitate was collected. The precipitate was washed three times with water and resuspended in PBS to obtain the nanoparticles with an average hydrated particle size of 118.2 nm and a Zeta potential of 12.06 mV.

[0055] Example 3 This embodiment provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of acetone to prepare solution 1; 0.1 nmol of VEGF-targeting siRNA was dissolved in 6 μL of nuclease-free water to prepare solution 2; 500 μg of lutein was dissolved in 10 μL of acetone to prepare solution 3; 350 μg of polyacetin was dissolved in 100 μL of acetone to prepare solution 4; 5.5 mg of myristoyl phosphatidylethanolamine-polyethylene glycol 2000 was dissolved in 5 mL of water to prepare solution 5; and chitosan quaternary ammonium salt was dissolved in 0.1 M acetic acid aqueous solution to prepare a 0.04% (w / v) polysaccharide solution.

[0056] Solution 1 and Solution 2 were mixed and shaken for 10 s. Then Solution 3 and Solution 4 were added and gently mixed. The mixture was then added dropwise to Solution 5. The mixture was stirred continuously at 1000 rpm for 30 min. After that, it was centrifuged at 800-1200 rpm for 15-30 min, and the precipitate was collected. The precipitate was washed three times with water and resuspended in PBS. Then, it was added dropwise to the polysaccharide solution and stirred continuously at 400 rpm for 60 min. The mixture was soaked at 4℃ for 8-12 h. After that, it was centrifuged at 800-1200 rpm for 15-30 min, and the precipitate was collected. The precipitate was washed three times with water and resuspended in PBS to obtain the nanoparticles with an average hydrated particle size of 162.4 nm and a Zeta potential of 22.5 mV.

[0057] Comparative Example 1 This comparative example provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of N,N-dimethylformamide to prepare solution 1; 0.4 nmol of VEGF-targeting siRNA was dissolved in 6 μL of nuclease-free water to prepare solution 2; 250 μg of polylactic acid-glycolic acid copolymer (lactic acid to glycolic acid molar ratio 50:50) was dissolved in 100 μL of N,N-dimethylformamide to prepare solution 4; 5 mg of distearate phosphatidylethanolamine-polyethylene glycol was dissolved in 5 mL of water to prepare solution 5.

[0058] Mix solutions 1 and 2, shake for 10 s, then add solution 4, mix gently, and add dropwise to solution 5. Stir continuously at 1000 rpm for 30 min, then centrifuge at 800-1200 rpm for 15-30 min, collect the precipitate, wash the precipitate 3 times with water, and resuspend it in PBS to obtain the nanoparticles (abbreviated as SiNPs).

[0059] Comparative Example 2 This comparative example provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of N,N-dimethylformamide to prepare solution 1; 50 μg of lutein was dissolved in 10 μL of N,N-dimethylformamide to prepare solution 3; 250 μg of polylactic acid-glycolic acid copolymer (lactic acid and glycolic acid molar ratio 50:50) was dissolved in 100 μL of N,N-dimethylformamide to prepare solution 4; 5 mg of distearate phosphatidylethanolamine-polyethylene glycol was dissolved in 5 mL of water to prepare solution 5; chitosan was dissolved in 0.1 M acetic acid aqueous solution to prepare a 0.03% (w / v) polysaccharide solution.

[0060] Add solutions 3 and 4 to solution 1, mix gently, and then add dropwise to solution 5. Stir continuously at 1000 rpm for 30 min, then centrifuge at 800-1200 rpm for 15-30 min, collect the precipitate, wash the precipitate 3 times with water, resuspend it in PBS, and then add it dropwise to the polysaccharide solution. Stir continuously at 400 rpm for 60 min, soak at 4℃ for 8-12 h, then centrifuge at 800-1200 rpm for 15-30 min, collect the precipitate, wash the precipitate 3 times with water, and resuspend it in PBS to obtain the nanoparticles (abbreviated as Chi@Lu NPs).

[0061] Comparative Example 3 This comparative example provides a corneal-penetrating nanoparticle, the preparation method of which includes: 50 μg of cationic liposome G2-C14 was dissolved in 20 μL of N,N-dimethylformamide to prepare solution 1; 0.4 nmol of VEGF-targeting siRNA was dissolved in 6 μL of nuclease-free water to prepare solution 2; 250 μg of polylactic acid-glycolic acid copolymer (lactic acid to glycolic acid molar ratio 50:50) was dissolved in 100 μL of N,N-dimethylformamide to prepare solution 4; 5 mg of distearate phosphatidylethanolamine-polyethylene glycol was dissolved in 5 mL of water to prepare solution 5; chitosan was dissolved in 0.1 M acetic acid aqueous solution to prepare a 0.03% (w / v) polysaccharide solution.

[0062] Solution 1 and Solution 2 were mixed and shaken for 10 s to form a complex through electrostatic interaction. Solution 4 was then added, and the mixture was gently mixed before being added dropwise to Solution 5. The mixture was stirred continuously at 1000 rpm for 30 min, followed by centrifugation at 800-1200 rpm for 15-30 min. The precipitate was collected, washed three times with water, and resuspended in PBS. The precipitate was then added dropwise to the polysaccharide solution and stirred continuously at 400 rpm for 60 min. The mixture was soaked at 4℃ for 8-12 h, followed by centrifugation at 800-1200 rpm for 15-30 min. The precipitate was collected, washed three times with water, and resuspended in PBS to obtain the nanoparticles (abbreviated as Chi@si NPs).

[0063] Test Example 1 A series of material characterizations were performed on the nanoparticles: The average hydrated particle size of the Si NPs nanoparticles in Comparative Example 1 under dynamic light scattering (DLS) was 70 ± 3.8 nm. Figure 2 (A) The average particle size under transmission electron microscopy (TEM) is 43.4 ± 4.5 nm. Figure 2 (B) The Zeta potential is -20.79 ± 2.4 mV. Figure 2 (C). Ultraviolet-visible absorption spectroscopy analysis showed ( Figure 3 The nanoparticle intermediate Lu / si NPs of Example 1 retained the characteristic absorption peak of lutein, indicating that lutein was successfully encapsulated in the nanoparticle system. The nanoparticle intermediate Lu / si NPs, chitosan, and Chi@Lu / si NPs of Example 1 were labeled with the fluorescent groups FITC and RhB, and fluorescence emission spectroscopy analysis was performed at an excitation wavelength of 495 nm. Figure 5 The nanoparticles possess an effective loading capacity for fluorescent functional molecules. In vitro release experiments showed that... Figure 7The nanoparticles Chi@Lu / siNPs in Example 1 exhibit a more sustained drug release behavior compared to Lu / si NPs, indicating that chitosan modification helps regulate the release kinetics of lutein.

[0064] Referring to the preparation method in Example 1, different lutein encapsulation efficiencies can be obtained by simply changing the mass ratio of cationic liposomes G2-C14 and lutein (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:10). Figure 4 (A) Average hydrated particle size ( Figure 4 Chi@Lu / si NPs (B) nanoparticles.

[0065] Following the preparation method of Example 1, only the concentration of the chitosan solution was changed (0.01% w / v, 0.02% w / v, 0.03% w / v, 0.06% w / v) to prepare Chi@Lu / si NPs nanoparticles. After chitosan modification, the particle size of the nanoparticles increased with increasing chitosan concentration. Figure 6 In A), the Zeta potential changes from a negative value to a positive value. Figure 6 Figure B shows that chitosan was successfully coated on the surface of the nanoparticles. When the chitosan concentration was 0.03% (w / v), the average hydrated particle size of the nanoparticles under dynamic light scattering (DLS) was 132.6 ± 2.8 nm, the Zeta potential was 17 mV, and the average particle size under transmission electron microscopy (TEM) was 51.5 ± 2.25 nm. Figure 6 (C)

[0066] Test Example 2 The cellular uptake capacity, endosomal escape capacity, biosafety, and gene silencing effect of the Chi@Lu / si NPs nanoparticles of Example 1, the nanoparticle intermediate Lu / si NPs, and the si NPs nanoparticles of Comparative Example 1 were verified as follows: Chi@Lu / si NPs were labeled with green fluorescent FAM (hereinafter referred to as FAM-siRNA). HCE cells (human corneal epithelial cells) were incubated with FAM-siRNA (siRNA concentration of 25 nM) for 4 h and 24 h. Cell nuclei were stained with blue fluorescent Hoechst 33342, and late endosomes / lysosomes were stained with red fluorescent LysoTracker Red. Observation was then performed using laser confocal microscopy (CLSM). Figure 8 (Center A, scale bar 10 μm) The yellow lines in the figure mark the areas used for fluorescence intensity distribution analysis. Fluorescence intensity profile analysis was performed on the areas marked by the yellow lines. Figure 8(B) The distribution of green and red fluorescence intensities is shown in Figure B, evaluating the endosome escape capability of the nanoparticles. The results show that the green fluorescence signal is distributed in the cytoplasm, and the intracellular fluorescence increases over time, indicating that the nanoparticles can effectively achieve endosome escape.

[0067] 293T cells ( Figure 9 (A) HCE cells ( Figure 9 (B) ARPE-19 cells ( Figure 9 Cell viability was detected after co-incubating Chi@Lu / siNPs, Lu / si NPs, and si NPs (siRNA concentrations of 0 nM, 5 nM, 10 nM, 25 nM, 50 nM, and 100 nM) for 24 h. The results showed that the nanoparticles all had good in vitro biocompatibility and low cytotoxicity.

[0068] Following the preparation method of Example 1, only the VEGF-targeting siRNA in Chi@Lu / si NPs was replaced with an equal amount of luciferase-targeting siRNA (its sequence is shown in SEQ ID NO.3~SEQ ID NO.4, abbreviated as si-Luci). 293T cells expressing luciferase were incubated with si-Luci (siRNA concentrations of 0 nM, 12.5 nM, 25 nM, and 50 nM) for 24 h before bioluminescence imaging was performed. Figure 10 (A), its average luminous intensity ( Figure 10 Quantification was performed using (B). Simultaneously, the siRNA in the siNPs was replaced with an equal amount of siRNA targeting luciferase, and the relative luminescence intensity of the nanoparticles at a concentration of 50 nM siRNA was measured. Figure 10 (C). The results showed that Chi@Lu / si NPs can effectively deliver siRNA and achieve efficient gene silencing.

[0069] Test Example 3 The ability of the Chi@Lu / si NPs nanoparticles from Example 1 to scavenge reactive oxygen species and inhibit inflammatory responses in vitro was verified, with lutein treatment alone serving as a control: ARPE-19 cells stimulated with 200 μM hydrogen peroxide for 2 h were incubated with lutein (50 μM) and Chi@Lu / si NPs (siRNA concentration of 50 nM) for 24 h, followed by DCFH-DA fluorescence staining. Figure 11 (B in the figure, scale bar is 20 μm) and quantification of relative fluorescence intensity of DCF ( Figure 11 (A) The mean fluorescence intensity of DCF was quantified by flow cytometry. Figure 12 ) Mitochondrial membrane potential was detected using confocal microscopy. Figure 13In Figure A (scale bar is 50 μm), JC-1 monomers (green fluorescence) and JC-1 aggregates (red fluorescence) were detected, and the red / green fluorescence intensity ratio was used for quantification. Figure 13 (B) The results showed that Chi@Lu / siNPs had good ability to scavenge intracellular ROS and resist oxidation. After treatment with Chi@Lu / siNPs, the red / green fluorescence ratio was significantly restored, indicating that it can effectively maintain mitochondrial functional stability and reduce oxidative stress damage.

[0070] RAW264.7 cells stimulated with 100 ng / mL LPS for 6 h were incubated with lutein (50 μM) and Chi@Lu / siNPs (siRNA concentration of 50 nM) for 24 h before being subjected to TNF-α ( Figure 14 (A), IL-6 ( Figure 14 IL-12 p70 (B) Figure 14 The expression of Chi@Lu / si NPs was detected. The results showed that Chi@Lu / si NPs could significantly reduce the secretion levels of TNF-α, IL-6 and IL-12 p70, and had a good anti-inflammatory effect.

[0071] Test Example 4 The in vitro inhibitory biological behavior of the Chi@Lu / si NPs nanoparticles from Example 1 was verified using free VEGF-targeting siRNA (abbreviated as Free si-VEGF) and VEGF-targeting siRNA via Beyotime Lipo8000. TM The transfected form introduced into cells (abbreviated as Lipo si-VEGF) serves as a control: HCE cells and HUVEC cells were incubated with Free si-VEGF, Lipo si-VEGF, and Chi@Lu / si NPs (siRNA concentration of 50 nM) for 24 h, respectively. The relative mRNA expression levels of vascular endothelial growth factor (VEGF) were then detected. Figure 15 Protein expression level detection (A~B) and Western blot ( Figure 15 (C~D). The results showed that VEGF mRNA was downregulated by 42.7% and 57.1% in HCE and HUVEC cells, respectively, after treatment with Chi@Lu / si NPs, which corroborated the protein expression results.

[0072] A scratch assay model was prepared using HUVEC cells. After treatment with Free si-VEGF, Lipo si-VEGF, and Chi@Lu / si NPs (siRNA concentration of 50 nM), representative migration images were recorded. Figure 16In the middle A (scale bar 500 μm), the wound healing results over 24 hours were quantified. Figure 16 (B) A Transwell migration model was established using HUVEC cells. After treatment with Freesi-VEGF, Lipo si-VEGF, and Chi@Lu / si NPs (siRNA concentration of 50 nM), representative migration images were recorded. Figure 17 In the middle A (scale bar 100 μm), the area of ​​migrating cells over 24 h was quantified. Figure 17 (B) The results showed that after treatment with Chi@Lu / si NPs, the migration ability of HUVECs was significantly weakened, the wound healing rate was slowed down, and the number of migrating cells was significantly reduced.

[0073] An in vitro lumen formation experimental model was prepared. After treatment with Free si-VEGF, Lipo si-VEGF, and Chi@Lu / si NPs (siRNA concentration of 50 nM), images of in vitro lumen formation were recorded for 6 hours. Figure 18 In section A, the scale is 100 μm), and the total branch length ( Figure 18 (B) and the number of network structures ( Figure 18 Quantitative analysis was performed using the C-type method. The results showed that Chi@Lu / siNPs significantly inhibited the formation of complete tubular structures in HUVECs, manifested as a significant reduction in the total branch length and the number of network structures.

[0074] HUVEC cells were incubated with Free si-VEGF, Lipo si-VEGF, and Chi@Lu / si NPs (siRNA concentration of 50 nM) for 24 h, followed by Alexa Fluor 488-labeled phalloidin staining, and confocal microscopy images of the cytoskeleton were recorded. Figure 19 (Scale bar: 20 μm). The results showed that the F-actin structure in the Chi@Lu / si NPs treatment group tended to be disordered, further inhibiting endothelial cell migration and angiogenesis.

[0075] Test Example 5 To further investigate the transmembrane permeation of nanoparticles in vitro and in vivo, DiO-labeled Chi@Lu / si NPs from Example 1 nanoparticles and Lu / si NPs intermediates were used. Fluorescence imaging of cells and mouse fundus sections was detected by confocal microscopy and a small animal in vivo imaging system.

[0076] HCE cells were treated with PBS, chitosan (0.5 mg / mL), Lu / siNPs (siRNA concentration of 50 nM), and Chi@Lu / siNPs (siRNA concentration of 50 nM) before immunofluorescence staining for tight junction protein ZO-1. Figure 20 (Scale bar: 10 μm). A Transwell model structure simulating the corneal epithelial barrier was constructed in vitro. Figure 21 In section A), the initial siRNA concentration in the upper chamber was 50 nM, and transepithelial electrical resistance (TEER) was measured to evaluate changes in corneal epithelial barrier integrity. Figure 21 (B) The relative fluorescence intensity of DiO in the lower chamber culture medium at different time points was quantified. Figure 21 In the middle chamber (C), ARPE-19 cells in the lower chamber were subjected to laser confocal imaging. Figure 21 (Medium D, scale bar 20 μm) The mean fluorescence intensity of ARPE-19 cells was quantitatively analyzed by flow cytometry. Figure 21 (E). The results showed that the ZO-1 distribution was remodeled to some extent after Chi@Lu / si NPs treatment, which could regulate the tight junction structure between cells and reduce the TEER value to a certain extent. In the in vitro transepithelial transport experiment, the fluorescence intensity of DiO-labeled Chi@Lu / si NPs in the lower chamber culture medium was significantly higher than that of Lu / si NPs, proving that chitosan modification enhanced the cross-barrier delivery efficiency of nanoparticles.

[0077] C57BL / 6J mice were treated with PBS, DiO-Lu / si NPs (siRNA concentration of 50 nM), and DiO-Chi@Lu / si NPs (siRNA concentration of 50 nM) for 6 h. Eye tissue sections were then subjected to confocal microscopy. Figure 22 A schematic diagram of the mechanism by which Chi@Lu / si NPs enhance transmucosal transport (scale bar 50 μm) is shown in Figure A. Figure 22 As shown in Figure B, DiO-Chi@Lu / si NPs exhibited significant green fluorescence signals in the retinal and choroidal regions, indicating that chitosan modification facilitates the delivery of nanoformulations to the posterior segment of the eye.

[0078] Test Example 6 Establishment and efficacy evaluation of a laser-induced CNV mouse model: Six- to eight-week-old C57BL / 6J mice were randomly divided into groups of five. Each group received 3 μL of one of the following nanoparticles (Example 1: Chi@Lu / si NPs), one of the following nanoparticle intermediates: Lu / si NPs; one of the following nanoparticles (Comparative Example 2: Chi@Lu NPs); or one of the following nanoparticles (Comparative Example 3: Chi@si NPs) (siRNA concentration 50 nM): twice daily for 14 consecutive days. The ocular surface was observed using a small animal fundus imaging system, and representative color photographs of the mouse fundus were recorded. Figure 23 (A) Collect eyeballs and capture representative optical coherence tomography (OCT) images. Figure 23 In the middle B (scale bar 200 μm), HE staining was performed. Figure 23 The safety of the nano-formulation was observed using a scale bar of 50 μm (C-C). Results showed that the fundus structure of the treated mice was clear, with no obvious abnormalities such as hemorrhage, edema, or inflammatory reactions. The retinal layers in all groups were intact and of uniform thickness, with no obvious structural disorder or pathological changes observed. The corneal and retinal tissue structures were intact and clearly layered, with no obvious inflammatory cell infiltration or tissue damage.

[0079] Test Example 7 CNV model was induced in 6-8 week old C57BL / 6J mice using laser photocoagulation at a wavelength of 532 nm, a spot diameter of 50 μm, and a laser power of 400 mW. After successful establishment of the CNV model, mice were randomly divided into groups of 5 mice each. Each group of mice received 3 μL of PBS, Chi@Lu / si NPs (Example 1), Lu / si NPs (nanoparticle intermediate), Chi@Lu NPs (Comparative Example 2), and Chi@si NPs (Comparative Example 3) (siRNA concentration of 50 nM) instilled into the eyes twice daily for 14 consecutive days. On day 14 after modeling, angiography was performed by injecting 10% sodium fluorescein solution (0.1 mL / 10 g body weight) via the tail vein. Representative fundus fluorescein angiography (FFA) images of individual CNV lesions were acquired 3-5 minutes after injection using a small animal fundus imaging system. Figure 24 (A) Quantitative analysis of the relative fluorescence intensity in CNV lesion areas of mice. Figure 24 (B) Quantitative analysis of the relative leakage area of ​​newly formed blood vessels. Figure 24 (C) Representative optical coherence tomography (OCT) images were collected from individual lesions in CNV mice. Figure 25 In the middle A region (scale bar 200 μm), the thickness of CNV lesions was quantified. Figure 25 (B); CNV mouse eye tissue was stained with hematoxylin-eosin (HE) ( Figure 26In the middle A, the scale bar is 50 μm, yellow indicates the thickness of CNV lesions, and green indicates the thickness of normal choroid (the thickness of CNV is quantified). Figure 26 (B)

[0080] The results showed that Chi@Lu / si NPs treatment significantly reduced fluorescence leakage in the lesion area, significantly decreasing fluorescence intensity and neovascularization leakage area. The elevation of CNV lesions was significantly reduced, and CNV thickness decreased significantly (to 58.84±7.67%). The tissue structure in the lesion area improved, with reduced neovascularization and inflammatory changes, and a relative decrease in CNV thickness. Chi@Lu / si NPs demonstrated significant anti-angiogenic and therapeutic effects in a laser-induced CNV mouse model.

[0081] In summary, the Chi@Lu / si NPs provided by this invention can effectively cross the blood-eye barrier and be delivered to the posterior segment lesion region of the eye. Figure 27 Hydrophobic drugs scavenge reactive oxygen species and inhibit the release of inflammatory factors, while siRNA targets and silences the expression of age-related macular degeneration pathogenic genes, synergistically reducing lesion thickness and treating age-related macular degeneration.

[0082] This invention illustrates a corneal-penetrating nanoparticle, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0083] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A corneal-penetrating nanoparticle, characterized in that, The nanoparticles comprise a core formed from cationic liposomes G2-C14, an aliphatic polyester, and an amphiphilic polymer, and a polysaccharide-modified shell; hydrophobic drugs and siRNA are loaded into the core.

2. The nanoparticles according to claim 1, characterized in that, The mass ratio of the cationic liposome G2-C14, aliphatic polyester, and amphiphilic polymer is 1:(3~7):(90~110); Preferably, the mass ratio of the cationic liposome G2-C14 to the polysaccharide is 1:(0.1~5); Preferably, the mass ratio of the cationic liposome G2-C14 to the hydrophobic drug is 1:(0.5~10); Preferably, the ratio of cationic liposome G2-C14 to siRNA is 1 μg:(0.001~0.01) nmol.

3. The nanoparticles according to claim 1 or 2, characterized in that, The aliphatic polyester includes any one or a combination of at least two of polylactic acid-glycolic acid copolymer, polylactic acid, or polyacetin; Preferably, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (50~75):(50~25); Preferably, the amphiphilic polymer comprises any one or a combination of at least two of the following: distearylphosphatidylethanolamine-polyethylene glycol, dimyristoylglycerol-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, or dioleoylphosphatidylethanolamine-polyethylene glycol. Preferably, the polysaccharide comprises chitosan and / or chitosan quaternary ammonium salt.

4. The nanoparticles according to any one of claims 1 to 3, characterized in that, The hydrophobic drug includes any one or a combination of at least two of lutein, astaxanthin, or curcumin; Preferably, the siRNA includes siRNA that targets genes causing age-related macular degeneration; Preferably, the age-related macular degeneration pathogenic gene includes any one or a combination of at least two of VEGF, complement factor H, complement factor B, ARMS2, or HTRA1; Preferably, the siRNA is a siRNA that targets VEGF.

5. The nanoparticles according to any one of claims 1 to 4, characterized in that, The average particle size of the nanoparticles is 50~200 nm; Preferably, the encapsulation efficiency of the hydrophobic drug and siRNA in the nanoparticles is independently not less than 40%.

6. The method for preparing nanoparticles according to any one of claims 1 to 5, characterized in that, The preparation method includes: An organic solvent containing cationic liposomes G2-C14 was mixed with an aqueous solution of siRNA, then mixed with an aliphatic polyester and a hydrophobic drug, and finally mixed with an aqueous solution of an amphiphilic polymer. The mixture was centrifuged, the precipitate was collected, soaked in a polysaccharide solution, centrifuged again, and the precipitate was collected to obtain the nanoparticles.

7. The preparation method according to claim 6, characterized in that, The organic solvent includes acetone and / or N,N-dimethylformamide; Preferably, the centrifugation speed is 800~1200 rpm and the time is 15~30 min; Preferably, the process of collecting the sediment further includes a water washing step; Preferably, the soaking time is 8 to 12 hours.

8. The use of the nanoparticles according to any one of claims 1 to 5 in the preparation of a medicament for treating age-related macular degeneration.

9. An ophthalmic preparation for treating age-related macular degeneration, characterized in that, The ophthalmic formulation comprises the nanoparticles as described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

10. The ophthalmic preparation according to claim 9, characterized in that, The dosage form of the ophthalmic preparation includes any one of eye drops, ophthalmic gels, or ophthalmic ointments; Preferably, the ophthalmic preparation is administered via ocular surface application.

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