Lipid nanoparticle for retina delivery of mRNA and preparation method and application thereof

Abstract

The invention discloses lipid nanoparticles for retina delivery of mRNA (messenger Ribonucleic Acid) as well as a preparation method and application of the lipid nanoparticles. The lipid nanoparticles comprise an ionizable lipid, neutral phospholipid, cholesterol and a PEGylated lipid; wherein the ionizable lipid is prepared from SM-102 and NT-O14B, and the ionizable lipid is prepared from the SM-102 and NT-O14B; the molar ratio of the SM-102 to the NT-O14B to the neutral phospholipid to the cholesterol to the PEGylated lipid is (59.5 to 63) to (7 to 10.5) to (8 to 12) to (16.4 to 20.6) to (1.4 to 1.6). Compared with a classical LNP formula, the LNP provided by the invention can efficiently deliver mRNA to the retina, and the mRNA is specifically ingested by the retinal pigment epithelial cells through an endocytosis and macropinocytosis pathway mediated by the caveolar protein. In-vivo and in-vitro experiments prove that the LNP can significantly improve the delivery efficiency of mRNA in the retina and the expression level of functional protein, and has good biocompatibility. The invention provides an efficient and safe non-virus delivery system for mRNA treatment of hereditary retina diseases (such as Leber congenital black armeng).

Description

Technical Field

[0001] This invention belongs to the field of gene therapy drug delivery systems, and in particular to a lipid nanoparticle for retinal delivery of mRNA, its preparation method and application. Background Technology

[0002] Hereditary retinal diseases (IRDs) are a group of neurodegenerative diseases caused by gene mutations, such as retinitis pigmentosa, Stargardt's disease, and Leber's congenital amaurosis, affecting approximately 1 in 2000 people worldwide. Currently, apart from Luxturna (AAV-mediated RPE65 gene therapy), there are no widely available and effective treatments.

[0003] The success of gene therapy heavily depends on an effective delivery system. Currently, adeno-associated virus (AAV) is a commonly used vector in ophthalmic gene therapy, but its application has limitations: first, AAV vectors are limited by their packaging capacity (approximately 4.8 kb), making it impossible to deliver large genes; second, this viral vector poses immunogenicity and long-term safety concerns.

[0004] Non-viral delivery systems based on lipid nanoparticles (LNPs) offer a potential solution to overcome the aforementioned bottlenecks. However, most existing LNPs are developed based on liver delivery (such as using standard formulations like SM-102 or MC3), which have low mRNA delivery efficiency to the eye, especially the retina, and struggle to overcome anatomical barriers such as the vitreous body and internal limiting membrane within the eye.

[0005] To improve ocular targeting, existing studies have made some attempts, such as modifying the LNP surface or regulating PEG density to improve retinal targeting. However, these attempts are limited, mostly involving single-variable optimization, and lack systematic formulation optimization strategies specifically for retinal mRNA delivery.

[0006] Therefore, gene therapy for hereditary retinal diseases (IRDs) still faces the core problem of insufficient delivery systems: AAV cannot meet the requirements for large gene delivery and safety, while conventional or simply modified LNPs are difficult to achieve efficient mRNA delivery to the retina. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing adeno-associated virus vectors, such as limited packaging capacity and high immunogenicity, as well as the low targeting efficiency of conventional lipid nanoparticles to the retina, and to provide a lipid nanoparticle for retinal mRNA delivery, its preparation method, and its application.

[0008] In a first aspect, the present invention provides a lipid nanoparticle for retinal mRNA delivery, the lipid nanoparticle comprising an ionizable lipid, a neutral phospholipid, cholesterol, and a PEGylated lipid; the ionizable lipid is composed of SM-102 and NT-O14B; the molar ratio of SM-102 to NT-O14B is (59.5-63):(7-10.5); and the N / P ratio of the lipid nanoparticle is 7 to 8.

[0009] Further, taking the total molar percentage of lipids in the lipid nanoparticles as 100%, the molar percentage of each component in the lipid nanoparticles is as follows: SM-102: 59.5-63%, NT-O14B: 7-10.5%, Neutral phospholipids: 8-12%, Cholesterol: 16.4-20.6% PEGylated lipids: 1.4-1.6%; The neutral phospholipid is DSPC; the PEGylated lipid is DMG-PEG2000.

[0010] Furthermore, the molar percentage of each component in the lipid nanoparticles is as follows: SM-102: 59.5%, NT-O14B: 10.5%, DSPC: 10%, Cholesterol: 18.5% DMG-PEG2000: 1.5%.

[0011] This specific formulation, named the B11 formulation, has been experimentally verified to achieve highly efficient mRNA delivery to retinal pigment epithelial cells both in vivo and in vitro.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned lipid nanoparticles, the method comprising the following steps: S1. Dissolve the ionizable lipids, neutral phospholipids, cholesterol, and PEGylated lipids in an organic solvent to form a lipid phase; S2. Dissolve the mRNA in an acidic aqueous buffer solution to form an aqueous mRNA solution; S3. In a microfluidic device, the aqueous mRNA solution and the lipid phase are mixed at a volume flow rate ratio of (5-7):1, thereby forming lipid nanoparticles encapsulating mRNA through self-assembly. S4. Dialyze the mixture to remove organic solvents and adjust the pH to 7.0-7.4.

[0013] Thirdly, the present invention provides the use of the above-mentioned lipid nanoparticles in the preparation of a medicament for treating hereditary retinal diseases.

[0014] Preferably, the hereditary retinal disease is selected from Leber congenital amaurosis, retinitis pigmentosa, or Stargardt disease.

[0015] The drug is preferably administered via intravitreal injection.

[0016] The lipid nanoparticles can specifically and efficiently deliver functional mRNA to retinal pigment epithelial cells.

[0017] Fourthly, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises lipid nanoparticles as described in any of the preceding claims and a pharmaceutically acceptable carrier. The lipid nanoparticles encapsulate therapeutic mRNA or mRNA encoding a gene-editing tool for treating corresponding eye diseases by compensating for mutated genes or performing gene editing.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: Highly efficient retinal targeting: The LNP formulations (such as B11) provided by this invention can achieve mRNA delivery efficiency several times higher than classic LNP formulations and specifically target the key therapeutic target of retinal pigment epithelium.

[0019] Functional integrity: The delivered mRNA can be successfully translated into a functional protein with full biological activity within the target cell, providing a reliable tool for gene therapy and gene editing.

[0020] Good safety: The LNP uses biocompatible materials, has no obvious cytotoxicity at effective doses, and as a non-viral carrier, it avoids the immunogenicity and long-term safety risks of viral carriers. Attached Figure Description

[0021] Figure 1 For the analysis of LNP cellular uptake and cytotoxicity, Figure 1 A is a histogram of fluorescence intensity of ARPE-19 cells after 6 hours of incubation with DiD-labeled LNPs (B11, S1, S2) or PBS control, as detected by flow cytometry. Figure 1 B represents the quantitative analysis of the average DiD fluorescence intensity. Figure 1 C represents the cell viability of ARPE-19 cells 24 hours after treatment with LNP B11, S1, or S2; Figure 2 For the study of the LNP B11 endocytic uptake mechanism, Figure 2 A shows the fluorescence intensity histogram of ARPE-19 cells after pretreatment with different endocytosis inhibitors (Dynasore, Nystatin, Blebbistatin) for 30 minutes and co-incubation with DiD-labeled LNP B11 for 6 hours, as detected by flow cytometry. Figure 2 B represents the quantitative results of the average DiD fluorescence intensity of each group; Figure 3 To analyze the in vitro transfection efficiency and cytotoxicity of LNP at different doses. Figure 3 A shows the luciferase activity of ARPE-19 cells after 24 hours of treatment with different doses (10, 50, 100, 200 ng mRNA / well) of Luc mRNA loaded with LNP B11, S1, and S2. Figure 3 B represents the effect of different doses of LNP treatment on the survival rate of ARPE-19 cells; Figure 4 To deliver mCherry mRNA to LNP for expression in the retina. Figure 4 A represents mouse eye tissue collected 24 hours after intravitreal injection of LNPs (B11, S1, S2) loaded with mCherry mRNA. Retinal sections were prepared and fluorescence imaging was performed. Red represents mCherry fluorescence signal, and blue represents DAPI-labeled cell nuclei. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; Figure 4 B represents a quantitative analysis of the proportion of mCherry-positive regions; Figure 5 Delivering Cre mRNA to Ai14 mice Figure 5 A represents mouse eye tissue collected after intravitreal injection of LNPs (B11, S1, S2) loaded with Cre mRNA, followed by retinal sections and fluorescence imaging. Red indicates tdTomato signal, and blue indicates DAPI-labeled cell nuclei. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; Figure 5 B represents the quantitative analysis of the average fluorescence intensity of tdTomato in retinal sections; Figure 5 C represents the preparation of retinal planus after intravitreal injection and fluorescence imaging; red represents tdTomato signal and blue represents DAPI-labeled cell nuclei. Figure 5D represents the quantitative analysis of the average fluorescence intensity of tdTomato in the retinal patch. Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0024] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0025] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0026] Example 1: Synthesis of Lipid Nanoparticles (LNPs) 1. Synthesis of LNPs Ionizable lipids (SM-102, NT-O14B), DSPC, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol at a predetermined molar ratio to form a lipid phase solution. Simultaneously, the mRNA encoding luciferase (Luc) was diluted in 50 mM, pH 4.0 citrate buffer to form the aqueous phase.

[0027] Calculation and control of N / P ratio The N / P ratio of LNP is controlled by the amount of feed, and its calculation formula is as follows: N / P = number of moles of amino groups in ionizable lipid feed / (mass of mRNA feed (g) / 330 (g / mol)). Among the ionizable lipids, SM-102 molecules contain one amino group and NT-O14B contains two amino groups.

[0028] Wherein, 330 (g / mol) is the average molecular weight of each nucleotide in the mRNA. In this invention, the ionizable lipids (SM-102 and NT-O14B) can be protonated under acidic buffer conditions, carrying a positive charge. Based on this formula, by precisely controlling the total molar number of ionizable lipids in the lipid phase and the mass of mRNA in the aqueous phase, the N / P ratio of the final LNP is made to fall within the range of 7 to 8. Dynamic light scattering and gel retardation experiments verified that the encapsulation efficiency of the LNPs formed at this N / P ratio is >95%, and the particle size distribution is uniform.

[0029] Subsequently, using a microfluidic mixer, the lipid and aqueous phases were mixed at a constant flow rate at a volume ratio of 1:5, allowing the lipids and mRNA to self-assemble into LNPs. The mixture was immediately dialyzed against 1× PBS to remove residual ethanol and adjust the pH to neutral. After dialyzing, the sample was concentrated to the target volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, as required for the experiment. The final LNP suspension was filtered through a 0.22 μm PES membrane, aliquoted into sterile centrifuge tubes, and stored at 4°C for later use.

[0030] 2. Synthesis of LNPs used for functional verification In the functional validation study, three formulations were used: the B11 formulation (molar ratio of SM-102 / NT-O14B / DSPC / Chol / DMG-PEG = 59.5 / 10.5 / 10 / 18.5 / 1.5), the classic SM-102 formulation (i.e., formulation S1, molar ratio of SM-102 / DSPC / Chol / DMG-PEG = 50 / 10 / 38.5 / 1.5), and the classic MC3 formulation (i.e., formulation S2, molar ratio of DLin-MC3-DMA / DSPC / Chol / DMG-PEG = 50 / 10 / 38.5 / 1.5). The encapsulated mRNA was selected according to specific experimental requirements, including Luc mRNA, mCherry mRNA, and Cre mRNA. The LNP preparation method was the same as the general synthesis method described above.

[0031] Example 2: In vitro performance evaluation of LNP 1. Cellular uptake experiment LNPs encapsulating Luc mRNA were synthesized according to the method in Example 1, and DiD fluorescent dye was added to the lipid fraction at a total molar ratio of 1% for labeling. The synthetic formulation included B11, S1, and S2. ARPE-19 cells were cultured at 1.6 × 10⁶ cells per well. 5Cells were seeded at a density of 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. Subsequently, DiD-labeled LNPs were added to each well at a dose of 200 ng mRNA, and the cells were incubated at 37°C for 6 h. After incubation, cells were thoroughly washed with PBS to remove untaken LNPs, then digested with trypsin and collected. Cells were collected by centrifugation at 500×g and washed twice with PBS. Finally, the cells were resuspended in PBS for later use. Flow cytometry was used to detect DiD fluorescence signals in the cells, with at least three biological replicates per group. The mean fluorescence intensity of DiD was analyzed using FlowJo 10.8.1.

[0032] Flow cytometry results showed that the PBS control group had almost no fluorescence signal, verifying the reliability of the experiment; compared with the classic formulations S1 and S2, the fluorescence peak of the B11 treatment group shifted significantly to the right, and the fluorescence intensity was significantly enhanced, indicating that B11 can be phagocytosed and accumulated by more cells, such as... Figure 1 A.

[0033] like Figure 1 As shown in Figure B, the average DiD fluorescence intensity of the B11 group was approximately twice that of S1 and S2, with a highly significant difference (p<0.001). This result demonstrates that the B11 formulation obtained through the system optimization of this invention can significantly enhance the endocytosis and accumulation of LNPs in retinal pigment epithelial cells.

[0034] In addition, the results of cytotoxicity detection by CCK-8 assay showed that ( Figure 1 (c) LNP B11 showed almost no toxicity to ARPE-19 cells within the working concentration range, and its cell viability was essentially the same as that of control formulations S1 and S2. This indicates that LNP B11 maintains good biocompatibility while retaining efficient cell uptake capacity.

[0035] 2. Research on the endocytic uptake mechanism of LNPs To investigate the endocytic uptake mechanism of LNP B11, three commonly used endocytosis inhibitors were used to intervene in the clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis pathways, respectively. ARPE-19 cells were inoculated at 1.6 × 10⁻⁶ cells per cell line. 5Cells were seeded at a density of 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. Before the experiment, various inhibitors were prepared into high-concentration stock solutions using DMSO as the solvent. These solutions were then diluted into preheated DMEM medium during the actual experiment to ensure that the same volume of inhibitor was used, minimizing the interference of DMSO toxicity on the experimental results. The control group was treated with DMEM medium containing an equal volume of PBS. Three endocytosis inhibitors were used: Dynasore (final working concentration 20 μM, inhibiting clathrin-mediated endocytosis), Nystatin (final working concentration 10 μM, inhibiting caveolin-mediated endocytosis), and Blebbistatin (final working concentration 10 μM, inhibiting macropinocytosis). Medium containing the respective inhibitor or PBS (control) was added to the corresponding wells and incubated at 37°C for 30 min to achieve the inhibitory effect. Cells were kept in the incubator during the treatment. After treatment, the inhibitor solution was removed, and culture medium containing DiD-labeled LNP B11 (200 ng mRNA per well based on lipid encapsulation) was added. The mixture was then incubated at 37°C for 6 h. Subsequent treatment and detection methods were the same as in Example 2.1.

[0036] Flow cytometry analysis results showed ( Figure 2 B), compared with the control group not treated with inhibitors, the fluorescence intensity change in the Dynasore-treated group was not significant (ns), suggesting that CME plays a limited role in the internalization of B11. Figure 2 (A, B). Conversely, the mean fluorescence intensity of the Nystatin and Blebbistatin treatment groups decreased significantly (***p<0.001, ****p<0.0001). Figure 2 (A, B). This discovery clarifies that the main pathway for LNP B11 to enter retinal pigment epithelial cells is caveolin-mediated endocytosis and macropinocytosis, providing a theoretical explanation for its efficient delivery mechanism.

[0037] 3. In vitro transfection of LNPs encapsulating Luc mRNA ARPE-19 cells were loaded at a rate of 2 × 10⁻⁶ 4Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 h at 37°C and 5% CO2 in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. LNPs B11, S1, and S2, encapsulating Luc mRNA, were synthesized using microfluidic synthesis. For transfection, LNPs containing different doses of Luc mRNA (10, 50, 100, and 200 ng per well) were diluted in serum-free DMEM medium, replacing the original medium, and incubated at 37°C for 24 h. After incubation, the medium was discarded, and the cells were gently washed once with PBS, followed by the addition of 100 μl of lysis buffer to lyse the cells. The lysis buffer was incubated at room temperature for 5 min, then centrifuged at 10000×g for 5 min, and the supernatant was collected for later use. Luciferase activity was detected using a firefly luciferase reporter gene assay kit. Add 50 μl of supernatant to a black 96-well plate, then add 100 μl of luciferase substrate, mix thoroughly, and immediately measure the luminescence intensity using a multi-mode microplate reader. Each experiment should contain at least three independent replicate wells.

[0038] 4. Cytotoxicity analysis ARPE-19 cells were loaded at a rate of 2 × 10⁻⁶ 4 Cells were seeded at a density of 10% fetal bovine serum and 1% penicillin-streptomycin in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Subsequently, LNP containing different doses of Luc mRNA (10, 50, 100, and 200 ng per well) was diluted in serum-free DMEM medium, replacing the original medium, and incubated at 37°C for 24 h. After incubation, 10 μl of CCK-8 reagent was added to each well, and the reaction was carried out at 37°C for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader. After subtracting the blank wells, the absorbance of untreated cell wells was used as a control. The relative fold change in cell viability for each treatment group was calculated to assess the effect of LNP treatment on cell viability.

[0039] The results of luciferase activity assay showed that ( Figure 3 (A) At all tested doses, B11 showed significantly higher delivery efficiency than S1 and S2 (p<0.0001). Particularly at low doses (10 ng and 50 ng / well), B11 was able to maintain relatively stable luciferase signaling, while its expression level showed a significant dose-dependent increase with increasing mRNA dose. At high doses (200 ng / well), the luciferase activity of the B11 delivery group was 2.6 times that of S1 and 2.5 times that of S2, highlighting its delivery advantage. Meanwhile, cytotoxicity assay results ( Figure 3B) indicates that no significant cytotoxicity was observed in any of the LNPs across all dosage groups, and cell viability remained above 90%. This demonstrates that the B11 formulation maintains good biocompatibility while significantly improving delivery efficiency.

[0040] Example 3: Evaluation of in vivo delivery efficacy of LNP 1. In vivo delivery of mCherry mRNA LNPs B11, S1, and S2, which encapsulate mCherry mRNA, were injected intravitreally into mouse eyes. Twenty-four hours after injection, the eyeballs were harvested and fixed in 4% paraformaldehyde at 4°C for approximately 12-16 hours. After fixation, the tissue was washed several times with PBS to remove residual paraformaldehyde, followed by a gradient dehydration process: the eyeballs were sequentially immersed in 70%, 80%, 95%, and 100% ethanol, each step for approximately 10-15 minutes. After dehydration, the tissue was cleared several times in xylene, each time for approximately 10 minutes. After complete clearing, the tissue was immersed in molten paraffin at 60°C for several hours until fully infiltrated. The tissue was then embedded in a paraffin block according to a predetermined orientation (marked as optic nerve or corneal direction) and allowed to solidify. After solidification, sections approximately 4 µm thick were cut using a rotary microtome and attached to glass slides. For immunofluorescence or fluorescent protein detection, slides required dewaxing and rehydration: slides were dewaxed twice in xylene for 5-10 minutes each time, followed by rehydration in a gradient of 100%, 95%, and 80% ethanol for approximately 2-3 minutes each, and finally rinsed with deionized water. After these steps, cell nuclei were stained with DAPI. The slides were scanned using a digital fluorescence scanner at excitation light of 580 nm and emission light of 610 nm, and the proportion of mCherry-positive areas to the total retinal area was analyzed using ImageJ.

[0041] Retinal slice fluorescence imaging showed ( Figure 4 Groups A and B11 produced strong mCherry red fluorescence in the retina, with signal specifically enriched in the retinal pigment epithelium (RPE), while almost no signal was observed in the adjacent neuroretinal layers (ONL, INL, GCL). Quantitative analysis showed that ( Figure 4In group B), the percentage of mCherry-positive area in group B11 was significantly higher than that in groups S1 and S2 (p<0.0001), being 3.7 times and 4.0 times higher, respectively. This result confirms that LNP B11 can efficiently cross the intraocular barrier and specifically deliver mRNA to retinal pigment epithelial cells (RPE cells), a key therapeutic target. This specificity in distribution suggests that B11 exhibits good targeting to RPE cells during ocular delivery. RPE plays a central role in the development and progression of retinal diseases; for example, in diseases such as Stargardt's disease and Leber congenital amaurosis, RPE dysfunction is often a crucial mechanism for disease progression. Therefore, the ability to efficiently and specifically deliver mRNA to RPE cells is of great significance for the future development of gene compensation or editing therapies. In summary, B11 not only demonstrates universality in mRNA delivery but also provides a potential technological basis for precision treatment of RPE-related diseases through its distribution specificity.

[0042] 2. In vivo delivery of functional Cre mRNA To further verify whether the mRNA delivered by LNP B11 can be effectively translated into a biologically active functional protein in vivo, LNPs encapsulating Cre mRNA were injected into the vitreous bodies of Ai14 transgenic mice. The Ai14 mouse genome contains a fluxed stop box upstream of the tdTomato fluorescent protein gene. The fluxed stop box blocks tdTomato expression, but when the Cre recombinase is successfully delivered and functions in the cells, this stop box is cleaved, thereby initiating tdTomato expression. One week after injection, eyeballs were harvested for the following analysis: Retinal sections: Sample preparation method is the same as in Example 3.1. Scanning was performed using a fluorescence digital scanner under excitation light of 554 nm and emission light of 581 nm, and the average fluorescence intensity of tdTomato fluorescent protein in the sections was analyzed using ImageJ.

[0043] Retinal Patches: After enucleation of the mouse eyeballs, they were immediately fixed in 4% paraformaldehyde solution for 30 min to ensure tissue integrity and reduce fluorescence signal attenuation. The eyeballs were then placed in 30% sucrose solution at 4°C overnight for dehydration to further protect the tissue structure. The eyeballs were dissected under a dissecting microscope. First, the cornea and lens were removed, and a small incision was made in the upper part of the eye cup as a directional marker. Then, starting from the optic disc head, the tissue was evenly cut radially into four quadrants, and the neuroretina was gently dissected to obtain a fully morphological RPE-retinal patch. The patch was flattened on a glass slide, with the four lobes of tissue unfolded in a petal-like shape, and the directional marker placed on top to ensure consistency between different samples. The tissue was then stained with DAPI to label the cell nuclei. After staining, the slide was mounted with an anti-quenching mounting medium, covered with a coverslip, and gently pressed to avoid air bubbles. Samples should be stored in the dark and imaging should be completed within a short period. A digital fluorescence scanner was used to scan samples at the set excitation / emission wavelengths (DAPI: 358 / 461 nm, tdTomato: 554 / 581 nm). All samples were acquired under the same parameters to ensure data comparability. Subsequent analysis used ImageJ software to analyze the mean fluorescence intensity of the tdTomato fluorescent protein; the data were used to quantitatively compare differences between different treatment groups.

[0044] Retinal section analysis showed that the B11 group exhibited significantly stronger tdTomato fluorescence signal than the S1 and S2 control groups (p<0.01), which was 1.7 times stronger than that of S1 and S2. Figure 5 A, B). This is consistent with the results of the aforementioned mCherry delivery experiment, further demonstrating the superiority of LNP B11 in in vivo mRNA delivery and expression. Furthermore, scanning imaging of retinal patches prepared from the same batch of eyeballs revealed that the fluorescence signal in the B11 group exhibited a wider distribution across the entire retinal surface, and the average fluorescence intensity was significantly higher than the control group, being 1.5 times that of S1 and 1.2 times that of S2 (****p<0.0001, **p<0.01). Figure 5 C, D).

[0045] These results demonstrate that the mRNA delivered by LNP B11 was successfully translated in retinal pigment epithelial cells, and its protein product possesses complete biological function, specifically mediating recombination events in the genome. In gene therapy research for IRDs, the expression of functional proteins is a crucial step in achieving gene repair and phenotypic improvement. Therefore, the results of this study highlight the potential of LNP B11 in therapeutic gene regulation and editing.

[0046] Example 4: Data Analysis Statistical differences between two groups were analyzed using unpaired Student's t-tests, while comparisons among multiple groups were performed using one-way ANOVA. All statistical analyses were performed using GraphPad Prism (version: 9.5.1). Data are expressed as mean ± standard error (mean ± SEM), and p < 0.05 was considered statistically significant. Significance levels in the figures are indicated by asterisks (*), representing *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, respectively. ns indicates no statistical difference between the two groups.

[0047] This invention yields an LNP formulation B11 with significantly optimized retinal delivery efficiency. This formulation can be efficiently taken up by RPE cells through caveolin-mediated endocytosis and macropinocytosis, and specifically achieves high levels of mRNA delivery and functional protein expression in the retinal RPE layer, while exhibiting good biocompatibility. It provides an excellent delivery tool for mRNA therapy of hereditary retinal diseases.

[0048] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

[0049] It should be particularly noted that the various embodiments listed in this specification and accompanying drawings are intended to illustrate the technical solutions and advantages of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core ideas and technical effects of the present invention, those skilled in the art can make any form of improvement, substitution, combination, or modification to the structural arrangement, process parameters, material selection, control logic, etc., of the described embodiments; any obvious changes based on the same concept should be considered equivalent solutions of the present invention and should be included within the scope of protection defined by the claims of the present invention. The actual scope of protection of the present invention is determined by the appended claims and should be correctly understood in conjunction with the specification and accompanying drawings.

Claims

1. A lipid nanoparticle for delivering mRNA to the retina, characterized in that, The lipid nanoparticle comprises an ionizable lipid, a neutral phospholipid, cholesterol, and a PEGylated lipid; The ionizable lipid consists of SM-102 and NT-O14B; The molar ratio of SM-102 to NT-O14B is (59.5-63):(7-10.5). The N / P ratio of the lipid nanoparticle is 7 to 8.

2. The lipid nanoparticle of claim 1, wherein, The molar percentage of each component, based on 100% of the total lipid moles in the lipid nanoparticle, is: SM-102: 59.5-63%, NT-O14B: 7-10.5%, neutral phospholipid: 8-12%, cholesterol: 16.4-20.6%, PEGylated lipid: 1.4-1.6%.

3. The lipid nanoparticle of claim 2, wherein, The neutral phospholipid is DSPC; the PEGylated lipid is DMG-PEG2000.

4. The lipid nanoparticle of claim 3, wherein, The molar percentage of each component in the lipid nanoparticle is: SM-102: 59.5%, NT-O14B: 10.5%, DSPC: 10%, cholesterol: 18.5%, DMG-PEG2000: 1.5%.

5. A method of preparing the lipid nanoparticle of any one of claims 1-4, characterized in that, The method comprises the following steps: S1, dissolving the ionizable lipid, neutral phospholipid, cholesterol, and PEGylated lipid in an organic solvent to form a lipid phase; S2, dissolving the mRNA in an acidic aqueous buffer to form an mRNA aqueous solution; S3, in a microfluidic device, mixing the mRNA aqueous solution and the lipid phase at a volume flow ratio of (5-7):1, thereby forming a lipid nanoparticle encapsulating mRNA through self-assembly; S4, dialyzing the mixture to remove the organic solvent and adjust the pH to 7.0-7.

4.

6. Use of the lipid nanoparticle of any one of claims 1-4 in the preparation of a medicament for treating a genetic retinal disease.

7. Use according to claim 6, characterized in that, The genetic retinal disease is selected from Leber congenital amaurosis, retinitis pigmentosa, or Stargardt disease.

8. Use according to claim 6, characterized in that, The medicament is administered by intravitreal injection.

9. Use according to claim 6, characterized in that, The lipid nanoparticle is used for delivering mRNA to retinal pigment epithelial cells.

10. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-4 and a pharmaceutically acceptable carrier, wherein the lipid nanoparticle encapsulates a therapeutic mRNA or an mRNA encoding a gene editing tool.

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