Biomimetic nanoplatform for treating retinal diseases

Abstract

A biomimetic nanoplatform for treating retinal diseases, a nanoplatform comprising an immune cell membrane-coated nanocarrier and a targeted drug delivery system comprising the nanoplatform and a therapeutic agent, as well as a method for preparing the nanoplatform and the targeted drug delivery system and use thereof in preparing a drug for preventing or treating retinal diseases and neurodegenerative diseases.

Description

Biomimetic nanoplatform for treating retinal diseases Technical Field

[0001] This invention generally relates to the field of ophthalmology. More specifically, this invention relates to a biomimetic nanoplatform for treating retinal diseases. Background Technology

[0002] According to WHO data, more than 300 million people worldwide suffer from retinal diseases such as glaucoma, optic atrophy, diabetic retinopathy, optic neuritis, and retinitis pigmentosa, which can lead to vision impairment and even irreversible blindness. With the aging population, the prevalence of these diseases will continue to rise. It is projected that the number of people with age-related retinal diseases globally will double within the next 30 years.

[0003] The main drug treatments include anti-vascular endothelial growth factor (VEGF) therapy, which uses VEGF inhibitors such as ranibizumab (Lucentis) and aflibercept (Eylea), widely used for diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. These drugs are administered via intravitreal injection to inhibit abnormal blood vessel proliferation and leakage. Laser photocoagulation is primarily used to manage diabetic retinopathy by closing leaking blood vessels to reduce retinal edema. Although topical eye drops are often explored for the treatment of retinal diseases due to their non-invasive nature, their efficacy is currently significantly limited by limitations such as the difficulty in achieving therapeutic concentrations in the retina. The sclera and other ocular barriers can hinder the penetration of therapeutic agents, making it difficult to achieve the desired treatment results. Other alternative therapies, such as periocular injections of anti-VEGF and surgical interventions, are invasive and carry risks of discomfort, infection, and other complications. Given the limitations of current retinal disease treatments, continued research to develop more effective, less invasive, and more readily available therapies is crucial.

[0004] With the development of nanotechnology, nanoplatforms have been applied to the diagnosis and treatment of various diseases. Nanoscale platforms aim to protect encapsulated diagnostic or therapeutic agents from degradation or leakage in the physiological environment and improve their targeted therapeutic capabilities and biodistribution. The relatively successful application of nanocarriers has significantly improved efficacy and safety. However, compared to traditional treatment methods, this technology still faces many challenges in clinical translation. One of the most critical issues is the difficulty for nanomaterials to cross physiological barriers, leading to reduced efficiency in delivering bioactive compounds to the target site. Furthermore, nanomaterials are recognized and cleared by phagocytosis after drug administration, which also poses a significant challenge in the development of nanodelivery systems.

[0005] Microglia, as resident immune cells of the retina, play a crucial role in maintaining homeostasis and responding to pathological conditions. Under normal physiological conditions, microglia perform important functions such as synaptic pruning, maintaining neuronal health, and immune surveillance. They continuously monitor the CNS microenvironment through their dynamic processes, responding to damage signals and potential threats. When faced with endogenous or exogenous damage, microglia are activated through various surface receptors that recognize damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This recognition triggers a multi-layered immune response, enabling microglia to internalize and degrade pathogens and cellular debris through mechanisms such as phagocytosis and receptor-mediated endocytosis. The anti-inflammatory effects of microglia are complex and context-dependent. While they can transform into a pro-inflammatory phenotype (M1) under certain conditions, characterized by the production of inflammatory cytokines and reactive oxygen species (ROS), they also exhibit anti-inflammatory (M2) properties, which are essential for tissue repair and inflammation resolution.

[0006] In recent years, a significant shift has occurred in the field of nanomaterials design. This shift involves utilizing biomimetic principles to construct nanosystems with higher biocompatibility and longer cycle life. These cell-based delivery systems are designed to replicate the biological characteristics of source cells while maintaining the physicochemical properties of nanoparticles. This top-down synthetic approach involves encapsulating nanostructures in thin layers using natural cell or organelle membranes (e.g., membranes extracted from platelets, leukocytes, erythrocytes, stem cells, cancer cells, bacteria, etc.). This membrane biomimetic technology achieves the complete protein and lipid composition and effective bioactive proteins of the parent cell, thereby endowing it with the desired functional properties of the source cell.

[0007] Baicalein (5,6,7-trihydroxyflavone) is a bioactive flavonoid compound extracted from the roots of Scutellaria baicalensis Georgi. Previous studies have demonstrated its safety profile and potent pharmacokinetic properties. Its significant lipophilicity allows it to cross the blood-brain barrier and enter the central nervous system. Baicalein exhibits a variety of therapeutic activities, including antibacterial, antiviral, antioxidant, anti-apoptotic, anticancer, and anti-inflammatory effects. Studies have confirmed that baicalein exerts a significant neuroprotective effect in a Parkinson's disease (PD) model, improving symptoms such as tremor, bradykinesia, and depression. The mechanisms by which baicalein reduces iron accumulation, inhibits neuronal apoptosis, and alleviates mitochondrial oxidative stress play a crucial role in addressing neurodegenerative diseases. It exerts these effects by regulating K-ATP channels to inhibit neuronal apoptosis. However, the poor water solubility of baicalein limits its clinical application. Therefore, improving the solubility, bioavailability, and stability of baicalin is crucial for expanding its therapeutic potential.

[0008] Therefore, the present invention aims to address these challenges existing in the prior art. Summary of the Invention

[0009] The technical problems to be solved by this invention include one or more of the following:

[0010] Current therapies lack effective targeting of activated microglia in the ischemic retina;

[0011] Current anti-inflammatory therapies cannot actively modulate the retinal immune microenvironment;

[0012] Small molecule antioxidants have a short residence time in the eye and poor bioavailability;

[0013] Existing cell membrane-coated nanoparticles have shown ineffectiveness in retinal applications;

[0014] Current treatments cannot prevent retinal neurodegeneration and vision loss;

[0015] Current treatments for neuroinflammation have limited efficacy and significant side effects;

[0016] Existing nanodelivery systems suffer from uncontrollable drug release and safety issues; and / or

[0017] Current therapies have limited applicability to various retinal diseases.

[0018] To address the aforementioned technical problems, the present invention provides the following technical solutions:

[0019] Option 1. A nanoplatform comprising an immune cell membrane-coated nanocarrier.

[0020] Option 2. According to the nanoplatform described in Option 1, wherein the cell membrane is derived from cells that participate in immune responses, inflammatory responses, antigen presentation or immune regulation in a disease state, or cells with immune or inflammatory regulatory functions obtained through genetic engineering, bioengineering or in vitro induction treatment, or cells in a pathologically activated state.

[0021] Option 3. The nanoplatform according to Option 2, wherein the cells are selected from microglia, macrophages derived from monocytes, dendritic cells, infiltrating leukocytes, Müller glial cells, astrocytes, and retinal pigment epithelial cells.

[0022] Option 4. The nanoplatform according to any one of Options 1 to 3, wherein the immune cell membrane is derived from immune cells in retinal tissue.

[0023] Option 5. The nanoplatform according to any one of Options 1 to 4, wherein the immune cell membrane is derived from microglia.

[0024] Option 6. The nanoplatform according to Option 5, wherein the microglia are microglia in retinal tissue.

[0025] Option 7. The nanoplatform according to any one of Options 1 to 6, wherein the immune cell membrane is derived from a single type of cell or from different types of cells.

[0026] Scheme 8. The nanoplatform according to any one of Schemes 1 to 7, wherein the immune cell membrane is a single layer or multiple layers.

[0027] Option 9. The nanoplatform according to any one of Options 1 to 8, wherein the cell membrane is designed to be modular and combinable.

[0028] Scheme 10. The nanoplatform according to any one of Schemes 1 to 9, wherein the nanocarrier has controlled release properties.

[0029] Scheme 11. The nanoplatform according to any one of Schemes 1 to 10, wherein the material of the nanocarrier is selected from biodegradable and / or biocompatible polymers, lipids, proteins, polysaccharides, inorganic or organic-inorganic hybrid materials.

[0030] Option 12. The nanoplatform according to Option 11, wherein the material of the nanocarrier is selected from polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol and its derivatives, chitosan, hyaluronic acid, albumin, liposomes, lipid nanoparticles, metal oxide nanoparticles, silicon-based nanomaterials, and any combination thereof.

[0031] Scheme 13. The nanoplatform according to any one of Schemes 1 to 11, wherein the material of the nanocarrier is polylactic-glycolic acid copolymer (PLGA).

[0032] Scheme 14. A biodegradable polymer nanoplatform coated with immune cell membrane.

[0033] Option 15. The nanoplatform according to Option 14, wherein the biodegradable polymer is selected from polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol and its derivatives.

[0034] Scheme 16. A PLGA nanoplatform coated with an immune cell membrane.

[0035] Scheme 17. A microglial cell membrane-coated PLGA nanoplatform.

[0036] Option 18. A targeted drug delivery system comprising:

[0037] Therapeutic agents; and

[0038] The nanoplatform of any one of Schemes 1 to 17

[0039] The therapeutic agent is encapsulated within a nanoplatform.

[0040] Option 19. The targeted drug delivery system according to Option 18, wherein the therapeutic agent is a small molecule drug, peptide, nucleic acid, antibody or protein drug.

[0041] Option 20. The targeted drug delivery system according to Option 19, wherein the therapeutic agent is a small molecule drug, peptide, nucleic acid, antibody, or protein drug for treating retinal diseases or neurodegenerative diseases.

[0042] Option 21. A targeted drug delivery system according to any one of Options 18 to 20, wherein the therapeutic agent is a drug having both anti-inflammatory and neuroprotective effects.

[0043] Option 22. The targeted drug delivery system according to Option 21, wherein the therapeutic agent is a natural flavonoid drug with dual anti-inflammatory and neuroprotective effects.

[0044] Option 23. The targeted drug delivery system according to any one of Options 18 to 22, wherein the therapeutic agent is baicalin.

[0045] Scheme 24. Use of the nanoplatform of any one of Schemes 1 to 17 or the targeted drug delivery system of any one of Schemes 18 to 23 in the preparation of a medicament for the prevention or treatment of retinal diseases.

[0046] Option 25. The use according to Option 24, wherein the retinal disease is a retinal disease involving ischemia-reperfusion (IR) injury.

[0047] Option 26. The use according to Option 24, wherein the retinal disease is a retinal disease involving inflammation.

[0048] Option 27. The use according to Option 24, wherein the retinal disease is a retinal degenerative disease involving neuroinflammation.

[0049] Option 28. The use according to Option 24, wherein the retinal disease is selected from retinal ischemic diseases, retinal inflammatory diseases, retinal degenerative diseases, and infectious or traumatic retinal diseases.

[0050] Option 29. The use according to Option 28, wherein the retinal degenerative disease is an autoimmune or hereditary retinal degenerative disease.

[0051] Option 30. The use according to any one of Options 24 to 29, wherein the retinal disease is selected from retinal ischemia or ischemia-reperfusion injury, retinal vascular occlusion, diabetic or hypertensive retinopathy, age-related macular degeneration and its associated choroidal neovascularization, glaucoma, uveitis, retinitis, retinal vasculitis and optic neuritis.

[0052] Option 31. Use of the nanoplatform of any one of Options 1 to 17 or the targeted drug delivery system of any one of Options 18 to 23 in the preparation of a medicament for the prevention or treatment of neuroinflammation.

[0053] Scheme 32. Use of the nanoplatform of any one of Schemes 1 to 17 or the targeted drug delivery system of any one of Schemes 18 to 23 in the preparation of a medicament for treating neurodegenerative diseases.

[0054] Option 33. The use according to Option 32, wherein the neurodegenerative disease is selected from Parkinson's disease (PD), Alzheimer's disease (AD), and multiple sclerosis (MS).

[0055] Scheme 34. A method for preparing a nanoplatform according to any one of Schemes 1 to 17, comprising:

[0056] Preparation of nanocarriers;

[0057] Nanocarriers were coated with immune cell membranes.

[0058] Scheme 35. A method for preparing a targeted drug delivery system according to any one of Schemes 18 to 23, comprising:

[0059] Preparation of nanocarriers;

[0060] Therapeutic agents are encapsulated using nanocarriers to obtain nanoparticles loaded with therapeutic agents;

[0061] Nanoparticles loaded with therapeutic agents are coated with immune cell membranes to obtain immune cell membrane-coated nanoparticles loaded with therapeutic agents, which can be used as a targeted drug delivery system.

[0062] Scheme 36. The method according to any one of Schemes 34 and 35, wherein the method further comprises isolating the cell membrane from the immune cells.

[0063] Option 37. The nanoplatform of any one of Options 1 to 17 or the targeted drug delivery system of any one of Options 18 to 23 for the prevention or treatment of retinal diseases, such as the retinal diseases described in Options 21 to 26.

[0064] Option 38. The nanoplatform of any one of Options 1 to 17 or the targeted drug delivery system of any one of Options 18 to 23, for the prevention or treatment of neuroinflammation.

[0065] Option 39. The nanoplatform of any one of Options 1 to 17 or the targeted drug delivery system of any one of Options 18 to 23 for the treatment of neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) and multiple sclerosis (MS).

[0066] Option 40. A method for preventing or treating retinal diseases in a subject of need, comprising:

[0067] The targeted drug delivery system for administration to the retina of the subject as described in any one of 18 to 23.

[0068] Option 41. A method for preventing or treating neuroinflammation in a subject of need, comprising:

[0069] The targeted drug delivery system described in any one of schemes 18 to 23 is administered to the subject.

[0070] Option 42. The method according to Option 40 or 41, wherein the administration is intravitreal injection, subretinal injection, subchoroidal injection, subconjunctival injection, subscleral injection, intra-anterior chamber injection, transscleral administration, transocular surface administration, eye drops, sustained-release implantation, or a combination thereof.

[0071] Option 43. A targeted drug delivery system for treating retinal degenerative diseases, comprising:

[0072] A nanoparticle platform comprising a polylactic-co-glycolic acid (PLGA) core encapsulating therapeutic agents; and

[0073] An immune cell membrane coating surrounding the PLGA core, wherein the coating enhances targeting of inflamed or diseased retinal tissue and neutralizes pro-inflammatory cytokines;

[0074] The system described therein provides a controlled and sustained release of therapeutic agents, thereby reducing neuroinflammation and improving therapeutic efficacy.

[0075] Option 44. The system according to Option 43, wherein the immune cell membrane coating is modular, allowing for customization by integrating different cell membrane types to target specific retinal diseases.

[0076] Option 45. The system according to Option 43, wherein the therapeutic agent is baicalin, which is encapsulated within a PLGA core to enhance its solubility, stability and bioavailability, thereby improving its anti-inflammatory and neuroprotective effects.

[0077] Option 46. The system according to Option 43, wherein the immune cell membrane coating is configured to neutralize pro-inflammatory cytokines selected from IL-6, IL-1 and TNF-α, thereby reducing neuroinflammation and preventing disease progression.

[0078] Option 47. A method for treating retinal degenerative diseases, comprising:

[0079] Administer the drug delivery system as described in scheme 43 to the subjects who require it;

[0080] Delivering therapeutic agents to inflamed or diseased retinal tissue via the homing ability of immune cell membrane coating; and

[0081] The therapeutic agent is released in a controlled and sustained manner to reduce neuroinflammation and improve the health of retinal tissue.

[0082] Scheme 48. A biomimetic drug delivery platform utilizing the BV2 microglia cell membrane.

[0083] Scheme 49. The biomimetic drug delivery platform according to Scheme 48, wherein the inherent characteristics of BV2 cells enable the platform to specifically target inflammatory sites, neutralize pro-inflammatory cytokines, regulate the release of encapsulated drugs, and prolong drug retention time, thereby significantly improving therapeutic efficacy compared with free drug delivery.

[0084] Option 50. A membrane-coated drug delivery platform. This platform significantly improves the stability and bioavailability of baicalin, thereby achieving improved anti-inflammatory and therapeutic outcomes both in vitro and in vivo (especially in neurodegenerative disease models).

[0085] Scheme 51. A method for preparing a biodegradable polylactic-coated glycolic acid copolymer (PLGA) nanoplatform coated with an immune cell membrane, the method comprising coating the nanoparticles with an immune cell membrane.

[0086] The significant features of the technical solution of this invention and the resulting technical effects are described below:

[0087] Immunocellular membrane coating: Nanocarriers such as PLGA nanoparticles are coated using immunocellular membranes. This feature enables drug delivery systems to mimic natural biological processes, enhancing targeting capabilities, particularly for inflamed or diseased tissues such as the retina. It can also neutralize pro-inflammatory cytokines, reduce neuroinflammation, and improve therapeutic efficacy for various diseases involving neuroinflammation, such as retinal diseases.

[0088] Nanoplatforms: Nanoplatforms are the core structure for drug encapsulation and delivery. By providing nanocarriers such as biocompatible and / or biodegradable polymer matrices, they ensure the controlled and sustained release of encapsulated drugs. This controlled-release mechanism effectively solves the problems of rapid drug clearance and low delivery efficiency commonly found in traditional delivery methods.

[0089] Modular and combinatorial design of cell membrane coating: This allows for the modification and combination of different types of cell membranes as coating materials for nanoparticles. This feature enables the selection of one or more cell membranes from different sources for monolayer or multilayer coating, based on the patient's disease pathology, immune-inflammatory characteristics, or molecular markers, such as retinal diseases or neurodegenerative diseases. This allows for precise regulation and treatment of individualized diseases like retinal diseases or neurodegenerative diseases. This feature also allows for the customization of drug delivery systems for different diseases, such as retinal diseases, by selecting the cell membrane type best suited to target specific conditions. It enhances the system's versatility, enabling its widespread application to various diseases, such as retinal diseases, simply by changing the cell membrane type.

[0090] Encapsulation and Stabilization of Flavonoids: Encapsulating the flavonoid baicalin within nanocarriers, such as biodegradable polymers like PLGA nanoparticles, improves its solubility and stability. This feature allows baicalin (a natural flavonoid with potent anti-inflammatory and neuroprotective effects) to be effectively used in clinical applications despite its poor solubility and stability in alkaline solutions. The nanoplatform enhances the bioavailability and therapeutic properties of baicalin in target tissues such as the retina.

[0091] These technological features work together to overcome the limitations of current treatments for retinal diseases and neurodegenerative diseases, providing a more targeted, efficient, and versatile drug delivery system with fewer side effects and better therapeutic outcomes.

[0092] The advantages of this invention compared to the prior art are as follows:

[0093] Targeted drug delivery: Immunocellular membrane-coated nanoplatforms allow for precise targeting of drugs to inflamed or diseased tissues, such as the retina. This targeted approach ensures that therapeutic agents are delivered directly to the disease site, improving efficacy and reducing the possibility of off-target effects. Current therapies, such as anti-VEGF injections, lack specificity and do not specifically target inflamed areas, potentially affecting healthy tissue and causing potential side effects. This invention minimizes these risks by focusing treatment on the sites most in need.

[0094] Providing controlled release of therapeutic agents: By selecting materials with controlled release properties, such as biodegradable polymers like PLGA, to prepare nanoplatforms, it is possible to achieve controlled and sustained release of encapsulated drugs over time. This reduces the need for frequent treatments and ensures stable therapeutic effects. Traditional therapies often require repeated injections, which can cause patient discomfort and increase the risk of complications. The controlled release mechanism in this invention improves patient compliance and the overall treatment experience by ensuring sustained therapeutic efficacy.

[0095] Addressing neuroinflammation: Immunocellular membrane coating neutralizes pro-inflammatory cytokines, directly resolving neuroinflammation, a key factor in the progression of retinal degenerative diseases. Current treatments primarily focus on symptoms or vascular issues, often neglecting the underlying neuroinflammation that drives disease progression. This invention provides a more comprehensive approach by simultaneously targeting both inflammation and the disease itself.

[0096] Versatility and Customizability: The modular and combinable nature of cell membrane coating allows for the customization of drug delivery systems for different retinal diseases by simply changing the type of membrane used. This adaptability makes the system of the present invention applicable to a wide range of retinal conditions. Existing technologies are often disease-specific and lack the flexibility to adapt to other conditions without significant modifications. The present invention's ability to provide customized solutions for multiple diseases with minimal modifications enhances its applicability, scope of application, and market potential.

[0097] Improving the stability and bioavailability of baicalin: By encapsulating baicalin in a nanoplatform, the system of this invention overcomes the problems of poor solubility and stability of baicalin, thereby improving its bioavailability and therapeutic efficacy. Traditional baicalin formulations have limited clinical application due to these issues. This invention enables this potent anti-inflammatory and neuroprotective agent to be used in the treatment of retinal diseases, providing a novel and previously underutilized therapeutic option.

[0098] Wider application potential: The technology of this invention has the potential to be extended beyond retinal diseases to other ocular and systemic diseases involving inflammation or requiring targeted drug delivery. Currently, most technologies have limited applicability. The broader applicability of this invention increases its potential impact, making it a more attractive option for future research and development.

[0099] Other advantages of this invention include: the components of the biomimetic membrane drug delivery platform (e.g., cell membranes and carrier materials such as PLGA) are readily available and inexpensive, and the preparation process is simple; by optimizing the synthesis parameters of the nanocarrier (e.g., biodegradable polymers such as PLGA nanocarriers), the size of the biomimetic membrane drug delivery platform can be controlled to meet different drug delivery needs; due to the presence of membrane proteins, the biomimetic membrane drug delivery platform can precisely target inflammatory sites, thereby improving the therapeutic efficacy of baicalein and avoiding its side effects; the biomimetic membrane drug delivery platform can deliver not only baicalein but also other therapeutic agents (e.g., nucleic acids, peptides, antibodies, proteins, and small molecule drugs).

[0100] In summary, this invention provides a more targeted, efficient, and customizable treatment option compared to existing technologies, addressing both the symptoms and root causes of related diseases. This holistic approach not only improves treatment outcomes but also enhances patient experience and safety, representing a significant advancement over current treatment methods. Attached Figure Description

[0101] Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, wherein:

[0102] Figures 1A-1C illustrate the conceptual design of membrane-coated baicalin-loaded nanoparticles (MBNPs) for treating retinal ischemia-reperfusion disorders involving ocular diseases. Figure 1A: Proteomics analysis revealing the molecular mechanism by which microglia attenuate inflammation and protect photoreceptors in a mouse model of IR injury. Figure 1B: Schematic diagram of MBNP synthesis. Figure 1C: Schematic diagram of the mechanism by which MBNPs inhibit inflammation and damage to retinal ganglion cells (RGCs).

[0103] Figures 2A-2L depict proteomic analyses revealing protein changes in IR-damaged retina following MEM treatment. Figures 2A and 2D show volcano plots of differential protein expression in the IR vs. CTL and MEM vs. IR groups, respectively; Figures 2B and 2E show hierarchical clustering heatmaps of significantly altered proteins in the IR vs. CTL and MEM vs. IR groups, respectively; Figures 2C and 2F show protein-protein interaction (PPI) networks of significantly regulatory proteins in the IR vs. CTL and MEM vs. IR groups, respectively; Figures 2G and 2J show gene ontology (GO) enrichment analyses of differentially expressed proteins in the IR vs. CTL and MEM vs. IR groups, respectively; Figures 2H and 2K show the distribution of quantitative protein variability in the IR vs. CTL and MEM vs. IR experiments, respectively; Figures 2I and 2L show violin plots illustrating the overall distribution of variability and fold change.

[0104] Figures 3A-3C depict the proteomic pathways and overlapping differentially expressed proteins in retinal IR damage and MEM treatment. Figure 3A: IR vs. Ctrl: KEGG pathway enrichment of proteins significantly dysregulated by IR damage (|log2FC|>1.3); Figure 3B: MEM vs. IR: KEGG enrichment of retina treated with MEM (|log2FC|>1.3); Figure 3C: Analysis of significantly differentially expressed proteins, highlighting representative proteins altered by IR and reversed by MEM.

[0105] Figures 4A-4L depict the characterization of MBNPs. Figures 4A-4C show representative TEM images (scale bar: 200 nm) of the nanocarrier, BNPs, and MBNPs, respectively; Figures 4D-4F show DLS analysis of the average hydrodynamic diameter and polydispersity index (PDI) of the nanocarrier, BNPs, and MBNPs, respectively; Figure 4G shows the drug loading efficiency of BAI and PLGA at different weight ratios; Figure 4H shows the cumulative release curves of free BAI vs. MBNPs in PBS at room temperature; Figure 4I shows the characteristic protein bands of BV2, BV2-derived membranes, and MBNPs resolved by SDS-PAGE; Figures 4J-4L show the size, zeta potential, and PDI of MBNPs over 7 days, respectively.

[0106] Figures 5A-5B: Figure 5A shows the particle size distribution of PLGA and DOTAP with different weight ratios, and Figure 5B shows the Zeta potential of PLGA and DOTAP with different weight ratios.

[0107] Figures 6A-6B: Figure 6A shows the cell viability of PLGA and DOTAP at different weight ratios and concentrations after 24 hours; Figure 6B shows the cell viability of PLGA and DOTAP at different weight ratios and concentrations after 48 hours.

[0108] Figures 7A-7B: Figure 7A depicts the particle size distribution of MEM and BNPs with different weight ratios; Figure 7B depicts the Zeta potential of MEM and BNPs with different weight ratios.

[0109] Figures 8A-8F depict the in vitro cell internalization kinetics of the membrane-coated nanoplatform. Figure 8A shows the cellular uptake behavior of MFNPs at 1, 4, 12, and 24 hours observed by confocal microscopy (CLSM), with MFNPs represented by green signals; Figure 8B shows the quantitative analysis of MFNP cellular uptake behavior by flow cytometry (FACS); Figure 8C shows the quantitative analysis of MFNPs (green fluorescence in Figure 8A); Figure 8D shows CLSM images of free FITC, FNPs, and MFNPs uptake in BV2 cells, with MFNPs represented by green signals; Figure 8E shows the quantitative analysis of MFNP cellular uptake by flow cytometry; Figure 8F shows the quantitative analysis of MFNPs (green fluorescence in Figure 8D). Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Experiments were independently repeated three times, with similar results. One-way ANOVA multiple comparison test was performed.

[0110] Figures 9A-9I depict the cellular selective uptake and antioxidant protective effects of MBNPs. Figures 9A-9C show the cell viability assessment of Raw 264.7, 661W, and BV2 cells after treatment with 1 μM, 10 μM, and 50 μM BAI or MBNPs for 24 hours, respectively; Figure 9D shows representative CLSM images of intracellular MFNPs in MC-3T3, Raw264.7, and BV2 cells, with MFNPs represented by green signals; Figure 9E shows the flow cytometry quantification of MFNP uptake (green fluorescence) in each cell type; Figure 9F shows the quantification of MFNP uptake (mean fluorescence) in Figure 9D; Figure 9G shows the intracellular baicalein content measured by LC-MS after treatment with equal doses of BAI and MBNPs; Figure 9H shows the ROS scavenging capacity of H2O2-induced oxidized BV2 cells after different treatments; Figure 9I shows the viability of H2O2-induced BV2 cells after different treatments. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The experiment was independently repeated three times, and the results were similar. One-way ANOVA multiple comparison test.

[0111] Figures 10A-10E depict the efficacy of MBNPs in inhibiting LPS-induced inflammation in vitro. Figure 10A shows the relative mRNA levels of IL-6, IL-1α, IL-1β, and TNF-α as measured by qPCR after LPS-stimulated BV2 cells were treated with increasing concentrations of MEM; Figure 10B shows the relative mRNA levels of IL-6, IL-1α, IL-1β, and TNF-α as measured by qPCR after LPS-stimulated BV2 cells were pretreated with equal doses of nanocarriers, BNPs, BAI, MEM, or MBNPs; Figure 10C shows representative immunofluorescence images of iNOS (green) in BV2 cells under each treatment; cell nuclei were stained with DAPI (blue); Figure 10D shows the quantification of iNOS fluorescence intensity; Figure 10E shows the ELISA assay of secreted IL-6, IL-1α, and TNF-α in the culture supernatant after treatment. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The experiment was independently repeated three times, and the results were similar. One-way ANOVA multiple comparison test.

[0112] Figure 11 depicts the relative mRNA levels of IL-4 and IL-10 measured by qPCR after pretreatment of LPS-stimulated BV2 cells with BAI-based nanoparticles and MEM.

[0113] Figure 12 illustrates a schematic diagram of a visual-motor response (OMR) apparatus used to measure visual acuity in mice. Mice are placed on a platform surrounded by rotating vertical gratings; the reflective head movements of the gratings are tracked to determine visual acuity (spatial frequency threshold, measured in cycles / degree).

[0114] Figures 13A-13B: Figure 13A shows a schematic diagram of the apparatus used to measure intraocular pressure (IOP) in mice using a tonometer. The tonometer probe (illustrated) is aligned with the mouse cornea to non-invasively record IOP at baseline and follow-up time points; Figure 13B shows IOP measurements (mmHg) for each group at baseline, 2 weeks after IR, and 4 weeks after IR.

[0115] Figures 14A-14D depict the protective and inflammatory modulatory effects of MBNPs on visual function in a mouse model of retinal irritation (IR). Figure 14A shows the experimental design and timeline of IR injury and treatment. Baseline measurements (ERG, OCT, OMR visual acuity, IOP) were performed first, followed by IR induction. One day after IR, intravitreal injections were administered: saline (IR control), baicalin (BAI), microglia membrane (MEM), or MBNPs. Two weeks after IR, functional assessments were repeated, followed by a second intravitreal injection. The final assessment was performed four weeks after IR; Figure 14B shows the ocular pharmacokinetics of free BAI and MBNPs over time; Figure 14C shows visual acuity (weeks / degree) in each treatment group (saline, BAI, MEM, MBNPs) at baseline, two weeks after IR injury, and four weeks after IR injury; Figure 14D shows the mRNA expression of retinal inflammatory markers one week after IR, showing fold changes relative to the uninjured control group. Data are mean ± SEM (n = 7 retinas per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. One-way ANOVA multiple comparison test.

[0116] Figures 15A-15H depict the role of MBNPs in maintaining retinal function after IR injury in vivo. Figure 15A shows representative pSTR waveforms of each group at baseline, 2 weeks, and 4 weeks; Figure 15B shows the quantitative analysis of peak STR amplitude over time for each group; Figure 15C shows the cross-sectional comparison of STR amplitude between groups at 2 weeks and 4 weeks; Figure 15D shows the statistical significance of pSTR differences between groups at each time point; Figure 15E shows representative a-waves and b-waves of each group at baseline, 2 weeks, and 4 weeks; Figure 15F shows the changes in a-wave and b-wave amplitude over time within each group; Figure 15G shows the inter-group comparison of a-wave and b-wave amplitude at 2 weeks and 4 weeks; Figure 15H shows the temporal progression of a-wave and b-wave amplitude for each group. Data are expressed as mean ± SEM (n=8). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. One-way ANOVA multiple comparison test.

[0117] Figures 16A-16D depict the protective effect of MBNPs on retinal structures and their promotion of RGC survival after IR injury. Figure 16A shows representative OCT images of the retina at baseline, 2 weeks and 4 weeks post-injury for each treatment group, including: IR group (saline), BAI group (baicalin), MEM group (membrane vesicles), and MBNPs group (membrane-coated nanoparticles loaded with baicalin). Figure 16B shows the quantification of the thickness of each retinal layer, including the retinal nerve fiber layer (RNFL), inner plexiform layer (IPL), retinal nerve fiber layer-inner nuclear layer complex (RNFL-INL), outer plexiform layer-outer nuclear layer complex (OPL-ONL), inner segment-retinal pigment epithelium complex (IS-RPE), and total retina (TRL). Figure 16C shows representative images of Brn3a immunofluorescence staining of retinal tilings from each group at week 4, showing retinal ganglion cells (RGCs). Figure 16D shows the quantification of Brn3a-positive RGC density / mm2. Data are expressed as mean ± SEM (OCT n=8, retinal twill staining n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. One-way ANOVA multiple comparison test.

[0118] Figures 17A-17C depict transcriptomic analysis and KEGG pathway enrichment among the treatment groups. Figure 17A is a volcano plot showing the distribution of differentially expressed genes (DEGs) in the group comparisons. The horizontal axis represents log2 fold change, and the vertical axis represents -log10 (P). Each point represents an independent gene; Figure 17B shows the gene expression profiles of significant changes in the comparisons between the IR group and the control group, and between the MBNPs group and the IR group; Figure 17C shows the KEGG pathway enrichment analysis of the DEGs obtained from the group comparisons; multiple signaling pathways were enriched in multiple comparisons, including neuroactive ligand-receptor interactions, the PI3K-Akt signaling pathway, the calcium signaling pathway, the cAMP signaling pathway, focal adhesion, axonal guidance, and glutamatergic synapses, reflecting molecular-level changes after treatment, consistent with the mechanisms of neuroinflammation and retinal damage.

[0119] Invention Details

[0120] In the following description, systems and / or methods are illustrated only as preferred examples. Those skilled in the art will understand that modifications, including additions and / or substitutions, can be made without departing from the scope and spirit of the invention. To avoid obscuring the essence of the invention, some specific details may be omitted; however, this disclosure is written to enable those skilled in the art to implement the teachings therein without excessive experimentation.

[0121] The core of this invention lies in providing a nanoplatform comprising a nanocarrier coated with an immune cell membrane. This platform represents a unique delivery mechanism that utilizes the biomimetic characteristics of the immune cell membrane to create a more efficient and safer method for directly delivering therapeutic drugs to target tissues, thereby improving drug delivery efficiency, controlled release, and therapeutic efficacy in target tissues.

[0122] This invention utilizes a bioactive immune cell membrane that can directly neutralize pro-inflammatory mediators and inhibit the activation of innate immunity in retinal tissue.

[0123] Immune cell membranes possess homing targeting capabilities, meaning they have a natural ability to target inflamed or diseased tissues, such as the retina. This targeting capability further enhances the precision of drug delivery systems, ensuring that therapeutic agents are delivered directly to the target tissue, such as the lesion site within the retina, improving therapeutic efficacy and reducing off-target effects, thereby minimizing potential side effects.

[0124] Immune cell membranes also have the ability to neutralize pro-inflammatory cytokines. By neutralizing pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, drug delivery systems can directly target underlying neuroinflammation associated with many diseases, such as retinal degeneration, thereby improving anti-inflammatory and neuroprotective effects.

[0125] Notably, the inventors discovered that immune cell membrane vesicles alone (without drugs) can inhibit ischemia-reperfusion (IR) injury-induced complement system activation, NLRP3 inflammasome activation, and cytokine release, effects that have never been reported in retinal applications of any immune cell-derived membrane.

[0126] The coating of immune cells on the nanoplatform mimics natural biological processes, significantly enhancing the system's targeting capabilities and its ability to neutralize pro-inflammatory cytokines. This innovative feature enables the drug delivery platform to precisely hom to inflamed or diseased tissues, such as the retina, while simultaneously neutralizing pro-inflammatory cytokines such as IL-6, IL-1, and TNF-α. This dual action precisely targets the intended tissue while simultaneously alleviating neuroinflammation, directly addressing the underlying pathological mechanisms of various diseases, including retinal degeneration.

[0127] In some embodiments, the immune cell membrane of the present invention is derived from cells involved in immune responses, inflammatory responses, antigen presentation, or immune regulation under disease conditions such as retinal diseases or neurodegenerative diseases. In some embodiments, the immune cell membrane of the present invention is derived from cells with immune or inflammatory regulatory functions obtained through genetic engineering, bioengineering, or in vitro induction treatment, and from cells in a pathologically activated state. In some embodiments, the cells include, but are not limited to, microglia, macrophages derived from monocytes, dendritic cells, infiltrating leukocytes, Müller glial cells, astrocytes, and retinal pigment epithelial cells. In some embodiments, the cells are immune cells in retinal tissue. In one specific embodiment, the immune cells that can be used as the source of the immune cell membrane of the present invention are microglia, particularly microglia in retinal tissue. The present invention achieves isotype targeting through microglia membrane coating, enabling specific delivery to inflammatory cell compartments. Membrane vesicles derived from immune cells possess inherent immunomodulatory functions when applied to ischemic or inflammatory tissues. Specifically, these immune cell membranes can bind to and neutralize pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6), regulate complement and Toll-like receptor (TLR) signaling pathways, and alter the phenotype of resident immune cells. This anti-inflammatory effect is not limited to a single cell source but reflects a universal mechanism inherent in immune cell-derived membrane vesicles, thus enabling interventional treatment of a range of ischemic, degenerative, and inflammatory diseases, such as retinal diseases.

[0128] A significant feature of this cell membrane coating invention is its modular and combinatorial design, characterized by the ability to modify and combine different types of cell membranes as coating materials for nanoparticles. This feature allows for the selection of one or more cell membranes from different sources for monolayer or multilayer coating, based on the pathological state, immune-inflammatory characteristics, or molecular markers of a patient's disease, such as retinal diseases or neurodegenerative diseases, to achieve precise regulation and treatment of individualized retinal or neurodegenerative diseases. This feature also allows for the customization of drug delivery systems for different diseases, such as retinal diseases, by selecting the cell membrane type best suited to target a specific condition. This adaptability enhances the versatility of the system / platform, enabling its widespread application to various diseases, such as retinal diseases, simply by changing the cell membrane type.

[0129] In some embodiments, the immune cell membrane of the present invention may be derived from a single type of cell or from different types of cells. In some embodiments, the immune cell membrane of the present invention may be derived from a single source of cells or from cells from different sources. In some embodiments, the coating of the immune cell membrane of the present invention may be single-layered or two or more layers.

[0130] Nanoplatforms are the core structure for drug encapsulation and delivery. By selecting carrier materials with controlled-release properties, such as biocompatible and / or biodegradable polymer matrices, they ensure the controlled and sustained release of encapsulated drugs. This controlled-release mechanism effectively solves the problems of rapid drug clearance and low delivery efficiency commonly found in traditional drug delivery methods.

[0131] The carrier materials of the nanoplatform of the present invention include, but are not limited to, biodegradable and / or biocompatible polymers, lipids, proteins, polysaccharides, inorganic or organic-inorganic hybrid materials, including but not limited to polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol and its derivatives, chitosan, hyaluronic acid, albumin, liposomes, lipid nanoparticles, metal oxide nanoparticles, silicon-based nanomaterials, and any combination thereof.

[0132] In some embodiments, biodegradable and / or biocompatible polymers are selected as carrier materials for the nanoplatform. In some embodiments, the biodegradable and / or biocompatible polymers include, but are not limited to, polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol, and their derivatives. In some embodiments, PLGA is selected as the carrier material for the nanoplatform. PLGA exhibits excellent biocompatibility and safety. PLGA also possesses unique degradability, undergoing ester bond hydrolysis in vivo, ultimately degrading into two natural metabolites, lactic acid and glycolic acid, which subsequently enter the tricarboxylic acid cycle and are ultimately completely excreted from the body as carbon dioxide and water, without systemic toxicity. PLGA also exhibits highly tunable release kinetics. By controlling the rate of drug diffusion from the polymer matrix and release during polymer degradation, PLGA can reduce peak-to-trough fluctuations in blood drug concentrations, maintaining a stable drug concentration within the therapeutic window. PLGA can enhance targeting and local efficacy, forming a drug "depot" at the lesion site (such as the joint cavity, tumor, and eye), continuously releasing high concentrations of drugs to act locally, while reducing systemic exposure and side effects.

[0133] In some embodiments, the present invention provides a biodegradable polymer nanoplatform coated with an immune cell membrane. In some embodiments, the biodegradable polymer is selected from polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol, and their derivatives. In some embodiments, the biodegradable polymer is PLGA.

[0134] In some embodiments, the present invention provides a PLGA nanoplatform coated with an immune cell membrane. In some embodiments, the present invention provides a PLGA nanoplatform coated with a microglia membrane. In some embodiments, the microglia are derived from microglia in retinal tissue.

[0135] This invention introduces a retinal-adapted nanoplatform with both targeting and modulating retinal inflammation capabilities. It provides a cutting-edge drug delivery system that integrates advanced biomimetic and nanoengineering technologies to address key challenges in the treatment of retinal diseases. At its core lies the integration of immune cell membranes with nanocarriers possessing drug-controlled release properties, such as biodegradable polymers like polylactic-co-glycolic acid (PLGA) nanoparticles, offering a breakthrough approach to achieving highly effective targeted therapies.

[0136] In one specific embodiment, the present invention provides a targeted drug delivery system comprising:

[0137] Therapeutic agents; and

[0138] The nanoplatform described in this article.

[0139] In one specific embodiment, the present invention provides a targeted drug delivery system comprising:

[0140] Therapeutic agents;

[0141] Nanocarriers for encapsulating therapeutic agents; and

[0142] Immunocellular membrane coating surrounding nanocarriers.

[0143] The nanocarriers and immune cell membrane coatings mentioned herein are as described in this article.

[0144] Those skilled in the art will understand that the nanoplatform of the present invention is suitable for delivering a variety of therapeutic agents, provided that the therapeutic agent is biocompatible with the nanoplatform.

[0145] In some embodiments, the therapeutic agent includes, but is not limited to, small molecule drugs, peptides, nucleic acids, antibodies, and protein drugs. In some embodiments, the therapeutic agent is a therapeutic agent for treating retinal diseases or neurodegenerative diseases. In some embodiments, the therapeutic agent is a small molecule drug, peptide, nucleic acid, antibody, or protein drug for treating retinal diseases or neurodegenerative diseases. In some embodiments, the therapeutic agent is a therapeutic agent with neuroprotective effects. In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the therapeutic agent is an antioxidant. In some embodiments, the therapeutic agent is a therapeutic agent with both anti-inflammatory and neuroprotective effects, such as natural flavonoids. In some embodiments, the therapeutic agent is baicalin.

[0146] In some embodiments, the delivery system of the present invention uses baicalein as a therapeutic agent. Baicalein is known to be a natural flavonoid compound with potent anti-inflammatory and neuroprotective effects; however, its solubility is limited and unstable. The system of the present invention significantly improves its solubility and stability, prolongs the drug's residence time in the eye, and enhances intracellular uptake by microglia by encapsulating baicalein in a controlled-release nanoplatform, thereby effectively enhancing its therapeutic effect.

[0147] The targeted drug delivery system of this invention has demonstrated in vivo dual protection of structure and function by simultaneously targeting immune and oxidative mechanisms, maintaining the survival of retinal ganglion cells and preserving vision.

[0148] The targeted drug delivery system described in this article enhances the effectiveness of existing therapies by improving drug targeting, controlled release capabilities, and therapeutic efficacy (especially in reducing neuroinflammation).

[0149] The present invention also provides a method for preparing the nanoplatform described herein, the method comprising: preparing a nanocarrier; and coating the nanocarrier with an immune cell membrane to obtain a nanoplatform.

[0150] The present invention also provides a method for preparing the biodegradable polymer nanoplatform coated with immune cell membrane as described in the present invention. The method includes: preparing a nanocarrier with a biodegradable polymer; and coating the nanocarrier with an immune cell membrane to obtain a biodegradable polymer nanoplatform coated with immune cell membrane.

[0151] The present invention also provides a method for preparing the targeted drug delivery system described herein, the method comprising: preparing a nanocarrier; encapsulating a therapeutic agent with the nanocarrier to obtain nanoparticles loaded with the therapeutic agent; and coating the nanoparticles loaded with the therapeutic agent with an immune cell membrane to obtain immune cell membrane-coated nanoparticles loaded with the therapeutic agent, which serve as the targeted drug delivery system.

[0152] In some embodiments, nanocarriers are prepared using biodegradable polymers. In some embodiments, the biodegradable polymer is selected from polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol, and their derivatives.

[0153] In some embodiments, PLGA is used to prepare the nanocarrier. In some embodiments, PLGA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) are used to prepare the nanocarrier. In some embodiments, the mass ratio of DOTAP to PLGA is 0-10:20. In some embodiments, the mass ratio of DOTAP to PLGA is 0:20, 1:20, 5:20, or 10:20. In one specific embodiment, the mass ratio of DOTAP to PLGA is 5:20 or 1:4.

[0154] In some embodiments, the mass ratio of the therapeutic agent to the biodegradable polymer is 0.5-4:10. In some embodiments, the therapeutic agent is baicalin (BAI), and the biodegradable polymer is PLGA. In some embodiments, the mass ratio of BAI to PLGA is 0.5-4:10, for example, 1:20, 1:10, 2:10, or 4:10. In one specific embodiment, the mass ratio of BAI to PLGA is 1:10.

[0155] In some embodiments, the method further includes isolating cell membranes from immune cells.

[0156] In some embodiments, after the target cells are cultured to a suitable density, the cells are washed with a cryogenic buffer solution and transferred to a hypotonic lysis buffer solution, which optionally contains a protease inhibitor. The cells are then lysed by repeated freeze-thaw cycles, mechanical disruption, or a combination thereof to obtain cell lysate. Subsequently, the cell lysate solution is centrifuged at low speed to remove cell nuclei and unlysed cell debris, and a supernatant containing cell membrane components is collected. This supernatant is then further centrifuged to enrich the cell membrane components, thereby obtaining the desired cell membrane material. Optionally, the obtained cell membrane can be quantitatively analyzed using protein quantification methods.

[0157] In some embodiments, the nanocarrier core is mixed with the cell membrane material at a predetermined mass ratio, which can be adjusted from no cell membrane coating to full coating to form membrane-core composite systems with different degrees of coating. Subsequently, the resulting mixture is subjected to ultrasound or other physical action to promote the fusion of the cell membrane with the nanocarrier surface, thereby obtaining cell membrane-coated nanoparticles. The cell membrane-coated nanoparticles are then purified and washed by centrifugation or equivalent separation methods and stored at low temperatures. The optimal cell membrane coating ratio for a specific retinal disease state is determined by characterizing the particle size, potential, or a combination thereof of the obtained nanoparticles.

[0158] The immune cell membrane-coated therapeutically loaded nanoparticles of the present invention have advantageous release characteristics. In one specific embodiment, the immune cell membrane-coated baicalein-loaded nanoparticles (MBNP) released ~50% of BAI within 24 hours at pH 7.4, while the free BAI formulation released approximately 90% of the free drug within 24 hours.

[0159] The immune cell membrane-coated nanoparticles loaded with therapeutic agents of the present invention have been shown to have good structural integrity and colloidal stability, indicating their suitability for in vitro and in vivo applications.

[0160] The immune cell membrane-coated nanoparticles loaded with therapeutic agents of the present invention have been shown to have no cytotoxicity in multiple cell types, indicating that they have good biocompatibility.

[0161] The experimental portion of this invention has sufficiently demonstrated that the cell membrane-coated nanoparticles loaded with therapeutic agents of this invention have one or more of the following effects: selective cellular uptake and antioxidant protection; effective inhibition of LPS-induced inflammation in vitro; visual function protection and inflammation regulation in retinal injury; retinal function salvage efficacy; preservation of retinal structure and enhancement of RGC survival rate after treatment; and immune regulation through multiple pathways.

[0162] This invention provides the use of the nanoplatform or targeted drug delivery system described herein in the preparation of medicaments for the prevention or treatment of retinal diseases.

[0163] This invention also provides the nanoplatform or targeted drug delivery system described herein for the prevention or treatment of retinal diseases.

[0164] The present invention also provides a method for preventing or treating retinal diseases in a subject in need, the method comprising administering the targeted drug delivery system described herein to the subject's retina.

[0165] In some embodiments, the retinal disease is a retinal disease involving ischemia-reperfusion injury. In some embodiments, the retinal disease is a retinal disease involving inflammation or neuroinflammation. In some embodiments, the retinal disease includes, but is limited to, ischemic retinal diseases, degenerative retinal diseases, inflammatory retinal diseases, and infectious or traumatic retinal diseases. In some specific embodiments, the retinal disease includes, but is not limited to, retinal ischemia or ischemia-reperfusion injury, retinal vascular occlusion, diabetic or hypertensive retinopathy, age-related macular degeneration and its associated choroidal neovascularization, glaucoma, uveitis, retinitis, retinal vasculitis, optic neuritis, autoimmune or hereditary degenerative retinal diseases, and infectious or traumatic retinal diseases.

[0166] In some embodiments, the administration methods include, but are not limited to, intravitreal injection, subretinal injection, subchoroidal injection, subconjunctival injection, subscleral injection, intra-anterior chamber injection, transscleral administration, transocular surface administration, eye drops, sustained-release implantation, and combinations thereof. In some embodiments, the administration is intravitreal injection.

[0167] The targeted drug delivery system described in this article helps slow disease progression and improve patient outcomes by delivering anti-inflammatory agents to the retina more effectively.

[0168] This invention provides the use of the nanoplatform or targeted drug delivery system described herein in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0169] This invention also provides the nanoplatform or targeted drug delivery system described herein for the prevention or treatment of neurodegenerative diseases.

[0170] The present invention also provides a method for preventing or treating neurodegenerative diseases in a subject in need, the method comprising administering the targeted drug delivery system described herein to the subject.

[0171] In some embodiments, the neurodegenerative disease is a neurodegenerative disease involving neuroinflammation. In some embodiments, the neurodegenerative disease is selected from Parkinson's disease (PD), Alzheimer's disease (AD), and multiple sclerosis (MS).

[0172] In some embodiments, the administration methods include, but are not limited to, intravitreal injection, subretinal injection, subchoroidal injection, subconjunctival injection, subscleral injection, intra-anterior chamber injection, transscleral administration, transocular surface administration, eye drops, sustained-release implantation, and combinations thereof.

[0173] This invention provides the use of the nanoplatform or targeted drug delivery system described herein in the preparation of medicaments for the management of neuroinflammation.

[0174] This invention also provides the nanoplatform or targeted drug delivery system described herein for the management of neuroinflammation.

[0175] The present invention also provides a method for managing neuroinflammation in a subject in need, the method comprising administering the targeted drug delivery system described herein to the inflamed tissue of the subject.

[0176] In some embodiments, administration methods include, but are not limited to, intravitreal injection, subretinal injection, subchoroidal injection, subconjunctival injection, subscleral injection, intra-anterior chamber injection, transscleral administration, transocular surface administration, eye drops, sustained-release implantation, and combinations thereof. In some embodiments, administration includes transocular surface administration and eye drops.

[0177] The innovative features / characteristics of this invention include one or more of the following:

[0178] Immune cell membrane coating: For the first time, membranes derived from immune cells, such as microglia, are used as coatings for nanoparticles, specifically designed to isomorphically target activated retinal immune cells, such as microglia, in a model of ischemia-reperfusion injury.

[0179] Providing nanoparticles with dual functions of immunomodulation and neuroprotection: Unlike existing membrane-coated systems (such as erythrocytes (RBCs) and neutrophils) that serve as passive carriers, this invention integrates a membrane that actively neutralizes inflammatory mediators with a core that provides sustained release of antioxidant drugs (baicalin), creating a synergistic immuno-drug delivery platform specific for retinal diseases.

[0180] Innovative Application: This system marks the first application of biomimetic methods for drug delivery in retinal diseases. Utilizing the natural properties of immune cell membranes, it creates a more efficient and safer method for directly delivering therapeutic drugs to the retina, overcoming the challenges of traditional delivery methods.

[0181] A novel effect of immune cell-derived membranes in retinal applications has been discovered: membrane vesicles alone (without drugs) can inhibit IR injury-induced complement system, NLRP3 inflammasome activation, and cytokine release.

[0182] Applicable to a wide range of retinal diseases: The system of the present invention is applicable to retinal diseases involving ischemia, inflammation, or neurodegeneration. Unlike early therapies limited to acute injury or vascular disease, the present invention aims to treat a variety of ischemic, inflammatory, or neurodegenerative retinopathy-related retinal diseases, such as diabetic retinopathy, age-related macular degeneration, and glaucoma, by modulating a microglia-driven immune cascade response;

[0183] Improved Drug Specificity: While baicalin is known for its anti-inflammatory and neuroprotective effects, its previous applications have been limited by its poor pharmacokinetic properties. The system of this invention encapsulates it in a nanocarrier, significantly improving its stability, solubility, and therapeutic efficacy in retinal ischemia / reperfusion injury. This property enhances the clinical potential of baicalin.

[0184] In summary, this invention addresses several key challenges and unmet needs in the treatment of neuroinflammatory diseases, such as retinal diseases (e.g., glaucoma, age-related macular degeneration, and diabetic retinopathy). Existing neuroinflammatory treatments often have limited efficacy and significant side effects, while the complex ocular structure and drug clearance mechanisms pose significant challenges to effective intraocular drug delivery. This biomimetic drug delivery system offers an innovative solution by improving drug delivery efficiency, enhancing treatment outcomes, and minimizing side effects.

[0185] The core of this invention lies in combining immune cell membranes with nanocarriers possessing controlled drug release properties, such as biodegradable polymers like polylactic-co-glycolic acid (PLGA) nanoparticles. This innovative method integrates the natural homing and immunomodulatory capabilities of immune cell membranes with the controlled-release properties of nanocarriers or PLGA nanoparticles, thereby solving the uncontrollable drug release and safety issues commonly found in existing nanodelivery systems. By utilizing the biomimetic properties of immune cell membranes, the system of this invention provides a more targeted and safer method for directly delivering therapeutic agents to retinal tissue, neutralizing pro-inflammatory cytokines, and alleviating neuroinflammation.

[0186] This invention also addresses the problems of poor solubility and low stability of baicalin. Baicalin is a compound with potent anti-inflammatory and neuroprotective effects, but its clinical application is limited due to its unsatisfactory pharmacokinetic properties. By integrating baicalin into an immune cell membrane-coated PLGA nanoplatform, its stability, solubility, and overall therapeutic potential are significantly improved, especially in the treatment of retinal ischemia / reperfusion injury.

[0187] Furthermore, this invention demonstrates excellent versatility and has broad application potential in a variety of retinal diseases. Its adaptable design allows it to be applied to a range of conditions, including retinal ischemia, age-related macular degeneration, and diabetic retinopathy, overcoming the limitations of existing therapies that typically target only specific diseases. By providing a robust, scalable, and efficient treatment platform, this invention represents a significant advancement in ophthalmic drug delivery, offering a targeted and effective solution for retinal diseases involving ischemia and neuroinflammation.

[0188] Experimental Section

[0189] Materials and methods

[0190] The main materials and reagents used in the experiments of this invention were sourced from the following sources: polylactic acid-glycolic acid copolymer (PLGA, weight average molecular weight: 66,000-107,000, purchased from Sigma-Aldrich, USA, catalog number: p1941); polyvinyl alcohol (PVA, purchased from Sigma-Aldrich, USA); fluorescein isothiocyanate (FITC, purchased from MedChemExpress, China); N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP, purchased from MedChemExpress, China); baicalein (purchased from Cayman Chemical, USA); lipopolysaccharide (LPS, purchased from Sigma-Aldrich, USA); acetonitrile (purchased from RCI Labscan Limited).

[0191] Reagents related to cell membranes and molecular biology include: cell membrane and cytoplasmic protein extraction kits (Beyotime, China); real-time quantitative PCR reagents (Takara); acrylamide gel preparation reagents (Bio-Rad); protein and peptide quantification reagents (Thermo Fisher Scientific); RNA extraction reagents (Fastagen); cell viability assay kits (Beyotime); reactive oxygen species assay kits (Thermo Fisher Scientific); and nuclear dyes and cytoskeleton dyes (Thermo Fisher Scientific).

[0192] The primary antibodies used included anti-iNOS antibody (Abcam) and anti-Brn3a antibody (Sigma), and the secondary antibodies included fluorescently labeled anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific). Cytokine detection was performed using an ELISA kit (Thermo Fisher Scientific) targeting mouse IL-6, TNF-α, and IL-1α.

[0193] The above-mentioned materials and reagents can be obtained and used by those skilled in the art from equivalent commercial sources.

[0194] Synthesis and characterization of nanocarriers

[0195] To synthesize and optimize PLGA-DOTAP nanocarriers, 20 mg of PLGA and different amounts (0, 1, 5, 10 mg) of DOTAP were dissolved in 1 ml of acetonitrile and sonicated for 5 minutes. Under magnetic stirring, the resulting mixture was added dropwise to 5 ml of 1% (w / v) PVA solution. After stirring for 4 hours to remove the organic solvent, the PLGA-DOTAP nanoparticles were collected by centrifugation at 12000 × g for 30 minutes at 4 °C, washed, and stored at 4 °C for later use. The hydrodynamic diameter, zeta potential, and cell viability of the nanoparticles were measured to determine the optimal PLGA to DOTAP ratio.

[0196] Cell membrane extraction and quantification

[0197] BV2 cells at approximately 80% confluence were washed with ice-cold PBS and then scraped into hypotonic buffer A (containing 1% PMSF) using a cell scraper. Cell membranes were separated using a Membrane and Cytosol Protein Extraction Kit. After five freeze-thaw cycles of liquid nitrogen in buffer A, the lysis buffer was centrifuged at 700×g, 4°C for 10 min to remove nuclei and debris. The supernatant was then ultracentrifuged (14,000×g, 30 min, 4°C) to collect the membrane precipitate. Total membrane proteins were quantified using the BCA protein assay.

[0198] Preparation of baicalin-loaded nanoparticles (BNPs)

[0199] Dissolve 20 mg PLGA and 5 mg DOTAP in 1 ml acetonitrile and sonicate for 5 minutes. Add 1 mg baicalein and sonicate again for 5 minutes. Add this mixture dropwise to 5 ml 1% PVA and stir for 4 hours to remove the organic solvent. Collect BNPs by centrifugation at 4 °C and 12000 × g for 30 minutes, wash, and store at 4 °C.

[0200] Preparation of membrane-coated BNPs (MBNPs)

[0201] BNPs were mixed with isolated cell membranes at nuclear membrane mass ratios of 1:0, 1:0.1, 1:0.5, 1:1, and 1:2. The suspensions were sonicated in an ice bath for 10 minutes to promote membrane fusion. MBNPs were then purified by centrifugation at 12000×g for 30 minutes at 4°C, washed, and stored at 4°C.

[0202] Encapsulation efficiency and release profile evaluation

[0203] MBNPs were synthesized according to a predetermined baicalin mass ratio. After centrifugation, the supernatant was collected, and the baicalin content was determined by ultraviolet spectrophotometry.

[0204] The release characteristics of baicalin from MBNPs were evaluated using a dialysis-based method. In short, a dialysis bag containing nanoparticles and free baicalin was immersed in 45 mL of PBS buffer (pH 7.4). The system was continuously stirred at 100 rpm at room temperature. At preset time points, 2 mL of sample was removed and 2 mL of fresh dissolution medium was added. The concentration of baicalin in the removed 2 mL sample was then analyzed using a UV spectrophotometer to determine the release characteristics.

[0205] Characterization of membrane proteins

[0206] The protein profile of the MBNP surface was analyzed by SDS-PAGE. Protein bands were stained using a Coomassie Brilliant Blue detection kit. A 12% SDS-PAGE gel was prepared using the TGX Stain-Free Fast Cast Acrylamide Kit. Samples (containing an equal amount of membrane proteins) were loaded onto the gel and subjected to electrophoresis. After electrophoresis, the gel was immersed in Coomassie Brilliant Blue staining buffer for 30 minutes, and then rinsed several times with water until the background was clear.

[0207] BAI's cellular uptake

[0208] 1×10 6 BV2 cells were seeded in 12-well plates and incubated at 37°C with MBNPs and BAI for 2 hours. Cells were then washed three times with pre-chilled PBS and collected. 200 μl of 50% acetonitrile was added to the cells, and the mixture was subjected to three freeze-thaw cycles and centrifuged at 4°C, 12000 rpm for 3 minutes. 100 μl of supernatant was collected. 400 μl of acetonitrile was added to the supernatant, and the mixture was sonicated for 10 minutes, then centrifuged at 4°C, 12000 rpm for 3 minutes. The supernatant was collected for LC-MS (Orbitrap) analysis.

[0209] In vitro cytotoxicity of nanocarriers and MBNPs

[0210] Cells were spaced at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated overnight to allow for adhesion. Once cell confluence reached approximately 70%, cells were incubated for 24 and 48 hours with different concentrations (0, 1, 10, 50, 100, 200, 300, 400, 500 μg / ml) of nanocarriers and MBNPs. Cell viability was quantitatively analyzed using the CCK-8 assay according to the manufacturer's instructions.

[0211] In vitro anti-inflammatory effects of MBNPs on BV2 cells

[0212] BV2 cells were loaded at 3 × 10 5 BV2 cells were seeded at a density of cells / well in 12-well plates and incubated overnight. BV2 cells were divided into seven groups: control group, LPS group, LPS + nanocarrier group, LPS + BNPs group, LPS + MBNPs group, LPS + BAI group, and LPS + cell membrane group. All LPS groups were pre-stimulated with 0.5 μg / ml LPS for 6 hours. Then, they were treated with 10 μMBAI and an equal amount of BAI containing BNPs and MBNPs for 42 hours, maintaining an LPS concentration of 0.5 μg / ml throughout the process. Total RNA was extracted using RNAfast200, and gene expression was quantitatively analyzed by q-PCR. This protocol is also applicable to membrane concentration anti-inflammatory measurements.

[0213] Cellular uptake of FITC-labeled nanoparticles FNPs and MFNPs

[0214] Fluorescein isothiocyanate (FITC) was used instead of baicalein as the fluorescent label. FITC-loaded nanoparticles (FNPs) and membrane-coated FITC-loaded nanoparticles (MFNPs) were prepared using the same preparation protocol as BNPs and MBNPs.

[0215] Uptake kinetics assay: BV2 cells were incubated at 5 × 10⁻⁶ cells per day. 4 MFNPs were seeded at a density of [number] cells / dish in confocal culture dishes, and then co-incubated with BV2 cells containing 1 μg / ml FITC for 1, 4, 12, and 24 hours, respectively. After incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 10 min, stained with phalloidin (35 min) and DAPI (10 min), and then imaged using a confocal microscope. Fluorescence intensity was quantitatively analyzed using ZEN software.

[0216] Formulation comparison: BV2 cells were incubated for 4 hours with free FITC, FNPs, or MFNPs (1 μg / ml FITC), respectively. Fixation, staining, and image analysis methods were the same as above.

[0217] Cell type specificity assay: To determine the optimal time for cell uptake, MC-3T3 cells (3 × 10⁻⁶) were subjected to cell type specificity assay. 5 (5 × 10⁻⁶ cells) 5 (5 × 10) and BV2 cells (5 × 10) 5 Cells were seeded in confocal culture dishes and exposed to MFNPs containing 1 μg / ml FITC for 4 hours. After washing with PBS, the cells were fixed with 4% PFA for 10 min, stained with phalloidin (35 min) and DAPI (10 min), and then imaged using confocal microscopy. Quantitative fluorescence intensity analysis was performed using ZEN software to assess differences in uptake among different cell types.

[0218] Animal research

[0219] Retinal ischemia / reperfusion injury model: A PBS bag was suspended 1 meter above the mice, connected to a glass microneedle via an extension tube. Adult C57BL / 6J mice (8-10 weeks old) were anesthetized by intraperitoneal injection of a sterile PBS solution (1:1:4) containing 120 mg / kg ketamine and 20 mg / kg xylazine. After pupil dilation with 1% tropicamide eye drops and corneal anesthesia with 0.5% proparacaine, a self-sealing corneal tunnel was created using a 30-gauge needle. This needle was replaced with a glass microneedle connected to a PBS reservoir via an extension tube, positioned 1 meter above the eye. The valve was opened to raise the intraocular pressure (IOP) to 75 mmHg and maintained for 1.5 hours. After this, the microneedle was removed, antibiotic eye drops were administered, and the anesthetized mice were placed on a warming pad until awakening. Retinal samples were acquired 1 week or 4 weeks after the injury for lateral analysis.

[0220] Mice were sacrificed 4 weeks after ischemia / reperfusion (I / R) injury for qPCR and IBA-1 testing. + Microglial cell immunostaining, and BRN3a staining of mice euthanized 4 weeks after injury. + Retinal ganglion cells (RGCs) and IBA-1 + Immunostaining of microglia. For quantifying morphological changes in microglia, N=5-7 mice per group; for qPCR measurement of microglia activation markers and pro-inflammatory cytokine mRNA levels, N=6 mice per group.

[0221] Intravitreal injection

[0222] Mice were randomly assigned to five groups: an IR control group (saline), a BAI group (free baicalein 100 μM, 2 μL), a MEM group (membrane protein 13.5 ng / μL, 2 μL), a nanocarrier group (27 ng / μL, 2 μL), and a MBNPs group (2 μL containing 100 μM baicalein + 27 ng / μL nanocarrier + 13.5 ng / μL membrane protein). Treatment was administered immediately after IR surgery via intravitreal injection, followed by weekly injections. Mice were anesthetized by intraperitoneal injection of a sterile PBS (1:1:4) solution of 120 mg / kg ketamine and 20 mg / kg toluenethiazide. Pupil dilation was achieved using 1% tropicamide eye drops, and corneal anesthesia was achieved using 0.5% promecaine. 2 μL of the treatment was delivered into the vitreous cavity using a 10 μL Hamilton syringe equipped with a 30-gauge needle via a RWD microinjector. Antibiotic eye drops were administered post-injection, and mice were placed on a warming pad until fully recovered.

[0223] Intraocular pharmacokinetics of baicalein

[0224] Mice with IR injury were randomly divided into two groups: a free BAI group and an MBNPs group. Each mouse received either 2 μL of 200 μM free baicalein or 2 μL of MBNPs (200 μM baicalein + 540 ng / μL nanocarrier + 270 ng / μL membrane protein). Mice were sacrificed at designated time points and their eyeballs were enucleated. The eyeballs were homogenized in 100 μL of 1% ascorbic acid solution using a tissue homogenizer (Servicebio.cn), followed by the addition of 400 μL of internal standard solution containing 10 μL of 25 ng / mL formononetin, and vortexed for 30 seconds. After centrifugation at 4°C and 12000 rpm for 15 minutes, the supernatant was collected, and the samples were dried overnight at 4°C in a vacuum centrifuge (Labconco, MO, USA). After drying, the sample was reconstituted with 100 μL of 50% acetonitrile, vortexed for 3 minutes, and then centrifuged again at 4 °C and 12,000 rpm for 15 minutes to collect the supernatant. 1 μL of the sample was injected into a QTRAP mass spectrometer (SCIEX Triple Quad 7500) for analysis.

[0225] Chromatographic separation was performed using an ACQUITY Premier BEH C18 column (1.7 μm, 2.1 × 100 mm, with VanGuard FIT guard column; Waters), maintained at 40 °C. Mobile phase A was HPLC-grade water, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. Isocratic elution was used, with a mobile phase ratio of 30% B / 70% A, and a flow rate of 0.30 mL / min.

[0226] The ion source parameters were set as follows: ion spray voltage +4,500V; ion source temperature 450℃; curtain gas pressure 46psi; ion source gas 1 pressure 55psi; ion source gas 2 pressure 70psi; collision gas in "medium" mode. Multiple reaction monitoring (MRM) mode was used, monitoring ion pairs at m / z 271→123 and 271→253. Typical compound-specific parameters were: declustering voltage 60V; inlet voltage 10V; collision energy 43eV (271→123) and 36eV (271→253); collision cell outlet voltage 10V; residence time 100ms / ion pair; switching interval 5ms. Data acquisition and peak integration were performed using Analyst software (SCIEX). The acceptance window for the ratio of qualitative to quantitative ion abundance was set to ±20% of the average ratio obtained from standard determination.

[0227] Electroretinography (ERG)

[0228] Mice were acclimatized to a dark room for 12 hours before measurement, and then anesthetized with a sterile PBS (1:1:4) solution of 120 mg / kg ketamine and 20 mg / kg toluenethiazide. Corneal anesthesia was performed using 0.5% promecaine, and the cornea was kept moist using hydrogel. Two gold-ringed active electrodes were placed on the corneal surface, two reference electrodes were subcutaneously inserted behind both ears, and one grounding electrode was subcutaneously placed at the base of the tail. Positive scotopic threshold response (pSTR) was recorded using the Roland RETIscan system, and the pSTR amplitude was quantified according to the manufacturer's protocol.

[0229] Intraocular pressure (IOP) measurement

[0230] Intraocular pressure (IOP) was assessed while the patient was awake. Measurements were obtained using a TonoLab rebound tonometer (Colonial Medical Supply). For each eye, six consecutive measurements were taken, and the average was recorded as a single data point. This process was repeated until at least six data points were collected. The final IOP was recorded as the average of these data points. All measurements were performed at the same time each day to minimize the impact of diurnal fluctuations.

[0231] Visual function measurement

[0232] Visual function was assessed using the optokinetic reflex paradigm, which can be used to quantify visual acuity (VA) and contrast sensitivity (CS). Mice were placed on an elevated platform in the center of a four-monitor optokinetic reflex field. Each test began with a uniform gray field of view, followed by computer-generated vertical gratings on all screens, rotated clockwise or counterclockwise as described in the literature (Xiao, J., et al., Visual Contrast Sensitivity Correlates to the Retinal Degeneration in Rhodopsin Knockout Mice. Invest Ophthalmol Vis Sci, 2019. 60(13): p. 4196-4204).

[0233] In the VA test, the spatial frequency of the black-and-white grating was systematically increased while the rotational speed was kept constant at 12 degrees / second. The highest spatial frequency that induced a stable head-tracking response was recorded as the VA threshold.

[0234] In the CS test, the raster was presented at a fixed spatial frequency (0.186 Hz / degree), while the Michelson contrast was progressively reduced. CS was calculated based on the threshold contrast that still induced tracking behavior, using the following formula:

[0235] Where Lmax and Lmin represent the brightness (cd / m²) of the white and black stripes, respectively. 2 All measurements were performed under the same ambient lighting conditions.

[0236] RNA sequencing

[0237] Total RNA from retinal tissues of different treatment groups was first homogenized using microbeads, followed by extraction using the Fast200 extraction kit. RNA sequencing was performed by Novogene Co., Ltd. All data were processed on DAVID and Jackson Laboratory platforms.

[0238] result

[0239] Figure 1 illustrates the mechanism of action and therapeutic effect of the microglia-coated nanoparticles of the present invention in retinal ischemia-reperfusion injury. Figure 1A shows the results obtained through proteomics analysis to elucidate the mechanism of action of microglia in inhibiting the inflammatory response and protecting retinal photoreceptors and retinal ganglion cells; Figure 1B shows a schematic diagram of the preparation of the cell membrane-coated nanoparticles; Figure 1C shows a schematic diagram of the mechanism of action of the cell membrane-coated nanoparticles in inhibiting the inflammatory response and alleviating retinal ganglion cell damage.

[0240] As shown in Figure 1C, under conditions of acute intraocular pressure elevation or ischemic injury, resting retinal microglia are activated and release pro-inflammatory cytokines, chemokines, and other inflammatory mediators, leading to damage to retinal ganglion cells (RGCs). The cell membrane-coated nanoparticles of the present invention bind to activated microglia through cell membrane-associated proteins retained on their surface and release active ingredients with anti-inflammatory or neuroprotective effects (such as baicalin) to them, thereby alleviating the inflammatory response. At the same time, the nanoparticles can also further alleviate ischemia-reperfusion-induced tissue damage by regulating complement cascade reactions and inflammasome signaling pathways, and by adsorbing or neutralizing inflammatory factors in the retinal microenvironment, thereby transforming microglia to a homeostatic phenotype and protecting retinal ganglion cells.

[0241] Proteomics reveals that microglia reduce inflammation while protecting photoreceptors.

[0242] The inventors discovered profound changes in retinal protein expression following irritation (IR) injury and observed that MEM treatment significantly modulated these changes. In the IR vs. Ctrl comparison, 5,878 proteins were quantified, including 152 upregulated proteins and 125 downregulated proteins (Figure 2A). In the MEM vs. IR comparison, 6,473 proteins were quantified, with 83 upregulated and 62 downregulated (Figure 2D). Clustering based on these differentially expressed proteins (DEPs) separated the MEM-treated samples from the IR group and showed that the global abundance pattern of MEM shifted towards a control-like profile, consistent with a partial rescue effect of IR-related proteomic states (Figures 2B, 2E).

[0243] In the comparison between IR and Ctrl, KEGG pathway enrichment analysis revealed significant changes in molecular pathways (Fig. 3A). Pathways related to innate immune activation and inflammatory signaling, phototransduction, and photoreceptor function were significantly enriched in proteins with altered IR (Figs. 2G and 2C, Fig. 3A). Interestingly, proteins affected by MEM were also associated with pathways related to innate immune activation and inflammatory signaling, and phototransduction (Figs. 2J and 2F, Fig. 3B).

[0244] To visualize the protein changes underlying these pathway alterations, a heatmap of the most significant changes in DEPs was plotted among the control, IR, and MEM-treated groups (Figure 3C). One protein cluster, including immune stress-related factors such as the MHC class I molecule H2-D1 and the glial cell activation marker Cd44, was strongly upregulated after IR injury and showed a significant decrease after MEM treatment. In contrast, another protein cluster containing photoreceptor-specific proteins (e.g., Abca4, Cnga1, and Rom1) was significantly downregulated in IR injury but remained at high abundance in the MEM-treated retina. These patterns suggest that many IR-dysregulated proteins, particularly those involved in immune signaling and photoreceptor function, were partially rescued by MEM therapy. These patterns indicate that MEM treatment exerts a neuroprotective effect by modulating key proteins, maintaining photoreceptor integrity while suppressing injury-induced inflammatory responses.

[0245] To assess the reliability and reproducibility of the quantitative proteomics data, the coefficient of variation (CV) was calculated for all 5878 quantified proteins, and their distribution was analyzed (Figures 2H and 2K). In the comparisons between the IR / CTL and MEM / IR groups, over 80% of the proteins had CVs below 20%, and over 95% had CVs below 30%, indicating high quantitative stability and biological reproducibility of the dataset. Further comparison of the overall variability levels between the two groups using CV violin plots (Figure 2I) showed a high degree of similarity in CV distributions between IR / CTL and MEM / IR, ruling out systematic noise caused by sample preparation, detection, or batch differences. Furthermore, distribution analysis of the log2 fold change (log2FC) of proteins showed that the distributions in both comparisons were centered around 0 (Figure 2L), and no systematic shift in overall proteomics expression was observed. This indicates that cell membrane treatment did not cause non-specific changes in global protein expression, and the observed protein changes mainly originated from the regulation of specific biological pathways. In summary, these quality control results validate the high reproducibility, low noise, and absence of systematic bias in the proteomics data of this study, providing a solid data foundation for subsequent differential protein screening and the elucidation of inflammation-immune pathway mechanisms.

[0246] Preparation and characterization of MBNPs

[0247] To construct membrane-coated nanocarriers with robust colloidal stability and surface charges conducive to cell-cell interactions, the formulation of cationic lipid / polymer hybrid nanoparticles composed of DOTAP and PLGA was optimized. Given the cytotoxicity of DOTAP, a series of formulations with increasing DOTAP:PLGA mass ratios were systematically evaluated based on hydrodynamic diameter, zeta potential, and cell viability. The addition of DOTAP resulted in a gradual decrease in particle size and a change in zeta potential from negative to positive (Figs. 5A, 5B). At DOTAP:PLGA ratios of 0:20, 1:20, and 5:20, BV2 microglia exhibited negligible cytotoxicity after 24 and 48 hours of exposure (Figs. 6A, 6B), supporting the selection of 5:20 as the optimized formulation for subsequent experiments.

[0248] The resulting baicalein-loaded nanoparticles (BNPs) were then coated with a membrane derived from BV2 cells using a series of differential centrifugation, freeze-thaw cycles, and sonication steps (Fig. 1B). Transmission electron microscopy (TEM) revealed that the MBNPs retained the spherical morphology of the core particles and had a visible membrane bilayer coating around the PLGA core (Figs. 4A-4C). Consistent with successful membrane fusion, dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter increased from ~183 nm (BNPs) to ~210 nm (MBNPs) (Figs. 4D-4F), and the MBNP particle size increased accordingly with increasing membrane coverage, while the surface potential decreased accordingly from +42 mV to -28 mV (Figs. 7A, 7B). The final MBNP formulation was cell membrane:PLGA:DOTAP:baicalein = 0.5:1:0.2:0.1, exhibiting a uniform size distribution (PDI = 0.175).

[0249] Encapsulation efficiency was also tested, reaching over 80% at a BAI:PLGA weight ratio of 1:10 (Figure 4G). Subsequently, the release characteristics of free BAI vs MBNPs were investigated at pH 7.4. Approximately 90% of the free BAI was released within 24 hours, while only ~50% of the BAI from MBNPs was released within 24 hours (Figure 4H).

[0250] SDS-PAGE analysis further confirmed the retention of key membrane proteins on the surface of MBNPs (Figure 4I). Notably, MBNPs maintained structural integrity and colloidal stability in physiological saline for 7 days, confirming their suitability for in vitro and in vivo applications (Figures 4J-4L).

[0251] In vitro cell internalization kinetics of membrane-coated nanoplatforms

[0252] To evaluate the cellular uptake kinetics of the membrane-coated nanoplatform, fluorescein isothiocyanate (FITC) was used as a fluorescent substitute for BAI to encapsulate FITC-loaded nanoparticles (MFNPs). Confocal laser scanning microscopy (CLSM) showed the time-dependent uptake of MFNPs by BV2 microglia over 24 hours (Fig. 8A, 8C). At 1 hour of incubation, the FITC signal was mainly localized to the cell membrane, indicating initial binding without internalization. Maximum intracellular accumulation occurred at 4 hours, followed by a decrease at 12 and 24 hours, reflecting endosome processing or degradation. Flow cytometry analysis confirmed the CLSM data, identifying a peak fluorescence signal at the 4-hour time point (Fig. 8B), which was selected for subsequent in vitro studies.

[0253] To further elucidate the promoting effect of membrane functionalization on nanoparticle uptake, BV2 cells were treated with free FITC, FITC-loaded naked nanoparticles (FNPs), and MFNPs for 4 hours (Figures 8D and 8E). Quantitative analysis by CLSM and flow cytometry showed that although the free FITC and FNP treatment groups produced only negligible intracellular signals, the fluorescence intensity of the MFNP treatment group increased by more than 10-fold (Figure 8F). These results confirm the role of BV2-derived membranes in enhancing cell adhesion and promoting nanoparticle internalization, consistent with previous reports that homomorphic membrane coatings promote uptake via membrane protein-mediated recognition and fusion.

[0254] Cellular selective uptake and antioxidant protection of MBNPs

[0255] BV2 cells, derived from mouse microglia, retain the immunophenotype and functional characteristics of primary microglia and were used as an in vitro model of retinal neuroinflammation. Meanwhile, 661W cells (an SV40-transformed photoreceptor-derived cell line) were used to evaluate photoreceptor toxicity due to their high sensitivity to light and oxidative stress. To assess safety, BV2, 661W, and RAW264.7 macrophages were incubated with baicalein and MBNPs at concentrations within a specified range. Results showed that none of the formulations exhibited cytotoxicity in any cell type within 24 hours, confirming the good biocompatibility of MBNPs (Figures 9A-9C).

[0256] Given the isotype-specific properties of microglia, the selective uptake of MBNPs was evaluated. CLSM and flow cytometry showed that after 4 hours of incubation, BV2 cells internalized significantly more MFNPs than MC-3T3 and RAW264.7 cells (Figs. 9D-9F). Quantitatively, BV2 cells uptake MFNPs 2.5 times that of MC3T3 cells and twice that of RAW264.7 cells. This preferential accumulation highlights the role of membrane-inherited recognition molecules in promoting isotype targeting.

[0257] To directly compare intracellular drug delivery, intracellular baicalein levels were measured after treatment with the same BAI dose. LC-MS quantification showed that the MBNPs group had significantly higher baicalein content: MBNP treatment increased the drug amount per cell by approximately 50-fold compared to free baicalein (Figure 9G).

[0258] To investigate the cytoprotective potential of MBNPs under oxidative stress, H2O2 was used to induce reactive oxygen species (ROS) production in BV2 cells. ROS-sensitive fluorescent probe detection showed that both free baicalein (BAI) and MBNPs significantly reduced intracellular ROS levels (Figure 9H). However, MBNPs provided better protection against oxidative damage: compared to the H2O2-treated control group, they salvaged approximately 60% of BV2 cell viability, superior to free baicalein and uncoated nanocarriers (Figure 9I). This enhanced efficacy stems from the selective uptake and microenvironment-responsiveness of MBNPs, enabling them to spatially focus free radical scavenging near ROS generation sites.

[0259] MBNPs potently inhibit LPS-induced inflammation in vitro.

[0260] This study evaluated the anti-inflammatory efficacy of MBNPs in LPS-activated microglia. BV2 cells were stimulated with LPS and treated with microglia membrane vesicles (MEM), free baicalein (BAI), or MBNPs (all at equivalent doses of membrane protein or baicalein). Quantitative PCR showed that MEM alone inhibited pro-inflammatory cytokine genes (IL-6, IL-1α, TNF-α) in a dose-dependent manner, almost normalizing IL-6 and TNF-α at the highest MEM dose (Fig. 10A). When comparing treatments with equivalent doses of baicalein, MBNPs induced the strongest inhibition of all measured cytokine mRNAs (IL-6, IL-1α, IL-1β, TNF-α) (Fig. 10B). Furthermore, the markers of M2 microglia, IL-4 and IL-10, were examined. Both showed an increasing trend after MBNP treatment, indicating microglia polarization from M1 to M2 (Fig. 11).

[0261] Immunofluorescence staining of the inflammatory enzyme iNOS validated these findings (Figs. 10C-10D). LPS induced a bright iNOS expression signal in BV2 cells; however, MBNP treatment almost eliminated this signal. MBNPs elicited the most potent anti-inflammatory response. Control experiments confirmed that the polymer core of the nanoparticles themselves did not possess anti-inflammatory activity (Fig. 10A). Notably, MEM vesicles attenuated several cytokine transcripts, particularly IL-6. This independent activity is consistent with evidence that immune cell membranes can bind to and neutralize cytokines (Zhou, Z., et al., Macrophage-Mimicking Cellular Nanoparticles Scavenge Proinflammatory Cytokines in Specimens of Patients with Inflammatory Disorders. Adv Sci (Weinh), 2024.11(31):p.e2401423; and Zhang, Q., et al., Neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis. Nat Nanotechnol, 2018.13(12):p.1182-1190), and also corroborates the above-mentioned proteomics results (Figure 2C).

[0262] Visual function protection and inflammation modulation of MBNPs in retinal injury

[0263] The neuroprotective efficacy of MBNPs was evaluated in a C57BL / 6 mouse model of intraretinal irritation (IR). All animals underwent baseline assessments prior to IR injury, including electroretinography (ERG), optical coherence tomography (OCT), optomotor reflex visual acuity (OMR), and intraocular pressure (IOP). One day after IR injury, mice received an intravitreal injection of one of four treatments: saline (IR), free baicalein (BAI), isolated BV2 membrane (MEM), or an MBNP formulation. Functional outcomes were measured 2 weeks after IR injury, as shown in the experimental timeline (Figure 14A), followed by a second intravitreal injection of the same treatment, and again at 4 weeks post-IR injury. Mice were sacrificed at 1 and 4 weeks post-treatment; retinal tissue was collected at 1 week for qPCR and at 4 weeks for whole-retinal patch staining.

[0264] First, ocular pharmacokinetics were assessed using LC-MS. Pharmacokinetic characteristics in mouse eyes showed that MBNPs exhibited higher early concentrations and significantly longer exposure compared to free baicalein (Figure 14B). Baicalein from MBNPs remained detectable throughout the 8-hour sampling window, while free drug rapidly declined, approaching the limit of detection at 4 hours. In summary, these data indicate that MBNPs enhance cellular delivery of baicalein and prolong its intraocular retention time.

[0265] Infrared retardation (IR) led to significant visual function loss in the saline-treated IR group, while MBNP treatment significantly maintained spatial visual acuity. Visual acuity was measured using the OMR test (Figure 12). Saline-treated IR eyes showed a sharp decline in visual acuity by week 2 and remained at a low level by week 4 (Figure 14C). In stark contrast, mice treated with MBNPs maintained significantly higher visual acuity at both weeks 2 and 4. By week 4, visual acuity in the MBNP group had almost recovered to pre-injury baseline levels, demonstrating robust functional protection. Statistical analysis confirmed that the visual function of MBNP-treated eyes was superior to all other groups, highlighting the remarkable efficacy of MBNP therapy.

[0266] IOP was monitored in all groups to assess intraocular homeostasis and the effect of treatment-induced secondary IOP elevation (Figures 13A, 13B). No significant IOP fluctuations were observed after IR injury or during treatment.

[0267] To investigate the potential mechanisms of neuroprotection, we quantified retinal mRNA levels of key inflammatory factors one week after IR (Fig. 14D). The saline-treated IR group showed significant upregulation of pro-inflammatory cytokines and innate immune receptors (including TNF-α, IL-6, IL-1α, IL-1β, Toll-like receptors 2 and 4 (TLR2, TLR4)) and the NLRP3 inflammasome component. MBNP treatment greatly suppressed this inflammatory surge. The expression of all measured inflammatory markers in mice receiving MBNPs was significantly lower than in the IR saline group. Notably, IL-4 and Pou4f1 showed an upregulation trend in the MEM and MBNP-treated groups compared to the IR group, indicating microglial polarization to the M2 type and protection of retinal ganglion cells. In conclusion, as a network of multiple regulatory systems, MBNPs demonstrate remarkable efficiency in reducing inflammation in vivo.

[0268] retinal function salvage efficacy of MBNPs

[0269] Visual function was evaluated using full-field electroretinography (ERG). Positive scotopic threshold response (pSTR) reflected the function of retinal ganglion cells. Representative pSTR waveforms for each treatment group are shown (Fig. 15A, 15B). At baseline, all groups showed comparable pSTR amplitudes. By 2 and 4 weeks post-IR injury, the pSTR amplitude in the saline-treated (IR) group decreased significantly. The MBNP group retained the largest pSTR amplitude, with only a slight decrease relative to baseline. pSTR amplitudes at 2 and 4 weeks were also compared among the groups (Fig. 15C, 15D). This retention in the MBNP group indicates effective protection of retinal neurons in the inner nuclear layer.

[0270] Representative a-wave (photoreceptor response) and b-wave (bipolar / Müller cell response) of each group at each time point are shown (Figs. 15E, 15F). All groups had similar a-wave and b-wave amplitudes at baseline. Following IR lesion, the b-wave amplitude in the IR group decreased significantly by week 4, and the a-wave amplitude also showed a partial decrease. The MBNP group maintained near-baseline a-wave and b-wave amplitudes even at week 4 (Figs. 15G, 15H). In summary, the ERG data indicate that MBNPs achieved the maximum neuroprotective effect.

[0271] Preservation of retinal structure and survival of RGCs after MBNPs treatment

[0272] Optical coherence tomography (OCT) was used to non-invasively monitor changes in retinal structural integrity over time. This high-resolution imaging modality allows for longitudinal assessment of retinal layer thickness, a sensitive indicator of neuronal survival and edema regression. Representative OCT images and quantitative analysis are shown (Figs. 16A, 16B).

[0273] At baseline, all groups exhibited comparable retinal morphology with clear layer structures and similar total retinal thickness. Following IR injury, the untreated eye (IR) showed progressive retinal thinning as early as 2 weeks post-injury, most notably in the inner retina. By week 4, significant disruption of the multilayered structure was observed, consistent with the known vulnerability of the inner retina to ischemic injury. In contrast, the eye treated with MBNPs retained near-baseline retinal thickness, with minimal thinning observed at both 2 and 4 weeks.

[0274] Consistent with the findings on retinal structure, whole-slice retinal staining confirmed significant differences in RGC survival among groups 4 weeks after IR injury. Brn3a immunostaining (Fig. 16C) showed significant RGC loss in the IR group, manifested as markedly sparse RGC cell bodies in the ganglion cell layer. Notably, retina treated with MBNPs showed the highest RGC density. Quantitative RGC counting (Fig. 16D) further validated these observations. IR injury leads to RGC density (cells / mm²). 2 The sharp decline confirmed significant cell loss compared to the MBNP treatment group.

[0275] Transcriptome analysis reveals that MBNPs play a multi-pathway role in immune regulation.

[0276] To elucidate the transcriptomic mechanism of MBNP-mediated protection, mRNA sequencing was performed on mouse retinas collected one week after IR injury. Differential gene expression and pathway enrichment analyses were performed among the following groups: IR vs. control, BAI vs. IR, MEM vs. IR, and MBNPs vs. IR.

[0277] Volcano plot analysis comparing IR versus control groups revealed widespread transcriptional dysregulation in response to ischemia-reperfusion (Fig. 17A). Numerous genes were significantly upregulated, including classic inflammation and immune-related mediators, consistent with the known roles of microglia activation, oxidative stress, and cytokine storm in the pathogenesis of retinal IR injury (Fig. 17B).

[0278] To further interpret the functional significance of the observed transcriptional alterations, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on differentially expressed genes (DEGs) (Figure 17C). A range of signaling pathways were enriched across multiple comparisons, including neuroactive ligand-receptor interactions, calcium signaling pathways, PI3K-Akt signaling pathways, cAMP signaling pathways, cell adhesion molecules, focal adhesion, glutamatergic synapses, and axonal guidance, reflecting molecular-level changes following treatment and consistent with mechanisms of neuroinflammation and retinal damage. These enriched pathways are broadly associated with neuroinflammation, synaptic remodeling, immune signaling, and neurovascular integrity.

[0279] In summary, these transcriptomic data demonstrate that retinal ischemia and subsequent treatment induce extensive molecular changes involving multiple biological networks functionally associated with retinal damage and repair. These findings provide molecular-level insights into potential therapeutic response mechanisms at the transcriptomic level.

[0280] The foregoing description of the invention is for illustrative purposes only and is not intended to be exhaustive or to limit the invention to the exact forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0281] These embodiments were chosen and described in order to best illustrate the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the different embodiments of the invention and to make various adaptive modifications according to the intended specific use.

Claims

1. A nanoplatform comprising an immune cell membrane-coated nanocarrier.

2. The nanoplatform according to claim 1, wherein the immune cell membrane is derived from cells participating in immune responses, inflammatory responses, antigen presentation, or immune regulation in disease states, cells with immune or inflammatory regulatory functions obtained through genetic engineering, bioengineering, or in vitro induction treatment, and cells in a pathologically activated state, or combinations thereof; more specifically, the cells are selected from microglia, macrophages derived from monocytes, dendritic cells, infiltrating leukocytes, Müller glial cells, astrocytes, and retinal pigment epithelial cells; more specifically, the cells are microglia.

3. The nanoplatform according to claim 2, wherein the immune cell membrane is derived from immune cells in retinal tissue; more specifically, the immune cell membrane is derived from microglia in retinal tissue.

4. The nanoplatform according to any one of claims 1 to 3, wherein the immune cell membrane is derived from the same or different sources; or the immune cell membrane is derived from the same or different cell types; or the immune cell membrane coating is single-layered or multi-layered.

5. The nanoplatform according to any one of claims 1 to 4, wherein the cell membrane is designed in a modular and combinable manner.

6. The nanoplatform according to any one of claims 1 to 5, wherein the material of the nanocarrier is selected from biodegradable and / or biocompatible polymers, lipids, proteins, polysaccharides, inorganic or organic-inorganic hybrid materials, more specifically, the material is selected from polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol and its derivatives, chitosan, hyaluronic acid, albumin, liposomes, lipid nanoparticles, metal oxide nanoparticles, silicon-based nanomaterials, and any combination thereof, more specifically, the material is PLGA.

7. A targeted drug delivery system, comprising: Therapeutic agents; as well as The nanoplatform according to any one of claims 1 to 6, The therapeutic agent is encapsulated within a nanoplatform.

8. The targeted drug delivery system of claim 7, wherein the therapeutic agent is a small molecule drug, peptide, nucleic acid, antibody or protein drug; or the therapeutic agent is a drug with dual anti-inflammatory and neuroprotective effects, such as a natural flavonoid drug, such as baicalin.

9. Use of the nanoplatform of any one of claims 1 to 6 or the targeted drug delivery system of any one of claims 7 to 8 in the preparation of a medicament for the prevention or treatment of retinal diseases or neurodegenerative diseases.

10. The use according to claim 9, wherein the retinal disease is a retinal disease involving ischemia-reperfusion (IR) injury; or the retinal disease is a retinal disease involving inflammation; or the retinal disease is selected from ischemic retinal diseases, inflammatory retinal diseases, degenerative retinal diseases, autoimmune or hereditary degenerative retinal diseases, and infectious or traumatic retinopathy; or the retinal disease is selected from retinal ischemia or ischemia-reperfusion injury, retinal vascular occlusion, diabetic or hypertensive retinopathy, age-related macular degeneration and its associated choroidal neovascularization, glaucoma, uveitis, retinitis, retinal vasculitis, and optic neuritis.

11. The use according to claim 9, wherein the neurodegenerative disease is selected from Parkinson's disease (PD), Alzheimer's disease (AD), and multiple sclerosis (MS).

12. Use of the nanoplatform of any one of claims 1 to 6 or the targeted drug delivery system of any one of claims 7 to 8 in the preparation of a medicament for managing neuroinflammation.

13. A method for preparing a nanoplatform according to any one of claims 1 to 6, comprising: Preparation of nanocarriers; Nanocarriers were coated with immune cell membranes.

14. A method for preparing a targeted drug delivery system according to any one of claims 7 to 8, comprising: Preparation of nanocarriers; Therapeutic agents are encapsulated using nanocarriers to obtain nanoparticles loaded with therapeutic agents; Nanoparticles loaded with therapeutic agents are coated with immune cell membranes to obtain immune cell membrane-coated nanoparticles loaded with therapeutic agents, which can be used as a targeted drug delivery system.

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