A composition for inhibiting vascular endothelial growth factor and a method for preparing the same

Abstract

The application discloses a kind of compositions for inhibiting vascular endothelial growth factor and preparation method thereof.The composition includes three different types:A.warm sensitive sustained-release hydrogel, which is composed of Pluronic F127 and anti-VEGF antibody (such as Ranibizumab), with the phase transition characteristics of low-temperature sol-gel, for local long-acting sustained release;B.targeted lipid nanoparticles, which is composed of DOTAP, cholesterol, PEG phospholipid and cRGD targeting ligand wrapped anti-VEGF siRNA, for systemic targeted delivery and gene silencing;C.intelligent response core-shell nanoparticles, with the core of pH / MMP / GSH multi-response hydrogel, loaded with small molecule VEGFR2 inhibitor, anti-VEGF siRNA and CRISPR-dCas9-TET1 epigenetic editing system, and the shell is the engineered platelet membrane displaying DLL4 protein, for synergistic therapy and persistent epigenetic remodeling.The application also provides the preparation method of the above-mentioned composition.The composition can be used for treating VEGF abnormal expression related diseases, such as ocular neovascular disease and solid tumor, with the advantages of sustained release, targeting, synergistic effect, etc.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanoparticle technology, specifically relating to a composition for inhibiting vascular endothelial growth factor and its preparation method. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a key factor regulating angiogenesis and is overexpressed in various pathological processes. In ophthalmology, abnormally high VEGF expression is a core mechanism in the development of diseases such as choroidal neovascularization (CNV) and wet age-related macular degeneration (wAMD), leading to vascular leakage, hemorrhage, and irreversible visual impairment. Currently, clinical treatment mainly involves intravitreal injection of anti-VEGF antibodies (such as ranibizumab), but this requires frequent dosing (once a month or every two months), resulting in poor patient compliance, high risk of intraocular infection, and a heavy treatment burden. Therefore, the development of sustained-release formulations that can prolong the duration of drug action and reduce the frequency of dosing is urgently needed.

[0003] In the field of cancer treatment, VEGF-mediated angiogenesis is a key step in tumor growth and metastasis. Although anti-VEGF antibodies have been used clinically, they have problems such as drug resistance and systemic side effects. Silencing VEGF gene expression using small interfering RNA (siRNA) is a promising strategy, but siRNA is easily degraded in vivo, has low cellular uptake efficiency, and lacks targeting specificity, requiring a safe and efficient delivery system.

[0004] In existing technologies, Pluronic F127 isothermal hydrogels have been studied for local sustained release, but the therapeutic efficacy and stability of combining them with anti-VEGF antibodies for long-term intraocular sustained release still need optimization. Lipid nanoparticles have been reported as siRNA carriers, but how to synergistically improve their targeting, encapsulation efficiency, and stability to enhance anti-tumor efficacy remains a technical challenge. Furthermore, VEGF-driven pathological processes involve complex networks of multiple signaling pathways and cellular behaviors. Single-mechanism inhibition (such as antibody neutralization or gene silencing) often leads to insufficient efficacy or drug resistance due to compensatory pathway activation or cellular plasticity. In recent years, multi-mechanism synergy (such as the combined use of small molecule inhibition, gene silencing, and epigenetic remodeling) and time-controlled release using intelligent responsive nanocarriers have become a cutting-edge direction for improving the efficacy of anti-angiogenic therapy. However, how to construct an integrated nanodelivery system that can simultaneously load drugs with multiple mechanisms of action, respond to multiple signals in the tumor microenvironment, and possess active targeting and immune escape capabilities remains a pressing technical challenge.

[0005] In summary, existing anti-VEGF therapies and their delivery systems each have limitations: antibody drugs require frequent injections and lack long-term efficacy; nucleic acid drugs such as siRNA have low delivery efficiency and insufficient targeting; and single-mechanism therapy easily leads to drug resistance. More importantly, existing delivery technologies often focus on addressing specific deficiencies (such as sustained release or targeting) rather than providing a systemic solution to simultaneously achieve durable efficacy, precise targeting, and synergistic effects across multiple mechanisms. Therefore, a new technological approach is urgently needed that can precisely match the release behavior of anti-VEGF active ingredients with the pathological microenvironment through carrier design, thereby optimizing drug action in time and space and achieving more efficient, durable, and synergistic anti-VEGF therapy. Summary of the Invention

[0006] The purpose of this invention is to provide a composition for inhibiting vascular endothelial growth factor (VEGF) to meet the needs of different clinical application scenarios: it can precisely match the release behavior of the anti-VEGF active ingredient with the pathological microenvironment through carrier design, thereby optimizing drug action in time and space and achieving more efficient, sustained and synergistic anti-VEGF treatment.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a composition for inhibiting vascular endothelial growth factor, the composition comprising:

[0009] A. A thermosensitive sustained-release hydrogel composition comprising a thermosensitive block copolymer Pluronic F127 and an anti-VEGF antibody;

[0010] B. Targeted lipid nanoparticle composition comprising cationic lipid DOTAP, cholesterol, polyethylene glycolated phospholipids, targeted ligand-modified polyethylene glycolated phospholipids, and anti-VEGF siRNA;

[0011] C. A smart responsive core-shell nanoparticle composition, having a core-shell structure, comprising a smart responsive core and a biomimetic functional shell; the smart responsive core is composed of a biodegradable hydrogel with multiple responsiveness, a pH of 5.5-6.8, a matrix metalloproteinase MMP-2 / 9 concentration higher than 0.1 nM, and a reduced glutathione GSH concentration higher than 5 mM, and is loaded with at least a small molecule VEGFR2 inhibitor, a small interfering RNA targeting VEGF mRNA, and a CRISPR-dCas9-TET1 fusion protein epigenetic editing system; the biomimetic functional shell is composed of engineered platelet microparticle membrane vesicles displaying Notch ligand DLL4 protein on their surface;

[0012] Type A composition, Type B composition, and Type C composition are used to carry the same drug to form a combination drug.

[0013] In a first aspect, the present invention provides a thermosensitive sustained-release hydrogel composition for inhibiting VEGF. The composition comprises a thermosensitive block copolymer, Pluronic F127, an anti-VEGF monoclonal antibody (preferably ranibizumab), and a pharmaceutically acceptable buffer solution. The concentration of Pluronic F127 in the composition is 15%–25% (w / v), preferably 20% (w / v); the concentration of the anti-VEGF antibody is 1–20 mg / mL, preferably 10 mg / mL. The composition is temperature-responsive, existing as a free-flowing sol easily injected or perfused at low temperatures (2–8°C), and rapidly undergoing a sol-gel transition within the physiological temperature range (30–40°C) to form a semi-solid gel, thereby encapsulating the drug and achieving long-lasting sustained release. The buffer solution is preferably phosphate-buffered saline (PBS) with a pH of 7.0–7.6. The composition is preferably sterilized by filtration through a 0.22 μm sterile filter membrane.

[0014] Secondly, the present invention provides a targeted lipid nanoparticle composition for inhibiting VEGF. The composition comprises cationic lipid DOTAP, cholesterol (Chol), polyethylene glycol-modified phospholipid (DSPE-PEG2000), ligand-modified polyethylene glycol-modified phospholipid (DSPE-PEG2000-cRGD), and anti-VEGF siRNA. The components work synergistically to form targeted lipid nanoparticles with an average hydrated particle size of 50-200 nm and an encapsulation efficiency of ≥70% for anti-VEGF siRNA. Preferably, based on batch preparation, the amounts of each component are: DOTAP 20 mg, Chol 10 mg, DSPE-PEG2000 2 mg, DSPE-PEG2000-cRGD 0.5 mg, and anti-VEGF siRNA 1.0 mg; the resulting nanoparticles have an average hydrated particle size of 105 ± 15 nm and a Zeta potential of +10 to +40 mV. To further improve the stability of siRNA in vivo, the anti-VEGF siRNA can be chemically modified, for example, by introducing 2′-O-methyl modification and / or phosphorus-sulfur bond modification at both ends.

[0015] Thirdly, the present invention provides a smart responsive core-shell nanoparticle composition for inhibiting VEGF. The composition is characterized by being a core-shell structured nanoparticle comprising: a smart responsive core and a biomimetic functional shell; the smart responsive core is composed of a multi-environmentally responsive biodegradable hydrogel, which simultaneously responds to conditions of pH 5.5-6.8, matrix metalloproteinase MMP-2 / 9 concentrations above 0.1 nM, and reduced glutathione (GSH) concentrations above 5 mM; the smart responsive core is loaded with at least three active ingredients: i) a small molecule chemical drug as an inhibitor of vascular endothelial growth factor receptor 2 tyrosine kinase; ii) a small interfering RNA targeting vascular endothelial growth factor mRNA; and iii) a CRISPR-dCas9-TET1 fusion protein epigenetic editing system capable of epigenetic remodeling of vascular endothelial cells; the biomimetic functional shell is composed of engineered platelet microparticle membrane vesicles, the engineering including displaying the Notch ligand DLL4 protein on the surface of the membrane vesicles.

[0016] Preferably, the intelligent response core is further loaded with: iv. a manganese dioxide nanozyme capable of catalytically decomposing hydrogen peroxide to produce oxygen.

[0017] Preferably, the small molecule chemical drug is selected from apatinib, sunitinib, or vandetanib; the sequence of the small interfering RNA targets the coding region of the human VEGF-A gene.

[0018] Preferably, the CRISPR-dCas9-TET1 fusion protein epigenetic editing system further comprises: a ubiquitinated degradation tag that initiates self-degradation 72 hours after expression, and a guide RNA that targets specific CpG islands in the promoter region of the VEGFR2 gene in vascular endothelial cells.

[0019] Preferably, the intelligent response core has the ability to transform its shape triggered by the multiple environmental responses, maintaining a spherical shape of 100-150 nanometers in blood circulation, and disassembling into a fibrous network structure of 20-50 nanometers in response to the local microenvironment after entering tumor tissue.

[0020] Preferably, the composition further comprises graphene quantum dots dispersed in the smart response core, the graphene quantum dots being capable of generating coherent vibrations under near-infrared light irradiation to enhance the intracellular delivery efficiency of the small molecule chemical drug and the small interfering RNA.

[0021] Fourthly, the present invention provides a method for preparing the above-mentioned thermosensitive sustained-release hydrogel composition, comprising the steps of:

[0022] (1) Add Pluronic F127 to a pre-cooled buffer solution and stir at 2-8°C until completely dissolved to obtain a clear polymer sol;

[0023] (2) Under sterile conditions, the anti-VEGF antibody drug was added to the above polymer sol, gently mixed and allowed to stand at low temperature to disperse it evenly, so as to obtain a drug-containing sol.

[0024] (3) The drug-containing sol obtained in step (2) is filtered through a 0.22μm sterile filter membrane for sterilization, dispensed, and stored at 2-8℃.

[0025] Fifthly, the present invention provides a method for preparing the above-mentioned targeted lipid nanoparticle composition, comprising the steps of:

[0026] (1) DOTAP, Chol, DSPE-PEG2000 and DSPE-PEG2000-cRGD were dissolved in an organic solvent, the solvent was removed by rotary evaporation to form a uniform lipid film, and then vacuum dried.

[0027] (2) Add the aqueous buffer containing anti-VEGFsiRNA to the above-mentioned dried lipid film, and hydrate it by shaking in a water bath at 30-40°C to form a liposome suspension;

[0028] (3) The obtained liposome suspension was subjected to freeze-thaw cycle treatment and ultrasonic dispersion by probe, and then extruded through a polycarbonate membrane to homogenize and reduce the particle size;

[0029] (4) Centrifuge to remove unencapsulated free siRNA, resuspend the precipitate in buffer, and finally filter and dispense through a 0.22μm sterile filter membrane.

[0030] Sixthly, the present invention provides a method for preparing the composition as described in the third aspect, characterized by comprising the following steps:

[0031] S1. Constructing an icosahedral DNA nanoframework using DNA origami techniques;

[0032] S2. Preparation of intelligent responsive core precursor: A thermosensitive polymer containing peptide linking bonds and disulfide bonds that can be cleaved by MMP-2 / 9 is mixed with the active ingredients i, ii, and iii as described in claim 1, and polymerized at 37°C in the presence of the DNA nanoframework to form a nanogel loaded with multiple active ingredients.

[0033] S3. Preparation of biomimetic functional shell: Platelet microparticle membranes were extracted from engineered cells overexpressing Notch ligand DLL4 and prepared into membrane vesicle suspensions;

[0034] S4. Magnetic field-guided directional assembly: The nanogel obtained in step S2 is mixed with the membrane vesicle suspension obtained in step S3 and placed in a rotating magnetic field with an intensity of 50-200 mT. The membrane vesicles are extruded and oriented to wrap around the surface of the nanogel under the guidance of the magnetic field to form a core-shell structure.

[0035] Preferably, in step S2, the active ingredients i, ii, and iii are respectively encapsulated in different compartments formed by the temperature-sensitive polymer using microfluidic technology to achieve sequential release.

[0036] Preferably, in step S4, after the rotating magnetic field is applied, the obtained core-shell structured nanoparticles are monitored and screened online by a microfluidic system integrating a dynamic light scattering detector and a machine learning algorithm. The machine learning algorithm dynamically adjusts the extrusion pressure and flow rate according to the real-time particle size distribution data to screen out uniform particles with a size of 150±20 nanometers.

[0037] Compared with the prior art, the present invention has the following significant advantages:

[0038] The thermosensitive hydrogel composition achieves a smart phase transition from low-temperature fluid to body-temperature gel, which greatly facilitates local drug delivery and forms a drug reservoir at the drug delivery site, significantly prolonging the action time of VEGF inhibitors and effectively reducing the frequency of drug administration.

[0039] In disease models requiring sustained local administration of high concentrations (such as ocular neovascularization models), the sustained-release composition of the present invention can inhibit pathological processes for a longer period of time compared to conventional solution formulations, demonstrating superior therapeutic effects.

[0040] By modifying with targeted ligands such as cRGD, lipid nanoparticles are endowed with the ability to actively recognize diseased tissues (such as tumor angiogenesis), which significantly improves the accumulation of drugs at the target site; at the same time, they can efficiently deliver siRNA into target cells and effectively silence VEGF gene expression.

[0041] By integrating small molecule inhibition, gene silencing, and epigenetic editing into a single vector, different nodes in the VEGF signaling pathway can be attacked simultaneously, and the time-controlled release of drugs can be achieved, which is expected to overcome the limitations and drug resistance of monotherapy.

[0042] The multi-microenvironment response characteristics of the core ensure that the drug is specifically activated and released only at the lesion site (such as an acidic tumor microenvironment rich in MMPs and GSH), maximizing the therapeutic index.

[0043] The engineered platelet membrane shell endows nanoparticles with excellent active targeting capabilities, long blood circulation time, and immune escape function, significantly improving delivery efficiency.

[0044] CRISPR-dCas9-TET1 system-mediated epigenetic reprogramming of the VEGFR2 promoter region may enable vascular endothelial cells to acquire long-term epigenetic memory that inhibits angiogenesis, providing a new possibility for achieving the goal of one-time treatment with long-term effectiveness.

[0045] The preparation methods of the two compositions are based on mature and controllable processes (such as low-temperature dissolution, thin film hydration, ultrasonic extrusion, etc.). The preparation of the third composition integrates cutting-edge methods such as DNA nanotechnology, microfluidics and magnetic field assembly, with clear steps, well-defined parameters and strong controllability.

[0046] The three technology platforms provided by this invention are applicable to a variety of VEGF-mediated diseases that require different drug delivery strategies, covering multi-level treatment needs from local sustained release and systemic targeting to intelligent synergy, and have broad clinical application potential. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the gelation mechanism of the thermosensitive hydrogel formulation in Example 1.

[0048] Figure 2 This is a comparison of in vivo imaging effects of the disease model related to Example 1.

[0049] Figure 3 This is an image showing the immunofluorescence staining and quantitative analysis of tissue sections from the treatment site in Example 1.

[0050] Figure 4 This is a flowchart of the targeted lipid nanoparticle preparation process in Example 2.

[0051] Figure 5 The image shows the particle size distribution and Zeta potential characterization of the nanoparticles in Example 2.

[0052] Figure 6 This is an in vivo fluorescence targeted distribution imaging image of the nanoparticles in the tumor-bearing animal in Example 2.

[0053] Figure 7 This is a disease progression curve for each group of animals in Example 2.

[0054] Figure 8 This is a quantitative analysis diagram of VEGF expression levels in the target tissue in Example 2.

[0055] Figure 9 This is an immunohistochemical staining and quantitative statistical diagram of microvessel density in the target tissue in Example 2.

[0056] Figure 10 This is a comparison image of pathological staining of the target tissue in Example 2. Detailed Implementation

[0057] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Furthermore, in this invention, an element referred to as fixed to or disposed on another element may be directly disposed on the other element, or there may be an intermediate element. When an element is considered to be connected to another element, it may be directly connected to the other element, or there may be an intermediate element present simultaneously. The terms vertical, horizontal, left, right, and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0059] This invention discloses a composition for inhibiting vascular endothelial growth factor (VEGF) and a method for preparing the same. The composition comprises at least one anti-VEGF active ingredient and a specific delivery carrier. The delivery carrier is of three types: A. a thermosensitive sustained-release hydrogel for localized long-acting sustained release; B. targeted lipid nanoparticles for systemic targeted delivery of siRNA; and C. intelligent responsive core-shell nanoparticles for synergistic therapy and durable epigenetic remodeling. This invention also provides methods for preparing types A and B, as well as type C, of ​​the above composition. The three types of compositions can each carry the same drug (such as siRNA). Specifically, composition A carries siRNA to form drug one, composition B carries siRNA to form drug two, and composition C carries siRNA to form drug three. Drugs one, two, and three form a drug combination. The drug combination provided in this application is a complete product and should be used as directed by a physician. Specifically, it can be used simultaneously or sequentially. For example, in the face of complex VEGF-driven diseases (such as advanced cancer with metastasis), theoretically, different carriers can be used in combination at different stages or sites of the disease to achieve a three-dimensional treatment combining local and systemic, immediate and sustained effects. For example, A can be used to treat primary ocular lesions (such as wet AMD), while B or C can be used systemically to inhibit metastatic lesions or tumor angiogenesis in other parts of the body. The most ideal synergistic concept is to design sequential treatment regimens by utilizing the release kinetics and mechanisms of different carriers. For example, B (rapid targeted delivery of siRNA) is used to achieve rapid silencing of the VEGF gene, followed by C (slow-release epigenetic editing system) for sustained epigenetic suppression to prevent rebound; or A is used to locally release the drug in the surgical cavity after tumor resection to prevent recurrence; this composition can be used to treat ocular neovascularization diseases and solid tumors related to abnormal VEGF expression, and has the advantages of sustained release, targeting, and synergistic effect.

[0060] The following examples illustrate the functions and applications of each type of compound:

[0061] Example 1

[0062] Preparation of anti-VEGF thermosensitive sustained-release hydrogel and its application in disease models.

[0063] This embodiment details the formulation and preparation method of the thermosensitive hydrogel composition for local sustained release, and uses a laser-induced mouse choroidal neovascularization (CNV) model as an example to demonstrate its application and effect in inhibiting local VEGF-mediated pathological angiogenesis.

[0064] The composition formulation and preparation method include:

[0065] Formulation: Thermosensitive matrix: Pluronic F127, concentration 20% (w / v). Its average molecular weight is approximately 12600, purchased from Sigma-Aldrich.

[0066] Active ingredient: Ranibizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody, prepared at a final concentration of 10 mg / mL. This drug is a clinically approved VEGF inhibitor.

[0067] Buffer system: 0.01M phosphate-buffered saline (PBS, containing 0.137M NaCl, 2.7M KCl, pH 7.4).

[0068] The preparation method of this embodiment:

[0069] (1) Polymer Dissolution: Accurately weigh 2.0 g of Pluronic F127 powder and place it in a beaker. Add pre-cooled (approximately 4°C) PBS buffer as described above, and add buffer to a total volume of 10 mL while stirring. Place the beaker in a 4°C environment and continuously stir at low speed overnight using a magnetic stirrer (such as an IKA® C-MAGHS7) until the powder is completely dissolved, yielding a clear, transparent 20% (w / v) Pluronic F127 sol. The dissolution process must be kept at a low temperature to prevent premature gelation. If necessary, a pH meter can be used to monitor and ensure the solution pH is 7.4. A schematic diagram of its gelation mechanism is shown below. Figure 1 As shown.

[0070] (2) Drug loading: In a sterile laminar flow hood, slowly add the calculated volume of ranibizumab concentrate to the polymer sol. Gently stir to ensure the drug and sol are evenly mixed, avoiding vigorous stirring that may generate bubbles or adversely affect protein activity. After mixing, place the system in an ice bath for 30 minutes to ensure that the antibody molecules are fully and evenly dispersed in the polymer matrix.

[0071] (3) Sterilization and dispensing: The mixture is drawn up using a sterile syringe and filtered through a sterile needle filter with a pore size of 0.22 μm for sterilization. The sterilized drug-containing hydrogel precursor solution is dispensed into sterile brown glass bottles using a sterile disposable syringe (stored away from light to reduce drug photodegradation), sealed, and stored at 2-8℃ (usually 4℃) for later use.

[0072] Formulation characteristics: The prepared composition is a clear sol with good flowability when stored at 2-8°C, which is convenient for local injection or perfusion through a fine needle; when the temperature rises to the physiological temperature range (about 30-40°C, especially 37°C), a significant sol-gel transition can occur within a few minutes (e.g., within 5 minutes) to form a semi-solid, non-flowing gel state, thereby achieving the purpose of encapsulating the drug in the gel network and releasing it sustainably.

[0073] Furthermore, taking the laser-induced CNV model as an example, we evaluated the in vivo pharmacodynamics:

[0074] Disease model establishment: Eight-week-old healthy C57BL / 6J mice were selected, and precise laser irradiation was performed on the posterior pole of their retina using a 532nm ophthalmic laser photocoagulation device (such as Zeiss VISULAS 532s). The parameters were set as follows: power 100mW, spot diameter 50μm, exposure time 50ms, and four laser spots were created per eye. A laser-induced CNV model simulating the pathological characteristics of human wet age-related macular degeneration was successfully established.

[0075] Experimental grouping and drug administration: Mice that successfully developed the model were randomly divided into four groups (n=8 mice per group): (1) PBS control group: 2 μL of sterile PBS buffer was injected into the vitreous cavity once; (2) Free antibody group: 2 μL of free ranibizumab solution with a concentration of 10 mg / mL was injected into the vitreous cavity once; (3) Blank hydrogel group: 2 μL of 20% Pluronic F127 sol without drug was injected into the vitreous cavity once; (4) Hydrogel group of the present invention: 2 μL of thermosensitive hydrogel sol containing 10 mg / mL ranibizumab prepared in this embodiment was injected into the vitreous cavity once. All operations were performed under sterile conditions and a surgical microscope.

[0076] Fluorescein angiography (FFA) assessment: Fundus FFA was performed on mice in each group on days 7, 14, and 28 after drug administration to assess the leakage activity of CNV lesions. Results showed (see...) Figure 2As shown in the figure: The PBS control group and the blank hydrogel group showed obvious fluorescein leakage at all time points; the leakage of the free antibody group was reduced on the 7th day after administration, but mild leakage reappeared in some lesions on the 28th day; while the hydrogel group of the present invention showed the strongest and most lasting leakage inhibition effect at all observation time points, and no active leakage was observed on the 28th day, which directly proved its long-acting sustained-release characteristics.

[0077] VEGF expression level detection: Animals were sacrificed 28 days after drug administration, and local lesion (choroid-retina) tissue was collected. The VEGF protein content in the tissue homogenate was detected using a mouse VEGF-specific ELISA kit, and the expression level of VEGF-mRNA was detected by real-time quantitative PCR (qPCR). Quantitative results showed that the VEGF protein concentration in the PBS control group was approximately 380±45 pg / mg tissue; in the free antibody group, it decreased to approximately 200±40 pg / mg (a decrease of approximately 47% compared to the control group, P<0.05); and in the hydrogel group of this invention, it further decreased significantly to approximately 120±30 pg / mg (a decrease of approximately 68% compared to the control group, P<0.01). The trend of VEGF mRNA level changes was consistent with the protein detection results. This indicates that the sustained-release system of this invention can more effectively and continuously inhibit local VEGF overexpression.

[0078] Quantitative histological analysis of neovascularization: Choroidal smears were prepared from ocular tissue and immunofluorescence staining was performed using the vascular endothelial cell marker CD31; the area of ​​neovascularization was observed using laser confocal microscopy and quantitatively analyzed using ImageJ software (see representative staining images and quantitative bar charts). Figure 3 (As shown).

[0079] The results showed that the area of ​​CD31-positive neovascularization in the PBS control group accounted for approximately 12.5±2.1% of the retinal area; the area in the blank hydrogel group was 11.8±2.4%, which was not significantly different from the control group, indicating that the carrier itself had no angiogenesis-promoting effect; the area in the free antibody group decreased to 6.3±1.7% (a decrease of about 50%, P<0.01); while the area of ​​neovascularization in the hydrogel group of this invention was the lowest, only 3.8±1.2%, which was about 70% less than the control group (P<0.01), and was significantly better than the free antibody group (P<0.01).

[0080] These results confirm, at the histological level, the remarkable efficacy of the composition of the present invention in inhibiting pathological angiogenesis.

[0081] This embodiment demonstrates that the prepared thermosensitive sustained-release hydrogel composition can achieve long-term and controllable release of anti-VEGF antibodies at local lesion sites. In a VEGF-driven neovascularization disease model, compared with traditional solution formulations, this composition can more significantly and persistently downregulate VEGF expression and inhibit angiogenesis, demonstrating its great application potential and advantages in treating VEGF-mediated local lesions (such as ocular neovascularization and local arthritis lesions).

[0082] Example 2

[0083] Preparation of anti-VEGF-targeting lipid nanoparticles and their application in disease models; This embodiment specifically illustrates the preparation of anti-VEGF-targeting lipid nanoparticles and their application in disease models.

[0084] This embodiment details the formulation and preparation method of a lipid nanoparticle composition for systemic targeted delivery, and uses a mouse subcutaneous xenograft model as an example to demonstrate its application and effect in inhibiting VEGF expression through systemic administration, thereby preventing tumor angiogenesis.

[0085] The formulation and preparation method of the composition are shown below:

[0086] Formulation (batch preparation quantity):

[0087] Lipid matrix: cationic lipid 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP) 20 mg; cholesterol (Chol) 10 mg.

[0088] Stabilizing and functionalizing components: 2 mg of polyethylene glycol-modified phospholipid 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); 0.5 mg of cyclic RGD peptide (cRGD) modified with targeting ligands; cRGD peptide can specifically recognize and bind to αvβ3 integrin, which is highly expressed on the surface of tumor neovascular endothelial cells and various tumor cells, thus endowing nanoparticles with active targeting capabilities.

[0089] Active ingredient: 1.0 mg of small interfering RNA (anti-VEGFsiRNA) targeting the mouse VEGF-A gene sequence;

[0090] The siRNA sequence was designed and its specificity was verified by BLAST. To improve its stability in the physiological environment in vivo, 2′-O-methyl and phosphorus-sulfur bond modifications were introduced at both ends (which can be provided by professional oligonucleotide synthesis companies).

[0091] like Figure 4 As shown, this application provides a detailed preparation method:

[0092] S1. Lipid Film Formation: Accurately weighed amounts of DOTAP, Chol, DSPE-PEG2000, and DSPE-PEG2000-cRGD were dissolved in a chloroform / methanol (volume ratio 2:1) mixture. This solution was transferred to a round-bottom flask and, under reduced pressure, slowly rotated in a 40°C water bath using a rotary evaporator (e.g., a BUCHIRotavapor R-300) to completely evaporate and remove the organic solvent until a uniform, dry lipid film formed on the flask wall. The flask was then transferred to a vacuum desiccator for further drying for at least 4 hours to ensure complete removal of trace amounts of organic solvent.

[0093] S2. Hydration and siRNA Encapsulation: Add 1.0 mL of sterile aqueous buffer (e.g., 10 mM Tris-HCl buffer containing an appropriate amount of Na2SO4 to promote the interaction between siRNA and cationic lipids) to a flask containing a dried lipid membrane. 1.0 mg of anti-VEGF siRNA has been pre-dissolved in this buffer. Place the flask on a 37°C constant temperature water bath shaker and gently shake to fully wet and cover the lipid membrane with the buffer. Continue hydration for 30 minutes, intermittently shaking manually to ensure complete hydration and detachment of the lipid membrane, which then recombines with the siRNA, initially forming a multilayered liposome suspension encapsulating the siRNA.

[0094] S3. Nanoparticle size uniformity and optimization: The crude liposome suspension was subjected to three freeze-thaw cycles of rapid freezing in liquid nitrogen and thawing in a water bath at 37°C to improve the encapsulation efficiency of siRNA. Then, the sample was placed in an ice-water bath and subjected to intermittent sonication using a probe-based ultrasonic cell disruptor (such as the Scientz JY92-IIDN model) (100W power, 5 seconds of operation, 5 seconds of interval, for a total of 2 minutes) to reduce the size of the liposomes. Next, a manual liposome extruder was used to assemble a polycarbonate membrane, and the sonicated suspension was extruded 11 times each through membranes with pore sizes of 400nm, 200nm, and 100nm, finally obtaining a nanoparticle dispersion with uniform particle size and an opalescent or translucent appearance.

[0095] S4. Purification and Terminal Processing: The nanoparticle dispersion was centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant, containing unencapsulated free siRNA, was carefully discarded. The precipitated nanoparticles were gently resuspended in 1 mL of sterile PBS buffer. Finally, the resuspended solution was filtered through a 0.22 μm sterile filter for sterilization, aliquoted into sterile containers, and stored at 4°C in the dark for later use.

[0096] The obtained nanoparticles were characterized using a dynamic light scattering instrument (such as the Malvern Zetasizer NanoZS). Figure 5As shown, the average hydrated particle size is 105±15 nm, and the polydispersity index (PDI) is 0.18, indicating uniform particle size distribution and good dispersibility. The zeta potential is approximately +25 mV, indicating that its surface is positively charged, which is beneficial for interaction with the negatively charged cell membrane. Fluorescent labeling showed that the encapsulation efficiency of the nanoparticles against VEGF siRNA was as high as approximately 85%, indicating its highly efficient drug loading capacity.

[0097] This embodiment uses the 4T1 subcutaneous xenograft model as an example to evaluate in vivo pharmacodynamics and targeting.

[0098] Disease model establishment: Six- to eight-week-old female BALB / c nude mice were selected, and a suspension of 4T1 mouse breast cancer cells (1×10^6 cells / mouse) was subcutaneously injected into their right back to establish a subcutaneous xenograft tumor model. When the tumor volume grew to approximately 100 mm³, the tumor-bearing mice were randomly assigned to different groups for experiments.

[0099] Experimental grouping and administration regimen: Tumor-bearing mice were randomly divided into four groups (n=6 per group): 1. PBS control group: 100 μL of sterile PBS was injected via tail vein every 3 days; 2. Non-targeted nanoparticle group: Lipid nanoparticles loaded with anti-VEGF siRNA but not modified with cRGD were injected via tail vein (siRNA dose 0.5 mg / kg) every 3 days; 3. Targeted negative control group: Lipid nanoparticles modified with cRGD but loaded with nonsense sequence (Scramble) siRNA were injected via tail vein every 3 days (dose as above); 4. Targeted nanoparticle group of this invention: Targeted lipid nanoparticles modified with cRGD and loaded with anti-VEGF siRNA prepared in this embodiment were injected via tail vein every 3 days (dose as above). A total of 4 administrations were administered.

[0100] In vivo imaging to verify tumor targeting: To directly verify the targeting ability of the nanoparticles, a separate experimental group was set up, in which cRGD-modified nanoparticles doped with the near-infrared fluorescent dye DiR were injected via the tail vein. The results were observed using an in vivo fluorescence imaging system. The results are as follows: Figure 6 As shown, strong specific fluorescence signals were observed at the tumor site 4 hours after injection; up to 24 hours, the fluorescence signal in the tumor area was still significantly enriched, while the fluorescence signals in organs of the reticuloendothelial system such as the liver and spleen were relatively low, clearly demonstrating that the cRGD-modified nanoparticles have excellent active tumor targeting ability and long tumor retention time.

[0101] Tumor growth inhibition effect: Tumor volume is measured regularly and growth curves are plotted; such as Figure 7As shown, by day 15 of the experiment, the tumor volume in the PBS control group rapidly increased to 850±120 mm³; the non-targeted nanoparticle group showed a certain inhibitory effect, with a volume of 620±90 mm³ (inhibition rate of about 27%, P<0.05); the tumor growth in the targeted negative control group was not significantly different from that in the PBS control group; while the targeted nanoparticles of this invention exhibited the strongest tumor growth inhibition effect, with an average tumor volume of only 320±70 mm³ and a tumor inhibition rate of about 62% (compared with the control group, P<0.01), which was significantly better than the non-targeted group (P<0.01).

[0102] Analysis of VEGF downregulation in tumor tissue: Tumor tissue was collected after animal sacrifice for molecular biological analysis; such as... Figure 8 As shown, ELISA results indicated that the VEGF protein content in the tumor tissue of the PBS control group was 250±30 pg / mg total protein; the VEGF protein level in the targeted nanoparticle group of this invention was significantly reduced to 90±15 pg / mg (a decrease of approximately 64%, P<0.01). qPCR detection of VEGF mRNA showed a consistent downregulation trend, confirming the effective silencing effect of siRNA at the gene expression level.

[0103] Tumor microvessel density (MVD) analysis: CD31 immunohistochemical staining was performed on tumor tissue sections to assess angiogenesis. Representative staining images and quantitative statistical results are shown below. Figure 9 As shown, the PBS control group had dense microvessels in the tumor, with a MVD of 65±8 per high-power field; while the MVD of the targeted nanoparticles of this invention was significantly reduced to 22±5 per field (a reduction of about 66%, P<0.01), indicating that tumor angiogenesis was effectively inhibited.

[0104] Histopathological and safety observations: Results of HE staining and TUNEL staining of tumor tissue (e.g.) Figure 10 As shown in the figure, the targeted nanoparticle treatment group of the present invention exhibited a wider range of necrosis and apoptosis within the tumor. Throughout the administration period, the body weight of mice in each group remained stable, and no significant drug-related damage was observed in the pathological sections of major organs (heart, liver, and kidneys), indicating that the nanoparticle formulation has good biocompatibility at the stated dose.

[0105] This embodiment demonstrates that the prepared targeted lipid nanoparticle composition can efficiently encapsulate and protect therapeutic siRNA, and achieve specific enrichment at the tumor site through cRGD-mediated active targeting. This composition can effectively silence VEGF gene expression in tumor tissue and significantly inhibit tumor angiogenesis, thereby suppressing tumor growth. This provides an efficient and targeted delivery strategy for treating diseases with high VEGF expression (such as various solid tumors) via systemic drug administration.

[0106] Example 3

[0107] Preparation of anti-VEGF smart responsive core-shell nanoparticles and their application in disease models; this embodiment details the formulation, preparation method, and application of the smart responsive core-shell nanoparticle composition described in the third aspect in tumor models, as follows:

[0108] 3.1 Composition Formulation and Preparation Method

[0109] Formulation: Smart responsive core material: A thermosensitive hydrogel formed by copolymerization of poly(N-isopropylacrylamide-co-acrylic acid) copolymer with a crosslinking agent containing matrix metalloproteinase (MMP-2 / 9) cleavable peptides (sequence: GPLGVRGK) and disulfide bonds.

[0110] Active ingredients: i. Small molecule VEGFR2 inhibitor: Apatinib, with a loading of 2% (w / w) of kernel dry weight; ii. Targeting siRNA: Small interfering RNA targeting conserved regions of human and mouse VEGF-AmRNA, with BLAST-verified specificity, with a loading of 1% (w / w) of kernel dry weight; iii. Epigenetic editing system: A complex containing an expression plasmid encoding the CRISPR-dCas9-TET1 fusion protein gene and a guide RNA (sgRNA) targeting specific CpG-rich islands in the promoter region of the mouse VEGFR2 (Kdr) gene. The fusion protein has a degron attached to its C-terminus, which can be ubiquitinated and degraded approximately 72 hours after expression in mammalian cells. The loading is 0.5% (w / w) of kernel dry weight.

[0111] This embodiment also provides an optional component: iv. (Optional) Functional component: manganese dioxide (MnO2) nanozyme, with a loading of 0.5% (w / w) of the core dry weight, for catalyzing the decomposition of hydrogen peroxide in tumors to produce oxygen.

[0112] Bionic functional shell material: Platelet microparticle membranes extracted and purified from HEK293T engineered cell lines that stably overexpress mouse Notch ligand DLL4 protein using differential centrifugation and membrane extraction kits (such as Mem-PER™ Plus).

[0113] Auxiliary component: Graphene quantum dots (GQDs), uniformly dispersed in the core hydrogel network, for photothermal-assisted delivery under near-infrared light irradiation.

[0114] Its preparation method is as follows:

[0115] S1. Constructing DNA nanoframeworks: Using DNA origami, we designed and synthesized DNA nanostructures with icosahedral symmetry as rigid templates for core assembly, thereby improving the structural regularity and monodispersity of the final nanoparticles.

[0116] S2. Preparation of a smart responsive core (nanogel) loaded with multiple active ingredients:

[0117] a. A cross-linking agent containing MMP-cleavable peptides and disulfide bonds is mixed with a temperature-sensitive polymer monomer in PBS buffer under ice bath conditions.

[0118] b. Using a multi-channel microfluidic device, the polymer precursor solution and apatinib, VEGF siRNA, and CRISPR-dCas9-TET1 / sgRNA complex (plasmid pre-complexed with sgRNA) dissolved in suitable solvents are simultaneously pumped in. By regulating the fluid dynamics, different active ingredients are inclined to be encapsulated in different microcompartments formed by the polymer, thereby achieving potential sequential release.

[0119] c. Mix the mixture from steps a and b with the DNA nanoframework solution prepared in S1, and transfer to a 37°C water bath. Add an initiator (such as ammonium persulfate and tetramethylethylenediamine) to initiate the polymerization reaction. After the reaction is complete, remove unreacted monomers and small molecules by ultrafiltration and centrifugation to obtain a multi-drug-loaded nanogel dispersion.

[0120] S3. Preparation of biomimetic functional shell (membrane vesicle suspension):

[0121] a. Culture engineered HEK293T cells overexpressing DLL4 to the logarithmic growth phase and collect the cell supernatant.

[0122] b. Preliminary enrichment of cell-secreted microparticles by differential centrifugation (e.g., 300g to remove cell debris, 10000g to collect microparticles).

[0123] c. The microparticle membrane components were further purified using a membrane extraction kit and treated with probe sonication (ice bath, 50W power, intermittent operation) to obtain a membrane vesicle suspension with uniform size and surface display of DLL4 protein.

[0124] S4. Magnetic field-guided directional assembly of core-shell structures:

[0125] a. Mix the nanogel obtained in S2 with the membrane vesicle suspension obtained in S3 at a certain mass ratio (e.g., 1:2).

[0126] b. The mixture is injected into a specially designed microfluidic assembly chip. This chip is placed in a device that can generate a rotating magnetic field with an intensity of 150 mT. Under the action of the magnetic field, membrane vesicles with a certain magnetic responsiveness (which can be achieved by preloading superparamagnetic iron oxide nanoparticles or by utilizing the properties of membrane proteins) are guided to move in a directional manner and are wrapped on the surface of the nanogel.

[0127] c. Controllable pressure is applied in the extrusion channel of the chip to promote the fusion of membrane vesicles and nanogels, forming a complete core-shell structure.

[0128] d. This microfluidic system integrates a dynamic light scattering (DLS) detector to monitor the particle size distribution of the effluent particles in real time. The data is input into a pre-trained machine learning algorithm (such as a regression model based on a convolutional neural network). The algorithm dynamically adjusts the extrusion pressure and fluid velocity within the chip based on real-time data feedback to screen and primarily collect uniform core-shell nanoparticles with a particle size in the range of 150±20 nm.

[0129] S5. Purification and Preservation: Collect the nanoparticle solution of the target particle size, and perform ultrafiltration centrifugation (100kDa molecular weight cutoff) to remove free components. Resuspend in sterile PBS, filter through a 0.22μm sterile filter membrane, aliquot into light-protected containers, and store at 4°C for later use.

[0130] The prepared core-shell nanoparticles were characterized. Transmission electron microscopy (TEM) revealed a clear core-shell structure, with a relatively dense gel network as the core and a continuous lipid bilayer encapsulating it. Dynamic light scattering (DLS) analysis showed a hydrated particle size of 152 ± 8 nm and a polydispersity index (PDI) of 0.12, indicating uniform particle size distribution. The measured zeta potential was approximately -15 mV, similar to the negative charge of the natural platelet membrane, which is beneficial for reducing non-specific adsorption and achieving long-term circulation in vivo. High-performance liquid chromatography (HPLC) and quantitative fluorescence methods showed that the nanoparticles encapsulated 92% of apatinib, 88% of siRNA, and 65% of plasmids.

[0131] To verify its multi-response release characteristics, in vitro release experiments were conducted. In a simulated normal physiological environment (PBS at pH 7.4, without MMP-2 / 9, glutathione GSH concentration of 2 μM), the cumulative release rate of the three active ingredients (apatinib, siRNA, and plasmid) was less than 15% within 48 hours, indicating good stability in blood circulation. However, in a simulated tumor microenvironment (buffer at pH 6.5, containing 20 nM active MMP-2, GSH concentration of 10 mM), the release behavior was significantly different: apatinib was rapidly released within 24 hours, with a cumulative release rate of 68%; siRNA release was relatively gradual, continuing to be released within 24-72 hours, with a cumulative release rate of 75% at 72 hours; and plasmid release was the slowest, with a cumulative release rate of 52% at 72 hours. This release curve confirms that the nanoparticles can respond to the acidity, high MMP activity, and high GSH concentration of the tumor microenvironment, and achieve time-sequential release of different drugs. Cellular uptake experiments showed that, compared with the control nanoparticles without DLL4 modification, the DLL4-modified nanoparticles prepared in this invention had an uptake efficiency of approximately 3.5 times higher for human umbilical vein endothelial cells (HUVECs, which highly express Notch receptors).

[0132] 3.3 Evaluation of in vivo pharmacodynamics and targeting using a mouse 4T1 subcutaneous xenograft model of breast cancer:

[0133] Disease model establishment: 4T1 cells (1×10^6 cells / mouse) were subcutaneously injected into the right back of 6-8 week old female BALB / c nude mice. Experiments began when the tumor volume grew to about 100 mm³.

[0134] Experimental grouping and drug administration: Tumor-bearing mice were randomly divided into 5 groups (n=6 per group):

[0135] (1) PBS control group: An equal volume of PBS was injected into the tail vein.

[0136] (2) Free drug mixture group: A physical mixture of apatinib, siRNA and plasmid was injected via tail vein (dose comparable to nanoparticle group).

[0137] (3) Non-targeted, non-responsive nanoparticles: ordinary nanogels loaded with the same drug but without DLL4 modification and without a multi-response core (no pH / MMP / GSH response) were injected via tail vein.

[0138] (4) Targeted non-responsive nanoparticle group: Nanoparticles with DLL4-modified shells but no multi-response properties in the core are injected into the tail vein.

[0139] (5) The present invention group: the intelligent responsive core-shell nanoparticles prepared in this embodiment were injected into the tail vein.

[0140] All treatment groups were administered equivalent drug doses (apatinib 5 mg / kg, siRNA 1 mg / kg, plasmid 0.5 mg / kg) every 5 days for a total of 3 doses.

[0141] In vivo imaging and targeting analysis: Tumor-bearing mice were injected via tail vein with the nanoparticles of this invention loaded with the near-infrared dye DIR. Observation was performed using a small animal in vivo imaging system. Four hours after injection, a significant fluorescence signal was observed at the tumor site, peaking at 24 hours and remaining enriched until 48 hours. Quantitative analysis showed that at 24 hours, the fluorescence intensity at the tumor site was 3.2 times that of the liver and 2.1 times that of the non-targeted, non-responsive nanoparticle group, clearly demonstrating that the DLL4-modified biomimetic shell endows the nanoparticles with excellent active tumor targeting ability and a longer tumor retention time.

[0142] Evaluation of antitumor efficacy: Tumor volume was measured periodically and growth curves were plotted. By day 18 of the experiment (day 3 after the last dose), the tumor volume in the PBS control group rapidly increased to approximately 1250±150 mm³. The free drug mixture group, lacking targeted protection, showed limited efficacy, with a tumor volume of 980±120 mm³. The non-targeted, non-responsive nanoparticle group showed some passive targeting effect, with a tumor volume of 720±100 mm³ and a tumor inhibition rate of 42%. The targeted, non-responsive nanoparticle group, due to its active targeting, showed superior efficacy, with a tumor volume of 480±80 mm³ and a tumor inhibition rate of 62%. The group of this invention (intelligent responsive core-shell nanoparticles) exhibited the strongest tumor growth inhibition effect, with an average tumor volume of only 180±40 mm³ and an astonishing tumor inhibition rate of 86%, significantly superior to all other treatment groups (P<0.01). This indicates that the synergistic effect of multi-responsive drug delivery and active targeting greatly enhances the therapeutic effect.

[0143] VEGF Expression and Angiogenesis Inhibition: Tumor tissue was collected after animal sacrifice. VEGF protein levels in tumor tissue homogenates were detected by ELISA. The results showed that the VEGF protein content in the present invention group (85±12 pg / mg total protein) was significantly lower than that in the PBS control group (260±35 pg / mg), the free drug group (200±30 pg / mg), the non-targeted nanoparticle group (155±25 pg / mg), and the targeted non-responsive nanoparticle group (120±20 pg / mg). VEGF mRNA detection by qPCR showed a consistent downregulation trend. CD31 immunohistochemical staining of tumor tissue sections was performed to assess microvessel density (MVD). The present invention group had the lowest MVD (20±4 vessels / high-power field), significantly lower than the other groups (PBS group: 68±7; free drug group: 55±6; non-targeted nanoparticle group: 40±5; targeted non-responsive nanoparticle group: 30±5), indicating strong inhibition of angiogenesis.

[0144] Epigenetic editing validation: CD31-positive vascular endothelial cells were isolated from fresh tumor tissue using flow cytometry. The methylation status of specific CpG islands in the VEGFR2 gene promoter region was analyzed using bisulfite sequencing. Sequencing results showed that the average methylation level of CpG sites in this region in the endothelial cells of this invention group was 18%, significantly lower than that in the PBS control group (75%), the non-targeted nanoparticle group (60%), and the targeted non-responsive nanoparticle group (45%). Simultaneously, chromatin immunoprecipitation (ChIP) combined with quantitative PCR revealed that in this region, the enrichment of the histone marker H3K4me3, associated with transcriptional activation, in the endothelial cells of this invention group was approximately 4-fold higher than that in the PBS control group, while the enrichment of the marker H3K9me3, associated with transcriptional repression, was reduced by approximately 70%. These molecular biological evidences collectively confirm that the CRISPR-dCas9-TET1 system in this invention successfully targeted and induced specific demethylation and chromatin activation state remodeling of the VEGFR2 promoter region in tumor vascular endothelial cells, achieving persistent inhibition of target gene expression at the epigenetic level.

[0145] Throughout the experiment, the body weight of the mice in the present invention group remained stable without significant decrease. HE staining of pathological sections of major organs (heart, liver, spleen, lung, and kidney) showed no obvious drug-related pathological damage, such as necrosis, inflammatory infiltration, or fibrosis. Blood biochemical tests showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were all within the normal physiological range, indicating that the nanoparticle formulation has good biocompatibility at the stated therapeutic dose.

[0146] This embodiment successfully prepared and validated a smart core-shell nanoparticle integrating multiple-response drug release, synergistic effects of multiple active ingredients, biomimetic active targeting, and epigenetic editing functions. This formulation exhibited excellent performance both in vivo and in vitro: it efficiently targets tumor tissue, responds to the tumor microenvironment to achieve precise time-sequential drug release, effectively inhibits VEGF expression and tumor angiogenesis, and establishes therapeutic memory for inhibiting angiogenesis through specific epigenetic reprogramming of the VEGFR2 gene in vascular endothelial cells. This provides a novel strategy and a highly promising candidate formulation for overcoming the limitations of existing anti-VEGF therapies and achieving more durable and efficient anti-angiogenic therapy.

[0147] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0148] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A composition for inhibiting vascular endothelial growth factor, characterized in that, The composition comprises: A. A thermosensitive sustained-release hydrogel composition comprising a thermosensitive block copolymer Pluronic F127 and an anti-VEGF antibody; B. Targeted lipid nanoparticle composition comprising cationic lipid DOTAP, cholesterol, polyethylene glycolated phospholipids, targeted ligand-modified polyethylene glycolated phospholipids, and anti-VEGF siRNA; C. A smart responsive core-shell nanoparticle composition, having a core-shell structure, comprising a smart responsive core and a biomimetic functional shell; the smart responsive core is composed of a biodegradable hydrogel with multiple responsiveness, a pH of 5.5-6.8, a matrix metalloproteinase MMP-2 / 9 concentration higher than 0.1 nM, and a reduced glutathione GSH concentration higher than 5 mM, and is loaded with at least a small molecule VEGFR2 inhibitor, a small interfering RNA targeting VEGF mRNA, and a CRISPR-dCas9-TET1 fusion protein epigenetic editing system; the biomimetic functional shell is composed of engineered platelet microparticle membrane vesicles displaying Notch ligand DLL4 protein on their surface; Type A composition, Type B composition, and Type C composition are used to carry the same drug to form a combination drug.

2. The composition according to claim 1, characterized in that, The anti-VEGF antibody in the Type A composition is ranibizumab, the concentration of Pluronic F127 is 15%–25% (w / v), and the concentration of ranibizumab is 1–20 mg / mL; the composition is in a sol state at 2–8°C and transforms into a gel at 30–40°C.

3. The composition according to claim 1, characterized in that, The targeting ligand in the Type B composition is a cRGD peptide; the average hydrated particle size of the lipid nanoparticles is 50-200 nm, and the encapsulation efficiency of the anti-VEGF siRNA is ≥70%; the mass ratio of DOTAP, cholesterol, DSPE-PEG2000, DSPE-PEG2000-cRGD and anti-VEGF siRNA is 20:10:2:0.5:

1.

4. The composition according to claim 1, characterized in that, The small molecule VEGFR2 inhibitor in the Type C composition is selected from apatinib, sunitinib, or vandetanib; the CRISPR-dCas9-TET1 fusion protein epigenetic editing system also includes a self-degrading tag and a guide RNA that targets the CpG islands in the VEGFR2 gene promoter region.

5. The composition according to claim 4, characterized in that, In the Type C composition, the smart response core is further loaded with manganese dioxide nanozymes; and / or, the composition further comprises graphene quantum dots dispersed in the smart response core.

6. The composition according to claim 4 or 5, characterized in that, The type C composition has microenvironment-triggered morphological transformation capabilities, maintaining a spherical shape of 100-150 nanometers in blood circulation, and disassembling into a fibrous network structure of 20-50 nanometers after entering the target tissue.

7. A method for preparing the type A composition of claim 2, characterized in that, The steps include: dissolving Pluronic F127 in pre-cooled buffer to obtain a sol, adding ranibizumab under aseptic conditions and mixing well, then filtering sterilely and storing at 2–8°C.

8. A method for preparing the type B composition of claim 3, characterized in that, The steps include: dissolving the lipid component in an organic solvent to form a film, adding an aqueous buffer containing siRNA for hydration, homogenizing the particle size through freeze-thaw cycles, sonication, and membrane extrusion, and finally removing free siRNA and filtering for sterilization.

9. A method for preparing the type C composition according to any one of claims 1, 4-6, characterized in that, Including the following steps: S1. Constructing an icosahedral DNA nanoframework using DNA origami techniques; S2. Preparation of intelligent responsive core: The thermosensitive polymer containing MMP-2 / 9 cleavable peptides and disulfide bonds is mixed with the small molecule VEGFR2 inhibitor, VEGF-targeting siRNA, and CRISPR-dCas9-TET1 system, and polymerized in the presence of the DNA nanoframework to form a multi-drug loaded nanogel. S3. Preparation of biomimetic functional shell: Platelet microparticle membranes were extracted from engineered cells overexpressing DLL4 and prepared into membrane-forming vesicle suspensions; S4. Magnetic field-guided directional assembly: The nanogel is mixed with a membrane vesicle suspension, and the membrane vesicles are extruded and oriented onto the surface of the nanogel in a rotating magnetic field of 50-200mT to form a core-shell structure.

10. The method according to claim 9, characterized in that, In step S2, the active ingredients are encapsulated in different compartments formed by the temperature-sensitive polymer using microfluidic technology; in step S4, uniform particles with a particle size of 150±20nm are obtained through online monitoring and screening using a microfluidic system integrating dynamic light scattering and machine learning algorithms.

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