Multi-targeting nanogel medicine and application thereof in relieving neuroinflammation and promoting spinal cord injury repair

Abstract

The invention relates to a multi-targeting nanogel drug and application thereof in relieving neuroinflammation and promoting spinal cord injury repair, the multi-targeting nanogel drug comprises quercetin-loaded liposome, the quercetin-loaded liposome comprises TAT peptide and CAQK peptide, the quercetin-loaded liposome is incorporated into double-network gel, and the double-network gel is used as a drug carrier. The dual-network gel is jointly formed by a dopamine grafted hyaluronic acid polymer and methacrylated gelatin. The multi-targeting nanogel drug can improve the dissolving property of quercetin, reduce toxic and side effects, promote targeting of an injured part, and contribute to passing through a blood spinal cord barrier, so that the integrity of the blood spinal cord barrier is protected, the spinal cord microenvironment is optimized, formation of glial scars is reduced, axon regeneration is promoted, and the recovery of a motor function is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a multi-targeted nanogel drug and its application in alleviating neuroinflammation and promoting spinal cord injury repair. Background Art

[0002] Spinal cord injury (SCI) can cause permanent damage to sensory, motor and autonomic nerves, resulting in lifelong paralysis. Primary SCI leads to vascular rupture, tissue ischemia and vasogenic edema. Within minutes, local microglia are recruited to the injured area, and these cells are activated into multiple phenotypes to regulate neuroinflammation. In addition, persistent inflammatory cell infiltration leads to microenvironmental destabilization, cystic cavities and neuronal death. Therefore, the inflammatory response is a core pathological process in SCI and plays a vital role in clinical prognosis. Effective control of secondary inflammatory responses can help stabilize the spinal cord lesion microenvironment, promote axon growth and improve injury repair.

[0003] Quercetin (Que), as a flavonoid, exhibits significant anti-inflammatory and antioxidant effects in the treatment of central nervous system injury. It has the effects of promoting microglia polarization to M2 type, regulating the microenvironment, protecting neurons, and enhancing the motor function of SCI mice, which indicates that quercetin has important application potential in the treatment of SCI. However, the effectiveness of quercetin in the treatment of SCI is often limited by poor solubility, low bioavailability, significant side effects, and the unique pathophysiological environment of the spinal cord.

[0004] A variety of drug delivery systems have been developed to enhance the solubility of low water-soluble compounds. Liposomal nanoparticles have unique advantages in enhancing drug solubility and have been widely used for drug encapsulation and delivery. However, they face some limitations in terms of effective delivery and targeted drug accumulation in vivo. For example, the blood-spinal cord barrier, a unique barrier of the spinal cord, prevents liposomes from entering the site of spinal cord injury. Studies have shown that surface modification of nanoparticles can significantly improve the permeability of drugs, and transcription-activating peptides (TAT) containing positively charged protein transduction domains can promote transmembrane penetration by interacting with negatively charged cell membranes. In addition, environmental factors of spinal cord injury (MMPs, CSPGs, ROS, etc.) have become effective targets for targeted therapy after SCI. Previous studies have shown that CAQK peptides can achieve precise targeted therapy by recognizing cartilage sulfate proteoglycans (CSPGs) that are secreted in large quantities in the damaged spinal cord area. The inclusion of membrane penetration and targeting functional molecules in nanoparticles is conducive to improving the therapeutic effect of drugs.

[0005] However, nanodrug formulations are adversely affected by poor specific distribution and insufficient drug accumulation during intravenous administration. In situ drug delivery at the SCI site can significantly reduce the systemic toxicity of liposomes and maximize the effectiveness of drugs. Due to the tunability, adhesion and excellent responsiveness of hydrogels, they have been widely used for in situ drug delivery in SCI. Various adhesive hydrogels are designed to achieve target adhesion, prevent premature drug loss, and improve therapeutic effects. Hydrogels containing nanomaterials can also enhance their drug loading capacity and functionality. The combination of transmembrane targeted liposomes and adhesive hydrogels is beneficial to maximize the therapeutic effect of drugs and exert multiple repair effects.

[0006] Although quercetin (Que) has the effects of promoting immune polarization and reducing inflammation, its low solubility, obvious side effects, and unique pathophysiological characteristics of the spinal cord limit its application. Therefore, how to design a multi-targeted nanogel delivery system for quercetin to enhance the solubility of quercetin, reduce toxic side effects, and promote targeting of injury sites is still a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0007] In order to solve the defects of the prior art, the present invention proposes a multi-targeted nanogel drug and its application in alleviating neuroinflammation and promoting spinal cord injury repair. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0008] On the one hand, the present invention relates to a multi-targeted nanogel drug, which includes quercetin-loaded liposomes, which have TAT peptides and CAQK peptides, and the quercetin-loaded liposomes are incorporated into a double network gel, and the double network gel is formed by dopamine-grafted hyaluronic acid polymers and methacryloyl gelatin. The present invention adopts a multi-targeting method to enhance the effectiveness of quercetin-loaded liposomes in regulating inflammatory responses and improving SCI repair. Liposome encapsulation improves the solubility of quercetin. By utilizing the interaction of TAT peptides with negatively charged cell membranes to promote transmembrane penetration, and the specific affinity of CAQK peptides to CSPGs secreted in large quantities at the SCI site, TAT peptides and CAQK peptides are grafted onto liposomes (LipTC) at the same time to promote drug crossing the blood-spinal cord barrier and targeted delivery to the damaged site. The drug-loaded liposomes (QLipTC) were further incorporated into a double network gel (HGM) formed by dopamine-grafted hyaluronic acid polymer (HADA) and methacryloyl gelatin (GelMA). By in situ administration at the SCI site, the therapeutic effect of quercetin can be more fully exerted. The results showed that QLipTC@HDM can effectively exert the anti-inflammatory effect of quercetin and promote SCI repair. This study shows that LipTC@HDM can become an effective drug delivery system for the treatment of SCI and be used in the biomedical field.

[0009] In a preferred embodiment of the present invention, the quercetin-loaded liposomes are made of soybean lecithin, cholesterol, DSPE-PEG, DSPE-PEG-TAT, DSPE-PEG-CAQK and quercetin.

[0010] In a preferred embodiment of the present invention, the molar ratio of soybean lecithin, cholesterol, DSPE-PEG, DSPE-PEG-TAT, and DSPE-PEG-CAQK in the quercetin-loaded liposomes is 60-80:20-30:8-12:4-6:4-6.

[0011] In a preferred embodiment of the present invention, the average particle size of the quercetin-loaded liposomes is 100-200 nm.

[0012] In a preferred embodiment of the present invention, the surface potential of the quercetin-loaded liposomes is a positive potential of 20 mV or more.

[0013] In a preferred embodiment of the present invention, the dopamine-grafted hyaluronic acid polymer is formed by cross-linking hyaluronic acid and dopamine.

[0014] In a preferred embodiment of the present invention, the double network gel is formed by cross-linking dopamine-grafted hyaluronic acid polymer and methacryloyl gelatin under 405nm light; preferably, the mass ratio of the dopamine-grafted hyaluronic acid polymer to the methacryloyl gelatin is 1-2:1-2.

[0015] Another aspect of the present invention also relates to the application of the above-mentioned multi-targeted nanogel drug, which is used for preparing a drug for treating spinal cord injury; preferably, the drug is used to relieve neuroinflammation and promote spinal cord injury repair.

[0016] Beneficial Effects

[0017] The present invention designs a novel multifunctional liposome gel delivery system (QLipTC@HDM), which is obtained by combining liposomes with the properties of penetrating the blood-spinal cord barrier and targeting the injury site with a double network adhesive gel (HDM). Experimental results show that encapsulating quercetin in LipTC can enhance its solubility, reduce quercetin toxicity, promote injury site targeting, and help cross the blood-spinal cord barrier. In addition, encapsulation in HDM significantly prolongs the retention time of QLipTC at the injury site after local administration. Crucially, the results show that QLipTC@HDM induces the transformation of microglia at the injury site of spinal cord injured mice to the M2 phenotype and reduces the inflammatory response. This intervention also protects the integrity of the blood-spinal cord barrier, optimizes the spinal cord microenvironment, reduces glial scar formation, promotes axonal regeneration, and enhances the recovery of motor function in SCI mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 . Schematic diagram of the preparation of transmembrane targeted nanogel drug delivery system (QLipTC@HDM) and its application in spinal cord injury repair. (A) Schematic diagram of transmembrane targeted nanodrug (QLipTC). (B) Synthesis route of dopamine-grafted hyaluronic acid (HADA). (C) Synthesis and dual network schematic diagram of multi-targeted nanogel drug (QLipTC@HDM). (D) Application of QLipTC@HDM in spinal cord injury repair, using HDM to prolong the retention time of drugs in the injured area, and using LipTC to promote the drug to cross the blood-spinal cord barrier to achieve targeted retention in the injured area, thereby exerting a strong anti-inflammatory effect and promoting nerve repair.

[0019] Figure 2 Preparation and characterization of QlipTC. (A) DSPE-PEG-CAQK, DSPE-PEG-TAT and DSPE-PEG-MAL 1H-NMR spectra. (B) FTIR spectra of DSPE-PEG-CAQK, DSPE-PEG-TAT and DSPE-PEG-MAL. (C) Transmission electron microscopy images of QLipTC, as well as its hydrodynamic radius and polydispersity index. (D) Statistical analysis of the zeta potential of Lip, QLip, QLipC, QLipT and QLipTC. (E) Particle size measurement of QLipTC for 21 consecutive days. (F) Encapsulation efficiency of quercetin in QLip and QLipTC. (G) Evaluation of cell viability of BV2 cells after 24 h of treatment with different concentrations of Que or QLipTC using the CCK-8 kit. (H) Fluorescence intensity analysis of FITC-labeled liposomes (FITC-Lip, FITC-LipC, FITC-LipT and FITC-LipTC) crossing from the upper chamber of Transwell to the lower chamber. (I) Flow cytometry analysis of the fluorescence intensity of FITC-labeled liposomes passing through the upper chamber of Transwell and entering the lower chamber of cells. (J) Statistical analysis of intracellular fluorescence intensity. (K) Representative fluorescence images of cells in which FITC-Lip, FITC-LipC, FITC-LipT or FITC-LipTC entered the lower chamber through the upper chamber of Transwell. The scale bar is 50 μm. (L) Statistical analysis of fluorescence intensity. * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0020] Figure 3 Preparation and characterization of QlipTC@HDM. (A) DA, HA and HADA 1 H-NMR spectra. (B) UV absorption spectra of DA, HA and HADA. (C) SEM images of GelMA, HADA and HDM. Scale bar is 100 μm. (D) Release curves of DiI in LipTC, LipTC@GelMA and LipTC@HDM. (E) Rheological properties of HDM. (F) Evaluation of biological tissue adhesion performance of HDM. (G) Gel was administered in situ after spinal cord injury in mice, and in vivo fluorescence imaging was detected at 6, 12, 24, 48 and 72 hours by small animal imaging system, and fluorescence imaging of isolated spinal cord was detected at 72 hours. (H) Statistical analysis of fluorescence intensity at the site of spinal cord injury. (I) Quantitative analysis of fluorescence intensity of isolated spinal cord tissue. (J) Survival rate of PC12 cells 24, 48 and 72 hours after treatment with each group of materials. (K) Survival rate of BV2 cells 24, 48 and 72 hours after treatment with each group of materials. (L) Calcein-AM / PI staining results of PC12 cells co-cultured with each group of materials for 24, 48 and 72 hours, scale bar is 50 μm. ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001.

[0021] Figure 4 Effects of QLipTC@HDM on BV2 cell viability and inflammation. (A,B) After 3 hours of OGD / R treatment, BV2 cells were treated with different concentrations of QLipTC and QLipTC@HDM for 24 hours, 48 ​​hours, and 72 hours for cell viability analysis. (C,D) After 3 hours of OGD / R treatment, BV2 cells were treated with different concentrations of QLipTC, and ELISA was used to analyze the expression levels of IL-6 and IL-10 in BV2 cells 24 hours later. (E,F) After 3 hours of OGD / R treatment, BV2 cells were treated with different concentrations of QLipTC@HDM, and ELISA was used to analyze the expression levels of IL-6 and IL-10 in BV2 cells 24 hours later. All experiments were repeated three times. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0022] Figure 5 .QLipTC@HDM promotes the transformation of microglia to M2 phenotype and inhibits inflammatory response. (A) Western blot analysis of IL-6, TNF-α, CD86 and Arginase protein expression in each group of cells. (BE) Quantitative analysis of IL-6, TNF-α, CD86 and Arginase protein levels. (FH) ELISA results show the concentrations of IL-6, TNF-α and IL-10 in BV-2 cells in each group. (I) Immunofluorescence images of CD86 protein and CD206 protein in BV2 cells in each group after treatment with different methods for 24 hours. Scale bar is 50μm. (J) Quantitative analysis of CD86 protein fluorescence intensity in cells in each group. (K) Quantitative analysis of CD206 protein fluorescence intensity in cells in each group. (L) Flow cytometry analysis of CD11b / CD86 positive cells and CD11b / CD206 positive cells. (M, N) Quantitative analysis of the proportion of CD86-positive cells in the CD11b-positive cell population and CD206-positive cells in the CD11b-positive cell population. * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0023] Figure 6 .QLipTC@HDM reduces the levels of inflammatory factors in the injured area of ​​SCI mice. (A, B, C) ELISA was used to quantitatively analyze the expression levels of IL-6, TNF-α, and IL-10 in the injured area of ​​the spinal cord 5 days after spinal cord injury. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0024] Figure 7.QLipTC@HDM promotes the transformation of microglia to M2 phenotype after spinal cord injury in mice and inhibits inflammatory response. (A) Western blot analysis of IL-6, TNF-α, CD86 and Arginase protein expression in the injured tissue 5 days after spinal cord injury. (BE) Quantitative analysis of IL-6, TNF-α, CD86 and Arginase protein levels. (FH) ELISA analysis of IL-6, TNF-α and IL-10 protein concentrations in mouse spinal cord tissue. (I) Immunofluorescence images of CD86 (red), Arg1 (green) and DAPI (blue) in spinal cord tissue. Scale bars are 500 μm (left) and 50 μm (right). Quantitative analysis of the mean fluorescence intensity of CD86 and Arg1 in the spinal cord injury core (J, K) and injury edge (L, M). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0025] Figure 8 .QLipTC@HDM improves the microenvironment of spinal cord injury lesions, reduces neuronal death, and inhibits glial scar formation. (A) Representative images of spinal cord tissue obtained 1 hour after injection of Evans blue (EB) dye via tail vein 24 hours after spinal cord injury. (B) Quantitative analysis of Evans blue content in spinal cord tissue. (C) Representative confocal images of spinal cord tissue obtained 1 hour after injection of EB dye via tail vein. Scale bar, 500 μm. (D) Western blot analysis of BAX, Bcl-2, MBP, Tuj1, NF200, and GFAP protein expression in tissues 28 days after spinal cord injury. (EJ) Quantitative analysis of BAX, Bcl-2, MBP, Tuj-1, NF200, and GFAP protein levels in each group. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0026] Fig. 9 .QLipTC@HDM improves damaged nerves and reduces glial scars. (A) Immunofluorescence images of MBP (red), NF200 (green), and DAPI (blue) in spinal cord tissue 28 days after SCI. The scale bars are 500 μm (left) and 50 μm (right). (B, C) Quantitative analysis of the mean fluorescence intensity of MBP and NF200 proteins. (D) Immunofluorescence images of GFAP (red), Tuj1 (green), and DAPI (blue) in spinal cord tissue 28 days after SCI. The scale bars are 500 μm (left) and 50 μm (right), respectively. (E, F) Quantitative analysis of the mean fluorescence intensity of GFAP and Tuj1. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0027] Fig.10 .QLipTC@HDM promotes motor function recovery and improves injured tissue morphology in mice after spinal cord injury. (A) BMS score of mice. (B) Representative footprint images of mice. (C) Representative images of hindlimb movement of mice. (D) Representative images of MEPs in gastrocnemius and hindlimb of mice. (E) Quantitative analysis of muscle weight. (F) Quantitative analysis of MEPs amplitude. (G) HE and Nissl staining images of spinal cord tissue 28 days after SCI. Scale bar is 500 μm. (H) Quantitative analysis of cavity area based on HE staining. (I) Representative images of bladder tissue 28 days after SCI. (J) Quantitative analysis of bladder tissue weight. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0028] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0030] Example 1

[0031] according to Figure 1 According to the scheme shown, a multifunctional liposome gel drug delivery system (QLipTC@HDM) was designed and synthesized to achieve the above objectives.

[0032] 1. Experimental methods

[0033] 1.1 Materials

[0034] Methacryloyl gelatin (GelMA) was purchased from Suzhou Yongquan Intelligent Equipment Co., Ltd. Quercetin (Que) and Evans blue were purchased from Sigma-Aldrich (Missouri, USA); hyaluronic acid (HA) was purchased from Rhawn (Shanghai, China). Dopamine (DA), phospholipids, and cholesterol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. DSPE-PEG3.4K, DSPE-PEG3.4K-Mal, and DSPE-PEG-FITC were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Peptides TAT ​​and CAQK were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. CSPGs (CC117) were purchased from Millipore (Massachusetts, USA). Enhanced CCK-8 reagent was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. DMEM, fetal bovine serum, penicillin-streptomycin solution, and 0.25% trypsin-EDTA were purchased from Gibco (New York, USA). Mouse TNF-a ELISA kit, mouse IL-10 ELISA kit, and mouse IL-6 ELISA kit were purchased from Beijing Solebold Technology Co., Ltd.

[0035] 1.2 Preparation and characterization of DSPE-PEG-TAT and DSPE-PEG-CAQK

[0036] DSPE-PEG-TAT and DSPE-PEG-CAQK were synthesized by the thiol-maleamide coupling method. TAT or CAQK was mixed with DSPE-PEG-Mal and reacted for 24 hours under nitrogen protection. The product mixture was dialyzed (MWCO: 3500) to eliminate any unreacted components and then frozen. The lyophilized product was dissolved in deuterated dimethyl sulfoxide and obtained using an Oxford NMRAS400 spectrometer. 1 H-NMR spectra. Infrared spectra were obtained using a Fourier transform infrared spectrometer.

[0037] 1.3 Synthesis and characterization of transmembrane targeted drug-loaded liposomes (QLipTC)

[0038] The preparation of QLipTC involves a solvent evaporation method. Soy lecithin, cholesterol, DSPE-PEG, DSPE-PEG-TAT, DSPE-PEG-CAQK (molar mass ratio of 60:20:8:4:4) and quercetin were mixed and dissolved in anhydrous ethanol. The mixture was then slowly injected into PBS at 50°C and the ethanol was evaporated while stirring. QLipTC was prepared by ultrasonic treatment (300 watts, 3 seconds on / 5 seconds off) for 5 minutes using a cell disruptor (JY92-IIN, Scientz) and then extruded through a 0.22 μm pore size membrane. To prepare fluorescent liposomes (FITC-LipTC), DSPE-PEG-FITC was used instead of DSPE-PEG. After high-speed centrifugation (12,000 rpm, 20 minutes) of the supernatant, the fluorescence intensity was detected using a multifunctional microplate reader to calculate the loading rate and encapsulation efficiency of quercetin. The encapsulation rate was calculated as (total quercetin content - free quercetin content) / total quercetin content, and the drug loading rate was calculated as (total quercetin content - free quercetin content) / liposome content. The particle size and zeta potential of the liposomes were evaluated by a laser nanoparticle size analyzer, and the stability of the liposomes was evaluated by monitoring the particle size and zeta potential during storage. Morphological analysis was performed using a transmission electron microscope (JEM 1200EX, JEOL). The liposomes were deposited on a 200-mesh copper grid, stained with 2% phosphotungstic acid for 10 minutes, and then observed under a transmission electron microscope.

[0039] 1.4 Synthesis and characterization of HDM and QLipTC@HDM

[0040] Double network gel (HDM) was formed by cross-linking and grafting of HADA and GelMA. HADA was cross-linked by hyaluronic acid (HA) and dopamine (DA). HA (c[COOH] = 3 mmol), EDC (1 mmol), NHS (1 mmol) and dopamine hydrochloride (3 mmol) were dissolved in MES buffer and reacted under nitrogen protection for 48 h. The product mixture was dialyzed through a dialysis bag with a MWCO of 10,000 and then freeze-dried. The synthesized HADA was characterized by nuclear magnetic resonance and ultraviolet spectroscopy. HDM was prepared by mixing 20% ​​HADA with 20% GelMA (1:2 volume ratio) and cross-linking with 405 nm light. QLipTC@HDM was obtained by incorporating QLipTC into the pre-gel solution of HDM. The microstructure of HDM was examined using a scanning electron microscope (Hitachi S-3000N), and the adhesion strength of HDM was analyzed.

[0041] 1.5 Barrier penetration and targeted detection

[0042] The barrier permeability of LipTC was evaluated in an in vitro blood-spinal cord barrier model using the Transwell system. Human umbilical vein endothelial cells (HUVECs) were cultured at 1x10 5 The cells were seeded in the wells of the Transwell at a density of 100 μL and cultured for 3 days to establish a tight monolayer. The bottom layer of the well plate was treated with chondroitin sulfate proteoglycans (CSPGs) for adhesion, and then 700 μL of culture medium was added. Then liposomes loaded with FITC were added to the upper chamber, and the fluorescence intensity of FITC in the lower chamber was measured using a multifunctional microplate reader after 3 hours. In addition, HUVEC cells were cultured in the upper chamber and SHSY5Y cells were cultured in the lower chamber. After adhesion, FITC-Lip was added to the upper chamber, and the SHSY5Y cells in the lower chamber were imaged using a laser confocal microscope (Leica STELLARIS 5). Image J software was used to quantify the FITC fluorescence intensity in the cell membrane. Subsequently, the SHSY5Y cells were trypsinized, and the FITC fluorescence was analyzed by flow cytometry to determine whether the liposomes passed through the double cell membrane and entered the cells in the lower chamber. The retention performance of LipTC@HDM in mice after SCI was evaluated using an in vivo small animal imaging system. ICG@GelMA, ICG@HDM, ICG-Lip@HDM and ICG-LipTC@HDM were administered after SCI, and in vivo fluorescence imaging (PerkinElmer IVIS Lumina XRMS Series III) was performed at 6, 12, 24, 48 and 72 hours. Ex vivo imaging of the spinal cord was performed after 72 hours.

[0043] 1.6 Drug release experiment

[0044] To evaluate the drug release profile, a fluorescent substance (DiI) was used instead of Que. Briefly, 2 μg of DiI was encapsulated into LipTC, LipTC@GelMA, and LipTC@HDM to prepare DiI-LipTC, DiI-LipTC@GelMA, and DiI-LipTC@HDM formulations. These samples were placed in plates containing 3 ml of PBS at 37°C. 300 μl of release medium was extracted for each sampling point, and 300 μl of new PBS was supplemented after each collection time point, which were 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, and 21 days. The fluorescence intensity of DiI was measured using a fluorescent microplate reader, and the cumulative release on the plate was calculated.

[0045] 1.7 Cell viability assay

[0046] Cell viability was assessed using a CCK-8 kit to determine the cytotoxicity of various components of Que and LipTC@HDM on PC12 and BV2 cells. Cell suspensions (1x 10^5 cells / mL) were seeded in 96-well plates and co-cultured with drugs for 24, 48, and 72 hours. Subsequently, the cell culture medium was replaced with a 10% CCK-8 solution, and the absorbance was measured at 450nm after 2 hours of incubation. In addition, Calcein-AM / PI staining was used to evaluate the effects of drugs on the active base morphology of PC12 and BV2 cells. Cells were cultured in 12-well plates for 24, 48, and 72 hours, then stained with Calcein-AM / PI, and images were acquired using a DMILLED inverted fluorescence microscope (Leica).

[0047] 1.8 Spinal cord injury model and treatment

[0048] One hundred and fifty female C57BL6 mice weighing between 18 and 20 g were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. and maintained in the Animal Center of Cixi Biomedical Research Institute, Wenzhou Medical University. The experimental protocol and animal handling procedures were carried out in accordance with the guidelines of the Experimental Animal Ethics Committee of Wenzhou Medical University. The mice were divided into five groups: sham operation (laminectomy only without spinal cord injury), spinal cord injury (SCI), SCI+HDM, SCI+QLipTC, and SCI+QLipTC@HDM. Before surgery, mice were fasted overnight and then anesthetized with 1% sodium pentobarbital (125 μL / 20 g). Using a precision spinal cord impactor (YHCI99, Reward Life Science Co., Ltd.), spinal cord injury was induced by impacting the spinal cord at a depth of 0.8 mm, a speed of 3.5 m / s, and a dwell time of 50 ms. After SCI, mice in the SCI group received 0.9% NaCl as a control. In contrast, the HDM group and the QLipTC@HDM group received treatment with 30 μL of pre-gel solution containing HDM and QLipTC@HDM, followed by irradiation with 405 nm light for 30 seconds to promote gelation, and the QLipTC group was administered by tail vein injection. The concentration of Que in the QLipTC and QLipTC@HDM groups was divided into three dose levels: low dose (250 μg / kg), medium dose (500 μg / kg) and high dose (1 mg / kg). After administration, the muscles and skin were sutured and maintained at 37°C until recovery. Intramuscular penicillin was given for the first three days after surgery. Bladder emptying was performed daily until spontaneous urination function was restored.

[0049] 1.9 Functional Behavioral Assessment

[0050] The Basso Mouse Scale (BMS) scoring system was used to evaluate the motor function of mice after SCI. Two observers evaluated the hindlimb motor function scores on days 1, 3, 7, 14, 21, and 28 after SCI. On day 28, the hindlimb gait was analyzed using the footprint analysis method. The mice were placed in a narrow lane (0.02m wide x 0.5m long) with their forelimbs marked with blue ink and their hindlimbs marked with red ink, and the hindlimb movements were recorded on day 28. Subsequently, neuroelectrophysiological testing was used to evaluate motor evoked potentials (MEPs). The mice were anesthetized before the examination, and after the electrodes were implanted, the spinal cord was stimulated and the MEP values ​​were recorded to reflect the ability to recover activities. Muscle and urinary function were further evaluated by analyzing the size and weight of the hindlimb gastrocnemius muscle and bladder.

[0051] 1.10 Immunoblotting

[0052] Western blot experiments were performed to quantify proteins. Protein concentrations in cells or spinal cord tissues were determined using a BCA protein assay kit (BioVision, P0010). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Then, the cells were incubated overnight with specific antibodies, including TNF-α antibody (Proteintech, 26405-1-AP), IL-6 antibody (Proteintech, 00097020), Arginase antibody (ZEN BIO, KK0819), CD86 antibody (Abcam, ab239075), β-actin antibody (ZEN BIO, KK1231), MBP antibody (Abcam, ab7349), Tuj1 antibody (Abcam, ab18207), Bax antibody (Proteintech, 50599-2-Ig), Bcl-2 antibody (Proteintech, 00113524), GFAP antibody (Affinity, DF6040) and NF200 antibody (Abcam, ab4680). After overnight incubation, the cells were reacted with secondary antibodies for 1 hour. The gray value of the target band was quantified using a chemiluminescence kit (ZEN BIO, 17047) and Image J.

[0053] 1.11 Immunofluorescence staining

[0054] Spinal cord tissues were embedded in paraffin and cut into 5 mm thick sections. Tissue sections were stained with HE and Nissl staining reagents and then examined using an optical microscope (Nikon, Japan). Cells and tissue sections were pretreated with 5% BSA solution for 1 hour and then incubated with primary antibodies at 4°C overnight, including CD86 antibody (1:500, Abcam, ad239075, for marking M1 microglia), CD206 antibody (1:50, Santa Cruz, sc-58986, for marking M2 microglia), GFAP antibody (1:200, Thermo Fisher Scientific, for marking GFP-expressing neural stem / progenitor cells), βIII tubulin antibody (Tuj1, 1:200, CST, for marking immature and mature neurons), NF200 antibody (1:5000, Abcam, for marking axonal neurons) and MBP antibody (1:200, Abcam, for marking myelin basic protein). Subsequently, the sections were incubated with Alexa 488, 594, or 647-labeled secondary antibodies (1:1000, Abcam) for 1 h at 37° C. Finally, the sections were mounted with DAPI-containing mounting solution (Solarbio, Beijing, China), and images were captured using a confocal laser microscope.

[0055] 1.12 Statistical analysis

[0056] Quantitative experimental results are expressed as mean ± standard deviation (SD). One-way ANOVA was used for significance assessment, followed by Tukey's multiple comparison test. GraphPad Prism software was used for inter-group comparisons, and statistical significance was defined as a p value less than 0.05. The experiment was repeated three times, and representative results are presented.

[0057] 2. Results and Discussion

[0058] 2.1 Preparation and characterization of barrier-penetrating targeted liposomes (QLipTC)

[0059] Liposomes are often used as drug carriers, but when used for spinal cord injury repair, their delivery is often limited by the blood-spinal cord barrier. In this study, barrier-penetrating targeted liposomes (QLipTC) were synthesized by solvent evaporation method, aiming to enhance the ability of drug delivery to the site of spinal cord injury. DSPE-PEG-TAT and DSPE-PEG-CAQK lipid materials were synthesized using the thiolmaleimide coupling method, which gave them barrier-penetrating and targeting capabilities. 1 In the H-NMR spectrum, there is no maleamide peak at 6.77 ppm ( Figure 2A), while the continued presence of the characteristic peaks of DSPE and PEG at 1.25ppm, 0.88ppm and 3.65ppm indicated successful coupling. The FTIR spectrum showed the disappearance of the maleamide peak at 1707cm^-1 and the presence of the amide peak at 1664cm^-1, further confirming the synthesis ( Figure 2 B). These lipids were combined with cholesterol, phospholipids and quercetin (Que) to prepare quercetin-loaded barrier-penetrating QLipTC. These liposomes were spherical in morphology with an average particle size of 160.142±3.25nm and a polydispersity index (PDI) of 0.194±0.512. It is worth noting that QLipTC was positively charged (29.44±2.03mV), while QLip was -10.37±2.03mV. The grafting of the membrane-penetrating targeting peptide enhanced the affinity with the negatively charged cell membrane and promoted cellular uptake ( Figure 2 D). The 21-day stability test showed that the particle size changed little ( Figure 2 E), indicating good stability. The encapsulation efficiency of quercetin was as high as 91.63±1.51% ( Figure 2 F). Toxicity tests using BV2 and PC12 cells showed that quercetin at concentrations exceeding 200 μmol / L was cytotoxic and poorly dispersed in the culture medium ( Figure 2 G). However, QLipTC encapsulated 200 μmol / L quercetin improved drug solubility and reduced cytotoxicity, enhancing cell survival ( Figure 2 G).

[0060] The barrier penetration and targeting effects of QLipTC were further tested in an in vitro blood-spinal cord barrier model. Fluorescence measurement showed that the FITC-LipTC group exhibited the strongest intensity, indicating that it has good barrier crossing ability ( Figure 2 H). SHSY5Y cell uptake experiments confirmed that the accumulation of fluorescence in the FITC-LipTC group was higher than that in the other groups, which was confirmed by flow cytometry and confocal microscopy ( Figure 2 IL).

[0061] 2.2 Preparation and characterization of LipTC@HDM

[0062] Encapsulating liposomes in a sticky gel can enhance their retention time at the site of injury and prolong the therapeutic effect. In this study, HADA sticky polymers and GelMA were cross-linked to form a double network sticky gel (HDM). 1 H-NMR spectrum showed that there was a hydrogen absorption peak of DA benzene ring between 6.8 and 7.2 ppm ( Figure 3 A), and UV-visible spectrophotometry detected the absorption peak of HADA at 280 nm ( Figure 3B), confirming the formation of HADA. Scanning electron microscopy (SEM) examination showed that HDM had a smaller pore size ( Figure 3 C), which is crucial for sustained release of drugs. To evaluate the effect of LipTC@HDM on drug release, DiI was loaded into liposomes as a drug. Release studies showed that LipTC released 91%±1.74 in 7 days, LipTC@GelMA released 85.63%±2.44 in 12 days, and LipTC@HDM released 82.57%±5.33 in 16 days, indicating that HDM has an enhanced ability to release drugs continuously ( Figure 3 D). Rheological tests showed that HDM formed a stable gel in the frequency range of 0-1rad / s ( Figure 3 E). Adhesion tests showed that HDM can effectively adhere to organs and spinal cord tissue in vitro ( Figure 3 F). Small animal fluorescence imaging showed that the ICG-LipTC@HDM group maintained strong fluorescence at the injured site for up to 72 hours, which was significantly better than the ICG-GelMA group, which had almost no detectable fluorescence at 72 hours. This may be due to the weak adhesion of GelMA, which caused transfer and drug diffusion after in vivo administration. In the in vitro imaging of spinal cord tissue, it was observed that the fluorescence intensity of ICG-LipTC@HDM was the highest in the injured tissue area, further confirming that HDM has excellent adhesion, retention and ability to reduce drug diffusion both in vivo and in vitro. Biocompatibility assessment showed that GelMA, HADA, HDM and QLipTC@HDM did not significantly change the activity and morphology of PC12 and BV2 cells ( Figure 3 JL). Overall, these findings suggest that QLipTC@HDM has good drug sustained release, effective retention at the injury site, and excellent biocompatibility, and has great potential for application in the treatment of spinal cord injury.

[0063] 2.3QLipTC@HDM promotes microglia polarization toward M2 phenotype and inhibits inflammatory response

[0064] Activated M2 subtype macrophages are known to inhibit inflammatory responses. Before evaluating the effect of QLipTC@HDM on BV-2 cell polarization, the concentration of quercetin at the cellular level was first examined. The study found that when the loading concentration of quercetin in QLipTC@HDM was 50μM, it had the most significant effect on enhancing cell survival under oxygen glucose deprivation / reperfusion (OGD / R) conditions ( Figure 4A, B). It is noteworthy that the best effect of increasing cell activity was observed in the QLipTC group when the quercetin loading concentration was 30 μM, which may be attributed to the sustained release characteristics of QLipTC after being encapsulated in HDM. The anti-inflammatory effect of QLipTC@HDM was then evaluated using an ELISA kit, and the results showed that IL-6 expression was significantly reduced and IL-10 expression was increased at 50 μM ( Figure 4 CF). Therefore, a concentration of 50 μM was selected for further cell studies. To elucidate the effect of QLipTC@HDM on microglial polarization, Western blot analysis and ELISA assays were used. The results showed that QLipTC@HDM significantly reduced the levels of M1 pro-inflammatory markers (CD86, IL-6, and TNF-α) induced by OGD / R stimulation, while enhancing the expression of M2 anti-inflammatory markers (IL-10 and Arginase) ( Figure 5 AH). Immunofluorescence analysis of CD86 and CD206 ( Figure 5 IK) and flow cytometry data ( Figure 5 These findings were further supported by the results of the QLipTC@HDM group (Figure 5A), which showed that the fluorescence intensity of CD86 was significantly reduced in the QLipTC@HDM group, while CD206 showed the highest fluorescence intensity. These results suggest that QLipTC@HDM treatment promotes the polarization of microglia toward the M2 phenotype and inhibits the polarization toward the M1 type.

[0065] 2.4QLipTC@HDM promotes the polarization of microglia to M2 phenotype and alleviates inflammatory response after spinal cord injury in mice

[0066] To determine the inhibitory effect of QLipTC@HDM on inflammatory response after SCI, the effects of different concentrations (250 μg / kg, 500 μg / kg, and 1 mg / kg) of quercetin were first evaluated at the animal level. Five days after SCI, inflammatory factors at the injury site were detected. The results showed that when the quercetin loading concentration was 500 μg / kg, the expression levels of IL-6 and TNF-α were the lowest, while the expression level of IL-10 was the highest ( Figure 6 AC). Subsequently, QLipTC@HDM loaded with 500 μg / kg quercetin was used in animal experiments. Western blot and ELISA assays showed that the QLipTC@HDM treatment group significantly reduced the levels of IL-6, TNF-α, and CD86 compared with the QLipTC group, while the levels of IL-10 and Arginase increased significantly (P<0.01)( Figure 7In addition, compared with the SCI group, the immunofluorescence intensity of the M1 microglial marker (CD86) in the injury center was significantly decreased, while the immunofluorescence intensity of the M2 microglial marker (Arg1) was significantly increased ( Figure 7 IM). The CD86 fluorescence intensity at the injury edge in the QLipTC@HDM-treated group was also significantly lower than that in the SCI group. Taken together, these findings suggest that QLipTC@HDM treatment can promote the transformation of microglia to the M2 phenotype after spinal cord injury in mice and alleviate the inflammatory response.

[0067] 2.5QLipTC@HDM improved the microenvironment of the spinal cord injury site after spinal cord injury in mice, reduced neuronal death and inhibited glial scar formation

[0068] The acute phase inflammatory response leads to increased blood-spinal cord barrier permeability, which damages the spinal cord environment and causes neuronal death. Evaluation of changes in blood-spinal cord barrier permeability showed that the spinal cord injury group had the highest Evans blue content at the spinal cord injury site, which was significantly reduced after QLipTC@HDM intervention ( Figure 8 AC). Western blot protein detection of the injured tissue showed that QLipTC@HDM treatment significantly reduced the level of pro-apoptotic protein (Bax) and increased the expression of anti-apoptotic protein (Bcl-2) ( Figure 8 DF). In addition, the expression of neuronal axon-related proteins (Tuj1, NF200) and myelin protein (MBP) increased significantly, while the expression of astrocyte marker protein (GFAP) decreased ( Figure 8 D, GJ). Immunofluorescence staining analysis showed that the fluorescence of NF200 and MBP was weak in the spinal cord injury group and HDM group, the fluorescence in the QLipTC group was enhanced, and the fluorescence in the QLipTC@HDM group was the strongest ( Fig. 9 AC).

[0069] To assess neural growth and glial scar formation, co-staining of GFAP and Tuj1 was performed ( Fig. 9 DF). The results showed that the fluorescence intensity of GFAP was higher in the SCI and HDM groups, accompanied by a decrease in Tuj1 fluorescence. After QLipTC@HDM treatment, the GFAP fluorescence level decreased, while the Tuj1 fluorescence intensity increased. Taken together, these findings suggest that QLipTC@HDM intervention improved blood-spinal cord barrier permeability, alleviated neuronal death, inhibited glial scar formation, and promoted axonal neuronal growth after SCI in mice.

[0070] 2.6QLipTC@HDM enhances motor function recovery in mice after spinal cord injury

[0071] The level of motor function and spinal cord tissue morphology were assessed in each group of mice. Fig.10 A shows that the BMS motor function score of the QLipTC@HDM-treated group increased significantly compared with the SCI group, and the flexibility, strength and coordination of the hind limb joints were better improved, which was also reflected in the hind limb footprint and joint activity assessment ( Fig.10 B and C). Neurophysiological tests and muscle analysis to evaluate motor evoked potentials (MEPs) and muscle function showed that QLipTC@HDM treatment was able to effectively alleviate muscle atrophy and had higher MEPs, showing significant recovery compared with the SCI group, which showed a decrease in muscle weight and a significant decrease in MEP amplitude ( Fig.10 DF). HE staining and Nissl staining were used to analyze the morphology of injured tissue and the function of neural Nissl bodies. The results showed that in the mice in the spinal cord injury group, obvious cavities appeared at the injured site, accompanied by a significant decrease and atrophy of Nissl bodies ( Fig.10 G). In contrast, the QLipTC@HDM-treated group showed the smallest lacunae. Evaluation of bladder function in mice after spinal cord injury to assess the recovery of urinary incontinence ( Fig.10 I, J), the results showed that the bladder weight was the highest in the spinal cord injury group, slightly decreased after QLipTC treatment, and the lowest in the QLipTC@HDM group. The results showed that the autonomous urination function was partially restored after QLipTC@HDM treatment. The above results indicate that QLipTC@HDM effectively enhances the recovery of motor function in mice after spinal cord injury, repairs spinal cord tissue morphology, and can best restore the autonomous urination function of the bladder.

[0072] 3. Conclusion

[0073] The present invention develops a transmembrane targeted liposome hydrogel drug delivery system (QLipTC@HDM) to enhance the therapeutic effect of Que in regulating neuroinflammation and improving neural repair. We developed dual-targeted liposomes (LipTC) for encapsulating Que (QLipTC), which enhances the solubility and stability of Que, reduces potential toxicity, and improves the targeting effect of Que on the site of injury and the property of crossing the blood-spinal cord barrier, significantly enhancing bioavailability. The hydrogel matrix (HDM) has a double network structure, and QLipTC is encapsulated in HDM. The residence time of QLipTC at the local site of injury is significantly prolonged by in situ delivery, which is of great significance for improving drug efficacy and reducing the frequency of administration. The QLipTC@HDM drug delivery platform has excellent effects in promoting microglial M2 polarization, enhancing the secretion of anti-inflammatory mediators, improving the permeability of the blood-spinal cord barrier, promoting axonal growth, reducing glial scar formation, and enhancing hindlimb motor function after spinal cord injury. In conclusion, our results suggest that QLipTC@HDM is a promising drug delivery system for the treatment of SCI and has great potential as a therapeutic strategy for the clinical management of SCI, showing broad prospects for clinical application.

[0074] The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A multi-targeted nanogel drug, comprising quercetin-loaded liposomes, wherein the quercetin-loaded liposomes have TAT peptides and CAQK peptides, and the quercetin-loaded liposomes are incorporated into a double network gel, wherein the double network gel is formed by dopamine-grafted hyaluronic acid polymer and methacryloyl gelatin.

2. The multi-targeted nanogel drug according to claim 1, wherein the quercetin-loaded liposome is made of soybean lecithin, cholesterol, DSPE-PEG, DSPE-PEG-TAT, DSPE-PEG-CAQK and quercetin.

3. The multi-targeted nanogel drug according to claim 2, wherein the molar ratio of soybean lecithin, cholesterol, DSPE-PEG, DSPE-PEG-TAT, and DSPE-PEG-CAQK in the quercetin-loaded liposome is 60-80:20-30:8-12:4-6:4-6.

4. The multi-targeted nanogel drug according to claim 2, wherein the average particle size of the quercetin-loaded liposomes is 100-200 nm. 5 . The multi-targeted nanogel drug according to claim 2 , wherein the surface potential of the quercetin-loaded liposome is a positive potential of more than 20 mV. 6 . The multi-targeted nanogel drug according to claim 1 , wherein the dopamine-grafted hyaluronic acid polymer is formed by cross-linking hyaluronic acid and dopamine.

7. The multi-targeted nanogel drug according to claim 1, wherein the double network gel is formed by cross-linking dopamine-grafted hyaluronic acid polymer and methacryloyl gelatin under 405nm light, and the mass ratio of the dopamine-grafted hyaluronic acid polymer to the methacryloyl gelatin is 1-2:1-2.

8. Use of the multi-targeted nanogel drug according to any one of claims 1 to 7, wherein the use is for preparing a drug for treating spinal cord injury; preferably, the drug is used to relieve neuroinflammation and promote spinal cord injury repair.

9. The use according to claim 8, wherein the drug is used to relieve neuroinflammation and promote the repair of spinal cord injury.

10. Use of the multi-targeted nanogel drug according to any one of claims 1 to 7, wherein the use is for preparing a blood-spinal cord injury penetrating and spinal cord injury site-targeted drug delivery system.

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