Functionalized targeted liposomes and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a functionalized targeted liposome and application thereof, comprising: lipids and a targeting ligand; wherein the targeting ligand can specifically bind to a TREM2 receptor. The liposome of the present application can specifically bind to the TREM2 receptor by means of the targeting ligand, realize precise targeting of cells containing the TREM2 receptor on the surface, ensure direct action on target cells, and further improve the precision of delivered substances. The liposome has targeting property, high stability, small side effects, and can be applied to the development of drug carriers, pharmaceutical compositions, etc., and has a wide application prospect. For example, the liposome of the present application is dynamically connected with a hydrogel molecule by a borate ester bond to prepare a hydrogel pharmaceutical composition containing the functionalized targeted liposome of the present application. The hydrogel pharmaceutical composition can release the liposome of the present application according to the change of pH and ROS in the surrounding environment in response to an inflammatory environment, and target cells containing the TREM2 receptor on the surface, i.e. microglial cells (having the TREM2 receptor on the surface), by virtue of the targeting ligand exposed on the surface, and regulate the polarization state of the cells, inhibit the polarization of the cells to the pro-inflammatory M1 type, and promote the polarization of the cells to the anti-inflammatory M2 type, thereby improving the local microenvironment of the spinal cord injury site and promoting nerve regeneration.

Description

Functionalized Targeted Liposomes and Their Applications Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to functionalized targeted liposomes and their applications. Background Technology

[0002] Spinal cord injury (SCI) is a severe central nervous system injury that often leads to motor dysfunction, sensory loss, and excretory dysfunction, and can even be life-threatening. Current treatments for SCI mainly include surgery, medication, and rehabilitation, but their effectiveness is usually limited. Besides surgery, methylprednisone is currently the only approved drug. Its main functions are to reduce secondary inflammation, repair the damaged blood-spinal cord barrier, improve spinal cord blood supply, scavenge free radicals, and promote the secretion of neurotrophic factors. However, the use of methylprednisone remains controversial. Some studies suggest that its use increases the risk of wound infection, elevated blood sugar, and gastrointestinal bleeding. Therefore, the latest guidelines only recommend its use within 8 hours of injury, and the duration of use should not exceed 24 hours. Hyperbaric oxygen therapy, as a traditional SCI rehabilitation treatment, involves administering high-concentration oxygen at pressures 1-3 times higher than atmospheric pressure to reduce cell apoptosis, alleviate inflammation, and reduce tissue edema. While it has some therapeutic effect, it cannot meet the comprehensive rehabilitation needs.

[0003] In recent years, the research and development of drugs for the treatment of spinal cord injury has mainly focused on neuroprotection, anti-inflammation, and nerve regeneration. However, there are two major difficulties in the application of related drugs in spinal cord injury: (1) It is difficult for drugs to form an effective concentration in the spinal cord injury area. Many neuroprotective agents and neurotrophic factors are easily inactivated in the body, and due to the existence of the blood-spinal cord barrier, drugs are difficult to enter the spinal cord injury area through the blood; (2) The side effects of drugs cannot be ignored. For example, vasoactive drugs may increase cardiovascular complications, hormone drugs pose a risk of lung infection, and minocycline may bring potential liver damage risks. Therefore, it is difficult for drugs for the treatment of spinal cord injury to work effectively in the injury area by simply injecting them intravenously or locally. There is an urgent need to develop new drug delivery systems for spinal cord injury.

[0004] Liposomes, as a drug delivery system, have good biocompatibility and targeting. In addition, liposomes are easily phagocytosed by the mononuclear macrophage system after entering the human body. Compared with traditional drug delivery methods, they have advantages such as fewer side effects, higher drug utilization, longer effective blood drug concentration duration, targeted drug delivery, and penetration of physiological barriers. However, traditional liposomes still have shortcomings in terms of targeting, controllable drug release, and in vivo stability.

[0005] Therefore, developing a targeted, highly stable, and low-side-effect drug liposome delivery system is of great significance for the treatment of spinal cord injury. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides functionalized targeted liposomes and their applications. The functionalized targeted liposomes of this invention, by utilizing the specific binding of a targeting ligand to the TREM2 receptor, achieve precise targeting of cells containing the TREM2 receptor on their surface, ensuring direct action on the target cells and further improving the accuracy of substance delivery. These liposomes possess targeting specificity, high stability, and few side effects, and can be applied to the development of drug carriers, pharmaceutical compositions, etc., showing broad application prospects. For example, by dynamically linking the functionalized targeted liposomes of this invention with hydrogel molecules via borate ester bonds, a hydrogel pharmaceutical composition containing the liposomes of this invention can be prepared. This composition can respond to changes in the pH and ROS of the surrounding environment, releasing the functionalized targeted liposomes of this invention in response to inflammatory environments. By exposing the targeting ligand on its surface, it targets cells containing the TREM2 receptor on their surface, namely microglia (which have the TREM2 receptor on their surface), and regulates their polarization state, inhibiting their pro-inflammatory M1 polarization and promoting their anti-inflammatory M2 polarization, thereby improving the local microenvironment at the site of spinal cord injury and promoting nerve regeneration.

[0007] This invention is based on the inventor's discoveries and understanding of the following problems:

[0008] Microglia originate from mononuclear cell lines in the bone marrow. They enter the brain and spinal cord through the bloodstream and colonize the central nervous system in the early stages of brain development. After spinal cord injury, damaged nerve cells release cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide (NO). The release of these cytokines induces the activation and proliferation of microglia. Activated microglia can recognize the site of injury and attract other immune cells by releasing cytokines and chemokines, thereby playing an important role in inflammatory responses and tissue repair.

[0009] Microglia, upon activation, differentiate into two polarization states: the M1 pro-inflammatory phenotype and the M2 anti-inflammatory phenotype. M1 microglia secrete various inflammatory factors, such as IL-1-α, IL-1β, and TNF-α, thereby exacerbating the inflammatory response. Activated M2 microglia exert their anti-inflammatory effects by phagocytizing cell debris or damaged neurons and secreting anti-inflammatory cytokines such as IL-4 and IL-10, reducing tissue inflammation, promoting axonal growth and spinal cord regeneration, and thus promoting tissue repair. Furthermore, related studies have shown that among the intrinsic cells of the nervous system, only microglia express TREM2 receptors in large quantities, and TREM2 receptor expression is upregulated in microglia under inflammatory conditions. Therefore, the inventors proposed developing a drug-targeted delivery system that targets microglia, which is of great significance for exploring new treatment strategies for spinal cord injury.

[0010] Based on the above understanding, the inventors innovatively selected TREM2 as the target and designed and prepared a functionalized drug-loaded liposome, FlipE. Utilizing the receptor-ligand interaction between fingolimod (FTY720) and the TREM2 receptor, the functionalized drug-loaded liposome, FlipE, is able to target microglia with TREM2 receptors on their surface. Furthermore, the inventors dynamically linked the functionalized drug-loaded liposome, FlipE, to a hydrogel via borate ester bonds to prepare the hydrogel FlipE@GelMA / HAPBA. This hydrogel can release the functionalized drug-loaded liposome, FlipE, in response to changes in the pH and ROS of the surrounding environment, thereby achieving the responsive release of drugs (such as isopropanol). Furthermore, by exposing the FTY720 on its surface, it targets microglia with TREM2 receptors on their surface, regulating the polarization state of microglia, inhibiting their pro-inflammatory M1 polarization, and promoting their anti-inflammatory M2 polarization, thereby improving the local microenvironment at the site of spinal cord injury and promoting nerve regeneration.

[0011] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0012] Therefore, in a first aspect, the present invention provides a liposome. According to embodiments of the invention, it comprises: lipids and a targeting ligand; wherein the targeting ligand specifically binds to the TREM2 receptor. The liposome according to embodiments of the invention, by utilizing the specific binding of the targeting ligand to the TREM2 receptor, achieves precise targeting of cells containing the TREM2 receptor on their surface, ensuring direct action on the target cells, namely microglia (which have the TREM2 receptor on their surface), further improving the accuracy of substance delivery. This liposome possesses targeting capability, high stability, and few side effects, and can be applied to the development of drug carriers, pharmaceutical compositions, etc., showing broad application prospects.

[0013] According to embodiments of the present invention, the liposomes described above may further have the following additional technical features:

[0014] According to embodiments of the present invention, the targeting ligand includes one or more of fingolimod, fingolimod hydrochloride, fingolimod phosphate, and sphingosine 1-phosphate.

[0015] According to embodiments of the present invention, the lipids include one or more of amphiphilic lipids, structural lipids, and PEG-lipids.

[0016] In a second aspect, the present invention provides a drug carrier. According to embodiments of the present invention, it includes the liposomes described in the first aspect. The drug carrier according to embodiments of the present invention utilizes the aforementioned liposomes for loading drugs, possessing advantages such as targeting, low cytotoxicity, high stability, and good biocompatibility. It can precisely and efficiently deliver drugs to target cells, facilitating better efficacy of the loaded drugs and enabling applications in the treatment of diseases such as central nervous system injury.

[0017] In a third aspect, the present invention provides a complex. According to embodiments of the invention, it comprises: the liposomes described in the first aspect or the drug carrier described in the second aspect; and a bioactive ingredient. The complex according to embodiments of the invention can be further prepared into a complex containing a bioactive ingredient using the aforementioned liposomes, and the bioactive ingredient can be precisely and efficiently delivered to target cells, which is beneficial for better performance of the loaded bioactive ingredient, and thus can be applied to the treatment of diseases such as central nervous system injury.

[0018] According to embodiments of the present invention, the above-described composite may further have the following additional technical features:

[0019] According to embodiments of the present invention, the bioactive ingredient includes one or more selected from DNA molecules, RNA molecules, proteins, polypeptides, and small molecule drugs.

[0020] In a fourth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, it comprises: the liposomes described in the first aspect, the drug carrier described in the second aspect, or the complex described in the third aspect. The pharmaceutical composition according to embodiments of the present invention, by incorporating the aforementioned liposomes, drug carriers, or complexes of the present invention, leverages their advantages such as targeting specific targets, low cytotoxicity, high stability, and good biocompatibility, thereby achieving multifunctionality and synergistic therapeutic effects, and can be applied to the treatment of central nervous system injuries such as spinal cord injuries.

[0021] According to embodiments of the present invention, the above-described pharmaceutical composition may further have the following additional technical features:

[0022] According to an embodiment of the present invention, the pharmaceutical composition is a drug-loaded hydrogel; the hydrogel further comprises a hydrogel; wherein the targeting ligand is linked to the hydrogel by a borate ester bond.

[0023] According to embodiments of the present invention, the pharmaceutical composition has at least one of the following uses: prevention, relief, adjunctive treatment and / or treatment of central nervous system injury; anti-inflammatory.

[0024] In a fifth aspect of the invention, the use of the liposomes described in the first aspect, the drug carriers described in the second aspect, the complexes described in the third aspect, or the pharmaceutical compositions described in the fourth aspect in the preparation of a medicament is provided.

[0025] Those skilled in the art will understand that the features and advantages described above for liposomes, drug carriers, complexes or pharmaceutical compositions also apply to this use, and will not be repeated here.

[0026] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0028] Figure 1 shows the TEM observation results of the targeted liposome FlipE in Example 1 of the present invention. In this figure, A is the overall TEM image of the targeted liposome FlipE with a scale bar of 200 nm, and B is the detailed TEM image of the targeted liposome FlipE with a scale bar of 100 nm.

[0029] Figure 2 shows the particle size determination results of the targeted liposome FlipE in Example 1 of the present invention;

[0030] Figure 3 shows the SEM observation results of the hydrogel FlipE@GelMA / HAPBA in Example 3 of the present invention. In this figure, A is the SEM observation image of the hydrogel FlipE@GelMA / HAPBA with a scale bar of 5 μm, and B is a magnified view of the part of the hydrogel FlipE@GelMA / HAPBA containing the targeted liposome FlipE with a scale bar of 500 nm.

[0031] Figure 4 shows the results of laser confocal microscopy observation after different cells engulfed fluorescent liposomes in Example 4 of the present invention. Among them, A is a representative image of astrocytes engulfing ordinary fluorescent liposome Cou6-Lip, B is a representative image of astrocytes engulfing targeted fluorescent liposome Cou6-Flip, C is a representative image of neuronal cells engulfing ordinary fluorescent liposome Cou6-Lip, D is a representative image of neuronal cells engulfing targeted fluorescent liposome Cou6-Flip, E is a representative image of microglia engulfing ordinary fluorescent liposome Cou6-Lip, and F is a representative image of microglia engulfing targeted fluorescent liposome Cou6-Flip.

[0032] Figure 5 shows the statistical analysis results of the average intracellular fluorescence intensity of different cells after phagocytizing fluorescent liposomes in Example 4 of the present invention, where ** means P<0.01 and *** means P<0.001;

[0033] Figure 6 shows the fluorescence imaging distribution of spinal cord tissue samples from rats in each group 24 h after injury in Example 5 of the present invention.

[0034] Figure 7 shows the immunofluorescence staining results of spinal cord tissue sections from rats in each group 24 h after injury in Example 5 of this invention. A, from top to bottom, shows the results of TREM2 antibody staining (red) + targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue) on the spinal cord transverse section of the Flip group, a magnified image of TREM2 antibody staining (red) + targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue), and a magnified image of targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue). B, from top to bottom, shows the results of TREM2 antibody staining (red) + ordinary fluorescent liposome Cou6-Lip (green) on the spinal cord transverse section of the lip group. The images show the results of TREM2 antibody staining (red), TREM2 antibody staining (red), TREM2 antibody staining (green), TREM2 antibody staining (red), TREM6 antibody staining (green ...

[0035] Figure 8 shows the drug release experiment results of the hydrogel FlipE@GelMA / HAPBA in Example 6 of the present invention;

[0036] Figure 9 shows the immunofluorescence staining results of CD86 receptor staining in microglia of each group in Example 7 of the present invention. From left to right, A shows the results of CD86 staining (red) and nuclear staining (blue) + mixed staining of CD86 and nuclear in the Con group; B shows the results of CD86 staining (red) and nuclear staining (blue) + mixed staining of CD86 and nuclear in the LPS group; C shows the results of CD86 staining (red) and nuclear staining (blue) + mixed staining of CD86 and nuclear in the LGH group; and D shows the results of CD86 staining (red) and nuclear staining (blue) + mixed staining of CD86 and nuclear in the FGH group.

[0037] Figure 10 shows the immunofluorescence staining results of CD206 receptor staining in microglia of each group in Example 7 of the present invention. From left to right, A shows the results of CD206 staining (green) and nuclear staining (blue) + mixed staining of CD206 and nuclear staining in the Con group; B shows the results of CD206 staining (green) and nuclear staining (blue) + mixed staining of CD206 and nuclear staining in the LPS group; C shows the results of CD206 staining (green) and nuclear staining (blue) + mixed staining of CD206 and nuclear staining in the LGH group; and D shows the results of CD206 staining (green) and nuclear staining (blue) + mixed staining of CD206 and nuclear staining in the FGH group.

[0038] Figure 11 shows the flow cytometry analysis results of CD86 receptor staining in microglia of each group in Example 7 of the present invention. Among them, A is the flow cytometry analysis of CD86 receptor staining in microglia of the Con group, B is the flow cytometry analysis of CD86 receptor staining in microglia of the LPS group, C is the flow cytometry analysis of CD86 receptor staining in microglia of the LGH group, and D is the flow cytometry analysis of CD86 receptor staining in microglia of the FGH group.

[0039] Figure 12 shows the results of gene expression detection of M1 type related microglia in each group in Example 7 of the present invention. In the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, A indicates CD86 gene expression in each group of microglia, B indicates IL1-β gene expression in each group of microglia, C indicates iNOS gene expression in each group of microglia, and D indicates TNF-α gene expression in each group of microglia.

[0040] Figure 13 shows the results of gene expression detection in microglia M2 type in each group in Example 7 of the present invention. In the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, A indicates ARG-1 gene expression in each group of microglia, B indicates IL-4 gene expression in each group of microglia, C indicates YM-1 gene expression in each group of microglia, and D indicates IL-10 gene expression in each group of microglia. Detailed Implementation

[0041] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0045] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0046] Terms and Definitions

[0047] In this article, the term "FTY720" refers to fingolimod, a potent immunosuppressant approved in 2010 for the treatment of relapsing-remitting multiple sclerosis, and is a sphingosine-1-phosphate (S1P) receptor modulator.

[0048] In this article, the term "TREM2 receptor" refers to a transmembrane receptor of an immunoglobulin superfamily, which is mainly expressed on myeloid cells, including microglia, macrophages, monocytes, dendritic cells, etc. TREM2 receptor transmits signals by binding to adaptor proteins DAP12 and DAP10, regulating the inflammatory response and phagocytic function of myeloid cells.

[0049] In this article, the term "ROS" refers to a series of highly reactive oxygen-containing substances in the body, such as superoxide anion (O2). - ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) - In inflammatory environments, ROS levels typically rise, and this invention utilizes this characteristic to design a drug release mechanism delivery system that can respond to changes in ROS.

[0050] In this article, the term "microglia" refers to the main immune cells in the central nervous system, which are responsible for monitoring the brain environment, clearing cellular debris and pathogens, etc. They play a key role in maintaining the homeostasis of the central nervous system and regulating the inflammatory response. Microglia express a variety of receptors, including the TREM2 receptor.

[0051] In this paper, the term "hydrogel" refers to a hydrophilic polymer network that can absorb a large amount of water and swell in water while maintaining its structural integrity. The three-dimensional network structure of hydrogels enables them to absorb and retain large amounts of water, forming a gel-like substance with good biocompatibility, degradability, and mechanical properties. It can be used as a drug carrier in drug delivery systems or as a tissue engineering scaffold to support cell growth and tissue repair. In the embodiments of this invention, the inventors dynamically linked the functionalized drug-loaded liposome FlipE to the hydrogel via borate ester bonds to prepare the hydrogel FlipE@GelMA / HAPBA.

[0052] In this document, the term "boronate bond" refers to a chemical bond formed by the reaction of boric acid with alcohol compounds. Boronate bonds play a crucial role in this invention by connecting targeted ligands (such as FTY720) to polymers (such as hydrogels). This connection enables the drug composition to release the drug in response to specific inflammatory conditions. Specifically, boronate bonds are easily broken in acidic environments (such as when the pH of the inflammatory region is typically low) and under conditions of highly reactive oxygen species (ROS), thereby achieving intelligent drug release.

[0053] In this paper, the term "LPS" refers to lipopolysaccharide, a complex glycolipid substance found in the outer membrane of Gram-negative bacteria. At the same time, LPS is a potent immunostimulant commonly used to induce inflammatory responses or polarization of cells. In this embodiment of the invention, the substance is used to stimulate microglia to induce them to polarize to the M1 type, thereby mimicking an inflammatory state.

[0054] Liposomes

[0055] This invention proposes a liposome. According to embodiments of the invention, it comprises: lipids and a targeting ligand; wherein the targeting ligand specifically binds to the TREM2 receptor. The liposome according to embodiments of the invention, by utilizing the specific binding of the targeting ligand to the TREM2 receptor, achieves precise targeting of cells containing the TREM2 receptor on their surface, ensuring direct action on the target cells, namely microglia (which have the TREM2 receptor on their surface), further improving the accuracy of substance delivery. This liposome exhibits targeting capability, high stability, and few side effects, and can be applied to the development of drug carriers, drug compositions, etc., showing broad application prospects.

[0056] According to embodiments of the present invention, the targeting ligand includes one or more of fingolimod, fingolimod hydrochloride, fingolimod phosphate, and sphingosine 1-phosphate. Thus, the ligand targeting the TREM2 receptor, including fingolimod, fingolimod hydrochloride, fingolimod phosphate, and sphingosine 1-phosphate, can be selected according to the specific application.

[0057] It should be noted that fingolimod is FTY720 in the embodiments of this invention. As a targeting ligand, it has the ability to specifically bind to the TREM2 receptor. Fingolimod hydrochloride, fingolimod phosphate, and sphingosine 1-phosphate are structurally similar to FTY720. They are chemically similar to fingolimod and can bind to the TREM2 receptor through a similar mechanism to FTY720, thereby achieving the targeted delivery effect of the liposomes of this invention. Therefore, tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs of FTY720, as well as the above-mentioned compounds, should all fall within the scope of protection of this invention. These compounds can all serve as targeting ligands for the TREM2 receptor, enabling the functionalized targeted liposomes of this invention to precisely target microglia (which have TREM2 receptors on their surface).

[0058] According to embodiments of the present invention, the lipids comprise one or more of amphiphilic lipids, structural lipids, and PEG-lipids. According to embodiments of the present invention, the amphiphilic lipids comprise those selected from 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline. 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1,2-diphytoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearateyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonic ... Acyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt, (S)-2-ammonium-3-((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxy)oxy)propionate sodium salt, dimyristoyl phosphatidylcholine, dimyristoyl phosphate ethanolamine, dimyristoyl phosphatidylglycerol, dioleoyl-phosphatidylethanolamine 4-(N-cis-butenyliminomethyl)-cyclohexane-1-carboxylate, dioleoyl phosphatidylglycerol, 1,2-dioleoyl-sn-glycerol Oil-3-(phospho-L-serine), cell fusion phospholipids, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylserine, distearylphosphatidylcholine, distearyl-phosphatidyl-ethanolamine, distearylphosphoethanolamine imidazole, 1,2-diundecanoyl-sn-glycerol-phosphocholine, lecithinylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphate, bis((S)-2-hydroxy-3-(oleoyloxy)propyl)ammonium phosphate, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-inositol), 1,2-distearyl-sn-glycerol-3-phosphate-L-serine, 1,2-Dilinoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine, and sphingomyelin; the structural lipids include those selected from cholesterol, coccosterol, sterol, and sphingomyelin. The PEG-lipid comprises one or more of the following: stigmasterol, stigmasterol, rapeseed sterol, tomatine, lycine, ursolic acid, and α-tocopherol; the PEG-lipid comprises one or more selected from PEG-DMG, 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, PEG-DSPE, PEG-DSG, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearate, PEG-DAG, PEG-DPPE, PEG-PE, PEG-S-DAG, PEG-ceramide, PEG-dialkoxypropylcarbamate, and PEG-c-DMA.

[0059] Drug carrier

[0060] This invention proposes a drug carrier. According to embodiments of the invention, it includes the aforementioned liposomes. The drug carrier according to embodiments of the invention utilizes the aforementioned liposomes for drug loading, possessing advantages such as targeting, low cytotoxicity, high stability, and good biocompatibility. It can precisely and efficiently deliver drugs to target cells, facilitating better efficacy of the loaded drug and enabling its application in the treatment of diseases such as central nervous system injury.

[0061] complex

[0062] This invention proposes a complex. According to embodiments of the invention, it comprises: the aforementioned liposomes or the aforementioned drug carriers; and a bioactive ingredient. The complex according to embodiments of the invention can be further prepared into a complex containing a bioactive ingredient using the aforementioned liposomes, and the bioactive ingredient can be precisely and efficiently delivered to target cells, which is beneficial for better performance of the loaded bioactive ingredient, and thus can be applied to the treatment of diseases such as central nervous system injury.

[0063] According to embodiments of the present invention, the bioactive ingredient includes one or more selected from DNA molecules, RNA molecules, proteins, peptides, and small molecule drugs. Therefore, the bioactive ingredient can be selected based on the specific application.

[0064] According to embodiments of the present invention, the replication of proteins and polypeptides is not particularly limited and can be peptide chains, proteins themselves, derivatives of the above substances, or complexes with other substances, such as Cas proteins or peptides of some of their structural domains, proteins loaded with radionuclides, antibodies, and other substances.

[0065] According to embodiments of the present invention, the bioactive component contains DNA, RNA, nucleic acid-protein complex, nucleic acid-lipid complex, nucleic acid-nuclein complex, etc., and the type of nucleic acid is not particularly limited. Specific examples of the nucleic acid may include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA, ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, triplex-forming oligonucleotide (TFO), or genes, etc.

[0066] Pharmaceutical Composition

[0067] This invention proposes a pharmaceutical composition. According to embodiments of the invention, it comprises: the aforementioned liposomes, the aforementioned drug carriers, or the aforementioned complexes. The pharmaceutical composition according to embodiments of the invention, by incorporating the aforementioned liposomes, drug carriers, or complexes, leverages their advantages such as targeting specific targets, low cytotoxicity, high stability, and good biocompatibility, thereby achieving multifunctionality and synergistic therapeutic effects, and can be applied to the treatment of central nervous system injuries such as spinal cord injuries.

[0068] According to an embodiment of the present invention, the pharmaceutical composition is a drug-loaded hydrogel; the drug-loaded hydrogel further comprises a biocompatible polymer matrix; wherein the targeting ligand is linked to the hydrogel via a borate ester bond. Thus, the pharmaceutical composition can be in the form of a drug-loaded hydrogel. The three-dimensional network structure of the hydrogel can effectively encapsulate and protect the functionalized targeting liposomes, drug carriers, complexes, and active pharmaceutical ingredients of the present invention, preventing premature degradation or clearance in vivo. It not only supports the stable release of the functionalized targeting liposomes, drug carriers, complexes, and active pharmaceutical ingredients of the present invention, but also allows for gradual degradation in vivo, avoiding potential side effects from long-term implantation, and has broad application prospects. The targeting ligand is linked to the hydrogel via a borate ester bond. This design enables the pharmaceutical composition to responsively release the functionalized targeting liposomes, drug carriers, complexes, and active pharmaceutical ingredients of the present invention under specific pH and ROS conditions in an inflammatory environment. This intelligent drug release mechanism ensures the precise release of key components in the inflammatory region. Through this responsive release, the functionalized targeting liposomes, drug carriers, complexes, and active pharmaceutical ingredients of the present invention can exert their maximum effect where needed, significantly improving therapeutic efficacy and safety. This also reduces the side effects of the functionalized targeted liposomes, drug carriers, complexes, and active pharmaceutical ingredients of the present invention being released in non-target areas.

[0069] According to embodiments of the present invention, the pharmaceutical composition has at least one of the following uses: prevention, relief, adjunctive treatment, and / or treatment of central nervous system injury; anti-inflammatory. Thus, the pharmaceutical composition of the present invention can be applied to the prevention, relief, adjunctive treatment, and / or treatment of central nervous system injury, and anti-inflammatory applications. Exemplarily, in the prevention and relief of spinal cord injury, the pharmaceutical composition of the present invention can protect nerve cells from further damage by continuously releasing the pharmaceutical composition. In the adjunctive treatment and treatment of spinal cord injury, the pharmaceutical composition can reduce inflammatory responses, improve the local microenvironment, and promote nerve regeneration by regulating the polarization state of microglia (which have TREM2 receptors on their surface).

[0070] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Thus, the addition of excipients further enhances the flexibility of the pharmaceutical composition in addressing different application scenarios.

[0071] In this document, the term "pharmaceuticalally acceptable excipient" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, all of which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329); except where any conventional carrier is incompatible with the active ingredient, it covers its use in therapeutic or pharmaceutical compositions.

[0072] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0073] The pharmaceutical compositions of the present invention can be administered by any acceptable method of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols, and current methods for preparing these dosage forms are known or readily apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a patient; furthermore, the pharmaceutical composition administered to the subject or patient is in the form of one or more dose units.

[0074] Uses in drug preparation

[0075] The present invention proposes the use of the aforementioned liposomes, the aforementioned drug carriers, the aforementioned complexes or the aforementioned pharmaceutical compositions in the preparation of pharmaceuticals.

[0076] Those skilled in the art will understand that the features and advantages described above for liposomes, drug carriers, complexes or pharmaceutical compositions also apply to this use, and will not be repeated here.

[0077] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Example 1: Preparation and observation of targeted liposome FlipE

[0079] 1. Preparation of Targeted Liposome FlipE

[0080] Take a round-bottom flask and dissolve 72 mg of soybean lecithin, 24 mg of cholesterol, 4 mg of FTY720, and 10 mg of isoenzyme flavonoids (EPT) in a chloroform-methanol mixture (5 mL chloroform + 3 mL methanol). Then, remove the solvent by rotary evaporation under vacuum at 40°C for 30 min, at which point a transparent liposome film forms at the bottom of the flask. Add 30 mL of phosphate-buffered saline (PBS) (10 mM, pH = 7.4), stir with magnetic beads for 15 min to completely dissolve the film, and let it stand for 10-30 min to hydrate, obtaining a liposome solution. Transfer the liposome solution to a 50 mL centrifuge tube, place it on ice, and intermittently sonicate with a probe for 3 min (30% intensity, 5 s on, 5 s off). Then centrifuge (4°C, 300-700 rpm) for 5 min. After centrifugation, quickly aspirate the supernatant into a new centrifuge tube to obtain the targeted liposome FlipE sample solution. Use a liposome extruder to sequentially pass the targeted liposome FlipE sample solution through a 400 mL atomizer. Polycarbonate membranes of 100 nm, 200 nm, and 100 nm were used to obtain targeted liposomes FlipE.

[0081] 2. TEM observation of targeted liposome FlipE

[0082] The microstructure of the obtained targeted liposome FlipE was observed using transmission electron microscopy (TEM, Tecnai Spirit, FEI, Czech Republic). The specific steps are as follows:

[0083] Place a 300-mesh copper mesh with a carbon film on filter paper, face up. Dilute the targeted liposome FlipE sample solution (3.6 mg / mL) obtained in step 1 by 20 times with PBS. Add 10 µL of the diluted targeted liposome FlipE sample solution to the surface of the copper mesh and let it stand for 2 min. Add 10 µL of 2% uranium acetate solution to the surface of the copper mesh for negative staining. After standing in the dark for 30 s, absorb the stain with filter paper. Add 10 µL of pure water to the surface of the copper mesh to wash away the excess stain. Then absorb the pure water with filter paper. Finally, place the copper mesh under the TEM observation stage for observation and photography.

[0084] The TEM observation results of the liposome FlipE target are shown in Figure 1.

[0085] The results showed that the targeted liposome FlipE exhibited a relatively uniform circular granular structure under the microscope, and the outer layer of the drug EPT could be seen in the magnified image.

[0086] 3. Particle size determination of targeted liposome FlipE

[0087] The targeted liposome FlipE sample solution (3.6 mg / mL) obtained in step 1 was diluted 10 times with PBS. 20 µL of the diluted targeted liposome FlipE sample solution was placed into the particle size cup of a dynamic light scattering instrument (DLS, Wyatt Technology Corporation, USA). The particle size of the targeted liposome FlipE was detected using the dynamic light scattering instrument.

[0088] The particle size determination results of the targeted liposome FlipE are shown in Figure 2.

[0089] The results showed that the targeted liposome FlipE had a uniform particle size in solution.

[0090] Example 2: Preparation of the targeted fluorescent liposome Cou6-Flip

[0091] In Example 2, the preparation process of the coumarin-targeted fluorescent liposome Cou6-Flip is the same as that of the preparation of the targeted liposome FlipE in Step 1 of Example 1, with the only difference being as follows:

[0092] Take a round-bottom flask and dissolve 72 mg of soybean lecithin, 24 mg of cholesterol, and 4 mg of FTY720 in a chloroform-methanol mixture (5 mL chloroform + 3 mL methanol), and add an additional 10 μL of coumarin 6-chloroform solution; then remove the solvent by rotary evaporation for 30 min under vacuum at 40°C in the dark; the remaining steps are the same as step 1 in Example 1.

[0093] The preparation steps for the coumarin 6-chloroform solution are as follows: Weigh 8 mg of coumarin 6 powder and dissolve it in 1 mL of chloroform to obtain the coumarin 6-chloroform solution.

[0094] Thus, the targeted fluorescent liposome Cou6-Flip was prepared.

[0095] Comparative Example 1: Preparation of ordinary liposomes (Lip) and ordinary fluorescent liposomes (Cou6-Lip)

[0096] (1) Preparation of ordinary liposomes (Lip)

[0097] The preparation process of ordinary liposomes Lip in Comparative Example 1 is the same as the preparation process of targeted liposomes FlipE in Step 1 of Example 1, with the only difference being as follows:

[0098] Take a round-bottom flask and dissolve 72 mg of soybean lecithin, 24 mg of cholesterol, and 10 mg of isopropanol (EPT) in a chloroform-methanol mixture (5 mL chloroform + 3 mL methanol). The remaining steps are the same as step 1 in Example 1. Thus, a common liposome Lip sample solution and a common liposome Lip are obtained.

[0099] (2) Preparation of conventional fluorescent liposome Cou6-Lip

[0100] The preparation process of the ordinary fluorescent liposome Cou6-Lip in Comparative Example 1 is the same as that of the targeted fluorescent liposome Cou6-Flip in Example 2, with the only difference being as follows:

[0101] Without adding FTY720 (4 mg), the remaining steps are the same as in Example 2.

[0102] Example 3: Preparation and observation of hydrogel FlipE@GelMA / HAPBA

[0103] 1. Preparation of FlipE@GelMA / HAPBA hydrogel

[0104] Weigh 160 mg of methacrylamide gelatin (GelMA), 20 mg of phenylboronic acid hyaluronic acid (HA-PBA, crosslinking agent), and 2 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) and dissolve them in 1 mL of PBS solution. Heat the solution in a 70°C water bath to completely dissolve all raw materials to obtain a photocrosslinked hydrogel precursor solution. Then, mix the photocrosslinked hydrogel precursor solution with 1 mL of the targeted liposome FlipE sample solution (3.6 mg / mL) prepared in Example 1. After thorough mixing, crosslink the solution with UV light for 200 s to obtain the hydrogel FlipE@GelMA / HAPBA (FGH).

[0105] 2. Observation of FlipE@GelMA / HAPBA hydrogel

[0106] The FlipE@GelMA / HAPBA hydrogel was placed in a 1.5 mL centrifuge tube, and an appropriate amount of liquid nitrogen was poured in to rapidly cool it down. Then, it was pre-frozen at -20°C for 24 h. After pre-freezing, it was freeze-dried using a vacuum dryer to obtain the FlipE@GelMA / HAPBA lyophilized sample. The FlipE@GelMA / HAPBA lyophilized sample was attached to the stage (fresh cut side up) using conductive tape. A 5 nm thick carbon film was sprayed onto the surface of the FlipE@GelMA / HAPBA lyophilized sample, and then it was placed in a scanning electron microscope (SEM, GEMINISEM 500, Zeiss, Germany) for observation.

[0107] The SEM observation results of the hydrogel FlipE@GelMA / HAPBA are shown in Figure 3.

[0108] The results showed that the prepared drug-loaded hydrogel FlipE@GelMA / HAPBA had a porous structure, and under magnification, regular spherical targeting liposomes FlipE could be observed connected to the hydrogel.

[0109] Example 4: Targeting microglia in vitro with the fluorescent liposome Cou6-Flip.

[0110] Neuronal cells (HT22), microglia (HAPI), and astrocytes were cultured adherently in 6-well plates, with an initial cell density of 500,000 cells / well in each well. After 12 h of seeding, the cell state was observed, and the culture medium was replaced with either the ordinary fluorescent liposome Cou6-Lip (3.6 mg / mL) prepared in Comparative Example 1 or the targeted fluorescent liposome Cou6-Flip (3.6 mg / mL) prepared in Example 2. The cells were cultured for 30 min in each medium, and then replaced with normal culture medium. The cells were observed using a laser confocal microscope, and the fluorescence intensity of each group was analyzed by flow cytometry. For the laser confocal microscope observation, bright field combined with the AF488 channel was used for observation and imaging.

[0111] Figure 4 shows the results of laser confocal microscopy observation of different cells after they engulfed fluorescent liposomes, and Figure 5 shows the statistical analysis results of the average intracellular fluorescence intensity of different cells after they engulfed fluorescent liposomes.

[0112] The results show:

[0113] (1) Observation results of laser confocal microscopy: The bright field results show the cell morphology. The green part represents the fluorescent particles phagocytosed by the cells (i.e., ordinary fluorescent liposome Cou6-Lip or targeted fluorescent liposome Cou6-Flip). Microglia showed the strongest fluorescence intensity in the three cell types, and the fluorescence of microglia in the targeted fluorescent liposome Cou6-Flip group was stronger than that in the ordinary fluorescent liposome Cou6-Lip group.

[0114] (2) Statistical analysis results of the average fluorescence intensity in cells: The fluorescence intensity of microglia that phagocytosed the fluorescently labeled targeted fluorescent liposome Cou6-Flip was not only significantly stronger than that of other cells treated under the same conditions, but also significantly stronger than that of microglia that phagocytosed the fluorescently labeled ordinary fluorescent liposome Cou6-Lip.

[0115] The above results indicate that the modification of FTY720 significantly enhances the affinity of liposomes for microglia.

[0116] Example 5: Targeting fluorescent liposome Cou6-Flip in vivo by binding to TREM2 receptor and targeting microglia.

[0117] 1. Constructing a rat spinal cord T9 segment 1 mm complete transverse transection model

[0118] After completely anesthetizing the rats, the hair on their backs was shaved. A scalpel was used to incise the skin and subcutaneous tissue along the midline of the back, and the subcutaneous fascia was separated. Muscle tissue was then separated along both sides of the spinous processes using a scalpel. A retractor was used to retract the skin and muscles. Ophthalmic scissors were used to cut the ligaments above and below the T9 segment. The T9 segment was held with toothed ophthalmic forceps, and the spine was lifted upwards to expose the intervertebral space. Bone forceps were used to remove bone along the intervertebral space to expose the spinal cord. The rats were then transferred to a microscope, and microscopic scissors were used to gently cut the dura mater on the surface of the spinal cord. Microscopic straight scissors were used to quickly sever the spinal cord, and a 1 mm length of spinal cord tissue was removed under a transparent ruler. The surgical area was then rinsed with physiological saline, and strict hemostasis was maintained. After confirming the absence of active bleeding, a 1 mm complete transverse section model of the T9 segment of the rat spinal cord was obtained. Each rat was then injected with 1 mL of Cou6-Lip (3.6 mg / mL) of the ordinary fluorescent liposome prepared in Comparative Example 1. After constructing the rat spinal cord T9 segment 1 mm full transverse model, the following solutions were prepared: Cou6-Flip (3.6 mg / mL, designated as Lip group), the targeted fluorescent liposome Cou6-Flip (3.6 mg / mL, designated as Flip group) prepared in Example 2, and SCI group. After observing for 15 min to confirm that there was no leakage of the solution, the back muscles and fascia of the rat were continuously sutured, and the skin was intermittently sutured.

[0119] 2. Material selection for grouting

[0120] After 24 hours of normal feeding, the rats in each group were completely anesthetized, fixed in a supine position on a foam board, and transferred to a negative pressure perfusion stage. The perfusion pump speed was adjusted to 50 rpm / s, and PBS solution was connected to the inlet and labeled. The abdominal cavity was exposed, and the skin was dissected upwards along the abdominal cavity to the costal margin. The diaphragm was punctured along the costal margin to cause lung collapse. The position of the heartbeat was observed, and the diaphragm was quickly cut open. The thoracic cavity was cut open and fixed in front of the heart to fully expose the heart. After making a small incision at the apex of the heart, the perfusion needle at the PBS end was quickly inserted into the aorta through the apex of the heart. After fixing the perfusion needle, the right atrial appendage was cut open to allow blood to flow out. The perfusion pump was turned on and timed for 15 minutes, and 250 mL of blood was perfused to fully replace the blood. After complete replacement, the inlet was replaced with 4% paraformaldehyde solution and perfusion was continued for 15 minutes. min; After perfusion, the rat spine was separated, the spine was transversely cut from the neck to the sacrum and the paravertebral tissue was separated, the lamina was bitten open along the ventral side with bone forceps, the spinal cord was separated and the spinal cord tissue was completely removed, soaked in 4% paraformaldehyde and labeled; then the spinal cord tissue and internal organs were soaked in 4% paraformaldehyde and stored at 4°C for 24 h to obtain spinal cord tissue samples and internal organ samples; then the spinal cord tissue samples and internal organ samples were sequentially transferred to 10%, 20% and 30% sucrose solutions (dissolved in PBS) for gradient dehydration, and after the spinal cord tissue samples and internal organ samples were completely dehydrated, they settled to the bottom.

[0121] 3. Fluorescence intensity detection in the spinal cord injury area

[0122] After the perfusion sampling was completed, the fluorescence intensity of the damaged area was detected using a live imaging system. First, the spinal cord tissue sample obtained in step 2 was wiped dry and placed on a black background paper before being placed in the live imaging system for detection. The parameters of the live imaging system were set as follows: exposure time: 0.5 s, detection wavelength range: 480 nm~520 nm.

[0123] The distribution of fluorescence imaging of spinal cord tissue samples from each group of rats 24 h after injury is shown in Figure 6.

[0124] The results showed that different colors represented the fluorescence intensity values ​​within their respective ranges, and the right side was a reference scale for fluorescence intensity values. After eliminating background fluorescence, no fluorescence distribution was observed in the spinal cord injury area of ​​rats in the SCI group, indicating that no exogenous fluorescence was detected in the spinal cord injury area. However, exogenous fluorescence was detected in the spinal cord injury areas of rats in the Flip and Lip groups, and the fluorescence distribution range in the spinal cord injury area of ​​the Flip group was significantly larger than that of the Lip group. At the same time, the fluorescence intensity (yellow) of the Flip group was also significantly higher than that of the Lip group (red).

[0125] The above results indicate that, compared to the Lip group, more liposomes accumulated in the spinal cord injury area of ​​the Flip group one day after spinal cord injury in rats.

[0126] 4. Spinal cord tissue sections

[0127] For the spinal cord tissue sections obtained in step 2, firstly, the spinal cord tissue is completely embedded using Optimal Cutting Temperature Compound (OCT). The embedding agent is checked and air bubbles are removed. The embedded spinal cord tissue sample is then placed on a quick-freezing stage to freeze. Next, the embedded spinal cord tissue sample is connected to the sample stage of the cryostat via the stage, and continuous slicing is performed using the crank handle. Each slice is 10 µm thick, resulting in spinal cord tissue section samples. The spinal cord tissue section samples are then laid flat and completely attached to an adhesive slide, paying attention to distinguishing the front and back sides. The slide is then air-dried in the dark and ventilated for 1-2 hours to obtain a slide containing spinal cord tissue sections. Finally, the slide containing spinal cord tissue sections is placed in a slide cassette and frozen at -80°C for later use.

[0128] 5. Immunofluorescence staining

[0129] After sectioning the spinal cord tissue, TREM2 immunofluorescence staining was performed. The specific steps are as follows:

[0130] Remove the glass slides containing spinal cord tissue sections that have been frozen at -80℃, and allow them to thaw at room temperature for 1.5 h. After thawing, place the spinal cord tissue sections in a wash box containing an appropriate amount of PBS and wash three times for 5 min each time. Then transfer the spinal cord tissue sections to a humidified chamber. First, wipe the liquid off the surface of the sections. Then, use an immunohistochemical pen to delineate the antibody incubation area around the tissue. Prepare a permeabilization and blocking solution of 0.3% Triton X-100 + 10% goat serum (diluted in PBS), and add it to the surface of the spinal cord tissue sections to completely submerge the tissue. Block at room temperature for 2 h. After blocking, shake off the blocking solution from the surface of the sections, and prepare a solution of 0.3% Triton X-100 + 5% goat serum (diluted in PBS) to dilute the TREM2 antibody. Dilute the antibody according to the appropriate ratio indicated in the kit instructions and incubate it on the tissue surface. Place the incubator at 4℃ for at least 16 h. Then, remove the spinal cord tissue sections, thaw at room temperature for 1 h, discard the antibody dilution solution, and wash three times in a wash box for 5 min each time. To thoroughly clean the antibody and wipe away any fluid around the tissue, place a mounting medium containing DAPI on the spinal cord tissue section, cover the mounting with a microscope coverslip, taking care to avoid air bubbles, and obtain the mounted spinal cord tissue section. Allow the mounted spinal cord tissue section to air dry in a ventilated area, checking for any uneven edges. Finally, use a fully automated slide scanner (Axio Scan. Z1, Zeiss, Germany) to photograph and observe the mounted spinal cord tissue section at a magnification of 10x.

[0131] The immunofluorescence staining results of spinal cord tissue sections from rats in each group 24 h after injury are shown in Figure 7.

[0132] The results showed that liposome fluorescence was observed in the spinal cord tissue of rats in both the Flip and Lip groups 24 h after injury, indicating that the liposomes were taken up locally by the spinal cord tissue. The fluorescence distribution range in the Flip group was significantly larger than that in the Lip group, indicating that the targeted fluorescent liposome Cou6-Flip in the Flip group could be taken up more by the spinal cord tissue. Furthermore, in the magnified field, the liposome fluorescence (green) and TREM2 antibody staining fluorescence (red) in the Flip group showed obvious colocalization (yellow), and the colocalization (yellow) range in the Flip group was significantly larger than that in the Lip group.

[0133] The above results indicate that the affinity between the targeted fluorescent liposome Cou6-Flip and spinal cord tissue cells is achieved through binding with the TREM2 antibody.

[0134] Example 6: Drug release experiment of hydrogel FlipE@GelMA / HAPBA

[0135] The hydrogel FlipE@GelMA / HAPBA prepared in Example 3 was mixed with 0.5 mL of phosphate buffer (pH=7.4), phosphate buffer (pH=5.0), and phosphate buffer containing H2O2 (100 μM) (pH=5.0), respectively. The release solution was taken out every 24 h, and the release solution was replenished with the corresponding buffer after each take-out. The taken-out release solution was dissolved in 100 μL of chloroform, and the drug concentration was detected by HPLC.

[0136] The drug release experiment results of the hydrogel FlipE@GelMA / HAPBA are shown in Figure 8.

[0137] The results showed that when the hydrogel FlipE@GelMA / HAPBA was in an environment of pH 7.4, the release curve of the drug isozyraxanthin was relatively flat, with a cumulative release of 10% of the total drug load by day 7; while when the hydrogel FlipE@

[0138] When GelMA / HAPBA is in an environment with pH=5.0, its drug release rate is higher than that with pH=7.4, and it releases a total of 35% of the total drug load by the 7th day. However, when the hydrogel FlipE@GelMA / HAPBA is in an environment with pH=5.0 and high ROS, it releases the drug at a high rate in the first 4 days, and the drug release rate slows down on the 6th and 7th days, and it can release a total of 44% of the total drug load by the end of the 7th day.

[0139] The above results demonstrate that the hydrogel FlipE@GelMA / HAPBA prepared in Example 3 can achieve responsive drug release under low pH and high ROS conditions.

[0140] Example 7: Characterization of the in vitro regulation of microglia polarization by hydrogel FlipE@GelMA / HAPBA

[0141] 1. Preparation of FlipE@GelMA / HAPBA hydrogel

[0142] Weigh 160 mg of methacrylamide gelatin (GelMA), 20 mg of hyaluronic acid phenylboronic acid (HA-PBA, crosslinking agent), and 2 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) and dissolve them in 1 mL of PBS solution. Heat the solution in a 70°C water bath to completely dissolve all raw materials to obtain a photocrosslinked hydrogel precursor solution. Then, mix the photocrosslinked hydrogel precursor solution with 1 mL of the targeted liposome FlipE sample solution (3.6 mg / mL) prepared in Example 1. After thorough mixing, crosslink the solution with UV light for 200 s to obtain the hydrogel FlipE@GelMA / HAPBA (numbered FGH).

[0143] 2. Preparation of hydrogel lipE@GelMA / HAPBA

[0144] Weigh 160 mg of methacrylamide gelatin (GelMA), 20 mg of phenylboronic acid hyaluronic acid (HA-PBA, crosslinking agent), and 2 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) and dissolve them in 1 mL of PBS solution. Heat in a 70°C water bath to completely dissolve all raw materials to obtain a photocrosslinked hydrogel precursor solution. Then, mix the photocrosslinked hydrogel precursor solution with 1 mL of the ordinary liposome Lip sample solution (3.6 mg / mL) prepared in Comparative Example 1. After thorough mixing, crosslink the sample with UV light for 200 s to obtain the hydrogel lipE@GelMA / HAPBA (designated LGH).

[0145] 3. Pretreatment of hydrogels

[0146] Under sterile conditions, FGH hydrogel and LGH hydrogel were incubated with culture medium simulating an inflammatory environment (purchased from Pronosei, catalog number PM150210B) for 4 days. After incubation, the supernatant of the two hydrogels was extracted to obtain extracts (FGH hydrogel and LGH hydrogel).

[0147] 4. Culture and polarization treatment of microglia

[0148] Microglia (HAPI cells, cell concentration 50,000 / cm³) were used. 2 Divided into 4 groups:

[0149] (1) Con group: Normal culture for 48 h, no treatment;

[0150] (2) LPS group: The cells in this group were stimulated with LPS (concentration of 1 μg / mL) and IFNγ (20 ng / mL) for 24 h to induce microglial cell polarization.

[0151] (3) LGH group: The cells in this group were stimulated with LPS (concentration of 1 μg / mL) and IFNγ (20 ng / mL) for 24 h to induce microglia polarization, and then the cells were treated with LGH hydrogel extract (LGH hydrogel) for 24 h.

[0152] (4) FGH group: The cells in this group were stimulated with LPS (concentration of 1 μg / mL) + IFNγ (20 ng / mL) for 24 h to induce microglia polarization, and then the cells were treated with FGH hydrogel extract (FGH hydrogel) for 24 h.

[0153] 5. Immunofluorescence staining

[0154] (1) CD86 receptor staining

[0155] CD86 receptor is a membrane surface antibody of microglia. When microglia M1 are polarized, the expression of CD86 receptor is upregulated. Therefore, CD86 antibody (purchased from Santa Cruz, catalog number sc28347) was used to label M1 type microglia. Immunofluorescence staining was performed on each group of microglia treated in step 4. Then, cell images were taken using a laser confocal microscope to record the staining intensity and distribution of CD86 in each group of microglia.

[0156] (2) CD206 receptor staining

[0157] CD206 antibody, also known as mannose receptor, is a membrane surface receptor for microglia. When microglia M2 are polarized, the expression of CD206 receptor is upregulated. Therefore, CD206 antibody (purchased from Abcam, catalog number ab64693) was used to label M2 type microglia. Immunofluorescence staining was performed on each group of microglia treated in step 4, and then cell images were taken using a laser confocal microscope to record the staining intensity and distribution of CD206 in each group of microglia.

[0158] The immunofluorescence staining results of CD86 receptor staining in microglia of each group are shown in Figure 9, and the immunofluorescence staining results of CD206 receptor staining in microglia of each group are shown in Figure 10.

[0159] The results show:

[0160] (1) CD86 (red) is distributed on the cell membrane. Cells with strong staining show a typical spindle-shaped appearance of M1 polarization. Among them, the CD86 staining intensity of the LPS group is significantly stronger than that of the Con group, indicating that HAPI cells are successfully polarized to the M1 state under the stimulation of LPS. The CD86 staining intensity of the LGH group is slightly weaker than that of the LPS group, but the difference is not obvious. The CD86 staining intensity of the FGH group is the lowest, which is not only significantly weaker than that of the LPS group, but also weaker than that of the LGH group. This indicates that under the inflammatory environment, due to the action of borate ester bonds and the targeting effect of the fluorescent liposome Cou6-Flip on microglia.

[0161] (2) CD206 (green) is distributed on the cell membrane. Cells with stronger staining show a typical M2 polarized round appearance. Among them, the CD206 staining intensity in the LPS group is significantly weaker than that in the Con group, indicating that HAPI cells reduce the generation of M2 state cells under LPS stimulation. The CD206 staining intensity in the LGH group is slightly stronger than that in the LPS group, but the difference is not significant. The CD206 staining intensity in the FGH group is the highest, indicating that in the inflammatory environment, due to the action of borate ester bonds and the targeting effect of the fluorescent liposome Cou6-Flip on microglia.

[0162] The above results indicate that FGH hydrogel has a significantly higher inhibitory effect on M1 polarization of microglia than LGH hydrogel.

[0163] 6. Flow cytometry analysis

[0164] After labeling each group of cells with CD86 antibody, the CD86 antibody content of single cells was detected by flow cytometry, and the proportion of CD86 positive cells in each group was calculated to assess the number of M1 microglia.

[0165] The flow cytometry results of CD86 receptor staining in microglia of each group are shown in Figure 11.

[0166] The results showed that: In Figure 11, AD represents the fluorescence distribution of microglia in each group, the horizontal axis represents fluorescence intensity, the vertical axis represents the number of cells at that fluorescence intensity, and the area under the curve represents the total number of cells. The fluorescence intensity of untreated cells fluoresced with CD86 was marked as a control in the statistical graph (vertical line position). The area under the curve to the right of the control reference line represents the number of CD86 positive cells. By calculating the proportion of CD86 positive cells in each group, it was found that the proportion of CD86 positive cells in the LPS group was 62.5%, which was significantly higher than that in the Con group (19.1%), indicating that LPS stimulation significantly increased the number of M1 polarized microglia. The proportions of CD86 positive cells in the LGH group and FGH group were 37.8% and 16.1%, respectively, which were lower than those in the LPS group.

[0167] The above results indicate that both LGH and FGH hydrogel extracts can inhibit M1 polarization in microglia, and FGH shows a significantly better inhibitory effect than LGH.

[0168] 7. Gene expression analysis

[0169] The expression levels of genes related to M1 and M2 microglia in each group of microglia were detected by qRT-PCR, including:

[0170] The M1-related genes detected include: CD86, IL1-β, iNOS, and TNF-α;

[0171] The M2-related genes detected include: ARG-1, IL-4, YM-1, and IL-10.

[0172] The results of gene expression detection for M1 type microglia in each group are shown in Figure 12, and the results of gene expression detection for M2 type microglia in each group are shown in Figure 13.

[0173] The results show:

[0174] (1) The expression level of M1-related genes in the LPS group was significantly higher than that in the Con group, while the relative expression level of M1-related genes in the LGH and FGH groups was lower than that in the LPS group, and the expression level in the FGH group was lower than that in the LGH group.

[0175] (2) The expression level of M2-related genes in the LPS group was significantly lower than that in the Con group, while the relative expression level of M1-related genes in the LGH and FGH groups was higher than that in the LPS group, and the expression level in the FGH group was higher than that in the LGH group.

[0176] The above results indicate that FGH hydrogel is significantly better than LGH hydrogel in inhibiting the expression of M1-related genes in vitro, while FGH hydrogel is significantly better than LGH hydrogel in promoting the expression of M2-related genes in vitro.

[0177] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0178] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of liposomes containing targeting ligands in the preparation of drug carriers for spinal cord injury, characterized in that, The targeting ligands include one or more of fingolimod, fingolimod hydrochloride, and fingolimod phosphate.

2. The application according to claim 1, characterized in that, The liposomes further comprise lipids, wherein the lipids include amphiphilic lipids and / or structural lipids.

3. The application according to claim 1, characterized in that, The drug carrier is loaded with bioactive components.

4. The application according to claim 3, characterized in that, The bioactive ingredients include one or more selected from small molecule drugs, nucleic acids, proteins or peptides.

Images