Functionalized targeted liposome and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a functionalized targeted liposome and application thereof, and the functionalized targeted liposome comprises lipid and a targeted ligand, wherein the targeting ligand can be specifically combined with a TREM2 receptor. According to the lipidosome, the targeting ligand can be specifically combined with the TREM2 receptor, accurate targeting of cells with the surface containing the TREM2 receptor is achieved, it is ensured that the cells directly act on target cells, the accuracy of a delivered substance is further improved, and the lipidosome has the advantages of being targeting, high in stability, small in side effect and high in bioavailability. The functionalized targeted liposome can be applied to the development of drug carriers, pharmaceutical compositions and the like, and has wide application prospects, for example, the liposome is dynamically connected with hydrogel molecules through a borate bond to prepare a hydrogel pharmaceutical composition containing the functionalized targeted liposome, and the application prospect is wide. The liposome disclosed by the invention can respond to an inflammatory environment according to pH and ROS changes of a surrounding environment to release the liposome disclosed by the invention, targets cells with TREM2 receptors on the surfaces, namely microglial cells (with the TREM2 receptors on the surfaces) by virtue of the target ligands exposed on the surfaces, regulates the polarization state of the cells, inhibits the polarization of the cells to a pro-inflammatory M1 type and promotes the polarization of the cells to an anti-inflammatory M2 type, so that the anti-inflammatory effect of the liposome is improved. Therefore, the local microenvironment of the spinal cord injury part is improved, and nerve regeneration is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to functionalized targeted liposomes and applications thereof. Background Art

[0002] Spinal cord injury (SCI) is a severe injury to the central nervous system that often leads to limb motor dysfunction, sensory loss, abnormal excretion function, and can even be life-threatening. Current treatments for SCI primarily include surgery, medication, and rehabilitation, but these treatments are generally ineffective. Besides surgery, methylprednisone is the only approved medication. Its primary effects are to reduce secondary inflammatory responses, repair damaged blood-spinal cord barriers, improve spinal cord blood supply, scavenge free radicals, and promote neurotrophic factor secretion. However, its use remains controversial. Some studies suggest that its use increases the risk of wound infection, elevated blood sugar, and gastrointestinal bleeding. Therefore, the latest guidelines recommend its use only within 8 hours of injury and for no more than 24 hours. Hyperbaric oxygen therapy, a traditional rehabilitation treatment for SCI, involves inhaling high-concentration oxygen at pressures 1-3 times higher than atmospheric pressure to reduce cell apoptosis, inflammation, and tissue edema. Although it has some therapeutic effects, it cannot meet comprehensive rehabilitation needs.

[0003] In recent years, the research and development of therapeutic drugs for spinal cord injury has mainly focused on neuroprotection, anti-inflammation and neuroregeneration. However, there are two major difficulties in the application of related therapeutic drugs in spinal cord injury: (1) It is difficult for drugs to form effective concentrations 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 have the risk of lung infection, and minocycline has the potential risk of liver damage. Therefore, it is difficult for spinal cord injury therapeutic drugs to work in the injury area by simple intravenous injection or local injection of drugs. There is an urgent need to develop new drug delivery systems for spinal cord injury.

[0004] As a drug delivery system, liposomes have good biocompatibility and targeting. In addition, after entering the human body, liposomes are easily phagocytosed by the mononuclear phagocyte system. Compared with traditional drug delivery methods, they have the advantages of fewer side effects, high drug utilization, long-term effective blood drug concentration, targeted drug delivery, and penetration of physiological barriers. However, traditional liposomes still have shortcomings in targeting, controllable drug release, and in vivo stability.

[0005] Therefore, the development of a drug liposome targeted delivery system with targeting, high stability and few side effects is of great significance for the treatment of spinal cord injury. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides functionalized targeted liposomes and their applications. The functionalized targeted liposomes of the present invention can specifically bind to the TREM2 receptor with the help of a targeting ligand, thereby achieving precise targeting of cells containing TREM2 receptors on the surface, ensuring that they act directly on the target cells, and further improving the accuracy of the delivered substance. The liposomes have targeting, high stability, and small side effects, and can be used to develop drug carriers, pharmaceutical compositions, etc., with broad application prospects. For example, the functionalized targeted liposomes of the present invention are dynamically connected to hydrogel molecules through a boronate bond to prepare a hydrogel pharmaceutical composition containing the liposomes of the present invention, which can release the functionalized targeted liposomes of the present invention in response to the inflammatory environment according to the pH and ROS changes of the surrounding environment, and target cells containing TREM2 receptors on the surface, i.e., microglia (with TREM2 receptors on the surface) by exposing the targeting ligand on the surface, and regulate their polarization state, inhibit their polarization to the pro-inflammatory M1 type, and promote their polarization to the anti-inflammatory M2 type, thereby improving the local microenvironment of the spinal cord injury site and promoting nerve regeneration.

[0007] The present invention is based on the inventor's discovery and understanding of the following problems:

[0008] Microglia originate from the mononuclear cell line in the bone marrow, enter the brain and spinal cord through the blood circulation, 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), nitric oxide (NO), etc. 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 response and tissue repair.

[0009] Upon activation, microglia differentiate into two polarized states: the M1 pro-inflammatory phenotype and the M2 anti-inflammatory phenotype. M1 microglia can secrete a variety of inflammatory factors, such as IL1-α, IL-1β, and TNF-α, thereby exacerbating the inflammatory response. Activated M2 microglia exert anti-inflammatory effects by phagocytosing cell debris or damaged neurons and secreting anti-inflammatory cytokines such as IL-4 and IL-10, thereby alleviating tissue inflammatory responses, promoting axonal growth and spinal cord regeneration, and thereby promoting tissue repair. Furthermore, relevant studies have shown that among the intrinsic cells of the nervous system, only microglia express TREM2 receptors in large quantities, and that TREM2 receptor expression in microglia is upregulated under inflammatory conditions. Therefore, the inventors proposed the development of a drug delivery system that can target 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, designed and prepared functionalized drug-loaded liposomes FlipE, and utilized the receptor ligand interaction between fingolimod (FTY720) and TREM2 receptors to enable the functionalized drug-loaded liposomes FlipE to target microglia with TREM2 receptors on their surface. Furthermore, the inventors dynamically connected the functionalized drug-loaded liposomes FlipE to the hydrogel through a boronate bond to prepare the hydrogel FlipE@GelMA / HAPBA. The hydrogel can release functionalized drug-loaded liposomes FlipE in response to the inflammatory environment according to the changes in pH and ROS in the surrounding environment, thereby achieving the responsive release of drugs (such as isoetzol). By exposing the FTY720 on the surface, it targets microglia with TREM2 receptors on the surface, regulates the polarization state of microglia, inhibits their polarization toward the pro-inflammatory M1 type, and promotes their polarization toward the anti-inflammatory M2 type, thereby improving the local microenvironment of the spinal cord injury site and promoting nerve regeneration.

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

[0012] Therefore, in the first aspect of the present invention, the present invention proposes a liposome. According to an embodiment of the present invention, it comprises: a lipid and a targeting ligand; wherein the targeting ligand can specifically bind to the TREM2 receptor. The liposome according to the embodiment of the present invention, with the help of the targeting ligand, can specifically bind to the TREM2 receptor, thereby achieving precise targeting of cells containing TREM2 receptors on the surface, ensuring that it directly acts on the target cells, i.e., microglia (with TREM2 receptors on the surface), further improving the accuracy of the delivered substance, and the liposome has targeting, high stability, and few side effects, and can be used to develop drug carriers, pharmaceutical compositions, etc., with broad application prospects.

[0013] According to an embodiment of the present invention, the liposome may further have the following additional technical features:

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

[0015] According to an embodiment of the present invention, the lipid comprises one or more of an amphiphilic lipid, a structural lipid and a PEG-lipid.

[0016] In a second aspect, the present invention provides a drug carrier. According to an embodiment of the present invention, the liposome described in the first aspect is included. The drug carrier according to an embodiment of the present invention utilizes the liposome to load drugs, has advantages such as targeting, low cytotoxicity, high stability, and good biocompatibility, and can accurately and efficiently deliver drugs to target cells, which is conducive to better exerting the efficacy of the loaded drugs, and can be used to treat diseases such as central nervous system damage.

[0017] In its third aspect, the present invention provides a complex. According to an embodiment of the present invention, the complex comprises: the liposome described in the first aspect or the drug carrier described in the second aspect; and a bioactive ingredient. The complex according to an embodiment of the present invention can be further prepared using the liposome to form a complex containing the bioactive ingredient. The complex can then accurately and efficiently deliver the bioactive ingredient to target cells, facilitating better utilization of the loaded bioactive ingredient and, consequently, being applicable to the treatment of diseases such as central nervous system damage.

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

[0019] According to an embodiment of the present invention, the bioactive components include 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 an embodiment of the present invention, the pharmaceutical composition comprises the liposome 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 an embodiment of the present invention, by including the aforementioned liposome, drug carrier, or complex of the present invention, leverages its advantages of targeting, low cytotoxicity, high stability, and good biocompatibility, thereby achieving the multifunctionality and synergistic therapeutic effects of the pharmaceutical composition, and is applied to the treatment of central nervous system injury diseases such as spinal cord injury.

[0021] According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may also 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 connected to the hydrogel via a boronate bond.

[0023] According to an embodiment of the present invention, the pharmaceutical composition has at least one of the following uses: prevention, relief, adjuvant treatment and / or treatment of central nervous system damage; anti-inflammation.

[0024] In the fifth aspect of the present invention, the present invention proposes the use of the liposome described in the first aspect, the drug carrier described in the second aspect, the complex described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a drug.

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

[0026] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 1 Figure 1 is a TEM observation result of the targeted liposome FlipE in Example 1 of the present invention, wherein A is the overall image of the targeted liposome FlipE under a TEM microscope, with a scale of 200 nm, and B is the detailed image of the targeted liposome FlipE under a TEM microscope, with a scale of 100 nm;

[0029] Figure 2 This is a graph showing the particle size measurement results of the targeted liposome FlipE in Example 1 of the present invention;

[0030] Figure 3 Figures 2 and 3 are SEM observation results of the hydrogel FlipE@GelMA / HAPBA in Example 3 of the present invention, wherein A is an SEM observation image of the hydrogel FlipE@GelMA / HAPBA, with a scale of 5 μm, and B is a local magnified image of the part of the hydrogel FlipE@GelMA / HAPBA containing the targeted liposome FlipE, with a scale of 500 nm;

[0031] Figure 4 These are the results of laser confocal microscopy observations of different cells after phagocytosis of fluorescent liposomes in Example 4 of the present invention, wherein A is a representative image of astrocytes phagocytosing ordinary fluorescent liposomes Cou6-Lip, B is a representative image of astrocytes phagocytosing targeted fluorescent liposomes Cou6-Flip, C is a representative image of neurons phagocytosing ordinary fluorescent liposomes Cou6-Lip, D is a representative image of neurons phagocytosing targeted fluorescent liposomes Cou6-Flip, E is a representative image of microglia phagocytosing ordinary fluorescent liposomes Cou6-Lip, and F is a representative image of microglia phagocytosing targeted fluorescent liposomes Cou6-Flip;

[0032] Figure 5 This is a statistical analysis result of the intracellular mean fluorescence intensity of different cells after phagocytosis of fluorescent liposomes in Example 4 of the present invention, where ** represents P < 0.01 and *** represents P < 0.001;

[0033] Figure 6 This is a graph showing the fluorescence imaging distribution results of spinal cord tissue samples of rats in each group 24 hours after injury in Example 5 of the present invention;

[0034] Figure 7 These are the immunofluorescence staining results of spinal cord tissue sections of rats in each group 24 hours after injury in Example 5 of the present invention, wherein A, from top to bottom, are TREM2 antibody staining (red) + targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue) of the cross section of the spinal cord in the Flip group, TREM2 antibody staining (red) + targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue) of the local magnified image, and targeted fluorescent liposome Cou6-Flip (green) + nuclear staining (blue) of the local magnified image. B, from top to bottom, are TREM2 antibody staining (red) + ordinary fluorescent liposome Cou6- Lip (green) + nuclear staining (blue), locally enlarged images of TREM2 antibody staining (red) + ordinary fluorescent liposome Cou6-Lip (green) + nuclear staining (blue), locally enlarged images of ordinary fluorescent liposome Cou6-Lip (green) + nuclear staining (blue) result graph, C from top to bottom are TREM2 antibody staining (red) + Cou6 fluorescent labeling (green) + nuclear staining (blue) of the spinal cord cross section of the SCI group, locally enlarged images of TREM2 antibody staining (red) + Cou6 fluorescent labeling (green) + nuclear staining (blue), locally enlarged images of Cou6 fluorescent labeling (green) + nuclear staining (blue);

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

[0036] Figure 9Figures 1 and 2 are immunofluorescence staining results of CD86 receptor staining of microglia in each group in Example 7 of the present invention, wherein A, from left to right, are CD86 staining (red), nuclear staining (blue) + mixed results of CD86 and nuclear staining of the Con group; B, from left to right, are CD86 staining (red), nuclear staining (blue) + mixed results of CD86 and nuclear staining of the LPS group; C, from left to right, are CD86 staining (red), nuclear staining (blue) + mixed results of CD86 and nuclear staining of the LGH group; and D, from left to right, are CD86 staining (red), nuclear staining (blue) + mixed results of CD86 and nuclear staining of the FGH group;

[0037] Figure 10 Figures 1 and 2 are immunofluorescence staining results of CD206 receptor staining of microglial cells in each group in Example 7 of the present invention, wherein A, from left to right, are CD206 staining (green), nuclear staining (blue) + mixed results of CD206 and nuclear staining of the Con group; B, from left to right, are CD206 staining (green), nuclear staining (blue) + mixed results of CD206 and nuclear staining of the LPS group; C, from left to right, are CD206 staining (green), nuclear staining (blue) + mixed results of CD206 and nuclear staining of the LGH group; and D, from left to right, are CD206 staining (green), nuclear staining (blue) + mixed results of CD206 and nuclear staining of the FGH group;

[0038] Figure 11 Figures 2 and 3 are flow cytometric analysis results of CD86 receptor staining of microglia in each group in Example 7 of the present invention, wherein A is a flow cytometric analysis graph of CD86 receptor staining of microglia in the Con group, B is a flow cytometric analysis graph of CD86 receptor staining of microglia in the LPS group, C is a flow cytometric analysis graph of CD86 receptor staining of microglia in the LGH group, and D is a flow cytometric analysis graph of CD86 receptor staining of microglia in the FGH group;

[0039] Figure 12 : The results of the detection of M1-type related genes in microglia in each group in Example 7 of the present invention are shown in Figure 1, where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. A is a graph showing the detection results of CD86 gene expression in microglia in each group, B is a graph showing the detection results of IL1-β gene expression in microglia in each group, C is a graph showing the detection results of iNOS gene expression in microglia in each group, and D is a graph showing the detection results of TNF-α gene expression in microglia in each group.

[0040] Figure 137 is a graph showing the expression detection results of M2-related genes in microglia in each group in Example 7 of the present invention, wherein * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. A is a graph showing the expression detection results of ARG-1 gene in microglia in each group, B is a graph showing the expression detection results of IL-4 gene in microglia in each group, C is a graph showing the expression detection results of YM-1 gene in microglia in each group, and D is a graph showing the expression detection results of IL-10 gene in microglia in each group. DETAILED DESCRIPTION

[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 understood 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 understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0043] The endpoints of the ranges and any values 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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0044] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0045] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[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 forms of multiple sclerosis and a sphingosine-1-phosphate (S1P) receptor modulator.

[0048] In this article, the term "TREM2 receptor" refers to a transmembrane receptor of the immunoglobulin superfamily, which is mainly expressed on myeloid cells, including microglia, macrophages, monocytes, dendritic cells, etc. The TREM2 receptor transmits signals by binding to the 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 species in the body, such as superoxide anions (O2 - ), hydrogen peroxide (HO) and hydroxyl radicals (OH-). In an inflammatory environment, ROS levels usually increase. The present invention utilizes this characteristic to design a drug release mechanism delivery system that can respond to ROS changes.

[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 cell debris and pathogens, etc. They play a key role in maintaining the homeostasis of the central nervous system and regulating inflammatory responses. Microglia express multiple receptors and specifically express TREM2 receptors, etc.

[0051] In this article, the term "hydrogel" is 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 the hydrogel enables it to absorb and retain a large amount of water to form a gel-like substance with good biocompatibility, degradability and mechanical properties. It can be used as a drug carrier for drug delivery systems, or as a tissue engineering scaffold to support cell growth and tissue repair, etc. In an embodiment of the present invention, the inventors dynamically connected the functionalized drug-loaded liposome FlipE to the hydrogel through a boronate bond to prepare the hydrogel FlipE@GelMA / HAPBA.

[0052] As used herein, the term "boronate bond" refers to a chemical bond formed by the reaction of boric acid with an alcohol compound. The boronate bond plays a crucial role in the present invention. It is used to connect a targeting ligand (such as FTY720) with a high molecular weight polymer (such as a hydrogel). This connection enables the pharmaceutical composition to responsively release the drug under specific inflammatory conditions. Specifically, the boronate bond is easily broken in an acidic environment (such as the pH value of the inflammatory area is generally low) and under conditions of high reactive oxygen species (ROS), thereby achieving intelligent drug release.

[0053] As used herein, the term "LPS" refers to lipopolysaccharide, a complex glycolipid present in the outer membrane of Gram-negative bacteria. LPS is a potent immunostimulant commonly used to induce inflammatory responses or polarization of cells. In an embodiment of the present invention, the substance is used to stimulate microglia to induce their polarization to the M1 type, thereby simulating an inflammatory state.

[0054] liposomes

[0055] The present invention proposes a liposome. According to an embodiment of the present invention, the liposome comprises: a lipid and a targeting ligand; wherein the targeting ligand can specifically bind to the TREM2 receptor. The liposome according to an embodiment of the present invention, with the help of the targeting ligand that can specifically bind to the TREM2 receptor, achieves precise targeting of cells containing TREM2 receptors on the surface, ensuring that it directly acts on the target cells, i.e., microglia (with TREM2 receptors on the surface), further improving the accuracy of the delivered substance. The liposome has targeting, high stability, and few side effects, and can be used to develop drug carriers, pharmaceutical compositions, etc., with broad application prospects.

[0056] According to an embodiment 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 includes fingolimod, fingolimod hydrochloride, fingolimod phosphate, and sphingosine 1-phosphate, which can be selected according to specific application conditions.

[0057] It should be noted that fingolimod is FTY720 in the embodiment of the present invention, which has the ability to specifically bind to the TREM2 receptor as a targeting ligand. Fingolimod hydrochloride, fingolimod phosphate and sphingosine 1-phosphate have structural similarities with FTY720. They are similar to fingolimod in chemical structure and can bind to the TREM2 receptor through a mechanism similar to FTY720, thereby achieving the effect of liposome targeted delivery of the present invention. Therefore, the tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs of FTY720 and the above compounds should all fall within the scope of protection of the present invention. These compounds can all be used as targeting ligands targeting the TREM2 receptor to achieve precise targeting of the functionalized targeted liposomes of the present invention to microglia (having TREM2 receptors on the surface).

[0058] According to an embodiment of the present invention, the lipid includes: one or more of an amphiphilic lipid, a structural lipid, and a PEG-lipid. According to an embodiment of the present invention, the amphiphilic lipid includes one or more selected from 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecene-sn-glycero-3-phosphocholine. , 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidoleonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, Acyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)phosphooxy)oxy)propionate, dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dimyristoylphosphatidylglycerol, dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dioleoylphosphatidylglycerol, 1,2-dioleoyl-sn-glycero Oil-3-(phospho-L-serine), cell fusion phospholipids, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylserine, distearoylphosphatidylcholine, distearoyl-phosphatidyl-ethanolamine, distearoylphosphoethanolamine imidazole, 1,2-diundecanoyl-sn-glycero-phosphocholine, egg phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphate, bis((S)-2-hydroxy-3-(oleoyloxy)propyl)ammonium phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-inositol), 1,2-distearoyl-sn-glycero-3-phospho-L-serine, 1,2-dilinoleoyl-sn-glycerol-3-phospho-L-serine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phospho-L-serine, 1-oleoyl-2-hydroxy-sn-glycerol-3-phospho-L-serine, 1-stearoyl-2-hydroxy-sn-glycerol-3-phospho-L-serine and one or more of sphingomyelin; the structural lipids include cholesterol, coprolol, maltosterol, maltose ... One or more of sterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid and α-tocopherol; the PEG-lipid includes one or more selected from PEG-DMG, 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide, PEG-DSPE, PEG-DSG, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-DAG, PEG-DPPE, PEG-PE, PEG-S-DAG, PEG-ceramide, PEG-dialkoxypropylcarbamate and PEG-c-DMA.

[0059] Drug carriers

[0060] The present invention provides a drug carrier. According to an embodiment of the present invention, the drug carrier includes the aforementioned liposome. The drug carrier according to the embodiment of the present invention utilizes the aforementioned liposome to load drugs, and has advantages such as targeting, low cytotoxicity, high stability, and good biocompatibility. It can accurately and efficiently deliver drugs to target cells, which is conducive to better exerting the efficacy of the loaded drugs, and can be used to treat diseases such as central nervous system damage.

[0061] Complex

[0062] The present invention provides a complex. According to an embodiment of the present invention, the complex comprises: the aforementioned liposome or the aforementioned drug carrier; and a bioactive ingredient. The complex according to an embodiment of the present invention can be further prepared using the aforementioned liposome to form a complex containing the bioactive ingredient. The complex can accurately and efficiently deliver the bioactive ingredient to target cells, facilitating better utilization of the loaded bioactive ingredient and, therefore, being applicable to the treatment of diseases such as central nervous system damage.

[0063] According to an embodiment of the present invention, the bioactive component includes one or more selected from DNA molecules, RNA molecules, proteins, polypeptides and small molecule drugs. Thus, the bioactive component can be selected according to actual application conditions.

[0064] According to an embodiment of the present invention, the replication of proteins and polypeptides is not particularly limited and can be a peptide chain, the protein itself, or a derivative of the above substances or a complex with other substances, for example, Cas protein or its partial domain peptide, protein loaded with nuclide, antibody and other substances.

[0065] According to an embodiment of the present invention, the bioactive component comprises DNA, RNA, a nucleic acid-protein complex, a nucleic acid-lipid complex, a nucleic acid-nuclide complex, etc., and the type of the nucleic acid is not particularly limited. Specific examples of the nucleic acid include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA, ribozymes, antisense oligonucleotides, plasmid DNA, peptide nucleic acids, triplex forming oligonucleotides (TFOs), or genes.

[0066] Pharmaceutical composition

[0067] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned liposome, the aforementioned drug carrier, or the aforementioned complex. The pharmaceutical composition according to an embodiment of the present invention, by including the aforementioned liposome, drug carrier, or complex of the present invention, leverages its advantages of targeting, low cytotoxicity, high stability, and good biocompatibility, thereby achieving multifunctionality and synergistic therapeutic effects, and is applicable to the treatment of central nervous system injuries such as spinal cord injury.

[0068] According to an embodiment of the present invention, the pharmaceutical composition is a drug-loaded hydrogel; the drug-loaded hydrogel further includes a biocompatible polymer matrix; wherein the targeting ligand is connected to the hydrogel via a boronate bond. Thus, the pharmaceutical composition can be in the form of a drug-loaded hydrogel, and the three-dimensional network structure of the hydrogel can well encapsulate and protect the functionalized targeted liposomes, drug carriers, complexes and active pharmaceutical ingredients of the present invention, preventing them from premature degradation or clearance in the body. It can not only support the stable release of the functionalized targeted liposomes, drug carriers, complexes and active pharmaceutical ingredients of the present invention, but also gradually degrade in the body, avoiding the side effects that may be caused by long-term implantation, and has broad application prospects. Wherein, the targeting ligand is connected to the hydrogel via a boronate bond. This design enables the pharmaceutical composition to responsively release the functionalized targeted 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 that the key ingredients can be accurately released in the inflammatory area. Through this responsive release, the functionalized targeted liposomes, drug carriers, complexes and active pharmaceutical ingredients of the present invention can play their maximum role where they are needed, significantly improving the therapeutic effect and safety. At the same time, the side effects of the functionalized targeted liposome, drug carrier, complex and active pharmaceutical ingredient released in non-target areas are reduced.

[0069] According to an embodiment of the present invention, the pharmaceutical composition has at least one of the following uses: prevention, alleviation, adjuvant therapy, and / or treatment of central nervous system injury; anti-inflammatory. Thus, the pharmaceutical composition of the present invention can be used in the prevention, alleviation, adjuvant therapy, and / or treatment of central nervous system injury, anti-inflammatory, and other aspects. For example, in the prevention and alleviation 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 adjuvant therapy 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 an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Thus, by adding the excipient, the flexibility of the pharmaceutical composition in coping with different application scenarios is further enhanced.

[0071] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring, coloring agent, or a combination thereof, which are known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329); except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is encompassed.

[0072] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0073] Administration of the pharmaceutical compositions of the present invention may be carried out by any acceptable mode of administration. The pharmaceutical compositions of the present invention may be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, and the current methods for preparing these dosage forms are known or obvious 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, buccal, 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 so as to allow the biologically active ingredients contained therein to be bioavailable after administration of the composition to the patient; in addition, the pharmaceutical composition administered to the subject or patient is in the form of one or more dosage units, and its use in the preparation of a medicament.

[0074] The present invention provides the use of the aforementioned liposome, the aforementioned drug carrier, the aforementioned complex or the aforementioned pharmaceutical composition in preparing drugs.

[0075] Those skilled in the art will appreciate that the features and advantages described above for liposomes, drug carriers, complexes or pharmaceutical compositions are also applicable to this use and will not be described in detail here.

[0076] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0077] Example 1: Preparation and observation of targeted liposome FlipE

[0078] 1. Preparation of targeted liposome FlipE

[0079] Take a round-bottom flask and dissolve 72 mg of soybean lecithin, 24 mg of cholesterol, 4 mg of FTY720 and 10 mg of isoelastin (EPT) in a chloroform-methanol mixture (5 mL of chloroform + 3 mL of methanol). Then, remove the solvent by rotary evaporation at 40°C under vacuum for 30 min. At this time, a transparent liposome film is formed at the bottom of the round-bottom flask. Then, add 30 mL of phosphate buffer PBS (10 mM, pH = 7.4) and stir with magnetic beads for 15 min to completely dissolve the film. Let it stand for 10 to 30 min to hydrate and obtain lipid The liposome solution was transferred to a 50 mL centrifuge tube, placed on ice, and intermittently treated with an ultrasonic probe for 3 minutes (intensity 30%, on for 5 seconds and off for 5 seconds), and then centrifuged in a centrifuge (4°C, 300-700 rpm) for 5 minutes. After centrifugation, the supernatant was quickly aspirated into a new centrifuge tube to obtain a targeted liposome FlipE sample solution; the targeted liposome FlipE sample solution was passed through 400 nm, 200 nm, and 100 nm polycarbonate membranes in sequence using a liposome extruder to obtain the targeted liposome FlipE.

[0080] 2. TEM observation of targeted liposome FlipE

[0081] The microscopic morphology of the obtained targeted liposome FlipE was observed using a transmission electron microscope (TEM, Tecnai Spirit, FEI, Czech Republic). The specific steps are as follows:

[0082] Place a 300-mesh copper grid with a carbon film on filter paper with the front side facing up; dilute the targeted liposome FlipE sample solution (3.6 mg / mL) obtained in step 1 20 times with PBS, take 10 μL of the diluted targeted liposome FlipE sample solution and drop it on the surface of the copper grid, let it stand for 2 minutes, add 10 μL of 2% uranyl acetate solution on the surface of the copper grid for negative staining, let it stand in the dark for 30 seconds, absorb the dye with filter paper, then add 10 μL of pure water to the surface of the copper grid to wash away the excess dye, then use filter paper to absorb the pure water, finally, place the copper grid under the TEM observation table for observation and photography.

[0083] The TEM observation results of the targeted liposome FlipE are shown in Figure 1 .

[0084] The results showed that the targeted liposome FlipE presented a relatively uniform round granular structure under the microscope, in which the magnified image showed that its outer layer was coated with the drug EPT.

[0085] 3. Particle size determination of targeted liposome FlipE

[0086] The targeted liposome FlipE sample solution (3.6 mg / mL) obtained in step 1 was diluted 10 times with PBS, and 20 μL of the diluted targeted liposome FlipE sample solution was placed in 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.

[0087] The particle size determination results of the targeted liposome FlipE are shown in Figure 2 .

[0088] The results showed that the targeted liposome FlipE had a uniform particle size in solution.

[0089] Example 2: Preparation of targeted fluorescent liposome Cou6-Flip

[0090] The preparation process of coumarin-targeted fluorescent liposome Cou6-Flip in Example 2 is the same as the preparation of targeted liposome FlipE in step 1 of Example 1, with the following differences:

[0091] In a round-bottom flask, 72 mg of soybean lecithin, 24 mg of cholesterol, and 4 mg of FTY720 were dissolved in a chloroform-methanol mixture (5 mL of chloroform + 3 mL of methanol), and 10 μL of coumarin 6-chloroform solution was added. The solvent was then removed by rotary evaporation under vacuum at 40°C for 30 min in the dark. The remaining steps were the same as step 1 in Example 1.

[0092] The preparation steps of coumarin 6-chloroform solution are as follows: 8 mg of coumarin 6 powder is quantitatively weighed and dissolved in 1 mL of chloroform to obtain coumarin 6-chloroform solution.

[0093] Thus, the targeted fluorescent liposome Cou6-Flip was prepared.

[0094] Comparative Example 1: Preparation of ordinary liposomes Lip and ordinary fluorescent liposomes Cou6-Lip

[0095] (1) Preparation of ordinary liposomes Lip

[0096] The preparation process of the common liposome Lip in Comparative Example 1 is the same as that of the preparation of the targeted liposome FlipE in step 1 of Example 1, except that:

[0097] Take a round-bottom flask and dissolve 72 mg of soybean lecithin, 24 mg of cholesterol, and 10 mg of isoelastin (EPT) in a chloroform-methanol mixture (5 mL of chloroform + 3 mL of methanol). The remaining steps are the same as step 1 in Example 1, thereby obtaining a common liposome Lip sample solution and common liposome Lip.

[0098] (2) Preparation of common fluorescent liposome Cou6-Lip

[0099] The preparation process of the common fluorescent liposome Cou6-Lip in Comparative Example 1 is the same as that of the targeted fluorescent liposome Cou6-Flip in Example 2, except that:

[0100] FTY720 (4 mg) was not added, and the remaining steps were the same as in Example 2.

[0101] Example 3: Preparation and Observation of Hydrogel FlipE@GelMA / HAPBA

[0102] 1. Preparation of hydrogel FlipE@GelMA / HAPBA

[0103] 160 mg of methacryloyl gelatin (GelMA), 20 mg of 4-hydroxybenzoic acid (HAPBA, cross-linking agent), and 2 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were weighed and dissolved in 1 mL of PBS solution. The raw materials were completely dissolved by heating in a 70°C water bath to obtain a photocrosslinked hydrogel precursor solution. The photocrosslinked hydrogel precursor solution was then mixed with 1 mL of the targeted liposome FlipE sample solution (3.6 mg / mL) prepared in Example 1. After thorough mixing, the solution was cross-linked under UV light for 200 s to obtain the hydrogel FlipE@GelMA / HAPBA (FGH).

[0104] 2. Observation of hydrogel FlipE@GelMA / HAPBA

[0105] The hydrogel FlipE@GelMA / HAPBA was placed in a 1.5 mL centrifuge tube, and an appropriate amount of liquid nitrogen was poured into it to quickly cool it down. It was then placed in a -20°C refrigerator for pre-freezing for 24 hours. After the pre-freezing was completed, it was freeze-dried in a vacuum dryer to obtain a freeze-dried sample of the hydrogel FlipE@GelMA / HAPBA. The freeze-dried sample of the hydrogel FlipE@GelMA / HAPBA was pasted on the stage (with the fresh cross-section facing up) using conductive tape, and a 5 nm thick carbon film was sprayed on the surface of the freeze-dried sample of the hydrogel FlipE@GelMA / HAPBA. It was then loaded into a scanning electron microscope (SEM, GEMINISEM 500, Zeiss, Germany) for on-machine observation.

[0106] The SEM observation results of hydrogel FlipE@GelMA / HAPBA are shown in Figure 3 .

[0107] The results showed that the prepared drug-loaded hydrogel FlipE@GelMA / HAPBA had a porous structure, and under magnified viewing angle, regular spherical targeted liposomes FlipE could be observed connected to the hydrogel.

[0108] Example 4: Targeted fluorescent liposome Cou6-Flip targets microglia in vitro

[0109] Neuronal cells (HT22), microglia (HAPI) and astrocytes were cultured adherently in 6-well plates, respectively. The initial density of cells in each well was 500,000 / well. The cell state was observed 12 hours after seeding, and the culture medium was replaced with 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 minutes each, then replaced with normal culture medium, and observed using a laser confocal microscope. The fluorescence intensity of the cells in each group was analyzed by flow cytometer, wherein the laser confocal microscope observation selected bright field combined with AF488 channel for observation and photography.

[0110] The results of laser confocal microscopy observation of different cells after phagocytosis of fluorescent liposomes are shown in Figure 4 The statistical analysis results of the intracellular average fluorescence intensity after different cells engulfed fluorescent liposomes are shown in Figure 5 .

[0111] The results show:

[0112] (1) Laser confocal microscopy observation results: Bright field results showed the cell morphology, in which the green part represented the fluorescent particles phagocytosed by the cells (i.e., ordinary fluorescent liposomes Cou6-Lip or targeted fluorescent liposomes Cou6-Flip). Among the three types of cells, microglia showed the strongest fluorescence intensity, and the fluorescence of microglia in the targeted fluorescent liposomes Cou6-Flip group was stronger than that in the ordinary fluorescent liposomes Cou6-Lip group;

[0113] (2) Statistical analysis results of the intracellular mean fluorescence intensity: The fluorescence intensity of microglia that engulfed 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 engulfed the fluorescently labeled ordinary fluorescent liposome Cou6-Lip.

[0114] The above results indicate that the modification of FTY720 significantly enhances the affinity of liposomes to microglia.

[0115] Example 5: Targeted fluorescent liposome Cou6-Flip binds to TREM2 receptors and targets microglia in vivo

[0116] 1. Construction of a 1mm complete transection model of the rat spinal cord T9 segment

[0117] After the rats were completely anesthetized, a shaver was used to remove the hair on the back. The skin and subcutaneous tissue were cut along the midline of the back with a scalpel to separate the subcutaneous fascia. The muscle tissue was separated along the spinous process on both sides with a scalpel. The skin and muscles were retracted with a retractor. The ligament tissue on both sides of the T9 segment was cut with ophthalmic scissors. The T9 segment was held with toothed ophthalmic forceps. The spine was lifted up to expose the intervertebral space. Bone rongeurs were used to bite off the bone along the intervertebral space to expose the spinal cord. The rats were then transferred under a microscope. Microscissors were used to gently cut the dura mater on the surface of the spinal cord. Microscissors were used to quickly cut the spinal cord. 1 mm long spinal cord tissue was cut under a transparent ruler. The surgical area was then rinsed with saline and hemostasis was strictly enforced. After confirming that there was no active bleeding, a 1 mm complete transection model of the rat spinal cord T9 segment was obtained; 1 mL of a solution containing the ordinary fluorescent liposome Cou6-Lip (3.6 mg / mL, numbered as the Lip group) prepared in Comparative Example 1, a solution containing the targeted fluorescent liposome Cou6-Flip (3.6 mg / mL, numbered as the Flip group) prepared in Example 2, and a 1 mm complete transection model of the rat spinal cord T9 segment were injected into the rats using a syringe. After the construction was completed, no treatment was performed (numbered as the SCI group). After waiting for 15 minutes to confirm that there was no leakage of the solution, the rat's back muscles and fascia were sutured continuously, and the skin was sutured intermittently.

[0118] 2. Infusion

[0119] After 24 h of normal feeding of rats in each group, the rats were completely anesthetized, fixed in supine position on a foam board, transferred to a negative pressure perfusion table, the perfusion pump speed was adjusted to 50 rpm / s, the inlet was connected to PBS solution and marked; the abdominal cavity was exposed, the skin was separated upward along the abdominal cavity to the rib margin, the diaphragm was punctured along the rib margin to cause the lung to shrink and collapse, the heart beating position was observed and the diaphragm was quickly cut open, the thorax was cut open and fixed while looking directly at the heart position to fully expose the heart; and after a small incision was made in the apex of the heart, the perfusion needle at the PBS end was quickly inserted into the aorta through the apex of the heart, and the right atrial appendage was cut open after the perfusion needle was fixed to allow blood to flow out; the perfusion pump was turned on and timed for 15 min, and 250 mL of perfusion was fully placed. Blood was exchanged, and after the replacement was complete, the input port was replaced with 4% paraformaldehyde solution and perfusion was continued for 15 minutes; after the perfusion was completed, the rat spine was separated, the spine was transected from the cervical to the sacral part, and the paraconical tissue was separated. The vertebral lamina was bitten open along the ventral side with bone forceps, the spinal cord was separated, and the spinal cord tissue was completely removed, immersed in 4% paraformaldehyde and labeled; then the spinal cord tissue and viscera were immersed in 4% paraformaldehyde and stored at 4°C for 24 hours to obtain spinal cord tissue samples and visceral samples; then the spinal cord tissue samples and visceral samples were transferred to 10%, 20% and 30% sucrose solutions (dissolved in PBS) for gradient dehydration, and the spinal cord tissue samples and visceral samples were sunk to the bottom after dehydration was completed.

[0120] 3. Fluorescence intensity detection of spinal cord injury area

[0121] After perfusion and sampling, the fluorescence intensity of the injured area was detected using a live imaging device. First, the spinal cord tissue sample obtained in step 2 was wiped dry, placed on a black background paper, and placed in the live imaging device for detection. The parameters of the live imaging device were set as follows: exposure time: 0.5 s, detection wavelength range: 480 nm to 520 nm.

[0122] The fluorescence imaging distribution results of the spinal cord tissue samples of rats in each group 24 hours after injury are shown in Figure 6 .

[0123] The results showed that different colors represent the numerical values of fluorescence intensity within the range, and the right side is the reference scale for the fluorescence intensity value. After eliminating the background fluorescence, no fluorescence distribution was observed in the spinal cord injury area of the rats in the SCI group, indicating that no exogenous fluorescence was detected in the spinal cord injury area; while exogenous fluorescence was detected in the spinal cord injury area of the rats in the Flip group and Lip group, and the fluorescence distribution range of the spinal cord injury area in the Flip group was significantly larger than that in the Lip group. At the same time, the fluorescence intensity of the Flip group (yellow) was also significantly higher than that of the Lip group (red).

[0124] The above results show that compared with the Lip group, more liposomes were enriched in the spinal cord injury area of the Flip group one day after spinal cord injury in rats.

[0125] 4. Spinal cord tissue sections

[0126] For the spinal cord tissue sections obtained in step 2, first use a tissue embedding agent (Optimal Cutting Temperature Compound, OCT) to completely embed the spinal cord tissue, check the embedding agent and remove bubbles in the embedding agent, and place the embedded spinal cord tissue sample on a quick-freezing table for quick freezing; then connect the embedded spinal cord tissue sample to the sample stage of the freezing microtome through the stage, and use a crank to perform continuous sectioning, each section with a thickness of 10 μm to obtain a spinal cord tissue section sample; flatten the spinal cord tissue section sample and completely attach it to an adhesive slide, paying attention to distinguishing the front and back sides, then dry the slide in the dark and ventilated for 1 to 2 hours to obtain a slide containing the spinal cord tissue section, and then place the slide containing the spinal cord tissue section in a section box and freeze it at -80°C for later use.

[0127] 5. Immunofluorescence staining

[0128] After the spinal cord tissue was sliced, TREM2 immunofluorescence staining was performed. The specific steps are as follows:

[0129] The slide containing the spinal cord tissue section stored at -80℃ was taken out and rewarmed at room temperature for 1.5h. The rewarmed spinal cord tissue section was placed in a rinse box containing an appropriate amount of PBS and washed three times for 5min each time. The spinal cord tissue section was then transferred to a wet box for operation. First, the liquid on the surface of the section was wiped dry. Then, the antibody incubation range was circled around the tissue using an immunohistochemistry pen, and 0.3% TritonX-100+10% goat serum (diluted with PBS) permeabilization + blocking solution was added dropwise to the surface of the spinal cord tissue slices to completely cover the tissue, and blocked at room temperature for 2 hours; after blocking was completed, the blocking solution on the surface of the slices was dried, and 0.3% TritonX-100+5% goat serum (diluted with PBS) was prepared to dilute the TREM2 antibody; the antibody was then diluted according to the corresponding proportion indicated in the kit instructions and incubated on the tissue surface, and placed in a 4°C refrigerator for more than 16 hours; the spinal cord tissue slices were then taken out and reheated at room temperature for 1 hour, the antibody dilution solution was discarded, and the slices were rinsed 3 times in the rinse box for 5 minutes each time to thoroughly clean the antibodies, and the liquid around the tissue was wiped dry, and a mounting medium containing DAPI was added to the spinal cord tissue slices, and the coverslips were covered with microscope coverslips, paying attention to avoid bubbles, and the spinal cord tissue slices with the sealing completed were obtained; the spinal cord tissue slices with the sealing completed were dried in a ventilated place, and the edges were checked for protrusions and unevenness, and finally an automatic slide scanner (Axio Scan.Z1, Zeiss, Germany) was used to photograph and observe the sealed spinal cord tissue sections, with a scanning magnification of 10 times.

[0130] The immunofluorescence staining results of spinal cord tissue sections of rats in each group 24 hours after injury are shown in Figure 7 .

[0131] The results showed that 24 hours after injury, liposome fluorescence was observed in the spinal cord tissue of rats in the Flip group and the Lip group, indicating that the liposomes were locally taken up by the spinal cord tissue; among them, the fluorescence distribution range of the Flip group was significantly larger than that of 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; further, in the magnified field of view, the liposome fluorescence (green) of the Flip group and the TREM2 antibody staining fluorescence (red) showed obvious colocalization (yellow), and at the same time, the colocalization (yellow) range of the Flip group was significantly larger than that of the Lip group.

[0132] The above results indicate that the affinity between the targeted fluorescent liposome Cou6-Flip and spinal cord tissue cells is achieved through binding to the TREM2 antibody.

[0133] Example 6: Drug release experiment of hydrogel FlipE@GelMA / HAPBA

[0134] 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 (pH = 5.0) containing H2O2 (100 μM), respectively. The release liquid was taken out every 24 h, and the corresponding buffer was replenished after each release liquid was taken out; the released liquid was dissolved in 100 uL of chloroform, and the drug concentration was detected by HPLC.

[0135] The drug release experimental results of hydrogel FlipE@GelMA / HAPBA are shown in Figure 8 .

[0136] The results showed that when the hydrogel FlipE@GelMA / HAPBA was in an environment of pH = 7.4, the release curve of the drug isoelastin was relatively flat, and 10% of the total drug loading was released on the 7th day; when the hydrogel FlipE@GelMA / HAPBA was in an environment of pH = 5.0, its drug release rate was higher than that of pH = 7.4, and 35% of the total drug loading was released on the 7th day; when the hydrogel FlipE@GelMA / HAPBA was in an environment of pH = 5.0 and high ROS, the drug was released at a high speed in the first 4 days, the release rate slowed down from the 6th to the 7th day, and 44% of the total drug loading was released at the end of the 7th day.

[0137] The above results indicate that the hydrogel FlipE@GelMA / HAPBA prepared in Example 3 can achieve responsive drug release in a low pH and high ROS environment.

[0138] Example 7: Characterization of the performance of hydrogel FlipE@GelMA / HAPBA in regulating microglial polarization in vitro

[0139] 1. Preparation of hydrogel FlipE@GelMA / HAPBA

[0140] 160 mg of methacryloyl gelatin (GelMA), 20 mg of 4-hydroxybenzoic acid (HAPBA, cross-linking agent), and 2 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were weighed and dissolved in 1 mL of PBS solution. The raw materials were completely dissolved by heating in a 70°C water bath to obtain a photocrosslinked hydrogel precursor solution. The photocrosslinked hydrogel precursor solution was then mixed with 1 mL of the targeted liposome FlipE sample solution (3.6 mg / mL) prepared in Example 1. After thorough mixing, the solution was cross-linked under UV light for 200 s to obtain the hydrogel FlipE@GelMA / HAPBA (numbered FGH).

[0141] 2. Preparation of hydrogel lipE@GelMA / HAPBA

[0142] 160 mg of methacryloyl gelatin (GelMA), 20 mg of 4-hydroxybenzoic acid (HAPBA, cross-linker), and 2 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were weighed and dissolved in 1 mL of PBS solution. The raw materials were completely dissolved by heating in a water bath at 70°C to obtain a photocross-linked hydrogel precursor solution. The photocross-linked hydrogel precursor solution was then mixed with 1 mL of the ordinary liposome Lip sample solution (3.6 mg / mL) prepared in Comparative Example 1. After thorough mixing, the mixture was cross-linked under UV light for 200 s to obtain the hydrogel lipE@GelMA / HAPBA (numbered LGH).

[0143] 3. Pretreatment of hydrogel

[0144] Under a sterile environment, FGH hydrogel and LGH hydrogel were incubated with culture medium simulating inflammatory environment (purchased from Pronocell, product number PM150210B) for 4 days. After the incubation was completed, the supernatants of the two hydrogel incubation treatments were extracted to obtain extracts (FGH hydrogel) and extracts (LGH hydrogel).

[0145] 4. Microglial cell culture and polarization treatment

[0146] Microglial cells (HAPI cells, cell concentration of 50,000 / cm 2 ) are divided into 4 groups:

[0147] (1) Con group: cultured normally for 48 h without any treatment;

[0148] (2) LPS group: cells in this group were stimulated with LPSLPS (concentration of 1 μg / mL) and IFNγ (20 ng / mL) for 24 h to induce microglial polarization;

[0149] (3) LGH group: LPS (concentration of 1 μg / mL) and IFNγ (20 ng / mL) were used to stimulate the cells for 24 h to induce polarization of microglial cells, and then the cells were treated with LGH hydrogel extract (LGH hydrogel) for 24 h;

[0150] (4) FGH group: LPS (concentration of 1 μg / mL) + IFNγ (20 ng / mL) was used to stimulate the cells in this group for 24 h to induce polarization of microglial cells in this group, and then the cells in this group were treated with FGH hydrogel extract (FGH hydrogel) for 24 h.

[0151] 5. Immunofluorescence staining

[0152] (1) CD86 receptor staining

[0153] The CD86 receptor is a membrane surface antibody of microglia. When microglia M1 polarize, the expression of CD86 receptor is upregulated. Therefore, CD86 antibody (purchased from Santa Cruz, catalog number sc28347) was used to label M1 microglia. Immunofluorescence staining was performed on each group of microglia treated in step 4, and then cell images were captured using a laser confocal microscope to record the staining intensity and distribution of CD86 in each group of microglia.

[0154] (2) CD206 receptor staining

[0155] CD206 antibody, also known as mannose receptor, is a membrane surface receptor of microglia. When microglia M2 polarize, it will upregulate the expression of CD206 receptor. Therefore, CD206 antibody (purchased from Abcam, catalog number ab64693) was used to label M2 microglia. Immunofluorescence staining was performed on each group of microglia treated in step 4, and then cell images were captured using a laser confocal microscope to record the staining intensity and distribution of CD206 in each group of microglia.

[0156] The immunofluorescence staining results of CD86 receptor staining of microglia in each group are shown in Figure 9 The immunofluorescence staining results of CD206 receptor staining of microglia in each group are shown in Figure 10 .

[0157] The results show:

[0158] (1) CD86 (red) is distributed on the cell membrane, and cells with stronger staining showed a typical spindle-shaped appearance of M1 polarization. Among them, the CD86 staining intensity in the LPS group was significantly stronger than that in the Con group, indicating that HAPI cells were successfully polarized to the M1 state under the stimulation of LPS; the CD86 staining intensity in the LGH group was slightly weaker than that in the LPS group, but the difference was not significant; and the CD86 staining intensity in the FGH group was the lowest, which was not only significantly weaker than that in the LPS group, but also weaker than that in the LGH group, indicating that under the inflammatory environment, due to the action of the boronate bond and the targeting effect of the targeted fluorescent liposome Cou6-Flip on microglia.

[0159] (2) CD206 (green) is distributed on the cell membrane, and cells with stronger staining exhibit a typical M2 polarized, round-shaped appearance. The CD206 staining intensity in the LPS group was significantly weaker than that in the Con group, indicating that HAPI cells reduced the generation of M2 cells under the stimulation of LPS; the CD206 staining intensity in the LGH group was slightly stronger than that in the LPS group, but the difference was not significant; and the CD206 staining intensity in the FGH group was the highest, indicating that under an inflammatory environment, due to the action of the boronate bond and the targeting effect of the targeted fluorescent liposome Cou6-Flip on microglia.

[0160] The above results show that the inhibitory effect of FGH hydrogel on microglial M1 polarization is significantly higher than that of LGH hydrogel.

[0161] 6. Flow cytometry analysis

[0162] After each group of cells was labeled with CD86 antibody, the single-cell CD86 antibody content was detected by flow cytometry, and the proportion of CD86-positive cells in each group of cells was calculated to evaluate the number of M1 microglia.

[0163] The results of flow cytometric analysis of CD86 receptor staining of microglia in each group are shown in Figure 11 .

[0164] The results show: Figure 11 AD in the middle are the fluorescence distribution of microglia in each group, the horizontal axis is the fluorescence intensity, the vertical axis is the cell number statistics under the fluorescence intensity, the area under the curve represents the total number of cells, and the fluorescence intensity of untreated cells exposed to CD86 fluorescence is marked in the statistical chart as the control (vertical line position), and the area under the curve to the right of the control reference line is the number of CD86-positive cells; by calculating the proportion of CD86-positive cells in the total 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 19.1% of the Con group, indicating that LPS stimulation significantly increased the number of M1-polarized microglia; while 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.

[0165] The above results show that the extracts of both LGH and FGH hydrogels can inhibit the M1 polarization of microglia. At the same time, the inhibitory effect of FGH is significantly better than that of LGH.

[0166] 7. Gene Expression Analysis

[0167] qRT-PCR was used to detect the expression levels of M1 and M2 microglia-related genes in microglia of each group, including:

[0168] The M1-related genes detected included: CD86, IL1-β, iNOS, and TNF-α;

[0169] The M2-related genes detected include: ARG-1, IL-4, YM-1, and IL-10.

[0170] The results of the expression of M1-related genes in microglia of each group are shown in Figure 12 The results of the expression of M2-related genes in microglia of each group are shown in Figure 13 .

[0171] The results show:

[0172] (1) The expression of M1-related genes in the LPS group was significantly higher than that in the Con group, while the relative expression of M1-related genes in the LGH and FGH groups was lower than that in the LPS group, and the FGH group was lower than the LGH group.

[0173] (2) The expression of M2-related genes in the LPS group was significantly lower than that in the Con group, while the relative expression of M1-related genes in the LGH and FGH groups was higher than that in the LPS group, and the expression in the FGH group was higher than that in the LGH group.

[0174] The above results indicate that FGH hydrogel is significantly better than LGH hydrogel in inhibiting the expression of M1-related genes in vitro. Meanwhile, FGH hydrogel is significantly better than LGH hydrogel in promoting the expression of M2-related genes in vitro.

[0175] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0176] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A liposome, characterized in that include: lipids and targeting ligands; Wherein, the targeting ligand can specifically bind to the TREM2 receptor.

2. The liposome according to claim 1, wherein The targeting ligand includes one or more of fingolimod, fingolimod hydrochloride, fingolimod phosphate and sphingosine 1-phosphate.

3. The liposome according to claim 1, wherein The lipids include: One or more of amphiphilic lipids, structured lipids and PEG-lipids.

4. A drug carrier, characterized in that The liposome comprises the liposome according to any one of claims 1 to 3.

5. A composite, characterized in that include: The liposome according to any one of claims 1 to 3 or the drug carrier according to claim 4; and bioactive ingredients.

6. The composite according to claim 5, characterized in that The bioactive components include one or more selected from DNA molecules, RNA molecules, proteins, polypeptides and small molecule drugs.

7. A pharmaceutical composition, characterized in that include: The liposome according to any one of claims 1 to 3, the drug carrier according to claim 4, or the complex according to claim 5 or 6.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is a drug-loaded hydrogel; The drug-loaded hydrogel further comprises a hydrogel; Wherein, the targeting ligand is connected to the hydrogel via a boronate bond.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition has at least one of the following uses: Prevent, alleviate, assist in treating and / or cure central nervous system damage; Anti-inflammatory.

10. Use of the liposome according to any one of claims 1 to 3, the drug carrier according to claim 4, the complex according to claim 5 or 6, or the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of a drug.

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