Liposome drug delivery system of targeted microglial cell Cx43 as well as preparation method and application of liposome drug delivery system

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a liposome drug delivery system of targeted microglial cells Cx43 as well as a preparation method and application of the liposome drug delivery system. The preparation method comprises the following steps: utilizing nano-liposome of a lipid bilayer formed by cationic lipid and neutral phospholipid, simultaneously adding methylacryloyl chitosan and TAT-CX43 polypeptide, encapsulating the methylacryloyl chitosan and the TAT-CX43 polypeptide in the nano-liposome, and finally performing crosslinking under an ultraviolet condition to obtain the nano-lipid colloid. The nano lipid colloid disclosed by the invention has better cell affinity; moreover, the TAT-CX43 polypeptide can enter cells, escape from decomposition of endolysosome and then slowly release the polypeptide in cytoplasm, so that the slow release performance of the medicine is improved. The nano lipid colloid targeting microglial cells Cx43 prepared by the invention can achieve the effect of slow release for 14 days in vivo, and the half-life period and bioavailability of drugs are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a liposome drug delivery system targeting microglial Cx43, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by slow and progressive memory dysfunction. It is the main cause of dementia in the elderly in the world today. With the increasing aging of society, the global incidence of AD has also increased dramatically. According to the latest statistics from the Lancet in 2019, AD has become the eighth leading cause of death in human society; and according to the latest epidemiological forecasts, more than 130 million people in the world will suffer from AD by 2050. However, to date, the pathological mechanism of AD is still unclear, which has directly led to no significant progress in the diagnosis and treatment of AD. For example, the long-term treatment strategy is still mainly to reduce Aβ deposition and neurofibrillary tangles, but related clinical trials have all ended in failure. Even if the FDA recently approved new drugs through the fast track, it is not enough to solve the fundamental clinical problems. Therefore, exploring the mechanism of AD and finding new therapeutic targets and developing new drugs will be of great significance to alleviate the burden of elderly diseases such as AD.

[0003] In AD-related studies, the use of amyloid β protein fragment 25-35 (Aβ25-35) to simulate AD pathology in vitro can cause microglial activation, and the Cx43 membrane potential of microglial cells is significantly increased, the hemichannels are opened, and the release of microglial proinflammatory factors such as TNFa and IL1b is promoted, causing neurotoxicity (Orellana, JA, et al., Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels. Journal of Neuroscience, 2011. 31 (13): p. 4962-4977.), suggesting that Cx43 can intervene in the treatment of AD by regulating microglial function. TAT-Cx43 polypeptide is modified based on the TAT peptide sequence of Cx43 protein, in which the TAT fragment is a cell permeable peptide segment. TAT-Cx43 peptide can inhibit the activity of Connexin43 (Cx43) glial hemichannels in glial cells, reduce the entry of cytotoxic substances in the extracellular environment into neurons, and thus protect neurons from damage. In addition, TAT-Cx43 can selectively inhibit the activity of Cx43 glial hemichannels in glial cells without affecting the functions of other channels or proteins. It has potential application prospects in the treatment of nervous system diseases and injuries, such as stroke, brain injury and neurodegenerative diseases. For example, Gangoso et al. found that TAT-Cx43 266-283 It can reduce c-Src-mediated hemichannel activity in astrocytes and exert neuroprotective effects (Ester Gangoso, Rocío Talaverón, Myriam Jaraíz-Rodríguez, et al. A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity[J]. Frontiers in Molecular Neuroscience, 2017, 10(10): 10).

[0004] However, in practical applications, although peptide drugs have the advantages of relatively stable in vitro properties (compared to proteins and some small molecule drugs), good biocompatibility, and low toxic side effects, after being injected into animals, they are easily decomposed by endopeptidases, resulting in a short half-life and low bioavailability. At the same time, peptide drugs have limited permeability to the blood-brain barrier and are difficult to be released in vivo to exert their effects. These shortcomings limit the practical application of peptide drugs in disease treatment, especially in neurological diseases. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art and provide a method for preparing nano lipid colloids targeting microglial Cx43.

[0006] The purpose of the present invention is to provide a nano lipid colloid targeting microglial Cx43.

[0007] Another object of the present invention is to provide the use of the nanolipid colloid targeting microglial Cx43 in the preparation of drugs for preventing and treating Alzheimer's disease.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing a nanolipid colloid targeting microglial Cx43, comprising the following steps:

[0010] S1. Preparation of lipid films of cationic lipids and neutral phospholipids;

[0011] S2. The methacrylated chitosan, the photoinitiator, the TAT-CX43 polypeptide and the acid-containing osmotic pressure regulator are mixed and dissolved to obtain a mixed solution, and the mixed solution is soaked in a lipid film;

[0012] S3. After soaking, the multilayer membrane structure is removed by filtration, centrifuged, the precipitate is resuspended, and cross-linked by ultraviolet light to obtain nano-lipid colloid;

[0013] Wherein, the amino acid sequence of the TAT-CX43 polypeptide is as shown in SEQ ID NO: 1 or has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1;

[0014] In step S2, the mass ratio of methacryloyl chitosan to photoinitiator in the mixed solution is 1:1-2.5.

[0015] The present invention delivers TAT-Cx43 polypeptide through nanolipid colloids to inhibit microglial Cx43 hemichannels, thereby exerting the effect of treating Alzheimer's disease (AD).

[0016] The present invention utilizes cationic lipids and neutral phospholipids to form nanoliposomes of lipid bilayers, and adds methacrylylated chitosan and TAT-CX43 polypeptides, which are encapsulated in nanoliposomes, and finally cross-linked under ultraviolet conditions to obtain nanoliposomes targeting microglia Cx43. Compared with nanoliposomes with only neutral phospholipids, the surface of the liposome drug delivery system of the present invention is positively charged in a neutral buffer, and this characteristic makes the nanoliposome colloid have better cell affinity. At the same time, the present invention adds methacrylylated chitosan (CMA) to the nanoliposome colloid, and uses ultraviolet rays to cross-link CMA, which can make the polypeptide enter the cell, escape the endolysosome, and then slowly release the polypeptide in the cytoplasm, thereby improving the sustained release performance of the drug. The nanoliposome targeting microglia Cx43 prepared by the present invention can achieve a sustained release effect of 14 days in vivo, greatly improving the drug half-life.

[0017] The nanolipid colloid provided by the present invention can alleviate cognitive impairment and hippocampal neuron dysfunction; promote microglial response to Abeta plaques and reduce neuroinflammation levels; can reduce the number of Abeta plaques, promote Abeta plaque morphology coagulation, reduce nerve axon damage around plaques, and reduce neuronal oxidative stress. The nanolipid colloid provided by the present invention has an effective intervention effect on Alzheimer's disease before and after the onset of the disease, and is of great significance in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

[0018] The mass ratio of methacryloyl chitosan to photoinitiator was 1:1-2.5, and the prepared nanolipid colloid had a good sustained release effect on TAT-Cx43 polypeptide, and the release rate was higher than 60% within 14 days. Reducing the amount of photoinitiator would lead to insufficient cross-linking of methacryloyl chitosan and poor sustained release of polypeptide; while increasing the amount of photoinitiator would lead to excessive cross-linking of methacryloyl chitosan, trapping the polypeptide in the nanolipid colloid and making it impossible to release.

[0019] Specifically, the amino acid sequence of the TAT-Cx43 polypeptide is shown in SEQ ID NO: 1, which is YGRKKRR QRRR-AYFNGCSSPTAPLSPMSP, wherein YGRKKRRQRRR is the amino acid sequence of the TAT polypeptide; and AYFNGCSSPTAPLSPMSP is a peptidomimetic of amino acids 266 to 283 of the Cx43 protein.

[0020] Further, the TAT-CX43 polypeptide has at least 85% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1. Further, the TAT-CX43 polypeptide has at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1. Further, the TAT-CX43 polypeptide has at least 95% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1. Further, the TAT-CX43 polypeptide has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1.

[0021] Preferably, the neutral phospholipid is one or more of phosphatidylcholine (EPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dimyristoylphosphatidylcholine.

[0022] Preferably, the cationic lipid is one or more of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), 1,2-dioleoyloxy-3-(dimethylamino)propane, 1,2-dioleyl-3-dimethylamino-propane or 1,2-dioleyl-3-dimethylamino-propane.

[0023] Furthermore, the specific operation of step S1 is: mixing neutral phospholipids, cationic lipids and organic solvents, and removing the organic solvent to obtain a lipid film.

[0024] Furthermore, in step S1, the neutral phospholipid, cationic lipid, and organic solvent are mixed according to (10-60) mg: (1-6) mg: 3 mL.

[0025] The inventors found in the study that the neutral phospholipids, cationic lipids and organic solvents are mixed according to the above ratios, and the yield of the prepared lipid bilayer nanolipid film is higher, while too high or too low mass concentration will lead to more multilayer lipid membrane structures, and less bilayer lipid membranes are obtained after filtration. Preferably, the neutral phospholipids, cationic lipids and organic solvents in the mixed solution are mixed according to (10-30) mg: (1-3) mg: 3mL. The yield of the lipid bilayer nanolipid film prepared within this range is higher. The bilayer nanolipid film is similar to the cell membrane structure. After fusion with the cell membrane, it can release the contents of the liposomes, while the multilayer membrane structure does not have this property. It will only release the inner layer of bilayer particles into the cell, which is not suitable for the release of polypeptides.

[0026] Furthermore, the organic solvent is chloroform or a mixed solution of chloroform and methanol. Preferably, the volume ratio of chloroform to methanol in the mixed solution of chloroform and methanol is 2 to 3:1.

[0027] Preferably, the removal of the organic solvent is carried out by rotary evaporation at 30-50°C.

[0028] Preferably, the osmotic pressure regulator is a sodium chloride solution with a mass concentration of 0.8-1.0%, a dextran solution with a mass concentration of 3-6%, or a sucrose solution with a mass concentration of 8-12%.

[0029] Preferably, the acid concentration in the acid-containing osmotic pressure regulator is 0.005 to 0.015 mol / L. More preferably, the acid is hydrochloric acid.

[0030] Preferably, the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator I2959) or a ketoester compound having a photoinitiating effect.

[0031] More preferably, the ketoester compounds having photoinitiating effect include but are not limited to ethyl pyruvate.

[0032] Preferably, the filtration is: filtering at least 3 times with a 0.4-0.6 μm filter membrane, and then filtering at least 5 times with a 0.2 μm filter membrane. The purpose of the filtration is to remove the multilayer membrane structure and obtain the liposome with a double-layer membrane structure.

[0033] The filter membrane may be made of polycarbonate, polysulfone, polyethersulfone or regenerated cellulose.

[0034] Preferably, the centrifugation is carried out at a centrifugal force of 150,000 to 400,000 g for 1 to 2 h. More preferably, the centrifugation is carried out at a centrifugal force of 300,000 g for 1 h.

[0035] Preferably, the precipitate is resuspended in PBS at pH 7.4. More preferably, the precipitate is resuspended in PBS at pH 7-7.8.

[0036] At the same time, the present invention also protects the nano lipid colloid targeting microglial cell Cx43 prepared by the preparation method.

[0037] Additionally, the present invention provides an application of the nanolipid colloid that protects and targets microglial Cx43 in the preparation of drugs for preventing and treating Alzheimer's disease.

[0038] The present invention has the following beneficial effects:

[0039] The present invention prepares a nanolipid colloid, uses cationic lipids and neutral phospholipids to form a nanolipid with a lipid bilayer, adds methacrylylated chitosan and TAT-CX43 polypeptide, encapsulates it in the nanolipid, and finally cross-links it under ultraviolet conditions to obtain a nanolipid colloid. Compared with nanolipids with only neutral phospholipids (such as lecithin), the surface of the nanolipid colloid of the present invention is positively charged in a neutral buffer, and this characteristic makes the nanolipid colloid have better cell affinity. At the same time, the present invention adds methacrylylated chitosan (CMA) to the nanolipid colloid, and uses ultraviolet light to cross-link CMA, so that the polypeptide can enter the cell, escape the endolysosome, and then slowly release the polypeptide in the cytoplasm, thereby improving the sustained release performance of the drug. The nanolipid colloid targeting microglia Cx43 prepared by the present invention can achieve a sustained release effect of 14 days in vivo, greatly improving the drug half-life and bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The physicochemical characteristics of TAT-CX43 nanolipid colloid packaging. Figure 1 Figure A in the figure is a schematic diagram of the nanolipid colloid packaging process; Figure 1 Figure B is a micrograph of nanolipid colloids under an electron microscope; Figure 1 Figure C is the particle size analysis chart of Malvern Zetasizer 2000; Figure 1 Figure D in the figure is the Malvern Zetasizer 2000 potential analysis diagram; Figure 1 Figure E in the figure is the particle size analysis diagram of Malvern Zetasizer 2000; Figure 1 Figure F shows the in vitro release effect of peptides packaged in different liposomes.

[0041] Figure 2 This is the experimental evidence diagram of glial cell junction protein Cx43 as a new target for AD prevention and treatment. Figure 2 A in the figure is a fluorescence micrograph of Cx43, Aβ plaques, and microglial marker IBA1 staining in hippocampal sections of AD patients and healthy controls; Figure 2 B is a microscopic image of Cx43, Aβ plaques, and microglial marker IBA1 staining in brain sections of AD model mice APP / PS1 and wild-type control mice; Figure 2 C in the figure is a fluorescence micrograph of the dye uptake experiment; Figure 2 D in the figure is a schematic diagram of knocking out Cx43 in microglia of APP / PS1 mice by transgenic mice; Figure 2E in the figure is a fluorescent micrograph of Aβ plaques and microglial marker IBA1 staining in brain sections of microglial Cx43 knockout APP / PS1 mice and related statistical data; Figure 2 F in the figure is the level of inflammatory factors TNFA and IL1B in the brain of each group detected by ELISA experiment; Figure 2 G in the figure is a low-magnification image of Aβ plaque staining in mouse sections; Figure 2 H in the figure is a high-magnification image of Aβ plaque staining in mouse sections; Figure 2 I in the figure is a microscopic image and statistical graph of RTN3 staining, a marker of Aβ plaques and axonal damage in mouse sections; Figure 2 J in the figure is a microscopic image and statistical graph of Homer1 staining, a marker of Aβ plaques and postsynaptic membranes in mouse sections; Figure 2 K in the figure is a statistical graph of the oxidative stress of neurons detected by MitoSox dye in living brain slices; Figure 2 L in the figure is a picture of the novel object behavior experiment to detect the cognitive function of mice; Figure 2 The M in the figure is an experimental diagram of the open field test to detect the movement and anxiety behavior of mice.

[0042] Figure 3 This is a diagram showing the inhibitory effect of TAT-CX43 on connexin hemichannels. Figure 3 Figure A is a micrograph of dye uptake in microglia in vitro and its related statistical data; Figure 3 Figure B is a micrograph of dye uptake in living brain slices of AD model mice and its related statistical data; Figure 3 Figure C is a micrograph of a fluorescence quenching recovery experiment in a living brain slice.

[0043] Figure 4 The in vivo distribution and sustained release characteristics of TAT-CX43 nanolipid colloid packaging are shown in Figure 2. Figure 4 Figure A is a fluorescence micrograph of the distribution of fluorescence signals in mice analyzed by a bioluminescence imaging instrument, and its local magnified image; Figure 4 Figure B is a microplate reader analyzing the Cy5.5 signal in brain homogenate and serum after drug treatment for different time periods; Figure 4 Figure C shows the Cy5.5 signal in different tissues one day after drug treatment.

[0044] Figure 5 This is a safety experiment diagram of TAT-CX43 nanolipid colloid packaging in mice. Figure 5 Figure A shows the weight changes of mice after intraperitoneal injection of TAT-Cx43 (TAT as control) nanoliposomes; Figure 5 Figures B and C in the figure are the electrocardiograms of mice before and after 6 weeks of treatment; Figure 5Figure D shows the serum biochemical analysis of the heart, liver, kidney, muscle, pancreas and other organs of mice before and after intraperitoneal injection.

[0045] Figure 6 This is a diagram showing the therapeutic effect of TAT-Cx43 nanolipid colloid on the Alzheimer's disease mouse model APP / PS1 mice. Figure 6 Figure A in the figure is a diagram of the drug treatment pattern; Figure 6 Figure B is the experimental diagram of the Barnes maze experiment; Figure 6 Figure C in the figure is a new object experiment diagram; Figure 6 Figure D in the figure is an open field experiment picture; Figure 6 Figure E in the figure is a picture of electrophysiological experiment of living brain slices; Figure 6 Figures FG in the figure are staining images of microglial marker IBA1 and Aβ plaques in mouse brain sections; Figure 6 Figure H in the figure is an ELISA experiment diagram; Figure 6 Figure I in the figure is a micrograph of Aβ plaque staining and related statistical data; Figure 6 Figure J in the figure is a microscopic image of Aβ plaques and RTN3, a marker of nerve axonal damage, and its related statistical data; Figure 6 K in the figure is the statistical chart of the relevant data of MitoSox dye staining.

[0046] Figure 7 This is an experimental diagram of the inhibitory effect of TAT-CX43 lipid colloid on connexin hemichannels. Figure 7 Figure A shows a micrograph of dye uptake in a living mouse brain slice and its related statistical data; Figure 7 Figure B is a micrograph of the fluorescence quenching recovery experiment in living brain slices.

[0047] Figure 8 This is a diagram showing the preventive effect of TAT-Cx43 nanolipid colloid on pathological changes in APP / PS1 mice.

[0048] in, Figure 8 Figure A in the figure is the experimental model diagram; Figure 8 Figure B is a micrograph of MitoSox staining experiment of living brain slices and its related statistical data; Figure 8 Figure C in the figure is a microscopic image and statistical graph of Aβ plaques and RTN3, a marker of nerve axonal damage; Figure 8 Figure D in the figure is a micrograph of Homer1 staining, a synaptic marker, and a statistical graph; Figure 8 Panel E in the figure is a micrograph of staining of microglia marker IBA1 and Aβ plaques; Figure 8 Figure F is a statistical graph showing the correlation between the number of Aβ plaques and the degree of aggregation; Figure 8 Figure G in the figure is a statistical diagram of microglia around plaques; Figure 8 The H in Figure 8 Figure E shows the three-dimensional reconstruction of the staining results and related data statistics. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] In the specific embodiment of the present invention, TAT-CX43 and TAT-CX43 266-283 They are the same polypeptide and have the same amino acid sequence SEQ ID NO:1.

[0051] In a specific embodiment of the present invention, the amino acid sequence of TAT is YGRKKRRQRRR.

[0052] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0053] Example 1 Preparation of TAT-CX43 Nanolipid Colloid

[0054] S1. 30 mg of egg phosphatidylcholine (EPC) and 3 mg of trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP) were dissolved in 3 mL of chloroform and rotary evaporated at 40 °C at 150 rpm for one hour to prepare a lipid film;

[0055] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9%, and the concentration of hydrochloric acid was 0.01 mol / L), and 60 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0056] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT-CX43 nanolipid colloid (DOTAP NLG).

[0057] Figure 1Figure A in the figure shows the general process of preparing TAT-CX43 nanolipid colloids. Peptides and methacryloyl chitosan (CMA) are mixed, then hydrated with the lipid layer, and finally UV cross-linked to obtain TAT-CX43 nanolipid colloids (DOTAP NLG).

[0058] Example 2 Preparation of TAT-CX43 Nanolipid Colloid

[0059] TAT-Cx43 is labeled with a Cy5.5 fluorescent group and packaged with a nanolipid colloid. The difference from Example 1 is that the TAT-CX43 polypeptide is first incubated with the Cy5.5 fluorescent group at room temperature for 30 minutes to achieve Cy5.5 labeling of the TAT-CX43 polypeptide, and unbound Cy5.5 is removed by dialysis, and then when performing step S2, the Cy5.5-labeled TAT-CX43 polypeptide is added, and the remaining steps are consistent with Example 1 to obtain a TAT-CX43 nanolipid colloid containing a Cy5.5 fluorescent group.

[0060] Example 3 Preparation of TAT-CX43 Nanolipid Colloid

[0061] S1. 60 mg of phosphatidylcholine (EPC) and 6 mg of DOTAP were dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0062] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9%, and the concentration of hydrochloric acid was 0.01 mol / L), and 60 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0063] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT-CX43 nanolipid colloid (DOTAP NLG).

[0064] Compared with Example 1, the difference is that in step S1, the added amounts of phosphatidylcholine (EPC) and DOTAP are different.

[0065] Example 4 Preparation of TAT-CX43 Nanoliposomes

[0066] S1. 10 mg of phosphatidylcholine (EPC) and 1 mg of DOTAP were dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0067] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9%, and the concentration of hydrochloric acid was 0.01 mol / L), and 60 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0068] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT-CX43 nanolipid colloid (DOTAP NLG).

[0069] Compared with Example 1, the difference is that in step S1, the added amounts of phosphatidylcholine (EPC) and DOTAP are different.

[0070] Comparative Example 1 Preparation of TAT-CX43 Nanoliposomes

[0071] S1. 30 mg of phosphatidylcholine (EPC) and 3 mg of DOTAP were dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0072] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9% and the concentration of hydrochloric acid was 0.01 mol / L), and 90 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0073] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT-CX43 nanolipid colloid (DOTAP NLG).

[0074] Compared with Example 1, the difference is that in step S2, the concentration of the photoinitiator is different.

[0075] In this comparative example, the amount of photoinitiator added was too high, resulting in excessive cross-linking of the methacrylylated chitosan. When conducting an in vitro sustained-release experiment, the experimental results showed that the sustained-release effect of the TAT-CX43 polypeptide was poor and it could basically not be released.

[0076] Comparative Example 2 Preparation of TAT-CX43 Nanoliposomes

[0077] S1. 30 mg of phosphatidylcholine (EPC) and 3 mg of DOTAP were dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0078] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9% and the concentration of hydrochloric acid was 0.01 mol / L), and 15 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0079] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT-CX43 nanolipid colloid (DOTAP NLG).

[0080] Compared with Example 1, the difference is that in step S2, the amount of photoinitiator added is low, the degree of CMA polymerization is low, and the sustained release effect of TAT-CX43 in the prepared TAT-CX43 nanoliposomes is poor.

[0081] Comparative Example 3 Preparation of EPC-NLG Encapsulating TAT-CX43

[0082] S1. 30 mg of phosphatidylcholine (EPC) was dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0083] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9%, and the concentration of hydrochloric acid was 0.01 mol / L), and 60 mg of a photoinitiator I2959 and 30 mg of a TAT-CX43 polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0084] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, centrifuge the supernatant in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, resuspend the precipitate in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain nanolipid colloid (EPC-NLG).

[0085] Compared with Example 1, no DOTAP was added to the prepared nanolipid colloid.

[0086] Comparative Example 4 Preparation of Nanolipid Colloids Encapsulating TAT

[0087] S1. 30 mg of phosphatidylcholine (EPC) and 3 mg of DOTAP were dissolved in 3 mL of chloroform and rotary evaporated at 150 rpm for one hour at 40 °C to prepare a lipid film;

[0088] S2. 30 mg of methacryloyl chitosan (CMA) was dissolved in 6 mL of a mixed solution (in the mixed solution, the mass concentration of sodium chloride was 0.9% and the concentration of hydrochloric acid was 0.01 mol / L), and 60 mg of a photoinitiator I2959 and 30 mg of a TAT polypeptide were added, and then the lipid film prepared in S1 was placed in the solution and soaked for 1 h;

[0089] S3. After soaking, filter using 0.4μm (3 times) and 0.2μm (5 times) polycarbonate membrane (Whatman GE) in turn, take the supernatant and centrifuge it in an ultrahigh speed centrifuge (300000g) for 1h, remove the supernatant, take the precipitate and resuspend it in 8mL pH7.4 PBS, and use 365nm ultraviolet polymerization and cross-linking to obtain TAT nanolipid colloids.

[0090] The difference from the example is that the TAT-CX43 polypeptide is replaced by the TAT polypeptide, and the other experimental conditions and operations are consistent with those of Example 1.

[0091] Experimental Example 1 Structural Characterization and In Vitro Sustained Release Effect

[0092] Experimental materials: TAT-CX43 nanolipid colloid (DOTAP NLG) prepared in Example 1 and EPC-NLG prepared in Comparative Example 3.

[0093] The microscopic morphology of liposomes was observed by electron microscopy; the particle size and potential of different nanoliposomes were analyzed by Malvern Zetasizer 2000; TAT-CX43 nanoliposomes were placed at 37°C for 7 days after preparation, and samples were taken every day to analyze the particle size of nanoliposomes using Malvern Zetasizer 2000; to evaluate the release of peptides in nanoliposomes, 1 ml of sample was added to the dialysis bag, which was then immersed in 14 ml of phosphate buffered saline (PBS) and shaken at 100 rpm and 37°C. Then samples were taken to measure the amount of peptides released into PBS and analyzed by spectrometer.

[0094] Figure 1 Figure B is a micrograph of different nanolipid colloids prepared under an electron microscope.

[0095] Figure 1 As can be seen from Figure C, the addition of DOTAP increases the surface potential of liposomes ( Figure 1 The volume of the TAT-CX43 nanolipid colloid (DOTAP NLG) prepared in Example 1 did not change significantly in vitro within 7 days ( Figure 1 Figure E in the figure).

[0096] The release effect of peptides packaged in different liposomes at 37°C in vitro. Figure 1 As can be seen from Figure F, the polypeptide release of Comparative Example 3 reached a plateau on the 9th day, that is, the polypeptide was no longer released, while the addition of DOTAP and CMA (Example 1) had a more significant effect on the sustained release of the polypeptide, and the polypeptide could still be slowly released in 7 to 14 days. This shows that DOTAP NLG nanocolloid improves the sustained release effect. TAT-CX43 nanolipid colloid significantly improves the sustained release effect within 14 days.

[0097] Experimental Example 2: Microglial junction protein Cx43 as a new target for the prevention and treatment of AD

[0098] Experimental methods:

[0099] (1) Five hippocampal paraffin sections of AD patients and healthy controls were obtained from the National Development and Function Human Brain Tissue Resource Bank. Five APP / PS1 mice and five wild-type (WT) mice were obtained and perfused at 4 and 9 months of age, and brain frozen sections were obtained. Immunofluorescence histochemistry was used to label IBA1 (a marker of microglia), Cx43, and Aβ plaques in hippocampal paraffin sections of AD patients and healthy controls and brain frozen sections of APP / PS1 mice and wild-type (WT) mice. Microscopic images were taken using an ultra-high-resolution microscope, and the Cx43 signal in IBA1-positive microglia was analyzed using IMARIS software.

[0100] (2) In vivo brain slice dye uptake experiment: 5 APP / PS1 mice and 5 wild-type mice were obtained. At 9 months of age, living brain slices were obtained using the shaking section method in artificial cerebrospinal fluid. Incubate in 5μg / mL EtBr (ethidium bromide) for 10 minutes, wash 3 times, fix the tissue with 4% PFA (paraformaldehyde), and perform immunofluorescence labeling of microglia. Among them, EtBr can enter cells through Cx43 hemichannels and bind to nuclear DNA to show color. The increase in EtBr brightness reflects the increase in Cx43 hemichannel function. In the living brain slices of APP / PS1 mice, 4 slices were taken from each mouse and incubated with 200μM GAP26 or CBX for 15 minutes before incubation with EtBr to block the Cx43 channel function.

[0101] (3) Obtain APP / PS1 mice, Cx3cr1-CreER mice, and Cx43-flox / flox mice, and breed 8 APP / PS1:Cx3cr1-CreER:Cx43-flox / flox mice through mating ( Figure 2 At 8 months of age, tamoxifen was administered orally to induce Cx43 knockout in microglia, resulting in microglial Cx43 knockout APP / PS1 mice (APP / PS1:Cx43 mgl -cKO ). Wild-type (WT) mice and APP / PS1 mice of the same age were obtained at the same time, with 14 mice in each group. Behavioral experiments were performed on the mice in the above groups when they were 9 months old. The novel object experiment detected the cognitive function of mice, and the open field experiment detected the motor function and anxiety-like behavior of mice. After the behavioral experiment, the samples were collected, the mice were killed by cervical dislocation, the brain was dissected, and the left brain was fixed with 4% PFA for frozen sections and immunofluorescence experiments. The right brain was subjected to a live brain slice experiment, and two brain slices were collected for ELISA experiments.

[0102] Experimental results:

[0103] from Figure 2 Figures A and B in the figure show that compared with healthy controls, the expression of microglial junction protein Cx43 was increased in AD patients (AD patients) and AD model mice (APP / PS1 mice), especially in microglia around Abeta plaques.

[0104] The function of Cx43 hemichannels was confirmed by using dye uptake experiments in living brain slices in APP / PS1 mice. Among them, EtBr can enter cells through Cx43 hemichannels and bind to nuclear DNA to show color. The increase in EtBr brightness reflects the increase in Cx43 hemichannel function. As controls, GAP26 (CAS No.: 197250-15-0) and CBX (carbenoxolone, carbenoxolone) are known Cx43 channel inhibitors that can inhibit the activation of Cx43 hemichannels. The results showed that the hemichannels of Cx43 in microglia in APP / PS1 mice were activated and opened ( Figure 2 Figure C in Figure 3). This suggests that the expression and function of Cx43 in microglia change in AD, which may be a target for AD intervention.

[0105] The microglial Cx43 knockout APP / PS1 mice were used to observe the effects on pathology and cognitive impairment in mice. Compared with APP / PS1 mice, microglial Cx43 knockout APP / PS1 mice (APP / PS1:Cx43 mgl-cKO ) Microglia upregulate their response to Abeta plaques ( Figure 2 Figure E in Figure 1). Figure 2 As shown in Figure F, compared with WT mice, inflammatory factors were upregulated in APP / PS1 mice, while APP / PS1:Cx43 mgl-cKO The secretion of inflammatory factors in mice in the APP / PS1:Cx43 mgl-cKO There was no significant change in the number of Abeta plaques in the mice of group Figure 2 G in Figure ), but the plaques are more aggregated ( Figure 2 The nerve damage around the Abeta plaques was reduced, as shown by the decrease in the number of RTN3-positive damaged axons ( Figure 2 I in Figure ), and an increase in Homer1-positive postsynaptic structures ( Figure 2 In addition, in vivo brain slice experiments also showed that APP / PS1:Cx43 mgl-cKO The oxidative stress in hippocampal neurons of the mice in the Figure 2 Behavioral experiments showed that APP / PS1:Cx43 mgl-cKO The cognitive function of the mice in the APP / PS1 group was improved compared with that of the APP / PS1 mice, but there was no significant difference between the mice in the WT group (novel object recognition test, Figure 2 L in the figure), while the emotions and motor abilities of mice were not affected (open field test, Figure 2 These evidences suggest that targeting microglial Cx43 hemichannels could be a target for the prevention and treatment of AD.

[0106] Experimental Example 3 Inhibitory effect of TAT-CX43 on connexin hemichannels

[0107] Experimental methods:

[0108] In order to explore the inhibitory effect of TAT-Cx43 on connexin hemichannels, we first isolated primary mouse microglia for culture, and used the dye uptake experiment (Dye Uptake) to detect the opening of connexin hemichannels. Specifically, the primary mouse microglia isolated and cultured above were divided into 8 groups, and different concentrations of TAT-CX43 peptides (0, 0.5, 1, 5, 10, 50, 100 μM) were added to the calcium-free HBSS buffer, the cell culture medium was removed, and the above-mentioned different concentrations of TAT-CX43 peptide solutions were added. At the same time, a negative control group was set up, and calcium-containing HBSS buffer was added, and the cells were incubated for 15 minutes; then EtBr was added to 5 μg / mL and incubated for 10 minutes; washed three times with calcium-containing HBSS, fixed with 4% PFA, and observed the brightness of cell EtBr under a microscope.

[0109] Dye Uptake was used in living brain slices of AD model mice (APP / PS1 mice) for verification. APP / PS1 mice were obtained, 5 in each group, and at 9 months of age, living brain slices were obtained using the oscillating slice method in artificial cerebrospinal fluid. The experimental group treated mouse brain slices with 50μM TAT-CX43 peptide, the control group 1 treated mouse brain slices with 50μM TAT peptide, and the control group 2 treated mouse brain slices with 200μM CBX (carbenoxolone); the above treatment incubated the brain slices for 15 minutes, and then incubated in 5μg / mL EtBr for 10 minutes. Wash 3 times, fix the tissue with 4% PFA, and perform immunofluorescence labeling of microglia. Use a microscope to observe the brightness of EtBr in microglia.

[0110] We further used the fluorescence quenching recovery experiment in living brain slices to detect the function of astrocyte Cx43 gap junction channels. The gap-FRAP experiment was used on living brain slices to detect the effect of TAT-Cx43 on connexin gap junction channels, and the pan-channel inhibitor CBX was used as a positive control. APP / PS1 mouse living brain slices were incubated at 37°C for 30 minutes with 1μM SR101 dye. After that, the experimental group treated the mouse brain slices with 50μM TAT-CX43, the control group 1 treated the mouse brain slices with 50μM TAT peptide, and the control group 2 treated the mouse brain slices with 200μM CBX; the above treatment was incubated for 15 minutes, and then the brain slices were transferred to a confocal microscope, SR101-stained positive cells were selected, and the fluorescence of the cells was quenched with a strong laser, and the fluorescence was observed to recover to the intensity of 9 minutes.

[0111] Experimental results:

[0112] Figure 3The results in Figure A show that in primary microglial cell culture, in a buffer solution without calcium and magnesium ions, the connexin hemichannels opened and the EtBr dye entered the cells; treatment with different concentrations of TAT-Cx43 achieved hemichannel opening inhibition (IC50 = 0.850uM).

[0113] Figure 3 The results in Figure B show that in the in vivo brain slice dye uptake experiment, TAT-Cx43 treatment can inhibit the opening of microglial hemichannels, and the inhibitory effect is comparable to that of the pan-channel inhibitor CBX.

[0114] Cx43 is also expressed in astrocytes, mainly playing the role of gap junction channels, and has important physiological effects. To verify that TAT-CX43 does not affect the function of gap junction channels, the fluorescence quenching recovery experiment of living brain slices was used to detect the function of astrocyte Cx43 gap junction channels. SR101 dye can enter cells through gap junction channels and transfer between cells. The speed and degree of fluorescence recovery reflect the function of gap junction channels. Figure 3 The results in Figure C show that in the gap-FRAP experiment in living brain slices, the results indicate that TAT-Cx43 does not affect the function of gap junction channels.

[0115] Experimental Example 4 In vivo distribution and sustained release characteristics of TAT-CX43 nanoliposome packaging

[0116] Experimental materials: TAT-CX43 nanoliposomes and TAT-CX43 polypeptide prepared in Example 2.

[0117] Experimental method: Wild-type mice were taken and adaptively fed for 7 days, then divided into two groups, 23 mice in each group. Normal water and feed were given during the feeding period:

[0118] Packaged peptide group: On day 0 of the experiment, the TAT-CX43 nanoliposomes prepared in Example 2 were intraperitoneally injected into mice at a dose of 8 nmol TAT-CX43 polypeptide / g;

[0119] Peptide-only group: On day 0 of the experiment, TAT-CX43 peptide was intraperitoneally injected into mice at a dose of 8 nmol TAT-CX43 peptide / g;

[0120] After the Packaged peptide group was injected as described above, the distribution of fluorescence signals in mice was analyzed using a bioluminescence imager on day 0, day 1, and day 7 of the experiment;

[0121] On day 0 of the experiment, the Packaged peptide group and the Peptide-only group were injected as described above, and three mice were selected from each group. Blood was collected from the eyeballs on day 1 of the experiment, and then the brain, heart, liver, and kidney were dissected;

[0122] After the Packaged peptide group and the Peptide-only group were injected as described above, 4 mice were taken from the eyeballs on the 0th day, the 1st day, the 3rd day, the 7th day, and the 14th day of the experiment for blood sampling, centrifugation to obtain serum, and then the brains were dissected and homogenized.

[0123] Experimental results:

[0124] from Figure 4 As shown in Figure A, the peptide TAT-CX43 in the nanoliposomes can be continuously released in the brain of mice within 7 days after intraperitoneal injection.

[0125] To compare the in vivo localization of the peptides by unpackaging and packaging, the fluorescence intensity of the mouse brain homogenates and serum isolated at different time points after injection was measured using a microplate reader. Figure 4 As can be seen from Figure B, after the TAT-CX43 polypeptide was packaged with nanoliposomes, the mouse brain homogenate still had strong fluorescence on the 14th day of the experiment, while the unpackaged TAT-CX43 polypeptide was directly injected into the mouse intraperitoneally, and it showed very weak fluorescence on the 7th day of the experiment; in the mouse serum, after the TAT-CX43 polypeptide packaged with nanoliposomes was injected intraperitoneally, there was still weak fluorescence on the 14th day of the experiment, but the unpackaged TAT-CX43 polypeptide could not detect fluorescence on the 7th day of the experiment. The results show that the nanoliposome packaging of the TAT-CX43 polypeptide of the present invention can significantly prolong the sustained release effect of the polypeptide in vivo.

[0126] On the first day after injection, the brain, heart, liver, and kidney were homogenized and the fluorescence intensity was measured using a microplate reader. Figure 4 As can be seen from Figure C, the nanoliposome packaging of the present invention can significantly increase the relative enrichment of TAT-CX43 polypeptide in the brain, which is more conducive to its application in the preparation of drugs for preventing and treating Alzheimer's disease.

[0127] Experimental Example 5 Biosafety of TAT-CX43 Nanoliposome Packaging

[0128] Experimental materials: TAT-CX43 nanoliposomes prepared in Example 1 and TAT nanoliposomes prepared in Comparative Example 4.

[0129] Experimental method: Wild-type mice were taken and adaptively fed for 7 days, then divided into two groups, 7 mice in each group, and given normal water and feed during the feeding period:

[0130] TAT-CX43 266～283 Group: The TAT-CX43 nanoliposomes prepared in Example 1 were intraperitoneally injected into mice at a dose of 8 nmol TAT-CX43 polypeptide / g and an injection frequency of once every 7 days (once every other week) for 6 weeks;

[0131] TAT group: The TAT nanoliposomes prepared in Comparative Example 6 were intraperitoneally injected into mice at a dose of 8 nmol TAT polypeptide / g and an injection frequency of once every 7 days (once every other week) for 6 weeks;

[0132] Before each weekly injection, the mice were weighed and recorded;

[0133] Before the first injection and one day after the last injection, mice were anesthetized with avertin, and the electrocardiogram of mice was measured using a small animal electrocardiogram recorder;

[0134] Before the first injection and one day after the last injection, blood was collected from the mouse orbits, serum was extracted, and biochemical indices of the mice were measured using a small animal blood biochemical kit.

[0135] Experimental results:

[0136] from Figure 5 As can be seen in Figure A, there was no significant change in the body weight of mice during the 6 weeks of treatment.

[0137] Since Cx43 is also highly expressed in the heart and plays an important physiological role, we detected the changes in the electrocardiogram of mice before and after drug administration. Figure 5 As shown in Figure B, TAT-Cx43 did not affect the electrocardiogram waveform of mice. Figure 5 Figure C also shows that TAT-Cx43 does not affect the heart rate, P wave, PR interval, QRS complex, QT interval and other characteristics of mice, indicating that TAT-Cx43 has no negative impact on the mouse heart.

[0138] Since Cx43 is also expressed in the liver and other organs, we used serum biochemical analysis to detect whether organs such as the heart, liver, kidney, muscle, pancreas, and immune and metabolic functions are affected by TAT-Cx43. Figure 5 As can be seen from Figure D, TAT-Cx43 does not cause abnormalities in blood biochemical indicators, all of which are within the normal reference value range.

[0139] Experimental Example 6 The therapeutic effect of TAT-Cx43 nanoliposomes on Alzheimer's disease mouse model APP / PS1 mice

[0140] Experimental materials: TAT-CX43 nanoliposomes prepared in Example 1 and TAT nanoliposomes prepared in Comparative Example 4.

[0141] Experimental methods: After 7 days of adaptive feeding, 8-month-old APP / PS1 mice were divided into 2 groups, 13 mice in each group; after 7 days of adaptive feeding, 8-month-old WT mice were divided into 2 groups, 9 mice in each group. Figure 6 The rats were grouped and fed in Figure A. Normal water and feed were given during the feeding period. The specific feeding methods for each group are as follows:

[0142] WT TAT group: The TAT nanoliposomes prepared in Comparative Example 6 were intraperitoneally injected into mice at a dose of 8 nmol TAT polypeptide / g and an injection frequency of once every 7 days (once every other week) for 6 weeks. Behavioral tests were performed on the seventh week of feeding.

[0143] WT TAT-CX43 266～283 Group: TAT-CX43 nanoliposomes prepared in Example 1 were intraperitoneally injected into mice at a dose of 8 nmol TAT-CX43 polypeptide / g. The injection frequency was once every 7 days (once every other week) for 6 weeks. Behavioral tests were performed on the seventh week of feeding.

[0144] APP / PS1 TAT group: The TAT nanoliposomes prepared in Comparative Example 6 were intraperitoneally injected into mice at a dose of 8 nmol TAT polypeptide / g and an injection frequency of once every 7 days (once every other week) for 6 weeks. Behavioral tests were performed on the seventh week of feeding.

[0145] APP / PS1 TAT-CX43 266～283 Group: TAT-CX43 nanoliposomes prepared in Example 1 were intraperitoneally injected into mice at a dose of 8 nmol TAT-CX43 polypeptide / g body weight, and the injection frequency was once every 7 days (once every other week) for 6 weeks. Behavioral tests were performed on the seventh week of feeding;

[0146] Specific behavioral experiments: Barnes maze, novel object experimental behavior, open field test.

[0147] After the above experiments, the mice in each group were dissected and samples were collected: the mice were killed by cervical dissection, and the brains were dissected. The left brain was fixed with 4% PFA for frozen sections and immunofluorescence experiments. The right brain was used for live brain slice experiments, and two brain slices were collected for ELISA experiments.

[0148] Experimental results:

[0149] For APP / PS1 mice, Figure 6 Figures B and C in the figure are the results of the Barnes maze and novel object test behavioral tests, respectively showing that compared with the APP / PS1 TAT group, APP / PS1TAT-CX43 266～283 The group spent more time exploring the target area in the Barnes maze and more time exploring the new object in the novel object test; both returned to levels close to the wild type (WT), that is, intraperitoneal injection of TAT-CX43 nanoliposomes can significantly improve the cognitive function of APP / PS1 mice. The open field test showed that the total movement distance of the mice and the movement distance in the middle area remained unchanged, that is, intraperitoneal injection of TAT-CX43 nanoliposomes did not affect the mice's motor ability or cause anxiety ( Figure 6 Figure D in the figure).

[0150] The brains of mice were dissected and electrophysiological experiments were performed on living brain slices. The results showed that in wild-type mice (WT mice), intraperitoneal injection of TAT-CX43 nanoliposomes had no significant effect on cognitive function and LTP of hippocampal neurons; while in APP / PS1 mice, intraperitoneal injection of TAT-CX43 nanoliposomes alleviated the LTP disorder of hippocampal neurons in APP / PS1 mice ( Figure 6 (E in Figure 1), indicating that the TAT-CX43 nanoliposomes prepared by the present invention significantly improved the synaptic plasticity of APP / PS1 mouse neurons.

[0151] At the histological level, immunostaining experiments on the microglial marker IBA1 and Aβ plaques in frozen sections of mouse brain revealed that intraperitoneal injection of TAT-CX43 nanoliposomes increased the number of microglia and their responsiveness to Abeta plaques ( Figure 6 (Figure FG in Figure 3).

[0152] At the same time, ELISA experiments on mouse brain showed that intraperitoneal injection of TAT-CX43 nanoliposomes reduced the release of inflammatory factors ( Figure 6 Figure H in Figure 1). Figure 6 As shown in Figures I to K, after intraperitoneal injection of TAT-CX43 nanoliposomes, the number of Abeta plaques did not change significantly, but the plaque cohesion was higher ( Figure 6 The RTN3-positive nerve damage around the plaque was reduced ( Figure 6 J in Figure 2), oxidative damage in hippocampal neurons was reduced ( Figure 6 These results demonstrate that the TAT-CX43 liposomes prepared in the present invention have a certain therapeutic effect on the pathology of AD and cognitive impairment.

[0153] Figure 7 For APP / PS1 TAT group and APP / PS1 TAT-CX43 266～283 The brains of mice in the group were subjected to dye uptake experiments (brain slices were taken and incubated in 5μg / mL EtBr for 10 minutes. Washed 3 times, the tissues were fixed with 4% PFA, and immunofluorescence was performed to label microglia) and fluorescence quenching recovery experiments in living brain slices (brain slices were taken and incubated with 1μM SR101 dye at 37°C for 30 minutes in living brain slices of APP / PS1 mice, and then the brain slices were transferred to a confocal microscope, SR101-stained positive cells were selected, and the fluorescence of the cells was quenched with a strong laser, and the fluorescence recovery was observed to 9 minutes). Among them, brain slices from the APP / PS1 TAT group were taken and treated with 200μM CBX (carbenoxolone) before the experiment as a negative control. Figure 7 The results in Figure A show that in the in vivo brain slice dye uptake experiment, TAT-Cx43 liposomes can inhibit the opening of microglial hemichannels after intraperitoneal injection, and the inhibitory effect is comparable to that of the pan-channel inhibitor CBX. Figure 7 The results of Figure B in the in vivo brain slice gap-FRAP experiment show that intraperitoneal injection of TAT-Cx43 liposomes does not affect the function of gap junction channels. The experimental results are consistent with the results of Experimental Example 3.

[0154] Experimental Example 7 Preventive effect of TAT-Cx43 nanoliposomes on pathological changes in APP / PS1 mice

[0155] Experimental materials: TAT-CX43 nanoliposomes prepared in Example 1 and TAT nanoliposomes prepared in Comparative Example 4.

[0156] Experimental methods: 3.5-month-old APP / PS1 mice (without Abeta pathology) were adaptively fed for 7 days and then divided into 2 groups, 5 mice in each group; 3.5-month-old WT mice were adaptively fed for 7 days and then divided into 2 groups, 5 mice in each group. Figure 8 The rats were grouped and fed in Figure A. Normal water and feed were given during the feeding period. The specific feeding methods for each group are as follows:

[0157] WT TAT group: mice were intraperitoneally injected with TAT nanoliposomes at a dose of 8 nmol TAT polypeptide / g once a week for 8 weeks, and histological examination was performed starting from the 9th week;

[0158] WT TAT-CX43 266～283 Group: mice were intraperitoneally injected with TAT-CX43 nanoliposomes at a dose of 8 nmol TAT-CX43 peptide / g, once a week for 8 weeks, and histological examination was performed starting from the 9th week;

[0159] APP / PS1 TAT group: mice were intraperitoneally injected with TAT nanoliposomes at a dose of 8 nmol TAT polypeptide / g, once a week for 8 weeks, and histological examination was performed starting from the 9th week;

[0160] APP / PS1 TAT-CX43 266～283 Group: Mice were intraperitoneally injected with TAT-CX43 nanoliposomes at a dose of 8 nmol TAT-CX43 polypeptide / g, once a week for 8 weeks, and histological examination was performed starting from the 9th week.

[0161] Experimental results:

[0162] The results showed that early treatment with TAT-CX43 nanoliposomes could effectively prevent neuronal oxidative stress ( Figure 8 Figure B) and axonal and synaptic damage around the plaque ( Figure 8 CD in Figure 2). Early treatment with TAT-CX43 nanoliposomes reduced the number of Abeta plaques and increased the aggregation of Abeta plaques ( Figure 8 EF in Figure 1), which increases the response and interaction of microglia to Abeta plaques ( Figure 8 The above data demonstrate the preventive effect of TAT-CX43 nanoliposomes on AD pathology.

[0163] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a nanolipid colloid targeting microglial Cx43, characterized in that: The following steps are involved: S1. Preparation of lipid films of cationic lipids and neutral phospholipids; S2. The methacrylated chitosan, the photoinitiator, the TAT-CX43 polypeptide and the acid-containing osmotic pressure regulator are mixed and dissolved to obtain a mixed solution, and the mixed solution is soaked in a lipid film; S3. After soaking, the multilayer membrane structure is removed by filtration, centrifuged, the precipitate is resuspended, and cross-linked by ultraviolet light to obtain nano-lipid colloid; Wherein, the amino acid sequence of the TAT-CX43 polypeptide is as shown in SEQ ID NO: 1 or has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1; In step S2, the mass ratio of methacryloyl chitosan to photoinitiator in the mixed solution is 1:1-2.

5.

2. The preparation method according to claim 1, characterized in that: The neutral phospholipids are one or more of phosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dimyristoylphosphatidylcholine.

3. The preparation method according to claim 1, characterized in that: The cationic lipid is one or more of trimethyl-2,3-dioleoyloxypropylammonium bromide, 1,2-dioleoyloxy-3-(dimethylamino)propane, 1,2-dioleyl-3-dimethylamino-propane or 1,2-dioleyl-3-dimethylamino-propane.

4. The preparation method according to claim 1, characterized in that: The specific operation of step S1 is: mixing neutral phospholipids, cationic lipids and organic solvents, and removing the organic solvents to obtain a lipid film.

5. The preparation method according to claim 4, characterized in that: The neutral phospholipid, cationic lipid and organic solvent are mixed according to (10-60) mg: (1-6) mg: 3 mL.

6. The preparation method according to claim 1, characterized in that: The osmotic pressure regulator is a sodium chloride solution with a mass concentration of 0.8-1.0%, a dextran glucose solution with a mass concentration of 3-6%, or a sucrose solution with a mass concentration of 8-12%.

7. The preparation method according to claim 1, characterized in that: The photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone or a ketoester compound with photoinitiating effect.

8. The preparation method according to claim 1, characterized in that: The centrifugation is carried out at a centrifugal force of 150,000 to 400,000 g for 1 to 2 hours.

9. The nanolipid colloid targeting microglial Cx43 prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nanolipid colloid targeting microglial Cx43 according to claim 9 in the preparation of drugs for preventing and treating Alzheimer's disease.

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