Adipose tissue targeting siRNA delivery system and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an adipose tissue targeted siRNA delivery system and a preparation method thereof. The preparation method comprises the following steps: forming a siRNA / PLL compound from polylysine and siRNA in DEPC water, then transferring the siRNA / PLL compound into dichloromethane containing a maleimide polyethylene glycol polylactic acid-glycolic acid copolymer, and carrying out ultrasonic emulsification to form a primary emulsion; dropwise adding the primary emulsion into a water phase containing polyvinyl alcohol, performing ultrasonic emulsification again to form a secondary emulsion, and stirring at room temperature to remove dichloromethane to form NP (at) siRNA; maleimide on the surface of NP (at) siRNA is used as a donor of a coupling reaction, and is stably crosslinked with sulfydryl provided by adipose tissue targeting peptide through a click reaction to form aNP (at) siRNA. Based on a polylactic acid-glycolic acid copolymer self-assembly strategy, a click chemistry method is adopted, the reaction conditions are mild, and the product can be used for treating fat-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a fat tissue-targeting siRNA delivery system and a preparation method thereof. BACKGROUND

[0002] Fat tissue, including white fat (subcutaneous and visceral fat, etc.) and brown fat, can participate in the regulation of various physiological functions of the body, including food intake, energy balance, insulin sensitivity, body temperature regulation and immune response, etc. High plasticity is an important feature of fat tissue, and external or physiological stimuli can lead to changes in its metabolism and structure. For example: ① rapid adjustment of energy metabolism to meet the fluctuation of body energy supply and demand; ② structural metabolic transformation in cold environment to quickly produce heat for insulation; ③ necessary dedifferentiation to participate in body damage repair. However, factors such as long-term energy excess, aging, and mental stress can lead to a decrease in the plasticity of fat tissue, accompanied by pathological conditions such as fibrosis, inflammation, progenitor cell aging, catecholamine resistance, and insulin resistance, ultimately leading to the occurrence and development of obesity and its related metabolic diseases. The core role of fat tissue dysfunction in diseases, as well as the strong plasticity of fat tissue, suggest that it can be used as a potential therapeutic target for metabolic diseases. However, the poor proliferation ability of mature adipocytes and the low drug uptake efficiency hinder the treatment of obesity, and the cytoplasm of mature adipocytes contains a large number of lipid droplets, making adipocytes relatively fragile and easily damaged by therapeutic operations and chemicals. Therefore, there is an urgent need to find a therapeutic drug that is biocompatible and easy to enter fat cells.

[0003] RNA interference technology is to introduce double-stranded RNA or artificially synthesized small interfering RNA (siRNA) into cells, and siRNA is degraded into small fragments of about 20 nucleotides by Dicer enzyme. Each small fragment of double-stranded RNA is divided into two single strands (guide strand and passenger strand), and the passenger strand is degraded, while the guide strand is integrated into the RNA-induced silencing complex. The guide strand can specifically base-pair with mRNA, inducing Argonayte2 protein to cut the formed double strand, thereby degrading mRNA and achieving silencing of the target gene. It has the following advantages: first, it uses base-pairing principle to make it have therapeutic specificity. Second, siRNA does not integrate into the genome, thereby avoiding the risk of host gene mutation and having high biological safety. Third, it only needs a few fragments to induce significant gene silencing effect in cells, and has high efficiency. Finally, according to the mRNA sequence of the target gene, siRNA can be designed reasonably to obtain siRNA that can silence any pathogenic gene, and has editability. Therefore, RNA interference technology is an ideal candidate for gene therapy.

[0004] Currently, siRNA delivery systems are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors can cause immune responses and toxicity, so they are not the first choice. Non-viral vectors mainly focus on two categories: lipid-based siRNA delivery vectors and polymer-based siRNA delivery vectors. The former mainly includes anionic liposomes, neutral liposomes, cationic liposomes, lipid particles, and lipid nanoparticles. Some delivery systems have been used in clinical disease treatment, but due to the instability of liposomes and their potential toxicity, safety and efficiency issues still exist. Polymer-based siRNA delivery vectors are a type of solid, biodegradable colloidal nanoparticles. Due to their high biosafety and strong plasticity, they have become a research hotspot for siRNA gene therapy. However, research on polymer-based siRNA delivery systems targeting adipose tissue is still limited. Summary of the Invention

[0005] To address the current lack of research on polymer-based siRNA delivery systems targeting adipose tissue, this invention provides an adipose tissue-targeted siRNA delivery system and its preparation method. Through a click chemistry method involving maleimide and thiol groups, an adipose tissue-targeting peptide is linked to a polylactic-co-glycolic acid copolymer, delivering siRNA to target adipose tissue and degrade specific mRNAs within the tissue. This provides a novel targeted intervention for the treatment of adipose tissue diseases. The siRNA delivery vector of this invention can simultaneously affect brown adipose tissue, subcutaneous fat, and visceral fat, without causing damage to tissues such as the liver, kidneys, lungs, heart, and brain.

[0006] In a first aspect, the present invention provides a method for preparing an adipose tissue-targeted siRNA delivery system, comprising: S1: Polylysine and siRNA are added to DEPC water to form an siRNA / PLL complex, which is then transferred to dichloromethane containing maleimide polyethylene glycol polylactic acid-glycolic acid copolymer and ultrasonically emulsified to form a primary emulsion. S2: The primary emulsion is added to an aqueous phase containing polyvinyl alcohol and then ultrasonically emulsified again to form a secondary emulsion. Polyvinyl alcohol is used as an emulsifier to produce a stable and uniform emulsion. Then, the dichloromethane is removed by stirring to form nanoparticles loaded with siRNA, denoted as NP@siRNA. S3: Using maleimide on the surface of NP@siRNA as a donor for the coupling reaction, it is stably cross-linked with the thiol group provided by the adipose tissue targeting peptide through a click reaction, and tris(2-carboxyethyl)phosphonic acid hydrochloride is used as a cross-linking agent to form NP@siRNA coupled with the adipose tissue targeting peptide, denoted as aNP@siRNA. The sequence of the adipose tissue targeting peptide is CKGGRAKDC.

[0007] Furthermore, the siRNA sequence is determined by the sequence of the target mRNA.

[0008] Furthermore, in S1, polylysine binds to siRNA and is added to DEPC water at a molar ratio of amino to phosphate groups of 1-8:1, forming an siRNA / PLL complex through electrostatic binding.

[0009] Furthermore, in S1, dichloromethane containing maleimide polyethylene glycol polylactic acid-glycolic acid copolymer is used as the main body of the nanodelivery system, and the concentration of maleimide polyethylene glycol polylactic acid-glycolic acid copolymer dissolved in dichloromethane is 30-90 mg / mL.

[0010] Furthermore, the mass ratio of maleimide polyethylene glycol polylactic acid-glycolic acid copolymer to siRNA / PLL complex is 375:11.

[0011] Furthermore, in S1, the siRNA / PLL complex is ultrasonically emulsified with dichloromethane containing maleimide-poly(lactic-co-hydroxyacetic acid) copolymer under ice bath conditions.

[0012] Furthermore, in S2, polyvinyl alcohol is used as an emulsifier to produce a stable and uniform secondary emulsion, and the aqueous phase containing polyvinyl alcohol has a concentration of 2-5 g / 100 mL.

[0013] Furthermore, in S2, the secondary emulsion is stirred at room temperature for 4-12 hours. If the stirring time is less than 4 hours, the organic solution cannot be completely evaporated, and if the stirring time is more than 12 hours, it will affect the stability of the nanoparticles.

[0014] Furthermore, in S2, the formed NP@siRNA is collected, washed, and resuspended in sterile PBS buffer.

[0015] Furthermore, in S3, tris(2-carboxyethyl)phosphonic acid hydrochloride and adipose tissue targeting peptide were added to sterile PBS buffer containing NP@siRNA and continuously stirred at 0-5°C. The molar ratio of adipose tissue targeting peptide to NP@siRNA was 1-2.4:1.

[0016] Furthermore, in S3, the reaction is carried out under reaction conditions with a pH of 7.0-7.4.

[0017] Furthermore, S3 also includes: adding the formed aNP@siRNA to a dialysis bag and dialyzing it in pure water for purification, with the dialysis bag having a molecular weight cutoff of 3500 Da.

[0018] Secondly, the present invention provides an adipose tissue-targeted siRNA delivery system obtained by the above preparation method.

[0019] The beneficial effects of this invention are as follows: (1) Based on the self-assembly strategy of polylactic acid-glycolic acid copolymer, the present invention designs and prepares an adipose tissue-targeting siRNA delivery system, which can specifically target and degrade the mRNA of adipose tissue for the treatment of fat-related diseases.

[0020] (2) The reaction can be carried out at room temperature without the need for metal catalysts and without the use of highly toxic organic solvents. The reaction conditions are mild, easy to control, and the product is easy to purify.

[0021] (3) The nanodelivery system can be functionalized as needed, such as by combining it with other drugs or target molecules, providing new ideas for the design of next-generation targeted therapies. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the construction of the adipose tissue-targeted siRNA delivery system of the present invention.

[0024] Figure 2 The image shows the dynamic light scattering data of the NP, NP@siRNA and aNP@siRNA nanocarriers in Test Example 1.

[0025] Figure 3 A bar chart comparing the cell activity of siRNA, NP@siRNA, and aNP@siRNA nanocarriers in Example 2.

[0026] Figure 4 The image shows the therapeutic effects in each group of mice in test case 3.

[0027] Figure 5 These are fluorescence imaging photographs (a) and fluorescence intensity comparison bar charts (b) of different tissues in each group of mice in test example 4.

[0028] Figure 4 and Figure 5 In the text, ﹡﹡﹡ represents P <0.001. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0030] The maleimide polyethylene glycol polylactic acid-glycolic acid copolymer (PLGA-PEG-Mal) used in the specific embodiments of the present invention is PLGA10k-PEG2k-Mal, which was purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0031] The sequence (Sequence 1) of the adipose tissue targeting peptide used in the specific embodiments of the present invention is CKGGRAKDC (Cys-Lys-Gly-Gly-Arg-Ala-Lys-Asp-Cys).

[0032] Example 1 (1) Add polylysine and siRNA to DEPC water at a ratio of amino:phosphate group = 2:1, mix gently, and let stand at room temperature for 30 minutes to form siRNA / PLL complex solution, wherein the concentration of polylysine is 1.76 mg / mL and the concentration of siRNA is 1.40 mg / mL. The siRNA sequence (Sequence 2) is GCAUUGGCGGAAUAUAGAA (Jiangsu Jima Gene). (2) Take 0.1 mL of the siRNA / PLL complex solution prepared in step (1) and add it dropwise to 1 mL of dichloromethane solution containing 30 mg PLGA-PEG-Mal. Emulsify the solution by sonication in an ice bath to produce a primary emulsion. (3) Add the primary emulsion dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution and then ultrasonically emulsify it again to form a secondary emulsion; (4) Stir the secondary emulsion at room temperature for 8 hours to evaporate the organic solvent dichloromethane to form a milky white clear liquid. At the same time, the nanoparticles harden during the solvent evaporation process, thereby producing stable and uniformly sized nanoparticles NP@siRNA. (5) Centrifuge the milky white clear liquid formed in step (4) at 13,000 rpm for 30 minutes to collect NP@siRNA, and wash it three times with DEPC water to remove excess polyvinyl alcohol and siRNA that was not captured or adsorbed onto the nanoparticle shell. (6) Resuspend the NP@siRNA in sterile PBS buffer solution (0.01M, pH=7.4) and mix thoroughly. If the subsequent adipose tissue targeting peptide ligation is not performed immediately, freeze-dry the PBS solution of the NP@siRNA and store the freeze-dried product at -20℃ for later use. When using, simply resuspend it in sterile PBS buffer solution. (7) Add 1 mL of PBS solution (1 mg / mL) of adipose tissue targeting peptide and 290 μL of PBS solution (1 mg / mL) of tris(2-carboxyethyl)phosphine hydrochloride to the PBS solution of NP@siRNA prepared in step (6). At this time, the pH value of the reaction system is in the range of 7.0-7.4. Stir gently for 12 hours at 4°C to form NP@siRNA coupled with adipose tissue targeting peptide and obtain nanoparticles aNP@siRNA. (8) The generated aNP@siRNA was added to a dialysis bag and purified by dialysis in a large amount of pure water. The molecular weight cutoff of the dialysis bag was 3500 Da.

[0033] Figure 1 The construction principle of the adipose tissue-targeted siRNA delivery system of the present invention is demonstrated as follows: The polylactic acid-glycolic acid copolymer (PLGA-PEG-Mal) exhibits excellent biodegradability and biocompatibility. The hydrophobic groups of the PLGA-glycolic acid copolymer self-assemble into spherical structures. These spherical structures can encapsulate positively charged cationic polymer polylysine and siRNA with specific sequences. Polylysine increases the encapsulation efficiency and drug loading of negatively charged siRNA through electrostatic interactions, while protecting the integrity of the siRNA itself and its function. The surface of the spherical structures formed by the PLGA-glycolic acid copolymer is modified with polyethylene glycol polymer, forming a hydrophilic layer on the particle surface. This system enhances biostability, increases the hydrophilicity of the delivery system, reduces immunogenicity, prolongs in vivo circulation time, and increases the chance of siRNA drugs entering cells. Maleimide groups are linked to polyethylene glycol polymers, and the maleimide groups and the thiol groups of adipose tissue targeting peptides form stable thioether bonds through click chemistry, thereby linking the adipose tissue targeting peptides to polyethylene glycol polylactic acid-glycolic acid copolymers. This allows for the specific delivery of therapeutic siRNA to adipose tissue. By utilizing RNA interference technology to specifically degrade pathogenic mRNA, an siRNA delivery system with adipose tissue targeting effect is constructed.

[0034] Example 2 The difference between Example 2 and Example 1 is that in step (1) of Example 2, polylysine and siRNA were added to DEPC water at a molar ratio of amino:phosphate group = 4:1, and the concentration of polylysine in the resulting siRNA / PLL complex solution was 3.52 mg / mL. The remaining steps and parameters were the same as in Example 1.

[0035] Example 3 The difference between Example 3 and Example 1 is that in step (2) of Example 3, 0.2 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 1 mL of dichloromethane solution containing 30 mg PLGA-PEG-Mal. The remaining steps and parameters are the same as in Example 1.

[0036] Example 4 The difference between Example 4 and Example 1 is that in step (2) of Example 4, 0.2 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 1 mL of dichloromethane solution containing 60 mg PLGA-PEG-Mal. The remaining steps and parameters are the same as in Example 1.

[0037] Example 5 The difference between Example 5 and Example 1 is that in step (1) of Example 5, polylysine and siRNA were added to DEPC water at a molar ratio of amino:phosphate group = 1:1, and the concentration of polylysine in the resulting siRNA / PLL complex solution was 0.89 mg / mL and the concentration of siRNA was 0.70 mg / mL. In step (2), 0.2 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 1 mL of dichloromethane solution containing 60 mg PLGA-PEG-Mal; The remaining steps and parameters are the same as in Example 1.

[0038] Example 6 The difference between Example 6 and Example 1 is that in step (1) of Example 6, polylysine and siRNA were added to DEPC water at a molar ratio of amino:phosphate group = 1:1, and the concentration of polylysine in the resulting siRNA / PLL complex solution was 0.89 mg / mL and the concentration of siRNA was 0.70 mg / mL. In step (2), 0.2 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 1 mL of dichloromethane solution containing 60 mg PLGA-PEG-Mal; In step (3), the primary emulsion is added dropwise to 4 mL of 2% polyvinyl alcohol aqueous solution; The remaining steps and parameters are the same as in Example 1.

[0039] Example 7 The difference between Example 7 and Example 1 is that in step (1) of Example 7, polylysine and siRNA were added to DEPC water at a molar ratio of amino:phosphate group = 1:1, and the concentration of polylysine in the resulting siRNA / PLL complex solution was 0.89 mg / mL and the concentration of siRNA was 0.70 mg / mL. In step (2), 0.2 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 1 mL of dichloromethane solution containing 60 mg PLGA-PEG-Mal; In step (3), the primary emulsion is added dropwise to 8 mL of 2% polyvinyl alcohol aqueous solution; The remaining steps and parameters are the same as in Example 1.

[0040] Example 8 The difference between Example 8 and Example 1 is that in step (1) of Example 8, polylysine and siRNA were added to DEPC water at a molar ratio of amino:phosphate group = 4:1, and the concentration of polylysine in the resulting siRNA / PLL complex solution was 1.78 mg / mL and the concentration of siRNA was 1.40 mg / mL. In step (2), 0.3 mL of the siRNA / PLL complex solution prepared in step (1) is added dropwise to 3 mL of dichloromethane solution containing 180 mg PLGA-PEG-Mal; In step (3), the primary emulsion is added dropwise to 30 mL of 2% polyvinyl alcohol aqueous solution; The remaining steps and parameters are the same as in Example 1.

[0041] Example 9 The difference between Example 9 and Example 1 is that the siRNA sequence used in step (1) of Example 9 is the example gene. Wdr The interference sequence siWDR6 ​​(Sequence 3), specifically AGGUGAAGCUCUUAGAGAA (Jiangsu Jima Gene), was used to name the nanoparticles finally prepared in Example 9 aNP@siWDR6.

[0042] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no siRNA / PLL complex was added during the preparation process, and no adipose tissue targeting peptide was coupled. The specific steps are as follows: (1) Add 1 mL of dichloromethane solution containing 30 mg PLGA-PEG-Mal to 10 mL of polyvinyl alcohol aqueous solution with a concentration of 2 g / mL, and emulsify by ultrasonication in an ice bath to form an emulsion; (2) Stir the emulsion from step (1) at room temperature for 8 hours to evaporate the organic solvent dichloromethane, then centrifuge at 13,000 rpm for 30 minutes using a high-speed centrifuge, and wash three times with DEPC water to obtain nanoparticles NP.

[0043] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that no adipose tissue-targeting peptide was coupled during the preparation process. The specific steps are as follows: (1) Add polylysine and siRNA to DEPC water at a molar ratio of amino:phosphate group = 2:1, mix gently, and let stand at room temperature for 30 minutes to form siRNA / PLL complex solution, wherein the concentration of polylysine is 1.76 mg / mL and the concentration of siRNA is 1.40 mg / mL. (2) Take 0.1 mL of the siRNA / PLL complex solution prepared in step (1) and add it dropwise to 1 mL of dichloromethane solution containing 30 mg PLGA-PEG-Mal. Emulsify the solution by sonication in an ice bath to produce a primary emulsion. (3) Add the primary emulsion dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution and then ultrasonically emulsify it again to form a secondary emulsion; (4) Stir the secondary emulsion at room temperature for 8 hours to evaporate the organic solvent dichloromethane to form a milky white clear liquid. At the same time, the nanoparticles harden during the solvent evaporation process, thereby producing stable and uniformly sized nanoparticles NP@siRNA. (5) Centrifuge the milky white clear liquid formed in step (4) at 13,000 rpm for 30 minutes to collect NP@siRNA, and wash it three times with DEPC water to remove excess polyvinyl alcohol and siRNA that was not captured or adsorbed onto the nanoparticle shell.

[0044] Comparative Example 3 The difference between Comparative Example 3 and Example 9 is that no adipose tissue-targeting peptide was coupled during the preparation process. The specific steps are as follows: (1) Add polylysine and siWDR6 ​​to DEPC water at a molar ratio of amino:phosphate group = 2:1, mix gently, and let stand at room temperature for 30 minutes to form a siWDR6 / PLL complex solution, wherein the concentration of polylysine is 1.76 mg / mL and the concentration of siWDR6 ​​is 1.40 mg / mL. (2) Take 0.1 mL of the siWDR6 / PLL complex solution prepared in step (1) and add it dropwise to 1 mL of dichloromethane solution containing 30 mg PLGA-PEG-Mal. Emulsify the solution in an ice bath to produce a primary emulsion. (3) Add the primary emulsion dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution and then ultrasonically emulsify it again to form a secondary emulsion; (4) Stir the secondary emulsion at room temperature for 8 hours to evaporate the organic solvent dichloromethane to form a milky white clear liquid. At the same time, the nanoparticles harden during the solvent evaporation process, thereby producing stable and uniformly sized nanoparticles NP@siWDR6. (5) Centrifuge the milky white clear liquid formed in step (4) at 13,000 rpm for 30 minutes to collect NP@siWDR6 ​​and wash it three times with DEPC water to remove excess polyvinyl alcohol and siWDR6 ​​that has not been captured or adsorbed onto the nanoparticle shell.

[0045] Test Example 1 The particle size and potential of the nanodelivery systems prepared in Example 1, Comparative Example 1, and Comparative Example 2 were measured using a nanoparticle size potentiometer, and the results are as follows: Figure 2 As shown. In terms of particle size, the uncoated siRNA nanoparticles (NP) prepared in Comparative Example 1 had a particle size of approximately 160 nm. The NP@siRNA prepared in Comparative Example 2 had a slightly larger particle size than the NP, between 160-180 nm, but the difference was not statistically significant. The particle size of aNP@siRNA further increased after coupling with adipose tissue targeting peptides, reaching approximately 230 nm, indicating successful attachment of adipose tissue targeting peptides to the nanoparticle surface. Meanwhile, from... Figure 2 It can also be seen that the adipose tissue-targeted siRNA delivery system of the present invention has a uniform distribution.

[0046] Test Example 2 The MTT assay was used to systematically evaluate the potential toxicity of aNP@siRNA, free siRNA (sequence 2, GCAUUGGCGGAAUAUAGAA) prepared in Example 1, and NP@siRNA prepared in Comparative Example 2 to 3T3-L1 cells. Specific experimental methods are as follows: First, 3T3-L1 cells were seeded into 96-well plates at a density of 1×10⁶ cells / well. 4 Cells were incubated at 100 μL / well at 37°C and 5% CO2 overnight. The old culture medium was then discarded, and 100 μL of fresh culture medium and 100 μL of free siRNA, NP@siRNA, and aNP@siRNA solutions with different siRNA concentrations (0.625, 1.25, 2.5, 5, and 10 μg / mL) were added to each well. Cells treated with PBS were used as a positive control. After co-incubation at 37°C for 24 h, 10 μL of MTT solution was added to each well, and incubation was continued for 4 h. The liquid in the wells was then removed, and 150 μL of DMSO was added to dissolve the formazan crystals formed at the bottom of the wells. The absorbance of each well at 490 nm was read using a microplate reader, and the percentage of absorbance relative to the positive control group was defined as the relative cell proliferation rate.

[0047] Experimental results show that... Figure 3 As shown, after treating cells with different concentrations of free siRNA, the cell viability gradually decreased with increasing concentration, slightly below 80% at 1.25 μg / mL, indicating that free siRNA itself has some toxicity to adipocytes, but the toxicity is low. When using PLGA-PEG-Mal to load siRNA, whether NP@siRNA or aNP@siRNA, the impact on cell viability was minimal. Cell viability showed a decreasing trend with increasing concentration, but at the highest concentration, cell viability remained at around 80%, which fully demonstrates that the siRNA delivery system of this invention has good biocompatibility.

[0048] Test Example 3 Male C57BL / 6 mice were fed a normal growth and reproduction diet until 8 weeks of age, then switched to a 60% high-fat diet for 6 weeks to maintain a weight of approximately 30g. They were then fed the same 60% high-fat diet again, while simultaneously receiving PBS, aNP@siRNA prepared in Example 1, aNP@siWDR6 ​​prepared in Example 9, NP@siRNA prepared in Comparative Example 2, and NP@siWDR6 ​​prepared in Comparative Example 3, depending on the group. Dosage was administered every 2 days, with the specific dosage calculated based on an siRNA concentration of 3.25 μg / g body weight. The PBS volume was determined based on the average volume administered to other groups. Mice were sacrificed at the end of the 6th week of administration, and subcutaneous fat, epididymal fat, and brown adipose tissue were collected, and changes in adipose tissue weight were recorded.

[0049] The results are as follows Figure 4 As shown, the total body fat content of mice in the NP@siWDR6 ​​and aNP@siWDR6 ​​groups was significantly reduced.

[0050] Test Example 4 Twenty-week-old male C57BL / 6 mice fed a normal growth and reproduction diet were randomly divided into PBS, NP, and aNP groups. Each group received an intraperitoneal injection of the corresponding PBS, indocyanine green-labeled NP@siWDR6, or indocyanine green-labeled aNP@siWDR6. The specific dosage was calculated based on an siRNA concentration of 3.25 μg / g body weight, and the PBS volume was determined based on the average administration volume of the other groups. aNP@siWDR6 ​​and NP@siWDR6 ​​were prepared according to the methods in Example 9 and Comparative Example 3, respectively. Various tissues were collected 24 hours after administration, and the fluorescence intensity of different tissues was detected using small animal in vivo imaging technology.

[0051] The results are as follows Figure 5As shown, the fluorescence intensity of brown adipose tissue, white adipose tissue in the groin, and white adipose tissue of indocyanine green-labeled NP@siWDR6 ​​and aNP@siWDR6 ​​mice was significantly stronger than that of the PBS group mice. The NP group also showed partial fluorescence in the liver, kidney, and lungs, while the aNP group showed no significant fluorescence in other tissues. This indicates that the siRNA delivery system of the present invention can specifically target adipose tissue, thereby enabling its use in the treatment of fat-related diseases.

[0052] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for preparing an adipose tissue-targeted siRNA delivery system, characterized in that, include: S1: Polylysine and siRNA are added to DEPC water to form an siRNA / PLL complex, which is then transferred to dichloromethane containing maleimide polyethylene glycol polylactic acid-glycolic acid copolymer and ultrasonically emulsified to form a primary emulsion. S2: The primary emulsion containing polyvinyl alcohol is ultrasonically emulsified again in the aqueous phase to form a secondary emulsion. Then, the dichloromethane is removed by stirring to form nanoparticles carrying siRNA, denoted as NP@siRNA. S3: Using maleimide on the surface of NP@siRNA as a donor for the coupling reaction, it is stably cross-linked with thiol groups provided by adipose tissue targeting peptides through a click reaction, and tris(2-carboxyethyl)phosphonic acid hydrochloride is used as a cross-linking agent to form aNP@siRNA; The sequence of the adipose tissue targeting peptide is CKGGRAKDC.

2. The preparation method according to claim 1, characterized in that, In S1, polylysine binds to siRNA and is added to DEPC water at a molar ratio of amino to phosphate groups of 1-8:1, forming an siRNA / PLL complex through electrostatic binding.

3. The preparation method according to claim 1, characterized in that, In S1, the concentration of maleimide-polyethylene glycol-polylactic acid-glycolic acid copolymer dissolved in dichloromethane is 30-90 mg / mL.

4. The preparation method according to claim 1, characterized in that, In S1, the mass ratio of maleimide polyethylene glycol polylactic acid-glycolic acid copolymer to siRNA / PLL complex is 375:

11.

5. The preparation method according to claim 1, characterized in that, In S2, the concentration of the aqueous phase containing polyvinyl alcohol is 2%-5%.

6. The preparation method according to claim 1, characterized in that, In S2, the secondary emulsion is stirred at room temperature for 4-12 hours.

7. The preparation method according to claim 1, characterized in that, In S3, tris(2-carboxyethyl)phosphonic acid hydrochloride and adipose tissue targeting peptide were added to sterile PBS buffer containing NP@siRNA and stirred continuously at 0-5°C. The molar ratio of adipose tissue targeting peptide to NP@siRNA was 1-2.4:

1.

8. The preparation method according to claim 7, characterized in that, In S3, the reaction is carried out under reaction conditions with a pH of 7.0-7.

4.

9. The preparation method according to claim 1, characterized in that, S3 also includes: adding the formed aNP@siRNA to a dialysis bag and dialyzing it in pure water for purification.

10. An siRNA delivery system obtained by the preparation method according to any one of claims 1-9.

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