Preparation method for micro-vehicle formed by enveloping sirna-loaded polypeptide dendrimer nanogel with a cell membrane of engineered mscs, and the use of same

Abstract

A preparation method for a micro-vehicle formed by enveloping siRNA-loaded polypeptide dendrimer nanogel with a cell membrane of engineered MSCs, and the use of same. The method comprises: using lentivirus-mediated gene transfection technology for constructing MSCs that highly express CXCR4 and PSGL-1; using cytokinin B for stimulating a cell to generate vesicles, and then performing centrifugation to collect the vesicles; by means of the electrostatic attraction between polypeptide dendrimer nanoparticles and siRNAs, attaching the siRNAs of IL-6 and HIF-1α into the polypeptide dendrimer nanoparticles; and by means of using a membrane extruder, enveloping the siRNA-loaded polypeptide dendrimer nanogel with the cell membrane. The prepared micro-vehicle exhibits a good property of aggregating at inflammatory blood vessel sites and a targeting function for joint cavities with rheumatoid arthritis. In addition, the loaded siRNAs can inhibit the expressions of IL-6 and HIF-1α in the joint cavities and inhibit the cell migration and angiogenesis. The micro-vehicle targeting inflammation sites has the advantages of excellent biocompatibility, ideal siRNA loading ratio, good inflammation targeting function, etc.

Description

Preparation method and application of engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a preparation method and application of an engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease with a complex pathophysiology characterized by synovial lining proliferation, angiogenesis, pannus formation, and cartilage and bone destruction. Clinically, nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DAMs), and glucocorticoids have been used to alleviate RA symptoms. However, given the potential for drug resistance and adverse reactions associated with prolonged, high-dose use, more effective and targeted treatments are under development. Monoclonal antibodies targeting proinflammatory cytokines, such as infliximab and tocilizumab, have demonstrated efficacy in RA patients. However, due to limited retention in target tissues, low bioavailability, and adverse reactions associated with systemic administration, monoclonal antibody therapy is less effective than conventional drugs. Compared to biologics, siRNAs (siRNAs) offer inherent advantages, such as interfering with mRNA translation and regulating downstream protein expression. However, their unsatisfactory stability limits their widespread clinical application.

[0003] To address this issue, the present invention developed a novel siRNA targeted delivery system. This system consists of engineered mesenchymal stem cells (MSCs) carrying siRNA-loaded peptide dendrimer nanogels (PDNPs-siRNAs) on their cell membranes for intravenous administration to treat rheumatoid arthritis. Compared to traditional linear polymers, peptide dendrimers possess a highly branched structure, a protein-like globular structure, and a large number of peripheral groups, making them ideal delivery vehicles. siRNAs are encapsulated within the PDNPs via electrostatic adsorption, enhancing their stability and enabling sustained release of therapeutic siRNAs. Furthermore, bioengineering techniques are employed to endow MSCs with chemotactic receptors and endothelial cell adhesion molecules, mediating their homing to inflamed and diseased tissues. P-selectin glycoprotein ligand-1 (PSGL-1), a glycoprotein expressed on the surface of all leukocytes, specifically targets the vasculature of damaged knee synovial tissue, which expresses high levels of the PSGL-1 ligands E- and P-selectin. In response to biological signals, the prepared microcarriers can cross the vascular barrier and reach diseased tissue. CXC chemokine receptor 4 (CXCR4) is a G protein-coupled transmembrane receptor. Its ligand, stromal cell-derived factor 1α (SDF-1α), is highly expressed on synovial cells and is a major regulator of synovial chemotaxis. Therefore, the present invention integrates MSC-derived cell membranes that highly express PSGL-1 and CXCR4, with peptide-based dendrimer nanoparticles and siRNAs targeting IL-6 and HIF1-α, to form a specific targeted delivery system for RA treatment. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the existing technology and provide a method for preparing and applying an engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs, thereby solving the problems of traditional drugs with multiple side effects, low drug absorption rate and lack of targeting.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] (1) The present invention provides a method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs, comprising:

[0007] S1, stimulate MSCs to produce vesicles and collect the vesicles by centrifugation;

[0008] S2, preparing polypeptide dendrimer nanogels loaded with siRNAs;

[0009] S3. The vesicles are mixed with the polypeptide dendrimer nanogel loaded with siRNAs and extruded to obtain engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs.

[0010] Furthermore, in S1, the MSCs cells are MSCs cells that overexpress CXCR4 and PSGL-1, and the preparation method thereof is: (1) constructing CXCR4 and PSGL-1 recombinant vectors using molecular biology technology; (2) packaging lentivirus in 293T cells using the CXCR4 and PSGL-1 recombinant vectors; (3) collecting lentivirus, infecting MSCs cells with the concentrated lentivirus, and screening positive cells that simultaneously express the two molecules using antibiotic labels and flow cytometry to obtain a stable MSCs cell line loaded with overexpressed CXCR4 and PSGL-1; the human CXCR4 sequence is SEQ ID NO: 1, and the human PSGL-1 sequence is SEQ ID NO: 2.

[0011] Furthermore, in S1, the method for preparing vesicles is: treating MSCs cells overexpressing CXCR4 and PSGL-1 with cytochalasin B, and obtaining MSCs cell vesicles overexpressing CXCR4 and PSGL-1 by ultrahigh-speed centrifugation.

[0012] Furthermore, in S2, the preparation method of the polypeptide dendrimer nanogel loaded with siRNAs is as follows: using double distilled water treated with diethyl pyrocarbonate to dissolve IL-6 interfering siRNA and HIF-1α interfering siRNA; dissolving the polypeptide dendrimer nanogel with double distilled water treated with diethyl pyrocarbonate to obtain a polypeptide dendrimer nanogel solution; and thoroughly mixing the above two solutions to obtain the polypeptide dendrimer nanogel loaded with siRNAs.

[0013] Furthermore, the weight ratio of the IL-6 interfering siRNA, HIF-1α interfering siRNA and polypeptide dendritic nanogel is 1:1:16-200; the two solutions are mixed for 30 minutes at room temperature.

[0014] Furthermore, the weight ratio of the IL-6 interfering siRNA, HIF-1α interfering siRNA and polypeptide dendritic nanogel is 1:1:80.

[0015] Furthermore, the sense strand of the IL-6 interfering siRNA is the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 4; the sense strand of the HIF-1α interfering siRNA is the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 6.

[0016] Furthermore, the preparation method of the polypeptide dendritic nanogel is as follows: under nitrogen, first, third-generation lysine (generation 3 poly (epsilon-lysine), G3K), benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (BPY), 1-hydroxybenzotriazole (HOBT) and O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) are mixed in anhydrous DMF (N,N-dimethylformamide), stirred at 0°C, and then, N,N-diisopropylethylamine (DIPEA) is added dropwise, and the mixture is further stirred at room temperature. After dialyzing for three days, polypeptide dendritic nanogel PDNPs are obtained.

[0017] Furthermore, the molar ratio of G3K, BPY, HOBT and HBTU is 1:(2-10):6:6; the mass volume ratio of G3K to anhydrous DMF is 1 g:5 ml; the stirring time at 0°C is 30 min, and the stirring time at room temperature is 24 h.

[0018] Furthermore, in S3, the vesicles and the polypeptide dendrimer nanogel loaded with siRNAs are evenly mixed, and the mixed solution is placed in a syringe on one side of a membrane extruder and squeezed back and forth several times to obtain engineered MSCs cell membrane-wrapped polypeptide dendrimer nanogel microcarriers M@PDNPs-siRNAs loaded with siRNAs.

[0019] Furthermore, the vesicles are mixed with the polypeptide dendrimer nanogel loaded with siRNAs in an equal amount.

[0020] Furthermore, the prepared M@PDNPs-siRNAs were passed through membranes with different pore sizes to obtain M@PDNPs-siRNAs of different particle sizes.

[0021] Furthermore, M@PDNPs-siRNAs can enter the target cells through cell membrane fusion to achieve cell transfection.

[0022] Furthermore, the polypeptide dendrimer nanogel loaded with siRNAs is also mixed with one or more of miRNAs, nucleic acid drugs, and nucleases.

[0023] (II) The present invention also provides the use of the above-mentioned engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs in the preparation of a drug for treating rheumatoid arthritis.

[0024] (1) The present invention uses MSCs cell vesicles that overexpress CXCR4 and PSGL-1 to enrich M@PDNPs-siRNAs at the site of inflammation, showing good inflammation targeting;

[0025] (2) The present invention designs a polypeptide dendrimer nanogel loaded with siRNAs. The polypeptide dendrimer nanogel is positively charged and can adsorb negatively charged nucleic acids or drugs, achieving long-term drug administration and sustained drug release, and is highly practical.

[0026] (3) The present invention prepares an engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs, which can be used for cell transfection and intravenous administration, achieving safe and targeted drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a schematic diagram of MSCs cell vesicles overexpressing CXCR4 and PSGL-1 prepared in Example 1;

[0028] FIG2 is an agarose gel electrophoresis image of the polypeptide dendrimer nanogel loaded with siRNAs prepared in Example 2;

[0029] FIG3 is a transmission electron micrograph of the engineered MSCs cell membrane-encapsulated siRNA-loaded polypeptide dendrimer nanogel microcarriers prepared in Example 3;

[0030] Figure 4 shows the effect of M@PDNPs-siRNAs on inhibiting target gene expression after transfection of synovial fibroblasts;

[0031] Figure 5 is a diagram showing the targeting verification effect of M@PDNPs-siRNAs. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the relevant drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The implementation conditions not specified in the embodiments are generally the conditions in routine experiments.

[0033] Example 1: Preparation of MSCs vesicles overexpressing CXCR4 and PSGL-1

[0034] (I) Preparation of MSCs overexpressing CXCR4 and PSGL-1:

[0035] (1) Construction of recombinant vectors for MSCs overexpressing CXCR4 and PSGL-1

[0036] Molecular biology techniques were used to construct recombinant vectors for overexpressing CXCR4 and PSGL-1. The vector Plv-IRES was selected, and the target sequence (CXCR4 or PSGL-1), three repeated FLAG tag sequences, EGFP sequence and puromycin resistance sequence were inserted into the open reading frame.

[0037] (2) Lentiviral packaging

[0038] Viruses were packaged in 293T cells using a lentiviral packaging system. The viral packaging system consisted of 20 μg of plv-IRES-CXCR4 or plv-IRES-PSGL-1, 15 μg of psPAX2, and 5 μg of PMD2G. 293T cells were cultured to 70% confluency. After changing the medium, the plasmid mixture and p3000 were mixed and allowed to stand at room temperature for 15 minutes. The mixture was then added to Lip3000 transfection reagent and the suspension was added dropwise to the 293T cells. After 48 hours of culture, the supernatant was collected.

[0039] (3) Virus infection of MSC cells

[0040] MSCs were cultured in 6-well plates to a cell density of 70%. 1 ml of the aforementioned lentiviral suspension was added, along with a transfection aid. After 48 hours of culture, puromycin was added for selection. Dead cells were removed, and the remaining puromycin-resistant cells were identified as MSCs overexpressing CXCR4 or PSGL-1.

[0041] (II) Preparation of vesicles:

[0042] MSCs overexpressing CXCR4 and PSGL-1 were cultured in complete culture medium. When the cell density reached approximately 80%, the culture medium was switched to serum-free medium and cytochalasin B was added at a working concentration of 10 μg / ml for 30 minutes. As shown in Figure 1, significant vesicle production was observed in the engineered MSCs after cytochalasin B treatment.

[0043] The treated cells were trypsinized, centrifuged at 1000 rpm for 5 minutes at room temperature, washed once with PBS, and centrifuged again at 1000 rpm for 5 minutes at room temperature. The supernatant was collected and centrifuged at 6000 rpm for 15 minutes at room temperature to precipitate the cell vesicles.

[0044] Example 2: Preparation of a Polypeptide Dendrimer Nanogel Loaded with siRNAs

[0045] (I) Preparation of polypeptide dendrimer nanogels (PDNPs):

[0046] Under nitrogen, G3K (1.00 g, 0.12 mmol), BPY (0.12 g / 0.6 mmol), HOBT (97 mg, 0.72 mmol), and HBTU (273 mg, 0.72 mmol) were mixed in 5 mL of anhydrous DMF and stirred at 0°C for 30 minutes. Then, a few drops of DIPEA were added dropwise. The mixture was further stirred at room temperature for 24 hours. The peptide dendrimer nanogels (PDNPs) were purified by dialysis against DMF and distilled water for 3 days.

[0047] (II) Preparation of polypeptide dendrimer nanogels loaded with siRNAs:

[0048] IL-6 and HIF-1α interference siRNAs were dissolved in double-distilled water treated with diethyl pyrocarbonate (DEPC), and 125 ng of each siRNA was taken and mixed evenly to 250 ng.

[0049] After drying, the peptide dendrimer nanogels (PDNPs) were dissolved in DEPC-treated double-distilled water. The weights of different PDNPs were: 0, 0.5, 1, 2, 5, 10, and 20 μg.

[0050] The two solutions were thoroughly mixed and reacted at room temperature for 30 minutes before analysis by agarose gel electrophoresis. As shown in Figure 2, when the siRNA concentration was 250 ng and the PDNP mass was 10 μg, the siRNAs were completely adsorbed, resulting in a peptide dendrimer nanogel loaded with siRNAs, namely PDNPs-siRNAs.

[0051] In this example, IL-6 interfering siRNAs and HIF-1α interfering siRNAs were synthesized by the company.

[0052] Among them, IL-6 interference siRNAs are composed of sense strands and antisense strands:

[0053] Sense strand 5′-CCUCUGGUCU UCUGGAGUATT-3′ (SEQ ID NO: 3);

[0054] Antisense strand 5′-UACUCCAGAAGACCAGAGGTT-3′ (SEQ ID NO: 4).

[0055] HIF-1α interference siRNAs are composed of sense and antisense strands:

[0056] Sense strand 5′- CUGAUAACGUGAACAAAUATT-3′ (SEQ ID NO: 5);

[0057] Antisense strand 5′- UAUUUGUUCACGUUAUCAGTT-3′ (SEQ ID NO: 6).

[0058] Example 3: Preparation of an engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs

[0059] The cell vesicles obtained in Example 1 were collected and their protein concentration was determined. Equal masses of the cell vesicles and the PDNPs-siRNAs from Example 2 were mixed and uniformly prepared to produce M@PDNPs-siRNAs. The mixture was placed in a syringe on one side of a membrane extruder and extruded back and forth 10 times to obtain MSCs membrane-encapsulated siRNA-loaded polypeptide dendrimer nanogel microcarriers, i.e., M@PDNPs-siRNAs.

[0060] The prepared M@PDNPs-siRNAs were analyzed by transmission electron microscopy. As shown in Figure 3, the cell membrane structure was clearly visible at the edge of the M@PDNPs-siRNAs.

[0061] Example 4: M@PDNPs-siRNAs transfection of synovial fibroblasts to detect transfection effect experimental steps

[0062] Synovial fibroblasts were cultured in complete medium. When the cells expanded to a density of about 60%, M@PDNPs-siRNAs were added. After 48 hours of co-culture, the cells were collected and the expression of the target gene was detected using real-time quantitative PCR technology, as shown in Figure 4.

[0063] As shown in Figure 4 , after synovial fibroblasts were treated with M@PDNPs-siRNAs, the expression of IL-6 and HIF-1α in the cells was significantly reduced.

[0064] Example 5: M@PDNPs-siRNAs targeting verification test

[0065] Using a collagen-induced RA mouse model, M@PDNPs-siRNAs (siRNAs carrying a Cy5.5 tag) were injected via the tail vein. Twenty-four hours later, small animal imaging technology was used to observe the accumulation of M@PDNPs-siRNAs at the site of inflammation. As shown in Figure 5, M@PDNPs-siRNAs were enriched in the validated joints, demonstrating the good targeting of M@PDNPs-siRNAs.

[0066] The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs, characterized in that: include: S1, stimulate MSCs to produce vesicles, and collect the vesicles by centrifugation; S2, preparing polypeptide dendrimer nanogel loaded with siRNAs; S3. The vesicles and the polypeptide dendrimer nanogel loaded with siRNAs are mixed and extruded to obtain engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs.

2. The method for preparing the engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs according to claim 1, characterized in that: In S1, the MSCs cells are MSCs cells that overexpress CXCR4 and PSGL-1, Its preparation method is: (1) Use molecular biology techniques to construct CXCR4 and PSGL-1 recombinant vectors; (2) Using CXCR4 and PSGL-1 recombinant vectors to package lentivirus in 293T cells; (3) Collecting lentivirus, infecting MSCs cells with the concentrated lentivirus, and using antibiotic labeling and flow cytometry to screen positive cells that simultaneously express two molecules to obtain a stable MSCs cell line overexpressing CXCR4 and PSGL-1.

3. The method for preparing the engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs according to claim 1, characterized in that: In S1, the method for preparing the vesicle is: MSCs cells overexpressing CXCR4 and PSGL-1 were treated with cytochalasin B, and MSCs cell vesicles overexpressing CXCR4 and PSGL-1 were obtained by ultra-high speed centrifugation.

4. The method for preparing the engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs according to claim 1, characterized in that: ­ In S2, the preparation method of the polypeptide dendrimer nanogel loaded with siRNAs is: Double distilled water treated with diethyl pyrocarbonate was used to dissolve IL-6 interference siRNA and HIF-1α interference siRNA; The polypeptide dendritic nanogel is dissolved in double distilled water treated with diethyl pyrocarbonate to obtain a polypeptide dendritic nanogel solution; The two solutions are fully mixed to obtain the polypeptide dendrimer nanogel loaded with siRNAs.

5. The method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs according to claim 4, characterized in that: The weight ratio of the IL-6 interfering siRNA, HIF-1α interfering siRNA and polypeptide dendritic nanogel is 1:1:16-200; the two solutions are mixed for 30 minutes at room temperature.

6. The method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs according to claim 4, characterized in that: The preparation method of the polypeptide dendritic nanogel is as follows: Under nitrogen, the third-generation lysine, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and O-benzotriazole-tetramethyluronium hexafluorophosphate were first mixed in anhydrous N,N-dimethylformamide and stirred at 0°C. Then, N,N-diisopropylethylamine was added dropwise, and the mixture was further stirred at room temperature. After dialysis, polypeptide dendritic nanogel PDNPs were obtained.

7. The method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs according to claim 6, characterized in that: The molar ratio of the third generation lysine, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and O-benzotriazole-tetramethyluronium hexafluorophosphate is 1:(2-10):6:6; The mass volume ratio of the third generation lysine to anhydrous N,N-dimethylformamide is 1g:5ml; The stirring time was 30 min at 0 °C and 24 h at room temperature.

8. The method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs according to claim 1, characterized in that: In S3, the vesicles and the polypeptide dendrimer nanogel loaded with siRNAs are mixed evenly, and the mixed solution is placed in a syringe on one side of a membrane extruder and squeezed back and forth several times to obtain engineered MSCs cell membrane-wrapped polypeptide dendrimer nanogel microcarriers M@PDNPs-siRNAs loaded with siRNAs.

9. The method for preparing engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarriers loaded with siRNAs according to claim 8, characterized in that: The vesicles and the polypeptide dendrimer nanogel loaded with siRNAs are mixed in equal amounts.

10. Use of the engineered MSCs cell membrane-encapsulated polypeptide dendrimer nanogel microcarrier loaded with siRNAs as claimed in any one of claims 1 to 9 in the preparation of a therapeutic drug for rheumatoid arthritis.

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