Stem cell miRNA modification and targeted delivery method based on space transcriptomics and application

Abstract

The invention relates to a stem cell miRNA modification and targeted delivery method based on space transcriptomics and application. The method comprises the following steps: screening high-expression miR-21 and miR-126 in a wound proliferation region by integrating Vision and Stereo-seq technologies; by adopting optimized liposome transfection and electroporation methods, the expression quantity of miR-21 is increased by 12.5 times, the expression quantity of miR-126 is increased by 15.7 times, and the cell activity is maintained at 90% or above. The surface of the hyaluronic acid nano-carrier with the particle size of 200-300 nanometers is used for modifying CD11b and ICAM-1 antibodies, so that accurate delivery is realized, and the stem cell migration capability is improved by 92%. In a rat skin wound model, the 7-day healing rate of an experimental group reaches 85.0 + / -3.2% which is obviously higher than 62.0 + / -4.5% of a control group; in a myocardial infarction model, the left ventricular ejection fraction of an experimental group is increased to 48.3 + / -5.2% which is obviously superior to 28.7 + / -4.1% of a model group, and meanwhile, the inflammatory factor level is reduced by 76% to 82%. The method provides an efficient and safe treatment strategy for tissue wound repair.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and precision medicine, specifically a method and application for stem cell miRNA modification and targeted delivery based on spatial transcriptomics, for the repair of wounds in tissues such as skin and myocardium. Background Technology

[0002] Wound repair is a complex physiological process involving multicellular collaboration and dynamic gene regulation. In current clinical practice, the low efficiency of chronic wound repair and the insufficient homing rate of stem cell therapy have become core bottlenecks in the field. Although traditional stem cell therapy has the potential for tissue regeneration, only 1%-5% of stem cells can migrate to the wound site after intravenous infusion [Hao Z, Haibo C, Wei W, et al. Cell survival and redistribution after transplantation into damaged myocardium.[J]. Journal of cellular and molecular medicine, 2010, 14(5): 1078-82. ], and the high concentration of inflammatory factors in the wound microenvironment can significantly inhibit the differentiation function of stem cells [Saeed K, Hamid K, Zahedi AA, et al. Inflammatory Microenvironment of Acute Myocardial Infarction Prevents Regeneration of Heart with Stem Cells Therapy.[J]. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacochemistry, 2019, 53(5): 887-909. ], leading to insufficient angiogenesis or abnormal fibrosis. This dual challenge of "low homing efficiency and microenvironment inhibition" significantly reduces the clinical translational effectiveness of traditional stem cell therapies.

[0003] MicroRNAs (miRNAs), as key regulatory molecules of gene expression, provide a new direction for enhancing the repair capacity of stem cells. Studies have shown that miR-126 can promote vascular endothelial cell differentiation by activating the PI3K / AKT pathway [Lingqiang C, Jing W, Bing W, et al. MiR-126 inhibits vascular endothelial cell apoptosis through targeting PI3K / Akt signaling. [J]. Annals of hematology, 2016, 95(3): 365-74.], while miR-21 can enhance the anti-apoptotic activity of cells by inhibiting the PTEN gene [Danish S, Minzhen H, Chull H, et al. MicroRNA-21 is a downstream effector of AKT that mediates its antiapoptotic effects via suppression of Fas ligand. [J]. The Journal of biological chemistry, 2010, 285(26): 20281-90.].

[0004] However, existing technologies have significant limitations in miRNA screening and transfection applications:

[0005] (1) Lack of spatial heterogeneity in miRNA screening: Traditional transcriptome analysis relies on tissue homogenate, which cannot capture the gene expression differences in the "inflammation-proliferation-remodeling" regions of wound tissue. For example, miR-155, which is highly expressed in macrophages in the inflammatory area of ​​skin wounds, is 3.2 times more expressed than in the proliferative area. It is functionally very different from the miR-29 family enriched in fibroblasts in the proliferative area. Homogenate analysis will completely mask this spatial specificity, resulting in the screening of miRNA targets that are out of sync with actual repair needs.

[0006] (2) The contradiction between efficiency and safety of transfection technology: Although viral vectors have high transfection efficiency, they have the risk of insertion mutation; liposome transfection often leads to a decrease of ≥30% in stem cell activity [Jonathan Y, Yanfeng Z, PNG, et al. Cationic, helical polypeptide-based gene delivery for IMR-90 fibroblasts and human embryonic stem cells. [J]. Biomaterials science, 2013, 1(7): 719-727.], neither method can take into account both transfection efficiency and cell function integrity.

[0007] The emergence of spatial transcriptomics technology has made it possible to analyze the spatial heterogeneity of wound tissue [Dominik S, Laura RK. Spatial transcriptomics herald a new era of transcriptome research.[J]. Clinical and translational medicine, 2023, 13(5):e1264-e1264.], but it has not yet been systematically combined with miRNA regulation technology. Existing research lacks a standardized translation process from spatial transcriptomics data to miRNA targets, and has not established a stem cell modification protocol based on the spatial characteristics of the microenvironment, resulting in a break in the technical chain of "spatial analysis-target screening-cell modification" [Iivari K, Paulina F, Tomi S, et al. Computational solutions for spatial transcriptomics[J]. Computational and Structural Biotechnology Journal, 2022, 204870-4884.]. These gaps make it difficult for stem cell therapy to achieve precise regulation of wound repair. Summary of the Invention

[0008] The purpose of this invention is to provide a method and application for stem cell miRNA modification and targeted delivery based on spatial transcriptomics. By integrating spatial transcriptomics with miRNA transfection technology, a complete technical path of "spatial feature analysis - region-specific miRNA screening - stem cell function enhancement - targeted delivery" is established for the first time. This not only solves the problem of the disconnect between miRNA targets and microenvironment requirements in traditional methods, but also improves stem cell activity by optimizing the transfection process, providing an innovative solution for wound repair that combines precision and safety.

[0009] The technical solution of this invention is: to provide a method for stem cell miRNA modification and targeted delivery based on spatial transcriptomics, comprising the following steps:

[0010] (1) Spatial transcriptomics analysis of the trauma microenvironment:

[0011] Spatial transcriptome sequencing of trauma tissue was performed using Visium or Stereo-seq technology to divide the tissue into inflammatory, proliferative, and remodeling zones. Region-specific miRNAs with a fold change of >2.0 relative to normal tissue and p<0.05 in each target region and targeting at least two repair-related genes were screened.

[0012] (2) Stem cell miRNA transfection: using mesenchymal stem cells or induced pluripotent stem cells as the target, employing...

[0013] (i) Liposome method: 50 nM miRNA mimic with 3 μL / mL Lipofectamine 3000, transfection efficiency ≥80%, or

[0014] (ii) Electroporation: 120 V, 20 ms pulse × 2 times, cell viability > 85%,

[0015] The expression level of the region-specific miRNA was upregulated by ≥5-fold.

[0016] (3) Targeted delivery:

[0017] HA-PEG nanocarriers with a particle size of 200-300 nm and a PDI < 0.2 were prepared by emulsification-solvent evaporation method. CD11b or ICAM-1 antibodies were covalently coupled to the surface of the carriers using the EDC / NHS method, with an antibody binding rate ≥ 85%. The stem cells obtained in step (2) were loaded onto the carriers and injected intravenously or locally, which increased the efficiency of stem cell enrichment at the wound site by more than 3 times compared with the unmodified carrier.

[0018] The region-specific miRNA is selected from at least one of miR-21, miR-126, miR-155, or miR-29a.

[0019] The traumatic tissue is skin trauma or myocardial injury tissue.

[0020] The repair-related genes targeted by the miRNA include VEGFA, TGF-β, PTEN, or combinations thereof.

[0021] The nanocarrier described in step (3) is pH responsive, with a cumulative release rate of ≥65% over 7 days at pH 6.5.

[0022] The use of miRNA-modified stem cells prepared by the method in the preparation of drugs for promoting the repair of skin or myocardial wounds.

[0023] A kit for carrying out the method comprises:

[0024] (i) Visium or Stereo-seq chips for spatial transcriptome sequencing;

[0025] (ii) Region-specific miRNAs (mimics);

[0026] (iii) Liposome transfection reagent or electroporation buffer;

[0027] (iv) HA-PEG nanocarriers;

[0028] (v) Anti-CD11b or anti-ICAM-1 antibody.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) Precise regulation of repair pathways: miR-21 and miR-126 screened under the guidance of spatial transcriptomics can specifically activate pathways such as VEGFA and PI3K / AKT, increasing vascular density by 40%-60% and collagen deposition uniformity by 30%.

[0031] (2) Enhanced repair efficacy in two dimensions: miRNA transfection increased the migration ability of stem cells by 2 to 3 times. The Transwell experiment showed that the number of migrating cells reached 215±18, which was significantly higher than 112±15 in the control group. At the same time, the proportion of stem cells differentiating into endothelial cells and fibroblasts increased by more than 50%. Flow cytometry showed that the proportion of CD31 positive cells was 35%, while that in the control group was 22%.

[0032] (3) Reduced risk of immune rejection: The nanocarrier exhibits excellent biocompatibility, which extends the survival time of stem cells in vivo to more than 21 days and reduces allogeneic transplant rejection by 80%. Serum test results showed that the TNF-α level in the experimental group was 68±15 pg / mL, which was significantly lower than that in the control group of 285±32 pg / mL.

[0033] (4) Clinical translation advantages: The non-viral transfection system used completely avoids the risk of gene insertion mutation. At the same time, the pH-responsive release characteristic achieves a release rate of 68% in an environment with a pH value of 6.5, which significantly enhances the microenvironment targeting. Attached Figure Description

[0034] Figure 1 This is a flowchart of the technical process (spatial transcriptome analysis → miRNA screening → stem cell transfection → targeted delivery).

[0035] Figure 2 This is a schematic diagram of the nanocarrier structure (HA-PEG nanocarrier surface modified with antibody). Detailed Implementation

[0036] Example 1

[0037] This embodiment describes the application of the stem cell miRNA modification and targeted delivery method based on spatial transcriptomics in skin wound repair, including the following steps:

[0038] 1. Animal Model Construction

[0039] Thirty-six healthy SD rats (half male and half female), aged 8 weeks, were used in the experiment. Their weight was strictly controlled within the range of 200-220g. They were acclimatized for one week in an SPF-grade animal facility under a 12-hour light / dark cycle, at a temperature of 22±1℃ and humidity of 50±5%. They were randomly divided into three groups of 12 rats each: a model group (no treatment), a control group injected with untransfected hUC-MSCs, and an experimental group injected with miR-21-transfected hUC-MSCs. Rats were fasted for 6 hours preoperatively but had free access to water. Anesthesia was induced and maintained using an isoflurane inhalation anesthesia system (VetFlo), with an induction concentration of 3% and a maintenance concentration of 1.5%. The skin preparation area is centered on the spine, with 2cm of operating space reserved on each side. The disinfection procedure involves wiping the area three times with 10% povidone-iodine solution, followed by deiodination with 75% ethanol. A specially made stainless steel circular skin punch with a diameter of 15mm is used to press vertically down to the fascia layer to create a full-thickness skin defect. A sterile cotton swab is used to gently press the wound for 30 seconds to ensure hemostasis. The wound is kept in a single cage after the operation and the condition of the wound is observed daily.

[0040] 2. Spatial Transcriptome Analysis

[0041] On the third day after modeling, areas containing newly formed granulation tissue at the wound edge were precisely collected and immediately flash-frozen in liquid nitrogen in OCT embedding medium, then stored at -80℃. 10μm thick sections were prepared using a Leica CM1950 cryostat with a blade temperature of -20℃ and a sample tip temperature of -18℃. Every five sections were stained with hematoxylin and eosin (HE) for microscopic examination to ensure intact tissue morphology. Sections were attached to Visium gene expression slides and sequentially fixed with 4% PFA for 10 minutes and permeabilized with 0.1% Triton X-100 for 18 minutes (optimal times determined through gradient experiments). Libraries were constructed strictly according to the 10x Genomics standard procedure. PE150 sequencing was performed using an Illumina NovaSeq 6000 S4 flow cell, yielding an average of 3.2 × 10^8 reads per sample, with a Q30 quality score > 90%. Data analysis was performed using Space Ranger software for sequence alignment and Seurat v4.0 for cell clustering. The MAST algorithm was used to screen for miR-21 with significantly high expression in the proliferation region with FDR < 0.05. Its expression level was upregulated by 2.8 times compared with normal tissue (p < 0.001), and its target gene PTEN was predicted by the TargetScan database.

[0042] Table 1. Expression characteristics of miRNAs screened by spatial transcriptomics

[0043]

[0044] As can be seen from the experimental results in Table 1, miR-21 is specifically highly expressed in the wound proliferation area, and its expression level is significantly upregulated by 2.8 times compared with normal tissue (p<0.001). It also plays a regulatory role by targeting the PTEN gene, providing a precise target for subsequent stem cell modification.

[0045] 3. Stem cell transfection and validation

[0046] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in DMEM / F12 medium containing 10% FBS. When the cell density reached 70%, the cells were gently washed with PBS containing 0.5 mM EDTA and then passaged by digestion with 0.25% trypsin containing 0.02% EDTA for 90 seconds. Flow cytometry analysis showed that the expression of CD73+ / CD90+ / CD105+ markers was >95%. 18-24 hours before transfection, cells from passages 3-5 were seeded at 1×10^5 / well in 6-well plates. The optimized transfection system consisted of 50 nM miR-21 mimic and 3 μL / mL Lipofectamine 3000 diluted separately with Opti-MEM and mixed in equal volumes. The mixture was allowed to stand at room temperature for 5 minutes to form a complex, and then replaced with complete medium after 6 hours. qPCR detection 48 hours after transfection showed that miR-21 expression was upregulated by 12.5-fold. Transwell migration assay showed that the number of migrating cells reached 215±18, while the control group had 112±15 (p<0.01). Western blot confirmed that PTEN protein expression was downregulated by 35% while the p-AKT / AKT ratio increased by 40%.

[0047] Table 2. Results of stem cell transfection parameter optimization

[0048]

[0049] As can be seen from the experimental results in Table 2, the transfection conditions of 50 nM miR-21 mimic combined with 3 μL / mL Lipofectamine 3000 can achieve a 12.5-fold upregulation of miRNA expression while ensuring a cell survival rate of over 93%, thus meeting the requirements for the preparation of functionally enhanced stem cells.

[0050] 4. Targeted therapy and efficacy evaluation

[0051] HA-PEG nanocarriers were prepared using an emulsification-solvent evaporation method. Malvern Zetasizer Nano ZS analysis showed an average particle size of 253±12 nm and a PDI of 0.12. Transmission electron microscopy revealed a regular spherical shape. CD11b antibody was coupled to the carrier surface via EDC / NHS activation, with a binding rate of 85.3% as determined by the BCA method. On day 3 post-modeling, four injections of 25 μL each were administered at 2 mm from the wound edge, using a Harvard Apparatus microinfusion pump at a flow rate of 10 μL / min. Each injection contained 2.5 × 10^5 transfected stem cells. Seven days post-surgery, ImageJ analysis showed that the wound healing rate in the experimental group was 85.0±3.2%, compared to 62.0±4.5% in the control group. The Masson staining score was 4.8±0.6, compared to 3.4±0.5 in the control group. CD31 immunofluorescence showed a vessel density of 32.5±4.1 vessels / mm², compared to 21.3±3.2 vessels / mm² in the control group. All procedures were performed in a biosafety cabinet and complete experimental records were maintained.

[0052] Table 3. Evaluation of the efficacy of skin wound repair

[0053]

[0054] As can be seen from the experimental results in Table 3, the wound healing rate of the miR-21 transfected stem cell treatment group was significantly increased to 85.0±3.2% (p<0.01), and the angiogenesis and collagen deposition indices were better than those of the control group, confirming that this method can simultaneously promote re-epithelialization and tissue remodeling.

[0055] Example 2

[0056] This embodiment describes the application of the stem cell miRNA modification and targeted delivery method based on spatial transcriptomics in myocardial injury repair, including the following steps:

[0057] 1. Establishment of a myocardial infarction model

[0058] Forty male C57BL / 6 mice aged 6-8 weeks, with their weight strictly controlled within the range of 20-25g, were randomly divided into a sham-operated group, a model group, a control group, and an experimental group, with 10 mice in each group. Preoperatively, mice were anesthetized with an intraperitoneal injection of 50 mg / kg sodium pentobarbital. They were then intubated and connected to a small animal ventilator (Harvard Apparatus) with a tidal volume of 0.3 mL and a respiratory rate of 120 breaths / min. A thoracotomy was performed at the left fourth intercostal space to expose the heart. The left anterior descending coronary artery (LAD) was located between the pulmonary conus and the left atrial appendage and ligated using 8-0 polypropylene sutures until the anterior myocardium showed significant pallor. A ST segment elevation >0.2 mV in lead II of the electrocardiogram confirmed successful model creation. In the sham-operated group, only sutures were threaded without ligation. The entire surgery was performed on a 37℃ constant-temperature operating table. All instruments were sterilized by autoclaving at 121℃ for 20 minutes. Postoperatively, mice received subcutaneous injections of 0.1 mg / kg buprenorphine for analgesia for three consecutive days.

[0059] 2. miRNA screening and validation

[0060] Seven days post-surgery, tissue samples from the infarct border region were precisely collected, embedded in OCT, and then prepared into 5μm frozen sections for Stereo-seq sequencing. An average of 5.8 × 10^8 reads were obtained per sample, with a resolution of 0.5μm. After removing spots with a UMI < 500, spatial hotspots were identified using the SPARK-X method. The results showed that miR-126 expression in the ischemic junction was upregulated 3.2-fold compared to normal myocardial tissue (p < 0.001). A psiCHECK2 reporter vector containing the wild-type 3'UTR sequence of the SPRED1 gene was constructed and co-transfected with miR-126 mimic into 293T cells. Using Lipofectamine 2000 (plamid:reagent = 1:2.5), 48 hours after transfection, a dual-luciferase assay showed a 58% decrease in fluorescence activity in the experimental group (p < 0.001), while no significant change was observed in the mutant vector group, confirming the direct targeting relationship between miR-126 and SPRED1.

[0061] Table 4. Spatial transcriptomic characteristics of myocardial tissue

[0062]

[0063] As can be seen from the experimental results in Table 4, the expression level of miR-126 in the ischemic junction was upregulated by 3.2 times compared with that in normal myocardial tissue (p<0.001), and it was significantly enriched in the VEGF signaling pathway, providing a region-specific regulatory target for myocardial repair.

[0064] 3. Stem cell preparation and delivery

[0065] Human embryonic stem cell-derived cardiomyocyte progenitor cells (hESC-CPCs) were maintained in mTeSR1 medium and digested with 1 mg / mL collagenase IV for 7 minutes before passage. Electroporation was performed using the Neon transfection system with optimized parameters of 120 V voltage, 20 ms pulses × 2 times, and Buffer R cell density adjusted to 1 × 10^7 / mL. qPCR confirmed a 15.7-fold upregulation of miR-126 expression after transfection. The nanocarrier was labeled with DiR fluorescence at 1 μg / mg of carrier. Dynamic light scattering analysis showed that the HA-CD11b nanocarrier had an average particle size of 250 ± 15 nm and a Zeta potential of -23.5 mV. In vitro release experiments confirmed a cumulative release rate of 68% after 7 days at pH 6.5 and 42% at pH 7.4. Three days post-surgery, 1×10^6 cells and a nanocarrier complex were injected into the infarct border area via epicardial injection at a cell-to-carrier mass ratio of 1:5. IVIS Spectrum in vivo imaging showed that the cells were mainly enriched in the cardiac region within 7 days.

[0066] Table 5. Characteristic parameters of nanocarriers

[0067]

[0068] As can be seen from the experimental results in Table 5, the HA-CD11b nanocarrier has a uniform particle size, an antibody binding rate of 85.3±4.2%, and a release rate of 68% under pH 6.5 conditions, which perfectly matches the microenvironmental requirements of myocardial ischemia.

[0069] 4. Evaluation of treatment effectiveness

[0070] Four weeks post-surgery, cardiac function was assessed using a VisualSonics Vevo 2100 ultrasound system with a probe frequency of 30 MHz. The left ventricular ejection fraction (LVEF) in the experimental group was 48.3±5.2%, compared to 28.7±4.1% in the model group. The left ventricular end-diastolic diameter (LVEDD) decreased to 4.5±0.4 mm, compared to 5.8±0.5 mm in the model group. After fixation with 4% PFA perfusion, 5 μm paraffin sections were prepared parallel to the long axis. Masson's trichrome staining showed an infarct area of ​​21.3±3.5% in the experimental group and 42.7±4.8% in the model group. α-SMA immunohistochemistry showed a vessel density of 45.2±5.6 vessels / field, compared to 18.6±3.1 vessels / field in the model group. Serum ELISA showed an 82% reduction in TNF-α levels in the experimental group compared to the model group (p<0.01). No pathological damage was observed in major organs after HE staining.

[0071] Table 6. Effects on Cardiac Function Improvement

[0072]

[0073] As can be seen from the experimental results in Table 6, the miR-126 transfection combined with targeted delivery system can reduce the myocardial infarction area by 50.1%, increase the left ventricular ejection fraction by 68.3%, and significantly improve the survival rate of transplanted stem cells, thus comprehensively reversing the process of myocardial remodeling.

Claims

1. A method for stem cell miRNA modification and targeted delivery based on spatial transcriptomics, characterized in that, Includes the following steps: (1) Spatial transcriptomics analysis of the trauma microenvironment: Spatial transcriptome sequencing of trauma tissue was performed using Visium or Stereo-seq technology to divide the tissue into inflammatory, proliferative, and remodeling zones. Region-specific miRNAs with a fold change of >2.0 relative to normal tissue and p<0.05 in each target region and targeting at least two repair-related genes were screened. (2) Stem cell miRNA transfection: using mesenchymal stem cells or induced pluripotent stem cells as the target, employing... (i) Liposome method: 50 nM miRNA mimic with 3 μL / mL Lipofectamine 3000, transfection efficiency ≥80%, or (ii) Electroporation: 120 V, 20 ms pulse × 2 times, cell viability > 85%, upregulating the expression level of the region-specific miRNA ≥ 5-fold; (3) Targeted delivery: HA-PEG nanocarriers with a particle size of 200-300 nm and a PDI < 0.2 were prepared by emulsification-solvent evaporation method. CD11b or ICAM-1 antibodies were covalently coupled to the surface of the carriers using the EDC / NHS method, with an antibody binding rate ≥ 85%. The stem cells obtained in step (2) were loaded onto the carriers and injected intravenously or locally, which increased the efficiency of stem cell enrichment at the wound site by more than 3 times compared with the unmodified carrier.

2. The method according to claim 1, characterized in that, The region-specific miRNA is selected from at least one of miR-21, miR-126, miR-155, or miR-29a.

3. The method according to claim 1 or 2, characterized in that, The traumatic tissue is skin trauma or myocardial injury tissue.

4. The method according to any one of claims 1-3, characterized in that, The repair-related genes targeted by the miRNA include VEGFA, TGF-β, PTEN, or combinations thereof.

5. The method according to any one of claims 1-4, characterized in that, The nanocarrier described in step (3) is pH responsive, with a cumulative release rate of ≥65% over 7 days at pH 6.

5.

6. Use of miRNA-modified stem cells prepared by the method according to any one of claims 1-5 in the preparation of medicaments for promoting the repair of skin or myocardial wounds.

7. A kit for carrying out the method according to any one of claims 1-5, comprising: (i) Visium or Stereo-seq chips for spatial transcriptome sequencing; (ii) Region-specific miRNAs (mimics); (iii) Liposome transfection reagent or electroporation buffer; (iv) HA-PEG nanocarriers; (v) Anti-CD11b or anti-ICAM-1 antibody.

Images