A plasmid, a delivery system for targeting senescent cells containing the plasmid, and a preparation method and application thereof

Abstract

A kind of plasmid, the preparation method and application of delivery system for targeting senescent cells containing the plasmid and its application, delivery system includes nanocomposite with core-shell structure, the nanocomposite includes plasmid complex wrapped by chimeric antigen receptor cell membrane for targeting senescent cells;The plasmid complex includes upconversion luminescent material and recombinant DNA plasmid.Delivery system for targeting senescent cells containing the plasmid delivers recombinant DNA plasmid into uPAR positive senescent cells, induces senescent cells to produce melanin, and the melanin is used to reduce ROS and generates photothermal effect under near-infrared light irradiation to drive temperature-sensitive hydrogel to shrink, and the same near-infrared light activates apoptosis effect module in plasmid through upconversion luminescent material to remove senescent cells, to realize the sequential regulation strategy of " first functional remodeling, then controlled removal".

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a plasmid, a delivery system containing the plasmid for targeting senescent cells, its preparation method, and its application. Background Technology

[0002] Chronic, non-healing wounds are often characterized by persistent inflammation, increased oxidative stress, extracellular matrix degradation, insufficient angiogenesis, and decreased tissue regeneration capacity. Recent studies have shown that the continuous accumulation of senescent cells in chronic wounds is a significant reason for their prolonged non-regenerative state. Although senescent cells have lost their normal proliferation and repair capabilities, they can still continuously secrete aging-related phenotypic factors, including inflammatory factors, chemokines, matrix-degrading enzymes, and pro-fibrotic factors, thereby maintaining local inflammation, oxidative stress, and tissue structural damage.

[0003] Existing therapeutic strategies targeting senescent cells mainly include small molecule senescent cell scavengers, senescence phenotype modulators, drug delivery systems targeting surface or intracellular markers of senescent cells, and immune cell-mediated senescent cell clearance strategies.

[0004] Among them, small molecule senescent cell scavengers can induce cell death by inhibiting senescent cell-dependent anti-apoptotic pathways. This type of scavenging strategy mainly aims to "directly eliminate senescent cells." Although it can reduce the number of senescent cells, it cannot restore the pathological microenvironment caused by long-term senescent cells, such as increased ROS, accumulation of inflammatory factors, and damage to the extracellular matrix. It also lacks the ability to remodel the wound microenvironment and initiate tissue regeneration.

[0005] Drug systems targeting intracellular markers such as lipofuscin and BCL-xL / BCL-2 can improve selectivity for senescent cells. However, some strategies targeting intracellular senescence markers require the drug or nanoparticles to first enter the cell before relying on the markers for selective action. These strategies may simultaneously enter both senescent and non-senescent cells, leading to reduced drug utilization efficiency and increased risk of unnecessary exposure to normal cells or tissues.

[0006] While immune cell-mediated senescent cell clearance methods, such as CAR-T cells (chimeric antigen receptor T cells) or CAR macrophages, can improve the recognition of surface markers on senescent cells, live cell therapy faces challenges including complex preparation, difficulty in batch-to-batch stability control, difficulty in maintaining cell viability in the local wound environment, limited tissue penetration, and potential immune-related adverse reactions. For local environments characterized by inflammation, hypoxia, and protease abundance, such as diabetic wounds, the stability and controllability of live cell therapy remain limited.

[0007] On the other hand, chronic wound repair also requires local material support and mechanical assistance. Existing hydrogel dressings can be used to maintain a moist environment, deliver drugs or growth factors, absorb exudate, and promote tissue repair. Some stimulus-responsive hydrogels can also respond to signals such as temperature, light, pH, or enzymes. However, most existing hydrogels only serve as passive dressings or drug carriers and cannot form an orderly linkage with the regulation of senescent cell fate. Summary of the Invention

[0008] This invention provides a plasmid, a delivery system for targeting senescent cells containing the plasmid, a method for preparing the plasmid, and its applications. The recombinant DNA plasmid includes a tyrosinase expression module for inducing melanin biosynthesis and an apoptosis effect module expressed under light response. The delivery system for targeting senescent cells containing the plasmid delivers the recombinant DNA plasmid into uPAR-positive senescent cells, inducing the senescent cells to produce melanin. The melanin is used to reduce ROS and generate a photothermal effect under near-infrared light irradiation to drive the contraction of a thermosensitive hydrogel. The same near-infrared light activates the apoptosis effect module in the plasmid through an upconversion luminescent material to clear senescent cells, thereby realizing a sequential regulatory strategy of "functional remodeling first, then controlled clearance". This makes melanin a key mediator in the continuous therapeutic sequence of "senescent cell functional remodeling - ROS microenvironment regulation - thermosensitive hydrogel contraction - subsequent light-controlled clearance".

[0009] The present invention provides a plasmid comprising a recombinant DNA plasmid containing an insert sequence, wherein the insert sequence comprises a constitutive expression unit and a light-responsive expression unit; the constitutive expression unit comprises a tyrosinase expression module driven by a constitutive promoter and a light-sensitive protein expression module, wherein the light-sensitive protein expression module forms a regulatory system with a light-sensitive dimer through light induction; the light-responsive expression unit comprises an inducible promoter that responds to the regulatory system and an apoptosis effector module located downstream of the inducible promoter.

[0010] This invention provides a plasmid in which the constitutive promoter is a CMV promoter. The tyrosinase expression module is used to induce melanin biosynthesis, and the tyrosinase expression gene in the tyrosinase expression module is Tyrosinase (TYR).

[0011] The present invention provides a plasmid in which the light-sensitive protein expression module includes a first light-sensitive protein expression gene, a second light-sensitive protein expression gene, a transcription factor, an enhancer, and a nuclear localization sequence. The first light-sensitive protein and the second light-sensitive protein are photoinduced to form a light-sensitive dimer. The light-responsive expression unit further includes a regulatory sequence that specifically binds to the transcription factor, an inducible promoter located downstream of the regulatory sequence, and a pause site located upstream of the regulatory sequence.

[0012] Specifically, the transcription factor is GAL4, the enhancer is VP64, the nuclear localization sequence is SV40 NLS, the regulatory sequence is 5×GAL4 upstream activating sequence (5×UAS), the inducible promoter is minimal CMVpromoter, and the pause site is pause site.

[0013] Specifically, the photosensitive dimer is selected from any one of CRY2 / CIB1, LOV2 / Zdk1, UVR8 / COP1, and PhyB / PIF.

[0014] This invention provides a plasmid in which the photosensitive dimer is CRY2 / CIB1. The photosensitive protein expression module includes a first expression module and a second expression module. The expression products of the first and second expression modules are photoinduced to form a regulatory system with CRY2 / CIB1 to activate the apoptosis effector module. The first expression module includes GAL4-SV40 NLS-CRY2, and the second expression module includes CIB1-SV40 NLS-VP64.

[0015] The present invention provides a plasmid in which the apoptosis factor in the apoptosis effector module is FADD / FAS or caspase8.

[0016] Specifically, the light-responsive expression unit also includes a reporter module located downstream of the apoptosis effect module, and the reporter module is IRES-mCherry.

[0017] The present invention provides a plasmid, wherein the inserted sequence includes the sequence shown in SEQ ID NO. 1.

[0018] This invention provides a plasmid that can be used as a mammalian expression vector. The mammalian expression vector is pcDNA3.1(+).

[0019] The present invention provides a delivery system for targeting senescent cells, the delivery system containing a recombinant DNA plasmid; the delivery system includes a core-shell structured nanocomposite (CMΦM-NPs), the nanocomposite comprising a plasmid complex encapsulated by a chimeric antigen receptor (CAR) cell membrane targeting senescent cells; the plasmid complex comprises an upconversion luminescent material and the recombinant DNA plasmid.

[0020] The present invention provides a delivery system for targeting senescent cells. The nanocomposite induces senescent cells to produce melanin through a tyrosinase expression module. The melanin generates a photothermal effect under near-infrared light irradiation. The near-infrared light activates an apoptosis-effect module through an upconversion luminescent material, thereby achieving controlled clearance of senescent cells.

[0021] Specifically, the chimeric antigen receptor cell membrane targeting senescent cells includes one or more of the following: anti-uPAR chimeric antigen receptor macrophage membrane, anti-uPAR chimeric antigen receptor T cell membrane, and anti-uPAR chimeric antigen receptor NK cell membrane. The chimeric antigen receptor recognition structure may also be selected from senescent cell surface markers such as DPP4 and NKG2D.

[0022] Specifically, the upconversion luminescent material is upconversion nanoparticles (UCNPs), which are combined with recombinant DNA plasmids via electrostatic interactions. The upconversion nanoparticles are modified with polyethyleneimine (PEI) and combined with the recombinant DNA plasmids to form a plasmid complex (UCNPs-DNA).

[0023] The present invention provides a delivery system for targeting senescent cells, the delivery system further comprising a thermosensitive hydrogel with thermally stimulated self-shrinking behavior; the thermosensitive hydrogel has an interconnected porous structure formed inside to deliver the nanocomposite.

[0024] Specifically, the thermosensitive hydrogel exhibits shrinkage behavior through a thermally induced phase transition during the heating process from 37 ℃ to 45 ℃, thereby promoting wound closure.

[0025] Specifically, the thermosensitive hydrogel is also loaded with melanin-binding peptides (MBPs). The melanin-binding peptides enhance melanin retention in the thermosensitive hydrogel, thereby strengthening the local photothermal and contraction effects.

[0026] The present invention provides a delivery system for targeting senescent cells, wherein the raw materials of the temperature-responsive hydrogel include a temperature-responsive polymer and thiolated hyaluronic acid (HA-SH), and the temperature-responsive polymer and thiolated hyaluronic acid form a stable hydrogel network through a Michael addition reaction; the temperature-responsive polymer is a PEG-based hyperbranched polymer prepared by RAFT polymerization.

[0027] The present invention provides a method for preparing a delivery system targeting senescent cells, comprising the following steps: 1) The upconversion luminescent material was mixed with the recombinant DNA plasmid to obtain a plasmid complex; 2) The plasmid complex was mixed with the chimeric antigen receptor cell membrane targeting senescent cells and extruded to obtain a nanocomposite.

[0028] The present invention provides a method for preparing a delivery system for targeting senescent cells, 2) followed by 3) the preparation of a thermosensitive composite hydrogel: mixing a nanocomposite, a temperature-responsive polymer, thiolated hyaluronic acid, a melanin-binding peptide, and a solvent to form a hydrogel precursor system; adjusting the pH of the hydrogel precursor system to form a stable hydrogel network through a Michael addition reaction, thereby obtaining a thermosensitive composite hydrogel loaded with nanocomposite.

[0029] Specifically, the solvent is sterile water or sterile phosphate buffer solution (PBS, pH 7.2-7.4).

[0030] This invention provides a method for preparing a delivery system targeting senescent cells. The final concentration of the nanocomposite in a thermosensitive composite hydrogel can be adjusted according to the wound size and treatment requirements. The concentration of upconversion nanoparticles in the thermosensitive composite hydrogel is 0.05–0.5 mg / mL, preferably 0.1–0.3 mg / mL. The concentration of recombinant DNA plasmid in the thermosensitive composite hydrogel is 0.003–0.05 mg / mL, preferably 0.006–0.03 mg / mL. The concentration of melanin-binding peptide in the thermosensitive composite hydrogel is 0.05–2 mg / mL, preferably 1–2 mg / mL.

[0031] This invention provides an application of a targeted delivery system for senescent cells, including the application of nanocomposites in the preparation of products for repairing chronic, difficult-to-heal wounds. These chronic, difficult-to-heal wounds include diabetic wounds, pressure injuries, chronic wounds in the elderly, radiation-induced wounds / radiation-induced skin damage, or other tissue damage associated with the accumulation of senescent cells.

[0032] The beneficial effects of this invention are: This invention proposes a sequential regulatory strategy of "functional remodeling followed by controlled clearance." The nanocomposite induces senescent cells to produce melanin through tyrosinase, temporarily transforming senescent cells into local microenvironment regulatory units before clearance. Utilizing the metabolic activity still present in senescent cells, functional remodeling occurs within a short window, thereby improving the pathological microenvironment associated with elevated ROS and chronic inflammation, before controlled clearance.

[0033] This invention employs a plasmid complex encapsulated in the anti-uPAR chimeric antigen receptor macrophage membrane to achieve specific recognition and delivery to uPAR-positive senescent cells, avoiding the issues of maintaining activity and immune risks associated with normal cell therapy in chronic, refractory wounds. It also exhibits better targeting selectivity than simple small molecules or ordinary nanoparticles.

[0034] This invention utilizes upconversion luminescent materials to convert near-infrared light into localized light signals, activating an apoptosis-effect module to achieve controlled clearance of senescent cells. Compared to uncontrollable or continuous clearance methods, this approach offers better temporal and spatial control, reducing the risk of non-target cells being affected.

[0035] This invention utilizes melanin produced by senescent cells as a bifunctional mediator. On one hand, melanin can reduce intracellular and local ROS levels at the wound site; on the other hand, melanin can act as a photothermal medium, driving the contraction of a thermosensitive hydrogel under near-infrared light irradiation, thereby coupling microscopic cellular regulation with macroscopic wound edge convergence.

[0036] The thermosensitive hydrogel selected in this invention serves not only as a local delivery reservoir for nanocomposites but also as a thermosensitive mechanical actuator. Experimental results show that the thermosensitive hydrogel provided by this invention exhibits significant shrinkage (projected area reduction of approximately 47.2%) during heating from 37 ℃ to 45 ℃, indicating its potential for assisting wound closure after photothermal triggering.

[0037] This invention demonstrated a clear wound-promoting effect in a diabetic wound model. Under near-infrared stimulation, the wound closure rate of the TRGel / CMΦM-NPs+NIR group was approximately 83.35% on day 14; this was accompanied by decreased p21 / uPAR signal, decreased IL-1β / TNF-α inflammatory signal, and improved histological repair.

[0038] The plasmids and delivery system provided by this invention have strong scalability. By changing the photoinduced dimer system, different light-controlled cell fate regulation can be achieved; by changing the CAR recognition structure, different senescent cell surface markers can be targeted; and by changing the thermosensitive hydrogel components, it can be adapted to different wound or tissue repair scenarios. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of the recombinant DNA plasmid (TYR-CRY2 / CIB1-FADD / FAS) of the present invention.

[0040] Figure 2 The image shows a TEM image of the nanoparticles in Example 2. Figure 2 In the image, A is a TEM image of UCNPs with a scale bar of 10 nm; Figure 2 B in the image is a TEM image of CMΦM-NPs, with a scale bar of 20 nm. Figure 2 In the magnified view, the red scale segment represents 5nm.

[0041] Figure 3 The results show the particle size distribution and zeta potential statistics of each nanoparticle in Example 2.

[0042] Figure 4 This is a schematic diagram of the RAFT polymerization reaction of TRHP according to the present invention.

[0043] Figure 5 For TRHP in Example 2 1 H nuclear magnetic resonance spectrum and ATR-FTIR spectrum.

[0044] Figure 6 This is the SEM image of TRGel in Example 2.

[0045] Figure 7This is a diagram showing the results of the thermal stimulation self-contraction experiment of TRGel in Example 2. Among them, Figure 7 A in the figure is an image of the thermal response shrinkage of TRGel in the temperature range of 37℃ to 45℃, with a scale bar of 10 mm; Figure 7 B in the figure represents the statistical result of the area shrinkage rate.

[0046] Figure 8 The results are statistical findings of the cell compatibility experiment of TRHP in Example 3.

[0047] Figure 9 The results are statistical results of the cell compatibility experiment of PEI-UCNPs in Example 3.

[0048] Figure 10 The results of the cell compatibility experiment of the UCNPs-DNA plasmid complex in Example 3 are statistical results.

[0049] Figure 11 The results of the in vitro targeting of uPAR by MΦM and CMΦM in Example 4 are shown. uPAR (red), MΦM and CMΦM (green), with a scale bar of 5 μm.

[0050] Figure 12 This is a diagram showing the experimental results of melanin targeting in Example 4. Among them, Figure 12 In the figure, A represents the staining result of the Masson-Fontana melanin staining kit, and the scale bar is 100 µm. Figure 12 B in the figure represents the statistical results of melanin production.

[0051] Figure 13 The results of immunofluorescence analysis of the activation of apoptosis signaling pathways in senescent cells in Example 4 are shown. In the figure, Did stains the cell membrane (pink), CMΦM is distributed on the cytoplasm (green), mCherry (red), and DAPI (blue). The scale bar is 20 μm.

[0052] Figure 14 The figure shows the experimental results of the Annexin V-APC / 7-AAD apoptosis detection method in Example 4.

[0053] Figure 15 This is a graph showing the experimental results of ROS expression levels in senescent cells in Example 4. Among them, Figure 15 In the image, A represents the DCFH-DA fluorescent probe detection image, with a scale bar of 100 μm. Figure 15 B in the figure represents the statistical results of the relative fluorescence intensity of ROS in each group of cells.

[0054] Figure 16 This is a schematic diagram of the animal experiment process in Example 5.

[0055] Figure 17 This presents the macroscopic statistical results of wound healing in the animal experiment of Example 5. Among them, Figure 17 In the image, A represents a photograph of the wounds of mice in each group; Figure 17 B in the figure represents the statistical result of the wound closure rate of the corresponding wound.

[0056] Figure 18 This is a diagram showing the results of the H&E staining experiment in Example 5. (The diagram shows the results of the H&E staining experiment in Example 5.) Figure 18 In the figure, A represents H&E stained images of wounds in each group to assess wound width (wound bed) and the morphology of newly formed epithelium. The scale bar is 1 mm. Figure 18 B in the figure represents the wound bed healing rate of each group based on H&E staining images.

[0057] Figure 19 The results are from the Masson trichrome staining experiment in Example 5. Figure 19 In the figure, A represents the Masson staining image of each group of samples, with a scale bar of 500 μm; Figure 19 B in the figure represents the statistical results of collagen deposition rate on day 14 for each group.

[0058] Figure 20 This image shows the co-localization staining results of uPAR (green) and p21 (red) in skin tissue from Example 5. Figure 20 In the image, A represents a co-localization staining image of uPAR (green) and p21 (red), with a scale bar of 200 μm. Figure 20 In the figure, B represents the result of the uPAR quantitative analysis; Figure 20 C in the figure represents the quantitative analysis result of p21.

[0059] Figure 21 Immunofluorescence staining images and quantitative analysis of IL-1β (red) and TNF-α (green) in skin tissue from Example 5. Figure 21 In the image, A represents an immunofluorescence staining image with a scale bar of 50 μm. Figure 21 B in the figure represents the quantitative analysis result of IL-1β; Figure 21 C in the figure represents the quantitative analysis result of TNF-α.

[0060] P<0.05, ** P<0.01, *** P<0.001 indicate that the difference is statistically significant. Detailed Implementation

[0061] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0062] All conventional reagents used in this invention were commercially available. The 3T3 cells (mouse embryonic fibroblast cell line), RAW 264.7 cell line, and HUVECs (human umbilical vein endothelial cells) used in this invention were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The Anti-uPAR-CAR plasmid (vector ID: VB241105-1043veh) used in this invention was purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., and the Anti-uPAR-CAR plasmid carries the green fluorescent protein EGFP.

[0063] The mice used in this invention were SPF-grade 6-8 week old male C57BL / 6J mice provided by the Experimental Animal Center of Wenzhou Medical University. All animal procedures were performed in accordance with the Guidelines for Laboratory Animal Care and Use of Wenzhou Medical University and were approved by the Animal Ethics Committee of Wenzhou Medical University.

[0064] Example 1: Preparation of Experimental Materials 1.1 Construction of recombinant plasmids Design and construction of recombinant DNA plasmid (TYR-CRY2 / CIB1-FADD / FAS): An insert sequence (SEQ ID NO. 1) of 7521 bp in length was designed according to a pre-defined expression framework. Figure 1 As shown, the insert sequence employs a dual expression unit design, comprising a constitutive expression unit and a light-responsive expression unit. The constitutive expression unit is driven by the CMV promoter and sequentially contains coding sequences for Tyrosinase (TYR), T2A, GAL4-SV40 NLS-CRY2, T2A, and CIB1-SV40 NLS-VP64. The light-responsive expression unit is regulated by a 5×GAL4 upstream activating sequence (5×UAS) and a minimal CMV promoter, downstream of which are the FADD-T2A-FAS apoptosis-effect module and the IRES-mCherry reporter module. The insert sequence was synthesized by Genscript Biotech Co., Ltd., and subcloned into a high-copy-count plasmid vector using the pcDNA3.1(+) plasmid vector via the CloneEZ cloning method. The recombinant plasmid was screened and amplified using ampicillin resistance bacteria to obtain the final recombinant DNA plasmid (TYR-CRY2 / CIB1-FADD / FAS).

[0065] 1.2 Preparation of UCNPs-DNA Positively charged PEI-UCNPs were prepared by modifying the surface of UCNPs with polyethyleneimine (PEI). 7.5 μL of PEI-UCNP dispersion (4 mg / mL, sterile PBS) was mixed with 2 μL of recombinant DNA plasmid solution (1 mg / mL, sterile PBS) to achieve a PEI-UCNP to recombinant DNA plasmid mass ratio of 15:1. The mixture was gently pipetted to homogenize and then allowed to stand at room temperature for 1 h to allow the positively charged PEI-UCNPs to recombine with the negatively charged recombinant DNA plasmid via electrostatic interaction, resulting in a UCNPs-DNA plasmid complex dispersion with a concentration of 3.37 mg / mL.

[0066] 1.3 Preparation of anti-uPAR chimeric antigen receptor macrophage membrane (CMΦM) RAW 264.7 cells were transfected using Lipofectamine 3000. RAW 264.7 cells were seeded in 6-well plates to a density of 60-80%. Before transfection, the original culture medium was aspirated, and 1 mL of Opti-MEM medium was added. Then, a DNA-liposome complex was prepared. Lipo3000 and P3000, the PiggyBac transposon vector, and the Anti-uPAR-CAR plasmid were diluted in 500 μL of Opti-MEM medium according to the manufacturer's instructions. After standing at room temperature for 15 min, the solutions were added to the wells. After 4-6 h, the original solution was aspirated, and fresh complete culture medium was added. The cells were cultured for another 48-72 h, and the fluorescence effect was observed and photographed under an inverted fluorescence microscope. The transfection rate was detected by flow cytometry. Once the fluorescence rate reached 60-70%, 2 μg / ml of puromycin was added to select target cells.

[0067] Expand the target cells to 14-16 plates using 100 mm cell culture dishes. Collect the cells by pipetting with sterile PBS. After washing the cells three times, use a cell membrane protein and cytoplasmic protein extraction kit (Beyotime, P0033) to precipitate cell membrane fragments according to the instructions. Discard the supernatant, add an appropriate amount of PBS to resuspend the precipitate, and prepare CMΦM. Store in a -80 ℃ freezer.

[0068] Untransfected RAW264.7 cell membranes were extracted using a cell membrane protein and cytoplasmic protein extraction kit (Beyotime, P0033) to obtain MΦM.

[0069] 1.4 Preparation of nanocomposite (CMΦM-NPs) The UCNPs-DNA plasmid dispersion prepared in step 1.2 was mixed with CMΦM prepared in step 1.3 at a mass ratio of 1:1 (UCNPs-DNA plasmid dispersion to CMΦM). The mixture was sonicated twice in an ice bath for 5 min each time. Then, in a clean bench, microporous membranes with pore sizes of 0.45 μm and 0.22 μm were squeezed sequentially using a 1 mL extruder. Each type of membrane was squeezed repeatedly 4-5 times to obtain a CMΦM-NPs dispersion with a concentration of 3.37 mg / mL, which was stored at 4 ℃.

[0070] MΦM-NPs dispersion was prepared according to the same method as CMΦM-NPs dispersion, except that CMΦM was replaced with an equal mass of MΦM. The concentration of MΦM-NPs dispersion was 3.37 mg / mL.

[0071] 1.5 Preparation of Thermosensitive Hydrogel A temperature-responsive polymer (TRHP) was synthesized using RAFT polymerization, with a MEO2MA to PEGDA molar ratio of 6:4. 1.69 g of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) was dispersed in 18 mL of dimethyl sulfoxide (DMSO) and mixed with 2.4 g of polyethylene glycol diacrylate (PEGDA, average molecular weight [MW] = 400 g / mol) to obtain a mixture. 177.6 mg of tetraethylthiuram disulfide (DS) and 137.8 mg of azobisisobutyronitrile (AIBN) were dissolved in 2 mL of DMSO, and this solution was then added dropwise to the mixture. The resulting solution was degassed using three freeze-thaw cycles and incubated at 70 °C for 6 hours. After the reaction was complete, the reaction was terminated by natural cooling to room temperature, yielding the temperature-responsive polymer TRHP, which was stored at -20 °C for later use.

[0072] TRHP was dissolved in sterile PBS to prepare a 10 wt% TRHP solution, denoted as solution A. Thioyl hyaluronic acid (HA-SH) was dissolved in sterile PBS to prepare a 1 wt% HA-SH solution, denoted as solution B. Melanin-binding peptide (MBP) powder was added to sterile PBS buffer, stirred to dissolve, and then brought to a final volume to obtain a 10 mg / mL solution C. The solution was then filtered through a 0.22 μm filter membrane to further ensure the sterility of the system.

[0073] Preparation of thermosensitive hydrogel (TRGel): First, add 20 μL of solution C to 30 μL of solution A and mix well. Then add 30 μL of solution B and add 20 μL of sodium hydroxide solution (0.1 M) to adjust the pH. Mix quickly and gently to allow TRHP and HA-SH to crosslink and trigger gelation, thus preparing a thermosensitive hydrogel containing MBP, denoted as TRGel.

[0074] Preparation of thermosensitive composite hydrogel (TRGel / CMΦM-NPs): First, add 20 μL of solution C to 30 μL of solution A and mix well. Then, add 19 μL of CMΦM-NPs dispersion (3.37 mg / mL), followed by 30 μL of solution B. Add 20 μL of sodium hydroxide solution (0.1M) to adjust the pH. Mix quickly and gently to change the pH of the hydrogel precursor system, thereby triggering crosslinking and gelation between TRHP and HA-SH to form a thermosensitive composite hydrogel containing CMΦM-NPs, denoted as TRGel / CMΦM-NPs.

[0075] 1.6 Preparation of thermosensitive hydrogel (TRGel / UCNPs-DNA) carrying plasmid complex A thermosensitive hydrogel carrying the plasmid complex was prepared according to the method for preparing thermosensitive composite hydrogels (TRGel / CMΦM-NPs). The difference from method 1.5 is that 9.5 μL of UCNPs-DNA dispersion and 9.5 μL of sterile PBS were used instead of 19 μL of CMΦM-NPs dispersion, and this was designated TRGel / UCNPs-DNA. The concentration of UCNPs-DNA in the TRGel / UCNPs-DNA precursor system was the same as that in the TRGel / CMΦM-NPs hydrogel precursor system.

[0076] Example 2 Characterization Experiment 2.1 Characterization of Nanoparticles The morphology and core-shell nanostructure of the materials were observed using a transmission electron microscope (TEM, JEM-F200) at an accelerating voltage of 100 kV. Aqueous suspensions of UCNPs and CMΦM-NPs were droplets added to a carbon-supported copper grid and allowed to dry completely at room temperature before testing and observation. TEM images are shown below. Figure 2 As shown, UCNPs are mainly spherical with an average particle size of about 20-40 nm, and CMΦM-NPs are covered with a thin and uniform cell membrane.

[0077] Using the Zetasizer Nano instrument, the hydrodynamic diameter (particle size distribution) and surface charge (Zeta potential) of nanoparticles (UCNPs, PEI-UCNPs, UCNPs-DNA, and CMΦM-NPs) obtained in each successive modification step were quantitatively analyzed by dynamic light scattering (DLS) and electrophoretic light scattering techniques. Samples were appropriately diluted in deionized water (or PBS) and measured at 25 °C. Figure 3As shown, dynamic light scattering (DLS) analysis revealed that the average hydrated particle size of UCNPs was approximately 30 nm, which increased to approximately 32 nm after PEI modification. After loading with recombinant DNA plasmids and further encapsulating with CMΦM, the average hydrated particle size of CMΦM-NPs increased to approximately 43 nm. Furthermore, the zeta potential at the membrane surface decreased from approximately −1 mV for UCNPs-DNA to approximately −20 mV. Combined with the core-shell structure observed by transmission electron microscopy, this demonstrates the successful preparation of CMΦM-NPs.

[0078] 2.2 Characterization of TRHP The RAFT polymerization reaction mixture was dialyzed against water using a dialysis membrane with a molecular weight cutoff of 3500 for 3 days, followed by freeze-drying at -60 °C to obtain a white, foamy product, which was then stored at -20 °C. The obtained TRHP was characterized using a 400 MHz nuclear magnetic resonance spectrometer (Avance 400, Bruker, Germany) and a Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific) in ATR mode. The molecular weight of the hyperbranched polymer was determined using a GPC gel permeation chromatography system (Waters 1525, Waters Corporation, USA).

[0079] Gel permeation chromatography (GPC) analysis showed that the molecular weight (Mn) of TRHP was 48,539 g / mol and the molecular weight (Mw) was 169,277 g / mol, indicating that the RAFT polymerization reaction was successful and that a hyperbranched polymer TRHP with controllable molecular weight was formed.

[0080] The chemical structure of TRHP with a molar ratio of 6:4 (MEO2MA:PEGDA) was confirmed by 1H-NMR and ATR-FTIR spectroscopy. Figure 5 As shown in Figure A, the characteristic vinyl proton signals of MEO2MA and PEGDA at 5.5–6.5 ppm (C=C) significantly decreased after polymerization, indicating that the double bonds were successfully consumed during RAFT polymerization. The broad signal at 3.3–4.2 ppm is attributed to the methoxy (-OCH3) and oxymethylene (-O-CH2-) protons of the MEO2MA and PEGDA segments, confirming their successful integration into the polymer backbone. Figure 5 As shown in B, the ATR-FTIR spectrum of TRHP exhibits typical absorption bands, which correspond to the stretching vibrations of the ester carbonyl group (approximately 1720 cm⁻¹). -1 COC stretching vibration (1100-1250 cm) -1 ) and alkyl CH stretching vibrations (2800-3000 cm) -1 The results, all derived from MEO2MA and PEGDA fragments, confirm the successful synthesis of TRHP.

[0081] 2.3 Characterization of TRGel The TRGel hydrogel samples were cut into 1-5 mm pieces. 3 Small pieces were frozen in liquid nitrogen and then rapidly fractured in the liquid nitrogen environment to obtain a fresh fracture cross section. The water in the sample was removed by freeze-drying, while the original three-dimensional microstructure of the hydrogel was preserved.

[0082] The freeze-dried sample was fixed on the SEM stage, and its surface was gold-plated. After gold plating, the sample stage was placed in the sample chamber of a field emission scanning electron microscope (Gemini SEM 300, Carl Zeiss) for observation. The cross-section of the hydrogel was scanned using SEM to observe its internal morphology and microstructure. Figure 6 As shown, the SEM images of the dehydrated hydrogel reveal a uniform and interconnected 3D porous structure, suitable for delivering encapsulated nanocomposites.

[0083] 2.4 Thermal Stimulation Self-Contraction Behavior of TRGel Disc-shaped TRGel was prepared in a disc mold according to the preparation method of Example 1. The hydrogel samples were incubated at 37 °C and 45 °C, respectively, to simulate the body temperature environment and the local heating environment after near-infrared photothermal triggering.

[0084] TRGel samples were placed in a constant-temperature heating platform or a constant-temperature water bath and equilibrated at 37 °C for 10 min. The initial morphology of the gel was photographed using a digital camera, and the surface temperature distribution was recorded using an infrared thermal imager. The temperature was then increased to 45 °C and incubated for another 10 min, after which the gel morphology was photographed again and the temperature distribution was recorded. To evaluate the shrinkage behavior of TRGel, top-view images of the gel before and after different temperature treatments were imported into ImageJ software. The projected area of ​​the gel was measured, and the area shrinkage rate was calculated using the following formula: Area shrinkage rate = (A... 37 -A 45 ) / A 37 ×100%; where A 37 A is the projected area of ​​the gel when treated at 37 °C. 45 The projected area of ​​the gel after treatment at 45 °C.

[0085] The results are as follows Figure 7 As shown, when the temperature rises from 37 ℃ to 45 ℃, TRGel undergoes significant thermal response shrinkage, with the projected area decreasing by approximately 47.2%. This indicates that TRGel has temperature-stimulated responsiveness and thermally induced self-shrinkage capability, and can be used as a local delivery reservoir and thermally responsive mechanical actuation material for wounds.

[0086] Example 3 Biocompatibility of Materials 3.1 Evaluation of the cellular compatibility of PEI-UCNPs, TRHP, and UCNPs-DNA plasmid complexes The in vitro cell compatibility of PEI-UCNPs, TRHP, and UCNPs-DNA in Example 1 was evaluated using 3T3 cells and HUVECs. PEI-UCNPs, TRHP, and UCNPs-DNA were diluted to different concentrations (0, 25, 50, 75, 100, 250, and 500 μg / mL) with sterile complete culture medium.

[0087] 3T3 cells and HUVECs were seeded into 96-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h to allow for full cell adhesion. The original culture medium was then discarded, and complete culture medium containing different concentrations of PEI-UCNPs, TRHP, and UCNPs-DNA was added, followed by further culture for 24 h or 48 h. After culture, the culture medium was discarded, CCK-8 working solution was added, and the plates were incubated at 37 °C for 30 min. The absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0088] The relative cell viability was calculated using the following formula: Relative cell viability (%) = (Absorbance value of experimental group / Absorbance value of control group) × 100%. The control group consisted of cells cultured in complete culture medium without the test material. A relative cell viability below 70% was used as a reference threshold for potential cytotoxicity; a relative cell viability above 70% but below the control group indicated a certain inhibitory effect on cell proliferation.

[0089] The results are as follows Figure 8 , Figure 9 , Figure 10 As shown, the co-culture results of 3T3 cells revealed that, compared to the control group, the relative survival rate of cells after co-culturing with various concentrations of TRHP for 24 h and 48 h was above 75%, without exhibiting potential cytotoxicity. Various concentrations of PEI-UCNPs showed inhibitory effects on cell proliferation, with relative survival rates exceeding 70% after 24 h and ranging from 50% to 70% after 48 h. Various concentrations of UCNPs-DNA showed some inhibitory effect on cell proliferation after 24 h of co-culturing, while at 48 h, low concentrations promoted proliferation, and high concentrations inhibited it. The co-culture results of HUVECs cells showed that, compared to the control group, various concentrations of TRHP did not exhibit potential cytotoxicity. Various concentrations of PEI-UCNPs showed promotion of proliferation at low concentrations and inhibition at high concentrations after 24 h and 48 h of co-culturing. Various concentrations of UCNPs-DNA did not show potential cytotoxicity after 24 h and 48 h of co-culturing, but low concentrations had a proliferative effect, and the proliferation activity increased in a time-dependent manner.

[0090] 3.2 Evaluation of live and dead cell staining using TRGel and TRGel / UCNPs-DNA plasmid complex systems The cell compatibility of the basic delivery materials (TRGel and TRGel / UCNPs-DNA from Example 1) was further evaluated using Calcein-AM / PI live / dead cell staining. 3T3 cells were seeded in 24-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h to allow for full cell adhesion. Three groups were then set up: a complete culture medium group as a control; a TRGel group; and a TRGel / UCNPs-DNA group. For the TRGel and TRGel / UCNPs-DNA groups, 30 μL of the corresponding hydrogel sample was added to each well, along with an appropriate amount of complete culture medium. The hydrogel samples and 3T3 cells were co-cultured for 24 h under near-infrared light-free conditions.

[0091] After culture, the culture medium was discarded, and the cells were gently washed once with PBS. Calcein-AM / PI live / dead cell double staining working solution was added, and the cells were incubated at 37 °C in the dark for 20 min. Subsequently, the cells were gently washed once with PBS, and green fluorescence, red fluorescence, and combined images were collected under a fluorescence microscope. Abundant green live cell fluorescence signals were observed in the control group, TRGel group, and TRGel / UCNPs-DNA group, while red dead cell fluorescence signals were less prominent. This indicates that under the conditions of adding 30 μL of hydrogel to each well and co-culturing for 24 h, neither the TRGel matrix nor the hydrogel system loaded with UCNPs-DNA caused significant baseline cytotoxicity, demonstrating good in vitro cell compatibility and making it a suitable base material system for subsequent cell delivery and local wound application.

[0092] Example 4 Targeted delivery function of nanocomposites 4.1 Inducing cellular senescence Wild-type 3T3 cells were induced with Dox solution (150 nM) and cultured for 2-3 days. Staining with a β-galactosidase kit revealed the formation of a deep blue product under the catalysis of senescence-specific β-galactosidase, indicating successful senescence of the 3T3 cells. Senescent cells were characterized by fluorescence staining to detect the cell surface marker uPAR and the mRNA of senescence surface markers in senescent 3T3 cells. Compared to wild-type 3T3 cells, senescence induction significantly increased the expression of uPAR on the cell surface, as well as the expression of senescence surface marker-related genes uPAR and p21. Senescent 3T3 cells with high uPAR expression were obtained and designated SnCs cells.

[0093] 4.2 CMΦM in vitro targeting uPAR MΦM and CMΦM were labeled with a DIO cell membrane green fluorescent probe, respectively. CMΦM and MΦM were co-cultured with SnCs cells and wild-type 3T3 cells (Norm) for 2 h, respectively, with a final concentration of 30 μg / mL of cell membrane protein in the culture system. Cell samples were fixed at room temperature with 4% paraformaldehyde solution, washed three times with PBS buffer, and then blocked with 5% BSA solution at room temperature for 1 h. Confocal dishes were then placed in a moistened dark box, and the corresponding primary antibody (uPAR) was added. The next day, the corresponding species-specific fluorescent secondary antibody was added, and the cells were incubated at 37 °C for 1 h. After washing three times with PBS, the cell nuclei were stained with DAPI for 30 minutes. Finally, the cells were observed and photographed under a focused laser scanning microscope.

[0094] The results are as follows Figure 11 As shown, CMΦM has the ability to target uPAR-overexpressing senescent 3T3 cells, but cannot target wild-type 3T3 cells. In contrast, MΦM has no targeting activity against either SnCs cells or wild-type 3T3 cells. This demonstrates the successful extraction of CMΦM and its ability to specifically target senescent cells in vitro.

[0095] 4.3 Targeted delivery of CMΦM-NPs 4.3.1 Targeted melanin production Wild-type 3T3 cells (Norm) and SnCs cells were plated separately, and each group of cells was divided into three groups (negative control group, MΦM-NPs group, and CMΦM-NPs group). Among them, MΦM-NPs and CMΦM-NPs were prepared according to the method described in Example 1.4, and the concentration of the dispersion was 3.37 mg / mL.

[0096] MΦM-NPs or CMΦM-NPs dispersions were added to complete culture medium to a final concentration of 60 μg / mL in the cell culture system, and co-cultured with wild-type 3T3 cells or SnCs cells for 48 h, respectively. A negative control group was prepared by adding an equal volume of sterile PBS and then topping up with complete culture medium. Fixed cells were stained using the Masson-Fontana melanin staining kit, observed and photographed under a confocal microscope, as shown below. Figure 12 As shown in A, it can be observed that only the CMΦM-NPs group in SnCs cells produces obvious black granules, while the normal cell group does not produce melanin.

[0097] Quantitative experiment on melanin production. Six groups of cells were seeded into 6-well plates with the same grouping. 2 mL of complete culture medium was added to each well, along with MΦM-NPs or CMΦM-NPs dispersion, and co-cultured for 48 h. The final concentration of MΦM-NPs or CMΦM-NPs in the culture system was 60 μg / mL (based on this system, the total amount of MΦM-NPs or CMΦM-NPs added to each well was 120 μg). After culture, cells were washed with PBS, and 500 μL of trypsin was added to each well to collect cells. Cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 500 μL of melanin lysis buffer (10% DMSO in 1 mol / L NaOH) was added. The plates were incubated at 80 ℃ for 60 min, centrifuged at 2000 rpm for 6 min, cell debris was removed, and the supernatant was retained. Blank wells (containing only NaOH) were added. After cooling, the cells were transferred to 96-well plates with 4 replicates. The relative absorbance was measured using a microplate reader at 450 / 475 nm. Figure 12 As shown in B, the SnCs+CMΦM-NPs group detected significantly more melanin than other groups, indicating that CMΦM-NPs successfully targeted senescent cells and produced melanin smoothly.

[0098] 4.3.2 Activation of apoptosis signaling pathways in senescent cells Under near-infrared light irradiation, UCNPs convert 980 nm near-infrared light into a light signal that can activate photosensitive protein modules, inducing CRY2 / CIB1 to bind in a photoresponsive manner to form a regulatory system, thereby activating the downstream FADD / FAS apoptosis effector module. mCherry, set in the recombinant DNA plasmid, serves as a fluorescent reporter signal for the activation of the apoptosis effector module, and its activation status can be reflected by red fluorescence.

[0099] Immunofluorescence was used to analyze the activation of apoptosis signaling pathways. Near-infrared laser parameters were 5 W and 980 nm. SnCs cells were divided into four groups (CMΦM-NPs+NIR, CMΦM-NPs, CMΦM+NIR, and NIR). The amount of cells added to each group was calculated at 1 mL per well. In the CMΦM-NPs+NIR and CMΦM-NPs groups, 17.8 μL of CMΦM-NPs dispersion (3.37 mg / mL) was added to each well, bringing the final concentration of CMΦM-NPs in the cell culture system to 60 μg / mL. In the CMΦM+NIR group, 8.9 μL of CMΦM stock solution was added to each well, followed by 8.9 μL of PBS, bringing the final concentration of CMΦM cell membrane protein in the culture system to 30 μg / mL. The NIR group received only 17.8 μL of PBS as an equal-volume control. The actual culture system volume was proportionally adjusted based on the final concentrations described above.

[0100] Each group of cells was cultured to 2×10⁻⁶. 5 Cells were cultured per well with the corresponding material for 2 days. Except for the CMΦM-NPs group, all other three groups were treated with near-infrared laser for 2 hours (10 seconds of continuous irradiation followed by a 50-second pause per minute). After incubation for 48 hours following near-infrared laser treatment, cells were stained with DiD, fixed with 4% paraformaldehyde solution, mounted, and observed under a confocal microscope. Figure 13 As shown, CMΦM is distributed on the cytoplasm (green). The CMΦM-NPs+NIR group showed obvious red fluorescence, which overlapped with the green cell membrane fluorescence, indicating successful expression of apoptosis factors. At the same time, it can be observed that the cells began to undergo apoptosis and decomposition, producing cell debris, and the cell nuclei were obviously condensed. The cell nuclei in other groups were round and plump. In the CMΦM-NPs group without NIR treatment, the apoptosis module was not activated, and no red fluorescence appeared. The CMΦM+NIR and NIR groups only added simple targeting of the cell membrane and NIR irradiation, without the participation of the regulatory system and apoptosis effector module, and did not cause significant damage to senescent cells.

[0101] SnCs cells were seeded into cell culture plates and cultured until they reached approximately 2 × 10⁶ cells / year. 5 After each well was filled with samples, the samples were divided into four groups: SnCs, STS, CMΦM-NPs, and CMΦM-NPs+NIR. The SnCs group received an equal volume of sterile PBS; the STS group received astrocytocin (STS) at a final concentration of 1 μM; the CMΦM-NPs and CMΦM-NPs+NIR groups each received 17.8 μL of CMΦM-NPs dispersion (3.37 mg / mL), and were brought to a final concentration of 60 μg / mL with complete culture medium. All groups were co-cultured for 48 h. Only the CMΦM-NPs+NIR group was treated with near-infrared laser for 2 h (980 nm, 5 W, continuous irradiation for 10 s per minute followed by a 50 s pause), and then cultured for another 48 h. Cell survival / death status was then observed using a Calcein-AM / PI double staining kit. The SnCs and CMΦM-NPs groups showed predominantly green live cell fluorescence, while the STS and CMΦM-NPs+NIR groups showed increased red PI positive signals, suggesting that CMΦM-NPs can induce SnCs cell death after near-infrared irradiation.

[0102] SnCs cells were treated with the same grouping, drug concentration, and near-infrared light conditions, and cultured for another 48 hours after light exposure. After treatment, cells were collected, stained according to the Annexin V-APC / 7-AAD apoptosis detection kit instructions, and flow cytometry was used to detect early apoptosis, late apoptosis / death, and the proportion of total apoptotic cells. Results are as follows: Figure 14As shown, the early apoptosis rate and the proportion of late apoptotic / dead cells in the CMΦM-NPs+NIR group were 28.5% and 4.22%, respectively, with a total apoptosis rate of 32.72%; the total apoptosis rate in the CMΦM-NPs group without NIR irradiation was 22.01%. These results indicate that CMΦM-NPs can increase the apoptosis level of SnCs cells under near-infrared irradiation.

[0103] The above results indicate that the near-infrared light response regulatory system can activate the apoptosis signaling pathway and induce cell death.

[0104] 4.3.3 Downregulating ROS expression levels in senescent cells A DCFH-DA-based reactive oxygen species (ROS) detection kit was used to detect ROS levels in 3T3 cells. 3T3 cells were seeded in 24-well plates and senescence was induced using the experimental method described in 4.1. Cells were randomly divided into four groups: SnCs (no treatment), NIR (near-infrared) laser irradiation, CMΦM-NPs (30 μg nanocomposite), and CMΦM-NPs+NIR (30 μg nanocomposite and NIR laser irradiation). Wild-type 3T3 cells (Norm group) served as the normal control. After 24 h of co-incubation with the corresponding materials, the NIR and CMΦM-NPs+NIR groups were irradiated with NIR lasers. The NIR laser parameters were the same as in 4.3.2: 980 nm, 5 W, with a total irradiation time of 2 h, using an intermittent irradiation method: continuous irradiation for 10 s followed by a 50 s pause per minute. The SnCs and CMΦM-NPs groups were not exposed to near-infrared light and were placed in a 37 °C, 5% CO2 incubator for the same time period as a light-free control. After the light exposure, the cells in each group were cultured in a 37 °C, 5% CO2 incubator for 24 h. The original culture medium was then aspirated, and the cells were washed three times with PBS. 200 μL of LCFH-DA working solution was added to each well, and the cells were incubated at 37 °C in the dark for 30 min. The working solution was then aspirated, and the cells were washed three times with PBS. 500 μL of PBS was added to each well. Finally, the cells were observed under an inverted fluorescence microscope, and the relative fluorescence intensity was calculated using ImageJ software.

[0105] The results are as follows Figure 15 As shown, compared with wild-type 3T3 cells, the microenvironment within SnCs cells is conducive to ROS accumulation, and the green fluorescence signal is significantly increased. Comparing the different senescent cell groups, compared with NIR irradiation alone, the CMΦM-NPs group produced melanin intracellularly, thereby inhibiting ROS production in senescent cells; simultaneously, compared with the CMΦM-NPs group, the CMΦM-NPs+NIR group had lower intracellular ROS expression levels.

[0106] Example 5 Animal Experiment 5.1 Construction of the skin lesion model and sample collection Diabetic wounds were constructed using healthy male C57 mice aged 6-8 weeks. On day -14, mice were intraperitoneally injected with streptozotocin (STZ) 110 mg / kg. Mice with blood glucose levels >16.7 mmol / L on days 3 and 7 post-injection were classified as type 1 diabetic mice. After all mice reached the target blood glucose level, they were randomly divided into 5 groups: PBS group, NPs+TRGel+NIR group, CMΦM-NPs+TRGel group, CMΦM-NPs+NIR group, and CMΦM-NPs+TRGel+NIR group, with n=15 in each group.

[0107] On day 0, mice were anesthetized by intraperitoneal injection of 1% (w / w) sodium pentobarbital solution. A full-thickness skin excision wound with a diameter of 8 mm was created on the back of the mice using a piercing instrument. 120 μL of the corresponding drug was added to the wound: PBS group received PBS; CMΦM-NPs+TRGel and CMΦM-NPs+TRGel+NIR groups received TRGel / CMΦM-NPs prepared in Example 1; CMΦM-NPs+NIR group received 0.538 mg / mL CMΦM-NPs diluent (the CMΦM-NPs dispersion prepared in Example 1 was diluted with PBS to ensure the concentration of the CMΦM-NPs diluent matched the concentration of CMΦM-NPs in TRGel / CMΦM-NPs); NPs+TRGel+NIR group received TRGel / UCNPs-DNA from Example 1.

[0108] Two days after drug administration, mice were anesthetized with isoflurane, and the dorsal wounds of mice in each NIR group were irradiated with near-infrared light (5W, 980 nm) for 15 minutes (30 seconds of irradiation followed by 1 minute of rest). The surface temperature was monitored in real time using an infrared thermal imaging system to ensure that the local temperature remained below 50 °C.

[0109] All wounds were photographed on days 0, 3, 7, 10, and 14 post-injury, and the wound closure rate was calculated using ImageJ software. The wound closure rate was calculated as (A0-At) / A0×100%, where A0 represents the wound area on day 0 and At represents the wound area at each time point after the injury.

[0110] Skin samples and tissue samples from the heart, liver, spleen, lungs and kidneys of each group were collected on days 3, 7, 10 and 14 post-injury. The samples were fixed in 4% paraformaldehyde solution at 4 ℃ for more than 24 hours, dehydrated, embedded in paraffin and prepared into 5 μm thick serial sections for subsequent staining observation and analysis.

[0111] 5.2 Experimental Results like Figure 17The skin wound images shown indicate that the CMΦM-NPs+TRGel+NIR group healed the fastest, followed by the CMΦM-NPs+NIR group. On postoperative day 10, the wound area in the CMΦM-NPs+TRGel+NIR group significantly decreased, and more black hair grew compared to other groups. By day 14, the wound closure rate in the CMΦM-NPs+TRGel+NIR group reached 83.35%, significantly higher than the PBS group (42%).

[0112] The skin temperature on the back of mice was recorded 1 minute after NIR irradiation, as shown in Table 1. In mice containing CMΦM-NPs, the melanin produced by senescent cells on the back of the body absorbed heat and increased in temperature under near-infrared irradiation, exerting photothermal therapeutic function while triggering the thermal response contraction behavior of the thermosensitive hydrogel. The CMΦM-NPs+NIR group showed a higher local temperature rise under NIR irradiation, while the average temperature of the TRGel / CMΦM-NPs+NIR group (44.8±1.0 ℃) was relatively lower. This indicates that the thermosensitive hydrogel exhibits a temperature buffering effect under the combined influence of CMΦM-NPs delivery efficiency regulation, the colloid's own thermal response contraction behavior, and the colloid's own endothermic properties, effectively preventing overheating damage to wound tissue.

[0113] Table 1. Statistical results of skin temperature on the back of mice in each group Skin sections taken on days 3, 7, 10, and 14 were used for H&E staining experiments, and the wound healing length was calculated using ImageJ software to statistically determine the wound bed healing rate. Figure 18 As shown, on day 14, the wound in the CMΦM-NPs+TRGel+NIR group showed new and neat tissue formation, with newly formed skin appendages and complete reepithelialization. The skin epithelialization and healing were slow in the treatment groups lacking CMΦM or TRGel. Quantitative analysis results showed that the wound bed healing rate was highest in the CMΦM-NPs+TRGel+NIR group, followed by the CMΦM-NPs+TRGel group.

[0114] Masson's trichrome staining experiment was performed on skin sections taken on days 3, 7, 10, and 14, and the collagen area percentage was calculated using ImageJ software. Figure 19 As shown, the blue collagen deposition in the CMΦM-NPs+TRGel+NIR group was significantly higher than that in other groups. On day 14, the collagen distribution and structure in this group were more mature, thicker, and more neatly arranged. Quantitative analysis results showed that the collagen deposition rate in the CMΦM-NPs+TRGel+NIR group reached 82%, effectively promoting collagen deposition in the wound.

[0115] Immunofluorescence staining was used to detect the expression levels of uPAR and p21 in skin tissue at days 3, 7, 10, and 14 to characterize their targeted clearance of senescent cells. The morphology of stained tissues was observed and photographed under a focused laser scanning microscope (red represents p21, green represents uPAR), and the relative fluorescence intensity was calculated using ImageJ. The expression levels of uPAR and p21 are shown below. Figure 20 As shown, on day 14, the expression levels of uPAR and p21 in the thermosensitive composite hydrogel group were significantly lower than those in the PBS group, verifying that the thermosensitive composite hydrogel promotes targeted apoptosis of senescent cells, indicating that this thermosensitive composite hydrogel system can effectively reduce the expression of senescence-related markers in the wound. Further comparison revealed that, compared with the TRGel / CMΦM-NPs group, the expression levels of uPAR and p21 in the TRGel / CMΦM-NPs+NIR group were further reduced, suggesting that the NIR-triggered photo-controlled apoptosis process helps enhance the clearance of senescent cells.

[0116] The expression levels of pro-inflammatory factors (TNF-α and IL-1β) in skin tissue on days 3 and 7 were detected using immunofluorescence staining. The morphology of the stained tissues was observed and photographed under a focused laser scanning microscope (red represents IL-1β, green represents TNF-α), and the relative fluorescence intensity was statistically analyzed using ImageJ. Figure 21 The immunofluorescence staining results of pro-inflammatory factors (TNF-α, IL-1β) in skin tissues of each group on days 3 and 7 are shown. On day 3, significant positive signals of TNF-α and IL-1β were observed in all experimental groups, indicating inflammatory cell infiltration in the wound. By day 7, the secretion and expression of pro-inflammatory factors (TNF-α, IL-1β) in the CMΦM-NPs+TRGel+NIR group were significantly reduced, while the expression of pro-inflammatory factors in the other four groups did not decrease but rather showed a tendency to worsen. Quantitative analysis showed that on day 7, the expression levels of TNF-α and IL-1β in the CMΦM-NPs+TRGel+NIR group were lower than those in the control group, showing a significant correlation with the immunofluorescence staining results. Simultaneously, the expression levels of pro-inflammatory factors in the CMΦM-NPs+TRGel+NIR group were significantly lower than those in the CMΦM-NPs+TRGel group.

[0117] The above results indicate that, under near-infrared stimulation, the wounds in the CMΦM-NPs+TRGel+NIR group successfully transitioned from a chronic inflammatory state to a controllable regenerative microenvironment.

[0118] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A plasmid, characterized in that, The invention includes a recombinant DNA plasmid containing an insert sequence, the insert sequence comprising a constitutive expression unit and a light-responsive expression unit; the constitutive expression unit comprises a tyrosinase expression module driven by a constitutive promoter and a light-sensitive protein expression module, the light-sensitive protein expression module being light-induced to form a regulatory system with a light-sensitive dimer; the light-responsive expression unit comprises an inducible promoter that responds to the regulatory system and an apoptosis effector module located downstream of the inducible promoter.

2. A delivery system for targeting senescent cells, characterized in that, The delivery system contains the recombinant DNA plasmid as described in claim 1; the delivery system includes a nanocomposite having a core-shell structure, the nanocomposite comprising a plasmid complex encapsulated by a chimeric antigen receptor cell membrane targeting senescent cells; the plasmid complex comprises an upconversion luminescent material and the recombinant DNA plasmid.

3. The delivery system for targeting senescent cells according to claim 2, characterized in that, The nanocomposite induces senescent cells to produce melanin through a tyrosinase expression module, and the melanin generates a photothermal effect under near-infrared light irradiation; the near-infrared light activates the apoptosis effect module through an upconversion luminescent material, thereby achieving controlled clearance of senescent cells.

4. The delivery system for targeting senescent cells according to claim 2, characterized in that, The chimeric antigen receptor cell membrane targeting senescent cells includes one or more of the following: anti-uPAR chimeric antigen receptor macrophage membrane, anti-uPAR chimeric antigen receptor T cell membrane, and anti-uPAR chimeric antigen receptor NK cell membrane.

5. The delivery system for targeting senescent cells according to claim 2, characterized in that, The upconversion luminescent material is an upconversion nanoparticle, which is combined with the recombinant DNA plasmid through electrostatic interaction.

6. The delivery system for targeting senescent cells according to claim 2, characterized in that, The delivery system also includes a thermosensitive hydrogel with thermally stimulating self-shrinking behavior; the thermosensitive hydrogel has an interconnected porous structure inside to deliver the nanocomposite.

7. The delivery system for targeting senescent cells according to claim 6, characterized in that, The thermosensitive hydrogel is also loaded with melanin-binding peptides.

8. The delivery system for targeting senescent cells according to claim 6, characterized in that, The raw materials of the temperature-sensitive hydrogel include a temperature-responsive polymer and thiolized hyaluronic acid. The temperature-responsive polymer and thiolized hyaluronic acid form a stable hydrogel network through a Michael addition reaction. The temperature-responsive polymer is a PEG-based hyperbranched polymer prepared by RAFT polymerization.

9. A method for preparing the delivery system according to any one of claims 2-8, characterized in that, The preparation method includes the following steps: 1) The upconversion luminescent material was mixed with the recombinant DNA plasmid to obtain a plasmid complex; 2) The plasmid complex was mixed with the chimeric antigen receptor cell membrane targeting senescent cells and extruded to obtain a nanocomposite.

10. The application of the delivery system according to any one of claims 2-8, characterized in that, Application of the nanocomposite in the preparation of products for repairing chronic, difficult-to-heal wounds.

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