Use of a targeted nanoparticle in diabetic foot

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a targeted nanoparticle in diabetic foot. The targeted nanoparticle comprises recombinant full-length human collagen type XVII, and the amino acid sequence of the recombinant full-length human collagen type XVII is shown as SEQ ID NO. 1. The targeted nanoparticle is guided by a sorting signal peptide (SEQ ID NO. 2) to enrich the recombinant full-length human collagen type XVII on the surface of the nanoparticle. The targeted nanoparticle can significantly accelerate wound healing of the diabetic foot, promote re-epithelialization, collagen deposition and orderly arrangement, angiogenesis and cell proliferation of the wound, and simultaneously inhibit local inflammatory reaction of the wound, so that the wound repair process is regulated through multiple targets, and the targeted nanoparticle can be effectively used for treatment of the diabetic foot.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of targeted nanoparticles in diabetic foot. Background Technology

[0002] Diabetic foot ulcers are a serious complication of diabetes and a leading cause of disability and death in diabetic patients. Due to long-term hyperglycemia, diabetic patients often suffer from peripheral neuropathy and lower extremity vascular disease. Peripheral neuropathy leads to decreased or lost sensation in the foot and abnormal pressure distribution on the sole, making ulcers easily triggered by minor injuries, friction from shoes and socks, or pressure. Lower extremity vascular disease results in reduced blood flow, tissue hypoxia, and insufficient nutrient supply, severely impairing wound healing. Diabetic foot ulcers are characterized by their persistent nature, high recurrence rate, high disability rate, long treatment period, and heavy medical burden, making them a challenging area for clinical treatment.

[0003] The healing impairment of diabetic foot ulcers involves complex pathological mechanisms that differ significantly from the normal wound healing process. Normal wound healing is a highly coordinated process, including the hemostatic, inflammatory, proliferative, and remodeling phases. However, in diabetic conditions, all of these phases are abnormal. Prolonged hyperglycemia promotes the formation of advanced glycation end products (AGEs). These products, after accumulating in tissues, can bind to their receptors, activating multiple damaging signaling pathways, increasing oxidative stress levels, and leading to damage to cell membranes, proteins, and DNA. Hyperglycemia can also impair the polarization of macrophages from a pro-inflammatory to a repair phenotype, keeping the wound in a chronic inflammatory state and hindering the transition from the inflammatory to the proliferative phase. Simultaneously, increased matrix metalloproteinase activity in diabetic wounds leads to excessive degradation of the extracellular matrix, preventing the stable deposition of new tissue. Furthermore, the high-glycemic environment directly reduces the proliferation and migration capacity of fibroblasts and keratinocytes, and endothelial dysfunction results in impaired angiogenesis, leaving the wound in a state of chronic ischemia and hypoxia. These multiple pathological factors are interconnected and mutually reinforcing, forming a vicious cycle that leads to delayed or even complete wound healing failure.

[0004] Currently, the main clinical treatments for diabetic foot ulcers include strict blood glucose control, thorough debridement, anti-infection therapy, improvement of lower limb blood circulation, application of various wound dressings, and growth factor therapy. However, existing treatments have limited effectiveness and cannot meet clinical needs. Exogenous growth factors have a short half-life and are easily inactivated in the high-glucose environment of diabetes, making it difficult to sustain their healing-promoting effects, and they also suffer from high costs and unstable efficacy. While wound dressings can provide a suitable healing environment, they lack the ability to actively promote tissue regeneration. For patients with severe lower limb vascular disease, revascularization surgery carries high risks and has limited indications. Therefore, developing novel, safe, and effective treatment strategies is of great significance for improving the treatment outcomes of diabetic foot ulcers.

[0005] In recent years, exosomes have attracted widespread attention as a novel cell-free therapeutic strategy. Exosomes are nanoscale vesicles secreted by cells, carrying various bioactive molecules such as proteins, lipids, and nucleic acids, and mediating intercellular communication. Exosomes possess low immunogenicity, high biocompatibility, and good tissue permeability, allowing them to cross biological barriers, making them ideal carriers for drug delivery. They also possess certain tissue repair functions. However, current exosome therapy technologies mainly employ exogenous loading methods to encapsulate therapeutic molecules into exosomes. This method suffers from drawbacks such as low loading efficiency, impaired exosome membrane integrity, potential immunogenicity, and poor batch-to-batch stability. Furthermore, existing exosome therapies typically target only a single site, making it difficult to simultaneously address the multiple pathological problems of diabetic foot, resulting in limited therapeutic efficacy.

[0006] Therefore, there is an urgent need in this field to develop a therapeutic strategy that can efficiently enrich therapeutic proteins, retain their natural activity, and have multi-target regulatory capabilities to meet the pressing clinical needs of diabetic foot ulcer treatment. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides an application of targeted nanoparticles in diabetic foot ulcers. These targeted nanoparticles are genetically engineered exosomes with a highly enriched surface of recombinant full-length human collagen type XVII. This invention employs an endogenous loading strategy, guiding the target protein to spontaneously accumulate on the exosome membrane via a sorting signal peptide, thus avoiding the drawbacks of exogenous loading. These targeted nanoparticles can regulate the wound repair process at multiple targets and can be effectively used for the treatment of diabetic foot ulcers.

[0008] The technical solution of this invention is as follows: On one hand, the present invention provides the application of targeted nanoparticles in the preparation of a drug for treating diabetic foot, wherein the targeted nanoparticles include recombinant type XVII full-length human collagen; The amino acid sequence of the recombinant XVII type full-length human collagen is shown in SEQ ID NO.1.

[0009] Specifically, the recombinant type XVII full-length human collagen is enriched on the surface of the targeted nanoparticles by a sorting signal peptide.

[0010] Preferably, the sorting signal peptide comprises the amino acid sequence shown in SEQ ID NO.2.

[0011] Specifically, the targeted nanoparticles may be selected from exosomes or microvesicles.

[0012] Specifically, the targeted nanoparticles are derived from engineered host cells that express the recombinant type XVII full-length human collagen.

[0013] More specifically, the host cell may be a eukaryotic cell.

[0014] Preferably, the host cell is a mammalian cell.

[0015] More preferably, the host cell is a 293T cell.

[0016] Specifically, the targeted nanoparticles are prepared by the following method: (1) Construct an expression vector, wherein the expression vector comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 and a sorting signal peptide encoding sequence; (2) Transfect the expression vector into host cells; (3) Cultivate the host cells and isolate and purify the targeted nanoparticles.

[0017] Specifically, the dosage form of the drug is selected from gel, spray, ointment, dressing, lyophilized powder for injection or injection.

[0018] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0019] More specifically, the pharmaceutically acceptable excipients are selected from excipients, buffers, stabilizers, preservatives, antioxidants, thickeners, emulsifiers, or solvents.

[0020] Preferably, the excipient is selected from mannitol, trehalose, sucrose, lactose, or glucose; The buffer is selected from phosphate buffer, citrate buffer or Tris buffer; The stabilizer is selected from human serum albumin, gelatin, or polyethylene glycol; The preservative is selected from benzyl alcohol, benzalkonium chloride or thimerosal; The antioxidant is selected from vitamin E, vitamin C, or sodium bisulfite; The thickener is selected from sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or carbomer.

[0021] In another aspect, the present invention provides a method for treating diabetic foot, comprising administering an effective amount of targeted nanoparticles to a subject; The surface of the targeted nanoparticles is enriched with recombinant type XVII full-length human collagen; The amino acid sequence of the recombinant XVII type full-length human collagen is shown in SEQ ID NO.1.

[0022] The beneficial effects of this invention are as follows: (1) The present invention provides a highly enriched recombinant XVII type full-length human collagen on the surface of the targeted nanoparticles. Guided by the sorting signal peptide, the recombinant XVII type full-length human collagen spontaneously accumulates on the nanoparticle membrane, avoiding the defects of low exogenous loading efficiency and impaired membrane integrity.

[0023] (2) The targeted nanoparticles of the present invention can significantly accelerate the healing of diabetic foot wounds. Experiments on a mouse model of full-thickness skin defects on the back showed that the wound healing speed of the mice treated with the targeted nanoparticles of the present invention was significantly better than that of the model control group, and the wounds were almost completely closed by day 14.

[0024] (3) The targeted nanoparticles of the present invention can promote wound re-epithelialization, collagen deposition and orderly arrangement, angiogenesis and cell proliferation. Histological staining results show that the targeted nanoparticles of the present invention can promote the formation of new epidermis, orderly arrangement of collagen fibers, induce microvascular reconstruction and promote cell proliferation.

[0025] (4) The targeted nanoparticles of the present invention can inhibit the local inflammatory response of the wound. Immunohistochemical results showed that the targeted nanoparticles of the present invention can significantly inhibit the expression of pro-inflammatory factors TNF-α and IL-6, and promote the transformation of the wound from the inflammatory phase to the repair phase. Attached Figure Description

[0026] Figure 1 The results of Western blotting for proteins derived from host cells.

[0027] Figure 2 Electron microscopy results for host cell-derived exosomes, scale bar = 100 nm.

[0028] Figure 3 The results of Western blot analysis of exosomes derived from host cells.

[0029] Figure 4 The data included the body weight and serum blood glucose levels of animals in different groups.

[0030] Figure 5 Images of wounds on the backs of mice at different stages.

[0031] Figure 6 HE, Sirius red, and Masson staining were used to stain mouse wound skin.

[0032] Figure 7 The expression of CD31, Ki67 and VEGF in mouse skin wounds.

[0033] Figure 8 The expression of inflammatory factors TNF-α and IL-6 in mouse skin wounds. Detailed Implementation

[0034] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0036] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0037] Example 1: Design of expression vector and construction of plasmid a. The target expression vector consists of: a protein expression sequence, an exosome sorting signal peptide (SEQ ID NO.2: MKFERQ), and recombinant full-length human collagen of type XVII (amino acid sequence as shown in SEQ ID NO.1, and encoding nucleotide sequence as shown in SEQ ID NO.3).

[0038] SEQ ID NO.1:

[0039] SEQ ID NO.3:

[0040] b. The composition of the control expression vector 1 includes: exosome sorting signal peptide 1 (SEQ ID NO.4:ATGCCGAGTGCCCCG) and recombinant type XVII full-length human collagen.

[0041] c. The control expression vector 2 consists of: exosome sorting signal peptide 2 (SEQ ID NO.5: ATGCTGTACCCTCTGCTG) and recombinant type XVII full-length human collagen.

[0042] d. Synthesis of Expression Sequences: The optimized exosome sorting signal peptide + recombinant full-length human collagen XVII sequence, exosome sorting signal peptide 1 + recombinant full-length human collagen XVII sequence, and exosome sorting signal peptide 2 recombinant full-length human collagen XVII sequence were synthesized into whole genes. Homologous recombination cloning was used to clone the recombinant full-length human collagen XVII expression sequence into the pcDNA3.1(+) plasmid, forming the basic vector for the genetically engineered exosome expression plasmid. Primers were as follows: Destination sequence: Forward amplification primer 5'-gctagcgtttaaacttaagcttATGAAGTTCGAGAGACAGATGG-3' (SEQ ID NO.6); Reverse amplification primer 5'- cgggtttaaacgggccctctagaTCAGGGCTTGACAGCAATGCT-3' (SEQ ID NO.7).

[0043] Control sequence 1: Forward amplification primer 5'-gctagcgtttaaacttaagcttATGCCGAGTGCCCCGATGG-3' (SEQ ID NO. 8); Reverse amplification primer 5'-cgggtttaaacgggccctctagaTCAGGGCTTGACAGCAATCGA-3' (SEQ ID NO.9).

[0044] Control sequence 2: Forward amplification primer 5'-gctagcgtttaaacttaagcttATGCTGTACCCTCTGCTGATG-3' (SEQ ID NO.10); Reverse amplification primer 5'-cgggtttaaacgggccctctagaTCAGGGCTTGACAGCAATCGA-3' (SEQ ID NO.11).

[0045] e. Plasmid Transformation and Extraction: Following standard plasmid transformation procedures, the constructed recombinant plasmid was transformed into DH5α *E. coli* competent cells. The next day, a single colony was picked and inoculated into 3 mL of LB medium, and amplified at 37°C and 200 rpm. After 12 hours, the bacterial culture was inoculated into 200 mL of LB medium and amplified a second time at 37°C and 200 rpm. After 12 hours, plasmid extraction was performed using a plasmid extraction kit (Tiangen Low Endotoxin Medium-Quantity Plasmid Purification Kit) according to the manufacturer's instructions. The concentration of the extracted plasmid was measured, and the sample was filtered through a 0.22 μm filter for sterilization.

[0046] Example 2: Host cell expression of recombinant type XVII full-length human collagen The correctly sequenced plasmid was transfected into 293T cells to detect the expression of recombinant full-length human collagen of type XVII. 48 h after transfection, the cells were lysed with pre-cooled RIPA to extract total cellular protein, and the expression of the target protein was detected by Western blotting using a recombinant full-length human collagen antibody of type XVII.

[0047] The results are as follows Figure 1 As shown, COL17A1 overexpression bands appeared in all host cells after transfection with the target expression vector (target sequence), control expression vector 1 (control sequence 1), and control expression vector 2 (control sequence 2).

[0048] Example 3: Enrichment and Identification of Exosomes Expressing Recombinant Type XVII Full-Length Human Collagen The correctly sequenced plasmid was transfected into 293T cells. Four days after transfection, the cell supernatant was collected for the isolation and purification of exosomes from the cell culture supernatant. The isolation and purification method was PEG precipitation combined with size exclusion chromatography.

[0049] The appearance, particle size, and surface COL17A1 protein of exosomes were characterized using immunoelectron microscopy. 5 μL of the exosome sample was mixed with 5 μL of 4% paraformaldehyde and added to a standard carbon-supported copper mesh (Zhongjing Keyi, 300 mesh BZ11023a). After standing for 15 min, excess sample was aspirated, and 10 μL of 1×PBS containing 0.2% BSA and 5% goat serum was added for blocking. After blocking for 15 min, excess blocking solution was aspirated, and the copper mesh was washed three times with 1×PBS containing 0.1% BSA. 10 μL of COL17A1 polyclonal antibody (proteintech, 31190-1-AP) (1:100) was added, and after incubation for 30 min, the copper mesh was washed three times with 1×PBS containing 0.1% BSA. Goat anti-Rabbit IgG H&L and Gold were added. The copper mesh was conjugated (Bioss, bs-0295G-Gold) (1:200) and incubated for 30 min. Then, the copper mesh was washed three times with 1×PBS containing 0.1% BSA, followed by three washes with deionized water. 2% uranyl acetate was added, and the mixture was allowed to stand for 20 min. Excess liquid was then removed, and the copper mesh was washed three times with deionized water. The structure of the exosomes and the distribution of recombinant type XVII full-length human collagen on the exosomes were observed under a 120 kV transmission electron microscope (JEM-1400Flash).

[0050] Immunoblot was used to detect the expression of exosome-specific proteins and recombinant type XVII full-length human collagen. An appropriate amount of the exosome sample was mixed with 5×SDS-PAGE loading buffer and heated in a boiling water bath for 5 min to denature the proteins. An SDS-PAGE gel was prepared and electrophoresis was performed at a constant voltage. Initially, the voltage was adjusted to 80V. When a narrow band formed at the boundary between the separating and stacking gels, the voltage was adjusted to 120V. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel first. A nitrocellulose membrane was prepared and soaked in transfer buffer. The soaked filter paper, nitrocellulose membrane, SDS-PAGE gel, and filter paper were stacked in a bottom-up order, carefully removing air bubbles between each layer. After electrophoresis, the membrane was immersed in 5% skim milk blocking buffer and blocked at room temperature for 2 h. The corresponding antibody was added, and the membrane was incubated overnight at 4℃. The membrane was washed three times with 0.5% PBST for 5 min each time. Add the corresponding HRP-labeled secondary antibody (diluted 1:5000 with washing buffer) and incubate at room temperature with gentle shaking for 1 h. Wash the membrane three times with 0.5% PBST, 5 min each time. Add a 1:1 mixture of chemiluminescent substrates A and B onto the nitrocellulose membrane, gently spreading the luminescent solution evenly with a micropipette to avoid air bubbles, incubate at room temperature for 5 min, and then place it in a chemiluminescence analyzer for imaging.

[0051] Electron microscopy results as follows Figure 2As shown, exosomes derived from 293T cells transfected with control expression vector 1 and control expression vector 2 also expressed COL17A1 protein. However, the exosomes secreted by 293T cells transfected with the target expression vector had denser gold particles on their surface, indicating a higher degree of enrichment of COL17A1 protein (target exosomes) on their surface.

[0052] Western blot results for proteins are as follows Figure 3 As shown, exosomes derived from 293T cells transfected with control expression vector 1 and control expression vector 2 also expressed COL17A1 protein, but the exosomes secreted by 293T cells transfected with the target expression vector were enriched with more COL17A1 protein (target sequence EV).

[0053] Example of effect 1 Forty 28-week-old SPF-grade male C57BL / 6J mice were selected and housed in a barrier-level animal room (temperature 20-26℃, humidity 30-70%), maintaining a 12-hour light-dark cycle. The experiment was divided into five groups: ① Normal control group (Control) (normal diet + blank hydrogel); ② Model control group (DIO model induced by high-fat, high-sugar diet (D12492) + blank hydrogel); ③ 293T exosome group (293T exo) (DIO model + 293T-derived exosome hydrogel); ④ Recombinant XVII type collagen group (COL17) (DIO model + pure protein hydrogel); ⑤ Recombinant XVII type collagen nanoparticle formulation group (Nano-COL17) (DIO model + Nanoparticle-based hydrogels). Hydrogel preparation method: Accurately weigh 30g of medical-grade poloxamer 188 and disperse it in about 100mL of double-distilled water. Place it in a 4℃ refrigerator and let it stand for 12-24 hours to prepare a 30% (w / w) pre-cooled matrix solution that is completely swollen and dissolved, forming a clear, transparent solution without dissolved particles. Add equal volumes of pre-cooled PBS, 40μg / mL of 293T exo, 40μg / mL of recombinant XVII collagen, and 40μg / mL of recombinant XVII collagen nanoparticles to the above 4℃ matrix solution to prepare blank hydrogels with final concentrations of 20μg / mL 293T exo hydrogel, 20μg / mL COL17 hydrogel, and 20μg / mL Nano-COL17 hydrogel. After uniform stirring, dispense and store at 4℃ for later use.

[0054] The model group and the drug-treated group were induced to develop obesity and hyperglycemia phenotypes through a 16-week high-fat, high-glycemic diet. After 16 weeks of high-fat, high-glycemic diet induction, the model group, Exo group, COL17 group, and Nano-COL17 group showed weight gain. Fasting blood glucose (FBG) test results showed that the mean fasting blood glucose values ​​of the high-fat, high-glycemic induction groups were higher than those of the normal control group mice. Figure 4 Before surgery, mice were anesthetized by isoflurane inhalation and given subcutaneous injection of buprenorphine (0.05-0.1 mg / kg) for analgesia. Two full-thickness skin defects, 8-10 mm in diameter and reaching the fascia layer, were created on either side of the midline of the mouse's back. A 37°C constant-temperature heating pad was used during the surgery to prevent hypothermia. From day 2 to day 12 post-modeling, 200 μL of the corresponding drug solution was evenly applied to the wound surface using a micropipette. Photos were taken on days 0, 3, 5, 7, 10, and 14 after drug administration. On day 14, mice were anesthetized and bled to death; blood from the heart and full-thickness skin tissue from the wound were collected.

[0055] Full-thickness skin tissue collected from the wound (including the wound center, edges, and some normal skin tissue) was immediately immersed in 4% paraformaldehyde fixative (PFA) and fixed at 4°C or room temperature for 24–48 hours. After fixation, the wound was incised along the midline of its maximum diameter with a sharp scalpel, ensuring the cut surface fully displays the wound center, the edge of new epithelial growth (granulation tissue), and the layers of normal skin on both sides. The tissue was then sequentially immersed in 75% ethanol (45 min), 85% ethanol (45 min), 95% ethanol (45 min), 95% ethanol (45 min), anhydrous ethanol (1 h), anhydrous ethanol (1 h), xylene (20 min), and xylene (20 min, until the tissue became clear). The sample was then sequentially immersed in soft paraffin (56°C, 1 h) and hard paraffin (58°C, 2 h). The tissue was placed flat with the cut side down in an embedding frame, injected into a paraffin base, and cooled to solidify on a cold stage. Serial sections were then prepared using a paraffin microtome, with a section thickness of 4–5 μm. After flattening the slices, attach them to a detachable glass slide (cationic glass slide), bake in a 60℃ oven for 2–3 hours or overnight, and store at room temperature for later use.

[0056] Before staining, the sections were dewaxed to water in a routine manner, and then placed in xylene (10 min), xylene (10 min), anhydrous ethanol (5 min), 95% ethanol (5 min), 85% ethanol (5 min), and 75% ethanol (5 min) in sequence, and finally washed with distilled water.

[0057] HE staining: After dewaxing to water, sections are stained with hematoxylin for 3–5 min, then rinsed with tap water to remove excess stain; immersed in 0.5%–1% hydrochloric acid-ethanol for a few seconds to differentiate; rinsed with tap water or warm running water for 10–15 min to regain blue color. Then stained with 0.5% eosin for 1–3 min. After rinsing with distilled water; rapidly dehydrated with 95% ethanol, then soaked in anhydrous ethanol for 2 min, then again in anhydrous ethanol for 2 min, then in xylene for 5 min, and finally again in xylene for 5 min. Add neutral resin and mount with a coverslip. Stain sections in a saturated solution of 0.1% Sirius red picric acid at room temperature in the dark for 60 min.

[0058] Sirius red staining: Immerse the slides in a saturated solution of 0.1% Sirius red picric acid and stain at room temperature in the dark for 60 min. Discard the staining solution and immediately wash twice with 0.5% acetic acid aqueous solution (approximately 1–2 min each time), vigorously shaking the slide to remove free dye from the surface. Quickly blot off excess water with filter paper; dehydrate three times with anhydrous ethanol, 1–2 min each time; clear twice with xylene (5 min each time), and mount with neutral resin.

[0059] Masson staining: After dewaxing to water, sections are placed in Bouin's solution and mordanted in a 56°C oven for 1 hour (or overnight at room temperature), then rinsed with running water until the yellow color disappears. Stain with azurite blue solution for 2–3 min, then rinse with water; stain with Weigert iron hematoxylin solution for 5–10 min, then rinse with running water. Differentiate with acidic ethanol solution for a few seconds, then rinse with water; stain with Ponceau S and acid fuchsin solution for 5–10 min, then rinse with distilled water. Differentiate in 1% phosphomolybdic acid solution for 3–5 min. Stain with 2% aniline blue (or brilliant green) solution for 5 min. Wash with 1% acetic acid solution for 1 min; dehydrate rapidly with anhydrous ethanol; clear with xylene, then mount with neutral resin.

[0060] Immunohistochemistry: After dewaxing, sections were immersed in 3% (hydrogen peroxide) methanol solution and incubated at room temperature in the dark for 10–15 min to inactivate endogenous peroxidase in the tissue. The sections were washed three times with PBS, 5 min each time. The sections were then immersed in antigen retrieval solution and microwaved on high to boiling, then reduced to medium-low heat and maintained at a gentle boil for 15–20 min, before naturally cooling to room temperature. The sections were washed three times with PBS, 5 min each time. Ki67, VEGF, and CD31 were treated with citrate buffer (pH 6.0); NF-α and IL-6 were treated with EDTA antigen retrieval solution (pH 9.0). Blocking was performed using 10% normal goat serum at 37°C for 30–60 min to inhibit non-specific binding sites. Dilute the following primary antibody solutions at a ratio of 1:400: anti-CD31 antibody (Seville, GB113151), anti-KI67 antibody (Seville, GB111141), anti-VEGF antibody (Seville, GB15165), anti-IL-6 antibody (Seville, GB11117), and anti-TNF-α antibody (Seville, GB115702). Apply the diluted primary antibody to the wound tissue and incubate overnight (12–16 h) at 4°C in a humidified chamber. The next day, remove the sections from 4°C and allow them to warm to room temperature for 20–30 min. Wash three times with PBS for 5 min each time. Add HRP (horseradish peroxidase)-labeled S-vision immunohistochemical polymeric secondary antibody (goat anti-rabbit) and incubate at 37°C in a humidified chamber for 30 min. Wash three times with PBS for 5 min each time. Prepare DAB chromogenic solution (1 mL buffer + 1 drop DAB). Concentrated solution, mixed well. Add the developing solution to the developing plate, and strictly control the development time under a microscope (usually 30 s to 5 min). When a clear yellowish-brown color appears at the positive site of the wound and there is no obvious background staining, immediately immerse in tap water to stop the reaction. Then, lightly counterstain the cell nuclei in hematoxylin staining solution for 30 s, and immediately rinse with tap water to remove excess stain; subsequently, immerse in 0.5% hydrochloric acid ethanol differentiation solution for rapid differentiation for 2 s, and quickly rinse with tap water to stop differentiation; then immerse in PBS buffer, 0.1% dilute ammonia water, or running water for 1–2 min to re-blue until the cell nuclei show a clear pale blue color, and rinse thoroughly with tap water. Next, sequentially push the sections into a gradient of ethanol for dehydration: 85% ethanol (2 min), 95% ethanol (2 min), anhydrous ethanol (2 min), anhydrous ethanol II (2 min); after dehydration, proceed to xylene for clearing: xylene (5 min), xylene (5 min), until the sections are completely clear.Finally, apply an appropriate amount of neutral resin to the center of the wound tissue, cover with a coverslip, lay flat in a ventilated place to dry, and then examine under a microscope.

[0061] The wounds on the backs of mice were continuously photographed and recorded on days 0, 3, 5, 7, 10, and 14 after modeling. Figure 5 The healing rate of full-thickness skin defects in mice was assessed in each group. The model control group (Model mice) exhibited significant delayed wound healing due to obesity and hyperglycemia phenotype induced by a high-fat, high-sugar diet. On days 3 and 5, the Model group wounds showed more severe inflammatory exudation, delayed scab formation, and significantly slower wound shrinkage from days 7 to 10 compared to the normal control group, suggesting that pathological hyperglycemia caused by diabetes severely inhibited the skin's self-repair ability. The 293T exosome group (Exo) and the recombinant XVII collagen group (COL17) both alleviated this delay to varying degrees, with the wound bed being drier on day 7 and the closure speed being improved compared to the Model group. The recombinant XVII collagen nanoparticle formulation group (Nano-COL17) showed the best therapeutic effect. From day 5 onwards, the wound area reduction in the Nano-COL17 group was significantly better than that in the Model group, and the scab formation was smoother than that in the pure protein COL17 group. By day 14, the wounds in the NC and Nano-COL17 groups were nearly completely closed, while the Model group still had obvious unhealed wounds. These macroscopic phenotypic results indicate that the nano-formulation can significantly enhance the bioavailability of recombinant type XVII collagen in the local environment of diabetic obese wounds and accelerate wound closure.

[0062] HE staining results ( Figure 6 The results showed that in the healthy mouse control group, a continuous and intact newly formed epithelial layer was visible, the epidermal tongue was fully differentiated, and the newly formed granulation tissue in the dermis was tightly arranged, with a small number of hair follicle primordia visible. In contrast, in the Model group, obvious epithelial defects or extremely thin and disordered newly formed epithelial cells were still visible at the center of the wound, the granulation tissue structure was loose, and there was a lot of residual inflammatory cell infiltration. The thickness of the newly formed epidermis and the granulation tissue in the COL17 group and the Nano-COL17 group tended to be more mature, with the dermal structure of the Nano-COL17 group being closest to normal skin (NC), and the fibroblasts were arranged in an orderly manner.

[0063] Masson trichrome staining results showed that the blue collagen fibers in the dermis of the Model group wound were sparse, fine, and extremely disordered. Collagen deposition in the Exo and COL17 groups showed a recovery compared to the Model group. The Nano-COL17 group wound exhibited a strong, dense blue collagen signal, with fibers displaying an orderly arrangement parallel to the epidermis, resembling long, wavy strips similar to normal tissue. This suggests that the nano-formulation not only promotes collagen synthesis but also optimizes its spatial arrangement.

[0064] Under a regular bright-field microscope, Sirius red specifically stained collagen fibers in the dermis of the wound a bright red, while the background appeared pale yellow. In the Model group, the distribution of red collagen signals in the wound was extremely uneven, with broken and weak fibers, and a low total positive staining area, indicating that the obese, hyperglycemic microenvironment severely inhibited total collagen deposition in the wound. The density of red collagen fibers in the COL17 and Exo groups increased compared to the Model group, but the distribution still showed some limitations. The Nano-COL17 group, on the other hand, exhibited large, continuous, deep red collagen fiber bundles; the fibers were large, densely packed, and regularly oriented. To elucidate the cellular mechanism by which recombinant type XVII collagen nanoparticles promote wound repair, immunohistochemical staining of wound tissue for CD31, Ki67, and VEGF was performed.

[0065] The results are as follows Figure 7 As shown, the CD31-positive microvessel count (MVD) and VEGF expression levels in the Model group wound were extremely low, confirming that hyperglycemia caused severe microvascular dysfunction. Vascular density improved in the Exo and COL17 groups. In the Nano-COL17 group, numerous clusters of CD31-positive endothelial cells were observed in the wound basal layer and granulation tissue, accompanied by strong VEGF secretion. This indicates that the nano-formulation, through continuous release of collagen signals, activated local endothelial cells, effectively inducing the reconstruction of the wound microvascular network and providing sufficient blood oxygenation support for tissue repair. In the newly formed epidermal basal layer and dermal granulation tissue at the wound edge, the Model group showed a sparse number of Ki67-positive proliferating cells. The Nano-COL17 group showed a significantly increased Ki67 positivity rate in basal keratinocytes and dermal fibroblasts, stimulating wound cell proliferation and migration, thereby accelerating the epidermal re-epithelialization process.

[0066] In DIO obese mice, due to systemic metabolic disorders, wounds are often in a state of pathological chronic hyperinflammatory. The changes in the local immune microenvironment were assessed by detecting the expression of pro-inflammatory factors TNF-α and IL-6. Results are as follows... Figure 8As shown, on day 14, the Model group still showed a large number of TNF-α positive signals (dark brown cytoplasm) secreted by macrophages and neutrophils in the dermis and subcutaneous tissue, indicating that the wound was still in the inflammatory phase. The TNF-α positive area in the Exo group and COL17 group was downregulated. The TNF-α positive infiltration in the Nano-COL17 group was extremely weak, basically returning to the level of the normal control group. The IL-6 expression results were highly consistent with the TNF-α trend. IL-6 showed extensive high expression in the Model group wound, and the positive expression of IL-6 in the wound tissue was inhibited after treatment with the Nano-COL17 nanoparticle formulation.

[0067] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a targeted nanoparticle in the preparation of a drug for treating diabetic foot, characterized in that, The targeted nanoparticles include recombinant type XVII full-length human collagen; The amino acid sequence of the recombinant XVII type full-length human collagen is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The recombinant XVII type full-length human collagen is enriched on the surface of the targeted nanoparticles by a sorting signal peptide.

3. The application according to claim 2, characterized in that, The sorting signal peptide contains the amino acid sequence shown in SEQ ID NO.

2.

4. The application according to claim 1, characterized in that, The targeted nanoparticles are selected from exosomes or microvesicles.

5. The application according to claim 1, characterized in that, The targeted nanoparticles are derived from engineered host cells that express the recombinant type XVII full-length human collagen.

6. The application according to claim 5, characterized in that, The host cell is a eukaryotic cell.

7. The application according to claim 6, characterized in that, The host cell is a mammalian cell.

8. The application according to claim 7, characterized in that, The host cell was a 293T cell.

9. The application according to claim 1, characterized in that, The targeted nanoparticles are prepared by the following method: (1) Construct an expression vector, wherein the expression vector comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 and a sorting signal peptide encoding sequence; (2) Transfect the expression vector into host cells; (3) Cultivate the host cells and isolate and purify the targeted nanoparticles.

10. The application according to claim 1, characterized in that, The dosage form of the drug is selected from gel, spray, ointment, dressing, lyophilized powder for injection or injection.

11. A method for treating diabetic foot, characterized in that, This includes administering an effective amount of targeted nanoparticles to the subject; The surface of the targeted nanoparticles is enriched with recombinant type XVII full-length human collagen; The amino acid sequence of the recombinant XVII type full-length human collagen is shown in SEQ ID NO.1.

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