Pigskin-derived recombinant lipid nanoparticles, composite hydrogel dressing as well as preparation method and application of pigskin-derived recombinant lipid nanoparticles and composite hydrogel dressing

Abstract

The invention discloses pigskin-derived recombinant lipid nanoparticles as well as a composite wound dressing and application of the pigskin-derived recombinant lipid nanoparticles. The nanoparticles are prepared by taking pigskin extracted lipid as a raw material and adopting an ethanol injection method, are rich in triglyceride and phosphatidylcholine, and have the biological activity of promoting fibroblast proliferation and migration. Furthermore, the active nano-particles are loaded in calcium ion cross-linked sodium alginate hydrogel to prepare the composite hydrogel dressing, and the dressing has injectability and shear thinning characteristics and can continuously release the active nano-particles. In-vitro experiments prove that the biocompatibility is good; animal experiments show that the traditional Chinese medicine composition can remarkably accelerate full-thickness skin defect healing and promote epidermis regeneration, ordered collagen deposition and angiogenesis. According to the invention, high-valued utilization of meat byproducts is realized, and a safe and efficient novel active dressing is provided for chronic wound treatment.

Description

Technical Field

[0001] This invention relates to biomedical materials, specifically to a porcine skin-derived recombinant lipid nanoparticle, a composite hydrogel dressing, its preparation method, and its application. Background Technology

[0002] Wound healing is a crucial physiological process for maintaining skin integrity and preventing fatal complications, but chronic, refractory wounds remain a global clinical challenge. Acute wound healing typically follows an ordered cascade process, but under the influence of factors such as diabetes and aging, it is prone to becoming chronic, characterized by prolonged inflammation, impaired angiogenesis, and increased susceptibility to infection, significantly increasing the difficulty and risk of treatment. Traditional dressings (such as gauze) can only provide basic coverage and absorption functions and cannot actively regulate the wound microenvironment to accelerate healing.

[0003] To overcome the limitations of traditional dressings, recent research has focused on using nanocarriers as core platforms for advanced wound dressings. Polymer nanoparticles, liposomes, and metal-organic frameworks have been extensively studied due to their customizable properties, and are used to load and deliver active substances such as antibacterial agents and growth factors. However, these synthetic nanosystems often suffer from insufficient biocompatibility, potentially leading to liver and kidney toxicity, nonspecific inflammatory reactions, or immunogenic risks, especially with long-term use, limiting their clinical translation. Meanwhile, naturally derived nanocarriers, such as mammalian cell exosomes, have attracted attention due to their excellent biocompatibility and inherent tissue repair functions. However, exosomes face industrialization bottlenecks such as low yield, complex separation and purification processes, high costs, and poor batch-to-batch uniformity, also making it difficult to meet the needs of large-scale clinical applications. It is worth noting that current research on naturally derived nanocarriers almost entirely focuses on natural vesicles such as exosomes. Researchers in this field generally limit their thinking to the optimization and improvement of exosomes, finding it difficult to break out of this inherent framework. Therefore, they rarely consider using porcine skin lipids to construct recombinant lipid nanoparticles as nanocarriers for wound repair. Porcine skin lipids, as a natural lipid component, have not yet received sufficient attention or exploration in the field of recombinant lipid nanoparticle construction and wound repair, making them an easily overlooked research direction with significant innovative exploratory value. Furthermore, even if researchers have occasionally considered the potential applications of porcine skin lipids, the technical difficulty in constructing recombinant lipid nanoparticles using them is significantly high, becoming a major bottleneck restricting the development of this direction. Porcine skin tissue naturally contains a large number of complex lipid components. These components are disordered and closely interacting. If conventional extraction and preparation methods are used, the resulting recombinant lipid nanoparticles are prone to aggregation, uneven particle size distribution, and poor structural stability, failing to meet the basic requirements for subsequent loading of active substances and wound dressings. To obtain porcine skin-derived recombinant lipid nanoparticles with stable performance and good uniformity, specific extraction and purification steps must be designed and optimized, and the separation, screening and recombination of lipid components must be precisely controlled. This technical requirement exceeds the scope of conventional nanoparticle preparation technology, and the control of process parameters requires extremely high precision, which further limits the exploration and practice of this technical route by those skilled in the art. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a porcine skin-derived recombinant lipid nanoparticle and composite hydrogel wound dressing that has good biocompatibility, active healing-promoting function, and is made from economically available raw materials; another purpose of this invention is to provide a method for preparing the above materials and their applications.

[0005] Technical solution: The porcine skin-derived recombinant lipid nanoparticles of the present invention are prepared from lipids extracted from porcine skin as raw materials; in the porcine skin-derived recombinant lipid nanoparticles, the relative content of triglycerides is 70%-85% and the relative content of phosphatidylcholine is 8%-15% based on the total mass of all lipids contained therein; the hydration kinetic particle size of the porcine skin-derived recombinant lipid nanoparticles is 80-120 nm.

[0006] More preferably, the hydration dynamic particle size of the nanoparticles is 80-100 nm.

[0007] The composite hydrogel dressing of the present invention comprises porcine skin-derived recombinant lipid nanoparticles and sodium alginate hydrogel, wherein the porcine skin-derived recombinant lipid nanoparticles are loaded in the sodium alginate hydrogel.

[0008] Preferably, the loading concentration of porcine skin-derived recombinant lipid nanoparticles is 500-2000 μg / mL.

[0009] Further preferred, the loading concentration of porcine skin-derived recombinant lipid nanoparticles is 2000 μg / mL.

[0010] Preferably, the sodium alginate hydrogel is a calcium ion crosslinked hydrogel.

[0011] The method for preparing porcine skin-derived recombinant lipid nanoparticles of the present invention includes extracting total lipids from porcine skin; dissolving the extracted total lipids in ethanol to obtain a lipid ethanol solution with a concentration of 10-20 mg / mL; and injecting the lipid ethanol solution into water under stirring conditions of 1000-2000 rpm to self-assemble the porcine skin-derived recombinant lipid nanoparticles.

[0012] More preferably, the concentration of the lipid ethanol solution is 15 mg / mL; the injection stirring speed is 1500 rpm.

[0013] Preferably, the method for extracting total lipids is as follows: extracting pig skin with a mixed solvent of methyl tert-butyl ether and methanol at 0-10°C, wherein the volume ratio of methyl tert-butyl ether to methanol in the mixed solvent is (2-4):1; then adding a mixed solution of water and methanol for separation, wherein the volume ratio of water to methanol is (2-4):1; collecting the supernatant, concentrating it, mixing it with dichloromethane, centrifuging to collect the precipitate, and drying it to obtain the total lipids.

[0014] Further preferred, the extraction temperature is 4°C, the volume ratio of methyl tert-butyl ether to methanol is 3:1, and the volume ratio of water to methanol is 3:1.

[0015] The preparation method of the composite hydrogel dressing of the present invention includes the following steps: (1) Preparation of the porcine skin-derived recombinant lipid nanoparticle dispersion; (2) Sodium alginate, guar gum and the nanoparticle dispersion are mixed to obtain mixed solution A; (3) Dissolve the calcium source and acidity regulator in water to obtain solution B; (4) Mix solution B with mixed solution A and cross-link the mixture to form the composite hydrogel dressing.

[0016] Preferably, in step (3), the calcium source is calcium carbonate, the acidity regulator is D-(+)-gluconic acid-δ-lactone, the mass ratio of the calcium source to the sodium alginate is (0.1-0.3):1, and the mass ratio of the acidity regulator to the calcium source is (1.5-3):1.

[0017] More preferably, the mass ratio of the calcium source to the sodium alginate is 0.2:1; and the mass ratio of the acidity regulator to the calcium source is 2:1.

[0018] The application of the porcine skin-derived recombinant lipid nanoparticles described in this invention in the preparation of drugs or biomaterials for promoting wound healing.

[0019] The application of the composite hydrogel dressing described in this invention in the preparation of drugs or biomaterials for skin repair.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. For the first time, bioactive nanoparticles were prepared using lipids extracted from pig skin, a byproduct of the meat processing industry, achieving high-value utilization of waste materials. The raw materials are widely available and inexpensive. The prepared pig skin-derived recombinant lipid nanoparticles (PS-rLNPs) are not inert carriers; their rich content of natural lipid components such as triglycerides and phosphatidylcholine endows them with intrinsic biological activity that promotes fibroblast proliferation and migration. Combining them with SA hydrogel, which provides a physical barrier and a humid environment, achieves a synergistic effect of "active treatment" and "passive protection."

[0021] 2. The sodium alginate composite hydrogel dressing (PS-rLNPs-SA) loaded with porcine skin-derived recombinant lipid nanoparticles (PS-rLNPs) possesses injectability and shear-thinning properties, making it easy to adhere to irregular wounds. The hydrogel matrix effectively loads and sustains the release of PS-rLNPs, overcoming the drawback of easily removed free nanoparticles and achieving long-lasting healing promotion. Animal experiments show that this composite dressing can significantly accelerate the healing of full-thickness skin defects, promote epidermal regeneration, orderly collagen deposition, and angiogenesis, with effects superior to single-component dressings.

[0022] 3. All components (pork skin lipids, sodium alginate) are of natural origin or recognized safe materials. In vitro and in vivo experiments have confirmed that the composite dressing is non-cytotoxic, non-hemolytic, and does not damage major organs, demonstrating high biocompatibility. Attached Figure Description

[0023] Figure 1 A pie chart showing the relative content of the main lipid components in PS-rLNPs.

[0024] Figure 2 Photograph of the prepared PS-rLNPs aqueous dispersion.

[0025] Figure 3 Transmission electron microscopy images of PS-rLNPs.

[0026] Figure 4 The hydrodynamic particle size distribution of PS-rLNPs is shown.

[0027] Figure 5 The graphs show the stability evaluation curves of PS-rLNPs in different media.

[0028] Figure 6 This is a schematic diagram of the Transwell experimental setup.

[0029] Figure 7 Fluorescence images of PS-rLNPs uptake by cells at different incubation times.

[0030] Figure 8 This is a statistical graph showing the change in average fluorescence intensity of cells over time.

[0031] Figure 9 A statistical graph showing the cellular uptake efficiency after treatment with different endocytosis inhibitors.

[0032] Figure 10 This is a colocalized fluorescence image of PS-rLNPs (green), lysosomes (red), and endoplasmic reticulum (red).

[0033] Figure 11 This is a schematic diagram of the PS-rLNPs-SA hydrogel preparation process.

[0034] Figure 12 This is a schematic diagram illustrating the injectability of hydrogels.

[0035] Figure 13 This is the viscosity-shear rate curve of the hydrogel.

[0036] Figure 14 The curves show the changes in storage modulus (G') and loss modulus (G") of the hydrogel as a function of angular velocity.

[0037] Figure 15Statistical graph of cell viability after treatment with extracts of different materials.

[0038] Figure 16 This is a fluorescent image of live cells stained with dye.

[0039] Figure 17 Macroscopic images of the wounds at different time points for different treatment groups.

[0040] Figure 18 This is a simulation diagram of the healing process.

[0041] Figure 19 This is a statistical chart of wound healing rate.

[0042] Figure 20 Representative images of H&E staining and Masson trichrome staining of wound tissue.

[0043] Figure 21 These are representative images of CD31 and Ki-67 immunohistochemical staining of wound tissue.

[0044] Figure 22 This is a statistical chart showing the relative collagen content of each group.

[0045] Figure 23 A statistical chart of CD31-positive regions in each group.

[0046] Figure 24 A statistical chart of Ki-67 positive areas in each group.

[0047] Figure 25 Images of hemolysis experiments with different concentrations of PS-rLNPs.

[0048] Figure 26 A statistical chart showing the hemolysis rate of different materials.

[0049] Figure 27 Images of H&E-stained tissue sections of major organs in mice.

[0050] Figure 28 This is a flowchart and application diagram of the preparation of porcine skin-derived recombinant lipid nanoparticles and composite hydrogel dressings according to the present invention. Detailed Implementation

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0052] Example 1: Preparation and characterization of porcine skin-derived recombinant lipid nanoparticles (PS-rLNPs) (1) Materials: Fresh pigskin was purchased from the local market. Methyl tert-butyl ether (MTBE) and methanol (MeOH) were purchased from Shanghai Titan Technology Co., Ltd. Anhydrous ethanol and dichloromethane (DCM) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0053] (2) Extraction of total lipids from pig skin: Fresh pig skin was cut into small pieces and mixed with MTBE:MeOH (3:1, v / v) and stirred in a water bath at 4°C. Then H2O:MeOH (3:1, v / v) was added, and the mixture was allowed to stand and separate into layers. The supernatant was collected, concentrated by rotary evaporation, and then mixed with DCM. After centrifugation, the supernatant was discarded, and the residue was dried under nitrogen and then vacuum dried to obtain the lipid product, which was stored at -80°C for later use.

[0054] (3) Preparation of PS-rLNPs: The solvent injection method was used. The total lipids extracted above were dissolved in anhydrous ethanol (15 mg / mL), sonicated (300 W, 5 min), and then filtered through a 0.22 μm membrane. Under vigorous stirring (1500 rpm), the ethanol solution was rapidly injected into pure water, and the nanoparticles were self-assembled to obtain the PS-rLNPs dispersion.

[0055] (4) Characterization results and discussion: Lipid composition: Analysis by liquid chromatography-mass spectrometry (LC-MS) showed that the main components of PS-rLNPs were triglycerides (77.9%) and phosphatidylcholine (11.6%), with the remainder including sphingomyelin (3.2%), phosphatidylethanolamine (2%), etc. Figure 1 (Pie chart showing the relative content of major lipid components).

[0056] Morphology and particle size: The prepared PS-rLNPs aqueous dispersion was a translucent milky white colloid exhibiting the Tyndall effect (…). Figure 2 Transmission electron microscopy (TEM) revealed that PS-rLNPs exhibited a monodisperse, spherical, dehydrated morphology with an average diameter of approximately 40 nm. Figure 3 Dynamic light scattering (DLS) determined its hydrated particle size to be 91.3 nm, and its polydispersity index (PDI) to be 0.225. Figure 4 (Hydrodynamic particle size distribution diagram).

[0057] Stability: After being placed at room temperature for 7 days in ultrapure water at pH 7.4 and cell culture medium containing 10% fetal bovine serum (FBS), the hydrated particle size fluctuation ranges were 75-102 nm and 91-103 nm, respectively, and the PDI values ​​were less than 0.25 and 0.30, respectively, indicating excellent colloidal stability. Figure 5 (Stability evaluation curves in different media).

[0058] Example 2: Evaluation of the in vitro biological effects of PS-rLNPs (1) Materials and cells: Mouse fibroblast cell line 3T3. Durbeco modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Service Biotechnology Co., Ltd. MTT reagent was purchased from Beyotime Biotechnology Co., Ltd.

[0059] (2) Cell proliferation assay (MTT method): 3T3 cells were seeded at 2×10³ cells / well in 96-well plates and cultured for 24 hours. The medium was then replaced with medium containing different concentrations of PS-rLNPs (100-500 μg / mL) or SB-rLNPs (nanoparticles prepared using soybean lecithin according to the same preparation method, 100-500 μg / mL). After culturing for another 24 hours, MTT solution was added and incubated for 4 hours. Formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm to calculate cell viability.

[0060] (3) Results and Discussion: PS-rLNPs showed no cytotoxicity in the concentration range of 0-500 μg / mL and promoted cell proliferation in a concentration-dependent manner. For 3T3 cells, the viability reached a maximum of 153.3% at 300 μg / mL; in contrast, SB-rLNPs had little effect on cell viability in all concentration ranges, with a viability of 100.2% at 300 μg / mL; suggesting that PS-rLNPs have an unexpected effect on promoting cell proliferation.

[0061] (4) Cell migration experiment: Scratch assay: 3T3 cells were cultured to confluence and scratched with a pipette tip. The experimental group was then treated with low-serum medium (2% FBS) containing 300 μg / mL nanoparticles, while the control group was treated with blank low-serum medium. Images were taken at 0 and 24 hours, and the scratch width was measured using ImageJ software. Results showed that after 24 hours of PS-rLNPs treatment, the scratch healing rate of 3T3 cells was 43.65%, significantly higher than the control group (20.75%). In contrast, the healing rate after treatment with SB-rLNPs at the same concentration was 21.38%, with no significant difference from the control group.

[0062] Transwell experiment: Schematic diagram of the experimental setup is shown below. Figure 6 As shown in the figure. Complete culture medium containing 10% FBS was added to the lower chamber, and serum-free cell suspension containing 300 μg / mL nanoparticles (experimental group) or suspension without nanoparticles (control group) was added to the upper chamber. After 24 hours of culture, cells were fixed, stained with crystal violet, and counted. The number of migrating 3T3 cells in the PS-rLNPs treatment group (510 per field of view) was significantly higher than that in the control group (350 per field of view) and the SB-rLNPs group (381 per field of view) (p<0.001), suggesting that PS-rLNPs have an unexpected effect on promoting cell migration.

[0063] Example 3: Study on the cellular uptake mechanism of PS-rLNPs (1) Materials: Coumarin 6, Chlorpromazine, Nystatin, Amiloride (EIPA), Lysosomal Red Fluorescent Probe (LysoTracker Red DND-99), Endoplasmic Reticulum Fluorescent Dye (ER-Tracker™ Red), 4′,6-Diamidin-2-phenylindole (DAPI).

[0064] (2) Time- and temperature-dependent cell uptake: 3T3 cells were incubated with 50 μg / mL coumarin 6-labeled PS-rLNPs at different times (0.5-4 h) or at 4℃, and the results were observed by confocal microscopy. Figure 7 Fluorescence images of PS-rLNPs uptake by cells at different incubation times (and flow cytometry analysis). Results showed that uptake increased in a time-dependent manner. Figure 8 (Statistical graph of the change in average fluorescence intensity of cells over time), and the uptake rate at 4℃ was 35% lower than that at 37℃, proving that it is an energy-dependent active process.

[0065] (3) Endocytosis pathway inhibitor experiment: Cells were pretreated with different inhibitors for 4 hours (chlorpromazine 30 μM inhibited clathrin-mediated endocytosis; nystatin 30 μM inhibited caveolin-mediated endocytosis; EIPA 100 μM inhibited macropinocytosis), and then incubated with fluorescently labeled PS-rLNPs for 0.5 hours. Flow cytometry analysis showed that the uptake inhibition rate in the chlorpromazine-only treatment group was 16.4%, suggesting that the endocytosis pathway mainly depended on clathrin-mediated endocytosis. Figure 9 (Statistical graph of cellular uptake efficiency after treatment with different endocytosis inhibitors).

[0066] (4) Intracellular localization: Cells were incubated with coumarin 6-labeled PS-rLNPs for 1 hour, and lysosomes or endoplasmic reticulum probes were added in the last 30 minutes. Confocal microscopy showed that PS-rLNPs (green) co-localized extensively with lysosomes and endoplasmic reticulum (red) markers (yellow signal), indicating that they associated with these organelles after entering the cell. Figure 10 (Image showing colocalization fluorescence of PS-rLNPs (green) with lysosomes (red) and endoplasmic reticulum (red).)

[0067] Example 4: Preparation and characterization of sodium alginate composite hydrogel loaded with PS-rLNPs (PS-rLNPs-SA) (1) Materials: Sodium alginate (SA), guar gum, calcium carbonate (CaCO3), D-(+)-gluconic acid-δ-lactone.

[0068] (2) Preparation of PS-rLNPs-SA: First, prepare PS-rLNPs stock solution (0.5-2 mg / mL). Solution A: Mix glycerol, SA, guar gum (mass ratio 5:1) with PS-rLNPs solution and stir until homogeneous. Solution B: Dissolve CaCO3 and GDL (mass ratio 1:2) in water and sonicate to clarify. Quickly pour solution B into solution A, stir for 1 minute, and let stand for 30 minutes to form a hydrogel. Figure 11 (Schematic diagram of the PS-rLNPs-SA hydrogel preparation process). The resulting hydrogel exhibits good injectability. Figure 12 (Schematic diagram of hydrogel injectability).

[0069] (3) Rheological characterization results and discussion: Tests were conducted using a rotational rheometer.

[0070] Shear-thinning behavior: Steady-state shear tests showed that all hydrogels exhibited shear-thinning behavior. As the PS-rLNPs concentration increased from 500 μg / mL to 2000 μg / mL, the initial viscosity at low shear rates significantly increased, while the viscosity decreased at high shear rates. The 2000 μg / mL group best met the requirements for application and retention of wound dressings. Figure 13 (Viscosity-shear rate curve of hydrogel).

[0071] Viscoelasticity: Dynamic oscillation frequency scanning showed that the storage modulus (G') of all hydrogels was greater than the loss modulus (G"), exhibiting solid-like elastic behavior. After the addition of PS-rLNPs, both G' and G" values ​​were lower than those of the pure SA hydrogel, indicating that the addition of nanoparticles increased the flexibility of the gel network. Figure 14 The curves showing the changes in storage modulus (G') and loss modulus (G') of the hydrogel as a function of angular velocity.

[0072] (4) In vitro biocompatibility evaluation: CCK-8 method: Extracts of SA hydrogel and PS-rLNPs-SA hydrogel were prepared (filtered after incubation at 37℃ for 24 hours) and added to culture medium at a 20% volume ratio to culture 3T3 cells for 24 hours. Results showed that neither extract was cytotoxic, and the PS-rLNPs-SA extract, like free PS-rLNPs, significantly promoted cell proliferation. Figure 15 (Statistical graph of cell viability after treatment with extracts from different materials).

[0073] Live / dead cell staining: After co-culturing cells with PS-rLNPs-SA extract for 24 hours, Calcein-AM staining was performed. Fluorescence microscopy showed a high proportion of live cells, further confirming its good biocompatibility. Figure 16 (live cell staining fluorescence image).

[0074] Example 5: In vivo wound healing experiment (1) Animal model establishment: Six-week-old female BALB / c mice were used in the experiment. After anesthesia, a full-thickness skin defect with a diameter of 8 mm was created on the back.

[0075] (2) Grouping and treatment: Mice were randomly divided into 4 groups: blank control group (untreated), blank SA hydrogel group, PS-rLNPs group (2 mg / mL), and PS-rLNPs-SA composite hydrogel group (2 mg / mL PS-rLNPs). The corresponding preparation was applied immediately after the operation and changed daily.

[0076] (3) Analysis results and discussion of healing rate: Photos were taken and recorded on days 0, 2, 6, 8, and 10. Figure 17 Macroscopic photographs of the wounds of different treatment groups at different time points were taken, and a simulation diagram of the healing process was drawn. Figure 18 Quantitative analysis showed that ( Figure 19 (Statistical chart of wound healing rate). The PS-rLNPs-SA group had the fastest healing speed, with a healing rate of 93.26% on day 10, significantly better than other groups. The free PS-rLNPs group had a better healing rate than the PS-rLNPs-SA group (55.54%) on day 6 (59.8%), but was subsequently surpassed, with a healing rate of 78.05% by day 10, demonstrating the sustained-release advantage of hydrogel.

[0077] (4) Histological and immunohistochemical analysis: Wound tissue was taken and stained on the 12th day after surgery.

[0078] H&E staining and Masson's trichrome staining: Representative images are shown below. Figure 20 As shown, the PS-rLNPs-SA group exhibited the most complete epidermal regeneration, the mildest inflammatory infiltration, the highest collagen deposition, and the most orderly fiber arrangement.

[0079] CD31 and Ki-67 immunohistochemical staining: Representative images are shown below. Figure 21 As shown, the PS-rLNPs-SA group had the highest angiogenesis density (CD31 positive area) and repair cell proliferation activity (Ki-67 positive area).

[0080] Quantitative statistics: such as Figure 22-24 As shown, the relative collagen content, CD31 positive area, and Ki-67 positive area of ​​the PS-rLNPs-SA group were significantly higher than those of the other groups.

[0081] Example 6: Biosafety Assessment (1) In vitro hemolysis experiment: Different concentrations of PS-rLNPs (0.1-1 mg / mL) were co-incubated with mouse erythrocytes, and the absorbance of the supernatant was measured after centrifugation to calculate the hemolysis rate. Figure 25(Photographs of hemolysis experiments with different concentrations of PS-rLNPs). The results showed that the hemolysis rates of each concentration of PS-rLNPs group, SA hydrogel group, and PS-rLNPs-SA group were similar to those of the PBS negative control group and much lower than those of the deionized water positive control group, indicating no significant hemolytic effect. Figure 26 (Statistical chart of hemolysis rates for different materials).

[0082] (2) In vivo organ toxicity assessment: Mice treated in Example 5 for 10 days were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were collected for H&E staining ( Figure 27 (H&E stained tissue sections of major organs of mice). Microscopic observation revealed no obvious pathological changes (such as inflammation or necrosis) in any of the major organs, indicating that PS-rLNPs-SA has good biocompatibility in vivo.

[0083] In summary, this invention successfully prepared a sodium alginate composite hydrogel dressing with porcine skin-derived recombinant lipid nanoparticles as the active ingredient. This dressing uses economical raw materials, has good biocompatibility, is easy to prepare, and possesses both injectable physical properties and the biological function of actively promoting wound healing, showing broad application prospects in the treatment of chronic wounds and other fields.

Claims

1. A porcine skin-derived recombinant lipid nanoparticle, characterized in that, The pigskin-derived reconstituted lipid nanoparticles are prepared from lipids extracted from pigskin; the relative content of triglyceride in the pigskin-derived reconstituted lipid nanoparticles is 70-85% by mass of the total lipids contained therein, and the relative content of phosphatidylcholine is 8-15%; and the hydration kinetic particle size of the pigskin-derived reconstituted lipid nanoparticles is 80-120 nm.

2. A composite hydrogel dressing, characterized by, The pigskin-derived reconstituted lipid nanoparticles are loaded in the sodium alginate hydrogel.

3. The composite hydrogel dressing of claim 2, wherein, The loading concentration of the pigskin-derived reconstituted lipid nanoparticles is 500-2000 μg / mL.

4. The composite hydrogel dressing of claim 2, wherein, The sodium alginate hydrogel is a calcium ion crosslinked hydrogel.

5. A method of preparing the porcine skin-derived recombinant lipid nanoparticle of claim 1, wherein, The pigskin-derived reconstituted lipid nanoparticles are prepared by extracting total lipids from pigskin; dissolving the extracted total lipids in ethanol to obtain a lipid ethanol solution with a concentration of 10-20 mg / mL; and self-assembling the lipid ethanol solution into the pigskin-derived reconstituted lipid nanoparticles under stirring at 1000-2000 rpm.

6. The production method according to claim 5, wherein The total lipids are extracted by extracting pigskin with a mixed solvent of methyl tert-butyl ether and methanol at 0-10℃, wherein the volume ratio of methyl tert-butyl ether to methanol in the mixed solvent is (2-4):1; then adding a mixed solution of water and methanol to separate the layers, wherein the volume ratio of water to methanol is (2-4):1; collecting the supernatant, mixing it with dichloromethane, centrifuging to obtain the precipitate, and drying to obtain the total lipids.

7. A method of preparing a composite hydrogel dressing according to any one of claims 2 to 4, characterised in that, The method comprises the following steps: (1) preparing the pigskin-derived reconstituted lipid nanoparticle dispersion liquid of claim 1; (2) mixing sodium alginate and guar gum with the nanoparticle dispersion liquid to obtain a mixed solution A; (3) dissolving a calcium source and an acidity regulator in water to obtain a solution B; (4) mixing solution B with the mixed solution A, and forming the composite hydrogel dressing through a crosslinking reaction.

8. The preparation method according to claim 7, characterized in that, In step (3), the calcium source is calcium carbonate, and the acidity regulator is D-(+)-glucono-δ-lactone; the mass ratio of the calcium source to the sodium alginate is (0.1-0.3):1; and the mass ratio of the acidity regulator to the calcium source is (1.5-3):

1.

9. Use of the pigskin-derived reconstituted lipid nanoparticles of claim 1 or the pigskin-derived reconstituted lipid nanoparticles prepared by the method of any one of claims 5-6 in the preparation of a drug or a biomaterial for promoting wound healing.

10. Use of the composite hydrogel dressing of any one of claims 2-4 or the composite hydrogel dressing prepared by the method of any one of claims 7-8 in the preparation of a drug or a biomaterial for skin repair.

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