Fibronectin nanogel for enhancing tissue permeation as well as preparation method and application of fibronectin nanogel

Abstract

The invention relates to a fibronectin nanogel for enhancing tissue permeation and a preparation method and application thereof. The fibronectin nanogel is prepared by carrying out Schiff base reaction on fibronectin and terephthalaldehyde and carrying out controllable crosslinking by adopting a reversed-phase microemulsion method to form a three-dimensional network structure. The fibronectin nanogel provided by the invention has excellent biocompatibility, permeability and biological activity, can significantly promote skin tissue repair, accelerates wound healing, and effectively inhibits the skin aging process by regulating and controlling extracellular matrix remodeling.

Description

Technical Field

[0001] The invention belongs to the fields of biomaterials, nanomaterials and nanomedicine, and particularly relates to a fibronectin nanogel for enhancing tissue penetration, a preparation method and an application thereof. Background Art

[0002] FN (fibronectin) plays multiple biological functions in the extracellular matrix. It not only supports cell adhesion and migration, but also plays a key role in tissue repair and wound healing (ACS Nano 2024, 18, 10625-10641). In addition, FN can regulate macrophage immunity and regulate the differentiation of macrophages into the anti-inflammatory M2 type (Bioact. Mater. 2024, 38, 45–54). During wound healing, FN can recruit fibroblasts and promote their migration to the wound surface, thereby effectively promoting tissue repair and regeneration (Trends in Cell Biology 2020, 30, 990-1013). In the early stages of wound healing, plasma fibronectin (pFN) plays a role by participating in platelet aggregation and adhesion. As the wound heals, FN regulates cell adhesion, migration, proliferation and apoptosis, reduces oxidative damage, and provides support for tissue recovery. In the later stages of healing, cellular fibronectin (cFN) is assembled by locally expressed FN and becomes a key participant in tissue remodeling, ensuring the integrity and functional recovery of wound healing (Science 2009, 326, 1216-1219). Research by Shamik Mascharak and his team revealed that during wound healing, inhibition of Yes-associated protein (YAP) can effectively prevent the activation of Engrailed-1, thereby producing more Engrailed-1-negative fibroblasts, promoting the recovery of skin ultrastructure and mechanical strength, and thus repairing wounds (Science 2021, 372, eaba2374). Therefore, FN may reduce scar formation and promote wound healing by inhibiting the expression of YAP and blocking the activation of Engrailed-1.

[0003] Long-term exposure to UV radiation damages the skin barrier and promotes inflammatory responses, which are the main causes of premature skin aging (also known as photoaging). Therefore, anti-inflammatory and extracellular matrix (ECM) remodeling are the main research directions of skin anti-aging (Sig. Transduct. Target. Ther. 2024, 9, 294). In addition, reactive oxygen species (ROS) generated by UV radiation activate signaling pathways such as MAPK and NF-κB, leading to collagen degradation, triggering local inflammation, and exacerbating oxidative stress (Adv. Mater. 2025, 2500552, DOI: https: / / doi.org / 10.1002 / adma.202500552). Therefore, the development of drugs that can inhibit inflammation, effectively scavenge ROS, and promote collagen remodeling is crucial for the treatment of photoaging. FN also plays an important role in inhibiting skin aging. FN can inhibit the NF-κB signaling pathway, promote the polarization of macrophages to an anti-inflammatory phenotype (M2 type), and increase the expression of intracellular antioxidant enzymes (Adv. Healthcare Mater. 2024, 13, 2401462), thereby alleviating the inflammatory infiltration and oxidative stress caused by photoaging.

[0004] Despite its excellent biological activity, FN's large molecular structure, complex three-dimensional morphology, and high hydrophilicity limit its permeability to cell membranes and tissues (Nat. Rev. Drug Discovery 2021, 20, 101-124), hindering its ability to act on relevant intracellular targets and its related pro-regenerative and anti-aging therapeutic effects. Therefore, research on how to enhance the permeability of FN within cells or tissues has become a crucial issue.

[0005] In recent years, researchers have successfully developed a variety of nanodelivery systems, such as liposomes, inorganic nanoparticles, nanogels (NGs), and polymers, for efficient intracellular delivery of proteins (Chemical Engineering Journal 2021, 425, 130498). Among them, NGs, as a three-dimensional network nanomaterial constructed by physical or chemical cross-linking, have shown significant advantages in the field of drug delivery due to their unique physicochemical properties, such as high drug loading capacity, surface modifiability, environmentally responsive drug release properties, excellent deformation ability, and outstanding biopenetration performance (Acta Biomater. 2019, 92, 1-18).

[0006] The currently developed nanoplatforms have not yet engineered FN into a nanogel form to break through the cell and tissue penetration barriers, thereby achieving the goal of enhancing cellular uptake efficiency and maintaining biological activity stability. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a fibronectin nanogel for enhancing tissue penetration and its preparation method and application, so as to solve the problem of FN tissue penetration, thereby achieving wound repair or inhibiting the process of skin photoaging.

[0008] The invention provides a fibronectin nanogel for enhancing tissue penetration. The fibronectin nanogel is prepared by Schiff base reaction between fibronectin and terephthalaldehyde and controllably cross-linking to form a three-dimensional network structure using an inverse microemulsion method.

[0009] The present invention provides a method for preparing a fibronectin nanogel for enhancing tissue penetration, comprising the following steps:

[0010] (1) Dissolve fibronectin and cross-linking agent terephthalaldehyde in organic solvent A to form a dispersed phase; dissolve Span 80 and Tween 80 in organic solvent B to form a continuous phase; then add the dispersed phase to the continuous phase and stir to form an emulsion in which the continuous phase covers the dispersed phase;

[0011] (2) The emulsion obtained in step (1) is sonicated, and then ethylenediamine is added and stirred for reaction; after the reaction is completed, the emulsion is centrifuged, the precipitate is resuspended and dispersed with ethanol, and the resuspended solution is dialyzed and purified to obtain fibronectin nanogel, i.e., FCN.

[0012] Preferably, in step (1), the mass ratio of fibronectin to terephthalaldehyde is 1:0.1-10, and the concentration of fibronectin in the dispersed phase is 0.2-5 mg / mL; the organic solvent A includes but is not limited to DMSO (dimethyl sulfoxide).

[0013] Preferably, in step (1), the mass ratio of Span 80 to Tween 80 is 1:0.1-0.3, and the concentration of Span 80 in the continuous phase is 5-40 mg / mL; the organic solvent B includes but is not limited to cyclohexane.

[0014] Preferably, the volume ratio of the dispersed phase to the continuous phase in step (1) is 1:5-20.

[0015] Preferably, the stirring temperature in step (1) is 8 to 37° C., and the stirring time is 10 to 20 minutes.

[0016] Preferably, the volume ratio of the ethylenediamine in step (2) to the emulsion in step (1) is 1:10-100.

[0017] Preferably, the ultrasound in step (2) is performed using an ultrasonic disruptor device, and the ultrasound time is 15 to 25 minutes; the stirring reaction time is 11 to 13 hours; the centrifugal speed is 10,000 to 14,000 rpm, and the centrifugal time is 10 to 20 minutes.

[0018] Preferably, the dialysis and purification process in step (2) is: using a dialysis membrane with a molecular weight cut-off (MWCO) of 8000 to 14000, dialysis in 2 to 2.5 L of water for 2 to 3 days, during which the water is changed 8 to 9 times; and freeze-drying after purification.

[0019] The present invention also provides an application of the fibronectin nanogel for enhancing tissue penetration in promoting regeneration / anti-skin aging.

[0020] Preferably, the application includes constructing fibronectin nanogel microneedles (FNG-MN) to increase the transdermal administration and drug sustained-release properties of FNG.

[0021] Preferably, the microneedle has a pyramid-shaped tip.

[0022] Preferably, the fibronectin nanogel microneedle preparation method comprises: uniformly mixing an aqueous solution of FNG nanogel and an aqueous solution of PVA-PVP, and then pouring the mixture into a microneedle mold, wherein the concentration of FNG in the mixed solution is 10-1000 μg / mL, and the concentration of PVA and PVP solutions are both 1-200 mg / mL. The microneedle mold containing the material solution is centrifuged (4000-6000 rpm, 10-20 minutes) to allow the material solution to reach the needle tip of the mold. The centrifuged microneedle mold is dried and then demolded, and the resulting microneedles are stored in a dry environment for future use.

[0023] Beneficial effects

[0024] (1) The synthesis method of the present invention is simple, the reaction conditions are mild, and the synthetic raw materials used are all environmentally friendly materials, which has the prospect of industrial implementation.

[0025] (2) The FNG prepared by the present invention has excellent cell compatibility. Compared with FN, FNG exhibits enhanced tissue penetration and is more easily internalized by cells.

[0026] (3) The FNG prepared by the present invention can better inhibit the expression of YAP, thereby effectively preventing the activation of Engrailed-1 and achieving scar-free wound healing.

[0027] (4) The FNG prepared by the present invention can better alleviate photoaging damage, promote collagen remodeling, reduce the expression of pro-inflammatory cytokines, increase the expression of anti-inflammatory cytokines, and improve the activity of antioxidant enzymes after being equipped with a microneedle delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation process and application of fibronectin nanogel in the present invention.

[0029] Figure 2IR spectra of terephthalaldehyde, FN and FNG prepared in Example 1 of the present invention.

[0030] Figure 3 (a) SEM image and (b) particle size distribution histogram of FNG prepared in Example 1 of the present invention.

[0031] Figure 4 The SDS-PAGE electrophoresis diagrams of FN and FNG prepared in Example 1 of the present invention.

[0032] Figure 5 The cell viabilities of L929 cells (a) and RAW264.7 cells (b) after treatment with different concentrations of FNG in Example 2 of the present invention are shown.

[0033] Figure 6 Flow cytometry was used to detect the phagocytosis of FN-Cy5.5 and FNG-Cy5.5 by L929 cells (a) and the mean fluorescence intensity of the cells (b) in Example 3 of the present invention.

[0034] Figure 7 The flow cytometry results for FNG-Cy5.5 phagocytosis by L929 cells after treatment with different inhibitors (a) and the mean fluorescence intensity of the cells (b) in Example 4 of the present invention are shown.

[0035] Figure 8 The scratch micrographs (a) and the quantitative results of cell migration area (b) of L929 cells treated with PBS, FN, and FNG at 0 h and 24 h in Example 5 of the present invention are shown.

[0036] Figure 9 These are CLSM bright field and slice scanning images of L929 cell spheres incubated with Cy5.5-FN and Cy5.5-FNG in Example 6 of the present invention.

[0037] Figure 10 The WB protein bands (a) and corresponding quantitative results (b) of YAP after L929 cells were treated with FN and FNG in Example 7 of the present invention.

[0038] Figure 11 The following are photos of wound sites (a) and quantitative results of wound area (b) of SD rats treated with PBS, FN, and FNG for 10 days in Example 8 of the present invention.

[0039] Figure 12 These are H&E and Masson staining images (a) and collagen volume fraction data (b) of SD rats treated with PBS, FN, and FNG for 10 days in Example 8 of the present invention.

[0040] Figure 13These are the YAP immunofluorescence staining images (a) and YAP expression quantification results (b) in tissues of SD rats treated with PBS, FN, and FNG for 10 days in Example 8 of the present invention.

[0041] Figure 14 (a) SEM image and (b) compressive strength performance data of the FNG microneedle in Example 9 of the present invention.

[0042] Figure 15 These are H&E, Masson, and EVG staining images (a) and the quantitative results of corresponding skin epidermal thickness, collagen volume fraction, and elastic fiber area (b) of Balb / c nude mice after treatment with PBS (negative control), photoaging modeling (positive control), FN, FNG, and FNG MN in Example 9 of the present invention.

[0043] Figure 16 These are the images (a) and corresponding quantitative results (bf) of immunohistochemical staining of TNF-α, IL-6, IL-1β, IL-10, and MMP3 in Balb / c nude mice treated with PBS (negative control), photoaging modeling (positive control), FN, FNG, and FNG MN in Example 9 of the present invention.

[0044] Figure 17 These are the quantitative results of SOD (a), CAT (b), GPX (c), MDA (d), and Hyp (e) in Balb / c nude mice after treatment with PBS (negative control), photoaging modeling (positive control), FN, FNG, and FNG MN in Example 9 of the present invention.

[0045] Figure 18 These are H&E staining images of the main organs of SD rats treated with PBS, FN, and FNG for 10 days in Example 10 of the present invention.

[0046] Figure 19 These are the test data of WBC (a), RBC (b), HGB (c), HCT (d), MCHC (e), and PLT (f) in the routine blood test / blood biochemistry analysis after 10 days of treatment in Example 10 of the present invention. DETAILED DESCRIPTION

[0047] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0048] The present invention characterized the synthesized nanogel using Fourier transform infrared spectroscopy (FITR), surface potential, dynamic light scattering (DLS), scanning electron microscopy (SEM), SDS-PAGE gel electrophoresis, and other means; verified the cell compatibility of FNG in vitro by CCK-8 experiment; studied the cellular internalization of FNG by flow cytometry; evaluated the cell migration-promoting effect of FNG by cell scratch test; established a 3D cell spheroid model, and evaluated the cell spheroid permeability of FNG by confocal laser scanning microscopy (CLSM); detected the expression of YAP in cells after FNG treatment by protein immunoblotting (Western Blot); evaluated the wound healing effect of FNG by full-thickness skin wound model in rats; evaluated the expression of YAP in FNG in vivo by immunofluorescence staining; established a nude mouse photoaging model, and verified the expression of anti-inflammatory and pro-inflammatory cytokines and antioxidant enzyme activity by combining immunohistochemistry and biochemical detection. The purpose is to explore the pro-regeneration and anti-skin aging effects of FNG and their molecular mechanisms. The biosafety of FNG was verified by hematoxylin-eosin (H&E) staining of major organs and blood routine / blood biochemistry tests.

[0049] Example 1

[0050] The preparation method of fibronectin nanogel (FNG) in this embodiment includes the following steps:

[0051] (1) 2.5 mg of FN and 25 mg of terephthalaldehyde were dissolved in 2 mL of DMSO to form a dispersed phase. The dispersed phase was then added dropwise to a continuous phase consisting of Span 80 / Tween 80 (468 mg / 92 mg, dissolved in 20 mL of cyclohexane) to initially form a cyclohexane-DMSO emulsion.

[0052] (2) The emulsion described in step (1) was homogenized by 20W ultrasound for 20 minutes, ethylenediamine (400 μL) was added to initiate the cross-linking reaction, and stirred for 12 hours; the reaction solution was centrifuged at 12000 rpm for 15 minutes, the supernatant was discarded, 5 mL of ethanol was added to resuspend the precipitate, and the resuspended solution was placed in a dialysis bag with a MWCO of 8000-14000, dialyzed in water for 3 days (2 L, 9 times), and finally freeze-dried to obtain FN nanogel, i.e., FNG.

[0053] The above FNG (FN / terephthalaldehyde mass ratio 1:10) was characterized. Figure 2 The following is the FITR infrared spectrum characterization of FNG. -1 An enhanced absorption peak appears near the surface of the nanostructured carbonyl group, which is attributed to the stretching vibration peak of N=C, indicating that FN and terephthalaldehyde are successfully cross-linked.

[0054] Table 1 shows that at different FN / terephthalaldehyde mass ratios, the hydrodynamic diameter of FNG ranged from 169.9 to 420.2 nm, and the zeta potential ranged from -12.4 to -18.7 mV. The FNG synthesized at an FN / crosslinker mass ratio of 1:10 had the smallest hydrodynamic diameter, 169.9 nm, and the lowest polydispersity index (PDI), 0.15. Therefore, an FN / terephthalaldehyde mass ratio of 1:10 was selected as the optimal FN nanogel synthesis ratio, and all subsequent cell experiments were performed using FN nanogels (FCN) synthesized at this ratio.

[0055] Table 1. Hydrodynamic diameter, PDI and surface potential of FNG at different FN / terephthalaldehyde mass ratios

[0056]

[0057] Scanning electron microscopy was used to characterize the morphology of FNG (FN / terephthalaldehyde mass ratio 1:10), as shown in Figure 2. Figure 3 As shown in the results of ab, FNG is spherical with a particle size of about 63.8nm, which is smaller than the test result of DLS. It is speculated that the particle size increases due to the swelling of FNG during the DLS test. At the same time, SDS-PAGE was used to qualitatively analyze the FN component in FNG, as shown in Figure 2. Figure 4 As shown, it can be clearly observed that FNG and FN have the same protein bands, proving that the FN molecule still maintains structural integrity after forming FNG.

[0058] Example 2

[0059] CCK-8 cytotoxicity assays were performed using mouse macrophages (RAW264.7) and fibroblasts (L929) as models to evaluate the cytotoxicity of FNG. The cells were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS) and 1% double-antibody in a constant temperature incubator at 37°C and 5% CO2. RAW264.7 and L929 cells were seeded in 96-well cell culture plates at a density of 1×10 4 Cells / well were cultured overnight. When about 80% of the cells were plated, fresh DMEM medium containing different concentrations of FNG (0, 5, 10, 20, 40, 60, 80, 100 μg / mL) was replaced and cultured for another 24 hours. Cell viability was detected using CCK-8. 100 μL of serum-free medium (containing 10% CCK-8) was added and incubated with the cells for 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader and the cell viability was calculated. The results are shown in Figure 2. Figure 5As shown in ab, even at an FNG concentration of 100 μg / mL, the viability of RAW264.7 and L929 cells still reached more than 100%, indicating that FNG has excellent cell compatibility.

[0060] Example 3

[0061] To evaluate the phagocytic ability of L929 cells to FNG, Cy5.5 was used to label FN and FNG. L929 cells were plated at 2×10 5 Cells were seeded at 400 μL of PBS at 400 μg / mL and cultured overnight. The next day, the culture medium was replaced with Cy5.5-FN or Cy5.5-FNG-containing medium and incubated for another 4 h. The cells were washed three times with PBS, digested, centrifuged, and resuspended in 300 μL of PBS. The fluorescence intensity of L929 cells after different treatments was analyzed by flow cytometry. Figure 6 As shown in ab, the mean fluorescence intensity in the FNG group was significantly stronger than that in the FN and PBS groups, indicating that FNG was more easily internalized by cells than FN. Figure 6 In b, *** represents p < 0.001. This standard was used in subsequent analyses unless otherwise specified.

[0062] Example 4

[0063] In order to study the cellular phagocytosis mechanism of FNG, FNG was labeled with Cy5.5. When exploring the cellular internalization mechanism of FNG, L929 cells were pre-incubated with endocytosis inhibitors for 2 hours, including methyl-β-cyclodextrin (10mM), chlorpromazine (20μM), amiloride hydrochloride (100μM) and genistein (700μM). Then, the culture medium was removed and the cells were treated with culture medium containing Cy5.5-FNG for 3 hours. The cells treated with the four inhibitors corresponded to Figure 7 FNG+M-β-CD, FNG+CP, FNG+Ami, and FNG+Gen in ab were tested with cells not treated with any inhibitors as controls. Finally, the fluorescence intensity of L929 cells was detected by flow cytometry. The results are shown in Figure 2. Figure 7 As shown in ab, the phagocytosis of FNG by L929 cells was reduced under the treatment of the four inhibitors, and the internalization amount of FNG was reduced the most after chlorpromazine treatment, which indicates that FNG enters L929 cells through multiple endocytic pathways, among which clathrin-mediated endocytosis is the most obvious. Figure 7 ** in b represents p < 0.01, and this standard was used in subsequent analyses unless otherwise specified.

[0064] Example 5

[0065] In order to investigate the cell migration promoting ability of FN or FNG, L929 cells were plated at 2×10 5 Cells were seeded at a density of 1 / 4 in 12-well plates and cultured overnight. Cells were then directly scratched with a 100 μL pipette tip to create a wound, washed twice with PBS, and imaged under a microscope (0 h). The culture medium was then replaced with medium containing FN or FNG. After a 3-h incubation, the 12-well plates were washed twice with PBS and the culture medium replaced with fresh complete medium. After an additional 24-h incubation, the plates were removed and imaged under a light microscope. Cell migration rates were calculated according to Equation 1.

[0066]

[0067] S0 and S1 represent the initial scratch area and the area occupied by cells growing in the scratch area after 24 h, respectively.

[0068] like Figure 8 As shown in a, after 24 hours of co-incubation with the material, cell migration in all experimental groups was clearly visible. In particular, the number of cells in the scratch area of ​​the cell group co-incubated with FNG increased significantly, and its cell migration rate was significantly improved compared with the PBS group and the FN group ( Figure 8 b).

[0069] Example 6

[0070] To evaluate the cell spheroid permeability of FN and FNG, Cy5.5 was used to label FN and FNG. A 1% (w / v) agarose solution was prepared in PBS, dissolved at high temperature, and sterilized in an autoclave. The agarose was then added to a 96-well plate while still hot, with 50 μL per well. After the agarose solidified, L929 cells were seeded in a 96-well cell culture plate containing agarose at a density of 2 × 10 4 Cells / well were placed in an incubator and cultured for about a week until visible cell spheres were formed. The culture medium was replaced with a medium containing Cy5.5-FN or Cy5.5-FNG. After incubation for 4 hours, the permeability was tracked using a confocal laser scanning microscope (CLSM). The cell sphere penetration ability of FN and FNG was evaluated by comparing the fluorescence intensity. The results are shown in Figure 2. Figure 9 As shown in the figure, the fluorescence intensity of the cell spheroids incubated with Cy5.5-FNG at different depths was much higher than that treated with Cy5.5-FN, indicating that the penetration ability of FNG was significantly stronger than that of FN.

[0071] Example 7

[0072] In order to explore the molecular mechanism by which FNG promotes scarless wound healing, L929 cells were cultured at 2 × 10 5The cells were seeded in a 12-well plate at a density of 100 μg / mL and cultured overnight. The culture medium was replaced with FN or FNG-containing culture medium and incubated for 24 h. The expression of YAP in the cells was then detected by Western Blot (WB). Figure 10 As shown in ab, WB quantitative analysis showed that compared with the PBS control group, both FN and FNG treatment groups significantly inhibited YAP protein expression (p < 0.001). Notably, FNG showed a stronger YAP downregulation effect, with its protein expression level significantly lower than that of the free FN group (p < 0.001).

[0073] Example 8

[0074] To investigate the wound healing effect of FNG in vivo, an 8 mm full-thickness circular biopsy wound was established on the dorsal side of SD rats. The drug was administered every 2 days and photographed. After 10 days, the rats were euthanized after the wounds reached the healing standard, and the tissues around the incision were obtained for tissue staining. Figure 11 As shown in ab, the wound healing speed of the FNG group was significantly faster than that of the FN group and the PBS group. The wound healing effect was evaluated by H&E staining (Hematoxylin-Eosin staining) and Masson's trichrome staining (Masson's trichrome staining). Figure 12 As shown in ab, FNG can better promote wound re-epithelialization and the formation of new hair follicles, while promoting collagen regeneration and tissue repair. The expression level of YAP protein in tissue samples was quantitatively evaluated by immunofluorescence staining. The results are shown in Figure 13 As shown in ab, compared with the control group, both the FN (p<0.01) and FNG treatment groups significantly downregulated YAP protein expression (p<0.001). Notably, the YAP fluorescence intensity in the FNG treatment group was still significantly lower than that in the free FN group (p<0.001), further confirming the significant advantage of the FNG nanodelivery system in enhancing therapeutic effects.

[0075] Example 9

[0076] In order to evaluate the anti-aging effect of FNG, a nude mouse skin photoaging model was established by irradiating the back skin of nude mice with medium-wave ultraviolet rays (280-315nm, UVB) for eight weeks. At the same time, in order to increase the transdermal delivery and drug release performance of FNG, fibronectin microneedles (FNG MN) were constructed for subsequent treatment. Figure 14 As shown in a, the FNG microneedle has a pyramidal tip and its compressive strength reaches 30.43N / 100 needles ( Figure 14b), sufficient to penetrate the skin. After modeling was completed, the drug was administered once every 2 days. After macroscopic observation showed that the photoaging symptoms of the skin of the nude mice in the treatment group were alleviated, euthanasia was performed and the back skin was excised for tissue staining. Skin thickening, collagen degradation and elastic fiber degeneration were evaluated by H&E, Masson and elastic fiber (Verhoeff's Van Gieson, EVG) staining. The results are shown in Figure 2. Figure 15 As shown in ab, the photoaging modeling group (positive control, Model) showed skin thickening, collagen degradation and elastic fiber degeneration accumulation, indicating that the modeling was successful. Compared with the FN and FNG groups, the characterization results of the FNG MN group showed that the skin tended to normalize, close to the negative control group (PBS, Control). Immunohistochemistry was used to evaluate the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) and skin aging biomarkers (MMP3) in the tissue. The results are shown in Figure 16 As shown in af, it can be seen that FNG MN treatment more effectively reduces the expression of pro-inflammatory cytokines, increases the expression of anti-inflammatory cytokines, and reduces the expression of MMP3. The biochemical detection of the content of antioxidant enzymes (SOD, CAT, GPX), lipid peroxidation byproducts (MDA) and collagen fiber components (Hyp) in tissues showed that Figure 17 As shown in ae, FNG MN treatment significantly increased antioxidant enzyme activity, decreased MDA content, and increased Hyp content. These results indicate that FNG MN treatment effectively alleviates photoaging damage, skin inflammation, and oxidative stress, and has a significant anti-aging effect on the skin.

[0077] Example 10

[0078] In order to evaluate the in vivo biosafety of FN and FNG, after the 10-day treatment cycle, the main organs (heart, liver, spleen, lung, and kidney) of the rats in each experimental group were dissected and fixed in 4% paraformaldehyde, then embedded in paraffin, sliced, and stained with H&E for histological observation. A PBS control group was also set up. At the same time, blood was extracted from the rats in each experimental group for routine blood tests / blood biochemistry tests, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean hemoglobin concentration (MCHC), and platelets (PLT). Figure 18 It can be seen that no pathological abnormalities were found in the staining results of the main organs of the mice in each experimental group, indicating that the FNG of the present invention has good biological safety. At the same time, the results of blood routine / blood biochemistry analysis showed that the WBC ( Figure 19 a) RBC( Figure 19 b) HGB( Figure 19 c) HCT ( Figure 19 d) MCHC( Figure 19e) and PLT( Figure 19 f) were all within the safe range, further verifying the biosafety of each research material.

[0079] The present invention synthesizes nanogel (FNG) based on FN. FN serves as both the main body of the gel and the bioactive material. The preparation process and function of FNG are as follows: Figure 1 Experimental results demonstrate that the nanogel possesses excellent biocompatibility. Compared to FN, FNG exhibits enhanced tissue penetration, is more readily internalized by cells, and can better inhibit YAP expression, thereby effectively preventing the activation of Engrailed-1 and achieving scarless wound healing. Furthermore, FNG delivered via a microneedle delivery system can more effectively alleviate photoaging-induced skin inflammation, collagen degradation, elastic fiber degeneration, and decreased antioxidant enzyme activity.

Claims

1. A fibronectin nanogel for enhancing tissue penetration, characterized in that: The fibronectin nanogel is prepared by Schiff base reaction between fibronectin and terephthalaldehyde, and controllably cross-linking is performed using a reverse microemulsion method to form a three-dimensional network structure.

2. A method for preparing a fibronectin nanogel for enhancing tissue penetration, comprising the following steps: (1) Dissolve fibronectin and cross-linking agent terephthalaldehyde in organic solvent A to form a dispersed phase; dissolve Span 80 and Tween 80 in organic solvent B to form a continuous phase; then add the dispersed phase to the continuous phase and stir to form an emulsion in which the continuous phase covers the dispersed phase; (2) The emulsion obtained in step (1) is sonicated, and then ethylenediamine is added and stirred for reaction; after the reaction is completed, the emulsion is centrifuged, the precipitate is resuspended and dispersed with ethanol, and the resuspended solution is dialyzed and purified to obtain fibronectin nanogel.

3. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: In step (1), the mass ratio of fibronectin to terephthalaldehyde is 1:0.1-10, the concentration of fibronectin in the dispersed phase is 0.2-5 mg / mL; and the organic solvent A includes dimethyl sulfoxide.

4. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: In step (1), the mass ratio of Span 80 to Tween 80 is 1:0.1-0.3, the concentration of Span 80 in the continuous phase is 5-40 mg / mL; and the organic solvent B comprises cyclohexane.

5. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: The volume ratio of the dispersed phase to the continuous phase in step (1) is 1:5 to 20.

6. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: The stirring temperature in step (1) is 8 to 37° C., and the stirring time is 10 to 20 minutes.

7. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: The volume ratio of the ethylenediamine in step (2) to the emulsion in step (1) is 1:10-100.

8. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: The ultrasonic time in step (2) is 15 to 25 minutes; the stirring reaction time is 11 to 13 hours; the centrifugal speed is 10,000 to 14,000 rpm, and the centrifugal time is 10 to 20 minutes.

9. The method for preparing fibronectin nanogel for enhancing tissue penetration according to claim 2, characterized in that: The dialysis and purification process of step (2) is as follows: using a dialysis membrane with a molecular weight cut-off of 8000 to 14000, dialysis in 2 to 2.5 L of water for 2 to 3 days, during which the water is changed 8 to 9 times; and freeze-drying after purification.

10. Use of the fibronectin nanogel for enhancing tissue penetration as claimed in claim 1 in promoting regeneration / anti-skin aging.

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