A squalene / calcium phosphotungsten oleogel, its preparation method and application

Abstract

This application discloses a squalene / calcium phosphotungsten oleogel, its preparation method, and its applications, relating to the field of biomedical technology. The squalene / calcium phosphotungsten oleogel is composed of squalene and calcium phosphotungsten nanowires; by mass percentage, the calcium phosphotungsten nanowire content is 2-5%, and the squalene content is 95-98%. The squalene / calcium phosphotungsten oleogel exerts anti-inflammatory and antioxidant effects by scavenging excess ROS, and can restore cell membrane fluidity, weaken mechanosensitive YAP pathway activation, and synergistically promote photoaging repair and scarless healing through multiple pathways. This comprehensive regulatory strategy provides a new solution for skin damage repair.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and more specifically, to a squalene / calcium phosphotungstic acid oleogel, its preparation method, and its application. Background Technology

[0002] Skin damage is a common health problem, mainly including chronic cumulative damage (such as photoaging) and acute traumatic damage (such as wounds and scar formation). Photoaging is primarily caused by UVB radiation, characterized by excessive accumulation of reactive oxygen species (ROS), collagen degradation, and chronic inflammation, clinically manifesting as wrinkles, pigmentation, and skin laxity. Statistics show that over 80% of skin aging is related to photodamage. Furthermore, skin wound healing is a complex, multi-stage process, including hemostasis, inflammation, proliferation, and remodeling phases. Globally, over 100 million people suffer from refractory wounds or pathological scars each year, affecting not only appearance but also potentially causing functional impairment.

[0003] In the field of photoaging prevention, existing strategies mainly include sunscreens and topical antioxidants. Although precious metal nanoparticles (such as palladium and platinum) and cerium oxide nanoparticles have shown some antioxidant potential, traditional formulations have significant limitations: physical sunscreen ingredients (such as zinc oxide) may generate free radicals under light exposure, and most antioxidants have insufficient bioavailability due to their high water solubility and poor skin permeability. More importantly, existing products are unable to effectively repair damaged skin barriers and collagen structures.

[0004] In wound healing and scar prevention, clinical practice mainly employs silicone patches, pressure therapy, or anti-inflammatory drugs. However, these methods often only partially inhibit scar formation and suffer from significant side effects and poor patient compliance. Recent studies have found a close correlation between scar formation and abnormal mechanotransmission: wound contraction activates the YAP signaling pathway, leading to excessive fibroblast activation and collagen deposition. Although YAP inhibitors such as verteporfen have shown anti-scarring effects in animal models, their phototoxicity limits clinical translation. Simultaneously, excessive ROS in the wound site exacerbates the inflammatory response, hinders epithelialization, and may indirectly promote YAP activation by reducing cell membrane fluidity and enhancing mechanotransmission; however, currently, there is a lack of effective strategies that can simultaneously clear ROS and regulate the biomechanical microenvironment.

[0005] Squalene (Sql), a natural triterpenoid lipid, is an important component of sebum and possesses excellent antioxidant, anti-inflammatory, and moisturizing properties, along with good biocompatibility. However, its oily liquid nature leads to poor skin adhesion, susceptibility to oxidation and deterioration, and incompatibility with traditional hydrogel dressings, limiting its application in skin repair. Summary of the Invention

[0006] The purpose of this application is to provide a squalene / calcium phosphotungstenate oil gel that exerts anti-inflammatory and antioxidant effects by scavenging excess ROS, and can restore cell membrane fluidity, weaken the activation of the mechanotropic YAP pathway, and promote photoaging repair and scarless healing through multiple pathways.

[0007] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows: On the one hand, this application provides a squalene / calcium phosphotungsten (Sql / PWC) oleogel, wherein the squalene / calcium phosphotungsten oleogel is composed of squalene and calcium phosphotungsten nanowires; by weight percentage, the content of calcium phosphotungsten nanowires is 2-5%, and the content of squalene is 95-98%.

[0008] On the other hand, this application provides a method for preparing squalene / calcium phosphotungsten oleogel, comprising the following steps: S1. Dissolve phosphotungstic acid and calcium nitrate tetrahydrate in water, stir until homogeneous, then add octadecene and oleylamine in sequence, stir to react, and then add ethanol. The white colloidal substance obtained by sedimentation is precipitated to obtain calcium phosphotungstic acid nanowires. S2. The calcium phosphotungsten nanowires and squalene obtained above are mixed and stirred in a certain proportion until uniform to obtain the squalene / calcium phosphotungsten oleogel.

[0009] In another aspect, this application provides the use of squalene / calcium phosphotungsten oleogel in the preparation of drugs or dressings for skin damage repair.

[0010] Compared with the prior art, the embodiments of this application have at least the following advantages or beneficial effects: This application proposes a novel nanocomposite material—squalene (Sql) / calcium phosphotungstenate (PWC) oleogel. This material achieves mild gelation (requiring only 2%-5% PWC addition) by forming a three-dimensional network structure within squalene using PWC subnanowires (approximately 1 nm in diameter and hundreds of nanometers in length) that perfectly preserve the bioactivity of squalene. It stabilizes squalene through the subnanowire network, enhancing its retention and sustained-release capabilities in the skin; efficiently scavenges ROS using squalene, reducing oxidative stress and inflammatory responses; and synergistically promotes photoaging repair and scarless healing through multiple pathways by restoring cell membrane fluidity and weakening mechanotropic YAP pathway activation. This comprehensive regulatory strategy provides a new solution for skin damage repair. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a schematic diagram illustrating the preparation of the oily gel of this application and its role in photoaging and wound healing.

[0013] Figure 2 The properties of Sql / PWC composites with different mass ratios in this application are characterized as follows: left: squalene solution, middle: Sql / PWC oleogel (99% squalene), right: Sql / PWC oleogel (98% squalene) and Sql / PWC oleogel (98% squalene).

[0014] Figure 3 This is an experimental diagram showing the determination of the total antioxidant capacity of squalene using the ABTS method in this application.

[0015] Figure 4 This is a diagram of the biocompatibility experiment of the Sql / PWC oleogel in this application.

[0016] Figure 5 This diagram illustrates the repair effect of squalene on UVB-induced photoaging of fibroblasts, as described in this application.

[0017] Figure 6 This diagram illustrates the experimental process of UVB-induced photoaging of the back skin of nude mice, and the repair effect of Sql / PWC oleogel on UVB-induced photoaging of nude mouse skin.

[0018] Figure 7 This is an experimental diagram illustrating the histological basis for the anti-photoaging effect of Sql / PWC oleogel in this application.

[0019] Figure 8 This is a graph showing the RNA sequencing analysis of photoaged mice treated with Sql / PWC oil gel according to this application.

[0020] Figure 9 This is an experimental diagram showing the changes in the levels of protein markers for skin inflammation caused by photoaging in this application.

[0021] Figure 10 This is an experimental diagram illustrating the effect of the Sql / PWC oleogel in promoting wound healing in a mouse skin trauma model.

[0022] Figure 11 This is an experimental diagram illustrating the histological effect of the Sql / PWC oleogel in promoting wound healing in a mouse skin trauma model.

[0023] Figure 12 This is an experimental diagram illustrating the effect of Sql / PWC oleogel in promoting collagen fiber regeneration and repair in a mouse skin trauma model.

[0024] Figure 13 This is an experimental diagram illustrating how the Sql / PWC oleogel promoted collagen remodeling and normalization in a mouse skin trauma model.

[0025] Figure 14 Experimental diagram showing how the Sql / PWC oleogel reduces oxidative stress in wound tissue according to this application.

[0026] Figure 15 This is an experimental diagram illustrating how Sql / PWC oleogel promotes angiogenesis and maturation in wounds, as presented in this application.

[0027] Figure 16 This is an experimental diagram illustrating the effect of Sql / PWC oleogel on promoting macrophage polarization in wounds, as presented in this application.

[0028] Figure 17 This is an experimental diagram showing the effect of Sql / PWC oleogel on the expression of inflammatory factors in wounds, as presented in this application.

[0029] Figure 18 This is an experimental diagram of downregulating keratin expression using Sql / PWC oleogel in this application.

[0030] Figure 19 This is an experimental diagram illustrating the enhancement of hair follicle stem cell lineage plasticity using Sql / PWC oleogel in this application.

[0031] Figure 20 The experimental diagram shows that Sql / PWC oleogel can reduce YAP expression and promote β-catenin expression.

[0032] Figure 21 This is an experimental diagram illustrating the Sql method used in this application to enhance cell membrane fluidity and reduce YAP expression in human dermal fibroblasts (HFb) after H2O2 damage. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to specific embodiments.

[0035] A squalene / calcium phosphotungsten oleogel, wherein the squalene / calcium phosphotungsten oleogel is composed of squalene and calcium phosphotungsten nanowires; by mass percentage, the content of calcium phosphotungsten nanowires is 2-5%, and the content of squalene is 95-98%.

[0036] A method for preparing a squalene / calcium phosphotungstic acid oleogel includes the following steps: S1. Dissolve phosphotungstic acid and calcium nitrate tetrahydrate in water, stir until homogeneous, then add octadecene and oleylamine in sequence, stir to react, and then add ethanol. The white colloidal substance obtained by sedimentation is precipitated to obtain calcium phosphotungstic acid nanowires. S2. The calcium phosphotungsten nanowires and squalene obtained above are mixed and stirred in a certain proportion until uniform to obtain the squalene / calcium phosphotungsten oleogel.

[0037] In some embodiments of this application, the ratio of phosphotungstic acid and calcium nitrate tetrahydrate in step S1 is 1:0.123, and the stirring reaction time is 8 hours.

[0038] In some embodiments of this application, the mixing and stirring time in step S2 above is 30 minutes.

[0039] The use of a squalene / calcium phosphotungstic acid oleogel in the preparation of a drug or dressing for the repair of skin damage.

[0040] In some embodiments of this application, the aforementioned skin damage includes photoaging damage, impaired wound healing, or scar formation.

[0041] In some embodiments of this application, the repair mechanism of the above-mentioned Sql / PWC oleogel includes scavenging ROS, inhibiting inflammatory responses, and enhancing cell membrane fluidity.

[0042] The features and performance of this application will be further described in detail below with reference to the embodiments. Example 1

[0043] This embodiment provides a squalene / calcium phosphotungstenate oil gel, its preparation method, and its role in photoaging and wound healing processes are illustrated in the following diagram. Figure 1 As shown. The specific preparation method is as follows: Synthesis of S1 and PWC: 2 g of phosphotungstic acid and 0.246 g of calcium nitrate tetrahydrate were dissolved in 32 ml of deionized water and stirred until homogeneous. 24 ml of octadecene and 8 ml of oleylamine were added sequentially, and the mixture was stirred at room temperature for 8 hours using a mechanical stirrer. After the reaction was completed, a large amount of ethanol was added to the reaction vessel, and the white colloidal precipitate obtained by sedimentation was PWC.

[0044] Preparation of S2, Sql / PWC oleogel: The obtained PWC was mixed with 100 ml Sql using mechanical stirring for 30 minutes to obtain a Sql / PWC oleogel containing approximately 98% Sql.

[0045] Other concentrations of Sql / PWC oleogels were obtained by adjusting the volume of squalene.

[0046] Regarding the raw material ratio of Sql / PWC gel, this embodiment found that when the PWC content is 1%, the mixture remains liquid; when the PWC content reaches 2%, a stable gel state can be formed (e.g., Figure 2 (As shown in A). Therefore, subsequent experiments all used a gel formulation with a PWC content of 2% (i.e., a squalene content of 98%).

[0047] As a control, Sql was replaced with an equal volume of paraffin oil to prepare PWC paraffin oil (Paraffin / PWC) gel, abbreviated as Par / PWC.

[0048] Transmission electron microscopy (TEM) imaging ( Figure 2 (B) shows the sub-nanometer linear structure of PWC. Compared to phosphotungstenate (PW), the XRD pattern of PWC does not reveal any crystal structure. Figure 2 C). Small-angle XRD ( Figure 2 D) shows that PWC has three peaks at 2.41°, 4.65°, and 7.33°, with an approximate ratio of 1:2:3, indicating a highly ordered layered structure within the material. These layers are periodically arranged at a fixed distance of 3.66 nm. Furthermore, the presence of Bragg reflection at 7.33° indicates a high degree of long-range order, with the periodicity of the layered structure well preserved over a wide range. XPS analysis shows that PWC contains P, W, and Ca, while PW contains only P and W. Figure 2 E). FTIR confirms the presence of CH bonds in PwC ( Figure 2 F), which is attributed to oleylamine. Finally, the rheological properties of the Sql / PWC olegel were evaluated. Due to the entanglement of the PWC nanowires, the storage modulus of the olegel is higher than its loss modulus (F). Figure 2 G). These findings confirm the successful synthesis of Sql / PWC oleogel ( Figure 2 Furthermore, the total antioxidant capacity of squalene was evaluated. With increasing squalene content, the absorbance of ABTS radicals at 734 nm decreased, indicating that squalene possesses antioxidant activity. Figure 3 ). Example 2

[0049] This embodiment investigates the safety of Sql / PWC oleogels.

[0050] Studies have reported that short-term, high-dose exposure to tungsten compounds can produce toxic effects. Oral or injectable administration of high doses of tungsten can cause symptoms such as growth retardation, loss of appetite, and ataxia in experimental animals (e.g., rats and mice). Extremely high doses may cause damage to multiple organs. High concentrations of tungsten ions may interfere with normal cellular metabolic processes and compete with or bind to certain important enzyme systems, thereby affecting their function. Epidemiological data on the direct toxicity of tungsten to humans are relatively limited, but some observations have found that workers in tungsten mining and smelting industries, if not properly protected, may experience respiratory irritation and increased health risks from long-term inhalation of tungsten dust.

[0051] To evaluate the safety of Sql / PWC oleogels in healthy mice and to explore the optimal mass ratio of Sql to PWC, we first prepared two types of Sql / PWC gels with PWC contents of 2% and 3%. Experiments showed that 3% Sql / PWC induced skin inflammation in mice (likely due to excessive oleylamine ligands), while 2% Sql / PWC did not cause significant inflammation. Figure 4 A). Therefore, subsequent experiments all used 2% Sql / PWC and 2% Par / PWC (abbreviated as Sql / PWC and Par / PWC, respectively).

[0052] In a mouse trauma model, Sql / PWC was applied to the skin wounds on the backs of mice. Heart, liver, spleen, lungs, and kidneys were collected on days 1, 7, 14, and 28 after treatment, and H&E sections were prepared to observe changes in the major organs after application. The experiment showed that Sql / PWC caused minimal damage to the major organs. Figure 4 B). After administration, most PWC remains in the wound area, while the remaining PWC absorbed through percutaneous penetration or oral administration can be eliminated by liver metabolism within one week. Figure 4 C). These data indicate that SQL / PWC has good biocompatibility. Example 3

[0053] This study systematically evaluated the regulatory effects of squalene (Sql) on cell proliferation, oxidative stress, aging, migration, and inflammatory responses by constructing a UVB-induced human fibroblast (HFb) photoaging model, aiming to elucidate the cellular mechanism by which squalene promotes skin photoaging repair.

[0054] The experimental methods are as follows: Human fibroblasts (HFb) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, and a photoaging model was established by UVB irradiation (80 mJ / cm², 10 min). The experiment was divided into a control group, a UVB group, a UVB + 50 μM Sql group, a UVB + 100 μM Sql group, and a UVB + 1000 μM Sql group. Cell viability and proliferation were detected using the CCK-8 assay; intracellular ROS levels were quantified by DCFH-DA staining combined with flow cytometry; cell senescence was assessed by SA-β-Gal staining; cell migration ability was analyzed by scratch assay; and the protein and gene expression of MMP-1, IL-6, and Col1A1 were detected by ELISA and qRT-PCR, respectively.

[0055] First, the safe concentration range of squalene was screened using cytotoxicity assays. The results showed that squalene had no significant toxicity to HFb at concentrations ranging from 50 to 1000 μM, while 100 μM squalene significantly promoted the proliferation of normal HFb cells. Figure 5 A). In the UVB damage model, UVB irradiation significantly inhibited cell proliferation (p<0.001), while treatment with 100 μM squalene effectively restored cell proliferation (p<0.05), indicating that squalene has a protective effect on photoaged cells. Figure 5 B).

[0056] UVB irradiation induced a significant increase in intracellular ROS levels, while the squalene-treated group showed a significant decrease in ROS fluorescence intensity. Figure 5 (C, E) suggests that squalene alleviates oxidative stress by scavenging excess ROS. Further SA-β-Gal staining revealed that the proportion of senescent cells in the UVB group increased to 45.68 ± 2.23%, significantly higher than the control group (13.78 ± 4.99%, p<0.001), while the proportion decreased to 17.99 ± 3.46% in the squalene-treated group (C, E). Figure 5 (D, F) demonstrates that squalene can effectively delay UVB-induced cell senescence.

[0057] Scratch assays showed that squalene significantly increased the migration rate of UVB-damaged cells (50.89 ± 10.04%), approaching the level of the control group (59.72 ± 5.44%), while the migration rate of the UVB group was only 22.02 ± 1.81%. Figure 5 (G, H) indicates that squalene promotes cell repair after injury. In terms of molecular mechanism, squalene upregulates Col1A1 expression (protein level recovered to 1490 ± 155.4 pg / mL, close to the control group's 1729 ± 105.5 pg / mL), while simultaneously inhibiting the gene and protein expression of MMP-1 (decreased to 221.3 ± 30.61 pg / mL) and IL-6. Figure 5(I, J), indicating that it exerts its repair function by balancing the synthesis and degradation of extracellular matrix and inhibiting inflammatory pathways.

[0058] In summary, this study demonstrates that squalene effectively alleviates UVB-induced photoaging damage by scavenging ROS, inhibiting cell senescence, promoting migration, and regulating collagen metabolism and inflammatory responses. Its mechanism of action is closely related to its antioxidant, anti-inflammatory, and matrix remodeling effects. This research provides experimental evidence for the clinical application of squalene in skin photoaging repair. Example 4

[0059] This embodiment establishes a UVB-induced photoaging model of nude mouse dorsal skin to systematically evaluate the biosafety, therapeutic efficacy, and molecular mechanism of Sql / PWC oleogel, focusing on its regulatory effects on skin barrier function, collagen metabolism, oxidative stress, and inflammatory response.

[0060] Experimental Methods: Twelve male nude mice aged 6-8 weeks were randomly divided into a control group, a UVB model group, and a UVB + Sql / PWC treatment group. A photoaging model was established by administering UVB irradiation for 8 weeks with a cumulative dose of 10.88 J / cm². The treatment group received topical application of 2% Sql / PWC oil gel for 9 days. Melanin index, erythema index, and sebum content were dynamically monitored using a skin analyzer. After sacrifice, skin tissue was collected for H&E, Masson's red, and Sirius red staining and immunohistochemical analysis. Differentially expressed genes were screened using RNA-Seq technology. Histological quantitative analysis was performed using ImageJ software.

[0061] Eight weeks after UVB irradiation, the model group mice showed significant photoaging characteristics: skin elasticity decreased by 47.3% (p<0.01), stratum corneum hydration decreased by 62.1% (p<0.001), accompanied by melanin deposition and increased TEWL values. Figure 6 AB). Nine days after Sql / PWC treatment, the skin elasticity in the treatment group recovered to 91.2% of that in the control group (p<0.05), the stratum corneum moisture content increased 2.3 times (p<0.01), and the erythema index significantly improved ( ). Figure 6 CD).

[0062] H&E staining, Masson's trichrome staining, Sirius red staining, and immunohistochemical staining for elastin and Col 1A1 showed that the Sql / PWC treatment group could effectively restore the UVB-induced photoaging phenotype. Figure 7A). Histological analysis showed that the epidermal thickness in the UVB model group increased to 2.1 times that of the normal group (p<0.001), the dermal collagen fiber arrangement was disordered, and the proportion of type I collagen decreased to 24.05±3.03%. Sql / PWC treatment restored the epidermal thickness to normal (p<0.01), and the dermal collagen content recovered to 43.59±3.44% (p<0.001). Sirius red polarized light showed that the ratio of type I / III collagen tended to normalize. Figure 7 BF). Immunofluorescence assay revealed that the expression levels of AQP3 and Flg in the skin of the treatment group were 3.2-fold and 2.8-fold higher than those in the model group, respectively (p<0.001). Figure 7 GI), DHE staining showed a 67.4% decrease in ROS levels (p<0.01). Figure 7 (G,J), indicating that Sql / PWC effectively enhances skin barrier function and alleviates oxidative damage.

[0063] Molecular mechanism studies showed that RNA-Seq analysis revealed 1655 genes upregulated and 2141 genes downregulated in the treatment group. Figure 8 AF). KEGG enrichment analysis showed significant inhibition of inflammation-related pathways such as the TNF signaling pathway and the NF-κB pathway. Figure 8 G). qPCR and Western blot validation revealed that MMP-1 expression in the treatment group decreased to 24.8% of the model group (p<0.001), and Col1A1 expression recovered to 91.5% of the normal level (p<0.01). Figure 9 A). ELISA testing showed a decrease in the levels of pro-inflammatory factors such as IL-1β and TNF-α by 72.3-85.6% (p<0.001). Figure 9 (BE) confirmed that Sql / PWC synergistically inhibits inflammatory responses and matrix degradation through multiple pathways.

[0064] In summary, this study demonstrates that Sql / PWC oleogel effectively reverses UVB-induced photoaging damage by scavenging ROS, regulating collagen metabolism, inhibiting inflammatory pathways, and repairing the skin barrier. Its multiple mechanisms of action include: ① restoring epidermal thickness and orderly arrangement of dermal collagen; ② enhancing barrier function by increasing AQP3 / Flg expression; and ③ maintaining extracellular matrix homeostasis by inhibiting MMP-1 and pro-inflammatory factor expression. This study provides systematic experimental evidence for the clinical translation of Sql / PWC oleogel. Example 5

[0065] This experiment systematically evaluated the regulatory effects of Sql / PWC oleogel on wound healing speed, healing quality, and scar formation by establishing a mouse model of full-thickness skin defects. It also explored the molecular mechanisms by which Sql / PWC oleogel promotes tissue regeneration and inhibits scarring by regulating oxidative stress, angiogenesis, immune microenvironment, and key signaling pathways (YAP / β-catenin).

[0066] Experimental Methods: Male C57BL / 6 mice aged 6-8 weeks were randomly divided into four groups: Control group (no treatment), Clinical Hydrogel group (carboxymethyl cellulose hydrogel), Paraffin / PWC group (paraffin oil gel), and Sql / PWC group (squalene oil gel). A full-thickness skin defect model with a diameter of 10 mm was created on the back, and the corresponding preparation was applied daily after surgery. The wound area was recorded on days 1, 3, 6, 9, and 14. Histological analysis (H&E, Masson, and Sirius red staining) was performed on days 5, 9, and 14. ROS levels were detected by DHE staining. Immunofluorescence was used to assess the expression of CD31 / α-SMA (angiogenesis), CD86 / CD206 (macrophage polarization), and YAP / β-catenin. Inflammatory factors such as TNF-α, IL-1β, and IL-10 were detected by ELISA. Differentially expressed genes were analyzed by RNA-Seq.

[0067] The healing speed of each group was assessed by dynamically monitoring changes in wound area. Figure 10 As shown in Figure A, the control group healed slowly, with significant defects still remaining on day 14; the Clinical Hydrogel group and the control group healed at similar rates; while the Sql / PWC group showed a significant healing advantage from day 3, with the wound essentially closed by day 9. Quantitative analysis showed ( Figure 10 B), the unhealed area in the Sql / PWC group on day 14 was only 21.3% of that in the control group (p<0.001), and the healing curve had the steepest slope, indicating that its healing-promoting effect was time-dependent. Figure 10 C). This result confirms that Sql / PWC oleogel effectively promotes epithelial cell migration and proliferation by providing a moist environment and bioactive components.

[0068] Histological analysis showed that Sql / PWC treatment significantly optimized tissue remodeling during the healing process. H&E staining on day 5 revealed that in the control group, the central wound was covered with a thick scab with no epidermal regeneration; while in the Sql / PWC group, continuous epidermal migration bands and dermal fibroblast activation were observed. Figure 11 A). By day 14, the epidermal thickness ratio in the Sql / PWC group was 0.96 ± 0.06, which was not different from the surrounding normal skin. Figure 11C), while the control group showed epidermal hyperplasia (ratio 1.68±0.20). Statistics on unhealed length ( Figure 11 B) Further, it was shown that the length of non-epithelialization in the Sql / PWC group on day 5 (4.47±0.34 mm) was significantly lower than that in the control group (5.55±0.30 mm), indicating that it shortens the healing cycle by promoting re-epithelialization.

[0069] Masson tricolor staining ( Figure 12 A) showed that on day 14, the density of blue collagen fibers in the dermis of the Sql / PWC group was significantly higher than that of the control group, and the fibers were arranged in an orderly manner. Quantitative analysis showed that ( Figure 12 B), the percentage of dermal collagen in the Sql / PwC group reached 43.59±3.44%, close to that of normal skin (52.29±5.32%), while the control group only had 24.05±3.03%. Hydroxyproline content was detected ( Figure 12 C) Further verification showed that the hydroxyproline level in the Sql / PWC group on day 14 (0.64±0.05 μg / mg) was 2.1 times that of the control group (p<0.001), indicating that it enhances dermal strength by promoting collagen synthesis.

[0070] Sirius red dyeing ( Figure 13 A) Under polarized light, the ratio of type I collagen (yellow-orange) to type III collagen (green) in the Sql / PWC group tends to be balanced. Statistical findings show that ( Figure 13 In the BD group, the type I / III collagen ratio on day 14 was 2.1±0.3, significantly lower than that in the control group (3.5±0.4, p<0.01), indicating that it inhibited scar formation by maintaining the proportion of type III collagen. As a major component in the early stages of repair, the retention of type III collagen helps improve tissue elasticity.

[0071] DHE fluorescent staining ( Figure 14 A) showed that the Sql / PWC group had the lowest ROS fluorescence intensity in the wound. Quantitative analysis ( Figure 14 B) indicates that on day 14, the ROS level in the Sql / PWC group decreased by 67.4% compared to the control group (p<0.001), and remained lower than other treatment groups. This result is consistent with the antioxidant properties of squalene, suggesting that Sql / PWC reduces oxidative damage by scavenging excess ROS, providing a favorable microenvironment for cell proliferation.

[0072] CD31 / α-SMA double staining ( Figure 15 A) shows that the Sql / PWC group had the highest density of neovascularization and the most intact angiogenesis. Statistical analysis revealed ( Figure 15The proportion of CD31+ area (6.58±0.90%) and α-SMA+ area (5.2±0.7%) in the Sql / PWC group were significantly higher than those in the control group (p<0.01), indicating that it simultaneously promotes angiogenesis and maturation. The increased proportion of mature vessels (α-SMA+) helps improve blood supply and accelerate tissue repair. CD86 (M1) / CD206 (M2) immunofluorescence (… Figure 16 A) showed that the proportion of M2 macrophages was significantly increased in the Sql / PWC group. The CD86 / CD206 ratio was statistically analyzed ( Figure 16 B) indicates that the Sql / PWC ratio (0.8±0.1) was lower than that of the control group (1.9±0.3, p<0.001), suggesting that it enhances anti-inflammatory and repair-promoting effects by inducing M2 polarization, thereby reducing the risk of fibrosis. Inflammatory factor detection ( Figure 17 The results showed that the Sql / PWC group had the lowest levels of pro-inflammatory factors TNF-α (33.54±3.85 μg / mL) and IL-1β (33.93±10.95 μg / mL), while the highest level of anti-inflammatory factor IL-10 (123.00±10.26 μg / mL) was observed (p<0.01). These results were consistent with macrophage polarization data, confirming that Sql / PWC promotes healing by regulating the inflammatory microenvironment.

[0073] RNA-Seq analysis ( Figure 18 AC analysis revealed that differentially expressed genes in the Sql / PWC group were enriched in the Wnt / β-catenin pathway. (Heatmap) Figure 18 B) showed downregulation of keratin genes (Krt25-28, 71-75) and upregulation of extracellular matrix genes. Wnt3a / Wnt5a ratio statistics ( Figure 18 C) showed that the Sql / PWC ratio increased by 2.3-fold (p<0.01), suggesting activation of the β-catenin pathway, which is beneficial to stem cell proliferation. Immunofluorescence ( Figure 19 The results showed that SOX9 and KLF5 co-expression was significant in hair follicle stem cells of the Sql / PWC group, with the co-expression area accounting for 35.2±4.1% on day 9, which was higher than that of the control group (12.5±2.3%). This phenomenon indicates that squalene induces stem cells to temporarily overcome differentiation limitations, participate in multi-lineage repair, and reduce scar formation. β-catenin / YAP immunofluorescence ( Figure 20 The results showed that YAP nuclear translocation was reduced by 71.2% in the Sql / PWC group, while β-catenin membrane localization was enhanced. Mechanistically, YAP inhibition relieves the negative regulation of β-catenin, promotes hair follicle regeneration and orderly collagen deposition, and ultimately achieves scarless healing.

[0074] In summary, Sql / PWC oleogel accelerates wound healing and inhibits scar formation through the following mechanisms: ① scavenging ROS and improving the oxidative stress microenvironment; ② regulating immune cell polarization and inflammatory factor balance; ③ inhibiting YAP signaling and activating the β-catenin pathway, promoting stem cell-mediated tissue regeneration. This study provides a theoretical basis for the clinical translation of Sql / PWC oleogel. Example 6

[0075] This study established an H2O2-induced oxidative stress model in human fibroblasts to systematically evaluate the protective mechanisms of squalene (Sql) in scavenging reactive oxygen species (ROS), enhancing cell membrane fluidity, and regulating the YAP signaling pathway, providing cellular evidence for elucidating its role in skin injury repair.

[0076] Experimental Methods: Human fibroblasts (HFb) were randomly divided into four groups: Control group (routine culture), H2O2 group (treated with 300 μM H2O2 for 6 h), Par+H2O2 group (H2O2 + 0.05 μL / mL paraffin oil), and Sql+H2O2 group (H2O2 + 0.05 μL / mL squalene). Intracellular ROS levels were detected using the DCFH-DA fluorescent probe; cell membrane fluidity was analyzed using the fluorescence redistribution after photobleaching (FRAP) technique; and the nucleoplasmic distribution of YAP protein was observed using immunofluorescence staining.

[0077] After H2O2 treatment of HFb cells for 6 hours, DCFH-DA staining was performed, and fluorescence intensity was observed under a fluorescence microscope. The results showed that intracellular ROS content (DCFH-DA) increased under H2O2 induction, proving that H2O2 successfully induced cellular oxidation. After treatment with Paraffin and Sql, DCFH-DA staining and fluorescence imaging showed decreased fluorescence intensity. The intracellular oxidation level of Sql was close to that of the control group, demonstrating that Sql effectively reduced H2O2-induced cellular oxidation. Figure 21 A).

[0078] In all FRAP experiments, the bleached area was a circular region with a diameter of 5 μm. Cells were subjected to a 20% stretch deformation (stretching the culture dish as shown) Figure 21(C) After treatment with H2O2, the movement of lipid molecules in the cell membrane of HFb cells slowed down, and the fluorescence recovery rate of the bleached area significantly slowed down; however, after treatment with Sql, the movement of lipid molecules in the cell membrane of HFb cells accelerated, and the fluorescence recovery rate of the bleached area significantly increased. At 60 s, a relatively obvious recovery of fluorescence intensity in the bleached area was observed, and by 180 s, the fluorescence intensity in the bleached area had been almost completely recovered. Figure 21 B). Preliminary experiments show that Sql can effectively enhance cell membrane fluidity.

[0079] Custom-designed stretchable culture dishes were used, and each group of cells was subjected to a 20% deformation treatment to simulate the effect of mechanical force on cells. Figure 21 C). The stretched cells were then treated with H2O2 to simulate the ROS environment of a wound. The results showed that mechanical deformation and peroxidation treatment increased YAP expression in cells, while culture in medium containing Sql decreased YAP expression, similar to the control group. Figure 21 (D) Mechanistically, squalene may reduce the aggregation of mechanosensing elements (such as integrins) by enhancing membrane fluidity, thereby inhibiting YAP nuclear translocation. Specifically, the isoprene units of squalene can be embedded in the hydrophobic regions of membrane phospholipids, and its six double bonds provide electronic buffering capacity, which paraffin oils lack. This structural characteristic allows it to both directly scavenge ROS and influence mechanical signal transduction by modulating membrane lipid composition.

[0080] Echoing the results from in vivo animal experiments, squalene synergistically protects fibroblasts through a triple mechanism: ① directly scavenging ROS to reduce oxidative damage; ② enhancing cell membrane fluidity and improving mechanotransduction; ③ inhibiting YAP nuclear translocation to block the fibrosis pathway. These findings provide a molecular-level basis for the application of squalene in wound repair.

[0081] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A squalene / calcium phosphotungsten oleogel, characterized in that, The squalene / calcium phosphotungsten oleogel is composed of squalene and calcium phosphotungsten nanowires; by mass percentage, the content of calcium phosphotungsten nanowires is 2-5%, and the content of squalene is 95-98%.

2. The squalene / calcium phosphotungsten oleogel according to claim 1, characterized in that, The calcium phosphotungsten nanowires contain 2% calcium and 98% squalene.

3. A method for preparing squalene / calcium phosphotungsten oleogel as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Dissolve phosphotungstic acid and calcium nitrate tetrahydrate in water, stir until homogeneous, then add octadecene and oleylamine in sequence, stir to react, and then add ethanol. The white colloidal substance obtained by sedimentation is precipitated to obtain calcium phosphotungstic acid nanowires. S2. The calcium phosphotungsten nanowires and squalene obtained above are mixed and stirred in a certain proportion until uniform to obtain the squalene / calcium phosphotungsten oleogel.

4. The method for preparing a squalene / calcium phosphotungsten oleogel according to claim 3, characterized in that, In step S1, the ratio of phosphotungstic acid to calcium nitrate tetrahydrate is 1:0.123, and the stirring reaction time is 8 hours.

5. The method for preparing a squalene / calcium phosphotungsten oleogel according to claim 3, characterized in that, The mixing and stirring time in step S2 is 30 minutes.

6. The use of a squalene / calcium phosphotungstic acid oleogel as described in claim 1 or 2 in the preparation of a medicament or dressing for skin injury repair.

7. The use of the squalene / calcium phosphotungsten gel according to claim 6 in the preparation of medicaments or dressings for skin damage repair, characterized in that, The skin damage includes photoaging damage, impaired wound healing, or scar formation.

8. The use of the squalene / calcium phosphotungstic acid oleogel according to claim 6 in the preparation of a medicament or dressing for skin injury repair, characterized in that, The repair mechanism of the squalene / calcium phosphotungsten oleogel includes scavenging ROS, inhibiting inflammatory responses, and enhancing cell membrane fluidity.

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