Enzyme-responsive nano-sulfur composite material, and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical nanomaterials, and particularly relates to an enzyme-responsive nano-sulfur composite material as well as a preparation method and application thereof. The enzyme-responsive nano-sulfur composite material comprises, from inside to outside, a core, an inner shell layer, an outer shell layer and a surface modification layer. The core is nano-sulfur. The inner shell layer is coated on the surface of the core and is composed of polyvinylpyrrolidone. The outer shell layer is coated on the surface of the inner shell layer and is composed of gelatin. The surface modification layer is adsorbed on the surface of the outer shell layer and comprises sodium lactate. The application has an ultra-small particle size and high hydrophilicity, can effectively penetrate a hair follicle biological membrane, can be specifically released in response to enzymes secreted by bacteria, does not damage normal cells, significantly improves treatment safety, has both antibacterial and barrier repair functions, and uses a green and mild synthesis process, uses a high-atomic-economy lactic acid-mediated disproportionation reaction and has mild reaction conditions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to an enzyme-responsive nano-sulfur composite material, its preparation method, and its application. Background Technology

[0002] Pathogenesis and Treatment Challenges of Propionibacterium acnes: Acne vulgaris is a chronic inflammatory skin disease affecting the pilosebaceous unit, with a complex pathogenesis. Propionibacterium acnes (… Propionibacterium acnes The English abbreviation is: P.acnes It has now been renamed Cutibacterium acnes It plays a central role in the development and worsening of acne.

[0003] Deep colonization and microenvironment: P.acnes It is a Gram-positive anaerobic bacterium that primarily colonizes deep within lipid-rich hair follicles. Hyperkeratosis of the follicular infundibulum leads to blockage of the opening, creating an anaerobic, lipid-rich microenvironment that highly favors the excessive proliferation of this bacterium. Inflammatory mechanism: Proliferation... P.acnes It secretes toxic factors such as lipase, protease, and hyaluronidase, which break down sebum to produce free fatty acids and stimulate the rupture of hair follicle walls. At the same time, it can also activate Toll-like receptor 2 (TLR2), induce keratinocytes to secrete inflammatory cytokines such as IL-1α and IL-8, and trigger a cascade of inflammatory responses.

[0004] The Biofilm Problem: Clinical Studies Have Revealed P.acnes It easily forms a stubborn biofilm within the hair follicle. This extracellular polysaccharide matrix forms a dense physical barrier that blocks antibiotic penetration, leading to increasingly serious drug resistance problems. Limitations of existing clinical drugs: Current first-line anti-acne therapies present a significant treatment paradox: antibiotics (such as clindamycin and tetracycline): long-term use has led to global... P.acnes Drug resistance rates soar, and it easily causes gut microbiota imbalance. Retinoic acid and benzoyl peroxide (BPO): Although their bactericidal and exfoliating effects are significant, their strong oxidizing and acidic properties easily damage the skin barrier function, leading to increased transepidermal water loss (TEWL) and triggering "retinoic acid dermatitis" or contact dermatitis, manifested as erythema, desquamation, stinging, and photosensitivity. This results in poor patient compliance and is unsuitable for sensitive skin. Lack of repair function: Most existing drugs focus on "killing" and "exfoliating," neglecting the "reconstruction" of the damaged skin barrier after inflammation. Without effective cell repair support during acne healing, atrophic scars (acne pits) due to dermal collagen destruction are very likely to remain.

[0005] The Application History and Materials Science Bottlenecks of Sulfur Preparations. Sulfur, a classic anti-acne ingredient, possesses multiple benefits including antibacterial, insecticidal, keratin-dissolving, and sebum-inhibiting effects, without inducing drug resistance. However, traditional sulfur preparations (such as sublimed sulfur and precipitated sulfur) face the following materials science bottlenecks in practical applications: Low bioavailability due to hydrophobicity: Sulfur has extremely high surface energy and strong hydrophobicity. In an aqueous matrix, sulfur particles tend to undergo severe aggregation, forming micron-sized or even larger aggregates. These large particles cannot penetrate the narrow hair follicle opening (typically only tens of micrometers in diameter), making it difficult to reach deeper tissues. P.acn es Infection foci. Ostwald Ripening: In liquid dispersions, small sulfur particles are extremely unstable, tending to dissolve and deposit on the surface of larger particles, leading to a continuous increase in particle size over time, poor formulation stability, and precipitation stratification. Single release mechanism and cytotoxicity: Traditional sulfur preparations often kill bacteria through indiscriminate oxidation, lacking responsive release capabilities to the lesion microenvironment. Chinese patent application CN105193945A discloses a lotion for the prevention and treatment of acne, comprising the following raw materials by weight: 1-10 parts boric acid, 2-9 parts resorcinol, 1-9 parts eucalyptus oil, 0.2-4 parts salicylic acid, 2.5-10 parts precipitated sulfur, 0.6-6 parts cetyl alcohol, 6-18 parts sorbitol, 0.2-3 parts potassium hydroxide, 5-15 parts white oil, 0.2-5 parts ethanol, and 50-60 parts distilled water. Although the acne-preventing wash developed by this invention is fast-acting, the high concentration of sulfur in direct contact with the skin, without sustained-release or encapsulation technology, can be toxic to normal keratinocytes, leading to dry skin and a damaged skin barrier.

[0006] Nanotechnology Attempts and Unresolved Issues. Although some studies have attempted to prepare nano-sulfur to improve its solubility, the existing technology still has the following shortcomings: (1) The synthesis route is not environmentally friendly: the common acid disproportionation method (such as hydrochloric acid + sodium thiosulfate) is violent, it is difficult to control the uniformity of particle size, and it not only produces irritating gas (SO2) but also leaves strong acid radical ions; (2) Surfactant residue: In order to maintain the stability of nano-sulfur, cationic surfactants such as CTAB or a large amount of organic solvents are often added. These additives themselves have high cytotoxicity, which limits their application on broken skin (such as broken pimples); (3) Lack of barrier repair function: existing nano-sulfur related patents mainly focus on "antibacterial efficiency", and there are few reports on "promoting skin tissue regeneration" or "repairing damaged stratum corneum".

[0007] In summary, the development of a novel delivery system for deep infection of Propionibacterium acnes is an urgent need in this field. The delivery system should simultaneously meet the following core requirements: (1) possessing ultra-small particle size and high hydrophilicity, enabling effective penetration of hair follicle biomembrane; (2) possessing environmentally responsive drug release characteristics, being environmentally responsive and able to specifically release enzymes (such as gelatinase) secreted by bacteria, significantly improving treatment safety; (3) possessing both antibacterial and barrier repair functions, significantly promoting the proliferation of keratinocytes (HaCaT) while killing bacteria, achieving synergistic effects of antibacterial-repair dual efficacy; (4) employing a green and mild synthesis process, with mild reaction conditions, and byproducts possessing skin-friendly and moisturizing benefits, meeting the safety and application requirements of topical preparations. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide an enzyme-responsive nano-sulfur composite material, its preparation method, and its applications. This invention achieves hydrophilic modification of sulfur, enabling it to be highly dispersed in physiological solutions, possessing ultra-small particle size and high hydrophilicity, allowing for effective penetration of hair follicle biomembranes and overcoming the low bioavailability caused by the hydrophobicity of traditional sulfur preparations. This invention exhibits environmentally responsive drug release characteristics, specifically releasing enzymes secreted by bacteria (such as gelatinase), enhancing adsorption through interfacial molecular bridging effects, and achieving specific killing of pathogens through an enzyme-responsive shell dissociation mechanism without damaging normal cells, significantly improving treatment safety. This invention combines antibacterial and barrier repair functions, significantly promoting keratinocyte (HaCaT) proliferation while killing bacteria, achieving a synergistic effect of antibacterial and repair. This invention employs a green and mild synthesis process, utilizing a lactic acid-mediated disproportionation reaction, resulting in high atom economy, mild reaction conditions, and bioactive byproducts (sodium lactate) with skin-friendly and moisturizing benefits, meeting the safety and application requirements of topical preparations.

[0009] The technical solution of this invention is: An enzyme-responsive nano-sulfur composite material, comprising, from the inside out, a core, an inner shell layer, an outer shell layer, and a surface modification layer, wherein the core is nano-sulfur; the inner shell layer is coated on the surface of the core and is composed of polyvinylpyrrolidone; the outer shell layer is coated on the surface of the inner shell layer and is composed of gelatin; and the surface modification layer is adsorbed on the surface of the outer shell layer and contains sodium lactate.

[0010] In this invention, the inner shell is an amphiphilic polymer, polyvinylpyrrolidone (PVP), used to disrupt the bacterial lipid bilayer membrane.

[0011] In this invention, the outer shell is gelatin, which serves as a protective layer and the outer shell of the enzyme-triggered barrier structure.

[0012] In this invention, the surface modification layer (Interface) consists of sodium lactate, a reaction byproduct, which is adsorbed onto the particle surface via non-covalent bonds to form an interfacial bonding layer.

[0013] Furthermore, the gelatin is type A gelatin or type B gelatin, serving as an enzyme response matrix.

[0014] Furthermore, the polyvinylpyrrolidone is one of PVP K15, PVP K17, PVP K25, PVP K30, PVP K60, and PVP K90, serving as a steric hindrance stabilizer.

[0015] Furthermore, the polyvinylpyrrolidone is PVP K30.

[0016] This invention also provides a method for preparing the enzyme-responsive nano-sulfur composite material, comprising the following steps: S1 prepares a polyvinylpyrrolidone solution, and adds the prepared polyvinylpyrrolidone solution to the raw material lactic acid solution to prepare a polyvinylpyrrolidone-lactic acid solution; S2. Add gelatin to the polyvinylpyrrolidone-lactic acid solution obtained in step S1 to prepare a polyvinylpyrrolidone-lactic acid-gelatin solution; S3 is used to prepare sodium thiosulfate (Na2S2O3) solution; S4 is used to prepare sodium hydroxide (NaOH) solution; S5. Add the sodium thiosulfate solution obtained in step S3 dropwise to the polyvinylpyrrolidone-lactic acid-gelatin solution obtained in step S2, while stirring during the dropwise addition. After the dropwise addition is complete, continue stirring for 2-4 hours, then add water, and then add the NaOH solution prepared in step S4 to adjust the pH, thus obtaining the final product.

[0017] Further, the mass concentration of the lactic acid solution used in step S1 is 50-98%. Lactic acid is a weak acid (pKa = 3.86), and its H2O content is low when its concentration is below 50%. + Insufficient supply cannot effectively drive the disproportionation reaction of sodium thiosulfate; when the lactic acid concentration is too high, although the degree of ionization of anhydrous lactic acid is low, the total amount is extremely high. When it comes into contact with sodium thiosulfate, it releases a large amount of SO2 gas instantly, causing local boiling of the system and destroying the newly formed PVP / gelatin shell; the pH drops sharply, causing the gelatin molecular chains to be acidified, losing gel properties and failing to form an enzyme-responsive shell.

[0018] Furthermore, in step S1, the mass concentration of polyvinylpyrrolidone (PVP) in the polyvinylpyrrolidone-lactic acid solution is 0.5-5%. If the PVP concentration is less than 0.5%, the number of molecular chains is insufficient to completely cover the surface of the newly formed sulfur nuclei. The hydrophobic sulfur nuclei undergo irreversible aggregation due to surface energy, forming micron-sized precipitates (>1000 nm), which cannot penetrate the hair follicle biomembrane (usually with a pore size <100 nm). If the PVP concentration is greater than 5%, the viscosity of the PVP solution increases exponentially with concentration, making it difficult to uniformly disperse sodium thiosulfate during dropwise addition. This results in excessively high local supersaturation, forming polydisperse particles (PDI>0.5).

[0019] Furthermore, in step S2, the mass concentration of gelatin in the polyvinylpyrrolidone-lactic acid-gelatin solution is 0.5-5%. If the concentration is too low, the gelatin network cannot form a complete enzyme-triggered barrier structure shell, exposing the nano-sulfur core and resulting in a lack of enzyme response specificity. If the concentration is too high, the gelatin forms a physical gel at room temperature (especially <28℃), causing a sharp increase in the system viscosity. The Na2S2O3 droplets cannot be uniformly dispersed, forming localized supersaturated reaction zones with extremely wide particle size distribution (PDI>0.6).

[0020] Furthermore, in step S3, the mass concentration of the Na2S2O3 solution is 20-40%. If the mass concentration of the Na2S2O3 solution is too low (<20%), the PVP / gelatin is relatively excessive, leading to excessive cross-linking of the polymer network, encapsulating too much water to form a dense hydration layer. The nano-sulfur is physically trapped inside the gel, making release difficult, and the effective sulfur content for antibacterial treatment is below the therapeutic threshold. If the mass concentration of sodium thiosulfate is too high (>40%), the local sulfur generation rate during dropwise addition far exceeds the PVP / gelatin coating rate, causing sulfur nuclei to collide and aggregate, disrupting steric stability, and making the particles prone to flocculation.

[0021] In step S4, the mass concentration of the NaOH solution is 10-40%. If the mass concentration of the NaOH solution is <10%, and pH needs to be adjusted from 4 to 7, a large amount of NaOH solution needs to be added, leading to excessive dilution of the system, a decrease in the concentration of nano-sulfur, and the need for an additional concentration step, violating the "purification-free" principle. The buffer system is diluted, resulting in decreased pH stability and easy acidification during storage (pH < 5), which can irritate the skin. When high-concentration NaOH solution (>40% is a highly corrosive solution) is added dropwise, the local pH momentarily exceeds 12, causing alkaline hydrolysis of gelatin (peptide bond breakage) and loss of enzymatic responsiveness. Nano-sulfur undergoes a disproportionation reaction under strong alkaline conditions, resulting in a loss of sulfur content and a decrease in antibacterial activity.

[0022] Further, in step S5, the volume ratio of Na2S2O3 solution to polyvinylpyrrolidone-lactic acid-gelatin solution is 1-4:6-9. If the volume ratio of Na2S2O3 solution to polyvinylpyrrolidone-lactic acid-gelatin solution is less than 1:9, the effective sulfur content in the final product is lower than the minimum concentration required for anti-acne treatment, and it cannot effectively inhibit the formation of Propionibacterium acnes biofilm. If the volume ratio of Na2S2O3 solution to polyvinylpyrrolidone-lactic acid-gelatin solution is greater than 4:6, the total amount of sulfur source exceeds the maximum coating capacity of PVP / gelatin polymer, resulting in a large number of free sulfur nuclei, PDI>0.8, and a bimodal particle size distribution (coated particles ~15 nm + free aggregates ~500 nm). The amount of SO2 generated is too large, the lactate-sodium lactate buffer pair is depleted, and the final pH is <4.5. A large amount of NaOH needs to be added for neutralization, introducing too many sodium ions, leading to hypertonic dehydration of the skin.

[0023] Furthermore, during the dropwise addition process in step S5, stirring is maintained at a speed of 400-600 rpm. After the dropwise addition is completed, stirring is continued at a speed of 150-350 rpm for 2-4 hours.

[0024] The system pH can be fine-tuned as needed. For example, for individuals needing acne treatment, the system pH can be fine-tuned to the skin's physiological pH (pH 6.5); for normal individuals, the system pH can be fine-tuned to the skin's physiological pH (pH 7.0-7.5). Due to the presence of excess lactic acid and the generated sodium lactate, a "lactic acid-sodium lactate buffer system" is naturally formed, which helps maintain the pH stability of the formulation. This step eliminates the purification operations such as dialysis, centrifugation, and washing required in traditional nano-formulations, and the resulting solution can be directly used as an aqueous matrix for the subsequent formulation of gels, emulsions, or serums.

[0025] The SNPs@PVP / Gelatin and its in-situ generated moisturizing buffer system described in this invention solve the problems of poor penetration, high irritation, and slow repair associated with traditional anti-acne drugs through the synergistic effect of the following three levels: 1. Physical level: Breaking down the biofilm defense barrier of bacteria (1) Hydrophilic modification: Traditional sulfur particles are highly hydrophobic, easily accumulating on the skin surface and struggling to penetrate deep into the hair follicle, which is filled with tissue fluid. This invention utilizes gelatin as a shell, endowing the nano-sulfur with excellent hydrophilicity. This surface hydrophilic modification layer allows the nanoparticles to slide smoothly down the hair follicle infundibulum like water molecules, reaching deep into the sebaceous gland. P.acnesColonization area. (2) Penetration of biofilm: Propionibacterium acnes often secretes extracellular polysaccharides to form a biofilm to resist drugs. The nanomaterials prepared in this invention have a particle size much smaller than the pores of the biofilm. Combined with the steric hindrance effect of PVP, they can penetrate through the biofilm matrix and contact the bacterial body at the bottom layer, breaking the bacterial biofilm defense barrier.

[0026] 2. Chemical / Biological Level: Response release of enzymes in the lesion microenvironment (1) Enzyme cleavage switch: P.acnes During metabolism, high concentrations of gelatinase and protease are secreted to decompose skin tissue. (2) On-demand release: The nanoparticles of this invention have a gelatin shell. When the nanoparticles come into contact with healthy skin, the gelatin shell remains intact, and the sulfur element does not leak out, avoiding stimulation of normal cells. Once it enters the bacterial-rich cyst, the gelatin shell is specifically degraded by the enzymes secreted by the bacteria, and the highly active nano-sulfur encapsulated inside is instantly exposed and released. (3) Sterilization principle: Due to its huge specific surface area, the exposed nano-sulfur quickly comes into contact with bacteria and is converted into pentasulfuric acid or hydrogen sulfide through an oxidation mechanism, interfering with the electron transport chain and energy metabolism of bacteria, leading to bacterial death. This lesion microenvironment-responsive activation mechanism maximizes the safety index of the drug.

[0027] 3. Physiological level: "Endogenous wet compress" and barrier remodeling This is the fundamental difference between the present invention and the prior art, thanks to the sodium lactate-lactic acid buffer pair deliberately retained in the reaction system: (1) Buffering and antibacterial: The weakly acidic environment (pH 4.5-5.5) maintained by the system simulates the acidic protective film of healthy skin, and this environment itself can inhibit the preference for neutral / alkaline environments. P. acnes Growth, while also facilitating the survival of resident probiotics (such as Staphylococcus epidermidis) on the skin, regulating the microecological balance. (2) Hydration repair (The “Wet Compress” Effect): The reaction byproduct sodium lactate, as a powerful NMF (natural moisturizing factor), provides continuous “hydration” for damaged and dry acne areas while killing bacteria. This not only alleviates the dryness caused by sulfur, but also provides an ideal moist environment for the migration and proliferation of keratinocytes (HaCaT). (3) Growth-promoting signal: Experimental data show that the vitality of HaCaT cells is as high as 133%±0.002. The mechanism is speculated to be: the polypeptide / amino acid fragments produced by gelatin degradation, combined with the energy metabolism substrate provided by sodium lactate, and trace sulfur as a cofactor for keratin synthesis, together activate the skin cell repair pathway, accelerate the synthesis of collagen and wound healing, thereby effectively preventing the formation of acne pits.

[0028] Another object of the present invention is to provide the application of the above-mentioned enzyme-responsive nano-sulfur composite material or the enzyme-responsive nano-sulfur composite material prepared according to the above-mentioned preparation method of enzyme-responsive nano-sulfur composite material in the preparation of anti-acne and skin barrier repair products.

[0029] Compared with the prior art, the present invention has the following advantages: (1) The preparation method of the enzyme-responsive nano-sulfur composite material provided by this invention is a "zero-waste" purification-free process, which significantly reduces production costs. Existing technologies for preparing nano-sulfur typically use strong acids (hydrochloric acid), producing ineffective salts such as sodium chloride, and leaving strong acid residues, requiring time-consuming and water-intensive dialysis or centrifugal washing steps. This invention cleverly utilizes lactic acid to drive the reaction, intentionally retaining reaction byproducts. This greatly simplifies the preparation process, realizing a single-reactor continuous synthesis process, and directly obtaining a usable formulation upon reaction termination, significantly shortening the production cycle and reducing industrialization costs.

[0030] (2) The preparation method of the enzyme-responsive nano-sulfur composite material provided by this invention transforms "byproducts" into "functional ingredients," constructing an endogenous moisturizing system. The core ingenuity of this invention lies in the fact that the reaction byproduct is sodium lactate. Sodium lactate is a core component of the natural moisturizing factor (NMF) of human skin and has extremely strong moisture absorption capacity. The biggest pain point of traditional sulfur preparations is "drying" and "peeling." In this invention, the retained sodium lactate directly constructs a highly moisturizing microenvironment around the nano-sulfur. When the preparation is applied to the affected area, the nano-sulfur exerts an antibacterial effect, while the coexisting sodium lactate simultaneously exerts a moisturizing and repairing effect. The two produce a synergistic effect, effectively alleviating the dryness and irritation of sulfur, and achieving "acne removal without damaging the skin."

[0031] (3) The preparation method of the enzyme-responsive nano-sulfur composite material provided by the present invention forms a self-buffering system, which improves the stability of the formulation. The retained unreacted lactic acid and the generated sodium lactate naturally form a buffer pair in the system. After the reaction, the system is weakly acidic (pH 4.5-5.5) due to excess lactic acid residue. NaOH solution needs to be added to adjust the pH to 6.5-7.5, which not only helps maintain the chemical stability of nano-sulfur, but also matches the physiological pH of healthy skin, which helps to inhibit alkaline-loving harmful bacteria and maintain the skin microecological balance. Attached Figure Description

[0032] Figure 1 These are images of the appearance of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention. Figure 2 This is a transmission electron microscope image of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention; Figure 3 This is a particle size distribution diagram of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention; Figure 4 This is a transmission electron microscope image of the control group sample after incubation for 0.5 hours; Figure 5 This is a transmission electron microscope image of the experimental group samples after incubation for 0.5 hours; Figure 6 This is a field emission electron microscope image of Staphylococcus aureus in the control group; Figure 7 Field emission electron microscopy image of Staphylococcus aureus treated with SNPs@PVP / Gelatin prepared in Example 1; Figure 8 This is a field emission electron microscope image of Pseudomonas aeruginosa from the control group; Figure 9 This is a field emission electron microscope image of Pseudomonas aeruginosa treated with SNPs@PVP / Gelatin prepared in Example 1. Figure 10 This is a field emission electron microscope image of Propionibacterium acnes in the control group; Figure 11 This is a field emission electron microscope image of Propionibacterium acnes treated with SNPs@PVP / Gelatin prepared in Example 1. Detailed Implementation

[0033] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0034] Example 1: Preparation of an enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) This embodiment demonstrates a method for preparing an enzyme-responsive nano-sulfur composite material, comprising the following steps: S1. Weigh an appropriate amount of PVP K30 and dissolve it in an appropriate amount of deionized water. Use an ultrasonic processor to sonicate the solution at a frequency of 40000 Hz for 10 minutes to ensure complete dissolution of PVP, thus obtaining a polyvinylpyrrolidone (PVP) solution. Add the obtained PVP solution to a raw material lactic acid solution with a mass concentration of 90%. Use a magnetic stirrer to stir at a speed of 500 rpm for 10 minutes to ensure thorough mixing of the PVP solution and the lactic acid solution, thus obtaining a PVP-lactic acid solution (PVP-lactic acid solution). The mass concentration of PVP in the PVP-lactic acid solution is 1%, and the mass concentration of lactic acid is 10%. S2. Weigh an appropriate amount of gelatin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS No.: 9000-70-8, G6317 gelatin, type A), and add it to the PVP-lactic acid solution obtained in step S1. Use a magnetic stirrer to stir at 60℃ and 500rpm for 10 minutes to ensure that the gelatin is completely dissolved. After stirring, allow it to stand and return to room temperature to obtain a PVP-lactic acid-gelatin solution. The mass concentration of gelatin in the PVP-lactic acid-gelatin solution is 1%. Preparation of S3 and Na2S2O3 solutions: Weigh an appropriate amount of Na2S2O3 and dissolve it in an appropriate amount of deionized water to prepare a Na2S2O3 solution with a mass concentration of 40%. S4. Preparation of NaOH solution: Weigh an appropriate amount of NaOH and dissolve it in an appropriate amount of deionized water to prepare a NaOH solution with a mass concentration of 40%. Preparation of S5 and SNPs@PVP / Gelatin: At room temperature, the Na2S2O3 solution prepared in step S3 was slowly added dropwise to the PVP-lactic acid-gelatin solution prepared in step S2, while stirring at 500 rpm. The volume ratio of the Na2S2O3 solution to the PVP-lactic acid-gelatin solution was 2:8. After the addition was complete, the mixture was stirred at 200 rpm for 3 hours at room temperature to promote the assembly of nano-sulfur (SNPs), PVP, and gelatin, and to remove SO2 gas. Then, an equal volume of deionized water was added. Finally, a 40% NaOH solution prepared in step S4 was added to adjust the pH to 7.2, thus obtaining the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin).

[0035] Furthermore, this invention unexpectedly revealed that the mass concentration of the Na2S2O3 solution and the volume ratio of the feed material must be synergistically controlled: when the mass concentration of the Na2S2O3 solution is higher than 40% and the volume ratio of the Na2S2O3 solution to the polyvinylpyrrolidone-lactic acid-gelatin solution is lower than 1:9, the system viscosity is too high, leading to uneven mixing; when the mass concentration of the Na2S2O3 solution is lower than 20% and the volume ratio of the Na2S2O3 solution to the polyvinylpyrrolidone-lactic acid-gelatin solution is higher than 4:6, the coating agent is diluted to the point that it cannot form a shell. Only when the mass concentration of the Na2S2O3 solution is between 20-40%, and the volume ratio of the Na2S2O3 solution to the polyvinylpyrrolidone-lactic acid-gelatin solution is within the coupling range of 1-4:6-9, can the dual conditions of "sufficient sulfur source" and "sufficient coating" be simultaneously satisfied, resulting in enzyme-responsive core-shell sulfur nanoparticles with a particle size <20 nm. This coupling relationship cannot be easily obtained by those skilled in the art through conventional experiments.

[0036] Comparative Example 1: Preparation of a nano-sulfur composite material This comparative example is similar to Example 1, except that: S1, Preparation of PVP-Na2S2O3 solution: Weigh an appropriate amount of Na2S2O3 and dissolve it in an appropriate amount of deionized water to prepare a Na2S2O3 solution with a mass concentration of 40%; add the prepared polyvinylpyrrolidone solution to the Na2S2O3 solution to prepare a PVP-Na2S2O3 solution; S2, Preparation of PVP-Na2S2O3-gelatin solution: Weigh an appropriate amount of gelatin and add it to the PVP-Na2S2O3 solution prepared in step S1 to prepare a PVP-Na2S2O3-gelatin solution with a mass concentration of 1%; S3, Preparation of lactic acid solution with a mass concentration of 10%; S5, At room temperature, slowly add the lactic acid solution prepared in step S3 to the PVP-Na2S2O3-gelatin solution prepared in step S2. The difference between this comparative example and Example 1 is that the order of adding sodium thiosulfate and lactic acid is reversed, while the other components and preparation methods are the same as in Example 1.

[0037] Comparative Example 2: Preparation of a nano-sulfur composite material This comparative example is similar to Example 1, except that it does not include the step of adding gelatin in step S2. The other components and preparation methods are the same as in Example 1.

[0038] Comparative Example 3: Preparation of a nano-sulfur composite material This comparative example is similar to Example 1, except that PVP does not occur throughout the entire process, and the other components and preparation methods are the same as in Example 1.

[0039] Example 1: Characterization of SNPs@PVP / Gelatin 1. Laser particle size analyzer (DLS) (1) Experimental method: Take a small amount of the enzyme-responsive nano-sulfur composite material prepared in Example 1, and the nano-sulfur composite materials prepared in Comparative Examples 1, 2, and 3 respectively, dilute them 10 times with ultrapure water, and measure the particle size, polymer dispersibility index (PDI, the larger the PDI, the wider the molecular weight distribution, and the smaller the PDI, the more uniform the molecular weight distribution) and potential by dynamic light scattering. (2) Experimental results: The average particle size and PDI of the nano-sulfur composite materials prepared in different groups are shown in Table 1.

[0040] Table 1. Comparison of average particle size and PDI of nano-sulfur composite materials prepared from different groups.

[0041] As shown in Table 1, the average particle size of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention is 15.54 ± 2.60 nm. The average particle size of the enzyme-responsive nano-sulfur composite materials prepared in Comparative Examples 1, 2, and 3 is significantly larger than that of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1, and the PDI also increases accordingly. Although the PDI increase of the enzyme-responsive nano-sulfur composite material prepared in Comparative Example 1 is not particularly large compared with that in Example 1, the particle size is significantly larger. This indicates that when the order of addition of sodium thiosulfate and lactic acid is reversed, an enzyme-responsive nano-sulfur composite material can still be prepared, but the particle size of the enzyme-responsive nano-sulfur composite material will increase. In Comparative Examples 1, 2, and 3, the particle size of the enzyme-responsive nano-sulfur composite material is significantly increased compared with that in Example 1, and the PDI also increases accordingly, resulting in uneven particle size distribution. This indicates that the order of addition of sodium thiosulfate and lactic acid has a significant impact on the particle size and dispersibility of the nano-sulfur composite material; the absence of gelatin or polyvinylpyrrolidone in the technical solution leads to increased particle size and decreased dispersibility. Therefore, the synergistic effect of the components in this invention effectively inhibits particle agglomeration, significantly reduces particle size, and improves dispersion uniformity.

[0042] 2. Transmission electron microscopy (TEM) The appearance image of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention is shown below. Figure 1 As shown. By Figure 1As can be seen, the SNPs@PVP / Gelatin prepared in Example 1 of this invention is a milky white solution. The morphology of the SNPs@PVP / Gelatin prepared in Example 1 was characterized using transmission electron microscopy. The transmission electron microscopy image of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention is shown below. Figure 2 As shown. By Figure 2 As can be seen, nanoparticles were observed under a transmission electron microscope.

[0043] The particle size distribution of the enzyme-responsive nano-sulfur composite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention is shown in the figure below. Figure 3 As shown. Figure 3 The test results show that the particle size of SNPs@PVP / Gelatin exhibits a unimodal normal distribution, with most particles having a size concentrated in the range of 10-15 nm, demonstrating good size uniformity and dispersibility.

[0044] Experimental Example 2: Biocompatibility of SNPs@PVP / Gelatin 1. Biocompatibility testing method for SNPs@PVP / Gelatin: (1) Take a small amount of the enzyme-responsive nano-sulfur composite material prepared in Example 1, and the nano-sulfur composite materials prepared in Comparative Examples 1, 2, and 3 respectively, dilute them 10 times with sterile culture medium, and perform filtration sterilization. (2) HaCaT cells were seeded into 96-well plates, with 10,000 cells seeded per well; (3) Add the sterilized nano-sulfur composite material from step (1) to the 96-well plate from step (2); make 3 replicates for each sample; make 3 replicates for the control wells prepared without the nano-sulfur composite material from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3; make 3 replicates for the blank wells using sterile culture medium. (4) After adding the sample for 24 hours, remove the cell supernatant and add 100 μL of 10% CCK8 reagent to each well; (5) Incubate the 96-well plate in an incubator at 37°C and 5% CO2 for 3 hours; (6) Use an ELISA reader to measure the absorbance (OD) of each well at 450 nm; (7) Formula for calculating cell viability: ×100% Among them, OD 样品 OD of each nano-sulfur composite material pore 450 Value, OD 空白孔 OD of blank wells containing only sterile culture medium 450Value, OD 对照孔 OD of control wells containing cells but without the addition of nano-sulfur composite material 450 value.

[0045] 2. Test Results: The effects of different samples on HaCaT cell viability are shown in Table 2.

[0046] Table 2 Effects of different samples on HaCaT cell viability

[0047] Table 2 shows that the enzyme-responsive sulfur nanocomposite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention achieved a HaCaT cell viability of 133% ± 0.002, which is significantly higher than that of Comparative Examples 1, 2, and 3. This demonstrates that the enzyme-responsive sulfur nanocomposite material (SNPs@PVP / Gelatin) prepared in Example 1 of this invention does not produce cytotoxicity and provides an ideal moist environment for the migration and proliferation of keratinocytes (HaCaT).

[0048] Experimental Example 3: Enzyme-responsive Degradation Test of SNPs@PVP / Gelatin This experimental case aims to verify the specific response release mechanism of enzyme-responsive sulfur nanocomposite materials to bacterial secreted enzymes.

[0049] The enzyme-responsive degradation assay method for SNPs@PVP / Gelatin is as follows: S1. Preparation of gelatinase: Weigh an appropriate amount of gelatinase (Shanghai Yuanye Biotechnology Co., Ltd., product number: S10053), dissolve it in deionized water to form a gelatinase solution with a concentration of 0.1 mg / mL. S2. Preparation of experimental group samples: Take an appropriate amount of the enzyme-responsive nano-sulfur composite material suspension prepared in Example 1, add the gelatinase solution with a concentration of 0.1 mg / mL obtained in step S1, the volume ratio of the enzyme-responsive nano-sulfur composite material suspension to the gelatinase solution is 10000:1, and incubate in a constant temperature shaker at 37℃ for 0.5 hours. S3. Preparation of control group samples: Take an equal amount of the enzyme-responsive nano-sulfur composite material prepared in Example 1 as in step S2, add an equal volume of PBS buffer (pH 7.4), and incubate in a 37°C constant temperature shaker for 0.5 hours; S4. Place the experimental group samples and the control group samples on a carbon-coated copper grid. After natural drying, image the morphology and structure of the nanoparticles under a transmission electron microscope.

[0050] Transmission electron microscope image of the control group sample after 0.5 h of incubation is shown below. Figure 4 As shown. By Figure 4 As can be seen, the nanoparticles exhibit a clear spherical structure with smooth edges and a thin gray halo (gelatin / PVP shell) around each particle. The particles are well dispersed and show no aggregation. The transmission electron microscope image of the experimental group samples after 0.5 hours of incubation is shown below. Figure 5 As shown. By Figure 5 It can be seen that after 0.5 hours of incubation, the polymer shell on the surface of the nanoparticles became blurred and rough.

[0051] The above enzyme-responsive degradation test of SNPs@PVP / Gelatin confirmed that the SNPs@PVP / Gelatin prepared in Example 1 of this invention has excellent enzyme-responsive characteristics and can specifically degrade the outer shell at the lesion site to achieve targeted drug release.

[0052] Test Example 4: In vitro broad-spectrum antibacterial activity test This experiment evaluated the inhibitory effect of the enzyme-responsive nano-sulfur composite material SNPs@PVP / Gelatin prepared in this invention on common pathogenic bacteria. The Staphylococcus aureus used in this experiment was ATCC 25923, purchased from the American Center for Type Culture Collection (ATCC); the Pseudomonas aeruginosa was ATCC 27853, purchased from the American Center for Type Culture Collection (ATCC); and the Propionibacterium acnes was ATCC 6919, purchased from the American Center for Type Culture Collection (ATCC).

[0053] The in vitro broad-spectrum antibacterial activity assay for SNPs@PVP / Gelatin is as follows: Dilute Staphylococcus aureus in the exponential growth phase with LB liquid medium. S.aureus ), Pseudomonas aeruginosa ( P. aeruginosa ), and Propionibacterium acnes ( P.acnes The solution was prepared to achieve a concentration of 3 × 10⁻⁶. 6CFU / mL. After treatment with the enzyme-responsive sulfur nanocomposite SNPs@PVP / Gelatin prepared in Example 1 for 24 hours, the bacterial cells were centrifuged at 8000 rpm for 3 minutes. The cells were then rinsed with sterile water and centrifuged again at 8000 rpm for 3 minutes to remove the supernatant. The cells were fixed overnight at 4°C with 2.5% glutaraldehyde, protected from light. Gradual dehydration was then performed using different concentrations of tert-butanol solution (30%, 50%, 70%, 85%, 95%). After each dehydration, the samples were centrifuged at 8000 rpm for 3–5 minutes. Subsequently, the samples were dehydrated twice with 100% tert-butanol, and the supernatant was discarded. Finally, tert-butanol was added to submerge the bacterial cells, and after drying using a CO2 critical point desiccator, the morphology was characterized using a field emission scanning electron microscope (FESEM, Ultra-55, Zeiss). A control group was also included, with only PBS solvent added, but no enzyme-responsive sulfur nanocomposite material was added.

[0054] Field emission electron microscopy (FESEM) observations: (1) Experimental method: The SNPs@PVP / Gelatin prepared in Example 1 were used to treat Staphylococcus aureus (SNPs@PVP / Gelatin) prepared above. S.aureus ), Pseudomonas aeruginosa ( P. aeruginosa ) and Propionibacterium acnes ( P.acnes After the bacterial cells are dried, use clean tweezers or a toothpick to apply a small amount of the dried bacterial powder onto the conductive tape, and then immediately place it into an ion sputtering instrument for gold sputtering. (2) Experimental results: Field emission electron microscopy image of Staphylococcus aureus in the control group is shown below. Figure 6 As shown; Field emission electron microscopy image of Staphylococcus aureus treated with SNPs@PVP / Gelatin prepared in Example 1. Figure 7 As shown; Field emission electron microscopy image of Pseudomonas aeruginosa in the control group is shown below. Figure 8 As shown; Field emission electron microscopy image of Pseudomonas aeruginosa treated with SNPs@PVP / Gelatin prepared in Example 1. Figure 9 As shown; Field emission electron microscopy image of Propionibacterium acnes in the control group is shown below. Figure 10 As shown; Field emission electron microscopy image of Propionibacterium acnes treated with SNPs@PVP / Gelatin prepared in Example 1. Figure 11 As shown. Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11In this context, "EHT" stands for "Extra High Tension," indicating accelerating voltage; "WD" stands for "Working Distance," indicating working distance; "Mag" stands for "Magnification," indicating magnification factor; "Signal A" indicates the signal type used by image channel A; "SE2" stands for "Secondary Electron 2," indicating secondary electronic signal 2; and "ZEISS" stands for "Carl Zeiss AG," indicating Carl Zeiss. For example, Figure 6 In the diagram, EHT=5.00 kV indicates that the accelerating voltage of the electron beam is 5.00 kV; WD=4.7 mm indicates that the distance from the sample surface to the objective lens is 4.7 mm; Mag=20.55 KX indicates that the image magnification is 20.55 kX; Signal A= SE2 indicates that a secondary electron detector is used to acquire the signal; and ZEISS indicates that the scanning electron microscope is manufactured by Carl Zeiss AG.

[0055] Depend on Figure 7 , Figure 9 , Figure 11 It can be seen that after treatment with SNPs@PVP / Gelatin, Staphylococcus aureus (… S.aureus ), Pseudomonas aeruginosa ( P. aeruginosa ) and Propionibacterium acnes ( P.acnes The cell membrane structure of the _____ underwent significant deformation and collapse, indicating that its integrity was severely compromised. In contrast, Figure 6 , Figure 8 , Figure 10 The cell membrane structure of the control group shown remains normal.

Claims

1. An enzyme-responsive nano-sulfur composite material, characterized in that, The enzyme-responsive nano-sulfur composite material comprises, from the inside out, a core, an inner shell layer, an outer shell layer, and a surface modification layer. The core is nano-sulfur. The inner shell layer, which is coated on the surface of the core, is composed of polyvinylpyrrolidone. The outer shell layer, which is coated on the surface of the inner shell layer, is composed of gelatin. The surface modification layer, which is adsorbed on the surface of the outer shell layer, contains sodium lactate.

2. The enzyme-responsive nano-sulfur composite material according to claim 1, characterized in that, The gelatin is either type A gelatin or type B gelatin.

3. The enzyme-responsive nano-sulfur composite material according to claim 1, characterized in that, The polyvinylpyrrolidone is one of PVP K15, PVP K17, PVP K25, PVP K30, PVP K60, and PVP K90.

4. The method for preparing the enzyme-responsive nano-sulfur composite material according to any one of claims 1-3, characterized in that, Includes the following steps: S1 prepares a polyvinylpyrrolidone solution, and adds the prepared polyvinylpyrrolidone solution to the raw material lactic acid solution to prepare a polyvinylpyrrolidone-lactic acid solution; S2. Add gelatin to the polyvinylpyrrolidone-lactic acid solution obtained in step S1 to prepare a polyvinylpyrrolidone-lactic acid-gelatin solution; S3 is used to prepare sodium thiosulfate solution; S4 is used to prepare sodium hydroxide solution; S5. Add the sodium thiosulfate solution obtained in step S3 dropwise to the polyvinylpyrrolidone-lactic acid-gelatin solution obtained in step S2, while stirring during the dropwise addition. After the dropwise addition is complete, continue stirring for 2-4 hours, then add water, and then add the sodium hydroxide solution prepared in step S4 to adjust the pH, thus obtaining the final product.

5. The method for preparing the enzyme-responsive nano-sulfur composite material according to claim 4, characterized in that, The mass concentration of the raw material lactic acid solution in step S1 is 50-98%.

6. The method for preparing the enzyme-responsive nano-sulfur composite material according to claim 4, characterized in that, In step S1, the mass concentration of polyvinylpyrrolidone in the polyvinylpyrrolidone-lactic acid solution is 0.5-5%.

7. The method for preparing the enzyme-responsive nano-sulfur composite material according to claim 4, characterized in that, In step S2, the mass concentration of gelatin in the polyvinylpyrrolidone-lactic acid-gelatin solution is 0.5-5%.

8. The method for preparing the enzyme-responsive nano-sulfur composite material according to claim 4, characterized in that, The mass concentration of the sodium thiosulfate solution in step S3 is 20-40%; the mass concentration of the sodium hydroxide solution in step S4 is 10-40%.

9. The method for preparing the enzyme-responsive nano-sulfur composite material according to claim 4, characterized in that, In step S5, the volume ratio of sodium thiosulfate solution to polyvinylpyrrolidone-lactic acid-gelatin solution is 1-4:6-9.

10. The application of the enzyme-responsive nano-sulfur composite material prepared according to any one of claims 1-3 or the preparation method of the enzyme-responsive nano-sulfur composite material according to any one of claims 4-9 in the preparation of anti-acne and skin barrier repair products.

Images