Drug-loaded liposome protein hydrogel compound as well as preparation method and application thereof

Abstract

The invention discloses a drug-loaded liposome protein hydrogel compound as well as a preparation method and application thereof. The compound disclosed by the invention is obtained by wrapping nano-liposome loaded with a fibroblast growth factor FGF21 by adopting BSA (Bovine Serum Albumin) modified hydrogel, and the BSA modified hydrogel is bovine serum albumin-based hydrogel modified by ICPS (Isocyanatopropyltriethoxysilane). The compound provided by the invention adopts a nano delivery system to effectively enhance the targeted therapeutic activity of FGF21, a synergistic delivery system of nano liposome and BSA hydrogel integrates the advantages of the nano liposome and the BSA hydrogel, overcomes the respective limitations, realizes drug controlled release and curative effect enhancement, and is pronounced in effect when applied to acne treatment through experimental verification.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a drug-loaded liposome protein hydrogel complex, its preparation method, and its application. Background Technology

[0002] Acne vulgaris, commonly known as acne, is a chronic inflammatory skin disease affecting the pilosebaceous unit. It primarily affects the pilosebaceous unit on the face, neck, back, and chest. The condition usually begins in adolescence, but in some cases it can persist into adulthood. Currently, there are various treatments for acne, but each method has its limitations. Topical medications such as retinoids and benzoyl peroxide are often irritating, affecting patient compliance, while topical antibiotics face the problem of drug resistance. Oral antibiotics also carry the risk of drug resistance and have many side effects; isotretinoin, in particular, requires strict monitoring due to its teratogenicity. In terms of physical therapy, red and blue light therapy has limited efficacy, chemical peels may cause irritation, laser treatments are expensive and carry the risk of pigmentation, and improper extraction of pimples can easily lead to infection and scarring. The challenges lie in the complexity of acne etiology, individual variability, poor patient compliance, antibiotic resistance, and the high cost of some treatments. Therefore, it is crucial to develop new treatment strategies that are safer, more effective, and can overcome the limitations of existing treatments.

[0003] The fibroblast growth factor (FGF) superfamily comprises a group of structure-associated signal transduction proteins with a conserved core of 150-300 amino acids and approximately 20 amino acids. The FGF superfamily consists of 22 members (FGF1-FGF23), divided into 7 subfamilies based on sequence and functional characteristics. FGF15 and FGF19 are homologous genes in rodents and humans, respectively. FGF21 is an important member of the FGF superfamily, playing a pleiotropic role in metabolic homeostasis, inflammation suppression, and tissue repair.

[0004] Fibroblast growth factor 21 (FGF21) has shown potential in regulating inflammatory responses and improving skin barrier function. However, the direct application of FGF21 faces several challenges. First, as a polypeptide molecule, FGF21 has inherent stability defects: its C-terminal region is susceptible to protease hydrolysis and is sensitive to temperature and pH, easily accumulating or degrading under normal storage conditions, leading to loss of activity. Second, FGF21 has an extremely short half-life in vivo (approximately 0.5-2 hours), which severely limits its bioavailability and dosing frequency, making it difficult to maintain effective therapeutic concentrations. Furthermore, as a large protein molecule, FGF21 has difficulty effectively penetrating the stratum corneum to reach deep targets in hair follicles, resulting in limited local bioavailability. Simultaneously, systemic administration may lead to non-specific distribution, while traditional topical application fails to achieve drug accumulation in acne lesions.

[0005] Nanoparticles are an effective means to improve drug bioavailability, prolong half-life, achieve controlled release, reduce off-target side effects, and enable targeted delivery. Liposomes are a mature therapeutic drug carrier system with over 50 years of history in drug delivery applications, possessing biodegradability, biocompatibility, low immunogenicity, and potential for controlled release and targeted delivery. Liposomes are mainly composed of amphipathic lipid molecules (usually phospholipids), which self-assemble into spherical structures in an aqueous environment to reduce the interaction between the hydrophobic portion and the aqueous environment. They can carry both hydrophobic and hydrophilic drugs, protecting them from degradation and allowing them to enter cells via endocytosis. However, liposomes also have some limitations, such as a tendency to fuse and leak, leading to premature drug release, and limited residence time on the skin surface, making it difficult to achieve sustained therapeutic effects. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a drug-loaded liposome protein hydrogel complex, its preparation method, and its applications.

[0007] To achieve its objective, the technical solution adopted by this invention is as follows: The first aspect of the present invention provides a drug-loaded liposome protein hydrogel complex, wherein the complex is obtained by encapsulating nanoliposomes loaded with fibroblast growth factor FGF21 in a BSA-modified hydrogel, wherein the BSA-modified hydrogel is a bovine serum albumin-based hydrogel modified with isopropyltriethoxysilane ICPS.

[0008] Preferably, the liposomes are soybean lecithin-cholesterol nanoliposomes.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned drug-loaded liposome protein hydrogel complex, comprising the following steps: S1. Prepare the nanoliposomes described above; S2. The drug FGF21 was loaded into nanoliposomes to obtain drug-encapsulated nanoliposomes FGF21-NLip; S3. Prepare the BSA-modified hydrogel solution; S4. Preparation of the drug-loaded liposome protein hydrogel complex: Disperse the FGF21-NLip obtained in step S2 into the BSA modified hydrogel solution prepared in step S3, and add an initiator to initiate a cross-linking reaction to obtain the drug-loaded liposome protein hydrogel complex.

[0010] In step S1, the nanoliposomes are prepared using a reverse-phase evaporation method, including the following steps: (1) Preparation of oil phase: Soybean lecithin and cholesterol were dissolved in chloroform at a mass ratio of 3~5:1 to obtain an oil phase solution; (2) Aqueous phase preparation: Prepare buffer A; preferably, buffer A is a citrate buffer with pH 4.6~4.8 and a concentration of 0.2~0.4mol / L; (3) Preparation of W / O emulsion: Add buffer A to the oil phase solution, mix and emulsify to obtain W / O emulsion; (4) Reverse phase evaporation and hydration: The W / O emulsion was transferred to a rotary evaporator to remove the organic solvent by rotary evaporation, which promoted the self-assembly of lipid molecules; then, buffer A was added to hydrate and reconstitute the hydrate. (5) Preparation of liposome suspension: The hydrate was removed by rotary evaporation and then subjected to a second vacuum evaporation to further remove residual organic solvent and promote the formation of liposomes, so as to obtain liposome suspension; (6) Particle size uniformization treatment: The liposomes in the liposome suspension are uniformly dispersed, and then the particle size is fined by microporous membrane filtration and liposome extruder in sequence to obtain nanoliposomes.

[0011] In step S2, the drug solution is mixed with the nanoliposomes prepared in step S1 and incubated at 35-40°C for 1-3 hours to achieve effective drug loading. The preferred mass ratio of FGF21 to liposomes is 1.6~2.0 mg: 700~800 mg; Preferably, the drug solution is an FGF21 solution with a concentration of 4.0~5.0 mg / mL, prepared by mixing 0.4 mL of FGF21 solution with 7~8 mL of nanoliposome suspension with a concentration of 90~110 mg / mL.

[0012] In step S3, the preparation method of the BSA modified hydrogel solution is as follows: Bovine serum albumin (BSA) is dissolved in buffer B, then ICPS is added, and the mixture is stirred at 35~40℃ to generate a BSA-ICPS conjugate solution, thus obtaining the BSA modified hydrogel solution. The preferred reaction time is 3.5~4.5h.

[0013] The buffer B is PBS buffer, and the mass-to-volume ratio of BSA, ICPS and buffer B is 100mg:90~110μL:1.4~1.6mL, preferably 100mg:100μL:1.5mL.

[0014] In step S4, the FGF21-NLip obtained in step S2 is dispersed into the BSA-modified hydrogel solution prepared in step S3, an initiator is added, and the mixture is stirred at 35-40°C and 350-450 rpm until the mixture self-assembles to form a hydrogel, and a cross-linking reaction is initiated to obtain a drug-loaded liposome protein hydrogel complex; preferably, the initiator is sodium fluoride.

[0015] In step S4, the ratio of FGF21-NLip, BSA-ICPS conjugate solution, and initiator obtained in step S2 is 700~800mg:8mL:30~34mg.

[0016] A third aspect of the present invention provides the use of the drug-loaded liposome protein hydrogel complex described above or the drug-loaded liposome protein hydrogel complex prepared by the above preparation method in the preparation of a medicament for treating acne.

[0017] The beneficial effects of this invention are: (1) Considering the complex pathological characteristics of acne, we selected widely used albumin—bovine serum albumin (BSA)—as the gel matrix. BSA not only possesses good biocompatibility, minimizing adverse reactions, but also exhibits excellent swelling, bioadhesion, stretching, and photothermal properties. These characteristics facilitate good adhesion between the gel and the acne lesion, promoting drug penetration and action. Using ICPS to modify BSA, we successfully prepared a hydrogel with an interconnected porous network structure. This hydrogel possesses excellent mechanical properties and good rheological characteristics, maintaining its morphology at the lesion site while being easy to inject, thus benefiting clinical application. The hydrogel has good moisturizing ability and controllable swelling properties, effectively releasing drugs and exhibiting different release kinetic characteristics.

[0018] (2) An innovative synergistic delivery system was constructed by combining nanoliposomes with BSA hydrogel. This system integrates the advantages of both, overcomes their respective limitations, and achieves controlled drug release and enhanced efficacy. On the one hand, BSA hydrogel can compensate for the mechanical defects of liposomes, prolong the residence time of drugs at the lesion site, and achieve sustained drug release. On the other hand, liposomes can improve the transdermal permeability of drugs, overcome the diffusion limitations of hydrogels, and accelerate the arrival of drugs at the target site. The working mechanism of this synergistic delivery system includes: liposomes encapsulate FGF21, protecting it from degradation and promoting its penetration through the skin barrier. Subsequently, the liposomes slowly release FGF21 in the BSA hydrogel, while BSA further promotes drug penetration by hydrating the skin. At the same time, its wound-healing effect can also accelerate the repair of acne lesions.

[0019] (3) The core innovations of this invention compared to the prior art are fourfold: a dual delivery system design, innovative breakthrough of liposome and hydrogel dual protection, sustained release for more than 72 hours; significantly improved acne treatment effect of FGF21, synergistic effect of multiple mechanisms, anti-inflammatory, repair and metabolic regulation; efficient encapsulation and improved stability, intelligent release and moisturizing synergy; clinical translation potential, injectability and viscoelasticity suitable for skin administration, moisturizing performance prolongs the duration of action, providing a "one-step" treatment strategy for acne, avoiding frequent administration, and providing a brand-new solution for acne treatment: ① This invention evaluated the biocompatibility of NLip, BSA@GEL and FGF21-NLip@B materials through in vitro experiments. The results showed that these materials have good cell compatibility and blood compatibility; compared with free FGF21, FGF21-NLip@B can significantly promote the proliferation and migration of HaCaT cells, indicating that it has excellent pro-repair ability. Moreover, the materials prepared in this study have potential hydroxyl radical scavenging ability. ② This invention demonstrated the most significant therapeutic effect on acne in a mouse model of acne, superior to the positive control group, the NLip@B group, and the FGF21 group, effectively alleviating inflammation, inhibiting sebaceous gland hyperplasia, and promoting skin repair. ③ This invention is safe at therapeutic doses. The establishment of the acne model and the intervention of drugs such as FGF21-NLip@B were mainly limited to local skin lesions, without causing systemic pathological damage to the major internal organs (heart, liver, spleen, lungs, and kidneys) of the mice. ④ This invention can significantly improve the pathological characteristics of acne (epidermal thickening, keratin plugging, and inflammatory infiltration). Its anti-inflammatory and repair effects are significantly better than those of the NLip@B group and the FGF21 group, while the latter two have relatively limited therapeutic effects, with only slight improvements observed and significant infiltration of inflammatory factors remaining. This indicates that the nanodelivery system can effectively enhance the targeted therapeutic activity of FGF21. Attached Figure Description

[0020] Figure 1 The stability study of FGF21-NLip includes: (A) changes in particle size and PDI of FGF21-NLip over 30 days; and (B) changes in zeta potential and encapsulation efficiency of FGF21-NLip over 30 days.

[0021] Figure 2 The following images are shown: (A) Fourier transform infrared spectra of BSA, BSA-ICPS and BSA@Gel (B@G); (B) SEM image structure of NLip-BSA hydrogel (scale bar = 50 μm); (C) SEM image structure of NLip-BSA hydrogel (scale bar = 20 μm).

[0022] Figure 3The following images are shown: (A) Frequency scans of B@G and NLip@B hydrogels; (B) Strain scans of B@G and NLip@B hydrogels; (CD) Injectable expression of the hydrogels and photographs of the “BSA” hydrogel.

[0023] Figure 4 The following data are shown: (A) the water retention of B@G and NLip@B hydrogels; (B) the swelling ratio of B@G and NLip@B hydrogels at different times; (C) the in vitro degradation curves of B@G and NLip@B hydrogels; and (D) the drug release rates of FGF21-NLip and FGF21-NLip@B (n=3).

[0024] Figure 5 The following are shown: (AC) Live / dead staining: fluorescence staining results of HaCaT cells co-cultured with each group of materials for 1 day (A), 3 days (B), and 7 days (C) (scale bar: 100 μm); (D) in vitro cell viability of each component at 1 day, 2 days, and 3 days (n=3); (E) photographs of hemolysis assays from water, saline, NLip, BSA@GEL, and FGF21-NLip@B; (F) hemolysis rates of water, saline, NLip, BSA@GEL, and FGF21-NLip@B hydrogels.

[0025] Figure 6 The following were shown: (A) morphology of human keratinocyte scratch marks at 0, 12, 24, and 36 hours; (B) growth of HaCaT at different concentrations of FGF21; (C) detection of the migration-promoting ability of different components on HaCaT. Compared to Con, n=3).

[0026] Figure 7 The in vitro antioxidant experiment of the hydrogel is shown: (A) the absorbance of methylene blue solution in different samples under Fenton system as a function of time, where the characteristic wavelength of methylene blue is 667 nm; (B) the color change of methylene blue under 5 different Fenton reactions.

[0027] Figure 8 A comparative diagram showing the pathological evolution and therapeutic effects of treatment in a mouse acne model.

[0028] Figure 9 Comparative images of HE staining pathological findings of heart, liver, spleen, lung, and kidney tissues from each group of mice; scale bar: 50 μm.

[0029] Figure 10 Dynamic pathological changes of back skin tissue stained with HE in a mouse acne model; scale bar: 250 μm.

[0030] Figure 11Masson staining of collagen fibers in skin tissue of a mouse acne model to assess the effect of treatment intervention; scale bar: 250 μm.

[0031] Figure 12 Immunohistochemical staining and pathological observation of skin tissue from mice in different treatment groups; scale bar: 100μm.

[0032] Figure 13 The results of serum inflammatory factor levels in mice in each group are shown (ELISA, Mean±SD, n=7). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] Unless otherwise specified, the methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials used are conventional reagents and materials in the art and can be obtained commercially.

[0035] Example 1: Preparation and characterization of FGF21 nanoliposome hydrogel 1. Main experimental reagents and their sources

[0036] 2 Experimental Methods 2.1 Preparation of nanoliposomes (1) Preparation of oil phase (O phase): Weigh 400 mg of soybean lecithin and 100 mg of cholesterol and dissolve them in 3 mL of chloroform. This step aims to form the framework of lipid nanoparticles and to use organic solvents to ensure that the lipid components are fully mixed and uniformly dispersed.

[0037] (2) Preparation of aqueous phase (W phase): Prepare a 0.3 mol / L citrate buffer solution and precisely adjust its pH value to 4.7. The low pH aqueous phase environment is the key to subsequent drug encapsulation, and FGF21 is driven into the liposomes through the pH gradient.

[0038] (3) Preparation of W / O emulsion: 1 mL of aqueous phase (W phase) was slowly added dropwise to 3 mL of oil phase (O phase) using a micropipette (volume ratio 1:3). Emulsification was performed using an ultrasonic disruptor, with the operating mode set to pulsed ultrasound for 3 seconds / interval for 3 seconds, for a total processing time of 3 minutes. This step aims to form a stable W / O emulsion, providing a prerequisite for subsequent reverse-phase evaporation. The phase stability of the W / O emulsion was greater than 30 minutes, indicating that the system has good dispersibility and stability.

[0039] (4) Reverse-phase evaporation and hydration: The W / O emulsion was transferred to a rotary evaporator and evaporated under reduced pressure in a constant temperature water bath at 55°C until the system reached a semi-transparent gel state. This process removed the organic solvent chloroform, promoting the self-assembly of lipid molecules. Then, 4 mL of citrate buffer solution (pH 4.7) with the same concentration as the initial aqueous phase was added for hydration reconstruction.

[0040] (5) Preparation of liposome suspension: Continue vacuum evaporation at 37°C to further remove residual organic solvent chloroform and promote the formation of liposomes to obtain blank liposome suspension.

[0041] (6) Particle size uniformization treatment: The liposome suspension was subjected to probe-type ultrasonic treatment at 25°C (5 seconds of ultrasonic treatment / 5 seconds of intermittent circulation, total duration of 5 minutes) to ensure uniform dispersion of liposomes and reduce aggregation. Subsequently, the particle size was finely treated by filtration through a 220nm microporous membrane and a liposome extruder to finally obtain a nano-sized liposome (NLip) suspension, ensuring the uniformity and controllability of the particle size of the nano-drug delivery system.

[0042] (7) Drug loading: 0.4 mL of FGF21 (amino acid sequence see UNIPROT ID: Q9NSA1) solution (4.5 mg / mL) with pH adjusted to 7.8 was mixed with 7.6 mL of NLip suspension (concentration of 100 mg / mL) and incubated in a 37°C incubator for 3 hours. The pH gradient was used to drive FGF21 into the liposomes to achieve effective drug loading, and finally the FGF21 nanodrug delivery system (FGF21-NLip) was obtained.

[0043] 2.2 Characterization of NLip and FGF21-NLip The particle size distribution, polydispersity index (PDI), and zeta potential of nanoliposomes were determined using a laser particle size analyzer (Litesizer 500, Anton Paar, South Korea). The specific procedure was as follows: First, the sample was diluted 5-fold, and 2 mL was placed in a four-way quartz cuvette. The particle size distribution and PDI values ​​of blank nanoliposomes (NLip and FGF21-NLip nanoliposomes loaded with FGF21) were measured using the laser particle size analyzer. Then, 1 mL of the sample was injected into an Ω-shaped zeta potential capillary tube, and the zeta potential values ​​of NLip and FGF21-NLip were measured inside the instrument. To ensure data reliability, each sample was measured in triplicate.

[0044] 2.3 Encapsulation efficiency test (1) Free FGF21 separation: Take 1.5 mL of FGF21-NLip suspension, centrifuge at 150,000×g at 4℃, carefully aspirate the supernatant (1.4 mL), label it as "free FGF21", and store it at -20℃.

[0045] (2) Total FGF21 release: The precipitate was resuspended in 1.4 mL PBS and sonicated (ice bath, 10% power, 5 seconds / cycle, 5-second interval, total 30 seconds). 56 μL of trypsin (2.5% w / v, final concentration 100 μg / mL) was added, and the mixture was incubated at 37 °C for 2 hours, vortexing once every 30 minutes. Then, 14 μL of Triton X-100 (10% v / v, final concentration 0.1%) was added, and the mixture was incubated at 37 °C for another 30 minutes. The mixture was centrifuged at 12,000 × g for 10 minutes, and 1.0 mL of the supernatant was ultrafiltered and concentrated to 200 μL, labeled "Total FGF21", and stored at -20 °C.

[0046] (3) FGF21 content determination (ELISA): Remove the ELISA kit from the -20°C freezer and allow it to equilibrate at room temperature (20-25°C) for 30 minutes. Prepare a standard curve with 7 concentration points using the provided standard diluent, following the kit instructions. Repeat each concentration point twice to ensure the accuracy of the standard curve.

[0047]

[0048] In the formula, C 总 Total FGF21 concentration; C 游离 : Free FGF21 concentration; V 上清液 Volume of supernatant (≈1.4 mL); V 总 Total volume of suspension (2.0 mL).

[0049] 2.4 Stability Study of Nanoliposomes Take an appropriate amount of FGF21-NLip suspension and store it in a refrigerator at 4°C. The particle size, zeta potential, and encapsulation efficiency of the FGF21 nanoliposomes were measured at 1, 3, 7, 14, and 30 days to evaluate their stability.

[0050] 2.5 Preparation of nanoliposome gels Preparation of bovine serum albumin (BSA)-based hydrogel: The following raw material ratios were used: 100 mg BSA was dissolved in 1.5 mL of phosphate-buffered saline (PBS), and sonication was used to ensure complete dissolution. Then, 100 μL of propyltriethoxysilane (ICPS) was added to the BSA solution, and the mixture was magnetically stirred at 37 °C for 4 hours to generate a BSA-ICPS conjugate solution (i.e., BSA-modified hydrogel solution). 4 mg of sodium fluoride (NaF) was added to 1 mL of the obtained BSA-ICPS conjugate solution as an initiator, and the mixture was stirred at 37 °C and 400 rpm for 3-4 hours until the cross-linking reaction was complete, thus self-assembling to form a bovine serum albumin protein hydrogel (BSA Based hydrogel, B@G).

[0051] To construct the supported hydrogel system, 1 mL of nanoliposomes (NLip) prepared in step (6) of “2.1 Preparation of nanoliposomes” or 1 mL of nanoliposomes loaded with FGF21 (FGF21-NLip) prepared in step (7) of “2.1 Preparation of nanoliposomes” was first dispersed in 8 mL of BSA-ICPS conjugate solution, and then 32 mg of sodium fluoride (NaF) was added and stirred at 37 °C and 400 rpm for 3-4 h until the crosslinking reaction was completed.

[0052] Through the above steps, protein hydrogel complexes encapsulated by nanoliposomes (NLip@B) and protein hydrogel complexes encapsulated by nanoliposomes loaded with FGF21 (FGF21-NLip@B) were prepared, thereby achieving efficient delivery of nanomedicines.

[0053] 2.6 Fourier Transform Infrared Spectroscopy Detection To verify the successful coupling of propyltriethoxysilane isocyanate (ICPS) and bovine serum albumin (BSA), this study employed Fourier transform infrared spectroscopy (FT-IR, PerkinElmer Frontier, USA) for characterization and analysis. The specific steps were as follows: the BSA-ICPS solution was freeze-dried to obtain BSA-ICPS lyophilized powder, which was then finely ground using a mortar and pestle. Subsequently, the finely ground powder was placed on the sample stage of a Fourier transform infrared spectrometer for spectral scanning analysis to determine whether ICPS was successfully coupled to the BS molecule.

[0054] 2.7 Rheological determination of NLip@B and B@G Dynamic strain scanning and dynamic frequency scanning tests were performed on the two types of gels. First, an appropriate amount of B@G gel was placed on the test pan of a rotational rheometer, the frequency was fixed at 1 Hz, and the strain range was set to 1% to 1000%, followed by dynamic strain scanning. During the scanning process, the elastic modulus G' and viscous modulus G" were used as the main observation indicators, and the changes of G' and G" over time were recorded in detail. The phase transition moment of the hydrogel was determined to be the point at which G' and G" values ​​were equal. Next, to investigate the variation of the hydrogel modulus with the oscillation frequency, another appropriate amount of B@G gel was placed on the test pan, and the oscillation frequency range was set to 0.1 Hz to 100 Hz for testing. Subsequently, the same experimental method was used to perform dynamic strain scanning and dynamic frequency scanning experiments on FGF21-NLip@B gel to comprehensively analyze its rheological properties.

[0055] 2.8 Determination of the moisturizing ability and in vitro degradation characteristics of NLip@B and B@G Accurately weigh NLip@B and B@G samples (100 mg / group) and place them in petri dishes, with three replicates per group. Transfer the petri dishes to a constant temperature and humidity chamber (32℃, 60% humidity) to simulate the temperature and humidity environment of the skin surface. Weigh the remaining mass of the samples at preset time points (0h, 1h, 2h, 4h, 6h, 8h, 12h, 24h) and record the data. The moisture retention rate is calculated using the following formula:

[0056] In the formula, W 0 Initial mass; W t The mass at time t.

[0057] To evaluate the in vitro degradation characteristics of the hydrogel, accurately weighed NLip@B and B@G samples (approximately 50 mg) were placed in centrifuge tubes, with three replicates per group. Sufficient PBS buffer was then added to each centrifuge tube to completely submerge the hydrogel, and an appropriate amount of trypsin was added to simulate the enzymatic degradation environment under physiological conditions. The centrifuge tubes were placed in a constant-temperature shaker set to 37°C and incubated at 100 rpm. At pre-set time points (days 0, 1, 2, 4, 7, 10, and 14), the centrifuge tubes were removed for further processing, and the supernatant was carefully removed after centrifugation at 5000 rpm for 5 minutes. To remove residual salts and enzymes, the precipitate was washed twice with deionized water. Finally, the precipitate was freeze-dried to constant weight, and the mass of the residue after drying was weighed and recorded. The in vitro degradation rate was calculated using the following formula: In vitro degradation rate (%) = ( W t / W 0) × 100%, of which W 0 For the initial mass, W t Let t be the residual mass after drying.

[0058] 2.9 Evaluation of in vitro release rate of drug-loaded nanoliposome-hydrogel To evaluate FGF21 release from FGF21-NLip and FGF21-NLip@B, regenerated cellulose dialysis bags with a MWCO of 30 kDa were used. Pretreatment of the dialysis bags included cutting them to approximately 10 cm, rinsing the inner and outer surfaces with deionized water, and soaking for at least 30 minutes to wet them. 1.0 mL of each sample was precisely loaded into the dialysis bag, air bubbles were removed, and the bags were sealed with dialysis clamps. The dialysis bags were then immersed in 50 mL centrifuge tubes containing 20 mL of PBS (pH 7.4) with 1% protease inhibitor and placed in a constant-temperature shaker at 37°C and 100 rpm. Based on the preliminary experimental results, sampling time points were set (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h). Each time, 1.0 mL of dialysate was carefully removed, transferred to a sterile low-protein adsorption centrifuge tube, and labeled. An equal volume of fresh PBS was immediately added to maintain a constant system volume. The collected samples were stored at -80℃ until ELISA detection was performed. All samples were measured at least three times.

[0059] 2.10 Injectability testing of NLip@B and FGF21-NLip@B First, we prepared the NLip@B hydrogel. Then, using a 5 ml syringe, we drew up an appropriate amount of the NLip@B hydrogel and continuously injected it into a culture dish, cleverly forming the letters "BSA". We evaluated its injectability by observing whether the hydrogel could maintain its gel state.

[0060] 2.11 Preparation of gel leachate To evaluate the cytotoxicity of hydrogels, extraction solutions of three protein hydrogels—B@G, NLip@B, and FGF21-NLip@B—were prepared according to ISO 10993-5:2009, the standard for biological evaluation of medical devices. The specific procedure was as follows: 0.5 mL of hydrogel sample was placed in a 15 mL sterile centrifuge tube, and 10 mL of cell culture medium containing 0.5% serum was added. The mixture was gently inverted to ensure the hydrogel was fully immersed in the medium. The centrifuge tube was then incubated at 37°C for 24 hours. After incubation, the original medium was transferred to a new 15 mL sterile centrifuge tube, resulting in a 1:20 volume ratio gel extraction solution. By varying the amount of 0.5% serum culture medium added to the centrifuge tube, hydrogel extraction solutions with different volume ratios could be prepared for subsequent cytotoxicity experiments.

[0061] 2.12 Recovery, culture, and cryopreservation of keratinocytes (HaCaT) Standard operating procedure.

[0062] 2.13 In vitro blood compatibility This experiment aimed to evaluate the effect of a gel material on hemolysis of mouse erythrocytes. First, blood was collected from the mouse orbital cavity and placed in a blood collection tube containing an anticoagulant. The blood was gently mixed to prevent clotting. To obtain a pure erythrocyte suspension, the blood was transferred to a 1.5 mL centrifuge tube, 1 mL of physiological saline was added, and the mixture was gently mixed and centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was carefully removed, and another 1 mL of physiological saline was added. This centrifugation and washing process was repeated 6 times until the supernatant was clear, indicating effective removal of plasma proteins. Subsequently, erythrocytes from the bottom of the centrifuge tube were collected and diluted with physiological saline to a concentration of 5% (v / v) to prepare a erythrocyte suspension. For the hemolysis experiment, 10 mg of the gel material was accurately weighed and placed in a 1.5 mL centrifuge tube. The experimental design was as follows: 500 μL of physiological saline and 500 μL of 5% erythrocyte suspension were added to centrifuge tubes containing gel, respectively; the positive control group consisted of 500 μL of sterile pure water and 500 μL of 5% erythrocyte suspension; the negative control group consisted of 500 μL of physiological saline and 500 μL of 5% erythrocyte suspension. All samples were incubated at 37°C for 1 h. After incubation, the samples were centrifuged at 3000 rpm for 5 min, and the state of each group after centrifugation was recorded by photograph. To quantitatively assess the degree of hemolysis, 200 μL of supernatant was carefully aspirated from each group and added to a 96-well plate. Subsequently, the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. According to the pre-set formula, the hemolysis rate could be calculated as follows: Hemolysis rate (%) = (Absorbance As of sample group - Absorbance Ab of negative control group) / (Absorbance Ap of positive control group - Absorbance Ab of negative control group) × 100%. Where As represents the absorbance value of the sample group, Ab represents the absorbance value of the negative control group, and Ap represents the absorbance value of the positive control group. Finally, to observe changes in erythrocyte morphology, 50 μL of erythrocyte suspension was added to 1 mL of physiological saline and gently mixed. 20 μL of the mixture was dropped onto a glass slide, covered with a coverslip, and the erythrocyte morphology was observed using an upright microscope, and the images were recorded.

[0063] 2.14 In vitro cytotoxicity assessment This study aimed to evaluate the cytotoxicity of hydrogel samples B@G extract, FGF21-NLip@B extract, and NLip.

[0064] First, 1×10 5Cells were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to observe cell status and quantity. When the cell density reached approximately 90%, the medium was replaced with 0.5% serum for 24 hours of serum starvation to reduce potential interference from serum on experimental results. Afterward, the original medium was discarded, and an equal volume of B@G extract, NLip@B extract, NLip solution, or medium containing only 0.5% serum was added to each well, and the plates were co-cultured for 6 hours. Subsequently, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for another 2 hours. Finally, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader, with cell-free wells serving as blank controls. Cell viability was calculated using the following formula: Cell viability (%) = ( As - A 0 ) / ( Ac - A 0 ) × 100%, of which As This represents the absorbance value of the hydrogel sample well at 450 nm. Ac The absorbance value at 450 nm represents the culture medium content in 0.5% serum culture medium. A 0 This represents the absorbance value of the blank aperture at 450nm.

[0065] To further investigate the effects of B@G leaching solution, FGF21-NLip@B leaching solution, and NLip on cell viability, we used the Calcein-AM / PI cell viability kit for staining experiments. 1×10 5 Cells were seeded into cell culture chambers of 12-well plates and cultured in a 37°C, 5% CO2 incubator. After cell attachment, NLip, B@G, and NLip@B were added to the top layer, and the cells were cultured for another 3 days. On days 1, 3, and 7, the cells were co-stained with Calcein-AM and PI dyes for 20 minutes. Then, cell growth at different time points was observed and photographed using a fluorescence inverted microscope to assess the effect of each sample on cell viability.

[0066] 2.15 In vitro determination of effective drug concentration of FGF21 HaCaT cell lines were cultured in a suitable medium containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator until 80-90% confluence. After trypsin digestion, cell density was adjusted, and single-cell suspensions were seeded into 9-well plates for subsequent cell proliferation experiments. After seeding, cells were cultured at 37°C in a 5% CO2 incubator for 24 hours to ensure full adhesion. The original medium was removed, and fresh medium containing different concentrations of FGF21 (100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL) was added, with three replicates per group. Cells were then cultured for another 24 hours at 37°C in a 5% CO2 incubator. After 24 or 48 hours of FGF21 treatment, follow the instructions of the CCK-8 kit, measure the absorbance value using an ELISA reader, and calculate the cell viability using the formula "Cell viability (%) = (OD sample group / OD blank control group) × 100%".

[0067] 2.16 HaCaT-induced migration detection HaCaT cells were used at a rate of 5 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured at 37°C in a 5% CO2 incubator until confluence reached over 90%. A 10 μL pipette tip was used to create a cross-shaped scratch, ensuring consistent scratch width across all wells. After washing three times with PBS to remove cell debris, serum-free medium containing 1% FBS was added to minimize interference from cell proliferation. Subsequently, according to experimental groups, medium containing different concentrations of FGF21-NLip, FGF21-NLip@B, or free FGF21 was added, with at least three replicates per group. Cells were further cultured at 37°C in a 5% CO2 incubator. At 0 h, 24 h, and 48 h post-scratching, microscopic photographs were taken at the same cell locations, ensuring consistent field of view, and the scratch width was recorded for subsequent analysis.

[0068] The scratch test was conducted in 6-well plates, initially seeded with 5 × 10⁶ cells / well. 5 Cells were collected and 6-well plates were incubated overnight in a cell culture incubator. After 24 hours, the cell status and number in the wells were observed. When the cells reached approximately 90% confluence, a 10 μL pipette tip was used to make a cross-shaped incision on the cell layer. The cells were then washed three times with PBS to completely remove any incisively incised cells. Next, an extract of FGF21 (100 mg / mL), FGF21-NLip (containing 100 mg / mL FGF21), and FGF21-NLip@B (containing 100 mg / mL FGF21) along with 0.5% serum medium were added to the wells, and the plates were returned to the cell culture incubator for further incubation. Cell migration was recorded and photographed at 0 h, 12 h, 24 h, and 36 h.

[0069] 3. Experimental Results 3.1 Characterization of FGF21-NLip Laser particle size analyzer analysis revealed that the average particle size of the NLip group was 71.09 nm ± 7.63 nm, the polydispersity index was 0.167 ± 0.02, and the zeta potential was -44.0 ± 1.25 mV. The average particle size of the FGF21-NLip group was 76.25 nm, with a standard deviation of 5.56 nm, a polydispersity index of 0.185 ± 0.01, a zeta potential of -46.7 ± 2.07 mV, and an encapsulation efficiency of 76.2 ± 1.8%.

[0070] The results showed that the particle size of the FGF21-NLip group was slightly larger than that of the NLip group, but both had relatively uniform particle size distribution and low polydispersity index, indicating a relatively uniform particle distribution. Regarding zeta potential, both groups exhibited high negative charges, indicating good stability of the nanoliposomes. The encapsulation efficiency of the FGF21-NLip group was 76.2%, indicating that FGF21 was effectively encapsulated within the liposomes. Overall, both groups of nanoliposomes exhibited good characteristics in terms of particle size, polydispersity index, and zeta potential, with the FGF21-NLip group achieving a higher encapsulation efficiency. TEM observation of the morphology of NLip and FGF21-NLip showed that the liposomes were spherical with a particle size of approximately 70-80 nm.

[0071] Table 1 Characterization of nanoliposomes

[0072] 3.2 Stability Study of FGF21-NLip Stability assessment of FGF21-NLip nanoliposomes showed that ( Figure 1 During the 30-day observation period, the physicochemical properties of the liposomes showed slight changes over time, but remained generally within acceptable limits. The particle size showed a slight increasing trend, and the PDI value also increased slightly, but the system maintained good dispersibility throughout. The absolute value of the Zeta potential decreased slightly, but remained above 40 mV, indicating acceptable electrostatic stability. However, the encapsulation efficiency gradually decreased, suggesting that the drug loading capacity of the liposomes decreased over time. Based on the above results, it can be concluded that the FGF21-NLip nanomedicine formulation successfully prepared in this embodiment has good stability, meeting the requirements for subsequent in vitro and in vivo experimental studies.

[0073] 3.3 Preparation and Characterization of FGF21-NLip@B 3.3.1 Preparation of FGF21-NLip@B Serum albumin, especially bovine serum albumin (BSA), is often used as a multifunctional platform to mimic the natural extracellular matrix (ECM) due to its crucial role in maintaining osmotic pressure and transporting various bioactive substances. In this study, BSA was modified with dimethyl silylation using ICPS. The reaction of ICPS with the -NH2 group in the BSA molecule endowed BSA with the potential to form hydrogels. Fourier transform infrared (FTIR) spectroscopy results are presented below. Figure 2 A shows that, compared to unmodified BSA, BSA-ICPS and BSA@Gel (B@G) show improvements in 1000-1100 cm⁻¹. -1 Stretching vibration peaks of Si-O-Si bonds appear within the range of 1640-1700 cm⁻¹. -1 Characteristic absorption peaks of the carbonyl group (C=O) in the urea bond were observed within the range. The appearance of these characteristic peaks further confirms that ICPS has been successfully modified onto the BSA molecule. Scanning electron microscopy results are as follows: Figure 2 As shown in BC, all NLip@B hydrogels have an interconnected porous network structure, and NLip can be uniformly dispersed inside the NLip@B hydrogel.

[0074] 3.3.2 Characterization of FGF21-NLip@B To evaluate the mechanical properties and stability of the FGF21-NLip@B hydrogel, amplitude scanning experiments were conducted, measuring the storage modulus (G') as a function of strain (Strain%) at two frequencies: 1 Hz and 10 Hz. Figure 3 As shown in Figure B, in the low strain region (1%-10%), G' remains relatively stable at both frequencies, indicating that the FGF21-NLip@B hydrogel possesses a distinct linear viscoelastic region. Within this region, the gel structure is not significantly disrupted, exhibiting inherent elasticity. To investigate the mechanical response of the BSA hydrogel at different timescales, we conducted frequency sweep experiments, measuring the storage modulus (G') and loss modulus (G'') of the gel in the frequency range of 0.1 Hz to 10 Hz. Figure 3 As shown in Figure A, G' is consistently higher than G'' across the entire frequency range, indicating that the BSA hydrogel exhibits pronounced gel properties, with elastic behavior being dominant. Furthermore, the frequency dependence of G' and G'' is relatively weak, with small variations across the entire frequency range. Within this region, the gel structure remains largely intact, exhibiting inherent elasticity. In summary, the gel demonstrates stable G' values ​​and frequency dependence over a wide strain range, confirming its excellent mechanical properties and favorable rheological characteristics. Furthermore, [the text abruptly ends here, likely due to an incomplete translation or source material]. Figure 3 As indicated by CD, the injectability of the gel was verified through a syringe extrusion test. All these results fully demonstrate the potential application value of this gel in the treatment of acne.

[0075] 3.4 Analysis of the properties and drug release behavior of FGF21-NLip@B hydrogel This study successfully constructed an FGF21 sustained-release delivery system based on NLip@B hydrogel and comprehensively evaluated its key performance characteristics. Figure 4 As shown in AB, both NLip@B and B@G hydrogels exhibit good moisturizing ability and controllable swelling properties, which helps maintain the moisture balance of the local microenvironment. Experimental results are as follows... Figure 4 As shown in Figure C, within a 14-day experimental period, the degradation rate of B@G was 80%, and the degradation rate of NLip@B was 92%. This indicates that both hydrogels can be effectively degraded to a high degree. A high degree of degradation usually means that the material can be effectively removed from the body, avoiding the potential risks of long-term residue. The results show that both delivery systems can effectively release FGF21 and exhibit different release kinetics. The FGF21-NLip group had a faster release rate in the early stages, reaching a cumulative release of 98% within 72 hours. In contrast, the FGF21-NLip@B group had a relatively slower release rate, but still achieved a cumulative release of 95% within 72 hours, making it more suitable for maintaining an effective concentration of FGF21 in the long term, promoting the repair of damaged skin and reducing scar formation. Figure 4 D). In summary, the study shows that the NLip@B hydrogel delivery system prepared in this study has excellent moisturizing properties, controllable degradation behavior, and sustained-release characteristics, which can effectively improve the treatment effect and reduce side effects, showing broad application prospects in the field of acne treatment.

[0076] 3.5 In vitro biocompatibility assessment of FGF21-NLip@B This study evaluated the biocompatibility of NLip, BSA@GEL, and FGF21-NLip@B materials using in vitro experiments. Live / dead staining results. Figure 5 AC indicates that after co-culturing with HaCaT cells for 1, 3, and 7 days, cells treated with different materials may exhibit varying degrees of green (live cells) and red (dead cells) fluorescence. The control group (culture medium containing 0.5% serum) showed higher green fluorescence intensity and lower red fluorescence, indicating good cell viability. The NLip, BSA@GEL, and FGF21-NLip@B groups showed relatively higher proportions of green fluorescence and relatively lower proportions of red fluorescence, indicating that they effectively maintained HaCaT cell viability and demonstrated good cell compatibility. CCK-8 assay results ( Figure 5D) The results also confirmed the above observations. Compared with the control group, the NLip, BSA@GEL, and FGF21-NLip@B groups all showed higher cell viability, indicating that these materials had no effect on cell activity and all had good cell compatibility. To further evaluate the blood compatibility of the materials, a hemolysis test was performed. Figure 5 (EF). Hemolysis test images of the negative control group (saline) and the BSA@GEL group showed clear, transparent supernatant, indicating no significant hemolysis; the positive control group (sterile pure water) showed a red supernatant, indicating complete lysis of red blood cells. The FGF21-NLip@B group is expected to be similar to the saline group, with a clear, transparent supernatant, indicating good blood compatibility of the hydrogel. Quantitative analysis of the hemolysis rate is expected to further verify the blood compatibility of each material. The hemolysis rate of the NLip group was significantly higher than that of the other groups (p<0.05), while the hemolysis rates of the BSA@GEL group and the FGF21-NLip@B group were both below 5%, meeting the standards for blood-compatible materials. Therefore, the hydrogel prepared in this study has good biocompatibility and can be used for the treatment of acne.

[0077] 3.6 Assay of FGF21-NLip@B promoting cell proliferation This study aimed to verify the proliferative and migration-promoting effects of FGF21 on HaCaT cells through in vitro experiments, and to evaluate the efficacy of different administration methods (free FGF21, FGF21-NLip, and FGF21-NLip@B), thereby providing a theoretical basis for acne treatment strategies based on FGF21-NLip@B hydrogel. First, to screen the effective dosage of FGF21, CCK-8 assay results showed that within the range of 50-100 mg / mL, FGF21 treatment of HaCaT cells for 24 h and 48 h resulted in a concentration-dependent increase in cell viability, peaking at 100 mg / mL. This concentration will be used in subsequent experiments. To further investigate its effect on HaCaT cell migration, a scratch assay was performed. Results showed that the scratch healing rate in the FGF21-NLip@B group was significantly higher than in other groups, indicating that FGF21-NLip@B can significantly promote HaCaT cell migration.

[0078] In conclusion, the results ( Figure 6The results indicate that FGF21-NLip@B can more effectively promote the proliferation and migration of HaCaT cells. Compared with direct application of FGF21, the acne treatment strategy of FGF21-NLip@B hydrogel may have a targeted delivery method, a longer duration of action, and lower toxicity. Then, the proliferative capacity of the gel was evaluated by CCK-8 assay. The expected results showed that the HaCaT cell viability of the FGF21, FGF21-NLip, and FGF21-NLip@B extract treatment groups was significantly higher than that of the control group (p<0.05), indicating that these treatments can promote HaCaT cell proliferation. More importantly, the cell viability of the FGF21-NLip@B extract group was expected to be significantly higher than that of the free FGF21 group and the FGF21-NLip group (p<0.05), which may be related to the protective effect of NLip on FGF21 and the sustained-release effect of the BSA hydrogel.

[0079] 3.7 In vitro antioxidant activity of NLip@B Acne is caused by a variety of factors, including inflammation, excessive sebum secretion, and keratinization of the pilosebaceous duct. Oxidative stress is a significant driving force behind these factors. Inflammation induces the production of reactive oxygen species (ROS), exacerbating inflammatory damage. Excess sebum is easily oxidized, forming irritating substances that further irritate the hair follicles. Abnormal keratinization is also related to oxidative stress. Excessive free radicals or slow clearance of them can activate inflammatory pathways, intensifying the inflammatory response and leading to clinical symptoms such as redness, papules, and pustules. Therefore, effectively eliminating free radicals is crucial for acne treatment.

[0080] To investigate the free radical scavenging ability of the materials, this study employed the Fenton reaction, using the fading of methylene blue (MB) as an indicator. In the Fenton reaction, H₂O₂ in Fe... 2+ Catalysis generates highly oxidizing hydroxyl radicals (•OH), which can oxidize MB, causing its color to lighten. The change in absorbance at 667 nm was detected using a microplate reader. Results ( Figure 7 A) indicates that although the scavenging ability of the BSA hydrogel decreased after crosslinking, it still exhibited a significant •OH scavenging effect compared to the blank Fenton system. These results suggest that the material prepared in this study possesses potential hydroxyl radical scavenging capabilities. Figure 7As shown in Figure B, the control group rapidly faded, confirming the formation of •OH. When BSA, GEL, and BSA-ICPS were added to the system, the fading process of the MB solution was significantly slowed, suggesting that these samples have the ability to inhibit •OH activity. To quantify the effect of ICPS coupling and cross-linking on the free radical scavenging effect of BSA, further investigation was conducted. In conclusion, considering the close relationship between acne occurrence and inflammatory response, and the crucial role of free radicals in the inflammatory response, the excellent free radical scavenging ability exhibited by this gel indicates its good adjunctive effect in acne treatment.

[0081] 4. Analysis and Summary: To overcome the problems of maintaining local drug concentration and significant side effects associated with traditional therapies, we innovatively encapsulated FGF21 in nanoliposomes and combined it with BSA hydrogel to construct a sustained-release, targeted, safe, and effective drug delivery system. This study conducted in-depth preparation, characterization, and performance studies focusing on the key aspects of this strategy. The main research results are summarized below: (1) FGF21-NLip nanoliposomes were successfully prepared. Laser particle size analysis determined the average particle size to be 76.25 nm, the PDI to be 0.185, the Zeta potential to be -46.7 mV, and the encapsulation efficiency to be 76.2%, indicating good particle size uniformity, high stability, and drug loading capacity. Stability studies showed that the physicochemical properties of FGF21-NLip changed only slightly during the observation period, demonstrating sufficient stability to meet the requirements of subsequent experiments.

[0082] (2) FGF21-NLip@B hydrogel with an interconnected porous network structure was successfully prepared by modifying BSA with ICPS. FTIR results confirmed that ICPS was successfully modified onto the BSA molecules. Rheological tests showed that the storage modulus G' of the hydrogel remained stable in the low strain region, and G' was always higher than the loss modulus G'', indicating that the hydrogel has excellent mechanical properties and good rheological characteristics, can maintain its morphology at the lesion site, and is easy to inject, which is beneficial for clinical application.

[0083] (3) The properties and drug release behavior of FGF21-NLip@B hydrogel were analyzed. The results showed that NLip@B hydrogel has good moisturizing ability and controllable swelling properties, can effectively release FGF21, and exhibits different release kinetic characteristics. Compared with the FGF21-NLip group, the release rate of the FGF21-NLip@B group is relatively slower, making it more suitable for maintaining the effective concentration of FGF21 in the long term, promoting the repair of damaged skin and reducing scar formation.

[0084] (4) The biocompatibility of NLip, BSA@GEL, and FGF21-NLip@B materials was evaluated through in vitro experiments. The results showed that these materials have good cell compatibility and blood compatibility, providing a safety guarantee for subsequent in vivo experiments.

[0085] (5) In vitro experiments confirmed that, compared with free FGF21, FGF21-NLip@B can significantly promote the proliferation and migration of HaCaT cells, indicating that it has excellent pro-repair ability. Compared with traditional acne treatment methods, this strategy based on FGF21-NLip@B hydrogel is expected to achieve targeted drug delivery, prolong the duration of action, and reduce toxic side effects.

[0086] (6) To investigate the antioxidant capacity of the material, the Fenton reaction was used in this study. The results showed that the material prepared in this study has the potential ability to scavenge hydroxyl radicals.

[0087] Example 2: Pharmacodynamic evaluation of FGF21 nanoliposome hydrogel for acne. 1. Main reagents and materials and their sources 1.1 Main Reagents and Materials As shown in Table 2.

[0088] Table 2 Main Experimental Reagents

[0089] 1.2 Laboratory Animals Forty-nine healthy male Kunming mice, weighing 30±2g, were purchased from Chengsibeifu (Beijing) Biotechnology Co., Ltd. The mice were 6-8 weeks old. The temperature was 24±2℃, RH 50%-70%, simulating a 12-hour day / night cycle, with normal diet. Each mouse was housed individually in its cage, allowing free movement within the cage. During the rearing period, the mice had free access to food and water. The experiment was conducted after a one-week acclimatization period. The animal experiments have passed the animal experiment ethics review, number WZU-2022-093.

[0090] 2 Experimental Methods 2.1 Preparation of Propionibacterium acnes bacterial suspension (1) Strains revive and proliferate: The freeze-dried Propionibacterium acnes was activated according to the standard procedure and then inoculated into Fluid Thioglycollate Medium (FTM). It was cultured at 37°C under anaerobic conditions for 48 hours to ensure that the strain entered the logarithmic growth phase.

[0091] (2) Collection of bacterial cells: The culture medium of Propionibacterium acnes in the logarithmic growth phase was centrifuged at low temperature at 5000 rpm for 20 minutes. After centrifugation, the supernatant was carefully collected and stored in a -20℃ refrigerator for subsequent experiments.

[0092] 2.2 Establishment of a mouse dorsal acne model (1) Preparation of experimental animals: 49 SPF-grade male mice were selected as experimental subjects.

[0093] (2) Hair removal on the back: Hair removal was performed on the back area of ​​all mice to facilitate subsequent operations and observation.

[0094] (3) Propionibacterium acnes infection: Use Propionibacterium acnes bacterial suspension in the logarithmic growth phase (concentration range: 107~109 CFU / mL).

[0095] 0.1 mL was injected subcutaneously into the back of each mouse. Injections were given every other day for a total of 7 injections.

[0096] (4) Oleic acid induction: Apply 100% oleic acid to the back of each mouse. The dosage is 0.05 mL per mouse per application. Apply once daily for a total of 14 applications. The entire modeling process lasts 14 days.

[0097] (5) Model evaluation: The success of the model construction is assessed by visual observation. A successful model should exhibit the following characteristics: localized epidermal thickening, hyperkeratosis, and inflammatory symptoms such as localized redness and swelling.

[0098] 2.3 Grouping and Administration Forty-two male mice with mild to moderate acne and seven healthy mice were selected. The healthy mice served as the blank control group. The remaining mice were randomly divided into six groups: model group, positive drug control group, NLip@B group, FGF21 (50 μg / mL) group, FGF21-NLip@B (50 μg / mL) group, and FGF21-NLip (50 μg / mL) group, with seven mice in each group. The specific drug administration for each group is as follows: (1) Blank control group: Apply 1mL of physiological saline to the hair removal area on the back once a day for 14 days.

[0099] (2) Model group: Apply 1 mL of physiological saline to the acne lesion area on the back once a day for 14 days.

[0100] (3) Positive drug control group: 100mg of 2% benzoyl peroxide gel was applied to the acne lesions on the back once a day for 14 days.

[0101] (4) FGF21 group: Apply 1 mL of physiological saline solution containing 50 μg / mL FGF21 to the acne lesion area on the back once a day for 14 days.

[0102] (5) FGF21-NLip group: Apply 1 mL of nanoliposome suspension containing 50 μg / mL FGF21 (FGF21-NLip) to the acne lesion area on the back once a day for 14 days.

[0103] (6) FGF21-NLip@B group: Apply 1 mL of nanoliposome hydrogel containing 50 μg / mL FGF21 (FGF21-NLip@B) to the acne lesion area on the back once a day for 14 days.

[0104] (7) NLip@B group: Apply 1 mL of FGF21-free nanoliposome-hydrogel (NLip@B) to the acne lesion area on the back once a day for 14 days.

[0105] 2.4 Morphological observation The changes in acne-affected skin of mice in each group were observed with the naked eye. During the experiment, after administration, the skin changes were directly observed with the naked eye and photographed every 7 days to record the changes.

[0106] 2.5 In vivo biosafety assays Fourteen days after administration, the mice were sacrificed, and their major organs, including the heart, liver, spleen, kidneys, and lungs, were collected and histologically evaluated using H&E staining to determine drug toxicity.

[0107] 2.6 Histological examination 2.6.1 Tissue paraffin embedding and sectioning For histological analysis, mice were sacrificed on day 14 after drug treatment, and skin, liver, and kidney tissues from acne lesions were collected. First, the skin tissue was washed with physiological saline and then laid flat on filter paper for fixation to prevent curling and deformation. The liver and kidney tissues were washed directly with physiological saline. Subsequently, the skin tissue was laid flat on filter paper and immersed in a 4% paraformaldehyde solution along with other tissues for fixation at 4°C for 24 hours. After fixation, the tissues were placed in an embedding cassette and rinsed overnight with running tap water to remove residual paraformaldehyde. Dehydration was performed using a gradient of ethanol solutions: the tissues were sequentially immersed in 70% ethanol for 12 hours, 80% ethanol for 1 hour, 95% ethanol for 45 minutes (repeated twice), and 100% ethanol for 30 minutes (repeated twice). For clearing, the tissues were immersed in xylene for 15 minutes each time, repeated twice. After clearing, the tissues were sequentially immersed in pre-melted soft wax and hard wax for 2 hours each. Finally, the tissue blocks were embedded in paraffin and sectioned to a thickness of 5 μm using a Leica microtome. After flattening, the sections were dried overnight in a preheated oven at 37°C for 12 hours in preparation for subsequent staining.

[0108] 2.6.2 Hematoxylin and eosin staining (H&E staining), Masson's trichrome staining, and immunohistochemical staining (IHC) Standard operating procedure.

[0109] 2.7 Detection and evaluation of serum inflammatory factor expression levels in a mouse acne model Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively analyze the levels of pro-inflammatory cytokines in the serum of mice in each group. Specifically, serum samples were collected by orbital blood sampling. The experimental groups included: blank group, model group, positive drug control group, NLip@B group, FGF21 group, FGF21-NLip@B group, and NLip@B group. All procedures were strictly performed in accordance with the operating manual provided by the ELISA kit manufacturer. The detected indicators included tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-6 (IL-6).

[0110] 2.8 Data Processing All data in this experiment were statistically analyzed using GraphPad Prism 8.0 software. All data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for statistical analysis. Compared with the control group, , , .

[0111] 3. Experimental Results 3.1 Morphological and pathological examination of acne in mice In the early stages of modeling (days 0-7), erythema, follicular dilation, and mild inflammation began to appear on the backs of mice in the model group, indicating the successful establishment of the acne model. As time progressed (days 7-14), the inflammatory response in the model group significantly worsened, with the appearance of papules, pustules, and sebaceous gland hyperplasia. Significant inflammatory cell infiltration in the dermis was evident, consistent with the pathological characteristics of mild to moderate acne. By days 14-21, the lesions in the model group reached their peak, with nodules and cysts appearing in some areas, and epidermal thickening.

[0112] In contrast, the control group mice showed no significant pathological changes in their skin. Among the treatment groups, the positive control group and the FGF21-NLip@B group showed significant inflammation relief, with significantly milder lesions than the model group. The FGF21-NLip@B group showed the most significant effect, with reduced inflammatory cell infiltration, inhibited sebaceous gland hyperplasia, and enhanced skin repair capacity. The NLip@B group and the FGF21 group also showed some improvement, but the effect was weaker than that of the FGF21-NLip@B group. Figure 8 ).

[0113] In summary, this study successfully established a mouse model of acne. The FGF21-NLip@B group showed the most significant acne treatment effect in the model mice, which was superior to the positive control group, the NLip@B group, and the FGF21 group. It could effectively relieve inflammation, inhibit sebaceous gland hyperplasia, and promote skin repair.

[0114] 3.2 In vivo biosafety study of FGF21-NLip@B In the model group and all treatment groups (including the positive drug control group, NLip@B group, FGF21 group, and FGF21-NLip@B group), no significant pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues. In the heart tissue, cardiomyocytes were neatly arranged, without fibrosis or inflammatory cell infiltration; in the liver tissue, hepatocytes were structurally intact, without fatty degeneration, necrosis, or inflammatory cell aggregation. In the spleen tissue, lymphocytes were evenly distributed, without abnormal proliferation or structural destruction. In the lung tissue, alveolar structures were clear, alveolar septa were normal, and no inflammatory cell infiltration or fibrosis was observed. In the kidney tissue, glomeruli and renal tubules were structurally intact, without swelling, inflammatory cell infiltration, or interstitial fibrosis. The organs and tissues of the blank control group mice also showed normal characteristics, with no significant differences from the model group and all treatment groups, indicating that the establishment of the acne model and drug treatment intervention did not significantly affect the heart, liver, spleen, lung, and kidneys of the mice.

[0115] In summary, the establishment of the acne model and the intervention of drugs such as FGF21-NLip@B in this experiment were mainly limited to local skin lesions and did not cause systemic pathological damage to the major internal organs (heart, liver, spleen, lungs, and kidneys) of the mice. Figure 9This result further confirms that FGF21-NLip@B is safe at therapeutic doses, providing important experimental evidence for its subsequent clinical application.

[0116] 3.3 HE staining pathological changes and evaluation of treatment intervention effects like Figure 10 As shown, in the skin tissue structure analysis, the blank group exhibited normal tissue morphology, with moderate epidermal thickness, no keratinization or inflammatory cell infiltration, and clearly visible adipose tissue, hair follicles, and sebaceous glands. In contrast, the model group's skin showed significant acne pathological characteristics, specifically: thickened epidermis, excessive keratinization of the stratum corneum forming numerous keratin plugs blocking hair follicles, accompanied by significant dilation of the hair follicle sebaceous glands, with a large accumulation of keratin and lipids inside. Furthermore, a large amount of inflammatory cell infiltration was observed in the dermis, mainly lymphocytes and neutrophils, primarily clustered around the hair follicles; dermal collagen fibers also showed disordered arrangement and loose structure. The treatment effects of the NLip@B group and the FGF21 group were relatively limited, with only slight improvement observed, and inflammatory factor infiltration remained significant. Notably, the FGF21-NLip@B group showed more significant pathological improvement, with relatively clear tissue structure, significantly reduced inflammatory cell infiltration, and a reduction in perifollicular inflammatory response. Epidermal continuity was also improved, with no obvious ulcers or erosions observed, and epidermal thickness gradually returned to normal. The treatment effect of the positive drug control group was similar to that of the FGF21-NLip@B group in terms of epidermal thickness and the degree of inflammatory cell infiltration; however, the FGF21-NLip@B group may show greater potential in restoring collagen fiber arrangement.

[0117] In summary, FGF21 nanoliposomes (FGF21-NLip@B) significantly improved acne pathological features (epidermal thickening, keratin plugging, and inflammatory infiltration), and their anti-inflammatory and repair effects were comparable to those of the positive control group, indicating that the nanodelivery system can effectively enhance the targeted therapeutic activity of FGF21.

[0118] 3.4 Masson staining of collagen fiber distribution and fibrosis assessment Acne lesions are often accompanied by the destruction or proliferation of collagen fibers in the dermis. Masson staining can clearly show the distribution and arrangement of collagen fibers, helping to assess whether acne leads to dermal fibrosis or scarring. Furthermore, Masson staining can compare the differences in collagen fiber arrangement and the degree of fibrosis between the model group and the treatment group, assessing whether the drug can inhibit fibrosis or promote tissue repair.

[0119] like Figure 11As shown, in the skin tissue of mice in the blank control group, collagen fibers were neatly arranged and evenly distributed, and the dermal structure was intact, with no obvious fibrosis or abnormal collagen deposition, indicating that collagen metabolism in normal skin tissue was in a balanced state. In contrast, the skin tissue of mice in the model group showed significant pathological changes: disordered collagen fiber arrangement, increased collagen deposition in local areas, and aggravated dermal fibrosis, suggesting that the inflammatory response of acne led to abnormal collagen metabolism. In the treatment group, the FGF21-NLip@B group showed the most significant improvement: collagen fibers were more regularly arranged, the fibrotic area was significantly reduced, and collagen deposition was close to normal levels, indicating that FGF21-NLip@B can effectively improve collagen fiber damage caused by acne and reduce dermal fibrosis. The NLip@B group and FGF21 showed no significant improvement. The improvement effect of the positive drug control group was similar to that of the FGF21-NLip@B group, further validating the therapeutic potential of FGF21-NLip@B.

[0120] 3.5 Immunohistochemical staining for assessment of inflammatory factor expression Immunohistochemical results ( Figure 12 The results showed that, compared with the control group, the positive expression levels of TNF-α, IL-1β, IL-6, IL-8, and C-10 in the skin tissue of the model group were significantly increased. Positive signals were mainly concentrated in the inflammatory cell infiltration areas of the epidermis and dermis, as well as around dilated hair follicles, with the increases in TNF-α and IL-6 being the most significant. In the positive drug control group, the expression levels of TNF-α, IL-1β, and IL-6 were significantly lower than those in the model group, but the decrease in IL-8 and C-10 was not significant. In the FGF21-NLip@B group, the expression levels of TNF-α, IL-1β, IL-6, and IL-8 were significantly lower than those in the model group and close to those in the control group. Positive signals were mainly distributed around hair follicles, while the decrease in the expression of chemokine C-10 was not significant. In conclusion, nanoliposome-encapsulated FGF21 combined with BSA hydrogel can effectively inhibit the inflammatory response, exert a synergistic effect, and effectively control acne inflammation.

[0121] 3.6 ELISA method was used to assess the effects of different treatment regimens on serum inflammatory factors in mice. TNF-α, IL-1β, IL-6, and IL-8 are all pro-inflammatory cytokines that play important roles in the inflammatory response of acne. ELISA results ( Figure 13The results showed that the serum levels of TNF-α, IL-1β, IL-6, and IL-8 in the model group mice were significantly elevated (p<0.001), indicating that the acne model induced a significant systemic inflammatory response. After 14 days of treatment, the expression levels of the four inflammatory factors in serum were significantly reduced in both the positive drug control group and the FGF21-NLip@B group (p<0.001). The levels of inflammatory factors in the FGF21 group, NLip@B group, and FGF21-NLipz group were not significantly different from those in the model group, suggesting that using FGF21 alone or simply adding FGF21 to liposomes may not effectively inhibit the inflammatory response. This may be due to FGF21 inactivation, indicating that using liposome-encapsulated FGF21 alone does not achieve a good therapeutic effect. These results are consistent with the immunohistochemical results, further confirming the potential of FGF21-NLip@B in acne treatment.

[0122] 4. Summary and Analysis This embodiment investigates the pharmacodynamics of FGF21-encapsulated in BSA hydrogel (FGF21-NLip@B) via nanoliposomes in a mouse acne model, aiming to evaluate its potential in acne treatment. Through successful acne model construction and comparison of the effects of different treatment groups, the significant anti-acne effect of FGF21-NLip@B was confirmed, specifically manifested in: (1) Improved pathological features of acne: Compared with the model group, the FGF21-NLip@B group can significantly reduce the morphological changes of skin erythema, papules, pustules caused by acne, reduce inflammatory response, inhibit sebaceous gland hyperplasia, and promote skin repair. The efficacy is better than that of the positive control group and the NLip@B group and FGF21 group used alone.

[0123] (2) Regulation of local microenvironment: Histopathological analysis (HE staining) demonstrated that FGF21-NLip@B can effectively improve pathological changes such as epidermal thickening, hyperkeratosis, hair follicle dilation and inflammatory cell infiltration, and significantly restore skin tissue structure. Masson staining further revealed that FGF21-NLip@B can effectively improve collagen fiber disorder and fibrosis, promote collagen structure repair and reconstruction, and reduce potential scar formation.

[0124] (3) Inhibition of inflammatory response: Immunohistochemical and ELISA results showed that FGF21-NLip@B could significantly reduce the expression levels of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-8 in local skin tissue and serum of model mice, effectively inhibiting the local and systemic inflammatory response caused by acne.

[0125] (4) Verification of in vivo safety: Pathological evaluation of important organs such as heart, liver, spleen, lung and kidney of mice in each treatment group was carried out. The results showed that FGF21-NLip@B and other drug interventions did not cause significant organ damage, which preliminarily verified the safety of the treatment plan and provided a guarantee for subsequent clinical application.

[0126] In summary, this study comprehensively evaluated the multiple pharmacological mechanisms of FGF21-NLip@B in acne treatment, including reducing inflammation, repairing skin structure, inhibiting fibrosis, and regulating the expression of inflammatory factors. The combined use of nanoliposomes and BSA hydrogel enhanced the stability and sustained-release properties of FGF21, enabling it to act more effectively on acne sites. These findings lay a solid foundation for the clinical translation of FGF21-NLip@B as a novel acne treatment drug and strategy, and hold promise for providing acne patients with safer and more effective treatment options.

Claims

1. A drug-loaded liposome protein hydrogel complex, characterized in that: The complex was obtained by encapsulating nanoliposomes loaded with fibroblast growth factor FGF21 in a BSA-modified hydrogel, wherein the BSA-modified hydrogel is a bovine serum albumin-based hydrogel modified with isopropyltriethoxysilane ICPS.

2. The drug-loaded liposome protein hydrogel complex according to claim 1, characterized in that: The liposomes are soybean lecithin-cholesterol nanoliposomes.

3. A method for preparing the drug-loaded liposome protein hydrogel complex according to claim 1 or 2, characterized in that, Includes the following steps: S1. Prepare the nanoliposomes described above; S2. The drug FGF21 was loaded into nanoliposomes to obtain drug-encapsulated nanoliposomes FGF21-NLip; S3. Prepare the BSA-modified hydrogel solution; S4. Preparation of the drug-loaded liposome protein hydrogel complex: Disperse the FGF21-NLip obtained in step S2 into the BSA modified hydrogel solution prepared in step S3, and add an initiator to initiate a cross-linking reaction to obtain the drug-loaded liposome protein hydrogel complex.

4. The preparation method according to claim 3, characterized in that: In step S1, the nanoliposomes are prepared using a reverse-phase evaporation method, including the following steps: (1) Preparation of oil phase: Soybean lecithin and cholesterol were dissolved in chloroform at a mass ratio of 3~5:1 to obtain an oil phase solution; (2) Aqueous phase preparation: Prepare buffer A; preferably, buffer A is a citrate buffer with pH 4.6~4.8 and a concentration of 0.2~0.4 mol / L; (3) Preparation of W / O emulsion: Add buffer A to the oil phase solution, mix and emulsify to obtain W / O emulsion; (4) Reverse phase evaporation and hydration: The W / O emulsion was transferred to a rotary evaporator to remove the organic solvent by rotary evaporation, which promoted the self-assembly of lipid molecules; then, buffer A was added to hydrate and reconstitute the hydrate. (5) Preparation of liposome suspension: The hydrate was removed by rotary evaporation and then subjected to a second vacuum evaporation to further remove residual organic solvent and promote the formation of liposomes, so as to obtain liposome suspension; (6) Particle size uniformization treatment: The liposomes in the liposome suspension are uniformly dispersed, and then the particle size is fined by microporous membrane filtration and liposome extruder in sequence to obtain nanoliposomes.

5. The preparation method according to claim 3 or 4, characterized in that: In step S2, the drug solution is mixed with the nanoliposomes prepared in step S1 and incubated at 35-40°C for 1-3 hours to achieve effective drug loading. The preferred mass ratio of FGF21 to liposomes is 1.6~2.0 mg: 700~800 mg; Preferably, the drug solution is an FGF21 solution with a concentration of 4.0~5.0 mg / mL, prepared by mixing 0.4 mL of FGF21 solution with 7~8 mL of nanoliposome suspension with a concentration of 90~110 mg / mL.

6. The preparation method according to claim 3 or 4, characterized in that: In step S3, the preparation method of the BSA modified hydrogel solution is as follows: Bovine serum albumin (BSA) is dissolved in buffer B, then ICPS is added, and the mixture is stirred at 35~40℃ to generate a BSA-ICPS conjugate solution, thus obtaining the BSA modified hydrogel solution. The preferred reaction time is 3.5~4.5h.

7. The preparation method according to claim 6, characterized in that: The buffer B is PBS buffer, and the mass-to-volume ratio of BSA, ICPS and buffer B is 100mg:90~110μL:1.4~1.6mL, preferably 100mg:100μL:1.5mL.

8. The preparation method according to claim 6 or 7, characterized in that: In step S4, the FGF21-NLip obtained in step S2 is dispersed into the BSA-modified hydrogel solution prepared in step S3, an initiator is added, and the mixture is stirred at 35-40°C and 350-450 rpm until the mixture self-assembles to form a hydrogel, and a cross-linking reaction is initiated to obtain a drug-loaded liposome protein hydrogel complex; preferably, the initiator is sodium fluoride.

9. The preparation method according to claim 8, characterized in that: In step S4, the ratio of FGF21-NLip, BSA-ICPS conjugate solution, and initiator obtained in step S2 is 700~800mg:8mL:30~34mg.

10. The use of the drug-loaded liposome protein hydrogel complex according to claim 1 or 2, or the drug-loaded liposome protein hydrogel complex prepared by the preparation method according to any one of claims 3 to 9, in the preparation of a medicament for treating acne.

Images