A supramolecular anti-hypertrophic scar material and its preparation and application

Abstract

The present invention discloses a supramolecular anti-hypertrophic scar material and its preparation and application, which relates to the technical field of nano-diagnostic and therapeutic materials. The material comprises amphiphilic vesicles formed by the assembly of water-soluble supramolecular macrocycles and hydrophobic Chinese herbal molecules through host-guest interactions, wherein the inner and outer layers of the vesicles are both supramolecular macrocycles, and the hydrophobic Chinese herbal molecules are located between the inner and outer layers of the vesicles; the interior of the amphiphilic vesicles is loaded with enriched metal nanoclusters. The present invention assembles hydrophilic supramolecular macrocycles and active ingredient hydrophobic Chinese herbal molecules into vesicles through supramolecular host-guest interactions, and then loads functional component metal nanoclusters, thereby constructing a high-performance supramolecular material for the treatment of hypertrophic scars based on the ferroptosis-apoptosis synergistic mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-diagnostic and therapeutic materials, and in particular to a supramolecular anti-hypertrophic scar material and its preparation and application. Background Art

[0002] Hypertrophic scarring is a fibroproliferative disease characterized by increased proliferation and decreased apoptosis of hypertrophic scar fibroblasts, leading to the deposition of a large amount of collagen-rich extracellular matrix. Hypertrophic scarring often presents with symptoms such as pain, itching, and even disfigurement, which seriously affect patients' quality of life. Current first-line treatment options, including cosmetic surgery, laser ablation, glucocorticoid injections, and silicone gel, are widely used in clinical practice. However, due to poor therapeutic efficacy and high recurrence rates, no treatment method has achieved satisfactory results, thus placing a high demand on the development of new and effective treatment strategies.

[0003] Supramolecular assembly has attracted widespread attention in the biological community due to its designability and controllability, high bioavailability and drug loading efficiency, and dynamic responsiveness. Water-soluble supramolecular macrocycles are cyclic organic compounds, such as cyclodextrins, cucurbiturils, and emerging functionalized pillararenes. They possess biocompatibility, unique host-guest properties, and specific recognition capabilities, providing favorable conditions for supramolecular assembly and are widely used to construct supramolecular nanomedicines. Although supramolecular macrocycles have made significant progress in nanodiagnosis and therapy and possess significant application potential, their application in anti-scarring is rarely reported.

[0004] Pathophysiological studies have shown that targeting apoptosis in hypertrophic scar fibroblasts by activating apoptotic pathways is one of the most effective approaches for treating hypertrophic scars. However, hematopoietic stem cells can develop resistance to chemotherapy-induced apoptosis because they excrete anti-hypertrophic scar drugs through resistance-associated transporters such as P-glycoprotein and multidrug resistance-associated protein. Therefore, introducing additional therapeutic modalities and apoptosis could target multiple signaling pathways and potentially overcome the apoptotic resistance of hypertrophic scar fibroblasts. Ferroptosis is an emerging form of cell death characterized by excessive reactive oxygen species and lipid peroxidation. Combining these two forms of cell death (ferroptosis and apoptosis) as an innovative therapeutic strategy for the treatment of hypertrophic scars holds great potential, but has yet to be reported. Hydrophobic Chinese herbal molecules are novel ferroptosis inducers, capable of generating free radicals to trigger oxidative stress and induce ferroptosis. Metal nanoclusters are a new class of nanotheranostics with well-defined structural composition, ultrasmall size, and tunable fluorescence properties, capable of inducing apoptosis through enzyme-like and photocatalytic properties. Hydrophobic herbal molecules and metal nanoclusters are two active ingredients that achieve synergistic anti-scarring effects through ferroptosis and apoptosis. However, the poor water solubility of herbal molecules significantly limits their therapeutic potential. The diagnostic and therapeutic properties of metal nanoclusters need to be improved, and their application in anti-scarring is under investigation. Furthermore, the therapeutic mechanism by which hydrophobic herbal molecules can be combined with metal nanoclusters to achieve synergistic enhancement of ferroptosis and apoptosis remains unclear. Summary of the Invention

[0005] In response to the deficiencies in the above-mentioned background technology, the present invention provides a supramolecular anti-hypertrophic scar material and its preparation and application. The scar material is a supramolecular material based on the ferroptosis-cell apoptosis synergistic mechanism to achieve high-performance anti-hypertrophic scar treatment. The present invention constructs supramolecular vesicles of hydrophilic supramolecular macrocycles and active ingredient hydrophobic Chinese herbal medicine molecules based on supramolecular host-guest interactions, which solves the problem of hydrophobicity of Chinese herbal medicine molecules and enhances the performance of hydrophobic Chinese herbal medicine molecules in inducing ferroptosis. The vesicles are loaded with enriched metal nanoclusters, which enhance their enzyme-like catalytic activity and efficiently induce cell apoptosis. They are further loaded into hydrogel microneedles to promote their transdermal delivery, showing superior therapeutic effects in shortening treatment time and improving the appearance of hypertrophic scars. A high-performance supramolecular material for the treatment of hypertrophic scars based on the ferroptosis-cell apoptosis synergistic mechanism is constructed, realizing the anti-hypertrophic scar treatment based on the ferroptosis-cell apoptosis synergistic strategy of supramolecular assembly. The preparation method provided by the present invention has simple process and strong operability. At the same time, the ferroptosis-cell apoptosis synergistic strategy based on supramolecular assembly provides innovative guidance for the effective treatment of hypertrophic scars and other diseases.

[0006] The first object of the present invention is to provide a supramolecular anti-hypertrophic scar material, characterized in that the material includes amphiphilic vesicles formed by the assembly of water-soluble supramolecular macrocycles and hydrophobic Chinese herbal molecules through host-guest interactions, wherein the inner and outer layers of the amphiphilic vesicles are both supramolecular macrocycles, and the hydrophobic Chinese herbal molecules are located between the inner and outer layers of the vesicles; the interior of the amphiphilic vesicles also includes metal nanoclusters.

[0007] Preferably, the supramolecular macrocycle is one or more of cyclodextrin, cucurbituril, calixarene, and pillararene.

[0008] Preferably, the hydrophobic Chinese herbal molecule is one or more of artemisinin, piperlongumamide, withaferin, trigonelline, matrine, and dihydroartemisinin.

[0009] Preferably, the metal nanoclusters are gold nanoclusters, silver nanoclusters, copper nanoclusters, or platinum nanoclusters.

[0010] Preferably, the molar ratio of the supramolecular macrocycle to the hydrophobic Chinese herbal medicine molecule is 20:1 to 1:20; the molar ratio of the supramolecular macrocycle in the amphiphilic vesicle to the metal in the metal nanoclusters is 5:1 to 1:100.

[0011] The second object of the present invention is to provide a method for preparing a supramolecular anti-hypertrophic scar material, comprising the following steps:

[0012] Preparation of metal nanoclusters;

[0013] The water-soluble supramolecular macrocycle aqueous solution and the hydrophobic Chinese herbal medicine molecule ethanol solution are uniformly mixed and stirred at room temperature for assembly to obtain an amphiphilic vesicle solution;

[0014] A metal nanocluster solution is added to a solution containing amphiphilic vesicles, mixed, and statically assembled at room temperature to obtain a supramolecular anti-hypertrophic scar material.

[0015] Preferably, the metal nanoclusters are prepared according to the following steps:

[0016] The metal precursor solution is added to the surface ligand aqueous solution and reacted at 25-100°C for 3-36 hours to obtain 1-2 nm metal nanoclusters protected by the surface ligand.

[0017] Preferably, the surface ligands of the metal nanoclusters include glutathione, L-cysteine, mercaptopropionic acid, lysozyme, 4-mercaptobenzoic acid, D-penicillamine, 11-mercaptoundecanoic acid, and bovine serum albumin.

[0018] Preferably, the concentration range of the water-soluble supramolecular macrocycle aqueous solution is 1-10 mM; the concentration range of the hydrophobic Chinese herbal medicine molecule ethanol solution is 1-10 mM; and the concentration range of the metal nanocluster solution is 1-10 mM.

[0019] The third object of the present invention is to provide a supramolecular anti-hypertrophic scar material for use in preparing a drug for treating hypertrophic scars.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a supramolecular anti-hypertrophic scar material and its preparation and application. The material comprises amphiphilic vesicles assembled by host-guest interactions between water-soluble supramolecular macrocycles and hydrophobic herbal molecules. The inner and outer layers of the vesicles are both supramolecular macrocycles, and the hydrophobic herbal molecules are located between the inner and outer layers of the vesicles. The amphiphilic vesicles are simply mixed with metal nanoclusters, and the metal nanoclusters are then loaded into the vesicles. The present invention synthesizes different types of hydrophilic supramolecular macrocycles (one or more of cyclodextrins, cucurbiturils, calixarene, pillararenes, and other water-soluble supramolecular macrocycles) and hydrophobic herbal molecules (one or more of artemisinin, piperlongumamide, withaferin, trigonelline, matrine, dihydroartemisinin, etc.) to construct supramolecular vesicles through supramolecular host-guest interactions. This addresses the hydrophobicity of the herbal molecules and enhances their ferroptosis-inducing properties.

[0022] The present invention further loads functional component metal nanoclusters on the basis of supramolecular vesicles, thereby enhancing the enzyme-like catalytic activity thereof and being able to efficiently induce cell apoptosis.

[0023] This study constructs a high-performance supramolecular material at the nanoscale for the treatment of hypertrophic scars based on a synergistic ferroptosis-apoptosis mechanism. This material is then loaded into hydrogel microneedles to facilitate transdermal delivery for treatment. This supramolecular assembly-based ferroptosis-apoptosis synergistic strategy provides innovative guidance for the effective treatment of hypertrophic scars and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the structure of the cucurbituril anti-hypertrophic scar material of Example 1.

[0025] Figure 2 This is the Fourier transform infrared spectrum of the amphiphilic vesicles formed by cucurbituril and dihydroartemisinin in Example 1.

[0026] Figure 3 This is the morphological characterization of the cucurbituril anti-hypertrophic scar material of Example 1.

[0027] Figure 4 The absorption spectra of the cucurbituril anti-hypertrophic scar material of Example 1 releasing hydrophobic Chinese herbal medicine molecular guests under different pH environments.

[0028] Figure 5 This is the protein characterization of the ferroptosis-apoptosis pathway induced by the cucurbituril anti-hypertrophic scar material in Example 1.

[0029] Figure 6 Schematic diagram of loading the cucurbituril anti-hypertrophic scar material of Example 1 onto the microneedles.

[0030] Figure 7 This is a diagram showing the wound surface treated with the cucurbituril anti-hypertrophic scar material of Example 1 in a rabbit ear hypertrophic scar model.

[0031] Figure 8 This is the immunological tissue staining characterization of the cucurbituril anti-hypertrophic scar material of Example 1 after in vivo treatment. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0033] In order to solve the problem of hydrophobicity of Chinese herbal medicine molecules, the present invention constructs supramolecular vesicles of hydrophilic supramolecular macrocycles and hydrophobic Chinese herbal medicine molecules based on supramolecular host-guest interactions, thereby solving the problem of hydrophobicity of Chinese herbal medicine molecules and enhancing the performance of hydrophobic Chinese herbal medicine molecules in inducing ferroptosis. Then, metal nanoclusters of functional components are loaded to enhance their enzyme-like catalytic activity, which can efficiently induce cell apoptosis, thereby constructing a high-performance supramolecular material for treating hypertrophic scars based on a synergistic mechanism of ferroptosis-cell apoptosis. The material is further loaded into hydrogel microneedles to promote its transdermal delivery and treatment, providing innovative guidance for the effective treatment of anti-hypertrophic scars and other diseases. The metal nanoclusters provided by the present invention have ultra-small size, excellent biocompatibility and unique enzyme-mimicking biocatalytic properties, and are ideal nanomedicines that trigger the process of cell apoptosis by promoting the production of reactive oxygen species.

[0034] To achieve the above objectives, the present invention provides a supramolecular anti-hypertrophic scar material in a first aspect. The material comprises amphiphilic vesicles assembled by a water-soluble supramolecular macrocycle and a hydrophobic Chinese herbal molecule through a host-guest interaction, wherein both the inner and outer layers of the amphiphilic vesicle are supramolecular macrocycles, and the hydrophobic Chinese herbal molecule is located between the inner and outer layers of the vesicle; the interior of the amphiphilic vesicle is loaded with enriched metal nanoclusters;

[0035] The supramolecular macrocycle is one or more of cyclodextrin, cucurbituril, calixarene, pillararene and other water-soluble supramolecular macrocycles.

[0036] The hydrophobic Chinese herbal molecule is one or more of artemisinin, piperlongumamide, withaferin, trigonelline, matrine, dihydroartemisinin, etc., which can induce ferroptosis or cell apoptosis.

[0037] See also Figure 1 As shown, the supramolecular anti-hypertrophic scar material provided by the present invention includes amphiphilic vesicles formed by the assembly of water-soluble supramolecular macrocycles and hydrophobic Chinese herbal molecules through host-guest interactions, wherein the inner and outer layers of the vesicles are both supramolecular macrocycles, and the hydrophobic Chinese herbal molecules are located between the inner and outer layers of the vesicles; after the amphiphilic vesicles are simply mixed with metal nanoclusters, the metal nanoclusters are loaded inside the vesicles.

[0038] The formation of the amphiphilic vesicles is mainly driven by the complexation between the hydrophilic supramolecular macrocycle and the hydrophobic Chinese herbal medicine molecules through host-guest interaction; the metal nanoclusters are incorporated into the amphiphilic vesicles through simple mixing and are evenly distributed inside the vesicles.

[0039] Wherein, the metal nanoclusters are gold nanoclusters, silver nanoclusters, copper nanoclusters, and platinum nanoclusters.

[0040] The molar ratio of the supramolecular macrocycle to the hydrophobic Chinese herbal medicine molecule is 20:1-1:20; the molar ratio of the supramolecular macrocycle in the amphiphilic vesicle to the metal in the metal nanocluster is 5:1-1:100.

[0041] The present invention assembles hydrophilic supramolecular macrocycles and hydrophobic Chinese herbal medicine molecules into vesicles through supramolecular host-guest interactions, and then loads functional component metal nanoclusters to construct a high-performance supramolecular material for treating hypertrophic scars based on the ferroptosis-cell apoptosis synergistic mechanism.

[0042] The supramolecular macrocycle is one or more of cyclodextrin, cucurbituril, calixarene, pillararene and other water-soluble supramolecular macrocycles, and has adjustable pore size, excellent biocompatibility and the ability to bind to a variety of biologically relevant guest molecules.

[0043] The hydrophobic Chinese herbal molecule is one or more of artemisinin, piperlongum amide, withaferin, trigonelline, matrine, dihydroartemisinin, etc., which can induce ferroptosis or cell apoptosis and have the ability to generate free radicals to trigger oxidative stress and induce ferroptosis.

[0044] The metal nanoclusters are gold nanoclusters, silver nanoclusters, copper nanoclusters, and platinum nanoclusters, which have enzyme-like catalytic activity and can effectively induce cell apoptosis.

[0045] Among them, supramolecular vesicles were constructed according to the molar ratio of supramolecular macrocycle to hydrophobic Chinese herbal medicine molecules of 20:1~1:20 and the concentration of 1-10 mM, and the reaction was carried out under stirring conditions at 10-50 °C for 5 min-36 h.

[0046] The loading is performed at a molar ratio of 5:1 to 1:100 between the supramolecular macrocycle in the amphiphilic vesicles and the metal in the metal nanoclusters and a concentration of 1-10 mM. The reaction is carried out at static conditions at 10-50 °C for 5 min-36 h.

[0047] A second aspect of the present invention provides a method for preparing a supramolecular anti-hypertrophic scar material, comprising the following steps:

[0048] Preparation of metal nanoclusters;

[0049] The water-soluble supramolecular macrocycle aqueous solution and the hydrophobic Chinese herbal medicine molecule ethanol solution are uniformly mixed and stirred at room temperature for assembly to obtain a solution containing amphiphilic vesicles;

[0050] A metal nanocluster solution is added to a solution containing amphiphilic vesicles, mixed, and statically assembled at room temperature to obtain a supramolecular anti-hypertrophic scar material.

[0051] The metal nanoclusters are prepared according to the following steps: adding a metal precursor solution such as a chloroauric acid aqueous solution, a silver nitrate aqueous solution, etc. to a ligand aqueous solution, reacting at 25-100°C for 3-36 hours to obtain surface ligand-protected metal nanoclusters of 1-2 nm.

[0052] The surface ligands of the metal nanoclusters include thiol molecules, selenol molecules, alkynyl molecules, phosphorus-containing molecules, nitrogen-containing molecules, dendrimers, amino acids, enzymes, polypeptides, proteins, etc., such as glutathione, L-cysteine, mercaptopropionic acid, lysozyme, 4-mercaptobenzoic acid, D-penicillamine, 11-mercaptoundecanoic acid, and bovine serum albumin.

[0053] The concentration range of the water-soluble supramolecular macrocycle aqueous solution is 1-10 mM; the concentration range of the hydrophobic Chinese herbal medicine molecule ethanol solution is 1-10 mM; and the concentration range of the metal nanocluster solution is 1-10 mM.

[0054] In one embodiment, a method for preparing a supramolecular anti-hypertrophic scar material comprises the following steps: assembling a water-soluble supramolecular macrocycle with a hydrophobic Chinese herbal medicine molecular guest through supramolecular interaction to generate amphiphilic vesicles, and further assembling metal nanoclusters to form an assembly having supramolecular assembly-activated ferroptosis-cell apoptosis synergistic anti-hypertrophic scar treatment.

[0055] The supramolecular macrocycle is one or more of cyclodextrin, cucurbituril, calixarene, pillararene and other water-soluble supramolecular macrocycles.

[0056] Cucurbituril is synthesized by mixing paraformaldehyde, glycoluril and hydrochloric acid solution and purified by centrifugation, rotary evaporation and recrystallization.

[0057] Carboxyl / amino / imidazole pillar arenes were synthesized by solvent dilution method using boron trifluoride etherate as catalyst and modification of water-soluble functional groups.

[0058] The hydrophobic Chinese herbal molecule is one or more of artemisinin, piperlongumamide, withaferin, trigonelline, matrine, dihydroartemisinin, etc., which can induce ferroptosis or cell apoptosis.

[0059] Preparation of aqueous solutions of supramolecular macrocycles and ethanol solutions of hydrophobic Chinese herbal medicine molecules.

[0060] The metal nanoclusters are 1-2 nm clusters protected by surface ligands obtained by reduction.

[0061] Amphiphilic vesicles were constructed with a molar ratio of supramolecular macrocycle to hydrophobic Chinese herbal medicine molecules of 20:1 to 1:20 and a concentration range of 1-10 mM. The reaction was carried out at 10-50 °C under stirring for 5 min-36 h.

[0062] The loading is performed according to the molar ratio of the supramolecular macrocycle in the amphiphilic vesicles to the metal in the metal nanoclusters of 5:1~1:100 and the concentration range is 1-10 mM. The reaction is carried out under static conditions at 10-50 ° C for 5 min-36 h.

[0063] A third aspect of the present invention provides a use of a supramolecular anti-hypertrophic scar material in the preparation of a material for treating hypertrophic scars.

[0064] During the application process, the supramolecular anti-hypertrophic scar material is added to the PDMS mold using hydrogel microneedles, centrifuged and then cross-linked under UV light. The microneedles are then peeled off from the PDMS mold after drying in the dark at room temperature.

[0065] Hypertrophic scars are caused by pathological changes or trauma. If they are caused by pathological changes, the material may also have antibacterial properties.

[0066] The mechanism of hypertrophic scarring is a synergistic ferroptosis-apoptosis mechanism. Hydrophobic herbal molecules can generate free radicals, triggering oxidative stress and inducing ferroptosis. The enzyme-like catalytic activity of metal nanoclusters can generate reactive oxygen species, inducing apoptosis. Supramolecular vesicles loaded with metal nanoclusters achieve this synergistic ferroptosis-apoptosis mechanism.

[0067] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0068] Example 1

[0069] A method for preparing a supramolecular anti-hypertrophic scar material comprises the following steps:

[0070] 1) Add 2.184 g of paraformaldehyde, 4.56 g of glycoluril, 18 mL of 12 M hydrochloric acid, and 6 mL of water to a 100 mL round-bottom flask placed in an oil bath. The mixture was reacted at 100 °C and 1500 rpm until a yellow clear solution formed, and then cooled naturally to room temperature. The supramolecular macrocyclic cucurbituril was purified by centrifugation, rotary evaporation, and recrystallization [7], and a high-purity product was finally obtained.

[0071] 2) Add 4.35 mL of ultrapure water to a 10 mL round-bottom flask placed in an oil bath. Add 0.15 mL of a 100 mM glutathione solution at room temperature (500 rpm) and stir for 5 minutes to evenly distribute the glutathione in the water. Then, add 0.5 mL of a 20 mM HAuCl₄ solution. Quickly place a condenser into the ground-jawed portion of the round-bottom flask and start the timer. When the solution turns colorless, remove the condenser and raise the stirrer to 70°C. Stop the reaction after 24 hours to obtain glutathione-protected gold nanoclusters. Store at 4°C in the dark.

[0072] 3) A 1 mM cucurbituril[7] aqueous solution and a dihydroartemisinin ethanol solution were mixed in a molar ratio of 1:1, and stirred at room temperature for 1 h to obtain a cucurbituril[7] / dihydroartemisinin supramolecular vesicle solution.

[0073] 4) The cucurbituril[7] / dihydroartemisinin supramolecular vesicle solution was mixed with glutathione-protected gold nanoclusters in a molar ratio of 1:1 and allowed to stand at room temperature for 24 h to assemble, thereby obtaining a supramolecular anti-hypertrophic scar material.

[0074] 5) Absorption spectroscopy was used to monitor the release of hydrophobic herbal molecules from the cucurbituril-based anti-hypertrophic scar material under different pH conditions. A 2 mL dialysis bag was placed in a 12-14 kDa dialysis bag, gently stirred at 150 rpm in a 5 mL centrifuge tube at room temperature, and immersed in 2 mL of PBS (pH 5.5 or 7.4) release medium. At each time point, 200 mL of dialysate was collected and an equal volume of PBS was added. The dialysate was then mixed with 100 L of 2% NaOH at 60°C for 30 minutes. The release capacity of the hydrophobic herbal molecule from the cucurbituril-based anti-hypertrophic scar material was monitored using a UV-visible spectrophotometer.

[0075] 6) The cucurbituril-based anti-hypertrophic scar material was co-incubated with cells. Hypertrophic scar cells treated with the cucurbituril-based anti-hypertrophic scar material for 48 hours were lysed with a buffer containing a protease inhibitor cocktail. The cell lysate was centrifuged at 12,000 rpm for 15 minutes at 4°C, and protein concentration was determined using a BCA protein quantification kit. The supernatant was denatured by mixing with sodium dodecyl sulfate polyacrylamide gel electrophoresis loading buffer at 100°C for 15 minutes. 40 μg of protein was separated by SDS-PAGE and transferred to a polyvinyl fluoride membrane. The membrane was blocked with 5% skim milk and incubated with α-SMA protein, Col-I and Col-III collagens at 4°C overnight. The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies at 37°C for 2 hours. Finally, protein bands were visualized using enhanced chemiluminescence detection reagents, and various ferroptosis-related protein pathways were detected using a chemiluminescence analyzer.

[0076] 7) The cucurbituril-based anti-hypertrophic scar material was diluted into a 10% GelMA solution to a final concentration of 50 M and then added to a PDMS mold. The mold was centrifuged and cross-linked with UV light for 30 seconds. After drying overnight in the dark at room temperature, the formed microneedles were released from the PDMS mold. The prepared cucurbituril-based anti-hypertrophic scar material microneedle patch was applied to the established scar model twice weekly for three weeks.

[0077] 8) After the experiment, scar tissue treated with the cucurbituril-based anti-hypertrophic scar material was fixed with 4% paraformaldehyde, dehydrated with graded ethanol, embedded in paraffin, and sectioned at 4 μm. Hematoxylin and eosin (H&E) staining was performed for histological analysis. Masson's trichrome was used to identify collagen fibers, which stain dark blue. For glutathione peroxidase 4 (GPX4) immunohistochemical staining, sections were quenched with 3% hydrogen peroxide for 10 minutes, blocked with 5% normal goat serum for 60 minutes, and incubated with a GPX4 primary antibody at a dilution of 1:200 overnight at 4°C. Detection was performed using an enzyme-linked secondary antibody and DAB substrate. This study characterized the therapeutic efficacy of the supramolecular anti-hypertrophic scar material in vivo.

[0078] Example 2

[0079] Same as Example 1, except that

[0080] Cucurbituril[7] aqueous solution and dihydroartemisinin ethanol solution were mixed at a molar ratio of 2:1;

[0081] The cucurbituril[7] / dihydroartemisinin supramolecular vesicle solution was mixed with mercaptopropionic acid-protected gold nanoclusters at a molar ratio of supramolecular to gold of 1:0.2.

[0082] Example 3

[0083] Same as Example 1, except that

[0084] Cucurbituril[7] aqueous solution and artemisinin ethanol solution were mixed at a molar ratio of 5:1;

[0085] The cucurbituril[7] / artemisinin supramolecular vesicle solution was mixed with mercaptopropionic acid-protected gold nanoclusters at a molar ratio of supramolecular to gold of 1:5.

[0086] Example 4

[0087] Same as Example 1, except that

[0088] Cucurbituril[7] aqueous solution and withaferin ethanol solution were mixed at a molar ratio of 10:1;

[0089] The cucurbituril[7] / withaferin supramolecular vesicle solution was mixed with 11-mercaptoundecanoic acid protected gold nanoclusters at a molar ratio of supramolecular to gold of 1:10.

[0090] Example 5

[0091] Same as Example 1, except that

[0092] Cucurbituril[7] aqueous solution and withaferin ethanol solution were mixed at a molar ratio of 1:10;

[0093] The cucurbituril[7] / withaferin supramolecular vesicle solution was mixed with 11-mercaptoundecanoic acid protected gold nanoclusters at a molar ratio of supramolecular to gold of 1:20.

[0094] Example 6

[0095] Same as Example 1, except that

[0096] The cyclodextrin aqueous solution and the artemisinin ethanol solution were mixed at a molar ratio of 1:20;

[0097] The cyclodextrin / artemisinin supramolecular vesicle solution was mixed with bovine serum albumin-protected gold nanoclusters at a molar ratio of supramolecular to gold of 1:20.

[0098] Example 7

[0099] Same as Example 1, except that

[0100] The cyclodextrin aqueous solution and the matrine ethanol solution were mixed at a molar ratio of 1:10;

[0101] The cyclodextrin / matrine supramolecular vesicle solution was mixed with D-penicillamine-protected copper nanoclusters at a molar ratio of supramolecular to gold of 1:20.

[0102] Example 8

[0103] Same as Example 1, except that

[0104] The cyclodextrin aqueous solution and the matrine ethanol solution were mixed at a molar ratio of 10:1;

[0105] The cyclodextrin / matrine supramolecular vesicle solution was mixed with D-penicillamine-protected copper nanoclusters at a molar ratio of supramolecular to gold of 1:50.

[0106] Example 9

[0107] A method for preparing a supramolecular anti-hypertrophic scar material comprises the following steps:

[0108] 1.22 g of pillar arene was dispersed in 60 mL of acetonitrile, 3.5 g of potassium carbonate was added, and after stirring at room temperature for 30 minutes, a small amount of potassium iodide and 5 mL of ethyl bromoacetate were added. The reaction mixture was heated under reflux for 18 hours under a nitrogen atmosphere. The filtrate was filtered and the solvent was removed by vacuum distillation. The crude product was redissolved in chloroform and slowly recrystallized by adding n-hexane to obtain product a. 1.47 g of compound a was dissolved in 60 mL of tetrahydrofuran and 30 mL of 20% sodium hydroxide solution was added. After reflux for 15 hours, the reaction mixture was cooled to room temperature and the solvent was removed by vacuum distillation. The crude product was dissolved in 100 mL of water, and dilute hydrochloric acid was gradually added until the precipitate reached a maximum. The filtered residue was washed with copious amounts of water to obtain product b. 0.24 g of compound b was dispersed in 3 mL of deionized water, 80 mg of sodium hydroxide was added, and the water was removed by vacuum distillation to obtain the carboxylated pillar arene.

[0109] 2) Dissolve AgNO₃ (6.8 mg, 40 mol) and L-cysteine ​​(36.8 mg, 120 mol) in 20 mL of ultrapure water and stir vigorously for 30 min. Then, add 1 mL of freshly dissolved NaBH₄ (15 mg, 400 mol) in cold water and stir for another 30 min to terminate the reaction. This yields L-cysteine-protected silver nanoclusters. Store at 4°C in the dark.

[0110] 3) A 1 mM carboxyl pillararene aqueous solution and a piperlongum amide ethanol solution were mixed in a 1:1 molar ratio and stirred at room temperature for 1 h to obtain a carboxyl pillararene / piperrlongum amide supramolecular vesicle solution.

[0111] 4) The carboxyl pillararene / piper longum amide supramolecular vesicle solution was mixed with L-cysteine-protected silver nanoclusters in a molar ratio of 1:1 and allowed to stand at room temperature for 24 h to assemble, thereby obtaining a supramolecular anti-hypertrophic scar material.

[0112] 5) The carboxyl pillararene-based anti-hypertrophic scar material was co-incubated with cells and lysed with a buffer containing a protease inhibitor cocktail. After treatment with the carboxyl pillararene-based anti-hypertrophic scar material for 24 hours, the hypertrophic scar cells were lysed. Polyacrylamide gel electrophoresis was used to visualize protein bands, and chemiluminescence was used to detect various ferroptosis-apoptosis pathway-related proteins.

[0113] 6) The carboxyl pillared arene-based anti-hypertrophic scar material was diluted into a 20% GelMA solution to a final concentration of 100 M and then added to a PDMS mold. The mold was centrifuged and then cross-linked with UV light for 30 seconds to prepare microneedles. The prepared microneedle patches of the carboxyl pillared arene-based anti-hypertrophic scar material were applied to the established scar model twice weekly for three weeks.

[0114] Example 10

[0115] Same as Example 9, except that

[0116] The carboxyl pillar aromatic hydrocarbon aqueous solution and the piperidine amide ethanol solution were mixed at a molar ratio of 1:0.1.

[0117] The carboxyl pillararene / piper longum amide supramolecular vesicle solution was mixed with L-cysteine ​​protected silver nanoclusters at a molar ratio of supramolecular to silver of 1:100.

[0118] Example 11

[0119] Same as Example 9, except that

[0120] The carboxyl pillar aromatic hydrocarbon aqueous solution and the piperlongum amide ethanol solution were mixed at a molar ratio of 1:0.5.

[0121] The carboxyl pillararene / piper longum amide supramolecular vesicle solution was mixed with L-cysteine ​​protected silver nanoclusters at a molar ratio of supramolecular to silver of 1:50.

[0122] Example 12

[0123] Same as Example 9, except that

[0124] The amino aromatic hydrocarbon aqueous solution and the piperlongum amide ethanol solution were mixed at a molar ratio of 1:5.

[0125] The aminopillararene / piperlongum amide supramolecular vesicle solution was mixed with lysozyme-protected silver nanoclusters at a molar ratio of supramolecular to silver of 1:10.

[0126] Example 13

[0127] Same as Example 9, except that

[0128] The calixarene aqueous solution and the trigonelline ethanol solution were mixed at a molar ratio of 1:10.

[0129] The calixarene / trigonelline supramolecular vesicle solution was mixed with lysozyme-protected silver nanoclusters at a molar ratio of supramolecular to silver of 1:20.

[0130] Example 14

[0131] Same as Example 9, except that

[0132] The calixarene aqueous solution and the trigonelline ethanol solution were mixed at a molar ratio of 1:15.

[0133] The calixarene / trigonelline supramolecular vesicle solution was mixed with 4-mercaptobenzoic acid-protected platinum nanoclusters at a molar ratio of supramolecular to silver of 1:5.

[0134] Example 15

[0135] Same as Example 9, except that

[0136] The calixarene aqueous solution and the trigonelline ethanol solution were mixed at a molar ratio of 1:20.

[0137] The calixarene / trigonelline supramolecular vesicle solution was mixed with 4-mercaptobenzoic acid-protected platinum nanoclusters at a molar ratio of supramolecular to silver of 1:0.5.

[0138] In order to illustrate the relevant properties of a supramolecular anti-hypertrophic scar material provided by the present invention, it is described in conjunction with the accompanying drawings.

[0139] Figure 2 This is the Fourier transform infrared spectrum of the amphiphilic vesicles formed by cucurbituril and dihydroartemisinin in Example 1.

[0140] Figure 2 It can be seen that -OH vibration is at 3364 cm -1 The displacement at 1709 cm and the C=O vibration at 1709 cm -1 The shift at 1021 cm further confirmed the interaction between the hydroxyl group of dihydroartemisinin and the carbonyl group of cucurbituril through hydrogen bonding. In addition, the CO vibration peak of cyclomethylene (1021 cm -1 ) has shifted significantly, and the CH vibration peak (1440 cm -1 These significant changes indicate that there is an interaction between dihydroartemisinin and cucurbituril.

[0141] Figure 3 This is the morphological characterization of the cucurbituril anti-hypertrophic scar material of Example 1.

[0142] Figure 3It can be seen that the high-angle annular dark field (HAADF) scanning TEM image shows that the cucurbituril anti-hypertrophic scar material is a spherical vesicle, and the EDS energy spectrum scanning shows that the S and Au elements are evenly distributed in the synthesized cucurbituril anti-hypertrophic scar material, which confirms the successful loading of gold nanoclusters.

[0143] Figure 4 The absorption spectra of the cucurbituril anti-hypertrophic scar material of Example 1 releasing hydrophobic Chinese herbal medicine molecular guests under different pH environments.

[0144] Figure 4 Absorption spectroscopy showed that at a pH of 7.4, the cucurbituril anti-hypertrophic scar material released less than 35% of dihydroartemisinin within 24 hours. In contrast, when the solution pH dropped to 5.0, the release of dihydroartemisinin increased significantly, indicating that the cucurbituril anti-hypertrophic scar material has good degradation behavior in an acidic environment.

[0145] Figure 5 Characterization of proteins in the ferroptosis-apoptosis pathway induced by the cucurbituril anti-hypertrophic scar material of Example 1. Control is a blank control group, CB[7] is a supramolecular macrocyclic cucurbituril[7], AuNCs are gold nanoclusters, DHA is dihydroartemisinin, CAD NPs are the final supramolecular anti-hypertrophic scar material; α-SMA is α-smooth muscle actin, Col-I and Col-III are excessive collagen deposition, collagen I and collagen III, GAPDH is an internal reference protein; XXKD is the molecular weight of the protein.

[0146] Figure 5 It can be seen that the expression of fibrosis-related proteins was analyzed by Western blot. Cucurbituril anti-hypertrophic scar material significantly reduced the protein expression of a-SMA, Col-I and Col-III.

[0147] Figure 6 Schematic diagram of loading the cucurbituril anti-hypertrophic scar material onto microneedles in Example 1. PDMS mold represents the PDMS mold; GelMA / CAD NPs represents loading the final supramolecular anti-hypertrophic scar material into GelMA-based microneedles; Centrifugation represents centrifugation; and UV crosslinking represents ultraviolet crosslinking.

[0148] Figure 6 It can be seen that in order to further improve the therapeutic efficiency of hypertrophic scars, we loaded cucurbituril anti-hypertrophic scar materials into GelMA-based microneedles to promote their transdermal delivery.

[0149] Figure 7This figure shows the wound surface of a rabbit ear hypertrophic scar model treated with the cucurbituril anti-hypertrophic scar material of Example 1. Here, Control is the blank control group, GelMA is the microneedle-only group, and GelMA / CAD NPs is the final supramolecular anti-hypertrophic scar material loaded in the microneedles.

[0150] Figure 7 It can be seen that the scars in the GelMA / CAD NPs microneedle patch group were relatively flat and the skin color was close to normal, while the scars in the other groups still showed a typical red color.

[0151] Figure 8 This is the immunohistochemical staining characterization of the in vivo treatment with the cucurbituril anti-hypertrophic scar material of Example 1. Control is the blank control group, GelMA is the microneedle-only group, and GelMA / CAD NPs is the final supramolecular anti-hypertrophic scar material loaded in the microneedles.

[0152] Figure 8 It can be seen that Figure 8 A is hematoxylin and eosin (H&E) staining, the results show that microneedles loaded with cucurbituril anti-hypertrophic scar material significantly inhibited the formation of hypertrophic scars, and the scar height and color were significantly reduced. Figure 8 B is the determination of collagen content by Masson's trichrome staining. It can be seen that the percentage of blue area representing collagen content was significantly inhibited in the microneedle patch group loaded with cucurbituril anti-hypertrophic scar material compared with the control group. Figure 8 C is the expression level of GPX4 detected by immunohistochemistry. The results showed that the number of GPX4-positive cells in the microneedle patch group loaded with cucurbituril anti-hypertrophic scar material was significantly decreased.

[0153] In summary, the present invention provides a supramolecular anti-hypertrophic scar material, its preparation method and application. The invention assembles hydrophilic supramolecular macrocycles with hydrophobic Chinese herbal medicine molecules to form vesicles through supramolecular host-guest interactions, and then loads functional component metal nanoclusters to construct a high-performance supramolecular material for treating hypertrophic scars based on a ferroptosis-cell apoptosis synergistic mechanism. The formed supramolecular vesicles solve the problem of hydrophobicity of Chinese herbal medicine molecules and enhance the performance of hydrophobic Chinese herbal medicine molecules in inducing ferroptosis. The vesicles are loaded with enriched metal nanoclusters, which enhance their enzyme-like catalytic activity and can efficiently induce cell apoptosis. They are further loaded into hydrogel microneedles to promote their transdermal delivery, showing superior therapeutic effects in shortening treatment time and improving the appearance of hypertrophic scars. The preparation method provided by the present invention is simple in process and highly operable. At the same time, the ferroptosis-cell apoptosis synergistic strategy based on supramolecular assembly provides innovative guidance for the effective treatment of anti-hypertrophic scars and other diseases.

[0154] The present invention describes preferred embodiments and their effects. However, those skilled in the art, once informed of the basic inventive concept, may make additional changes and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the invention.

[0155] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A supramolecular anti-hypertrophic scar material, characterized in that: The material includes amphiphilic vesicles formed by the assembly of water-soluble supramolecular macrocycles and hydrophobic Chinese herbal medicine molecules through host-guest interactions, wherein the inner and outer layers of the amphiphilic vesicles are both supramolecular macrocycles, and the hydrophobic Chinese herbal medicine molecules are located between the inner and outer layers of the vesicles; the interior of the amphiphilic vesicles also includes metal nanoclusters; The supramolecular macrocycle is a cucurbituril[7]; The hydrophobic Chinese herbal molecule is dihydroartemisinin; The metal nanoclusters are gold nanoclusters; The molar ratio of the supramolecular macrocycle to the hydrophobic Chinese herbal medicine molecule is 20:1-1:20; the molar ratio of the supramolecular macrocycle in the amphiphilic vesicle to the metal in the metal nanocluster is 5:1-1:

100.

2. A method for preparing the supramolecular anti-hypertrophic scar material according to claim 1, characterized in that: The following steps are involved: Preparation of metal nanoclusters; The water-soluble supramolecular macrocycle aqueous solution and the hydrophobic Chinese herbal medicine molecule ethanol solution are uniformly mixed and stirred at room temperature for assembly to obtain an amphiphilic vesicle solution; A metal nanocluster solution is added to a solution containing amphiphilic vesicles, the mixture is allowed to stand and assemble at room temperature, and thus a supramolecular anti-hypertrophic scar material is obtained.

3. The method for preparing the supramolecular anti-hypertrophic scar material according to claim 2, characterized in that: The metal nanoclusters are prepared according to the following steps: Add the metal precursor solution to the surface ligand aqueous solution and react at 25-100°C for 3-36 hours to obtain 1-2 nm metal nanoclusters protected by the surface ligands; The surface ligand of the metal nanoclusters is glutathione.

4. The method for preparing the supramolecular anti-hypertrophic scar material according to claim 2, characterized in that: The concentration range of the water-soluble supramolecular macrocycle aqueous solution is 1-10 mM; the concentration range of the hydrophobic Chinese herbal medicine molecule ethanol solution is 1-10 mM; and the concentration range of the metal nanocluster solution is 1-10 mM.

5. Use of the supramolecular anti-hypertrophic scar material according to claim 1 in the preparation of a drug for treating hypertrophic scars.

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