Isoliquiritigenin fullerene hydrocolloid dressing and preparation process thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, and discloses an isoliquiritigenin fullerene hydrocolloid dressing and a preparation process thereof. The dressing comprises a fullerene-isoliquiritigenin nano compound serving as an active core, and a pH sensitive hydrogel matrix coating the nano compound. The hydrogel matrix is formed by in-situ crosslinking of hydrazide hyaluronic acid and an aldehyde polyethylene glycol cross-linking agent through an acid-cleavable hydrazone bond. The preparation method comprises the following steps: preparing fullerene and isoliquiritigenin into a nano compound through a nano precipitation method; dispersing the nano-composite in a hydrazide hyaluronic acid solution to obtain a first precursor; according to the invention, the delivery problem of hydrophobic components is solved by constructing the nano-composite, and the synergistic interaction of the two active components is realized; meanwhile, the dressing is endowed with intelligent responsiveness to the inflammation microenvironment by utilizing an acid-sensitive hydrazone bond, on-demand targeted release of active ingredients can be realized, and the dressing has remarkable clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, in particular to an isoliquiritigenin fullerene hydrocolloid dressing and a preparation process thereof. Background Art

[0002] Local inflammation, especially inflammation occurring in the skin and other surface tissues, is a very common pathological condition in clinical practice. The development of efficient and safe topical preparations to control local inflammation and promote tissue repair has always been a research hotspot in the medical field. Natural products have attracted much attention due to their wide sources and diverse activities. For example, isoliquiritigenin, a natural flavonoid compound, has been shown to have significant anti-inflammatory activity. However, its clinical application is greatly limited, mainly due to the strong hydrophobicity brought about by its molecular structure, which results in extremely low solubility in aqueous systems and is difficult to prepare into topical preparations with high bioavailability and the ability to effectively penetrate into the site of inflammation.

[0003] During the development of inflammation, oxidative stress damage caused by excessive production of reactive oxygen free radicals is a key factor in exacerbating tissue damage and delaying the repair process. Therefore, the introduction of antioxidant ingredients in anti-inflammatory treatment has become an important auxiliary strategy. Fullerene, as an all-carbon molecule, exhibits superb free radical scavenging ability due to its unique cage-like conjugated structure and is considered to be a highly promising nano-antioxidant. However, similar to isoliquiritigenin, fullerene also faces the same severe application bottleneck, that is, it is almost completely insoluble in water and most biocompatible solvents, which makes its application in the biomedical field, especially the preparation of uniform and stable topical preparations, full of technical challenges.

[0004] To achieve localized, sustained drug release, hydrogel dressings have been extensively studied as an ideal drug carrier. They can provide a moist healing environment for the wound surface and be loaded with active drugs. However, existing hydrogel technologies have significant shortcomings in delivering these hydrophobic drugs. Simply physically mixing insoluble drug powders into a hydrophilic gel matrix can easily lead to aggregation and uneven distribution of drug particles, compromising the stability of the formulation and failing to ensure accurate and reproducible dosing. Furthermore, traditional hydrogel dressings typically release drugs passively and at a constant rate, lacking the ability to sense and respond to changes in the lesion microenvironment. They are unable to intelligently adjust drug release rates based on dynamic changes in the severity of inflammation (such as a decrease in local pH), making it difficult to achieve "on-demand drug delivery." This, to a certain extent, limits their therapeutic efficacy and may result in drug waste. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides an isoliquiritigenin fullerene hydrocolloid dressing and its preparation process, which aims to efficiently and uniformly load hydrophobic anti-inflammatory and antioxidant components into a topical external preparation, and enable it to respond to the acidic microenvironment signals unique to the inflammatory site to achieve targeted and on-demand release of drugs.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides an isoliquiritigenin fullerene hydrocolloid dressing.

[0007] The dressing comprises: a nanocomposite formed by non-covalent interaction of fullerene and isoliquiritigenin; and a pH-sensitive hydrogel matrix. The nanocomposite serves as an active core and is uniformly dispersed in the network structure of the pH-sensitive hydrogel matrix.

[0008] In one embodiment of the present invention, the pH-sensitive hydrogel matrix is ​​formed by cross-linking two precursors: the first precursor is hydrazide-modified hyaluronic acid at a concentration of 0.5% to 2.0% (w / v) of the total dressing volume; the second precursor is an aldehyde-modified polyethylene glycol crosslinker at a concentration of 0.5% to 2.0% (w / v) of the total dressing volume. The combination of fullerene and isoliquiritigenin not only addresses the hydrophobicity and dispersibility issues of isoliquiritigenin by utilizing fullerene as a nanocarrier, but also leverages the fullerene's inherent free radical scavenging ability with isoliquiritigenin's biosignaling inhibitory effects, creating a synergistic and complementary anti-inflammatory mechanism. Furthermore, through a specific chemical cross-linking mechanism, the hydrogel matrix constructs an intelligent response system. Under normal physiological pH conditions, the hydrogel structure is stable and slowly releases the active ingredient. However, under the acidic microenvironment specific to inflammatory sites, the internal chemical bonds break, causing changes in the gel network structure, thereby accelerating the release of the nanocomplex, achieving drug accumulation and efficient action at the lesion site.

[0009] As a preferred technical solution, the cross-linked network of the pH-sensitive hydrogel matrix is ​​composed of acid-cleavable hydrazone bonds formed by the reaction of hydrazide groups on the hydrazide hyaluronic acid molecular chain and aldehyde groups on the aldehyde-modified polyethylene glycol cross-linker molecular chain.

[0010] As a preferred technical solution, the aldehyde-modified polyethylene glycol cross-linking agent is a multi-arm aldehyde-modified polyethylene glycol, such as a four-arm aldehyde-modified polyethylene glycol, to form a structurally stable and uniform three-dimensional network.

[0011] As a preferred technical solution, in the nanocomposite, the molar ratio of fullerene to isoliquiritigenin is 1:1 to 1:5, which helps to achieve effective loading and stable dispersion of isoliquiritigenin.

[0012] The second aspect of the present invention provides a preparation process for the above-mentioned isoliquiritigenin fullerene hydrocolloid dressing.

[0013] The core of this process is to gently embed the pre-prepared active nanocomplex into the smart hydrogel network through in situ cross-linking. It specifically includes the following steps: a. First, an aqueous dispersion of fullerene-isoliquiritigenin nanocomposite was prepared; b. Secondly, the first precursor containing a hydrazide group is dissolved in the aqueous dispersion of the nanocomposite prepared in step a to obtain a homogeneous active precursor solution; c. At the same time, dissolving the second precursor containing an aldehyde group in a buffer solution to obtain a crosslinker solution; d. Finally, the active precursor solution is mixed with the crosslinker solution. The two precursors react chemically through the functional groups (hydrazide and aldehyde) at the ends of the molecular chains to form a hydrogel in situ at room temperature.

[0014] As a further limitation of step (a), the specific operation includes: first dissolving fullerene and isoliquiritigenin in an organic solvent such as tetrahydrofuran, and complexing them by using π-π stacking and hydrophobic interaction; then injecting the organic phase solution into the aqueous phase by nanoprecipitation to form a suspension of the nanocomposite; finally, removing the organic solvent and unbound free drug by a dialysis process to obtain a pure nanocomposite aqueous dispersion.

[0015] As a further limitation on the first precursor used in step b, the first precursor is hydrazide-modified hyaluronic acid. Its preparation method comprises first chemically activating the carboxyl groups of the hyaluronic acid using a carbodiimide (e.g., EDC·HCl), then reacting the activated hyaluronic acid with an excess of adipic acid dihydrazide to introduce hydrazide functional groups into the hyaluronic acid molecular chain.

[0016] As a preferred technical solution, in step b, the final concentration of the first precursor (hydrazide hyaluronic acid) in the active precursor solution is 1.0% to 4.0% (w / v).

[0017] As a preferred technical solution, in step (c), the final concentration of the second precursor (aldehyde-modified polyethylene glycol) in the cross-linking agent solution is 1.0% to 4.0% (w / v).

[0018] As a preferred technical solution, in the mixing process of step (d), the active precursor solution and the crosslinker solution are mixed in equal volumes, and the total molar ratio of hydrazide groups to aldehyde groups in the mixed system is ensured to be in the range of 0.9:1 to 1.1:1 to achieve sufficient and efficient crosslinking.

[0019] The present invention provides an isoliquiritigenin fullerene hydrocolloid dressing and its preparation process, which has the following beneficial effects: 1. This invention constructs a nanocomposite of fullerene and isoliquiritigenin, not simply physically mixing the two drugs. Instead, it leverages their molecular structural properties to form a close bond, achieving functional complementarity. Fullerene, a broad-spectrum free radical scavenger, combines with isoliquiritigenin, which regulates biological signaling pathways, to jointly combat multiple pathogenic factors in the inflammatory microenvironment, resulting in a synergistic therapeutic effect (1+1>2), significantly enhancing the overall anti-inflammatory activity of the dressing.

[0020] 2. The core of the pH-sensitive hydrogel matrix designed in this invention is the utilization of acid-cleavable hydrazone bonds as crosslinking points. This enables the dressing to "sense" the unique acidic microenvironmental signals at the site of inflammation. Under normal physiological conditions, the gel structure is stable, slowly releasing the drug to maintain basic treatment. However, when inflammation occurs, the acid triggers the gel network to disintegrate rapidly, enabling the concentrated and rapid release of active ingredients at the lesion site, improving drug utilization and targeting of the treatment.

[0021] 3. To address the technical challenge of both isoliquiritigenin and fullerene being insoluble in water, the present invention innovatively utilizes a nanoprecipitation method to pre-prepare them into a nanocomposite that can be stably dispersed in an aqueous phase. This key step effectively transforms the hydrophobic active core into an aqueous dispersion, enabling it to be evenly embedded within the hydrogel network. This fundamentally prevents aggregation and sedimentation of drug particles within the matrix, ensuring the physical stability of the final dressing product and the accuracy of dosing.

[0022] 4. The dressing of this invention forms an in-situ gel at the target site within minutes by mixing two flowable precursor solutions during use. This "mix-and-use" feature not only simplifies the application process but also allows the dressing to perfectly conform to irregular wound surfaces or tissue cavities, forming a dense protective barrier. This in-situ gel formation avoids the inconvenience of cutting and fitting preformed dressings, significantly improving the convenience and compliance of clinical use.

[0023] 5. The present invention uses hyaluronic acid and polyethylene glycol, two widely used clinical polymers with excellent biocompatibility and biodegradability, as the hydrogel backbone. These two materials are inherently non-toxic and non-immunogenic, and their degradation products are safely absorbed and metabolized by the body. This fundamentally ensures the safety of the dressing when in contact with human tissue, reduces the risk of secondary irritation or allergic reactions, and provides a safe and reliable guarantee for long-term and repeated use. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0025] Example 1 This embodiment provides a method for preparing an isoliquiritigenin fullerene hydrocolloid dressing.

[0026] 1. Preparation of Fullerene-Isoliquiritigenin (C60-ISL) Nanocomplexes Fullerene (C60) and isoliquiritigenin (ISL) were weighed and dissolved in tetrahydrofuran (THF) to prepare stock solutions of 1.2 mg / mL each. The two stock solutions were mixed at a molar ratio of 1:3 between C60 and ISL to form an organic phase mixed solution. This mixed solution was stirred at 450 rpm using a magnetic stirrer for 4 hours at 25°C in the dark.

[0027] The organic phase solution was then injected into a 15-fold volume of ultrapure water, which was being vigorously stirred at 1000 rpm, using a microsyringe pump at a constant rate of 0.6 mL / min. The resulting nanocomposite aqueous dispersion was transferred to a dialysis bag with a molecular weight cutoff of 7000 Da and dialyzed against ultrapure water at 4°C for 36 hours, with the external ultrapure water replaced every 5 hours. After dialysis, the liquid in the bag was collected to obtain the C60-ISL nanocomposite aqueous dispersion.

[0028] 2. Preparation of Hydrazide-Conjugated Hyaluronic Acid (HA-ADH) Sodium hyaluronate (HA) with a molecular weight of 120 kDa was dissolved in 100 mM MES buffer, pH 6.0, to prepare a 7 mg / mL solution. EDC·HCl and NHS were added to this solution to achieve a molar ratio of HA carboxyl groups, EDC, and NHS of 1:3:3, and the solution was stirred at room temperature for 45 minutes. Subsequently, adipic acid dihydrazide (ADH) was added to achieve a molar ratio of HA carboxyl groups to ADH of 1:15, and the reaction was stirred continuously for 24 hours. After the reaction, the solution was placed in a dialysis bag with a molecular weight cutoff of 14,000 Da and dialyzed against 100 mM NaCl for 24 hours and then against ultrapure water for 48 hours. The purified product was freeze-dried to obtain the HA-ADH precursor.

[0029] 3. Preparation of Drug-Loaded pH-Responsive Hydrogels Solution A: The HA-ADH freeze-dried powder obtained in step 2 was weighed and dissolved in the C60-ISL nanocomposite aqueous dispersion prepared in step 1 to prepare a solution A with a final HA-ADH concentration of 2.5% (w / v).

[0030] Solution B: Weigh 10 kDa four-arm aldehyde-modified polyethylene glycol (4-arm PEG-CHO) and dissolve it in pH 7.4 PBS buffer to prepare a 2.5% (w / v) solution B.

[0031] When in use, quickly mix equal volumes of solution A and solution B and let them stand at 25°C for about 4 minutes to form a hydrocolloid dressing.

[0032] Example 2 This embodiment provides a method for preparing an isoliquiritigenin fullerene hydrocolloid dressing using relatively low parameter values.

[0033] 1. Preparation of Fullerene-Isoliquiritigenin (C60-ISL) Nanocomplexes Weigh C60 and ISL and dissolve them separately in THF to prepare a stock solution of 0.5 mg / mL each. Mix C60 and ISL in a 1:1 molar ratio. Stir at 300 rpm for 2 hours at 20°C in the dark. Subsequently, inject the organic phase into a 10-fold volume of ultrapure water at a rate of 0.2 mL / min and stir at 800 rpm. Place the product in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze against ultrapure water at 4°C for 24 hours, changing the water every 6 hours.

[0034] 2. Preparation of Hydrazide-Conjugated Hyaluronic Acid (HA-ADH) HA (50 kDa) was dissolved in 100 mM MES buffer (pH 5.5) to a 5 mg / mL solution. EDC·HCl and NHS were added to a molar ratio of HA carboxyl groups, EDC, and NHS of 1:2:2, and the mixture was stirred for 30 minutes. ADH was added to a molar ratio of HA carboxyl groups to ADH of 1:10, and the reaction was continued for 24 hours. The reaction solution was purified using a dialysis bag with a molecular weight cutoff of 12,000 Da and lyophilized.

[0035] 3. Preparation of Drug-Loaded pH-Responsive Hydrogels Solution A: The above-mentioned HA-ADH freeze-dried powder was dissolved in the prepared C60-ISL nanocomposite aqueous dispersion to prepare solution A with a final HA-ADH concentration of 1.0% (w / v).

[0036] Solution B: Weigh 5 kDa 4-arm PEG-CHO and dissolve it in PBS buffer (pH 7.4) to prepare a 1.0% (w / v) solution B. The molar ratio of hydrazide to aldehyde groups after mixing is 1:0.9.

[0037] Equal volumes of solution A and solution B were mixed and allowed to stand at 20°C for about 8 minutes to form a hydrocolloid dressing.

[0038] Example 3 This embodiment provides a method for preparing an isoliquiritigenin fullerene hydrocolloid dressing, using relatively high parameter values.

[0039] 1. Preparation of Fullerene-Isoliquiritigenin (C60-ISL) Nanocomplexes Weigh C60 and ISL and dissolve them separately in THF to prepare a stock solution of 2.0 mg / mL each. Mix C60 and ISL in a molar ratio of 1:5. Stir at 600 rpm for 6 hours at 25°C in the dark. Subsequently, inject the organic phase into a 20-fold volume of ultrapure water at a rate of 1.0 mL / min and stir at 1200 rpm. Place the product in a dialysis bag with a molecular weight cutoff of 7000 Da and dialyze against ultrapure water at 4°C for 48 hours, changing the water every 4 hours.

[0040] 2. Preparation of Hydrazide-Conjugated Hyaluronic Acid (HA-ADH) 200 kDa HA was dissolved in 100 mM MES buffer (pH 6.5) to a 10 mg / mL solution. EDC·HCl and NHS were added to a molar ratio of HA carboxyl groups, EDC, and NHS of 1:4:4, and the mixture was stirred for 60 minutes. ADH was added to a molar ratio of HA carboxyl groups to ADH of 1:20, and the reaction was continued for 24 hours. The reaction solution was purified using a dialysis bag with a molecular weight cutoff of 14,000 Da and lyophilized.

[0041] 3. Preparation of Drug-Loaded pH-Responsive Hydrogels Solution A: The above-mentioned HA-ADH freeze-dried powder was dissolved in the prepared C60-ISL nanocomposite aqueous dispersion to prepare solution A with a final HA-ADH concentration of 4.0% (w / v).

[0042] Solution B: Weigh 20 kDa 4-arm PEG-CHO and dissolve it in PBS buffer (pH 7.4) to prepare a 4.0% (w / v) solution B. The molar ratio of hydrazide to aldehyde groups after mixing is 1:1.1.

[0043] Equal volumes of solution A and solution B were mixed and allowed to stand at 25°C for about 1 minute to form a hydrocolloid dressing.

[0044] Comparative Example 1 Compared with Example 1, the difference is that in step 1, "Preparation of Fullerene-Isoliquiritigenin (C60-ISL) Nanocomposite," fullerene (C60) is not added. Instead, isoliquiritigenin (ISL) is dissolved in tetrahydrofuran (THF) at the same concentration. The same nanoprecipitation method is used to prepare an isoliquiritigenin nanosuspension. This suspension is used in place of the C60-ISL nanocomposite aqueous dispersion in all subsequent steps. The remaining steps and parameters are the same.

[0045] Comparative Example 2 Compared with Example 1, the difference is that in step 1, "Preparation of Fullerene-Isoliquiritigenin (C60-ISL) Nanocomposite," isoliquiritigenin (ISL) is not added. Instead, fullerene (C60) is dissolved in tetrahydrofuran (THF) at the same concentration. The same nanoprecipitation method is used to prepare a fullerene nanoparticle aqueous dispersion. This dispersion is used in place of the C60-ISL nanocomposite aqueous dispersion in all subsequent steps. The remaining steps and parameters remain the same.

[0046] Comparative Example 3 Compared with Example 1, the difference is that step 2, "Preparation of hydrazide-modified hyaluronic acid (HA-ADH)," is omitted. In step 3, solution A is prepared by directly dissolving unmodified sodium hyaluronate (HA) powder of the same molecular weight as in Example 1 in the C60-ISL nanocomposite aqueous dispersion prepared in step 1, at the same concentration of HA-ADH as in Example 1. The remaining steps and parameters are the same.

[0047] Comparative Example 4 Compared with Example 1, the difference is that in step 3, "Preparation of Drug-Loaded pH-Responsive Hydrogel," solution A and solution B are mixed at a volume ratio of 10:1 (solution A:solution B), rather than equal volumes. The remaining steps and parameters remain the same.

[0048] Comparative Example 5 Compared with Example 1, the differences are: Step 1, "Preparation of the fullerene-isoliquiritigenin (C60-ISL) nanocomposite," was omitted. In Step 3, the preparation of Solution A was modified to: when preparing the HA-ADH solution, untreated fullerene (C60) and isoliquiritigenin (ISL) solid powders were directly added to the HA-ADH aqueous solution and stirred to mix. The remaining steps and parameters remained the same.

[0049] Test Example 1: Comparative Test of Stability of Preparations Experimental Description This test aims to verify the necessity of the process step of pre-preparing fullerene and isoliquiritigenin into a nanocomposite in order to obtain a uniform and stable hydrocolloid dressing.

[0050] Experimental Materials: Sample A: isoliquiritigenin fullerene hydrocolloid dressing prepared according to the complete process of Example 1.

[0051] Sample B: A hydrocolloid dressing prepared according to the process of Comparative Example 5, except that untreated fullerene and isoliquiritigenin solid powders are directly mixed with the hydrazide hyaluronic acid aqueous solution.

[0052] Experimental steps: Take 5 ml of freshly prepared sample A and sample B respectively and place them in transparent, sealed glass sample bottles.

[0053] Place the two sample bottles vertically at room temperature (25°C) in the dark.

[0054] The macroscopic state of the hydrogels in the two sample bottles was directly observed and recorded by the naked eye at the time of preparation (0 hours) and after standing for 24 hours, focusing on its color uniformity, transparency, and whether there was visible particle aggregation or precipitation stratification.

[0055] Experimental data Table 1 Comparative observation results of the stability of hydrogels prepared in Example 1 and Comparative Example 5

[0056] Summary of experimental results The above test results demonstrate that the dressing prepared according to the technical solution of the present invention exhibits excellent macroscopic uniformity and physical stability. As shown in Table 1, the product of Example 1 was a homogeneous, translucent gel upon completion and retained its original uniform morphology after 24 hours of standing, with no observed aggregation or precipitation of any components. In contrast, the product of Comparative Example 5, prepared using simple physical mixing, exhibited significant component unevenness from the outset, and experienced severe phase separation and sedimentation within a short period of time.

[0057] The underlying mechanism for this significant difference lies in the key pretreatment step employed in the present invention: a nanoprecipitation method is used to prepare a stable aqueous nanocomposite of the hydrophobic fullerene and isoliquiritigenin. This step exploits the non-covalent interaction between the fullerene's large π-conjugated surface and the isoliquiritigenin molecules, effectively converting the two water-insoluble active ingredients into nanounits that are stably dispersed in the aqueous phase. These nanounits are evenly distributed and physically embedded in the subsequently formed three-dimensional hydrogel network, thus ensuring the macroscopic homogeneity of the final product.

[0058] This testing demonstrates that pre-preparing the active ingredient into a nanocomposite is crucial for addressing the challenge of uneven dispersion of hydrophobic components within the hydrogel matrix. This is not simply a matter of mixing components, but rather a creative process design that ensures uniform quality and performance of the final product. This high degree of uniformity is the foundation and prerequisite for achieving controlled and reproducible drug release and ensuring the synergistic therapeutic effects of fullerene and isoliquiritigenin at the site of action.

[0059] Test Example 2: Hydrogel Matrix Gel Formation Ability Test Experimental Description This test is intended to verify the necessity and effectiveness of the hydrogel matrix formed by the specific chemical reaction designed in the present invention, and to verify the importance of the key component distribution ratio in forming a stable gel structure.

[0060] Experimental Materials: Sample A was prepared according to the complete process of Example 1, wherein the active precursor solution and the cross-linker solution were mixed in equal volumes.

[0061] Sample C: prepared according to the process of Comparative Example 3, except that unchemically modified hyaluronic acid was used instead of hydrazide hyaluronic acid.

[0062] Sample D: prepared according to the process of Comparative Example 4, except that the mixing volume ratio of the active precursor solution to the cross-linking agent solution is 10:1.

[0063] Experimental steps: Take three clean, dry transparent glass bottles and label them A, C, and D respectively.

[0064] The two precursor solutions corresponding to sample A, sample C, and sample D are mixed according to their prescribed proportions, and the timer is started immediately.

[0065] The gel formation condition was determined by the inverted test tube method, which involves inverting the sample bottle 180 degrees every 30 seconds and observing the state of the mixed liquid in the bottle.

[0066] When the mixture no longer exhibits macroscopic flow after 30 seconds of inversion, the time at this point is recorded as the "gelation time." If it still does not gel after 5 minutes, it is recorded as "no gelation."

[0067] After mixing for 30 minutes, the final macroscopic state of all samples was observed and recorded.

[0068] Experimental data Table 2 Comparative test results of hydrogel gelling ability of Example 1 and Comparative Examples 3 and 4.

[0069]

[0070] Summary of experimental results The above test results intuitively demonstrate the effectiveness and accuracy of the chemical cross-linking system proposed in this invention. As shown in Table 2, only Sample A, prepared according to Example 1, successfully transformed into a stable gel with a fixed morphology and elasticity within minutes. However, the samples from Comparative Examples 3 and 4 failed to form a self-supporting gel structure under the same conditions, indicating that they were incapable of forming the desired dressing.

[0071] The underlying mechanism of this result lies in the core design of the present invention: the construction of a three-dimensional network through a specific chemical reaction. In Example 1, the hydrazide groups (-NHNH2) introduced into the molecular chain of hydrazide-modified hyaluronic acid (the first precursor) and the aldehyde groups (-CHO) on the molecular chain of aldehyde-modified polyethylene glycol (the second precursor) undergo a highly efficient and specific condensation reaction under mild physiological conditions, forming chemically stable hydrazone bonds (-C=N-NH-). When the two precursors are mixed at a functional group molar ratio close to 1:1, these newly formed hydrazone bonds serve as crosslinking points, connecting the linear polymer chains into a complete, pervasive three-dimensional network, thereby transforming the entire system from a liquid to a solid gel.

[0072] This test has effectively confirmed that the formation of the dressing is not derived from the physical entanglement of polymer chains. Comparative Example 3, due to the use of chemically unmodified hyaluronic acid, does not have a hydrazide group that can react with an aldehyde group on its molecular chain, and therefore cannot start the chemical crosslinking process, and the mixture always remains liquid. Meanwhile, the result of Comparative Example 4 shows that even if both precursors have corresponding reactive groups, the ratio of functional groups is seriously unbalanced, resulting in insufficient crosslinking reaction, and it is impossible to form an effective network structure, which only shows an increase in viscosity. Therefore, the crosslinking system proposed by the present invention, based on specific chemical modification and accurate proportioning, is the indispensable technical basis for building this functional hydrocolloid dressing, which fully reflects the purposefulness and creativity of its design.

[0073] Test Example 3: In vitro pH-responsive release behavior test Experimental Description This test aims to verify whether the hydrocolloid dressing described in the present invention has the ability to respond to a simulated inflammatory acidic microenvironment and achieve on-demand accelerated release of active ingredients.

[0074] Experimental Materials: Sample A: a hydrocolloid dressing prepared according to the complete process of Example 1.

[0075] Sample C: a product prepared according to the process of Comparative Example 3, wherein the matrix thereof is composed of hyaluronic acid that has not been chemically modified.

[0076] Release media: Phosphate buffered saline (PBS) with pH 7.4 and pH 6.0 were prepared respectively.

[0077] Experimental steps: Accurately weigh four samples, each weighing 1 gram, two of which are sample A and two are sample C.

[0078] Place each sample in a separate dialysis bag (molecular weight cutoff 8000 Da) and tie the bag tightly.

[0079] Prepare four beakers, each containing 100 ml of release medium at different pH values. Immerse one dialysis bag containing sample A and one dialysis bag containing sample C in PBS at pH 7.4. Immerse the other two samples (one sample A and one sample C) in PBS at pH 6.0.

[0080] All four beakers were placed in a thermostatic water bath shaker at 37°C and shaken continuously at 100 rpm.

[0081] At predetermined time points (2 h, 8 h, 24 h), 1 ml of release medium was removed from each beaker and immediately added to the beaker with 1 ml of fresh PBS medium of the corresponding pH.

[0082] The concentration of isoliquiritigenin (ISL) in the samples was determined by high performance liquid chromatography (HPLC), and the cumulative release percentage at different time points was calculated based on the initial drug loading.

[0083] Experimental data Table 3 Comparison of cumulative release rates between Example 1 and Comparative Example 3 under different pH conditions

[0084] Summary of experimental results The above test results clearly reveal the unique pH-responsive drug release characteristics of the dressing of the present invention. As shown in Table 3, the sample prepared in Example 1 exhibited a slow and sustained release behavior in a simulated normal physiological environment (pH 7.4), releasing only about a quarter of the active ingredient within 24 hours. However, when placed in an acidic environment (pH 6.0) simulating inflammation, the release rate significantly accelerated, with the cumulative release over 24 hours reaching 76.2%, more than three times that at pH 7.4. In contrast, the sample in Comparative Example 3 showed almost no difference in release profile regardless of pH, consistently exhibiting a slow release pattern.

[0085] The fundamental mechanism of this phenomenon lies in the intelligent chemical design of the dressing matrix of the present invention. The three-dimensional network of the dressing is cross-linked via hydrazone bonds formed by the reaction of hydrazide groups with aldehyde groups. This hydrazone bond is acid-sensitive, meaning that its chemical structure is stable in a neutral environment of pH 7.4, effectively constraining the gel network and thus limiting the diffusion of the nanocomposite loaded internally. When the external environment becomes acidic, the acid catalyzes the hydrolysis and cleavage of the hydrazone bonds, resulting in a reduction in the cross-linking points of the gel network, an increase in the network pore size, and even partial disintegration. This macroscopic structural change greatly reduces the physical barrier to the nanocomposite, allowing it to be rapidly released into the surrounding medium.

[0086] These test results strongly confirm that the dressing's intelligent release capability stems not from simple physical mixing but directly from its unique cross-linking system based on acid-cleavable chemical bonds. Comparative Example 3, lacking this chemical cross-linking structure, exhibits release behavior solely driven by simple physical diffusion, making it insensitive to pH changes. This design of the present invention enables the dressing to sense and respond to pathological signals at the site of inflammation, releasing high concentrations of the active ingredient precisely where it is needed most. This is the core innovation that distinguishes the present invention from traditional sustained-release formulations and enables targeted, on-demand treatment.

[0087] Test Example 4: In vitro synergistic antioxidant capacity test Experimental Description This test aims to verify whether the two active ingredients of the dressing described in the present invention, fullerene and isoliquiritigenin, exhibit a synergistic effect in their core antioxidant function after being combined through the formation of a nanocomposite.

[0088] Experimental Materials: Sample A: a hydrocolloid dressing (containing a C60-ISL complex) prepared according to the complete process of Example 1.

[0089] Sample E: a hydrocolloid dressing (containing only ISL) prepared according to the process of Comparative Example 1.

[0090] Sample F: a hydrocolloid dressing (containing only C60) prepared according to the process of Comparative Example 2.

[0091] Reagent: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) ethanol solution.

[0092] Experimental steps: Sample Pretreatment: Accurately weigh equal masses of Sample A, Sample E, and Sample F. Add sufficient pH 6.0 PBS buffer to each suspension and thoroughly sonicate and vortex until the gel is completely disintegrated, forming a uniform suspension. Adjust the final volume of each suspension so that the concentrations of C60 and ISL in Sample A correspond to and are equal to the concentrations of ISL in Sample E and C60 in Sample F.

[0093] Reaction system construction: Prepare three sets of test tubes. Add 2 ml of the suspension of sample A, sample E, and sample F to each set of test tubes.

[0094] Reaction and Measurement: Quickly add 2 ml of freshly prepared DPPH ethanol solution (0.1 mM) to each test tube. Mix thoroughly and allow to react at room temperature (25°C) in the dark for 30 minutes.

[0095] After the reaction was completed, the absorbance of each reaction solution was immediately measured at a wavelength of 517 nm using a UV-visible spectrophotometer.

[0096] A blank control group (2 mL ethanol + 2 mL DPPH solution) and a sample background control group (2 mL sample suspension + 2 mL ethanol) were set up to correct the results.

[0097] The free radical scavenging rate of each sample was calculated according to the formula: DPPH free radical scavenging rate (%) = [1 - (A_sample - A_sample background) / A_blank control] × 100%.

[0098] Experimental data Table 4 Comparison of DPPH radical scavenging ability between Example 1 and Comparative Examples 1 and 2

[0099] Summary of experimental results These test results strongly demonstrate that the present invention's combination of fullerene and isoliquiritigenin produces a synergistic effect on core functions. As shown in Table 4, both Comparative Example 1, containing isoliquiritigenin alone, and Comparative Example 2, containing fullerene alone, exhibited some free radical scavenging ability. However, the free radical scavenging rate of Example 1, in which the two were combined to form a nanocomposite and encapsulated in a hydrogel, was far higher than the simple sum of the scavenging rates of the first two (35.8% + 21.3% = 57.1%), demonstrating a significant synergistic antioxidant effect.

[0100] The underlying mechanism of this synergistic effect stems from the unique molecular-level binding and complementary functions of fullerene and isoliquiritigenin. In the nanocomplex of this invention, fullerene acts as a highly efficient "free radical sponge," rapidly and broadly capturing and neutralizing a wide range of reactive oxygen species. Simultaneously, isoliquiritigenin, with its phenolic hydroxyl structure, scavenges free radicals by donating hydrogen atoms. When the two are tightly bound together through π-π stacking, the large conjugated system of the fullerene effectively stabilizes the semiquinone radical formed by isoliquiritigenin after donating hydrogen atoms, promoting its antioxidant cycle and thereby enhancing overall antioxidant capacity.

[0101] Therefore, this invention goes beyond simply physically mixing two active ingredients. Instead, through the key technical feature of constructing a nanocomposite, it creatively achieves the therapeutic potential of "1+1>2." This synergistic effect enables the dressing to not only modulate biological signaling pathways at the site of inflammation through isoliquiritigenin, but also more effectively scavenge free radicals that cause oxidative stress and tissue damage, fundamentally strengthening its ability to combat the inflammatory microenvironment. This design fully demonstrates the invention's innovative combination of active ingredients and delivery strategy, and is the core guarantee for achieving highly effective anti-inflammatory effects.

[0102] 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. An isoliquiritigenin fullerene hydrocolloid dressing, characterized in that: include: Nanocomplexes formed by fullerene and isoliquiritigenin; and pH-sensitive hydrogel matrices; Among them, the nanocomplex is dispersed in a pH-sensitive hydrogel matrix; The pH-sensitive hydrogel matrix contains hydrazide-modified hyaluronic acid at a concentration of 0.5-2.0% and aldehyde-modified polyethylene glycol cross-linker at a concentration of 0.5-2.0%.

2. The dressing according to claim 1, characterized in that The pH-sensitive hydrogel matrix is ​​cross-linked by acid-cleavable hydrazone bonds formed by the reaction of the hydrazide groups on the hydrazide-modified hyaluronic acid and the aldehyde groups on the aldehyde-modified polyethylene glycol cross-linker.

3. The dressing according to claim 2, characterized in that The aldehyde-modified polyethylene glycol cross-linking agent is multi-arm aldehyde-modified polyethylene glycol.

4. The dressing according to claim 1, characterized in that In the nanocomposite, the molar ratio of fullerene to isoliquiritigenin is 1:1 to 1:

5.

5. A process for preparing the isoliquiritigenin fullerene hydrocolloid dressing according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Preparation of fullerene-isoliquiritigenin nanocomposite aqueous dispersion; b. dissolving the first precursor containing a hydrazide group in the aqueous dispersion of the nanocomposite of step a to obtain an active precursor solution; c. dissolving the second precursor containing an aldehyde group in a buffer solution to obtain a crosslinker solution; d. The active precursor solution is mixed with the crosslinker solution, and the hydrazide groups react with the aldehyde groups to form a hydrogel by in situ crosslinking.

6. The process according to claim 5, characterized in that Step a specifically includes: dissolving fullerene and isoliquiritigenin in an organic solvent for complexation; injecting the organic phase solution containing the complex into the aqueous phase through a nanoprecipitation method to form a nanosuspension; and removing the organic solvent and free drugs through dialysis to obtain the nanocomplex aqueous dispersion.

7. The process according to claim 5, characterized in that The first precursor is hydrazide hyaluronic acid, and its preparation steps include: activating the carboxyl group of hyaluronic acid by carbodiimide chemical method; reacting the activated hyaluronic acid with excess adipic acid dihydrazide to introduce hydrazide groups.

8. The process according to claim 5, characterized in that In the active precursor solution in step b, the concentration of the first precursor is 1.0-4.0%.

9. The process according to claim 5, characterized in that In the cross-linking agent solution in step c, the concentration of the second precursor is 1.0-4.0%.

10. The process according to claim 5, characterized in that In step d, the active precursor solution and the cross-linking agent solution are mixed in equal volumes, and the molar ratio of hydrazide groups to aldehyde groups after mixing is between 0.9:1 and 1.1:1.

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