Antifungal nanogels, methods of making and antifungal products thereof

Abstract

The application discloses an antifungal nanogel and a preparation method and antifungal product thereof, relates to the technical field of antifungal materials, and discloses an antifungal nanogel which comprises the following preparation raw materials: a cell-penetrating peptide modified quaternary ammonium polysaccharide derivative and a polyphenol crosslinking agent. The antifungal nanogel of the application realizes the high-efficiency antifungal effect through deep penetration mediated by the cell-penetrating peptide and the synergistic effect of the quaternary ammonium polysaccharide derivative and the polyphenol.

Description

Technical Field

[0001] This application relates to the field of antifungal materials technology, and in particular to an antifungal nanogel, its preparation method, and an antifungal product. Background Technology

[0002] Fungal infections, especially deep infections caused by opportunistic pathogens such as Candida and Aspergillus, have become a serious public health problem threatening human health. Clinically, most refractory and recurrent fungal infections are closely related to the formation of biofilms. Fungal biofilms are dense three-dimensional structures composed of fungal cells, extracellular polymers, and hyphal networks. They act as a physical barrier, significantly hindering the penetration of traditional antifungal drugs (such as azoles and polyenes), while providing a drug-resistant microenvironment for the internal fungi. This results in the drug dosage required for conventional treatment being much higher than that for airborne bacteria, and also makes them highly susceptible to developing drug resistance.

[0003] Quaternized polysaccharides have been extensively studied due to their good water solubility and broad-spectrum antibacterial activity. However, their effectiveness against certain pathogenic fungi that easily form biofilms (such as Candida albicans) remains challenging. Fungal biofilms are complex community structures encapsulated by their own secreted extracellular polymers. Their dense physical barrier severely restricts the penetration of antibacterial substances, and quaternized polysaccharides have limited penetration ability into mature fungal biofilms, making it difficult to effectively act on the deep cells within the biofilm. Summary of the Invention

[0004] The main objective of this application is to provide an antifungal nanogel, its preparation method, and an antifungal product. The antifungal nanogel of this application achieves a highly efficient antifungal effect through deep penetration mediated by membrane-penetrating peptides and the synergistic effect of quaternized polysaccharide derivatives and polyphenols.

[0005] To achieve the above objectives, this application provides an antifungal nanogel comprising the following raw materials: Quaternized polysaccharide derivatives modified with transmembrane peptides and polyphenol crosslinking agents.

[0006] In one embodiment, the concentration ratio between the membrane-penetrating peptide-modified quaternized polysaccharide derivative and the polyphenol crosslinking agent is 0.8 to 6.

[0007] In one embodiment, the polyphenol crosslinking agent includes at least one of tannic acid, gallic acid, catechin, protocatechuic acid, and gentianic acid.

[0008] In one embodiment, the membrane-penetrating peptide-modified quaternized polysaccharide derivative comprises the following raw materials: Modified transmembrane peptides with complementary functional group pairs and quaternized polysaccharides.

[0009] In one embodiment, the polysaccharide includes chitin and / or chitosan.

[0010] In one embodiment, the membrane-penetrating peptide includes at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0011] In one embodiment, the complementary functional group pair includes any one of the following: Azide and alkynyl groups; Carboxyl and amino groups; Maleimide group and thiol group; Aldehyde and amino groups; Thiol group and thiol group.

[0012] In one embodiment, the degree of quaternization of the quaternized polysaccharide is 0.25 to 1.25.

[0013] In one embodiment, the quaternized polysaccharide has a weight-average molecular weight of 1.5 × 10⁻⁶. 3 ~9×10 5 g / mol.

[0014] In one embodiment, the degree of deacetylation of the quaternized polysaccharide is 25-99%.

[0015] In one embodiment, the grafting rate of the transmembrane peptide is 0.1 to 0.8.

[0016] In one embodiment, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0:5 to 5:1.

[0017] To achieve the above objectives, this application provides a method for preparing an antifungal nanogel, comprising the following steps: Antifungal nanogels were prepared by mixing membrane-penetrating peptide-modified quaternized polysaccharide derivatives with polyphenol crosslinking agents.

[0018] To achieve the above objectives, this application provides an antifungal product comprising the antifungal nanogel as described above, or the antifungal nanogel prepared by the antifungal nanogel preparation method described above.

[0019] This application provides an antifungal nanogel, the raw materials of which include: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide and a polyphenolic crosslinking agent. This application embodiment covalently modifies the membrane-penetrating peptide onto the quaternized polysaccharide backbone, allowing the membrane-penetrating peptide component in the resulting nanogel to retain its inherent cell-penetrating ability. This effectively overcomes the dense extracellular polymer barrier of fungal biofilms, solving the technical problem that traditional quaternized polysaccharides, due to their large molecular weight and dense positive charge, are difficult to autonomously penetrate into the biofilm. This nanogel exhibits excellent penetration ability against mature fungal biofilms, and can efficiently deliver the quaternized polysaccharide backbone to the deep layers of the biofilm through the "molecular anchoring" and carrying effect of the membrane-penetrating peptide. Upon reaching the deep layers, the high-density positive charge on the backbone interacts strongly with the negatively charged components on the surface of the fungal cell membrane, disrupting membrane integrity and causing intracellular leakage, thereby achieving efficient killing of fungal cells in a deeply encapsulated state. Furthermore, by introducing polyphenol cross-linking agents, they were successfully cross-linked with membrane-penetrating peptide-modified quaternized polysaccharide derivatives through hydrogen bonding to form nanogels. Notably, the polyphenol cross-linking agents themselves possess broad-spectrum antifungal activity. Their phenolic hydroxyl groups can chelate metal ions, interfere with fungal enzyme systems, and disrupt cell wall structures. Therefore, they produce a synergistic effect with the membrane-penetrating peptide-modified quaternized polysaccharides. The quaternized polysaccharides are responsible for electrostatically disrupting cell membranes, while the polyphenol cross-linking agents synergistically attack at the metabolic and cell wall levels. The antifungal activity of the two when used together is significantly better than the sum of their individual effects, achieving a highly efficient antifungal effect. Attached Figure Description

[0020] Figure 1 The Fourier transform infrared spectrum of the sample involved in the embodiments of this application; Figure 2 This is a schematic diagram of the transmission electron microscopy test results of sample NG7 involved in the embodiments of this application; Figure 3 This is a schematic diagram showing the determination results of the minimum inhibitory concentration against Candida albicans in the samples involved in the embodiments of this application; Figure 4 The cell survival rate is the rate of the sample and L929 cells after incubation in the embodiments of this application.

[0021] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] The following detailed description, with appropriate reference to the accompanying drawings, discloses the antifungal nanogel, its preparation method, and embodiments of the antifungal product of this application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for the purpose of enabling those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0024] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0025] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0026] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, preferably sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0027] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the technical solution of this application is further described below in conjunction with the accompanying drawings and embodiments. However, this application is not limited to the listed embodiments, but should also include any other well-known modifications within the scope of the claims made in this application.

[0028] To better understand the technical solution of this application, a detailed description will be provided below in conjunction with the accompanying drawings and specific implementation methods.

[0029] In conventional techniques, although quaternized polysaccharides perform well in inhibiting fungal growth, their ability to penetrate mature fungal biofilms is limited when dealing with certain pathogenic fungi that easily form biofilms (such as Candida albicans), making it difficult to effectively act on the deep cells inside the biofilm.

[0030] This application provides an antifungal nanogel, the raw materials of which include: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide and a polyphenolic crosslinking agent. This application embodiment covalently modifies the membrane-penetrating peptide onto the quaternized polysaccharide backbone, allowing the membrane-penetrating peptide component in the resulting nanogel to retain its inherent cell-penetrating ability. This effectively overcomes the dense extracellular polymer barrier of fungal biofilms, solving the technical problem that traditional quaternized polysaccharides, due to their large molecular weight and dense positive charge, are difficult to autonomously penetrate into the biofilm. This nanogel exhibits excellent penetration ability against mature fungal biofilms, and can efficiently deliver the quaternized polysaccharide backbone to the deep layers of the biofilm through the "molecular anchoring" and carrying effect of the membrane-penetrating peptide. Upon reaching the deep layers, the high-density positive charge on the backbone interacts strongly with the negatively charged components on the surface of the fungal cell membrane, disrupting membrane integrity and causing intracellular leakage, thereby achieving efficient killing of fungal cells in a deeply encapsulated state. Furthermore, by introducing polyphenol cross-linking agents, they were successfully cross-linked with membrane-penetrating peptide-modified quaternized polysaccharide derivatives through hydrogen bonding to form nanogels. Notably, the polyphenol cross-linking agents themselves possess broad-spectrum antifungal activity. Their phenolic hydroxyl groups can chelate metal ions, interfere with fungal enzyme systems, and disrupt cell wall structures. Therefore, they produce a synergistic effect with the membrane-penetrating peptide-modified quaternized polysaccharides. The quaternized polysaccharides are responsible for electrostatically disrupting cell membranes, while the polyphenol cross-linking agents synergistically attack at the metabolic and cell wall levels. The antifungal activity of the two when used together is significantly better than the sum of their individual effects, achieving a highly efficient antifungal effect.

[0031] The first embodiment of this application provides an antifungal nanogel comprising the following raw materials: a quaternized polysaccharide derivative modified with a transmembrane peptide and a polyphenol crosslinking agent.

[0032] In one feasible embodiment, the polyphenol crosslinking agent includes at least one of tannic acid, gallic acid, catechin, protocatechuic acid, and gentianic acid.

[0033] In one feasible embodiment, the polyphenol crosslinking agent is a polyphenolic substance with phenolic hydroxyl groups. It successfully crosslinks with the quaternized polysaccharide derivative modified with the membrane-penetrating peptide through hydrogen bonding to form a nanogel. At the same time, the polyphenol crosslinking agent itself has broad-spectrum antifungal activity. Its phenolic hydroxyl groups can chelate metal ions, interfere with the fungal enzyme system, and destroy the cell wall structure. Therefore, it has a synergistic effect with the quaternized polysaccharide modified with the membrane-penetrating peptide. The quaternized polysaccharide is responsible for electrostatically destroying the cell membrane, while the polyphenol crosslinking agent attacks the cell membrane synergistically from the metabolic and cell wall levels. The antifungal activity of the two when used together is significantly better than the sum of the effects of using them alone, achieving a highly efficient antifungal effect.

[0034] In one feasible embodiment, the concentration ratio between the membrane-penetrating peptide-modified quaternized polysaccharide derivative and the polyphenol crosslinking agent is 0.8 to 6.

[0035] Optionally, the concentration ratio between the membrane-penetrating peptide-modified quaternized polysaccharide derivative and the polyphenol crosslinking agent can be 0.8, 0.9, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc. In this embodiment, by controlling the concentration ratio of the membrane-penetrating peptide-modified quaternized polysaccharide derivative to the polyphenol crosslinking agent within an appropriate range, it is possible to ensure that the two form a stable and uniform nanogel network through hydrogen bonds, avoiding loose structure or drug leakage due to insufficient crosslinking, and agglomeration and precipitation due to excessive crosslinking.

[0036] In one feasible embodiment, the membrane-penetrating peptide-modified quaternized polysaccharide derivative comprises the following preparation materials: a membrane-penetrating peptide modified with complementary functional group pairs and a quaternized polysaccharide.

[0037] In one feasible embodiment, the membrane-penetrating peptide-modified quaternized polysaccharide derivative is formed by the membrane-penetrating peptide being coupled to the quaternized polysaccharide backbone via a covalent bond or linker arm.

[0038] Optionally, the membrane-penetrating peptide-modified quaternized polysaccharide derivative is a membrane-penetrating peptide-coupled quaternized polysaccharide derivative.

[0039] In one feasible embodiment, the polysaccharide includes: chitin (QC) and / or chitosan (QCS).

[0040] Optionally, chitin and chitosan, as natural amino polysaccharides, inherently possess certain antifungal activities. Chitosan's antifungal activity primarily stems from its free amino groups. Under slightly acidic conditions, these amino groups protonate to form positively charged ammonium ions, making them polycationic electrolytes. These positive charges can electrostatically adsorb onto negatively charged components on the fungal cell membrane surface (such as phospholipids, proteins, and teichoic acid), leading to cell membrane structural disorder, increased permeability, leakage of intracellular electrolytes and proteins, and ultimately, fungal cell death. Chitin, as an insoluble fiber or nanocrystal, can encapsulate fungal hyphae or spores through physical entanglement and adsorption, interfering with their uptake and attachment of nutrients, thereby inhibiting fungal growth and reproduction.

[0041] Optionally, quaternization modification of the chitin or chitosan backbone introduces hydrophilic quaternary ammonium groups, solving the solubility bottleneck of chitin and endowing it with strong polycationic properties; at the same time, it also enhances the persistence of the positive charge of chitosan. This makes quaternized chitin and quaternized chitosan high-performance materials that combine water solubility, strong positive charge, and highly efficient antifungal activity.

[0042] In one feasible embodiment, the membrane-penetrating peptide includes at least one of: octaarginine (R8), TAT (trans-transcription activator membrane-penetrating peptide), Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0043] Optionally, fungal biofilms (such as Candida albicans biofilms) are dense three-dimensional structures composed of extracellular polymers with small pore sizes and negative charges, making them susceptible to interception by traditional antibacterial macromolecules on their surface. R8 is rich in arginine (guanidino), which can form bidentate hydrogen bonds, resulting in strong but reversible binding with anionic components such as phosphate and sulfate groups in the cell membrane and extracellular polymers. This dynamic "binding-dissociation" process endows them with an internalization ability like a "shuttle," enabling them to actively carry quaternized polysaccharides across the dense barrier of the biofilm, reaching the fungal cells deep within the biofilm. Furthermore, R8 can carry quaternized polysaccharides into the cell interior through endocytosis or direct membrane penetration. Once inside the cell, the positive charge of the quaternized polysaccharides can interfere with mitochondrial function and disrupt nucleic acid metabolism, thus killing fungi from within.

[0044] Optionally, the general structural formulas of membrane-penetrating peptide-modified quaternized polysaccharide derivatives include: QC-R8 and QCS-R8.

[0045] Optionally, the membrane-penetrating peptide and the quaternized polysaccharide are coupled via a coupling reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, wherein the coupling reaction includes any one of the following: click chemistry reaction, amidation reaction, Michael addition reaction, reductive amination reaction, and disulfide bond exchange reaction.

[0046] In one feasible embodiment, the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs, which include any one of the following: azide and alkynyl; carboxyl and amino; maleimide and thiol; aldehyde and amino; thiol and thiol.

[0047] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated by amidation reaction, then a carboxyl group needs to be modified on one of the membrane-penetrating peptide and the quaternized polysaccharide, while an amino group needs to be modified on the other.

[0048] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated via Michael addition reaction, then maleimide groups need to be modified on one of the membrane-penetrating peptides and the quaternized polysaccharide, while thiol groups are modified on the other.

[0049] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated by a reductive amination reaction, then an aldehyde group needs to be modified on one of the membrane-penetrating peptide and the quaternized polysaccharide, while an amino group needs to be modified on the other.

[0050] Optionally, if a transmembrane peptide-modified quaternized polysaccharide derivative is generated through a disulfide bond exchange reaction, then one of the transmembrane peptide and the quaternized polysaccharide needs to be modified with a thiol group, while the other is modified with a disulfide bond structure (thiol group).

[0051] Optionally, if the membrane-penetrating peptide and the quaternized polysaccharide are reacted through a click chemical reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, the quaternized polysaccharide can be an azide-quaternized polysaccharide, and the membrane-penetrating peptide can be a terminally alkyne-modified membrane-penetrating peptide.

[0052] Optionally, the terminally alkyne-terminated transmembrane peptides include R8-Pra and / or TAT-Pra.

[0053] Optionally, membrane-penetrating peptides contain multiple active groups such as amino and carboxyl groups. If other reaction methods are used, the membrane-penetrating peptides may randomly attach to the polysaccharide backbone at multiple sites, causing some of the membrane-penetrating peptides to lose their penetrating activity. Click chemistry (azide-alkynyl cycloaddition) is an orthogonal reaction; the alkynyl group only reacts with the azide group and does not react at all with the natural functional groups such as amino and guanidine groups on the membrane-penetrating peptide. Therefore, by using terminally alkyne-modified membrane-penetrating peptides, it is possible to ensure that the membrane-penetrating peptide is fixed in an "upright, outward" manner, maximizing the exposure of its active penetrating domain. At the same time, click chemistry can be carried out under physiological conditions (room temperature, aqueous phase, neutral pH), which can protect the tertiary structure and membrane-penetrating activity of the membrane-penetrating peptide and avoid peptide chain denaturation or inactivation that may occur in traditional organic synthesis.

[0054] Alternatively, the quaternized polysaccharide can be azidolated to enable click chemistry. By introducing an azido group first, the membrane-penetrating peptide (terminal alkyneation) can only undergo a click reaction with the azido group at its terminal. This "terminal-to-backbone" linkage ensures that each membrane-penetrating peptide molecule is fixed to the polysaccharide in a defined, outwardly extended conformation, maximizing its activity for penetrating biological membranes.

[0055] In one feasible embodiment, the degree of quaternization substitution of the quaternized polysaccharide is 0.25–1.25. For example, the degree of quaternization substitution of the quaternized polysaccharide is 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.25, etc. In this embodiment, by controlling the degree of quaternization substitution, an optimal balance is found between the solubility, bactericidal activity, linkage efficiency, and biosafety of the polysaccharide derivative, enabling the bactericidal polysaccharide and the penetrating peptide to synergistically enhance each other.

[0056] In one feasible embodiment, the weight-average molecular weight of the quaternized polysaccharide is 1.5 × 10⁻⁶. 3 ~9×10 5 g / mol; for example, the weight-average molecular weight of quaternized polysaccharides is 1.5 × 10⁻⁶ g / mol. 3 g / mol, 5×10 3 g / mol, 1×10 4 g / mol, 5×10 4 g / mol, 1×10 5 g / mol, 2×10 5 g / mol, 3×10 5 g / mol, 4×10 5 g / mol, 5×10 5 g / mol, 6×10 5 g / mol, 7×10 5 g / mol, 8×10 5 g / mol, 9×10 5 g / mol, etc. Weight-average molecular weight determines the chain length and hydrodynamic volume of the polysaccharide backbone; in the final product of membrane-penetrating peptide modification, it mainly affects penetration ability, in vivo circulation time, and dosage form selection. If the weight-average molecular weight is too low, the efficiency of the membrane-penetrating peptide decreases; if the weight-average molecular weight is too high, the polysaccharide derivative faces severe steric hindrance when moving in the dense extracellular polymeric network, making it difficult to penetrate deep into the biological membrane even with the traction of the membrane-penetrating peptide.

[0057] In one feasible embodiment, the degree of deacetylation of the quaternized polysaccharide is 25%–99%. For example, the degree of deacetylation of the quaternized polysaccharide is 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, etc. By maintaining the degree of deacetylation within a suitable range, on the one hand, a sufficient number of free amino groups on the polysaccharide backbone are ensured as active sites for subsequent quaternization reactions, thereby introducing sufficient quaternary ammonium groups to endow the polysaccharide derivative with a high density of positive charge and excellent water solubility, ensuring antifungal activity; on the other hand, a moderate degree of deacetylation retains an appropriate amount of acetylation units, allowing the polysaccharide derivative to still be recognized and gradually degraded by lysozyme in vivo, avoiding the risk of long-term retention caused by slow degradation due to excessively high deacetylation, ultimately achieving a synergistic balance between highly efficient bactericidal activity and good biodegradability.

[0058] In one feasible embodiment, the grafting rate of the membrane-penetrating peptide is 0.1–0.8. For example, the grafting rate of the membrane-penetrating peptide is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, etc. By controlling the grafting rate of the membrane-penetrating peptide, on the one hand, it ensures that a sufficient number of membrane-penetrating peptides synergistically act on the fungal biofilm to generate a highly efficient penetration driving force, enabling the quaternized polysaccharide backbone to successfully reach the deep layers of the biofilm; on the other hand, it avoids that an excessively high grafting rate would cause the membrane-penetrating peptides to become too crowded on the polysaccharide backbone, mutually obscuring the active domains or failing to fully extend due to steric hindrance, thereby preserving the independent penetration activity of each membrane-penetrating peptide molecule.

[0059] In one feasible embodiment, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0.5 to 5:1. For example, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc. By maintaining the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide within a suitable range, it is ensured that a sufficient number of membrane-penetrating peptides are covalently linked to the polysaccharide backbone, enabling them to generate a synergistic penetration effect and efficiently drive the complex to cross the dense barrier of the fungal biofilm. On the other hand, it avoids excessive crowding of the polysaccharide backbone surface due to an excessively high proportion of membrane-penetrating peptides, preventing the membrane-penetrating peptide molecules from interfering with each other or losing activity due to insufficient extension caused by steric hindrance. At the same time, it also prevents unreacted free membrane-penetrating peptide residues from increasing potential toxicity and preparation costs.

[0060] Optionally, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 1 to 3:1.

[0061] In this embodiment, the raw materials for preparing the antifungal nanogel include: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide and a polyphenolic crosslinking agent. This embodiment covalently modifies the membrane-penetrating peptide onto the quaternized polysaccharide backbone, allowing the membrane-penetrating peptide component in the resulting nanogel to retain its inherent cell-penetrating ability. This effectively overcomes the dense extracellular polymer barrier of fungal biofilms, solving the technical problem that traditional quaternized polysaccharides, due to their large molecular weight and dense positive charge, are difficult to autonomously penetrate into the biofilm. This nanogel exhibits excellent penetration ability against mature fungal biofilms, and can efficiently deliver the quaternized polysaccharide backbone to the deep layers of the biofilm through the "molecular anchoring" and carrying effect of the membrane-penetrating peptide. Upon reaching the deep layers, the high-density positive charge on the backbone interacts strongly with the negatively charged components on the surface of the fungal cell membrane, disrupting membrane integrity and causing intracellular leakage, thereby achieving efficient killing of fungal cells in a deeply encapsulated state. Furthermore, by introducing polyphenol cross-linking agents, they were successfully cross-linked with membrane-penetrating peptide-modified quaternized polysaccharide derivatives through hydrogen bonding to form nanogels. Notably, the polyphenol cross-linking agents themselves possess broad-spectrum antifungal activity. Their phenolic hydroxyl groups can chelate metal ions, interfere with fungal enzyme systems, and disrupt cell wall structures. Therefore, they produce a synergistic effect with the membrane-penetrating peptide-modified quaternized polysaccharides. The quaternized polysaccharides are responsible for electrostatically disrupting cell membranes, while the polyphenol cross-linking agents synergistically attack at the metabolic and cell wall levels. The antifungal activity of the two when used together is significantly better than the sum of their individual effects, achieving a highly efficient antifungal effect.

[0062] The second embodiment of this application provides a method for preparing an antifungal nanogel, which is used to prepare the antifungal nanogel as described above. The method includes the following steps: Step S10: Mix the membrane-penetrating peptide-modified quaternized polysaccharide derivative with a polyphenol crosslinking agent to prepare an antifungal nanogel.

[0063] In one feasible embodiment, microfluidic preparation of nanogels can be employed. For example, a membrane-penetrating peptide-modified quaternized polysaccharide derivative and a polyphenol crosslinking agent are dissolved in deionized water to prepare a membrane-penetrating peptide-modified quaternized polysaccharide derivative solution and a polyphenol crosslinking agent solution, respectively. The membrane-penetrating peptide-modified quaternized polysaccharide derivative solution and the polyphenol crosslinking agent solution are then aspirated into microfluidic phases A and B, respectively. The flow rate is set, and the injection volume ratio is controlled to mix the two solutions in the microfluidic chip, thereby preparing an antifungal nanogel.

[0064] Optionally, the flow rate can be set to 5 mL / h.

[0065] Optionally, the injection volume ratio can be 1:1.

[0066] In one feasible embodiment, the preparation method of the membrane-penetrating peptide-modified quaternized polysaccharide derivative includes: Step S11 provides quaternized polysaccharides and membrane-penetrating peptides.

[0067] The quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional groups.

[0068] In one feasible embodiment, a quaternized polysaccharide and a membrane-penetrating peptide are provided, wherein the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs to achieve a coupling reaction between the two.

[0069] Optionally, the complementary functional group pair includes any one of the following: azide and alkynyl; carboxyl and amino; maleimide and thiol; aldehyde and amino; thiol and thiol.

[0070] Optionally, the membrane-penetrating peptide includes at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0071] Optionally, if the membrane-penetrating peptide-modified quaternized polysaccharide derivative is prepared by click chemistry, a terminally alkyne-modified membrane-penetrating peptide can be provided, and the quaternized polysaccharide can be azidated to obtain an azidated quaternized polysaccharide.

[0072] Optionally, the quaternized polysaccharide can be modified by azidation through a diazo transfer reaction to obtain azidated quaternized polysaccharide.

[0073] Alternatively, a diazo transfer reagent (such as imidazole-1-sulfonyl azide, trifluoromethanesulfonyl azide, etc.) can be used to react with the primary amino groups on the quaternized polysaccharide backbone to convert these amino groups into azide groups.

[0074] Step S12: The membrane-penetrating peptide is coupled to the quaternized polysaccharide backbone through a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0075] In one feasible embodiment, the membrane-penetrating peptide is coupled to the quaternized polysaccharide backbone by a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0076] Optionally, the coupling reaction includes any one of the following: click chemistry, amidation, Michael addition, reductive amination, and disulfide exchange reaction.

[0077] Optionally, the coupling chemical bonds in the membrane-penetrating peptide-modified quaternized polysaccharide derivatives include: single bonds, amide bonds (-CO-NH-), thioether bonds (-S-), disulfide bonds (-SS-), secondary amine bonds (-CH2-NH-), or linking groups containing 1,2,3-triazole rings.

[0078] Optionally, if the membrane-penetrating peptide and the quaternized polysaccharide are reacted through a click chemical reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, the quaternized polysaccharide can be an azide-quaternized polysaccharide, and the membrane-penetrating peptide can be a terminally alkyne-modified membrane-penetrating peptide.

[0079] Optionally, the click chemical reaction is a copper-catalyzed azido-alkynyl cycloaddition reaction; the reaction is carried out in a copper salt / reducing agent catalytic system, where the copper salt provides the catalyst source and the reducing agent is used to reduce divalent copper to monovalent copper in situ to initiate the catalytic cycle.

[0080] Optionally, to optimize reaction efficiency and product purity, the catalytic system may also include ligands (such as nitrogen-containing ligands) to stabilize monovalent copper and accelerate the reaction, as well as free radical or side reaction inhibitors to suppress by-products or oxidative degradation that may occur during the reaction.

[0081] Optionally, the click reaction is carried out under mild conditions, and the reaction system is an aqueous system or a mixture of water and organic solvents to ensure that the reactants are fully dissolved and to maintain the bioactivity of the transmembrane peptides.

[0082] In one feasible embodiment, the reaction temperature of the click chemical reaction is 0~60 ℃, for example, 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, etc.

[0083] In one feasible implementation, the reaction time of the click chemical reaction is 1 to 72 h, for example, 1 h, 5 h, 10 h, 20 h, 30 h, 40 h, 50 h, 60 h, 70 h, 72 h, etc.

[0084] Optionally, the product obtained from the coupling reaction is purified to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0085] Optionally, the purification process includes at least one of complexation copper removal, dialysis, ultrafiltration, precipitation separation, washing, freeze drying, and spray drying.

[0086] Exemplarily, the steps for preparing octaargine-modified quaternized chitosan via amidation include: accurately weighing 1.0 g of quaternized chitosan and dissolving it in 50 mL of MES buffer (0.1 M, pH 5.5) to prepare a homogeneous solution with a concentration of 20 mg / mL. Subsequently, 0.15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.09 g of N-hydroxysuccinimide (NHS) are added sequentially to the solution, wherein the molar ratio between the amino group of the quaternized chitosan, EDC, and NHS is controlled at 1:2:2, and the mixture is activated at 25 °C with stirring in the dark for 2 h. After activation, a pre-prepared octaargine solution is slowly added dropwise, wherein the octaargine solution is prepared by dissolving 0.5 g of Fmoc-R8-COOH with an N-terminal Fmoc protecting group in 10 mL of deionized water; the pH of the reaction system is adjusted to 7.2 using 0.1 M NaOH solution. The reaction was carried out under constant temperature and magnetic stirring at 25°C for 24 h. After the reaction, a 20% (v / v) piperidine solution was added to the system, and the reaction was carried out at room temperature for 2 h to completely remove the Fmoc protecting group. The final reaction mixture was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da, and dialyzed continuously in deionized water in the dark for 72 h, with the deionized water being replaced every 8 h to remove unreacted small molecules and inorganic salts. Finally, the dialysate was pre-frozen at -80°C for 12 h and then freeze-dried in a freeze dryer for 48 h to obtain pure octameric arginine-quaternized chitosan peptide polysaccharide modified with amide bond acid. It was a pale yellow flocculent solid with a yield of approximately 78%.

[0087] Exemplarily, the steps for preparing octaargine-modified quaternized chitosan via thiol-maleimide Michael addition include: accurately weighing 0.8 g of pre-synthesized maleimide-modified quaternized chitosan (Mal-QC, maleimide substitution degree approximately 15%), dissolving it in 40 mL of deoxyphosphate buffer (PBS, 0.1 M, pH 6.5), and purging with high-purity nitrogen for 30 minutes to prevent subsequent thiol oxidation. Separately, accurately weighing 0.2 g of cysteine-terminated octaargine (Cys-R8, containing free thiol groups as detected by Ellman's reagent) and dissolving it in 10 mL of the same deoxyphosphate buffer. Under nitrogen protection and at room temperature (25 °C), the Cys-R8 solution is slowly added dropwise to the Mal-QC solution using a constant-pressure dropping funnel, with the addition time controlled at 30 min; after the addition is complete, the system is kept at 25 °C and magnetically stirred for 6 h in the dark. Due to the high specificity and conversion rate of this reaction, the reaction solution was directly transferred to a dialysis bag with a molecular weight cutoff of 3500 Da after the reaction, and dialyzed with deionized water for 48 h. Subsequently, it was freeze-dried at -50 ℃ and a vacuum degree of less than 10 Pa for 36 h to finally obtain octameric arginine-modified quaternized chitosan peptide polysaccharide linked by thioether bonds, which was a white powder solid with a mass of about 0.85 g.

[0088] Exemplarily, the steps for preparing responsive octaargine-modified quaternized chitosan via disulfide bond oxidative crosslinking include: accurately weighing 1.0 g of thiolized quaternized chitosan and dissolving it in 50 mL of Tris-HCl buffer (0.05 M, pH 8.0) to prepare a homogeneous polymer solution. Subsequently, 0.3 g of cysteine-terminated octaargine (Cys-R8) is added to the system. After the short peptide is completely dissolved and mixed evenly, sterile air is continuously and slowly introduced into the reaction system at a flow rate of approximately 50 mL / min to provide a mild oxidative environment. The reaction is carried out under constant temperature water bath conditions of 30 °C with continuous magnetic stirring for 12 h, promoting oxidative crosslinking between the thiol groups of the QC-SH side chain and the thiol groups of the Cys-R8 terminal group to form stable disulfide bonds. After the reaction is completed, the pH of the system is neutralized to 7.0 using 0.1 M HCl solution to terminate the reaction. The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water at 4 °C in the dark for 72 h. After sterile filtration and freeze-drying, the dialysate yielded a disulfide-modified octameric arginine-quaternized chitosan peptide polysaccharide derivative with tumor microenvironment reduction responsiveness. It was a sponge-like solid with a mass of approximately 1.1 g.

[0089] In this embodiment, the raw materials for preparing the antifungal nanogel include: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide and a polyphenolic crosslinking agent. This embodiment covalently modifies the membrane-penetrating peptide onto the quaternized polysaccharide backbone, allowing the membrane-penetrating peptide component in the resulting nanogel to retain its inherent cell-penetrating ability. This effectively overcomes the dense extracellular polymer barrier of fungal biofilms, solving the technical problem that traditional quaternized polysaccharides, due to their large molecular weight and dense positive charge, are difficult to autonomously penetrate into the biofilm. This nanogel exhibits excellent penetration ability against mature fungal biofilms, and can efficiently deliver the quaternized polysaccharide backbone to the deep layers of the biofilm through the "molecular anchoring" and carrying effect of the membrane-penetrating peptide. Upon reaching the deep layers, the high-density positive charge on the backbone interacts strongly with the negatively charged components on the surface of the fungal cell membrane, disrupting membrane integrity and causing intracellular leakage, thereby achieving efficient killing of fungal cells in a deeply encapsulated state. Furthermore, by introducing polyphenol cross-linking agents, they were successfully cross-linked with membrane-penetrating peptide-modified quaternized polysaccharide derivatives through hydrogen bonding to form nanogels. Notably, the polyphenol cross-linking agents themselves possess broad-spectrum antifungal activity. Their phenolic hydroxyl groups can chelate metal ions, interfere with fungal enzyme systems, and disrupt cell wall structures. Therefore, they produce a synergistic effect with the membrane-penetrating peptide-modified quaternized polysaccharides. The quaternized polysaccharides are responsible for electrostatically disrupting cell membranes, while the polyphenol cross-linking agents synergistically attack at the metabolic and cell wall levels. The antifungal activity of the two when used together is significantly better than the sum of their individual effects, achieving a highly efficient antifungal effect.

[0090] The third embodiment of this application provides an antifungal product, comprising the antifungal nanogel described above.

[0091] Optionally, antifungal products are used to inhibit fungal growth and / or kill fungi.

[0092] Alternatively, fungi include Candida albicans.

[0093] Optionally, antifungal products are used to inhibit fungal biofilm formation and / or remove mature fungal biofilms.

[0094] Optionally, antifungal products include at least one of the following: antifungal coatings, dressings, gels, lotions, sprays, membrane materials, granules, and compound formulations.

[0095] Compared with conventional technologies, the beneficial effects of the antifungal product provided in the embodiments of the present invention are the same as those of the antifungal nanogel provided in the above embodiments, and other technical features of the antifungal product are the same as those disclosed in the methods of the above embodiments, and will not be repeated here.

[0096] In order to enable those skilled in the art to clearly understand the details and operations of the above embodiments of this application, and to demonstrate the significant improvement in performance of the embodiments of this application, the above technical solutions are illustrated below through multiple embodiments.

[0097] Example 1 A certain amount of quaternized chitosan (QCS) was weighed and dissolved in HCl solution, and NaHCO3 was added. The mixture was stirred vigorously for 30 min. Then, imidazole-1-sulfonyl azidohydrochloride and NaHCO3 were slowly added. CuSO4·5H2O was dissolved in water and methanol, and the resulting solution was added to the above reaction system. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for more than one week until the dialysis was clean. The solution was then freeze-dried to obtain the quaternized chitosan azidohydrochloride intermediate (QCS-N3).

[0098] A certain amount of CuSO4·5H2O and sodium ascorbate were weighed and dissolved in deionized water. QCS-N3 and terminally alkydinated octameric arginine (R8-Pra) were added to the solution, along with THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) ligand and aminoguanidine hydrochloride. The reaction was carried out at room temperature for 48 h. After the reaction was completed, MEDTA / Na solution was added to complex and remove copper, and stirring was continued for 1 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for more than one week until the dialysis was complete. After freeze-drying, a transmembrane peptide-modified azidoquamated chitosan derivative, QCS5-R8, was obtained. The molar ratio of the polysaccharide structural unit of QCS5-R8 to the quaternizing agent was 1:20, the molar ratio of the azidoquamated polysaccharide intermediate to the terminally alkydinated transmembrane peptide was 1:2, the degree of deacetylation was 95%, and the weight-average molecular weight was 2.094 × 10⁻⁶. 4 g / mol, grafting rate 32%.

[0099] QCS5-R8 and tannic acid (TA) were dissolved in deionized water to prepare a series of QCS5-R8 solutions and TA solutions with different concentration ratios. The QCS5-R8 solution and TA solution were aspirated into the microfluidic phases A and B, respectively, with a flow rate of 5 mL / h and an injection volume ratio of 1:1, so that the two solutions were mixed in the microfluidic chip to prepare nanogels.

[0100] The nanogels prepared by the method described in Example 1 above are shown in Table 1 below: Table 1:

[0101] The structural characterization of the samples involved in Example 1 was performed using Fourier transform infrared spectroscopy (FTIR), and the results are as follows:Figure 1 As shown, the horizontal axis represents wavenumber, and the vertical axis represents transmittance. The test samples include QCS5-R8, TA, and NG7. Figure 1 The display shows TA at 3366 cm. -1 The broad absorption peak originates from the stretching vibration of -OH, 1715 cm⁻¹. -1 The absorption peak is attributed to the stretching vibration of the carbonyl group (C=O), and the C=C stretching vibration peak of the benzene ring is located at 1611 cm⁻¹. -1 The QCS5-R8 is 3415cm tall. -1 The broad absorption peak at 1697 cm⁻¹ corresponds to the OH / NH stretching vibration peak. -1 The characteristic absorption peak of amide I with carbonyl group (C=O) appears. Compared with QCS5-R8, the -OH stretching vibration peak of the nanogel NG7 with added TA shifts to a lower wavenumber, and the C=O stretching vibration peak also redshifts, proving that there is an intermolecular hydrogen bonding between QCS5-R8 and TA. FTIR characterization results show that QCS5-R8 and TA have successfully crosslinked to form a nanogel through hydrogen bonding interactions.

[0102] Furthermore, the NG7 sample from Example 1 was subjected to transmission electron microscopy testing, and the results are as follows: Figure 2 As shown, NG7 exhibits a spherical structure with clear boundaries.

[0103] Furthermore, the antifungal activity of the examples was evaluated using the minimum inhibitory concentration (MIC) method for Candida albicans, and the results are as follows: Figure 3 As shown, where, Figure 3 The tested samples include: TA, QCS5-R8, NG3, NG4, NG5, NG7, and NG8; see [link / reference] Figure 3 It can be seen that the MIC values ​​of TA and QCS5-R8 are 100 μg / mL and 50 μg / mL, respectively. The antifungal activity of a series of nanogels prepared by TA and QCS5-R8 was improved, with MIC values ​​ranging from 12.5 to 25 μg / mL. Among them, NG4, NG5 and NG7 had the lowest MIC values.

[0104] Furthermore, the in vitro cytotoxicity of NG4, NG5, and NG7, which exhibited superior antibacterial activity, was evaluated using cell viability assays. The results were referenced from [reference needed]. Figure 4It is evident that at all tested concentrations (0–1600 μg / mL), the survival rate of L929 cells decreased with increasing nanogel concentration. Within the concentration range of 12.5–100 μg / mL, NG4, NG5, and NG7 cells all exhibited high cell compatibility with L929 cells, with cell viability exceeding 80%. Furthermore, the HC50 values ​​within the concentration range of 400–800 μg / mL indicate good cell compatibility of the nanogel.

[0105] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the patent protection scope of this application.

Claims

1. An antifungal nanogel, characterized in that, The following raw materials are included in the preparation: Quaternized polysaccharide derivatives modified with transmembrane peptides and polyphenol crosslinking agents.

2. The antifungal nanogel as described in claim 1, characterized in that, The concentration ratio between the membrane-penetrating peptide-modified quaternized polysaccharide derivative and the polyphenol crosslinking agent is 0.8 to 6.

3. The antifungal nanogel as described in claim 1, characterized in that, The polyphenol crosslinking agent includes at least one of tannic acid, gallic acid, catechin, protocatechuic acid, and gentianic acid.

4. The antifungal nanogel as described in claim 1, characterized in that, The membrane-penetrating peptide-modified quaternized polysaccharide derivative comprises the following raw materials: Modified transmembrane peptides with complementary functional group pairs and quaternized polysaccharides.

5. The antifungal nanogel as described in claim 4, characterized in that, The polysaccharide includes: chitin and / or chitosan; And / or, the membrane-penetrating peptide comprises at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

6. The antifungal nanogel as described in claim 4, characterized in that, The complementary functional group pair includes any one of the following: Azide and alkynyl groups; Carboxyl and amino groups; Maleimide group and thiol group; Aldehyde and amino groups; Thiol group and thiol group.

7. The antifungal nanogel as described in claim 4, characterized in that, The degree of quaternization of the quaternized polysaccharide is 0.25–1.25; And / or, the weight-average molecular weight of the quaternized polysaccharide is 1.5 × 10⁻⁶. 3 ~9×10 5 g / mol; And / or, the degree of deacetylation of the quaternized polysaccharide is 25-99%.

8. The antifungal nanogel as described in claim 4, characterized in that, The grafting rate of the membrane-penetrating peptide is 0.1–0.8; And / or, the molar ratio between the transmembrane peptide and the quaternized polysaccharide is 0:5 to 5:

1.

9. A method for preparing an antifungal nanogel, characterized in that, The method is applied to the preparation of antifungal nanogels as described in any one of claims 1 to 8, and the method comprises the following steps: Antifungal nanogels were prepared by mixing membrane-penetrating peptide-modified quaternized polysaccharide derivatives with polyphenol crosslinking agents.

10. An antifungal product, characterized in that, The antifungal product includes the antifungal nanogel as described in any one of claims 1 to 8, or the antifungal nanogel prepared by the method for preparing the antifungal nanogel as described in claim 9.

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