Nystatin microspheres with enhanced bacteriostatic function and preparation method and application thereof

Abstract

This invention discloses a nystatin microsphere with targeted enhanced antifungal function, its preparation method, and its application. The microsphere uses sodium alginate as a carrier, internally encapsulating the antifungal drug nystatin. The surface is covalently modified with the β-glucan binding domain of the Dectin-1 protein, constructing a smart drug delivery system with fungal targeting function. The microsphere preparation process is simple and reproducible, and can be scalable using an emulsion-ionic crosslinking method combined with protein coupling technology. In vitro characterization shows that the microspheres have uniform particle size and stable dispersion, and Dectin-1 modification significantly enhances their specific binding ability against Candida albicans. This targeted delivery system, while successfully preserving drug activity, significantly improves the antifungal efficiency of nystatin through Dectin-1-mediated targeting, achieving efficacy close to that of free drug, and providing a new strategy for constructing highly efficient and long-acting antifungal agents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nystatin microsphere with targeted enhanced antibacterial function, its preparation method, and its application. Background Technology

[0002] Intestinal fungal infections, especially those caused by Candida albicans, are among the most common opportunistic infections in clinical practice, frequently occurring in immunocompromised patients, individuals on long-term broad-spectrum antibiotic use, or those undergoing intestinal surgery. Under specific conditions, Candida albicans can proliferate excessively and invade the intestinal mucosa, leading to candidal colitis, which can even cause systemic dissemination and has a high mortality rate in severe cases. Currently, the main drugs used clinically to treat intestinal fungal infections include azoles (such as fluconazole), polyenes (such as nystatin and amphotericin B), and echinocandins. Among these, nystatin, as a non-absorbable polyene antifungal drug, is widely used to treat gastrointestinal Candida infections due to its local action in the intestine and its low systemic circulation. However, traditional nystatin preparations (such as oral suspensions) still suffer from insufficient drug concentrations, lack of targeting, biofilm-related resistance, and dosage form limitations. In recent years, targeted drug delivery systems for fungal infections have gradually become a research hotspot. Among these, strategies utilizing innate immune receptors to achieve targeted drug delivery show promising promise. Dectin-1 is a pattern recognition receptor expressed on the surface of immune cells such as macrophages and neutrophils. It can recognize β-1,3-glucan components in fungal cell walls with high affinity and specificity, a recognition mechanism that plays a crucial role in fungal immune surveillance. Based on this characteristic, modifying drug carriers with Dectin-1 can achieve active targeted delivery of antifungal drugs, improve drug accumulation at the fungal infection site, enhance efficacy, and reduce systemic side effects. In terms of dosage form, nanospheres, as a commonly used drug carrier, possess good biocompatibility, controlled-release properties, and surface modifiability, making them suitable for oral drug delivery systems. Encapsulating nystatin in nanospheres can improve its stability, prolong intestinal retention time, and achieve local sustained release of the drug. Further modifying the surface of nanospheres with Dectin-1 could potentially endow them with the ability to actively target intestinal fungi, thereby overcoming the shortcomings of existing nystatin formulations. Currently, although some studies have reported the application of Dectin-1-based targeted drug delivery systems in the treatment of systemic fungal infections, research on targeted oral delivery, especially in combination with microsphere carriers, for localized intestinal fungal infections remains relatively limited. Therefore, developing a novel nystatin formulation that is fungal-targeting, effectively inhibits biofilms, and is suitable for intestinal administration has significant clinical importance and application value. Summary of the Invention

[0003] To address the problems of insufficient local drug concentration, lack of targeting, difficulty in effectively clearing fungal biofilms, and poor stability of traditional oral formulations in the gastrointestinal environment, existing technologies for the treatment of intestinal fungal infections using nystatin, this invention provides a nystatin microsphere with targeted enhanced antibacterial function, its preparation method, and its application. This invention comprises Dectin-1 modified nystatin nanospheres. Through a strategy combining drug loading with receptor targeting, nystatin achieves precise delivery and efficient accumulation at the site of intestinal fungal infection, thereby improving therapeutic efficacy, reducing dosage, and minimizing side effects. This enables precise targeted delivery and efficient treatment of intestinal fungal infections such as Candida albicans, and provides a new dosage form option for targeted therapy of intestinal fungal infections.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A nystatin microsphere with targeted enhanced antibacterial function includes: a microsphere carrier matrix, wherein the microsphere carrier matrix encapsulates nystatin and Dectin-1 protein, wherein the Dectin-1 protein is covalently linked to the surface of the microsphere carrier matrix.

[0005] This microsphere, using sodium alginate as a carrier, encapsulates the antifungal drug nystatin and covalently modifies the surface of the Dectin-1 protein (whose functional domain specifically recognizes β-glucan), constructing a smart drug delivery system with fungal targeting capabilities. The microsphere preparation process is simple and reproducible, and can be scalable using an emulsion-ionic crosslinking method combined with protein coupling technology. In vitro characterization showed that the microspheres have uniform particle size and stable dispersion, and Dectin-1 modification significantly enhanced their specific binding ability to Candida albicans. In a simulated gastrointestinal environment, the microspheres effectively protected the drug and exhibited sustained-release properties. Further antibacterial experiments showed that, compared with unmodified microspheres and free drug, Dectin-1 modified microspheres had stronger inhibitory and clearance effects on Candida albicans and significantly reduced the effective therapeutic dose. This formulation increases the drug concentration at the lesion site through an active targeting mechanism, potentially enhancing efficacy while reducing systemic toxicity, providing a new formulation strategy for the clinical treatment of refractory fungal infections. In the evaluation of antibacterial performance, the agar diffusion method showed that the diameter of the inhibition zone formed by the Dectin-1 modified microspheres after 48 hours was comparable to that of the free drug, confirming that the released drug has the same inherent antibacterial activity as the active pharmaceutical ingredient. More importantly, time-inhibition rate kinetic analysis showed that the antibacterial effect of the Dectin-1 modified microspheres was significantly better than that of the unmodified ordinary microspheres, and its inhibition rate approached or even reached the level of the free drug in the later stages of the experiment. These results collectively demonstrate that this targeted delivery system, while successfully preserving drug activity, significantly improves the antibacterial efficiency of nystatin through Dectin-1-mediated targeting, achieving a level of efficacy approaching that of the free drug, and providing a new strategy for constructing highly effective and long-acting antifungal agents.

[0006] The microsphere carrier matrix is ​​sodium alginate or its derivative.

[0007] The microsphere carrier matrix is ​​sodium alginate, and the Dectin-1 protein receptor is covalently linked to the carboxyl group of sodium alginate via a coupling reaction mediated by carbodiimide and N-hydroxysuccinimide.

[0008] The ligand for the Dectin-1 protein is β-glucan.

[0009] Sodium alginate, Dectin-1 protein, and β-glucan synergistically construct a three-tiered intelligent antifungal system: Sodium alginate microspheres, acting as a multifunctional platform, not only directionally immobilize and protect the activity of Dectin-1 protein through their gel network but also serve as a responsive carrier to achieve targeted drug release. Dectin-1 protein is transformed from a natural immune receptor into an engineered trap, specifically recognizing β-glucan on the fungal surface and driving the microspheres to complete multivalent anchoring. β-glucan, as a target signal, initiates the entire recognition and response cascade. Its unique advantage lies in the deep complementarity of the three components: Dectin-1 endows the system with highly precise antibody recognition capabilities, sodium alginate provides a microenvironment-responsive intelligent drug release and immunomodulatory interface, and β-glucan ensures the specificity and biological relevance of the target. The three components work together to achieve an integrated closed loop of recognition, anchoring, release, and regulation. While precisely killing fungi, the microspheres can be phagocytosed by immune cells, thereby enhancing their clearance ability. They also regulate immune homeostasis by adsorbing free β-glucan, thus improving treatment efficiency while reducing toxic side effects and inflammatory risks. This demonstrates the deep integration advantages of material properties, biorecognition, and immune regulation.

[0010] A method for preparing nystatin microspheres includes the following steps: S1: Provides a microsphere carrier matrix loaded with nystatin; S2: In the presence of an activator, the Dectin-1 ligand is co-incubated with the microsphere carrier matrix loaded with nystatin obtained in step S1, so that the Dectin-1 ligand is covalently linked to the surface of the microsphere carrier matrix to obtain nystatin microspheres.

[0011] In step S1, the microsphere carrier matrix loaded with nystatin is prepared by emulsification crosslinking method; in step S2, the activator is a combination of carbodiimide and N-hydroxysuccinimide.

[0012] The use of the nystatin microspheres in the preparation of a medicament for the prevention and / or treatment of fungal infections. The fungal infection is Candida albicans infection. The medicament is formulated as an oral dosage form for the prevention and / or treatment of intestinal fungal infections.

[0013] An antifungal pharmaceutical composition comprising a therapeutically effective amount of the nystatin microspheres, and a pharmaceutically acceptable carrier or excipient.

[0014] Compared with the prior art, the present invention has the following significant advantages: (1) Precise targeting and enhanced efficacy: By utilizing Dectin-1’s specific recognition of fungal β-glucan, the drug is actively enriched at the site of infection, significantly increasing the local drug concentration and enhancing the antifungal effect.

[0015] (2) Overcoming biofilm drug resistance: Microspheres can attach to the surface of biofilm and gradually release drugs, improve the penetration and effect of drugs on fungi in biofilm, effectively inhibit biofilm formation and remove existing biofilms.

[0016] (3) Reduce systemic toxicity: The drug mainly exerts its effects locally in the intestines, reducing systemic absorption and thus reducing the systemic toxic side effects that nystatin may cause.

[0017] (4) Improve stability and compliance: Microsphere carriers protect nystatin from the gastrointestinal environment and improve oral stability; at the same time, they can regulate the release rate, prolong the duration of action, and reduce the frequency of administration.

[0018] (5) The preparation process is controllable and easy to scale up production: The microsphere preparation and surface modification technology used is mature, the process has good repeatability, and it has the potential for industrial production.

[0019] This invention provides a novel drug delivery system that is targeted, efficient, and safe for the treatment of intestinal fungal infections, and has promising clinical application prospects. Attached Figure Description

[0020] Figure 1 SEM images of Dectin-1-Nys-MS.

[0021] Figure 2 TEM image of Dectin-1-Nys-MS.

[0022] Figure 3 Particle size distribution of Dectin-1-Nys-MS.

[0023] Figure 4 Potential distribution of Dectin-1-Nys-MS.

[0024] Figure 5 Infrared spectral analysis of Dectin-1-Nys-MS.

[0025] Figure 6 Effect of different storage temperatures on the encapsulation efficiency of Dectin-1-Nys-MS.

[0026] Figure 7 Effect of different storage temperatures on the particle size of Dectin-1-Nys-MS.

[0027] Figure 8 Effects of Dectin-1-Nys-MS on in vitro release.

[0028] Figure 9 Effects of Dectin-1-Nys-MS on gastrointestinal digestion.

[0029] Figure 10 Comparison of inhibition zone diameters of different formulations against Candida albicans.

[0030] Figure 11-12 Long-term antibacterial kinetics of different formulations against Candida albicans. Detailed Implementation

[0031] Preparation Example A Dectin-1 modified nystatin microsphere, comprising: (1) Preparation of drug-loaded microspheres Preparation of sodium alginate drug-loaded aqueous phase: Weigh out sodium alginate powder, dissolve it in an appropriate amount of deionized water, and prepare a 1.0% (w / v) solution.

[0032] Place the above solution in a 60 ℃ water bath and stir continuously with magnetic force (300 rpm) until it is completely dissolved and clear.

[0033] After the solution has cooled to room temperature, filter it using a 0.45 μm nylon membrane to remove undissolved particles or impurities, and keep the filtrate for later use.

[0034] Accurately weigh nystatin and dissolve it in an 80% (v / v) ethanol solution, using sonication to aid dissolution, to prepare a suspension with a concentration of 10 mg / mL. Slowly add the nystatin suspension to the prepared sodium alginate solution at a ratio of nystatin:sodium alginate = 1:4 (w / w).

[0035] The two were thoroughly mixed using a vortex mixer to obtain a drug-loaded sodium alginate mixture. The mixture was then filtered again using a 0.45 μm nylon membrane to obtain the final aqueous phase, which was stored on ice or in a 4 °C freezer protected from light until use.

[0036] Oil phase preparation: Take an appropriate amount of MCT oil (medium-chain triglyceride oil). The volume ratio of the oil phase to the internal aqueous phase should be strictly controlled at V(oil phase):V(aqueous phase) = 4.624:1.

[0037] Add Span 80 (sorbitan monooleate) emulsifier to the MCT oil at a concentration of 4% (w / v). Stir with a magnetic stirrer (500 rpm) for 10 minutes at room temperature until the Span 80 is completely dissolved and the oil phase is clear and transparent.

[0038] Preparation of W / O (oil-in-water) colostrum: The prepared drug-loaded aqueous phase was added dropwise and slowly to the prepared oil phase using a syringe connected to a fine-hole burette needle and under magnetic stirring (800 rpm).

[0039] After the addition is complete, transfer the mixture to an ice bath and emulsify it using an ultrasonic cell disruptor (200 W, 10 minutes, ice bath throughout to prevent sample overheating and drug degradation). After emulsification, a uniform, milky-white W / O type colostrum should be obtained, without obvious stratification.

[0040] Ion crosslinking and curing (forming microspheres): Prepare a 3.62% (w / v) aqueous solution of calcium chloride (CaCl2) as a crosslinking agent and pre-cool it to 4 °C. The amount of crosslinking agent should be twice the total volume of the W / O primary emulsion.

[0041] With magnetic stirring (800 rpm), the W / O colostrum was added dropwise to the pre-cooled CaCl2 solution through a titration needle.

[0042] After the addition is complete, continue magnetic stirring at room temperature for 30 minutes to ensure that the sodium alginate droplets and calcium ions are fully cross-linked and solidified to form solid microspheres.

[0043] Microsphere collection and purification: Transfer the cross-linked mixture to a centrifuge tube and centrifuge at 4 °C and 12,000 rpm for 10 minutes. Carefully discard the supernatant; a pale yellow precipitate will be visible at the bottom of the tube.

[0044] Resuspend the precipitate in an appropriate amount of anhydrous ethanol and gently vortex for 30 seconds to elute any residual oil phase and Span 80. Centrifuge again under the same conditions and discard the supernatant. This step can be repeated 1-2 times.

[0045] Resuspend the precipitate in pre-cooled PBS buffer (pH 7.4), centrifuge, and discard the supernatant. Repeat this step twice to thoroughly remove ethanol and free calcium ions.

[0046] The final precipitate is sodium alginate microspheres loaded with nystatin. Depending on subsequent experimental needs, it can be resuspended in an appropriate amount of PBS buffer (for Dectin-1 modification) or freeze-dried to obtain a solid powder.

[0047] (2) Standard operating procedure for Dectin-1 protein covalently coupled modified microspheres Preparation of activator working solution (EDC / NHS activator solution): Weigh out 7.68 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 11.54 mg of N-hydroxysuccinimide.

[0048] Dissolve the weighed EDC and NHS powders together in 1.2 mL of MES buffer solution, and gently blow them to dissolve and clarify them completely.

[0049] Microsphere surface carboxyl group activation: Take 1.0 mL of the prepared drug-loaded microsphere suspension and place it in a 1.5 mL centrifuge tube. Centrifuge at 4°C and 12,000 rpm for 10 minutes, and carefully discard the supernatant.

[0050] Add 1.0 mL of freshly prepared EDC / NHS activation working solution to the microsphere precipitate. Gently pipette or vortex to completely resuspend the microspheres in the activation solution.

[0051] Place the reaction system on a shaker and gently shake at 60 rpm for 30 minutes at room temperature. After the reaction is complete, immediately proceed to the next step of cleaning; do not allow it to stand for an extended period.

[0052] Weigh out 61.8 mg of boric acid and 400 mg of sodium chloride. Dissolve both in approximately 7 mL of deionized water, stirring until completely dissolved. Slowly add 1 M sodium hydroxide solution dropwise to precisely adjust the pH of the solution to 8.3. Make up to a final volume of 10 mL with deionized water and mix well. This is the 0.1 M borate buffer solution.

[0053] Accurately weigh or measure a certain amount of Dectin-1 protein according to the desired final concentration. Dissolve the protein in 0.5 mL of prepared borate buffer, mix gently, avoiding foaming (the protein solution should be kept on ice).

[0054] Protein coupling reaction: The activated microsphere suspension was centrifuged at 4 °C and 12,000 rpm for 10 minutes, and the supernatant containing EDC / NHS was carefully discarded.

[0055] The microsphere precipitate was quickly washed once with 1 mL of pre-cooled borate buffer (resuspended, centrifuged, and the supernatant discarded) to remove residual activator and byproducts.

[0056] Immediately resuspend the washed microsphere precipitate in 0.5 mL of Dectin-1 protein solution.

[0057] The reaction system was placed on a shaker and gently shaken at 60 rpm for 2 hours at 4°C.

[0058] After the reaction was complete, 50 μL of 1 M Tris-HCl buffer was added directly to the reaction system.

[0059] Gently shake or pipette to mix, and let stand at room temperature for 5 minutes to quench any residual active esters.

[0060] Dilute the reaction mixture with 2 mL of pre-cooled PBS buffer, incubate at 4°C, 12,000 × g for 10 min, and carefully discard the supernatant.

[0061] This washing step (resuspended in PBS and then centrifuged) should be repeated 2-3 times to thoroughly remove uncoupled free proteins and chemical reagents.

[0062] The final precipitate is the Dectin-1 modified nystatin-targeting microspheres.

[0063] Resuspend it in an appropriate amount of sterile, pyrogen-free PBS buffer and store it at 4°C in the dark, or freeze-dry it.

[0064] Example 1: SEM of Dectin-1-Nys-MS The surface morphology and three-dimensional structure of the microspheres were characterized at high resolution using scanning electron microscopy. Lyophilized Dectin-1-Nys-MS powder was uniformly dispersed on conductive tape, followed by sputtering of a gold-palladium alloy layer approximately 10 nm thick in a high-vacuum coating apparatus to enhance the sample's conductivity. The treated sample was then placed on the scanning electron microscope stage, and secondary electron images were observed and captured at different magnifications under accelerating voltages of 5–15 kV. Figure 1 SEM images show that the prepared drug-loaded microspheres have a regular spherical morphology, uniform nanoscale particle size, and good dispersibility.

[0065] Example 2: TEM of Dectin-1-Nys-MS The microstructure of the microspheres was characterized using transmission electron microscopy (TEM). First, the Dectin-1-Nys-MS suspension was diluted 10-fold with ultrapure water and sonicated at 200 W for several minutes to ensure uniform dispersion. Then, a small amount of the dispersion was dropwise onto a copper grid coated with a carbon film using a microcapillary tube. After adsorption, the sample was negatively stained with 2% phosphotungstic acid solution (pH 7.0), and after removing excess stain, it was dried at room temperature. The prepared sample was then placed in the TEM sample holder and observed and imaged at an accelerating voltage of 120 kV. Figure 2 TEM observation results showed that the prepared drug-loaded microspheres had regular morphology and uniform size, with a diameter of approximately 180-220 nm, consistent with the results measured by scanning electron microscopy (SEM).

[0066] Examples 3-4: Particle size and potential of Dectin-1-Nys-MS The particle size distribution, dispersity, and surface charge of Dectin-1-Nys-MS were systematically characterized using a Malvern laser particle size analyzer. 1.0 mg of lyophilized Dectin-1-Nys-MS powder was accurately weighed and dissolved in 1.0 mL of anhydrous ethanol. A homogeneous dispersion was obtained by vortexing and ultrasonic treatment in an ice bath. An appropriate amount of sample was added to a clean cuvette, and the determination was repeated three times, recording the average hydrodynamic particle size and polydispersity index. Subsequently, a dedicated Zeta potential sample cell was used, and the same batch of sample suspension was injected, ensuring no air bubble interference. The Zeta potential values ​​were then determined three times again using phase-analytical light scattering at the same temperature. The average particle size of Dectin-1-Nys-MS was 176 nm. The absolute value of the Zeta potential was greater than 30 mV, indicating that the microspheres carried a high charge density on their surface and that there was strong electrostatic repulsion between the particles, which could effectively inhibit aggregation and improve the physical stability of the system.

[0067] Example 5: Infrared spectrum of Dectin-1-Nys-MS Fourier transform infrared spectroscopy (FTIR) was used to characterize the chemical structure of nystatin raw material, blank sodium alginate microspheres, drug-loaded microspheres, and Dectin-1 modified drug-loaded microspheres. All samples were vacuum-dried, mixed with potassium bromide, ground, and pressed into transparent sheets, and then subjected to 4000-500 cm⁻¹ spectroscopy. -1 Scanning was performed within the wavelength range. By comparing and analyzing the characteristic peaks in the spectra, the characteristic carbonyl peak of nystatin (~1700 cm⁻¹) could be observed simultaneously in the spectrum of the drug-loaded microspheres. -1 ) and the characteristic peak of sodium alginate carboxylate (~1600 cm⁻¹) -1 and ~1410 cm -1 This confirms successful drug encapsulation; a protein amide I band (~1650 cm⁻¹) further appears in the spectrum of Dectin-1 modified microspheres. -1 ) and amide II band (~1540 cm) -1 The characteristic absorption provided key chemical structural evidence for the successful coupling of the Dectin-1 protein to the surface of the microspheres.

[0068] Examples 6-7: Effect of storage temperature on the stability of Dectin-1-Nys-MS The effect of storage temperature on the stability of Dectin-1-Nys-MS was evaluated by setting five temperature gradients (-20°C, 4°C, 28°C, 37°C, and 60°C) for long-term stability studies. Microsphere samples from the same batch were aliquoted and stored in the above-mentioned temperature conditions in the dark, with samples taken for analysis after 24 hours. The average particle size of the microspheres was determined by dynamic light scattering to evaluate their physical stability, while the total and free drug content were determined by high-performance liquid chromatography (HPLC), and the encapsulation efficiency was calculated to evaluate their chemical stability.

[0069] As temperature increased, the encapsulation efficiency of Dectin-1-Nys-MS decreased significantly, while the particle size continued to increase: at -20 ℃, both the encapsulation efficiency and particle size remained stable, indicating that the microsphere membrane structure was in a gel state and drug leakage was minimal; when the temperature rose to 4 ℃, the encapsulation efficiency decreased to 58.93%, and the particle size increased to 238.56 nm, suggesting an increase in membrane permeability; when the temperature continued to rise to 28 ℃ and 37 ℃, drug loss accelerated, and the encapsulation efficiency further decreased to 48.87% and 40.55%, with particle sizes reaching 310.7 nm and 461.93 nm, respectively; at a high temperature of 60 ℃, the encapsulation efficiency plummeted to 36.16%, and the particle size significantly expanded to 806.93 nm, reflecting that high temperature caused significant relaxation or even destruction of the membrane structure, exacerbating drug leakage.

[0070] Example 8: In vitro release The in vitro drug release characteristics of Dectin-1-Nys-MS and Nys-MS were determined by reverse dialysis. Five mL samples (Dectin-1-Nys-MS, Nys-MS, and an equal volume of Nys control solution) were accurately measured and placed in a pretreated dialysis bag. The bag was then immersed in 100 mL of PBS release medium (pH 7.4) at 37 °C under constant temperature and magnetic stirring conditions. One mL samples were taken at time points of 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, and 72 h, and an equal volume of fresh release medium was immediately added. After filtration through a 0.22 μm filter, the drug concentration was determined by HPLC. Using the total release of nystatin in Nys-MS at 0 h as the 100% release baseline, the cumulative release percentage (Q, %) at each time point was calculated using a cumulative correction formula. In vitro release curves were plotted with Q as the ordinate and time (h) as the abscissa to systematically compare the release kinetics differences between Dectin-1 modified microspheres, unmodified microspheres, and free drug. Figure 8 As shown, Dectin-1 targeting significantly altered the release kinetics of the microspheres. Throughout the 72-hour release cycle, the cumulative release rate of the targeted microspheres was significantly higher than that of the ordinary microspheres at all time points. Ultimately, the total cumulative release rate of the targeted microspheres reached 62.49%, an increase of 16.83% compared to the ordinary microspheres (45.66%). Dectin-1 modification not only accelerated the early release of the drug but also comprehensively improved the total amount and efficiency of drug release from the microspheres. This provides clear and strong kinetic evidence for its enhanced antibacterial activity in in vitro pharmacodynamic studies.

[0071] Example 9: Evaluation of gastrointestinal digestion using Dectin-1-Nys-MS simulation To evaluate the protective effect of Dectin-1 modification on microspheres in the gastrointestinal environment, this study simulated a complete gastrointestinal digestion process. 10 mg of Dectin-1-Nys-MS and Nys-MS were accurately weighed and mixed with 4 mL of preheated 37°C simulated gastric fluid, respectively. The mixtures were incubated at 37°C and 200 rpm for 6 hours, with samples taken at 0.5, 1, 2, 3, 4, 5, 6, 12, and 24 hours. Subsequently, the samples were vortexed and sonicated with 1 mL of 10% Triton X-100 solution, then frozen at -80°C for 10 minutes to completely inactivate the enzymes. After centrifugation at 12000 rpm for 10 minutes, the supernatant was collected, and the nystatin content was quantitatively determined by HPLC. The drug residue rate was calculated using the 0-hour sampling result as the initial drug loading baseline. After gastric digestion, an equal volume of microspheres was digested with 4 mL of simulated intestinal fluid under the same conditions for 24 hours, with the sampling and detection procedures as before. Figure 9 As shown, both Dectin-1-Nys-MS and Nys-MS exhibited significant pH-dependent release characteristics in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8). In the SIF environment, both formulations displayed typical biphasic release patterns. Dectin-1-Nys-MS showed a drug leakage rate of 90.37% within 24 h, while Nys-MS had a cumulative release rate of 83.49%, lower than the unmodified group. This is attributed to the additional diffusion barrier formed by Dectin-1 protein on the microsphere surface. In contrast, in the SGF environment, the leakage rates of both formulations were significantly reduced, reaching only 11.75% and 10.64% respectively at 24 h, and their release curves largely overlapped, indicating that Dectin-1 modification did not alter the stability of the microspheres under acidic conditions. The above results indicate that the microsphere system maintains stability in acidic environments and exhibits a pH-responsive controlled-release mode that allows for rapid release in near-neutral environments, making it a potential oral intestinal-targeted drug delivery system that can improve drug bioavailability and reduce gastric irritation.

[0072] Example 10: Characterization of inhibition zones by Dectin-1-Nys-MS Evaluation was performed using the agar diffusion method (inhibition zone test). A suspension of the logarithmic growth phase Candida albicans standard strain (HBJZDL068-1, Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) was evenly spread on the surface of Sabouraud agar medium. Sterile antimicrobial susceptibility testing discs, each soaked in equal masses (based on nystatin) of free nystatin solution, nystatin microsphere suspension, Dectin-1 modified nystatin microsphere suspension, and blank microsphere suspension, were placed on the inoculated medium surface. The plates were incubated at 37 ℃ for 48 hours, and the diameter of the transparent inhibition zone formed around each disc was observed and measured. Figure 10The results showed that no inhibition zone formed around the blank microspheres (Blank-MS), and the bacterial growth was uniform, proving that the carrier material itself had no antibacterial activity and ruling out carrier interference. Drug-loaded microspheres (Nys-MS) produced a distinct but slightly smaller inhibition zone than free nystatin (Nys), suggesting that drug release was somewhat hindered after microsphere encapsulation. However, the inhibition zone diameter formed by the Dectin-1 modified drug-loaded microspheres (Dectin-1-Nys-MS) after 48 hours was comparable to that of the free drug, confirming that the released drug had the same inherent antibacterial activity as the active pharmaceutical ingredient. The synergistic mechanism of Dectin-1-Nys-MS is attributed to the specific recognition of fungal cell walls by Dectin-1 as a β-glucan receptor, promoting microsphere adhesion to fungi and local drug accumulation, and potentially producing a synergistic antifungal effect. Experimental results confirm that the microsphere encapsulation technology successfully preserved the antifungal activity of nystatin, and that surface modification of Dectin-1 enhanced the antibacterial effect, supporting the further development value of Dectin-1-Nys-MS as a targeted antifungal agent.

[0073] Examples 11-12: Kinetic Study of the Bactericidal Effect of Dectin-1 Coupled with Nystatin Microspheres on Candida albicans in Simulated Intestinal Fluid To evaluate the targeted synergistic effect of microspheres in simulated intestinal fluid, we conducted a time-bactericidal kinetic experiment. Candida albicans culture in the logarithmic growth phase (1×10⁻⁶) was used. 6 CFU·mL -1 The drug was mixed with equal amounts of nystatin-containing ordinary microspheres (Nys-MS) and Dectin-1 targeted microspheres (Dectin-1-Nys-MS) in simulated intestinal fluid to achieve a final drug concentration equal to its respective minimum inhibitory concentration (MIC). A growth control without the drug was also included. The mixture was incubated in a shaker at 35°C, and samples were taken at 0, 2, 4, 6, 8, 12, and 24 hours. To ensure that the count accurately reflects the number of viable bacteria rather than the sustained inhibitory effect of the drug, the samples were serially diluted immediately after sampling, then plated on Sabouraud agar plates, and colony-forming units were counted after incubation.

[0074] Inhibition rate (%) = (1 - CFUexp / CFUcon) × 100%.

[0075] In the formula: CFUexp is the number of colonies in the sample group; CFUcon is the number of colonies in the control group.

[0076] Figure 11-12The time-inhibition rate kinetic analysis showed that the antibacterial effect of Dectin-1 modified microspheres was significantly better than that of unmodified ordinary microspheres, and its inhibition rate approached or even reached the level of free drug in the later stages of the experiment. This targeted delivery system, while successfully preserving drug activity, significantly improved the antibacterial efficiency of nystatin through Dectin-1-mediated targeting, achieving efficacy close to that of free drug and providing a new strategy for constructing highly effective and long-acting antifungal agents.

Claims

1. A nystatin microsphere with targeted enhanced antibacterial function, characterized in that, include: The microsphere carrier matrix contains nystatin and Dectin-1 ligand, the Dectin-1 ligand being covalently linked to the surface of the microsphere carrier matrix.

2. The nystatin microspheres according to claim 1, characterized in that, The microsphere carrier matrix is ​​sodium alginate or its derivative.

3. The nystatin microspheres according to claim 2, characterized in that, The microsphere carrier matrix is ​​sodium alginate, and the Dectin-1 ligand is covalently linked to the carboxyl group of sodium alginate via a coupling reaction mediated by carbodiimide and N-hydroxysuccinimide.

4. The nystatin microspheres according to any one of claims 1-3, characterized in that, The Dectin-1 ligand is β-glucan.

5. A method for preparing nystatin microspheres according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Provides a microsphere carrier matrix loaded with nystatin; S2: In the presence of an activator, the Dectin-1 ligand is co-incubated with the microsphere carrier matrix loaded with nystatin obtained in step S1, so that the Dectin-1 ligand is covalently linked to the surface of the microsphere carrier matrix to obtain nystatin microspheres.

6. The preparation method according to claim 5, characterized in that, In step S1, the microsphere carrier matrix loaded with nystatin is prepared by emulsification crosslinking method; in step S2, the activator is a combination of carbodiimide and N-hydroxysuccinimide.

7. The use of the nystatin microspheres according to any one of claims 1-4 in the preparation of medicaments for the prevention and / or treatment of fungal infections.

8. The application according to claim 7, characterized in that, The fungal infection is Candida albicans infection.

9. The application according to claim 7 or 8, characterized in that, The drug is formulated as an oral preparation for the prevention and / or treatment of intestinal fungal infections.

10. An antifungal pharmaceutical composition, characterized in that, The product comprises a therapeutically effective amount of nystatin microspheres as described in any one of claims 1-4, and a pharmaceutically acceptable carrier or excipient.

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