Applications of nanocomposites with core-shell structures

Abstract

The present application provides the use of a nanocomposite with a core-shell structure. Specifically, the present application provides the use of a nanocomposite with a core-shell structure in the preparation of a drug for preventing and / or treating Clostridium difficile infection and recurrent diseases caused thereby, wherein the nanocomposite comprises: ZnO nanoparticles as a core; mesoporous silica as a shell coated on the surface of the ZnO nanoparticles; an antibiotic loaded in the pore channel of the mesoporous silica; hyperbranched polylysine modified on the surface of the mesoporous silica; and a pH-responsive coating material as the outermost layer. The nanocomposite provided by the present application can achieve precise targeted release of the drug in the colon, and through the sequential and synergistic mechanism of "membrane breaking-sterilization-spore inhibition-repair", it can simultaneously solve multiple factors leading to CDI recurrence such as biofilm protection, spore storage, bacterial population destruction, mucosal damage, etc., and improve the treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials and the prevention and treatment of infectious diseases. Specifically, this invention relates to the use of nanocomposite materials with a core-shell structure. Background Technology

[0002] Clostridium difficile (CD) is a Gram-positive anaerobic spore-forming bacillus and a major pathogen causing nosocomial infections and antibiotic-associated diarrhea. In recent years, due to the widespread use of broad-spectrum antibiotics, the incidence of Clostridium difficile infection (CDI) has been rising globally, even surpassing that of methicillin-resistant Staphylococcus aureus in some parts of the United States. The clinical symptoms of CDI range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon, making it a significant public health concern worldwide.

[0003] Currently, the first-line treatments for CDI are primarily metronidazole and vancomycin. Depending on the severity of the disease, mild to moderate infections can be treated with oral or injectable metronidazole, while severe infections are best treated with oral vancomycin. However, existing treatment regimens have the following prominent issues:

[0004] (1) High relapse rate, leading to refractory and relapsed CDI

[0005] Regardless of whether metronidazole or vancomycin is used for treatment, there is a risk of recurrence. Clinical data shows that the recurrence rate of CDI is approximately 20%, and for patients with multiple relapses, the risk of subsequent recurrences can be further increased. The core reason for recurrence is that antibiotics such as vancomycin mainly kill the vegetative cells of Clostridium difficile, but are ineffective against spores. Residual spores can germinate after antibiotic treatment ends, leading to infection recurrence. In addition, antibiotic treatment further disrupts the intestinal flora homeostasis, reducing the patient's resistance to Clostridium difficile colonization and creating a vicious cycle of "infection-treatment-recurrence".

[0006] (2) Biofilms protect bacteria and reduce the effectiveness of antibiotics.

[0007] Clostridium difficile can form biofilms in the gut, embedding itself in its own secreted extracellular polymer matrix, significantly increasing its resistance to antibiotics. The reduced metabolic activity of bacteria within the biofilm and the biofilm barrier hindering drug penetration make it difficult for conventional antibiotics to completely eliminate them. Current research confirms that the quorum sensing (QS) system plays a crucial regulatory role in bacterial biofilm formation and virulence factor expression. Therefore, relying solely on antibiotics is insufficient to overcome biofilm defenses.

[0008] (3) Antibiotic treatment further disrupts the gut microbiota homeostasis and may promote the colonization of drug-resistant bacteria.

[0009] While oral vancomycin primarily exerts its effects locally in the gut, some patients may experience systemic exposure, particularly ICU patients, those receiving high doses (>500 mg / day), or those with gastrointestinal inflammatory conditions. More importantly, vancomycin use may promote intestinal colonization by vancomycin-resistant enterococci (VREs). Although fecal microbiota transplantation (FMT) has been shown to restore gut microbiota diversity, thereby preventing CDI recurrence, its long-term safety remains unknown, and it faces challenges such as complex donor selection and limited acceptability.

[0010] (4) Limitations of existing nanotechnology solutions

[0011] In recent years, nanomaterials have shown great promise in the field of anti-infection, but existing technologies still have significant shortcomings:

[0012] ZnO nanoparticles: possess broad-spectrum antibacterial activity, which can be achieved by releasing ZnO nanoparticles. 2+ The generation of reactive oxygen species (ROS) disrupts bacterial membrane structures. However, ZnO nanoparticles suffer from problems such as easy aggregation, poor stability in aqueous environments, and limited antibacterial activity. Studies have shown that surface modification can improve their stability and antibacterial properties, but existing research mainly focuses on single modification strategies, such as doping with transition metals or encapsulation with biomolecules.

[0013] Mesoporous silica nanoparticles (MSNs) possess a large specific surface area, tunable pore size, and good biocompatibility, making them ideal drug carriers. MSN surfaces are easily modified to load various drugs or biomolecules. However, MSNs alone lack the ability to actively disrupt biofilms, making it difficult to solve the problem of biofilm protection for bacteria.

[0014] Hyperbranched polylysine (HBPL): As a synthetic cationic antimicrobial peptide analog, HBPL exhibits excellent biocompatibility and antimicrobial activity. Recent studies have shown that HBPL can block bacterial quorum sensing systems, inhibit biofilm formation, reduce the expression of bacterial virulence factors, and is less likely to induce drug resistance. Furthermore, HBPL can capture planktonic bacteria through electrostatic interactions, preventing them from adhering to tissues. However, HBPL is sensitive to environmental factors (hydrolysis, oxidation, pH changes, proteases, etc.), and its stability and delivery efficiency need further improvement.

[0015] In summary, current CDI treatment strategies face multiple challenges, including high relapse rates, biofilm protection, spore reservoirs, and microbial disruption. Single antibiotics are insufficient to eliminate bacteria within biofilms, cannot disrupt quorum sensing networks, and cannot inhibit early sporulation processes. While nanomaterials for antimicrobial purposes have been studied, they are mostly simple additives, lacking colon-directed release control and time-sequential synergistic design. Summary of the Invention

[0016] The present invention aims to overcome the above-mentioned shortcomings in the treatment and recurrence control of Clostridium difficile infection (CDI) in the prior art, and to provide a novel nanocomposite material that can simultaneously solve multiple key obstacles in the treatment of CDI.

[0017] The purpose of this invention is to provide the use of the core-shell structured nanocomposite material of this invention in the preparation of medicaments for the prevention and / or treatment of Clostridium difficile infection and its recurrent diseases. The nanocomposite material provided by this invention can achieve precise targeted drug release in the colon, and through a sequential synergistic mechanism of "membrane disruption-bactericidal-spore inhibition-repair," it simultaneously addresses multiple factors leading to CDI recurrence, such as biofilm protection, spore storage, microbial community disruption, and mucosal damage, significantly reducing the recurrence rate and improving treatment efficacy.

[0018] Existing first-line treatments (vancomycin) primarily kill the vegetative cells of Clostridium difficile, but are ineffective against spores. Residual spores can germinate after drug withdrawal, leading to infection recurrence. Simultaneously, antibiotic treatment further disrupts gut microbiota homeostasis, weakening the host's resistance to Clostridium difficile colonization and creating a vicious cycle of "infection-treatment-recurrence." The nanocomposite material of this invention provides an intervention strategy that acts simultaneously on both vegetative cells and spores without further disrupting gut microbiota homeostasis.

[0019] The preparation process of the core-shell structured nanocomposite material provided by this invention is stable and controllable, including steps such as HBPL synthesis, ZnO@MSN construction, antibiotic loading, HBPL surface modification, and pH-responsive coating, which enables the nanocomposite material to have the potential for large-scale production.

[0020] The above-mentioned objective of the present invention is achieved through the following technical solution.

[0021] Use of core-shell structured nanocomposites in the preparation of medicaments for the prevention and / or treatment of Clostridium difficile infection and its recurrent diseases, wherein said nanocomposites comprise:

[0022] ZnO nanoparticles as the core;

[0023] Mesoporous silica, which acts as a shell, coats the surface of ZnO nanoparticles;

[0024] Antibiotics loaded within the pores of the mesoporous silica;

[0025] Hyperbranched polylysine modified on the surface of the mesoporous silica; and

[0026] As the outermost pH-responsive coating material.

[0027] Preferably, in the uses described in this invention, the drug is used for at least one of the following therapeutic purposes:

[0028] (a) Disruption of Clostridium difficile biofilm;

[0029] (b) Inhibits Clostridium difficile toxin expression;

[0030] (c) Reduce the number of Clostridium difficile spores;

[0031] (d) Reduce the recurrence rate of Clostridium difficile infection;

[0032] (e) Reduces colitis and tissue damage caused by Clostridium difficile infection.

[0033] Preferably, in the use described in this invention, the drug is a pharmaceutical composition, and the pharmaceutical composition comprises an effective amount of the nanocomposite material and pharmaceutically acceptable excipients.

[0034] Preferably, in the use described in this invention, the pharmaceutical composition is an oral formulation.

[0035] Preferably, in the use described in this invention, the oral preparation is a solution, suspension, capsule, or tablet.

[0036] Preferably, in the use described in this invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, binders, disintegrants, lubricants, and flow aids.

[0037] In the applications described in this invention, the nanocomposite material of this invention uses ZnO nanoparticles as the core and mesoporous silica as the shell to form a core-shell structure (ZnO@MSN). The large specific surface area of ​​MSN is used to load antibiotics (such as vancomycin), and hyperbranched polylysine (HBPL) is modified on the surface of MSN. The outermost layer is coated with a pH-responsive material (such as Eudragit S100) to form a structurally complete and functionally integrated nanocomposite material.

[0038] Preferably, in the application described in this invention, the pH-responsive coating material is a pH-sensitive polymer that dissolves or degrades in an environment with pH ≥ 7.0.

[0039] This invention utilizes a pH-responsive outer coating to enable the nanoplatform to safely pass through the stomach and small intestine (low pH environment) after oral administration, triggering release only in the colon (pH≥7.0), achieving colon-limited delivery and reducing disruption to the gut microbiota.

[0040] Preferably, in the use described in this invention, the pH-sensitive polymer is selected from synthetic polymeric pH-sensitive materials or natural polymeric pH-sensitive materials.

[0041] Preferably, in the application described in this invention, the synthesized polymeric pH-sensitive material is a methacrylic acid copolymer.

[0042] Preferably, in the use described in this invention, the synthesized polymeric pH-sensitive material is a copolymer of methacrylic acid and methyl methacrylate (poly(methacrylic acid-co-methyl methacrylate)), more preferably a copolymer of methacrylic acid and methyl methacrylate in a molar ratio of 1:4, such as Eudragit S100.

[0043] Preferably, in the application described in this invention, the natural polymer pH-sensitive material is resistant starch.

[0044] Preferably, in the use described in this invention, the antibiotic is an antibiotic against Gram-positive bacteria.

[0045] Preferably, in the use described in this invention, the antibiotic is a glycopeptide antibiotic or a nitroimidazole antibiotic.

[0046] In this invention, the selected antibiotics are mainly antibiotics that inhibit Clostridium difficile.

[0047] Preferably, in the use described in this invention, the glycopeptide antibiotic is selected from at least one of vancomycin, norvancomycin, and teicoplanin.

[0048] Preferably, in the use described in this invention, the nitroimidazole antibiotic is metronidazole.

[0049] Preferably, in the application described in this invention, the hyperbranched polylysine is modified on the surface of the mesoporous silica by electrostatic adsorption.

[0050] Hyperbranched polylysine (HBPL) contains numerous terminal amino groups in its molecular structure, giving it a positive charge under weakly acidic to neutral conditions. Mesoporous silica (MSN) surfaces are rich in silanol groups (Si-OH), which can undergo protonation or deprotonation in aqueous solutions, resulting in a negative charge or neutrality. Based on these chemical properties, HBPL can be modified onto the MSN surface through electrostatic adsorption. Specifically, the silanol groups on the MSN surface are partially deprotonated and become negatively charged, while the amino groups of HBPL are protonated and become positively charged. These two groups spontaneously combine through electrostatic interactions to form stable hyperbranched polylysine modified on the mesoporous silica surface. This method is simple, rapid, requires no additional chemical reagents, and maintains the native conformation and biological activity of HBPL.

[0051] Preferably, in the use described in this invention, the hyperbranched polylysine is prepared by heating and polymerizing L-lysine or its salt in the presence of an alkaline substance, a catalyst and urea in an inert atmosphere.

[0052] Preferably, in the use described in this invention, the number-average molecular weight of the hyperbranched polylysine is 3-10 kDa.

[0053] Preferably, in the application described in this invention, the average particle size of the ZnO nanoparticles is 100-300 nm.

[0054] Preferably, in the use described in this invention, the mass ratio of ZnO, mesoporous silica, antibiotic, hyperbranched polylysine and pH-responsive coating material in the nanocomposite material is (0.15-0.45): (0.55-0.75): (0.6-0.8): (0.5-0.7): (2-4).

[0055] The method for preparing a core-shell structured nanocomposite material provided by this invention includes the following steps:

[0056] (1) Using ZnO nanoparticles as templates, a mesoporous silica precursor is coated on their surface. After template removal and calcination, composite particles with ZnO nanoparticles as the core and mesoporous silica as the shell are obtained (denoted as ZnO@MSN).

[0057] (2) The core-shell composite particles obtained in step (1) are mixed with antibiotics and hyperbranched polylysine in a solvent, shaken and incubated, and then separated by solid-liquid separation to obtain composite particles (denoted as ZMHV) with antibiotics loaded in the mesoporous silica channels and hyperbranched polylysine modified on the surface of the mesoporous silica.

[0058] (3) The composite particles loaded with antibiotics and modified with hyperbranched polylysine obtained in step (2) are dispersed in a coating solution containing pH-responsive coating material and coated by ultrasonic vibration to obtain a nanocomposite material with pH-responsive coating material on the outermost layer (denoted as ZMHVS).

[0059] Preferably, in the method described in this invention, step (1) includes the following steps:

[0060] First, zinc oxide nanoparticles coated with polyvinylpyrrolidone (ZnO@PVP) were synthesized using zinc source and polyvinylpyrrolidone as raw materials. Then, they were dispersed in a mixed system containing hexadecyltrimethylammonium bromide, ammonia, and tetraethyl orthosilicate. The precipitate obtained by centrifugation was then calcined to remove the template, thus obtaining the core-shell composite particles (ZnO@MSN).

[0061] Preferably, in the method described in this invention, the coating solution in step (3) is an ethanol-water solution of a pH-responsive coating material.

[0062] Preferably, in the method described in this invention, the solvent in step (2) is deionized water.

[0063] Preferably, in the method described in this invention, the preparation of hyperbranched polylysine in step (2) includes the following steps:

[0064] L-lysine or its salt is mixed with an alkaline substance, a catalyst, and urea, and heated under inert gas protection to carry out a polycondensation reaction.

[0065] In the preparation of hyperbranched polylysine, this invention adds urea as a hydrogen bond regulator and chain growth promoter. This effectively regulates the amino-carboxyl reaction rate in the local reaction environment, reduces side reactions, and increases the number of amino active end groups. This improves the composite efficiency with inorganic nanoparticles (HMSN (i.e., HBPL-modified MSN surface) and ZnO), significantly enhancing the construction stability of the nano-drug delivery system. Furthermore, it gives the drug delivery system higher drug loading efficiency, better stability, and stronger bacterial membrane penetration ability, making it particularly suitable for local drug delivery in infectious diseases.

[0066] Preferably, in the method described in this invention, the L-lysine or its salt is L-lysine hydrochloride.

[0067] Preferably, in the method described in this invention, the alkaline substance is potassium hydroxide.

[0068] Preferably, in the method described in this invention, the catalyst is dibutyltin dilaurate.

[0069] Preferably, in the method described in this invention, the polycondensation reaction is carried out at 140-160°C for 10-14 hours.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] (1) Beneficial effects at the structural design level

[0072] (1.1) The unique core-shell structure endows composite materials with multifunctional integration capabilities.

[0073] This invention uses a ZnO@MSN core-shell structure as the core carrier, wherein:

[0074] ZnO core: serves not only as structural support but also as a Zn 2+ It serves as a storage depot and a reactive oxygen species (ROS) generation center, participating in synergistic sterilization.

[0075] Mesoporous silica shells provide a large specific surface area and regular mesoporous channels, enabling efficient loading of antibiotics (such as vancomycin). They can also be modified with hyperbranched polylysine (HBPL) to achieve partitioned drug loading, where antibiotics are loaded within the pores and functional molecules are modified on the surface, thus avoiding interference between drugs.

[0076] (1.2) Partitioned load balancing enables functional independence and collaboration

[0077] The antibiotic is loaded into the MSN pores, and HBPL is modified on the MSN surface. The two are spatially separated but functionally synergistic: the antibiotic is protected in the pores and its stability is improved; HBPL is exposed on the surface and can contact the biofilm immediately to perform the function of membrane rupture; during release, HBPL is released first (on the surface) and the antibiotic is released later (in the pores), naturally forming a sequential release order, without the need for additional controlled release design.

[0078] (1.3) pH-responsive outer coating enables precise colon targeting

[0079] The outermost coating, Eudragit S100 (or other colonic pH-responsive material), remains insoluble at pH < 7.0, protecting the nanoplatform as it passes intact through the stomach and small intestine. Dissolution triggers release only in the colon (pH ≥ 7.0), achieving colon-specific delivery. In vivo animal tracing experiments confirmed that the nanocomposite material of this invention specifically aggregates and remains in the colon, significantly reducing non-specific drug release in the early gastrointestinal tract, thereby maximizing the protection of intestinal flora homeostasis.

[0080] (2) Beneficial effects at the functional mechanism level

[0081] (2.1) Sequential and synergistic mechanism for systematic intervention in key aspects of CDI relapse

[0082] This invention designs a sequential activation mechanism of "first breaking the membrane - then sterilizing - then inhibiting spores - then repairing," and experiments have shown that:

[0083] Phase 1: HBPL disrupts biofilms

[0084] In vitro experiments showed that ZMHVS could significantly disrupt existing Clostridium difficile biofilms and inhibit the formation of new biofilms; the reduction of extracellular polymeric substances (EPS) and the decrease in biofilm thickness opened up channels for subsequent sterilization.

[0085] Phase Two: Antibiotics such as vancomycin and zinc 2+ Synergistic sterilization

[0086] Antibiotics such as vancomycin inhibit cell wall synthesis, leading to the release of ZnO and Zn. 2+ The disruption of bacterial membrane structure and the generation of reactive oxygen species (ROS) work synergistically, resulting in a significantly better bactericidal effect than single drugs. The ROS generation capacity of ZnO is extended due to the protection of the MSN shell, enhancing its antibacterial durability.

[0087] Phase 3: Inhibition of virulence expression and sporulation program

[0088] Gene expression analysis showed that the Clostridium difficile toxin genes tcdA and tcdB were significantly downregulated in the ZMHVS treatment group; the expression of sporulation-related genes was suppressed, and the early sporulation program was blocked; the core redox maintenance pathway was negatively enriched, indicating that the bacteria were in a state of oxidative stress and had difficulty initiating the sporulation program.

[0089] Phase 4: Promoting Mucosal Repair

[0090] Animal experiments showed that the ZMHVS treatment group had significantly reduced colonic mucosal damage, restored epithelial structure, and allowed HBPL to exert its repair potential.

[0091] (2.2) Achieve effective removal of spores

[0092] Spores are a "reservoir" for CDI recurrence. This invention significantly reduces spore numbers through the combined action of the following mechanisms: efficient removal of vegetative cells during the bactericidal phase, blocking the transformation of vegetative cells into spores; inhibition of sporulation-related gene expression, reducing spore formation at the source; animal experiments have confirmed that the number of spores in the colonic contents of the ZMHVS treatment group was significantly reduced, an effect that is difficult to achieve with existing antibiotics.

[0093] (3) Beneficial effects at the level of in vivo therapeutic effects

[0094] (3.1) Significantly reduces recurrence rate and improves survival rate

[0095] In animal models of CDI relapse, compared to the vancomycin monotherapy group:

[0096] Reduced weight loss: Mice in the ZMHVS treatment group maintained more stable weight and recovered faster;

[0097] Improved survival rate: The survival rate of mice in the ZMHVS treatment group was significantly higher than that in the vancomycin monotherapy group;

[0098] Reduced recurrence rate: During the follow-up period after treatment, the recurrence rate in the ZMHVS group was significantly lower than that in the control group, demonstrating its systemic intervention effect on recurrence.

[0099] (3.2) Reduce colon inflammation and restore tissue integrity

[0100] Colon length is an indirect indicator of the severity of inflammation. The colon length in the ZMHVS treatment group was significantly longer than that in the infected control group, approaching the level of the healthy control group.

[0101] Inflammatory factor detection showed that the levels of pro-inflammatory factors such as TNF-α and IL-6 returned to normal after ZMHVS treatment, indicating that intestinal inflammation was effectively controlled.

[0102] Histopathological examination (HE staining) showed that the colonic mucosa structure of the ZMHVS group was intact and the infiltration of inflammatory cells was reduced, which was in line with the design expectation of "promoting mucosal repair".

[0103] (3.3) Protecting gut microbiota homeostasis

[0104] By employing colonic pH-responsive coating to achieve confined release, the nanoplatform remains intact in the stomach and small intestine without releasing drugs, thereby avoiding damage to the normal flora of the stomach and small intestine; maintaining the diversity of the intestinal flora and preserving colonization resistance; and reducing the risk of colonization of drug-resistant bacteria such as VRE caused by exposure to antibiotics such as vancomycin.

[0105] (4) Beneficial effects at the preparation and application levels

[0106] The preparation method provided by this invention has clear, simple, and controllable steps, making it suitable for industrialization: HBPL synthesis adopts conventional thermal polycondensation, with readily available raw materials and stable yield; the ZnO@MSN core-shell structure is prepared by template method combined with calcination process, which can precisely control particle size and shell thickness; antibiotic loading and HBPL modification are carried out in aqueous phase or under mild conditions, without damaging the active ingredients; pH-responsive coating adopts conventional coating process, which is suitable for industrial scale-up. Attached Figure Description

[0107] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0108] Figure 1 The accompanying photographs and Fourier transform infrared (FT-IR) spectra of L-lysine hydrochloride and the prepared HBPL from Example 1 of the present invention are shown, illustrating the structural transformation of lysine monomer into HBPL.

[0109] Figure 2 The image shows a physical picture of ZnO@MSN located in a test tube and a particle size distribution diagram of ZnO@MSN.

[0110] Figure 3 Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of ZnO@MSN and their corresponding energy dispersive spectroscopy elemental mappings (Zn, Si, O) are shown to characterize its morphology and elemental distribution.

[0111] Figure 4 The nitrogen adsorption-desorption isotherm and pore size distribution curve of ZnO@MSN are shown.

[0112] Figure 5 The zeta potentials of HBPL, ZnO@MSN, ZMHV, and ZMHVS are shown.

[0113] Figure 6Electron microscope images of ZMHV and ZMHVS and their corresponding energy spectrum elemental mappings (Zn, Si, O, N) are shown.

[0114] Figure 7 The full X-ray photoelectron spectroscopy (XPS) spectra of HBPL, MSN, and ZnO@MSN are shown to analyze the surface elemental composition of each material; and the full XPS spectra of ZnO, ZnO@MSN, ZMH, ZMHV, and ZMHVS are shown to compare and analyze the changes in elemental composition during the composite process. It should be noted that "ZMH (i.e., HBPL modified on the surface of ZnO@MSN)" is only for comparison with "ZMHV" to verify that Van and HBPL are present in ZnO@MSN. Its preparation method is the same as "ZMHV", except that only "HBPL" is added and Van is not added.

[0115] Figure 8 The pH-responsive release verification diagram and in vivo tracer diagram of the composite material are shown. It should be noted that "ZMCVS" in the figure represents ZM@Eudragit S100 containing CV (crystal violet); "ICG" represents indocyanine green; "ZM@ICG@Eudragit S100" represents ZM@Eudragit S100 containing indocyanine green.

[0116] Figure 9 The diagram shows the effect of the composite material on Clostridium difficile biofilm; the left diagram verifies the destructive effect of the composite material on the already formed biofilm, and the right diagram verifies the inhibitory effect of the composite material on biofilm maturation.

[0117] Figure 10 This demonstrates how FITC-labeled HBPL penetrates the Clostridium difficile biofilm after being co-cultured with a mature Clostridium difficile biofilm for 4 hours.

[0118] Figure 11 This demonstrates the use of SYTO 9 (for live bacteria) and PI (for dead bacteria) for double staining of biofilms, and the quantification of biofilm thickness using z-stack scanning with a laser confocal microscope.

[0119] Figure 12 The ROS generation capability of ZnO@MSN was evaluated using a DCFH-DA fluorescent probe.

[0120] Figure 13 The results show that the expression levels of virulence-related genes (such as tcdA, tcdB, tcdR, etc.) in Clostridium difficile treated with ZMHV are significantly reduced; this conclusion is consistent with the results of qRT-PCR (left figure) and transcriptome analysis (right figure) (the genes in the figure are Gene IDs from the NCBI database).

[0121] Figure 14 The results of GSEA enrichment analysis based on a custom “early / mid / late sporulation” gene set are shown; the analysis shows that ZMHV treatment mainly inhibits the early sporulation program of Clostridium difficile.

[0122] Figure 15 The transcriptomic enrichment analysis results are shown, demonstrating the enrichment signal (NES = -1.87, padj = 2.70 × 10⁻⁶) in the core redox maintenance module within the custom "redox / oxidative stress" gene set. -5 );

[0123] Figure 16 This illustrates the restorative effect of the nanocomposite material of this invention on the intestinal epithelial barrier function. Top image: Laser confocal microscopy images showing the expression (green) of tight junction protein ZO-1 and nuclear DAPI staining (blue) in Caco-2 cells after different treatments, scale bar = 60 μm. Bottom image: HE staining results of colon tissue from a CDI mouse model, scale bar = 250 μm;

[0124] Figure 17 The curves showing weight change and survival rate are displayed.

[0125] Figure 18 The results of colon length statistics are shown;

[0126] Figure 19 The results of spore counts in colonic contents are shown; where C. difficile represents Clostridium difficile; Clostridium innocuum represents Clostridium innocuum; and Enterocloster clostridioformis represents Clostridium clostridioformis.

[0127] Figure 20 The results of inflammatory factor detection are shown. Detailed Implementation

[0128] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0129] It should be noted that all raw materials used in the embodiments of this invention are commercially available. All reagents were of analytical grade, and the experimental water was deionized water.

[0130] Example 1

[0131] Preparation and characterization of ZMHVS nanocomposite material

[0132] 1. Synthesis of hyperbranched polylysine (HBPL)

[0133] Weigh out L-lysine hydrochloride (13.725 g, 0.075 mol), potassium hydroxide (4.2 g, 0.075 mol), dibutyltin dilaurate catalyst (0.05 g), and urea (0.1 g). Grind the solid components (L-lysine hydrochloride and potassium hydroxide) into powder and add the catalyst, stirring until homogeneous. Place the powder mixture in a reactor and heat to 150°C under nitrogen protection for 12 hours. After the reaction is complete, cool the reactor to room temperature to obtain a brown reaction product.

[0134] The product was dissolved in methanol, and the precipitated potassium chloride was removed by filtration. After evaporating the solvent from the filtrate, the residue was dissolved in deionized water and transferred to a dialysis bag (MWCO 3000 g / mol) for dialyzing for 48 hours to remove excess salts and unreacted monomers. The dialyzed solution was then ultrafiltered using an ultrafiltration tube (10,000 NMWL), and fractions with a molecular weight less than 10 kDa were collected. The collected solution was freeze-dried to obtain a pale yellow solid hyperbranched polylysine (HBPL) in approximately 50% (w / w) yield.

[0135] Structural characterization: Small amounts of L-lysine hydrochloride and the above-mentioned HBPL product were analyzed by Fourier transform infrared spectroscopy (FT-IR). The results showed that HBPL was at 3277.24 cm⁻¹. -1 A broad peak appears at 1636.19 cm⁻¹, corresponding to the -NH / OH stretching vibration; -1 and 1535.88 cm -1 Characteristic absorption peaks of amide I and amide II bands appeared at the specified locations, while the corresponding characteristic peaks of L-lysine hydrochloride were significantly weakened or shifted (see [reference]). Figure 1 ).

[0136] 2. Preparation of ZnO@MSN core-shell structure

[0137] (1) Preparation of ZnO nanoparticles

[0138] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 0.64 g) and polyvinylpyrrolidone (PVP, 0.7 g) were weighed and dissolved in 50 mL of deionized water, and stirred until completely dissolved. The pH of the solution was adjusted to approximately 9.1 with ammonia, and the reaction was carried out in a 70℃ water bath with stirring for 3 hours. After the reaction was completed, the precipitate was separated by centrifugation at 10,000 rpm, washed three times each with deionized water and ethanol, and dried at 60℃ for 24 hours to obtain ZnO@PVP nanoparticles.

[0139] (2) MSN shell covering

[0140] The ZnO@PVP nanoparticles (300 mg) were dispersed in a mixed solvent of deionized water (100 mL) and ethanol (60 mL). Ammonia (2.2 mL) and hexadecyltrimethylammonium bromide (CTAB, 300 mg) were added, and the mixture was stirred at room temperature for 30 minutes. Then, tetraethyl orthosilicate (TEOS, 0.5 mL) was slowly added dropwise, and the reaction was stirred at room temperature for 6 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm, washed three times with deionized water and ethanol, and dried at 60 °C.

[0141] The dried product was placed in a muffle furnace and heated to 550°C at a heating rate of 2°C / min for 6 hours to remove the PVP and CTAB templates, thus obtaining ZnO@MSN core-shell structured nanoparticles.

[0142] Structural characterization:

[0143] Particle size analysis: Dynamic light scattering (DLS) measurements showed that the hydrated particle size of ZnO@MSN was mainly distributed in the range of 100-300 nm, with a concentrated particle size distribution (see [link to relevant documentation]). Figure 2 ).

[0144] Morphological observation: Transmission electron microscopy (TEM) revealed that the product has a typical core-shell structure, with a dark ZnO core (approximately 100-150 nm in diameter) located inside, completely encapsulated by a light-colored mesoporous silica shell (approximately 30-50 nm thick) (see [link]). Figure 3 Scanning electron microscopy (SEM) revealed that the particles were spherical, with smooth surfaces and good dispersion. Energy dispersive spectroscopy (EDS) mapping showed that Zn was concentrated in the core region, while Si and O were uniformly distributed in the shell, confirming the formation of a core-shell structure (see [link to relevant documentation]). Figure 3 ).

[0145] Pore ​​structure analysis: The nitrogen adsorption-desorption isotherm shows typical type IV isotherm characteristics, and the BET specific surface area is approximately 550 m². 2 / g. The pore size distribution curve (BJH method) shows that the pore size is concentrated in the range of 2-4 nm, confirming the existence of mesoporous structure (see [link]). Figure 4 ).

[0146] 3. Simultaneous loading of vancomycin and hyperbranched polylysine (preparation of ZMHV)

[0147] ZnO@MSN nanoparticles (1 mg) were dispersed in ultrapure water (1 mL), and vancomycin (Van, 1 mg) and hyperbranched polylysine (HBPL, 1 mg) were added. The mixture was incubated overnight (12 hours) at 4 °C with shaking. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. The precipitate was washed three times with 1 mL of deionized water to remove unloaded vancomycin and unadsorbed HBPL. The mixture was freeze-dried to obtain a composite material with vancomycin loaded in the pores and HBPL adsorbed on the surface, denoted as ZMHV.

[0148] Drug loading determination: Van was detected using a spectrophotometer (210 nm), and HBPL was detected using gel permeation chromatography. Specifically, standard solutions of different concentrations (0-1000 μg / mL) were prepared using vancomycin standards, with deionized water as the solvent. The absorbance of each standard solution was measured at 210 nm using a UV spectrophotometer. A standard curve was plotted with vancomycin concentration as the x-axis and absorbance as the y-axis, and linear regression analysis was performed. Under the same conditions, the absorbance of the test sample (supernatant after reaction) was measured at 210 nm, and the concentration of vancomycin in the sample was calculated based on the standard curve.

[0149] Gel permeation chromatography (GPC) was performed by dissolving polycations at a concentration of 5 mg / mL in 0.1 M NaHCO3. For each analysis, 100 μL of sample solution was injected and analyzed on a VISCOTEK TDA 300 instrument, which was kept at 25 °C and equipped with Shodex OHpak SB-803 HQ and SB-804 HQ columns, as well as an HB-100 column. A G guard column was used, with 0.1 M NaHCO3 as the eluent, and a flow rate of 0.5 mL / min. Sample elution was monitored simultaneously using an online viscometer detector and a differential refractive index detector. Elution time was converted to molecular weight using a universal calibration curve constructed using narrow polydisperse poly(ethylene oxide) standards.

[0150] Modification verification:

[0151] The zeta potential results showed that HBPL was significantly positively charged, with a zeta potential of approximately +27 mV. The unmodified ZnO@MSN surface was negatively charged, with a zeta potential of approximately -16 mV. When HBPL was simultaneously loaded with vancomycin to form ZMHV, its zeta potential changed from negative to +9 mV, indicating a significant reversal of the surface charge, suggesting that the positively charged HBPL had been successfully adsorbed and coated onto the ZnO@MSN surface. Further introduction of Eudragit S100 yielded ZMHVS, whose zeta potential decreased again to approximately -14 mV, indicating that the surface charge underwent partial neutralization or redistribution after drug loading. These results demonstrate that the surface charge of the material undergoes a regular change during the stepwise assembly process, proving that the functional components were successfully loaded onto the nanoparticle surface (see [link to documentation]). Figure 5 ).

[0152] Transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS) revealed the following elemental mapping results: ZMHV nanoparticles exhibit an irregular spherical structure, as shown in the figure. The corresponding elemental mapping results show that Zn, Si, and O elements are uniformly distributed within the particles, indicating the successful construction of the ZnO@MSN core-shell structure. Simultaneously, a certain degree of N element signal was detected in ZMHV, mainly originating from the nitrogen-containing groups in HBPL and vancomycin molecules. Further encapsulation with Eudragit S100 resulted in ZMHVS, which maintained a complete spherical structure overall, but a relatively uniform outer coating structure was observed at the particle edges. Elemental mapping results showed that Zn, Si, and O elements remained uniformly distributed, while the N element signal was significantly enhanced in the particle surface region, suggesting that nitrogen-containing Eudragit S100 was successfully encapsulated on the ZMHV surface. These results demonstrate the successful encapsulation and stable modification of ZMHV by Eudragit S100 from both morphological and elemental distribution perspectives (see [link to relevant documentation]). Figure 6 ).

[0153] X-ray photoelectron spectroscopy (XPS): XPS results further validated the stepwise construction process of the nanoparticles. A distinct Zn 2p characteristic peak was observed in the ZnO sample, while a Si 2p peak appeared in ZnO@MSN, confirming the successful formation of the mesoporous silica shell. HBPL modification resulted in ZMH, with a distinct N1s peak at approximately 400 eV, indicating successful modification of the particle surface by amino-rich HBPL. Further loading with vancomycin to form ZMHV further enhanced the N1s signal, indicating the successful introduction of nitrogen-containing organic molecules. Considering the surface-sensitive properties of XPS, it can be inferred that vancomycin was mainly loaded into the pore structure of the mesoporous silica, while HBPL was mainly modified on the nanoparticle surface. Subsequent coating with Eudragit S100 yielded ZMHVS (see next section), whose XPS spectrum still retained characteristic elemental signals such as Zn, Si, O, C, and N, further demonstrating the structural stability of the material after Eudragit S100 coating. Meanwhile, the overall signal intensity of characteristic peaks such as Zn2p, Si2p, O1s, C1s, and N1s all showed varying degrees of attenuation. This is mainly attributed to the outer coating structure formed by Eudragit S100 on the particle surface, which shields the internal structure and related elemental signals to some extent. Since XPS is a surface-sensitive technology, this overall attenuation of elemental signals indicates that Eudragit S100 has successfully coated the outer layer of nanoparticles, thus further verifying the layer-by-layer assembly structure of ZMHVS (see [link to XPS]). Figure 7 ).

[0154] 4. pH-responsive outer coating (preparation of ZMHVS)

[0155] Eudragit S100 (1 g) was weighed and dissolved in an ethanol / water mixture (19:1, volume / volume, 20 mL) to prepare a coating solution (50 mg / mL). ZMHV composite material (1 mg) was dispersed in the coating solution (100 μL) and ultrasonically vibrated for 2 hours to ensure homogeneity. The mixture was then dried in a 60°C oven to evaporate the solvent, yielding the final product coated with Eudragit S100, denoted as ZMHVS.

[0156] Coating verification:

[0157] Electron microscopy revealed a blurred coating layer at the edges of the ZMHVS particles after coating. Elemental mapping showed that Zn, Si, and O elements remained uniformly distributed, while the N element signal was significantly enhanced on the particle surface, indicating that nitrogen-containing Eudragit S100 was successfully coated onto the ZMHV surface (see [link to study]). Figure 6 ).

[0158] XPS analysis revealed a further increase in the intensity of the C 1s peak in the full XPS spectrum of ZMHVS, along with the appearance of a characteristic peak of Eudragit S100. This confirms the successful coating of Eudragit S100. In the XPS spectrum of ZMHVS, the overall signal intensity of characteristic peaks such as Zn2p, Si2p, O1s, C1s, and N1s all decreased to varying degrees. This is mainly attributed to the formation of an outer coating structure of Eudragit S100 on the particle surface, which shields the internal structure and related elemental signals to some extent. Since XPS is a surface-sensitive technique, this overall attenuation of elemental signals indicates that Eudragit S100 was successfully coated onto the outer layer of the nanoparticles, further validating the layer-by-layer assembly structure of ZMHVS (see [link to relevant documentation]). Figure 7 ).

[0159] 5. Composition analysis of the final product

[0160] As described above, Van was detected using a spectrophotometer (210 nm), HBPL was detected using gel chromatography, and other substances were analyzed by TEM / SEM. The following content composition can be obtained.

[0161] Based on a total mass of 100% composite materials:

[0162] ZnO core: 6.2%

[0163] Mesoporous silica shell: 12.6%

[0164] Vancomycin (intra-channel): 13.2%

[0165] Hyperbranched polylysine (surface): 11.3%

[0166] Eudragit S100 coating layer: 56.6%

[0167] Converted to mass ratio, ZnO: MSN: Van: HBPL: Eudragit S100 ≈ 0.33 : 0.67 : 0.70 : 0.60 : 3.00.

[0168] Example 2

[0169] Evaluation of the sequential synergistic antibacterial properties of the nanocomposite ZMHVS

[0170] This embodiment verifies the colonic pH-responsive release characteristics, sequential synergistic antibacterial mechanism, and therapeutic effect on Clostridium difficile infection (CDI) of ZMHVS through a series of in vitro and in vivo experiments.

[0171] 1. pH-responsive release experiment

[0172] ZnO@MSN was labeled purple with crystal violet (CV) and coated with Eudragit S100. ZMCVS (1 mg) was dispersed in buffer solutions of different pH values ​​(pH 2.0, 8.5, and 11, 1 mL each) and incubated with shaking at 37°C. Color changes and sedimentation were observed during incubation to assess the stability and response behavior of the material under different pH conditions. Appearance changes of each group of samples were recorded after 1 h of incubation.

[0173] 2. In vivo fluorescence imaging experiment

[0174] To assess the distribution and retention of the nanosystem in vivo, the nanomaterial ZM@ICG@Eudragit S100, labeled with the fluorescent dye (ICG), was administered to mice via gavage. In vivo fluorescence imaging was performed at 3h, 12h, 24h, and 48h to observe changes in its distribution. Free ICG was used as a control group.

[0175] Result: As Figure 8 As shown in Figure A, ZMCVS exhibited significant pH-responsive characteristics under different pH conditions. At pH 2.0, the system color lightened considerably, and the supernatant was almost colorless, indicating that the nanomaterial structure remained stable and there was no significant release. However, at pH 8.5 and pH 11, the supernatant turned purple, indicating that the nanomaterial released its internally loaded ZMCV. This trend became more pronounced after 1 hour of incubation, demonstrating that Eudragit S100 possesses significant pH-responsive release characteristics.

[0176] like Figure 8 As shown in Figure B, free ICG was mainly distributed in the upper gastrointestinal tract 3 hours after administration, and its concentration decreased rapidly after 12 hours. A significant fluorescence signal was observed during excretion, which weakened considerably at 24 hours and essentially disappeared at 48 hours, indicating a short residence time in the gastrointestinal tract. In contrast, the ZM@ICG@Eudragit S100 group showed a relatively stable fluorescence signal in the abdomen from 3 to 24 hours, with enrichment in the colon region, indicating that this nanosystem has good stability and retention capacity in the gastrointestinal tract. The fluorescence signal essentially disappeared by 48 hours, suggesting that the material was eventually completely excreted. These results demonstrate that the Eudragit S100-encapsulated nanosystem can increase the material's residence time in the intestine and promote its delivery to the colon.

[0177] 3. Biomembrane disruption and inhibition experiments

[0178] (1) Disruption of existing biofilms

[0179] Clostridium difficile R20291 bacterial culture was inoculated into 24-well plates and anaerobic cultured for 48 hours to form a mature biofilm. The culture medium was then discarded, and samples from different treatment groups (PBS control group (Ctrl), ZMHV group, ZnO@MSN group, and HBPL group) were added, and cultured for another 24 hours. The amount of residual biofilm was determined using crystal violet staining.

[0180] Result: As Figure 9 The left figure shows the pre-formed biofilms treated with different concentrations of HBPL (32-512 μg / mL), ZnO@MSN (25-200 μg / mL), Van (4 μg / mL), and ZMHV (5-60 μg / mL). Crystal violet quantitative results showed that HBPL significantly reduced biofilm mass at concentrations ≥256 μg / mL; ZnO@MSN exhibited significant biofilm scavenging activity at concentrations ≥150 μg / mL; and ZMHV produced a strong biofilm disruption effect at concentrations ≥5 μg / mL. As controls, the biofilm structures of the control group and the vancomycin-treated group remained intact (see [reference needed]). Figure 9 (Left image).

[0181] To evaluate the biofilm permeability of nanoparticles, FITC-labeled HBPL was loaded onto ZnO@MSN to obtain ZMHV, which was then co-incubated with Clostridium difficile biofilm for 4 hours. Confocal microscopy showed that the FITC fluorescence signal was distributed throughout the entire biofilm depth, confirming that HBPL actively permeated the entire biofilm. Figure 10 ).

[0182] (2) Inhibition of biofilm formation

[0183] Clostridium difficile culture and samples from different treatment groups (as above) were co-inoculated into 24-well plates and anaerobic incubated for 48 hours. The amount of biofilm formed was determined by crystal violet staining, and the biofilm thickness was observed using a laser confocal microscope.

[0184] Result: As Figure 9 As shown in the right figure, in the biofilm formation inhibition experiment, the above-mentioned gradient concentrations of each component were added at the initial stage of bacterial inoculation. The results showed that within the tested concentration range, HBPL, ZnO@MSN, and sub-inhibitory concentrations of vancomycin failed to effectively inhibit the formation of new biofilms. In stark contrast, ZMHV almost completely blocked biofilm formation across the entire concentration range of 5-60 μg / mL (see [reference missing]). Figure 9 (The image on the right).

[0185] (3) The biofilm was stained with live / dead bacteria using the SYTO 9 / PI kit. After incubation in the dark for 30 min, the biofilm was washed twice with sterile PBS. Then, the biofilm was imaged using a laser confocal microscope (CLSM). Green fluorescence indicates live bacteria with intact cell membranes, and red fluorescence indicates bacteria with damaged membranes.

[0186] Figure 11 This study demonstrates the use of SYTO 9 (for live bacteria) and PI (for dead bacteria) for double staining of biofilms, and the quantification of biofilm thickness using z-stack scanning. Results showed that the control group had the thickest biofilm and the strongest SYTO 9 fluorescence. The HBPL and ZnO@MSN treatment groups showed decreased biofilm thickness and SYTO 9 fluorescence intensity compared to the control group. The ZMHV treatment group had the lowest biofilm thickness and SYTO 9 fluorescence intensity.

[0187] 4. Reactive Oxygen Species (ROS) Generation Experiment

[0188] 0.5 mL and 1 mM of the DCFH-DA probe (Shanghai Maokang, MX4802-10 mg) were reacted with 2 mL of NaOH aqueous solution (1 mM) at room temperature for 30 min, and then neutralized with 7.5 mL of PBS to obtain a 50 μM DCFH stock solution. The final concentration of 5 μM DCFH was mixed with PBS, ZnO (nanoparticles), and ZnO@MSN to monitor the fluorescence signal of the indicator. The excitation wavelength was 488 nm, and the fluorescence intensity of DCFH at 525 nm was measured to detect ROS generation.

[0189] Result: As Figure 12 As shown, the generation of reactive oxygen species (ROS) in ZnO nanoparticles and ZnO@MSN over time was evaluated by fluorescence detection. The ROS probe detection showed that ZnO@MSN could continuously generate significant ROS signals for 72 hours, and the intensity was higher than that of pure ZnO nanoparticles under the same conditions.

[0190] 5. Analysis of Virulence and Sporulation Gene Expression

[0191] Clostridium difficile and ZMHV were co-cultured for 12 hours, and total RNA was extracted from the bacteria. The expression levels of toxin genes and sporulation-related genes were detected by qRT-PCR. Prokaryotic transcriptomics was used to analyze the transcriptional levels of toxin genes and sporulation-related genes.

[0192] Result: As Figure 13 As shown, after ZMHV treatment, the expression levels of tcdA, tcdB, and tcdR genes were downregulated to 0.1-fold, 0.08-fold, and 0.08-fold respectively compared to the control group (see [reference]). Figure 13 (See the left image). The transcriptome heatmap also shows that ZMHV significantly inhibits the expression of the toxin gene (see the left image). Figure 13 (See the right figure). This confirms that the expression of sporulation-related genes is significantly downregulated (see the figure on the right). Figure 13 (Left), while GSEA enrichment analysis of transcriptome results confirmed that ZMHV can inhibit the early sporulation program of Clostridium difficile (see left). Figure 14 (See right figure). Transcriptome enrichment analysis showed that the custom "redox / oxidative stress" gene set was significantly negatively enriched in the ZMHV group. Further analysis of this gene set revealed a highly significant negative enrichment signal in its core redox maintenance module (NES = -1.87, padj = 2.70 × 10⁻⁶). -5 While the stress-induced detoxification module showed no significant changes, ZMHV primarily weakened Clostridium difficile's ability to maintain basal redox homeostasis without inducing a typical stress-induced detoxification response. This indicates that ZMHV did not induce a typical oxidative stress response characterized by significant activation of detoxification pathways, but rather led to a systematic downshift of Clostridium difficile's core redox maintenance module. This suggests that ZMHV may fundamentally disrupt bacterial energy metabolism and redox homeostasis by disrupting the electron transport chain, depleting intracellular reducing power, or damaging iron-sulfur cluster proteins. Simultaneously, the overall metabolic instability of the bacteria also makes it difficult to initiate the normal sporulation program (see...). Figure 15 ).

[0193] 6. Promotes mucosal repair

[0194] Caco-2 cells were placed in 24-well plates containing cell slides and allowed to adhere completely for one day. After discarding the supernatant, fresh culture medium was added. The experimental groups were incubated with concentrated Clostridium difficile supernatant (toxin Tcd) and Tcd+HBPL, respectively, and then incubated at 37°C for 4 hours. The culture medium was discarded, and the cells were fixed, permeabilized for 10 minutes, blocked, incubated with primary antibody, incubated with fluorescent secondary antibody, incubated with DAPI, and protected against fluorescence quenching. The slides were then removed with forceps, inverted onto glass slides, and mounted. Finally, the slides were photographed using a CLSM scanner.

[0195] Colons were harvested from CDI-infected mice on day 3 post-infection, fixed in 4% paraformaldehyde for 24 hours, routinely dehydrated, cleared, and then embedded in paraffin to prepare 10 μm thick serial sections. After dewaxing in xylene and rehydration with graded ethanol, the sections were stained with hematoxylin-eosin (HE). Following staining, the sections were dehydrated with graded ethanol, cleared in xylene, and mounted. Finally, the pathological morphological changes of the colon tissue were observed and photographed under an optical microscope.

[0196] At the cellular level, Caco-2 cells were co-cultured with Clostridium difficile supernatant (toxin). Caco-2 cells treated with HBPL showed a more continuous barrier protein (ZO-1) (appearing green under a laser confocal microscope), similar to the control group. However, Caco-2 cells treated with only the toxin, without HBPL, lacked the barrier protein (see...). Figure 16 (See the image above). In the CDI mouse model, colon tissue was taken on day 3 post-infection for histological analysis. HE staining results showed that the colonic mucosa structure in the CDI model group was severely damaged, manifested as extensive mucosal erosion, disordered crypt structure, and extensive lymphocyte infiltration. In the vancomycin treatment group, the mucosal continuity was partially restored, but significant inflammatory cell infiltration and irregular crypt structure remained. In contrast, the colonic mucosa structure in the ZMHVS treatment group was intact, the crypts were regularly arranged, and only a few scattered inflammatory cell infiltrations were observed. Its histological morphology was similar to that of the unmodeled healthy control group (see [link to relevant documentation]). Figure 16 (See the image below).

[0197] 7. In vivo distribution and colon targeting evaluation

[0198] To assess the distribution and retention of the nanosystem in vivo, the nanomaterial ZM@ICG@Eudragit S100, labeled with the fluorescent dye (ICG), was administered to mice via gavage. In vivo fluorescence imaging was performed at 3h, 12h, 24h, and 48h to observe changes in its distribution. Free ICG was used as a control group.

[0199] Result: As Figure 8 As shown in Figure B, free ICG was mainly distributed in the upper gastrointestinal tract 3 hours after administration, and its fluorescence intensity decreased rapidly after 12 hours, with a significant fluorescence signal observed during excretion. The signal weakened significantly at 24 hours and essentially disappeared at 48 hours, indicating a short residence time in the gastrointestinal tract. In contrast, the ZM@ICG@Eudragit S100 group showed a relatively stable fluorescence signal in the abdomen from 3 to 24 hours, and was enriched in the colon region, indicating that this nanosystem has good stability and retention capacity in the gastrointestinal tract. The fluorescence signal essentially disappeared by 48 hours, indicating that the material was eventually completely excreted. These results demonstrate that the Eudragit S100-encapsulated nanosystem can increase the retention time of the material in the intestine and promote its delivery to the colon.

[0200] 8. Evaluation of the therapeutic effect in CDI mouse model

[0201] (1) Establishment of CDI model

[0202] A CDI model was established using a mouse model of Clostridium difficile infection and recurrence induced by a combination of antibiotics and clindamycin. Seven days prior to inoculation, mice were fed a combination of antibiotics for five days (including kanamycin (40 mg / kg), gentamicin (3.5 mg / kg), colistin (4.2 mg / kg), metronidazole (21.5 mg / kg), and vancomycin (4.5 mg / kg)). One day before inoculation, clindamycin was injected intraperitoneally to disrupt the intestinal flora. On day 0, Clostridium difficile was administered via gavage to establish the infection model (1 × 10⁻⁶). 8 CFU / mL) was administered via drug intervention on days 1–4. The control group received vancomycin, while the experimental group received ZMHVS. On day 5, after collecting stool samples, clindamycin was injected intraperitoneally again to induce relapse, followed by observation until day 14. Body weight and clinical symptoms were recorded daily during the experiment, and tissue and blood samples were collected on day 3 for histological, inflammatory factor, and Western blot analysis, respectively.

[0203] (2) Treatment plan

[0204] The control group dose of vancomycin was 0.4 mg / mL, and the ZMHVS dose was 1.2 mg / mL, administered orally.

[0205] (3) Observation indicators

[0206] Weight change: The weight of mice was recorded daily, and a weight change curve was plotted.

[0207] Survival rate: The mortality of mice in each group was recorded, and Kaplan-Meier survival curves were plotted.

[0208] Colon length: Mice were sacrificed on day 3 and colon length was measured.

[0209] Spore counting: Colon contents were collected, treated with ethanol, and then plated to count the number of spores.

[0210] Inflammatory factor detection: Colon tissue homogenate was taken and the levels of inflammatory factors such as TNF-α and IL-6 were detected by ELISA.

[0211] Histopathology: Colon tissue was stained with hematoxylin and eosin (HE) and pathological changes were observed under a light microscope.

[0212] (4) Results

[0213] Weight and survival rate: such as Figure 17As shown, dynamic monitoring of body weight changes revealed that mice in the CDI model group experienced significant weight loss during both the infection and relapse phases. The vancomycin treatment group showed a more moderate weight loss after the initial infection, but experienced a significant weight decrease during the relapse phase, with a similar magnitude to the CDI group. The ZMHVS treatment group showed only slight weight loss during both infections, and its weight change curve was significantly better than that of the CDI model group and the vancomycin treatment group. Survival analysis showed that by day 14, the survival rate of healthy control mice was 100%, compared to 40% in the CDI model group, 60% in the vancomycin treatment group, and 90% in the ZMHVS treatment group.

[0214] Colon length: as Figure 18 As shown, the colon length of mice in the CDI model group was significantly shorter than that in the unmodeled healthy group, approximately 5.13 cm. Although the colon length in the vancomycin treatment group was slightly longer than that in the CDI model group, it was still significantly shorter than that in the healthy group, approximately 7.23 cm. In contrast, the colon length in the ZMHVS treatment group was not significantly different from that in the healthy group, approximately 7.84 cm, and was significantly longer than both the CDI model group and the vancomycin treatment group.

[0215] Number of spores: such as Figure 19 As shown, the number of spores in the colon contents of the CDI model group was as high as 10. 6 CFU / g, vancomycin group spore count approximately 10 5 CFU / g, the number of spores in the ZMHVS group decreased to 10. 2 The CFU / g level was significantly lower than that of other groups.

[0216] Inflammatory factors: such as Figure 20 As shown, the levels of IFN-γ, IL-1α, IL-1β, IL-6, and TNF-α in the CDI model group were significantly higher than those in the other three groups. The levels of various inflammatory factors in the ZMHVS treatment group were similar to those in the healthy control group, with most indicators showing no significant differences. The levels of IFN-γ, IL-6, and TNF-α in the vancomycin treatment group remained higher than those in the healthy control group, while the levels of other inflammatory factors showed no significant differences compared to the healthy control group.

[0217] Histopathology: such as Figure 16 As shown, the CDI model group exhibited severe damage to the colonic mucosal structure, characterized by extensive mucosal erosion, disordered crypt structure, and abundant lymphocyte infiltration. The vancomycin treatment group showed partial restoration of mucosal continuity, but still exhibited significant inflammatory cell infiltration and irregular crypt structure. In contrast, the ZMHVS treatment group showed intact colonic mucosal structure, regular crypt arrangement, and only a small number of scattered inflammatory cell infiltrations; its histological morphology was similar to that of the unmodeled healthy control group.

[0218] 9. Conclusion

[0219] Based on the above experimental results, the ZMHVS nanocomposite material prepared in this invention achieves colonic confinement release through pH-responsive coating, disrupts the biofilm barrier through surface HBPL, and releases Zn through vancomycin and ZnO within the pores. 2+ It synergistically kills bacteria while inhibiting toxin expression and sporulation, ultimately significantly reducing the recurrence rate of CDI, improving survival rate, alleviating inflammation, and promoting mucosal repair, achieving a sequential synergistic treatment of "first breaking the membrane - then killing bacteria - then inhibiting sporulation - then repairing".

Claims

1. Use of a core-shell structured nanocomposite material in the preparation of a medicament for the prevention and / or treatment of Clostridium difficile infection and its recurrent diseases, wherein the nanocomposite material comprises: ZnO nanoparticles as the core; Mesoporous silica, which acts as a shell, coats the surface of ZnO nanoparticles; Antibiotics loaded within the pores of the mesoporous silica; Hyperbranched polylysine modified on the surface of the mesoporous silica; as well as As the outermost pH-responsive coating material.

2. The use according to claim 1, wherein, The drug is used for at least one of the following therapeutic purposes: (a) Disruption of Clostridium difficile biofilm; (b) Inhibits Clostridium difficile toxin expression; (c) Reduce the number of Clostridium difficile spores; (d) Reduce the recurrence rate of Clostridium difficile infection; (e) Reduces colitis and tissue damage caused by Clostridium difficile infection.

3. The use according to claim 1, wherein, The drug is a pharmaceutical composition, and the pharmaceutical composition comprises an effective amount of the nanocomposite material and pharmaceutically acceptable excipients.

4. The use according to claim 3, wherein, The pharmaceutical composition is an oral preparation.

5. The use according to claim 4, wherein, The oral preparation is a solution, suspension, capsule, or tablet.

6. The use according to claim 3, wherein, The pharmaceutically acceptable excipient is selected from at least one of fillers, binders, disintegrants, lubricants, and flow aids.

7. The use according to claim 1, wherein, The pH-responsive coating material is a pH-sensitive polymer that dissolves or degrades in an environment with pH ≥ 7.0; Preferably, the pH-sensitive polymer is selected from synthetic polymeric pH-sensitive materials or natural polymeric pH-sensitive materials; More preferably, the synthesized polymeric pH-sensitive material is a methacrylic acid copolymer; More preferably, the synthesized polymeric pH-sensitive material is a copolymer of methacrylic acid and methyl methacrylate; More preferably, the synthesized polymeric pH-sensitive material is a copolymer of methacrylic acid and methyl methacrylate in a molar ratio of 1:4; More preferably, the synthesized polymeric pH-sensitive material is Eudragit S100; More preferably, the natural polymeric pH-sensitive material is resistant starch; Preferably, the antibiotic is an antibiotic effective against Gram-positive bacteria; Preferably, the antibiotic is a glycopeptide antibiotic or a nitroimidazole antibiotic; More preferably, the glycopeptide antibiotic is selected from at least one of vancomycin, teicoplanin, and teicoplanin; More preferably, the nitroimidazole antibiotic is metronidazole; Preferably, the hyperbranched polylysine is modified on the surface of the mesoporous silica by electrostatic adsorption; Preferably, the hyperbranched polylysine is prepared by heating and polymerizing L-lysine or its salt in the presence of an alkaline substance, a catalyst and urea in an inert atmosphere.

8. The use according to claim 1, wherein, The number-average molecular weight of the hyperbranched polylysine is 3-10 kDa.

9. The use according to claim 1, wherein, The average particle size of the ZnO nanoparticles is 100-300 nm.

10. The use according to claim 1, wherein, In the nanocomposite material, the mass ratio of ZnO, mesoporous silica, antibiotic, hyperbranched polylysine and pH-responsive coating material is (0.15-0.45): (0.55-0.75): (0.6-0.8): (0.5-0.7): (2-4).

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