VCZ-ZIF-8 nanoparticles and application thereof as antifungal drugs

Abstract

The invention relates to VCZ-ZIF-8 nanoparticles and application of the VCZ-ZIF-8 nanoparticles as antifungal drugs, and belongs to the technical field of medicines.The VCZ-ZIF-8 nanoparticles are used as the antifungal drugs for intraocular inflammation treatment, the half-life period is effectively prolonged, the VCZ-ZIF-8 nanoparticles have an obvious slow-release effect, and the effective concentration time gt is maintained through single injection; after 21 days, repeated administration is not needed, good biological membrane penetrability is achieved, and drug resistance caused by fungal biological membranes can be reduced or overcome; the VCZ-ZIF-8 nano-particles are injected through rabbit eyes, and the result shows that compared with injection of VCZ, the VCZ-ZIF-8 nano-particles can remarkably improve the survival rate of retinal microvascular endothelial cells and retinal pigment epithelial cells and have good safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, in particular, relates to VCZ@ZIF-8 nanoparticles and application thereof as antifungal drugs. BACKGROUND

[0002] The incidence of fungal endophthalmitis is increasing year by year, and the incidence of fungal endophthalmitis accounts for 12.7% of postoperative endophthalmitis in China. According to the cause of infection, fungal endophthalmitis can be exogenous (directly from keratitis, trauma or intraocular surgery, etc. external source), or endogenous (related to systemic fungal disease). Due to the long incubation period of fungal endophthalmitis, early diagnosis is difficult, treatment is tricky, and visual prognosis is poor, with a high rate of blindness (more than half of the patients with fungal endophthalmitis have a final visual acuity of <0.05) and even possible eye atrophy; although researchers have made many attempts, the effect is still limited.

[0003] In the Chinese expert consensus on prevention and treatment of infectious endophthalmitis after cataract extraction surgery, it is recommended that when considering fungal infectious endophthalmitis, vitreous cavity injection of antifungal drugs is recommended. However, the antifungal drug for vitreous cavity injection should have high safety. Voriconazole (VCZ) is a triazole antifungal drug, a derivative of fluconazole, which has a wider antibacterial spectrum than fluconazole and higher safety than amphotericin B, the earliest antifungal drug used in ophthalmology. The half-life of voriconazole is only 2.5 hours, so vitreous cavity injection of voriconazole for treatment of candidal endophthalmitis often requires multiple vitreous cavity injections, and repeated vitreous cavity injection can easily cause serious complications such as vitreous hemorrhage, proliferative and organized, and retinal detachment. At the same time, multiple injections cause great physical and psychological pain and economic pressure to patients. Although a large amount of drugs can prolong the effective concentration of intraocular drugs, the retinal toxicity of large doses of drugs will cause damage to the only vision of the patient.

[0004] One of the defining features of biofilm is the extracellular matrix (ECM), which is produced by microorganisms and encapsulates the microorganisms, rich in proteins, polysaccharides, lipids, nucleic acids and other molecules form a bio-polymer, these molecules can interact and form a strong protective network with the bacterial surface. Functionally, ECM acts as a barrier to prevent antifungal drugs from diffusing into the interior of the biofilm, which is one of the factors of fungal drug resistance. Studies have shown that on the surface of implanted artificial particles, although there is no clinical or subclinical symptoms, biofilms can still be found on the artificial lenses removed from the long-term non-complication eyes after cataract surgery. The formation of biofilms has limited the use and design of ocular devices, such as artificial particles, scleral buckling, conjunctival plugs, and orbital implants. Even in non-biological material implants such as capsular bag and corneal stroma, biofilms can be observed. Since there is evidence that microbial biofilms are involved in many eye infections, the formation of biofilms makes the treatment of endophthalmitis more difficult. In order to solve the problem of drug resistance caused by biofilm, researchers have made many attempts, such as developing new antifungal drugs, or by modifying existing antifungal drugs, so that the synthesized drugs have better biofilm elimination effect.

[0005] In summary, it is still a difficult problem to be solved to achieve sustained release of antibacterial drugs at the infection site and to overcome drug resistance caused by fungal biofilm. SUMMARY

[0006] In order to overcome the problems in the background art, the present application provides a VCZ@ZIF-8 nanoparticle and its application as an antifungal drug. The VCZ@ZIF-8 nanoparticle of the present application is used as an antifungal drug for the treatment of endophthalmitis, the half-life is effectively prolonged, has obvious sustained release effect, the effective concentration time is maintained >21 days after single injection, repeated administration is not required, and has good biofilm penetration, which can reduce or overcome the drug resistance caused by fungal biofilm.

[0007] To achieve the above purpose, the present application is realized by the following technical scheme: The VCZ@ZIF-8 nanoparticle takes ZIF-8 as a carrier and loads voriconazole VCZ.

[0008] Further, the nanoparticle has a particle size of 100-200 nm and a surface potential of +15 to +25 mV.

[0009] The nanoparticle is applied in the preparation of an antifungal drug.

[0010] Further, the nanoparticle releases the drug in response to a pH of 5.0-7.4.

[0011] Further, the VCZ@ZIF-8 nanoparticles are applied to preparation of treatment of endophthalmitis caused by fungal infection.

[0012] The application further provides an antifungal drug delivery system comprising the VCZ@ZIF-8 nanoparticles.

[0013] The application further provides a preparation method of the VCZ@ZIF-8 nanoparticles, comprising the following steps: (1) Preparation of ZIF-8 The methanol solution of Zn(NO3)2·6H2O is mixed with CTAB, the 2-MeIM methanol solution is added, and after sufficient stirring, centrifugal washing and vacuum drying, ZIF-8 is obtained. (2) Drug loading of VCZ@ZIF-8 The ZIF-8 is dissolved in ethanol with a purity of ≥99.5% with VCZ, stirred for 24 hours, centrifugally washed and vacuum dried, and the VCZ@ZIF-8 nanoparticles are obtained.

[0014] Further, in step (1), the amount of CTAB is 0.1-0.2 g per millimole of Zn(NO3)2·6H2O; and the molar ratio of 2-MeIM to Zn(NO3)2·6H2O is 4-8:1.

[0015] Further, in step (2), the ZIF-8 and VCZ are dissolved in ethanol with equal mass.

[0016] The application has the following beneficial effects: The nanoparticles of the application are used for treatment of endophthalmitis caused by fungi, have a sustained-release effect, an effective concentration of a single injection is greater than 21 days, the drug efficacy time is greatly prolonged, repeated administration is not needed, and thus complications such as vitreous hemorrhage and retinal detachment caused by repeated injection are effectively avoided, and the nanoparticles have good stability under physiological conditions (pH 7.4), and unnecessary drug leakage during in vivo transportation of VCZ can be avoided.

[0017] The VCZ@ZIF-8 nanoparticles exhibit excellent biofilm penetration and clearance capacity, and can reduce drug resistance caused by fungal biofilms.

[0018] The rabbit eye injection safety analysis shows that the VCZ@ZIF-8 can significantly reduce the toxicity of voriconazole, can significantly improve the survival rate of retinal microvascular endothelial cells and retinal pigment epithelial cells relative to injection of VCZ, and has good safety. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a TEM morphology of ZIF-8 and VCZ@ZIF-8 nanoparticles of the application; Figure 2DLS particle size of ZIF-8 and VCZ@ZIF-8 nanoparticles of the present application; Figure 3 XRD crystal form of VCZ@ZIF-8 nanoparticles of the present application; Figure 4 X-ray diffraction pattern of ZIF-8 and VCZ@ZIF-8 nanoparticles of the present application; Figure 5 TGA pattern of VCZ@ZIF-8 nanoparticles of the present application; Figure 6 pH-responsive drug release curve of VCZ@ZIF-8 nanoparticles of the present application; Figure 7 Comparison of cell activity of retinal vascular endothelial cells (RMEC) and retinal pigment epithelial cells (PRE) under different drug conditions of the present application; Figure 8 Calcein-AM / PI staining results of VCZ@ZIF-8 nanoparticles of the present application in retinal vascular endothelial cells (RMEC) and retinal pigment epithelial cells (PRE); Figure 9 Slit lamp photographs of different drug injections into rabbit eyes in the embodiments of the present application; Figure 10 ERG electrophysiological results (waveform) after injection of different drugs into the vitreous cavity of rabbits in the embodiments of the present application; Figure 11 ERG electrophysiological results (amplitude) after injection of different drugs into the vitreous cavity of rabbits in the embodiments of the present application; Figure 12 Retinal morphological results after injection of different drugs into the vitreous cavity of rabbits for 21 days in the embodiments of the present application; Figure 13 H&E staining results of important organs (heart, liver, spleen, lung, kidney) of the whole body after injection of different drugs into the vitreous cavity of rabbits in the embodiments of the present application; Figure 14 Pictures of VCZ@ZIF-8 nanoparticle biomembrane penetration observed under a confocal microscope in the present application; Figure 15 Biomembrane elimination effect of VCZ@ZIF-8 nanoparticles of the present application; wherein, A and C are flat plate experiments and statistical analysis, B and D are fungal dead and live staining and fluorescence intensity analysis; Figure 16 Slit lamp observation pictures of different drug treatments for fungal endophthalmitis at different times in the embodiments of the present application; Figure 17 Comparison of rabbit eye histopathological examination results under different drug conditions. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention will be described in detail below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0021] In order to illustrate the present invention more clearly, the following examples are provided for detailed description. Example 1

[0022] (1) Preparation of ZIF-8 Synthesis of ZIF-8: 9.6 mL of a 0.070 M solution of Zn(NO₃)₂-6H₂O in methanol was mixed with 0.15 g of cetyltrimethylammonium bromide and stirred at room temperature for 5 minutes. Then, 9.6 mL of a 0.43 M solution of 2-methylimidazole in methanol was added to the mixture and stirred for an additional 5 minutes. The mixture was then allowed to stand at room temperature for 6 hours. After centrifugation and washing with 20.0 mL of methanol, this process was repeated twice. The product was collected and dried under vacuum to yield ZIF-8.

[0023] (2) VCZ@ZIF-8 drug delivery 10 mg of ZIF-8 and 10 mg of voriconazole were added to 20 mL of anhydrous ethanol and shaken for 24 hours. The mixture was centrifuged and washed twice with 20.0 mL of ethanol. Finally, the product was vacuum-dried to obtain VCZ@ZIF-8 nanoparticles.

[0024] The TEM morphology of the prepared VCZ@ZIF-8 nanoparticles and ZIF-8 is shown in the attached figure. Figure 1 As shown, through the attached Figure 1 It can be seen that the synthesized ZIF-8 presents a relatively regular polyhedron shape and is relatively uniform in size ( Figure 1 Left). After loading VCZ, the morphology of the nanoparticles did not change significantly ( Figure 1 This indicates that ZIF-8 can maintain its original structure without changes after loading with VCZ.

[0025] Dynamic light scattering was used to measure the -3 The diameter of VCZ@ZIF-8 in 10% potassium phosphate buffer (PB) and 10% fetal bovine serum was measured. Figure 2 The average particle size of VCZ@ZIF-8 is close to that of ZIF-8, which is consistent with the size observed by transmission electron microscopy.

[0026] The dried VCZ@ZIF-8 powder was analyzed by X-ray diffraction analyzer, and the X-ray diffraction pattern is shown in FIG. 1. Figure 3 As shown in FIG. 1, the XRD of VCZ@ZIF-8 can be observed at 2θ = 10.38°, 12.74°, 14.7°, 16.44°, 18.06° and 26.72°, which is consistent with the typical characteristic diffraction peaks of ZIF-8. It is shown that the loading of voriconazole does not change the structure of ZIF-8, which is consistent with the result observed by transmission electron microscopy.

[0027] According to the BET method, the influence of the loading of voriconazole on the pore size and specific surface area of ZIF-8 MOFs was studied by N2 adsorption, and the BET curve is shown in FIG. 2. Figure 4 The BET curve shows that the surface area of ZIF-8 is large, and the pore volume is also large. Compared with ZIF-8, the specific surface area and pore volume of VCZ@ZIF-8 decrease, which shows that VCZ is successfully loaded into the internal pores of ZIF-8, and VCZ@ZIF-8 also has rich micropores, so that VCZ can be released from these micropores.

[0028] The thermodynamic properties of VCZ@ZIF-8 were explored by TGA in N2 environment, and the result is shown in FIG. 3. Figure 5 VCZ@ZIF-8 also has a part of weight loss when the temperature is increased to about 200°C, which is mainly caused by the partial degradation of VCZ encapsulated in the internal pores of nanoparticles, because previous studies show that VCZ starts to degrade when the temperature is increased to 172.41°C, and voriconazole is completely degraded when the temperature is higher than 250°C. The weight loss at about 340°C is consistent with the descending rate of the TGA curve of ZIF-8, and it is speculated that it is mainly caused by the degradation of ZIF-8, which is consistent with the previous research results. When the temperature is further increased to above 400°C, the weight loss of ZIF-8 slows down, and the weight loss trend of VCZ@ZIF-8 is greater than that of ZIF-8. At this time, we speculate that the weight loss of VCZ@ZIF-8 is caused by the further degradation of VCZ. It can be seen that the VCZ is built into ZIF-8, and ZIF-8 also plays a role in protecting the built-in drug as an organic metal framework.

[0029] The prepared 0.1 mol / l KH2PO4 and 0.1 mol / l Na2HP4 were mixed as a mother liquor, and the pH was measured after mixing, and finally the pH value was adjusted using NaOH solution, so that the pH of the solution was 7.4, 6.5 and 5.6, respectively. In order to study the release of VCZ, 10 mg of VCZ@ZIF-8 was placed in a dialysis bag, and then immersed in a centrifuge tube containing 10 mL of phosphate buffered saline (PBS) with pH values of 7.4, 6.5 and 5.6, respectively, and shaken gently (150 rpm) at 37°C. After different time intervals, 1 mL aliquots were collected, and 1 mL of PBS was added to the centrifuge tube to ensure that the volume of liquid in the centrifuge tube was constant, and finally the UV-vis absorbance at 256 nm was measured and the release rate of voriconazole was calculated, and the release curve was plotted, as shown in FIG. 6. Figure 6 Release rate = mass of released voriconazole / mass of voriconazole carried in VCZ@ZIF-8. The in vitro release curve results show that the release amount of VCZ@ZIF-8 is small at pH 7.4 (i.e. physiological state), and it has stability under physiological conditions, which can avoid unnecessary drug leakage of VCZ during in vivo transportation. Under acidic conditions, the release amount increases and can reach 90%.

[0030] In vitro cytotoxicity test: (1) CCK-8 method for detecting cell activity Logarithmic phase growth of retinal vascular endothelial cells (RMEC) and retinal pigment epithelial cells (RPE) were inoculated in 96-well plates, 100 μl of cell suspension (about 5000 cells / well) was added to each well, and placed in an incubator (37°C, 5% CO2) for culture to the appropriate density (60-80%).

[0031] The original cell culture solution was discarded, and prepared VCZ@ZIF-8, VCZ, PBS without fetal bovine serum (FBS) were added, and the drug concentration was 0 as the control group, and no treatment as the blank group, and continued to culture for 24 hours.

[0032] The supernatant was discarded, the cells were washed with PBS, and finally 100 μl of cell culture solution containing 10% CCK8 was added to each well, and incubated for 1-2 hours.

[0033] The absorbance (OD) value of each well at 450 nm was detected by a fully automatic enzyme marker, and the following formula was used to calculate the cell survival rate of different groups: Cell survival rate (%) = (experimental group OD value-blank group OD value) / (control group OD value-blank group OD value) x 100%. For example, Figure 7As shown in A, when the concentration of VCZ is 100 μg / ml, the RMEC activity is significantly decreased, but when the concentration of VCZ@ZIF-8 (calculated based on the equivalent concentration of VCZ) is 100 μg / ml, the RMEC activity does not show significant changes, and until the concentration of VCZ@ZIF-8 (calculated based on the equivalent concentration of VCZ) reaches 300 μg / ml, the RMEC activity shows a significant decrease. However, 300 μg / ml of ZIF-8 does not show any toxicity. As shown in Figure 7 As shown in B, when the concentration of VCZ is 250 μg / ml, the RPE activity is significantly decreased, but when the concentration of VCZ@ZIF-8 (calculated based on the equivalent concentration of VCZ) is 250 μg / ml, the RPE activity does not show significant changes, and until the concentration of VCZ@ZIF-8 (calculated based on the equivalent concentration of VCZ) reaches 300 μg / ml, and the RPE activity of the ZIF-8 group at the equivalent concentration does not show a significant decrease. This indicates that the VCZ@ZIF-8 constructed by loading VCZ on ZIF-8 significantly reduces the toxicity of voriconazole.

[0034] (2) Calcein-AM / PI double staining method for detecting cell death and activity A. Experimental grouping: same as the CCK-8 experiment grouping described above B. Retinal vascular endothelial cells (RMEC) and retinal pigment epithelial cells (RPE) were inoculated in 96-well confocal culture dishes at 5000 cells / well and placed in an incubator for 24 hours of continuous culture C. The culture medium in the culture dish was poured out, and VCZ@ZIF-8, VCZ, ZIF-8 with the same concentration as the CCK-8 experiment were added according to the above grouping, and the incubation was continued for 24 hours D. Preparation of Calcein-AM / PI double staining solution: 10 μL of Calcein-AM and 20 μL of PI were added to a centrifuge tube containing 10 mL of cell culture solution E. 100 μL of the above double staining solution was added to each well, and the staining was performed for 10 minutes F. The survival of cells in each group was observed under a laser confocal microscope, and the results are shown in Figure 8 As shown in C, when the concentration of VCZ is 100 μg / ml, the number of RMEC live cells is significantly reduced, but when the concentration of VCZ@ZIF-8 (calculated based on the equivalent concentration of VCZ) and ZIF-8 is 100 μg / ml, the number of RMEC live cells does not show significant changes. As shown in Figure 8 D, compared with Figure 8 Similar to C, the RPE was stained by Calcein-AM / PI, as shown in Figures 2-4As shown, when VCZ is 250 μg / ml, the number of RPE live cells is significantly reduced, but at 300 μg / ml VCZ@ZIF-8 (calculated at the same concentration of VCZ) and ZIF-8, the number of RPE live cells does not change significantly.

[0035] Rabbit eye safety experiment: Experimental grouping: healthy New Zealand white rabbits were randomly divided into 2 experimental groups and 1 negative control group. The 2 experimental groups respectively received intravitreal injection of 100 μg / 0.1 mL VCZ@ZIF-8 suspension (calculated at the equivalent of voriconazole) 0.1 mL, ZIF-8 (equal to the concentration of ZIF-8 in VCZ@ZIF-8) suspension 0.1 mL, and the negative control group was injected with 100 μL of sterile normal saline in the same way.

[0036] Surgical method: after the animals were anesthetized with compound tropicamide eye drops, xylazine + su-tai intramuscular injection, binox eye drops were used for surface anesthesia, in order to avoid high intraocular pressure, 1 ml needle was used for anterior chamber puncture in advance, and 0.1 mL aqueous humor was extracted, then the needle was inserted at 2 mm behind the corneal limbus (note to avoid the lens), 100 μL of drug or normal saline was injected into the vitreous center, and after the operation, kelebi tu eye drops were used for eye drops.

[0037] Observation index: 1) Live examination: 3 weeks after injection, the eye condition was observed under a slit lamp microscope (such as attached Figure 9 ), the slit lamp observation showed that 21 days after intravitreal injection, compared with the control group, there was no obvious inflammation in the eye, the cornea was transparent, no KP and Tyn phenomenon was observed, and the vitreous light transmittance did not change significantly.

[0038] 2) The results of electroretinogram (ERG) examination are shown in Figure 10 and 11 , the waveforms are shown in Figure 10 , the amplitudes are shown in Figure 11 , and it can be seen from Figure 10 that VCZ@ZIF-8 is safer at the same concentration than voriconazole, and it can be seen from Figure 11 that VCZ@ZIF-8 is safer at the same concentration.

[0039] After 3 weeks of model establishment, ERG examination was performed using Espion Diagnosys LLC, Littleton, MA, USA). The rabbits were dark adapted for at least 30 minutes, and the examination was performed in a dark room with thick curtains in the ERG special room. The preparation was performed under red light. After the rabbits were anesthetized, they were given “Mydriatic” to dilate the pupils. Under dark red light, the rabbits were fixed on the animal test platform, and the rabbits were required to lie flat during the whole process, that is, the height of the left and right eyes was consistent relative to the stimulation port of the flash stimulator, and the rabbits were fully exposed. Under dark red light, the electrodes were connected: the ground electrode was connected to the tail, the negative electrode needle was inserted into the subcutaneous tissue of the back of the rabbit’s neck (approximately in the middle of the two ears), and then the “negative” interfaces of the two channels were connected; after the corneal electrode was coated with carbomer gel, it was attached to the cornea of the rabbit. The positive electrode of one channel was connected to the right eye, and the positive electrode of the second channel was connected to the left eye. Then, the ERG examination was performed according to the set program.

[0040] Histopathological examination: A tissue fixation: After 3 weeks of surgery, one rabbit was randomly sacrificed, and the eyeball was taken out and fixed in FAS eyeball fixing solution for 24 hours to ensure that there was enough fixing solution covering the tissue.

[0041] B Dehydration: 70% ethanol, 15-30 min; 80% ethanol, change once, 2-4 h; 95% ethanol, change once, 2-4 h; 100% ethanol (to ensure that the 100% ethanol is water-free, the ethanol can be placed in a copper sulfate container to absorb water).

[0042] C Transparency: The dehydrated eyeball tissue was placed in dimethylbenzene I solution for 30 minutes, and then placed in dimethylbenzene II solution for 30 minutes.

[0043] D Tissue embedding: The dehydrated eyeball was immersed in melted paraffin, and the tissue was fully immersed in paraffin (about 2 hours were required), and then embedding was performed E Tissue sectioning: The paraffin block was trimmed as needed, the section thickness was 5 um, and the blade angle was 20-30 degrees. The section was attached to a glass slide, and the section was obtained after being placed in a 60°C incubator for 1-2 hours.

[0044] F De-waxing: The paraffin section was placed in dimethylbenzene I solution and dimethylbenzene II solution for 5 minutes each.

[0045] G Hydration: The paraffin section was placed in 100%, 95%, 80%, and 75% ethanol solutions in sequence, each for 3 min, and distilled water was washed for 5 min.

[0046] H Hematoxylin staining: Hematoxylin staining was performed at 30°C for about 10 min, distilled water was washed for 15 min, and the water was drained.

[0047] I Differentiation: Place the paraffin sections in 1% hydrochloric acid ethanol differentiation solution for 5-30 s until the sections turn red, then rinse with water for about 15 minutes until the sections turn blue; J Dehydration: Place the paraffin sections in 75%, 95%, l00% ethanol solutions for 5 min each.

[0048] K Eosin re-staining: Eosin dye re-staining for 2 min, distilled water rinse for 1 min; 0.5% eosin alcohol solution for 1-2 minutes.

[0049] L Dehydration: Place the paraffin sections in 95% and 100% ethanol solutions for 5 min each→ 95% ethanol dehydration for 2 min→ 100% ethanol dehydration for 1 min M Transparency: Place the paraffin sections in xylene I solution and xylene II solution for 5 min each.

[0050] N Mounting: Drop 1-2 drops of neutral gum on the dry section, cover with a cover glass (do not leave air bubbles). Finally, observe under a Leica microscope and take a photo under the microscope (as shown in the Figure 12 ), the results show that the intravitreal injection of VCZ@ZIF-8 is safe, and the retinal H&E staining shows that the retinal tissue is arranged in order, the structure of each layer is clear, the morphology is normal, and no inflammatory cell infiltration is observed.

[0051] Systemic toxicity experiment Three weeks after the operation, one rabbit was sacrificed from each group, and its important organs (heart, liver, spleen, lung, kidney) were collected and fixed with 4% paraformaldehyde for 24 hours, followed by H&E staining (the same as above).

[0052] The results are shown in the Figure 13 , the results show that the central muscle cells in the control group, VCZ@ZIF-8 group and ZIF-8 group are arranged in order, the cell nuclei are uniform in size, and the cytoplasm and extracellular matrix are uniformly stained and uniform in size; the liver tissue structure is complete, and the liver cells and liver blood sinuses can be seen arranged in a radial pattern around the central vein, and no obvious inflammatory cell infiltration is observed; the liver cells are regular in shape, the nuclei are large and round, and the cytoplasm is abundant. The spleen tissue structure is clear, and the deep red red pulp and the scattered gray-white punctate white pulp can be seen, and the irregular cord-like spleen cords and the blood passage spleen sinuses located between the spleen cords can be seen. The lung bronchial mucosa and alveoli are not deformed, no cells are shed, and no inflammatory cells are infiltrated and aggregated; the alveolar wall is continuous, the interstitium is not edematous, and no inflammatory cells are infiltrated. The kidney tissue structure is clear, the cortical glomeruli and renal tubules are arranged in order, the glomeruli and renal cysts are normal in size, and the proximal tubular epithelial cells are normal in structure. It shows that VCZ@ZIF-8 does not cause any obvious damage to the important organs in vivo.

[0053] VCZ@ZIF-8 penetration test on Candida albicans biofilm Candida albicans biofilm culture in vitro ① Candida albicans single colony from Sabouraud medium, inoculated into conical flask sterile glass tube containing BHI culture solution, 1000 rpm, 30℃ shaking bed culture overnight; ② Turbidity meter adjusted to 0.5 Mclntyre turbidity; ③ The prepared Candida albicans suspension was inoculated into 96-well cell culture plate (100 μL / well); ④ Placed in 28℃ incubator for 24 hours, after Candida albicans adhered to the 96-well plate, discarded the normal saline, added BHI 200 μL, 28℃ cultured for 48 hours.

[0054] ⑤ The 48-hour biofilm in the 96-well plate was exposed to 25 μg / mL of VCZ, VCZ@ZIF-8 (equivalent voriconazole concentration) for 6 hours; According to the dye instruction, each group of samples was double-fluorescent dyed with acridine orange (AO) and ethidium bromide (EB) for 30 min; the VCZ@ZIF-8 penetration on Candida albicans biofilm was observed under confocal microscope, and the results were shown in Figure 14 VCZ could only cause membrane damage to the Candida albicans on the surface, but could not penetrate the biofilm, and VCZ almost could not cause membrane damage to the Candida albicans more than 8 μm. VCZ@ZIF-8 could penetrate the biofilm and cause membrane damage to the whole layer of biofilm, and compared with the condition of pH 7.4, VCZ@ZIF-8 could cause more membrane damage to Candida albicans under the condition of pH 5.0.

[0055] Evaluation of biofilm elimination effect of sustained-release system (fungal plate counting method) ① The 48-hour Candida albicans biofilm in the 96-well plate was exposed to 10, 20 and 50 μg / mL of VCZ, VCZ@ZIF-8 (equivalent voriconazole concentration) for 24 hours respectively; ② The 96-well plate was ultrasonically treated for 8 minutes to further disperse the biofilm; ③ The suspension was continuously diluted in PBS; ④ 100 μL of the above bacterial suspension diluent was spread on BHI agar plate; ⑤ Incubated at 37℃ for 24 hours; ⑥ The number of colony forming units (CFU) was calculated and expressed as CFU / mL.

[0056] ② Fungal plate counting results, the results were shown in Figure 15As shown in Figures A and C of the VCZ@ZIF-8 assay, Figure A shows a plate assay. At pH 7.4 and pH 5.0, the number of surviving C. albicans cells in the VCZ@ZIF-8 group was significantly lower than that in the VCZ group at all three concentrations (10, 20, and 50 µg / ml) (all P < 0.05). Furthermore, the number of surviving C. albicans cells after treatment with VCZ@ZIF-8 at pH 5.0 was also significantly lower than that after treatment at pH 7.4 (Figures 15A and 15C).

[0057] Elimination effect of VCZ@ZIF-8 on Candida albicans biofilm (fungal live / dead staining method): ① 48-hour-old C. albicans biofilms in 96-well plates were exposed to 10, 20, and 50 μg / mL VCZ, VCZ@ZIF-8 (equivalent to voriconazole concentrations) for 24 hours; ② The 96-well plate was sonicated for 8 minutes to further disperse the biofilm; ③ Serially dilute the suspension in PBS; ④ Take 100 μL of the above bacterial suspension dilution and spread it on a BHI agar plate; ⑤ Incubate at 37°C for 24 hours; ⑥ According to the dye instructions, the above-mentioned different concentrations of each group of specimens were dyed with LIVE / DEAD FungaLight TM Fluorescence staining was performed with Yeast Viability Kit for 30 s; ⑦ Image J image analysis software counted the red and green fluorescence values, calculated the dead bacteria / live bacteria ratio and the percentage of dead bacteria. The results are shown in the attached figure. Figure 15 As shown in Figures B and D, Figure B shows a live-or-dead staining image of the fungus, and Figure D shows fluorescence intensity analysis. At all three concentrations (10, 20, and 50 µg / ml) at pH 7.4 and pH 5.0, green fluorescence (live fungal cells) in biofilms treated with VCZ@ZIF-8 was significantly reduced compared to the VCZ group. At pH 5.0, green fluorescence in the VCZ@ZIF-8-treated group was also significantly weaker than that in the same group treated at pH 7.4 (Figures 15B and 15D).

[0058] Figure 15The results showed that VCZ@ZIF-8 was more effective than voriconazole in eliminating fungal biofilms under the same pH conditions. Under acidic conditions, VCZ@ZIF-8 exhibited superior biofilm elimination efficacy compared to normal conditions. VCZ@ZIF-8 demonstrated excellent biofilm penetration and removal capabilities, possibly driven by the following mechanism: First, VCZ@ZIF-8 possesses a positive charge (+20.87 mV), which attracts the negatively charged surface of C. albicans cells. This allows VCZ@ZIF-8 to penetrate the biofilm matrix and bind to the C. albicans surface. Subsequently, the acidic microenvironment within the C. albicans biofilm promotes the release of VCZ from VCZ@ZIF-8, resulting in high concentrations of VCZ within the biofilm.

[0059] Observation on the efficacy of voriconazole sustained-release system in the treatment of Candida albicans endophthalmitis: Thirty-six rabbits were used in the experiment. The right eyes of all rabbits were selected to establish the Candida albicans endophthalmitis model according to the above method. The experimental animals were randomly divided into three groups, with 12 eyes in each group.

[0060] Group A: blank control group, sterile PBS was injected into the vitreous cavity; Group B: intravitreal injection of VCZ@ZIF-8 100 μg / 0.1 mL (calculated based on the equivalent voriconazole concentration), 100 μL; Group C: intravitreal injection of VCZ 100 μg / 0.1 mL, 100 μL; The patients were observed for 3 weeks after surgery. The anterior segment and vitreous cavity were observed with a slit lamp on days 1, 3, 5, 7, 10, 14, and 21, and endophthalmitis was clinically scored. The scoring criteria are shown in the following table.

[0061]

[0062] It was observed that up to 21 days later, VCZ@ZIF-8 had a better therapeutic effect than voriconazole and a more obvious sustained-release effect. Figure 16 As shown, over time, the PBS group experienced progressively worsening conjunctival congestion and corneal edema, progressively increased vitreous opacity, and the appearance of iris neovascularization. In the VCZ and VCZ@ZIF-8 treatment groups, mild conjunctival congestion and mild corneal edema were observed on days 1-3, with no significant vitreous opacity. On day 5, the symptoms and inflammation in the VCZ@ZIF-8 group were slightly more severe than those in the VCZ group. From day 7 to day 21, the symptoms and inflammation in the VCZ@ZIF-8 group were milder than those in the VCZ group. From day 1 to day 21, the symptoms and inflammation in the VCZ and VCZ@ZIF-8 treatment groups were milder than those in the PBS group.

[0063] Histopathological examination 21 days after intravitreal injection of the drug, rabbit eyes were taken for H&E staining. Figure 17As shown: PBS group and voriconazole treatment group retinal structure damage, vitreous cavity can be seen a large number of inflammatory cell infiltration. VCZ@ZIF-8 injection group vitreous cavity no inflammatory cell infiltration, retinal structure can be seen, retinal surface no inflammatory cell infiltration.

[0064] Finally, it should be explained that the above preferred embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope defined by the appended claims.

Claims

1. VCZ@ZIF-8 nanoparticles, characterized in that ZIF-8 was used as a carrier to load voriconazole VCZ.

2. The nanoparticles according to claim 1, characterized in that The nanoparticles have a particle size of 100-200 nm and a surface potential of +15 to +25 mV.

3. Use of the nanoparticles according to claim 1 or 2 in the preparation of antifungal drugs.

4. The use according to claim 3, characterized in that The VCZ@ZIF-8 nanoparticles are used in the preparation of treatment for intraocular inflammation caused by fungal infection.

5. An antifungal drug delivery system, characterized in that: Comprising the VCZ@ZIF-8 nanoparticles as claimed in claim 1 or 2.

6. The method for preparing nanoparticles according to claim 1 or 2, wherein: The following steps are involved: (1) Preparation of ZIF-8 Mix the methanol solution of Zn(NO3)2·6H2O with CTAB, add 2-MeIM methanol solution, stir thoroughly, centrifuge and wash, and then vacuum dry to obtain ZIF-8; (2) VCZ@ZIF-8 drug delivery ZIF-8 and VCZ were dissolved in ethanol with a purity of ≥99.5%, stirred for 24 h, washed by centrifugation, and then vacuum-dried to obtain VCZ@ZIF-8 nanoparticles.

7. The method for preparing nanoparticles according to claim 6, characterized in that: In step (1), the amount of CTAB used is 0.1-0.2 g / mmol Zn(NO3)2·6H2O; the molar ratio of 2-MeIM to Zn(NO3)2·6H2O is 4-8:

1.

8. The method for preparing nanoparticles according to claim 6 or 7, characterized in that: In step (2), equal amounts of ZIF-8 and VCZ are dissolved in ethanol.

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