Nanoparticles for treating pseudomonas aeruginosa keratitis and preparation method thereof
By loading antibiotics onto AuNR@vTSMS nanoparticles and utilizing the photothermal effect, the drug resistance problem of multidrug-resistant Pseudomonas aeruginosa was solved, achieving significant antibacterial effects and corneal inflammation recovery.
Patent Information
- Application Number
- CN202511597994.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-02-27
AI Technical Summary
Multidrug-resistant Pseudomonas aeruginosa exhibits high resistance to commonly used antibiotics, making traditional treatment strategies ineffective. Furthermore, it prevents drugs from entering cells through osmotic regulation and biofilm formation.
Using AuNR@vTSMS nanoparticles as a carrier, antibiotics are loaded and glutathione is depleted through tetrasulfide cleavage. Combined with photothermal effects, this targets the core metabolic weaknesses of Pseudomonas aeruginosa, enhancing the antibacterial effect.
It significantly reduces the minimum inhibitory concentration of antibiotics, restores antibacterial efficacy, reverses bacterial resistance, and restores corneal transparency and health within five days.
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Figure CN121570587A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of pharmaceutical carriers and ocular therapeutic drugs, and particularly relates to a nanoparticle for treating multi-drug resistant Pseudomonas aeruginosa keratitis and a preparation method thereof. BACKGROUND
[0002] Bacterial keratitis is a serious ocular infectious disease, and its treatment usually faces severe challenges due to the emergence of multi-drug resistant Pseudomonas aeruginosa. Multi-drug resistant Pseudomonas aeruginosa shows extremely high resistance to a variety of commonly used antibacterial drugs, such as a 100% resistance rate to penicillin, and a resistance rate of more than 30% to carbapenems, fluoroquinolones and cephalosporins. This extensive drug resistance makes it difficult for traditional antibacterial treatment strategies to work. The drug resistance mechanism of multi-drug resistant Pseudomonas aeruginosa is complex and diverse, and its drug resistance not only depends on its own structural barrier, but also is regulated by metabolic adaptability. They maintain a low oxidative stress environment through permeability regulation, membrane repair and biofilm formation, thereby stabilizing the intracellular environment, making it difficult for antibiotics to contact the protected targets and affecting the treatment effect. SUMMARY
[0003] The present application aims to overcome the defects of the prior art and provide a nanoparticle for treating Pseudomonas aeruginosa keratitis.
[0004] The technical solution of the present application is as follows:
[0005] A nanoparticle for treating Pseudomonas aeruginosa keratitis, taking gold nanorods (AuNR) as the core, externally coated with a nano-shell modified with tetrathiomolybdate (vTSMS), forming an AuNR@vTSMS core-shell nanoparticle structure; the shell surface has a radial spike structure and is loaded with an antibiotic (AB).
[0006] Further, the antibiotic (AB) is at least one of penicillin, ciprofloxacin, ceftazidime and imipenem.
[0007] Further, the mass ratio of the gold nanorods and the nano-shell vTSMS is 1 : (0.5 ~ 5), and the loading amount of the antibiotic is 20 ~ 30%.
[0008] Further, the size of the nanoparticle ranges from 75 to 150 nm.
[0009] Further, after the nanoparticle enters the bacterial cell, it targets the core metabolic weakness of Pseudomonas aeruginosa, induces glutathione depletion through tetrathiol cleavage, and further produces a sulfur overload effect to reverse the drug resistance of the bacteria.
[0010] Further, the nanoparticles can absorb 808 nm near-infrared light to generate heat, and can reach a temperature of 45℃ within 10 minutes of irradiation of 0.5 W / cm 2 of 808 nm near-infrared light.
[0011] Further, the nanoparticles can significantly enhance the antibacterial effect by combining the photothermal effect with the loaded antibiotics to synergistically treat bacterial keratitis.
[0012] The application also provides use of the nanoparticles in the preparation of a drug for treating Pseudomonas aeruginosa keratitis.
[0013] The application also provides a preparation method of the nanoparticles for treating Pseudomonas aeruginosa keratitis, comprising the following steps:
[0014] Step 1. Preparation of gold nanorods;
[0015] First, a chemical mixture of gold nanorods, CTAB and NaOH solution is added to deionized water to prepare a dispersion; wherein the ratio range between gold nanorods, CTAB, NaOH and deionized water is (1.4~1.8) g: (0.15~0.25) g: (0.000005~0.0005) mol: (10~15) ml;
[0016] Subsequently, 1-5 ml of TEOS, 1-5 ml of BTES and 10-30 ml of cyclohexane are added to the previously prepared dispersion, and stirred vigorously at 60-80℃ for 60-72 h, and then centrifuged at 5000 -30000 rpm for 15-30 min; after removing the supernatant, the precipitate is washed thoroughly to remove impurities, and AuNR@vTSMS core-shell nanoparticles are prepared;
[0017] Step 3. Synthesis of AuNR@vTSMS-AB nanoparticles
[0018] First, AuNR@vTSMS and antibiotics (AB) are added to a phosphate buffer solution, and then stirred overnight, and the mass ratio of AuNR@vTSMS to antibiotics (AB) is 1: (5-20); Subsequently, the mixture is centrifuged at a speed of 12000-15000 rpm for 10-15 min; after removing the supernatant, the precipitate is washed to remove impurities, and AuNR@vTSMS-AB is obtained.
[0019] Further, in step one, the seed-mediated growth method is used to synthesize gold nanorods: first, 50-70 volumes of NaBH4 solution is mixed with 1 volume of aqueous solution containing HAuCl4 (0.5-1.5 mM) and CTAB (0.05-0.15 M) to prepare Au solution; then, 1 volume of Au solution is added to 10-20 volumes of Au growth solution to prepare gold nanorods after stirring. The Au growth solution includes 1-2 volumes of HAuCl4, 6-10 volumes of AgNO3, 5-7 volumes of HCl and 0.3-0.7 volumes of ascorbic acid.
[0020] The nanoparticle for treating Pseudomonas aeruginosa keratitis provided by the application is mainly prepared from components such as cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES), chloroauric acid (HAuCl4), sodium borohydride (NaBH4), silver nitrate (AgNO3), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), glutathione, ethanol solution, crystal violet and ascorbic acid. First, gold nanorods (AuNR) are prepared as a core by using a seed-mediated growth method. Then, in a biphasic oil-water phase composed of cyclohexane and water, CTAB and NaOH are introduced into the water phase as a template and alkali respectively, and TEOS and BTES are introduced into the oil phase and then gradually into the water phase, and AuNR@vTSMS core-shell nanoparticles are synthesized by epitaxial growth on the gold nanorods. Finally, an antibiotic is loaded into the mesoporous nanoparticles by using a negative pressure adsorption strategy to prepare the nanoparticle for treating Pseudomonas aeruginosa keratitis.
[0021] Specifically, the AuNR@vTSMS-AB nanoparticle has obvious radially distributed thorn-like nanotubes on the surface, and the gold nanorod is encapsulated at the center position of the AuNR@vTSMS-AB.
[0022] In multidrug-resistant Pseudomonas aeruginosa, glutathione plays a crucial role as a main regulator of redox and sulfur homeostasis, which not only neutralizes antibiotic-induced oxidative damage, but also tightly integrates with central metabolic pathways. For example, glutathione synthesis glutamate-pantothenic acid axis enhances chloramphenicol resistance. In addition, glutathione enhances the structural integrity of the bacterial cell wall by coordinating sulfur flow. Previous studies have shown that disrupting glutathione metabolism triggers a metabolic cascade, while increasing oxidative stress and preventing the production of cysteine, thereby eliminating the drug resistance of Pseudomonas aeruginosa by disrupting its redox and cell wall structure. However, Pseudomonas aeruginosa prevents drug entry by forming a biofilm and reducing membrane permeability, and how to efficiently deliver therapeutic drugs targeting glutathione metabolism to the intracellular environment becomes another key challenge.
[0023] To solve the above problems, the AuNR@vTSMS-AB nanoparticle provided by the application has a rough surface with radial spikes, exhibits superior cell invasiveness and a unique intracellular transport pathway compared with traditional spherical particles, and can effectively improve the drug delivery efficiency in Pseudomonas aeruginosa cells. After the AuNR@vTSMS-AB nanoparticle enters the bacterial cells, the four-sulfide is cleaved to induce the depletion of glutathione, and further produce a sulfur overload effect to reverse the drug resistance of the bacteria, and target the destruction of glutathione metabolism. In addition, the AuNR@vTSMS-AB nanoparticle can also be combined with photothermal therapy, and synergize with the antibiotic load to further enhance the antibacterial effect, and only five days are needed to restore the corneal transparency and health in a mouse keratitis model.
[0024] The beneficial effects of the application are:
[0025] 1. The nanoparticle AuNR@vTSMS-AB provided by the application can significantly reduce the minimum inhibitory concentration of four antibiotics (penicillin, ciprofloxacin, ceftazidime and imipenem), and restore their antibacterial efficacy.
[0026] 2. The nanoparticle AuNR@vTSMS-AB provided by the application innovatively targets the core metabolic weakness of Pseudomonas aeruginosa through glutathione depletion and sulfur overload effect, and provides a safer and more sustainable means to reverse antibiotic resistance.
[0027] 3. The nanoparticle AuNR@vTSMS-AB provided by the application can also be combined with photothermal therapy, and synergize with the loaded antibiotic to further enhance the antibacterial effect. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 The preparation process of the AuNR@vTSMS-AB nanoparticle and the mechanism diagram of the effect of reversing Pseudomonas aeruginosa drug resistance are shown.
[0029] Figure 2 The transmission electron microscope images of the gold nanorod, AuNR@vTSMS core-shell nanoparticle and AuNR@vTSMS-AB nanoparticle of the application.
[0030] Figure 3 The scanning transmission electron microscope image and Mapping image of the AuNR@vTSMS-AB nanoparticle of the application. Scale: 20 nm.
[0031] Figure 4 The evaluation of the glutathione depletion capacity of the AuNR@vTSMS-AB nanoparticle in Example 1 of the application is shown.
[0032] Figure 5 shows the photothermal effect of AuNR@vTSMS-AB nanoparticles evaluated in Example 2 of the present application.
[0033] Figure 6 shows the high-resolution transmission electron microscopy images of the 4-stage section of the AuNR@vTSMS-AB nanoparticles treating P. aeruginosa and the corresponding Mapping images in Example 3 of the present application. Scale bar: 20 nm.
[0034] Figure 7 shows the in vivo therapeutic effect of AuNR@vTSMS-AB nanoparticles on P. aeruginosa keratitis evaluated in Example 4 of the present application. Scale bar: 1 mm.
[0035] Figure 8 shows the frozen section (IF staining) images of the corneas of the mice in the blank group, AuNR@vTSMS-AB nanoparticles and AuNR@vTSMS-AB nanoparticles combined with photothermal effect treatment in Example 4 of the present application. Scale bar: 100 μm. DETAILED DESCRIPTION
[0036] The technical solutions of the present application are further described and explained in the following detailed description with reference to the accompanying drawings.
[0037] In the following examples, the C57BL / 6 mice used were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and the feeding and animal experiments of the mice were carried out in accordance with the requirements of the Ethics Committee of the Experimental Animal Center of Shandong First Medical University. The following steps were used to establish the C57BL / 6 mouse bacterial keratitis model: first, the C57BL / 6 mice were anesthetized by intraperitoneal injection of ready-to-use tribromoethanol solution, and then placed under a dissecting microscope (magnification = x30). Then, a 25-gauge needle was used to make three ~1 mm long incisions in parallel on the central cornea of the left eye, destroying the corneal epithelial barrier. Subsequently, 5 μL of drug-resistant PA suspension (1 x 10 5 CFU / mL) was dropped onto the ocular surface. After 24 hours of infection, keratitis was successfully induced.
[0038] As shown in Figure 1 and 2 In the following examples, the preparation method of the main material is as follows:
[0039] Preparation Example 1
[0040] (1) Gold nanorods: First, an Au solution was prepared by mixing 120 mL of ice-cooled NaBH4 solution with 2 mL of an aqueous solution containing HAuCl4 (1 M) and CTAB (0.1 M, hexadecyltrimethylammonium bromide). Then, 2.0 mL of the Au solution was added to the Au growth solution (prepared by mixing 2.4 mL of 0.1 M HAuCl4, 16 mL of 0.1 M AgNO3, 12 mL of 0.15 M HCl and 1.2 mL of 0.1 M ascorbic acid), and stirred for 12 hours to obtain gold nanorods. The synthesized AuNR cores were thoroughly washed with deionized water and ethanol to remove impurities.
[0041] (2) AuNR@vTSMS: First, a chemical mixture of 1.5 g gold nanorods, 0.2 g CTAB, and 100 μL 0.1 M NaOH solution prepared in (1) was added to 12 mL of deionized water to prepare a dispersion. Subsequently, 3.2 mL TEOS (tetraethyl orthosilicate), 3.2 mL BTES (bis[3-(triethoxysilyl)propyl]tetrasulfide), and 40 mL cyclohexane were added to the previously prepared dispersion, and the mixture was stirred vigorously at 65 °C for 72 h, followed by centrifugation at approximately 10,000 rpm for 15 min. After removing the supernatant, the precipitate was washed three times with deionized water and ethanol to remove impurities, thus obtaining AuNR@vTSMS core-shell nanoparticles.
[0042] (3) vTSMS: Remove the gold nanorods in the above preparation process and repeat the remaining steps in (2) to prepare vTSMS nanoparticles.
[0043] (4) AuNR@vTSMS-AB / CIP: 1 mg of AuNR@vTSMS prepared in (2) above and 10 mg of any antibiotic (AB) were added separately to phosphate buffer solution (pH 7.4) and stirred overnight. Subsequently, the mixture was centrifuged at 15,000 rpm for 10 min. After removing the supernatant, the precipitate was washed to remove impurities to obtain AuNR@vTSMS-AB.
[0044] In this preparation example, when the antibiotic (AB) is ciprofloxacin (CIP), AuNR@vTSMS-CIP is prepared.
[0045] Specifically, such as Figure 3 As shown, the prepared AuNR@vTSMS-AB / CIP has distinct radially distributed spiky nanotubes, with the gold nanorods serving as the core encapsulated at the center of the AuNR@vTSMS-AB / CIP.
[0046] Preparation Example 2
[0047] (1) Gold nanorods: First, 120 mL of ice-cooled NaBH4 solution was mixed with 2 mL of an aqueous solution containing HAuCl4(1.1 M) and CTAB (0.1 M) to prepare an Au solution. Subsequently, 2.0 mL of the Au solution was added to an Au growth solution (prepared by mixing 2.4 mL of 0.1 M HAuCl4, 16 mL of 0.15 M AgNO3, 12 mL of 0.08 M HCl, and 1.2 mL of 0.1 M ascorbic acid) to prepare gold nanorods after stirring for 12 hours. The synthesized AuNR core was washed thoroughly with deionized water and ethanol to remove impurities.
[0048] (2) AuNR@vTSMS: First, a chemical mixture of 1.5 g of gold nanorods prepared in (1) above, 0.2 g of CTAB, and 100 μL of 0.1 M NaOH solution was added to 12 mL of deionized water to prepare a dispersion solution. Subsequently, 3.2 mL of TEOS (tetraethyl orthosilicate), 3.2 mL of BTES (bis[3-(triethoxysilyl)propyl]tetrasulfide), and 40 mL of cyclohexane were added to the previously prepared dispersion solution, and the mixture was stirred vigorously at 80°C for 60 hours, and then centrifuged at about 8000 rpm for 30 min. After removing the supernatant, the precipitate was washed three times with deionized water and ethanol to remove impurities, thereby preparing AuNR@vTSMS core-shell nanoparticles.
[0049] (3) vTSMS: The gold nanorods were removed during the preparation process described above, and the remaining steps in (2) above were repeated to prepare vTSMS nanoparticles.
[0050] (4) AuNR@vTSMS-AB: 1 mg of AuNR@vTSMS prepared in (2) above and 10 mg of an arbitrary antibiotic (AB) were each added to a phosphate buffer solution (pH 7.4), and then stirred overnight. Subsequently, the mixture was centrifuged at 13000 rpm for 12 min. After removing the supernatant, the precipitate was washed to remove impurities, thereby obtaining AuNR@vTSMS-AB.
[0051] In the present preparation example, when the antibiotic (AB) is penicillin (PCN), AuNR@vTSMS-PCN is prepared.
[0052] Preparation Example 3
[0053] (1) Au nanorods: First, 120 mL of ice-cooled NaBH4 solution was mixed with 2 mL of an aqueous solution containing HAuCl4(0.9 M) and CTAB (0.15 M) to prepare an Au solution. Subsequently, 2.0 mL of the Au solution was added to an Au growth solution (prepared by mixing 2.4 mL of 0.1 M HAuCl4, 16 mL of 0.12 M AgNO3, 12 mL of 0.2 M HCl, and 1.2 mL of 0.1 M ascorbic acid) to prepare Au nanorods after stirring for 12 hours. The synthesized Au NR core was washed thoroughly with deionized water and ethanol to remove impurities.
[0054] (2) AuNR@vTSMS: First, a chemical mixture of 1.5 g of the Au nanorods prepared in (1) above, 0.2 g of CTAB, and 100 μL of 0.1 M NaOH solution was added to 12 mL of deionized water to prepare a dispersion solution. Subsequently, 3.2 mL of TEOS (tetraethyl orthosilicate), 3.2 mL of BTES (bis[3-(triethoxysilyl)propyl]tetrasulfide), and 40 mL of cyclohexane were added to the previously prepared dispersion solution, which was then stirred vigorously at 60°C for 65 hours, followed by centrifugation at about 12,000 rpm for 15 min. After removing the supernatant, the precipitate was washed three times with deionized water and ethanol to remove impurities, thereby preparing AuNR@vTSMS core-shell nanoparticles.
[0055] (3) vTSMS: The Au nanorods were removed during the preparation process described above, and the remaining steps in (2) above were repeated to prepare vTSMS nanoparticles.
[0056] (4) AuNR@vTSMS-AB: 1 mg of AuNR@vTSMS prepared in (2) above and 10 mg of an arbitrary antibiotic (AB) were each added to a phosphate buffer solution (pH 7.4), which was then stirred overnight. Subsequently, the mixture was centrifuged at 12,000 rpm for 15 min. After removing the supernatant, the precipitate was washed to remove impurities, thereby obtaining AuNR@vTSMS-AB.
[0057] In the present preparation example, when the antibiotic (AB) is ceftazidime (CAZ), AuNR@vTSMS-CAZ is prepared.
[0058] In each of the following examples, the sample AuNR@vTSMS-AB / CIP was prepared using the preparation example 1.
[0059] Example 1
[0060] The nanoparticles for treating Pseudomonas aeruginosa keratitis proposed in the application can induce glutathione depletion through tetrasulfide cleavage, thereby reversing the drug resistance of bacteria. The AuNR@vTSMS-AB nanoparticles were used to evaluate the ability of glutathione depletion by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and experimental groups, control groups and blank groups were set up for comparison.
[0061] Experimental group: First, prepare a 10 mM DTNB probe solution and store it in the dark for further testing. Then, prepare AuNR@vTSMS-AB solutions with concentrations of 2.5, 5.0, 7.5 and 10 μg / mL, and mix them with 10 mM glutathione solutions, respectively. These mixed solutions are then incubated at room temperature for 0.5, 1, 2, 4 and 8 hours, respectively. Mix 500 μL of the mixed solutions with different incubation times with 500 μL of the DTNB probe solution, and incubate them in the dark for 20 min. Finally, measure the absorbance at 412 nm.
[0062] Control group: Prepare AuNR@vTSMS-AB solutions with concentrations of 2.5, 5.0, 7.5 and 10 μg / mL, and mix them with PBS solutions (pH 7.4), respectively. After incubating these mixed solutions at room temperature for 0.5, 1, 2, 4 and 8 hours, respectively, measure the absorbance at 412 nm.
[0063] Blank group: Prepare a 10 mM DTNB probe solution, and mix it with a PBS solution (pH 5.5) and measure the absorbance at 412 nm.
[0064] As Figure 4 The calculated glutathione depletion rate UV-visible absorption spectrum is shown, which shows a decrease in absorbance at ~415 nm of AuNR@vTSMS over time, indicating that tetrasulfide cleavage induces GSH depletion. The glutathione depletion rate is calculated by the following formula:
[0065] Glutathione depletion rate (%) = 1 -
[0066] Where A1, A0, A are the absorbances of the experimental group, the control group and the blank group, respectively. The calculation results show that the glutathione degradation efficiency induced by 10 µg / mL AuNR@vTSMS-AB can reach 89% within 8 hours.
[0067] Example 2
[0068] The nanoparticles for treating pseudomonas aeruginosa keratitis provided by the application also have excellent photothermal effect. In order to investigate the photothermal performance of the AuNR@vTSMS-AB nanoparticles, 808 nm infrared light (NIR, 0.5 W / cm 2 ) was used to irradiate vTSMS, gold nanorods and AuNR@vTSMS-AB for 10 minutes respectively, and the temperature of the sample was recorded every 30 seconds by an infrared thermal imager, and the infrared thermal image was collected every 1 minute. The results are shown in Figure 5 As shown in the drawings, under NIR irradiation, both the gold nanorods and the AuNR@vTSMS-AB exhibit excellent photothermal effect, and the photothermal effect of the gold nanorods under 808 nm laser irradiation is as high as 60℃. Although the vTSMS shell partially hinders the photothermal performance of the gold nanorods, the AuNR@vTSMS-AB still reaches a temperature of 45℃ within 10 minutes of NIR irradiation.
[0069] Example 3
[0070] The nanoparticles for treating pseudomonas aeruginosa keratitis provided by the application can enhance the ability to kill pseudomonas aeruginosa by combining photothermal effect and cooperating with antibiotics. In order to evaluate the in vitro antibacterial effect of the AuNR@vTSMS-AB nanoparticles, first, PBS, antibiotic CIP, AuNR@vTSMS, AuNR@vTSMS-AB, AuNR@vTSMS-AB +NIR (808 nm, 0.5 W / cm 2 ) were selected as samples and added to the pseudomonas aeruginosa solution. As shown in Figure 6 , high-resolution transmission electron microscopy and Mapping were used to observe the sections of the four stages of the treatment of pseudomonas aeruginosa by AuNR@vTSMS-AB nanoparticles. Initially, when the pseudomonas aeruginosa was co-cultured with AuNR@vTSMS-AB, the aggregation of AuNR@vTSMS-AB led to the enrichment of the bacterial cell wall and internal sulfur (S) and oxygen (O) elements, and the cell morphology remained intact (stage I). Subsequently, AuNR@vTSMS-AB entered the pseudomonas aeruginosa cells, consumed intracellular glutathione, and released gold nanorods and CIP. This process was accompanied by the breaking of the four sulfur bonds, resulting in a large number of dispersed S and O elements, which indicated a significant increase in thiol compounds and oxidized glutathione (stage II). Next, under the combined action of CIP and photothermal effect, the pseudomonas aeruginosa cells were significantly deformed, showing cytoplasm leakage and a decrease in O and S elements, confirming that the bacterial cells were highly sensitive to CIP (stage III). Finally, the destruction of bacterial metabolism further enhanced the sensitivity to CIP, leading to complete rupture of the bacterial cells, massive leakage of cytoplasm, and death of the bacteria (stage IV).
[0071] Example 4
[0072] The AuNR@vTSMS-CIP nanoparticles for treating Pseudomonas aeruginosa keratitis proposed by the application can combine with the photothermal effect to restore the corneal transparency and health of C57BL / 6 mice (suffering from Pseudomonas aeruginosa keratitis) within five days. In order to explore the therapeutic effect of AuNR@vTSMS-CIP nanoparticles on bacterial keratitis C57BL / 6 mice:
[0073] Evaluation of the therapeutic effect of AuNR@vTSMS-CIP nanoparticles on bacterial keratitis C57BL / 6 mice
[0074] The mice successfully induced with keratitis were randomly divided into five groups, namely blank, CIP, AuNR@vTSMS, AuNR@vTSMS-CIP and AuNR@vTSMS-CIP + NIR. The blank group was not given treatment, and the last three groups were respectively given CIP, AuNR@vTSMS, AuNR@vTSMS-CIP solution treatment, 3 times a day, 10 μL each time. The last group AuNR@vTSMS-CIP + NIR group was given NIR (808 nm, 0.5 W / cm 2 ) irradiation and AuNR@vTSMS-CIP solution treatment, 3 times a day, 10 μL each time. All treatments lasted for 5 days, and the treatment effect of the infected eyes of the mice in each group was ophthalmically evaluated by a slit lamp microscope on the first day, the third day and the fifth day, respectively. As shown in Figure 7 , the treatment effects of the CIP group and the AuNR@vTSMS group were equivalent to those of the blank control group, and there was no statistical difference in the corneal clinical score. The AuNR@vTSMS-CIP eye drops had significant anti-drug resistant bacteria and anti-inflammatory effects, and the corneal transparency was obviously restored compared with the blank control group.
[0075] In order to further determine the anti-inflammatory effect of AuNR@vTSMS-CIP on bacterial keratitis, the mice were euthanized after 5 days of treatment, and the euthanized mice were taken out of the left cervical vertebra, and the eyeballs were taken out and frozen sectioned (8 μm) to observe the expression of inflammatory cytokines TNF-α and IL-6. The sections were blocked in PBS containing 5% bovine serum albumin (BSA) for 1 hour, and then incubated with TNF-α (1:400, CST) and IL-6 (1:200, Proteintech) antibodies at 4°C overnight. The next day, the sections were incubated with the corresponding two antibodies at room temperature for 1 hour, and then the cell nuclei were stained with 4,6-diamino-2-phenylindole (DAPI), and the sections were mounted with an anti-fading fluorescent mounting medium. Finally, the sections were observed under a fluorescence microscope. As shown in Figure 8As shown, the fluorescence expression of TNF-α and IL-6 in the cornea of mice in the AuNR@vTSMS-CIP group and the AuNR@vTSMS-CIP + NIR group was significantly reduced, in contrast, the CIP group and the AuNR@vTSMS group did not produce strong anti-inflammatory effect.
[0076] The above description is merely preferred embodiments of the present application, and thus is not intended to limit the scope of the present application. Any equivalent changes and modifications made in accordance with the spirit of the present application should still fall within the scope of the present application.
Claims
1. A nanoparticle for treating Pseudomonas aeruginosa keratitis, characterized in that: The structure consists of AuNR@vTSMS core-shell nanoparticles with gold nanorods AuNR as the core and tetrathiosulfate-modified nanoshells vTSMS as the outer shell. The shell surface has radial spikes and is loaded with antibiotics.
2. The nanoparticles for treating Pseudomonas aeruginosa keratitis as described in claim 1, characterized in that: The antibiotic is at least one of penicillin, ciprofloxacin, ceftazidime, and imipenem.
3. The nanoparticles for treating Pseudomonas aeruginosa keratitis as described in claim 1, characterized in that: The mass ratio of gold nanorods to nanoshells vTSMS is 1:(0.5~5), and the loading of the antibiotic is 20~30% by mass.
4. The nanoparticles for treating Pseudomonas aeruginosa keratitis as described in claim 1, characterized in that: The size range of the nanoparticles is 75-150 nm.
5. The nanoparticles for treating Pseudomonas aeruginosa keratitis as described in claim 1, characterized in that: Once inside the bacterial cell, the nanoparticles target the core metabolic weakness of Pseudomonas aeruginosa, inducing glutathione depletion through tetrasulfide cleavage and further generating a sulfur overload effect to reverse bacterial drug resistance.
6. The nanoparticles for treating Pseudomonas aeruginosa keratitis as described in claim 1, characterized in that: The nanoparticles can absorb near-infrared light at 808 nm to generate heat, at a rate of 0.5 W / cm². 2 Irradiation with 808 nm near-infrared light for 10 minutes can reach a temperature of 45°C.
7. A nanoparticle for treating Pseudomonas aeruginosa keratitis as described in any one of claims 1 to 6, characterized in that: These nanoparticles, through the synergistic treatment of bacterial keratitis by loading antibiotics and combining them with photothermal effects, can significantly enhance antibacterial efficacy.
8. The use of the nanoparticles as described in any one of claims 1 to 6 in the preparation of a medicament for treating Pseudomonas aeruginosa keratitis.
9. A method for preparing nanoparticles for treating Pseudomonas aeruginosa keratitis as described in any one of claims 1 to 6, characterized in that, Includes the following steps: Step 1. Prepare gold nanorods AuNR; Step 2. Synthesis of AuNR@vTSMS core-shell nanoparticles First, a chemical mixture of gold nanorods, CTAB, and NaOH solution was added to deionized water to prepare a dispersion; wherein the ratio of gold nanorods, CTAB, NaOH, and deionized water ranged from (1.4~1.8) g: (0.15~0.25) g: (0.000005~0.0005) mol: (10~15) ml; Subsequently, 1-5 ml TEOS, 1-5 ml BTES and 10-30 ml cyclohexane were added to the previously prepared dispersion, and the mixture was stirred vigorously at 60-80 °C for 60-72 h. Then, it was centrifuged at 5000-30000 rpm for 15-30 min. After removing the supernatant, the precipitate was thoroughly washed to remove impurities, and AuNR@vTSMS core-shell nanoparticles were obtained. Step 3. Synthesis of AuNR@vTSMS-AB nanoparticles First, AuNR@vTSMS and antibiotic AB were added to a phosphate buffer solution and stirred overnight. The mass ratio of AuNR@vTSMS to antibiotic was 1:(5-20). Then, the mixture was centrifuged at 12000-15000 rpm for 10-15 min. After removing the supernatant, the precipitate was washed to remove impurities, yielding AuNR@vTSMS-AB.
10. The preparation method according to claim 9, characterized in that: In step one, gold nanorods are synthesized using a seed-mediated growth method: First, 50-70 volumes of NaBH4 solution are mixed with 1 volume of an aqueous solution containing 0.5-1.5 mM HAuCl4 and 0.05-0.15 MCTAB to prepare an Au solution; then, 1 volume of the Au solution is added to 10-20 volumes of Au growth solution, which is prepared by mixing 1-2 volumes of HAuCl4, 6-10 volumes of AgNO3, 5-7 volumes of HCl and 0.3-0.7 volumes of ascorbic acid. After stirring thoroughly for 6-24 h, gold nanorods are obtained.