Photo-thermal and photodynamic integrated antibacterial nano-drug, preparation method thereof and application of nano-drug in preparation of biofilm-related keratitis drugs

Abstract

The invention provides a photo-thermal and photo-dynamic integrated antibacterial nano-drug as well as a preparation method and application thereof in preparation of a biological membrane related keratitis drug, and belongs to the technical field of nano-drugs. The complete biological membrane is sheared and cracked through DNase I, and Mn < 2 + > can further enhance the biological membrane cracking effect of DNase I at the same time. Aiming at free bacteria released after a biological membrane is cracked, the nano-drug disclosed by the invention shows good capabilities of heating and generating ROS (reactive oxygen species) under the irradiation of near-infrared light, a short-term sterilization effect is achieved by utilizing photo-thermal and photodynamic force, and in addition, the loaded ciprofloxacin can be slowly released, so that a long-acting and efficient sterilization function is achieved. In the later healing stage of bacterial keratitis, Mn < 2 + > decomposes H2O2 in a microenvironment to generate O2, so that an anoxic microenvironment in a biological membrane can be relieved to promote healing of damaged corneas, and phenotypic transformation of macrophages can be regulated to a certain extent to promote disease outcome.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicines, and in particular to a photothermal-photodynamic integrated antibacterial nanomedicine, a preparation method thereof, and an application thereof in the preparation of a biofilm-related keratitis drug. Background Art

[0002] Bacterial keratitis is one of the most important blinding eye diseases in the world. In recent years, the proportion of Gram-positive cocci has been on the rise, while the proportion of Gram-negative bacilli has been gradually decreasing. Currently, coagulase-negative Staphylococci and Pseudomonas aeruginosa are common pathogens of bacterial keratitis in my country, and the sensitivity of various genera to commonly used antibiotics has been declining. The treatment of bacterial keratitis should be controlled by drugs as early as possible to avoid corneal transplantation surgery, but the current drug treatment faces two major challenges:

[0003] (1) The types of antibacterial drugs are limited and the drug sensitivity is weak;

[0004] (2) Antibacterial drugs cannot effectively treat the biofilm protective barrier formed by long-term bacterial infection; therefore, the development of new antibacterial drugs for biofilm-related keratitis caused by bacterial infection is of great significance for the treatment of bacterial membrane keratitis.

[0005] Studies have shown that biofilms formed by the long-term infection of bacteria can be wrapped in an extracellular polymer (EPS) matrix, providing an isolated living environment for free bacteria, acting as a shield to protect parasitic bacteria from penetration and diffusion of antibiotic attacks, making treatment fundamentally face huge challenges. In addition, the formation of biofilms caused by more than 80% of bacterial infections requires a very high dose of antibiotics, up to 1,000 times the usual dose to be cured. Therefore, new antibacterial strategies are urgently needed to not only minimize the possibility of drug resistance, but also to effectively combat bacterial biofilm-related infections.

[0006] Photothermal therapy (PTT) is a promising treatment method with the advantages of non-invasiveness, high controllability, and broad-spectrum bactericidal properties. PTT converts absorbed light energy into heat energy, and the superheated environment (PTAs) generated by the photothermal agent can separate stubborn biofilms, thereby promoting the penetration of antimicrobial drugs. In addition, the local hyperthermic effect causes PTA, which can subsequently destroy the integrity of bacteria and interfere with the physiological functions of bacteria, leading to bacterial death. It is worth noting that PTT does not rely on the cell internalization of antibiotics to kill bacteria, so the possibility of drug resistance is low. All of the above advantages make PTT stand out among the methods for ablating bacterial biofilm-related infections. However, considering that PTT therapy needs to reach a higher temperature to have anti-biofilm and bactericidal effects, excessively high temperatures may cause a certain degree of corneal damage. How to balance efficacy and biosafety is a difficult problem faced by photothermal therapy. Therefore, the single use of photodynamic therapy to treat biofilm-related keratitis caused by bacterial infection still faces many challenges.

[0007] Photodynamic therapy (PDT) has the potential for clinical application with high specificity and low side effects, which can utilize reactive oxygen species (ROS) or singlet oxygen ( 1 O2) thus has the potential to kill bacteria in a short-term manner, which is produced by the combined effects of appropriate light irradiation, photosensitizer molecules and environmental oxygen. However, during PDT treatment, due to the hypoxic conditions in the biofilm microenvironment, insufficient oxygen supply may lead to 1 The O2 production is low and the PDT efficacy is reduced. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide a photothermal-photodynamic integrated antibacterial nanomedicine and its preparation method and application in the preparation of biofilm-related keratitis drugs. The photothermal-photodynamic integrated antibacterial nanomedicine provided by the present invention has a good therapeutic effect on biofilm-related keratitis, while enhancing the bactericidal efficacy and ensuring the biological safety during the drug application process.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a photothermal-photodynamic integrated antibacterial nanomedicine, comprising hollow manganese dioxide nanoparticles, a photosensitizer and an antibacterial drug loaded inside the hollow manganese dioxide nanoparticles, and DNase I modified on the surface of the hollow manganese dioxide nanoparticles;

[0011] The antibacterial drugs include ciprofloxacin and / or gatifloxacin.

[0012] Preferably, the photosensitizer comprises indocyanine green.

[0013] Preferably, the mass ratio of the hollow manganese dioxide nanoparticles, the photosensitizer and the antibacterial drug is 2:(1-2):1.

[0014] Preferably, the particle size of the photothermal and photodynamic integrated antibacterial nanomedicine is 100 to 150 nm.

[0015] The present invention provides a method for preparing the above-mentioned photothermal and photodynamic integrated antibacterial nanomedicine, comprising the following steps:

[0016] The silica nanospheres, potassium permanganate and water are mixed and subjected to a hydrothermal reaction to obtain MnO2@SiO2 nanospheres;

[0017] The aqueous dispersion of MnO2@SiO2 nanospheres is mixed with a sodium carbonate solution and subjected to an etching reaction to obtain hollow manganese dioxide nanoparticles;

[0018] Mixing the hollow manganese dioxide nanoparticles, polyacrylamine hydrochloride and water to obtain PAH-encapsulated hollow manganese dioxide;

[0019] Mixing the PAH-encapsulated hollow manganese dioxide, polyacrylic acid and water to obtain PAH- and PAA-encapsulated hollow manganese dioxide;

[0020] The hollow manganese dioxide wrapped by PAH and PAA, a carboxyl activator and a buffer solution are mixed to perform a carboxyl activation reaction to obtain a carboxyl activation reaction solution;

[0021] Adding DNaseI to the carboxyl activation reaction solution to perform a coupling reaction to obtain DNaseI-modified hollow manganese dioxide;

[0022] The DNaseI-modified hollow manganese dioxide is mixed with a photosensitizer, an antibacterial drug and water, and pumped and stirred under vacuum conditions to obtain a photothermal and photodynamic integrated antibacterial nanomedicine.

[0023] Preferably, the particle size of the silica nanospheres is 100 to 150 nm;

[0024] The mass ratio of the silicon dioxide nanospheres to potassium permanganate is 1:1-2;

[0025] The temperature of the hydrothermal reaction is 100-150° C. and the time is 20-24 hours.

[0026] Preferably, the concentration of the sodium carbonate solution is 0.5 g / mL;

[0027] The temperature of the etching reaction is 50-60° C. and the time is 10-12 hours.

[0028] Preferably, the mass ratio of the hollow manganese dioxide nanoparticles to polyacrylamine hydrochloride is 2:1;

[0029] The mass ratio of the hollow manganese dioxide nanoparticles to polyacrylic acid is 5 to 6:1.

[0030] Preferably, the mass ratio of the hollow manganese dioxide encapsulated by PAH and PAA to DNaseI is 4 to 5:1;

[0031] The coupling reaction time is 10 to 12 hours;

[0032] The pumping and stirring time is 20 to 24 hours.

[0033] The present invention provides the use of the above-mentioned photothermal and photodynamic integrated antibacterial nanomedicine in the preparation of biofilm-related keratitis drugs.

[0034] The present invention provides a photothermal-photodynamic integrated antibacterial nanomedicine (denoted as DNaseI-HMnO2@ICG@CIPNPs), comprising hollow manganese dioxide nanoparticles, a photosensitizer and an antibacterial drug loaded inside the hollow manganese dioxide nanoparticles, and DNase I modified on the surface of the hollow manganese dioxide nanoparticles; the antibacterial drug includes ciprofloxacin. The nanomedicine provided by the present invention uses hollow manganese dioxide nanoparticles (HMnO2) as a carrier, modifies DNase I on the surface of the hollow manganese dioxide nanoparticles to obtain HMnO2-DNase I, and loads photosensitizer and ciprofloxacin at the same time. DNase I is an enzyme that destroys phosphodiester bonds by hydrolysis and non-specifically cuts DNA. The present invention shears and cleaves the intact biofilm by DNase I, and at the same time, Mn 2+ The effect of DNaseI on cleaving biofilm can be further enhanced to achieve the effect of 1+1>2. In addition, for the free bacteria released after cleaving the biofilm, the present invention uses photothermal therapy (PTT) and photodynamic therapy (PDT) combined with antibiotics to exert a bactericidal effect. Specifically, under near-infrared light, the nanomedicine of the present invention exhibits good ability to increase temperature and generate ROS, and uses photothermal and photodynamic therapy to achieve a short-term bactericidal effect. In addition, the loaded ciprofloxacin can be sustained-released to achieve a long-term and efficient bactericidal function. In the late healing stage of bacterial keratitis, Mn 2+ Decomposing H2O2 in the microenvironment to produce O2 can not only alleviate the hypoxic microenvironment in the biofilm and promote the healing of damaged cornea, but also 2+ It can also regulate the phenotypic transformation of macrophages to a certain extent, from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and promote disease recovery.

[0035] The nanomedicine provided by the present invention can realize the integrated sterilization therapy of photothermal and photodynamic therapy, which can not only alleviate the problem that traditional antibiotics are prone to drug resistance, but also solve the problem that clinical antibacterial drugs cannot effectively treat the biofilm protective barrier formed by long-term bacterial infections. At the same time, nanomedicine can achieve a certain degree of promoting healing and anti-inflammatory effects by regulating the biofilm infection microenvironment.

[0036] The present invention provides a method for preparing the above-mentioned photothermal and photodynamic integrated antibacterial nanomedicine, which has simple operation, low cost and is easy to realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Transmission electron microscopy images of MnO2@SiO2 nanospheres and hollow manganese dioxide nanoparticles;

[0038] Figure 2 The microstructure diagram of DnaseI-HMnO2@ICG@CIP;

[0039] Figure 3 This is the XPS spectrum of DnaseI-HMnO2@ICG@CIP;

[0040] Figure 4 The photothermal effect of samples with different concentrations under near-infrared light irradiation;

[0041] Figure 5 Results of reactive oxygen production of DnaseI-HMnO2@ICG@CIP nanoparticles under near-infrared radiation;

[0042] Figure 6 The anti-biofilm performance of nanomedicine was verified;

[0043] Figure 7 To verify the bactericidal properties of nanomedicine;

[0044] Figure 8 Results are from animal testing. DETAILED DESCRIPTION

[0045] The present invention provides a photothermal-photodynamic integrated antibacterial nanomedicine, characterized in that it comprises hollow manganese dioxide nanoparticles, a photosensitizer and an antibacterial drug loaded inside the hollow manganese dioxide nanoparticles, and DNase I modified on the surface of the hollow manganese dioxide nanoparticles.

[0046] In the present invention, the antibacterial drug includes ciprofloxacin and / or gatifloxacin, preferably ciprofloxacin. In the present invention, the photosensitizer preferably includes indocyanine green. In the present invention, the mass ratio of the hollow manganese dioxide nanoparticles, the photosensitizer and the antibacterial drug is preferably 2:(1-2):1, more preferably 2:1:1.

[0047] In the present invention, the DNase I is preferably chemically bonded to the surface of the hollow manganese dioxide nanoparticles; specifically, the surface of the hollow manganese dioxide nanoparticles is preferably coated with polyacrylamine hydrochloride and polyacrylic acid, and the amino group of the DNase I is connected to the carboxyl group of the polyacrylic acid through -NH-CO-.

[0048] In the present invention, the particle size of the photothermal and photodynamic integrated antibacterial nanomedicine is preferably 100 to 150 nm.

[0049] The present invention provides a method for preparing the above-mentioned photothermal and photodynamic integrated antibacterial nanomedicine, comprising the following steps:

[0050] The silica nanospheres, potassium permanganate and water are mixed and subjected to a hydrothermal reaction to obtain MnO2@SiO2 nanospheres;

[0051] The aqueous dispersion of MnO2@SiO2 nanospheres is mixed with a sodium carbonate solution and subjected to an etching reaction to obtain hollow manganese dioxide nanoparticles;

[0052] Mixing the hollow manganese dioxide nanoparticles, polyacrylamine hydrochloride and water to obtain PAH-encapsulated hollow manganese dioxide;

[0053] Mixing the PAH-encapsulated hollow manganese dioxide, polyacrylic acid and water to obtain PAH- and PAA-encapsulated hollow manganese dioxide;

[0054] The hollow manganese dioxide wrapped by PAH and PAA, a carboxyl activator and a buffer solution are mixed to perform a carboxyl activation reaction to obtain a carboxyl activation reaction solution;

[0055] Adding DNaseI to the carboxyl activation reaction solution to perform a coupling reaction to obtain DNaseI-modified hollow manganese dioxide;

[0056] The DNaseI-modified hollow manganese dioxide is mixed with a photosensitizer, an antibacterial drug and water, and pumped and stirred under vacuum conditions to obtain a photothermal and photodynamic integrated antibacterial nanomedicine.

[0057] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0058] The present invention mixes silicon dioxide nanospheres, potassium permanganate and water, and performs a hydrothermal reaction to obtain MnO2@SiO2 nanospheres (manganese dioxide encapsulated silicon dioxide nanospheres). In the present invention, the particle size of the MnO2@SiO2 nanospheres is preferably 100-150 nm.

[0059] In the present invention, the method for preparing the silicon dioxide nanospheres preferably comprises the following steps:

[0060] Tetraethyl silicate, ammonia water and ethanol are mixed to carry out a hydrolysis reaction to obtain a hydrolysis reaction liquid;

[0061] The hydrolysis reaction liquid is washed with alcohol and dried to obtain silicon dioxide nanospheres.

[0062] In the present invention, the mass concentration of the ammonia water is preferably 25-28%; the volume ratio of the tetraethyl silicate, ammonia water and ethanol is preferably 15:7.5:350. In the present invention, the hydrolysis reaction is preferably carried out under stirring, the temperature of the hydrolysis reaction is preferably room temperature, and the time is preferably 5-6 hours.

[0063] In the present invention, the alcohol reagent used in the alcohol washing is preferably methanol, and the alcohol washing is preferably centrifugal washing. In the present invention, the drying is preferably freeze drying.

[0064] In the present invention, the particle size of the silica nanospheres is preferably 100 to 150 nm.

[0065] In the present invention, the mass ratio of the silica nanospheres to potassium permanganate is preferably 1:1-2, more preferably 1:1; the mass ratio of the silica nanospheres to water is preferably 0.1 mg:1 mL.

[0066] In the present invention, the hydrothermal reaction is preferably carried out in a high-pressure reactor, and the temperature of the hydrothermal reaction is preferably 100-150°C, more preferably 120-140°C; the time is preferably 20-24h. In the present invention, after the hydrothermal reaction, the present invention preferably cools, centrifuges and dries the obtained hydrothermal reaction liquid to obtain MnO2@SiO2 nanosphere solid. In the present invention, the cooling is preferably natural cooling, and the time is preferably 12h; the washing liquid used in the centrifugal washing is preferably water, and the drying method is preferably freeze-drying.

[0067] After obtaining the MnO2@SiO2 nanospheres, the present invention mixes the aqueous dispersion of the MnO2@SiO2 nanospheres with a sodium carbonate solution, performs an etching reaction, and obtains hollow manganese dioxide nanoparticles (denoted as HMnO2). In the present invention, the dosage ratio of the MnO2@SiO2 nanospheres to water in the aqueous dispersion of the MnO2@SiO2 nanospheres is preferably 0.5g:30mL, and the concentration of the sodium carbonate solution is preferably 0.5g / mL. In the present invention, the volume ratio of the aqueous dispersion of the MnO2@SiO2 nanospheres to the sodium carbonate solution is preferably 3:2.

[0068] The present invention preferably performs the etching reaction in an oil bath. In the present invention, the temperature of the etching reaction is preferably 50-60°C, more preferably 55-60°C; the time is preferably 10-12h, more preferably 11-12h. After the etching reaction, the present invention preferably centrifuges and washes the obtained etching reaction solution and dries it. In the present invention, the drying is preferably vacuum freeze drying.

[0069] After obtaining the hollow manganese dioxide nanoparticles, the present invention mixes the hollow manganese dioxide nanoparticles, polyacrylamine hydrochloride and water to obtain PAH-wrapped hollow manganese dioxide. In the present invention, the mass ratio of the hollow manganese dioxide to polyacrylamine hydrochloride is preferably 2:1; in the present invention, the dosage ratio of the hollow manganese dioxide to water is preferably 0.3 mg: 40 mL. In the present invention, the mixing method is preferably stirring and mixing, and the stirring and mixing time is preferably 2 hours. In the present invention, after the mixing, the present invention preferably centrifuges and washes the obtained mixed solution and dries it. In the present invention, the drying is preferably vacuum freeze-drying. In the present invention, the role of the polyacrylamine hydrochloride is to undergo self-assembly with PAA.

[0070] After obtaining the PAH-encapsulated hollow manganese dioxide, the present invention mixes the PAH-encapsulated hollow manganese dioxide, polyacrylic acid and water to obtain the PAH- and PAA-encapsulated hollow manganese dioxide. In the present invention, the mass ratio of the hollow manganese dioxide to polyacrylic acid is preferably 5 to 6:1, and the amount ratio of the hollow manganese dioxide to water is preferably 0.3 mg: 40 mL. In the present invention, the mixing method is preferably stirring and mixing, and the stirring and mixing time is preferably 2 hours. In the present invention, after the mixing, the present invention preferably centrifuges and washes the obtained mixed solution and dries it. In the present invention, the drying is preferably vacuum freeze-drying. In the present invention, the role of the polyacrylic acid is to react with DNaseI to modify DNaseI to the surface of the hollow manganese dioxide nanoparticles.

[0071] After obtaining the hollow manganese dioxide wrapped by PAH and PAA, the present invention mixes the hollow manganese dioxide wrapped by PAH and PAA, a carboxyl activator and a buffer solution, performs a carboxyl activation reaction, and obtains a carboxyl activation reaction solution. In the present invention, the carboxyl activator is preferably EDC and NHS, and the buffer solution is preferably a 2-morpholineethanesulfonic acid solution (EMS). In the present invention, after the mixing, the concentration of the hollow manganese dioxide wrapped by PAH and PAA in the obtained mixed solution is preferably 1.25 mg / mL, the concentration of the EDC is preferably 0.4M, and the concentration of the NHS is preferably 0.1M. In the present invention, the carboxyl activation reaction is preferably carried out under stirring, the temperature of the carboxyl activation reaction is preferably room temperature, and the time is preferably 2 to 4 hours.

[0072] After obtaining the carboxyl activation reaction solution, the present invention adds DNaseI to the carboxyl activation reaction solution to perform a coupling reaction to obtain a DNaseI-modified hollow manganese dioxide. In the present invention, the mass ratio of the hollow manganese dioxide encapsulated by PAH and PAA to DNaseI is 4 to 5:1. In the present invention, the DNaseI is preferably added in the form of a solution, and the concentration of the DNaseI solution is preferably 0.4 to 0.5 mg / mL.

[0073] In the present invention, the coupling reaction is preferably carried out under stirring, the coupling reaction temperature is preferably room temperature, and the time is preferably 12 hours. After the coupling reaction, the present invention preferably centrifuges and washes the obtained coupling reaction solution and dries it, and the drying is preferably freeze-drying.

[0074] After obtaining the DNaseI-modified hollow manganese dioxide, the present invention mixes the DNaseI-modified hollow manganese dioxide with a photosensitizer, an antibacterial drug and water, and performs pumping and stirring under vacuum conditions to obtain a photothermal and photodynamic integrated antibacterial nanomedicine. In the present invention, the photosensitizer is preferably indocyanine green, and the antibacterial drug preferably includes ciprofloxacin and / or gatifloxacin, more preferably ciprofloxacin.

[0075] In the present invention, the mass ratio of the DNaseI-modified hollow manganese dioxide to the photosensitizer and antibacterial drug is preferably 2:1:1. In the present invention, the temperature of the pumping and stirring is preferably room temperature, the time is preferably 24h, and the speed of the pumping and stirring is preferably 500rpm.

[0076] After the loading, the present invention preferably performs centrifugal washing and drying on the obtained product. In the present invention, the drying is preferably freeze drying.

[0077] The present invention provides the use of the above-mentioned photothermal-photodynamic integrated antibacterial nanomedicine in the preparation of a biofilm-related keratitis drug. The photothermal-photodynamic integrated antibacterial nanomedicine provided by the present invention destroys the biofilm through DNA lyase, and can perform short-term sterilization through photothermal combined with photodynamic therapy, and can also achieve long-term sterilization through sustained-release ciprofloxacin. And for biofilm-related keratitis caused by bacterial infection, the nanomedicine can achieve a certain degree of healing and anti-inflammatory effects by regulating the hypoxic environment of the biofilm infection microenvironment, providing a new solution to overcome biofilm keratitis caused by multidrug-resistant antibiotic bacteria infection.

[0078] The photothermal and photodynamic integrated antibacterial nanomedicine provided by the present invention, its preparation method and its application in the preparation of biofilm-related keratitis drugs are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0079] Example 1

[0080] The preparation method of the photothermal and photodynamic integrated antibacterial nanomedicine adopts the following steps:

[0081] 350 mL of ethanol solution was mixed with 7.5 mL of ammonia water and 15 mL of tetraethyl silicate solution and stirred for 5 to 6 hours. The sample was obtained by centrifugation, and methanol was used as a solvent. After high-speed centrifugation washing, the sample was freeze-dried in a vacuum to obtain silica nanospheres (SiO2) with a particle size of 100 nm.

[0082] Potassium permanganate and 0.3 g of silica nanospheres were dissolved in 30 mL of ultrapure water at a mass ratio of 1:1, reacted in an autoclave at 150°C for 24 hours, cooled for 12 hours, washed three times by high-speed centrifugation, and freeze-dried to obtain MnO2@SiO2 nanospheres.

[0083] 0.5 g of MnO2@SiO2 was first dissolved in 30 mL of ultrapure water and then dissolved in 20 mL of 0.5 g / mL sodium carbonate solution. The two were evenly mixed and stirred in an oil bath at 60°C for 12 hours, washed three times by high-speed centrifugation, and freeze-vacuum dried to obtain hollow manganese dioxide nanoparticles (HMnO2).

[0084] 0.3 g of hollow manganese dioxide and polyacrylamine hydrochloride (PAH) were mixed in a mass ratio of 2:1 in 40 mL of ultrapure water, stirred for 2 hours, and then centrifuged and washed at high speed. The synthesized PAH-HMnO2 was resuspended in 40 mL of ultrapure water, and then 50 mg of polyacrylic acid (PAA) was added and stirred for 2 hours, centrifuged and washed at high speed, and freeze-vacuum dried to obtain HMnO2-PAH / PAA. 50 mg of HMnO2-PAH / PAA was mixed with 0.4 M EDC and 0.1 M NHS in 40 mL of 2-morpholineethanesulfonic acid solution (EMS), stirred for 2 hours, and then 20 mL of 0.5 mg / mL DNase I solution was added. After stirring for 12 hours, it was centrifuged, collected, washed, and freeze-dried to obtain the sample HMnO2-DNaseI. The hollow manganese dioxide connected with DNase I was stirred with photosensitizer indocyanine green and ciprofloxacin in a mass ratio of 2:1:1 in an aqueous solution system under vacuum conditions for 24 hours to load indocyanine green and ciprofloxacin inside the hollow manganese dioxide. After high-speed centrifugal washing and freeze-drying, the photothermal and photodynamic integrated antibacterial nanomedicine (DNaseI-HMnO2@ICG@CIP) was obtained.

[0085] The microstructures of MnO2@SiO2 and hollow manganese dioxide nanoparticles were verified by transmission electron microscopy and scanning electron microscopy. Figure 1 As shown, Figure 1 The successful preparation of MnO2@SiO2 and hollow manganese dioxide nanoparticles was demonstrated.

[0086] The microstructure of the obtained DnaseI-HMnO2@ICG@CIP is shown in the figure Figure 2 shown.

[0087] The XPS spectrum of the obtained DnaseI-HMnO2@ICG@CIP is as follows Figure 3 The successful modification of ICG can be proved by the peak of sulfur element in XPS.

[0088] Test Example 1: Photothermal and photodynamic performance verification of nanomedicine

[0089] (1) Study the photothermal performance of DnaseI-HMnO2@ICG@CIP nanoparticles under near-infrared radiation: Nanoparticles of different concentrations (50, 100, 200 μg / mL) were dispersed in phosphate buffered saline and exposed to near-infrared light (1 W / cm 2 ) for 10 minutes, and a thermal imager was used to observe the photothermal effects of samples with different concentrations under near-infrared light irradiation.

[0090] The photothermal effects of samples with different concentrations under near-infrared light irradiation are shown in Figure 2. Figure 4 As shown. Figure 4 As can be seen from a in the figure, under infrared light, DnaseI-HMnO2@ICG@CIP shows concentration dependence. As the concentration increases, the temperature at the same time shows an increasing trend.

[0091] To further clarify the specific components that produce the PDT effect, experiments with different samples were conducted. Figure 4 As shown in b, compared with the control group, both increased under infrared irradiation. This is because HMnO2 has a certain photothermal effect. In addition, due to the loading of photosensitizer, DnaseI-HMnO2@ICG@CIP shows better photothermal efficiency than simple DnaseI-HMnO2 and HMnO2.

[0092] The on / off cycle heating scheme of DnaseI-HMnO2@ICG@CIP (100 μg / mL) is as follows Figure 4 As shown in c, it can be seen that DnaseI-HMnO2@ICG@CIP shows photothermal reversibility and recycling performance.

[0093] (2) Study on the generation of reactive oxygen species by DnaseI-HMnO2@ICG@CIP nanoparticles under near-infrared radiation:

[0094] 1,3-Diphenylisobenzofuran (DPBF) probe was used to detect 1O2 generation. Generally, DPBF has a characteristic absorption peak at 410 nm, and the intensity will be irreversibly reduced in the presence of 1O2. DPBF solution (1 mM, dissolved in ethanol) was mixed with 100 μM H2O2-containing DNaseI-HMnO2@ICGNPs ethanol solution (100 μg / mL) in a 1:1 volume ratio. The mixture was illuminated by NIR laser (808 nm, 1 W cm -2 ) and continuously irradiate the mixed solution at 410 nm every 2 minutes until the characteristic absorption peak almost disappears. The absorption spectra at different times are shown in Figure 2. Figure 5 As shown in a. It can be seen that when DPBF is co-incubated with the final nanodrug (DnaseI-HMnO2@ICG@CIP) suspension containing 100μM H2O2, the absorbance gradually decreases with time, indicating that 1O2 is produced, which is time-dependent.

[0095] Then, ethanol solutions of control group HMnO2 and DnaseI-HMnO2 were prepared, mixed with DPBF solution and irradiated under near-infrared light. The absorption peak at 410 nm was measured every two minutes. The absorption spectra at different times are shown in Figure 2. Figure 5 As shown in b.

[0096] Comparison of the consumption rates of DNaseI-HMnO2@ICG@CIP+H2O2 and DNaseI-HMnO2@ICG@CIP Figure 5 As shown in c. The DPBF consumption rate of DNaseI-HMnO2@ICG@CIP+H2O2 is faster than that of DNaseI-HMnO2@ICG@CIP. This phenomenon is attributed to the production of O2, which promotes the production of 1O2 by ICG.

[0097] By detecting the absorption spectra of different nanodrugs reacting with DPBF probes under near-infrared light, it can be proved that the nanodrug (DnaseI-HMnO2@ICG@CIP) has good photodynamic properties; by monitoring the heating effect of different samples under near-infrared light with a thermal imager, it can be proved that the nanodrug has excellent photothermal properties and photothermal stability.

[0098] Test Example 2: The lysis effect of nanomedicine on biofilm

[0099] Staphylococcus aureus was selected as a typical test bacterium. The bacterial strain was grown in a liquid culture medium at 37°C for 14 h. The bacteria were then collected by centrifugation (3000 rpm, 6 minutes) and washed three times with phosphate buffered saline. The obtained bacteria were redispersed in phosphate buffered saline, and the bacterial concentration was adjusted by an ELISA instrument to obtain a bacterial solution for the biofilm lysis effect test. The bacterial solution concentration was about 107 CFU / mL -1 Then the bacterial solution (10 7 CFU / mL -1 , Staphylococcus aureus, 600μL) were cultured in 6-well plates for 24 hours to form bacterial biofilms. The liquid culture medium containing bacteria was aspirated from the wells and rinsed once with phosphate buffer to prevent the adhesion of the biofilm. Then different nanomedicines (HMnO2-DNaseI, DnaseI-HMnO2@ICG@CIP, NIR+DnaseI-HMnO2@ICG@CIP, all at a concentration of 300μg / mL) were added to co-culture with the biofilm at 37°C for 12 hours. After washing 2 to 3 times with phosphate buffered saline, 100μL of methanol solution was added to each well for fixation, followed by staining with 1mL of crystal violet dye, incubated at room temperature for 30min, and gently washed with sterile phosphate buffered saline to remove excess staining. 1.0mL of 95% ethanol was added, and the optical density (OD) value of the homogenate was measured at 590nm to quantify the amount of remaining crystal violet-stained biofilm.

[0100] The anti-biofilm performance of nanomedicines is verified as follows Figure 6 shown. Figure 6 The crystal violet-stained microscope images clearly show that DnaseI-HMnO2@ICG@CIP has excellent biofilm lysis ability. Compared with the PBS group, the biofilm residues of HMnO2, HMnO2-DNaseI, DnaseI-HMnO2@ICG@CIP, and NIR+DnaseI-HMnO2@ICG@CIP all showed a downward trend, and NIR+DnaseI-HMnO2@ICG@CIP had the best effect. This is because DnaseI can still kill bacteria with light, heat, and drugs while lysing the biofilm.

[0101] Test Example 3: Verification of the bactericidal performance of nanomedicine

[0102] Staphylococcus aureus was selected as a typical test bacterium. The bacterial strain was grown in a liquid culture medium at 37°C for 14 h. The bacteria were then collected by centrifugation (3000 rpm, 6 minutes) and washed three times with phosphate buffered saline. The obtained bacteria were redispersed in phosphate buffered saline, and the bacterial concentration was adjusted by an ELISA instrument to obtain a bacterial solution for antibacterial effect testing. The bacterial solution concentration was about 10 7 CFU / mL -1 .

[0103] Before testing, all samples (control group, HMnO2, HMnO2-DNaseI, DnaseI-HMnO2@ICG@CIP, NIR+DnaseI-HMnO2@ICG@CIP) were sterilized.7 CFU / mL -1 , Staphylococcus aureus, 100 μL) were co-cultured with the nanodrug for 4 h for plating experiments.

[0104] The bactericidal performance verification results of nanomedicine are as follows Figure 7 As shown. Figure 7 It can be seen that by comparing the number of colonies on the bacterial culture plate, NIR+DnaseI-HMnO2@ICG@CIP has a significant decrease compared to the PBS group. This is because DnaseI has multiple functions, including photothermal and drug sterilization, while lysing the biofilm. The bacterial plating concentration gradient experiment proves that the final synthesized nanomedicine has good bactericidal efficacy at 50μg / mL.

[0105] Test Example 4 Animal Experiment

[0106] Under sterile conditions, 20 μL of Pseudomonas aeruginosa containing 10 to the eighth power colony forming units (CFU) was injected into the corneal stroma of C57 mice. The prognosis was analyzed by taking slit lamp and corneal transparency (ulcer area) comparison photos. The results are as follows: Figure 8 shown.

[0107] In the slit lamp image, the left image shows the scope and area of ​​corneal opacity and the regularity of the surface, while the right image is stained with sodium fluorescein to show the degree of corneal defect. Based on the clinical evaluation of the severity of BK in the above images, the following conclusions can be drawn: the cornea of ​​the control group was severely infected, with opacity and purulent changes in the central cornea. In addition, the slit lamp examination score and corneal thickness value increased rapidly, and obvious corneal opacity, stromal edema and other infection manifestations were seen, as well as a certain degree of corneal defect.

[0108] In the NIR+DnaseI-HMnO2@ICG@CIP treatment group, after seven days of treatment (eye drops three times a day, morning, noon and evening, 5 μL each time), the cornea was clear and there was no obvious corneal ulcer, showing an excellent therapeutic effect.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A photothermal and photodynamic integrated antibacterial nanomedicine, characterized in that: The method comprises hollow manganese dioxide nanoparticles, a photosensitizer and an antibacterial drug loaded inside the hollow manganese dioxide nanoparticles, and DNase I modified on the surface of the hollow manganese dioxide nanoparticles; The antibacterial drugs include ciprofloxacin and / or gatifloxacin.

2. The photothermal and photodynamic integrated antibacterial nanomedicine according to claim 1, characterized in that: The photosensitizer includes indocyanine green.

3. The photothermal and photodynamic integrated antibacterial nanomedicine according to claim 1 or 2, characterized in that: The mass ratio of the hollow manganese dioxide nanoparticles, the photosensitizer and the antibacterial drug is 2:(1-2):

1.

4. The photothermal and photodynamic integrated antibacterial nanomedicine according to claim 1, characterized in that: The particle size of the photothermal and photodynamic integrated antibacterial nanomedicine is 100-150 nm.

5. The method for preparing the photothermal and photodynamic integrated antibacterial nanomedicine according to any one of claims 1 to 4, characterized in that: The following steps are involved: The silica nanospheres, potassium permanganate and water are mixed and subjected to a hydrothermal reaction to obtain MnO2@SiO2 nanospheres; The aqueous dispersion of MnO2@SiO2 nanospheres is mixed with a sodium carbonate solution and subjected to an etching reaction to obtain hollow manganese dioxide nanoparticles; Mixing the hollow manganese dioxide nanoparticles, polyacrylamine hydrochloride and water to obtain PAH-encapsulated hollow manganese dioxide; Mixing the PAH-encapsulated hollow manganese dioxide, polyacrylic acid and water to obtain PAH- and PAA-encapsulated hollow manganese dioxide; The hollow manganese dioxide wrapped by PAH and PAA, a carboxyl activator and a buffer solution are mixed to perform a carboxyl activation reaction to obtain a carboxyl activation reaction solution; Adding DNaseI to the carboxyl activation reaction solution to perform a coupling reaction to obtain DNaseI-modified hollow manganese dioxide; The DNaseI-modified hollow manganese dioxide is mixed with a photosensitizer, an antibacterial drug and water, and pumped and stirred under vacuum conditions to obtain a photothermal and photodynamic integrated antibacterial nanomedicine.

6. The preparation method according to claim 5, characterized in that: The particle size of the silicon dioxide nanospheres is 100 to 150 nm; The mass ratio of the silicon dioxide nanospheres to potassium permanganate is 1:1-2; The temperature of the hydrothermal reaction is 100-150° C. and the time is 20-24 hours.

7. The preparation method according to claim 5, characterized in that: The concentration of the sodium carbonate solution is 0.5 g / mL; The temperature of the etching reaction is 50-60° C. and the time is 10-12 hours.

8. The preparation method according to claim 5, characterized in that: The mass ratio of the hollow manganese dioxide nanoparticles to polyacrylamine hydrochloride is 2:1; The mass ratio of the hollow manganese dioxide nanoparticles to polyacrylic acid is 5 to 6:

1.

9. The preparation method according to claim 5, characterized in that: The mass ratio of the PAH and PAA-encapsulated hollow manganese dioxide to DNaseI is 4 to 5:1; The coupling reaction time is 10 to 12 hours; The pumping and stirring time is 20 to 24 hours.

10. Use of the photothermal-photodynamic integrated antibacterial nanomedicine according to any one of claims 1 to 4 or the photothermal-photodynamic integrated antibacterial nanomedicine prepared by the preparation method according to any one of claims 5 to 9 in the preparation of drugs for biofilm-related keratitis.

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