Preparation and application of viral physical antibacterial nano preparation

Abstract

The invention discloses preparation and application of a viral physical antibacterial nano preparation, and belongs to the technical field of novel materials. The preparation method comprises the following steps: preparing viral silicon oxide nano-particles in an oil / water two-phase reaction system by adopting a single micelle epitaxial growth method, modifying the viral silicon oxide nano-particles by virtue of amino and carboxylic acid-polyethylene glycol-phenylboronic acid, and finally preparing a self-thermophoresis nano-motor by adopting an electron beam evaporation manner, so as to obtain the viral physical antibacterial nano-preparation. According to the invention, a physical antibacterial mode and a nano motor technology are combined to construct a physical antibacterial nano preparation, so that efficient removal of drug-resistant bacteria and destruction of biofilms can be realized, high biological safety is maintained, and key problems in treatment of drug-resistant bacteria infectious keratitis are solved.

Description

Technical Field

[0001] The invention relates to the technical field of new materials, and in particular to the preparation and application of a virus-like physical antibacterial nano preparation. Background Art

[0002] Infectious keratitis refers to corneal inflammation caused by bacteria and other pathogenic microorganisms. If not treated in time or the effect is poor, the infection can infiltrate deep into the corneal stroma, destroy the corneal structure, cause corneal perforation, and cause permanent visual dysfunction.

[0003] In 2012, Professor Crawford of Swinburne University of Technology in Australia found that the surface of cicada wings and dragonfly wings with special micro-nano structures (such as nanopillars, nanoneedles, etc.) can cause mechanical rupture of bacterial cell membranes attached to them, thereby causing bacterial death. Further research shows that the main mechanism is that bacteria and micro-nano structures interact under gravity, shear force or surface tension. Once the elastic limit of the interaction between the two is reached, the micro-nano structure will physically tear or directly pierce the bacterial cell membrane, causing the bacterial cell structure to rupture and die. This new method of sterilization completely through physical and mechanical sterilization rather than biochemical mechanisms shows unique advantages in removing drug-resistant bacteria. Inspired by this, Academician Fan Chunhai of Shanghai Jiaotong University reported that graphene oxide with sharp edges can be inserted into bacteria to cut the cell membrane, resulting in cell membrane damage, leakage of contents and death. Professor Mahmoud of Hamad Bin Khalifa University in Qatar and Professor Soroush of Drexel University in the United States have also successively proved that blade-shaped MXenes have physical antibacterial properties. However, physical antibacterial nanoparticles are usually used for in vitro antibacterial activities and have not been used in the repair of tissue infections in vivo. There are two main reasons for this. First, physical antibacterial nanoparticles generally lack initiative and can only interact with bacteria through Brownian motion or passive diffusion. Their antibacterial activity is significantly dependent on time and they are unable to efficiently remove bacteria and destroy biofilms. Second, they lack specific targeting of bacteria and can harm normal cells and tissues. Therefore, giving physical antibacterial nanoparticles autonomous motion properties and achieving targeted recognition of bacteria is a prerequisite for their application in the treatment of drug-resistant bacterial infectious keratitis.

[0004] Nanomotors are nanomaterials that convert other forms of energy (such as magnetic energy, light energy, chemical energy, etc.) into their own driving force. Based on their autonomous motion performance, the construction of physical antibacterial nanoformulations through nanomotors is the most direct and effective strategy to promote bacterial removal and biofilm destruction. At present, research on nanomotors in physical antibacterial has made some progress. For example, Truong et al. from the Royal Melbourne Institute of Technology in Australia reported that magnetically responsive gallium-based liquid metal droplets exposed to a rotating magnetic field will be physically driven and transform their shape to form sharp edges. When in contact with bacterial biofilms, the magnetic field drives the particle movement, and the presence of nano-sharp edges achieves physical antibacterial and destroys the dense biofilm matrix. Professor Zhang Li's team at the Chinese University of Hong Kong used natural sunflower pollen particles to load magnetic liquid metal droplets to prepare a magnetic sea urchin-shaped nanomotors. The natural micron spikes inherent in pollen particles and the sharp edges of magnetic liquid metal actively destroy dense biofilm matrices and a variety of bacteria through mechanical force. Subsequently, they used the spiral Fe2O3 nanomotor as a basis, combined with the robotic arm-assisted magnetically controlled remote drive and endoscopic delivery and observation, to achieve physical removal of biofilm in the middle ear catheter. However, both the magnetic liquid metal droplets and the spiral Fe2O3 have problems such as being too large in size (>10μm) and having a very high magnetic field intensity (T level) for induced motion, which severely restricts their in vivo application. Compared with the use of magnetic field drive, the eye, as the only photosensitive organ in the human body, is closer to in vivo application in light-driven nanomotors. Professor Kong Biao of Fudan University classified the driving strategies of nanomotors and pointed out that near-infrared light-driven self-thermophoretic nanomotors have the unique advantages of controllable size and easy manipulation. Self-thermophoresis is based on the temperature gradient generated at the nanoscale to drive the movement of the nanomotor, which is usually achieved by asymmetrically modifying the near-infrared light photothermal conversion material of the nanomotor. It can move under mild photothermal conditions (<45°C) and can avoid damage to tissues caused by excessive local temperature. In addition, the self-thermophoretic nanomotors reported so far are usually Janus spherical structures, and the pressure acting on the bacterial cell membrane is usually smaller than the corresponding cell membrane modulus. It is difficult to destroy the cell membrane by the physical mechanical action of the nanomotors themselves, resulting in a lower-than-expected efficiency in removing drug-resistant bacteria. Therefore, combining physical antibacterial methods with nanomotor technology to construct physical antibacterial nanoformulations can achieve efficient removal of drug-resistant bacteria and destruction of biofilms, solving the key problems faced in the treatment of drug-resistant bacterial infectious keratitis. Summary of the invention

[0005] The purpose of the present invention is to prepare and apply a virus-like physical antibacterial nanoformulation to solve the problems existing in the above-mentioned prior art. The virus-like physical antibacterial nanoformulation has the ability to move autonomously under NIR light, has obvious antibacterial effect, and has obvious therapeutic effect on drug-resistant bacterial infectious keratitis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a virus-like physical antibacterial nano preparation, comprising the following steps:

[0008] Preparation of virus-like silica nanoparticles;

[0009] The virus-like silicon oxide nanoparticles are amino-modified and then modified with carboxylic acid-polyethylene glycol-phenylboronic acid, and then the self-thermophoretic nanomotor is prepared by electron beam evaporation of Au target to obtain the virus-like physical antibacterial nanopreparation.

[0010] Preferably, the method for preparing virus-like silicon oxide nanoparticles comprises the following steps:

[0011] Hexadecyltrimethylammonium bromide, sodium hydroxide and water are mixed and stirred for reaction, and then tetraethoxysilane is added to the mixture and stirred continuously to obtain a product, which is then centrifuged, washed and treated with a methanol solution of hydrogen chloride to obtain virus-like silica nanoparticles.

[0012] Preferably, the ratio of hexadecyltrimethylammonium bromide, sodium hydroxide and water is 1 g:1 g:(20-30) mL, and the stirring is at 60° C. for 2 hours;

[0013] The ratio of the hexadecyltrimethylammonium bromide to the tetraethoxysilane is 1 g:(5-15) mL; and the continued stirring is continuous stirring at 60° C. for 72 hours.

[0014] Preferably, the amino modification of the virus-like silicon oxide nanoparticles comprises the following steps:

[0015] The virus-like silicon oxide nanoparticles are dispersed in ethanol, and then aminopropyltriethoxysilane is added, refluxed and centrifuged, and the product is collected, washed with ethanol, and stored in ethanol to obtain amino-modified virus-like silicon oxide nanoparticles.

[0016] Preferably, the ratio of the virus-like silicon oxide nanoparticles, the ethanol and the aminopropyltriethoxysilane is 100 mg: (20-40) mL: (100-200) μL;

[0017] The reflux time is 15-20 hours.

[0018] Preferably, the carboxylic acid-polyethylene glycol-phenylboronic acid modification comprises the following steps:

[0019] Carboxylic acid-polyethylene glycol-phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester are dissolved in ethanol and stirred evenly, and then amino-modified virus-like silica nanoparticles are added, stirred for reaction, and then the product is collected by centrifugation, washed and dispersed in water.

[0020] Preferably, the mass ratio of the carboxylic acid-polyethylene glycol-phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and amino-modified viral-like silica nanoparticles is 2:1:1:2, and the stirring reaction time is 20-30 hours.

[0021] Preferably, the self-thermophoretic nanomotor is prepared by electron beam evaporation of Au target, comprising the following steps:

[0022] Nanoparticles modified with carboxylic acid-polyethylene glycol-phenylboronic acid are evenly dropped on a glass slide and vacuum dried to form a single-layer structure. Gold is then sprayed and deposited on the surface of the nanoparticles using a sputtering coating technique. The nanoparticles are then ultrasonically stripped in deionized water and the product is collected to obtain a self-thermophoretic nanomotor.

[0023] The present invention also provides a virus-like physical antibacterial nano preparation, which is prepared by the preparation method.

[0024] The present invention also provides the use of the virus-like physical antibacterial nano preparation in the preparation of an antibacterial nano preparation for treating drug-resistant bacterial infectious keratitis.

[0025] The present invention discloses the following technical effects:

[0026] The present invention provides a virus-like physical antibacterial nanoparticle (VMSNT), which is prepared by a single micelle epitaxial growth method in an oil / water two-phase reaction system. Virus-like silicon oxide nanoparticles (VMSN) are then modified by a modification method. In order to make the prepared nanoparticles biocompatible and bacterial targeting, the nanoparticles are modified with carboxylic acid-polyethylene glycol-phenylboronic acid; finally, electron beam evaporation is used to synthesize self-thermophoretic nanomotors, which have a spike morphology and autonomous motion function under NIR light. Compared with traditional nanoparticles, the "virus-like" nanoparticles provided by the present invention can effectively reduce the contact area with the bacterial cell membrane. In addition, the "virus-like" nanoparticles have better near-infrared absorption due to their rich branched structure and high specific surface area. The self-thermophoretic nanomotors constructed based on the "virus-like" nanoparticles can generate a larger temperature gradient around the nanoparticles, thereby generating a stronger self-driving force. Drive VMSNT to physically destroy the cell membrane and deeply penetrate the biomembrane through self-thermal penetration. Through the synergistic effect of the topological geometric structure of the spikes and the self-thermal electrophoresis effect, physical antibacterial shows high performance in eradicating drug-resistant bacteria and biofilms. The present invention combines physical antibacterial methods with nanomotor technology to construct physical antibacterial nanoformulations, which can achieve efficient removal of drug-resistant bacteria and destruction of biofilms while retaining a high degree of biosafety, solving the key problem faced in the treatment of drug-resistant bacterial infectious keratitis.

[0027] Animal experiments have shown that the novel virus-like physical antibacterial nanomotor constructed by the present invention has a good antibacterial effect on drug-resistant bacterial infectious keratitis, which has obvious advantages over the traditional single treatment mode and has little toxic side effects. The present invention provides a cascade targeting strategy of "autonomous movement-active targeting", which enhances the ability of nanomotor to target bacteria and improves the safety of physical antibacterial nanoformulations for the treatment of drug-resistant bacterial infectious keratitis. Therefore, the design provided by the present invention introduces a new way of physical antibacterial action into conventional nanomotor and paves the way for its practical application in vivo, which has broad application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 FT-IR spectra of VMSN, VMSN-NH2 and VMSN-NH2-PBA;

[0030] Figure 2 Elemental mapping image of VMSNT, scale bar: 100 nm;

[0031] Figure 3 Results of the biocompatibility evaluation of VMSNT; A: Cytotoxicity evaluation on healthy cells; B: Effect on the structure of mouse cornea;

[0032] Figure 4 Results of the efficacy evaluation of VMSNT in treating keratitis caused by drug-resistant bacteria; (A) Slit lamp examination and AS-OCT images of MRSA-infected mouse eyes from day 0 to day 7 after various treatments on the ocular surface of C57BL / 6J mice; (B) Mannitol salt agar plate culture results of in vitro corneal tissue immersion solution in different treatment groups; (C) H&E staining images of corneal tissue sections after 7 days of different treatments; Scale bar: 200 μm. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1 Preparation method of a virus-like physical antibacterial nanosystem

[0039] 1. Synthesis of virus-like mesoporous silica (VMSN) nanoparticles

[0040] VMSN was synthesized in a two-phase system using a cationic surfactant as a template and TEOS as a silicon source. First, 1 g of hexadecyltrimethylammonium bromide (CTAB) and 1 g of sodium hydroxide were mixed in 25 mL of deionized water and stirred at 60 °C for 2 hours. Then 10 mL of TEOS (20% v / v) was added to the mixture, which was then stirred continuously at 60 °C for 72 hours. The final product was separated by centrifugation at 1000 g for 8 minutes and then washed three times with water and ethanol. The product was treated with 0.25 M HCl-CH3OH solution to remove the surfactant template and obtain VMSN. The resulting VMSN was washed with ethanol and dispersed in ethanol.

[0041] 2. Synthesis of VMSN-NH2 by post-synthesis modification method

[0042] 100 mg of the prepared VMSN was uniformly dispersed in 30 mL of ethanol, and then 200 μL of aminopropyltriethoxysilane (APTES) was added to the solution and refluxed at 60° C. for 18 h. Then, the VMSN-NH2 nanomaterials were collected by centrifugation, washed with ethanol, and then dispersed and stored in ethanol.

[0043] 3. Synthesis of VMSN-NH2-PBA by amide reaction method:

[0044] 10 mg of carboxylic acid-polyethylene glycol-phenylboronic acid (COOH-PEG-PBA), 5 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 5 mg of N-hydroxysuccinimide ester (NHS) were dissolved in 5 mL of ethanol and stirred. Then, 10 mg of VMSN-NH2 was introduced into the above solution, and the resulting mixture was stirred for 24 hours. After centrifugation, the final product was gently rinsed in deionized water for several times and then dispersed in water to obtain the products VMSN-NH2-PBA and SMSN-NH2-PBA.

[0045] 4. Synthesis of VMSNT

[0046] The VMSN-NH2-PBA solution was evenly dropped onto a freshly cleaned glass slide and dried by vacuum to form a monolayer structure. Subsequently, Au was applied to the glass slide using a sputter coating technique. Finally, the VMSNTs were collected from the substrate by mild sonication in deionized water.

[0047] Example 2 FT-IR detection

[0048] The spectra of VMSN, VMSN-NH2 and VMSN-NH2-PBA prepared in Example 1 were detected by FT-IR. Figure 1 As shown, virus-like silica nanoparticles were successfully synthesized into nanomotor VMSN-NH2-PBA through amino modification and carboxylic acid-polyethylene glycol-phenylboronic acid.

[0049] Example 3 Elemental Analysis of VMSNT

[0050] The elemental mapping image of VMSNT was measured by EDS. Figure 2 As shown, elemental analysis clearly revealed the presence of expected elements in VMSNT, including B (boron), Si (silicon), and Au (gold).

[0051] Example 4 Biocompatibility Evaluation

[0052] The cell counting kit-8 (CCK-8) test was used to verify the cytotoxicity of VMSNT. Figure 3 As shown in Figure 2, even when incubated with a high concentration of 120 μg / mL VMSNT, the relative cell survival rate remained above 90%, indicating that the cytotoxicity of VMSNT to healthy cells was negligible ( Figure 3 Middle A). Further observation of the changes in the mouse corneal structure after topical application of VMSNT was performed using a slit lamp microscope. The images showed that the mouse cornea was in good condition with no inflammatory response ( Figure 3 (middle B).

[0053] Example 5 Establishment and treatment of MRSA-infected keratitis

[0054] 10 μL of methicillin-resistant Staphylococcus aureus (MRSA) suspension (10 7 CFU / mL) was injected into the cornea of ​​C57BL / 6J mice for mechanical injury to establish the C57BL / 6J mouse model of MRSA-infected keratitis. Mice were randomly divided into PBS group and VMSNT group, and the samples were administered 3 times a day, 10 μL each time. After topical administration of the sample suspension, C57BL / 6J mice were exposed to 808 nm NIR laser (0.25 W / cm 2 ) for 3 minutes. On days 0, 1, 3, 5, and 7, the mouse corneas were evaluated by anterior segment optical coherence tomography and slit lamp microscopy. After 7 days, the corneas were removed and ground. The diluted suspension (5 μL) was spread on a standard agar plate and incubated at 37°C for 24 hours. The colonies were counted to assess the number of bacteria in the cornea. In addition, the present invention also collected tissue samples for pathological histological studies.

[0055] The results are as follows Figure 4 As shown in the figure, on day 0, all corneas were cloudy and edematous, and there was no significant difference between the two groups, indicating that the bacterial infection level in the two groups was consistent at the beginning of retreatment, and the model was successfully established. In the PBS treatment group, inflammation gradually worsened over time, and large areas of ulcer lesions appeared. However, after 7 days of treatment in the VMSNT group, the corneas of the infected eyes almost returned to normal levels, and the inflammation also subsided rapidly, indicating a significant treatment effect ( Figure 4 The bactericidal activity of VMSNT and PBS was investigated by agar plate counting method. In comparison, the antibacterial performance of VMSNT was significantly improved ( Figure 4 Further analysis of corneal tissue by HE staining clearly showed that the corneal structure was intact after VMSNT treatment, with almost no inflammatory cell infiltration ( Figure 4 Middle C).

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a virus-like physical antibacterial nanoparticle preparation, characterized in that: The steps include: Preparation of virus-like silica nanoparticles; The virus-like silicon oxide nanoparticles are amino-modified and then modified with carboxylic acid-polyethylene glycol-phenylboronic acid, and then the self-thermophoretic nanomotor is prepared by electron beam evaporation of Au target to obtain the virus-like physical antibacterial nanoparticle.

2. The preparation method according to claim 1, characterized in that The method for preparing virus-like silicon oxide nanoparticles comprises the following steps: Hexadecyltrimethylammonium bromide, sodium hydroxide and water are mixed and stirred for reaction, and then tetraethoxysilane is added to the mixture and stirred continuously to obtain a product, which is then centrifuged, washed and treated with a methanol solution of hydrogen chloride to obtain virus-like silica nanoparticles.

3. The preparation method according to claim 2, characterized in that: The ratio of hexadecyltrimethylammonium bromide, sodium hydroxide and water is 1 g: 1 g: (20-30) mL, and the stirring is 60° C. for 2 hours; The ratio of the hexadecyltrimethylammonium bromide to the tetraethoxysilane is 1 g:(5-15) mL; and the continued stirring is continuous stirring at 60° C. for 72 hours.

4. The preparation method according to claim 1, characterized in that: The amino modification of the virus-like silicon oxide nanoparticles comprises the following steps: The virus-like silicon oxide nanoparticles are dispersed in ethanol, and then aminopropyltriethoxysilane is added, refluxed and centrifuged, and the product is collected, washed with ethanol, and stored in ethanol to obtain amino-modified virus-like silicon oxide nanoparticles.

5. The preparation method according to claim 4, characterized in that: The ratio of the virus-like silicon oxide nanoparticles, the ethanol and the aminopropyltriethoxysilane is 100 mg: (20-40) mL: (100-200) μL; The reflux time is 15-20 hours.

6. The preparation method according to claim 1, characterized in that: The carboxylic acid-polyethylene glycol-phenylboronic acid modification comprises the following steps: Carboxylic acid-polyethylene glycol-phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester are dissolved in ethanol and stirred evenly, and then amino-modified virus-like silica nanoparticles are added, stirred for reaction, and then the product is collected by centrifugation, washed and dispersed in water.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the carboxylic acid-polyethylene glycol-phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and amino-modified virus-like silicon oxide nanoparticles is 2:1:1:2, and the stirring reaction time is 20-30 hours.

8. The preparation method according to claim 1, characterized in that: The method of preparing a self-thermophoretic nanomotor by electron beam evaporation of an Au target comprises the following steps: Nanoparticles modified with carboxylic acid-polyethylene glycol-phenylboronic acid are evenly dropped on a glass slide and vacuum dried to form a single-layer structure. Gold is then sprayed and deposited on the surface of the nanoparticles using a sputtering coating technique. The nanoparticles are then ultrasonically stripped in deionized water and the product is collected to obtain a self-thermophoretic nanomotor.

9. A virus-like physical antibacterial nanoparticle preparation, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the virus-like physical antibacterial nanoformulation as claimed in claim 9 in the preparation of an antibacterial nanoformulation for treating drug-resistant bacterial infectious keratitis.

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