An antibacterial artificial nanocell and its preparation method and application

Abstract

The present invention discloses an antibacterial artificial nanocell and its preparation method and application. The antibacterial artificial nanocell is used for preparing a drug for treating osteomyelitis, and the antibacterial artificial nanocell is prepared through the following steps: mixing polyvinylpyrrolidone or its aqueous solution, CuCl2·2H2O or its aqueous solution, FeCl2·4H2O or its aqueous solution, elesclomol or its DMSO solution, NaOH or its aqueous solution, and H2O2 or its aqueous solution, stirring evenly, washing, ultrafiltering, and centrifuging to obtain nanoparticles; preparing cell membranes by using bone marrow stromal stem cells; mixing the nanoparticles and the cell membranes evenly, and incubating and extruding them by ultrasound to obtain the antibacterial artificial nanocell; wherein, the mass ratio of the protein of the nanoparticles to the cell membranes is 1:(0.9-1.1). The antibacterial artificial nanocell provided by the present invention effectively avoids the two major clinical problems of large side effects caused by long-term and high-dose use of antibiotics and high risk of debridement surgery in the traditional treatment of osteomyelitis, and kills bacteria from the perspectives of mediating Fenton reaction and cuproptosis, two non-antibiotic principles, so as to achieve excellent osteomyelitis treatment effect.

Description

Technical Field

[0001] The invention belongs to the technical field of osteomyelitis therapeutic drugs, and specifically relates to an antibacterial artificial nanocell and a preparation method and application thereof. Background Art

[0002] Osteomyelitis is an infectious bone disease that mainly affects children and the elderly. In recent years, its incidence and the proportion of resistant bacteria infections have increased year by year. Traditional treatment is long-term high-dose antibiotics combined with surgical debridement, which mainly has the following two problems: ① Side effects / adverse reactions and induced bacterial resistance caused by long-term high-dose antibiotics; ② Risks of anesthesia / reinfection / disability associated with debridement surgery. Introducing new non-antibiotic treatment strategies from the perspective of other treatment principles is of great significance to further improve the efficacy of osteomyelitis. Summary of the invention

[0003] The present invention provides an antibacterial artificial nanocell, which effectively avoids the two major clinical problems of large side effects of long-term and large-dose use of antibiotics in traditional osteomyelitis treatment and high risk of debridement surgery. It kills bacteria from the perspective of two non-antibiotic principles, namely, mediating Fenton reaction and copper death, to achieve excellent osteomyelitis treatment effect. In addition, the present invention also provides a preparation method of antibacterial artificial nanocells and applications thereof.

[0004] The first aspect of the present invention provides a method for preparing an antibacterial artificial nanocell, wherein the antibacterial artificial nanocell is used to prepare a drug for treating osteomyelitis, and the antibacterial artificial nanocell is prepared by the following steps:

[0005] Polyvinyl pyrrolidone or its aqueous solution, CuCl2·2H2O or its aqueous solution, FeCl2·4H2O or its aqueous solution, ilisimol or its DMSO solution, NaOH or its aqueous solution, H2O2 or its aqueous solution are mixed, stirred evenly, washed, ultrafiltered and centrifuged to obtain nanoparticles;

[0006] Bone marrow stromal stem cells were used to prepare cell membranes;

[0007] The nanoparticles and cell membranes are mixed evenly, and then ultrasonically incubated and extruded to obtain antibacterial artificial nanocells; wherein the protein mass ratio of the nanoparticles to the cell membrane is 1:(0.9-1.1).

[0008] The antibacterial artificial nanocells provided by the present invention provide a new idea for the treatment of osteomyelitis. The antibacterial artificial nanocells provided by the present invention are a new non-antibiotic strategy for the treatment of osteomyelitis, which effectively avoids the two major pain points in the traditional treatment of osteomyelitis. One is the side effects / adverse reactions and induced bacterial resistance caused by long-term and large-dose use of antibiotics, and the other is the risks of anesthesia / reinfection / disability associated with debridement surgery.

[0009] In one embodiment of the present invention, the preparation of the nanoparticles comprises the following steps:

[0010] Mix the polyvinyl pyrrolidone aqueous solution, CuCl2·2H2O aqueous solution and FeCl2·4H2O aqueous solution, stir evenly, then add the DMSO solution containing ilisimol, stir evenly, then add the NaOH aqueous solution and the H2O2 aqueous solution in sequence, stir and react for 30 to 35 minutes, wash with distilled water and ultrafilter and centrifuge for at least 3 times to obtain nanoparticles.

[0011] In one embodiment of the present invention, the concentration of the polyvinyl pyrrolidone aqueous solution is 202-247 mg / mL, the concentration of the CuCl2·2H2O aqueous solution is 36-44 mg / mL, the concentration of the FeCl2·4H2O aqueous solution is 18-22 mg / mL, the concentration of ilisimol in the DMSO solution containing ilisimol is 168-206 mg / mL, the concentration of the NaOH aqueous solution is 0.018-0.022 M, and the concentration of the H2O2 aqueous solution is 9-10 M.

[0012] In one embodiment of the present invention, the volume ratio of polyvinyl pyrrolidone aqueous solution, CuCl2·2H2O aqueous solution, FeCl2·4H2O aqueous solution, DMSO solution containing ilisimol, NaOH aqueous solution, and H2O2 is (1323~1617):(90~110):(90~110):(4.5~5.5):(2970~3630):(60~74).

[0013] In one embodiment of the present invention, the pore size of the ultrafiltration membrane is 9-11 KDa.

[0014] In one embodiment of the present invention, the preparation of the cell membrane comprises the following steps:

[0015] When the bone marrow stromal stem cells were cultured to a cell density of 80-90%, the cells were scraped off with a cell scraper, washed three times with PBS, and a cell membrane lysis solution was added to lyse the cells on ice for 13-15 minutes, and then repeatedly frozen and thawed with liquid nitrogen for 3-5 cycles, and the cell membranes were collected after centrifugation at 4°C and 13000-14000g for 30-35 minutes to obtain cell membranes.

[0016] In one embodiment of the present invention, the nanoparticles are mixed evenly with the cell membrane, and after ultrasonic treatment at 35-37°C for 15-20 minutes, they are first squeezed through a polycarbonate membrane with a pore size of 180-220 nm for 12-15 times, and then squeezed through a polycarbonate membrane with a pore size of 90-110 nm for 12-15 times, and centrifuged at 13000-14000g for 3-5 minutes, and purified and collected to obtain antibacterial artificial nanocells.

[0017] The second aspect of the present invention provides an antibacterial artificial nanocell, which is prepared by the above-mentioned preparation method.

[0018] The third aspect of the present invention provides the use of the above-mentioned antibacterial artificial nanocells in the preparation of a drug for treating osteomyelitis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The antibacterial artificial nanocells provided by the present invention are a new non-antibiotic strategy for the treatment of osteomyelitis, which effectively avoids the two major pain points in the traditional treatment of osteomyelitis. One is the side effects / adverse reactions and induced bacterial resistance caused by long-term and large-dose use of antibiotics, and the other is the risks of anesthesia / reinfection / disability associated with debridement surgery.

[0021] 2. The antibacterial artificial nanocells provided in the embodiment of the present invention disintegrate to release copper and iron ions, copper ion carrier ilisimol and hydrogen peroxide under the weakly acidic conditions of the osteomyelitis infection site, and sterilize from the perspective of two non-antibiotic principles, namely, mediating Fenton reaction and copper death, to achieve excellent osteomyelitis treatment effect, and provide a new idea for the treatment of osteomyelitis. Specifically, referring to Figure 4 As shown, in the antibacterial artificial nanocell provided by the embodiment of the present invention, copper and iron ions are catalyzed by the copper-iron catalytic loop (Cu + -to-Cu 2+ and Fe 3+ -to-Fe 2+ ) enhances the Fenton reaction to kill bacteria. In addition, the copper ion carrier ilisimol transports excess copper ions into bacteria, inducing their copper death. Furthermore, bone marrow stromal stem cell membrane modification is used to improve the biocompatibility and bone marrow targeting of the nanosystem.

[0022] 3. The drug for treating osteomyelitis prepared by using the antibacterial artificial nanocells provided in the embodiment of the present invention is a composite drug system that combines chemodynamic therapy and inducing copper death, thereby achieving an excellent effect on the treatment of osteomyelitis. The drug for treating osteomyelitis prepared by using the antibacterial artificial nanocells provided in the embodiment of the present invention is administered by intravenous injection when used, thereby exerting the effect of antibacterial treatment of osteomyelitis.

[0023] 4. The method for preparing antibacterial artificial nanocells provided in the embodiment of the present invention has a wide source of raw materials and a simple and safe process.

[0024] Instruction Manual

[0025] Figure 1 This is a schematic diagram of the preparation process of antibacterial artificial nanocells in an embodiment of the present invention;

[0026] Figure 2 This is a transmission electron microscopy photograph of the antibacterial artificial nanocells prepared in Example 1 (scale bar, 100 nm);

[0027] Figure 3 The in vitro antibacterial effect of the artificial nanocells prepared in Example 1 against S. aureus and MRSA bacteria (**p < 0.01 relative to the CFE@CM group);

[0028] Figure 4 Schematic diagram of the sterilization mechanism of antibacterial artificial nanocells in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0030] In this application, the use of "or" means "and / or" unless otherwise stated. In the case of multiple dependent claims, use of "or" refers to more than one of the preceding independent or dependent claims only in the alternative.

[0031] Unless otherwise indicated, as used in accordance with this disclosure, the following terms shall be understood to have the following meanings:

[0032] It should be noted that the scientific and technical terms and abbreviations used in the present invention have the meanings commonly understood by those skilled in the art. The following lists some of the terms and abbreviations used in the present invention:

[0033] The term "treat" when used in connection with the treatment of, for example, a pathology or disease, refers to alleviating and / or eliminating one or more symptoms of the pathology or disease, and / or delaying the progression of one or more symptoms of the pathology or disease and / or reducing the incidence or severity of one or more symptoms of the pathology or disease, and / or preventing the pathology or disease. The term "treat" may refer to prophylactic treatment, which includes delaying the onset of a pathology or disease or preventing the onset of a pathology or disease.

[0034] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art in practical applications according to the present invention still belong to the scope of protection of the present invention.

[0035] Example 1

[0036] This embodiment provides an antibacterial artificial nanocell, which is CFE@CM. The preparation of the antibacterial artificial nanocell in this embodiment includes the following steps:

[0037] Preparation of CFE: 330 mg of polyvinyl pyrrolidone was dissolved in 1.47 mL of distilled water; then, 100 μL of CuCl2·2H2O (39.7 mg / mL) and 100 μL of FeCl2·4H2O (20 mg / mL) aqueous solution were added thereto and stirred for 5 min; then, 5 μL of ilisimol solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution and stirred for 5 min; subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H2O2 (10 M) were added in sequence and stirred rapidly for 30 min. Afterwards, the nanoparticles were washed with distilled water and centrifuged three times by ultrafiltration (10 kDa), and the obtained nanoparticles were CFE;

[0038] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronocell, Wuhan, China) were cultured in a 10-cm cell culture dish. When the cell density reached 90%, the cells were scraped off with a cell scraper and washed three times with PBS. Then, 1 mL of cell membrane lysis buffer was added, and the cells were lysed on ice for 14 min, followed by four cycles of repeated freezing and thawing with liquid nitrogen. The cell membranes were collected after centrifugation at 14,000 g for 30 min at 4°C.

[0039] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and ultrasonically treated at 36°C for 15 min; then, the mixture was extruded through 200 nm and 100 nm polycarbonate membranes for 14 times respectively, centrifuged at 14000 g for 5 min, and CFE@CM artificial nanocells were purified and collected, i.e., the antibacterial artificial nanocells in this embodiment.

[0040] Example 2

[0041] This embodiment provides an antibacterial artificial nanocell, which is CFE@CM. The preparation of the antibacterial artificial nanocell in this embodiment includes the following steps:

[0042] Preparation of CFE: 297 mg of polyvinyl pyrrolidone was dissolved in 1.47 mL of distilled water; then, 90 μL of CuCl2·2H2O (39.7 mg / mL) and 90 μL of FeCl2·4H2O (20 mg / mL) aqueous solution were added thereto and stirred for 5 min; then, 4.5 μL of ilisimol solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution and stirred for 5 min; subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H2O2 (10 M) were added in sequence and stirred rapidly for 30 min. Afterwards, the nanoparticles were washed with distilled water and centrifuged three times by ultrafiltration (10 kDa), and the obtained nanoparticles were CFE;

[0043] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronocell, Wuhan, China) were cultured in a 10-cm cell culture dish. When the cell density reached 90%, the cells were scraped off with a cell scraper and washed three times with PBS. Then, 1 mL of cell membrane lysis buffer was added, and the cells were lysed on ice for 14 min, followed by four cycles of repeated freezing and thawing with liquid nitrogen. The cell membranes were collected after centrifugation at 14,000 g for 30 min at 4°C.

[0044] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and ultrasonically treated at 36°C for 15 min; then, the mixture was extruded through 200 nm and 100 nm polycarbonate membranes for 14 times respectively, centrifuged at 14000 g for 5 min, and CFE@CM artificial nanocells were purified and collected, i.e., the antibacterial artificial nanocells in this embodiment.

[0045] Example 3

[0046] This embodiment provides an antibacterial artificial nanocell, which is CFE@CM. The preparation of the antibacterial artificial nanocell in this embodiment includes the following steps:

[0047] Preparation of CFE: 363 mg of polyvinyl pyrrolidone was dissolved in 1.47 mL of distilled water; then, 110 μL of CuCl2·2H2O (39.7 mg / mL) and 110 μL of FeCl2·4H2O (20 mg / mL) aqueous solution were added thereto and stirred for 5 min; then, 5.5 μL of ilisimol solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution and stirred for 5 min; subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H2O2 (10 M) were added in sequence and stirred rapidly for 30 min. Afterwards, the nanoparticles were washed with distilled water and centrifuged three times by ultrafiltration (10 kDa), and the obtained nanoparticles were CFE;

[0048] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronocell, Wuhan, China) were cultured in a 10-cm cell culture dish. When the cell density reached 90%, the cells were scraped off with a cell scraper and washed three times with PBS. Then, 1 mL of cell membrane lysis buffer was added, and the cells were lysed on ice for 14 min, followed by four cycles of repeated freezing and thawing with liquid nitrogen. The cell membranes were collected after centrifugation at 14,000 g for 30 min at 4°C.

[0049] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and ultrasonically treated at 36°C for 15 min; then, the mixture was extruded through 200 nm and 100 nm polycarbonate membranes for 14 times respectively, centrifuged at 14000 g for 5 min, and CFE@CM artificial nanocells were purified and collected, i.e., the antibacterial artificial nanocells in this embodiment.

[0050] Experimental Example 1

[0051] In this experimental example, the CFE@CM artificial nanocells prepared in Example 1 were photographed using a transmission electron microscope, which specifically includes the following steps:

[0052] (1) Take 5 μL of the CFE@CM solution prepared in Example 1 and drop it on the copper grid for transmission electron microscopy observation, let it stand at room temperature for 5 min, and absorb the excess solution with filter paper;

[0053] (2) Take 5 μL of 2% phosphotungstic acid solution and drop it on the copper grid for transmission electron microscopy observation, let it stand at room temperature for 45-60 seconds, and then use filter paper to absorb the excess solution;

[0054] (3) Allow to stand at room temperature for 10 min. After the liquid on the copper mesh evaporates, you can observe and take photos using a transmission electron microscope.

[0055] The transmission electron microscopy image of CFE@CM prepared in Example 1 is as follows: Figure 2 As shown by Figure 2It can be seen that the antibacterial artificial nanocells CFE@CM prepared in Example 1 were successfully prepared, with a diameter of about 100 nm, uniform size and good dispersibility.

[0056] Experimental Example 2

[0057] In this experimental example, the antibacterial effect of the CFE@CM artificial nanocells prepared in Example 1 was verified, including the following steps:

[0058] (1) Add 10 7 CFU of S. aureus were added to each well of another 96-well cell culture plate. 7 CFU of MRSA bacteria;

[0059] (2) Add a medium containing CM at pH 6.0, a medium containing CF@CM at pH 6.0, a medium containing ELC at pH 6.0, a medium containing CFE@CM at pH 6.0, a medium containing CM at pH 7.4, a medium containing CF@CM at pH 7.4, a medium containing ELC at pH 7.4, and a medium containing CFE@CM at pH 7.4 to the wells of two 96-well cell culture plates, respectively. After culturing at 37°C for 3 hours, measure the absorbance of each well at 600 nm, which is the bacterial activity density. Note: The CM in the culture medium is the cell membrane prepared in Example 1, the ELC in the culture medium is ilisimol, and the CFE@CM in the culture medium is the antibacterial artificial nanocell prepared in Example 1; the preparation method of CF@CM in the culture medium is the same as the preparation method of CFE@CM in Example 1, the only difference is that ilisimol or DMSO solution containing ilisimol is not added during the preparation of CF@CM.

[0060] The results of the test are as follows Figure 3 As shown in the figure, both CF@CM and CFE@CM artificial nanocells have antibacterial effects at pH 6.0 and pH 7.4, and the effect of CFE@CM is more obvious. In addition, the antibacterial effects of CF@CM and CFE@CM artificial nanocells at pH 6.0 are stronger than those at pH 7.0, indicating that both CF@CM and CFE@CM artificial nanocells have weak acid responsiveness.

[0061] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is limited only by the claims and their full scope and equivalents.

[0062] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A method for preparing antibacterial artificial nanocells, characterized in that: The antibacterial artificial nanocells are used to prepare a drug for treating osteomyelitis, and the antibacterial artificial nanocells are prepared by the following steps: Polyvinyl pyrrolidone or its aqueous solution, CuCl2•2H2O or its aqueous solution, FeCl2•4H2O or its aqueous solution, ilisimol or its DMSO solution, NaOH or its aqueous solution, H2O2 or its aqueous solution are mixed, stirred evenly, washed, ultrafiltered and centrifuged to obtain nanoparticles; Bone marrow stromal stem cells were used to prepare cell membranes; The nanoparticles and cell membranes are mixed evenly, and then ultrasonically incubated and extruded to obtain antibacterial artificial nanocells; wherein the protein mass ratio of the nanoparticles to the cell membrane is 1: (0.9-1.1).

2. The method for preparing antibacterial artificial nanocells according to claim 1, characterized in that: The preparation of the nanoparticles comprises the following steps: Mix the polyvinyl pyrrolidone aqueous solution, CuCl2•2H2O aqueous solution and FeCl2•4H2O aqueous solution, stir evenly, then add the DMSO solution containing ilisimol, stir evenly, then add the NaOH aqueous solution and the H2O2 aqueous solution in sequence, stir and react for 30 to 35 minutes, wash with distilled water, and ultrafilter and centrifuge for at least 3 times to obtain nanoparticles.

3. The method for preparing antibacterial artificial nanocells according to claim 2, characterized in that: The concentration of polyvinyl pyrrolidone aqueous solution is 202-247 mg / mL, the concentration of CuCl2•2H2O aqueous solution is 36-44 mg / mL, the concentration of FeCl2•4H2O aqueous solution is 18-22 mg / mL, the concentration of elisemomol in DMSO solution containing elisemomol is 168-206 mg / mL, the concentration of NaOH aqueous solution is 0.018-0.022 M, and the concentration of H2O2 aqueous solution is 9-10 M.

4. The method for preparing antibacterial artificial nanocells according to claim 3, characterized in that: The volume ratios of polyvinyl pyrrolidone aqueous solution, CuCl2•2H2O aqueous solution, FeCl2•4H2O aqueous solution, DMSO solution containing ilisimol, NaOH aqueous solution, and H2O2 are (1323~1617): (90~110): (90~110): (4.5~5.5): (2970~3630): (60~74).

5. The method for preparing antibacterial artificial nanocells according to claim 2, characterized in that: The pore size of the ultrafiltration membrane is 9 to 11 KDa.

6. The method for preparing antibacterial artificial nanocells according to claim 1, characterized in that: The preparation of cell membranes includes the following steps: When the bone marrow stromal stem cells were cultured to a cell density of 80-90%, the cells were scraped off with a cell scraper, washed three times with PBS, and cell membrane lysis buffer was added to lyse the cells on ice for 13-15 min. The cells were then repeatedly frozen and thawed with liquid nitrogen for 3-5 cycles, and the cell membranes were collected after centrifugation at 4°C and 13000-14000 g for 30-35 min to obtain cell membranes.

7. The method for preparing antibacterial artificial nanocells according to claim 1, characterized in that: The nanoparticles are mixed evenly with the cell membrane, and after ultrasonic treatment at 35-37°C for 15-20 min, they are first squeezed through a polycarbonate membrane with a pore size of 180-220 nm for 12-15 times, and then squeezed through a polycarbonate membrane with a pore size of 90-110 nm for 12-15 times, and centrifuged at 13000-14000 g for 3-5 min, and purified and collected to obtain antibacterial artificial nanocells.

8. An antibacterial artificial nanocell, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

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