Antibacterial artificial nano-cell, and preparation method therefor and use thereof

Abstract

An antibacterial artificial nano-cell and a preparation method therefor. The antibacterial artificial nano-cell is prepared by the following steps: mixing polyvinylpyrrolidone or an aqueous solution thereof, CuCl2·2H2O or an aqueous solution thereof, FeCl2·4H2O or an aqueous solution thereof, Elesclomol or a DMSO solution thereof, NaOH or an aqueous solution thereof, and H2O2 or an aqueous solution thereof, uniformly stirring the mixture, followed by washing, ultrafiltration and centrifugation to obtain nanoparticles, uniformly mixing the nanoparticles with cell membranes prepared from bone marrow stromal cells, and carrying out ultrasonic incubation and extrusion to obtain the antibacterial artificial nano-cells.

Description

An antibacterial artificial nanocell, its preparation method and application Technical Field

[0001] This invention belongs to the field of osteomyelitis treatment drug technology, specifically relating to an antibacterial artificial nanocell, its preparation method, and its application. Background Technology

[0002] Osteomyelitis is an infectious bone disease that primarily affects children and the elderly. In recent years, its incidence and the proportion of infections caused by drug-resistant bacteria have been increasing annually. Traditional treatment involves long-term, high-dose antibiotic use combined with surgical debridement, which presents two main challenges: ① the side effects / adverse reactions and induction of bacterial resistance caused by long-term, high-dose antibiotic use; ② the risks associated with debridement surgery, such as anesthesia complications, reinfection, and disability. Introducing novel non-antibiotic treatment strategies from the perspective of other treatment principles is of great significance for further improving the efficacy of osteomyelitis treatment. Summary of the Invention

[0003] This invention provides an antibacterial artificial nanocell that effectively avoids two major clinical problems in the treatment of osteomyelitis: the large side effects of long-term, high-dose antibiotic use and the high risk of debridement surgery. It kills bacteria from the perspective of mediating the Fenton reaction and copper death, two non-antibiotic principles, and achieves excellent therapeutic effects for osteomyelitis. In addition, this invention also provides a method for preparing antibacterial artificial nanocells and their applications.

[0004] The first aspect of this invention provides a method for preparing antibacterial artificial nanocells, which are used to prepare drugs for treating osteomyelitis. The antibacterial artificial nanocells are prepared through the following steps:

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

[0006] Cell membranes were prepared using bone marrow stromal stem cells;

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

[0008] The antibacterial artificial nanocells provided by this invention offer a new approach to the treatment of osteomyelitis. As a novel non-antibiotic strategy for treating osteomyelitis, the antibacterial artificial nanocells provided by this invention effectively avoid two major pain points in the traditional osteomyelitis treatment process: first, the side effects / adverse reactions and induction of bacterial resistance caused by long-term high-dose use of antibiotics; and second, the risks of anesthesia / reinfection / disability associated with debridement surgery.

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

[0010] After mixing polyvinylpyrrolidone aqueous solution, CuCl2·2H2O aqueous solution, and FeCl2·4H2O aqueous solution and stirring until homogeneous, DMSO solution containing illismol is added and stirred until homogeneous. Then, NaOH aqueous solution and H2O2 aqueous solution are added in sequence and stirred for 30-35 minutes. After washing with distilled water and ultrafiltration centrifugation at least 3 times, nanoparticles are obtained.

[0011] In one embodiment of the present invention, the concentration of the polyvinylpyrrolidone 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 ilismo in the DMSO solution containing ilismo 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 polyvinylpyrrolidone aqueous solution, CuCl2·2H2O aqueous solution, FeCl2·4H2O aqueous solution, DMSO solution containing irismo, 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 to 11 kDa.

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

[0015] When bone marrow stromal stem cells are cultured to a cell density of 80-90%, the cells are scraped off with a cell scraper, washed three times with PBS, and cell membrane lysis buffer is added. The cells are lysed on ice for 13-15 minutes, and then subjected to 3-5 cycles of freeze-thaw in liquid nitrogen. After centrifugation at 4°C and 13000-14000g for 30-35 minutes, the cell membrane is collected.

[0016] In one embodiment of the present invention, the nanoparticles are mixed evenly with a cell membrane, and then sonicated at 35-37°C for 15-20 min. The mixture is then extruded 12-15 times through a polycarbonate membrane with a pore size of 180-220 nm, and then extruded 12-15 times through a polycarbonate membrane with a pore size of 90-110 nm. The mixture is then centrifuged at 13000-14000 g for 3-5 min, and the resulting purified artificial nanocells for antibacterial use are collected.

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

[0018] A third aspect of the present invention provides the application of the above-mentioned antibacterial artificial nanocells in the preparation of a medicament for treating osteomyelitis.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The antibacterial artificial nanocells provided by this invention are a novel non-antibiotic strategy for the treatment of osteomyelitis, which effectively avoids two major pain points in the traditional osteomyelitis treatment process: first, the side effects / adverse reactions and the induction of bacterial resistance caused by long-term high-dose use of antibiotics; and second, the risks of anesthesia / reinfection / disability associated with debridement surgery.

[0021] 2. The antibacterial artificial nanocells provided in this embodiment of the invention, under the weakly acidic conditions of the osteomyelitis infection site, disintegrate and release copper and iron ions, the copper ion carrier irismo, and hydrogen peroxide. From the perspective of mediating the Fenton reaction and copper death—two non-antibiotic principles—they kill bacteria, achieving excellent therapeutic effects for osteomyelitis and providing a new approach to the treatment of osteomyelitis. Specifically, referring to Figure 4, in the antibacterial artificial nanocells provided in this embodiment of the invention, copper and iron ions pass through a copper-iron catalytic loop (Cu... + -to-Cu 2+ and Fe 3+ -to-Fe 2+ This enhances the Fenton reaction for sterilization. In addition, the copper ion carrier irismo transports excess copper ions into bacteria, inducing copper death. Furthermore, the use of bone marrow stromal stem cell membrane modification improves the biocompatibility and bone marrow targeting of the nanosystem.

[0022] 3. The drug for treating osteomyelitis prepared using the antibacterial artificial nanocells provided in this embodiment of the invention is a composite drug system that combines chemodynamic therapy and copper apoptosis induction, achieving excellent therapeutic effects for osteomyelitis. When used, the drug for treating osteomyelitis prepared using the antibacterial artificial nanocells provided in this embodiment of the invention is administered via intravenous injection to exert its antibacterial therapeutic effect on osteomyelitis.

[0023] 4. The method for preparing antibacterial artificial nanocells provided in this embodiment of the invention has a wide range of raw material sources and is simple and safe in process.

[0024] Instruction manual illustrations

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

[0026] Figure 2 is a transmission electron microscope image (scale bar, 100 nm) of the antibacterial artificial nanocells prepared in Example 1.

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

[0028] Figure 4 is a schematic diagram of the bactericidal mechanism of the antibacterial artificial nanocells in an embodiment of the present invention. Detailed Implementation

[0029] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0030] In this application, unless otherwise stated, the use of "or" means "and / or". In the case of multiple dependent claims, "or" is used only in alternatives to refer to more than one of the aforementioned independent or dependent claims.

[0031] Unless otherwise specified, 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 their abbreviations used in this invention have meanings commonly understood by those skilled in the art. The following is a list of some of the terms and abbreviations used in this invention:

[0033] The term "treatment" when used in relation to the treatment of a lesion or disease refers to the relief and / or elimination of one or more symptoms of that lesion or disease, and / or the delay in the progression of one or more symptoms of that lesion or disease and / or the reduction in the incidence or severity of one or more symptoms of that lesion or disease, and / or the prevention of that lesion or disease. The term "treatment" can also refer to preventative treatment, which includes delaying the onset of a lesion or disease or preventing the onset of a lesion or disease.

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art based on the present invention in practical applications still fall within 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 polyvinylpyrrolidone was dissolved in 1.47 mL of distilled water. Next, 100 μL of an aqueous solution of CuCl₂·2H₂O (39.7 mg / mL) and 100 μL of an aqueous solution of FeCl₂·4H₂O (20 mg / mL) were added, and the mixture was stirred for 5 min. Then, 5 μL of ilimismol solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution, and the mixture was stirred for 5 min. Subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H₂O₂ (10 M) were added sequentially, and the mixture was stirred rapidly for 30 min. Afterward, the nanoparticles were washed with distilled water, ultrafiltered (10 kDa), and centrifuged three times. The obtained nanoparticles were CFE.

[0038] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronosel, Wuhan, China) were cultured in 10 cm cell culture dishes. When the cell density reached 90%, the cells were scraped off with a cell scraper, washed three times with PBS, and then 1 mL of cell membrane lysis buffer was added. The cells were lysed on ice for 14 min, and then subjected to four cycles of freeze-thaw in liquid nitrogen. After centrifugation at 14000g for 30 min at 4°C, the cell membrane was collected.

[0039] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and sonicated at 36°C for 15 min. Then, the mixture was squeezed through 200 nm and 100 nm polycarbonate membranes 14 times each, centrifuged at 14000 g for 5 min, and purified to obtain CFE@CM artificial nanocells, which are the antibacterial artificial nanocells in this example.

[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 polyvinylpyrrolidone was dissolved in 1.47 mL of distilled water. Next, 90 μL of an aqueous solution of CuCl₂·2H₂O (39.7 mg / mL) and 90 μL of an aqueous solution of FeCl₂·4H₂O (20 mg / mL) were added, and the mixture was stirred for 5 min. Then, 4.5 μL of ilimismol solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution, and the mixture was stirred for 5 min. Subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H₂O₂ (10 M) were added sequentially, and the mixture was stirred rapidly for 30 min. Afterward, the nanoparticles were washed with distilled water, ultrafiltered (10 kDa), and centrifuged three times. The obtained nanoparticles were CFE.

[0043] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronosel, Wuhan, China) were cultured in 10 cm cell culture dishes. When the cell density reached 90%, the cells were scraped off with a cell scraper, washed three times with PBS, and then 1 mL of cell membrane lysis buffer was added. The cells were lysed on ice for 14 min, and then subjected to four cycles of freeze-thaw in liquid nitrogen. After centrifugation at 14000g for 30 min at 4°C, the cell membrane was collected.

[0044] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and sonicated at 36°C for 15 min. Then, the mixture was squeezed through 200 nm and 100 nm polycarbonate membranes 14 times each, centrifuged at 14000 g for 5 min, and purified to obtain CFE@CM artificial nanocells, which are the antibacterial artificial nanocells in this example.

[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 polyvinylpyrrolidone was dissolved in 1.47 mL of distilled water. Next, 110 μL of an aqueous solution of CuCl₂·2H₂O (39.7 mg / mL) and 110 μL of an aqueous solution of FeCl₂·4H₂O (20 mg / mL) were added, and the mixture was stirred for 5 min. Then, 5.5 μL of ilisimor solution (ELC, CAS: 488832-69-5, 187.2 mg / mL dissolved in DMSO) was added to the above solution, and the mixture was stirred for 5 min. Subsequently, 3.3 mL of NaOH aqueous solution (0.02 M) and 67 μL of H₂O₂ (10 M) were added sequentially, and the mixture was stirred rapidly for 30 min. Afterward, the nanoparticles were washed with distilled water, ultrafiltered (10 kDa), and centrifuged three times. The obtained nanoparticles were CFE.

[0048] Preparation of cell membrane (CM): Bone marrow stromal stem cells (purchased from Pronosel, Wuhan, China) were cultured in 10 cm cell culture dishes. When the cell density reached 90%, the cells were scraped off with a cell scraper, washed three times with PBS, and then 1 mL of cell membrane lysis buffer was added. The cells were lysed on ice for 14 min, and then subjected to four cycles of freeze-thaw in liquid nitrogen. After centrifugation at 14000g for 30 min at 4°C, the cell membrane was collected.

[0049] Preparation of CFE@CM: CFE and CM were mixed (1:1 cell membrane protein mass ratio) and sonicated at 36°C for 15 min. Then, the mixture was squeezed through 200 nm and 100 nm polycarbonate membranes 14 times each, centrifuged at 14000 g for 5 min, and purified to obtain CFE@CM artificial nanocells, which are the antibacterial artificial nanocells in this example.

[0050] Experimental Example 1

[0051] In this experimental example, the CFE@CM artificial nanocells prepared in Example 1 were photographed using transmission electron microscopy. The specific steps included:

[0052] (1) Take 5 μL of the CFE@CM solution prepared in Example 1 and drop it onto the copper grid for transmission electron microscopy. Let it stand at room temperature for 5 min and then use filter paper to absorb the excess solution.

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

[0054] (3) Continue to let it stand at room temperature for 10 minutes. After the liquid on the copper grid has evaporated, it can be observed and photographed by transmission electron microscopy.

[0055] Figure 2 shows a transmission electron microscope image of the CFE@CM prepared in Example 1. As can be seen from Figure 2, the antibacterial artificial nanocells CFE@CM prepared in Example 1 were successfully prepared. The diameter is about 100 nm, the size is uniform, and the dispersion is good.

[0056] Experiment Example 2

[0057] This experimental example verifies the antibacterial effect of the CFE@CM artificial nanocells prepared in Example 1, including the following steps:

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

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

[0060] The results are shown in Figure 3. Both CF@CM and CFE@CM artificial nanocells exhibited antibacterial effects at pH 6.0 and pH 7.4, with CFE@CM showing a more pronounced effect. Furthermore, the antibacterial effects of both CF@CM and CFE@CM artificial nanocells were stronger at pH 6.0 than at pH 7.0, indicating that both CF@CM and CFE@CM artificial nanocells possess weakly acid-responsive properties.

[0061] The above description discloses only preferred embodiments of the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to make good use of the 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 embodiments, those skilled in the art will readily foresee that all the raw materials or their equivalents, processing methods or their equivalents listed or exemplified in the present invention can achieve the present invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve the present invention. Examples 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 drugs for treating osteomyelitis, and are prepared through the following steps: Polyvinylpyrrolidone or its aqueous solution, CuCl2·2H2O or its aqueous solution, FeCl2·4H2O or its aqueous solution, elixmol or its DMSO solution, NaOH or its aqueous solution, and H2O2 or its aqueous solution are mixed, stirred evenly, washed, ultrafiltered and centrifuged to obtain nanoparticles. Cell membranes were prepared using bone marrow stromal stem cells; The nanoparticles are mixed evenly with the cell membrane, and then extruded after ultrasonic incubation to obtain antibacterial artificial nanocells; wherein the mass ratio of the nanoparticles to the cell membrane protein 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 includes the following steps: After mixing polyvinylpyrrolidone aqueous solution, CuCl2·2H2O aqueous solution, and FeCl2·4H2O aqueous solution and stirring until homogeneous, DMSO solution containing illismol is added and stirred until homogeneous. Then, NaOH aqueous solution and H2O2 aqueous solution are added in sequence and stirred for 30-35 minutes. After washing with distilled water and ultrafiltration centrifugation at least 3 times, nanoparticles are obtained.

3. The method for preparing antibacterial artificial nanocells according to claim 2, characterized in that, The concentrations of the polyvinylpyrrolidone aqueous solution were 202–247 mg / mL, the concentrations of the CuCl2·2H2O aqueous solution were 36–44 mg / mL, the concentrations of the FeCl2·4H2O aqueous solution were 18–22 mg / mL, the concentrations of ilismo in the DMSO solution containing ilismo were 168–206 mg / mL, the concentrations of the NaOH aqueous solution were 0.018–0.022 M, and the concentrations of the H2O2 aqueous solution were 9–10 M.

4. The method for preparing antibacterial artificial nanocells according to claim 3, characterized in that, The volume ratio of polyvinylpyrrolidone aqueous solution, CuCl2·2H2O aqueous solution, FeCl2·4H2O aqueous solution, DMSO solution containing irismol, NaOH aqueous solution, and H2O2 is (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 9–11 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 bone marrow stromal stem cells are cultured to a cell density of 80-90%, the cells are scraped off with a cell scraper, washed three times with PBS, and cell membrane lysis buffer is added. The cells are lysed on ice for 13-15 minutes, and then subjected to 3-5 cycles of freeze-thaw in liquid nitrogen. After centrifugation at 4°C and 13000-14000g for 30-35 minutes, the cell membrane is collected.

7. The method for preparing antibacterial artificial nanocells according to claim 1, characterized in that, The nanoparticles were mixed evenly with the cell membrane and ultrasonically treated at 35–37°C for 15–20 min. Then, the mixture was extruded through a polycarbonate membrane with a pore size of 180–220 nm 12–15 times, and then extruded through a polycarbonate membrane with a pore size of 90–110 nm 12–15 times. The mixture was then centrifuged at 13,000–14,000 g for 3–5 min and purified to obtain antibacterial artificial nanocells.

8. An antibacterial artificial nanocell, characterized in that, It is prepared by any one of the preparation methods of claims 1-7.

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