Acid-activated anti-infective nanovesicles, and methods of making and using the same

Abstract

The application discloses an acid-activated anti-infection nanovesicle and a preparation method and application thereof, which is prepared by loading an acid-activated photosensitizer BODIPY molecule with antibacterial function into human skin fibroblast-derived nanovesicles, and is named as BODIPY@FNVs; the BODIPY molecule in the BODIPY@FNVs can be activated in an acidic environment.The advantages of the application include that the BODIPY is combined with human skin fibroblast-derived nanovesicles (FNVs), the preparation method is simple and low in cost, the antibacterial deficiency of the FNVs is compensated, and the tissue repair and infection control effects are enhanced; in an acidic environment caused by bacterial infection, the BODIPY@FNVs generates singlet oxygen under light irradiation, and releases biological active ingredients such as mRNA and proteins in the vesicle interior, and jointly plays the roles of sterilization, bacteriostasis and promotion of tissue repair, accelerates wound healing, has good biological safety, has wide application prospects and popularization value in the treatment of various infectious diseases, and has wide applicability.

Description

Technical Field

[0001] This invention relates to the field of anti-infective drug technology, and more particularly to anti-infective vesicle preparation technology. Background Technology

[0002] Infected wounds refer to tissue damage caused by pathogenic microorganisms, commonly seen in burn wounds and postoperative wound infections. These wounds not only delay healing but can also lead to serious complications such as sepsis, tissue necrosis, and even amputation, placing a significant burden on patients' quality of life and the healthcare system. Currently, the treatment of infected wounds mainly relies on wound debridement and local and systemic antibiotic therapy. However, with the widespread use of antibiotics, the emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and drug-resistant Gram-negative bacteria, is becoming increasingly serious, greatly increasing the difficulty of treatment. Therefore, developing innovative treatment strategies for the intervention of infected wounds to address the challenge of antibiotic-resistant bacteria and improve clinical efficacy is urgently needed.

[0003] Extracellular vesicles (EVs) are nanoparticles (30-200 nm in diameter) encapsulated in a lipid bilayer, carrying specific molecular information from donor cells. They possess advantages such as high stability, strong biocompatibility, and low likelihood of triggering immune responses, making them a more promising cell-free therapeutic strategy than cell therapy. While mesenchymal stem cell-derived EVs (MSC-EVs) can promote wound healing, their extraction process is complex, and they suffer from issues such as red blood cell contamination and long culture times. Furthermore, MSC-EVs have limited antibacterial effects, insufficient for infection control. Summary of the Invention

[0004] The purpose of this invention is to provide an acid-activated anti-infective nanovesicle, its preparation method, and its application, in order to solve the problem of the lack of efficient anti-infective drugs for repairing infected wounds with antibiotic-resistant bacteria in the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An acid-activated anti-infective nanovesicle, named BODIPY@FNVs, is prepared by encapsulating BODIPY molecules, an acid-activated photosensitizer with antibacterial function, onto nanovesicles derived from human skin fibroblasts. The BODIPY molecule is shown below:

[0007]

[0008] R is selected from Cl, Br, and I.

[0009] Furthermore, the BODIPY@FNVs are less than 200 nm.

[0010] The present invention also provides the application of the acid-activated anti-infective nanovesicles described above in the preparation of drugs for repairing infectious wounds.

[0011] The present invention also provides a method for preparing the acid-activated anti-infective nanovesicles, the process comprising: synthesizing BODIPY molecules, preparing nanovesicles derived from human skin fibroblasts, co-incubating BODIPY molecules with nanovesicles derived from human skin fibroblasts, and obtaining a nanovesicle solution loaded with BODIPY molecules by extrusion method, wherein the nanovesicles loaded with BODIPY molecules are BODIPY@FNVs.

[0012] Furthermore, the mass ratio of the BODIPY molecules to the nanovesicles ranges from 1:1 to 55.

[0013] Furthermore, within the nanovesicle solution system containing BODIPY molecules, the concentration of BODIPY molecules in BODIPY@FNVs does not exceed 425 μmol / L.

[0014] Furthermore, the preparation process of the nanovesicles derived from human skin fibroblasts includes: [the following steps are described in the original text, but the translation is incomplete and requires further context.] 6 Human skin fibroblasts were mixed and resuspended with 1 mL of lysis buffer at a ratio of 1 cell to 1 mL of lysis buffer. The mixture was lysed at 4 °C for 12 h. After centrifugation at 3200 g for 5 min, the cell supernatant was extruded through a gradient using a liposome extruder to obtain nanovesicles smaller than 200 nm.

[0015] Furthermore, the gradient extrusion operation involves sequentially extruding the cell supernatant using 800nm, 400nm, and 200nm polycarbonate membranes.

[0016] Furthermore, after co-incubating BODIPY molecules with nanovesicles derived from human skin fibroblasts for 30 min, BODIPY@FNVs were obtained by reciprocating extrusion using a 200 nm polycarbonate membrane.

[0017] The advantages of this invention are: Human skin fibroblast (HSF)-derived nanovesicles (FNVs) carry specific molecular information from the donor cells, resulting in higher yields and making them more suitable for skin repair. The photosensitizer BODIPY molecules, in an acidic environment, combine with photodynamic therapy (PDT) to efficiently generate reactive oxygen species (ROS), inducing bacterial lysis and exhibiting excellent bactericidal effects with almost no induction of drug resistance. Combining BODIPY with human skin fibroblast-derived nanovesicles (FNVs) is a simple and low-cost preparation method that not only compensates for the insufficient antibacterial activity of FNVs but also enhances tissue repair and infection control effects. In the acidic environment induced by bacterial infection, BODIPY@FNVs generate singlet oxygen (ROS) under light irradiation. 1O2) simultaneously releases bioactive components such as mRNA and proteins from the vesicles, which work together to kill bacteria, inhibit bacteria, and promote tissue repair, accelerating wound healing. It exhibits good biocompatibility and has broad application prospects and promotional value in the treatment of various infectious diseases, with wide applicability. It provides an ideal strategy for treating infected wounds, effectively addressing the acidified wound microenvironment caused by bacterial infection. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 This is the preparation equation for the BODIPY molecule in Example 1.

[0020] Figure 2 This is a diagram showing the protein expression of the BODIPY molecule-encapsulated nanovesicles and human skin fibroblasts according to the present invention.

[0021] Figure 3 Transmission electron microscopy images of the inventors' skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs).

[0022] Figure 4 This is an experimental diagram showing the antibacterial activity of the BODIPY nanovesicles (BODIPY@FNVs) of this invention against Staphylococcus aureus and Escherichia coli.

[0023] Figure 5 The images show scratch test results of the inventors' skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs).

[0024] Figure 6 The image shows the repair effect of the inventors' skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs) on infected wounds in rats.

[0025] Figure 7 This is a biotoxic tissue section of the inventors' skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs). Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0027] Example 1: Preparation of acid-activated anti-infection nanovesicles with anti-infection and healing-promoting functions

[0028] (1) Synthesis of BODIPY molecules:

[0029] 1) Dissolve 4-bromobenzaldehyde (1.85 g, 10 mmol) and 1-(4-(diethylamino)phenyl)ethyl ketone (1.91 g, 10 mmol) in ethanol (10 mL). Slowly add 10% NaOH (10 mL) to the solution while stirring at room temperature until a large amount of precipitate forms, which generally takes about 6 hours. Collect the precipitate on a filter and wash with cold MeOH to obtain yellow compound 1;

[0030] 2) Compound 1 (1.44 g, 4 mmol), nitromethane (4.88 g, 80 mmol), and NaOH (32 mg, 0.8 mmol) were heated in ethanol (3 mL) solution at 60 °C for 12 hours. After cooling to room temperature, the solvent was removed, and the resulting oily residue was dissolved in ethyl acetate and washed with water. The organic layer was washed with brine, dried with sodium sulfate, and concentrated to give a yellow oily residue, which was compound 2.

[0031] 3) Compound 2 (1.26 g, 3 mmol), ammonium acetate (8.08 g, 105 mmol), and n-butanol (30 mL) were placed in a 50 mL round-bottom flask and heated under reflux for 24 hours. After cooling to room temperature, the precipitate was filtered and washed with cold ethanol and diethyl ether to give a blue solid compound 3.

[0032] 4) Compound 3 (112 mg, 0.15 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) in the dark under argon atmosphere, followed by the addition of N,N-diisopropylethylamine (0.55 mL, 3 mmol). After stirring the solution for 10 minutes, BF3Et2O (0.37 mL, 3 mmol) was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was extracted with chloroform and washed with water and brine. The organic layer was dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with CH2Cl2 / petroleum ether (1 / 1) to give the purple solid product BODIPY molecules. The preparation equation for BODIPY molecules is as follows: Figure 1 As shown.

[0033] The BODIPY molecule prepared above is shown in formula (I). It will be activated in an acidic environment, and the structure of the activated BODIPY molecule is shown in formula (II).

[0034]

[0035] Where R represents Br.

[0036] Of course, in addition to using 4-bromobenzaldehyde as a raw material, other raw materials can be used to replace it, so that R in formulas (I) and (II) represents one of Br, Cl, and I.

[0037] (2) Preparation of human skin fibroblast-derived nanovesicles (FNVs): Human skin fibroblasts were lysed to obtain a membrane suspension, which was then extruded using a liposome extruder to obtain nanovesicles. The specific preparation steps are as follows: 1) Cell culture and collection: Human skin fibroblasts were cultured in conditioned medium, and the cell pellet was collected. 2) Cell lysis: The pellet was added to a hypotonic cell lysis buffer (approximately 1 mL of lysis buffer was used for every 1 × 10⁻⁶ cells). 6 1) Cells were incubated overnight at 4°C to fully lyse the cells. 2) Mechanical grinding: The cell suspension was ground 20 times using a tissue grinding pestle to ensure complete cell lysis. 3) Centrifugation: The cell suspension was centrifuged at 3200g for 5 minutes at 4°C, the precipitate was discarded, and the supernatant was retained. 4) Nanovesicle extrusion: The supernatant was extruded sequentially through Millipore polycarbonate (PCTE) membranes with pore sizes of 800nm, 400nm, and 200nm using a liposome extruder. Each size of PCTE membrane was extruded 20-40 times to achieve preliminary reconstruction of the cell membrane and remove impurities. 5) Nanovesicle collection: The final extruded solution was collected to obtain nanovesicles. This process ensured the successful preparation of nanovesicles and the integrity of the membrane structure.

[0038] (3) Preparation of BODIPY nanovesicles: After co-incubating the BODIPY molecules of formula (I) obtained in step (1) and the nanovesicles obtained in step (2), the BODIPY nanovesicles were extruded through a liposome extruder. The specific preparation steps are as follows: 1) Addition of BODIPY solution: The DMSO solution of the BODIPY molecules of formula (I) obtained in step (1) was added to the nanovesicle solution derived from human skin fibroblasts obtained in step (2). The mass ratio of BODIPY molecules to nanovesicles was controlled to be 1:4. 2) Incubation: The mixture was incubated at room temperature for 30 minutes to ensure that the BODIPY molecules were fully bound to the nanovesicles. 3) Extrusion reconstruction: The incubated mixture was extruded through a Millipore polycarbonate (PCTE) membrane with a pore size of 200 nm using a liposome extruder, and the mixture was reciprocated 20-40 times to complete the final reconstruction of the nanovesicles. 4) Purification: The reconstructed nanovesicles were purified by gradient centrifugation to remove free BODIPY molecules and other impurities that were not encapsulated in the nanovesicles, resulting in a pure acid-activated anti-infective nanovesicle solution. The final concentration of BODIPY in the BODIPY nanovesicles (BODIPY@FNVs) was measured to be approximately 170 μmol / L.

[0039] Example 2

[0040] The difference between this embodiment and embodiment 1 is that in step (3), the mass ratio of BODIPY molecules to nanovesicles is 1:55.

[0041] Example 3

[0042] The difference between this embodiment and Example 1 is that in step (3), the mass ratio of BODIPY molecules to nanovesicles is 1:1. In the acid-activated anti-infection nanovesicle solution system, the final measured concentration of BODIPY in BODIPY nanovesicles (BODIPY@FNVs) is approximately 425 μmol / L.

[0043] Test Example 1

[0044] Western blotting was used to detect protein expression in human skin fibroblasts (step 2) and BODIPY nanovesicles (step 3) of Example 1. Figure 2 As shown, both HSFs and BODIPY@FNVs express the protein markers CD9 and TSG101, while the endoplasmic reticulum protein marker Calnexin expressed by HSFs was not detected in BODIPY@FNVs, indicating that the preparation of FNVs was successful. (CD9: leukocyte differentiation antigen 9; TSG101: recombinant protein of tumor susceptibility gene 101; Calnexin: recombinant human calcinin).

[0045] The morphology and structure of the human skin fibroblast-derived nanovesicles from step (2) and the BODIPY nanovesicles from step (3) of Example 1 were characterized using transmission electron microscopy. Figure 3 As shown, FNVs exhibit a typical cup-shaped structure of 100-200 nm, indicating that FNVs were successfully prepared. Meanwhile, BODIPY@FNVs also maintained the cup-shaped structure, but the surface showed expansion characteristics, further confirming that BODIPY molecules were successfully encapsulated in FNVs.

[0046] Test Example 2: Antibacterial Test of Acid-Activated Anti-Infective Nanovesicles with Antibacterial and Healing-Promoting Functions

[0047] The antibacterial properties of BODIPY nanovesicles in step (3) of Example 1 were tested. The specific operation was as follows: BODIPY nanovesicles and 10 6 A mixed solution of CFU / mL bacterial culture at pH 5.5 was incubated for 30 min, followed by incubation with 1.5 W / cm² solution. 2 The bacterial culture was irradiated with an 808nm laser for 15 minutes, then diluted 100-fold, and 100μL was plated on LB agar plates to observe colony growth. Figure 4As shown, compared with the control group, BODIPY@FNVs only exhibited significant bactericidal effects against Escherichia coli and Staphylococcus aureus under acidic conditions of pH 5.5 after irradiation with an 808nm laser. This also indicates that nanovesicles themselves do not possess antibacterial function, but their antibacterial ability is significantly enhanced after encapsulating BODIPY molecules and undergoing 808nm laser irradiation.

[0048] Test Example 3: Scratch test of acid-activated anti-infective nanovesicles with antibacterial and healing-promoting functions.

[0049] The migration ability of BODIPY nanovesicles in step (3) of Example 1 was tested. The specific operation was as follows: The migration performance of the vesicles in step (2) and BODIPY vesicles in step (3) of Example 1 was tested. The specific operation was as follows: The wound healing effect of the vesicles in step (2) and BODIPY nanovesicles in step (3) of Example 1 was evaluated in vitro using a scratch test. Human umbilical vein endothelial cells (HUVECs) were used at 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 90% in 6-well plates. Parallel scratches were made in each well using the tip of a 200 μL pipette. Cells were washed once with PBS and then treated with the vesicles from step (2) of Example 1, the BODIPY nanovesicles from step (3), or PBS, respectively. Images were taken using an optical microscope after 12 hours and 24 hours. Figure 5 As shown, compared with the control group, FNVs and BODIPY@FNVs significantly promoted cell migration, and the addition of BODIPY molecules did not have an adverse effect on cell migration.

[0050] Test 4: Treatment of infected wounds with acid-activated antibacterial nanovesicles possessing antibacterial and healing-promoting functions.

[0051] The in vivo therapeutic effect of BODIPY nanovesicles in step (3) of Example 1 was evaluated. The specific procedures for constructing infected wounds in rats were as follows: On day 0, a full-thickness skin defect was created on the dorsal side of the rat using surgical scissors, and 20 μL of mixed bacterial solution was instilled to induce infection. The infected wounds were divided into three groups. Except for the control group, which was treated only with physiological saline, the other two groups were instilled with nanovesicles FNVs and BODIPY nanovesicles BODIPY@FNVs prepared in Example 1, respectively; after 24 hours, 1.5 W / cm² of saline solution was used. 2 The wound was irradiated with an 808nm laser for 15 minutes for photodynamic sterilization. Afterwards, appropriate PBS, FNV nanovesicles, or BODIPY@FNV nanovesicles were instilled daily in conjunction with photodynamic therapy, and wound healing was recorded on days 3, 7, and 14. Figure 6As shown, compared with the control group, the wound healing speed of both the FNVs and BODIPY@FNVs treatment groups was significantly improved, with the BODIPY@FNVs treatment group exhibiting the fastest wound healing speed. This indicates that BODIPY@FNVs can effectively accelerate wound healing through its antibacterial and healing-promoting functions.

[0052] The acid-activated anti-infective nanovesicles prepared in Examples 2-4 were used in the same infectious wound treatment experiment and showed the same effect.

[0053] Test 5: Biocompatibility test of acid-activated antibacterial nanovesicles with antibacterial and healing-promoting functions.

[0054] HE staining was performed on the heart, liver, spleen, lungs, and kidneys of rats treated with PBS, FNVs, and BODIPY@FNVs. Figure 7 As shown, no obvious pathological changes or inflammatory infiltration were observed in the visceral organs after treatment with PBS, FNVs, and BODIPY@FNVs, indicating that the prepared BODIPY@FNVs have good biocompatibility.

[0055] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. An acid-activated nanovesicle that kills bacteria and fights infection through photodynamic therapy, characterized in that: It was prepared by encapsulating the acid-activated photosensitizer BODIPY molecule with photodynamic antibacterial function in nanovesicles derived from human skin fibroblasts, and named BODIPY@FNVs. The BODIPY molecule is shown below: ； R is selected from Cl, Br, and I.

2. The nanovesicles according to claim 1, characterized in that: The BODIPY@FNVs are less than 200 nm.

3. The use of nanovesicles as described in any one of claims 1-2 in the preparation of photodynamic therapy drugs for infected wounds.

4. A method for preparing nanovesicles as described in any one of claims 1-2, characterized in that: The process includes: synthesizing BODIPY molecules, preparing nanovesicles derived from human skin fibroblasts, co-incubating BODIPY molecules with nanovesicles derived from human skin fibroblasts, and obtaining a nanovesicle solution loaded with BODIPY molecules by extrusion. The nanovesicles loaded with BODIPY molecules are called BODIPY@FNVs.

5. The method for preparing nanovesicles according to claim 4, characterized in that: The mass ratio of the BODIPY molecules to the nanovesicles ranges from 1:1 to 55.

6. The method for preparing nanovesicles according to claim 4, characterized in that: Within the BODIPY molecule-encapsulated nanovesicle solution system, the concentration of BODIPY molecules in BODIPY@FNVs does not exceed 425 μmol / L.

7. The method for preparing nanovesicles according to claim 5, characterized in that: The preparation process of the nanovesicles derived from human skin fibroblasts includes: [the following steps are described in the original text, but the translation is incomplete and requires further context.] 6 Human skin fibroblasts were mixed and resuspended with 1 mL of lysis buffer at a ratio of 1 cell to 1 mL of lysis buffer. The mixture was lysed at 4 °C for 12 h. After centrifugation at 3200 g for 5 min, the cell supernatant was extruded through a gradient extrusion device to obtain nanovesicles smaller than 200 nm.

8. The method for preparing nanovesicles according to claim 7, characterized in that: The gradient extrusion operation involves sequentially extruding the cell supernatant using 800 nm, 400 nm, and 200 nm polycarbonate membranes.

9. The method for preparing nanovesicles according to claim 5, characterized in that: BODIPY molecules were co-incubated with nanovesicles derived from human skin fibroblasts for 30 min, and then BODIPY@FNVs were obtained by reciprocating extrusion using a 200 nm polycarbonate membrane.

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