Acid-activated anti-infection nano vesicle as well as preparation method and application thereof

Abstract

The invention discloses an acid-activated anti-infection nano-vesicle and a preparation method and application thereof, the acid-activated anti-infection nano-vesicle is prepared by entrapping an acid-activated photosensitizer BODIPY molecule with an antibacterial function by a nano-vesicle derived from human skin fibroblast, the acid-activated anti-infection nano-vesicle is named as BODIPY at FNVs, and when the BODIPY at FNVs is in an acid environment, the BODIPY molecule can be activated. The preparation method has the advantages that the BODIPY is combined with the human skin fibroblast-derived nano-vesicles (FNVs), the preparation method is simple and low in cost, not only is the defect of the antibacterial ability of the FNVs made up, but also the tissue repair and infection control effects are enhanced, in the acid environment caused by bacterial infection, the BODIPY-coated FNVs generate singlet oxygen under the illumination condition, and the antibacterial activity of the human skin fibroblast-derived nano-vesicles is improved. Meanwhile, bioactive components such as mRNA and protein in the vesicles are released, the effects of sterilization, bacteriostasis and tissue repair promotion are jointly achieved, wound healing is accelerated, good biological safety is achieved, wide application prospects and popularization value are achieved in treatment of various infectious diseases, and the application range is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-infection drugs, and in particular to the technology for preparing anti-infection vesicles. Background Art

[0002] Infected wounds refer to tissue damage caused by infection with pathogenic microorganisms, which are common in burn wounds, postoperative wound infections, etc. This type of wound not only delays healing, but may also cause serious complications such as sepsis, tissue necrosis, and even amputation, which imposes a huge burden on patients' quality of life and the medical system. At present, the treatment of infected wounds mainly relies on wound debridement, local and systemic antibiotic treatment, etc. However, with the widespread use of antibiotics, the emergence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and drug-resistant Gram-negative bacteria has become more and more serious, and the difficulty of treatment has been greatly increased. Therefore, it is urgent to develop innovative treatment strategies for the intervention of infected wounds to meet the challenges of antibiotic-resistant bacteria and improve clinical efficacy.

[0003] Extracellular vesicles (EVs) are nanoparticles (30-200nm in diameter) wrapped by a lipid bilayer. They carry specific molecular information of donor cells and have advantages such as high stability, strong biocompatibility, and low immune response, making them a more promising cell-free treatment strategy than cell therapy. Although mesenchymal stem cell-derived EVs (MSC-EVs) can promote wound healing, their extraction process is complicated and there are problems such as red blood cell contamination and long culture time. At the same time, the antibacterial effect of MSC-EVs is limited and is not enough to cope with infection control. Summary of the invention

[0004] The purpose of the present invention is to provide an acid-activated anti-infective nanovesicle and a preparation method and application thereof, so as to solve the problem that there is a lack of anti-infective drugs for efficiently repairing infectious wounds with antibiotic-resistant bacteria in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An acid-activated anti-infection nanovesicle, which is prepared by encapsulating the acid-activated photosensitizer BODIPY molecule with antibacterial function in nanovesicles derived from human skin fibroblasts, is named BODIPY@FNVs. The BODIPY molecule is as follows:

[0007]

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

[0009] Furthermore, the BODIPY@FNVs is smaller than 200 nm.

[0010] The present invention also provides an application of the acid-activated anti-infective nanovesicles in preparing infectious wound repair drugs.

[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 the BODIPY molecules with the nanovesicles derived from human skin fibroblasts, and obtaining a nanovesicle solution encapsulating the BODIPY molecules by an extrusion method, wherein the nanovesicles encapsulating the BODIPY molecules are BODIPY@FNVs.

[0012] Furthermore, the mass ratio of the BODIPY molecule to the nanovesicle is in the range of 1:1-55.

[0013] Furthermore, in the nanovesicle solution system encapsulating 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: 6 The human skin fibroblasts and lysis buffer were mixed and resuspended at a ratio of 1 cell to 1 mL of lysis buffer, and 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 less than 200 nm.

[0015] Furthermore, the gradient extrusion operation is to reciprocately extrude the cell supernatant using 800nm, 400nm, and 200nm polycarbonate membranes in sequence.

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

[0017] The advantages of the present invention are that the nanovesicles (FNVs) prepared from human skin fibroblasts (HSFs) carry specific molecular information of donor cells, have higher yields and are more suitable for skin repair. The photosensitizer BODIPY molecules efficiently produce reactive oxygen species (ROS) in an acidic environment combined with photodynamic therapy (PDT), triggering bacterial lysis, showing excellent bactericidal effects and almost no drug resistance. Combining BODIPY with nanovesicles (FNVs) derived from human skin fibroblasts has a simple preparation method and low cost. It not only makes up for the lack of antibacterial power of FNVs, but also enhances the effects of tissue repair and infection control. In the acidic environment caused by bacterial infection, BODIPY@FNVs produces singlet oxygen ( 1O2), while releasing the bioactive components such as mRNA and protein inside the vesicles, which play a role in sterilization, antibacterial and promoting tissue repair, accelerate wound healing, have good biosafety, and have broad application prospects and promotion value in the treatment of various infectious diseases, and have wide applicability. It provides an ideal strategy for treating infected wounds and has a good therapeutic effect on the acidified wound microenvironment formed by bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0019] Figure 1 It is the preparation formula of BODIPY molecule in Example 1.

[0020] Figure 2 This is a graph showing protein expression in the nanovesicles encapsulating BODIPY molecules and human skin fibroblasts of the present invention.

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

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

[0023] Figure 5 The scratch test diagram of the human skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs) of the present invention.

[0024] Figure 6 This is a diagram showing the repair effect of the infectious wounds of rats treated with the human skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs) of the present invention.

[0025] Figure 7 The biotoxicity tissue sections of human skin fibroblast-derived nanovesicles (FNVs) and BODIPY nanovesicles (BODIPY@FNVs) of the present invention are shown. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary 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)ethanone (1.91 g, 10 mmol) in ethanol (10 mL), slowly drip 10% NaOH (10 mL) into the above solution and stir at room temperature until a large amount of precipitate is formed, which usually takes about 6 hours. Collect the precipitate on the filter and wash it 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 an ethanol (3 mL) solution at 60°C for 12 hours. After cooling to room temperature, the solvent was removed, and the obtained oily residue was dissolved in ethyl acetate and washed with water. The organic layer was washed with brine, dried over sodium sulfate, and concentrated to obtain 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 formed precipitate was filtered and washed with cold ethanol and ether to obtain a blue solid compound 3;

[0032] 4) Under argon conditions, compound 3 (112 mg, 0.15 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) in the dark, and then N, N-diisopropylethylamine (0.55 mL, 3 mmol) was added. After the solution was stirred for 10 minutes, BF3Et2O (0.37 mL, 3 mmol) was added and 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 and eluted with CH2Cl2 / petroleum ether (1 / 1) to obtain a purple solid product BODIPY molecule. The preparation formula of BODIPY molecule is as follows Figure 1 shown.

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

[0034]

[0035] Wherein R represents Br.

[0036] Of course, in addition to using 4-bromobenzaldehyde as the raw material, other raw materials can also be used as substitutes, so that R in formula (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 extruded through a liposome extruder to obtain nanovesicles. The specific preparation steps are as follows: 1) Cell culture and collection: Human skin fibroblasts were cultured with conditioned medium and cell pellets were collected. 2) Cell lysis: The pellet was added with hypotonic cell lysis buffer (about 1 mL of lysis buffer for every 1×10 6 cells), and let stand overnight at 4°C to fully lyse the cells. 3) Mechanical grinding: Use a tissue grinding pestle to grind the cell suspension 20 times to ensure complete cell lysis. 4) Centrifugation: Centrifuge at 3200g for 5 minutes at 4°C, discard the precipitate, and retain the supernatant. 5) Nanovesicle extrusion: Use a liposome extruder to pass the supernatant through a Millipore polycarbonate (PCTE) membrane with a pore size of 800nm, 400nm and 200nm in sequence. Each specification of PCTE membrane is reciprocated and extruded 20-40 times to achieve preliminary reconstruction of the cell membrane and remove impurities. 6) Nanovesicle collection: Collect the final extruded solution to obtain nanovesicles. This process ensures the successful preparation of nanovesicles and the integrity of the membrane structure.

[0038] (3) Preparation of BODIPY nanovesicles: After the BODIPY molecule of formula (I) obtained in step (1) and the nanovesicles obtained in step (2) are co-incubated, they are extruded through a liposome extruder to obtain BODIPY nanovesicles. The specific preparation steps are as follows: 1) Addition of BODIPY solution: Add the DMSO solution of the BODIPY molecule of formula (I) obtained in step (1) to the nanovesicle solution derived from human skin fibroblasts obtained in step (2). The mass ratio of BODIPY molecules to nanovesicles is controlled to be 1:4. 2) Incubation: The mixture is incubated at room temperature for 30 minutes to ensure that the BODIPY molecules are fully combined with the nanovesicles. 3) Extrusion reconstruction: Use a liposome extruder to pass the incubated mixture through a Millipore polycarbonate (PCTE) membrane with a pore size of 200 nm, and reciprocate for 20-40 times to complete the final reconstruction of the nanovesicles. 4) Purification: The reconstructed nanovesicles were purified using gradient centrifugation to remove free BODIPY molecules and other impurities that were not loaded into the nanovesicles, thereby obtaining 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 embodiment 1 is that in step (3), the mass ratio of BODIPY molecules to nanovesicles is 1:1, and in the acid-activated anti-infective nanovesicle solution system, the actual measured final concentration of BODIPY in the BODIPY nanovesicles (BODIPY@FNVs) is about 425 μmol / L.

[0043] Test Example 1

[0044] The protein expression of human skin fibroblasts in step (2) and BODIPY nanovesicles in step (3) of Example 1 was detected by Western Blot experiment. Figure 2 As shown, both HSFs and BODIPY@FNVs expressed 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: tumor susceptibility gene 101 recombinant protein; Calnexin: recombinant human calnexin).

[0045] The morphological structures of the human skin fibroblast-derived nanovesicles in step (2) of Example 1 and the BODIPY nanovesicles in step (3) were characterized using transmission electron microscopy. Figure 3 As shown, it can be observed that FNVs present a typical cup-shaped structure of 100 to 200 nm, indicating that FNVs were successfully prepared; at the same time, BODIPY@FNVs also maintained a cup-shaped structure, but showed swelling characteristics on the surface, further confirming that the BODIPY molecules were successfully encapsulated in FNVs.

[0046] Test Example 2 Antibacterial test of acid-activated anti-infection nanovesicles with antibacterial and healing functions

[0047] The antibacterial properties of the BODIPY nanovesicles in step (3) of Example 1 were tested as follows: BODIPY nanovesicles and 10 6 CFU / mL bacterial solution with pH = 5.5, incubate for 30 min and then use 1.5W / cm 2 The bacterial solution was then diluted 100 times and 100 μL was applied to the LB plate to observe the growth of the colonies. Figure 4As shown in the figure, compared with the control group, BODIPY@FNVs showed significant bactericidal effects on Escherichia coli and Staphylococcus aureus only after 808nm laser irradiation in an acidic environment of pH 5.5. At the same time, this also shows that the nanovesicles themselves do not have antibacterial function, but after encapsulating BODIPY molecules and irradiating with 808nm laser, their antibacterial ability is significantly improved.

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

[0049] The migration ability of the BODIPY nanovesicles in step (3) of Example 1 was tested, and the specific operation was as follows: The vesicles in step (2) of Example 1 and the BODIPY nanovesicles in step (3) were tested for migration performance, and the specific operation was as follows: The wound healing effect of the vesicles in step (2) of Example 1 and the BODIPY nanovesicles in step (3) was evaluated in vitro using a scratch test. Human umbilical vein endothelial cells (HUVEC) were cultured at 2.0×10 5 The cells were seeded in a 6-well plate at a density of 90% per well, and parallel scratches were made in each well using a 200 μL pipette tip. The wells were washed once with PBS and then treated with the vesicles in step (2) of Example 1, the BODIPY nanovesicles in step (3), or PBS. Photos were taken using an optical microscope after 12 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 adversely affect cell migration.

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

[0051] The in vivo therapeutic effect of BODIPY nanovesicles in step (3) of Example 1 was evaluated. The specific operation of constructing infectious wounds in rats is as follows: On day 0, surgical scissors were used to create a full-thickness skin defect on the back of the rat, and 20 μL of mixed bacterial solution was dripped into the wound to induce infection. The infectious wounds were divided into 3 groups. Except for the control group treated with only normal saline, the nanovesicles FNVs and BODIPY nanovesicles BODIPY@FNVs prepared in Example 1 were dripped into the other 2 groups respectively; after 24 hours, 1.5W / cm 2 The wound was irradiated with 808nm laser for 15min for photodynamic sterilization. Then, the corresponding PBS, nanovesicles FNVs or BODIPY nanovesicles BODIPY@FNVs were dripped every day, combined with photodynamic therapy, and the wound recovery was recorded on the 3rd, 7th and 14th days. Figure 6As shown in the figure, compared with the control group, the wound healing speed of the FNVs and BODIPY@FNVs treatment groups was significantly improved, among which the wound healing speed of the BODIPY@FNVs treatment group was the fastest. This shows that BODIPY@FNVs can effectively accelerate the healing of wounds through its antibacterial and healing-promoting functions.

[0052] The same infectious wound treatment test was carried out using the acid-activated anti-infective nanovesicles prepared in Examples 2 to 4, and the same effect was achieved.

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

[0054] HE staining was performed on the heart, liver, spleen, lung and kidney of rats treated with PBS, FNVs and BODIPY@FNVs. Figure 7 As shown, there were no obvious pathological changes and inflammatory infiltrations in the internal organs after treatment with PBS, FNVs and BODIPY@FNVs, which indicated that the prepared BODIPY@FNVs had good biocompatibility.

[0055] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. An acid-activated anti-infective nanovesicle, characterized in that: It is prepared by encapsulating the acid-activated photosensitizer BODIPY molecule with antibacterial function in nanovesicles derived from human skin fibroblasts, named BODIPY@FNVs. The BODIPY molecule is as follows: R is selected from Cl, Br, I.

2. The acid-activated anti-infective nanovesicle according to claim 1, characterized in that: The BODIPY@FNVs are smaller than 200 nm.

3. Use of the acid-activated anti-infective nanovesicles as described in any one of claims 1 to 2 in the preparation of infectious wound repair drugs.

4. A method for preparing the acid-activated anti-infective nanovesicles according to 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 nanovesicle solution encapsulating BODIPY molecules by extrusion method. The nanovesicles encapsulating BODIPY molecules are BODIPY@FNVs.

5. The method for preparing an acid-activated anti-infective nanovesicle according to claim 4, characterized in that: The mass ratio of the BODIPY molecule to the nanovesicle is in the range of 1:1-55.

6. The method for preparing an acid-activated anti-infective nanovesicle according to claim 4, characterized in that: In the nanovesicle solution system encapsulating BODIPY molecules, the concentration of BODIPY molecules in BODIPY@FNVs does not exceed 425 μmol / L.

7. The method for preparing an acid-activated anti-infective nanovesicle according to claim 5, characterized in that: The preparation process of the nanovesicles derived from human skin fibroblasts comprises: 6 The human skin fibroblasts and lysis buffer were mixed and resuspended at a ratio of 1 cell to 1 mL of lysis buffer, and 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 less than 200 nm.

8. The method for preparing an acid-activated anti-infective nanovesicle according to claim 7, characterized in that: The gradient extrusion operation is to reciprocately extrude the cell supernatant using 800 nm, 400 nm, and 200 nm polycarbonate membranes in sequence.

9. The method for preparing an acid-activated anti-infective nanovesicle according to claim 5, characterized in that: After BODIPY molecules were co-incubated with nanovesicles derived from human skin fibroblasts for 30 minutes, BODIPY@FNVs were obtained by reciprocating extrusion using a 200 nm polycarbonate membrane.

Images