CN116236456A - Targeting endothelial cell delivery vehicle and application thereof in promoting wound healing - Google Patents
Targeting endothelial cell delivery vehicle and application thereof in promoting wound healing Download PDFInfo
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Abstract
A targeted endothelial cell delivery vehicle prepared by the steps of: the method comprises the steps of obtaining ABs through chemical induction of fat stem cell apoptosis, and then sequentially carrying out the steps of hypotonic, ultrasonic, active molecule mixing for promoting healing, extrusion and the like to obtain the functionalized apoptotic body nano vesicle carrying active molecule for promoting wound healing, wherein the prepared ABs surface has CX3CL1 protein. Proved by verification, the prepared ABs nano vesicle has good biocompatibility, and can be used for realizing 'finding me-eating me' of endothelial cells in an anoxic microenvironment by inducing CX3CL1/CX3CR1, thereby promoting proliferation, migration and tube forming capacity of the endothelial cells. In vivo experiments, ABs nanometer vesicles can promote rapid wound closure, and simultaneously can realize slow release of vascular-promoting drugs and promote neovascularization of wound surfaces of diabetic rats by releasing signals of finding me-eating me to target endothelial cells.
Description
Technical Field
The invention relates to a medical product made of biological materials, in particular to an extracellular vesicle for removing unknown cell contents, which has safety and is used as a delivery carrier, thereby being beneficial to promoting wound repair.
Background
Neovascularization is a critical process driving ischemic tissue repair, however for chronic (difficult to heal) wounds, the long-term hypoxic microenvironment results in insufficient vascularization and thus delayed wound healing. Endothelial cells, which are key cells constituting new blood vessels, clearly play a central role in the vascularization process.
CN202211118736.X discloses a magnesium hydride loaded microneedle and its application in wound healing, wherein the microneedle patch comprises a support layer and a microneedle, the needle body of the microneedle is made of biodegradable material, and the needle tip contains MgH 2 . In vitro and in vivo verification shows that MgH is loaded 2 The microneedle patches of (a) can promote the wound healing process, promote the polarization of M2, enhance the proliferation and migration of cells, improve angiogenesis and reduce ROS production.
CN202211169998.9 discloses a silver-containing microcarrier comprising ascorbic acid, ag + And a coating agent, which is bonded toConfigured as a "flower-like" with a surface area of 106.5m 3 /g±20m 3 And/g, the mesoporous material has mesoporous characteristic, and the pore diameter range is 5 nm-225 nm. Silver-containing microcarriers were verified to release Ag in response to environmental ROS + The exosome is loaded, so that the resistance of the exosome to oxidative denaturation can be improved, the stability of the exosome is improved, and the exosome is prevented from being rapidly damaged to be inactivated. Exosome-loaded microcarriers are delivered to the wound surface, such as: after the diabetes wound surface, bacteria can be effectively eliminated, and the apoptosis of damaged oxidized cells is promoted, so that the regeneration microenvironment is improved, exosomes are delivered and continuously released at target sites, the biological functions of fibroblasts and endothelial cells are regulated, the angiogenesis is promoted, and the wound surface healing is accelerated.
Delivery strategies targeting endothelial cells have gained widespread attention in recent years (Science, 2018, 359, 1335-1336), delivery vehicles mainly including liposomes (Biomaterials, 2020, 232, 119706) and Extracellular Vesicles (EVs). Liposome-targeted endothelial cell delivery strategies are common, for example: kowalski et al (European Journal of Pharmaceutics and Biopharmaceutics,2015, 89, 40-47) intervene in inflammation by integrin receptor-ligand mediated liposome delivery of drugs into microvascular endothelial cells at the site of inflammation. As synthetic nanoparticles, liposomes have the advantage of being easy to modify and prepare, but this also inevitably leads to inherent toxicity. EVs are natural cell products, and therefore have low immunogenicity and low cytotoxicity compared to liposomes. In addition, the lipid membrane composition makes EVs an ideal choice for fusion with target cells, and can avoid degradation while loading the drug. Therefore, EVs have stronger application value as drug carriers compared with liposomes.
Currently, EVs-based drug loading methods include both endogenous and exogenous drug loading. Endogenous drug delivery is the delivery of drugs into cells via dendrimers/cell membrane penetrating peptides and the like, which then induce the cells to produce drug-encapsulated EVs. For example, zhuang et al (nanoscales, 2020, 12:173-188) prepared cell penetrating peptides and tumor necrosis factor-alpha (TNF-alpha) -anchored exosomes coupled to superparamagnetic iron oxide nanoparticles with membrane-targeted anti-cancer activity for inhibiting tumor growth. But this approach is complex to prepare and has low drug encapsulation efficiency. Exogenous drug carriers include electroporation (Neurological Research,2008, 30, 288-293), osmotic shock (Adv health Mater.2022,11 (5): e 2100047), extrusion (Int J pharm.2020,5;573:118802;J Extracell Vesicles.2021,10 (13): e 12163), freeze thawing (Micromachines (Basel), 2019,1,10 (11): 750; jnanobiliotechnology.2021, 30,19 (1): 459), surfactants (am.j. Trans.res, 2020, 12:6302-6313), ultrasound (Cancer Res,2017,77:3-13; theranostics,2022, 12:1247-1266), and the like, but the following problems still remain: (1) Failure to remove residual components that are not relevant to the therapeutic purpose can present a potential threat; (2) Physical and chemical treatments on the cell membrane surface may destroy the structure and function; (3) May affect the loading stability, for example electroporation may lead to the production of nucleic acid aggregates (J.tissue.Eng.regen, 2016,10: E167-E176).
Disclosure of Invention
It is an object of the present invention to provide a method for preparing extracellular vesicles to remove unknown cellular contents and to improve the safety of application in humans.
Another object of the present invention is to provide a delivery vehicle for targeting endothelial cells, which uses the prepared extracellular vesicles as delivery vehicles, and is beneficial for promoting (such as chronic diabetes) wound healing.
It is a further object of the present invention to provide vesicles that are targeted to endothelial cells for promoting (e.g., chronic diabetes) wound healing.
It is a further object of the present invention to provide the use of a targeted endothelial cell delivery vehicle for the manufacture of a medicament for promoting (e.g. chronic diabetes) wound healing.
A fifth object of the present invention is to provide a use of a targeted endothelial cell delivery vehicle for the preparation of a medical device for promoting (e.g. chronic diabetes) wound healing.
Apoptotic Bodies (ABs) are a major class of EVs that are formed by the breakdown of apoptotic processes formed following programmed cell death (Nature Reviews Drug Discovery,2022, 21, 379-399). In contrast to exosomes and microvesicles which can only function by pinocytosis-endocytosis, ABs itself can send out a "find me" signal and a "eat me" signal, attracting phagocytes for phagocytosis. Furthermore, the high surface-to-volume ratio of ABs promotes surface-specific interactions with target cells (Journal of Controlled Release,2022, 351, 394-406). Thus, targeting the delivery of specific pro-vascularization drugs to endothelial cells via ABs is a breakthrough delivery strategy.
The CX3CR1 protein is expressed on the surface of endothelial cells through hypoxia induction, so that specific receptor-ligand mediation with apoptotic body surface CX3CL1 is realized. The "eat me" signal is mediated by phosphatidylserine receptor-ligands on the endothelial cell membrane and apoptotic body membrane surfaces. Under the dual signal effect, specific targeted delivery of drugs to endothelial cells is achieved.
A process for preparing extracellular vesicle includes such steps as chemically inducing the apoptosis of fat stem cells to obtain ABs (diameter is 600-1000 nm), and sequentially mixing and squeezing the active molecules for promoting wound healing.
The prepared ABs surface has CX3CL1 protein, can be identified and combined with CX3CR1 on endothelial cells, and realizes targeted delivery of the endothelial cells. The active molecules with the function of promoting wound healing are loaded into ABs by taking the active molecules as a carrier, and the targeted delivery of the active molecules into endothelial cells can be realized, thereby being beneficial to (for example, diabetes) wound repair
Active molecules for promoting wound healing such as: GLP-1 (7-36) and its metabolites, liraglutide and its metabolites, semraglutide and its metabolites, and Deferoxamine (DFO). GLP-1 (7-36) and its metabolite have effect in promoting healing of diabetic wound surface. DFO acts as an angiogenic agent directly on endothelial cells to up-regulate VEGF expression and promote vascular growth (Journal of Tissue Engineering and Regenerative Medicine,2016, 10, E167-E176).
The obtained DFO-carrying nABs (DFO-nABs) were confirmed to have particle diameters of 300nm to 500nm and surface charges of-15 mV.
Another method for preparing functionalized nano vesicles comprises the steps of firstly, re-suspending ABs in a hypotonic dissolution buffer solution at 4+/-0.5 ℃ for 1 hour+/-0.2 hour to obtain cellular membrane porous ABs (pABs); then, the mixed solution of ABs and the active molecules for promoting wound healing is subjected to ultrasonic treatment for 10 minutes plus or minus 2 minutes, 3000g plus or minus 200g is centrifuged for 10 minutes plus or minus 2 minutes, the supernatant is removed and then washed for a plurality of times (such as 2 times, 3 times or more), and the active molecules for promoting wound healing on the surface of ABs are removed; then, ABs loaded with the healing promoting active molecules are respectively put into a squeezer and repeatedly squeezed for 10 to 15 times.
Another method for preparing the functionalized nano vesicles comprises the steps of firstly, re-suspending ABs in a hypotonic dissolution buffer solution at 4 ℃ for 1 hour to obtain pABs; the mixture of ABs and DFO was sonicated for an additional 10 minutes. Centrifuging at 3000g for 10min, removing supernatant, and washing for several times (such as 2 times, 3 times or more), and removing DFO on ABs surface; the ABs loaded with DFO is then fed into an extruder (e.g., avanti, USA) and repeatedly extruded 10-15 times.
The method of the invention adopts a hypotonic dissolving buffer solution of 10mM of Tris with pH7.4 and containing 10mM of MgCl 2 And 1mM phenylmethylsulfonyl fluoride.
In the method of the invention, a polycarbonate filter membrane is used as an extruder, and the suitable pore sizes are as follows: 1000nm or 425nm.
The dosage ratio of the method of the invention, ABs and the healing promoting active molecule is as follows: 10wt%, 20wt% or 30wt%.
In vitro experiments show that ABs nano vesicles have good biocompatibility, and can be used for inducing endothelial cells in an anoxic microenvironment to find me and eat me through CX3CL1/CX3CR1, so that proliferation, migration and tube forming capacity of the endothelial cells are promoted. In vivo experiments, ABs nanometer vesicles can promote rapid wound closure, and simultaneously can realize slow release of vascular-promoting drugs and promote neovascularization of wound surfaces of diabetic rats by releasing signals of finding me-eating me to target endothelial cells.
The functional nano vesicle prepared by the invention is used as an active ingredient for targeting endothelial cells and promoting (such as chronic diabetes) wound healing. Mixing the vesicle with other adjuvants, and making into medicine (preparation) for repairing diabetic wound.
These pharmaceutical excipients may be used conventionally in various formulations, such as: but are not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, and the like; may also be selected for use in response to a substance, such as: the auxiliary materials can effectively improve the stability and the solubility of the compounds contained in the composition or change the release rate, the absorption rate and the like of the compounds, thereby improving the metabolism of various compounds in organisms and further enhancing the administration effect of the composition.
In aqueous injection solutions, the auxiliary materials generally comprise isotonic agents and buffers, and necessary emulsifying agents (such as Tween-80, pluronic, and Poloxamer), solubilizers, and bacteriostats. In addition, the composition also comprises other pharmaceutically acceptable pharmaceutical excipients, such as: antioxidants, pH adjusters, analgesics, and the like.
Adjuvants used in preparing liquid preparations generally include solvents, water, oils (e.g., fatty acids), emulsifiers, and optionally preservatives.
Various excipients and vesicles of the invention are formulated into dosage forms useful for administration (drug delivery), such as: but not limited to, aqueous injection, powder for injection, powder, patch, suppository, emulsion, cream, gel, aerosol, spray, powder spray, sustained release agent, controlled release agent, etc. In addition, specific purposes or modes of administration may be achieved, such as: sustained release administration, controlled release administration, pulse administration, etc., and auxiliary materials used, such as: but are not limited to, gelatin, albumin, chitosan, polyethers and polyesters such as: but are not limited to, polyethylene glycol, polyurethane, polycarbonate, copolymers thereof, and the like. The main expression "advantageous administration" is referred to as: but not limited to, improving therapeutic effect, improving bioavailability, reducing toxic side effects, improving patient compliance, and the like.
Drug-containing medical devices that combine drugs with medical devices have also become common, such as: dressing comprising vesicles according to the invention. The vesicle of the invention is also used as an active ingredient to be loaded or coated on a material for preparing a medical device for repairing the diabetic wound. Common scaffold materials are: PLA, PLGA, metals, and the like. And mixing with biocompatible degradable material to obtain micropins and their micropin array, or loading in metal micropins to obtain micropin chip. When the micro-needle is inserted into the skin, the vesicle is released in the epithelial tissue, so that the healing capacity of the diabetic wound surface is improved.
Drawings
FIG. 1 is a graph showing the results of preparation and characterization of desferrioxamine-loaded apoptotic-body nanovesicles (DFO-nANBs), wherein a is a schematic representation of the preparation of DFO-nANBs; b is a fluorescent staining image of the apoptosis markers Annexin V and C1q on the ABs surface; c is an image of nuclear content (blue in visual field) transferred to cell membrane boundaries during nABs preparation, indicating removal of nuclear content; d is an SEM image of ABs; e is SEM image of porous apoptotic bodies (pABs) after hypotonic treatment; f is SEM image of apoptotic body nanovesicles (nABs) after extruder treatment; g is SEM of DFO-nABs; h is a fluorescence diagram of water-soluble drug molecules entering the cell membrane; i is a fluorescence image of the drug molecules successfully loaded into the apoptotic body nano vesicles; j is a diameter comparison graph of the vesicles of each group; k is a Zeta potential comparison graph of each group; l is in vitro release profile of nABs loaded with DFO at different concentration gradients;
FIG. 2 is a graph showing the results of cell uptake and cell compatibility assays of nABs with DFO-nABs; wherein a is a schematic diagram of ABs binding to endothelial cell surface CX3CL1/CX3CR1 receptor-ligand to achieve "find me" signal release; b is WB detection of CX3XR1 expression level of endothelial cells after 24h of normoxic and anoxic pretreatment; c is the statistical analysis of CX3XR1 expression quantity of endothelial cells under normoxic and anoxic environments; d is a typical fluorescent image of nABs phagocytized after 24 hours of co-culture with endothelial cells after 24 hours of normoxic/hypoxic pretreatment released by the "eat me" signal; e is a typical fluorescent image of nABs phagocytized after 24 hours of co-culture with normoxic/hypoxic pretreated endothelial cells released by "eat me" signals; f is a representative image of live-dead staining of different concentrations of nABs after co-culturing with endothelial cells after 24 hours of hypoxia pretreatment; g is a representative image of live and dead staining after co-culturing DFO at different concentrations with endothelial cells after 24 hours of hypoxia pretreatment; h is a graph of cell viability results at 24, 48 and 72 hours of co-culture of different concentrations of nABs with endothelial cells after 24 hours of hypoxic pretreatment; i is a graph of cell viability results at 24, 48 and 72 hours of co-culture of DFO at different concentrations with endothelial cells after 24 hours of hypoxic pretreatment; j is a graph of the percentage of living cells to dead cells after co-culturing different concentrations of nABs with endothelial cells after 24 hours of hypoxia pretreatment for 72 hours; k is a graph of the percentage of living cells to dead cells after co-culturing DFO with different concentrations and endothelial cells after 24 hours of anoxic pretreatment for 72 hours;
FIG. 3 is a graph of experimental results of different concentrations of DFO on endothelial cell vascularization and in vitro wound healing capacity after 24h of hypoxic pretreatment; wherein a is a graph of the effect of DFO with different concentrations on the endothelial cell tube forming experiment after hypoxia pretreatment for 24 hours; b is a graph of the effect of different concentrations of DFO on the scratch migration ability of endothelial cells after anoxic pretreatment for 24 hours; c is a connection quantity statistical graph of the new blood vessels; d is a statistical chart of the number of the new blood vessel network; e is a statistical chart of the length of the new blood vessel; f is a statistical diagram of total area of the new blood vessel network; g is a cell mobility statistical graph;
FIG. 4 is a graph showing the effect of different concentrations of DFO-nABs on endothelial cell migration ability after 24h of hypoxic pretreatment; wherein a is a schematic diagram of nABs, which can induce endothelial cells after hypoxia pretreatment for 24 hours through releasing double signals to find and phagocytize themselves so as to promote migration capacity; b is a typical image of endothelial cells Transwell after hypoxia pretreatment for 24 hours at different concentrations of DFO-nABs; c is a statistical plot of cell numbers per field of view after treatment of endothelial cells at different concentrations of DFO-nABs following a 24h hypoxic pretreatment; d is an OD value comparison result graph of each group after eluting with glacial acetic acid;
FIG. 5 is a graph showing the results of verification of the effect of different treatment modes on wound healing of diabetes mellitus in rats; a is a schematic diagram of constructing a rat diabetes wound model; b is a typical image of the control, DFO, nABs and DFO-nABs treated wounds at days 3, 7 and 14; c is a software simulated image of the control, DFO, nABs, DFO-nABs treated wound on days 3, 7 and 14; d is a comparison result graph of the wound area percentage of each group; e is a comparison result graph of the area of each group of wounds;
FIG. 6 is a graph showing the effect of different treatments on wound healing of rats facing diabetes in the tissue layer, wherein a is a typical image of HE staining of each group; b is a representative image of each group of Masson trichromatic stains; c is a comparison result graph of the wound lengths of each group; d is a comparison result graph of the thickness of each group of epidermis; e is a comparison result graph of the collagen deposition areas of each group;
FIG. 7 is a graph showing the effect of different treatments on vascularization and collagen deposition of diabetic wounds in rats; wherein a is a representative image of each set of CD31 fluorescent stains; b is a representative image of each group of α -SMA fluorescent stains; c is a representative image of each set of COL-I fluorescent staining; d is a representative image of each set of COL-III fluorescent staining; e is a comparison result graph of the fluorescence density of each group of CD 31; f is a comparison result graph of the fluorescence densities of the alpha-SMA of each group; g is a comparison result graph of COL-I fluorescence densities of each group; h is a comparison of the fluorescence densities of the COL-III groups.
Detailed Description
The technical scheme of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only for illustrating the technical scheme of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical scheme of the present invention, which is intended to be covered by the scope of the claims of the present invention.
The test methods used in the following examples of the present invention are specifically described below:
1) Characterization of ABs
Protein concentration was determined according to BCA protein assay kit (abin, shanghai) instructions. DLS diameter analysis and Zeta potential analysis were performed by Zetasizer Nano ZSE (malvern, uk). .
2) Characterization of DFO-nABs
With Dil marker ABs, the residual cell content was observed in Dil-marked ABs by Hoechst nuclear staining after hypotonic-sonication. According to the literature (Sci adv.2020,22;6 (30): eaba 2987), the nuclear contents of pABs migrate from the cell center to the cell membrane border or outer edge after a series of treatments, indicating successful removal of the nuclear contents. Apoptosis-related markers Annexin V (Absin, shanghai) and C1q (Absin, shanghai) were verified by fluorescent staining, and adipose stem cell surface markers CD9 and CD108, apoptosis-related protein caspase-3,cleaved caspase-3, were detected by Western Blot. Morphology characterization of Abs, pABs, nABs and DFO-nABs was examined by TEM. And the diameters of the groups under different fields of view were statistically analyzed by Image J software. DFO was labeled with 5,6-FAM NHS (ruixi organism, western china) because 5,6-FAM NHS contains carboxylic acid, green fluorescent labeling can be achieved by carbodiimide activation of carboxylic acid and reaction with primary amine in DFO, and thus it was observed whether DFO was successfully encapsulated in ABs and apoptotic small-body nanovesicles (nABs) after hypotonic-ultrasonic treatment.
3) Drug encapsulation and in vitro release capabilities of DFO-nABs
The ability of the nABs to encapsulate DFOs is calculated as follows:
Encapsulation efficiency＝(Weight of drug in nABs)/(Initial weight of drug)
the in vitro release capacity was measured by means of an ultraviolet spectrophotometer. Specifically, 10mg of DFO-nABs were dispersed in 10ml of PBS with shaking at 37℃in which DFO was grafted with 5 (6) -FAM fluorophore, and then the supernatants were collected by centrifugation at various time points. The supernatant was tested for fluorescence intensity to obtain in vitro release results.
4) Cell biocompatibility detection
The biocompatibility of different concentrations of nABs with endothelial cells was first examined. Endothelial cell-specific media containing different concentrations of nABs served as growth media for endothelial cells. Endothelial cell viability was determined with CCK-8 (Duojin channel, japan). The OD of the solution was then recorded by a microplate reader (Varioskan Flash 3001, thermo, finland) at a wavelength of 450 nm. After 3 days of incubation, the inoculated cells were stained with a live/dead cell double staining kit (Sigma-Aldrich, usa) for 30 minutes at room temperature and examined under an olympus IX71 optical microscope (olympus, japan). Similarly, the biocompatibility of DFO-ABs with endothelial cells was also examined.
5) Endothelial cell function experiment
Dil marker ABs and DFO-nABs were co-cultured in endothelial cells at a concentration of 1. Mu.g/ml for 24h, medium was removed and washed 3 times with PBS, 4% paraformaldehyde was fixed for 20min, cytoskeleton was stained with FITC-phalloidin, nuclei were stained with DAPI, and the process of phagocytizing DFO-nABs by endothelial cells was characterized under a fluorescence microscope.
The effect of different concentrations of DFO-nABs on endothelial cell migration activity was characterized by scoring experiments. First, cells were inoculated in 6-well dishes, scratched with 200. Mu.L plastic needles, washed with PBS, and then added with cell culture media having different concentrations of DFO-nAs. Optical images were obtained at 0h, 24h and 48h and mobility was analyzed by Image J.
Endothelial cell vascularization capacity was characterized by a tube-forming experiment. Slowly thawing Matrigel in a refrigerator at 4deg.C, pre-cooling in 24 Kong Gongju coke plate, adding 50 μl/hole Matrigel matrix gel into pore plate, placing in cell incubator for 60min to solidify, digesting different groups of cells, and adjusting cell density to 5×10 5 mu.L of cell suspension was added per well at each/mL, placed in a cell incubator for 6h, washed three times with PBS, fixed with paraformaldehyde for 20min, cytoskeleton stained with PE-phalloidin, nuclei stained with DAPI, and angiogenesis of each group under different fields was analyzed by Image J.
Migration capacity of endothelial cells after different groups of treatments was characterized by Transwell migration experiments. Preparation of a Density of 5X 10 5 mu.L of each cell suspension was taken and added to a Transwell chamber, 600. Mu.L of DFO-nABs and pure medium groups of different concentrations were added to a lower chamber, the cells were incubated in an incubator for 24 hours, the medium was removed, PBS was discarded, washed 3 times, paraformaldehyde was fixed for 20min,0.1% crystal violet was stained for 20min, the bottom surface of the cells was gently rinsed with PBS, observed under a light microscope, and analyzed by Image J software.
6) Hypoxia microenvironment enhances the "me" ability of endothelial cells
According to previously published literature, hypoxia induces increased expression of CX3CR1 on the surface of endothelial cells in the sustained hypoxic microenvironment of diabetic wounds, thereby promoting binding to apoptotic bodies that surface express CX3CL 1. The CX3CL1 level on the surface of endothelial cells in an anaerobic microenvironment was detected by Western Blot as compared to CX3CR1 expression levels in normoxic environments, while the CX3CL1 level on the surface of ABs, DFO-nABs was detected.
7) DFO-nABs promote VEGF expression in vitro
Four groups were paired using Western Blot: blank, DFO, nABs, and DFO-nABs were co-cultured with endothelial cells for 24 hours, respectively, and HIF-1. Alpha. And VEGF protein was detected on the endothelial cells.
8) In vivo experiments
All in vivo protocols were performed according to institutional animal care guidelines. All procedures involved in animal experiments were approved by the Shanghai university of traffic animal research Committee. 180 g-250 g male Sprague-Dawley (SD) rats were fasted 8h prior to surgery and injected with Streptozotocin (STZ) (50 mg/kg, i.p.) to rapidly induce a model of type 1 diabetes. After 1 week, blood glucose levels reached above 16.7m by measuring the tail veins of the rats. Rats were randomly divided into 6 groups (control group, hypoxic apoptotic body group, short fiber group, hypoxic apoptotic body short fiber group), respectively, were anesthetized by intraperitoneal injection of pentobarbital sodium, and the backs were treated with an electric razor and depilatory cream. Creating three circular full-thickness skin wounds on the back of rats
The wound covering covers the wound to avoid infection. Changes in wound repair were recorded using a digital camera on days 0, 3, 7, 14 and 21, respectively. The following formula was used to evaluate wound Closure Rate (CR):
CR(％)＝(A0-Ac)/A0×100％
a0 is the wound closure area on day 0 and Ac is the wound closure area on that day.
Tissue samples were obtained on days 3, 7, 14 and 21 and fixed in 4% paraformaldehyde for 48 hours. After paraffin embedding, the tissue samples were cut into 7 μm sections and treated according to our previous work. Briefly, the histological analysis of the sections was performed using HE staining according to the manufacturer's instructions. CD31, α -SMA, COl-I and COl-III immunostaining was then detected. The number of new blood vessels on days 3, 7, 14 and 21 was calculated and the diameters of the new blood vessels of the three regions were randomly calculated according to the immunofluorescence staining software Image J for CD31, α -SMA.
9) Statistical analysis
All values are expressed as mean ± standard deviation. Differences between groups were analyzed using Student t-test. p (w < 0.05) is significance.
Example 1 extraction and characterization of 1ABs
First, at 37 ℃,5% CO 2 Fat stem cells (Anas organism, shanghai, china) were cultured under the condition of (A). Apoptosis is induced chemically by adding staurosporine (STS) (Sigma, USA) to the medium for 5 μm,12h, and the rupture of the nuclear membrane is seen under the microscope, and many bubbles and bud-like protrusions are formed on the cell surface and gradually separated from the cells, and fall off to the cell interstitium, i.e. apoptotic bodies. After trypsin digestion, 300g,10min, the supernatant was carefully aspirated and the pellet removed. The supernatant was centrifuged at 3000g for 10 minutes to obtain a mixture of apoptotic bodies and characterized accordingly. ABs was suspended in 1 XPBS and stored at-80℃for subsequent experiments.
The apoptosis of adipose mesenchymal stem cells was induced by the literature reported staurosporine (STS) and then apoptotic bodies were obtained by differential centrifugation (see fig. 1 a). Apoptosis-related markers were characterized by immunofluorescent staining and Western Blot. As shown in FIG. 1b, apoptosis-related markers Annexin V and C1q are both positive, and Western Blot results show that the adipose-derived stem cell markers CD105 and CD45 are highly expressed, and apoptosis-related proteins caspase 3 and clean caspase 3 are both highly expressed. TEM shows that apoptotic bodies have double-layer cell membranes and complete structure, and the diameter is 600 nm-1000 nm.
EXAMPLE 2 preparation of DFO-nABs
ABs was first dissolved in hypotonic lysis buffer (10 mM pH7.4 Tris, 10mM MgCl) according to the literature (ACS Nano,2020, 14, 5818-35) 2 And 1mM phenylmethylsulfonyl fluoride), at 4℃for 1 hour, to obtain cell membrane porous ABs (pABs). The mixture of ABs and DFO (10%, 20% and 30% by weight) was sonicated in a sonicator for a further 10min. Centrifuging at 3000g for 10min, and removing supernatant; gently using DD waterThe surface was rinsed 3 times to remove the DFO from the ABs surface. The ABs loaded with deferoxamine was put into an Avanti extruder (USA), and repeatedly extruded 10 to 15 times through a polycarbonate filter membrane with a diameter of 1000nm and 425nm to obtain DFO-nABs with a uniform diameter.
In this example, the permeability of the cell membrane was increased by hypotonic treatment, called pABs, and the cell membrane contents were discharged outside the cell membrane by gentle sonication, as shown in fig. 1c, using host to label DNA in the cell membrane, and the membrane contents migrated to the edge of the cell membrane, indicating successful removal of the cell membrane contents. The drug deferoxamine is added while the cell membrane contents are expelled, and under the action of ultrasound, water-soluble drug molecules enter the interior through the cell membrane with increased permeability. This process was visualized by 5 (6) -FAM labelling of primary amine bonds in deferoxamine, as shown in fig. 1h, almost all of the content of the pABs core loaded with deferoxamine was cleared, whereas drug molecules were successfully loaded into apoptotic bodies. In order to further increase the stability of the loaded apoptotic bodies, an Avanti extruder with 1000nm and 450nm filter membranes is adopted to repeatedly extrude pABs back and forth, the permeable cell membrane is remodeled into a compact cell membrane, and the diameter of the apoptotic bodies is reduced to 300-500 nm, namely the remodeled apoptotic bodies and nAbs. This process allows for a more stable encapsulation of the drug in the nABs, resulting in DFO-loaded apoptotic body nanovesicles (DFO-nABs). As shown in fig. 1i, similar to pABs, drug molecules were successfully loaded into apoptotic body nanovesicles. TEM images of pABs, nABs, DFO-nABS are shown in FIGS. 1e, 1f and 1g, respectively. In FIG. 1g, it can be clearly seen that the drug molecules were perfectly entrapped in the nAs.
Further, the diameter distribution and zeta potential of each group were counted, and as shown in FIG. 1j, the diameters of the nABs and DFO-nABs after the extruder treatment were significantly reduced to 300nm to 500nm, and the zeta potential showed the maximum surface charge of the DFO-nABs, reaching-15 mV, as compared with ABs and pABs.
By setting three different mixed concentrations of DFO and nABs, low/middle/High (Low/middle/High) drug loading of nABs was prepared, and as shown in fig. 1L, the three groups all achieved slow release in PBS over time, and the High group increased in vitro release concentration over time to 50 μm at day seven.
Example 3 verifies that nABs release "find me" and "eat me" signals attract endothelial cells to target phagocytosis.
There are a number of ways currently used to enhance targeting of EVs, and they fall into two general categories: active targeting and passive targeting 15. Active targeting refers to altering the transcriptome of EVs-derived cells, such as transfecting cells with an overexpression or recombinant expression strategy targeting vector. This approach is mainly directed to the case of insufficient expression of the target protein of interest, but the preparation process is complicated and there are problems associated with unstable expression levels of the target protein. Passive targeting refers to the fact that the transcriptome is not introduced, and the medicine is naturally distributed by utilizing the structural characteristics of specific tissues and organs, so that targeted treatment is realized. In this example, endothelial cells in the hypoxic microenvironment highly express CX3CR1, thereby amplifying the "Find" signal, so that the prepared DFO-nABs can be better recognized and phagocytized (see fig. 2 a), thereby exerting the effect of targeting endothelial cells to achieve drug release. The CX3CR1 receptor expression levels of endothelial cells in normoxic and anoxic environments are found to be greatly different through Western Blot, and the anoxic pretreatment ensures that the endothelial cells highly express CX3CR1, which is consistent with our expectations. Images of endothelial cell phagocytosis ABs and DFO-nABs in normoxic conditions are shown in FIG. 2 d. Images of endothelial cell phagocytosis ABs and DFO-nABs in hypoxic environments are shown in FIG. 2 e. As shown in the fluorescent image, endothelial cells that mimic the hypoxic microenvironment of a chronic wound after hypoxia treatment can "find/attract" more DFO-nABs, and thus more phagocytosis, mainly mediated by phosphatidylserine receptor/ligand binding.
Example 4 cell biocompatibility and cell function assays
Cell biocompatibility testing can be used to predict whether a material will constitute a potential risk to a patient. In this implementation, HUVEC cells and varying concentrations of nABs and DFO were selected for CCK8 and live-dead staining to obtain the safest concentration of nABs and DFO. The OD values were measured using the Cell Counting Kit (CCK 8, dojindo) kit and the results showed (see fig. 2 d) that there was a significant difference over the group of less than 1 μg/ml when the concentration of nABs was greater than 1 μg/ml over 3 days, indicating that high concentration of nABs resulted in reduced cell viability.
Meanwhile, at 1. Mu.g/ml, the cell viability was highest. Different concentrations of nABs were co-cultured with endothelial cells for 72 hours, then examined by live/dead staining and observed by fluorescence microscopy (fig. 2 c). The results show that the proportion of dead cells (red) to living cells (green) in the 2.5 μg/ml and 5 μg/ml groups was significantly different from the other three groups (FIG. 2 e). Similarly, CCK8 and live/dead staining were also used in different concentrations of DFO and endothelial cell culture groups. The results showed that 50. Mu.M DFO had the highest cell compatibility (see FIG. 2f, FIG. 2g and FIG. 2 h). At a concentration of 100 μm, cell viability was significantly reduced. Thus, in the cell function experiments, 10. Mu.M, 25. Mu.M and 50. Mu.M were selected so that endothelial cells remained viable and were able to continue to proliferate.
Example 5 Effect of different concentrations of DFO on the in vitro angiogenic process by the tube test
Angiogenesis is a key factor in the healing process of chronic diabetic wounds, and therefore the effect of different concentrations of DFO on the in vitro angiogenic process was assessed by a tube-forming test. As shown in fig. 3a, 3c, 3d and 3e, human Umbilical Vein Endothelial Cells (HUVECs) after 50 μm DFO treatment showed more connections, more vessel numbers and vessel areas, and longer vessel lengths than control and other concentrations of DFO. It is currently known that deferoxamine's ability to promote angiogenesis is mainly through induction of transcriptional activation of hypoxia inducible factor-1 alpha, promoting downstream VEGF secretion, and thus angiogenesis, whereas good action concentrations of 50. Mu.M are similar to literature reports.
Cell scratch tests are the most common method of determining the migration and repair capacity of cells, and as shown in fig. 3b, migration of four groups of cells on the wound surface was increased after 0 hours, 24 hours and 48 hours (scratch test). At 48 hours, the closure rate reached 57% for the 50. Mu.M group, which is significantly higher than for the other three groups (see FIG. 3 g). This further demonstrates the ability of the 50 μm DFO group to promote endothelial cell migration.
Example 6 demonstration of the Effect of DFO-nANBs on endothelial cell migration ability after hypoxia pretreatment
Since the nABs can release both "find me" and "eat me" signals, transwell migration experiments were performed with endothelial cells treated with DFO at different concentrations in order to further characterize the ability of the nABs to promote endothelial cell migration (see fig. 4 a). FIGS. 4b and 4c show that the 50. Mu.M DFO group has the strongest migration of endothelial cells into the Transwell lower chamber compared to the other three groups, and the number of cells per field can reach 145. The OD value was detected after elution of the cell-bound crystal violet dye with acetic acid. FIG. 4d shows that the OD of the 50. Mu.M DFO group is highest, further illustrating the strongest number of migrating cells. In summary, in vivo experiments we selected 1 μg/ml nAs loaded with DFO at a concentration of 50 μM released, achieving optimal biofunction effects while guaranteeing biosafety.
Example 7 validation of the effect of DFO-nABs on wound healing in rats with diabetes
A diabetic rat wound model was constructed as shown in FIG. 5 a. Time was selected as 0 day, 3 days, 7 days and 14 days as observation time points, and pictures were taken. In addition, rats were sacrificed at day 14 and histologically stained, including HE staining and Masson trichromatography. As shown in fig. 5b and 5c, the other three experimental groups each showed an enhancement of wound healing compared to the control group, in which DFO-nABs group reached the optimal wound healing effect on day 14. After the wound area is counted by Image J software, the relative wound area percentage (see FIG. 5 d) and scar area (see FIG. 5 e) of each group are obtained, and it can be seen that the three experimental groups also show better treatment effect compared with the control group, and the relative wound area percentage of the DFO-nABs group is only about 1% at the 14 th day, and the scar area is only 0.02cm 2 。
Example 8 demonstration of the Effect of DFO-nANBs on tissue layer facing wound healing of diabetes in rats
In this example, histological staining analysis was performed on diabetic wounds. As shown in fig. 6a, the wound length of the control group was longest compared to the other three groups at day 14, with statistical differences. Whereas the DFO-nABs group had the shortest wound length (see fig. 6 c) and the thickest epidermal layer thickness (fig. 6 d), which was reported to promote prognosis of diabetic wound healing (Biochem Biophys Res commun.2018,10;505 (4): 966-972). As shown by Masson trichromatic staining in fig. 6b, the nABs, DFO and DFO-nABs groups all showed more collagen (blue in view) deposition than the control group, with statistical differences (see fig. 6 e).
Example 9 demonstration of the effects of DFO-nABs on vascularization and collagen deposition in diabetic wounds in rats
Immunofluorescent staining of endothelial cell marker CD31 and smooth muscle cell marker α -SMA was used to examine the effect of different treatments applied in vivo on vascularization of diabetic wounds in rats. As shown in FIG. 7a, on day 14, the control group showed almost no neovascularization, while the other three groups had more neovascularization, with the most number of neovascularization of DFO-nANBs and the highest CD31 density (FIGS. 7a and 7 e). The alpha-SMA immunofluorescence also showed similar results (fig. 7b and 7 f). Thus, the ability of DFO-nABs to deliver pro-vascularized drugs by releasing dual signals to target endothelial cells in the hypoxic microenvironment of the diabetic wound is further demonstrated by in vivo experiments. COL-I and COL-III are closely related to chronic wound repair, wherein COL-I provides support structures and forces to the skin, maintaining the toughness and stiffness of the skin; COL-III provides elasticity to the skin and has good repairing promoting effect. It has been reported that DFO can accelerate diabetic wound healing by promoting collagen deposition, as also demonstrated by the in vivo experimental results of this study, as shown in FIGS. 7c and 7g, that the DFO-nABs group had more COL-I deposition than the control group. As shown in FIGS. 7d and 7h, the DFO-nABs groups had more COL-III deposition than the control groups. Thus, this further demonstrates the ability of DFO-nABs to promote wound healing.
Claims (10)
1. A method for preparing extracellular vesicles, comprising the steps of:
firstly, obtaining ABs through chemical induction of fat stem cell apoptosis, and then sequentially carrying out the steps of hypotonic, ultrasonic, active molecule mixing for promoting healing, extrusion and the like to obtain the functionalized apoptotic body nano vesicle carrying the active molecule for promoting wound healing;
the ABs surface produced had CX3CL1 protein.
2. The method of claim 1, wherein the active molecule for promoting wound healing is DFO.
3. The method for preparing extracellular vesicles according to claim 2, wherein the particle size of the functionalized apoptotic body nanovesicles carrying DFO is 300nm to 500nm and the surface charge is up to-15 mV.
4. The method for preparing extracellular vesicles according to claim 1, wherein ABs is resuspended in hypotonic lysis buffer at 4 ℃ ± 0.5 ℃ for 1 hour ± 0.2 hours to obtain ABs cellular membranes.
5. The method for preparing extracellular vesicles according to claim 1, wherein the mixture of ABs and the active molecules for promoting wound healing is sonicated for 10min ± 2 min and then centrifuged for 10min ± 2 min at 3000g ± 200 g.
6. The method for preparing extracellular vesicles according to claim 1, wherein ABs loaded with active molecules for promoting wound healing is put into an extruder and repeatedly extruded 10 to 15 times.
7. The method of preparing extracellular vesicles according to claim 1, wherein the hypotonic solution is 10mM pH7.4 Tris buffer containing 10mM MgCl 2 And 1mM phenylmethylsulfonyl fluoride.
8. A functionalized nanovesicle capable of targeting endothelial cells, characterized in that it is obtainable by a process according to any one of claims 1 to 7.
9. The use of the functionalized nano-vesicles according to claim 8 for preparing a medicament for promoting wound healing.
10. The use of the functionalized nanovesicles according to claim 8 for the preparation of medical devices for promoting wound healing.
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