| As the largest organ of the body,skin is the first defensive line for the external stimuli with the function of protection,metabolism,regulation of body temperature and perception of external stimuli.Because the maxillofacial skin is in the exposed part,it is vulnerable to traumatic factors that can cause lacerations,burns,and exfoliation injuries.At the same time,there still remains the clinical problem of skin tissue defect after maxillofacial malignant tumor resection.It is essential to restore the abnormal tissue structure and function in tissue repair and regeneration.Unlike other skin on the body surface,restoring the maxillofacial skin is of great importance for aesthetic appearance,pronunciation,and psychological health.Therefore,tissue repair and regeneration after maxillofacial skin defect is a clinical problem that needs to be solved urgently.In the current researches and explorations,immunomodulatory therapy based on mesenchymal stem cells(MSCs)and extracellular vesicles(EVs)has shown great potential.A large number of studies have shown that stem cells can regulate the local immune microenvironment to facilitate cell proliferation,improve the vascularization level,and thereby promote tissue repair and regeneration mediated by EVs.EVs,the independent biological units with a double-layer lipid membrane structure produced by cells,exist in various tissues,body fluids and circulation of the body,which are regarded as the main mediator of intercellular communication.Studies have shown that EVs regulate the function of receptor cells by transfer of bioactive molecules between cells,which plays an important role in multiple physiological processes such as tissue development and regeneration,immune regulation and homeostasis maintenance.However,the disadvantages of natural EVs in application,including heterogeneity,insufficient targeting ability and lysosomal degradation,hinders the further transformation and application of EVs in clinical treatment.In order to effectively improve the treatment efficiency and exert the potential of EVs,this study intends to construct a functionally specific engineered EVs to improve the local immune microenvironment and promote tissue repair and regeneration through inflammation regulation.Based on the previous researches of our group,apoptotic bodies(ABs)are a kind of EVs formed by outward blebbing of the plasma membrane during cell apoptosis,which can induce the polarization of macrophages through efferocytosis and regulate the immune microenvironment.Given that,this study fabricates the engineered extracellular vesicles by coating the membrane derived from apoptotic bodies on the drug-preloaded nanoparticles.Through in vivo and in vitro experiments,the ability of engineered EVs to target macrophages and then to regulate the immune microenvironment was verified,and its efficacy on promoting tissue repair and regeneration was tested in a skin defect mouse model.Based on the above scientific issues and conception,the following experiments were designed and carried out:Ⅰ.Construction and characterization of chimeric apoptotic bodies1.Isolation and characterization of apoptotic bodies derived from T cellsT cells were isolated from 6-8-week-old female C57BL/6J mice.They were activated in vitro for 48 h followed by induction of apoptosis.The apoptotic bodies secreted by T cells are separated by differential centrifugation and identified by:(1)The morphology of ABs was determined by scanning electron microscopy(SEM);(2)The size distribution of apoptotic bodies was detected by dynamic light scattering(DLS);(3)The expression of apoptosis molecular markers on the surface of ABs(Annexin V,Clq)was detected by flow cytometry and fluorescence imaging;(4)The expression of apoptosis molecular markers on ABs(cleaved caspase-3)was detected by Western blot.2.Fabrication and characterization of chimeric apoptotic bodiesABs undergoes hypotonic and ultrasonic treatment to remove the vesicle contents to generate the AB membrane(ABM).ABM is coated on the surface of mesoporous silica nanoparticles(MSNs)to construct chimeric apoptotic bodies(cABs).Characterization and performance test of cAB s were conducted as follows:(1)The morphology and structure of cABs were observed by transmission electron microscope(TEM);(2)The size distribution and zeta potential of cAB s were detected by DLS;(3)The retainment of the membrane protein of cAB s was determined by Western blot;(4)The short-term and long-term storage stability of cABs were determined by particle size analysis;(5)The serum stability,circulation time and blood compatibility of cABs were analyzed.Ⅱ.Studies on the targeted ability of chimeric apoptotic bodies1.Studies on inflammation site targeting abilityTNF-α was used to stimulate human umbilical vein endothelial cells(HUVECs)in vitro to simulate inflammatory endothelium.HUVECs were treated with equivalent amounts of RhB-labeled cABs and MSNs,the adhesion of cABs and MSNs on the surface of HUVECs was observed by confocal microscope.The full-thickness dorsal skin defect model of mice was constructed,150 μl of DiR-labeled cABs,ABM and MSNs suspension(2 mg/ml)were injected into the mice intravenously.The accumulation of the therapeutic agents on the back of the mice was observed by the in vivo imaging system(IVIS).2.Studies on specific phagocytosis of cABs by macrophagesBone marrow derived macrophages(BMDMs)were cultured and treated with 50μg/ml RhB-labeled cABs,PKH26-labeled ABM and RhB-labeled MSNs for 3 h.The uptake of different agents by BMDMs was observed by confocal microscope.Then,BMDMs were treated with RhB-labeled cABs with different time(0,3,6,12,24,48 h)or different concentrations(0,5,10,25,50,100 μg/ml)to determine the uptake of cABs using confocal microscope and flow cytometry.In addition,10 μg/ml RhB-labeled cABs were added to BMDMs,fibroblasts(FBs),bone marrow mesenchymal stem cells(BMMSCs)and T cells,respectively.The uptake of cABs by different cells was detected by confocal microscope and flow cytometry.Finally,a co-culture system of BMDMs and fibroblasts was established,and 10 μg/ml RhB-labeled cABs were added for 2 h to detect the uptake of cABs.The full-thickness dorsal skin defect model of mice was constructed,100 μl of RhB-labeled cABs,PKH26-labeled ABM and RhB-labeled MSNs suspension(1 mg/ml)were intravenously injected into the mice.After 6 h,the skin tissues were harvested and the slices were prepared to detect the cellular distribution and uptake of cAB s via immunofluorescence staining.Ⅲ,Construction of drug loading and intracellular release system in cABs1.Loading and intracellular release of microRNAmiR-21 is conjugated to the MSN surface by disulfide bonds through electrostatic interaction,the encapsulation efficiency was calculated.Glutathione(GSH)-contained solution was used to simulate the intracellular microenvironment.The same amount of cy5-labeled MSNmiR-21 was resuspended in PBS and GSH solutions,and the fluorescence intensity of miR-21 released in the supernatant was measured at different timepoints.PKH67-labeled ABM was coated on the surface of cy5-labeled MSNmiR-21 to generate cABsmiR-21.The generated cABsmiR-21 was applied to BMDMs to detect the intracellular delivery and release of miR-21 at different time points.2.Loading and intracellular release of small molecule drugsCurcumin(Cur)was loaded in the MSN through physical adsorption and the surface is encapsulated with polyester,the encapsulation efficiency was calculated.The PBS solution added with esterase was used in vitro to simulate the intracellular microenvironment.The same amount of MSNCur was resuspended in PBS solution with or without esterase,and the fluorescence intensity of curcumin released in the supernatant was measured at different timepoints.PKH26-labeled ABM was coated on the surface of MSNCur to generate cABsCur.The generated cABsCur was applied to BMDMs to detect the intracellular delivery and release of curcumin at different timepoints.Ⅳ.Studies on the inflammation-regulatory ability of cABs in vitro1.Phenotypic regulation of macrophage by cABs in vitroLipopolysaccharide(LPS)is used to treat BMDMs to simulate inflammatory macrophages in vitro.MSNs,cABsnull(cABs without drug loading),cABsmiR-21,and cABsCur are applied to treat BMDMs respectively,the phenotypic distribution of macrophages and the inflammatory microenvironment were detected as follows.(1)Immunofluorescence staining was conducted to detect the iNOS/CD206-positive population in macrophages;(2)Western blot was conducted to detect the expression levels of iNOS,CD206 and Arg-1 in macrophages;(3)ELISA was conducted to detect the secretion level of pro-inflammatory factors(TNF-α,IL-6)and anti-inflammatory factors(TGF-β,IL-10)by macrophages.2.Detection of the function of fibroblastsFibroblasts were treated with the conditioned medium from macrophages for 24 h.(1)Immunofluorescence staining was performed to detect the expression level of the Ki-67 in fibroblasts;(2)Scratch test was performed to detect the migration ability of fibroblasts.3.Detection of the function of endothelial cellsHUVECs were treated with the conditioned medium from macrophages for 4 h,and the tube formation assay was carried out to detect the tube formation ability of HUVECs.Ⅴ.Studies on the therapeutic effects of cABs in cutaneous wound mice model1.Establishment and treatment of cutaneous wound mice modelThe full-thickness dorsal skin defect model was established in 6-8-week-old female C57BL/6J mice to investigate the ability of cABs to promote tissue repair and regeneration.The experimental groups were set up as follows:PBS group,cABsnull group,cABsmiR-21 group,cABsCur group.The treatment started on the first day,and a total of 5 doses were administered intravenously every other day.On the 12th day,local skin tissues were harvested for further histological analysis.The therapeutic efficacy is evaluated as follows:(1)Pictures of the wound were taken on day 0,day 3,day 5,day 7,day 10 and day 12,wound healing rate and body weight were recorded and calculated;(2)H&E staining was performed to observe the healing of skin tissue;(3)Immunofluorescent staining was carried out to detect the local macrophage phenotype distribution(iNOS,CD206),the expression level of cytokeratin 14(CK14),Ki67 and CD31 and the level of inflammatory factors(TNF-α,TGF-β)in healed skin;(4)ELISA was conducted to detect the level of pro-inflammatory factors(TNF-α,IL6)and anti-inflammatory factors(TGF-β,IL-10).2.Biocompatibility evaluation of cABs in animal experimentsWild-type 6-8-week-old female C57BL/6J mice were injected with PBS,cABsmiR-21,and cABsCur according to the above treatment regimen.On the 12th day,major organs were harvested for H&E staining,and whole blood and serum were collected for blood cell count analysis and serum biochemical detection.The results are as follows:Ⅰ.Construction and characterization of chimeric apoptotic bodies1.Isolation and characterization of apoptotic bodies derived from T cellsThe spleen-derived T cells isolated in this study highly expressed CD45 and CD3.The isolated ABs by in vitro apoptosis induction exhibited spherical vesicles with a particle size of 1 μm and high expression of Annexin V and Clq.Western blot showed that the surface molecules of T cells(CD3,CD44)are also expressed,but the expression level of cleaved caspase-3 is significantly increased in ABs,indicating that ABs was successfully isolated and could be used in subsequent experiments.2.Fabrication and characterization of chimeric apoptotic bodiesThe cABs showed a typical "membrane-core" structure of natural membrane-coated nanoparticles by TEM.After the coating of ABM,the diameter of the cABs increased by 20 nm and the potential was-17.7±0.4 mV.SDS-PAGE and Western blot results showed that the membrane protein composition was retained during cABs construction.Compared with MSNs,cABs displayed good storage stability in PBS and water,the well serum stability and the significantly longer circulation time.The blood compatibility assay showed no significant hemolysis caused by cABs.e3.Studies on the targeted ability of chimeric apoptotic bodies1.Studies on inflammation site targeting abilityIn vitro experiments showed that compared with MSNs,cAB s highly expressed Mac1 and CD44,which mediated the recruitment and adhesion of cABs on the surface of HUVEC.In vivo imaging system showed that ABM and cABs were specifically enriched in the skin defect on the back of the mice compared with MSNs.2.Studies on specific phagocytosis of cABs by macrophagesIn vitro experiments results showed that compared with the MSNs group,there is a significantly high uptake efficiency in cABs and ABM groups,and cABs were engulfed by macrophages in a time-and concentration-dependent manner.Different from the high phagocytosis rate of BMDMs,FBs,BMMSCs and T cells seldom internalized cABs.In the co-culture system of BMDMs and FBs,cABs were engulfed by macrophages specifically accompanied by almost no cABs engulfed by fibroblasts.In vivo immunofluorescence staining showed that cABs were mainly uptaked by macrophages specifically.Ⅲ.Construction of drug loading and intracellular release system in cABs1.Loading and intracellular release of microRNAmiR-21 is conjugated to the MSN by disulfide bonds with an encapsulation efficiency of more than 90%.In the in vitro release experiment,the release of miR-21 from MSNmiR21 markedly increased over time in the presence of GSH,finally reaching 80%after 5 days.The fluorescence images showed that free miR-21 could not enter the cell,and cABsmiR-21 could achieve the intracellular delivery of miR-21.At 6 h,miR-21 was released from the cABsmiR-21 and dispersed evenly in the cytoplasm.2.Loading and intracellular release of small molecule drugsCurcumin is conjugated to the MSN by physical absorption and polyester encapsulation with an encapsulation efficiency of more than 80%.The organic polyester layer on the surface of MSNCur was observed under the TEM.In vitro release experiments showed that under the action of esterase,curcumin is responsively released from MSNCur to reach 70%at 36 h.The fluorescence images showed that cABsCur could achieve the intracellular delivery of curcumin,and curcumin was released from cABsCur and evenly dispersed in the cytoplasm at 6 h.Ⅳ.Studies on the inflammation-regulatory ability of cABs in vitro1.Phenotypic regulation of macrophage by cABs in vitroAfter the treatment of cABsmiR-21 and cABsCur,inflammatory macrophages transformed from a pro-inflammatory phenotype to an anti-inflammatory phenotype.Immunofluorescence results showed that the ratio of iNOS-positive cells was significantly reduced,and the ratio of CD206-positive cells was significantly increased.Western blot showed that the expression level of iNOS decreased,accompanied with the increased expression level of CD206 and Arg-1.The level of pro-inflammatory factor TNF-α and IL-6 decreased,while the level of anti-inflammatory factors TGF-β and IL-10 increased in the supernatant.2.Detection of the function of fibroblastsThe conditioned medium of BMDMs treated with cABsmiR-21 and cABsCur can significantly increase the rate of Ki-67-positive cells in fibroblasts and the healing rate of cell scratches.3.Detection of the function of endothelial cellsThe conditioned medium of BMDMs treated with cABsmiR-21 and cABsCur can significantly promote the tube formation ability of HUVECs.Ⅴ.Studies on the therapeutic effects of cABs in cutaneous wound mice model1.Establishment and treatment of cutaneous wound mice modelThe results of in vivo experiments showed that compared with the PBS group and cABsnull group,the cABsmiR-21 and cABsCur groups significantly enhanced the wound healing rate.H&E staining showed the better healed skin structure and reduced subcutaneous inflammatory cell infiltration level in the cABsmiR-21 and cABsCur groups.Immunofluorescence staining results showed that the number of iNOS-positive cells was decreased,and the number of CD206-positive cells was increased in the cABsmiR-21 and cABsCur groups.At the same time,cABsmiR-21 and cABsCur obviously promoted the expression levels of CK14,Ki-67 and CD31 in the healed skin tissue.The immunofluorescence and ELISA results indicated that treatment of cABsmiR-21 and cABsCur decreased the level of pro-inflammatory factors(TNF-α,IL-6),and increased the level of anti-inflammatory factors(TGF-p,IL-10)in the skin tissue.2.Biocompatibility evaluation of cABs in animal experimentsAfter treatment with PBS,cABsmiR-21 and cABsCur,H&E staining of major organs showed no acute inflammatory injury.Blood cell count analysis and serum biochemical tests showed that the administration of cABsmiR-21 and cABsCur had no significant influence on blood cell count,liver and kidney function indexes.Based on the above results,the following conclusions are drawn:1.Chimeric apoptotic bodies were successfully constructed based on natural extracellular vesicles,which have good stability,biocompatibility and excellent drug loading efficiency and intracellular release property.2.Chimeric apoptotic bodies can target to inflammatory endothelium mediated by surface chemotactic molecules in vitro and be specifically engulfed by macrophages.Through efferocytosis,effective intracellular delivery and responsive release of nucleic acids and small molecule drugs,chimeric apoptotic bodies realized the targeted regulation of macrophage phenotype and function.3.Chimeric apoptotic bodies are specifically enriched in the inflammatory site and engulfed by macrophages in the cutaneous defect model,which regulated macrophages phenotype to anti-inflammatory pathway,thereby modulating the immune microenvironment to promote skin tissue repair and regeneration.Collectively,we established a modular construction strategy for functionally specific engineered extracellular vesicles.It not only inherits the chemotaxis ability and biocompatibility of natural extracellular vesicles,but also integrates the excellent loading efficiency and the intracellular responsive release system of nanoparticles.It specifically delivers therapeutics with proved curative effects to the macrophages distributed in the damage site to play an immunomodulatory role for promotion of tissue repair and regeneration.The strategy to construct functionally specific engineered extracellular vesicles present in this study effectively improves the treatment efficiency and exerts the potential of extracellular vesicles.It breaks through the current bottleneck problems of extracellular vesicles as a standardized delivery system,and promotes the transformational application of extracellular vesicles in clinical management. |
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