Engineering Bacteria Activated Multi-functionalized Hydrogel For Promoting Chronic Wound Healing

BackgroundChronic wounds have become a long-term medical problem,and their incidence and accompanying medical costs have been increasing year by year.Although a large number of studies have been conducted on how to promote the wound repair,the clinical treatment effect of chronic wounds is still poor.Therefore,it is of great clinical significance to solve the key problems in the process of chronic wound healing and to develop new strategies to promote wound healing.Vascular injury or angiogenesis deficiency is the pathological basis of most refractory wounds caused by diabetes mellitus and peripheral vascular disease.Macrovascular and microvascular disease impairment causing insufficient blood supply to the wound,which cannot supply sufficient oxygen and nutrients for the wound repair process,leading to impaired inflammatory response and increased risk of infection.Delivery of exogenous angiogenic growth factors,such as vascular endothelial growth factor(VEGF),is beneficial for wound closure and angiogenesis.However,direct application of VEGF has not shown clear benefits in clinical trials,possibly due to the insufficient VEGF or its instability in highly proteolytic and oxidative environment of the chronic wound.Therefore,we sought to develop a delivery system that can continuously produce and protect VEGF,which can directly stimulate angiogenesis and subsequent regeneration of the impaired diabetic wound.A massive local inflammatory response impedes angiogenesis in diabetic wounds.The wound microenvironment is characterized by the diverse and sequential biological activity of macrophages,which polarize from a classically activated M1 phenotype with pro-inflammatory properties to alternatively activated M2 phenotype exhibiting anti-inflammatory and tissue repair functions.In diabetic wounds,macrophages exhibit a reduced capability to induce the phenotypic switch from M1 to M2 due to hyperglycemia and presence of excessive glycosylation residues,resulting in a sustained influx and activation of pro-inflammatory cells.This helps to accumulation of M1 macrophages,promoting the harsh microenvironment of prolonged inflammation,strong proteolysis,and excessive oxidative stress.Therefore,a successful strategy for diabetic wound healing would simultaneously stimulate angiogenesis and modulate the macrophage polarization to reduce local inflammation.Our study aimed to elucidate the etiology and microenvironment of diabetic wounds and develop a promising avenue for promoting angiogenesis in diabetic wounds.This was achieved by utilizing engineered probiotics encapsulated in hydrogel to locally produce and deliver pro-angiogenetic factor(VEGF)and macrophages-regulated mediator(lactic acid).In the bacteria-supporting and GF protecting HP hydrogel,engineered L.lactis equipped with an artificially designed gene circuit continuously secretes VEGF,thereby providing continuous angiogenic growth signal.L.lactis alters the phenotype shift of macrophages by producing lactic acid.The polarization of accumulated M1 macrophages to M2 breaks the hostile status of pro-inflammatory,proteolytic and apoptotic wound microenvironment.these effects improved the bioavailability of VEGF and reinforced the angiogenic signals.Therefore,our strategy drove the functional reconstruction of vessel networks and promoted the rapid regeneration of chronic wounds.Objective:This study intends to incorporated living engineered probiotics and heparin-modified poloxamer to explore its local administration for promoting angiogenesis and wound repair,which could provide a new direction of the development of new functional materials.Method:1 Design and characterizations of engineered probiotics transformed with plasmid for secreting VEGF1.1 Engineering design and modification of L.lactis:Using the probiotics L.lactis as the chassis,engineering plasmids were assembled with functional gene elements such as coding signal peptide(Usp45),biological effector protein(Vegf),inducer tolerance factor(nsr),etc.,which were transformed into L.lactis by electric shock,and an engineered probiotics(LL＿VEGF)that could secrete VEGF induced by food-grade antimicrobial peptide Nisin was constructed.1.2 Biological characterization of the secretion of VEGF protein and lactic acid by LL＿VEGF:The optimal induction concentration was determined by fitting the growth curves at different concentrations of nisin.The ability of LL＿VEGF to secrete VEGF was determined by Western blot and ELISA.Lactate dehydrogenase was knocked out based on thermal-sensitive plasmid and homologous recombinant double exchange method,and the capacity of LL＿VEGF to produce lactic acid by lactate dehydrogenase was investigated by measuring the p H lactic acid concentration.2 Preparation and material characterization of HP@LL＿VEGF2.1 Synthesis and characterization of HP copolymer:The HP copolymer was prepared by modifying heparin onto poloxamer by EDC/NHS method using terminally ammoniated poloxam polymer as the framework.The structure of copolymers and the formation of chemical bonds were characterized by NMR spectroscopy and Fourier transform infrared spectroscopy.2.2 Preparation and characterization of HP@LL＿VEGF:LL＿VEGF was loaded into HP hydrogel by low temperature mixing method,and the temperature sensitivity of HP@LL＿VEGF was tested by rheometer.The growth and distribution of engineered probiotics in hydrogel were analyzed by scanning electron microscope and confocal laser scanning microscope.The dynamic production and secretion of VEGF and lactic acid were characterized by ELISA and determination of lactic acid concentration.3 The role of HP@LL＿VEGF in promoting angiogenesis and regulating macrophage phenotype3.1 HP@LL＿VEGF promote angiogenesis in vitro:CCK-8 proliferative activity assay,Ki67 immunofluorescence assay,scratch assay,and vascular catheterization assay were used to evaluate the effect of HP@LL＿VEGF on the proliferation,migration and vascular structure formation of HUVECs in vitro.3.2 HP@LL＿VEGF to promote the transformation of macrophages from M1 to M2phenotype:Flow cytometry,immunofluorescence staining,RT-q PCR and Western blot were used to investigate the changes in the proportion of M2-type macrophages and the expression of M2-type related markers in M1-type polarized BMDMs after HP@LL＿VEGF treatment.4 Study of HP@LL＿VEGF’s effect of promoting wound repair4.1 Establishment of diabetic wound model in mice:The diabetic mouse model was induced by STZ combined with high sugar and high fat diet.Full-thickness skin defect model was made with skin perforator.4.2 Biosafety assessment of local in situ use of HP@LL＿VEGF:local congestion induced by inflammation was evaluated by laser speckle blood flow imager;The expression of inflammation-related genes was detected by RT-q PCR to evaluate the local inflammatory response of the wound after HP@LL＿VEGF use.Systemic inflammation of mice after HP@LL＿VEGF was evaluated by counting inflammatory cells in peripheral blood.The effect of HP@LL＿VEGF on the systemic acid-base balance of mice was evaluated by measuring the serum lactic acid concentration.4.3 Local residence and distribution of engineering probiotics on the wound:using HP@LL＿m Cheery as the reporting carrier,fluorescence images of the wound at different time points were collected by the small animal imaging system to analyze the growth and activity maintenance of engineering probiotics on the wound.The distribution of engineering probiotics in the wound was observed by collecting frozen slices of the wound tissue at different time points.4.4 HP@LL＿VEGF’effect of promoting diabetic wound healing:After HP@LL＿VEGF was applied to the wound of diabetes,the changes of the wound were collected and counted continuously.The effect of HP@LL＿VEGF on promoting granulation tissue regeneration and collagen regeneration was evaluated by H&E staining and Masson staining of wound tissue sections.The effect of HP@LL＿VEGF on promoting angiogenesis was evaluated by laser speckle flow imaging and CD31immunofluorescence staining.Immunofluorescence staining of i NOS,M1-type macrophage markers,CD206 and Arg1 was used to evaluate the effect of HP@LL＿VEGF in reducing M1-type macrophage infiltration and increasing the proportion of M2-type macrophages in wound tissue.5 Study on the mechanism of promoting wound healing of HP@LL＿VEGF5.1 Transcriptome sequencing and screening of differentially expressed genes:Transcriptome sequencing was performed on the wound tissues of HP@LL＿VEGF treatment and the control group using Illumina’s Hi Seq X platform,and the differentially expressed genes of the two groups were screened using the mouse reference genome as mapping.5.2 Enrichment analysis of DEGs:Gene Ontology database was used to analysis the functional enrichment;KEGG database was used to enrich the signal pathways of the selected differentially expressed genes.The STRING algorithm is used to mine the information of protein-protein interaction.Results:1 Characterizations of engineered probiotics transformed with plasmid for secreting VEGF1.1 Nisin can induce and enhance the secretion of VEGF protein by LL＿VEGF,and the concentration of 10μg/m L has no effect on the growth of LL＿VEGF,but it can significantly inhibit the growth of wild-type LL and Staphylococcus aureus,forming a relatively bacteriostatic environment.1.2 The VEGF protein with the right structure was detected in the supernatant of LL＿VEGF culture,and the dynamic secretion of VEGF was positively correlated with the concentration of Nisin.On LL＿VEGF,the production of VEGF was maximized after 3 h of Nisin induction(about 6 ng per 10~9 cells),but gradually decreased within 24 h due to its short half-life.1.3 After 12 h of culture,the lactic acid concentration in the supernatant of wild and engineering strains was about 60 mmol/L,while the lactate dehydrogenase knockout strains was about 12 mmol/L.2 Material characterization of HP@LL＿VEGF2.1 The results of NMR showed that there was a characteristic chemical shift between heparin(H)and poloxam(P)in the HP copolymer.2.2 FTIR result showed the characteristic amide bond structure in HP copolymer.2.3 HP@LL＿VEGF has typical reverse temperature-sensitive properties.HP@LL＿VEGF has typical reverse temperature-sensitive properties.It keeps the sol state during preparation and preservation.After applied to the skin wound,the gel is quickly formed to cover the wound,the gelation temperature is 25-30,and the modulus after gelation is 9 k Pa.2.4 HP hydrogel could maintain high growth and plasmid activity of engineered probiotics.The engineered probiotics were confined in the hydrogel and distributed uniformly in space.With the extension of time,the number of bacteria gradually increased.2.5 HP@LL＿VEGF VEGF protein can be continuously produced and released within 24 h,and the degradation of VEGF is reduced by the modification of heparin.2.6 HP@LL＿VEGF can continue to produce and release lactic acid molecules for 24 h.3 The role of HP@LL＿VEGF in promoting angiogenesis and regulating macrophage phenotype3.1 The proliferation experiment showed that HP@LL＿VEGF could promote the proliferation of HUVECs and increase the proportion of Ki67-positive cells in vitro.3.2 The results of scratch test showed that HP@LL＿VEGF increased the migration rate of HUVECs in vitro and promoted the intercellular closure;3.3 Tubulation experiments suggest that HP@LL＿VEGF improves the ability of vascular endothelial cells to form vascular structures in vitro;3.4 Flow cytometric analysis showed that HP@LL＿VEGF increased the proportion of M2-type macrophages in M1-type BMDMs in vitro,and the effect was derived from lactic acid;3.5 The results of immunofluorescence staining showed that HP@LL＿VEGF could induce the expression of CD206 in cell membrane and Arg1 in cytoplasm related to M2-like macrophages phenotype,and inhibit the expression of CD86 in M1-like macrophages;3.6 HP@LL＿VEGF can reduce the expression of inflammatory mediators and proteases(TNF-αi NOS MMP9)and promote the expression of endogenous growth factors(IL-10 Arg1 CD206 VEGF)and other pro-repair signaling factors(IL-10 Arg1CD206 VEGF)by producing lactic acid.4 Study of HP@LL＿VEGF’s effect of promoting wound repair4.1 After local application of HP@LL and a functional hydrogel,the inflamation-induced hyperemia around the wound was decreased,and the expression of the inflammation-related gene IL-1βNF-κB NOS2 TNF-αwas not significantly increased;4.2 The number of WBC,LYM and MON in peripheral blood of mice treated with HP@LL＿VEGF had no significant changes compared with the wound control group;4.3 The serum lactic acid concentration of diabetic mice has no significant difference among all groups;4.4 After combined with hydrogel,the engineered bacteria were confined to the local area of the wound,and the fluorescence intensity reached its peak at 12 h and maintained at a high intensity within 24 h.From the frozen section results,it was observed that the engineered bacteria were confined to the surface of the wound and isolated from the wound tissue;4.5 The wound healing rate of mice treated with HP@LL＿VEGF was significantly faster,and the healing rate was close to 50%on the 6th day.On the 12th day,the wound healing rate of the ALI-group was about 90%,the wound healing rate of the control group and the HP group was about 30-50%,and the wound healing rate of the AC-group and the YL group was about 70-80%;4.6 Compared with the other groups,the granulation tissue of the wound in HP@LL＿VEGF group was thicker and the collagen deposition was higher;4.7 The proportion of i NOS~+M1 macrophages was significantly decreased in HP@LL and HP@LL＿VEGF groups,while the infiltration of CD206~+and Arg1~+M2macrophages was increased;4.8 The blood perfusion of wound area was significantly increased and the ratio of CD31 positive area was higher in HP@LL＿VEGF treatment group.5 Study on the mechanism of promoting wound healing of HP@LL＿VEGF5.1 A total of 3080 differentially expressed genes were identified by using HP@LL＿VEGF,including 1026 up-regulated genes and 2054 down-regulated genes;5.2 After stratified cluster analysis and screening,after HP@LL＿VEGF treatment,the expression of Vegfa,Fgf1,Vegfb,Egf,Notch4 and other genes related to angiogenesis and wound healing were significantly up-regulated,while the expression of inflammation-related genes,such as Tnf,Ccl2,Il-18,Il-1r1,My D88,proteolysis related genes and apoptosis related genes were significantly down-regulated;5.3 KEGG enrichment analysis indicated that the functions of the up-regulated genes were mainly concentrated in the cellular pathways that positively regulate angiogenesis,wound healing and energy metabolism,while the functions of the down-regulated genes were mainly concentrated in the signaling pathways related to the reduction of inflammatory cell infiltration,apoptosis,hyperglycemia injury,and proteolysis in the wound;5.4 GO enrichment analysis results suggest,increase the function of the gene set mainly enriched in the oxidation reduction process The oxygen-containing substances cells response steady tissue blood circulation regulation Cell response of the growth factor of vascular formation,etc.,cut the function of the gene set main enrichment in the protein hydrolysis process positively regulate apoptosis pathway of TNF cell response Leucocyte migration lymphocyte activation of IL-6 in the inflammatory response is activated Chronic inflammation,etc5.5 The results of protein interaction network analysis showed that the core network functions of VEGF,EGF,FLT1 and FGF1 were up-regulated genes,while TNF dominated the protein interaction network in down-regulated genes.Conclusion:This topic proposed a new living engineering probiotic hydrogel materials in treatment of chronic wound,we successfully design and preparation of the load reactive engineering probiotics multi-function hydrogel(HP@LL＿VEGF)the functional materials through curing will be able to secrete VEGF and lactic acid bacteria wrapped inside the hydrogel to support the growth of the bacteria,continuous production and delivery by in situ VEGF and lactic acid,stimulate the migration of endothelial proliferation.At the same time,life activities such as angiogenesis regulate the transformation of M1-type macrophages to M2-type macrophages,and reshape the local immune response of the wound surface.Both of them jointly promote the wound healing microenvironment conducive to the regeneration of blood vessels and tissues,thus promoting the process of functional reconstruction of the vascular network,and further the rapid repair of the chronic wound.