Study On Composite Materials Based On Carbon Quantum Dots To Promote Wound Repair

BackgroundCarbon quantum dots(CQDs)have great potential in the field of wound repair due to their significant antibacterial,anti-inflammatory and anti-reactive oxygen species(ROS)effects and potential to promote angiogenesis.Recombinant human collagen Ⅲ(Col Ⅲ)is widely used in wound repair due to its excellent properties of low antigenicity and high biocompatibility,therefore it is considered as a candidate material for supporting CQDs.Previous researches on the application of CQDs in wound repair mainly focused on infected wounds,the effect of its application on burn wounds and diabetic wounds and the related mechanisms are still being obscure.Besides,accurate releasing of drugs in time and sequence and to match the period of wound repair remains a huge challenge of precision medicine at present.ObjectiveThis study aims to explore the multiple applications of CQDs and their composites in improving the therapeutic effect of deep partial-thickness burn wounds and diabetic wounds and analyze their potential mechanisms.Part one of this study intends to prepare and explore the characterization of CQDs-NO,and to verify its effect on deep partialthickness burn wound repair in rats and its potential mechanism.The objective in Part two is to prepare photo-crosslinking type Ⅲ collagen hydrogel loaded with CQDs(C-ColⅢ-MA)to identify the potential synergistic effect between type Ⅲ collagen and CQDs in this novel hydrogel.To explore the matching mechanism between drug releasing during the degradation process of C-ColⅢ-MA and the healing process of diabetic wound repair.MethodPart One:CQDs were prepared in two steps including the hyperthermia-intermittent and ultrasonic cracking method,further tested the NO-releasing rate and biocompatibility of CQDS-NO.The biological function of CQDs-NO was determined by the scratch test,Western blotting,and transcriptome sequencing.Finally,CQDs were used to treat deep partial-thickness burn wounds in rats to observe the therapeutic effect.Part Two:Photo-crosslinking Type Ⅲ collagen(Col Ⅲ-MA)was prepared by modifying Col Ⅲ with methacrylate anhydride group,and a novel photosensitive collagen hydrogel loaded with carbon quantum dots(C-col Ⅲ-MA)was assembled by Col Ⅲ-MA and CQDs at a certain ratio was prepared and characterized.The effects of C-col Ⅲ-MA on the adhesion,proliferation and migration of human dermal fibroblast(HFB)were investigated by cell live/dead staining,cytoskeleton staining,CCK-8 assay and transwell assay.Superserum of HFB cultured on hydrogel was used to stimulate Human Microvascular Endothelial Cell(HMEC),and its effect on HMEC tubeformation was observed.The effects of C-ColⅢ-MA on the biological function of HFB and its possible molecular mechanism were analyzed by transcriptome sequencing.The protein expressions of platelet-derived growth factor D(PDGFD)and platelet-derived growth factor receptor β(PDGFRβ)were detected by immunofluorescence staining.HFB was cultured on hydrogel for longer days and its supernatant was taken for Elisa detection to observe the PDGFD production of HFB stimulated by the hydrogel.The effects of two hydrogels on the secretion and assembly of Type I Collagen Alpha 1(COL1A1)of HFB were also observed.C-col Ⅲ-MA and Col Ⅲ-MA were applied to a diabetic mouse wound model to observe their effect on wound healing.Wound tissue samples were collected and stained by HE and Masson to observe the epithelialization and dermal thickness under the intervention of hydrogels.The wound tissue sections were immunohistochemistry stained with CD31 and Ki67 to observe effects of hydrogels on promoting vascularization and cell proliferation.ResultsPart I:The particle size of CQDs-NO was 1.6 ± 0.3 nm and the Zeta potential was45.37±3.73 m V.CQDs-NO was prepared by hyperthermia method and ultrasonic cracking method.Under ultraviolet excitation,CQDs-NO showed green fluorescence.The possible chemical groups on the surface of CQDS-NO were analyzed by FTIR and MS.The molecular formula may be C7H17N3 O corresponding to the peak value of m/z = 158.FITR and MS results indicate that C-N=O groups may exist on the surface of CQDS-NO.XRD results confirm the amorphous lattice structure.In vitro experiments CQDs-NO showed no cytotoxicity at concentrations of 1 μg/m L and 5 μg/m L.NO was released by CQDs-NO when co-cultured with cells or glutathione peroxidase.Transcriptome sequencing indicates CQDs-NO can promote biological function of human umbilical vein endothelial cell(HUVEC)such as angiogenesis,cellsubstrate adhesion,extracellular matrix organization,cell migration and wound healing.CQDs-NO can promote deep partial-thickness burn wounds by promoting vascularization,matrix deposition and regulating wound inflammatory response.Part Two:Col Ⅲ-MA emits purple-blue fluorescence,C-Col Ⅲ-MA emits purple-red fluorescence Under the excitation of ultraviolet light(wavelength =365 nm),UV absorption spectrum and visible light transmission spectrum were measured.ColⅢ-MA and C-ColⅢ-MA showed homogeneous and porous spongy structures under field emission electron microscopy.The hydroge bond between ColⅢ-MA and CQDs was found by FTIR spectra.After loading CQD,the elastic modulus of the hydrogel was significantly increased by four times.In onditions at 37℃ and 0.0025‰(w/v)concentration of type Ⅲcollagenase,300 μL C-ColⅢ-MA hydrogel was completely degraded after 132 h.During the degradation process,the release rate of CQDs increased over time,and the release rate of CQDs reached 98.2% after the totaly degradation of C-col Ⅲ-MA,which realized the spatio-temporal matching between the degradation of slow-release system and wound healing.The results of cell live/death staining,cytoskeleton staining,CCK-8 assay,scratches assay and transwell assay confirmed that compared with ColⅢ-MA,C-ColⅢ-MA can promote the adhesion,proliferation and migration of HFB,and the HFB supernatant cultured on C-Col Ⅲ-MA hydrogel can promote the tube formation of HMEC.Transcriptome sequencing and GO enrichment analysis of up-regulated DEGs showed that,compared with Control,the C-ColⅢ-MA group was significantly enriched in promoting cytoskeleton regulation,local cell adhesion,fibroblast proliferation,cell growth factor binding,and extracellular matrix assembly and interaction.PEGFD(0.86 times),PDGFDRβ(1.75 times)and Col12A1(3.75 times),Col6A3(3.71 times),Col4A4(3.78times),Col1A1(2.98 times),Col7A1(2.55 times),Col5A1(2.50 times),AKT3(1.64 times),LAMB1(2.16 times)and HIF1A(1.20 times)were significantly up-regulated among them,.The results of immunofluorescence(IF)suggest that protein expressions of PDGFD and PDGFDRβ of HFB cutrued on C-ColⅢ-MA are increased compared with those in the ColⅢ-MA and Control groups,which is consistent with sequencing results.ELISA results suggested that compared with the Control group,the releasing PDGFD in the supernatant of HFB tends be more in the two hydrogel groups than the Control group,and the capacity of releasing PDGFD in C-Col Ⅲ-MA group was the strongest.Cell Immunofluorescence(IF)technique suggested that the average fluorescence intensity of COL1A1 expressed in HFB cultured on the two hydrogels was much stronger than the Control group,and the fluorescence signal of C-ColⅢ-MA group was the strongest.Compared with the Control and ColⅢ-MA,the C-ColⅢ-MA group can effectively promote the healing rate of diabetic wounds in the wound model of diabetic mice.HE and Masson staining of pathological sections showed that the collagen deposition was increased and the local dermal thickness was thickened after wound healing in the C-ColⅢ-MA group,immunohistochemical staining also showed that the expressions of CD31 and Ki67 were significantly increased.ConclusionCQDs-NO with good biocompatibility and can promote a variety of biological functions of HUVEC in vitro experiments.CQDs-NO can stimulate deep partial-thickness burn wound healing by upregulating vascularization,matrix deposition and regulating wound inflammatory response in vivo.Therefore it can be used as a substitute strategy for deep partial-thickness burn healing.Compared with ColⅢ,C-ColⅢ-MA has equally good photoluminescence and optical properties,but more stable intermolecular binding force,more substantial mechanical properties,and can release CQDs stably.C-Col Ⅲ-MA can effectively enhance the adhesion,proliferation and migration of HFB with good biocompatibility.This study demonstrated that,C-ColⅢ-MA can significantly promote the synthesis,secretion and expression of PDGFD,PDGFDRβ and COL1A1 of HFB,and its supernatant can promote the long-term secretion of PDGFD and significantly promote the tube formation of HMEC.Besides,In vivo experiments,C-Col Ⅲ-MA significantly enhanced the local angiogenesis capacity of the wound,cell proliferation ability,and the secretion and deposition ability of the wound through the synergistic effect of CQDs and ColⅢ,which can significantly improve the healing of diabetic wounds.