| BackgroundWound healing is a complex biological process that involves the action of many cells,cytokines,and inflammatory mediators,and a series of changes at the cellular and molecular levels to restore the skin’s barrier function.Bone marrow mesenchymal stem cells(BMSCs)possess multi-lineage differentiation potential.They can participate in the vascularization and epithelialization processes during skin wound healing.The mobilization,chemotaxis,aggregation,and differentiation of BMSCs into endothelial progenitor cells(EPCs)are mainly induced by the chemotactic function of the stromal cell derived factor 1(SDF-1).The expression of SDF-1 is up-regulated locally in damaged tissues and during ischemia and hypoxia,leading to the chemotaxis and homing of BMSCs to the site of injury,where they promote vascularization and wound repair.In this way,the presence of SDF-1 locally at high concentrations is necessary for recruiting stem cells for wound repair.However,creating a SDF-1 concentration gradient has proven challenging.Due to their unique properties,polymeric nanoparticles(Nanoparticle,NP)have great potential for medical applications such as drug delivery and medical imaging..For clinical treatment,the specific physical,chemical,or biological features at disease sites can trigger responses in nanoparticles.Excessive amounts of ROS are found in the lesions of many diseases such as wounds and inflammatory lesions and are considered one of the causative factors.The development of ROS-responsive polymeric drug delivery systems,in which high concentrations of ROS cause the degradation of the polymers and the release of the carried drug,whereas physiological concentrations of ROS cannot trigger the degradation of polymers,provides an efficient and accurate method for targeted therapies.In this study,we used PPADT as a vehicle to package SDF-1 into nanoparticles that were administered intravenously.These nanoparticles were here shown to target wounds and form an effective and stable SDF-1 gradient in those wounds and surrounding tissues.We sought to determine how the response of PPADT nanoparticles to ROS affected the release of the drug whether nanoparticles could increase the stability of SDF-1and form targeted site localization of macromolecular drugs,thereby enhancing its function in promoting BMSC chemotaxis,wound vascularization,and wound healing.Methods1.Preparation and characterization of SDF-1α-PPADT nanoparticles(1)Synthesis of poly-(1,4-phenyleneacetone dimethylene thioketal)(PPADT)Synthetize the poly-(1,4-phenyleneacetone dimethylene thioketal)(PPADT),determine the molecular weight by Gel permeation chromatography(GPC)and verify its molecular structure by 1H-NMR and 13C-NMR.(2)Preparation of SDF-1α-PPADT nanoparticlesPrepared SDF-1α-PPADT nanoparticles by double emulsion solvent evaporation.Use transmission electron microscopy(TEM),particle size analyzer to observe the characterization of nanoparticles.(3)Drug-loading capacity of SDF-1α-PPADT nanoparticlesBCA protein content measured nanoparticles calculated SDF-1α-PPADT drug loading.(4)Analysis the cytotoxicity of SDF-1α-PPADT nanoparticles in vitro(CCK8).(5)Evaluate the release of SDF-1α from SDF-1α-PPADT nanoparticles in vitro.(6)Evaluate the activity of SDF-1α in the SDF-1α-PPADT nanoparticles in vitro.Transwell chamber experiments evaluate the biological activity of SDF-1α nanoparticles;2.In vivo study SDF-1α-PPADT nanoparticles on acute wounds in mice(1)Analysis the toxicity of the SDF-1α-PPADT nanoparticles in vivo.Calculate the mortality of mouses.Measure the changes of enzyme in serum and evaluate the damage of each organ(CK,ALT,AST).Observe the pathological changes of major organs by H&E staining.(2)Evaluate the wound-targeting of SDF-1α-PPADT nanoparticles in vivo.(3)Evaluate the release of SDF-1α from SDF-1α-PPADT nanoparticles in vivo.(4)SDF-1α-PPADT nanoparticles and BMSC chemotaxis and homingObserve the number of GFP+CD31+ cells in the tissue of wounds surrounding by laser scanning confocal microscopy and evaluate the chemotactic effect of SDF-1α-PPADT on BMSCs.(5)SDF-1α-PPADT nanoparticles and wound revascularization,and wound healingEvaluate the tissue vascularization by immunohistochemistry(the number of CD31+ cells).Observe the wound healing and record the time of wound healing.ResultsNuclear magnetic resonance(NMR)was used to determine the molecular structure of the synthesized products.1H-NMR results were as follows: Per repeating unit,(600 MHz,CDCl3)δ ppm 7.30(4H),3.87(4H),and 1.62(6H).13C-NMR results were as follows:(600 MHz,CDCl3)δ ppm 129.304,77.231,77.019,76.807,34.806,and 30.787.The molecular structure was consistent with that reported in the literature(14).Gel permeation chromatography(GPC)was used to determine molecular weight.Current results show the average molecular weight to be 11,544 Da with a dispersity(PDI)of 1.974,suitable for further preparation of nanoparticles.The diameter of the SDF-1α-PPADT nanoparticles was 124.7 ± 27.47 nm.Observed under a transmission electron microscope,the nanoparticles appeared uniform in size with no adhesion.The drug-loading capacity of the nanoparticles was 1.8%.To determine whether the SDF-1α-PPADT nanoparticles were cytotoxic,a CCK8 assay was performed to determine the effect of various concentrations of SDF-1α-PPADT nanoparticles on RAW264.7 cell proliferation.When RAW264.7 cells were cultured in the presence of different concentrations of SDF-1α-PPADT nanoparticles for 24 h,the cell proliferation rate was not significantly different from that of the control cells(P > 0.05),suggesting that SDF-1α-PPADT nanoparticles had little effect on cell proliferation and minimal cytotoxicity.When put into PBS or the Fenton’s reagent,the nanoparticles showed a significant burst release within 1 h in both buffers.In PBS,the nanoparticles released 11% of the loaded drug from 1 to 48 h.They showed a more steady release in the Fenton’s reagent.By 8 h,60% of the drug had been released.By 48 h,the drug release was nearly complete.This indicates that the SDF-1α-PPADT nanoparticles were relatively stable in a normal environment without ROS,but the presence of ROS triggered a stable and complete release of the carried drug within 48 h.To determine whether the process of nanoparticle preparation affects the biological activity of SDF-1α,we used a transwell system to determine the chemotactic activity of SDF-1α released from the nanoparticles and compared it with that of the SDF-1α standard.When the lower chamber of the transwell system contained only low glucose DMEM medium,nearly no BMSCs migrated from the upper chambers into the lower chambers after 6 h incubation.When the medium in the lower chambers was supplemented with 100 ng/ml of SDF-1α standard or 100 ng/ml of SDF-1α extracted from the nanoparticles,many BMSCs migrated to the lower chambers.The numbers of migrated cells from these two conditions showed a ratio of 1:0.73,indicating that SDF-1α released from the nanoparticles retained significant chemotactic activity.To determine the biological toxicity of SDF-1α-PPADT nanoparticles,various concentrations of SDF-1α-PPADT nanoparticles were injected into mice through the tail vein,and the survival of these mice was monitored for one week.Liver and kidney functions and the morphology of major organs were analyzed after one week.All mice injected with the nanoparticles survived.CK,AST,and ALT were not significantly changed(P> 0.05).Histological examination of the major organs and tissues showed no morphological abnormalities or infiltration of inflammatory cells,indicating that the nanoparticles had a good biological compatibility.12 h after tail vein injection of Cy5-SDF-1α-PPADT nanoparticles into mice with full-thickness skin defects,the wounds and the surrounding tissues became fluorescent,while the other parts of the bodies were not fluorescent,suggesting that the PPADT nanoparticles responded to the high levels of ROS in the wounds,resulting in wound-targeted drug delivery.To determine whether the accelerated wound healing was due to SDF-1’s function in promoting homing of BMSCs or EPCs to the wounds,thereby inducing wound vascularization,exogenous BMSCs labeled with GFP were infused into wounded mice.Three days later,GFP+CD31+ cells could be found in the wounds.Mice in the SDF-1α-PPADT group had significantly more GFP+CD31+ cells accumulated in their wounds than those in the blank nanoparticles,pure SDF-1α or blank nanoparticles combined with pure SDF-1α groups,suggesting that SDF-1α-PPADT nanoparticles promoted BMSC homing to the wounds.Conclusions:1.The synthesized ROS-reactive organic polymer PPADT had a molecular weight of about 11 k Da with a dispersity of 1.97.ROS-responsive nanoparticles containing SDF-1α were prepared.The diameter of these nanoparticles was about 110 nm,and it’s drug loading rate was 1.8%.2.SDF-1α encapsulated in nanoparticles had a slightly lower activity,and the PPADT nanoparticles can target the encapsulated drugs to lesions with high concenrations of ROS.3.In mice with full-thickness skin defects,the concentration of SDF-1α in the serum increased significantly and was maintained long time after nanoparticle injection.The PPADT nanoparticles responded to the high levels of ROS in the wounds,resulting in wound-targeted drug delivery and SDF-1α-PPADT nanoparticles caused more GFP-BMSCs to localize to the wounds,leading to increased wound vascularization and accelerated wound healing. |
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