The Development Of Caspase-3-targeted Nir-? Fluorescence Probe And Application Of The Probe In Early Diagnosis Of Necrosis Region In Skin Avulsion

Part1Objective: Skin avulsion always occurs in the extremities and the skin as well as subcutaneous tissues were detached from the underlying fasciae and musculoskeletal structures due to the heavy shearing forces.At the same time,it is accompanied by different degrees of soft tissue damage.The major myocutaneous perforating vessels usually accompany skin avulsion so that tissue biological changes in the early stages of the flaps were induced by ischemia and(or)hypoxia.However,such covered changes do not immediately show obvious clinical symptoms.Therefore,a new strategy is requested to realize the early definition of skin avulsion and necrotic area,which could provide the possibility for early and effective removal of skin necrotic area.In this way the risk of secondary necrosis and infection could be induced.Near infrared fluorescence imaging technology(NIR)can be divided into first window(NIR-I)and second window(NIR-II)according to its fluorescence emission.In comparison,the NIR-II window has dramatically improved imaging quality with higher resolution and sensitivity.According to these advantages,the NIR-II imaging technology has been widely used in medical molecular imaging in recent years,which may provide technical support for early accurate definition of avulsed skin necrotic area.In the first part,a fluorescent probe was synthesized by fluorophore CH1055 and a caspase-3 targeting peptide(GRRRDEVDK)and its chemical properties was also characterized.Methods: According to our previous work,we similarly synthesized NIR-II molecular probe CH1055-GK by fluorophore CH1055 and caspase-3 targeting peptide GRRRDEVDK in the experimental group.CH1055-PEG was also used as the control probe.molecular weight of each probe was then determined by matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).Results: The molecular weight of the probe CH1055-GK was 2.064 KDa and the control probe CH1055 PEG was 1.958 KDa.Conclusions: The NIR-II fluorescent probe CH1055-GK synthesized by the fluorescent group CH1055 and the caspase-3 targeting peptide GRRRDEVDK also has the advantage of small molecular weight.Part2Objective: The assessment of the binding capacity of CH1055-GK to Caspase-3 was detected in apoptotic cells and ex vivo skin tissue.Furthermore,the cytotoxicity of this probe in vitro was also discovered.Methods: Apoptotic cells were incubated with the probes,and then the binding capacity of CH1055-GK to Caspase-3 was evaluated by probe uptake ability and probe affinity;Similarly,the immunofluorescence of the probe and the cells were used to test the binding capacity as well;In addition,NIR-II imaging of avulsed skin in ex vivo was also used to assess the binding properties of the probe to Caspase-3.The CCK-8 assay was used to detect the cytotoxicity of the probe CH1055-GK in fibroblast cell(HSF),vascular endothelial cell(HUVEC),and epidermal cell(HACAT).Results: The probe CH1055-GK could bind to avulsed skin to visualize the necrotic in the NIR-II window with specific and highly efficient binding ability,which could be blocked by the excess peptide GRRRDEVDK(P<0.001).The combination of CH1055-GK and avulsed skin showed a significant high fluorescence signal within 2 hours,and the fluorescence intensity reached saturation within 6 hours and continued for 24 hours.CH1055-GK at different concentrations had no obvious cytotoxicity to HSF,HUVEC and HACAT.Conclusions: CH1055-GK could efficiently and specifically bound to avulsed skin tissue to image necrotic area without showing obvious cytotoxicity.Part3Objective: Assessing the binding ability of CH1055-GK to avulsed skin in vivo aimed to explore the possibility of early diagnosis of avulsed skin and definition of necrotic skin.In addition,the characteristics of NIR-II window imaging technology were discussed according to results of MRI imaging.Finally,H & E staining was used to detect the toxicity of probe in vivo.Methods: CH1055-GK was administrated into the mice through the tail vein as the experimental group and CH1055-PEG was also injected into the mice as the control group.The blocking group was injected within 2 hours before the probe injection.Three groups chose the same time point for imaging detection(2h,6h,12 h,24h).Moreover,MRI was used to detect normal mice and skin avulsion mice.For in vivo cytotoxicity,the probe CH1055-GK was injected into mice through the tail vein as the experimental group,and an equal volume of PBS was administrated to the control group,and then their major organs(Heart,Liver,Spleen,Stomach,Lung,Kidney,Brain,and Intestine)were harvested 24 h post-injection,and H&E staining was performed to evaluate toxicity in vivo.Results: NIR-II imaging: a region with high fluorescence intensity could be detected within 2h post-injection of CH1055-GK and the area of high fluorescence intensity was larger than the necrosis of the dorsal skin observed by our eyes.In Comparison with the results in 12 h and 24 h,we found that the former visible area of skin necrosis observed by our eyes was much smaller than the latter.However,the results of the NIRII imaging did not show the same difference,which showed the same range of high fluorescence intensity,indicating that the accurately determine of the necrotic area can be detected by NIR-II imaging.While injecting the contrast agent CH1055-PEG into the mice,there was no high fluorescence intensity appeared at all time points except extremely low fluorescence intensity(P <0.001).In the blocking group,most of the probe binding sites were occupied by competitive inhibitory polypeptides,resulting in the probe's free tissue space being finally eliminated in vitro(P <0.001).For the MRI imaging results,we could discover some more obvious changes including tissue edema and ruptured skin,but it was difficult to find the necrotic area of the avulsion skin in early stage.On the contrary,NIR-II imaging could detect directly the area with the high fluorescence signal of the necrotic parts in avulsed skin,indicating that the NIR-II imaging could distinguish the area of necrosis in skin avulsion.Finally,H & E staining results showed that the mice in the probe group did not have acute injury and inflammation.Conclusions: The probe CH1055-GK can specifically bind to avulsed skin in vivo and presented skin necrosis areas.Furthermore,NIR-II small molecular probe CH1055-GK was a safe biological probe.