Bionic Photothermal Hydrogel Based On 1-monocaffeoyl Glycerides Promotes Wound Healing In Diabetic Rats

Diabetic wound is a chronic,difficult-to-heal complication of diabetes.Diabetic wound has three major pathological bases:vascular lesions,neuropathy and infection,which form a vicious circle and lead to prolonged healing of the wounds.Presently,different drugs are mainly administered orally to control infection,vascular lesions and neuropathy,but oral administration of drugs for wound healing has problems such as inability to target all three pathologies at the same time,low bioavailability and high side effects.There is a lack of effective topical drugs for infected diabetic wounds in clinical practice.Therefore,we expect to obtain a therapeutic drug that can target all three pathological bases simultaneously and design an effective delivery treatment for them.Caffeic acid（CA）is the main active constituent of the Chinese herbal medicine C.minor,which has many pharmacological effects such as antioxidant,anti-inflammatory,antibacterial,anti-drug resistance and promoting nerve repair.To address the disadvantages of CA such as low amphipathicity and low bioavailability,we previously successfully synthesized 1-monocaffeoyl glycerol（1-MCG）,which has been shown to have excellent amphipathic,biocompatible and antioxidant properties.We propose to apply 1-MCG as a functional component in the topical treatment of diabetic wounds,to investigate its physiological activity and feasibility of in vivo application,and to design a multifunctional hydrogel for the treatment of diabetic wounds with its typical symptoms.The main contents and results of this thesis are as follows:（1）The physiological activity and mechanism of 1-MCG and CA in vitroFirstly,1-MCG was confirmed to have good biocompatibility with RAW 264.7,SH-SY5Y and Ha Cat cells.With the help of H₂O₂-damaged RAW 264.7,SH-SY5Y and Ha Cat,the antioxidant property of 1-MCG to protect cells was verified,and the results showed that 1-MCG-treated cells exhibited superior cell viability,cell morphology,cell proliferation and migration ability than CA-treated cells.These could be attributed to the ability of 1-MCG to reduce ROS levels and its effect on intracellular JNK pathway of JNK,NF-κB,IL-8,TNF-α,COX-2 and i NOS,etc.Subsequently,the expression levels of PI3K,p53,ERK,VEGF,TGF-βand CAM in cells were measured to demonstrate that 1-MCG could regulate cell proliferation and differentiation through PI3K and MAPK signaling pathways.In addition,1-MCG could effectively reduce the ratio of MMP-9/TIMP-1,which suggested that 1-MCG could better inhibit the balance of degradation and deposition of ECM in wounds to promote wound repair.The results of CAM expression levels and membrane potential indicated that 1-MCG could maintain the membrane potential and signaling homeostasis of cells and reduce apoptosis.（2）Non-covalent interaction between 1-MCG/CA and BSA/LYS and the biological properties of their binding complexesThe interaction between 1-MCG and BSA/LYS were investigated by fluorescence spectroscopy,simultaneous fluorescence spectroscopy,UV spectroscopy,circular dichroism spectroscopy,molecular docking and multifunctional wave function（Multiwfn）,followed by H₂O₂-damaged RAW 264.7,SH-SY5Y and Ha Cat cells to investigate the antioxidant properties of the 1-MCG-BSA complex The inhibition and bactericidal ability of 1-MCG-LYS was determined by measuring MIC and MBC values.The results showed that 1-MCG could spontaneously bind to BSA and LYS to form the complex and the caffeoyl and glycerol skeletons were identified as the main binding regions,and the main binding force was hydrophobic interaction.The activity results showed that 1-MCG-BSA has good biocompatibility and better antioxidant activity,and it can maintain the intact and normal morphology of cells.The results of anti-bacterial assays showed that 1-MCG and LYS had synergistic effects on the inhibition and fungicidal activities against S.aureus and Enterococcus faecalis.（3）Preparation and characterization of 1-MCG-based biomimetic photothermal hydrogelWe successfully prepared a multifunctional 1-MCG-based hydrogel with photothermal effect,excellent adhesion ability,and controlled release ability by the"one-pot method".The FTIR,SEM,rheological scan,NIR response,adhesion ability,gelation time,swelling,controlled release properties and safety of the hydrogels were investigated.The results showed that the hydrogel could transform into a flowable gel under NIR irradiation to match different depths and shapes of wounds.1-MCG-PDP hydrogel has a more regular,orderly and uniformly dense pore structure,and gels mainly throughπ-πand hydrogen bonding interactions.This structure results in a higher LVR region for 1-MCG-PDP hydrogel.1-MCG-PDP hydrogel has a higher solution absorption capacity,its volume is more easily expanded by absorption,and the local pressure on the wounds is lower.This hydrogel is able to adhere tightly to the skin surface and meet the movement of the joint without shedding.In addition,1-MCG-PDP hydrogel has both NIR and collagenase response.Finally,the results of the safety study of the hydrogel showed that the cell survival rate was>85%in the presence of different hydrogel extracts.（4）1-MCG-PDP hydrogel promotes the recovery of diabetic rat wounds infected with S.aureusAfter constructing diabetic rat wounds infected with S.aureus,different groups of hydrogels were applied to the rat wounds,and the results showed that 1-MCG-PDP hydrogel significantly accelerated the healing of the wounds,exhibiting superior epithelialization,angiogenesis and collagen deposition than CA-PDP hydrogel.1-MCG-PDP hydrogel exhibited the best antibacterial ability.1-MCG-PDP hydrogel was able to modulate the levels of inflammatory factors such as TNF-αand IL-8 in wounds and polarize macrophages to M2 anti-inflammatory phenotype to regulate inflammation and immune response in wounds.1-MCG-PDP hydrogel promoted vascular growth by elevating the level of VEGF.1-MCG-PDP hydrogel could also improve the collagen deposition and cell growth in wounds by decreasing the MMP-9/TIMP-1 ratio.1-MCG-PDP hydrogel can provide higher levels of neurotrophic factors and more bipolar neuronal cells by upregulating the expression of neurological factors such as GDNF,GFAP and PGP 9.5.The hydrogel promotes the healing of diabetic wounds by promoting vascular regeneration,nerve repair and antibacterial effects.