Synthesis Of Four Porous Composite Drugs And Their Therapeutic Performance On Mustard/Sarin Poisoned Mice

As two categories of chemical warfare agents,erosive agents and nerve agents pose a major threat to the country,environment and human health and safety.Sulfur mustard（HD）is a typical representative of erosive poisons,erosion damage caused by skin exposure to HD is generally treated as burns and scalds,antibiotics,antioxidants,anti-inflammatory,antibacterial and other drugs should be used for symptomatic treatment,there are problems in the treatment such as the short drug effect time which requires repeated administration,and a single drug effect requires the combined use of multiple drugs.Therefore,it is of great significance to develop a multifunctional composite drug with sustained release properties.In this paper,two sustained-release composite drugs aiming to treat skin damage caused by HD were prepared based on the porous metal-organic frameworks（MOFs）Bio-MOF-13and medi-MOF-1,and their characterization and biological evaluation were carried out.Nerve agents mainly include sarin,tabun,Viex and Soman,etc.,it enters the human body inactivating the acetylcholinesterase（ACh E）in body and causes people to be poisoned.At present,2-PAM antidote is commonly used for treatment.However,the current clinical administration methods are difficult to maintain a sustained and stable blood concentration,and the drugs cannot effectively cross the blood-brain barrier and cannot restore the ACh E activity in central nervous system.To solve this problem our research group designed and synthesized two porous sustained-release composite drugs（2-PAM@MCM-41）-LINKER-ACh E and 2-PAM@VB1-MIL-101-NH₂（Fe）in the early stage,which was characterized and biologically evaluated in this paper.The main research results are as follows:1.For the treatment of chronic erosive skin lesions caused by HD,bacterial infection and the formation of bacterial biofilms are lethal to the healing of erosive wounds.In this paper,the porous MOFs material Bio-MOF-13 with antibacterial function was used as the drug-carrying material.Bio-MOF-13-COOH was synthesized by reaction of the amino group on surface of Bio-MOF-13 with succinic anhydride.It is negatively charged in deionized water at p H6.0.The antibacterial and anti-inflammatory drug cefazolin sodium（Ancef）,which is clinically used for the treatment of HD toxicity,was loaded into the pores of Bio-MOF-13-COOH in p H 6.0 deionized water.Then the positively charged under p H 9.0 alkaline protease（Alcalase 2.4 L FG）with biofilm scavenging function was loaded onto the surface of the material by electrostatic action,and a composite drug Alcalase@（Ancef@Bio-MOF-13-COOH）was successfully synthesized.The composite drug was characterized by IR,P-XRD,SEM,BET,Zeta potential and particle size analysis.The drug loading rate of the composite drug to Ancef was 17.0 wt%,and the loading rate of Alcalase protease was 20.6 wt%.The activity of Alcalase protease modified on the surface of the composite drug was determined,and it was reduced by 9.8%compared with the free Alcalase protease activity.The Ancef release from composite drug in different p H simulated body fluid PBS solutions was investigated.After 30 h,the drug release in p H 4.0,p H 6.0 and p H 7.4 PBS solutions was 85.8%,82.2%and 64.0%,respectively.The composite drug showed rapid drug release in acidic environment and slow drug release in alkaline environment.The inflammatory site of eroded skin is acidic,and as the inflammation decrease,the p H gradually returns to near neutrality,so that the drug can be released on demand.The guinea pig was used as a biological model,the skin was poisoned with HD,and the eroded skin was smeared with the composite drug.The composite drug showed a good effect of promoting the healing of the eroded skin.2.In this chapter,medi-MOF-1 was used as the base material,which is a MOFs compound with the drug molecule curcumin as the organic linker and Zn²⁺as the metal node.Dopamine（PA）and Ag NO₃ were sequentially added to the medi-MOF-1 suspension to make PA self-aggregate on the surface of MOFs to form polydopamine（PDA）,Ag NO₃ was reduced in situ by the phenolic hydroxyl group of PDA to silver nanoparticles（Ag NPs）with broad-spectrum antibacterial properties.Then polyethylene glycol amine,a PEG derivative with hydrophilic properties,was added to covalently linking its amino group with the phenolic hydroxyl group of PDA to improve the poor water solubility of curcumin,thus,a composite drug PEG@Ag NPs@PDA@medi-MOF-1 was prepared.The composite drug was characterized by IR、P-XRD,BET,SEM and particle size analysis.The decomposition of the composite drug in phosphate buffered saline（PBS）with different p H was investigated.In PBS with p H 7.4,p H 6.0 and p H 4.0,the curcumin released rate could reach to11.02%、9.35%and 8.01%after 300 h,respectively.Explore the therapeutic effect of the composite drug on erosion damage caused by HD exposure to guinea pig skin.With the increase of days after skin HD poisoned,compared with the poisoned group without any treatment,the skin erosion in the group treated with the composite drug was smaller,after the third day of treatment,the erosion range was significantly reduced and scabs appeared,after the fifth day,the skin basically returned to normal.The composite drug showed a good effect of promoting the healing of eroded skin.3.Based on the previous research work of our group,the composite drug（2-PAM@MCM-41）-LINKER-ACh E was characterized by IR and SEM-EDS.The loss of 2-PAM during the post-synthetic modification of the composite drug was accounted.The drug loading rate of 2-PAM@MCM-41 to 2-PAM was determined to be 28.2%.In the process of 2-PAM@MCM-41 modifying LINKER to synthesize（2-PAM@MCM-41）-LINKER,no 2-PAM leakage losses.During the further modification of acetylcholinesterase（ACh E）to synthesize（2-PAM@MCM-41）-LINKER-ACh E,3.9%of 2-PAM was lost,and the loading rate of 2-PAM by the composite drug was 21.1%.The therapeutic performance of the composite drug in mice exposed to the nerve agent sarin was evaluated.After the mice were administered 2.16 mg/kg sarin,under atropine assist the composite drug was administered by gavage.All the mice survived within 24 hours,while the sarin infected groups without any treatment all died.The ACh E reactivation rate in mice was tested,and the ACh E activity in treatment group was significantly higher than that in sarin poisoned without any treatment group,and the ACh E reactivation rate reached 79.9%at 120 min.After the composite drug is exposed to the nerve agent sarin,the gate valve ACh E is opened,and 2-PAM is slowly and continuously released,which effectively solves the problem that the activity of ACh E cannot be effectively revived due to too little 2-PAM and the activity of ACh E in body is inhibited by excessive 2-PAM.Composite drug achieve the purpose of on-demand drug release through gated drug release,which is expected to effectively solve the problem of hard control of blood drug concentration in treatment of nerve agent poisoning,and provide a simpler treatment method.4.Based on the previous research work of our group,the brain targeting function of the composite drug 2-PAM@VB1-MIL-101-NH₂（Fe）was verified.The composite drug was fluorescently labeled with fluorescein isothiocyanate（FITC）,and the fluorescently labeled composite drug was microinjected into the heart of zebrafish,and the brain fluorescence of zebrafish was observed by confocal microscope.The result showed,zebrafish which injected fluorescently labeled composite drug FITC-VB1-MIL-101-NH₂（Fe）had obvious fluorescence in brain,indicating that the composite drug had a good brain targeting function.In order to further verify the brain-targeting function,mice were used as biological models,the composite drug was injected into mice intragastric poisoned by nerve agent sarin through tail vein,and the ACh E activity in the brain of mice was tested at a specific time.Compared with mice injected the composite drug 2-PAM@MIL-101-NH₂（Fe）without VB1modification,the ACh E activity in brain of mice which injected the composite drug 2-PAM@VB1-MIL-101-NH₂（Fe）linked with the brain targeting functional group VB1 was significantly higher.The ACh E reactivation rate in the brain could reach 88.9%after 4.75 h,indicating that the composite drug could successfully cross the blood-brain barrier.The composite drug has good sustained-release performance and good brain targeting function,which can effectively solve the problems of difficulty in controlling the blood drug concentration and the inability of the drug to cross the blood-brain barrier in the treatment of nerve agent poisoning using 2-PAM.