Preparation Of Tumor Microenvironmentresponsive Nanomicelles For Co-delivery Of Antitumor Prodrug And Enzyme

Cancer is one of the leading causes of death in the world.Up to date,various cancer treatment approaches including chemotherapy,phototherapy,radiotherapy,etc.have been widely studied by researchers.“Enzyme–prodrug” therapy(EPT)as a novel therapeutic approach has shown a broad application prospect for global healthcare.It is a two-step strategy: initially,a foreign prodrug-activating enzyme is delivered to the tumor cells using various targeting approaches;then,a prodrug is introduced as a nontoxic precursor of an active drug into the targeted cells,where the prodrug can be specifically activated into an anticancer drug through enzymatic activity.Although “enzyme–antitumor prodrug” therapy has shown a broad application prospect and brings a new way of thinking,there are still many key issues to be solved.“Enzyme–prodrug” therapy is a two-step delivery strategy that increases the patient's pain and treatment costs.Besides,the second drug delivery time is not easily graspable(for antibody-directed enzyme prodrug therapy,coupling of the enzyme and an antibody could allow it to be targeted selectively to the tumor surface with the characteristics of the antibodies to identify tumor surface antigen after intravenous injection,and enzyme was taken to the targeted tumor.After the coupling of antibody and enzyme was fully removed from the blood,the prodrug without anticancer activities or low activity in normal tissues and organs can be injected intravenously.Otherwise,the enzyme stranded in normal tissues and organs in the body can catalyze prodrug and cause damage to normal tissue.So the second drug delivery time is not easily graspable).Prodrug doses in “enzyme–prodrug” therapy is relatively high,because only drug of reaching a certain concentration in the lesion sites can play a role of treatment.But there is a stranded phenomenon in drug delivery process.If the prodrug dose is too small,the drug concentration at tumor tissue is not enough.But prodrug dose is too big,it may cause the accumulation of drug and certain side effects to the body.Moreover,it increases the treatment cost.If enzymes and prodrugs are transmitted to the tumor cell at the same time,many disadvantages caused by a two-step EPT can be avoided.Therefore,the exploitation of an efficient delivery vehicle that can simultaneously carry prodrugs and enzymes to the target sites is vitally important.The combination of horseradish peroxidase and 3-indoleacetic acid(HRP-IAA)has recently been extensively studied “enzyme–prodrug” system.IAA which is a powerful plant growth phytohormone can be activated by HRP to produce a series of free radicals(ROS),including indolyl radical,skatole radical and peroxyl radical.It can lead to cell lipid peroxidation,cell membrane structure change,DNA damage,reduced the Bcl-2 activity and activation of related apoptosis gene.However,the influence of the same concentration IAA is not significant for normal cells growth,and IAA is not easily oxidized by mammals peroxidase.Esters are the most common linkages in prodrug design.They can be readily hydrolyzed by esterases which are ubiquitously distributed in the body,leading to the release of the parent drugs.Ethyl 3-indoleacetate(EIA)is a hydrophobic prodrug that,once transmitted to the tumor location,can be quickly hydrolyzed by the double action of esterases and low pH condition of the tumor location to produce IAA.Polymeric micelles consisting of amphiphilic polymers formed by self-assembly has demonstrated great potential in delivering anticancer drugs,siRNA,and other therapeutic agents.Nanomicelles have priority to enter the tumor tissue rather than the normal tissue by an enhanced permeability and retention(EPR)effect,which reduces the side effects of chemotherapy.In addition,micelles have a longer blood circulation time and improve the utilization rate of the drug.Furthermore,micelles have many design abilities for specific stimulation in the tumor microenvironment.For instance,tumor tissues(pH 6.5–6.8),endosomes(pH 5.5–6.5),and lysosomes(pH 4.5–5.5)are the slightly acidic microenvironments different from the bloodstream and normal tissues(pH 7.4),so pH-sensitive micelles can be designed to achieve the intelligent control release of anticancer drugs in tumor sites.In addition,the intracellular concentrations of reductive glutathione(GSH)is 100–1000 times higher than the extracellular concentrations of GSH.Reduction-sensitive polymeric micelles with a disulfide bond which rapidly cleaved inside cancer cells,can realize the pinpointed control release of drug.Consequently,polymeric micelles are considered ideal carriers for co-delivery of enzymes and prodrugs for tumor therapy.This paper mainly designed different tumor microenvironment-sensitive polymer micelle to realize the co-delivery of enzymes and prodrugs,so as to avoid the defects of the two-step enzyme prodrug therapy.The concrete content includes:(1)we synthesize the amphiphilic copolymer PEG-PAsp(-AED)-CA,which forms nanomicelles through self-assembly,and co-loads the HRP and EIA.PEG forms a shell layer,the HRP enzymes can be loaded on the amino groups of PAsp(AED)by electrostatic adsorption.EIA can be coated in a micellar nucleus formed by the hydrophobic building block CA which exists in the human body and has been successfully applied in drug delivery.In the structural design,an interlayered GSHsensitive disulfide bond module was engineered into the PAsp(AED)of polymeric micelles to turn the drug and enzyme release once the micelle is up to tumor cells which have a high GSH concentration.EIA can be rapidly hydrolyzed to produce IAA by the double action of low pH and esterases in the tumor.Then IAA can be activated by HRP to produce a large number of ROS to kill tumor cells.TEM photographs show that the polymer micelle is around 40 nm,and the cell survival rate test shows that the EIA/HRP polymer micelle is highly lethal to human lung adenocarcinoma cells.Thus,co-delivery of EIA and HRP demonstrates great potential in cancer therapy.(2)we synthesize the pH-sensitive copolymer mPEG-PAsp(THA)-AcMal7,which forms nanomicelles through self-assembly,and co-loads the HRP and EIA.PEG forms a shell layer,the HRP enzymes can be loaded on the ammonium groups of PAsp(THA)by electrostatic adsorption,AcMal7 as pH-sensitive hydrophobic chain segment is used to form hydrophobic micellar cores to coate EIA.Once the micelle is up to tumor cells,pH-sensitive hydrophobic AcMal7 is transformed to hydrophilic Mal7 under low pH of tumor site.At this point,the micellar structure was damaged.Then,the EIA and HRP was released,and produced a large number of ROS to kill tumor cells.TEM photographs show that the polymer micelle is successfully prepared,and the cell survival rate test shows that pH-sensitive copolymer micelle demonstrates great potential in the co-delivery of enzyme and prodrug for cancer therapy.Compared with the first work,this work avoid reducing amino for the consumption of free radicals and improve the treatment efficiency.