Preparation Of PH-Sensitive Pluronic L61 Modified Chitosan Nanoparticles And Evaluation Of Its Antitumor Effect

The treatment of cancer today mainly relies on chemotherapy,but small molecule chemotherapy drugs have some defects such as strong toxicity,poor water solubility and lack of tumor specificity.In addition,chemotherapy drugs induce multiple drug resistance(MDR)in tumor tissue,severely limiting the therapeutic effect.The reason of MDR generation relies on the ATP-binding cassette transporters(ABC).Therefore,the MDR effect of tumor cells can be reversed by inhibiting the production of ATP and the activity of ATPase.Nano-drug delivery system can effectively improve the solubility,stability and circulation time of small molecule chemotherapeutic drugs in Vivo.It is very difficult for nano-drug delivery systems to achieve optimal drug concentration in tumor cells by enhancing the Enhanced permeability and retention effect(EPR),the reason is that the nano-drug delivery system can not break and release the drug in a short time after it is taken up by tumor cells.A novel drug delivery system based on tumor-specific micro-environment with stimulation response(such as p H-sensitive,reductive-sensitive,enzyme-sensitive)can achieve accurate and rapid drug release in tumor sites.In this paper,macromolecule crosslinker(L61-OE)was obtained by modifying pluronic L61 with Ortho ester(OE),then L61-OE was reacted with Chitosan to form nanoparticles,and anti-tumor drug doxorubicin(DOX)was loaded on it,a p H-sensitive nano-drug delivery system(L61-OE-CS/DOX)that reverses tumor multiple drug resistance was thus constructed.Pluronic L61 has the characteristics of inhibiting ATPase activity and reducing ATP production in tumor cells.The Ortho ester bond is hydrolyzed rapidly in the environment of Tumor micro-acid,which causes the depolymerization of particles and the rapid release of drugs.A series of in vivo experiments were carried out to evaluate the physicochemical properties and antitumor effects of the drug delivery system:(1)On the basis of the reported synthesis of Ortho ester(OE),the OE is connected to the two ends of pluronic L61 to synthesize L61-OE monomer,and then the active ester DSC is connected to both ends of the monomer.The L61-OE-SC crosslinking agent was obtained.Using the same method to make L61 react with DSC,the control group L61-SC crosslinker was obtained.The structure of L61-OE-SC and L61-SC was determined by Nuclear Magnetic Resonance Spectroscopy.(2)The synthesized L61-OE-SC and L61-SC crosslinkers were dissolved in acetone and crosslinked with chitosan(CS)to prepare p H-sensitive L61-OE-CS and control L61-CS nanoparticles,and the particle size,morphology,and stability of the two nano-transport systems are measured by using the Dynamic laser light scattering(DLS)and Scanning Electron Microscope(SEM).(3)DOX is encapsulated in the prepared nanoparticles to obtain L61-OE-CS/DOX and L61-CS/DOX drug-loaded nanoparticles.DLS and Transmission Electron Microscope(TEM)were used to detect the size,morphology,drug loading,and drug release rate of drug-loaded nanoparticles in different environments.At the same time,Laser Scanning Confocal Microscopy and Flow Cytometry were used to determine the uptake of two kinds of nanoparticles by tumor cells.In order to prove the effect of L61 on mitochondria,we used a fluorescence microscope to observe the ROS production of free drugs,two blank nanoparticles and their drug-loaded particles.At the same time,we use the MTT method to explore the cytotoxicity of the developed nanoparticles.(4)Next,a two-week in vivo anti-tumor experiment was carried out on tumor-bearing mice.After two weeks,the results of the changes in the size of the tumor and the weight of the mouse and the final tumor weight and tumor photos were obtained: p H-sensitive drug-loaded nanoparticles can better control tumor growth and improve the quality of life of mice.Both the drug distribution and tissue section photos show that p H-sensitive drug-loaded nanoparticles can effectively reduce the damage of drugs to normal tissues.In conclusion,p H-sensitive drug-loaded nanoparticles based on Ortho ester and pluronic L61 have good antitumor effect and can significantly prolong the life of tumor-bearing mice.