Preparation Of SiRNA-Drug Self-assembly For Its Application In Cancer Therapy

In recent years,more and more diseases have been treated by combination therapy.It can not only enhance the therapeutic effect but also improve the drug resistance.Paclitaxel can be used to treat many diseases,such as advanced ovarian cancer,breast cancer,non-small cell lung cancer.However,due to its poor water solubility,it is necessary to add other cosolvents or carriers to improve the water solubility in clinical application.Unfortunately,using cosolvents will increase the practical operation difficulty and risk factor.For example,special devices are required(such as infusion equipment with filters)of the application of polyoxyethylene castor oil dissolve paclitaxel,and patients have to be injected with anti-allergic drugs in advance.To overcome this shortcoming,researchers use a variety of carriers to delivery paclitaxel.For instance,the use of albumin-loaded paclitaxel can not only significantly promote the water solubility,but also greatly improve the bioavailability of paclitaxel with the better targeting of albumin.Therefore,it is a great significance to develop novel,efficient and low-toxic carriers to improve the water solubility of paclitaxel.Nucleic acids(such as DNA,siRNA)are endogenous substances with good biocompatibility and targeting(e.g.aptamers).siRNA is one of the effector molecules in gene therapy,which can specifically recognize the target gene and prevent protein being translated by its mRNA,to achieve gene silence effects.However,many obstacles are hinder the ultimate functionality of siRNAs in clinic.These barriers are including enzyme degradation,recognition by the immune system,renal clearance,impediments to tumor tissue penetration and uptake into tumor cells,endosomal trap once in tumor cells,and off-target effects.Recently,researchers have developed a variety of vectors for deliver siRNA or chemically modifying siRNA to overcome the deficiencies of siRNA,and further expanded their applications.In this thesis,three hydrophobic compounds were designed and synthesized and their interaction with DNA or siRNA was studied.The specific research contents are as follows:(1)The terminally functionalized paclitaxel derivatives TM2 was designed and synthesized and its reactivity with DNA was studied.Compound TM2 was composed of alkynyl group and paclitaxel.The alkynyl group can be click-reacted with DNA-N3.However,the experimental result show that this reaction was failed due to low efficiency and difficult to purify.Therefore,the scheme was improved in subsequent experiments,through using a copper-free click.(2)The paclitaxel derivative TM3 was designed and synthesized.Azide group is one of three end groups in TM3,it can be copper-free click reaction with cyclooctyne on siRNA or DNA strand,and other two groups are paclitaxel.Compared to compound TM2,the compound TM3 has higher reactivity,this improvement may increase the amount of drugs and enhance hydrophobicity,and easier self-assembly.(3)The linker compound TM4 was designed and synthesized and successfully bridged the compound TM3 and DNA.TM4 contains three terminal groups,wherein the two ends are modified with cyclooctyne,and the other end is modified with a p-nitro group.The p-nitro group can reaction with siRNA-NH2 or DNA-NH2 to introduce cyclooctyne on siRNA or DNA strand.It lays the foundation for subsequent reactions with azide compounds.(4)The nanomicelle Y formed by self-assembly of compound TM3 and DNA.Firstly,the nanomicelle Y was verified by gel electrophoresis,then its morphology and endocytosis were observed by microscope.The experimental results show that the nanomicelle Y is a round particle and can be endocytosed by cells which distributed in the cytoplasm.Next,siRNA can be hybridized to the DNA strand for activity assay studies.