Construction And In Vitro Evaluation Of Folic Acid Modified SN38 Prodrug Self-assembled Micelle Delivery System

According to the"Global Cancer Report"from the International Health Organization（WHO）,the number of cancer patients and deaths caused by tumors are rising rapidly around the world.One of the most conventional tumor treatment is chemotherapy,however,many chemotherapy drugs have some shortcomings,such as poor water-solubility and low-bioavailability.Therefore,the chemotherapeutic drug delivery system based on nanocarriers has gradually entered people’s vision.1.Preparation and characterization of micellar TAT-PEG₂₀₀₀-SN38This chapter used hydrophilic polymer polyethylene glycol（PEG）to connect SN38 and TAT（GRKKRRQRRRQC）,determined the synthetic route of TAT-PEG₂₀₀₀-SN38.We designed that the PEG connected SN38 by ester bond,the ester bond can help SN38 release under slightly acidic environment stimulation in the tumor site.The other end of PEG connected the cell-membrane peptide TAT to increase the ability into cells.The dipeptide with GC sequence（without cell penetrating effect）was selected as the control to study the cellular uptake of TAT-PEG₂₀₀₀-SN38.We used ¹H NMR,IR and UV to ensure that the product was successfully synthesized.The uniform micellar TAT-PEG₂₀₀₀-SN38 was prepared by ethanol injection method,the particle size of TAT-PEG₂₀₀₀-SN38 was135.24±2.36 nm,PDI was 0.129±0.047,and Zeta potential was+12.33m V by laser particle size analyzer.2.Anti-tumor activity evaluation of micellar TAT-PEG₂₀₀₀-SN38 in vitroIn this chapter,the micelles prepared above have been used for anti-tumor activity evaluation in vitro.We observed the inhibitory effects of SN38,GC-PEG₂₀₀₀-SN38 and TAT-PEG₂₀₀₀-SN38 on A549 and He La cells.The inhibitory effects of the three groups of drugs on tumor cells increased with the increase of the drug concentration from 2 nmol/L to 20000nmol/L（the same concentration of SN38）,but compared with SN38 and GC-PEG₂₀₀₀-SN38,the TAT-PEG₂₀₀₀-SN38 group showed significantly higher cell inhibition rate.Though the observation and analysis of qualitative and quantitative,the uptake of TAT-PEG₂₀₀₀-SN38 group was significantly increased more than GC-PEG₂₀₀₀-SN38 group.On A549 and He La cells,the uptake of the TAT-PEG₂₀₀₀-SN38 group was 4.7 and 3.6 times higher than GC-PEG₂₀₀₀-SN38 group,respectively.3.Establishment of FA/TAT-PEG₂₀₀₀-SN38 delivery system and investigation of its propertiesFolate receptor（FRs）expressed in many human tumor cells,and low expression or not in normal cells,and folic acid and folate receptors with high affinity.Drugs can enter cells via a pathway mediated by folate receptors.So this chapter used folic acid（FA）modified micellar TAT-PEG₂₀₀₀-SN38 to obtain FA/TAT-PEG₂₀₀₀-SN38 delivery system by electrostatic adsorption.The particle size and Zeta potential of the delivery system changed with the concentration of folic acid.By investigating the stability and release capacity of the delivery system in vitro,we found that the delivery system could remain stable for a certain period of time under physiological conditions and release SN38 under acidic conditions（p H5.6）.4.Anti-tumor evaluation of FA/TAT-PEG₂₀₀₀-SN38 delivery system in vitroIn this chapter,we used the FA/TAT-PEG₂₀₀₀-SN38 delivery system on the A549 cells with low folate receptor expression and the He La cells with high folate receptor expression.The research results showed that the growth viability of the He La cells were related to the concentration of folic acid in the range of FA5 to FA20,the tumor growth viability decreased with the increase of the folic acid concentration（from 71.50%to 42.70%）.However,on the A549 cells,the increase of folic acid concentration did not cause the decrease in tumor cell viability.Additionally,confocal laser observation and flow cytometry measurement also showed that combination of folic acid and TAT-PEG₂₀₀₀-SN38 can increase the uptake of FA/TAT-PEG₂₀₀₀-SN38 delivery system on the He La cells,and flow cytometry showed that the fluorescence uptake of FA20 was 2.2 times of FA0.But on the A549 cells with low folate receptor expression,increaed the folic acid concentration did not increase the uptake of FA/TAT-PEG₂₀₀₀-SN38 delivery system.At last,we have studied the mechanism of FA/TAT-PEG₂₀₀₀-SN38 delivery system in two cells,and found that both cells uptake the delivery system mainly through the clathrin-mediated endocytic pathway,followed by lipid raft.The uptake of He La cells also affected by high-concentration folic acid solution（0.1M）,and its inhibition rate is 34.55%,but the uptake of A549 cells was not change,indicated that the FA/TAT-PEG₂₀₀₀-SN38 delivery system can enter cells through a folate receptor-mediated pathway,which is related to the expression of folate receptors on the cell surface.This result showed that the FA/TAT-PEG₂₀₀₀-SN38 delivery system can effectively target tumor cells with high folate receptor expression,and thereby reducing the toxicity to normal cells.