Nano-micellar Delivery System Based On PepT1/ER Target:Design,Synthesis And Bioactivity Studies

Peptide transporter 1(PepT1)and estrogen receptor(ER)are highly expressed in some tumor tissues,which are closely related to the occurrence and development of tumor.In our previous study,we found that PepTl can specifically recognize and absorbe L-valine-L-Valine(L-Val-L-Val)dipeptide prodrugs,and raloxifene has a high affinity for ER and has antagonistic activity.Based on this,this paper attempts to introduce the L-Val-L-Val dipeptide fragment and raloxifene structure fragment at the hydrophilic end of the amphiphilic polymer respectively.Then,the ring-opening polymerization of N-carboxy-?-amino acid-cyclic anhydride(NCA)of amino acid(alanine or histidine)was initiated by macromolecular primary amine compound.Two series of block polymerization were designed and synthesized based on PepT1 and ER-targeting respectively.The results of IR and 1H NMR characterization and analysis demonstrate that we successfully synthesized the target compound.Doxorubicin(DOX)was used to obtain PepT1 and ER-targeted nanometer micelles drug delivery system,and their physicochemical,appearance and bioactivity were studied.In this thesis,four polymers based on PepT1 receptor targeting were designed and analyzed.The particle size was within 200 nm by DLS,and it was spherical by transmission electron microscopy(TEM).The drug loading and encapsulation efficiency of the drug-loaded micelles were measured by UV spectroscopy.Four polymer micelles could effectively load DOX.The drug loading rate of polymer L-Val-L-Val-PEG-PHis was 8.5%and the encapsulation efficiency was 89.5%.And the release of DOX could reach 85%at 24 h after the introduction of acid-sensitive poly-histosine block at pH 5.5,which was significantly higher than 40%of normal physiological conditions,showed the characteristics of the acidic environment sensitive.In vitro antitumor experiments,four drug-loaded micelles showed activity comparable to free DOX in human hepatoma cell line(HepG2),human gastric cancer cell line(SGC7901)and human breast cancer cell(MCF-7).In this paper,the cytotoxicity of polymer micelles to Caco-2 cells was studied.The experimental results show that the polymer micelles are substantially non-toxic to the cells and exhibit comparable toxicity to DOX after loading on DOX.In vitro cell uptake assay showed that the cell uptake of polymer micelles was significantly higher than that of free DOX.And the cellular uptake of DOX of the target drug L-Val-L-Val-PEG-PAla-DOX and L-Val-L-Val-PEG-PHis-DOX was also significantly higher than that of non-target drug Ac-PEG-PAla-DOX and Ac-PEG-PHis-DOX.Competitive experime-nts further demonstrated the targeting effect of PepTl.Four polymers based on ER receptor targeting were also designed and synthesized.The nanostructures were confinned by DLS and TEM.The particle size distribution(<200 nm)and morphology(spherical)were also detected.UV test showed that 4 polymers could effectively load DOX.The drug loading rate of polymer Rlx-PEG4000-PHis was 9.07%,the encapsulation efficiency was 91.43%,and the loading effect was very good.In vitro cytotoxicity experiments showed that the polymer material was non-toxic to SGC7901,MCF-7,human non-small cell lung cancer(A549)and human normal cells(LO2),and the polymer material itself was safe and non-toxic.Four polymer micelles showed good cytotoxicity to SGC7901,A549 and MCF-7 cells comparable to free DOX after loading DOX,and had good prospects for further study.