Study On The Construction Of Tumor Targeting PH/reduction-responsive Non-anticoagulant Heparin ES2-paclitaxel Nanodelivery System And Its Anti-tumor Effect

The formation of angiogenesis is crucial to the growth and metastasis of tumors and can provide nutrients for the occurrence and development of tumors.Our research group screened a polypeptide sequence(ES2,IVRRADRAAVP)in the previous study,and found that this sequence can significantly inhibit the formation of tumor neovascularization.Based on previous research results,the ES2 as the research object,chemical modification of it with non-anticoagulant heparin(GSHP)has been adopted to improve its existing problems such as short half-life and poor stability.In order to increase the tumor targeting effect,firstly using phenylboronic acid,which has a high affinity for the highly expressed sialic acid on the surface of tumor cells,as the target group,phenylboronic acid molecules are connected to the sugar chain using a condensation reaction between the amino group on 3-aminophenylboronic acid(PBA)and the carboxyl group on GSHP,and the sample GSHP-PBA(GP)was obtained.Secondly,in order to increase the balling ability of the nanoparticles,D-α-Tocopherol succinate(TOS)is connected to GSHP in the form of ester bonds to obtain a sample GSHP-PBA～TOS(GPT).Finally,using cystamine(Cys)as a connecting arm to connect GSHP and ES2,sample ES2-GSHP-PBA～TOS(EGPT)was obtained.In addition,in order to increase the anti-tumor activity of nanoparticles,the broad-spectrum anti-tumor drug paclitaxel(PTX)was encapsulated inside the nanoparticles to obtain the final nanoparticles PTX/EGPT NPs.PTX/EGPT NPs nanoparticles have the effect of "one drug and two targets",under the action of PBA,the drug reaches the tumor site,and under the stimulation of the tumor microenvironment(low pH,high GSH),the nanoparticles disintegrates and release ES2 and PTX,thus inhibiting the proliferation of endothelial cells and killing tumor cells at the same time,thus achieving the tumor inhibitory effect.The main contents and results of this study are as follows:(1)Synthesis and Characterization of PTX/EGPT NPsFirst,non-anticoagulant heparin GSHP was prepared by sodium periodate oxidation and sodium borohydride reduction,using EDCI and NHS as catalysts,PBA is connected to the sugar chain GSHP through amide bonds,subsequently,EDCI,NHS,and DMAP were used as catalysts to connect TOS to the sugar chain through ester bonds.Then,connect ES2 to the sugar chain using Cys as a link to obtain EGPT.The structure of the polymer in each step was characterized by 1H NMR,and the final characterization results showed that EGPT was successfully prepared.Finally,PTX was wrapped inside the nanoparticles by physical encapsulation to obtain PTX/EGPT NPs,the drug loading and encapsulation rate of PTX were studied by high performance liquid chromatography(HPLC).Dynamic light scatterometer was used to investigate the particle size of nanoparticles,and transmission electron microscopy was used to observe the morphology of nanoparticles.Finally,the drug release behavior of nanoparticles was studied by simulating the tumor microenvironment.The results showed the drug loading and encapsulation rate of PTX were 4.39%±0.56%and 70.11%±8.66%,respectively.The nanoparticles were spherical in aqueous solution with a particle diameter of 202.32 ± 4.32 nm.In addition,PTX and ES2 can achieve rapid release in weak acidic environments and high glutathione environments,respectively,indicating that nanoparticles have the characteristics of tumor microenvironment sensitivity.(2)In vitro biological evaluation of PTX/EGPT NPsUsing EAhy926 cells and B16F10 cells as research objects,the anti-angiogenesis and anti-tumor abilities effects of PTX/EGPT NPs in vitro were evaluated using CCK-8,migration,invasion,lumen,and apoptosis experiments,in vitro tumor targeting of PTX/EGPT NPs was studied by laser confocal and flow cytometry.The results showed that PTX/EGPT NPs could significantly inhibit the proliferation,migration and invasion of the two types of cells,due to the presence of ES2,nanoparticles can significantly inhibit the lumen formation of endothelial cells.In addition,the results showed that PTX/EGPT NPs can induce apoptosis of endothelial cells and melanoma cells.It was found that PTX/EGPT NPs had stronger targeting effect on melanoma cells compared with ES2 by targeting experiments.(3)In vivo biological evaluation of PTX/EGPT NPsUsing small animal in vivo imaging system investigate the tissue distribution of PTX/EGPT NPs in mice,tumor bearing mouse model was used to evaluate the anti-tumor ability of nanoparticles in vivo,at the same time,the expression of apoptosis factor and vascular growth factor in mouse tumor tissue was studied through immunohistochemistry experiments.The results of in vivo distribution indicate that nanoparticles have significant tumor targeting and can remain in tumor sites for longer periods of time.The results of anti-tumor experiments in vivo indicate that nanoparticles have a strong inhibitory effect on solid tumors in vivo,with an inhibitory rate of 76.56%,and have no significant toxic and side effects on mice.In addition,immunohistochemical results showed that nanoparticles could significantly inhibit the expression of Bcl-2,COX-2,MMP2/9,VEGF and TGF-β,and significantly promote the expression of Cyt-C and Caspase-3.In conclusion,PTX/EGPT NPs showed good anti-angiogenesis and anti-tumor activity.In addition,this study successfully applied tumor targeting agents,anti-angiogenesis drugs,anti-tumor transfer drugs and chemotherapy drugs in the same drug delivery system,which has a good application prospect.