Nano-drug Delivery System Based On Thiophene Derivatives And Their Antitumor Activity

Globally,the death caused by malignant tumors ranks only second to cardiovascular and cerebrovascular diseases,therefore,malignant tumors are serious threats to human health.At present,chemotherapy is one of the most commonly used methods in the clinical treatment of malignant tumors.Thiophene derivatives(TPs)were widely found in nature.Moreover,a thiophene ring skeleton was also contained in effective molecular structures of anti-tumor drugs with good antitumor activity.Therefore,TPs had attracted researchers' extensive attentions in the field of biological medicine.The antitumor activities of six TPs were also investigated in this paper,showing that they all possessed certain antitumor activity.However,the advantages,including poor solubility,high toxicity and no selectivity still need to be overcome.The novel drug delivery system can delivery TPs to lesion tissue with the help of water-soluble drug carrier.Meanwhile,abundant functional groups were contained on the surface of the drug carriers,which was easy to be modified by targeted receptors and endowed their targeting.Therefore,the disadvantages including poor solubility,high toxicity and no selectivity will hopefully to be solved by drug delivery system.Under such background,this paper focuses on the screening and evaluation of the anti-tumor activity of TPs,and the establishment of a drug delivery system.The detailed conclusions are as follow:1.The anti-tumor activity screening and physical and chemical properties of TPs.Firstly,the antitumor activity of six TPs in HepG2 and SMMC-7721 cell lines were investigated by MTT assay.TP 5 displayed higher antitumor activity than other TPs including paclitaxel when the concentration of TPs was 30.0 ?g/mL.And TP 5 had a good recovery,precision,and stability.The solubility of TP 5 was only 26.36 ?g/mL in deionized water and 27.64 ?g/mL in PBS(pH 7.4),showing low solubility in aqueous solution.The solubility of TP 5 in organic solvents was relative higher,but organic solvents are toxic and not suitable as pharmaceutical vehicles.2.The evaluation of anti-tumor activity of TP 5.In order to further evaluate the anti-tumor activity of TP 5,the anti-tumor mechanism of TP 5was studied in vitro and in vivo using cellular and molecular biological methods.The results showed that TP 5 could increase LDH release in hepatocellular carcinoma cells(HepG2 and SMMC-7721),inhibit tumor cell migration,induce apoptosis,enhance caspase-3,-8,-9 activity,increase ROS content and decrease mitochondrial membrane potential.Western Blot assay results showed that TP 5 could activate PARP,caspase-3,-8,-9,increase the expression of apoptotic proteins Bad and Bax related to mitochondrial pathway,and inhibit the expression of apoptotic protein Bcl-2.Finally,HepG2-and SMMC-7721-xenograft tumor model were established by injecting cancer cells into the back of nude mice.After 14 days of treatment,the tumor volume was dramatically decreased.The western blot analysis results of apoptosis-related proteins in tumor tissues are consistented with the experimental results in vitro,which further demonstrated that TP 5 can induce apoptosis of liver cancer cells.3.Study on folate-targeted PLGA drug delivery system.In order to improve the solubility of TP 5 and endowed it targeting,TP 5 was loaded in PLGA nanoparticles and folate was modified on their surface.And folate-targeted PLGA drug delivery system(TP 5-FPNPs)were obtained.Their structure,drug-loading capacity,encapsulation efficiency,stability,release behavior in vitro,antitumor activity and uptake were investigated.TP 5-FPNPs was spherical and the particle size was 170 nm,drug-loading capacity and encapsulation efficiency was 2.90% and 94.64% ± 0.73,respectively.The stability was satisfactory.TP 5 was released ~80.0% from TP 5-FPNPs at 48.0 h.Meanwhile,TP 5-FPNPs had a better anti-tumor effect and higher uptake than TP 5-PNPs.4.The establishment of albumin nanoparticle drug delivery system.In order to futher improve the drug-loading capacity of TP 5,HSA based nanoparticles(TP 5-NPs)was synthesized.The optimum conditions for preparing TP 5-NPs was obtained by adjusting the temperature and concentration of TP 5.The TP 5-NPs had an encapsulation efficiency of 99.59% and drug-loading capacity of 3.70%.TP 5 was slowly released from TP 5-NPs in vitro over 120.0 h.TP 5-NPs possessed high antitumor activity for HepG2 and SMMC-7721 cell lines.ROS levels were elevated and mitochondrial membrane potentials reversed when the two cell lines were treated with TP 5-NPs for 12 h.Cellular uptake of fluorescence-labeled TP 5-NPs in vitro was analyzed by flow cytometry and laser confocal scanning microscopy.Fluorescence intensity increased over time,suggesting that TP 5-NPs were efficiently taken up by tumor cells.In conclusion,TP 5-NPs showed great promise as an anticancer therapeutic agent.5.The evaluation of antitumor activity of TP 5-NPs in vivo.The antitumor activity and mechanism of TP 5-NPs in vivo were investigated by establishing BALB/c nude mice and BALB/c mice models of heterotopic tumors.The results showed that after treatment with TP 5-NPs,the weight of nude mice had no significant change,the liver,spleen and kidney had no obvious damage,and the tumor volume of nude mice decreased significantly.Western Blot results showed that cleaved PARP,cleaved caspase-3,cleaved caspase-8,cleaved caspase-9,pro-apoptotic protein Bax and Bad increased significantly,while anti-apoptotic protein Bcl-2 decreased.It is further demonstrated that TP 5-NPs can inhibit the growth of tumors in nude mice in vivo and induce apoptosis of hepatocellular carcinoma cells through caspase-dependent mitochondrial pathway.In BALB/c mouse model of heterotopic tumors,TP 5-NPs can enhance splenic index and induce changes of inflammatory factors including matrix metalloproteinase,interleukin and chemokine,which are related to immune regulation,in spleen and serum.TP 5-NPs can also regulate the expression of NF-kappa B and Nrf2 and their downstream antioxidant proteins,indicating that oxidative stress-mediated immune response plays a role in the anti-tumor process of TP 5-NPs.