Tumor Targeting And Stimuli-Responsive Liposome For Cancer Therapy

In recent decades,the incidence and mortality of malignant tumors have been on a continuous rise.Due to the high malignancy of tumors,the difficulty of surgery and the poor prognosis,the high recurrence and metastasis rates have been the difficulties of cancer treatment.Chemotherapy is one of the most widely used therapeutic methods in cancer patients.Chemotherapeutic drugs mainly kill cancer cells through drug toxicity,but traditional chemotherapy is facing increasing challenges due to problems such as high side effects,poor targeting and frequent drug administration that generates drug resistance.In this thesis,multifunctional liposomes were constructed based on a combination chemotherapy strategy to address the problems of low effective concentration of drugs at tumor sites,poor stability of liposomes for clinical use and limited efficacy of single-acting drugs,in order to achieve high efficiency and low toxicity against tumors while improving the overall safety of nanoliposomes.In the second chapter of this thesis,we address the problems of intra-tumor heterogeneity of hepatocellular carcinoma,low levels of therapeutic agents released by nanocarriers in tumor tissues leading to the inability of treatment to reach the therapeutic level of drugs and the ease of tumor recurrence.Firstly,DSEG-PBA polymer was successfully synthesized and incorporated into the liposome base material,and then a novel multifunctional DSEG-PBA liposome was prepared by thin film hydration-extrusion method.This system realized the simultaneous loading of hydrophilic doxorubicin hydrochloride(DOX·HCl)in the hydrophilic inner cavity and hydrophobic doxorubicin(DOX)in the lipid bilayer.The structure/morphology of the liposomes was observed by transmission electron microscopy(TEM),dynamic light scattering measurement(DLS)and confocal laser scanning microscopy(CLSM),and the particle size of the liposomes was approximately 150 nm with uniform particle size.The drug loading capacity(LC)and encapsulation efficiency(EE)of DSEG-PBA liposomes were 9.2% and 93.4%,respectively,as measured by UV-vis.The long-term stability of DSEG-PBA liposomes was demonstrated by 90-day stability evaluation.In vitro release results showed that DSEG-PBA liposomes were stable under physiological conditions while responding to and rapidly releasing the drug in a slightly acidic(pH=5.0)lysosomal environment in tumor cells.Cellular phagocytosis assay data confirmed that cellular uptake increased with time.Quantitative analysis by fluorescence-activated cell sorting(FACS)further demonstrated that PBA coupling increased the cellular uptake of DSEG-PBA liposomes through SA-mediated endocytosis.Alamar blue(AB)assay demonstrated that DSEG-PBA liposomes had optimal tumor cell killing effect.In vivo imaging results in nude mice showed high fluorescence intensity in tumor sites and weak fluorescence intensity in normal tissues 12 h after drug administration,indicating that DSEG-PBA liposomes could successfully target tumor tissues.Biodistribution and pharmacokinetic results demonstrated longer retention time in blood and slower systemic clearance,which improved the enrichment of DSEG-PBA liposomes in tumor tissues.In vivo antitumor results showed that DSEG-PBA liposomes significantly inhibited tumor growth in mice,with complete disappearance of tumors by day 60 and mouse survival rate remaining at 70% after 365 days with no tumor recurrence,demonstrating the excellent antitumor efficacy of this targeted long-circulating liposome.In addition,immunological analysis of tumor tissues after 17 and 45 days showed that the signal of tumor cell apoptosis was significantly increased on day 45 of treatment.The results showed that the treatment strategy was safe and effective.In the third chapter of this thesis,we address the aggressive characteristic of breast cancer,tumor angiogenesis that promotes cancer progression and metastasis,and the limited effectiveness of single drug chemotherapy.A targeted,reduction-sensitive nanoliposome PPP@(D+A)was successfully developed using PBA-PEG-SS-PCL polymer with egg phosphatidylcholine(e PC)to inhibit breast tumor growth and prevent tumor metastasis.This PPP@(D+A)liposome was coated with Apatinib(APA)between phospholipid bilayers,which could effectively increase the solubility of lipid-soluble drug APA.Meanwhile,its hydrophilic inner cavity was loaded with DOX·HCl to increase the drug loading capacity.The TEM and CLSM characterization confirmed that the liposomes had a rounded,bilayered spherical structure.And the high drug loading capacity(9.16% for DOX·HCl and 3.34% for APA,respectively)of PPP@(D+A)liposomes was confirmed by UV-vis assay,which provided a basis for the effective guarantee of intracellular drug concentration.The in vitro stability study demonstrated that the PPP@(D+A)liposomes were stable in the in vitro blood circulation.In vitro evaluation of reduction-sensitivity and drug release behavior confirmed that PPP@(D+A)liposomes responded rapidly to the disulfide bonds on the polymer chain and released DOX·HCl and APA in response to 10 mM GSH.Cytotoxicity assays confirmed that PPP@(D+A)liposomes enhanced the growth inhibition of 4T1 tumor cells and also effectively inhibited the growth of vascular endothelial cells(EC).Biodistribution results showed that the uptake of PPP@(D+A)liposomes at tumor sites reached 12.9% ID g-1,suggesting that high uptake of PPP@(D+A)liposomes by tumor cells would greatly enhance the efficacy of cancer treatment.In vivo antitumor studies confirmed that PPP@(D+A)liposomes effectively suppressed 4T1 tumor growth and and the survival rate of the mice was 70.0% after 60 days.The tumor cells showed obvious apoptotic features by H&E,Ki67 and TUNEL analysis of tumor tissues.And the results of CD31 antibody staining of vascular images showed that almost no intact tumor vessels could be seen marked by any fluorescence.Immunohistochemical staining(IHC)did not show any tumor lung metastasis,Bouin’s fixative staining of pulmonary nodules proved that the lung metastasis inhibition rate was up to 97.5%.The results of in vivo antitumor study showed that the dual drug synergistic liposomes played a positive role in antitumor therapy and inhibition of tumor metastasis.The results of body weight and survival rate of mice and histological staining of main organs confirmed the optimal dual drug synergy of PPP@(D+A)liposomes with low systemic toxicity and good safety profile.