LyP-1-conjugated Liposome Drug Delivery System For Targeting Lymphatic Metastatic Tumor

Lymphatic metastasis, one of the most important spread pathways for many types of solid tumor, has been believed to be a key prognostic factor for survival of patients. Currently surgical removal of primary tumor and metastatic lymph nodes (LNs) and conventional chemotherapy are the two major treatment means. But lymphatic metastases are hardly completely removed by surgery, and conventional chemotherapy often causes severe systemic toxicity. Obviously an effective drug delivery system for targeting lymphatic metastatic tumor will enhance the treatment of lymphatic metastatic tumors. Liposomes were widely used as lymphatic system-targeted drug carrier since they could be selectively absorbed by lymphatics and captured by LNs after subcutaneous (s.c.) injections. However, the application of liposomes in lymphatic metastatic tumor-targeted drug delivery has been hampered by their limitation of low targeting rate of plain liposomes to metastatic LNs. There is increasing evidence that tumor lymphatics and tumor-associated macrophages in LNs play an importance role in lymphatic metastasis. LyP-1is a peptide that can specifically bind to tumor cells, tumor lymphatics and tumor-associated macrophages. Therefore, this paper aims to construct a LyP-1-conjugated liposome drug delivery system for targeting lymphatic metastatic tumors, in order to enhance the inhibition effect of loaded antineoplastic agents on lymphatic metastatic tumors, and to repress lymphatic metastasis by destroying tumor lymphatics and tumor-associated macrophages.In Chapter1, LyP-1was synthesized and LyP-1-conjugated liposome drug delivery system was constructed. LyP-1and fluorescein labeled LyP-1were synthesized by solid phase peptide synthesis technique and characterized by HPLC and MS. LyP-1-PEG-DSPE was synthesized by the reaction between hydrosulfide group and maleimide group and iodine oxidation method, and characterized by HPLC,1H-NMR and FTIR techniques. LyP-1-conjugated PEGylated liposomes (L-P-LS) loaded with doxorubicin (DOX), FAM and DiR were prepared, respectively, and characterized of their vesicle sizes and size distributions, physical appearances, encapsulated efficiency and in vitro leakage rate. The results showed that fluorescein labeled LyP-1and LyP-1-PEG-DSPE were successfully synthesized. The prepared liposomes have narrowly distributed vesicle sizes of around90nm, which were suitable for lymphatic system-targeted delivery. The DOX-loaded liposomes were spherical particles as characterized by TEM and AFM. The leakage rates of all kinds of liposomes could meet the requirements of subsequent experiments. LyP-1modification has no obvious influence on the properties of liposomes.In Chapter2, the in vitro and in vivo targeting abilities of LyP-1and L-P-LS were investigated. The in vitro targeting ability of LyP-1to tumor cells including MDA-MB-435melanoma cells, SPC-A1lung adenocarcinoma cells, NCI-H292lung cancer cells and BxPC-3pancreatic cancer cells was assessed by cellular uptake test. Near infrared in vivo fluorescence imaging technique was used to study the in vivo targeting ability of LyP-1to metastatic LNs of MDA-MB-435melanoma. Cellular uptake test was used to investigate the in vitro targeting ability of L-P-LS to MDA-MB-435, SPC-A1, NCI-H292and BxPC-3cells. Nude mice models of lymphatic metastatic tumors were constructed by s.c. inoculation of MDA-MB-435, NCI-H292, SPC-A1and GFP-tagged SPC-A1cells, and based on these models, the in vivo targeting ability of L-P-LS to lymphatic metastatic tumors was investigated. The results showed that LyP-1could be specifically taken up by the four cell lines and metastatic LNs of MDA-MB-435melanoma, exhibited good in vitro and in vivo targeting ability. L-P-LS showed good in vitro targeting ability to the four tumor cell lines. The LN sections of MDA-MB-435melanoma and NCI-H292lung cancer animal models exhibited obvious lymphatic metastases in popliteal and iliac LNs at the inoculated side, and not in those at the uninoculated side and other major organs. The LN sections of SPC-A1lung adenocarcinoma animal model did not exhibit obvious lymphatic metastases in all LNs, but obvious isolated tumor cells could be observed in popliteal and iliac LNs at the inoculated side on GFP-tagged SPC-A1lung adenocarcinoma animal model. L-P-LS exhibited good in vivo targeting ability to lymphatic metastatic tumors of MDA-MB-435melanoma, NCI-H292lung cancer and SPC-A1lung adenocarcinoma.In Chapter3, the pharmacodynamic study of inhibition effect of LyP-1-conjugated DOX-loaded liposomes on tumor cells and lymphatic metastatic tumors was carried out. The in vitro growth inhibition of L-P-LS/DOX on MDA-MB-435, SPC-A1and NCI-H292tumor cells was investigated by MTT assay. On lymphatic metastatic tumor models of MDA-MB-435melanoma and SPC-A1lung adenocarcinoma, the inhibition effect of L-P-LS/DOX on lymphatic metastatic tumor was studies. The results showed that LyP-1modification enhanced the growth inhibition effect of DOX liposomes on tumor cells in vitro. The pharmacological results on MDA-MB-435melanoma animal model showed that compared with P-LS/DOX group, L-P-LS/DOX group exhibited significantly enhanced inhibition effect on lymphatic metastatic tumor in popliteal and iliac LNs, and decreased metastases rate in popliteal and iliac LNs. The pharmacological results on SPC-A1lung adenocarcinoma animal model showed that L-P-LS/DOX demonstrated significant inhibition effect on lymphatic metastatic tumors in both popliteal and iliac LNs, whereas P-LS/DOX only on those in popliteal LNs.In Chapter4, the in vitro and in vivo safety of LyP-1-conjugated DOX-loaded liposomes was assessed. The cytotoxicity of blank L-P-LS and P-LS on MDA-MB-435, SPC-A1and NCI-H292tumor cells was investigated by MTT assay. The tissue damage to rat foot pad and nude mice heart caused by long-term s.c injection of L-P-LS/DOX and P-LS/DOX was investigated. The results showed that blank L-P-LS and P-LS showed no obvious cytotoxicity on these tumor cell lines. Injection of DOX caused severe necrosis to skin of injection site, where as L-P-LS/DOX and P-LS/DOX caused slight local tissue edema. Injection of DOX caused severe myocardial cells denaturation and myocardial fibers necrosis to nude mice heart, whereas L-P-LS/DOX and P-LS/DOX caused no obvious abnormality.In Chapter5, the targeting mechanism and pharmacodynamic mechanism of LyP-1-conjugated liposomes to lymphatic metastatic tumors were investigated. The immunofluorescence staining method was used to verify the specific binding of LyP-1to tumor lymphatics in metastatic LNs. The near infrared fluorescence in vivo imaging technique was used to investigate the uptake of L-P-LS and P-LS by normal LNs in vivo. The lymphatic metastatic tumor nude mice model was established by inoculation of GFP-tagged NCI-H292tumor cells, based on which the specific binding of L-P-LS to tumor metastases in LNs was investigated. The immunofluorescence staining method was used to study the specific binding of L-P-LS to tumor lymphatics and tumor-associated macrophages in metastatic LNs of MDA-MB-435melanoma and SPC-A1lung adenocarcinoma animal models. Following treatment of lymphatic metastatic tumors by different DOX formulations, the lymphatic vessel density (LVD) was quantified using immunofluorescence staining method to elucidate the pharmacodynamic mechanism of L-P-LS/DOX on lymphatic metastatic tumors. The results showed that LyP-1could specifically bind to tumor lymphatics in metastatic LNs. LyP-1modification demonstrated no influence on liposome uptake by normal LNs, indicating that it had no influence on nonspecific uptake of liposome by LNs. L-P-LS could specifically distributed adjacent to GFP-tagged NCI-H292tumor metastases in LNs, whereas P-LS could not. L-P-LS could specifically bind to tumor lymphatics and tumor-associated macrophages following being captured by metastatic LNs. Compared with P-LS/DOX group, L-P-LS/DOX group showed significantly decreased LVD in popliteal LNs, indicating that L-P-LS/DOX repressed lymphatic metastasis by destroying tumor lymphatics and decreasing LVD.In summary, in this study we successfully constructed LyP-1-conjugated PEGylated liposome drug delivery system for targeting lymphatic metastatic tumors. The results indicated that L-P-LS showed good targeting ability to lymphatic metastatic tumors following s.c. injection. Due to the specific binding of L-P-LS to tumor metastases, tumor lymphatics and tumor-associated macrophages, LyP-1modification not only enhanced the inhibition effect on lymphatic metastases of DOX liposomes, but also enhanced the repression effect on lymphatic metastasis. LyP-1-conjugated liposome drug delivery system is a promising active targeting delivery system for treatment of lymphatic metastatic tumors.