| Objective:This study was conducted to design and prepare a new drug carrier called LyP-1-MWNTs（LyP-1-conjugated Multi-walled Carbon Nanotubes） for lymphatic targeted chemotherapy in pancreatic cancer, and detect their physicochemical properties by methods of TGA, TEM techniques. The anti-cancer effects of GEM-loaded LyP-1-MWNTs on lymphatic metastasis of pancreatic cancer were also investigated in vitro and in vivo.Methods:（1）LyP-1peptide and5-FAM labeled LyP-1were synthesized by means of Fmoc/tbu solid phase peptide synthesis, and characterized by HPLC and MS methods. Carboxylic Cut-MWNTs was synthesized through mixed acid process, followed by the covalent conjugation with Maleimide-PEG-NH2by the condensation reaction between carboxyl group and amino group. The synthesis process of LyP-1-MWNTs was mainly counted on the specific reaction between hydrosulfide group and maleimide group followed by iodine oxidation procedure, after which the LyP-1-MWNTs complex was characterized by TGA, TEM techniques, its dispersion and stability in suspended liquid were also observed.（2） Single5-FAM and LyP-1pretreatment groups served as controls, the ability of targeted uptaking5-FAM labeled LyP-1in vitro of human pancreatic cancer cells BxPC-3and BxPC-3-LN5（with high metastatic capacity to the lymph nodes） were detected by fluorescence microscope and flow cytometry. The anti-cancer effects of GEM-loaded LyP-1-MWNTs on the BxPC-3-LN5cell were evaluated by means of MTT assay, and cell cycle redistribution, proliferation index, and apoptosis rate were also determined through flow cytometry.（3）The nude mice model of lymphatic metastatic tumor of pancreatic cancer was established by s.c. inoculation of BxPC-3-LN5cells, and the metastatic tumor in bilateral popliteal, inguinal, para-iliac, renal hilar lymph nodes were observed.5-FAM-LyP-1and5-FAM-LyP-1-MWNTs were delivered by tail vein and foot pad injection respectively in the nude mice model, the specific affinity of LyP-1contained to tumor lymphatics in metastatic LNs was investigated using immunofiuorescence staining method. The lymph node staining property of LyP-1-MWNTs was evaluated by observing the degree of black dyeing in above mentioned lymph nodes, combined with pathological diagnosis through HE staining. Meanwhile, the anti-cancer effects of GEM-loaded LyP-1-MWNTs on tumor metastases in LNs were assessed by measuring volume and weight of the popliteal lymph nodes in different treatment groups. All extracted lymph nodes were treated with En Vision immunohistochemical staining and TUNEL method for confirming the proliferation and apoptosis of tumor cells in metastatic lymph nodes. Finally, the toxicity of LyP-1-MWNTs complexes was evaluated:the vein blood was collected to analyze the variation of WBC, ALT and Scr; vital organ tissue including heart, liver, spleen, lung, kidney were extracted for pathological analysis by HE staining in order to detect the tissue injury.Results:（1）Design and preparation of LyP-1-MWNTs:Pristine MWNTs were firstly cut into short tubes by mixed acid process, meanwhile, carboxylic groups were covalently conjugated to the cut carbon nanotubes（Cut-MWNTs）. The Cut-MWNTs still exhibited long tubular structures of100-200nm with diameter of30～40nm in characterization of TEM. Compared with pristine MWNTs, Cut-MWNTs could be stably well-dispersed in water, and not easy to aggregate to form obvious precipitation even3months later. LyP-1peptide and5-FAM labeled LyP-1, synthesized by Fmoc/tbu solid phase peptide synthesis technique, were successfully prepared, and identified with the purity of more than98%of both two peptides. The synthesis process of LyP-1-MWNTs was mainly counted on the covalent conjugation between LyP-1and Cut-MWNTs through Maleimide-PEG-NH2. The grafting ratio of LyP-1polymer on Cut-MWNTs could reach10.2%by TGA analysis. Under the transmission electron microscope, the surface of MWNTs was wrapped by polymers, which resulted to slight winding of carbon nanotubes. Nevertheless, compared with Cut-MWNTs, there was no obvious effect on the dispertion and stability of LyP-1-MWNTs in water.（2） Targeted effect of LyP-1-MWNTs in vitro:There was no significant uptaking difference of LyP-1in vitro between BxPC-3and BxPC-3-LN5, though they both had high targeted endocytosis of5-FAM-LyP-1. In addition, when pretreated with free LyP-1peptide, the uptaking amount of5-FAM-LyP-1obviously decreased, possibly because of competitive binding to its specific receptor located on the surface of cell membrane of LyP-1.Blank MWNTs had good biocompatibility, and had very mild inhibition on cell proliferation. The GEM loading process of MWNTs did not cause any pharmacodynamic destruction, GEM-loaded MWNTs had much higher inhibitory effect on BxPC-3-LN5proliferation than GEM did （P<0.05）. LyP-1peptide showed active targeted efficiency in the concentration-and time-dependent manner. The inhibition effect of LyP-1-MWNTs-GEM was significantly higher than that of MWNTs-GEM and GEM at relatively higher drug concentrations and after long-duration response, with apparent arrest of cancer cells in G0/G1phases, increase of cell apoptosis and decrease of cell proliferation.（3） Targeted effect of LyP-1-MWNTs in vivo:The nude mice model of lymphatic metastatic tumor of pancreatic cnacer was established by s.c. inoculation of BxPC-3-LN5cells in foot pad. The LN sections showed lymphatic metastases occurred in popliteal, para-iliac lymph nodes at inoculated side, and in inguinal LNs at bilateral sides. After administration of5-FAM-LyP-1（tail vein injection） and5-FAM-LyP-1-MWNTs（foot pad injection） respectively, LyP-1could specifically bind to tumor lymphatics in metastatic LNs. LyP-1-MWNTs had better lymph node tracing property than MWNTs. The popliteal, inguinal, para-iliac, renal hilar lymph nodes along drainage pathway were all apparently dyed black. A large amount of MWNTs nanoparticles were found gathering around metastatic foci under microscope.With smallest LN volume, lowest immunohistochemical positive index and highest apoptosis index（AI）, the targeted anti-tumor effects of lymph node metastasis in vivo were significantly better in LyP-1-MWNTs-GEM grouop than in MWNTs-GEM and GEM group respectively （P<0.05）. Although skin of foot pad of injection-site was dyed black, all the mice in the MWNTs-contained groups maintained a good health status, and had no significant variations in body weight, blood routine, hepatic and renal function examinations compared with the other groups, besides, the HE staining of vital organs tissue showed no obvious tissue injury or MWNTs deposition, the above detected results revealed that LyP-1-MWNTs had good biocompatibility and safety.Conclusions:LyP-1-MWNTs is a novel targeted drug carrier, possessing excellent lymphatic affinity and fluidity with good biocompatibility. GEM-loaded LyP-1-MWNTs exhibited more effective antitumor activity against pancreatic cancer than GEM and MWNTs-GEM both in vitro and in vivo, which would have potential applications in active targeted chemotherapy of lymphatic metastasis in pancreatic cancer. |
PDF Full Text Request