Magnetic Nanoparticles As Drug Carrier And Their Applications In Lymphatic Targeting Chemotherapy For Pancreatic Cancer

Objective Effectively lymphatic targeting drug delivery systems using magnetic multi-walled carbon nanotube(mMWNT) and magnetic activated carbon(mCH40) were presented.Physical testing applied to the particles was described.Feasibility of delivering mMWNT-Gemcitabine(mMWNT-GEM) and mCH40-Gemcitabine (mCH40-GEM) to the regional lymph nodes via intra-lymphatic infusion in rats was evaluated.The anti-cancer effects of mMWNT-GEM and mCH40-GEM on cell growth,apoptosis and lymph node metastasis of pancreatic cancer were investigated in vitro and in vivo.Methods(1) mMWNT was synthesized with a layer of magnetite Fe₃O₄ nanoparticles on the surface of the multi-walled carbon nanotube(MWNT) through chemical methods,mCH40 were prepared in a ball mill using Fe₃O₄ nanoparticles and activated carbon.Particles were characterized visually by transmission electron microscopy (TEM),scanning electron microscopy(SEM),x-ray diffraction(XRD),and Vibrating Sample Magnetometer(VSM).Subcutaneous(s.c.) injection ofmMWNT and mCH40 into the left footpad of the rats was carried out to identify their feasibility of lymph node mapping.(2) In the study of pharmacokinetics,rats were randomly allocated to five groups:GEM alone group,mMWNT-GEM(with/without constant magnetic field) group,and mCH40-GEM(with/without constant magnetic field) group.The plasma and lymph node GEM levels were measured by high performance liquid chromatography(HPLC) at 6,12,24,48,72,120,192,and 240 h after final administration of the drugs.The effects of GEM,mMWNT,mCH40,mMWNT-GEM, and mCH40-GEM on pancreatic cancer cell lines BxPC-3 and SW1990 were investigated and compared using MTT assay and flow cytometry.Cell growth inhibition,cell cycle redistribution,proliferation index,and apoptosis rate were determined.(3) Highly metastatic sublines(BxPC-3-LN3) were selected in vivo by serial transplantation,lymph node metastasis formation and culturing the tumor cells in vitro.Lymph node metastatic model of pancreatic cancer was established in nude mice.Five weeks after tumor transplantation,mice were separated into ten groups with each group comprising six mice:control group,mCH40 alone group,mMWNT alone group,GEM alone group,mCH40-GEM without constant magnetic field(CMF) group,mMWNT-GEM without CMF group,mCH40-GEM(low-dose) with CMF group,mMWNT-GEM(low-dose) with CMF group,mCH40-GEM(high-dose) with CMF group,and mMWNT-GEM(high-dose) with CMF group.The drugs were injected on day 1 and 8,and the mice were sacrificed on the 15th day after treatment. Volume and weight of the popliteal lymph nodes were determined,and WBC,ALT and SCr were examined.All lymph nodes were examined by H&E staining and CK immunohistochemical staining.Apoptosis of tumor cells in metastatic lymph nodes was examined by TUNEL method.Results(1) The dispersibility and stability of the dispersions of mMWNT and mCH40 in aqueous liquids were good.mMWNT had exhibits long tubular structures of 1～2μm with diameter of 40～60nm,and mCH40 with spherical morphologies had diameter of 25～30nm.Both of them had high magnetic field strength,with mMWNT superior to mCH40.Both mMWNT and mCH40 were good at lymph node staining. However,mMWNT had a lower degree of staining when its concentration level below 6.25mg/ml.The magnetic nanoparticles travelled in the lymph fluid with distribution mainly in the cortical zone of the lymph nodes.Transmission electron microscopy (TEM) showed that some mCH40 particles in the lymph nodes were phagocytosed by macrophages.No evidence of local toxicity such as red swelling,drug allergy, ulceration or erosion of skin was observed from mMWNT and mCH40 injection, except that skin was dyed black because of particles' gathering,mMWNT and mCH40 were not identified in the liver,spleen,kidney,heart and lung under histological examination,and nor had these organs had pathological changes,such as inflammation,fibrosis and granuloma.(2) mMWNT and mCH40 could absorb GEM effectively,and were guided to the targeted lymph nodes by their properties and magnetic field.GEM was released slowly in the lymph nodes under the effects of the carriers,and maintained high concentration for a relatively long duration of time, while the concentrations in plasma were lower.Retention of GEM to targeted lymph nodes was more in mMWNT group versus mCH40 group.Both mMWNT and mCH40 alone showed low cytotoxicity toward pancreatic cancer cells,however the carriers enhanced GEM injury to the malignant cells,mMWNT was able to penetrate into cells and could transport GEM across cell membranes.Both mMWNT and GEM were able to affect cell cycle distribution of pancreatic cancer cells.GEM caused a reduction in G2/M phase due to an efficient arrest in G0/G1 phases,while mMWNT caused accumulation of cells in S and G0/G1 phases,which displayed their synergistic cytotoxic activity.(3) After three rounds of serial transplantation,highly metastatic sublines(BxPC-3-LN3) were established.The lymph node metastatic rate was 100%(6/6) following footpad injection with BxPC-3-LN3 cells.Tumor cells were identified in popliteal,inguinal,para-iliac,para-aortic and renal hilar lymph nodes,and lymph node micrometastasis was detected.Both mMWNT and mCH40 were good at metastatic lymph node staining,and metastatic lesions in the lymph node did not affect the carriers' travelling.The magnetic nanoparticles were found gathering around metastatic foci under microscope.Inhibition of lymph node metastasis by mMWNT-GEM and mCH40-GEM was increased under the effect of constant magnetic field,with mMWNT-GEM superior to mCH40-GEM,and more effectively in the high-dose versus low-dose(mMWNT/mCH40)-GEM group. Suppression of lymph node metastasis by GEM was also increased under the effect of mMWNT and mCH40.However,mMWNT and mCH40 alone did not enhance therapeutic effects of anti metastasis compared with the control group.Although injection-site skin was dyed black because of particles' gathering two weeks after administration of the drugs,no evidence of local toxicity such as red swelling,drug allergy,ulceration or erosion of skin was observed.All the mice in the medicated groups maintained a good health status,with blood routine examination,hepatic and renal function within the normal ranges,however myelosuppression occurred in the GEM alone group.Conclusions mMWNT and mCH40,both with properties of lymphatic affinity and magnetic targeting,belong to novel drug carriers in magnetic targeted drug delivery system.In addition to tracing lymph nodes,they can enhance effects of chemotherapeutic drugs for lymph node metastasis and decrease toxic side effects. mMWNT,with the unique characteristics of carbon nanotube(CNT),has more effective anti-metastasis activity than mCH40,and broad application prospect and research development.However,many further studies are necessary to determine the safety of this novel nanometer material-mMWNT before it applies to clinics.