Inhalable Chemotherapy Drugs And Bacterial Preparations For The Treatment Of Primary Lung Cancer

Lung cancer dominates the top position of morbidity and mortality among all the malignant tumors in China.Systemic chemotherapy is usually applied to clinical therapy of lung cancer,probably leading to serious toxic and side effects due to wide drug distribution in the body to difficultly increase the dose and weaken therapeutic effect.Melatonin(MLT)is an endogenous small molecule produced by the human pineal gland.It shows anti-tumor effect at a high dose level,although low oral bioavailability is given.Therefore,MLT is an active pharmacological ingredient with poor druggability.Paclitaxel(PTX)is a commonly used anti-tumor drug in clinic and only the intravenous dosage form is available.Bacteria show anti-tumor effects with the tumor targeting effect.In this study,liposomal melatonin dry powder inhalers(LMD)and liposomal paclitaxel-loaded bacterial formulations were prepared.Their therapeutic effects of on primary lung cancer after intratracheal administration were explored and the mechanisms were discussed.1.Liposomal melatonin dry powder inhalers(LMD)Liposomal melatonin was prepared with the ethanol injection method.A optimal formulation was screened out with the orthogonal tests.The encapsulation efficiency of LMD was high to 98.9%after melatonin was determined with the HPLC.LMD was obtained after lyophilization of liposomal melatonin added with mannitol.The optimal formulation of liposomal melatonin included soybean phospholipid/cholesterol/MLT(10:1:5,mol/mol).LMD was composed of mannitol/liposome(4:1,w/w).LMD appeared as spherical particles under a scanning electron microscope and the powders had good fluidity.The rehydrated LMD had a small size of 65.15 nm and the zeta potential of-14.2 mV with still high entrapment efficiency and the drug loading efficiency of 6.2%.The median aerodynamic diameter of LMD was 6.73?m and the ratio of fine particles(FPF<8.06?m)was 22.2%so that LMD was suitable for pulmonary delivery.LMD showed the similar anti-A549 lung cancer cell effect to MLT and gemcitabine at low concentrations(<0.5 mM)but significantly higher effect at higher concentrations(>1 mM).Therefore,MLT had anti-lung cancer cell activity and liposomes enhanced this effect.At low concentrations(<1 mM),LMD,MLT and gemcitabine made most have normal bronchial cells(BEAS-2B)survival(>80%).However,at the high concentrations(5 mM),the survival rates with LMD and MLT were still high(>70%)while gemcitabine showed obvious cytotoxicity with the survival rate of 48.17%.Therefore,MLT is safe at whether low or high doses.Rat primary lung cancer models were established 45 days post-intratracheal administration of 3-methylcholanthrene and N-N-dimethylnitrosamine.MLT,gemcitabine and LMD were intratracheally administered into the lungs of rats,respectively.All of them showed significant anticancer effects with few tumor nodules and infiltrated inflammatory cells and LMD's effect was the highest.The level of some tumor markers further demonstrated the anticancer effects with decreasing of NF-?B p65,enhancement of apoptosis(tunel staining)and reduction of malondialdehyde levels,where LMD was the highest medication compared to the other ones.2.Liposomal paclitaxel-loaded bacteria formulationsLiposomal paclitaxel(LM)was prepared with the film dispersion method and liposomal paclitaxel dry powder inhalers(LPD)were obtained after mannitol was added and lyophilization.The optimal formulation of LP included paclitaxel/cholesterol/phospholipid(1:1.5:15,w/w)with a high encapsulation efficiency of 97.2%,the particle size of 64.26 nm and the zate potential of-9.96 mV.Liposomal paclitaxel-loaded bacteria formulations were prepared by electroporation afterLPD was added into the bacterial suspension(E.coli or L.casei).Electroporation produce distinct pores in the bacterial cell wall,promoting the entry of LP and leading to a encapsulation efficiency of 95%.Both LP and electroporation had no effect on the growth of the bacteria.The bacteria had significant cytotoxicity on A549 cells,depending on concentrations and time,wherein E.coli was stronger than L.casei.The survival rates of A549 cells were 45.6%and 51.9%for 10~6CFU/mL E.coli and L.casei,respectively.LP showed higher cytotoxicity than paclitaxel.The mixture of LP and bacteria showed higher cytotoxicity than LP,but lower than the liposomal paclitaxel-loaded bacteria formulations.Apoptosis of A549 cells was detected with the flow cytometry.The apoptotic rates for E.coli,L.casei and LP were 7.7%,3.8%and 13.6%,respectively,which greatly increased for the mixture of LP and bacteria;and the liposomal paclitaxel-loaded bacteria formulations showed the highest effect.Therefore,the bacteria serve as carriers to deliver LPto cells and the synergistic effect was shown.Although the co-cultivation of bacteria and A549 cells made a little bacteria death,no significant change was shown comparing to the original bacteria.E.coli,L.casei,LP,bacteria and the mixture of LP and bacteria,and the liposomal paclitaxel-loaded bacteria formulations were intratracheally administered to the lungs of lung cancer rats(1 mg PTX and/or10~6 CFU/mL bacterial).All the medications down-regulated the expressions of Caspase-3 and Bcl-2,reduced the expressions of VEGF,and enhanced the anti-tumor effect,wherein the liposomal paclitaxel-loaded bacteria formulations were the highest.The expressions of inflammatory factors such as TNF-?,IL-4,HIF-1?,and IFN-?were enhanced by the bacterial formulations.Therefore,the liposomal paclitaxel-loaded bacteria formulations showed high anticancer effects both direct action and immune enhancement.Liposomal paclitaxel-loaded bacteria formulations provide a new solution for the clinical treatment of lung cancer.In this study,a pharmacological active human endogenous substance,MLT,was used for perparation of liposomal formulations and powder inhalers that were directly delivered to the lung enhance the anti-lung cancer effect.The hydrophobic drug paclitaxel was prepared to its liposomal formulation and then loaded into the bacteria.The high anticancer effect of the liposomal paclitaxel-loaded bacteria formulations was confirmed after intratracheal administration with low side effects.The novel formulations are a promising anticancer dosage form.