| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 96 === Lung cancer is one of the most harmful forms of cancer. The long-term survival rate of lung cancer patients treated by conventional modalities remains far from satisfactory. Encapsulated anticancer drugs in nanocariers, can protect not only the integrity of drugs during transport in circulation but also the normal tissues from the toxicity. To develop an effective drug delivery system for lung cancer therapy, gelatin nanoparticles (GPs) were modified with NeutrAvidinFITC-biotinylated epidermal growth factor (bEGF) to form EGF receptor (EGFR)-seeking nanoparticles (GP-Av-bEGF). And cisplatin (cis-dichlorodiammine- platinum (II), CDDP) was incorporated in nanocarriers for active targeting. Aerosol droplets of nanocarriers were generated by using a nebulizer and delivered to mice model of lung cancer via aerosol delivery.
Firstly, preparing the EGF modified nanoparticle is required. The results showed that the modification process had no significant influence on particle size (220 nm) and zeta potential (- 9.3 mV). By the in vitro test, GP-Av-bEGF resulted in higher entrance efficiency on adenocarcinoma cells (A549) than that on normal lung cells (HFL1) because A549 possessed greater amounts of EGFR. We also found that uptake of GP-Av-bEGF by A549 cells was time and dose dependent. Confocal microscopy confirmed the cellular internalization of GP-Av-bEGF, and more fluorescent spots of these nanoparticles were obviously observed as well as lysosomal entrapment in A549.
Secondly, the effect of aerosol delivery was confirmed. Analysis of the aerosol size revealed that 99 % of the nanoparticles after nebulization had a mass median aerodynamic diameter (MMAD) within the suitable range (0.5–5µm) for lower airway deposition. The safety of inhaled nanoparticles was examined by lung edema and myeloperoxidase (MPO) activity assay. There’s no finding suggestive of acute lung inflammation following inhalation. The fluorescence images obtained from live mice with lung cancer showed the GP-Av-bEGF could target the cancerous lungs in a more specific manner. Fluorescence analysis of the organs revealed that the GP-Av-bEGF was mainly distributed in cancerous lungs. In contrast, nanoparticle accumulation was lower in normal lungs. Results of theses two parts revealed that GP-Av-bEGF could target to the EGFR-over expression cancer cells in vitro and vivo.
The anticancer activities were studied by cisplatin (CDDP) incorporated in gelatin nanoparticles with or without EGF modification. The in vitro anticancer study showed GP-CDDP-bEGF was more potent than free CDDP and GP-CDDP, due to a rapid onset of action to cell cycle and with the lower IC 50 at 4.23 uM for the inhibition of A549 cell growth. Cells treated by GP-CDDP-bEGF for only 4 hr had the highest sub-G1 phase from PI stain. The result of intracellular Pt accumulation shows that higher amount of Pt was accumulated in cells treated by GP-CDDP-bEGF.
The CDDP formulations: free CDDP, GP-CDDP and GP-CDDP-bEGF were given as 4 doses at an equivalent CDDP dose of 12 mg/kg with 4-day intervals between inhalation to A549 lung meta mice. This treatment showed GP-CDDP-bEGF was less toxic and also had been concentrated in the EGFR overexpressed cells according to the higher Pt accumulation in cancerous lung. The anticancer effect of the cancerous mice treated by these CDDP formulations via inhalation, unfortunately it had no significant difference in vivo. It may result from the insufficient dosage deposition to the lungs.
To summarize, the gelatin nanoparticles with CDDP incorporated and decorated with EGF tumor specific ligands were successfully development. The in vitro targeting and anticancer effect is confirmed. And the aerosol delivery of nanodrug carrier was set up and may prove to be beneficial drug carriers when administered by simple aerosol delivery for the treatment of lung cancer patients for clinical using.
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