吸入給藥方式遞送以EGFR為標的之奈米明膠-順鉑藥物載體於肺癌治療之探討

Abstract

近年來癌症一直是國內十大死因排名的首位，且肺癌更居國人癌症死亡之冠。儘管醫學已有相當進展，目前肺癌的長期存活率甚低，且傳統化療之副作用多，因此尋找有效的治療方式實為當務之急。奈米藥物載體經由適當的表面修飾，可達到穩定藥物且具有傳送藥物至癌症區域的功能。奈米藥物載體以呼吸給藥之方式近幾年才開始發展。其給藥效果比一般口服式的藥物來的有效率且直接，因此本研究製備可標的癌細胞表面表皮生長因子受器(EGF receptor, EGFR)之導向奈米明膠顆粒 (gelatin nanoparticles, GPs)，並結合肺癌第一線化療用藥-順鉑 (cisplatin, CDDP)，以達到標靶治療的效果。並選擇吸入方式給藥，將藥遞送至肺癌老鼠，以評估其治療效果。 本研究的第一部分為製備具標的EGFR功能之明膠粒子，並確認其體外( in vitro)與體內(in vivo)之標的功能。奈米明膠顆粒(GPs)表面與卵白素(NeutrAvidin)鍵結後(GP-Av)，再結合以生物素修飾之表皮生長因子(GP-Av-bEGF)，以達到標的EGFR過度表現之癌細胞。經過多層修飾後的明膠奈米顆粒並無顯著差異，其顆粒大小為220 nm、表面電位為 - 9.3 mV。比較不同EGFR表現量之細胞: 由於A549細胞表面有較高EGFR表現，因此其細胞內累積量明顯高於其他細胞；且A549 細胞內之奈米明膠顆粒累積量與劑量及時間有依存性。以共軛焦顯微鏡觀察顆粒於細胞內之分布，可觀察A549細胞以GP-Av-bEGF顆粒培養有較多的綠色螢光分佈，同時顆粒位於溶小體(lysosome)內。於此確認於細胞階段可有效標的EGFR過度表現細胞。 接著，建立老鼠肺癌模式，使用霧化器以吸入方式給予奈米顆粒之體內模式。明膠奈米溶液霧化噴出之粒子，其霧化顆粒99 % 位於0.5–5µm，此粒徑可確保顆粒有效達到肺泡並直接由人體吸收。吸入給藥的安全性以肺水腫 (lung edema)及肺部髓質過氧化酶 (myeloperoxdiase, MPO)分析，確認無急性肺部發炎情形。體內螢光分佈分析，觀察到以EGF修飾之顆粒(GP-Av-bEGF)集中於癌症誘發之老鼠肺部，顯示其可有效標的癌症細胞；反之未以EGF修飾之顆粒(GP-Av)並無此功能。此明膠奈米顆粒表面修飾EGF並以霧化器吸入方式，仍可保有EGF之活性，並於動物體內可標的EGFR過度表現之癌細胞。 本研究的第二部分則進行明膠奈米顆粒包覆順鉑(CDDP)之抗癌測試。明膠顆粒包覆順鉑，再加以EGF表面修飾之奈米顆粒(GP-CDDP-bEGF)進行體外抗癌測試，顯示其較未經表面修飾之明膠順鉑載體(GP-CDDP)及順鉑溶液 (free CDDP)於低劑量時即可有效殺死癌效胞。A549細胞內之順鉑累積量以GP-CDDP-bEGF組為最高。而PI染色及流式細胞儀分析結果顯示: GP-CDDP-bEGF作用四小時後，細胞sub-G1 peak有顯著增加，顯示明膠顆粒包覆順鉑並以bEGF修飾表面 (GP-CDDP-bEGF)之載體，於細胞培養階段可快速影響癌細胞周期，且達到抗癌效果。 於抗癌動物實驗，不同劑型之順鉑載體(free CDDP,GP-CDD, GP-CDDP-bEGF)皆以CDDP濃度12 mg/kg給藥，於治療四次後進行評估。肺癌老鼠的肺部順鉑累計量，以GP-CDDP-bEGF組最高，而於腎之累計量低，可降低使用此藥之副作用；且由血液分析肝腎功能顯示 GP-CDDP-bEGF除了可提高藥於肺部的累積量之外，且較無毒性。而現階段不同劑型之順鉑載體應用於動物實驗，其抗癌效果並無明顯差異。檢討之可能因素為實際肺部之累計藥量尚未達到有效治療之劑量所致。 綜合以上之結果，明膠奈米顆粒包覆順鉑且經表面修飾EGF(GP-CDDP-bEGF)之載體，於體外測試顯示具有效標的及毒殺EGFR過度表現之癌細胞，具抗癌效果。於動物實驗顯示以霧化吸入給藥方式，並不會損害EGF的活性，表面修飾EGF之奈米載體仍具有於體內標的EGFR表現之細胞功能；而體內抗癌效果則需再更進一部確認。此研究初步建立了將標靶式奈米藥物載體以吸入方式傳遞之給藥模式，未來若可進一步應用此簡易且非侵入式之給藥方式，對癌症病患而言是一大福音。

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.