雙胜肽修飾奈米劑型應用於運鐵蛋白受體過度表現細胞之抗癌藥物遞送

Abstract

奈米顆粒運用在癌症上的標靶遞輸系統在近年來被廣泛的研究，除了依靠奈米顆粒本身的高滲透長滯留效應 (Enhanced permeability and retention effect, EPR effect)能夠使其被動的累積在腫瘤組織中，還可以透過接枝特殊的配體 (ligand)達到辨認癌細胞表面所擁有的特殊受體，藉此達到主動標靶的效果。 運鐵蛋白受體 (Transferrin receptor, TfR)為一細胞膜上之醣蛋白，主要功能為調控鐵的攝取、運送與儲存，因此生長快速的癌細胞，其運鐵蛋白的表現量甚至能比正常細胞高出100倍，使得運鐵蛋白受體成為癌症治療的標靶之一。此外，除了藉由特殊的配體與受體達到標靶的效果，細胞穿透胜肽 (Cell penetrating peptides, CPPs)也被廣泛的應用在抗癌藥物的遞送策略中，有研究指出，結合腫瘤標靶胜肽 (Tumor targeting peptide, TTP)與細胞穿透胜肽，更能增加對腫瘤細胞的遞送效率，也可以解決細胞穿透胜肽選擇性較低的問題。 本篇研究使用聚乳酸-甘醇酸 (Poly(lactide-co-glycolide), PLGA)接枝不同分子量之聚乙二醇二胺 (Poly(ethylene) bis(amine), PEG diamine)形成具有不同PEG鍊長之PLGA-PEG2k、PLGA-PEG5k共聚物，並於PLGA-PEG2k末端接枝細胞穿透胜肽R9，PLGA-PEG5k則接枝具有運鐵蛋白受體標靶能力之T7胜肽，並使用溶媒揮發法將兩種聚合物製備成雙胜肽修飾之奈米顆粒，比較劑型中混合細胞穿透胜肽對於運鐵蛋白受體過度表現乳癌細胞株MDA-MB-231之遞送效率影響，並包覆歐洲紫杉醇 (Docetaxel, DTX)形成含藥奈米顆粒，監測其粒徑、表面電位、載藥率以及包覆率，同時進行不同劑型間之體外釋放實驗，以確認奈米顆粒之控釋效果，並比較不同酸鹼值以及酯酶對於奈米顆粒釋放藥物的影響。此外，為確認奈米顆粒透過胞吞作用進入細胞後能脫離溶酶體 (Lysosome)使藥物能作用於細胞核，本實驗使用溶酶體專一性之螢光染劑LysoBriteTM與螢光奈米顆粒進行細胞內追蹤試驗，觀察奈米顆粒脫離溶酶體之效果。最後以MTT試驗比較空白載體對於運鐵蛋白表現量不同之細胞毒性，並使用含藥奈米顆粒比較歐洲紫杉醇各種劑型對於MDA-MB-231之細胞毒殺效果，以確認其抗癌藥物之遞送效果。 實驗結果顯示，奈米顆粒製備部分，所有劑型之粒徑接落在141.2±3.8-159.9±10.1 nm之範圍，且PDI值皆小於0.3，顯示奈米顆粒具有狹窄之粒徑分布，表面電位則因接枝胜肽的不同而有所區別，接枝T7胜肽之表面電位落在-13.5±0.4 mV左右，接枝R9胜肽則接近-7.7±0.7 mV，雙胜肽混合之奈米顆粒則約-9.6±1.5 mV。在包覆歐洲紫杉醇後，粒徑大小介於149.2±3.5-192.1±5.7 nm之間，藥物包覆率介於五到七成，載藥率約為6.4±0.8-9.7±0.6%。空白載體的細胞吞噬實驗中，在相同T7胜肽濃度之條件下 (50 µg/mL)，MDA-MB-231細胞對於接枝T7胜肽之奈米顆粒吞噬程度為60.14±1.24%，T7與R9雙胜肽修飾奈米顆粒之吞噬程度為95.94±14.19%；MCF-7細胞對於接枝T7胜肽之奈米顆粒吞噬程度為4.83±1.27%，T7與R9雙胜肽修飾奈米顆粒之吞噬程度為22.44±4.84%，說明在劑型中加入細胞穿透胜肽R9可增加奈米顆粒被細胞吞噬的效果，另外，雙胜肽修飾之奈米顆粒可藉由PLGA-PEG5k-T7對R9胜肽造成的屏蔽效應，減少R9胜肽與運鐵蛋白受體低表現細胞之細胞膜接觸，進而提高對運鐵蛋白受體過度表現之細胞之選擇性。在細胞內追蹤試驗中也可以發現，接枝R9胜肽之奈米顆粒與T7/R9雙胜肽修飾奈米顆粒在60分鐘時就能觀察到大部分之奈米顆粒已脫離溶酶體，證明劑型中若添加帶有正電之物質有較佳的溶酶體脫離效果。在細胞毒性試驗中，給予相同聚合物濃度 (1 mg/mL)之雙胜肽修飾空白載體，MDA-MB-231細胞之細胞存活率為60.90±3.01%，MCF細胞之細胞存活率為70.66±2.86%，兩者具有顯著差異，顯示藉由不同鍊長接枝不同胜肽之遞送策略，的確能夠減少載體對於運鐵蛋白受體低度表現細胞所造成的毒性，而含藥奈米顆粒之細胞毒殺實驗中，比較DTX之DMSO溶液、未接枝胜肽、單獨接枝T7、單獨接枝R9以及雙胜肽修飾之奈米顆粒，其IC50分別為156.33±9.61、123.05±14.43、85.53±8.64、71.57±3.13、34.76±3.67 ng/mL，雙胜肽接枝奈米顆粒之細胞毒殺效果有顯著的提升。

The targeting drug delivery system of nanoparticle for cancer therapy has been widely studied in recent years. The enhanced permeability and retention effect (EPR effect) make nanoparticles tend to passively accumulate to the tumor site. The specific receptors on the surface of cancer cells are identified astumor targeting ligands to achieve the active targeting function. Transferrin receptor (TfR) is a glycoprotein on the cell membrane. It regulates the uptake, transport, and storage of iron. The transferrin receptor expression can even be 100 times higher than normal cells, which makes the transferrin receptor as one of the targets for cancer therapy. In addition to targeting the specific receptors and facilitating the receptor-mediated endocytosis, cell penetrating peptides (CPPs) have also been widely used in the delivery of anticancer drugs many decades. Studies have shown that the combination of tumors targeting peptides (TTP) and cell penetrating peptides can increase the efficiency of drug delivery, and can also improve the selectivity of cell penetrating peptides. Poly(lactide-co-glycolide (PLGA) was conjugated with different molecular weights of poly(ethylene bis(amine) (PEG diamine). The PLGA-PEG2k and PLGA-PEG5k copolymers with different chain lengths of PEG were grafted with the cell-penetrating peptide R9 and the transferrin receptor targeting peptide T7 respectively. Two kinds of copolymer were mixed and prepared dual-peptide conjugated nanoparticles by solvent evaporation method. The cellular uptake efficiency of different formulations was measured in transferrin receptor overexpressed MDA-MB-231 and low expressed MCF-7 breast cancer cell lines. The docetaxel loaded nanoparticles were further prepared and characterized its particle size, zetapotential, drug loading, and encapsulation efficiency. In vitro release experiments were performed at pH 7.4 and pH 5.0 with or without lipase. In addition, in order to confirm that the nanoparticles were able to escape from lysosomes after endocytosis, we used lysosomal-specific fluorescent dye LysoBriteTM and fluorescent nanoparticles to evaluate the intracellular distribution of nanoparticles. Finally, the cytotoxicity of the blank nanoparticles and docetaxel loaded nanoparticles were measured by MTT assay. IC50 of different formulations were calculated to compare their anti-cancer effect. The experimental results showed that the particle sizes of all formulations fall within the range of 121.7±0.7-159.9±10.1 nm, and the PDI values are less than 0.3, indicating that the nanoparticles have a narrow particle size distribution. The zeta potential was different due to the grafted peptides. PP5kT7* NPs fell to approximately -13.5±0.4 mV, that of PP2kR9* NPs were approximately -7.7±0.7 mV, and that of PP5kT7*/PP2kR9* NPs was approximately -9.6±1.5 mV. After encapsulation of docetaxel, the particle sizes were between 149.2±3.5 and 192.1±5.7 nm, and the encapsulation efficiency was between 49.5±3.7% and 71.4±6.2%, and the drug loading was approximately 6.4±0.8-9.7±0.6% In the cellular uptake study, the cellular uptake efficiency of PP5kT7* NPs and PP5kT7*/PP2kR9 NPs in MDA-MB-231 cell line was 60.14±1.24% and 95.94±14.19% respectively. The cellular uptake efficiency of PP5kT7* NPs and PP5kT7*/PP2kR9 NPs in MCF-7 cell line was 4.83±1.27% and 22.44±4.84% respectively. The results showed that PP5kT7*/PP2kR9 NPs had better cellular uptake efficiency due to the additional R9 peptide. Moreover, PP5kT7*/PP2kR9 NPs remained the selectivity to transferrin receptor overexpressed cells because PLGA-PEG5k-T7* provided shielding effects and reduced the interaction between R9 peptide and the cell membrane of transferrin receptor low expression cells. In intracellular trafficking experiments, almost all PP2kR9* NPs and PP5kT7*/PP2kR9 NPs escaped from lysosome in 60 minutes, indicating that the addition of positive charged R9 peptide in formulations provided better lysosomal escape effects. In the cytotoxicity assay, at the same polymer concentration (1 mg/mL) of the PP5kT7*/PP2kR9 NPs, the cell of MDA-MB-231 cells was 60.90±3.01%, and the cell viability of MCF-7 cells was 70.66± 2.86%. The cytotoxicity of PP5kT7*/PP2kR9 NPs significantly decreased in transferrin low expression cells, indicating that the strategy of shielding CPP with long chain PEG5k can reduce the cytotoxicity of nanoparticles in transferrin low expression cell line. Moreover, the IC50 values of free DTX, DTX@PP5k NPs, DTX@PP5kT7* NPs, DTX@PP2kR9* NPs and DTX@PP5kT7*/PP2kR9* NPs in MDA-MB-231 cells were 156.33±9.61, 123.05±14.43, and 85.53±8.64, 71.57±3.13, and 34.76±3.67 ng/mL respectively, indicating the dual-peptide modified nanoparticles had best anti-cancer effect.