胜肽修飾之奈米顆粒包覆細胞週期素激酶抑制劑於癌症治療及神經保護之研究

Abstract

本研究選用之細胞週期素激酶抑制劑seliciclib (CDK1, 2, 5, 7, and 9 inhibitor)根據文獻報導具備抗癌以及神經保護作用，利用癌細胞、腦內皮細胞以及分化後神經母細胞瘤細胞過度表現運鐵蛋白受體（Transferrin receptor, Tf）性質，將藥物包覆於表面以標靶該受體的T7胜肽修飾之奈米顆粒，以促進奈米顆粒被癌細胞吞噬能力以及通過血腦障壁（Blood-brain barrier, BBB）到達目標細胞產生神經保護之作用。 本研究以聚乳酸-甘醇酸 （poly(D, L-lactide-co-glycolide), PLGA）作為奈米顆粒骨架，表面接枝馬來醯胺-聚乙二醇-胺（maleimide poly(ethylene glycol) amine, Mal-PEG-NH2），接枝率為60.6±4.0 mol%，再以溶媒揮發法製備奈米顆粒，並透過Cys*-T7上的硫醇基與馬來醯胺上的碳雙鍵反應，進行奈米顆粒表面修飾，T7的接枝率為32.7±3.2 mol%。奈米顆粒產率皆可達70%以上，未修飾以及T7修飾之奈米顆粒粒徑分別為95.8±2.0 nm與98.8±1.9 nm，皆呈單一峰粒徑分佈（PdI < 0.1），表面電荷因T7的修飾由-25.0±3.0 mV上升為0.8±2.2 mV。 為探討以T7修飾之奈米顆粒對於不同癌細胞的標靶能力，於各細胞株進行吞噬實驗，結果顯示在T7胜肽的修飾下，奈米顆粒的吞噬效率在運鐵蛋白受體過度表達的癌細胞有顯著的提升，其中MDA-MB-231乳癌細胞可提升吞噬效率13.3倍，其次依序為SKOV-3卵巢癌細胞提升6.3倍、U87-MG神經膠質瘤細胞提升4.5倍；但在運鐵蛋白受體低表現量之A549非小細胞肺癌細胞，其吞噬效率僅提升2.5倍。 同樣的，在神經保護的研究中，T7修飾之奈米顆粒對於bEnd.3腦內皮細胞以及分化前後SH-SY5Y神經母細胞瘤細胞的標靶能力，也以流式細胞儀進行定量，結果發現在bEnd.3細胞中，有T7修飾相較於未修飾之奈米顆粒，其吞噬效率提升4.9倍；相對於L929運鐵蛋白受體低表現量之對照細胞僅提升1.2倍，顯示T7修飾之奈米顆粒可藉由標靶運鐵蛋白受體提升細胞之吞噬量。另外，分化前後SH-SY5Y細胞的吞噬效率提升分別為10.9倍以及26.6倍。 為探討奈米顆粒對於血腦障壁的穿透能力，採用穿透式細胞培養盤（Transwell plate）建立bEnd.3腦內皮細胞以及分化後SH-SY5Y擬神經細胞的共同培養系統，給予未修飾以及胜肽修飾奈米顆粒，發現T7修飾有助於奈米顆粒穿透體外血腦障壁並進入目標神經細胞，除此之外，在小鼠體內分佈試驗中，也發現胜肽修飾的奈米顆粒能夠進入腦中並累積。 進一步將奈米顆粒包覆seliciclib，未修飾以及T7修飾奈米顆粒粒徑分別為115.7±5.5 nm與127.3±0.7 nm（PdI < 0.2），表面電荷分別為-30.8±9.2 mV和-20.0±4.2 mV；藥物包覆率皆可達60%以上，且藥物負載率可達12.3%；分別於4°C水溶液以及冷凍乾燥後保存於-20°C的4週安定性試驗顯示其粒徑無顯著變化。此外，在體外釋放試驗中，在pH 5.5環境的奈米載體釋放速率較pH 7.4的環境快。 此外，48小時之癌細胞毒殺試驗結果顯示，U87-MG、MDA-MB-231以及SKOV-3細胞在給予經T7修飾的包藥奈米顆粒組別，其IC50分別降為無胜肽修飾的包藥奈米顆粒的0.3、0.5以及0.7倍；而在A549細胞，有無胜肽修飾對於其IC50並無顯著差異。 在評估seliciclib對神經保護的療效方面，本研究採用MPP+神經毒素誘發分化後SH-SY5Y細胞產生神經毒性，在預先給予分化後SH-SY5Y細胞T7修飾的包藥奈米顆粒組別兩小時後再給予MPP+，使原先對MPP+的IC50（0.75±0.06 µg/mL）有顯著之提升，IC50分別為預先給seliciclib純藥：1.11±0.19 µg/mL、seliciclib@PPM NPs：0.85±0.12 µg/mL、seliciclib@PPM NPs-Cys*-T7：3.15±0.69 µg/mL。 根據以上結果，可支持使用運鐵蛋白受質標靶胜肽於遞送藥物至上述目標細胞並產生癌細胞毒殺以及神經保護的潛力。

Seliciclib, a broad CDKs inhibitor (including CDK1, CDK2, CDK5, CDK7 and CDK9), exerted its potential role in cancer treatment and neuroprotection for neurodegenerative diseases. In addition, taking advantage of overexpressive transferrin receptor (TfR) on most cancer cells, bEnd.3 endothelial cells and differentiated SH-SY5Y neuroblastoma cells, T7 peptide, a TfR targeting ligand, was served to facilitate the NPs internalized by targeting cells for better anti-cancer efficacy and blood-brain barrier (BBB) penetrating ability promising the potential in neuroprotection effect. In this study, poly(D, L-lactide-co-glycolide) (PLGA) firstly conjugated with maleimide poly(ethylene glycol) amine (Mal-PEG-NH2) was served as the backbone of the nanoparticles (NPs). All NPs were prepared by solvent evaporation method, further conjugated with Cys*-T7 peptide through formation of thioether bond with maleimide on NPs. The yields of all NPs were above 70% and particle sizes ranged from 95.8±2.0 nm to 98.8±1.9 nm after Cys*-T7 peptide-conjugation with narrow size distribution (PdI<0.1); the zeta potentials of NPs increased from -25.0±3.0 to 0.8±2.2 mV attributed to the positively charged amino acids of T7 peptide. To investigate the targeting ability of T7 peptide, the cellular uptake of NPs study was proceeded. The efficiency of NPs uptaken by cells was elevated in T7-modified NPs compared to unmodified NPs among highly TfR-expressive cancer cells, especially for MDA-MB-231 breast cancer cells followed by SKOV-3 ovarian cancer cells and U87-MG glioma cells. In contrast, A549 non-small cell lung cancer cells served as negative control of TfR-expression, displayed the lowest improvement of uptake efficiency after T7-conjugated. Furthermore, for bEnd.3 cells and SH-SY5Y cells, the higher uptake efficiency of T7-modified NPs compared to unmodified ones was observed; on the contrary, only slight change was observed in L929 negative cells. There findings implied that Cys*-T7 peptide strengthened the cellular uptake ability through recognition of TfR. The in vitro BBB model was established with co-cultured bEnd.3 cells and differentiated SH-SY5Y cells to evaluate the penetrating ability of NPs. Higher amount of NPs internalized by both bEnd.3 and differentiated SH-SY5Y cells was observed with T7-conjugated NPs compared to unconjugated ones. Further encapsulating seliciclib in the NPs, the particles size increased approximately 20 nm, with narrow size distribution; the zeta potentials of NPs increased from -30.8±9.2 to -20.0±4.2 mV. The drug encapsulation efficiency reached 60.0±1.2% and drug loading was approximate 12.3±0.5%. The 4-week stability study demonstrated the great stability of drug-loaded NPs. The in vitro release study showed that seliciclib released faster from NPs in pH 5.5 than pH 7.4 release medium. In 48-hour cytotoxicity assay, the IC50 of seliciclib@NPs-T7 showed decrease by approximately two folds than seliciclib@NPs in U87-MG, MDA-MB-231 and SKOV-3 cells. However, there was no significant difference in IC50 between seliciclib@NPs-peptide and seliciclib@NPs in A549 cells. The neuroprotection efficacy of seliciclib@NPs against MPP+-induced neurotoxicity was evaluated by treatment with seliciclib or seliciclib@NPs prior to MPP+ exposure, the IC50 of MPP+ was rescued by 1.48-fold, 1.13-fold and 4.2-fold for raw seliciclib, seliciclib@PPM NPs and seliciclib@PPM NPs-Cys*-T7, respectively. In conclusion, Cys*-T7 peptide-modified NPs provided the targeting delivering platform for either cancer cells or BBB-penetration and also promised the potentials of seliciclib for cancer therapy and neuroprotection.