雙胜肽修飾之奈米載體包覆帕博西尼應用於神經膠質瘤治療

Abstract

神經膠質瘤 (Glioma)為目前最常見的原發性腦腫瘤，然而血腦障壁 (Blood brain barrier, BBB)限制了大部分藥物的遞輸，成為目前臨床治療一大困境。本篇研究以聚乳酸-甘醇酸 (poly(lactide-co-glycolide), PLGA)作為藥物載體骨架，於上接枝聚乙二醇二胺 (poly(ethylene glycol) bis(amine), PEG-diamine)以增加奈米顆粒安定性，利用腦內皮細胞及神經膠質瘤細胞上高度表現運鐵蛋白受體 (Transferrin receptor, TfR )特性，選用對運鐵蛋白受體具標靶能力的T7胜肽及有助於載體穿膜的R9胜肽接枝於PLGA-PEG上，由於R9為非選擇性之細胞穿膜胜肽，為了降低其對於正常之毒殺性，利用PEG鏈長之差異性，使R9接枝於短鏈PEG並包埋於長鏈PEG中，降低其全身循環時與正常細胞直接接觸造成毒殺作用。

本實驗所使用之奈米顆粒皆以溶媒揮發法製備而得，在空的載體中，產率可達70%以上，其粒徑大小落於162.6 nm至173.5 nm之間，且呈現單一粒徑分布 (PDI<0.2)，表面電荷隨著R9胜肽修飾而增加，落於-18.6 mV至7.9 mV之間。

除了對物化性質分析之外，為了探討不同胜肽接枝奈米顆粒對於細胞的標靶能力，針對bEnd.3腦內皮細胞及U87-MG神經膠質瘤細胞進行細胞吞噬實驗。實驗結果顯示，首先，無論在bEnd.3腦內皮細胞或U87-MG神經膠質瘤細胞中，T7修飾可增加細胞對奈米顆粒吞噬，但在L929低表現之對照細胞株中，T7修飾對於奈米顆粒吞噬並無明顯影響。接著，進一步探討雙胜肽修飾之奈米顆粒對於細胞吞噬效率影響，流式細胞儀定量分析結果顯示，在bEnd.3細胞中，相較於未修飾之奈米載體，T7、R9及雙胜肽修飾之奈米顆粒可提升細胞吞噬量分別達2.1、4.1及2.8倍；而在U87-MG細胞中，可分別達1.7、4.6及2.2倍。由以上結果可知T7修飾之奈米顆粒可藉由標靶運鐵蛋白受體以提升細胞吞噬量，而R9修飾可藉由穿膜增加細胞吞噬量，雖然R9胜肽修飾能最有效提升奈米顆粒進入細胞效率，但其非選擇性限制了臨床應用性。

為了進一步探討奈米顆粒對於血腦障壁的穿透效率，本實驗利用穿透式細胞培養盤 (Transwell plate)共同培養bEnd.3腦內皮細胞及U87-MG神經膠質瘤細胞以建立了體外血腦障壁模型，其中，在4小時及12小時穿透試驗結果皆顯示，bEnd.3腦內皮細胞中對於不同胜肽修飾之奈米顆粒具有不同吞噬效率，由高到低依序為R9>雙胜肽>T7>未修飾，而在U87-MG細胞中亦得到相同趨勢，此結果可說明藉由雙胜肽修飾，不但可提升奈米顆粒穿透血腦障壁能力，且能增加其進入神經膠質瘤細胞之能力。除此之外，本研究亦利用體內試驗觀察奈米顆粒之全身循環分布，結果顯示T7修飾相較於未修飾及R9修飾之奈米顆粒更能累積於腦部，且雙胜肽修飾能更進一步提升奈米顆粒於腦部累積量。

接著，將奈米粒子進一步包覆Palbociclib (PBC)作為治療藥物探討其對於神經膠質瘤治療之助益，PBC為一種週期蛋白依賴性激酶4/6抑制劑 (Cyclin dependent kinase 4/6 inhibitors, CDK4/6 inhibitors)，能專一性阻斷癌細胞中高度表現的CDK4/6蛋白生成，進而遏止下游細胞週期調控相關路徑，以抑腫瘤細胞的增生。所有包覆藥物之奈米顆粒產率可達65%以上，其粒徑大小落於168.4 nm至185.8 nm之間，且呈現單一粒徑分布 (PDI<0.2)，其表面電荷落於-17.8mV至-10.0 mV之間，藥物包覆率可達60%，且負載率可達8%。於4˚Ｃ水中及冷凍乾燥後回溶於水之4週安定性試驗顯示奈米顆粒之粒徑皆可維持，並無明顯凝集現象。此外，在體外釋放試驗中，可知於pH 5.5環境下的奈米載體釋放藥物速度，較pH 7.4環境下為快。在細胞毒殺試驗中，可發現胜肽修飾之奈米顆粒可提升PBC對於細胞的毒殺效果，其純藥及包覆於各種不同胜肽修飾之奈米顆粒之IC50分別為PBC純藥：18.9 ± 1.3 μg/mL、未修飾：8.2 ± 0.8 μg/mL、T7修飾：6.0 ± 0.7 μg/mL、雙胜肽修飾：2.9 ± 0.5 μg/mL及R9修飾：2.4 ± 0.1 μg/mL。

以上結果證明雙胜肽修飾之奈米顆粒能藉由胜肽之標靶及穿透能力提升PBC遞輸到腦部效率，將有潛力作為針對神經膠質瘤的治療方法。

Treatment of glioma remains a critical challenge worldwide, since therapeutic effect is greatly hindered by the poor transportation across blood brain barrier (BBB) and the low penetration into tumor cells. To overcome this hurdle, we investigated the potential of dual-ligand polymeric vector to deliver the desired therapeutic agent into glioma cells. In this study, poly(lactic-co-glycolic acid) (PLGA) was served as the main polymer, and poly(ethylene glycol) bis(amine) (PEG-diamine) was conjugated to improve their stability. In addition, the transferrin receptor targeting ligand (T7) and cell-penetrating peptide (R9) were decorated for improving transport of anticancer drugs across BBB and into tumor tissues. Since R9 was characterized as a ligand without specificity, PEG with shorter chain lengths (PEG2k) was chose for conjugation of R9, while T7 was conjugated on long chain of PEG5k. By this design, R9 could be shielded during circulation.

All the blank nanoparticles (NPs) were prepared by solvent evaporation method, with all the yields up to 70%. The particle sizes were at the range of 162.6 nm to 173.5 nm with mono-distribution (PDI<0.2), and the zeta potentials were ranged from -18.6 mV to 7.9 mV, showing increasing manner with the modification of R9 peptide.

The targeting effects of these NPs were evaluated by in vitro cellular uptake study. A higher intracellular internalization of NPs could be seen in bEnd.3 and U87-MG cells when treated with T7-NPs, compared to unconjugated NPs. However, it showed no difference in L929 negative cells. The quantitative analysis by flow cytometry illustrated that the cellular uptakes of T7-, R9- and dual peptide-NPs were respectively increased by 2.1-, 4.1-, and 2.8- times in bEnd.3 cells and 1.7-, 4.6-, and 2.2- times in U87-MG cells (compared to unconjugated NPs). Although R9-NPs exhibited the prominent increase in cellular uptake, the non-specific property limited their use in clinic.

To further explore the transport efficiency of these NPs, the in vitro BBB model co-cultured with bEnd.3 and U87-MG cells was established. After 4 hr and 12 hr incubation with different formulations, the internalization of NPs in bEnd.3 displayed a clear tendency as follows: R9-NPs > dual peptide-NPs > T7-NPs > unconjugated NPs, showing statistical difference between each other. Meanwhile, the same results could also be seen in U87-MG cells, exploiting the BBB-crossing and glioma-penetrating ability of dual peptide-NPs. In vivo imaging also revealed that T7-NPs accumulated more specifically in brain tissues than non-targeted and R9-NPs, and the dual peptide- NPs could further enhance accumulation in the brain, showing the success of overcoming the in vivo BBB by dual targeting mechanism.

These NPs were further loaded with palbociclib (PBC), a potent cell cycle inhibitor selective for CDK4/6, to exert anti-glioma effect. They presented the sizes of 168.4 nm to 185.8 nm with narrow size distribution (PDI<0.2). The zeta potentials were in the range of -17.8 mV to -10.0 mV. The encapsulation efficiency achieved 60%, and the drug loading was approximately 8%. All these formulations exhibited great stability at 4℃ in ddH2O and after lyophilized for 28 days. In vitro release study demonstrated PBC released faster in pH 5.5 than pH 7.4. The in vitro cytotoxicity study showed that the anti-glioma effect of PBC was significantly elevated by encapsulation of NPs, especially for those with peptide modification. (IC50 of free PBC: 18.9 μg/mL, PBC@NPs: 8.2 μg/mL, PBC@T7-NPs: 6.0 μg/mL, PBC@dual peptide NPs: 2.9 μg/mL and PBC@R9-NPs: 2.4 μg/mL)

In conclusion, this dual peptide functionalized NPs served as great glioma targeting systems to promote the penetration and accumulation of PBC into tumor sites, which would be of pronounced significance for the therapy of glioma.