Demirgöz Döne, Garg Ashish, Kokkoli Efrosini
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Langmuir. 2008 Dec 2;24(23):13518-24. doi: 10.1021/la801961r.
In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.
近年来，人们付出了巨大努力来设计改进的递送系统，通过引入位点特异性表面配体来进一步增强其选择性靶向性。本研究的目的是构建靶向α5β1的隐形脂质体（覆盖有聚乙二醇（PEG）的纳米颗粒），使其能够与表达α5β1的LNCaP人前列腺癌细胞结合，并在细胞内有效释放包封的物质。为此，用纤连蛋白模拟肽（PR_b）对脂质体（有或没有PEG2000）进行功能化修饰，并递送至LNCaP细胞。通过1H NMR表征PEG2000和其他脂质体成分的量，通过二辛可宁酸蛋白测定法测定肽的量。纤连蛋白是α5β1的天然配体，一种有前景的纤连蛋白模拟肽设计包括通过连接体连接的主要结合位点（RGD）和协同位点（PHSRN），并通过间隔基团从表面延伸出来。我们之前设计了一种肽两亲分子PRb，它采用疏水尾部，连接到由间隔基团、协同位点序列、模拟天然蛋白纤连蛋白中存在的距离和疏水性/亲水性的连接体（从而呈现出整体“中性”连接体）以及最终的主要结合序列组成的肽头基的N端。我们研究了仅用PR_b或用PR_b和PEG2000功能化修饰的不同脂质体制剂。对于PR_b靶向的聚乙二醇化脂质体，当肽浓度为2 mol%及更高时，观察到有效的细胞结合。与GRGDSP靶向的隐形脂质体相比，PR_b功能化在LNCaP结合、内化效率以及用包封肿瘤坏死因子-α（TNFα）的脂质体孵育LNCaP细胞后的细胞毒性方面均优于GRGDSP。更重要的是，PR_b是α5β1特异性的，而许多整合素与小的RGD肽结合。因此，所提出的PR_b靶向递送系统有潜力以高效且特异的方式将治疗性物质递送至前列腺癌细胞。
