Nanostructured materials designed for cell binding and transduction
Nanostructured materials designed for cell binding and transduction
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DOI：
10.1021/bm015515c
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发表时间：
2001-06-01
影响因子：
6.2
通讯作者：
Wooley, KL
中科院分区：
文献类型：
--
作者：
Liu, JQ;Zhang, Q;Wooley, KL
介绍了壳交联型(SCK)纳米粒子与来自人类免疫缺陷病毒TAT蛋白的蛋白转导结构域(PTD)序列YGRKKRRQRRR的表面功能化，并证明了这些纳米生物结合物表现出的细胞结合作用。采用了一种收敛的合成策略，即SCK纳米颗粒和PTD分别制备，然后在将PTD组分固定在固体载体上的过程中偶联在一起。通过胶束化聚己内酯-b-丙烯酸两亲嵌段共聚物制备了SCK纳米粒子，然后将胶束电晕内的丙烯酸残基进行酰胺化交联，PTD序列构建在固体载体上，从C端到N端，然后延伸四个甘氨酸残基，留下氨基链末端用于随后与SCK表面剩余的丙烯酸官能团偶联。最后，从固体载体上进行切割，这也促进了多肽侧链官能团的去保护以及组成SCK核心域的聚(epsilon-己内酯)片段的水解，得到PTD衍生的纳米笼结构(PTD-Nanocage)。SCK前体与荧光素-5-氨基硫脲的共价标记提供了荧光标记的PTD-纳米笼纳米生物结合物，使其能够通过荧光显微镜进行检测。发现荧光PTD-纳米笼生物偶联物能与CHO细胞和HeLa细胞相互作用，而缺乏PTD成分的类似结构不能。用流式细胞仪和荧光激活细胞分选技术(FACS)分析与荧光PTD-纳米笼生物结合物结合的CHO细胞。分离的生物结合CHO细胞的荧光共聚焦显微镜显示，生物结合的纳米颗粒主要位于细胞外围；然而，纳米颗粒也被转导到细胞内。
The surface-functionalization of shell cross-linked (SCK) nanoparticles with the oligomeric peptide sequence YGRKKRRQRRR, the protein transduction domain (PTD) from the human immunodeficiency virus TAT protein, is described, and the cell binding interactions these nanobioconjugates exhibit are demonstrated. A convergent synthetic strategy was employed, whereby the SCK nanoparticles and the PTD were prepared independently and then coupled together during immobilization of the PTD component on a solid support. The SCK nanoparticles were prepared by the micellization of amphiphilic block copolymers of poly(epsilon-caprolactone-b-acrylic acid), followed by amidation-based cross-linking of the acrylic acid residues located within the micellar corona, The PTD sequence was constructed upon a solid support, from C-terminus to N-terminus, followed by extension with four glycine residues, leaving the amino chain end for subsequent coupling with remaining acrylic acid functionalities present on the surface of the SCK. Finally, cleavage from the solid support was performed, which also facilitated deprotection of the peptide side chain functionalities as well as hydrolysis of the poly(epsilon-caprolactone) segments composing the SCK core domain, to yield PTD-derivatized nanocage structures (PTD-nanocage). Covalent labeling of the SCK precursor with fluorescein-5-thiosemicarbazide provided fluorescently tagged PTD-nanocage nanobioconjugates to allow for their detection by fluorescence microscopy. The fluorescent PTD-nanocage bioconjugates were found to interact with CHO cells and HeLa cells, whereas the analogous structure lacking the PTD component did not. CHO cells bound with fluorescent PTD-nanocage bioconjugates were analyzed using flow cytometry and fluorescence activated cell sorting (FACS). Fluorescence confocal microscopy of isolated bioconjugate-bound CHO cells indicated that the bioconjugated nanoparticles were primarily located near the cell periphery; however, transduction of the nanoparticle into the cells also occurred.