Golani-Armon Adi, Golan Moran, Shamay Yosi, Raviv Lior, David Ayelet
Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, and ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer-Sheva, Israel 84105.
Bioconjug Chem. 2015 Feb 18;26(2):213-24. doi: 10.1021/bc500529d. Epub 2015 Feb 9.
Dendritic cells (DCs) are a family of specialized antigen presenting cells (APCs) that detect antigens and initiate a wide spectrum of immune responses against them. These characteristics make them promising candidates for immunotherapy manipulations. In this study we designed and synthesized DC-targeted block copolymers composed of linear polyethylenimine (PEI) conjugated to hydrophilic polyethylene glycol (PEG) installed with a DC-targeting peptide (DC3, primary sequence FYPSYHSTPQRP). Two different conjugation procedures (basic and modified) were employed to synthesize the DC3-PEG-b-PEI and the control SCRM-PEG-b-PEI (with a scrambled DC3 peptide sequence) by one-pot synthesis, in two steps. In the first, basic conjugation procedure, PEG with N-hydroxysuccinimide (NHS) ester and maleimide (MAL) groups (NHS-PEG-MAL, 3.5 kDa) was first coupled to linear PEI (25 kDa) via the NHS group to yield the intermediate MAL-PEG-b-PEI, that was then conjugated to N-terminus-cysteine harboring peptides DC3 or SCRM via the MAL double bond to yield the final DC3-PEG-b-PEI or SCRM-PEG-b-PEI copolymers, respectively. In the second, modified conjugation procedure, Fmoc-cysteine harboring DC3 or SCRM peptides were first conjugated to NHS-PEG-MAL via the MAL group followed by coupling to linear PEI via the NHS functional group. Fmoc cleavage yielded the same final product as in the basic procedure. The modified conjugation procedure was capable of yielding block copolymers richer with peptides, as determined by (1)H NMR analysis. Self-assembly of DC3-PEG-b-PEI copolymers and DNA molecules yielded nanosized polyion complexes (polyplexes), with lower surface charge and limited cytotoxicity when compared to the PEI building block. Significant transfection efficiency of the DC-targeted polyplexes by murine dendritic DC2.4 cells was observed only in DC3-PEG-b-PEI/DNA polyplexes synthesized by the modified conjugation procedure. These polyplexes, with higher peptide-load, showed greater transfection capability in DC2.4 cells relative to the control nontargeted SCRM-PEG-b-PEI/DNA polyplexes, but not in endothelial cells. The transfection efficiency was comparable to or higher than that of the PEI/DNA positive control. The results indicate that PEGylated-PEI polyplexes show significant transfection efficiency into DCs when decorated with DC3 peptide, and are attractive candidates for immunotherapy via DCs.
树突状细胞（DCs）是一类特殊的抗原呈递细胞（APCs），可检测抗原并引发针对这些抗原的广泛免疫反应。这些特性使其成为免疫治疗操作的理想候选者。在本研究中，我们设计并合成了靶向DC的嵌段共聚物，该共聚物由与亲水性聚乙二醇（PEG）共轭的线性聚乙烯亚胺（PEI）组成，并安装了DC靶向肽（DC3，一级序列为FYPSYHSTPQRP）。采用两种不同的共轭方法（基本方法和改进方法），通过一锅法分两步合成DC3-PEG-b-PEI和对照SCRM-PEG-b-PEI（具有打乱的DC3肽序列）。在第一种基本共轭方法中，首先将带有N-羟基琥珀酰亚胺（NHS）酯和马来酰亚胺（MAL）基团的PEG（NHS-PEG-MAL，3.5 kDa）通过NHS基团与线性PEI（25 kDa）偶联，得到中间体MAL-PEG-b-PEI，然后通过MAL双键将其与带有N端半胱氨酸的肽DC3或SCRM偶联，分别得到最终的DC3-PEG-b-PEI或SCRM-PEG-b-PEI共聚物。在第二种改进共轭方法中，首先将带有Fmoc-半胱氨酸的DC3或SCRM肽通过MAL基团与NHS-PEG-MAL偶联，然后通过NHS官能团与线性PEI偶联。Fmoc裂解产生与基本方法相同的最终产物。如通过（1）H NMR分析所确定的，改进的共轭方法能够产生富含肽的嵌段共聚物。DC3-PEG-b-PEI共聚物与DNA分子的自组装产生了纳米级聚离子复合物（多聚体），与PEI构建块相比，其表面电荷更低且细胞毒性有限。仅在通过改进共轭方法合成的DC3-PEG-b-PEI/DNA多聚体中观察到小鼠树突状DC2.4细胞对DC靶向多聚体的显著转染效率。这些具有更高肽负载量的多聚体在DC2.4细胞中相对于对照非靶向SCRM-PEG-b-PEI/DNA多聚体显示出更高的转染能力，但在内皮细胞中则不然。转染效率与PEI/DNA阳性对照相当或更高。结果表明，用DC3肽修饰的聚乙二醇化PEI多聚体对DC显示出显著的转染效率，是通过DC进行免疫治疗的有吸引力的候选者。
