Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington 98195, USA.
J Am Chem Soc. 2012 Oct 10;134(40):16554-7. doi: 10.1021/ja3085803. Epub 2012 Oct 1.
Adaptation of in vitro optimized polymeric gene delivery systems for in vivo use remains a significant challenge. Most in vivo applications require particles that are sterically stabilized, which significantly compromises transfection efficiency of materials shown to be effective in vitro. We present a multifunctional well-defined block copolymer that forms particles useful for cell targeting, reversible shielding, endosomal release, and DNA condensation. We show that targeted and stabilized particles retain transfection efficiencies comparable to the nonstabilized formulations. A novel, double-head agent that combines a reversible addition-fragmentation chain transfer agent and an atom transfer radical polymerization initiator through a disulfide linkage is used to synthesize a well-defined cationic block copolymer containing a hydrophilic oligoethyleneglycol and a tetraethylenepentamine-grafted polycation. This material effectively condenses plasmid DNA into salt-stable particles that deshield under intracellular reducing conditions. In vitro transfection studies show that the reversibly shielded polyplexes afford up to 10-fold higher transfection efficiencies than the analogous stably shielded polymer in four different mammalian cell lines. To compensate for reduced cell uptake caused by the hydrophilic particle shell, a neuron-targeting peptide is further conjugated to the terminus of the block copolymer. Transfection of neuron-like, differentiated PC-12 cells demonstrates that combining both targeting and deshielding in stabilized particles yields formulations that are suitable for in vivo delivery without compromising in vitro transfection efficiency and are thus promising carriers for in vivo gene delivery applications.
将体外优化的聚合物基因传递系统适用于体内仍然是一个重大的挑战。大多数体内应用需要颗粒具有空间稳定性，这大大降低了在体外有效的材料的转染效率。我们提出了一种多功能的、结构明确的嵌段共聚物，它可以形成用于细胞靶向、可逆屏蔽、内涵体释放和 DNA 凝聚的颗粒。我们表明，靶向和稳定的颗粒保留了与非稳定配方相当的转染效率。一种新型的双头试剂，通过二硫键将可逆加成-断裂链转移试剂和原子转移自由基聚合引发剂结合在一起，用于合成一种含有亲水性聚乙二醇和接枝有四亚乙基五胺的聚阳离子的结构明确的阳离子嵌段共聚物。这种材料有效地将质粒 DNA 凝聚成盐稳定的颗粒，在细胞内还原条件下解屏蔽。体外转染研究表明，与类似的稳定屏蔽聚合物相比，可逆屏蔽的聚集体在四种不同的哺乳动物细胞系中提供高达 10 倍的转染效率。为了补偿由于亲水性颗粒壳引起的细胞摄取减少，进一步将神经元靶向肽连接到嵌段共聚物的末端。对神经元样分化的 PC-12 细胞的转染表明，将靶向和屏蔽同时结合到稳定的颗粒中，可以得到适合体内递送的配方，而不会降低体外转染效率，因此是体内基因传递应用的有前途的载体。
