Lemieux P, Vinogradov S V, Gebhart C L, Guérin N, Paradis G, Nguyen H K, Ochietti B, Suzdaltseva Y G, Bartakova E V, Bronich T K, St-Pierre Y, Alakhov V Y, Kabanov A V
Supratek Pharma Inc, Armand-Frappier Institute, Laval, PQ, Canada.
J Drug Target. 2000;8(2):91-105. doi: 10.3109/10611860008996855.
Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic. block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes. which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes ("stealth effect"). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mixing with NanoGel suspension, which results in the formation of small particles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion. Further. loaded NanoGel particles cross-polarized monolayers of intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.
对由核酸和合成聚合物形成的自组装复合物进行了质粒和寡核苷酸（oligo）递送方面的评估。在这些研究中使用了具有线性、支化、树枝状、嵌段或接枝共聚物结构的聚阳离子。所有这些分子都能与核酸结合，这是由于聚阳离子的阳离子基团与DNA的磷酸基团之间形成了盐键协同体系。为了提高DNA/聚阳离子复合物的溶解度，开发了含有聚阳离子链段和非离子可溶性聚合物（例如聚环氧乙烷（PEO））链段的阳离子嵌段和接枝共聚物。这些共聚物与短DNA链（如寡核苷酸）结合，会形成含有由中和的DNA - 聚阳离子复合物产生的疏水位点和由PEO产生的亲水位点的物种。这些物种会自发缔合形成聚离子复合胶束，其具有由中和的聚离子形成的疏水核心和由PEO形成的亲水外壳。这种复合物非常小（10 - 40纳米），并且尽管电荷完全中和但在溶液中稳定。它们在体外和体内对寡核苷酸都显示出显著活性。阳离子共聚物与质粒DNA结合会形成更大（70 - 200纳米）的复合物，这些复合物在细胞转染研究中实际上没有活性。很可能是PEO阻止了这些复合物与细胞膜结合（“隐身效应”）。然而，将特定配体连接到PEO冠层上可以产生在溶液中稳定且能与靶细胞结合的复合物。当阳离子共聚物中的PEO被膜活性的PEO - b - 聚环氧丙烷 - b - PEO分子（普朗尼克123）取代时，获得了最有效的复合物。在小鼠全身给药后，这种复合物在肝脏中表现出转基因表达水平升高。为了提高复合物的稳定性，开发了纳米凝胶载体，其是通过使用乳化/溶剂蒸发技术将PEI与双端活化的PEO交联合成的小水凝胶颗粒。通过与纳米凝胶悬浮液混合固定寡核苷酸，这会导致形成小颗粒（80纳米）。掺入纳米凝胶中的寡核苷酸能够到达细胞内的靶点并以序列特异性方式抑制基因表达。此外，负载的纳米凝胶颗粒能穿过肠细胞（Caco - 2）的交叉极化单层，表明这些系统在寡核苷酸口服给药方面具有潜在用途。总之，使用聚阳离子进行基因递送的方法可用于设计具有非常广泛的物理化学和生物学特性的基因转移复合物，这对于设计新一代更有效的非病毒基因递送系统至关重要。
