UCL School of Pharmacy, University College London, London, UK.
Curr Top Med Chem. 2014;14(9):1172-81. doi: 10.2174/1568026614666140329231718.
We have previously demonstrated in a therapeutic study that a single systemic course of DAB-Am16 dendriplexes loaded with plasmid expressing TNFα over a period of time of 10 days led to a regression of 100% of tumours and to long term cures of up to 80% of animals. However, the formulation had a relatively low colloidal stability requiring administration soon after nanoparticle preparation. Similar to other cationic polyplex and dendrimer DNA delivery systems, DAB-AM16 dendrimer formulations contained a substantial proportion of free polymer; this free polymer is present independently of the specific polymer:DNA ratio and increases with increasing proportion of polymer (N:P charge ratio) in the formulation. It has previously been shown for this and other systems that the excess of polymer plays a role in promoting the transfection efficiency of synthetic vectors. This has been linked to effects of the polymer on the efficiency of intracellular processing, e.g. endosomal release. However, the free polymer may have additional effects that are relevant to the efficiency of the formulation. This study therefore considered the effect of free dendrimer on the colloidal stability of the complexes, the interaction of the complex with the formulation medium, and with biological components, i.e. electrolytes and serum proteins after administration. Analysis of the total potential of interaction shows that, even at high N:P ratios, the excess of free dendrimer in the medium is not enough to induce the aggregation of the formulation due to depletion forces. This finding is unusual and can be attributed to the particularly low Mw of these dendrimers (1.6 kDa). On the other hand, formulations are highly sensitive to the strength of the dendrimer:DNA interactions. These can be controlled by the degree of protonation (α) of the dendrimer which is strongly dependent on bulk pH. Modulation of the protonation level to α≥0.4 allows reproducible production of colloidally stable particles. Finally, we have demonstrated that electrolytes and proteins present in physiological media play a crucial role to favour the efficiency of these synthetic vectors reducing the toxicity associated with their cationic groups.
我们之前在一项治疗研究中证明，在 10 天的时间内，全身性给予负载 TNFα 表达质粒的 DAB-Am16 树枝状聚合物胶束可使 100%的肿瘤消退，并使 80%的动物获得长期治愈。然而，该制剂的胶体稳定性相对较低，需要在纳米颗粒制备后不久给药。与其他阳离子多聚物和树枝状聚合物 DNA 传递系统类似，DAB-AM16 树枝状聚合物制剂含有大量游离聚合物；这种游离聚合物独立于特定的聚合物：DNA 比存在，并随着制剂中聚合物比例（N：P 电荷比）的增加而增加。先前已经表明，对于这种和其他系统，聚合物的过剩在促进合成载体的转染效率方面起着作用。这与聚合物对细胞内处理效率的影响有关，例如内体释放。然而，游离聚合物可能具有与制剂效率相关的其他影响。因此，本研究考虑了游离树枝状聚合物对复合物胶体稳定性的影响、复合物与制剂介质以及给药后与生物成分（即电解质和血清蛋白）的相互作用。总相互作用势能分析表明，即使在高 N：P 比下，由于耗竭力，介质中游离树枝状聚合物的过剩不足以诱导制剂聚集。这一发现是不寻常的，可以归因于这些树枝状聚合物的分子量特别低（1.6 kDa）。另一方面，制剂对树枝状聚合物：DNA 相互作用的强度非常敏感。这些可以通过树枝状聚合物的质子化程度（α）来控制，而质子化程度强烈依赖于体相 pH 值。调节质子化水平至α≥0.4，可重复性地产生胶体稳定的颗粒。最后，我们证明了生理介质中存在的电解质和蛋白质在提高这些合成载体的效率方面起着至关重要的作用，降低了与其阳离子基团相关的毒性。
