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20.2.2 Formulation of polyplexes
Polyplex formulation effects transfection efficiency and stability. It is a kinetically controlled process, where successive addition of components, polymer and DNA effects the polyplex size and its transfection efficiency. PEI interacts electrostatically with DNA and RNA to form a stable complex when condensed. Polymer characteristics like molecular weight, density of charges and the composition of complex influence the process of complex formation and condensation. For instance, low molecular weight and lower charge density decreases condensation. The medium composition also affects the complex formation process. Ionic strength of saline solution controls the size of complex formed, higher the concentration larger the complex and less its binding efficiency. Sodium phosphate is a better carrier when compared to NaCl or phosphate buffered saline (PBS). Higher the ratio of positive to negative charge on the cationic polymer, better is the compaction process. At charge ratio 1, DNA is bound completely with the complex while at neutral state it aggregates and shows low solubility.
20.2.3 Structure of polyplexes
DNA-PEI condensates are spherical, globular or rod like in the presence of polycations. Toroid shaped structures are formed when unmodified polylysine condenses DNA. The nature of complex formed depends on the degree of branching, as linear structures have lower rate of transfection compared to branched structures. For example PEIs having high branching form small structure with high transfection ability but are toxic. More branching with secondary and tertiary amine groups enhance transfection efficiency and decrease toxicity.
20.3 Charge and stability of polyplexes
Solubility of polyplexes increases on increasing the number of polycationic charge because of the formation of hydrophilic cationic core around the polyplex. At neutral charge polyplexes based on PEI and DNA show no solubility. The final structure and transfection capacity depends on the storage conditions employed for polyplexes. For instance, storage for 3 weeks improves the transfection of polyplexes by highly branched PEI derivative due to stronger electrostatic interaction. Salt causes a charge shielding effect when used at high concentration and thus decreases binding between DNA and polymer used. Polymer size is an important factor in determining transfection efficiency and is also considered for biocompatibility and extravasation when used for targeting cells outside vasculature. The size factor is itself based on the structure and the complex nature of polyplexes. Fully complexed structures are condensed completely. Condensation rate is high for high molecular weight, longer, and branched chain polymer. Completely condensed structures are protected from degradation in extracellular environment and are easily taken up by the cell through electrostatic interaction. Large PEI-DNA polyplexes consist of aggregates of smaller units. As time increases positively charged polyplexes aggregates more rapidly. Surface charge, molecular weight and ionic strength of the medium also influence the aggregation. The components which shield the charge, decrease interaction and hence aggregation, thus inhibiting rapid elimination by the reticulo-endothelial system. The N/P ratio controls the aggregation of complexes. Hydrophobic interaction and van-der waal forces cause aggregation in complexes with low N/P value while high N/P ratio decreases aggregation because of electrostatic repulsion between positive charge surface present on the complex.