Endo-lysosomal escape strategies of cationic polymer-mediated gene delivery at a glance.

In 2012, the first gene therapy agent was approved by the Europe Medicines Agency leading to increased interest in this research field. Beside viruses, non-viral agents based on lipids or polymers represent aspiring alternatives to deliver the genetic material. Different hurdles have to be overcome depending on the kind of nucleic acid used, where plasmid DNA (pDNA) and small interfering RNA represent the common ones. The main challenge for transfection agents, in particular for pDNA delivery, is the transfer to the cell and into the cell nuclei. Within the group of transfection vesicles, cationic polymers show promising features and variability, as they can be synthesized with tailor-made physical and chemical properties (architectures and functionalisation). In the field of polymer-based gene delivery, the tuning potential of polymers by using different architectures like graft and star-shaped polymers as well as self-assembled block copolymers is immense. In particular, in the last few years numerous new polymer designs showed enhanced transfection properties in combination with good biocompatibility. Furthermore, new insights into the transfection mechanism demonstrated the continuous progress in this field. Polymer architecture influences the polyplex characteristics and the latter has an impact on the transfection mechanism, e.g. the interaction with the cellular membrane depends on the polyplex shape. Moreover, polyplex dissociation can be easily influenced by the polymer chemistry, thus biodegradable linkers lead to well suited polymers with reduced toxicity and high delivery potential, and are also promising for in vivo applications. This review focuses on the influence of polymer architectures for pDNA transfection in vitro, showing recent developments and insights. The theoretical background concerning the biological challenges for cationic polymers and the impact of graft-or star-shaped architectures as well as self-assembled structures will be presented in detail.

Asymmetric flow field-flow fractionation (AF4) is a widely used and versatile technique in the family of field-flow fractionations, indicated by a rapidly increasing number of publications. It represents a gentle separation and characterization method, where nonspecific interactions are reduced to a minimum, allows a broad separation range from several nano- up to micrometers and enables a superior characterization of homo- and heterogenic systems. In particular, coupling to multiangle light scattering provides detailed access to sample properties. Information about molar mass, polydispersity, size, shape/conformation, or density can be obtained nearly independent of the used material. In this Perspective, the application and progress of AF4 for (bio)macromolecules and colloids, relevant for "nano" medical and pharmaceutical issues, will be presented. The characterization of different nanosized drug or gene delivery systems, e.g., polymers, nanoparticles, micelles, dendrimers, liposomes, polyplexes, and virus-like-particles (VLP), as well as therapeutic relevant proteins, antibodies, and nanoparticles for diagnostic usage will be discussed. Thereby, the variety of obtained information, the advantages and pitfalls of this emerging technique will be highlighted. Additionally, the influence of different fractionation parameters in the separation process is discussed in detail. Moreover, a comprehensive overview is given, concerning the investigated samples, fractionation parameters as membrane types and buffers used as well as the chosen detectors and the corresponding references. The perspective ends up with an outlook to the future.

To date, cationic polymers with high transfection efficiencies (TE) often have a high cytotoxicity. By screening an 18-membered library of cationic 2-oxazoline-based polymers, a polymer with similar TE as linear poly(ethylene imine) but no detectable cytotoxicity at the investigated concentrations could be identified. The influence of the polymer side chain hydrophobicity and the type and content of amino groups on the pDNA condensation, the TE, the cytotoxicity, the cellular membrane interaction as well as the size, charge, and stability of the polyplexes was studied. Primary amines and an amine content of at least 40% were required for an efficient TE. While polymers with short side chains were non-toxic up to an amine content of 40%, long hydrophobic side chains induced a high cytotoxicity.