Polymer gene delivery: overcoming the obstacles

doi:10.1016/j.drudis.2013.06.014

Drug Discovery Today

Volume 18, Issues 21–22, November 2013, Pages 1090-1098

https://doi.org/10.1016/j.drudis.2013.06.014 Get rights and content

Highlights

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Minor modifications to polymer structure determine transfection properties.
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Efficient gene delivery requires timely release of DNA.
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Serum stability is influenced by charge density and packaging efficiency.
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Cellular uptake pathway is polymer and cell type dependant.

Recent progress in gene therapy has opened doors for the development of new and multifunctional delivery agents based on the tailored synthesis of polymers. These polymers are in their infancy compared with viral agents, which have been optimised during millions of years of evolution, making viral vectors naturally efficient transfection agents. To improve the efficiency of polymer gene delivery to the level seen in viral vectors, it is necessary to understand the challenges faced by polymer gene delivery vectors both in vitro and in vivo. In this review, we analyse and discuss those obstacles that scientists have to overcome to design a highly efficient synthetic transfection agent.

Section snippets

Frequently used polymers in gene delivery

Polymers used for gene delivery vary in molecular weight, structure and molecular composition. Having linear, branched or dendritic structures, these polymers bind DNA electrostatically, reducing the size of the DNA and/or RNA into nano-sized particles (polyplexes) providing protection and promoting cellular uptake. Electrostatic binding to nucleic acids is prompted by cationic primary, secondary, tertiary and quaternary amines residing on the backbone, pendant groups or grafted monomers.

Polymer structure

The molecular weight and chain length of a polymer have a significant effect on its cellular uptake, endosomal escape, DNA unpackaging and nuclear internalisation. High-molecular-weight (HM_W) polymers show better DNA binding, cellular uptake and transfection efficiency, whereas low-molecular-weight (LM_W) polymers show less cytotoxicity and better DNA unpackaging 10, 11. Artursson et al. studied the effect of LM_W chitosan (⩽5 kDa) related to its physical shape and stability for gene delivery in

DNA packaging

The efficient packaging of DNA before delivery into cells is a major step for successful transfection using polymer-based delivery vectors. Polymers have to have DNA-binding properties to bind to DNA and prevent it from enzymatic degradation and promote cellular uptake. It is well established that binding usually occurs by hydrophobic and electrostatic interactions between the phosphate groups (anionic) along the DNA backbone and cationic groups (usually amine groups) of the polymer agent [19].

Serum stability

Polymers used for gene delivery have the important role of protecting DNA from degradation by serum enzymes. This role is maintained as long as the DNA is tightly bound to the polymer and can travel freely to its target cell. Disassembly and release of DNA from the polyplex can occur after interaction of the latter with negatively charged serum proteins. Rapid blood elimination of polycation–DNA complexes results from their binding to serum albumin and other proteins owing to the aggregation

Cellular uptake

Endocytosis is a process by which cells engulf extracellular molecules by forming invaginations in the cell membrane. This is energy dependent and is the main process by which most polyplexes are taken up by the cell. Endocytosis is an umbrella term that comprises macropinocytosis, phagocytosis and receptor-mediated endocytosis. Phagocytosis is generally carried out by specialised cells, such as monocytes, macrophages and neutrophils [30]; however, is not an ideal endocytic pathway of polyplex

Polymer buffering capacity and the ‘proton sponge’ theory

Cationic polymers can induce endosomal escape because of their net positive charge [40]. Endosomes maintain a certain pH that can be destabilised by protonatable polymers, such as PEI. As the polyplexes enter the cell and become trapped in endosomal vesicles, each endosome has membrane-bound ATPase ion channels that pump protons into the endosome. The polymers become protonated and prevent the acidification of the endosome. This resistance leads to the continuous influx of protons and the

DNA release

Polyplexes are required not only to have high stability outside the cell to ensure that the DNA is protected from serum enzymes, but also to disassemble upon entry into the cell to enable the release and efficient integration of DNA into the host genome. Vector unpacking and DNA release from polyplexes have been identified as barriers to efficient polymer gene delivery [50]. Shorter polycations have a higher probability of dissociating from DNA, enabling higher gene expression over a short

Nuclear internalisation

The nucleus is the control centre of the cell and contains the genomic information required for protein synthesis. It is enclosed by two membranes that enable the passage of small particles (⩽10 nm) freely into and out of the cytoplasm [55]. Larger particles are transported by NLS [56] via nuclear pore complexes embedded in the nuclear membranes. It has been established that proliferating cells are easier to transfect because they undergo mitosis regularly. During the process of cell division,

Future directions

Polymer composition has been shown to have a vital role in regulating transfection, uptake and cell viability [62]. Parameters, including the amount and type of amine groups, charge density, and hydrophilic and hydrophobic content, have direct effects on the strength of the polyplex–cell membrane interaction, serum stability, DNA release, endosomal escape and nuclear localisation. For the future development of polycations, it is important to examine the best combination of elements that make an

Concluding remarks

The combination of cell- and nuclear-penetrating peptides, cationic endosomal buffering polymers and hydrophilic functionality (for improved serum stability) in one delivery vector seems to overcome the significant barriers associated with conventional cationic polymer-based gene delivery vectors. However, many of these delivery vectors fail to cross all the barriers and only work in well-studied and well-characterised cell lines. In addition, questions still remain surrounding the fate of DNA

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ReviewPost screenPolymer gene delivery: overcoming the obstacles

Highlights

Section snippets

Frequently used polymers in gene delivery

Polymer structure

DNA packaging

Serum stability

Cellular uptake

Polymer buffering capacity and the ‘proton sponge’ theory

DNA release

Nuclear internalisation

Future directions

Concluding remarks

Mol. Ther.

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Polymer gene delivery: overcoming the obstacles