Polyplex transfection

Cohen RN, van der Aa MA, Macaraeg N, Lee AP, Szoka FC Jr (2009) Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection. J Control Release 135 166-174... 

The size of polyplexes plays a role in the endosomal uptake, the cytoplasmic transport and the migration through the nucleopore complexes which mediate bidirectional transport between the cytoplasm and nucleus. The transfection efficiency of the small, stable polyplexes was found to be 100-500 times lower compared with the larger, aggregated polyplexes. Similarly, a lower in vitro transfection efficiency was observed in cases where the polyplexes were prepared with a lower amount of DNA, which may be due to limited contact of the small polyplex with the cells. Indeed, smaller polyplexes transfected efficiently when either the transfection volume or the transfection time was increased. The smaller particles are less able to destabilize the endosomes, resulting in a lower transfection. ... 

In addition to covalently modified ROMP polymers, noncovalent poly-plexes of cationic ROMP polymers with DNA have been used as transfection agents [218, 219]. Breitenkamp and Emrick showed that PCOE functionalized with cationic polylysine grafts complexed with a plasmid that expresses GFP. This polyplex transfected COS-1 and HeLa cells at comparable or superior levels to commercially available jetPEI (linear polyethyleneimine), SuperFect (dendrimer), and Lipofectamine 2000 (liposomal carrier) transfection vehicles [220]. The ROMP-derived carrier was nontoxic to cells, while all three commercially available reagents led to significant levels of cell death. 

The neoangiogenic tumor vasculature overexpresses certain integrins and other surface markers, which can also be used for targeting of polyplexes. The RGD peptide motif has been successfully applied for integrin-targeted pDNA [125-128] and siRNA [129, 130] delivery. In many cases, the PEG motif-containing peptide was attached to the polycation via a PEG spacer. For RGD-PEG-PEI/pDNA polyplexes, an optimum grafting with RGD-PEG was required because transfection... 

First clinical human gene therapy trials with polyplexes were performed using cancer vaccines based on autologous patient tumor cells. These were modified ex vivo with interleukin-2 pDNA. To obtain high level transfection rates of patient s primary tumor cells, Tf-PLL/pDNA polyplexes linked with inactivated endosomolytic adenovirus particles were applied [221]. Polymer-based in vivo human gene transfer studies were performed with PEGylated PLL polyplexes, delivering CFTR pDNA to the airway epithelium of cystic fibrosis patients [222],... 

Boeckle S, Fahrmeir J, Roedl W, Ogris M, Wagner E (2006) Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes. J Control Release 112 240-248... 

Poly-L-lysine was one of the original reagents used to condense DNA into a transfecting particle (21) and numerous studies have been widely reported with these reagents. Oligolysine peptides as short as (K)i6 will also package pDNA, alone (polyplex) or in combination with lipids (lipopolyplex) (22-26). In both lipopolyplex and polyplex formulations, the DNA is condensed... 

The assembly of NLS in peptide-based gene delivery systems has been achieved by the non-covalent binding of plasmid to either free NLS embedded with polyplexes or to NLS linked to a cationic sequence, such as (PKKKRKV)4-K2o (Table 16.7), AKRARLSTSFNPVYPYEDES-K20 (Table 16.7) or H9-2 sequence (nls-H9-2) (Table 16.4). With nls-H9-2, the transfection efficiency with a formulation containing... 

Van de Wetering, P., Schuurmans-Nieuwenbroek, N.M.E., Hennink, W.E. and Storm, G. (1999b) Comparative transfection studies of human ovarian carcinoma cells in vitro, ex vivo and in vivo with poly(2-(dimethylamino) ethyl methacrylate)-based polyplexes. J. Gene Med., 1, 156-165. 

Fig. 1 Proposed mechanism of gene transfection [117]. (1) Formation of the DNA/polymer complex (polyplex), (2) endocytosis of the polyplex, (3) fusion of endosome and lysosome, (4) release of the polyplex into the cytosol, (5a) incorporation of the polyplex into the nucleus, (5b) release of the siRNA into the cytosol, (6) transcription of the DNA into mRNA followed by release of the polyamine back into the cytosol, (7a) translation of mRNA, and (7b) mRNA degradation. The metabolism of the polyamine is still unclear. Reproduced with permission from [117]. Copyright 2002 Elsevier...

Transfection activity was measured in B16F10 mouse melanoma cells. The transfection efficiency of all RHB/DNA complexes was 3-30 times higher compared to PEI (25 kDa) polyplexes. Investigation on transfection activity on the... 

Abstract Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific dehvery is also briefly... 

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