Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms.
Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical "proton sponge mechanism". PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.
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- "A caveolae-mediated mode of endocytosis has been suggested to traffic polymer–DNA complexes to the Golgi network  . In order to check whether the R 16 polyplexes localize to the Golgi, we performed immunostaining of giantin, a Golgi marker, after incubation of CHO-K1 cells with R 16 polyplexes for 1 h. "
ABSTRACT: Arginine-rich peptides have been used extensively as efficient cellular transporters. However, gene delivery with such peptides requires development of strategies to improve their efficiency. We had earlier demonstrated that addition of small amounts of exogenous glycosaminoglycans (GAGs) like heparan sulfate or chondroitin sulfate to different arginine-rich peptide-DNA complexes (polyplexes) led to an increase in their gene delivery efficiency. This was possibly due to the formation of a 'GAG coat' on the polyplex surface through electrostatic interactions which improved their extracellular stability and subsequent cellular entry. In this report, we have attempted to elucidate the differences in intracellular processing of the chondroitin sulfate (CS)-coated polyplexes in comparison to the native polyplexes by using a combination of endocytic inhibitors and co-localization with endosomal markers in various cell lines. We observed that both the native and CS-coated polyplexes are internalized by multiple endocytic pathways although in some cell lines, the coated polyplexes are taken up primarily by caveolae mediated endocytosis. In addition, the CS-coat improves the endosomal escape of the polyplexes as compared to the native polyplexes. Interestingly, during these intracellular events, exogenous CS is retained with the polyplexes until their accumulation near the nucleus. Thus we show for the first time that exogenous GAGs in small amounts improve intracellular routing and nuclear accumulation of arginine-based polyplexes. Therefore, addition of exogenous GAGs is a promising strategy to enhance the transfection efficiency of cationic arginine-rich peptides in multiple cell types. Copyright © 2014 Elsevier B.V. All rights reserved.Biochimica et Biophysica Acta (BBA) - Biomembranes 01/2015; 1848(4). DOI:10.1016/j.bbamem.2015.01.012 · 3.84 Impact Factor
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- "Transfection efficiencies were decreased in the presence of a small-molecule inhibitor for caveolae-mediated endocytosis, a pathway that circumvents the acidification process necessary for endosomal buffering. These results suggest that transfection may be more productive when HPMA-oligolysine polyplexes are routed via a non-acidifying endocytic route, similarly to other polycation systems [60,79,80]. Therefore, understanding the uptake pathway of various polymer formulations can aid in the rationale design of improved materials. "
ABSTRACT: The complex nature of in vivo gene transfer establishes the need for multifunctional delivery vectors capable of meeting these challenges. An additional consideration for clinical translation of synthetic delivery formulations is reproducibility and scale-up of materials. In this review, we summarize our work over the last five years in developing a modular approach for synthesizing peptide-based polymers. In these materials, bioactive peptides that address various barriers to gene delivery are copolymerized with a hydrophilic backbone of N-(2-hydroxypropyl)methacrylamide (HPMA) using reversible-addition fragmentation chain-transfer (RAFT) polymerization. We demonstrate that this synthetic approach results in well-defined, narrowly-disperse polymers with controllable composition and molecular weight. To date, we have investigated the effectiveness of various bioactive peptides for DNA condensation, endosomal escape, cell targeting, and degradability on gene transfer, as well as the impact of multivalency and polymer architecture on peptide bioactivity.Journal of Biological Engineering 10/2013; 7(1):25. DOI:10.1186/1754-1611-7-25 · 2.48 Impact Factor
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ABSTRACT: The work employs carbon dot (CD) which has been emerging as a fluorescent nanomaterial with excellent biocompatibility and perceived as a promising alternative to quantum dot (QD), to monitor the association/dissociation of polymeric carrier/plasmid DNA (pDNA) complex during transfection. To shed light on the underlying post-endosomal events and provide the insight to design rational and efficient gene delivery vector, the adopted strategy exploited the quenching of the fluorescence of CD by Au nanoparticles. The surface of CD and Au was modified with highly cationic polymer, polyethylenimine (PEI) and subsequent treatment with non-labeled pDNA gave rise to quenched delivery complex. High salt concentration triggered the dissociation of the complex with accompanied fluorescence recovery arising due to the increase in distance between CD and Au. The studies revealed the potential of the developed CD-PEI/Au-PEI/pDNA ternary nano-assembly as a highly efficient hybrid transfecting agent with high cell viability under the optimum condition. The changes occurred at the intracellular level during transfection especially post-endosomal step were monitored by fluorescence measurement using fluorescence microscope. This nano-assembly system was found to be very effective at monitoring the carrier/pDNA dissociation in a non-labeled manner, thus provides efficient strategy to study the mechanistic aspect of polymer-mediated pDNA delivery.Biomaterials 06/2013; 34(29). DOI:10.1016/j.biomaterials.2013.05.072 · 8.56 Impact Factor
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