PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles.
TL;DR: In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.
About: This article is published in European Journal of Cell Biology.The article was published on 2004-01-01. It has received 684 citations till now. The article focuses on the topics: Gene delivery & PEGylation.
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TL;DR: The history of the development of PEGylated nanoparticle formulations for systemic administration is described, including how factors such as PEG molecular weight, PEG surface density, nanoparticle core properties, and repeated administration impact circulation time.
2,465 citations
Cites background from "PEGylation significantly affects ce..."
...PEGylation has been shown to sterically interfere with membrane fusion of lipid-based NPs, resulting in the poor endosome escape [90, 196]....
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...uptake of small (≤100 nm) PEGylated polymeric gene vectors was found to be indistinguishable from that of non-PEGylated cationic polymeric gene vectors [90, 171]....
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...For example, IgM production against PEG was decreased when doxorubicinloaded liposomes were administered compared to empty PEGylated liposomes [90, 98]....
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...Laverman and Dams demonstrated complement activation and a dramatic reduction in halflife after the second injection of PEGylated liposomes, which they later showed involved hepatosplenic macrophages [89, 90]....
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TL;DR: The biological barriers to gene delivery in vivo are introduced and recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems are discussed, some of which are currently undergoing testing in clinical trials.
Abstract: Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches
2,460 citations
TL;DR: With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-Delivery systems will become an important tool for human gene therapy.
Abstract: The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure–function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
2,361 citations
TL;DR: It is anticipated that precisely engineered nanoparticles will emerge as the next-generation platform for cancer therapy and many other biomedical applications.
Abstract: In medicine, nanotechnology has sparked a rapidly growing interest as it promises to solve a number of issues associated with conventional therapeutic agents, including their poor water solubility (at least, for most anticancer drugs), lack of targeting capability, nonspecific distribution, systemic toxicity, and low therapeutic index. Over the past several decades, remarkable progress has been made in the development and application of engineered nanoparticles to treat cancer more effectively. For example, therapeutic agents have been integrated with nanoparticles engineered with optimal sizes, shapes, and surface properties to increase their solubility, prolong their circulation half-life, improve their biodistribution, and reduce their immunogenicity. Nanoparticles and their payloads have also been favorably delivered into tumors by taking advantage of the pathophysiological conditions, such as the enhanced permeability and retention effect, and the spatial variations in the pH value. Additionally, targeting ligands (e.g., small organic molecules, peptides, antibodies, and nucleic acids) have been added to the surface of nanoparticles to specifically target cancerous cells through selective binding to the receptors overexpressed on their surface. Furthermore, it has been demonstrated that multiple types of therapeutic drugs and/or diagnostic agents (e.g., contrast agents) could be delivered through the same carrier to enable combination therapy with a potential to overcome multidrug resistance, and real-time readout on the treatment efficacy. It is anticipated that precisely engineered nanoparticles will emerge as the next-generation platform for cancer therapy and many other biomedical applications.
1,603 citations
TL;DR: An introduction to the biological challenges that siRNA delivery materials aim to overcome is provided, as well as a discussion of the way that the most effective and clinically advanced classes of si RNA delivery systems are designed to surmount these challenges.
Abstract: RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges. The systems that we discuss are diverse in their approaches to the delivery problem, and provide valuable insight to guide the design of future siRNA delivery materials.
1,489 citations
References
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TL;DR: The new stereological methods for correct and efficient sampling and sizing of cells and other particles are reviewed and practical examples of applications to a wide range of histological entities are illustrated.
Abstract: The new stereological methods for correct and efficient sampling and sizing of cells and other particles are reviewed. There is a hierarchy of methods starting from the simplest where even the microscopic magnification may be unknown to the most complex where typically both section thickness and the magnification must be known. Optical sections in suitably modified microscopes can be used to improve the ease and speed with which even the most demanding of these methods are performed. The methods are illustrated by practical examples of applications to a wide range of histological entities including synapses, neurons and cancer cells, glomerular corpuscles and ovarian follicles.
2,471 citations
TL;DR: In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice, while PEGylation of the complexes mediated reporter gene transfer to the tumor without significant toxicity.
Abstract: We investigated the in vitro and in vivo properties of DNA/transferrin-polyethylenimine (800 kDa) complexes before and after covalent coupling of poly(ethylene glycol) (PEG). Upon incubation with plasma, the positively charged non-PEGylated DNA complexes form aggregates. Plasma proteins such as IgM, fibrinogen, fibronectin and complement C3 were found to bind to non-PEGylated DNA complexes. At DNA concentrations relevant for in vivo gene delivery a strong aggregation of erythrocytes was also observed. PEGylation of the complexes strongly reduces plasma protein binding and erythrocyte aggregation. Furthermore, PEGylated complex size was stabilized and had a reduced surface charge. Prolonged circulation in the blood of the PEGylated complexes was also observed when injected intravenously. In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice. In contrast, PEGylated complexes mediated reporter gene transfer to the tumor without significant toxicity.
1,188 citations
"PEGylation significantly affects ce..." refers background or methods in this paper
...bPEI polyplexes were PEGylated through grafting of a reactive PEGmolecule to free amine groups (Ogris et al., 1999) while CDP polyplexes were PEGylated through formation of inclusion complexes between a PEG-containing conjugate and the cyclodextrin moieties of the polymer (Pun andDavis, 2002) (Fig....
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...(a) bPEI polyplexes were PEGylated through grafting of a succinimidyl propionate (SPA)-PEG5000 conjugate to free amines of bPEI (Ogris et al., 1999)....
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..., 2003) or after (Ogris et al., 1999; Pun and Davis, 2002) vector complexation with the nucleic acid....
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...bPEI polyplexes were PEGylated by addition of 4 g methoxy-PEG5000-succinimidyl propionate (Nektar, San Carlos, CA; 20 mg/ml in dH2O) per microgram pDNA (Ogris et al., 1999)....
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TL;DR: The results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.
1,051 citations
"PEGylation significantly affects ce..." refers background in this paper
...PEGylation and/or reaction to primary amines on the surface of bPEI polyplexes may impair the TMproton sponge∫ effect (Behr, 1994; Sonawane et al., 2003), a significant factor in the effectiveness of bPEI as a delivery vector....
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TL;DR: The results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding.
771 citations
"PEGylation significantly affects ce..." refers background in this paper
...Diffusion of pDNA in cytoplasm is severely restricted (Lukacs et al., 2000; Lechardeur and Lukacs, 2002)....
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TL;DR: Application of the small particles in more concentrated form and over extended periods of time improves transfection activity, and analysis of gene expression at the cellular level revealed that the differences in overall gene expression largely result from different intensities per expressing cell, while the difference in the percentage of expressing cells is less substantial.
Abstract: Under physiological salt concentration, plasmid DNA complexed with transferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) forms huge (up to > 1000 nm) aggregates, unless the individual components are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio. At low ionic strengths, however, small particles with an average size of 40 nm are formed over a broad range of N/P ratios. Interestingly, in transfection experiments these small particles result in a 10-fold (B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduced luciferase gene expression efficiency in comparison to the large complexes formed in physiological salt solutions. Limited transport of the small particles to the cell surfaces is one possible reason for this effect. Application of the small particles in more concentrated form and over extended periods of time improves transfection activity. Reduced intracellular release may be another explanation for the decreased transfection efficiency; incubation with chloroquine or incorporation of the endosomolytic peptide INF5 into the small complexes enhances gene expression approximately 10-fold. Analysis of gene expression at the cellular level using a green fluorescence protein reporter gene and flow cytometry revealed that the differences in overall gene expression largely result from different intensities per expressing cell, while the difference in the percentage of expressing cells is less substantial.
594 citations
"PEGylation significantly affects ce..." refers background in this paper
...067 mg/ml in 20 mM HEPES, 5% glucose) (Ogris et al., 1998) and CDP (1....
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...Thus, it is likely that unmodified polyplexes transfect adherent cells more successfully than suspension cells in vitro because the aggregates of unmodified polyplexes can simply sediment onto adherent cells (Boussif et al., 1996; Ogris et al., 1998; Zou et al., 2000), a scenario far removed from that which polyplexes would encounter in vivo....
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