Enhancing in vivo circulation and siRNA delivery with biodegradable polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) copolymers.
ABSTRACT The purpose of this study was to enhance the in vivo blood circulation time and siRNA delivery efficiency of biodegradable copolymers polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hy-PEI-g-PCL-b-PEG) by introducing high graft densities of PCL-PEG chains. SYBR(®) Gold and heparin assays indicated improved stability of siRNA/copolymer-complexes with a graft density of 5. At N/P 1, only 40% siRNA condensation was achieved with non-grafted polymer, but 95% siRNA was condensed with copolymer PEI25k-(PCL570-PEG5k)(5). Intracellular uptake studies with confocal laser scanning microscopy and flow cytometry showed that the cellular uptake was increased with graft density, and copolymer PEI25k-(PCL570-PEG5k)(5) was able to deliver siRNA much more efficiently into the cytosol than into the nucleus. The in vitro knockdown effect of siRNA/hyPEI-g-PCL-b-PEG was also significantly improved with increasing graft density, and the most potent copolymer PEI25k-(PCL570-PEG5k)(5) knocked down 84.43% of the GAPDH expression. Complexes of both the copolymers with graft density 3 and 5 circulated much longer than unmodified PEI25 kDa and free siRNA, leading to a longer elimination half-life, a slower clearance and a three- or fourfold increase of the AUC compared to free siRNA, respectively. We demonstrated that the graft density of the amphiphilic chains can enhance the siRNA delivery efficiency and blood circulation, which highlights the development of safe and efficient non-viral polymeric siRNA nanocarriers that are especially stable and provide longer circulation in vivo.
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ABSTRACT: Polyethylenimines (PEI)s are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as authophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25 kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
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ABSTRACT: Theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease that is as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive "fingerprint" of each tumor. With such information, theranostic agents can be designed to personalize treatment and minimize damage to normal tissue. Here we have developed a nanoplex platform for theranostic imaging of prostate cancer (PCa). In these proof-of-principle studies, a therapeutic nanoplex containing multimodal imaging reporters was targeted to prostate-specific membrane antigen (PSMA), which is expressed on the cell surface of castrate-resistant PCa. The nanoplex was designed to deliver small interfering RNA (siRNA) along with a prodrug enzyme to PSMA-expressing tumors. Each component of the nanoplex was carefully selected to evaluate its diagnostic aspect of PSMA imaging and its therapeutic aspects of siRNA-mediated down-regulation of a target gene and the conversion of a prodrug to cytotoxic drug, using noninvasive multimodality imaging. Studies performed using two variants of human PC3-PCa cells and tumors, one with high PSMA expression level and another with negligible expression levels, demonstrated PSMA-specific uptake. In addition, down-regulation of the selected siRNA target, choline kinase (Chk), and the conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) were also demonstrated with noninvasive imaging. The nanoplex was well-tolerated and did not induce liver or kidney toxicity or a significant immune response. The nanoplex platform described can be easily modified and applied to different cancers, receptors, and pathways to achieve theranostic imaging, as a single agent or in combination with other treatment modalities.ACS Nano 08/2012; 6(9):7752-62. · 12.03 Impact Factor
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ABSTRACT: The discovery of RNA interference (RNAi) as a naturally occurring mechanism for gene knockdown has attracted considerable attention toward the use of small interfering RNAs (siRNAs) for therapeutic purposes. Likewise, microRNAs (miRNAs) have emerged as important cellular regulators of gene expression, and their pathological underexpression allows for novel therapeutic strategies ('miRNA replacement therapy'). To address issues related to the instability, charge, and molecular weight of small RNA molecules, nanoparticle formulations have been explored for their in vivo application. Polyethylenimines (PEIs) are positively charged, linear, or branched polymers that are able to form nanoscale complexes with small RNAs, leading to RNA protection, cellular delivery, and intracellular release. This review highlights the important properties of various PEIs with regard to their use for in vivo RNA delivery. PEI modifications for increased efficacy, altered pharmacokinetic properties, improved biocompatibility and, upon covalent coupling of ligands, targeted delivery are described. An overview of various modified PEIs and a comprehensive list of representative studies using PEI-based siRNA or miRNA delivery in vivo are given. WIREs Nanomed Nanobiotechnol 2013. doi: 10.1002/wnan.1228 For further resources related to this article, please visit the WIREs website.Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 05/2013; · 5.68 Impact Factor