Acetylation of PAMAM dendrimers for cellular delivery of siRNA

Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ, USA.
BMC Biotechnology (Impact Factor: 2.03). 05/2009; 9(1):38. DOI: 10.1186/1472-6750-9-38
Source: PubMed


The advancement of gene silencing via RNA interference is limited by the lack of effective short interfering RNA (siRNA) delivery vectors. Rational design of polymeric carriers has been complicated by the fact that most chemical modifications affect multiple aspects of the delivery process. In this work, the extent of primary amine acetylation of generation 5 poly(amidoamine) (PAMAM) dendrimers was studied as a modification for the delivery of siRNA to U87 malignant glioma cells.
PAMAM dendrimers were reacted with acetic anhydride to obtain controlled extents of primary amine acetylation. Acetylated dendrimers were complexed with siRNA, and physical properties of the complexes were studied. Dendrimers with up to 60% of primary amines acetylated formed approximately 200 nm complexes with siRNA. Increasing amine acetylation resulted in reduced polymer cytotoxicity to U87 cells, as well as enhanced dissociation of dendrimer/siRNA complexes. Acetylation of dendrimers reduced the cellular delivery of siRNA which correlated with a reduction in the buffering capacity of dendrimers upon amine acetylation. Confocal microscopy confirmed that escape from endosomes is a major barrier to siRNA delivery in this system.
Primary amine acetylation of PAMAM dendrimers reduced their cytotoxicity to U87 cells, and promoted the release of siRNA from dendrimer/siRNA complexes. A modest fraction (approximately 20%) of primary amines of PAMAM can be modified while maintaining the siRNA delivery efficiency of unmodified PAMAM, but higher degrees of amine neutralization reduced the gene silencing efficiency of PAMAM/siRNA delivery vectors.

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Available from: Kathryn E Uhrich, Jan 03, 2014
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    • "Surface acetylation of PAMAM dendrimers reduced their cytotoxicity to U87 cells and promoted the release of siRNA from dendrimer/siRNA complexes. Twenty percent acetylation of primary amines of PAMAM could maintain the siRNA delivery efficiency of unmodified PAMAM, but higher degrees of amine neutralization decreased the gene silencing efficiency of PAMAM dendrimer vectors [23]. These researchers also modified G5 PAMAM dendrimer with cyclic RGD targeting peptides, studied the effect of RGD density on cell-free binding affinities and cellular internalization, and evaluated their siRNA delivery ability [24, 25]. "
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    ABSTRACT: RNA interference (RNAi) was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA) are used to specifically downregulate the expression of the targeted gene, known as the term "gene silencing." One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc.) of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system.
    Full-text · Article · Oct 2013 · The Scientific World Journal
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    • "The rationale to undertake the present investigation was to address following issues, viz., toxicity, transfection efficiency and surface charge of the modified polymers. It was speculated that grafting of EDAC onto bPEI would (i) decrease the density of the primary amines, which are responsible for toxicity in cationic polymers [32] [33] [34] [35] [36], (ii) improve the interactions of the polymers with cell membranes, as alkyl chains in EDAC would impart hydrophobicity to the polymers, (iii) facilitate the uptake and internalization of the pDNA complexes, and (iv) weaken the vector/pDNA complex, which would result in the easy disassembly of the complexes leading to marked improvement in the transfection efficiency. Keeping these points into consideration, we conjugated N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide onto bPEI to yield trisubstituted guanidinyl-modified PEI polymers, which were then evaluated for their transfection efficiency, cell viability and buffering capacity. "
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    ABSTRACT: Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treatment for various acquired and inherited diseases. Here, we report on the synthesis and characterization of N-ethyl-N'-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine (sGP) polymers and investigation of their capability to carry DNA and siRNA in vitro. Zinc triflate-mediated activation of primary amines of branched polyethylenimine (bPEI) followed by reaction with varying amounts of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC) resulted in the generation of a small series of trisubstituted guanidinyl-modified polyethylenimine polymers. Determination of primary amines on modified polymers by TNBS assay revealed 62-84% of the attempted conjugation of EDAC onto bPEI. These modified polymers were shown to condense plasmid DNA and retard its mobility on 0.8% agarose gel. Further, these polymers were evaluated for their capability to carry pDNA into the cells by performing transfection assay on various mammalian cells. All the modified polymer/pDNA complexes exhibited significantly higher levels of gene expression with one of the complexes, sGP3/pDNA complex, displayed ~1.45 to 3.0 orders of magnitude higher transfection efficiency than that observed in the native bPEI and the commercial transfection reagent, Lipofectamine™. The efficacy of sGP3 polymer was further assessed by siRNA delivery, which resulted in ~81% suppression of the target gene. In conclusion, these studies demonstrate the potential of these substituted guanidinyl-modified PEIs as efficient gene delivery vectors.
    Full-text · Article · Apr 2013 · Colloids and surfaces B: Biointerfaces
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    • "Cationic dendrimers were reported to destroy mitochondrial membranes, leading to the generation of reactive oxygen species causing oxidative stress, DNA damage, and apoptosis.35,36 The removal of surface amine groups by acetylation or PEGylation can effectively decrease the cytotoxicity of PAMAM and PPI dendrimers,23,37 indicating that surface charge plays an important role in the cytotoxicity of both dendrimers.34 Generation 3 PAMAM and generation 4 PPI have equivalent numbers of primary amine groups (pKa about 10) which are fully protonated under physiological conditions (pH about 7.4). "
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    ABSTRACT: Polyamidoamine (PAMAM) and polypropylenimine (PPI) dendrimers are the commercially available and most widely used dendrimers in pharmaceutical sciences and biomedical engineering. In the present study, the loading and release behaviors of generation 3 PAMAM and generation 4 PPI dendrimers with the same amount of surface amine groups (32 per dendrimer) were compared using phenylbutazone as a model drug. The dendrimer-phenylbutazone complexes were characterized by (1)H nuclear magnetic resonance and nuclear Overhauser effect techniques, and the cytotoxicity of each dendrimer was evaluated. Aqueous solubility results suggest that the generation 3 PAMAM dendrimer has a much higher loading ability towards phenylbutazone in comparison with the generation 4 PPI dendrimer at high phenylbutazone-dendrimer feeding ratios. Drug release was much slower from the generation 3 PAMAM matrix than from the generation 4 PPI dendrimer. In addition, the generation 3 PAMAM dendrimer is at least 50-fold less toxic than generation 4 PPI dendrimer on MCF-7 and A549 cell lines. Although the nuclear Overhauser effect nuclear magnetic resonance results reveal that the generation 4 PPI dendrimer with a more hydrophobic interior encapsulates more phenylbutazone, the PPI dendrimer-phenylbutazone inclusion is not stable in aqueous solution, which poses a great challenge during drug development.
    Full-text · Article · Dec 2011 · International Journal of Nanomedicine
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