mPEG-PAMAM-G4 Nucleic Acid Nanocomplexes: Enhanced Stability, RNase Protection, and Activity of Splice Switching Oligomer and Poly I:C RNA

Abstract

Dendrimer chemistries have virtually exploded in recent years with increasing interest in this class of Polymers as gene delivery vehicles. An effective nucleic acid delivery vehicle must efficiently bind its cargo and form physically stable complexes. Most importantly, the nucleic acid must be protected in biological fluids and tissues, as RNA is extremely susceptible to nuclease degradation. Here, we characterized the association of nucleic acids with generation 4 PEGylated Poly(amidoamine)dendrimer (mPEG-PAMAM-G4). We investigated the formation, size, and stability over time of the nanoplexes at various N/P ratios by gel shift and dynamic light scatter spectroscopy (DLS). Further characterization of the mPEG-PAMAM-G4:nucleic acid association was provided by atomic force microscopy (AFM) and by circular dichroism (CD). Importantly, mPEG-PAMAM-G4 complexation protected RNA from treatment with RNase A, degradation in serum and various tissue homogenates. mPEG-PAMAM-G4 complexation also significantly enhanced the functional delivery of RNA in a novel engineered human melanoma cell line with splice-switching oligonucleotides (SSOs) targeting a recombinant luciferase transcript. mPEG-PAMAM-G4 triconjugates formed between gold nanoparticle (GNP) and particularly manganese oxide (MnO) nanorods, Poly IC, an anti-cancer RNA, showed enhanced cancer-killing activity by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction

“…Nevertheless, the general A-form pattern of CD spectra was maintained, suggesting that dendrimers do not seriously alter the structure of siRNAs. These results accord with previous findings showing interactions of dendrimers with RNA (Reyes- Reveles et al, 2013;Suzuki et al, 1997). Similarly, as in the EB assay, heparin disassociated siRNA from complexes formed with PAMAM and CBD, but did not cause so pronounced siRNA release form CPD/siRNA dendriplexes (Fig.…”

Rapid Exchange Between Free and Bound States in RNA–Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release

“…For example, as the closest comparison, CD spectra of polyplexes of PEGylated PAMAM G4 and a double stranded RNA showed little changes at N:P of 5 and above whereas polyplexes with a 20-nucleotide antisense DNA showed significant spectral changes at same N:P ratios, suggesting conformational changes in the antisense DNA. 26 …”

Dendrimers as Drug Nanocarriers: The Future of Gene Therapy and Targeted Therapies in Cancer

“…cell membranes causing their disruption [101,102]. This disadvantage can be diminishing by surface modification with different targeting or shielding moieties providing with not only low toxicity but also enhance the cell uptake and specific accumulation of nucleic acid molecules inside cells [103][104][105][106]. For example, novel targeted nanoparticle system consisting of FLT3 ligandconjugated PAMAM G7 encapsulating a pivotal tumor suppressor and negative regulator of FLT3 miRNA-miR-150, was developed by Jiang et al [107] to treat FLT3-overexpressing acute myeloid leukemia (AML), a leukemia associated with unfavorable prognosis.…”