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 anticancer RNA, showed enhanced cancer-killing activity by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

Figure 1

Molecular structure of mPEG-PAMAM-G4.

Figure 2

(A) Gel retardation assay of mPEG-PAMAM-G4/RNA, (B) Bromophenol blue color and direction shift as a function of mPEG-PAMAM-G4 interaction. N/P ratio: (1) 10, (2) 5, (3) 1, (4) 0.4, (5) 0.2, (D) mPEG-PAMAM-G4 only, (C) Control RNA only.

Figure 3

Representative size measurements using DLS. (A) mPEG-PAMAM-G4 is complexed with RNA at various N/P ratios 0.2, 0.4, 1, 5, 10. (B) A representative average size peak of N/P ratio 10. Data is depicted as Intensity of size in nm. Inset table depicts average diameters using Number, Intensity, and Volume parameters. These are also shown in nm, and the prominent peak was chosen as the representative size.

Figure 4

AFM images of the mPEG-PAMAM-G4:DNA nanocomplexes. (A) Plasmid DNA only (B) mPEG-PAMAM-G4complexed with plasmid DNA at a 1:1 ratio.

Figure 5

Circular dichroism spectrum of RNA (A) or DNA (B) +/− mPEG-PAMAM-G4 at 5:1 or 10:1 N/P ratios. Y axis is F (mdeg) and x axis wavelength in nm.

Figure 6

Size stability over time. DLS was utilized to analyze aggregation of nanoplexes using N/P ratios over a 72-hour time period. Experiments were done in triplicate and repeated twice and the error bars represent the standard deviation.

Figure 7

RNA protection in fetal bovine serum and RNase A. mPEG-PAMAM-G4 was complexed with RNA at N/P ratios of 10 (Row A), 5 (Row B), 1 (Row C), 0.4 (Row D), and 0.2 (Row E). Lanes 1, 3, 5, 7, and 9 have mPEG-PAMAM-G4, and lanes 2, 4, 6, 8, and 10 lack mPEG-PAMAM-G4. FBS was added: (lanes 1–2) 2 µL, (lanes 3–4) 4 µL, (lanes 5–6) 6 µL, (lanes 7–8) 8 µL, (lanes 9–10) 10 µL. Lane 11 is a blank. Lanes 12 and 13 show FBS-less N/P and RNA-only controls, respectively. Row F: mPEG-PAMAM-G4 was complexed with RNA at N/P of 5:1. RNase A (0.6 mg/mL) were added: (Lanes 1–2) 3 µL, (3–4) 5 µL, (5–6) 10 µL, (7–8) 15 µL, (9–10) 20 µL, (11–12) 0 µL.

Figure 8

RNA protection against heart (lanes 1–2), lung (lanes 3–4) and eye (lanes 5–6) mouse tissue homogenates. mPEG-PAMAM-G4 was complexed with RNA at an N/P ratio of 10 (lanes 1, 3, and 5). Lanes 2, 4, and 6 have RNA only. Lane 7 is blank. Lanes 8 and 9 show tissue-less N/P and RNA-only controls, respectively. All samples were incubated at 37°C for 72 hours. Aliquots were removed at: 2 hours (Row A), 12 hours (Row B), 18 hours (Row C), 24 hours (Row D), 36 hours (Row E), 48 hours (Row F), 60 hours (Row G), and 72 hours (Row H).

Figure 9

Enhanced splice-switching in an engineered human cancer cell line (A375 pLuc) enhances SSO delivery. SSO in nanocomplexes with mPEG-PAMAM-G4 and lipofectamine (10:1/Lipo or 5:1/Lipo) provided more splicing correction and hence cell luminescence, in comparison to Lipofectamine (Lipo) or mPEG-PAMAM-G4 controls at either 10:1 or 5:1 N/P ratios. RLU: Relative light units. P-value of <.0001***. P-value of <.005**, indicates significance of SSO delivery for 10:1/Lipo or 5:1/Lipo in comparison to Lipo only control.

Figure 10

Enhanced cancer-killing effects of Poly IC complexed with mPEG-PAMAM-G4 and various nanomaterials. A375 human melanoma cells were treated with an anti-cancer RNA either complexed with dendrimer at various ratios, or conjugated to various nanomaterials using the mPEG-PAMAM-G4 as a stabilizing intermediate, to enhance the cancer-killing effects observed with Poly IC alone. Cells were treated with either Poly IC (100 nM), mPEG-PAMAM-G4 alone (9.5 µg/mL), N/P 1:1 and 5:1 ratios (Poly IC at 100nM, dendrimer at 9.5 µg/mL and 30 µg/mL), or nanoconjugates formed between MnO nanoparticles (0.025 µg/µL) or GNP (5% v/v of 3X stock) and N/P 1:1, +/− Lipofectamine, With Lipofectamine, the mPEG-PAMAM-G4 increased the cancer cell killing effects of Poly IC with increasing N/P ratio. MnO- and GNP-conjugated complexes demonstrated an increased potency of the 1:1 N/P ratio that was more effective than even the N/P 5:1 complex.