Association of poly I:C RNA and plasmid DNA onto MnO nanorods mediated by PAMAM

Abstract

In this study, manganese oxide (MnO) nanorods and its association with polyamidoamine dendrimer (PAMAM) and macromolecular RNA were analyzed. Because manganese is found naturally in cells and tissues and binds proteins and nucleic acids, nanomaterials derived from manganese, such as first generation MnO, may have potential as a biocompatible delivery agent for therapeutic or diagnostic biomedical applications. Nucleic acids have a powerful influence over cell processes, such as gene transcription and RNA processing; however, macromolecular RNA is particularly difficult to stabilize as a nanoparticle and to transport across cell membranes while maintaining structure and function. PAMAM is a cationic, branching dendrimer known to form strong complexes with nucleic acids and to protect them from degradation and is also considered to be a cell penetrating material. There is currently much interest in polyinosinic:polycytidylic RNA (poly I:C) because of its potent and specific immunogenic properties and as a solo or combination therapy. In order to address this potential, here, as a first step, we used PAMAM to attach poly I:C onto MnO nanorods. Morphology of the MnO nanorods was examined by field emission scanning electron microscopy (FESEM) and their composition by energy dispersive X-ray microanalysis (EDX). Evidence was generated for RNA:PAMAM:MnO nanorod binding by a gel shift assay using gel electrophoresis, a sedimentation assay using UV spectroscopy, and zeta potential shifts using dynamic laser light scattering. The data suggest that RNA was successfully attached to the MnO nanorods using PAMAM, and this suggestion was supported by direct visualization of the ternary complexes with FESEM characterizations. In order to confirm that the associations were biocompatible and taken up by cells, MTT assays were carried out to assess the metabolic activity of HeLa cells after incubation with the complexes and appropriate controls. Subsequently, we performed transfection assays using PAMAM:MnO complexes with pDNA encoding a green fluorescent protein reporter gene instead of RNA. The results suggest that the complexes had minimal impact on metabolic activity and were readily taken up by cells, and the fluorescent protein was expressed. From the evidence, we conclude that complexes of PAMAM:MnO interact with nucleic acids to form associations that are well-tolerated and readily taken up by cells.

Figure 1

Morphology of MnO nanorods. (a) SE image of unwashed MnO powder. Bar, 2 µm. (b) SE image of washed MnO nanorods dispersed on a silicon nitride substratum. Arrow indicates a large spatula; arrowheads indicate filaments. Bar, 2 µm. (c) STEM-HAADF image of washed nanorods dispersed on a silicon nitride substratum. Arrow indicates a large spatula; double arrow indicates a small spatula; arrowheads indicate filaments. Bar, 500 nm. (d) STEM-BF image of washed filaments at high magnification dispersed on a silicon nitride substratum. Bar, 200 nm. STEM, scanning transmission electron microscopy; HAADF, high-angle annular dark-field image mode; BF, bright field image mode.

Figure 2

EDX of MnO nanorods. (a) SE image of washed MnO nanorods dispersed on a silicon nitride substratum. Bar, 1 µm. (b) EDX qualitative spectrum generated from the point on a single nanorod indicated by an asterisk in (a). Energy peaks labeled Mn and O are derived from the designated nanorod; Al, specimen holder; Si and N (not labeled), support substratum. Mn, manganese; O, oxygen; Al, aluminum; Si, silicon.

Figure 3

Morphology of ethanol-washed, ternary RNA:PAMAM:MnO conjugates. (a) SE image of washed MnO nanorods embedded in the association matrix. Bar, 1 µm. (b) SE image of washed complex at high magnification ; arrow indicates discrete association matrix. Bar, 500 nm. SE, secondary electron image mode.

Figure 4

RNA:PAMAM:MnO binding indicated by shifts in zeta potential. Controls were either MnO alone or PAMAM:MnO. The samples with PAMAM, RNA and RNA:MnO showed no shift, and were removed from the figure for simplicity.

Figure 5

UV spectra of (a) Poly I:C concentration gradient showing inosine UV absorbance peak at 248 nm and cytidine peak near 270 nm; (b) MnO nanorods showing no UV absorbance. PAMAM and PAMAM:MnO UV spectra showing PAMAM absorbance peak around 210 nm and PAMAM:MnO absorbance peak circa 205 nm.

Figure 6

UV spectroscopy of sedimentation assay. (a) A decrease in RNA UV absorbance after centrifugation indicates binding to, and sedimentation with, other molecules. A greater decrease in RNA absorbance is observed when MnO is present, indicating the RNA:PAMAM is forming a complex with the nanorods. Poly = poly I:C RNA. (b) A decrease in pDNA UV absorbance after centrifugation also indicates binding to, and sedimentation with, other molecules. A greater decrease in pDNA absorbance is observed when MnO is present, indicating the pDNA:PAMAM is forming a complex with the nanorods. The UV spectrum from RNA, pDNA, and PAMAM alone are the same before and after centrifugation, therefore they were excluded from Figure 6 for simplicity.

Figure 7

Gel shift assay using 2% agarose gel electrophoresis showing that RNA migration is inhibited when combined with PAMAM and PAMAM:MnO complexes. Lanes (left to right): 1, RNA; 2, PAMAM; 3, RNA:PAMAM; 4, RNA:PAMAM; 5, RNA:PAMAM:MnO; 6, PAMAM:MnO.

Figure 8

MTT assay to determine ability of HeLa cells to reduce yellow MTT into purple formazan (a) after 24 hour incubation with MnO only, RNA:PAMAM:MnO, and pDNA:PAMAM:MnO; (b) after incubation with MnO only for 12, 24, 36, and 48 hours. For both assays, there were only minor decreases in metabolic activity, as compared to the cell only control. Error bars for both figures were plotted using standard error.

Figure 9

MTT to analyze the effects of varying concentrations of PAMAM on the metabolic activity of HeLa cells. The cells incubated with PAMAM only showed the least amount of metabolic activity, while a definite increase was observed in those cells incubated with PAMAM:MnO. The cells incubated with the RNA: PAMAM:MnO and pDNA:PAMAM:MnO complexes proved to be significantly more metabolically active than the other two samples.

Figure 10

Transfection assay using pDNA expressing GFP reporter gene. The transfection of HeLa cells was performed with the following samples: PAMAM:MnO, which served as the negative control and demonstrated no fluorescent cells and was therefore excluded from this figure; (a) pDNA:PAMAM only, which displayed only minor fluorescence; (b) Lipofectamine:pDNA, which served as the positive control; and (c) pDNA:PAMAM:MnO, which showed significantly more fluorescence than the other samples. Representative images from each group are shown here.