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Elucidating the RNA Nano-bio Interface: Mechanisms of AntiCancer Poly I:C RNA and Zinc Oxide Nanoparticle Interaction Meghana Ramani, Tuyen Duong Thanh Nguyen, Santosh Aryal, Kartik C. Ghosh, and Robert K. DeLong J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b02954 • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on June 5, 2017
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Elucidating the RNA Nano-bio Interface: Mechanisms of Anti-Cancer Poly I:C RNA and Zinc Oxide Nanoparticle Interaction
Meghana Ramania,b, Tuyen Duong Thanh Nguyena,c, Santosh Aryala,c, Kartik C. Ghoshd, Robert K DeLonga,b* a
Nanotechnology Innovation Center of Kansas State, bDepartment of Anatomy and Physiology,
c
Department of Chemistry, Kansas State University, Manhattan, Kansas 66502, USA.
d
Department of Physics, Astronomy and Materials Science, Missouri State University,
Springfield, MO 65897, USA.
*Corresponding author email id:
[email protected] 1 ACS Paragon Plus Environment
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Abstract: Understanding the RNA nano-bio interface is critical to advance RNA based therapeutics. As a clinically relevant RNA polyinosinic:cytidilic acid (poly I:C) is perhaps the best studied in clinical trials and is now considered an anti-metastatic RNA targeting agent. So too, zinc oxide nanoparticle (ZnO-NP) has well known anti-cancer activity. In this work, we explore the RNAnano-bio interface of poly I:C, its mononucleotides and homopolymers with ZnO NP by UV, fluorescence and Fourier Transform Infrared (FT-IR) spectroscopies. The loading method and ionic concentration (1.0M Na+) were optimized for greater physical association of RNA with the NP, providing greater payload (150 µg/mg NP). The physical parameters of RNA nano-bio interaction, denoting the degree of association, was quantified by modified Stern-Volmer equations (Kb = 329.6 g-1L). This interface was further studied by two dimensional fluorescence difference spectroscopy (2D-FDS), where greater interaction was indicated by considerable quenching of the fluorescent “hot-spot”. The mononucleotides and homopolymers of inosine had higher payload, binding constants and 2D-FDS quenching, implicating the purine ring in ZnOpIC interaction due to its greater electron density. X-ray photoelectron spectroscopy indicates the presence of RNA on the NP surface. Infrared spectral studies confirm that pIC interacts directly through inosine with the positive surface of ZnO via the carboxyl group and aromatic ring, and indirectly via the phosphate group.
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Introduction The anti-metastatic RNA targeting agent polyinosinic:cytidilic acid (poly I:C/pIC), is currently under clinical trials for cancer treatment as a combinational chemo- and immunotherapy.1–4 With the emergence of RNA nanotechnology, researchers are investigating the biomedical potential of poly I:C RNA loaded organic and inorganic nanoparticles (NP) for enhanced therapy.5–9 Zinc oxide (ZnO) is a biologically relevant NP due to its immunogenicity and preferential cancer cell selectivity, and is being studied as a potential anti-cancer agent10–12. The isoelectric charge of these NP (9–10) renders them with strong positive surface charge under physiological conditions13. In nucleic acid therapies, such cationic NP offer advantage of being able to directly bind nucleic acids by electrostatic interactions, overcoming the requirement of surface functionalization14. Further, a detailed investigation of the nature of these molecular interactions is lacking in literature and the kinematics yet to be determined. Most importantly, RNA interaction to ZnO NP is poorly understood. Such a combination primarily generates a largely unknown and complicated ‘nano-bio interface’ comprising various physicochemical interactions, kinetics and thermodynamical exchanges.15–17 This interface fundamentally affects the structure, function, stability and ultimately the bioactivity of these complexes. Due to these reasons, there is a growing interest in the study of the nature and strength of interactions of RNA and its components with NP. An understanding of this interface is essential for the advancement of such RNA-NP based therapeutics as they offer prospects of tuning the NP surface or the RNA nucleotides for improved binding, higher loading, stability and functional delivery. The strength of RNA-NP interaction decides the fate of these complexes in cellular environment. Fluorescence spectroscopy is a commonly used tool for biomolecule-NP interaction studies. Various adsorption models like the Frisch–Simha–Eirich model, Langmuir 3 ACS Paragon Plus Environment
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isotherm, Hills equation and the Stern-Volmer kinetics have been used to evaluate the adsorption kinetics.17–21 We recently compared the BSAI model based on Langmuir isotherm and SternVolmer kinetics to determine the binding constants and observed that both the techniques provided similar values. 14 Here, we thus employ the most commonly used Stern-Volmer kinetics using the inherent NP fluorescence, to determine the NP-RNA binding constants which provides a reliable quantitative information of the nano-bio interaction. 14,22–24 However, nucleic acids present a variety of different functional groups to the surface of the NP which could potentially contribute to stabilize the nano-bio interaction. Most of the work to date has been done on understanding the DNA-NP interaction using spectroscopic tools such as FTIR, XPS, circular dichroism, Raman, and NMR.21,25–30 A few computational studies indicate a direct interaction of the ZnO via the hexagonal ring of nucleobases or with the ring nitrogen having a lone pair of electron.15,31,32 We have reported direct ring-mediated binding of adenine in ATP RNA to ZnO NP by Raman spectroscopy.26 Here, we confirm the binding of poly I:C RNA to the surface of ZnO NP by XPS by monitoring the Zn 2p, N 1s, and O 1s peaks of ZnO, RNA and the nanoconjugates. To understand the mechanism of interaction, FT-IR is a very useful tool for qualitative and quantitative analyses of the structure and interaction of nucleic acids33. Using FT-IR, Arora et al have shown strong interaction of ZnO NP with ribose nucleotides mainly via the nucleobases, lone pair of nitrogen atom as well as the two available protons of the phosphate groups.34 Computational studies along with FTIR and FT-Raman analyses has shown that ZnO mainly interaction with guanine via the ring N1-atom through physisorption.35 Similar interactions with oxygen and nitrogen sites of DNA with other semiconducting nanostructures is investigated by Wang et al. 36 ZnO surface has been reported to be a good binding substrate for cytidine monophosphate, a ribonucleotide of pIC.34 However, the interaction of inosine and pIC with ZnO NP is not yet studied. 4 ACS Paragon Plus Environment
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The design of our study was to compare the binding of mononucleotides, homopolymers and the dsRNA poly I:C to ZnO NP. Though nucleic acid-NP interactions have been studied vastly, this manuscript extends prior basic work to therapeutically relevant RNA-NP complexes, leading to innovative approaches to increase the RNA payload with enhanced binding and stability for better therapeutics. In the present work, we systematically study the interactions of pIC RNA, the mononucleotides of inosine and cytidine and their homopolymers with ZnO NP by spectroscopic techniques. Surface analyses of ZnO NP, RNA and the complexes by XPS confirm the binding of RNA to ZnO surface. The shifts in FT-IR vibrations of pIC and fluorescence quenching of ZnO, together discriminate the interaction mechanism. It is conclusive from these multiple studies that inosine is what drives this nano-bio interaction. These gateway results provide promising ramifications in the field of RNA nano-bio interaction and more broadly RNA binding, delivery and therapeutics by ZnO NP. Materials and Methods ZnO-NP (size
