RNA Loading on Nano-Structured Hyperbranched β-Cyclodextrin.

Original Article RNA Loading on Nano-Structured Hyperbranched β-Cyclodextrin Sorina Hirbod 1, Shohreh Nafisi 1,2*, Howard I Maibach 2* 1. Department of Chemistry, Central Tehran Branch, Islamic Azad University, Tehran, Iran 2. Department of Dermatology, University of California, San Francisco, CA, USA Abstract * Corresponding authors: Shohreh Nafisi, Ph.D., Department of Chemistry, Central Tehran Branch, Islamic Azad University, Tehran, Iran Howard I Maibach, Ph.D., Department of Dermatology, University of California, San Francisco, USA Tel: +98 21 22426554, +1 415 7535304 E-mail: , Received: 18 Nov 2016 Accepted: 13 Feb 2017 Background: β-Cyclodextrin functionalized hyper-branched polyglycerol (HBCD: βCD-g-PG), a biocompatible polymer, has recently been proposed for delivery of poorly water soluble compounds. Methods: The present study examines the interaction of HBCD with RNA, utilizing a constant concentration of RNA and different HBCD/RNA ratios of 1/16 to 1/1, at physiological condition in an aqueous solution. Circular Dichroism (CD), UV-visible, FTIR spectroscopic methods, zeta potential and Dynamic Light Scattering (DLS) were used to analyze the particle formation, particle charge, particle size, aggregation, RNA conformation, binding constant and mode, and the effect of polymer complexation on RNA stability. Results: The results indicate that the interaction of RNA with HBCD leads to the formation of a linear dendritic supramolecule biopolymer with an overall binding constant of KHBCD/RNA= 1.25 × 103. Conclusion: The small sized synthesized polymer can be considered as an appropriate system for preventing RNA aggregation and protecting the gene by host-guest interaction. Avicenna J Med Biotech 2018; 10(1): 15-21 Keywords: Aggregation, Gene delivery, Particle size, Polymer Introduction Recently, a variety of macromolecule structures such as Cyclodextrins (CDs) and dendrimers have been examined for the formation of stable complexes with genes 1-6 . CDs, cyclic oligosaccharides are well-known structures in supramolecular polymers in which the monomers are held together by reversible and highly directional non-covalent bonds 7,8. These biocompatible and nontoxic compounds 9 possess a cage-like supramolecular structure, with hydrophilic outer surfaces and lipophilic inner cavities. Various hydrophobic drugs can be embedded in their hydrophobic cavities via non-covalent interactions 10-14. However, CDs can only cause a minor enhancement in the solubility of hydrophobic drugs 15,16. Recent advances have been focused on functionalizing CDs to obtain small sized macromolecules with higher free cavities and water solubility 17-19. Polyglycerol dendron functionalized cyclodextrins (β-CD-gPG) are novel carriers which can be synthesized by controlled anionic polymerization of glycidol. The Hyperbranched Cyclodextrins (HBCD) are small sized, well-defined polymers with higher water solubility, oxidative stability and biocompability. They exhibit high potential of encapsulating hydrophobic guest mol- ecules due to the molecules binding to interior or exterior part of their structure 20-22. Tao et al studied the host-guest interaction between β-CD-g-PG with long alkyl chain adamantines 23. The incorporation of insulin into HBCD polymers significantly caused enhanced drug absorbance across the nasal barrier and decreased blood glucose concentration 24 . Paclitaxel was effectively encapsulated by HBCD for targeted drug delivery 25. Recently, β-cyclodextrine-polyethyleneimine polymers (PEI-CDs) were examined for in vitro transport of miRNA(microRNA) and siRNA (small interfering RNA) 6,26. CDs have also been studied as topical drug delivery systems. Application of CDs derivatives in transdermal drug delivery has improved drug release/permeation, decreased druginduced local irritationand hasoptimized systemic and local dermal drug delivery. Recently, Loftsson et al reviewed the role of cyclodextrins on drug delivery through biological membranes 27. Although several experiments report the interaction of CDs with guest molecules 28,29, there is no precise information on the molecular aspects of interaction between HBCD and RNA in aqueous solution. Thus, HBCD interaction with RNA was studied in aqueous Copyright © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved. Vol. 10, No. 1, January-March 2018 15 RNA Delivery solutions with HBCD/RNA molar ratios of 1/16 to1/1 at pH=6.5-7.5 utilizing CD, UV, and FTIR measurements. The complexes were analyzed to study the particles size and charge parameters. The structural analyses of RNA secondary structures, binding site of HBCD to RNA, and HBCD-RNA binding constant were provided by spectroscopic results. Materials and Methods Materials Sodium methoxide, methanol and acetone were purchased from Merck. β-cyclodextrin, glycidol and Baker’syeast RNA from Sigma Chemical (St. Louis, MO) were employed without further purification. The absorbance band at 280 nm was utilized to check the protein content of RNA solutions. The A260/A280 ratio for RNA was 2.10, indicating that the RNA samples were nearly free from protein 30. Other chemicals were of reagent grade and used without further purification. Synthesis of HBCD polymer HBCD (β-CD-g-PG) was synthesized by anionic ring opening multi branching polymerization method 20 . Briefly, β-CD (0.5 g) was deprotonated by dissolving sodium methoxide (14.4 mmol) in dried methanol. Mixture was stirred at room temperature using a polymerization ampule equipped with a magnetic stirrer for 1 hr. Methanol was vaporized via vacuum oven at 60°C for 2 hr. Glycidol (92 mmol) was added to deprotonated β-CD and temperature was gradually increased up to 120°C. The mixture was stirred at 120°C for 12 hr and allowed to cool. It was dissolved in pure methanol, and precipitated upon addition of aceton. It was neutralized by filtration over cation-exchange resin. The precipitate was dried via vacuum oven at 80°C. Finally, a yellow viscous product was obtained with 87% yield. Preparation of stock solutions Homogeneous sodium-RNA solutions (1% w/w: 10 mg/ml) were prepared by dissolving RNA in 10 ml of phosphate buffer (pH=7.4) with occasional stirring at 5°C for 24 hr. Using the molar extinction coefficient of 6600 cm-1 M-1 which was represented as molarity of phosphate groups, the final concentration of RNA stock solution was assessed spectrophotometrically at 260 nm 31,32 . UV absorbance of the diluted RNA solution (40 μM) at 260 nm was 0.11 (with the path length of 1 cm), and the final concentration of RNA stock solution was 25 mM in phosphate. For infrared spectroscopic measurements, various amounts of HBCD in phosphate buffer (pH=7.4) were added dropwise to RNA solutions to achieve favorable HBCD contents of 0.625, 1.25, 2.5, 5 and 10 mM with an ultimate RNA concentration of 10 mM. Infrared spectra were recorded 1 hr after the mixing of HBCD solutions with RNA solutions. For UV measurements, (...truncated)