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Reversible Covalent-Crosslinked Polycations with Enhanced Stability and ATP-Responsive Behavior for Improved siRNA Delivery Zhanwei Zhou, Minghua Zhang, Yadong Liu, Chenzi Li, Qingyan Zhang, David Oupicky, and Minjie Sun Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00922 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018
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Biomacromolecules
Reversible Covalent-Crosslinked Polycations with Enhanced Stability and ATP-Responsive Behavior for Improved siRNA Delivery
Zhanwei Zhou a, ‡, Minghua Zhang a, ‡, Yadong Liu a, Chenzi Li a, Qingyan Zhang a, David Oupicky a, b, Minjie Sun a, *
a State Key Laboratory of Natural Medicines and Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China b Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha NE 68198, USA
Keywords: Reversible Covalent Crosslinking, Enhanced Stability, ATP-Responsive, Hyaluronic Acid, siRNA Delivery
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ABSTRACT Cationic polyplex as commonly used nucleic acid carriers faced several shortcomings, such as high cytotoxicity, low serum stability and slow cargo release at the target site. The traditional solution is covering a negative charged layer (e.g. hyaluronic acid, HA) via electrostatic interaction. However, it was far from satisfactory for the de-shielding by physiological anions in circulation (e.g. serum proteins, phosphate). In this study, we proposed a new strategy of reversible-covalent crosslinking to enhance stability in circulation and enable stimulidisassembly of polyplexes in tumor cells. Here, 25k polyethyleneimine (PEI) was chosen as model polycations for verifying the hypothesis. HA-PEI conjugation was formed by the crosslinking of adenosine triphosphate grafted HA (HA-ATP) with phenylboronic acid grafted PEI (PEI-PBA), via the chemical reaction between PBA and ATP. Compared with non-covalent polyplex by electrostatic interaction (HA/PEI), HA-PEI exhibited much better colloidal stability and serum stability. The covered HA-ATP layer on PEI-PBA could maintain stable in the absence of physiological anions, while HA layer on PEI in HA/PEI group showed obvious detachment after anion’s competition. More importantly, the covalent crosslinking polyplex could selectively release siRNA in the ATP rich environment of cytosol and significantly improve siRNA silence. Besides, the covalent crosslinking with HA-ATP could effectively reduce the cytotoxicity of cationic polyplex, improve the uptake by B61F10 cells and promote the endosomal escape. Consequently, this strategy of HA-PEI conjugation significantly enhanced the siRNA transfection in the absence or presence of FBS (fetal bovine serum) on B16F10 cells and CHO cells. Taken together, the reversible covalent crosslinking approach shows obvious superiority compared with the non-covalent absorption strategy. It held great potential to be
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Biomacromolecules
developed to polish up the performance of cationic polyplex on reducing the toxicity, enhancing the serum tolerance and achieving controlled release of siRNA at target site. INTRODUCTION Cationic polyplex has been proved to promote the transfection of nucleic acid.1-2 However, the positive charged surface and nonspecific binding with anionic agent in the blood circulation limited its widespread utility.3 Recently, many strategies have been developed to improve the performance of the polyplex. Among them, using HA or PEG to shield the positive charge seem to be the most effective methods.4-6 But the assembly method of coating HA layer on polyplex by physical adsorption attended to the replaced by physiological anions, such as serum proteins, phosphate, etc., resulting in poor serum stability and exposure of the inner polycations.7 Hence, physical assembly strategy is thought to be not effective for conquering the drawbacks of cationic polyplexes. In this study, we tried to use covalent assembly strategy to solve this problem. Polyethyleneimine (PEI) with molecular weight at 25 kDa (PEI 25k) was chosen as the model polycations and PEI/siRNA as the model cationic gene polyplex. PEI 25 k exhibits high transfection efficiency and has been applied as golden standard for DNA/RNA transfection. However, its properties of strong positive charge and significant cytotoxicity greatly limits the widespread employment.8-9 Therefore, the properties of PEI 25k showed the importance to be decorated and meet up the hypothesis of the current research. Hyaluronic acid (HA), with high negative ionic charge density,10 was brought in the outer layer of siRNA delivery systems. HA, as the main component of the extracellular matrix (ECM) and ubiquitously distributed in synovial fluids, has been developed for various biomedical
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applications due to its biocompatibility, biodegradability and non-immunogenicity.11-12 Owing to the negative charge of HA, the blood stability of the siRNA delivery systems has been enhanced for electrostatic repulsion between HA and components with negative charge surface.13 Moreover, HA has been widely used in tumor-targeted delivery via binding to CD44 receptors which overexpresses on various cancer cells specifically.10-13 Nevertheless, the plasma stability of HA modified complex nanoparticles was still low due to weak electrostatic force formed by negative HA and positive polyplexes.7 Stronger binding was reqiured to ensure HA firmly anchored on the surface of cationic polyplex, such as covalent binding, coordination binding. Phenylboronic acid (PBA), a Lewis acid, is capable of forming reversible borate esters with cis-diol-containing compounds including carbohydrates, ribonucleotides.14-15 Herein, the reaction between PBA and sugar was suitable to be used in this study to enhance the binding between HA and cationic polyplex. Moreover, the stability of covalent boronic acid-diol coupling depends on the environmental concentration of other sugars, such as glucose, fructose and ATP.14 The reversible covalent conjugation is naturally based on the dynamic chemical reaction between PBA and sugar. Therefore, enrichment of sugar in tumor environment could be utilized as trigger for breakage of boronic acid-diol coupling. Notably, the intracellular ATP concentration is 1-10 mM, significantly higher than extracellular concentration (
