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Acid degradable cationic galactose-based hyperbranched polymers as nano-therapeutic vehicles for Epidermal Growth Factor Receptor (EGFR) knockdown in Cervical Carcinoma Yi-Yang Peng, Diana Diaz-Dussan, Piyush Kumar, and Ravin Narain Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01066 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018
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Biomacromolecules
Acid degradable cationic galactose-based hyperbranched polymers as nano-therapeutic vehicles for Epidermal Growth Factor Receptor (EGFR) knockdown in Cervical Carcinoma Yi-Yang Peng†, Diana Diaz-Dussan†, Piyush Kumar‡, and Ravin Narain
＊, †
†Department ‡
of Chemical and Materials Engineering, University of Alberta, Edmonton T6G 1H9, Alberta Canada Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, T6G 1Z2, Alberta, Canada
KEYWORDS: Acid Degradable Glycopolymer, siRNA delivery, EGFR knockdown, Cervical Cancer therapy, Targeted Release.
ABSTRACT: Strong signalling cascades derive from upregulation and overexpression of growth factors such as the EGF-family (epidermal growth factors) has been crucially related to cancer pathogenesis. Gene silencing techniques to modulate the expression of oncogenes and tumor suppresor genes, is a strategy that shows great promise for cancer management but still faces some limitations in the design of biocompatible and effective vectors. In this study we synthetized by reversible additionfragmentation chain transfer (RAFT) polymerization, several acid degradable galactose-based hyperbranched cationic polymers with varying molecular weights (10 to 20 kDa) and compositions with 2-lactobioamidoethyl methacrylamide [LAEMA] and 2- aminoethyl methacrylamide hydrochloride [AEMA] at different ratios (2.0, 1.0 & 0.5) and evaluated their ability to enhance Epidermal Growth Factor Receptor (EGFR) knockdown in cervical carcinoma. All the polymer constructs have enhanced capabilities to condensate siRNA (small interfering RNA), showing low toxicity at higher LAEMA:AEMA ratios (1.0 and 2.0). Western blot assays were conducted to quantify the EGFR expression of each treatment group demonstrating superior gene knockdown efficiency for the polymers having a LAEMA:AEMA ratio of 2.0 than the lower ratio counterparts; while maintaining low toxicity levels. Gene silencing of EGFR of up to 60% was achieved with acid degradable polymers having 10 kDa molecular weight and a LAEMA:AEMA ratio of 2.0. The superior stability of the polyplexes under physiological conditions and the low cytotoxicity observed in the 48 h post-transfection, demonstrated the high potential of these acid degradable galactose-based hyperbranched cationic polymers for EGFR silencing treatment applications at the clinical level.
INTRODUCTION The role of growth factors in driven strong cascade signaling in the development and
maintenance of cancer has been known since 1985, where Sporn and Roberts demonstrated that cancer exhibited a lower requirement of exogenous growth factors to maintain high proliferation rates1. Since then, a great amount of studies have confirmed a direct correlation of growth factors as oncogenes2-3. Different families of growth factors like the EGF-family have a role in the pathogenesis of certain carcinomas; overexpression and aberrant activation of EGFR (epidermal growth factor receptors) which, is a hallmark of various cancers such as lung, breast, and ovarian carcinomas, leads to uncontrolled cell proliferation and evasion of apoptotic pathways4-7. Thus it is desired to regulate this key protein expression. It has been reported that chemotherapeutic sensitivity has been increased by silencing the EGFR gene, resulting in tumor regression8-9. After the discovery of the efficient gene silencing ability of
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interfering ribonucleic acids (siRNAs) in 1998, two decades of intensive investigation on gene therapy has demonstrated that a number of malignant and gene-related diseases can be treated by using external gene sources to regulate oncogene expressions10-12. Small interfering RNAs (siRNA) are 21-30 nucleotides duplexes which have the capability of cancer suppression through inhibition of gene expression of the causing oncogenes and/or activation of tumor suppressor genes13-14. This provides an alternative to cancer treatment from conventional surgery, radiotherapy, and chemotherapy15-17. However, many limitations still exist that prevent its clinical application. Due to the presence of ribonucleases, siRNA is vulnerable to degradation in the blood stream if there is no protection18. The small size of siRNAs (less than 5 nm) allows the rapid removal of siRNAs from the blood stream through renal filtration and clearance19, making reaching of the targeted cells quite a challenge. Naked siRNA cannot easily penetrate through the cell membrane to reach the cytoplasm due to repulsive forces which lead to low cellular uptake and results in ineffective transfection efficiencies20. Prior research has confirmed that these defies can be addressed and the outcomes of treatment can be greatly improved by using non-viral vectors such as lipids21-22 proteins23-24, polymers25-26, and carbohydrate-based polymers27-30. Combination therapies have also been used and provided a better therapeutic efficacy than a single therapy42. Thus, several studies have been conducted with the hope to develop a versatile nano-carrier that can effectively deliver and release siRNA into the cytoplasm of the targeted cells to enhance the therapeutic efficacy of this technology, for instance towards the treatment of malignant tumors.
Scheme 1. (a) Synthesis of acid degradable and cationic galactose-based hyperbranched polymers (P(LAEMA-st-AEMA-st-DMP)) via RAFT polymerization; (b) Polyplexes formation by the electrostatic interactions between siRNA and cationic glycopolymers; (c) siRNAs were released into the cytoplasm via polyplex degradation from the hydrolysis of ketal bond in the acidic environment of the endosome
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Biomacromolecules
Furthermore, this strategy should not lead to adverse effects on the normal cells as the cell viability would not be affected significantly by the knockdown of overexpressed EGFR on the cellular surface of normal cells28,31-32. However, siRNA treatment with no toxicity and high transfection efficiency is not yet achievable because of drawbacks in the delivery carrier32. Synthetic carbohydrate-based polymers, also known as glycopolymers, have the potential of overcoming some of the challenges associated with siRNA delivery. Glycopolymers have many unique properties such as, high stability in physiological environment28,33, enhanced blood biocompatibility34, and induced carbohydrate-specific recognition in cell-cell communications35 that support their suitability to become a leading nano-carrier. Previous studies have shown that cationic glycopolymers could form stable polyplexes with siRNA and high internalization in cells leading to high epidermal growth factor receptors (EGFR) knockdown in cervical carcinoma as compared to using siRNA alone3637
. However, these findings have not been clinically viable, as the required high knockdown efficiency,
could not be achieved without sacrificing cell viability since more cationic moieties are needed to achieve the target outcome. In this study, we aimed to reduce or eliminate the cytotoxicity of these gene carriers by incorporating acid degradability into the system while maintaining high gene knockdown. Once degraded, the small polymer fragments are expected to be less toxic as compared to the hyperbranched polymers.
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Table 1. Characterization of acid degradable glycopolymers by Gel Permeation Chromatography, Zeta Potential Analysis, and MTT Assay. Polymer
GPC Mn (kDa)
Mw/Mn
% Crosslnker
Polymer Composit ion
Zeta Potential (mV)
IC50 (mg/mL)
HGA 1
21.6
1.26
3.5
(LAEMA27-stAEMA54-stDEP)
53.8±2.8
