Cationic Galactose-Conjugated Copolymers for ... - ACS Publications

Subscriber access provided by Weizmann Institute of Science

Article

Cationic Galactose-Conjugated Copolymers for Epidermal Growth Factor (EGFR) Knockdown in Cervical Adenocarcinoma Stephen Quan, Piyush Kumar, and Ravin Narain ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00085 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 14, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Cationic Galactose-Conjugated Copolymers for Epidermal Growth Factor (EGFR) Knockdown in Cervical Adenocarcinoma Stephen Quan1, Piyush Kumar2, Ravin Narain1* 1

Donadeo Innovation Centre in Engineering, Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada

2

Department of Oncology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada

Abstract Glycopolymers

of

lactobionamidoethyl

statistical

and

methacrylamide

block

configurations

(LAEMA)

and

were

synthesized

2-aminoethyl

from

2-

methacrylamide

hydrochloride (AEMA) by the reversible addition-fragmentation chain transfer (RAFT) polymerization. These cationic glycopolymers were found to form very stable polyplexes with EGFR siRNA as determined by dynamic light scattering and agarose gel electrophoresis. The polyplexes revealed to be very stable even in the presence of serum proteins. Transfection studies of the glycopolymer-EGFR siRNA polyplexes were achieved in HeLa cells to determine the EGFR knockdown efficiency, cellular uptake and cytotoxicity. In this study, the block copolymer with the shortest AEMA segment was the most effective in EGFR gene silencing, however this block copolymer revealed to be slightly more toxic as compared to the statistical copolymers studied at higher w/w ratios. In addition, gene silencing of up to 80-85 % was achieved with this low molecular weight block copolymer. Keywords: Glycopolymers, siRNA, gene knockdown, RAFT

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

Introduction Over the past decade, the development of gene therapeutics for the systemic disruption of gene expression to treat various malignant and genetic diseases has been extensively pursued1-3. Small interfering RNA (siRNA) therapy has the capacity to interfere with critical cellular pathways by knocking down and silencing gene expression of essential proteins, thus opening new pathways for alternative treatments of chemotherapy resistance diseases4-6. However, the effectiveness of siRNA therapies in vivo have been limited since siRNA are prone to degradation by Ribonuclease (RNase) enzymes in the blood stream leading to low efficacy, resulting in transient expression with low tissue selectivity and relatively poor cellular uptake reaffirmed that the design of effective non-viral

7-9

. Reports have

gene carriers with lipids, polymers,

carbohydrates, proteins and viral vectors can significantly improve the outcome of oligonucleotide therapies10-11

12

. Therefore, significant effort has been dedicated towards the

preparation of versatile nanocarriers which can improve the pharmacokinetics and potential applications of these innovative gene therapeutic tools. Recently, synthetic glycopolymers have drawn enormous attention due to their unique capabilities of imitating naturally occurring polysaccharides, increasing blood biocompatibility of gene delivery vehicles and promoting carbohydrate-specific recognition in cell-cell communication13-16.

It has been reported that the carbohydrate-protein interactions are

significantly improved with multivalent display of carbohydrate ligands, referred to as the “glycosidic cluster effect”

17-18

. However, the strength and affinity of these interactions are

largely governed by their pendant display density and relative spatial arrangement

19

. In many

studies, glycopolymer-based delivery systems were shown to be stable in physiological salt and serum conditions, to provide active modes of cellular targeting for delivery, to decrease


Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


degradation of DNA/siRNA and other non-specific interactions in the blood stream

4-6

. Thus,

glycopolymer-conjugated gene delivery vehicles could potentially exploit these properties to improve the knockdown efficacy and biocompatibility, typically associated with many toxic commercially available transfection agents. In several types of cancers such as lung, breast and ovarian carcinomas, epidermal growth factor receptor (ErBb1/EGFR) is largely overexpressed, and is responsible for uncontrolled cell proliferation and evading apoptotic pathways.20-23 Reports provide evidences that silencing the EGFR gene blocks EGFR-mediated cell proliferation pathways and inhibits downstream cellsignalling pathways between other ErBb family members, which can subsequently increase chemotherapeutic sensitivity and induce tumor regression

24-25

. The researchers have shown that

HeLa cells overexpressing EGFR on the cell surface can be knocked down effectively with no adverse effect on the cell viability

26-27

. Herein, we present the RAFT synthesis of a family of

cationic statistical and block glycopolymers for the complexation and delivery of EGFR siRNA in HeLa cells to examine the gene knockdown efficiency, cellular uptake and cytotoxicity. Materials and Methods Materials. 2-Aminoethyl methacrylamide hydrochloride (AEMA), 2-Lactobionamidoethyl methacrylamide (LAEMA), and the chain transfer agent, cyanopentanoic acid dithiobenzoate (CTP) were synthesized in the laboratory according to previously reported protocols13. The initiator, 4,4′-Azobis-(cyanovaleric acid) (ACVA), was obtained from Sigma-Aldrich (Oakville, Canada). Branched Polyethyleneimine, PEI (Mw = 25 kDa) was obtained from Polysciences Inc. Streptomycin (10 mg mL−1), penicillin (10000 U mL−1), DMEM/F12 media, Opti-MEM (OMEM), 0.25% trypsin–EDTA, and Fetal Bovine Serum (FBS) were obtained from Gibco. Control EGFR siRNA–FITC conjugate, human EGFR-specific small interfering RNA



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

(EGFR siRNA), and primary antibody (rabbit polyclonal EGFR specific IgG) were purchased from Santa Cruz Biotechnology. Human EGFR–siRNA consisting of 19–25 nucleotides is specific for chromosomal locus 7p11.2. The FITC-conjugated control siRNA consisting of 19-25 nucleoptides is a non-specific scrambled siRNA. HRP-stabilized (3,3′, 5,5′-tetramethylbenzidine) (TMB) substrate and Horseradish peroxidase (HRP)-conjugated secondary antibody (Anti-rabbit IgG) were purchased from Promega Corporation. SYBRSafe DNA gel stain was obtained from Fisher Scientific. The organic solvents were purchased from Caledon Laboratories Ltd (Georgetown, Canada), and were used without further purification.

Scheme

1.

Structures

of

2-aminoethylmethacrylamide

hydrochloride

(AEMA),

2-

lactobionamidoethyl methacrylamide (LAEMA), 4,4’-azobis(4-cyanovaleric acid, (ACVA) and 4-cyanopentanoic acid dithiobenzoate, (CTP).13 Cationic Glycopolymers Synthesis. The cationic glycopolymers were prepared as described in a previously reported procedure16. In a typical synthesis of a block copolymer, the AEMA macroCTA is synthesized using 4,4’-azobis(4-cyanovaleric acid) (ACVA) as the initiator and 4-


Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent (CTA) (Scheme 1). AEMA monomer (591 mg or 3.5 mmol) was first dissolved in double distilled water (4 mL) followed by the addition of 1 mL CTP (10 mg or 0.035 mmol, target DPn = 50) and ACVA (2 mg or 0.0047 mmol) in N,N’-Dimethylformamide (DMF) solution. After degassing with nitrogen for 30 mins, the polymerization was carried out at 70 °C for 6 h. The reaction was stopped in liquid nitrogen and the resulting polymer was isolated after precipitation in acetone. The residual monomer was removed by washing twice the solids with 2-propanol. The macroCTA, P(AEMA58), was used to prepare the P(AEMA58-b-LAEMA56) copolymer. LAEMA (1.22 g or 2.6 mmol) was first mixed with P(AEMA58) solution (0.5 g or 0.052 mmol) in double distilled water and 1 mL ACVA (2.9 mg) in DMF solution was subsequently added. After purging with nitrogen, the polymerization was achieved in an oil bath at 70 °C for 24 h. The diblock copolymer P(AEMA58-b-LAEMA56) was obtained after precipitation in acetone followed by three washes with methanol to remove residual monomer. Preparation of Glycopolymer-siRNA Polyplexes EGFR-siRNA (250 ng) was complexed with cationic glycopolymers (in either water or OMEM media) at different weight/weight ratios and the mixture was incubated at room temperature (23 °C) for 30 minutes. Characterization of Polymers and Cationic Glycopolymer-siRNA complexes Dynamic Light Scattering (DLS) and Zeta Potential Measurements. The hydrodynamic size and charge of the cationic glycopolymer-siRNA complexes were determined using ZetaPlus-Zeta Potential Analyzer (Brookhaven Instruments Corporation) at a scattering angle θ = 90 °C. The cationic glycopolymer-siRNA complexes were formulated at w/w ratio of 15 in water and OMEM media. The aggregation of glycopolymer-siRNA polyplexes was also evaluated in the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

presence of serum proteins in OMEM. The net charge of the polyplexes was determined in deionized water. Gel Permeation Chromatography. Average molecular weights and polydispersity were determined by conventional gel permeation chromatography (GPC) system using two WAT011545 Waters columns at a flow rate of 1.0 mL/min using a 0.5 M sodium acetate / 0.5 M acetic acid buffer as eluent. Monodisperse Pullulan standards (Mw - 5900-404,000 g mol-1) were used for the conventional GPC system. Agarose Gel Electrophoresis. The complexes were prepared in water at varying w/w ratios as described previously.[REF] The complexes were added to 1% garose gel 1:10000 dilution SYBRsafe DNA gel stain in 1X TAE buffer. Cell Culture. HeLa cells were cultured in DMEM medium containing with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (100 units of penicillin, 100 µg streptomycin and 0.0085% fungizone) in a humidified atmosphere at 37 °C and 5% CO2. At about 80% confluency, the cells were sub-cultured by dissociating with 0.25% trypsin-EDTA, and were cultured twice per week.13

Fluorescent Labelling of Glycopolymers. The glycopolymers are fluorescently labelled with rhodamine isothiocyanate (RITC) using the hydroxyl groups on the carbohydrate residues, as previously reported28. Briefly, a glycopolymer solution at a concentration of 5 mg/mL in 4% NaHCO3 at pH 8.5 was prepared. RITC was dissolved in DMSO at a concentration of 1 mg/mL which was then added dropwise to the glycopolymer solution (100 µL/mL of glycopolymer), and


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the mixture was stirred in the dark for 5 days. Residual free RITC was removed by dialysis and the solution was subsequently lyophilized to obtain a bright orange powder. Flow Cytometry. The HeLa cells were trypsinized and subsequently seeded into 6 well plate at 500,000 cells per well. After incubation overnight, the HeLa cells were treated with the cationic glycopolymer-FITC-labelled control EGFR siRNA complexes for 4 h at a w/w ratio of 100 in OMEM containing 10% FBS. After the media was removed, and washed with 1×PBS at pH 7.4 (three times), the cells were trypsinized. Then, the cells were centrifuged at 300 rpm and the pellet was re-suspended in FCS buffer (1×PBS pH 7.4, 0.5% FBS, 2 mM EDTA, 0.05% w/v sodium azide). The cells were characterized using a BD FACS dual laser flow cytometer (488 and 635 nm) (Cross Cancer Institute). Transfection of EGFR-siRNA. HeLa cells were seeded into 96 well plates at a cell density of 10000 cells per well. The polyplexes were prepared in OMEM (with and without serum proteins), and 100 µL of the polyplexes mixture (siRNA or control siRNA) was added per well. After incubation for 6 h, the media was removed from the well and replaced with 100 µL of DMEM media containing with 10% fetal bovine serum (FBS). The EGFR knockdown efficacy was characterized after 48 h cell growth. In-Cell Enzyme-Linked Immunosorbent Assay (ELISA). All the buffers were prepared in house as previously reported26-27. The cells were allowed to grow for 48 h, then the media was removed and the cells were fixed in wells using 3.7% formalin in 1× TBS at 25 °C for 10 min. The cells were subsequently washed twice with 1×TBS and treated with the permeabilization buffer for 15 min. After washing twice with 1×TBS, the cells



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

were quenched for 15 min. The cells were then incubated with the blocking solution for an additional 30 min and washed twice with the wash buffer. Diluted primary antibody rabbit polyclonal EGFR-IgG buffer solution (1: 250 dilutions) (50 µL) was added to each well. The plate was then incubated at 25 °C for 1 h, followed by the removal of the primary antibody solution and after washing three times with of wash buffer (200 µL), HRP-conjugated secondary antibody solution (1:10000 dilution in wash buffer) (100 µL) was added to each well, and the plate was incubated at 25 °C for 1 h. After removal of the secondary antibody solution and washing with the wash buffer (200 µL), TMB substrate (100 µL) was added to each well. After incubation in the dark for 20 min, the blue precipitates were dissolved with 0.1 M phosphoric acid solution (100 µL per well) and the absorbance (of the yellow color formed indicating the presence of EGFR receptors) was measured using a TECAN microplate reader at λ = 420 nm. The percentage cell surface EGFR receptors on are calculated :

%

100

Janus Green Assay for Cell Viability. Cell viability was determined by the Janus green assay and a typical procedure for the assay is as followed.26 A solution of Janus green was made by solubilizing 0.3% w/v Janus green dye in distilled deionized water. The plate content (after siRNA ELISA analysis) was removed, the plate was washed with 1×PBS pH 7.4 three times, and 100 µL of Janus green solution was added per well. The plate was then incubated at 25 °C for 30 min. The Janus green solution was removed and after washing five times with 1×PBS pH 7.4, HCl solution (0.5 M)_was added for 10 min. The absorbance was read using a TECAN microplate reader at λ = 570 nm. The cell viability after transfection was determined by:


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


%

!

Confocal Fluorescence Microscopy. After treatment, the cells were imaged following a standard procedure previously reported.13 HeLa cells were cultured as mentioned above, trypsinized and seeded onto glass cover slips in 6 well plate at 100,000 cells per well. After incubation overnight, the media was removed and replaced with RITC-labelled glycopolymerFITC control EGFR siRNA polyplexes at a w/w ratio of 100 in OMEM and subsequently incubated for 4 h in a humidified atmosphere at 37 °C and 5 % CO2. After removal of the media and washing with 1×PBS (three times), the cells were treated with 1 µg/mL of DAPI dissolved in PBS for 10 min. After that, the solution was removed and washed with 1×PBS (three times), the cells were fixed with 3.7% formalin in 1xPBS for 10 min. The formalin was then removed, washed with 1xPBS (three times) and the glass coverslip was fixed on a microscope slide with nitrocellulose (30% isopropyl alcohol) dissolved in ethyl acetate. The cells were visualized using a confocal microscope at 490 and 570 nm emission spectra for FITC and RITC, respectively (Olympus Fluoview FV10i)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Scheme 2. Cationic block glycopolymer synthesis carried out by the reversible additionfragmentation chain transfer process (RAFT).13b

Results and Discussion

Cationic Polymers Syntheses. Homopolymers and statistical copolymers were prepared by the reversible addition-fragmentation chain transfer polymerization method (RAFT) (Supporting Information Table 1). The synthesized homopolymer, P(AEMA), were used as macro-CTAs to copolymerize LAEMA monomer using water/2-propanol solvent system (Scheme 2). After dialysis and lyophilisation, the polymers were characterized by GPC and 1H NMR for their molecular weights and compositions respectively (Supporting Information Figure S1).


Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Characterization of the homopolymer, statistical and diblock glycopolymers by size exclusion chromatography. Polymer Composition AEMA40 P(AEMA17-b-LAEMA17) P(AEMA22-st-LAEMA22) P(AEMA58-b-LAEMA56) P(AEMA50-st-LAEMA45)

Mn (gmol-1) 6,500 10,230 14,600 38,550 35,850

Mw/Mn 1.39 1.22 1.33 1.30 1.15

Figure 1. Agarose gel electrophoresis showing the polyplex formation at various weight/weight ratios of cationic glycopolymers with EGFR siRNA (250 ng).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

Figure 2. Characterization of the glycopolymer-siRNA polyplexes in deionized water for their size and charges by dynamic light scattering and zeta potential instrumentation. Cationic Glycopolymer-siRNA Polyplex formation. The efficiency of glycopolymer binding with EGFR control siRNA was evaluated at different weight-to-weight (w/w) ratios by agarose gel electrophoresis (Figure 1). All the glycopolymer-siRNA (Scheme 3) formed stable polyplexes at a w/w ratio greater than 1, however, P(AEMA17-b-LAEMA17) was able to fully condense with siRNA at w/w ratio lower than 1, indicating that the cationic glycopolymers demonstrated a very high capacity for binding with the negatively charged EGFR siRNA. Additionally, branched PEI (25 kDa) and linear homopolymer, P(AEMA)40, formed stable polyplexes at w/w ratio of 5. Analysis by dynamic light scattering (DLS) showed that P(AEMA17-b-LAEMA17) formed the smallest stable polyplexes with EGFR siRNA that were approximately 300 nm, as compared to P(AEMA22-st-LAEMA22) which formed complexes that were approximately 500 nm at 15 w/w ratio in deionized water (Figure 2). As expected, the low molecular weight copolymers formed smaller polyplexes with EGFR siRNA in water, whereas


Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the higher molecular weight copolymers P(AEMA58-b-LAEMA56) and P(AEMA50-st-LAEMA45) formed complexes that were approximately 800 and 600 nm, respectively (Figure 2 and Supporting Information Figure S4). Interestingly, addition of 10% fetal bovine serum to the solution created much smaller glycopolymer complexes which were 150-200 nm in size, a phenomenon also reported in previous findings

26

. The addition of serum also affected the

surface charges of the glycopolymer polyplexes with the most dramatic decrease from 13 to 2 mV with the P(AEMA50-st-LAEMA45)-siRNA complexes (Figure 2 and Supporting Information Figure S2). This may be due to the binding of serum proteins neutralizing the cationic charge of the complexes and creating smaller and more robust, compact nanoparticles.

Scheme 3. Formation of polyplexes between EGFR siRNA with statistical and block glycopolymers



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

Figure 3. Cellular uptake of RITC-labelled-P(AEMA17-b-LAEMA17) complexed with FITCcontrol EGFR SiRNA at w/w ratio of 100 after 4 h incubation; imaged using confocal fluorescence microscopy. In Vitro Uptake of Glycopolymer-siRNA Complexes. The binding interaction and uptake of RITC-labelled-P(AEMA17-b-LAEMA17) complex with FITC-control EGFR siRNA in HeLa cells is studied by confocal fluorescence microscopy 4 h post-transfection (Figure 3). The fluorescence images show that the RITC-glycopolymer and FITC-control siRNA has a strong binding affinity and forms stable complexes at w/w ratio of 100. Interestingly, the polyplexes localized towards the outer wall of the cell nucleus in the HeLa cells, thus demonstrating the capacity for endosomal escape and exogenous macromolecule trafficking towards cell nucleus


Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for siRNA gene silencing without dissociation or degradation of the glycopolymer delivery vesicle. These results are also consistent with previously published data showing that complexes having a Fusogenic peptide containing a DNA condensing motif, a Nuclear localization agent and a Targeting motif (FDNT) biopolymer and pEGFR-GFP are internalized rapidly (less than 80 min) and are localized close to the nucleus.29 This may be a suitable method for in vivo delivery of siRNA from RNase degradation while being transported in the blood stream to the tumor site; further in vivo studies should be conducted.

Figure 4. Flow cytometry data of cellular uptake of control FITC-EGFR siRNA-glycopolymer polyplexes in HeLa cells at a w/w ratio of 100. Percent of gated cells and fluorescence intensities for all treated samples in the gated region defined by the negative control marker (M1). Internalization of Fluorescently Labelled Glycopolymers in HeLa Cells. The cellular internalization of control FITC-EGFR siRNA glycopolymer polyplexes in the presence of serum proteins in HeLa cells was studied (Figure 4). The analysis of FITC fluorescence intensity indicates that P(AEMA17-b-LAEMA17) complexes demonstrated the highest uptake ability with and MFI of 6.39 and 20.51% positive fluorescent cells within the gated region (Figure 4). Both low molecular weight glycopolymers displayed higher uptake compared to the high molecular



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

weight counterparts, with P(AEMA22-st-LAEMA22) reported 6.2% positive fluorescent cells within the gated region and MFI of 3.67. The highest molecular weight glycopolymer-FITC siRNA complexes demonstrated poor uptake in comparison to the P(AEMA17-b-LAEMA17) complexes Overall, as revealed by flow cytometry, the uptake of glycopolymer-siRNA complexes demonstrates that the lower molecular weight diblock species P(AEMA17-bLAEMA17) performs the best transfection efficiency as compared to the statistical conformation and the higher molecular weight counterparts.


Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. A) Relative percent of HeLa cells surface EGFR expression 48 h post transfection with either EGFR or control siRNA (250 pM). Gene knockdown efficacies of EGFR siRNA were significantly different than the control siRNA for each treatment (p 0.05) Knockdown of EGFR in HeLa cells. The overexpression of EGFR in cancer leads to constant ligand binding and activation of downstream genetic events, such as angiogenesis and metastasis and uncontrolled cellular proliferation.30 HeLa cells were selected in this study due to the prevalence and overexpression of EGFR on the cell surface, which can be knocked down effectively without compromising the cell viability26-27. Previous studies by Lyon et al. demonstrated a clear improvement in chemotherapeutics sensitivity from EGFR knockdown in HeLa cells31-32. The gene knockdown efficacies of the cationic glycopolymer-EGFR siRNA complexes were studied at different w/w ratios in the presence and absence of serums by In-Cell ELISA, at a dosage of 250 pM EGFR siRNA per treatment (Figure 5A). Using PEI above a w/w



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

ratio of 5 causes noticeable apoptosis and cell death with samples treated with or without siRNA/DNA plasmids. Previous studies with PEI as gene delivery vectors show considerable toxicity at higher w/w ratios, therefore providing the motivation to develop non-toxic and biocompatible alternatives to PEI as gene transfection delivery systems.33-34 Branched 25kDa PEI-siRNA complexes formed at w/w of 5 showed approximately 18-20% knockdown efficiency as compared to the control. Higher w/w ratio above 5 with branched PEI revealed higher cytotoxicity, in accordance with previous reports35-37. The low molecular weight diblock glycopolymer P(AEMA17-b-LAEMA17) showed efficient EGFR knockdown of approximately 20-22% at a w/w ratio of 15 with relatively high cell viability, as compared to the control siRNA treatment. However, the toxicity of the system drastically increased with an increase of the w/w ratios, as the results indicate less than 50% cell viability 72 h post-transfection at a w/w of 100 with approximately 80-85% EGFR knockdown. The gene knockdown efficiency and cell viability were assessed for the high molecular weight glycopolymer-complexes and results indicated minimal knockdown efficacy, but relatively high cell viability at a w/w ratio of 60 (Supporting Information Figure S3). These findings are consistent with results from Reineke et al. which showed that the toxicity and cell viability of the system decreased as the AEMA block lengths of the glycopolymer increased.15 In contrast, P(AEMA22-st-LAEMA22) demonstrated relatively low gene knockdown efficiency of approximately 5-7% and 10-12% at of 15 and 200 w/w ratios, respectively (Figure 5B). Interestingly, even at w/w ratios up to 200, the toxicity of the system was very low in comparison to the diblock copolymer, P(AEMA17-b-LAEMA17). It is hypothesized that the higher cell viability may be associated with charge distribution along the polymer chain; with a high local charge distribution in a diblock polymer compared to a dispersed charge distribution in a statistical configuration. In addition, the cell growth is


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


observed to enter into a senescent where the cells are still viable, however, the rate of proliferation has drastically slowed down. Experiments past the 48 h time point should be evaluated. These findings indicate that the lower molecular weight diblock polymer P(AEMA17b-LAEMA17) is an acceptable carrier for gene transfection at low w/w ratios of 15 with a EGFR knockdown efficacy similar to PEI, however, increasing the w/w ratios to 100 or above drastically increases toxicity of the system thus causing apoptosis. Conversely, the lower molecular weight statistical polymer P(AEMA22-st-LAEMA22) demonstrated slightly lower gene knockdown efficacy compared to PEI and P(AEMA17-b-LAEMA)17, but the overall biocompatibility of the system is tolerable to cells even at w/w ratios of 200. Conclusion Herein we have described the preparation of cationic glycopolymers for siRNA delivery. These copolymers demonstrated the capability to form stable nanoparticles with EGFR siRNA with or without serum proteins. The polyplexes demonstrated effective gene knockdown in HeLa cells, however diblock polymers contributed to a higher cellular toxicity at high w/w ratios as compared to statistical conformations. Furthermore, the incorporation of carbohydrate residues on the polymer chain increased biocompatibility of the delivery system. Future studies will focus on synthesizing different copolymer architectures and evaluate the biological response of siRNA delivery in vivo. Corresponding Author *Prof. Ravin Narain, Donadeo Innovation Centre for Engineering, Department of Chemical and Materials Engineering, University of Alberta, 116 St and 86 Ave, Edmonton, AB, T6G 2G6, Canada. Email: [email protected]. Tel: 780 492 1736. Fax: 780 492 2881.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Notes The authors declare no competing financial interest. Acknowledgements This work was supported by funding from Natural Sciences and Engineering Research Council of Canada, CREATE Molecular Imaging Probes (NSERC cMIP grant #371050-2010) and Alberta Innovates Health Solutions (AIHS; grant 201201164). Supporting Information NMR, GPC, DLS and EGFR expression data. This material is available free of charge via the internet at http://pubs.acs.org. References (1) Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004, 432, 173-178. (2) de Fougerolles, A.; Vornlocher, H.; Maraganore, J.; Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 2007, 6, 443-453. (3) Dykxhoorn, D.; Palliser, D.; Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 2006, 13, 541-552. (4) Creixell, M.; Peppas, N. Co-delivery of siRNA and therapeutic agents using nanocarriers to overcome cancer resistance. Nano Today. 2012, 7, 367-379. (5) Kim, S.; Jeong, J.; Lee, S.; Kim, S.; Park, T. PEG conjugated VEGF siRNA for antiangiogenic gene therapy. J. Control. Release 2006, 116, 123-129. (6) Huang, C.; Li, M.; Chen, C.; Yao, Q. Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications. Expert Opin. Ther. Targets 2008. 12, 637-645. (7) Ryther, R.; Flynt, A.; Phillips, J.; Patton, J. siRNA therapeutics: big potential from small RNAs. Gene Ther. 2005, 12, 5-11. (8) Whitehead, K.; Langer, R.; Anderson, D. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129-138.


Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Chen, S.; Zhaori, G. Potential clinical applications of siRNA technique: benefits and limitations. Eur. J. Clin. Invest. 2011, 41, 221-232. (10) Wang, J.; Lu, Z.; Wientjes, M.; Au, J. Delivery of siRNA therapeutics: barriers and carriers. J.AAPS. 2010, 12, 492-503. (11) Gao, Y.; Liu, X.; Li, X. Research progress on siRNA delivery with nonviral carriers. Int. J. Nanomedicine 2011, 6, 1017-1025. (12) Mintzer, M.; Simanek, E. Nonviral vectors for gene delivery. Chem. Rev. 2008, 109, 259302. (13) (a) Quan, S.; Wang, Y.; Zhou, A.; Kumar, P.; Narain, R. Galactose-based Thermosensitive Nanogels for Targeted Drug Delivery of Iodoazomycin Arabinofuranoside (IAZA) for Theranostic Management of Hypoxic Hepatocellular Carcinoma. Biomacromolecules 2015, 16, 1978-1986. (b) Thapa, B.; Kumar, P.; Zeng, H.; Narain, R. Asialoglycoprotein ReceptorMediated Gene Delivery to Hepatocytes Using Galactosylated Polymers. Biomacromolecules 2015, 16, 3008-3020 (14) Li, J.; Zacharek, S.; Chen, X.; Wang, J.; Zhang, W.; Janczuk, A.; Wang, P. Bacteria targeted by human natural antibodies using α-Gal conjugated receptor-specific glycopolymers. Bioorg. Med. Chem. 1999, 7, 1549-1558. (15) Smith, A.; Sizovs, A.; Grandinetti, G.; Xue, L.; Reineke, T. Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions. Biomacromolecules 2011, 12, 3015-3022. (16) Adokoh, C.; Quan, S.; Hitt, M.; Darkwa J.; Kumar P.; Narain R. Synthesis and Evaluation of Glycopolymeric Decorated Gold Nanoparticles Functionalized with Gold-Triphenyl Phosphine as Anti-Cancer Agents. Biomacromolecules 2014, 15, 3802-3810. (17) Lundquist, J.; Toone, E. The cluster glycoside effect. Chem. Rev. 2002, 102, 555-578. (18) Narla, S.; Nie, H.; Li, Y.; Sun, X. Recent advances in the synthesis and biomedical applications of chain-end functionalized glycopolymers. J. Carbohydr. Chem. 2012, 31, 67-92. (19) Ahmed, M.; Narain, R. Glycopolymer bioconjugates. in Engineered Carbohydrate-based Materials for Biomedical Applications. Wiley, Hoboken, 2011, 167-188. (20) De Luca, A.; Carotenuto, A.; Rachiglio, A.; Gallo, M.; Maiello, M.; Aldinucci, D.; Pinto, A.; Normanno, N. The role of the EGFR signaling in tumor microenvironment. J. Cell. Phys. 2008, 214, 559-567.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(21) Larsen, A.; Ouaret, D.; El Ouadrani, K.; Petitprez, A. Targeting EGFR and VEGF (R) pathway cross-talk in tumor survival and angiogenesis. Pharma. Thera. 2011, 131, 80-90. (22) Steichen, S.; Caldorera-Moore, M.; Peppas, N. A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics. Eur. J. Pharma. Sci. 2013, 48 , 416427. (23) Diaz-Padilla, I.; Monk, B.; Mackay, H.; Oaknin, A. Treatment of metastatic cervical cancer: future directions involving targeted agents. Crit. Rev. Oncol. Hematol. 2013, 85, 303314. (24) MacEwan, S.; Chilkoti, A. Harnessing the power of cell‐penetrating peptides: activatable carriers for targeting systemic delivery of cancer therapeutics and imaging agents. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2013, 5, 31-48. (25) Kigawa, J. New strategy for overcoming resistance to chemotherapy of ovarian cancer. Yonago Acta Medica 2013, 56, 43-50. (26) Ahmed, M.; Wattanaarsakit, P.; Narain, R. Cationic glyco-nanogels for epidermal growth factor receptor (EGFR) specific siRNA delivery in ovarian cancer cells. Poly. Chem. 2013, 4, 3829-3836. (27) Ahmed, M.; Ishihara, K.; Narain, R. Calcium mediated formation of phosphorylcholinebased polyplexes for efficient knockdown of epidermal growth factor receptors (EGFR) in HeLa cells. Chem. Comm. 2014, 50, 2943-2946. (28) Ahmed, M.; Narain, R. Intracellular delivery of DNA and enzyme in active form using degradable carbohydrate-based nanogels. Mole. Pharm. 2012, 9, 3160-3170. (29) Canine, B.; Wang, Y.; Ouyang, W.; Hatefi, A. Development of targeted recombinant polymers that can deliver siRNA to the cytoplasm and plasmid DNA to the cell nucleus. J. Control. Release 2011, 151, 95-101. (30) Sebastian, S.; Settleman, J.; Reshkin, S.; Azzariti, A.; Bellizzi, A.; Paradiso, A. The complexity of targeting EGFR signalling in cancer: from expression to turnover. Biochem. Biophy. Acta. Rev. Can. 2006, 1766, 120-139. (31) Dickerson, E.; Blackburn, W.; Smith, M.; Kapa, L.; Lyon, L. Chemosensitization of cancer cells by siRNA using targeted nanogel delivery. BMC Can. 2010, 10, 1-11. (32) Smith, M.; Lyon, L. Multifunctional nanogels for siRNA delivery. Acc. Chem. Res. 2011, 45, 985-993.


Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(33) Urban, K.; Werth, S.; Abuharbeid, S.; Czubayko, F.; Aigner, A. RNAi-mediated genetargeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 2005, 12, 461-466. (34) Forrest, M.; Koerber, J.; Pack, D. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjugate Chem. 2003, 14, 934-940. (35) Zintchenko, A.; Philipp, A.; Dehshahri, A.; Wagner, E. Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity. Bioconjugate Chem. 2008, 19, 1448-1455. (36) Beyerle, A.; Irmler, M.; Beckers, J.; Kissel, T.; Stoeger, T. Toxicity pathway focused gene expression profiling of PEI-based polymers for pulmonary applications. Mole. Pharm. 2010, 7, 727-737.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical Abstract

Cationic Galactose-Conjugated Copolymers for Epidermal Growth Factor (EGFR) Knockdown in Cervical Adenocarcinoma Stephen Quan1, Piyush Kumar2, Ravin Narain1* 1

Donadeo Innovation Centre in Engineering, Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada

2

Department of Oncology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada


Page 24 of 24

Cationic Galactose-Conjugated Copolymers for ... - ACS Publications

Recommend Documents