Synthesis and characterization of quaternized poly(Î²-amino ester) for

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Synthesis and characterization of quaternized poly(#-amino ester) for highly efficient delivery of small interfering RNA Yun Liu, Jing Chen, Yue Tang, Shuhan Li, Yushun Dou, and Jiewen Zheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00549 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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Molecular Pharmaceutics

Synthesis and characterization of quaternized poly(β-amino ester) for highly efficient delivery of small interfering RNA

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Yun Liu1, Jing Chen1, Yue Tang†, Shuhan Li, Yushun Dou and Jiewen Zheng Department of Pharmacy, China Pharmaceutical University, Nanjing 211198, PR China Corresponding author: Yue Tang, Tel: +8613851713608. Fax: +862583271355. E-mail address: [email protected]. 1 These authors contributed equally to this work and should be regarded as co-first authors.

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Abstract

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The development of non-viral vectors for gene delivery has gained attention over the past decades.

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Specifically, poly(β-amino ester) (PBAE) has shown great potential in improving the delivery of

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gene therapeutics. It has been observed that low molecular weight PBAE displayed low

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transfection activities while quaternization could enhance the transgene expression efficacy of

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PBAE. Herein, PBAE quaternary ammonium salt (PBAEQAS) was synthesized to increase the

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positive charge of the polymers, which resulted in an increase in siRNA binding efficiency based

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on self-assembly electrostatic interaction. Specifically, the nanoparticle surface was positively

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charged, which increased the uptake ability of siRNA. Compared with acrylate-PBAEQAS/siNC

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nanoparticles

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nanoparticles and amine-PBAEQAS/siSurvivin nanoparticles induced more efficient cell apoptosis

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and gene silencing. All these results suggested that PBAEQAS would be a promising gene delivery

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vector for cancer treatment.

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Keywords: Poly(β-amino ester); quaternization; nanoparticles; siRNA; transfection

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1. Introduction

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and

amine-PBAEQAS/siNC

nanoparticles,

acrylate-PBAEQAS/siSurvivin

Gene therapy is an important part of cancer treatment, and it can be achieved by substitution

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of altered genes, inhibition of oncogene or insertion of new genes [1]. A successful gene delivery

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system promises efficient gene transfection. The carriers for the gene delivery are divided into

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viral vectors and non-viral vectors. Viral vectors pose high gene transfer efficiency in vivo, but

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they are more likely to cause immunogenicity because of their strong innate immune response [2],

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and the viral vectors also have virus-associated toxic side effects [3]. While non-viral vectors have

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the advantages of reduced cytotoxicity, targeting property and high transfection efficacy [4-6].

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Non-viral vectors usually refer to liposomes, cationic polymers, peptides and inorganic materials

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[7-9].

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In the past decades, a number of polymeric materials have been utilized in formulating gene

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delivery systems, such as poly(lactic-co-glycolicacid) (PLGA), chitosan, poly(amidoamine)

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(PAMAM) and poly(L-lysine) (PLL), etc [10-12]. Poly(β-amino esters)(PBAE) are promising

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materials due to their biodegradable and biocompatible nature. The special chemical structure

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endows PBAE with availability for further modification [5,13,14]. To date, a library of PBAEs had

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been synthesized. Among them, some types of PBAE own higher in vitro gene transfection

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efficiencies than polyethyleneimine (PEI), however, there still exists a part of PBAE owning low

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transfection ability before modification. Thus, this polymeric material is complicated and pending

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further study [15-17]. It has been observed that PBAE with high molecular weight were able to

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mediate effective intracellular gene delivery, but they also showed considerable toxicities [18].

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While low molecular weight PBAE posed low transfection efficiency, for the low positive charge

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density of PBAE may reduce its ability to compact siRNA in physiological conditions [19]. When

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the molecular weight of PBAE is less than 10 kD, it can not effectively condense siRNA [20, 21],

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but lower molecular weight PBAE reacted more quickly than higher molecular weight PBAE in

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the case of being able to bind to siRNA [22]. And it had been found that PBAE/siRNA

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nanoparticles are generally prepared in the acidic buffer solution, the particle size will increase

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significantly when the nanoparticles were transferred into physiological conditions (pH 7.4) [23].


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Scheme .1. Synthesis of PBAEQAS and the delivery of PBAEQAS/siRNA nanoparticles for cell

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transfection.

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Recently, most studies focused on various derivatives of polymers to improve the efficiency

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of gene delivery. For example, It had been found that grafting polyethylene glycol (PEG) in PBAE

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could enhance the stability of PBAE/DNA complexes [6, 24]. Highly branched PBAE had been

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deeply studied, and it has been proved that branched PBAE could significantly improve the

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transfection efficiency[25-28]. Low molecular weight polyethyleneimine-terminated PBAE could

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improve the gene delivery efficiency than that of the polymer before modification [13]. Moreover,

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structural modifications, such as changing the quantity and types of amines, have been

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implemented to enhance the siRNA delivery efficiency [29, 30]. Bhavani Miryala et al. had proved

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that quaternization of aminoglycoside-derived polymers could enhance the gene delivery

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efficiency [31]. By quaternary ammonium reaction, the positive charge density of small molecular

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weight PBAE is increased and the combination efficiency of siRNA is improved at the same time.

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Therefore, quaternizing the ammine groups of PBAE may be a new way to improve the gene

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transfection ability of different types of PBAE.

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The objective of this study was to explore a novel kind of vector based on PBAE with

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enhanced siRNA delivery capability by increasing the positive charge density through

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quaternization. Scheme. 1 shown the synthesis of PBAE quaternary ammonium salt (PBAEQAS),

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the preparation of nanoparticles and the delivery of nanoparticles for cell transfection. Firstly,

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PBAE was synthesized by a typical Michael addition reaction, and quaternary ammonium reaction

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was applied to prepare PBAEQAS. Secondly, the siRNA retardation assay was performed and the

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stability of the formulations were evaluated. Thirdly, the impact of intracellular delivery of such



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polymers on the cellular uptake and mechanisms, apoptosis and gene silencing of A549 was

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assessed. We then developed these vectors to deliver survivin siRNA (siSurvivin) to inhibit the

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expression of survivin genes in cancer cell. And the influence of amine end-modified PBAEQAS

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and acrylate end-modified PBAEQAS on transfection efficiency was investigated as well.

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2. Materials and methods

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2.1. Materials and cell culture

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1,4-butanediol diacrylate, 4-amino-1-butanol and thiazolylbluetetrazolium bromide (MTT)

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were purchased from D&B Biological Science and Technology Co.Ltd (Shanghai, China). Agarose

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was obtained from Solarbio Biotech Co. Ltd. (Beijing, China). JJ Red was purchased from Biolite

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Biotech Co. Ltd. (Tianjing, China). Ribonuclease A (RNase A), Diethyl pyrocarbonate (DEPC),

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Chlorpromazine, Genestin and Amiloride were purchased from Aldrich Chemical Co. (Shanghai,

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China). Filipin III was obtained from Cayman Chemical Co. (Ann Arbor, MI). Fetal bovine serum

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(FBS, Sijiqing) was purchased from Zhejiang Tianhang Biotechnology Co. Ltd (Hangzhou, China).

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Trypsin, penicillin, streptomycin, RPMI 1640 cell culture medium, Hoechst 33342 and Trizol total

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RNA extraction reagent were obtained from KeyGen BioTech (Nanjing, China). LysoTracker Red

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was purchased from the Beyotime Institute of Biotechnology (Haimen, China). Annexin V-FITC

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and propidium iodide (PI) apoptosis assays kit was purchased from Xinle Biotech Co. Ltd.

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(Shanghai, China). The quantitative real time reverse transcription polymerase chain reaction

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(qRT-PCR) quantitation kit were purchased from GenePharm(Shanghai, China) . The siRNA

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targeting survivin mRNA (siSurvivin), FAM-labeled siRNA and negative control siRNA (siNC)

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were synthesized by GenePharm (Shanghai, China). The sequences of siRNA are as follows:

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siSurvivin:

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5’-UGUAGAGAUGCGGUGGUCCdTdT-3’.

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5’-UUCUCCGAACGUGUCACGUTT-3’; antisense: 5’-ACGUGACACGUUCGGAGAATT-3’.

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All the reagents and chemicals were of analytical grade.

sense:

5’-GGACCACCGCAUCUCUACAdTdT-3’; siNC:

antisense: sense:

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A549 cells (human lung adenocarcinoma cells) were obtained from American Type Culture

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Collection (ATCC) and were cultured at 37℃ in 5% CO2 incubator supplemented with complete


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RPMI 1640 medium (containing 10% FBS, 100 units/ml penicillin and 100µg/ml streptomycin),

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allowed to attach overnight for subsequent experimentation.

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Scheme. 2. Synthesis of PBAE (A) and PBAEQAS (B).

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2.2. Synthesis of PBAE and PBAE quaternary ammonium salt(PBAEQAS)

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PBAE were synthesized following a two-step procedure described previously [13, 32, 33].

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Briefly, 1,4-butanediol diacrylate and 4-amino-1-butanol (at 1:1 molar ratio of amine:diacrylate)



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were added into a round-bottom flask and the reaction solution was stirred at 90℃ for 24 h under

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the protection of nitrogen. To obtain amine end-capping modified PBAE, additional amine

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monomers were added and the final solution was stirred for another 1 h at room temperature.

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Tertiary amine groups of PBAE were converted to quaternary ammonium (QA) by reaction with

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iodomethane (Scheme.2) [34].

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A certain amount of PBAE solution was dissolved in 10 ml of isopropyl alcohol, and then

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iodomethane (at 2:1 molar ratio of iodomethane/PBAE) was added dropwise. The reaction mixture

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was stirred at 60℃ for 16 h. The solution was then removed and the retained solid was washed

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three times with ethyl alcohol, followed by lyophilization for 24 h. The structures of the

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synthesized polymers were confirmed by 1H NMR and FT-IR analysis. The 1H NMR was

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obtained on a Bruker AVANCE 500MHz Spectrometer. All measurements were performed at 300K,

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using pulse accumulating of 64 scans and the LB parameter of 0.30 Hz. D2O was used as the

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solvents for dissolving of polymers. The average molecular weight of the polymers were

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determined by a gel permeation chromatography(GPC, Shimadzu, Japan) equipped with a

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refractive index detector (RID-10A, SHIMADZU, Tokyo, Japan). The SEC-300 column (300×7.8

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mm, 5 µm) was eluted with dd H2O at the flow rate of 1 mL/min 40 ℃ . Samples of

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PEG(polyethylene glycol) with average molecular weights in the range of 3070-21600 Da were

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used as standards for calibration.

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2.3. Preparation and characterization of nanoparticles

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The polyplexes were formulated by mixing PBAEQAS and siRNA at different weight ratios.

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RNase-free diethyl pyrocarbonate (DEPC)-treated ultrapure water was used as solvent for all

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solutions of siRNA. Briefly, the PBAEQAS stock solutions in DMSO (100 mg/ml) were diluted

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with ultrapure water to 1mg/ml. The above-mentioned solution was then mixed with a solution of

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siRNA (100 ng/µl), vortexed for 10 s and incubated at room temperature for 30 min. Analysis of

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particle size distribution and zeta potential was performed in a Zetasizer Nano ZS (Malvern

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Instruments, Malvern, United Kingdom). To assess siRNA retardation, the resulting nanoparticles

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were characterized by agarose gel electrophoresis. The nanoparticles were added to wells of


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agarose gel (1%, containing 0.01% JJ Red). The samples were run at 80 V for 30 min, visualized

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by UV illumination. In order to investigate the stability of the formulation, the nanoparticles (each

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group contained 50 nM of siRNA respectively) were incubated in presence of 25 mg/ml RNase A

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at 37℃ [35]. After 20 min the samples were heated in water bath at 80℃ to stop reaction, then the

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resulting solution were treated with heparin and investigated by agarose gel electrophoresis.

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2.4. Cytotoxicity assay

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Cell cytotoxicity assays of A549 cells were performed via the MTT assay. A549 cells were

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seeded on 96-well plates at 2×104 cells/ml, cultured in complete medium RPMI 1640 for 24 h. The

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medium was replaced with serum-free RPMI 1640 (200 µl) which containing various levels of

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nanoparticles. After incubation at 37℃ for 24 h, the medium was changed with MTT solution (180

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µl serum-free RPMI 1640 and 20 µl MTT (1mg/ml)) and further incubated at 37℃ for 4 h. Finally,

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the medium was removed and 200 µl DMSO was added and then incubated for 10 min.

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Absorbance was measured by a Microplate Reader (Multiskan FC, USA) at 490 nm. Cell viability

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was expressed as a relative percentage compared with untreated cells.

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2.5. Cellular uptake and mechanisms

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A549 cells were seeded in 6-well plates at a density of 2×105 cells/ml and incubated for 12

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h. Then the cells were washed with PBS and incubated with serum-free RPMI 1640 containing

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nanoparticles for 2 h, 4 h, 6 h, 24 h. Nanoparticles were prepared as described previously. Briefly,

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FAM labeled siRNA (50nM) was mixed with PBAEQAS (1mg/ml) at a weight ratio of 50:1,

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vortexed for 10 s and incubated at room temperature for 30 min in the dark. The cells were then

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washed with PBS for three times, and then the cells were analyzed by flow cytometry

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(MACSQuant Analyzer 10, Miltenyi, Germany). Furthermore, fluorescence microscopy (Olympus

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IX53, Japan) was used to qualitatively observe the cells uptake. A549 cells were transfected with

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the nanoparticles for 6 h, fixed with 4% paraformaldehyde, and stained with Hoechst 33342.

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The mechanistic studies of FAM-labled siRNA loaded nanoparticles on cellular uptake were

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performed at 4℃ for 30 min or incubated with various endocytic inhibitors (sodium azide (1

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mg/ml), chlorpromazine (10 mg/mL), hypertonic sucrose (450 mM), genistein (200 µM), filipin (5



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µg/ml), amiloride (13.3 µg/ml) ) within a period of 30 min at 37℃ prior to the addition of

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polyplexes and then the cell were incubated with the nanoparticles for 4 h at 37℃ for the uptake

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mechanistic studies. Cells incubated with nanoparticles in the absence of endocytic inhibitors at

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37℃ for 4 h were set as control [36,37].

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2.6. Apoptosis of A549 cells

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A549 cells were seeded on 6-well plates and cultured overnight. Then the medium was

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replaced with serum-free RPMI 1640. The nanoparticles were added to each test well. After 6 h

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incubation at 37℃, the medium was replaced with fresh medium containing 10% FBS, and

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cultured for another 42 h. The cells were washed twice with PBS and harvested by EDTA-free

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trypsin, re-suspended with 300 µl binding buffer and 5 µl annexin V-FITC, and incubated at room

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temperature for 15 min. 5 µl PI was added before the cells were analyzed by flow cytometry

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instrument. All the operations needed carrying out under protection from light. The cells incubated

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with PBAEQAS/siNC nanoparticles and the cells incubated with naked siSurvivin were set as the

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control.

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2.7. In vitro gene silencing

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A549 cells were seeded in 6-well plates with 2 ml RPMI 1640 medium containing 10% FBS.

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After the cells showed 70% to 80% confluence, the culture medium was changed into fresh

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complete medium containing mocks, siNC-loaded nanoparticles, and siSurvivin-loaded

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nanoparticles respectively. After 6 h incubation, the medium contained samples were replaced with

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fresh serum-free medium and further cultured for another 42 h. Then the cells were washed with

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PBS and lysed by Trizol reagent (KeyGEN BioTECH). The isolated total RNA were quantified by

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micro UV Spectrophotometer Nano 100 (All for life science, China) at 260 nm. qRT-PCR was

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performed by using the SYBR Green I dye method according to the manufacturer's instructions.

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qRT-PCR analysis was performed by using QuantStudio™ 3 Real-Time PCR System, The data

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were analyzed using the comparative CT(2-△△CT) method [38].

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2.8. Statistical analysis

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All experiments were carried out in triplicate as the means±SD. The Student’s t-test was


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performed to determine the statistical significance of the difference group means at p < 0.005,

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0.005

Synthesis and characterization of quaternized poly(Î²-amino ester) for

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