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Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery
International Journal of Biological Macromolecules xxx (xxxx) xxx
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery Zhiyu Su, Tseyenkhorloo Erdene-Ochir, Tsogzolmaa Ganbold ⇑, Huricha Baigude * School of Chemistry & Chemical Engineering, Inner Mongolia Key Laboratory of Mongolian Medicinal Chemistry, Inner Mongolia University, 235 West College Road, Hohhot, Inner Mongolia 010021, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2019 Received in revised form 13 October 2019 Accepted 14 October 2019 Available online xxxx Keywords: siRNA delivery Curdlan Nanoparticle Endosome release Cancer cells
a b s t r a c t Developing nucleic acid-based tools to control disease-relevant gene expression in human disorders, such as siRNAs, opens up potential opportunities for therapeutics. Because of their high molecular weight and polyanionic nature, synthetic siRNAs fail to cross biological membranes by passive diffusion and therefore, generally require transmembrane siRNA delivery technologies to access the cytoplasm of target cells. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan derivative 6AC-100 in a regioselective manner to introduce different ratios of imidazole rings in the amino units (denoted as Curimi) and evaluated their siRNA binding ability, cytotoxicity, endosome buffering capacity and siRNA transfection efficiency. The novel curdlan based Curimi polymers formed nanoparticles with siRNA at pH 7.4 in range of 85–105 nm and their size distribution increased along with decreasing pH condition. The zeta potential increased by lowering pH value as well. Curimi polymers showed lower toxicity and higher buffering capacity compared to 6AC-100, and efficiently delivered siRNA against to PLK1 into cancer cells, and subsequently, significantly inhibited target mRNA level. Our result suggested that novel curdlan based Curimi polymers may be used as efficient siRNA carrier for cancer therapy. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction A number of polymeric nanoparticles for the intracellular delivery of siRNA have been previously studied. But lack of efficient siRNA release due to the endosomal trap, which could provide sufficient siRNA concentration and allows efficient down-regulation of specific gene transcription at mRNA level, can be another obstacle [1,2]. The interference of mRNA translation is proportional to the amount of delivered siRNA in the cytoplasm and maximum gene silencing efficiency is only obtained with delivery of siRNA above certain threshold. Therefore, siRNA should be dissociated from carrier in the cytoplasm for efficient gene silencing because only free forms of siRNA can specially bind to RISC in the cytoplasm [3]. Nanoparticles can be internalized into the cells via an endocytotic pathway through either specific (e.g., receptor-mediated endocytosis) or non-specific interactions (e.g., adsorptive endocytosis). Unfortunately, endocytosis generally guides the nanoparticles into the endosome, an intracellular membrane where they are fated to eventual degradation in lysosome [4–6]. An efficient ⇑ Corresponding author. E-mail addresses: [email protected] (T. Ganbold), [email protected] (H. Baigude).
polymeric siRNA carrier should be able to induce endosomal escape and rapidly release the maximum number of siRNA into the cytoplasm [3,7]. According to the endocytic pathway, a clathrin-coated vesicle containing nanoparticles will sequentially mature into an endosome with a pH of ~7–5.2, then into a lysosome with a pH of ~5.2–4.5. Thus it is important to develop a delivery system that is able to release siRNA just within the endosome and undergo subsequent endosomal escape and avoid from lysosomal digestion [2,5]. Facilitating endosome escape has long been the focus of siRNA delivery research. Depending on the type of nanoparticles, different strategies are used to enhance endosomal escape. Although external stimulations, such as photochemical internalization (PCI), are also used to improve endosome escape ability of nanoparticles, the common strategies are based on the acidic endosomal micro-environment, which is to improve pH buffering capacity and enhance ‘‘proton sponge” effect to destabilize the endosomal membrane [7,8]. The ‘‘Proton sponge” effect mostly occurs in certain cationic polymers with a high buffering capacity over a wide range of pH. These polymers usually contain protonatable secondary and/or tertiary amine groups with pKa close to endosomal/lysosomal pH [9]. In addition, several types of membrane disruptive polymers and imidazole-containing polymers have been investigated to facilitate
https://doi.org/10.1016/j.ijbiomac.2019.10.129 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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escape from the endosomes and achieve efficient transportation of genetic materials into the cytosol. In particular, imidazolecontaining polymers have gained much attention recently, as they exhibit pKa ~ 6 and can undergo protonation in the acidic endosomal environment leading, eventually, to efficient endosomal disruption [10]. The polar imidazole ring has serves as a pH sensitive fusogen because the imidazole has an electron lone pair on the unsaturated nitrogen. The polar imidazole ring, which contains two nitrogen separated with a methylene, hydrogen bonds through the amino hydrogen as the donor and the imino nitrogen as the acceptor [6,11,12]. The pKa of imidazole group is around 6, and therefore this group is protonated in slightly acidic milieu. Since the cationic polymer nanoparticles interact with negatively charged endosomal membranes, induces influx of water and ions, and eventually brings about endosomal destabilization and siRNA release [5]. In addition, pH sensitive polymer is focused on cancer therapy due to their pH dependent release. This strategy could not only stabilize the therapeutic system (polymer complex with anticancer drug) at neutral and tumor extracellular pH (~6.8), but also facilitate the disintegration of therapeutic system and the resultant quick drug release in response to the low endocytic pH (<6.0) upon 2C5-mediated internalization [1]. Based on these reports, in this study, we modified 6AC-100 with pH sensitive Imidazole group to improve buffering capacity and enhance endosomal escape of curdlan derivative 6AC-100. 2. Materials and methods 2.1. Materials Curdlan was purchased from Wako Pure Chemical Industries, Itd. (Osaka, Japan). Imidazole-4(5)-acetic acid hydrochloride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from TCI chemicals (Tokyo, Japan) and Aladdin (Shanghai, China), respectively. PLK1 gene silencing siRNA, PLK1 and b-actin primers, and fluorescein isothiocyanate (FITC)-siRNA were synthesized by Takara Biotechnology co., Ltd (Dalian, Liaoning, China). 2.2. Preparation of Curimi polymers 6-amino-6-dexoy-curdlan (6AC-100) was prepared as a previously reported method [13]. 40 mg 6AC-100 was dissolved in 2 mL of PBS and added 0.1 M HCl by pipetting until 6AC-100 was completely dissolved, and pH of the solution to pH ~ 9.0 was adjusted by 0.5 M NaOH. 1 mL mixture of imidazole-4(5)-acetic acid hydrochloride (4.8 or 12,1 or 24,2 mg) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (7,1 or 17,8 or 35,7 mg) in 0.1 M MES buffer (pH = 6.0) was previously prepared and then added into the 6AC-100 solution. The mixed solution was rotated continuously at room temperature for overnight. The new products (denoted as Curimi-1, Curimi-2 and Curimi-3) were purified by dialyzing (MWCO 3500) against deionized water to remove any trace of monomer for 2 days. The dialysis membrane was pre-wetted in water for 30 min before use. After the Curimi polymers were freeze-dried, the characteristic of Curimi polymers (solubilized in D2O) was analyzed by carbon nuclear magnetic resonance (13C NMR) analysis (500 MHz Bruker Avance III, Germany). 2.3. Measurements of particle size and zeta potential of Curimi nanoparticles The size and the zeta potential of the nanoparticles were determined in triplicates by dynamic light scattering (DLS) using
Zetasizer, Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). siRNAs were mixed with Curimi nanoparticles at a 6/1 ratio (N/P) in 100 lL ddH2O and the solutions were incubated at RT for 20 min. The samples were diluted 1:100 with double distilled water before measuring. 2.4. Transmission electron microscopy (TEM) Shape and surface morphological examination of Curimi/siRNA nanoparticles at N/P ratio 6/1 in aqueous solution was investigated by transmission electron microscopy (TEM). After Curimi nanoparticles and siRNA was mixed at a 6/1 ratio of N/P and incubated at RT for 20 min, two drops of sample were placed on a copper grid and air dried for 10 min, and then negatively stained with 2% phosphomolybdic acid solution for 10 min. The grid was air-dried for 10 min and examined under the TEM. 2.5. Buffering capacity measurements The endosome buffering capacities of Curimi polymers were measured by acid-base titration method. Each 10 mg of Curimi polymers was dissolved in 10 mL of dH2O and pH value of the solution was adjusted to pH 10 using 0.1 M NaOH. Then the solution was titrated by adding 10 mL of 0.1 M HCl until the pH dropped to ~2. 2.6. Electrophoretic mobility shift assay An electrophoretic mobility shift assay was used to investigate the integration of Curimi polymers and siRNA by electrostatic interaction. Curimi and siRNA complexes were prepared in aqueous solution at various N/P ratios and incubated at RT for 20 min, and then run into a 2% (w/v) agarose gel in 1X TBE buffer at 100 V for 20 min. The gel was stained with 0.5 mg/mL ethidium bromide for 20 min at RT and the RNA band was visualized on a UV illuminator using a Gel Logic 212 PRO imaging system (Carestream, Toronto, Canada). To evaluate the serum stability, Curimi nanoparticles (8 lg) and siRNA (100 lM) were incubated with an equal volume of FBS (final concentration 50% v/v) at 37 . After the samples were kept at 2, 6, 12, 24 and 48 h, 1 lL of 0.5 M EDTA was immediately added to stop any nuclease activity. Then, 1 lL of heparin (50 lg) was added to each sample to release the siRNA from each complex and the samples were subjected to gel electrophoresis. 2.7. Cell culture Human epitheloid cervix carcinoma (Hela) and Human liver hepatocellular carcinoma (HepG2) cells were maintained in high glucose Dulbecco’s modified eagle’s medium (DMEM) (BI, USA) supplemented with 5% (v/v) fetal bovine serum (FBS) (BI, Israel) and 1% (v/v) penicillin/streptomycin mixture (Gibco, Life technologies, USA) in culture dish (Corning, USA), and the cell cultures were grown in cell culture incubator (Nuaire, USA), under the cell culturing condition (atmosphere of 5% CO2 at 37 °C and 70–80% humidity). The cells were sub-cultured every two days. After the cell cultures were washed with 1X PBS (pH 7.2–7.4) (Biotopped, Life technologies) twice, added 3 mL of Trypsin (Gibco, Life technologies, USA) to detach the cells from the cell culture dish, and then incubated in cell culture incubator for 3–5 min. After 5 mL of pre-warmed DMEM medium was added to the detached cells, the cell suspension was removed into 15 mL centrifuge tube (Corning, Life technologies, USA), and centrifuged at 1000 rpm for 5 min using high-speed micro centrifuge (Hitachi, CF16RN, Japan). The upper layer was discarded and the cell pellet was resuspended in DMED medium. The cell suspension was poured into cell culture dish and cultured in cell culture incubator. The cell passaging
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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procedure was carried out in laminar flow hoods (Thermo scientific, USA). 2.8. Cellular toxicity assay To evaluate cellular toxicity of Curimi nanoparticles, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was carried out. Hela or HepG2 cells were seeded in a 96-well plate (Corning-Coaster, Tokyo, Japan) at a density of 1 104 cells/well in 100 mL DMEM medium and the cells were incubated in cell culture incubator under cell culturing condition for 24 h. The next day, the pre-cultured cells were treated with various final concentration of Curimi nanoparticles (20, 50, 80 and 120 mg/mL) in 100 mL of DMEM medium. 6AC-100 was used for comparison group. After the treated cells were incubated for another 24 h, previously prepared 50 mL of 1X MTT solution (final concertation; 0.2–0.5 mg/ml) was added to each well, and incubated at 37 °C for 4 h. After the viable cells with active metabolism convert MTT into a purple colored formazan product, the cell medium was removed, and 100 mL of DMSO was added to dissolve the formazan product. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer (FilterMax F5, Molecular Devices, USA). Filter-sterilize the MTT solution through a 0.2 mM filter into a sterile 2.9. Cellular uptake and lysosome tracking HepG2 cells were seeded in 24-well culture plates (CorningCoaster, Tokyo, Japan). When achieving 70–80% confluence, the cells were treated with the FITC-siRNA complexed with Curimi nanoparticles at the N/P ratio of 6 in 500 mL of serum-free optimem medium. The mixture (Curimi/FITC-siRNA) was incubated at RT in 100 mL of serum-free optimem medium for 20 min to condense Curimi polymer and siRNA in nanoparticle formation prior to transfection and another 400 mL of optimem medium was added to the transfection solution. After 30 min, 2 h, 4 h and 12 h incubation respectively, the cells were stained with Lyso-Tracker Red (dilution at 1: 5000, Solarbio Life Science, China) at RT for 2 h. After washed with 1X PBS (pH 7.2–7.4) 3 times, the cells were fixed in 4% paraformaldehyde for 15 min, RT. Then washed with 1X PBS 3 times, cells were permeabilized with 0.2% Triton X-100 in PBS, 5 min at RT. After washed with 1X PBS, nucleus was stained with DAPI (40 ,6-diamidino-2-phenylindole) at a final concentration of 0.5 mg/mL for 5 min at RT by protecting from light. The stained cells were rinsed in 1X PBS 3 times and dried the coverslips in air. The coverslips were mounted using mounting medium and then stored at 4 °C until capturing cell images for about 1–3 days. The cell images were captured under confocal laser-scanning microscopy (Olympus, Fluoview FV 1000). 2.10. Western blotting assay HepG2 cells were seeded in 6-well plate (4 105 per well). After 24 h, either siControl or siPlk1 (final concentration at 50 nM) were transfected with Curimi-1, -2 and -3, respectively. 24 h later, the cell lysates were harvested and total protein was extracted with Mammalian Protein Extraction Kit (KWbiotech, Beijing, China). The concentration of total proteins was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (25 mg total proteins) either from siControl or siPLK1 treated cells was loaded and separated on SDS-PAGE gel electrophoresis. Proteins were transferred to membrane and incubated with antibodies (PLK1 Rabbit mAb, 1:1000, #4513; Phospho-Histone H2A.X (Ser139) Rabbit mAb, 1:1000, #9718;
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GAPDH Rabbit mAb, 1:1000, #2118; Cell Signaling Technology, Danvers, MA, USA). After incubation with secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), 1:4000, ab97051, Abcam), images were acquired by using chemiluminesence detection. 2.11. Transfection with chloroquine or nigericin In order to investigate the endosomal escape mechanism of Curimi nanoparticles, we used two kinds of chemical reagents, chloroquine or nigericin. HepG2 Cells were seeded in a 12-well plate in DMEM medium 5% FBS and 1% penicillin/streptomycin. When the cell were grown to reach 70–80% confluence, the cells were pre-incubated in serum-free Opti-mem medium containing 100 mM chloroquine or 10 mM nigericin for 30 min, and the cells were treated with Curimi nanoparticles complexed with the green florescence protein reporter plasmid DNA (pDNA) in 500 mL of serum free optimem medium. After 4 h of incubation, the medium was exchanged with fresh DMEM medium containing 5% FBS and 1% penicillin/streptomycin and the cells were incubated for further 2 days at 37 °C in 5% CO2. Subsequently, medium was removed and the cells were rinsed with 1X PBS. After the wells were trypsinised, the cell fluorescence intensity was measured on a luminometer (FilterMax F5, Molecular Devices, USA). 3. Results and discussions To explore potential of curdlan, curdlan modified derivative 6AC-100 polymer was conjugated with a layer of imidazole groups at three different ratios of imidazole molecule (Scheme 1; designated as Curimi-1, Curimi-2 and Curimi-3). The conjugated imidazole rings on 6AC-100 was determined using 13C NMR analysis and the molecular weight distribution of the polymers was measured by gel permeation chromatography (GPC). In 13C NMR, the newly attached imidazole moiety showed a signal at 174.32 ppm (C = O), confirming the imidazole ring successfully connected on the curdlan backbone (Fig. 1). The molecular weight distribution of Curimi-1 was Mn of 23.22 kDa and Mw of 45.61. Curimi-2 and Curimi-3 polymers gave Mn of 22.71 kDa and Mw of 53.95 and, Mn of 23.07 kDa and Mw of 61.72 kDa, respectively, by GPC analysis. The molecular weight of Curimi polymers was similar because the conjugation of moieties on the 6AC-100 backbone resulted in minor differences in the final products. The degree of substitution (DS) of Curimi nanoparticles were 5,2% (Curimi-1), 10.1% (Curimi2) and 24,7% (Curimi-3) by elemental analysis. 3.1. RNA binding assay and particle size measurement by TEM The positive charge of Curimi polymers and negative charge on nucleic acid have tendency to enforce electrostatic interactions between them to form Curimi/siRNA nanoparticles, which was evaluated by gel shift assay using 2.0% agarose gel electrophoresis (Fig. 2A). The migration of Curimi-1/siRNA and Curimi-2/siRNA complexes in gel were completely retarded at N/P ratio of 3 and 4, respectively, while Curimi-3/siRNA complexes were totally retarded at N/P ratio 6. The siRNA condensing ability of Curimi polymers was decreased when more imidazole was incorporated to 6AC-100 due to their positive charge ratio on the polymers, as well as sterical hindrance. Moreover, the morphology imaging of the polymers was captured under transmission electron microscopy (TEM). The result showed spherical particles encapsulating siRNA have diameters about 55 nm (Fig. 2B), suggesting that Curimi polymers have a great ability to bind siRNA via electrostatic interaction. Moreover, Curimi polymers protected siRNA from degradation by serum nuclease, indicating that Curimi/siRNA complexes have similar stability with the positive control (6AC-100) in serum (Fig. 2C).
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Scheme 1. Synthesis of imidazole modified 6-amino-6-deoxy-curdlan (Curimi polymers).
Fig. 1.
13
C NMR spectrum of Curimi-3 polymer in D2O.
Fig. 2. A. siRNA condensing ability of Curimi polymers. (A) The siRNA condensing ability was decreased when more imidazole groups were applied to positively charged amino group of 6AC-100. (B) TEM image of complex of Curimi nanoparticles and siRNAs. (C) Serum stability test. Curimi-3/siRNA complex was incubated in 50% serum at 37 °C for 30 min, followed by RNA extraction and agarose gel electrophoresis.
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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The particle size and zeta potential of the Curimi polymers complexed with siRNA were measured in neutral solution. The average size distribution of individual Curimi was in range of 84–105 nm at pH 7.4. However, the size of nanoparticles was increased as pH decreased (Table 1). When pH drops to 6.5, Curimi nanoparticles become partly protonated and its hydrophilicity increased. As a result, the nanoparticles began to swell to balance the increasing electrostatic repulsions. As pH goes down to 5.5 (endosomal pH), there was a significant nanoparticle size increased. The hydrodynamic diameter was enlarged to 600–800 nm. Because further disruption of the nanoparticle core forces ionized Curimi to dissociate from the particles, making nanoparticle disintegrate and aggregate into bigger formation. The size of the particles obtained by DLS measurement was bigger than that of TEM, which were consistent between the three different ratios of Curimi polymers. It can be explained that the particles may have shrank during the sample preparation process. At the same time, Zeta potential of the Curimi nanoparticles were observed at different pH value as same as the size distributions and the nanoparticles showed good stability (Table 1). The charges of the complexed nanoparticles increased by lowering pH value from 7.4 to 5.5, where ionization of the imidazole group occurred. The positive surface charge of nano-aggregates increased with decreasing pH due to enhanced ionization of imidazole groups, an effect more pronounced when more imidazole was introduced into the polymers. These results indicated that the nanoparticles could be stable at the tumor pH and would disintegrate at endosomal pH.
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3.3. Endosome buffering capacity measurements Acid-base titration was performed to assess endosome buffering capacity of Curimi polymers. Many works have demonstrated the important role of the endosome buffering capacities of polymeric gene delivery carriers regarding efficient endosomal escape and transfection. Fig. 3 presents the acid-base titration curves of Curimi polymers. All the polymers were shown a strong buffering effects and strength correlation of buffering effect was occurred with increasing imidazole rings on the polymers. Moreover, Curimi-3 had the strongest titration effect, which is modified with more imidazole rings, while 6AC-100 showed slightly proton sponging effect compared to Curimi polymers. The polar imidazole ring has serves as a pH sensitive fusogen because the imidazole has an electron lone pair on the unsaturated nitrogen. 3.4. Cytotoxicity assay Cytotoxicity assay was conducted to evaluate the effect of the Curimi polymer concentration on cell viability. MTT assay was performed to determine the cytotoxicity of Curimi polymers in two different cell lines, that is, HepG2 cells and HeLa cells, over a wide range of concentrations (20, 50, 80, 120 mg/mL). The 6AC-100 were used comparison groups (Fig. 4). The cell viability in the presence of 6AC-100 was decreased more with increasing
pH value
3.2. Particle size and zeta potential measurements by DLS
H2O 6AC-100 Curimi-1 Curimi-2 Curimi-3
8 6 4 2 0
0.1
0.2
0.3
0.4
0.5
0.6
0.1 M HCl (mL) Fig. 3. Buffering capacity of Curimi polymers. The increase in endosome buffering capacities was occurred in Curimi nanoparticles with the increase of imidazole rings.
polymer concentrations compared to Curimi polymers. At the highest concentration of 6AC-100 (120 mg/mL), the cell viability was around 65% compared to non-treated group in two cell lines. Whereas, slight changes in cell viability was observed in presence of Curimi polymers. Even with exposure to the highest concentration of Curimi polymers, more than ~80–90% of cells were remained viable in two cell lines, which suggested the all Curimi polymers exert negligible toxicity and they are highly compatible with living cells. Interestingly, the cell viability was slightly increased with increasing DS of Imidazole rings. It is plausible due to decreasing amount of free amine group on the polymer chains.
3.5. Cellular uptake and transfection efficiency of Curimi polymers The cellular uptake of FITC labelled siRNA by Curimi polymers were investigated using HepG2 cells by confocal laser scanning microscopy following the incubation of HepG2 cells with Curimi/ FITC-siRNA complexes for 6 h at 37 °C. As shown in Fig. 5, increased flourescence emission in the cells displayed higher cellular uptake of the complexes. Curimi polymers clearly exhibit an increasing cellular uptake in HepG2 cells compared to control 6AC-100, suggesting that those polymers show a significant enhancement of siRNA delivery. Moreover, to evaluate plasmid DNA delivering ability of Curimi polymers, 293 T cells were treated with GFP reporting plasmid DNA complexed with Curimi polymers for 24 h and flourescence emission in the cells was captured under electron microscopy (Fig. 6A). Quantification of GFP expressing cells by flow cytometry indicated that the plasmid DNA delivering efficiency of Curimi polymers increased with increasing degree of substitute of imidozole ring, with Curimi-3 showing the highest transfection efficiency of 55% GFP positive cells (Fig. 6B), suggesting endosome releasing ability of the polymers was enhanced by increasing imidazole rings on the polymers.
Table 1 Particle size and zeta potential of the siRNA-loaded Curimi nanoparticles at different pH (7.4, 5.5, 4.5), reported as mean ± SD (n = 3). Samples
Curimi-1 Curimi-2 Curimi-3
Particle size (d nm) +DNA (PDI)
Zeta potential (mV) +DNA
pH 7.4
pH 6.5
pH 5.5
pH 7.4
pH 6.5
pH 5.5
83.8 (0.143) 104.7 (0.259) 96.6 (0.415)
222.1 (0.450) 243.7 (0.337) 272.2 (0.194)
638.5 (0.421) 684.0 (0.273) 867.9 (0.252)
26.9 ± 2.55 25.1 ± 2.43 25.3 ± 2.75
28.9 ± 3.74 27.2 ± 4.43 29.0 ± 4.23
30.6 ± 3.54 31.6 ± 2.53 32.5 ± 3.63
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Fig. 4. Cell Viability studies of Curimi polymers at different concentrations in HepG2 and GES-1 cells. 6AC-100 was used for comparison group. Each value represents the mean ± SEM (n = 4). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT).
Fig. 5. Cellular uptake of Curimi nanoparticles with FITC-siRNA. (A) Confocal images of HepG2 cells treated with 6AC-100 or curimi nanoparticles complexed to FITC-siRNA; (B) Quantification of fluorescence signal of HepG2 cells transfected with 6AC-100- or curimi-siRNA complexes.
3.6. Endosome escape and transfection efficiency of Curimi polymers To investigates endosomal escape mechanism of Curimi polymers, we used two chemical reagents, chloroquine and nigericin. Chloroquine is an endosome disruptive agent by pH buffering in endosome [14]. Nigericin is a carboxylic ionophore which inhibits endosomal acidification by monovalent cation exchange [15]. HepG2 cells were pre-treated with the two chemicals in independent manner and the cells were co-transfected with pGFP complexed with Curimi polymers (Fig. 7A and B). The transfection efficiency of Curimi polymers and 6AC-100 in the presence of chloroquine were significantly decreased while the nigericin promotes the transfection efficiency of Curimi polymers. It was reported that chloroquine can support transfection efficiency of polyplexes having no endosome buffering ability [16]. In this experiment, the transfection efficiency of Curimi polymers in chloroquine condition decreased, revealing that Curimi polymers may have their own ability for endosome escape such as endosome buffering. In the presence of nigericin, the transfection efficiency of Curimi polymers and 6AC-100 increased to ~80% values in comparison with transfection without nigericin (Fig. 7B). It was reported
that cationic polymers having endosome buffering capacity such as PEI25k show reduced transfection efficiency in the presence of nigericin [16]. To monitor the process of endosome escape and evaluate the siRNA releasing ability of Curimi polymers, we used lysotracker to stain the lysosome in HepG2 cells by confocal laser scanning microscopy following the incubation of the cells with Curimi/ FITC-siRNA complexes for different incubation times at 37 °C. As shown in Fig. 7C, Curimi polymers clearly exhibit an increasing endosome escape, evidenced by disintegration of green (siRNA) and red dots (lysosome) 12 h after transfection, suggesting that those Curimi polymers show a significant enhancement of siRNA delivery. 3.7. siRNA delivery effect of Curimi polymers To test in vitro siRNA delivery effect of Curimi nanoparticles on HepG2 cells, we treated the cells with siRNA against Polo-like kinase-1 (Plk1) complexed to Curimi nanoparticles (Fig. 8). Plk1 plays a key role in driving mitotic events, including centrosome disjunction and movement, and is frequently over-expressed in
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Fig. 6. Plasmid transfection efficiency of Curimi polymers. 293T cells were treated with pcDNA3-eGFP complexed to Curimi-1, Curimi-2 or Curimi-3, respectively. 6AC-1-00 was used as a comparison group. (A) GFP expressing cells were observed under fluorescence microscope. (B) Quantification of GFP expression by flow cytometry.
Fig. 7. Transfection efficiency in HepG2 cells in the presence of 100 lM chloroquine (A) or 10 mM nigericin (B). Each value represents the mean ± SEM (n = 3). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT). (C) Staining of lysosome (lysotracker, red color) to monitor the process of endosome escape and release of FITC-siRNA (green) from Curimi and 6AC-100. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Fig. 8. Transfection efficiency of Curimi polymers in HepG2 cells. Curimi mediated delivery of siRNA against Plk1 induces decreased mRNA (A) and protein level (B) of Plk1. Each value represents the mean ± SEM (n = 4). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT).
human cancers [17]. Plk1 inhibition results in cell growth inhibition and DNA damage along with arresting cells in mitotic phase and leads to apoptosis [18,19,20]. Therefore, knockdown of Plk1 have been considered to be a promising therapeutic strategy. As shown in Fig. 8A, treatment of HepG2 cells with siPlk1 complex to Curimi polymers showed 55%–60% of gene silencing effect on mRNA level (Fig. 8A). Moreover, Western blot analysis showed that protein level of Plk1 was significantly decreased in the cells transfected with siPlk1/Curimi complexes, confirming the DNA damage induced by Plk1 knockdown (Fig. 8B).
4. Conclusion Novel pH-sensitive curdlan-based Curimi polymers were successfully synthesized and their siRNA condensing ability, buffering capacity, cellular toxicity and transfection efficiency were evaluated. The curdlan based novel Curimi polymers enhance the endosomal escape through increasing buffering capacity and efficiently deliver siRNA into cancer cells. Our results prove that adequate modification of 6AC-100 with different ratio of imidazole rings is an efficient approach to convert the polymer into a nucleic acid carrier. Acknowledgements This research was kindly supported by the National Natural Science Foundation of China (Grant number: 81560568, 21875124). References [1] H. Wu, L. Zhu, V.P. Torchilin, pH-sensitive poly(histidine)-PEG/DSPE-PEG copolymer micelles for cytosolic drug delivery, Biomaterials 34 (4) (2013) 1213– 1222. [2] Y. Wu, W. Gu, J. Tang, Z.P. Xu, Devising new lipid-coated calcium phosphate/carbonate hybrid nanoparticles for controlled release in endosomes for efficient gene delivery, J. Mater. Chem. B 5 (34) (2017) 7194– 7203.
[3] Y.J. Kwon, Before and after endosomal escape: Roles of stimuli-converting siRNA/polymer interactions in determining gene silencing efficiency, Acc. Chem. Res. 45 (7) (2012) 1077–1088. [4] D.C. Drummond, M. Zignani, J. Leroux, Current status of pH-sensitive liposomes in drug delivery, Prog. Lipid Res. 39 (5) (2000) 409–460. [5] J.S. Park, T.H. Han, K.Y. Lee, S.S. Han, J.J. Hwang, D.H. Moon, S.Y. Kim, Y.W. Cho, N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release, J. Control Release 115 (1) (2006) 37–45. [6] K. Seo, D. Kim, pH-dependent hemolysis of biocompatible imidazole-grafted polyaspartamide derivatives, Acta Biomater. 6 (6) (2010) 2157–2164. [7] D. Ma, Enhancing endosomal escape for nanoparticle mediated siRNA delivery, Nanoscale 6 (12) (2014) 6415–6425. [8] Y. Matsushita-Ishiodori, T. Ohtsuki, Photoinduced RNA interference, Acc. Chem. Res. 45 (7) (2012) 1039–1047. [9] W. Liang, J.K.W. Lam, Endosomal escape pathways for non-viral nucleic acid delivery systems, Mol. Regul. Endocytosis (2012). [10] R. Shrestha, M. Elsabahy, S. Florez-Malaver, S. Samarajeewa, K.L. Wooley, Endosomal escape and siRNA delivery with cationic shell crosslinked knedellike nanoparticles with tunable buffering capacities, Biomaterials 33 (33) (2012) 8557–8568. [11] Y. Wang, P. Li, F. Chen, L. Jia, Q. Xu, X. Gai, Y. Yu, Y. Di, Z. Zhu, Y. Liang, M. Liu, W. Pan, X. Yang, A novel pH-sensitive carrier for the delivery of antitumor drugs: histidine-modified auricularia auricular polysaccharide nano-micelles, Sci. Rep. 7 (1) (2017) 4751. [12] E.B. Anderson, T.E. Long, Imidazole- and imidazolium-containing polymers for biology and material science applications, Polymer 51 (12) (2010) 2447–2454. [13] J. Han, J. Cai, W. Borjihan, T. Ganbold, T.M. Rana, H. Baigude, Preparation of novel curdlan nanoparticles for intracellular siRNA delivery, Carbohydr. Polym. 117 (2015) 324–330. [14] M.A. Wolfert, L.W. Seymour, Chloroquine and amphipathic peptide helices show synergistic transfection in vitro, Gene Ther. 5 (1998) 409. [15] C. Uherek, J. Fominaya, W. Wels, A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery, J. Biol. Chem. 273 (15) (1998) 8835–8841. [16] Y.-b. Lim, S.-m. Kim, H. Suh, J.-s. Park, Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier, 2002. [17] L. Smith, R. Farzan, S. Ali, L. Buluwela, A. Saurin, D.W. Meek, The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation, Sci. Rep. 7 (1) (2017) 16115. [18] M.A. van Vugt, R.H. Medema, Getting in and out of mitosis with Polo-like kinase-1, Oncogene 24 (17) (2005) 2844–2859. [19] K. Strebhardt, Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy, Nat. Rev. Drug Discovery 9 (8) (2010) 643–660. [20] R. van der Meer, H.Y. Song, S.H. Park, S.A. Abdulkadir, M. Roh, RNAi screen identifies a synthetic lethal interaction between PIM1 overexpression and PLK1 inhibition, Clin. Cancer Res.: An Official J. Am. Assoc. Cancer Res. 20 (12) (2014) 3211–3221.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery Zhiyu Su, Tseyenkhorloo Erdene-Ochir, Tsogzolmaa Ganbold ⇑, Huricha Baigude * School of Chemistry & Chemical Engineering, Inner Mongolia Key Laboratory of Mongolian Medicinal Chemistry, Inner Mongolia University, 235 West College Road, Hohhot, Inner Mongolia 010021, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2019 Received in revised form 13 October 2019 Accepted 14 October 2019 Available online xxxx Keywords: siRNA delivery Curdlan Nanoparticle Endosome release Cancer cells
a b s t r a c t Developing nucleic acid-based tools to control disease-relevant gene expression in human disorders, such as siRNAs, opens up potential opportunities for therapeutics. Because of their high molecular weight and polyanionic nature, synthetic siRNAs fail to cross biological membranes by passive diffusion and therefore, generally require transmembrane siRNA delivery technologies to access the cytoplasm of target cells. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan derivative 6AC-100 in a regioselective manner to introduce different ratios of imidazole rings in the amino units (denoted as Curimi) and evaluated their siRNA binding ability, cytotoxicity, endosome buffering capacity and siRNA transfection efficiency. The novel curdlan based Curimi polymers formed nanoparticles with siRNA at pH 7.4 in range of 85–105 nm and their size distribution increased along with decreasing pH condition. The zeta potential increased by lowering pH value as well. Curimi polymers showed lower toxicity and higher buffering capacity compared to 6AC-100, and efficiently delivered siRNA against to PLK1 into cancer cells, and subsequently, significantly inhibited target mRNA level. Our result suggested that novel curdlan based Curimi polymers may be used as efficient siRNA carrier for cancer therapy. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction A number of polymeric nanoparticles for the intracellular delivery of siRNA have been previously studied. But lack of efficient siRNA release due to the endosomal trap, which could provide sufficient siRNA concentration and allows efficient down-regulation of specific gene transcription at mRNA level, can be another obstacle [1,2]. The interference of mRNA translation is proportional to the amount of delivered siRNA in the cytoplasm and maximum gene silencing efficiency is only obtained with delivery of siRNA above certain threshold. Therefore, siRNA should be dissociated from carrier in the cytoplasm for efficient gene silencing because only free forms of siRNA can specially bind to RISC in the cytoplasm [3]. Nanoparticles can be internalized into the cells via an endocytotic pathway through either specific (e.g., receptor-mediated endocytosis) or non-specific interactions (e.g., adsorptive endocytosis). Unfortunately, endocytosis generally guides the nanoparticles into the endosome, an intracellular membrane where they are fated to eventual degradation in lysosome [4–6]. An efficient ⇑ Corresponding author. E-mail addresses: [email protected] (T. Ganbold), [email protected] (H. Baigude).
polymeric siRNA carrier should be able to induce endosomal escape and rapidly release the maximum number of siRNA into the cytoplasm [3,7]. According to the endocytic pathway, a clathrin-coated vesicle containing nanoparticles will sequentially mature into an endosome with a pH of ~7–5.2, then into a lysosome with a pH of ~5.2–4.5. Thus it is important to develop a delivery system that is able to release siRNA just within the endosome and undergo subsequent endosomal escape and avoid from lysosomal digestion [2,5]. Facilitating endosome escape has long been the focus of siRNA delivery research. Depending on the type of nanoparticles, different strategies are used to enhance endosomal escape. Although external stimulations, such as photochemical internalization (PCI), are also used to improve endosome escape ability of nanoparticles, the common strategies are based on the acidic endosomal micro-environment, which is to improve pH buffering capacity and enhance ‘‘proton sponge” effect to destabilize the endosomal membrane [7,8]. The ‘‘Proton sponge” effect mostly occurs in certain cationic polymers with a high buffering capacity over a wide range of pH. These polymers usually contain protonatable secondary and/or tertiary amine groups with pKa close to endosomal/lysosomal pH [9]. In addition, several types of membrane disruptive polymers and imidazole-containing polymers have been investigated to facilitate
https://doi.org/10.1016/j.ijbiomac.2019.10.129 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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escape from the endosomes and achieve efficient transportation of genetic materials into the cytosol. In particular, imidazolecontaining polymers have gained much attention recently, as they exhibit pKa ~ 6 and can undergo protonation in the acidic endosomal environment leading, eventually, to efficient endosomal disruption [10]. The polar imidazole ring has serves as a pH sensitive fusogen because the imidazole has an electron lone pair on the unsaturated nitrogen. The polar imidazole ring, which contains two nitrogen separated with a methylene, hydrogen bonds through the amino hydrogen as the donor and the imino nitrogen as the acceptor [6,11,12]. The pKa of imidazole group is around 6, and therefore this group is protonated in slightly acidic milieu. Since the cationic polymer nanoparticles interact with negatively charged endosomal membranes, induces influx of water and ions, and eventually brings about endosomal destabilization and siRNA release [5]. In addition, pH sensitive polymer is focused on cancer therapy due to their pH dependent release. This strategy could not only stabilize the therapeutic system (polymer complex with anticancer drug) at neutral and tumor extracellular pH (~6.8), but also facilitate the disintegration of therapeutic system and the resultant quick drug release in response to the low endocytic pH (<6.0) upon 2C5-mediated internalization [1]. Based on these reports, in this study, we modified 6AC-100 with pH sensitive Imidazole group to improve buffering capacity and enhance endosomal escape of curdlan derivative 6AC-100. 2. Materials and methods 2.1. Materials Curdlan was purchased from Wako Pure Chemical Industries, Itd. (Osaka, Japan). Imidazole-4(5)-acetic acid hydrochloride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from TCI chemicals (Tokyo, Japan) and Aladdin (Shanghai, China), respectively. PLK1 gene silencing siRNA, PLK1 and b-actin primers, and fluorescein isothiocyanate (FITC)-siRNA were synthesized by Takara Biotechnology co., Ltd (Dalian, Liaoning, China). 2.2. Preparation of Curimi polymers 6-amino-6-dexoy-curdlan (6AC-100) was prepared as a previously reported method [13]. 40 mg 6AC-100 was dissolved in 2 mL of PBS and added 0.1 M HCl by pipetting until 6AC-100 was completely dissolved, and pH of the solution to pH ~ 9.0 was adjusted by 0.5 M NaOH. 1 mL mixture of imidazole-4(5)-acetic acid hydrochloride (4.8 or 12,1 or 24,2 mg) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (7,1 or 17,8 or 35,7 mg) in 0.1 M MES buffer (pH = 6.0) was previously prepared and then added into the 6AC-100 solution. The mixed solution was rotated continuously at room temperature for overnight. The new products (denoted as Curimi-1, Curimi-2 and Curimi-3) were purified by dialyzing (MWCO 3500) against deionized water to remove any trace of monomer for 2 days. The dialysis membrane was pre-wetted in water for 30 min before use. After the Curimi polymers were freeze-dried, the characteristic of Curimi polymers (solubilized in D2O) was analyzed by carbon nuclear magnetic resonance (13C NMR) analysis (500 MHz Bruker Avance III, Germany). 2.3. Measurements of particle size and zeta potential of Curimi nanoparticles The size and the zeta potential of the nanoparticles were determined in triplicates by dynamic light scattering (DLS) using
Zetasizer, Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). siRNAs were mixed with Curimi nanoparticles at a 6/1 ratio (N/P) in 100 lL ddH2O and the solutions were incubated at RT for 20 min. The samples were diluted 1:100 with double distilled water before measuring. 2.4. Transmission electron microscopy (TEM) Shape and surface morphological examination of Curimi/siRNA nanoparticles at N/P ratio 6/1 in aqueous solution was investigated by transmission electron microscopy (TEM). After Curimi nanoparticles and siRNA was mixed at a 6/1 ratio of N/P and incubated at RT for 20 min, two drops of sample were placed on a copper grid and air dried for 10 min, and then negatively stained with 2% phosphomolybdic acid solution for 10 min. The grid was air-dried for 10 min and examined under the TEM. 2.5. Buffering capacity measurements The endosome buffering capacities of Curimi polymers were measured by acid-base titration method. Each 10 mg of Curimi polymers was dissolved in 10 mL of dH2O and pH value of the solution was adjusted to pH 10 using 0.1 M NaOH. Then the solution was titrated by adding 10 mL of 0.1 M HCl until the pH dropped to ~2. 2.6. Electrophoretic mobility shift assay An electrophoretic mobility shift assay was used to investigate the integration of Curimi polymers and siRNA by electrostatic interaction. Curimi and siRNA complexes were prepared in aqueous solution at various N/P ratios and incubated at RT for 20 min, and then run into a 2% (w/v) agarose gel in 1X TBE buffer at 100 V for 20 min. The gel was stained with 0.5 mg/mL ethidium bromide for 20 min at RT and the RNA band was visualized on a UV illuminator using a Gel Logic 212 PRO imaging system (Carestream, Toronto, Canada). To evaluate the serum stability, Curimi nanoparticles (8 lg) and siRNA (100 lM) were incubated with an equal volume of FBS (final concentration 50% v/v) at 37 . After the samples were kept at 2, 6, 12, 24 and 48 h, 1 lL of 0.5 M EDTA was immediately added to stop any nuclease activity. Then, 1 lL of heparin (50 lg) was added to each sample to release the siRNA from each complex and the samples were subjected to gel electrophoresis. 2.7. Cell culture Human epitheloid cervix carcinoma (Hela) and Human liver hepatocellular carcinoma (HepG2) cells were maintained in high glucose Dulbecco’s modified eagle’s medium (DMEM) (BI, USA) supplemented with 5% (v/v) fetal bovine serum (FBS) (BI, Israel) and 1% (v/v) penicillin/streptomycin mixture (Gibco, Life technologies, USA) in culture dish (Corning, USA), and the cell cultures were grown in cell culture incubator (Nuaire, USA), under the cell culturing condition (atmosphere of 5% CO2 at 37 °C and 70–80% humidity). The cells were sub-cultured every two days. After the cell cultures were washed with 1X PBS (pH 7.2–7.4) (Biotopped, Life technologies) twice, added 3 mL of Trypsin (Gibco, Life technologies, USA) to detach the cells from the cell culture dish, and then incubated in cell culture incubator for 3–5 min. After 5 mL of pre-warmed DMEM medium was added to the detached cells, the cell suspension was removed into 15 mL centrifuge tube (Corning, Life technologies, USA), and centrifuged at 1000 rpm for 5 min using high-speed micro centrifuge (Hitachi, CF16RN, Japan). The upper layer was discarded and the cell pellet was resuspended in DMED medium. The cell suspension was poured into cell culture dish and cultured in cell culture incubator. The cell passaging
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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procedure was carried out in laminar flow hoods (Thermo scientific, USA). 2.8. Cellular toxicity assay To evaluate cellular toxicity of Curimi nanoparticles, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was carried out. Hela or HepG2 cells were seeded in a 96-well plate (Corning-Coaster, Tokyo, Japan) at a density of 1 104 cells/well in 100 mL DMEM medium and the cells were incubated in cell culture incubator under cell culturing condition for 24 h. The next day, the pre-cultured cells were treated with various final concentration of Curimi nanoparticles (20, 50, 80 and 120 mg/mL) in 100 mL of DMEM medium. 6AC-100 was used for comparison group. After the treated cells were incubated for another 24 h, previously prepared 50 mL of 1X MTT solution (final concertation; 0.2–0.5 mg/ml) was added to each well, and incubated at 37 °C for 4 h. After the viable cells with active metabolism convert MTT into a purple colored formazan product, the cell medium was removed, and 100 mL of DMSO was added to dissolve the formazan product. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer (FilterMax F5, Molecular Devices, USA). Filter-sterilize the MTT solution through a 0.2 mM filter into a sterile 2.9. Cellular uptake and lysosome tracking HepG2 cells were seeded in 24-well culture plates (CorningCoaster, Tokyo, Japan). When achieving 70–80% confluence, the cells were treated with the FITC-siRNA complexed with Curimi nanoparticles at the N/P ratio of 6 in 500 mL of serum-free optimem medium. The mixture (Curimi/FITC-siRNA) was incubated at RT in 100 mL of serum-free optimem medium for 20 min to condense Curimi polymer and siRNA in nanoparticle formation prior to transfection and another 400 mL of optimem medium was added to the transfection solution. After 30 min, 2 h, 4 h and 12 h incubation respectively, the cells were stained with Lyso-Tracker Red (dilution at 1: 5000, Solarbio Life Science, China) at RT for 2 h. After washed with 1X PBS (pH 7.2–7.4) 3 times, the cells were fixed in 4% paraformaldehyde for 15 min, RT. Then washed with 1X PBS 3 times, cells were permeabilized with 0.2% Triton X-100 in PBS, 5 min at RT. After washed with 1X PBS, nucleus was stained with DAPI (40 ,6-diamidino-2-phenylindole) at a final concentration of 0.5 mg/mL for 5 min at RT by protecting from light. The stained cells were rinsed in 1X PBS 3 times and dried the coverslips in air. The coverslips were mounted using mounting medium and then stored at 4 °C until capturing cell images for about 1–3 days. The cell images were captured under confocal laser-scanning microscopy (Olympus, Fluoview FV 1000). 2.10. Western blotting assay HepG2 cells were seeded in 6-well plate (4 105 per well). After 24 h, either siControl or siPlk1 (final concentration at 50 nM) were transfected with Curimi-1, -2 and -3, respectively. 24 h later, the cell lysates were harvested and total protein was extracted with Mammalian Protein Extraction Kit (KWbiotech, Beijing, China). The concentration of total proteins was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (25 mg total proteins) either from siControl or siPLK1 treated cells was loaded and separated on SDS-PAGE gel electrophoresis. Proteins were transferred to membrane and incubated with antibodies (PLK1 Rabbit mAb, 1:1000, #4513; Phospho-Histone H2A.X (Ser139) Rabbit mAb, 1:1000, #9718;
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GAPDH Rabbit mAb, 1:1000, #2118; Cell Signaling Technology, Danvers, MA, USA). After incubation with secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), 1:4000, ab97051, Abcam), images were acquired by using chemiluminesence detection. 2.11. Transfection with chloroquine or nigericin In order to investigate the endosomal escape mechanism of Curimi nanoparticles, we used two kinds of chemical reagents, chloroquine or nigericin. HepG2 Cells were seeded in a 12-well plate in DMEM medium 5% FBS and 1% penicillin/streptomycin. When the cell were grown to reach 70–80% confluence, the cells were pre-incubated in serum-free Opti-mem medium containing 100 mM chloroquine or 10 mM nigericin for 30 min, and the cells were treated with Curimi nanoparticles complexed with the green florescence protein reporter plasmid DNA (pDNA) in 500 mL of serum free optimem medium. After 4 h of incubation, the medium was exchanged with fresh DMEM medium containing 5% FBS and 1% penicillin/streptomycin and the cells were incubated for further 2 days at 37 °C in 5% CO2. Subsequently, medium was removed and the cells were rinsed with 1X PBS. After the wells were trypsinised, the cell fluorescence intensity was measured on a luminometer (FilterMax F5, Molecular Devices, USA). 3. Results and discussions To explore potential of curdlan, curdlan modified derivative 6AC-100 polymer was conjugated with a layer of imidazole groups at three different ratios of imidazole molecule (Scheme 1; designated as Curimi-1, Curimi-2 and Curimi-3). The conjugated imidazole rings on 6AC-100 was determined using 13C NMR analysis and the molecular weight distribution of the polymers was measured by gel permeation chromatography (GPC). In 13C NMR, the newly attached imidazole moiety showed a signal at 174.32 ppm (C = O), confirming the imidazole ring successfully connected on the curdlan backbone (Fig. 1). The molecular weight distribution of Curimi-1 was Mn of 23.22 kDa and Mw of 45.61. Curimi-2 and Curimi-3 polymers gave Mn of 22.71 kDa and Mw of 53.95 and, Mn of 23.07 kDa and Mw of 61.72 kDa, respectively, by GPC analysis. The molecular weight of Curimi polymers was similar because the conjugation of moieties on the 6AC-100 backbone resulted in minor differences in the final products. The degree of substitution (DS) of Curimi nanoparticles were 5,2% (Curimi-1), 10.1% (Curimi2) and 24,7% (Curimi-3) by elemental analysis. 3.1. RNA binding assay and particle size measurement by TEM The positive charge of Curimi polymers and negative charge on nucleic acid have tendency to enforce electrostatic interactions between them to form Curimi/siRNA nanoparticles, which was evaluated by gel shift assay using 2.0% agarose gel electrophoresis (Fig. 2A). The migration of Curimi-1/siRNA and Curimi-2/siRNA complexes in gel were completely retarded at N/P ratio of 3 and 4, respectively, while Curimi-3/siRNA complexes were totally retarded at N/P ratio 6. The siRNA condensing ability of Curimi polymers was decreased when more imidazole was incorporated to 6AC-100 due to their positive charge ratio on the polymers, as well as sterical hindrance. Moreover, the morphology imaging of the polymers was captured under transmission electron microscopy (TEM). The result showed spherical particles encapsulating siRNA have diameters about 55 nm (Fig. 2B), suggesting that Curimi polymers have a great ability to bind siRNA via electrostatic interaction. Moreover, Curimi polymers protected siRNA from degradation by serum nuclease, indicating that Curimi/siRNA complexes have similar stability with the positive control (6AC-100) in serum (Fig. 2C).
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Scheme 1. Synthesis of imidazole modified 6-amino-6-deoxy-curdlan (Curimi polymers).
Fig. 1.
13
C NMR spectrum of Curimi-3 polymer in D2O.
Fig. 2. A. siRNA condensing ability of Curimi polymers. (A) The siRNA condensing ability was decreased when more imidazole groups were applied to positively charged amino group of 6AC-100. (B) TEM image of complex of Curimi nanoparticles and siRNAs. (C) Serum stability test. Curimi-3/siRNA complex was incubated in 50% serum at 37 °C for 30 min, followed by RNA extraction and agarose gel electrophoresis.
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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The particle size and zeta potential of the Curimi polymers complexed with siRNA were measured in neutral solution. The average size distribution of individual Curimi was in range of 84–105 nm at pH 7.4. However, the size of nanoparticles was increased as pH decreased (Table 1). When pH drops to 6.5, Curimi nanoparticles become partly protonated and its hydrophilicity increased. As a result, the nanoparticles began to swell to balance the increasing electrostatic repulsions. As pH goes down to 5.5 (endosomal pH), there was a significant nanoparticle size increased. The hydrodynamic diameter was enlarged to 600–800 nm. Because further disruption of the nanoparticle core forces ionized Curimi to dissociate from the particles, making nanoparticle disintegrate and aggregate into bigger formation. The size of the particles obtained by DLS measurement was bigger than that of TEM, which were consistent between the three different ratios of Curimi polymers. It can be explained that the particles may have shrank during the sample preparation process. At the same time, Zeta potential of the Curimi nanoparticles were observed at different pH value as same as the size distributions and the nanoparticles showed good stability (Table 1). The charges of the complexed nanoparticles increased by lowering pH value from 7.4 to 5.5, where ionization of the imidazole group occurred. The positive surface charge of nano-aggregates increased with decreasing pH due to enhanced ionization of imidazole groups, an effect more pronounced when more imidazole was introduced into the polymers. These results indicated that the nanoparticles could be stable at the tumor pH and would disintegrate at endosomal pH.
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3.3. Endosome buffering capacity measurements Acid-base titration was performed to assess endosome buffering capacity of Curimi polymers. Many works have demonstrated the important role of the endosome buffering capacities of polymeric gene delivery carriers regarding efficient endosomal escape and transfection. Fig. 3 presents the acid-base titration curves of Curimi polymers. All the polymers were shown a strong buffering effects and strength correlation of buffering effect was occurred with increasing imidazole rings on the polymers. Moreover, Curimi-3 had the strongest titration effect, which is modified with more imidazole rings, while 6AC-100 showed slightly proton sponging effect compared to Curimi polymers. The polar imidazole ring has serves as a pH sensitive fusogen because the imidazole has an electron lone pair on the unsaturated nitrogen. 3.4. Cytotoxicity assay Cytotoxicity assay was conducted to evaluate the effect of the Curimi polymer concentration on cell viability. MTT assay was performed to determine the cytotoxicity of Curimi polymers in two different cell lines, that is, HepG2 cells and HeLa cells, over a wide range of concentrations (20, 50, 80, 120 mg/mL). The 6AC-100 were used comparison groups (Fig. 4). The cell viability in the presence of 6AC-100 was decreased more with increasing
pH value
3.2. Particle size and zeta potential measurements by DLS
H2O 6AC-100 Curimi-1 Curimi-2 Curimi-3
8 6 4 2 0
0.1
0.2
0.3
0.4
0.5
0.6
0.1 M HCl (mL) Fig. 3. Buffering capacity of Curimi polymers. The increase in endosome buffering capacities was occurred in Curimi nanoparticles with the increase of imidazole rings.
polymer concentrations compared to Curimi polymers. At the highest concentration of 6AC-100 (120 mg/mL), the cell viability was around 65% compared to non-treated group in two cell lines. Whereas, slight changes in cell viability was observed in presence of Curimi polymers. Even with exposure to the highest concentration of Curimi polymers, more than ~80–90% of cells were remained viable in two cell lines, which suggested the all Curimi polymers exert negligible toxicity and they are highly compatible with living cells. Interestingly, the cell viability was slightly increased with increasing DS of Imidazole rings. It is plausible due to decreasing amount of free amine group on the polymer chains.
3.5. Cellular uptake and transfection efficiency of Curimi polymers The cellular uptake of FITC labelled siRNA by Curimi polymers were investigated using HepG2 cells by confocal laser scanning microscopy following the incubation of HepG2 cells with Curimi/ FITC-siRNA complexes for 6 h at 37 °C. As shown in Fig. 5, increased flourescence emission in the cells displayed higher cellular uptake of the complexes. Curimi polymers clearly exhibit an increasing cellular uptake in HepG2 cells compared to control 6AC-100, suggesting that those polymers show a significant enhancement of siRNA delivery. Moreover, to evaluate plasmid DNA delivering ability of Curimi polymers, 293 T cells were treated with GFP reporting plasmid DNA complexed with Curimi polymers for 24 h and flourescence emission in the cells was captured under electron microscopy (Fig. 6A). Quantification of GFP expressing cells by flow cytometry indicated that the plasmid DNA delivering efficiency of Curimi polymers increased with increasing degree of substitute of imidozole ring, with Curimi-3 showing the highest transfection efficiency of 55% GFP positive cells (Fig. 6B), suggesting endosome releasing ability of the polymers was enhanced by increasing imidazole rings on the polymers.
Table 1 Particle size and zeta potential of the siRNA-loaded Curimi nanoparticles at different pH (7.4, 5.5, 4.5), reported as mean ± SD (n = 3). Samples
Curimi-1 Curimi-2 Curimi-3
Particle size (d nm) +DNA (PDI)
Zeta potential (mV) +DNA
pH 7.4
pH 6.5
pH 5.5
pH 7.4
pH 6.5
pH 5.5
83.8 (0.143) 104.7 (0.259) 96.6 (0.415)
222.1 (0.450) 243.7 (0.337) 272.2 (0.194)
638.5 (0.421) 684.0 (0.273) 867.9 (0.252)
26.9 ± 2.55 25.1 ± 2.43 25.3 ± 2.75
28.9 ± 3.74 27.2 ± 4.43 29.0 ± 4.23
30.6 ± 3.54 31.6 ± 2.53 32.5 ± 3.63
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Fig. 4. Cell Viability studies of Curimi polymers at different concentrations in HepG2 and GES-1 cells. 6AC-100 was used for comparison group. Each value represents the mean ± SEM (n = 4). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT).
Fig. 5. Cellular uptake of Curimi nanoparticles with FITC-siRNA. (A) Confocal images of HepG2 cells treated with 6AC-100 or curimi nanoparticles complexed to FITC-siRNA; (B) Quantification of fluorescence signal of HepG2 cells transfected with 6AC-100- or curimi-siRNA complexes.
3.6. Endosome escape and transfection efficiency of Curimi polymers To investigates endosomal escape mechanism of Curimi polymers, we used two chemical reagents, chloroquine and nigericin. Chloroquine is an endosome disruptive agent by pH buffering in endosome [14]. Nigericin is a carboxylic ionophore which inhibits endosomal acidification by monovalent cation exchange [15]. HepG2 cells were pre-treated with the two chemicals in independent manner and the cells were co-transfected with pGFP complexed with Curimi polymers (Fig. 7A and B). The transfection efficiency of Curimi polymers and 6AC-100 in the presence of chloroquine were significantly decreased while the nigericin promotes the transfection efficiency of Curimi polymers. It was reported that chloroquine can support transfection efficiency of polyplexes having no endosome buffering ability [16]. In this experiment, the transfection efficiency of Curimi polymers in chloroquine condition decreased, revealing that Curimi polymers may have their own ability for endosome escape such as endosome buffering. In the presence of nigericin, the transfection efficiency of Curimi polymers and 6AC-100 increased to ~80% values in comparison with transfection without nigericin (Fig. 7B). It was reported
that cationic polymers having endosome buffering capacity such as PEI25k show reduced transfection efficiency in the presence of nigericin [16]. To monitor the process of endosome escape and evaluate the siRNA releasing ability of Curimi polymers, we used lysotracker to stain the lysosome in HepG2 cells by confocal laser scanning microscopy following the incubation of the cells with Curimi/ FITC-siRNA complexes for different incubation times at 37 °C. As shown in Fig. 7C, Curimi polymers clearly exhibit an increasing endosome escape, evidenced by disintegration of green (siRNA) and red dots (lysosome) 12 h after transfection, suggesting that those Curimi polymers show a significant enhancement of siRNA delivery. 3.7. siRNA delivery effect of Curimi polymers To test in vitro siRNA delivery effect of Curimi nanoparticles on HepG2 cells, we treated the cells with siRNA against Polo-like kinase-1 (Plk1) complexed to Curimi nanoparticles (Fig. 8). Plk1 plays a key role in driving mitotic events, including centrosome disjunction and movement, and is frequently over-expressed in
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Fig. 6. Plasmid transfection efficiency of Curimi polymers. 293T cells were treated with pcDNA3-eGFP complexed to Curimi-1, Curimi-2 or Curimi-3, respectively. 6AC-1-00 was used as a comparison group. (A) GFP expressing cells were observed under fluorescence microscope. (B) Quantification of GFP expression by flow cytometry.
Fig. 7. Transfection efficiency in HepG2 cells in the presence of 100 lM chloroquine (A) or 10 mM nigericin (B). Each value represents the mean ± SEM (n = 3). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT). (C) Staining of lysosome (lysotracker, red color) to monitor the process of endosome escape and release of FITC-siRNA (green) from Curimi and 6AC-100. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129
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Fig. 8. Transfection efficiency of Curimi polymers in HepG2 cells. Curimi mediated delivery of siRNA against Plk1 induces decreased mRNA (A) and protein level (B) of Plk1. Each value represents the mean ± SEM (n = 4). The symbol ‘‘*” indicates significant different (p < 0.05) compared to non-treated (NT).
human cancers [17]. Plk1 inhibition results in cell growth inhibition and DNA damage along with arresting cells in mitotic phase and leads to apoptosis [18,19,20]. Therefore, knockdown of Plk1 have been considered to be a promising therapeutic strategy. As shown in Fig. 8A, treatment of HepG2 cells with siPlk1 complex to Curimi polymers showed 55%–60% of gene silencing effect on mRNA level (Fig. 8A). Moreover, Western blot analysis showed that protein level of Plk1 was significantly decreased in the cells transfected with siPlk1/Curimi complexes, confirming the DNA damage induced by Plk1 knockdown (Fig. 8B).
4. Conclusion Novel pH-sensitive curdlan-based Curimi polymers were successfully synthesized and their siRNA condensing ability, buffering capacity, cellular toxicity and transfection efficiency were evaluated. The curdlan based novel Curimi polymers enhance the endosomal escape through increasing buffering capacity and efficiently deliver siRNA into cancer cells. Our results prove that adequate modification of 6AC-100 with different ratio of imidazole rings is an efficient approach to convert the polymer into a nucleic acid carrier. Acknowledgements This research was kindly supported by the National Natural Science Foundation of China (Grant number: 81560568, 21875124). References [1] H. Wu, L. Zhu, V.P. Torchilin, pH-sensitive poly(histidine)-PEG/DSPE-PEG copolymer micelles for cytosolic drug delivery, Biomaterials 34 (4) (2013) 1213– 1222. [2] Y. Wu, W. Gu, J. Tang, Z.P. Xu, Devising new lipid-coated calcium phosphate/carbonate hybrid nanoparticles for controlled release in endosomes for efficient gene delivery, J. Mater. Chem. B 5 (34) (2017) 7194– 7203.
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Please cite this article as: Z. Su, T. Erdene-Ochir, T. Ganbold et al., Design of curdlan-based pH-sensitive polymers with endosome buffering functionality for siRNA delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.10.129