Redox-Sensitive Gelatin/Silica-Aptamer Nanogels for Targeted siRNA Delivery
RNA interference (RNAi) has potential advantages over other gene therapy approaches due to its high specificity and the ability to inhibit target gene expression. However, the stability and tissue-specific delivery of siRNA remain as the biggest obstacles for RNAi therapeutics. Here, we developed such a system by conjugating gelatin-based nanogels with the nucleolin-targeted AS1411 aptamer and deoxynucleotide-substituted siRNA together (Apt-GS/siRNA) via a disulfide linker to achieve transient docking of siRNA. These Apt-GS/siRNA nanogels demonstrated favorable release of siRNA under reducing conditions owing to disulfide cleavage. Furthermore, this smart system could electively release siRNA into the cytosol in nucleolin-positive cells (A549) by a glutathione-triggered disassembly and subsequently efficient RNAi for luciferase. Besides, disulfide-equipped Apt-GS nanogels showed good biocompatibility in vitro. Taken together, this redox-responsive, tumor-targeting smart nanogels display great potential in exploiting functionalized siRNA delivery and tumor therapy.
KeywordssiRNA delivery Anti-nucleolin aptamer AS1411 Nucleolin targeting Gene therapy RNA interference
Dulbecco’s modified Eagle’s medium
- GS NGs
Siloxane-crosslinked gelatin nanogels
Small interfering RNAs
Negative DNA oligonucleotide
RNA interference (RNAi) is a sequence-specific silencing of genes and also currently being evaluated as a most promising method in the treatment of a wide range of diseases including genetic disorders, cancers, and infectious diseases, owing to its high specificity and low toxicity . Small interfering RNAs (siRNAs) with double-stranded RNA molecules are more resistant to nuclease degradation than antisense oligonucleotides and therefore show advantages over antisense therapy. Incorporation of chemically modified nucleotides into siRNAs was used to increase or decrease the efficacy and the duration of RNAi. Replacing the ribonucleotides of sense strand with deoxynucleotides has been proved could increase the stability of sense strand and keep ∼ 40% efficacy of RNAi [2, 3]. However, tissue-specific delivery of siRNA remains to be a big obstacle for its applications despite that the great gene knockdown potencies observed in the in vitro studies [4, 5].
Nanoparticle-based delivery has potential advantages in effective siRNA stabilization and further modification for site-specific delivery [6, 7, 8]. Productive site-directed delivery of siRNA can enhance transfection and reduce off-target effects, which are required by most practical applications . Nucleolin is associated with diverse biological processes and wildly expressed in the nucleus and cytoplasm of various normal cells. Moreover, nucleolin is highly expressed on the plasma membranes of actively proliferating cancer cells relative to their normal counterparts and hence used as an attractive target for antineoplastic treatments. Aptamer AS1411 (also known as AGRO100), a G-quartet DNA aptamer, can strongly bind to cell surface nucleolin and block the antiapoptotic pathway in cancer cells by combining with nuclear factor-kB essential modulator, which has reach phase II clinical trials as the first nucleic acid aptamer drug for the treatment of cancer in humans [10, 11]. Thus far, AS1411 has been successfully conjugated to various nanoparticles and internalized them into cancer cells [12, 13, 14, 15]. Gelatin has been employed for siRNA encapsulation due to its excellent biocompatibility, biodegradability, and gelation properties [16, 17, 18]. Our group has explored a series of siloxane-crosslinked gelatin nanogels (GS NGs) with controlled size and surface charge, demonstrating the transfection efficiency of GS NGs in vitro and in vivo [19, 20].
Besides intracellular barriers, intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape equally remain challenging. Conjugation of siRNA to a carrier through labile or nonlabile bonds is a promising method for overcoming this delivery challenge. Stimuli-responsive release under acidic pH and redox potential has received great interest. Disulfide cross-link displays great potential in a burst of drug release via cleaved linkages in tumor cells due to higher glutathione (GSH) level in the extracellular environment [21, 22]. It has reported that poly (lactic-co-glycolic acid) (PLGA)/siRNA conjugates formed via a disulfide bond exhibit an enhanced encapsulation and delivery efficiency .
Gelatin (bloom number: 240-270, pH 4.5–5.5) was purchased from BBI Company Inc. (USA). N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), 3-glycidoxypropyl-trimethoxysilane (GPSM), and 3-aminopropyl-trimethoxysilane (APTMS) were purchased from Sigma-Aldrich Co. (USA). 3-(4, 5-Dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Amresco Co. (USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin, and streptomycin were obtained from Hyclone Co. (USA). AS1411 aptamer, 5′-FAM-labeled AS1411, and negative control DNA (TDO) were synthesized by Sangon Biotech Co. (China). Thiol sense strand of the Luc-siRNA, 5′-SH-(CH2)6-CTTACGCTGAGTACTTCGATT-3′ (deoxynucleotides substitute for ribonucleotides) and anti-sense strand, 3′-TTGAAUGCGACUCAUGAAGCU-5′, and FAM-labeled anti-sense strand at the 3′ end were synthesized and purified with HPLC by Gene Pharma Co. (China). Lipofectamine 2000, luciferase plasmid pGL3, and luciferase assay system were purchased from Promega Co. (USA). The malignant human lung adenocarcinoma A549 cells and normal NIH 3T3 fibroblasts were used and provided by the Biomedical Engineering Centre of Xiamen University (China). All materials used were of analytical grade and without further purification. The glassware was thoroughly cleaned and rinsed with deionized water.
DNA sequences are as follows:
AS1411 aptamer: 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTTTTTTTTT-3′, Control TDO: 5′-CACCGGGAGGATAGTTCGGTGGCTGTTTTTTTTTTTTT-3′
Preparation and Characterization of Apt-GS/siRNA Complexes
Amino-functionalized gelatin/silica nanogels (GS NGs) were first prepared by a sol-gel procedure, as previously described . Typically, 0.2 g of GPSM was added to 20 mL of 0.75% gelatin solution in HCl solution (pH = 3) at 60 °C under 30 min of stirring, and subsequently adding 0.08 g APTMS and incubating for another 24 h. The obtained GS NGs were purified by centrifugation (14,000 rpm, 25 °C, 12 min) for three times. Secondly, GS NGs (0.5 mL, 5 g/L in PBS) were treated with 25 uL SPDP (20 mM in DMSO) for 60 min at room temperature, followed by the addition of thiolated AS1411 (1 mg/mL) and stirring for 12 h. Apt-GS nanogels were obtained by centrifugation (14,000 rpm, 25 °C, 12 min) and purified with deionized water thrice. Thirdly, the obtained SPDP-activated Apt-GS NG suspension (80 mL, 5 mg/mL) was directly mixed with siRNA sense strand overnight at 4 °C. After purification by centrifugation, the siRNA anti-sense strand was added and incubated for 5 min at 94 °C, then annealed for 20 min at 47 °C to form Apt-GS/siRNA complexes.
The surface morphologies of AS1411-GS and AS1411-GS/siRNA complexes were examined by transmission electron microscopy (TEM) (Hitachi S-4300, Japan). The particle size and zeta potentials of each sample were measured by a Nano-ZS Zetasizer dynamic light scattering detector (Malvern Instruments, UK). The effective particle diameters were calculated from the autocorrelation function using the Malvern Zetasizer software assuming a log-normal distribution. The conjugations of GS and AS1411 or siRNA were determined by collecting and measuring the concentration of unattached FAM-labeled DNA using F-7000 fluorescence microscopy (Olympus-IX73, Japan). Total amino group (-NH2) levels on the surface of GS nanogels were quantitatively determined by using the ninhydrin colorimetric reaction. The amount was about 0.642 mmol/g.
Gel Electrophoresis Analysis
Gel electrophoresis was conducted at 100 V for 20–30 min using 2% (w/v) agarose gel in TBE buffer. Images were observed by irradiation with a gel documentation system (Tanon GIS-2008, China). In the Apt-GS/siRNA encapsulation assay, the naked siRNA, GS/siRNA, and Apt-GS/siRNA complexes were loaded into the gel without any additives. In redox-responsive assays, GS/siRNA and Apt-GS/siRNA complex were incubated with 10 mM GSH solution in PBS for 2 h before measurement.
In Vitro Cytotoxicity
The cytotoxicity against human lung adenocarcinoma A549 cells was evaluated by MTT assay. Briefly, A549 cells (1 × 104 cells/well) were seeded in polystyrene 96-well culture plates and incubated for 24 h till the 70% confluence. After removing the culture medium, 100 μL of serum-free DMEM medium containing nanogels (100–600 mg/mL) was added to each well. Cells treated with medium only served as a negative control group. After 24 h co-incubation, 100 μL of fresh medium with 20 μL of MTT solution (5 mg/mL in PBS buffer) was added and cultured for another 4 h. Then, MTT solution was removed and 100 μL of dimethyl sulfoxide (DMSO) was added. After oscillating for 30 min in the dark, the absorbance of each well was measured by a micro-plate reader (TECAN DNA export, Swiss) at the 490 nm wavelength. All experiments were conducted in triplicate. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells.
The cells are co-cultured with 25 μL of FAM-labeled Apt-GS and TDO-GS nanogels (2 mg/mL) for 10 h in serum-free medium at 37 °C. After washing three times with PBS, cells were fixed with paraformaldehyde (4% in PBS) for 30 min and then observed with confocal laser scanning microscope (Leica, Germany). To quantify the cellular uptake, the cells were treated with 25 μL of FAM-labeled AS1411-GS and TDO-GS (2 mg/mL) for a period (0.5–16 h) in serum-free medium at 37 °C. To evaluate the role of nucleolin in the cellular uptake of the Apt-GS NGs, the mixture of the prepared Apt-GS NGs and free AS1411 aptamers of varied concentrations (0.5, 5, and 50 μM) was incubated with the A549 cells for 8 h. Then, cells were re-suspended in ice-cold PBS, and the fluorescent cells with FAM-labeled nanogels were counted from 10,000 cells by using an EPICS XL flow cytometer (Beckman Coulter, USA). Data analysis was performed with EPICS XL flow cytometer software, and analytical gates were chosen as 1% of control cells falling within the positive region.
The ability to silence genes was examined using the combination of siRNA for luciferase and the luciferase-expressing cells. Briefly, luciferase-expressing A549 (2 × 105 cells/well) and NIH 3 T3 fibroblasts (4 × 105 cells/well) were seeded in 12-well plate and cultured overnight and then treated with 100 μL of test complexes (80 μg/mL) containing LUC-siRNA (or control siRNA) for 8 h. The samples were divided into three groups: (a) only Apt-GS/siRNA complexes for 8 h, (b) Apt-GS/siRNA complexes for 8 h followed by liposomes for further 1 h (named as A-GS/si→Lip group), and (c) co-incubation of Apt-GS/siRNA complexes and liposome for 8 h (A-GS/si+Lip group). The cells without any treatment and those transfected with luciferase plasmid pGL3 (Madison, WI, USA) were as controls. After further 40 h incubation, gene knockdown efficiency was investigated by quantifying the luciferase expression in cells using a luciferase assay system according to the manufacturer’s protocol. Experiments were carried out in triplicate, and data are shown as means ± standard errors of the means.
Results and Discussions
Synthesis and Characterization of GS-AS1411/siRNA Complexes
siRNA Loading and Release
Cytotoxicity and Nucleolin Targeting
Next, we examined the concentration effect of nanogels on the RNAi. As shown in Fig. 7b, gene interference efficiency reached the maximum at the complex concentration of 80 μg/ml. The decreased KD efficiency at a concentration of 120 μg/ml may due to an increased cytotoxicity from aptamer. It is also noted that addition of an endosome escape agent liposome can increase RNA interference efficiency at low concentrations of Apt-GS/siRNA nanogels (40 μg/ml and 80 μg/ml) but did not lead to a reduction at high concentrations (120 μg/ml). Thus, it is believed that Apt-GS can successfully deliver siRNA to the tumor cells and subsequently result in specific RNAi.
The stability and tissue-specific delivery of siRNA remain as the biggest obstacles for RNAi therapeutics. Here, deoxynucleotide-substituted siRNA is used to improve stability of siRNA, and GS core was further coated with AS1411 aptamer for tumor targeting and disulfide linker for redox-responsive delivery of siRNA. The vehicle has a spherical structure with a size of about 200 nm and possesses positive charges at the surface. These as-prepared Apt-GS/siRNA nanogels could protect the cargo and exhibited accelerated siRNA release under DTT adding by disulfide cleavage. Moreover, with the targeting aptamer AS1411, the new Apt-GS nanogels Apt-GS could effectively deliver and release more cargo to the cytoplasm, leading to a significant (~ 5 folds in vitro improvement in silence activity in nucleolin-overexpressing A549 cells). Overall, these findings suggest that delivery of deoxynucleotide-substituted siRNA is an innovative strategy and these redox-responsive, tumor-targeting smart nanogels hold great promise for siRNA delivery and tumor therapy.
ZX and XY performed the experiments and prepared the manuscript. XY and ZY participated in the cell access of siRNA carrier. WQ and MR participated in the synthesis and characterization of nanoparticles in the study coordination discussion of the results. ZB and RL supervised the whole work and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by the Zhejiang Provincial Natural Science Foundation of China (No. LY18C100002) and National Natural Science Foundation of China (Nos. 31371012 and 31400797).
The authors declare that they have no competing interests.
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