1. Introduction
RNA interference (RNAi), triggered by small interfering RNA (siRNA), is a sequence-specific post–transcriptional gene silencing process that provides a powerful approach to interfere with the function of any disease–associated protein in a highly selective manner [
1,
2]. Therefore, siRNAs have been emerging as promising therapeutic reagents for the treatment of inherited and acquired diseases [
2]. So far, six siRNA drugs have been approved for marketing worldwide [
3]. A growing number of clinical trials of RNAi–based solid tumors treatment further demonstrate the potential of siRNA therapeutics in treating cancer patients by specifically turning off gene expression that promotes cancer cell survival, migration, and tumorigenicity [
4]. However, the therapeutic outcomes extremely rely on the safe and efficient delivery of siRNA therapeutics in target cells, as they are susceptible to nuclease degradation and renal clearance [
5]. Moreover, siRNA molecules are hydrophilic biomacromolecules with strongly negative charge, which prevents them from spontaneously crossing cell membranes [
6,
7]. During the past decades, tremendous efforts have been dedicated to exploit delivery vehicles for siRNA therapeutics [
5,
6,
7]. However, there is still an unmet need for the specific delivery of siRNA therapeutics to disease–related cells [
7,
8]. Given this, the development of on–demand delivery vehicles capable of transporting siRNA specifically to cancer cells is highly desirable and also of paramount importance for RNAi–based cancer therapy.
Lipids and polymers are the two most advanced vectors for siRNA therapeutics in clinical and preclinical studies [
9]. For instance, the first siRNA drug approved by FDA, Onpattro [
10], is a lipid nanoparticle formulation, while the first clinic trial of siRNA therapeutics is a polymer–based formulation [
11]. Dendrimers, a special type of polymers, are emerging as promising delivery vehicles for siRNA therapeutics by virtue of their well-defined molecular structure and unique multivalent cooperativity [
12,
13]. In particular, amphiphilic dendrimers, a kind of lipid/dendrimer hybrid with judiciously designed hydrophilic and hydrophobic components, exhibit excellent performance on siRNA delivery thanks to the combined advantageous of lipid and polymer vectors as well as unique characteristics of dendrimer vectors [
14,
15]. We and others have recently established several types of amphiphilic dendrimers, including amphiphilic poly(amidoamine) (PAMAM) dendrimers [
16,
17], amphiphilic peptide dendrimers [
18], amphiphilic poly(aminoester) dendrimers [
19], and amphiphilic polyglycerol dendrimers [
20], all of which possess the excellent capability of delivering siRNA therapeutics for the treatment of different diseases. Therefore, amphiphilic dendrimers are able to be a promising platform for elaborating on–demand vectors for cancer cell–specific siRNA delivery.
The tumor microenvironment has a variety of atypical hallmarks, such as hypoxia, abnormal redox environment, low pH, upregulated secretion of specific enzymes, overexpression of inflammatory meditators, etc. [
21]. Notably, the hypoxia environment within the tumor tissues leads to the continuous production of reactive oxygen species (ROS) within cancer cells [
22,
23]. Thus, high levels of ROS are one of the intrinsic characteristics specifically associated with cancer cells [
23,
24]. Harnessing this factor to exploit on–demand vectors that are capable of ROS–triggered siRNA release in cancer cells constitute a promising strategy to achieve cancer cell–specific siRNA delivery for RNAi–based cancer therapy.
Herein, we report a class of ROS–responsive amphiphilic dendrimers (
Fc-AmDs) for on–demand siRNA delivery upon the exposure to the elevated ROS level in cancer cells (
Scheme 1). These dendrimers are composed of hydrophilic PAMAM dendron and hydrophobic alkyl chains with different chain lengths containing ROS–sensitive ferrocene groups (
Scheme 1A). The hydrophilic PAMAM dendrons, which feature with amine groups and are positively charged at physiological pH, have been designed to interact with the negatively charged siRNA through electrostatic interactions. Meanwhile, the tertiary amine groups presented in PAMAM dendrons can be further protonated in acidic environments (e.g., pH 5.0 in endosome), thus promoting endosome escape via the proton sponge effect [
15,
25,
26]. Moreover, the hydrophobic alkyl chains with ferrocene groups endow
Fc-AmDs with good assembly properties. Particularly, the hydrophobic ferrocene is converted to the hydrophilic ferrocenium cation triggered by elevated ROS in cancer cells [
27,
28]. This hydrophobic–to–hydrophilic transition is anticipated to disrupt the hydrophobic/hydrophilic balance of
Fc-AmD nanoassemblies and cause their complete disintegration, eventually initiating siRNA release for effective gene silencing within cancer cells (
Scheme 1B).
2. Materials and Methods
2.1. Materials
The human AKT2 siRNA (forward primer: 5′–GCUCCUUCAUUGGGUACAAdTdT–3′; reverse primer: 5′–UAAUGUGCCCGUCCUUGUCdTdT–3′) and GAPDH (forward primer: 5′–AATCCCATCACCATCTTCCA–3′; reverse primer: 5′–TGGACTCCACGACGTACTCA–3′) were purchased from GenScript Biotech Corp. (Nanjing, China). The scramble siRNA (forward primer: 5′–ACGUGACACGUUCGGAGAAdTdT–3′; reverse primer: 5′–UUCUCCGAACGUGUCACGUdTdT–3′) was purchased from Biosyntech Co., Ltd. (Suzhou, China). All the biochemical reagents are suitable for cell culture and were purchased from Sigma–Aldrich (Shanghai, China), Servicebio (Wuhan, China), Cell signaling Technology (Danvers, MA, USA), Vazyme Biotech Co., Ltd. (Nanjing, China) and Thermo Fisher Scientific Inc. (Carlsbad, CA, USA). All the reagents were used without any further purification from commercial sources.
All the chemical reagents are analytical grade and were purchased from Aladdin Ltd. (Shanghai, China), Energy Chemical Ltd. (Shanghai, China), J&K Scientific (Beijing, China) or Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methyl acrylate and ethylenediamine were distilled before use. The compounds
8E and
8A were synthesized according to the reported literature [
17]. All the other chemicals were purchased from commercial sources and used as received. All the solvents used were freshly distilled. Dialysis tubes were purchased from Yuanye Bio–Technology Co., Ltd. (Shanghai, China).
A Bruker AC (300 MHz) spectrometer was employed to determine the 1H NMR and 13C NMR spectra of compounds. All the chemical shifts were recorded in parts per million (δ, ppm) with reference to tetramethylsilane (TMS). Mass spectra were measured by using the Agilent 6230 time–of–flight LC/MS (LC/TOF) system. The infrared spectra were recorded by using a Bruker ALPHA FT–IR spectrophotometer (Bruker, Ettlingen, Germany) in the range of 400–4000 cm−1.
2.2. Synthesis and Characterization of Fc-AmDs
Fc-C6-AmD 8A: 1H NMR (300 MHz, CD3OD/CDCl3) δ 7.89 (s, 1H), 4.79 (br, 2H), 4.47–4.34 (m, 4H), 4.18 (s, 5H), 3.81 (s, 2H), 3.50–3.15 (m, 30H), 2.96–2.67 (m, 44H), 2.66–2.50 (m, 12H), 2.49–2.25 (m, 28H), 2.05–1.89 (m, 2H), 1.67–1.55 (m, 2H), 1.52–1.34 (m, 4H). 13C NMR (75 MHz, CD3OD/CDCl3) δ 174.04, 173.36, 173.18, 171.93, 143.24, 123.85, 75.58, 70.43, 69.54, 68.05, 52.12, 49.81, 49.25,47.87, 40.39, 39.26, 37.26, 33.47, 30.05, 29.48, 29.01, 28.79, 26.70, 26.23. ESI–HRMS (m/z): calcd for C90H167FeN33O15, [M+2H]2+ 1004.6424, found 1004.6393. HPLC (RT = 16.1 min). IR (cm−1): υ 1630.50 and 1542.97 (–NH(CO)–).
Fc-C8-AmD 8A: 1H NMR (300 MHz, CD3OD/CDCl3) δ 7.87 (s, 1H), 4.79 (br, 2H), 4.46–4.33 (m, 4H), 4.18 (s, 5H), 3.82 (s, 2H), 3.47–3.14 (m, 30H), 2.98–2.69 (m, 44H), 2.68–2.53 (m, 12H), 2.49–2.28 (m, 28H), 1.98–1.86 (m, 2H), 1.67–1.52 (m, 2H), 1.45–1.27 (m, 8H). 13C NMR (75 MHz, CD3OD/CDCl3) δ 173.86, 173.36, 173.17, 171.93, 143.35, 123.84, 75.56, 70.42, 69.52, 68.05, 52.12, 49.80, 49.05, 47.68, 41.26, 40.61, 39.10, 37.26, 33.46, 30.00, 29.31, 26.13, 25.97. ESI–HRMS (m/z): calcd for C92H171FeN33O15, [M+2H]2+ 1018.6581, found 1018.6541. HPLC (RT = 19.1 min). IR (cm−1): υ 1631.17 and 1541.60 (–NH(CO)–).
Fc-C10-AmD 8A: 1H NMR (300 MHz, CD3OD/CDCl3) δ 7.87 (s, 1H), 4.79(br, 2H), 4.44–4.32 (m, 4H), 4.19 (s, 5H), 3.82 (s, 2H), 3.45–3.16 (m, 30H), 2.93–2.68 (m, 44H), 2.66–2.53 (m, 12H), 2.50–2.16 (m, 28H), 1.97–1.85 (m, 2H), 1.66–1.53 (m, 2H), 1.46–1.23(m, 12H). 13C NMR (75 MHz, CD3OD/CDCl3): δ 175.52, 174.94, 145.36, 125.29, 72.07, 71.26, 69.78, 54.08, 51.77, 43.41, 42.54, 42.54, 40.90, 39.13, 35.48, 35.25, 31.68, 31.21, 31.04, 31.04, 30.65, 30.43, 28.40, 27.71. ESI-HRMS (m/z): calcd for C94H175FeN33O15, [M+2H]2+ 1032.6737, found 1032.6722. HPLC (RT = 20.2 min). IR (cm−1): υ 1630.66 and 1542.24 (–NH(CO)–).
2.3. HPLC of Fc-AmDs
A high–performance liquid chromatography (HPLC) analysis was conducted on a Waters Empower system (Waters 1525, binary HPLC pump, Waters Corp., The Capricorn, Singapore) equipped with a photodiode array detector (Waters 2998, Waters Corp., The Capricorn, Singapore) and a SinoChrom C8 column (5 μm, 4.6 mm × 250 mm). A gradient elution mode of 10–50% acetonitrile in water was employed over 40 min. The mobile phases consisted of acetonitrile and water, both of which contained 0.04% TFA. The flow rate was 0.8 mL/min, with an injection volume of 20 μL. Peaks were detected at 210 nm. The retention times (RTs) were recorded in minutes. All reagents were HPLC–gradient grade and purchased from Anhui Tedia High Purity Solvents Co., Ltd. (Anqing, China) and Aladdin Ltd. (Shanghai, China).
2.4. Critical Aggregation Concentration (CAC)
CAC was determined using pyrene as a fluorescent probe. The solutions of Fc-AmDs at different concentrations varying from 1.0 × 10−7 to 2.0 × 10−4 mol/L were prepared, and the final pyrene concentration was 3 × 10−7 mol/L in water. The solutions were sonicated for 30 min and kept at room temperature for 2 h to promote the micelle formation prior to fluorescence measurement. Fluorescence spectra were recorded at the emission wavelength of 334 nm on FL8500 fluorescence spectrophotometer at room temperature. The excitation bandwidth was 20 nm, and the emission bandwidth was 1 nm. The fluorescence intensity ratio of I373/I384 was analyzed as a function of dendrimer concentration.
2.5. Potentiometric pH Titration
The solution of Fc-AmDs (5 mg in 5 mL) was adjusted to a pH of around 2–3 with 1.0 M HCl. The pH titration was performed with 0.05 M NaOH using METTLER TOLEDO FE28–Bio pH meter (Mettler–Toledo International Inc., Shanghai, China).
2.6. ROS Response Measurement of Fc-AmDs
The oxidation process of Fc-AmDs was detected using UV–Vis spectrophotometry. Fc-AmDs were added to H2O2–containing acetate buffer solution and incubated at 37 °C with gentle shaking. Then, the UV absorption of the solution was measured by UV–1900i UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan).
The hydroxyl radical (·OH) produced by Fenton–like reaction was detected using TMB. Briefly, Fc-AmDs and TMB were added to H2O2–containing acetate buffer solution and incubated at 37 °C with gentle shaking. Then, the UV absorption of the mixed solution was measured by UV–Vis spectrophotometry.
2.7. Dynamic Light Scattering (DLS)
2.7.1. DLS Analysis of Fc-C8-AmD 8A
A 200 μM solution of Fc-C8-AmD 8A was prepared by dissolving the compound in ultrapure water. The sizes of Fc-C8-AmD 8A solutions were measured using a NanoBrookOmni (Brookhaven, Holtsville, NY, USA) equipped with a standard 633 nm laser at 25 °C.
2.7.2. DLS Analysis of siRNA/Fc-C8-AmD 8A Nanoparticles
The solution of siRNA/Fc-C8-AmD 8A complexes was prepared via mixing the siRNA solution and Fc-C8-AmD 8A solution at N/P ratio of 10, resulting in a final concentration of 1.0 μM for the siRNA. Ultrapure water was used for the solvent. The sizes of the siRNA/Fc-C8-AmD 8A complexes solutions were measured by the same DLS method as described above.
2.7.3. DLS Analysis of the ROS–Triggered Disassembly of siRNA/Fc-C8-AmD 8A Nanoparticles
The solutions of siRNA/Fc-C8-AmD 8A complexes were prepared as described above. PB solution (pH 5.0) was used for the solvent. The sizes of the siRNA/Fc-C8-AmD 8A complexes in the absence and presence of H2O2 were measured at different time points (0, 1, 2, 4, 6, 8, 12, and 24 h) using the same DLS method as described above.
2.8. Gel Retardation Analysis
The solutions of Fc-AmDs and siRNA were mixed at different N/P ratios ranging from 0.2 to 10 and incubated at 37 °C for 30 min. The final concentration of siRNA in each sample was 200 ng/well. The siRNA/Fc-AmDs complexes were analyzed via electrophoretic mobility–shift assays conducted using 2% agarose gels in standard TAE buffer for 15 min. Following staining with GoodView nucleic acid dyes, the siRNA bands were imaged using an automatic chemiluminescence imaging system (5200 Multi) (Tanon, Shanghai, China).
2.9. Stability of siRNA/Fc-C8-AmD 8A Complex against RNase
An aliquot containing 200 ng of siRNA and the indicated amounts of Fc-C8-AmD 8A solution at N/P ratio of 10 was incubated in the presence of RNase A (1.0 μg/mL) at 37 °C for different times (0, 10, 30, 60, 90, and 120 min). Afterwards, aliquots (10.0 μL) of the corresponding solution were added 1.0 μL of 2% SDS solution on ice. The mixtures were analyzed by gel retardation as described above. Naked siRNA served as a control.
2.10. RNA Dissociation Assay
A solution containing 0.1 μg ethidium bromide (EB), 1.32 μg siRNA, and an appropriate amount of Fc-AmDs at a N/P ratio of 10 in PB buffer at pH 5.0 was added to the black 96–well plate and incubated at 25 °C for 30 min. Then, to 60 μL of the above siRNA/Fc-AmDs complexes was added the 40 μL of heparin solution in PB at different concentrations varying from 0 to 50 U/mL to achieve a total volume of 100 μL. After a further incubation of 30 min at 25 °C, the mixture was measured at 590 nm using a Cytation5 Microplate Reader (BioTek, Winooski, VT, USA) with 360 nm fluorescence excitation. The fluorescence values were normalized to wells containing EB siRNA solution only. All samples were assayed in triplicate.
2.11. Cell Culture
Mouse fibroblast L929 cells, madin-darby canine kidney MDCK cells, human normal liver L02 cells, and human ovarian cancer SKOV–3 cells were purchased from Tongpai Biotechnology Co., Ltd. (Shanghai, China). L929 cells were maintained in DMEM (Hyclone, Logan, UT, USA), supplemented with 10% FBS. MDCK cells were maintained in MEM (Hyclone, Logan, UT, USA) with 10% FBS. Human ovarian cancer SKOV–3 cells were cultured in McCOY’S 5A (Hyclone, Logan, UT, USA) with 10% FBS. L02 cells were maintained in RMPI–1640 (Hyclone, Logan, UT, USA) with 10% FBS. All cells were cultured in an incubator with a humidified environment of 5% CO2 and a constant temperature of 37 °C.
2.12. MTT(3–(4,5–Dimethylthiazol–2–yl)–2,5–Diphenyltetrazolium Bromide) Assay
Cancer cells (SKOV–3 cells) and normal cells (MDCK cells, L929 cells, and L02 cells) (8 × 103) were seeded in 96–well plates and cultured for 24 h. Cells were then treated with Fc-AmDs at different concentrations, ranging from 0.25 to 50 μM, for 8 h. After transfection, the medium was replaced with fresh medium. Then, 48 h later, the cells were treated with MTT solution and incubated for a further 4 h. After removing the solution, the cells were re–suspended in DMSO. Cytation5 (BioTek, Winooski, VT, USA) was used to measure the optical density (OD) of DMSO solutions, which was read at 570 nm. The viability of cells was assessed by the difference between the OD values of treated and untreated cells. All samples were assayed in triplicate.
2.13. Flow Cytometry
One day prior to the transfection, SKOV–3 cells were seeded at 8.0 × 104 cells/well in a 24–well plate. The cells were incubated with Cy5–labeled siRNA/Fc-C8-AmD 8A complex (50 nM Cy5–siRNA, N/P ratio 10) for 1, 2, 4, 6, and 8 h. After three washes with cold PBS, the cells were analyzed by flow cytometry (Attune NxT, Thermo Fisher Scientific). All samples were assayed in triplicate.
2.14. Confocal Microscopy
One day prior to the transfection, SKOV–3 cells were seeded in 2.5 dishes (8.0 × 104 cells/dish). The cells were incubated with Cy5–labeled siRNA/Fc-C8-AmD 8A complex (50 nM Cy5–siRNA, N/P ratio of 10) for 1, 2, 4, 6, and 8 h at 37 °C. After three washes with cold PBS, the cells were stained by PBS mixed with Hoechst33342 (10 μg/mL) and Lyso-Tracker Red (0.10 μM) for 10 min at 37 °C. Images were acquired with a Zeiss LSM880 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany) using ZEN2.3 pro software (Carl Zeiss GmbH).
2.15. ROS Level Measurement
SKOV–3 and NAC–treated SKOV–3 cells (1.5 × 105 cells/group) were treated with the CellROX® orange Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) at a final concentration of 1.0 μM and incubated at 37 °C for 30 min. Following three washes with PBS, the mean fluorescence intensity of the cells was quantified by flow cytometry (Attune NxT, Thermo Fisher Scientific). Each assay was performed in triplicate.
2.16. In Vitro Transfection
SKOV–3 cells and NAC–pretreated SKOV–3 cells were seeded in 6–well plates and grown for 24 h. The solutions of siAKT2/Fc-AmDs complexes with 50 nM siRNA at an N/P ratio of 10 were prepared before transfection. After incubation with siAKT2/Fc-AmDs complexes for 8 h, the transfection mixture was replaced with the complete medium. And then, the cells were incubated for an additional 48 h for qRT–PCR assay and 72 h for Western blot assay.
2.16.1. Effect of Dioleoylphosphatidylethanolamine (DOPE)
The effect of DOPE was assessed by the transfection experiments as described above. Before transfection, the solutions of siAKT2/Fc-C8-AmD 8A complexes were prepared with DOPE at mole ratio of 1.0 (DOPE/dendrimer).
2.16.2. Effect of Bafilomycin A1
After preincubation of SKOV–3 cells with 200 nM bafilomycin A1 at 37 °C for 1 h, the effect of bafilomycin A1 was assessed by the transfection experiments as described above.
2.17. Quantitative Real-Time (qRT)–PCR Analysis
The transfection experiments were performed in SKOV–3 cells as described above. After isolation with the Trizol method (Vazyme Biotech Co., Ltd., Nanjing, China), the total RNAs of SKOV–3 cells were reverse–transcribed by a Reverse Transcription Kit (Vazyme Biotech Co., Ltd., Nanjing, China). The expression of AKT2 was analyzed by quantitative real–time PCR performed on the QuantStudio3™ real–time PCR System (Applied Bisystems, Thermo Fisher Scientific) using 2 × SYBR Green (Vazyme Biotech Co., Ltd., Nanjing, China). The expression of GAPDH was used for normalization of the qPCR data.
2.18. Western Blot Analysis
Samples containing equal amounts of protein (15 μg) from SKOV–3 cells were separated by SDS–PAGE gradient gel and transferred to the PVDF membrane after electrophoresis. After being blocked in 5% skimmed milk, the PVDF membranes were incubated with anti–human vinculin rabbit polyclonal antibody (Cell signaling Technology, Danvers, MA, USA, 1:5000) or anti–human AKT2 rabbit polyclonal antibody (Cell signaling Technology, Danvers, MA, USA, 1:1000) at 4 °C for 12 h. Then, the membranes were washed and incubated with anti–rabbit or anti-mouse monoclonal secondary antibodies (Invitrogen, Boston, MA, USA, 1:5000) for 2 h at 25 °C. Specific proteins were recorded by an enhanced-chemiluminescence Western blotting analysis system (Tanon, Shanghai, China).
2.19. Statistical Tests
All data are presented as mean ± SD unless otherwise indicated. One–way analysis of variance (ANOVA), two–way ANOVA, unpaired Student’s t-test or Mann–Whitney test (GraphPad Prism 8.01) were used for statistical analysis. A p value < 0.05 was considered statistically significant, whereby all significant values in various figures are indicated as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.