Penetration and distribution of drug through the avascular regions of human solid tumors after extravasation are crucial concerns for antitumor efficacy. To address this issue, an
RNA interference (RNAi) was first identified in
Stability in biological fluids and effective delivery constitute the main challenges for RNA-based therapeutics [
Human malignancies are target diseases for siRNA-based therapeutics, with a long list of potential targets related to aberrant signaling pathways in cancer cells [
In the present study, DsiRNA was successfully loaded into CS nanoparticles using the ionic gelation method. DsiRNA-CS nanoparticles were characterized by their particle size, zeta potential, morphology, binding and entrapment efficiency,
Low-molecular-weight (192 kDa) CS with a 75–85% degree of deacetylation (DD) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and pentasodium tripolyphosphate (TPP) was obtained from Merck (Darmstadt, Germany). DsiRNA targeting the VEGF gene [5′-rGrGrArGrUrArCrCrCrUrGrArUrGrArGrArUrCrGrArGrUAC-3′ (sense strand) and 5′-rGrUrArCrUrCrGrArUrCrUrCrArUrCrArGrG rGrUrA rCrUrCrCrCrA-3′ (antisense strand)] of 27 bp in length was purchased from Integrated DNA Technologies (IDT) (USA). Chinese hamster lung fibroblasts (V79) and human colorectal adenocarcinoma cells (DLD-1) were obtained from American Type Culture Collection (ATCC, USA). Human umbilical vein endothelial cells (HUVECs), Medium 200, low serum growth supplement (LSGS), Roswell Park Memorial Institute medium (RPMI) 1640, and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco (USA).
PureLink RNA Mini Kit, Lipofectamine RNAiMAX, PureLink DNase, SuperScript VILO MasterMix First Strand Synthesis SuperMix for RT-PCR, and PlatinumTaq DNA Polymerase were purchased from Life Technologies (Carlsbad, USA). Hoechsct 33342 stain was obtained from Thermo Scientific Dharmacon (USA). The live/dead cell viability assay kit and human VEGF Elisa kit were purchased from Invitrogen (USA). Fluorescein-labeled siRNA and DsiRNA were purchased from IDT (USA). The collagen-coated microporous membrane (0.4
CS nanoparticles were prepared via the ionic gelation method with some modifications [
To incorporate DsiRNA into CS-TPP nanoparticles, 15
DLD-1 cells were maintained in RPMI-1640 and supplemented with 5% pen-strep and 10% heat-inactivated FBS in a humidified, 5% (v/v) CO2 atmosphere at 37°C. Cells were grown on collagen-coated microporous (0.4
The transwell inserts containing 2 mL of complete RPMI-1640 medium in each insert were placed in a 6-well plate containing 2.5 mL of complete RPMI-1640 medium in each well, followed by occasional agitation [
The release profile of the DsiRNA-CS nanoparticles was studied in PBS at pH 7.4. Samples (4 mL) were centrifuged at 35,000 rpm for 30 min at 25°C, and the pellets were resuspended in PBS (3 mL) at pH 7.4. The mixture was placed on magnetic stirrer with a stirring speed of 100 rpm at 37°C for 15 days. At predetermined time intervals, samples were centrifuged at 35,000 rpm for 30 min at 25°C. Then, a whole volume of supernatant was taken for analysis and replaced with an equivalent volume of fresh buffer solution. The amount of DsiRNA released in the supernatant was analyzed by a UV-vis spectrophotometer (Shimadzu 1800) at a wavelength of 260 nm.
The V79 cell line (ATCC, Manassas, VA, USA) was cultured in DMEM supplemented with 10% FBS and 1% pen-strep at a seeding density of 2 × 104 cells per well. The DLD-1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% pen-strep at a seeding density of 4 × 104 cells per well. Moreover, the HUVECs (Gibco, USA) were cultured in medium 200 at a seeding density of 3 × 104 cells per well. The HUVECs were supplemented with 10% LSGS and 1% pen-strep. All cells were incubated with DsiRNA-CS nanoparticles for 24 and 48 h.
After 24 and 48 h incubation of samples with cells, a final dilution of 1/10 per cell volume of alamarBlue reagent was added to the treated cells, followed by incubation for 4 h prior to analysis. The absorbance of each sample at 570 nm (A570) was measured using a microplate reader (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). All cells were maintained at 37°C in a humidified 5% CO2/95% air atmosphere.
Cell viability of all the samples was determined using the following equation:
A cell viability assay was performed to measure the functional status of the cells by detecting cytoplasmic esterase activity using the LIVE/DEAD Viability/Cytotoxicity kit for mammalian cells (Invitrogen, Carlsbad, CA, USA). The kit contains calcein, which stains living cells (green), and ethidium bromide, which stains dead cells (red). This assay was performed in 96-well plates. Briefly, DLD-1 cells were plated at a seeding density of 4 × 104 cells per well. The cells were supplemented with 10% FBS and 1% pen-strep and maintained at 37°C in a humidified 5% CO2/95% air atmosphere. The cells were treated with DsiRNA-CS nanoparticles for 24 and 48 h. Subsequently, the cells were rinsed twice with PBS, followed by adding fluorochromes (calcein/ethidium bromide). The cells were treated with DsiRNA-CS treated with DsiRNA-CS nanoparticles for 24 and 48 h and incubating for 45 min. The reagents were removed by rinsing with PBS followed by analysis using a Floid Cell Imaging Station (Molecular Probes Life Technology, France).
DLD-1 cells (4 × 104) were seeded in a 6-well tissue culture plate and incubated for 24 h (80% confluence). The cells in each well were incubated for 4 h in a medium with 10% FBS at 37°C with free 6-FAM-DsiRNA or with DsiRNA-CS nanoparticles. After incubation, the cells were washed twice with PBS and then stained with 1
After obtaining RNA from the cells, the quality of RNA was assessed by 1% w/v agarose gel electrophoresis at 90 V for 60 min in Tris-acetate-EDTA (TAE) buffer and visualized by ethidium bromide staining. RNA was quantified using the Infinite 200 PRO NanoQuant (Tecan, Switzerland). The gel images were taken by an image reader (Fujifilm LAS-3000, Tokyo, Japan). In the next step, complementary DNA (cDNA) was prepared from 1.5
Data was presented as mean ± SD. The data were analyzed using independent
The mean particle size of DsiRNA-CS nanoparticles was significantly increased by increasing the CS concentration from 0.1% to 0.4% w/v (Table
Particle size, PDI, and zeta potential of DsiRNA-CS nanoparticles prepared at different CS concentrations,
CS concentration (% w/v) | Particle size (nm) ± SD | PDI ± SD | Zeta potential (mV) ± SD |
---|---|---|---|
0.1 | 126.37 ± 15.52 | 0.30 ± 0.05 | +30.50 ± 2.55 |
0.2 | 180.00 ± 18.78 | 0.35 ± 0.18 | +37.13 ± 2.01 |
0.3 | 230.23 ± 13.80 | 0.40 ± 0.05 | +40.27 ± 4.07 |
0.4 | 336.50 ± 11.38 | 0.55 ± 0.02 | +55.30 ± 3.91 |
SD, standard deviation;
The zeta potential of DsiRNA-CS nanoparticles increased with increasing CS concentration at a constant DsiRNA concentration as shown in Table
AFM micrographs of DsiRNA-CS nanoparticles prepared from 0.3% w/v CS revealed a spherical morphology (Figures
AFM micrographs of DsiRNA-CS nanoparticles (prepared from 0.3% w/v CS) of 2-dimensional (a) and 3-dimensional (b). TEM micrographs of DsiRNA-CS nanoparticles prepared from 0.1% (c), 0.2% (d), 0.3% (e), and 0.4% (f) w/v CS at different magnifications (9900x, 60 kx, 26500x).
A higher DsiRNA entrapment efficiency was obtained (92% to 100%) for CS nanoparticles when CS concentration was increased from 0.1% to 0.4% w/v as measured by UV-vis spectrophotometry and shown in Figure
Encapsulation efficiency (a) of DsiRNA-CS nanoparticles prepared from different CS concentrations (0.1% to 0.4% w/v CS),
Strong binding between DsiRNA and CS nanoparticles was observed (due to the absence of a trailing band), as shown in Figure
In heparin displacement assay experiment, heparin was used to displace DsiRNA from CS nanoparticles prepared with different CS concentrations (0.1%, 0.2%, 0.3%, and 0.4% w/v), as shown in Figure
The ability of a carrier to protect its payload from nuclease degradation is an important property for efficient gene delivery. Similar to siRNA, DsiRNA must also be protected from nuclease digestion for maximal activity in the cells. To address this, a serum protection test was carried out for CS nanoparticles in 10% FBS. Naked DsiRNA started to degrade as early as 0 min due to degradation during mixing and freezing steps as shown in Figure
Electrophoretic mobility (a) of naked DsiRNA and DsiRNA-CS nanoparticles, following incubation in RPMI medium containing 10% FBS for 48 h. Relative density (b) of gel electrophoresis bands of serum protection assay (FBS), by imageJ software.
Currently, to our knowledge, there is no information regarding the stability and integrity of DsiRNA-carriers in human serum. Knowledge about the effects of human serum on the stability of DsiRNA-carriers may help in the development of new, more promising DsiRNA-carriers. This experiment was conducted using DsiRNA-CS nanoparticles. Naked DsiRNA started to degrade at as early as 0 min incubation as shown in Figure
Electrophoretic mobility of naked DsiRNA and DsiRNA-CS nanoparticles following incubation in human serum for 48 h.
The study was performed in deionized distilled water or PBS (pH 7.4) for 15 days. The storage stability of DsiRNA-CS nanoparticles (CS concentration of 0.3% w/v) in deionized distilled water at 4°C and 25°C is shown in Figures
Storage stability of DsiRNA-CS nanoparticles: in deionized distilled water at 4°C (a) and 25°C (b) and in PBS at 4°C (c) and 25°C (d), for 15 days,
The
The release profile of DsiRNA-CS nanoparticles at pH 7.4 for 15 days,
The cytotoxic effect of DsiRNA-CS nanoparticles was investigated in V79, DLD-1, and HUVECs cell lines by an alamarBlue cell viability assay. In V79 cells, only a 5–11% loss of cell viability was observed for DsiRNA-CS nanoparticles as shown in Figure
Cytotoxicity effects of DsiRNA-CS nanoparticles after 24 and 48 h incubation in V79 (a), DLD-1 (b), and HUVECs (c) cell lines,
In the DLD-1 cell line, DsiRNA-CS nanoparticles caused a cytotoxic effect after 24 and 48 h incubation as shown in Figure
HUVECs are isolated from the vein of the umbilical cord and are commonly used for physiological and pharmacological investigations involving macromolecule transport, blood coagulation, angiogenesis, and fibrinolysis. In addition, HUVECs are also available which are prescreened for VEGF response. VEGF is an important signaling protein involved in both vasculogenesis and angiogenesis.
For LIVE/DEAD cell viability assay, the results showed that untreated cells did not produce any loss of cell viability (Figure
(a) Live/dead cell viability assay of DsiRNA-CS nanoparticles in DLD-1 cells after 24 h and 48 h incubation. Untreated cells (A, D), naked DsiRNA (B, E), and DsiRNA-CS nanoparticles (C, F) after 24 h and 48 h incubation, respectively (green and red colors represent viable and dead cells, resp.). (b) Internalization and localization of 6-FAM-labeled DsiRNA-CS nanoparticles in DLD-1 cells after 6 h incubation. Scale bar represents 10
After incubation of DLD-1 cells with 6-FAM-DsiRNA entrapped within CS nanoparticles for 6 h, fluorescence could be detected within the cells. The fluorescence was mainly distributed in the cytoplasm, which is in agreement with previous observations for siRNA cellular uptake and an earlier report (Figures 8(b)(D)–(F)) [
In order to investigate the silencing activity of
Analysis of
The morphology of the collagen layer and 6 days of growth of MCLs was examined using SEM (Figures
Across a wide range of human tumors and/or cell lines, expression of
The VEGF levels in MCLs were determined by ELISA after exposure to naked DsiRNA, CS nanoparticles (unloaded), and DsiRNA-CS nanoparticles for 24 and 48 h. A DsiRNA concentration of 80 pmol was used in all formulations. The results showed that naked DsiRNA did not exert any significant reduction in the VEGF level in MCLs when compared to untreated cells (Figure
(a) VEGF mRNA expression in MCLs of DLD-1 cells of untreated MCLs (A), MCLs exposed to naked DsiRNA (B), DsiRNA-CS nanoparticles at 20 pmol (C and D), and 80 pmol (E and F) for 24 and 48 h incubations, respectively. (b) The level of VEGF protein in MCLs after exposure to DsiRNA-CS nanoparticles for 24 and 48 h.
DsiRNA distribution in MCLs was evaluated upon exposure of 6-FAM-labeled naked DsiRNA entrapped into CS nanoparticles (80 pmol) for 24 and 48 h. The naked DsiRNA (control) only showed some fluorescence on the surface of MCLs with no penetration observed up to 48 h incubation (Figures
DsiRNA distribution in MCLs after 24 and 48 h incubation with naked 6-FAM DsiRNA (control) and 6-FAM DsiRNA-CS nanoparticles. Scale bar represents 50
DsiRNA-CS nanoparticles that had a small particle size, positive surface charge, regular morphology, high encapsulation efficiency, and exhibited sustained release of DsiRNA were successfully prepared by the ionic gelation method. Moreover, these nanoparticles provided maximal protection of DsiRNA in 10% FBS and human serum for up to 48 h incubation. Cytotoxic effects were determined to be dependent on the concentration of CS in the case of V79 and DLD-1 cells and otherwise in the case of HUVECs. Moreover, DsiRNA-CS nanoparticles were successfully internalized into DLD-1 cells. RT-PCR studies revealed that DsiRNA entrapped within CS nanoparticles could highly downregulate
The authors report no competing interests in this work.
The authors gratefully acknowledge all the members of Centre for Drug Delivery Research Group for their technical support and advice and the Ministry of Education, Malaysia, for financially supporting the project (ERGS/1/2011/SKK/UKM/02/11).