The clinical utility of siRNA therapy has been hampered due to poor cell penetration, nonspecific effects, rapid degradation, and short half-life. We herewith proposed the formulation development of STAT6 siRNA (S6S) nanotherapeutic agent by encapsulating them within gelatin nanocarriers (GNC). The prepared nanoformulation was characterized for size, charge, loading efficiency, release kinetics, stability, cytotoxicity, and gene silencing assay. The stability of S6S-GNC was also assessed under conditions of varying pH, serum level, and using electrophoretic assays.
RNAi is a naturally occurring gene silencing process that has the advantages of a high degree of specificity and the potential to silence genes of interest [
The signal transducer and activator of transcription 6 (STAT6) is one of the most prominent transcription factors that regulate gene expression in response to extracellular polypeptides that lead to cellular proliferation, differentiation, and apoptosis [
The clinical utility of siRNA has been limited to its inherent properties; for example, naked siRNA is prone to degradation and has a shorter plasma half-life, rapid renal clearance, and limited permeability across cell membranes [
Cellular uptake and intracellular mechanism of action (MOA) of targeted S6S-GNC.
Gelatin (type A; 175 g Bloom Strength; with isoelectric point of 8-9, and average molecular weight of 40–50 kDa; GELITA, USA) was graciously provided as a gift from the manufacturer. Glutaraldehyde (GTA) was purchased from Alfa Aesar (Heysham, Lancaster) as a 25% aqueous solution. Genipin (GEN) was kindly provided as a gift sample from Wilshire Technologies, Inc. (Princeton, NJ, USA). Acetaminophen (APAP) was purchased from Sigma Aldrich. STAT6 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Ethanol, dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and lactose monohydrate were purchased from VWR International (Radnor, PA, USA). Spectra/Por Dialysis membranes (MWCO 25 kDa and 100 kDa) were obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). Distilled deionized and 0.22
The A549 (human lung adenocarcinoma epithelial cell line, CCL-185, ATCC, Manassas, VA) cells were grown as monolayers in 75 cm2 tissue culture flasks (Greiner Bio-one, Monroe, NC, USA) at 37°C under 5% CO2 in F12-K supplemented medium (Life Technologies, Grand Island, NY, USA) with 10% v/v fetal bovine serum (FBS) and an antibiotic antimycotic solution of penicillin (5000 U/mL), streptomycin (0.1 mg/mL), and neomycin (0.2 mg/mL) (PSN). Cell culture media and PSN stock solutions were purchased from Cellgro (Herndon, VA, USA). Heat inactivated FBS was purchased from Atlanta Biologicals (Lawrenceville, GA, USA).
The GNCs formed from the whole gelatin fraction were prepared by one step desolvation technique. Briefly, a 1% w/v gelatin type “A” solution was prepared by dissolving gelatin in distilled deionized H2O at 50°C under gentle stirring at 400 rpm. When the gelatin solution became homogeneous and transparent, the temperature of the solution was reduced to 35°C and 19.98 mg acetaminophen (model drug engaged to optimize formulation conditions), added, and dissolved. Then, the desolvation step was accomplished, wherein 80% (v/v) ethanol was added at a rate of 1 mL/min under constant stirring at 600 rpm. Following this, 150
The high molecular weight (HMW) fraction was prepared by the classical 2-step desolvation technique, where 5% (w/v) gelatin type “A” was first desolvated with an equal volume of acetone for 12 minutes under gentle stirring. After 12 minutes, the supernatant that contained the low molecular weight (LMW) gelatin fraction, water, and acetone was decanted and discarded. The HMW fraction sediment was allowed to dry and underwent mass reconciliation. The HMW gelatin was redissolved in distilled deionized H2O 1% (w/v) solution at 50°C under gentle stirring. When the gelatin solution became homogeneous and transparent, the temperature of the solution was reduced to 35°C and 19.80 mg acetaminophen was added and dissolved. Then, a second desolvation step commenced, where 80% v/v pure ethanol was added dropwise at a rate of 1 mL/min under a constant stirring rate of 600 rpm. Five minutes after the ethanol addition ended, 150
The MMW fraction was prepared by a modified 2-step desolvation technique, where 5% w/v gelatin type “A” was first desolvated with an equal volume of acetone for 5 seconds, quickly decanted into another beaker, and then allowed to desolvate for another 12 minutes where the LMW fraction was decanted and discarded. The first contains HMW fraction, while the LMW gelatin in water and acetone supernatant was discarded. The MWW fraction sediment was allowed to dry and underwent mass reconciliation.
The MMW gelatin was redissolved in distilled deionized H2O to make a 1% w/v solution at 50°C under gentle stirring at 400 rpm. When the gelatin solution became homogeneous and transparent, the temperature of the solution was reduced to 35°C, and 22.92 mg acetaminophen was added and dissolved. Then, a second desolvation step commenced, where 80% pure ethanol was added dropwise at a rate of 1 mL/min under constant stirring at 600 rpm. Five minutes after the ethanol addition ended, 150
The whole, HMW, and MMW gelatin fractions were compared for their resultant nanocarrier particle size, polydispersity index, and entrapment efficiency (EE%).
Type A gelatin-based nanocarriers were prepared using the 2-step desolvation technique with slight modifications (Figure
Preparation of S6S-GNC. The 1% w/v aqueous gelatin solution is incubated with the STAT6 siRNA for 10 min at 35°C, and then ethanol and crosslinker are added dropwise at a stirring rate of 600 rpm at 35°C for 1 hr, at which point the stirring rate is reduced to 200 rpm. After approximately 4 h, the ethanol is completely evaporated, and STAT6 siRNA loaded gelatin nanocarriers remain in a colloidal suspension in water or PBS pH 7.4. The resultant nanoparticles were collected by centrifugation and resuspended for subsequent characterization or lyophilization in the presence of 1% w/w lactose monohydrate.
S6S-GNC was formulated by employing the optimized 2-step desolvation methodology (Figure
Particle size analysis report for GNC formulated at 600 rpm stir rate (magnetic stir bar method). Data pertains to nanocarrier formulation at stir rates of 300 rpm and 700 rpm which were not included because the particle sizes obtained therein were outside acceptance range. The bars, dots, and error bars represent the mean ± standard deviation
The particle size of the prepared S6S-GNC was determined by dynamic light scattering using a NICOMP ZLS 380 analyzer (PSS-NICOMP, Santa Barbara, USA) [
The entrapment efficiency was determined by employing Vivaspin500 ultracentrifuge filters (MWCO100 kDa, Viva Products, Inc., Littleton, MA, USA) using UV spectrophotometry to quantify the free siRNA in a sample. Briefly, S6S-GNC formulation was placed on the top of the Vivaspin filter membrane and centrifuged at 16,200 ×g for 10 min. The aqueous filtrate was then subjected to UV spectrophotometric analysis to determine the free S6S content using a BioSpek-nano Micro-volume UV-Vis Spectrophotometer (Shimadzu, Columbia, MD, USA). Sample sizes of 2
S6S release from S6S-GNC formulation was assessed under physiological pH employing phosphate-buffered saline (PBS; pH 7.4) as release milieu. Briefly, two mL of S6S-GNC formulation was placed inside a dialysis bag (MWCO 100 kDa, Fisher Scientific, USA). The membrane bags were placed in 50 mL of PBS pH 7.4 under constant agitation condition (300 rpm) at 37°C. At predetermined time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 24, 48, 72 hr), 0.5 mL of dissolution medium was collected, and equal volume of fresh dissolution medium was replaced to simulate perfect sink conditions. The samples were analyzed at each time interval using the S6S from the developed formulations.
Stability of S6S-GNC under conditions of varying pH and serum level was also assessed to investigate the stability of developed formulation under different environments [
Stability of encapsulated S6S was also assessed by agarose gel electrophoresis as described previously [
The effect of S6S-GNC on viability of A549 cell lines was measured using established MTT assay protocol [
The cell internalization was evaluated by treating Nile red dye loaded GNC in A549 cells [
In order to assess the efficiency of STAT6 silencing by various formulations under investigation, expression levels of STAT6 protein were monitored following nanoformulation treatment with appropriate controls. Briefly, untreated control, S6S + lipofectamine complex, and S6S-GNC-treated A549 cells were lysed using RIPA buffer, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as per previously described [
The experiments were conducted in triplicate with data reported as mean ± standard deviation. Experimental statistics were analyzed using Minitab 16 Statistical Software (State College, PA, USA). The significance level was set at
According to a recent report by American Cancer Society, cancer is a leading cause of death in the United States, and by end of year 2013, approximately half a million Americans are anticipated to succumb to cancer [
The aim of this this investigation was to provide proof of concept that gelatin polymer (FDA approved polymer) based nanocarrier formulations of S6S will provide alternate mode to attain therapeutic benefit of siRNA in cancer therapy [
Gelatin is a biodegradable/biocompatible polymer approved by FDA for I.V. administration. Gelatin-based nanoparticles represent an attractive strategy, since a significant amount of bioactive can be incorporated into the protein-based nanoparticle matrix [
For the preparation of GNC’s, a two-step desolvation technique was utilized, wherein in first step, the gelatin type A was fractionated to remove the LMW fraction using acetone as a desolvating agent, and then the second step was performed to form the nanocarriers [
The effect of varying gelatin molecular weight on formulation of GNC was also studied by Coester et al. in 2000, wherein molecular weight of gelatin was reported to be greatly influencing the stability as well as particle size of the developed gelatin nanocarriers [
GNC formulations were optimized using a 33 Taguchi orthogonal array design with the independent variables being stirring rate, ethanol volume, and GEN concentration and the dependent variable of particle size (Table
Taguchi orthogonal array design for the optimization of S6S-GNC using APAP as a model drug.
Factor | Levels | ||
---|---|---|---|
1 | 2 | 3 | |
Stirring rate (rpm) | 300 | 600 | 700 |
Ethanol proportions (% v/v) | 70 | 80 | 90 |
10% GTA ( |
100 | 150 | 300 |
Interaction plot for the dependent variable particle size in the Taguchi orthogonal array experimental design for the formulation development of GNC.
We have also utilized a modified two-step desolvation technique to prepare the GNC as a colloidal delivery system, and the key factors effecting formulation of GNC were considered in the preparation of the nanoformulation. Particle size is a highly influential dependent variable that influences the cellular uptake of nanoparticles and the tissue and organ distribution of nanoparticles [
Also, body distribution studies have shown that nanoparticles >230 nm will accumulate in the spleen because of the capillary diameter within this organ [
Particle size and zeta potential of the S6S-GNC batches and placebo-GNC. The bars represent particle size (nm), the square markers represent the zeta potential (mV), and the error bars represent standard deviation.
The
It was widely reported that encapsulation of bioactive agents in the nanoparticles significantly ameliorates as well as prevent degradation of loaded bioactivities [
Stability of S6S-GNC formulation. Outcome is as expressed by size (nm) and zeta potential (mV) under the influence of varying pH between 5.4 and 8.4 and 10% v/v FBS at physiological pH 7.4 to mimic the serum found in human blood. Results are represented as mean ± SD
Additionally, agarose gel electrophoretic mobility shift assay was also performed to assess the stability of entrapped siRNA during formulation conditions and exposure to RNAse. Figure
Agarose gel electrophoretic mobility shift assay. The scrambled siRNA control, scrambled siRNA treated with RNAse control, S6S-GNC, S6S-GNC treated with RNAse, filtrate, filtrate treated with RNAse, and the placebo-GNC treated with RNAse were loaded onto a 1% w/v agarose gel and electrophoresed at a constant voltage of 70 V until the bromophenol blue marker bands were well separated. This study was performed to examine the stability of the encapsulated siRNA due to preparation conditions, as well as the stability in the presence of RNAse. The arrow head indicates the distance traveled by the cleaved siRNA fragments.
After physiochemical characterization of GNC formulation, we have evaluated the
Cytotoxicity of the developed S6S-GNC compared to placebo, native S6S and S6S with lipofectamine on A549 lung cancer cells. The graph shows the % cell viability observed after 24 and 48 hr following treatment. Cell viability was performed using
The cell internalization of nanoparticles plays an important role in eliciting therapeutic effect [
Fluorescence images of control and Nile red loaded GNC (Nile-GNC) taken after 15 min and 1 h of treatment in A549 cells (at 40x magnification). For this assay, lung cancer A549 cells
STAT6 protein is involved in tumor progression and resistance and was reported to be activated by phosphorylation; therefore, downstream expression of pSTAT6, in addition to other downstream proteins, IFN
Measurement of STAT6 protein expression by western blot. The effect of STAT6 siRNA-GNC on the expression of STAT6 in A549-treated lung cancer cells was shown. A549 cells were preincubated with S6S-GNC and S6S-lipofectamine complex and without any treatment (control). The cells were lysed, and STAT6 protein expression was analyzed by western blot of whole cell lysates. The
Stable and effective S6S-GNC formulation with small particle size of <80 nm and encapsulation efficiency of >85% was successfully developed. In addition, the formulation was found to be stable in presence of buffers solutions, serum solution, and RNAase. The S6S-GNC formulation showed sustained release of S6S, which is highly desirable considering long-term effect of formulation with reduced dosing interval. S6S loaded GNC evaluated in A549 lung cancer cell line inferred significantly (
The authors report that they have no conflict of interests.
The authors thankfully acknowledge the financial support provided by the Leahi Fund to Treat & Prevent Pulmonary Disease of the Hawai’i Community Foundation, Honolulu, HI, USA. The authors also gratefully acknowledge the research support of the Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo and the Research Corporation of the University of Hawai’i at Hilo, HI, USA.