rHDL Nanoparticle Preparation and siRNA Incorporation
SiRNA was incorporated into rHDL nanoparticles [36
] and stored at -20°C. Briefly, apolipoprotein A-I (Apo A-I), which is the major core protein in the rHDL particle, was isolated and purified using a plasmid vector for the production of protein [37
]. Next, a mixture of lipids (cholesterol [C]/cholesteryl oleate [CE]/egg yolk phosphatidylcholine [PC], molar ratio of 1:5:1.3:115) was dried under a stream of nitrogen. Five micrograms of siRNA was preincubated with 25 µg of oligolysine (mean MWt, 500–2000) at 30°C, for 30 minutes, and then added to the lipid ingredients. The oligolysine/siRNA mixture was then combined with lipids and dispersed in 60 µl of DMSO and 1.4 ml of buffer (10 mM Tris, 0.1 M KCl, 1 mM EDTA pH 8.0). Sodium cholate, 140 µl (100 mg/ml stock in 0.15 M NaCl, 0.003 M KCl, 0.15 M KH2
, pH 7.4 [designated as PBS]) was added to produce a final PC-to-cholate molar ratio of ~1:1.6. Apo A-I (12.7 mg/ml) in 0.4 ml of PBS was added to the mixture, and the final volume was adjusted to 2 ml with PBS. The lipid-protein-cholate mixture was then incubated for 12 hours at 4°C, followed by dialysis against 2 L of PBS, for 2 days, with three buffer changes. Stability of the formulation was determined using RiboGreen assay (Quant-iT Ribogreen Kit, Cat no. R11490; Invitrogen, Carlsbad, CA) in subsequent gel exclusion chromatography. The rHDL/targeted siRNA solution was then diluted in 0.9% normal saline to a concentration of 0.2 mg/kg siRNA/0.2 ml of rHDL solution. In addition, zeta potential of the rHDL nanoparticle was measured using Zeta Plus (Brookhaven Instrument Co, Novato, CA). Briefly, 1 ml of nanoparticles was added to a 1-ml cuvette for the assessment of zeta potential of nanoparticles.
Transmission Electron Microscopy
After dialysis against 0.125 M ammonium acetate, 2.6 mM ammonium carbonate, 0.26 mM EDTA, pH 7.4, the rHDL samples were negatively stained with 2% sodium phosphotungstate, pH 7.2, and placed on Formvar/carbon-coated 200-mesh nickel grid support films (Ted Pella, Inc, Redding, CA). The particles were visualized (magnification of 50,000) using a Zeiss 910 transmission electron microscope (Carl Zeiss MicroImaging, LLC, Thornwood, NY). The photographs obtained were enhanced, and the particle diameter was determined with Adobe Imageready CS2 software (Adobe Systems, Inc, San Jose, CA).
Cell Lines and Culture Conditions
The derivation and source of human epithelial ovarian cancer cell lines HeyA8, SKOV3ip1, and HeyA8-MDR have been reported previously [7–9,38
]. Human colorectal cancer cell line (HCT116) was a generous gift from Dr Lee Ellis (University of Texas MD Anderson Cancer Center, Houston, TX). All cell lines used in this study were authenticated by the characterized cell line core at the University of Texas MD Anderson Cancer Center. Briefly, the HeyA8 and SKOV3ip1 cells were maintained in RPMI 1640 supplemented with 15% fetal bovine serum (FBS) and 0.1% gentamicin sulfate (Gemini Bio-Products, Woodland, CA). HeyA8-MDR cells were maintained in RPMI 1640 supplemented with 15% FBS, 300 ng/ml paclitaxel (Abraxis BioScience, Los Angeles, CA), and 0.1% gentamicin sulfate. HCT116 cells were kept in culture using Dulbecco modified Eagle medium supplemented with 10% FBS and 0.1% gentamicin sulfate. All cells were kept in 5% CO2
/95% air at 37°C [39
In Vitro Gene Silencing
STAT3 (target sequence 5′-GCCUCUCUGCA GAAUUCAA-3′), FAK (target sequence 5′CCACCUGGGCCAGUAUUAU-3′), and a nontargeted control sequence (target sequence 5′-UUCUCCGAACGUGUCACGU-3′) were purchased from Sigma-Aldrich Corporation (Woodland, TX), and RNAiFect transfection reagent was used (Qiagen, Valencia, CA) as per the manufacturer's recommendations. Briefly, SKOV3ip1 and HeyA8-MDR ovarian cancer cells were transfected with 1.33 µg of specific siRNA/well in six-well plates in triplicates, at 70% confluence. Transfection was performed using the 1:3.75 of siRNA and transfection reagent, respectively, in serum-free medium for 6 hours with the following treatment groups: control siRNA and STAT3 siRNA.
RNA Extraction and Complementary DNA Preparation
Cells were homogenized with Trizol (Invitrogen, Carlsbad, CA). RNA was extracted (chloroform), precipitated (isopropanol), and purified (75% ethanol). cDNA was generated with 2.0 µg of high-quality RNA using SuperScript-II reverse transcriptase kit (Invitrogen) [40
cDNA microarray was performed using the Illumina platform (Illumina Inc, San Diego, CA) on SKOV3 cells that were treated in 10-cm cell culture plates with either STAT3 (8.0 µg) or control siRNA (8.0 µg) in triplicate using RNAiFect transfection reagent (Qiagen) as per the manufacturer's recommendations. STAT3 gene silencing was confirmed at the protein level using Western blot before the microarray analysis. Gene expression data from microarray analysis were then loaded into the IPA Ingenuity pathway database, and differentially expressed genes related to apoptosis were selected for validation.
Polymerase Chain Reaction Assay
Reverse transcription-polymerase chain reaction (RT-PCR) was preformed using cDNA from normal human organs and cancer cell lines. cDNA from human liver was used as a positive control for SR-B1 expression. Quantitative real-time PCR was performed in Applied Biosystems 9500 series using conditions that have been previously described [41
] using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in triplicate. β-Actin was used as an endogenous control. Mean fold change is reported.
Western Blot Analysis
Cell lysates were prepared with modified radioimmune precipitation lysis buffer [42
]. Proteins were separated on 8% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Philadelphia, PA), followed by incubation with STAT3 (1:2500) or FAK (1:5000) antibodies (BD Biosciences, San Diego, CA) overnight at 4°C. Primary antibody was detected using antimouse immunoglobulin G (GE Healthcare, Buckinghamshire, England, UK) and developed with a chemiluminescence detection kit (Perkin-Elmer, Covina, CA). β-Actin (1:2000; Sigma) or vinculin (1:3000; Cell Signaling Technology, Inc, Danvers, MA) confirmed equal loading.
Apoptosis Assay in Ovarian Cancer Cell
Twenty-four hours after treatment with control siRNA or STAT3 siRNA, ovarian cancer cells (SKOV3ip1 and HeyA8-MDR) cells were treated with docetaxel (1 or 500 nM, respectively) for 72 hours (triplicate), washed, and incubated with 5 µl of Annexin V/PE and 7AAD antibodies (BD Pharmingen, San Diego, CA) for 30 minutes. Flow cytometry was then performed to analyze the samples as previously described [43
Fresh-frozen (OCT) sections were stained for CD31 (1:800 dilution; Pharmingen) [7,44
], and Ki-67 staining was performed on 5-µm-thick formalin-fixed paraffin-embedded specimens [8
]. To quantify microvessel density (MVD), cell proliferation index (Ki-67), and cleaved caspase-3, five random 0.159-mm2
fields were studied at 100x magnification for each tumor, and microvessel density percent Ki-67 and percent cleaved caspase-3-positive cells were counted [9
]. SR-B1 expression in human epithelial ovarian tumors (n
= 50) was determined [38
] using paraffin-embedded human ovarian cancer specimens from the University of Texas MD Anderson Cancer Center's tumor bank after institutional review board approval. High versus
low expression was determined by a gynecologic pathologist using the following procedures: intensity was scored from 1 to 3, and distribution was scored from 1 to 4. The product of the two values was used to determine the extent of the expression, and a score greater than 4 was considered a high expression.
Fresh-frozen sections of tumor tissues from therapy experiments (SKOV3ip1 model) were stained by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL; green; Promega, Madison, WI) [38
] and counterstained with Hoechst 1:10,000 [44
]. An apoptotic body was represented by green fluorescence. To quantify apoptotic cells, TUNEL-positive cells were calculated in 10 random fields at 200x from five separate slides per group. Average values are presented.
Selective Delivery of siRNA
Female nude mice bearing SKOV3ip1 tumors were injected intravenously (intravenously [IV] through the tail vein) or intraperitoneally (IP) with 0.2 mg/kg of fluorescently tagged (Alexa555) control siRNA (Sigma-Aldrich) or untagged control siRNA (n = 3 per group). Forty-eight hours later, mice were killed, and tumors and organs (brain, heart, lung, liver, kidney, and spleen) were collected and frozen in OCT.
Fresh-frozen tumor tissues from orthotopic ovarian cancer models (SKOV3ip1) were fixed in acetone, washed with PBS, and counterstained with Hoechst (1:10,000). Fluorescence microscopy was used to analyze slides at 400x. Ten high-power fields were analyzed per slide, and a mean is reported.
Orthotopic Ovarian Cancer Models
Female nude mice (10–12 weeks old) were obtained from the US National Cancer Institute. All experiments were approved by the Institutional Animal Care and Use Committee of the MD Anderson Cancer Center.
FAK Targeting In Vivo
For FAK targeting experiments (SKOV3ip1 model), treatment was given in the following groups (n = 10 per group): 1) empty rHDL nanoparticles, 2) control siRNA/rHDL, 3) FAK siRNA/rHDL, 4) control siRNA + docetaxel, and 5) FAK siRNA/rHDL + docetaxel.
STAT3 Targeting In Vivo
For STAT3 silencing experiments, treatment was given according to the following groups (n = 10 per group): 1) control siRNA/rHDL, 2) control siRNA/rHDL + docetaxel, 3) STAT3 siRNA/rHDL, and 4) STAT3 siRNA/rHDL plus docetaxel. Tumor cells from appropriate ovarian cancer mouse models (HeyA8, 2.5 x 105; SKOV3ip1 and HeyA8-MDR, 1.0 x 106) were injected intraperitoneally into mice on day 0. Mice were randomized and treatment was started on day 7. About 3 (HeyA8) or 5 weeks (SKOV3ip1 and HEYA8-MDR) later, mice were subjected to necropsy, and tumors were harvested.
Colorectal Cancer Metastasis Model
For colorectal cancer metastasis model, HCT116 cells (1.0 x 106
) were injected into the spleen (n
= 10 per treatment group) of female nude mice. Two weeks later, treatment with oxaliplatin was started as previously described [39
], according to the groups shown in . After 4 weeks, mice were killed, and tumors were harvested.
Figure 2 Systemic delivery of siRNA using rHDL nanoparticles. (A) Uptake of Alexa555-labeled siRNA/rHDL in SKOV3ip1 tumors. A single IV or IP injection of 0.2 mg/kg of Alexa555 labeled or 0.2 mg/kg of IV and IP injection of untagged siRNA/rHDL was administered (more ...)
In Vivo Dose-Finding Experiment
An effective dose required to silence STAT3 or FAK genes in vivo was determined by injecting doses ranging from 0.1 to 0.2 mg/kg of STAT3 siRNA/rHDL or FAK siRNA/rHDL in SKOV3ip1 tumor-bearing mice. Mice were subjected to necropsy on day 2, 4, or 6. Tumors were harvested, and protein expression was determined using Western blot analysis as described above.
Continuous variables were compared using the Student's t test (between two groups) or analysis of variance (for all groups) if normally distributed. In nonparametric values, continuous variables were compared with the use of the Mann-Whitney rank sum test or Kruskal-Wallis test (for all groups). For in vivo rHDL therapy studies, 10 mice per treatment group were randomly assigned. Thus, our sample size provided 80% power to detect a 50% reduction in tumor weight at 5% level of statistical significance. We performed all statistical tests using SPSS (SPSS, Inc, Chicago, IL) and GraphPad Prism 5 software (GraphPad software, Inc, La Jolla, CA). We considered P < .05 to be significant. All statistical tests performed in this study were two-tailed.