Preparation of wt-p53 plasmid loaded gelatin nanoparticles
Thiolated gelatin was synthesized and purified using an established method that conjugates 2-iminothiolane to primary amine groups on type B gelatin [23, 25]. Lyophilized purified thiolated gelatin was used for nanoparticle preparation and encapsulation of plasmid by desolvation method developed and optimized in our lab [23, 25, 29]. Typically, 1% (w/v) thiolated gelatin solution was prepared in deionized distilled water at 37°C and pH was adjusted to 7 using 0.2 M NaOH. 1 mg plasmid DNA was gently mixed in the gelatin solution followed by slow addition of chilled ethanol with continuous stirring at 600 rpm. Gelatin nanoparticles are formed when the solvent composition changes to 75% hydro-alcoholic solution following which 0.5 mL 8% (v/v) glyoxal solution was added drop-wise to crosslink the thiol group. The particles were purified and concentrated by tangential flow filtration, freeze-dried and stored at 4°C until used.
SH-Gel-PEG and SH-Gel-PEG-peptide nanoparticles were prepared by a method described before [23, 29]. Briefly, freeze-dried nanoparticles (10 mg/mL) were suspended in 0.1 M phosphate buffer (pH 7.4) and incubated with methoxy-PEG-succinimidylcarbosyl methyl ester (mPEG-PEG-SCM, MW 2000 Da) or maleimide-PEG-SCM (MAL-PEG-SCM, MW 2000 Da) for 2 h at room temperature with slow stirring to form SH-Gel-PEG and SH-Gel-PEG-MAL particles respectively. The particles were purified by ultra-centrifugation at 18,800 g for 30 min (Beckman Coulter Optima™ LE-80 k; rotor 70Ti; Brea, CA), washed twice in deionized water and freeze-dried. SH-Gel-PEG-MAL particles (10 mg/mL) were suspended in 0.1 M phosphate buffer (pH 6.5) with 10% weight of 12 amino acid EGFR binding peptide flanked with four glycine spacer and a terminal cysteine (i.e., Y-H-W-Y-G-Y-T-P-Q-N-V-I-G-G-G-G-C) for 6 h at room temperature to facilitate binding of the sulfhydryl group of cysteine to maleimide group on PEG. The peptide modified nanoparticles were purified by ultra-centrifugation at 16,000 rpm for 30 minutes, washed twice in deionized water, freeze-dried and stored at 4°C until used. The physico-chemical properties, plasmid loading efficiency and stability of the particles were characterized, details of which have been published elsewhere . The typical average size of gelatin nanoparticles was found to be between 130-230 nm with SH-Gel being the smallest in size (~ 130 nm). PEG modification of the SH-Gel particles increased the size to nearly 180 nm and subsequent peptide functionalization further increased the size to nearly 230 nm. The average surface charge of the nanoparticles was found to be around -20 mV and the p53 gene loading efficiency was found to be around 95%.
Preparation of gemcitabine loaded gelatin nanoparticles
10 mg base form of gemcitabine was dissolved in 5 mL methanol with 100 mg succinimidyl 3-[2-pyridyldithio]-propionate) (SPDP) at 80°C under reflux for 48 hours. The reaction was monitored by thin-layer chromatography (TLC) (R
0.67 (CHCl3/MeOH, 8:2)). Solvent was removed in vacuo with rotary evaporator IKA RV10 at 60°C, and the residue was purified by silica gel chromatography (200 mL, CHCl3 and 200 mL CHCl3/MeOH, 9:1) to give gemcitabine-SPDP. UV spectrometer was used to monitor elute at λ = 268 nm.
Purified gemcitabine-SPDP was dried in vacuo and then dissolved in 1 mL dimethyl sulfoxide (DMSO). For conjugation with gemcitabine-SPDP, thiolated gelatin (10 mg/mL) was dissolved in 0.1 M PBS/EDTA (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, pH 7.5). Gemcitabine-SPDP was added to thiolated gelatin solution and stirred overnight at room temperature. Formed gemcitabine-gelatin disulfide conjugates were dialyzed against DI water overnight and then purified polymers were used for nanoparticle synthesis. The conjugation of gemcitabine-SPDP and gemcitabine-gelatin were confirmed by reverse phase HPLC, using a C18 column (Thermo-Fisher Scientific, MA), with the UV detector set at 268 nm. The mobile phase was composed of 20% of MeOH/H2O (5:5) and 80% 0.5 M ammonium acetate solution. The elution was performed by isocratic flow and flow rate was 1 mL/min. Standard curve was established with pure gemcitabine and release of drug was determined based on standard curve. Gemcitabine loaded gelatin nanoparticles were synthesized following the protocol similar to one used for p53 gene loaded nanoparticle synthesis (described above). The size and charge measurement of gemcitabine loaded SH-Gel, SH-Gel-PEG and SH-Gel-PEG-peptide nanoparticles were found to be consistent with that observed for p53 gene loaded nanoparticles. The average particle size was found to be between 130-230 nm and the average surface charge for all the different nanoparticle systems was found to be around -20 mV.
In vitro release of gemcitabine from the nanoparticles was performed in the presence of proteolytic enzyme (0.2 mg/mL) and glutathione to mimic the intracellular (5 mM) and extracellular (0.1 mM) environment in the tumor . The drug release studies were carried out at 37°C with PBS solution as control. 20 mg of gemcitabine-loaded nanoparticles were weighed into microcentrifuge tubes and dissolved in 1.5 ml of buffer containing glutathione and/or protease. Samples were incubated in temperature controlled shaker and 0.5 ml of supernatant was drained at specified intervals (15, 30, 45, 60, 120, 240 and 360 minutes). Sink conditions were maintained by replacing an equal volume of release medium each time. Collected samples were centrifuged at 13,000 rpm for 15 min, filtered through 0.2 μm filters and analyzed by reverse phase HPLC, using a C18 column using assay condition described above. Additional file 1: Figure S1 shows the drug release profile of SH-Gel, SH-Gel-PEG and SH-Gel-PEG-peptide nanoparticles.
Subcutaneous pancreatic tumor model development
Panc-1 human pancreatic adenocarcinoma cells were obtained from American Type Culture Collection ATCC, Manassas, VA) (Manassas, VA) and were grown in DMEM media supplemented with 10% FBS and 1% Pen-Strep. Animal handling and procedures were performed according to an approved protocol by Northeastern University, Institutional Animal Care and Use Committee (NU-IACUC) and the Radiation Safety Committee within the office of Environmental Health and Safety. Six weeks old female SCID Beige mice, weighing approximately 20 g, were purchased from Charles River Laboratories (Wilmington, MA) and were used for efficacy studies.
To inoculate subcutaneous tumors, animals were mildly anesthetized by inhalation of 2% Isoflurane (St. Joseph, MO) in 100% oxygen and approximately 3 million Panc-1 cells in 100 μl of PBS and Matrigel mixture (1:1) was injected subcutaneously into the left flanks of female SCID Beige mice. Tumors were allowed to grow and reach a palpable volume and during this period, the animals were monitored for food/water intake, body weight and any signs of discomfort. Any animals that seemed lethargic were sacrificed.
In vivo nanoparticle administration and dosing schedule
The animals were randomized into different treatment groups for efficacy studies when the tumor volume reached 200 mm3. The dosing schedule and treatment groups for different formulations of wt-p53 alone, gemcitabine alone and wt-p53/gemcitabine in combination have been outlined in Additional file 1: Figure S2. The plasmid treatment group mice were each administered with 20 μg plasmid at day 0, 2 and 4. From the 12 mice per treatment group, 3 mice were euthanized at day 7 and 18 for in vivo transfection analysis while remaining 6 mice were sacrificed after completion of the study (day 33). Similarly, mice receiving gemcitabine treatment were intravenously administered with 4 doses of free or formulated drug at a dose of 5 mg/kg at day 0, 7, 14 and 21.
The wt-p53 plasmid and gemcitabine combination efficacy study was performed where plasmid loaded particles were dosed at day 0, 2 and 4 followed by gemcitabine loaded particle administration at day 5, 12, 19 and 26. In vitro studies of gelatin nanoparticle loaded p53 gene transfection followed by assessment of its expression and effect on downstream apoptosis markers revealed that apoptotic activity is maximum 96 hour post-transfection . The first dose of gemcitabine was therefore administered at day 5 when the apoptotic effect of p53 gene would have taken effect. All doses for efficacy studies were administered to the animals intravenously via tail vein injection. Tumor volumes were measured and recorded every 3 days for all treatment groups and all the mice were sacrificed at the completion of the study (day 33). The mice were sacrificed by isoflurane inhalation followed by cervical dislocation, tumor mass was weighed and flash frozen in liquid nitrogen for analysis of protein, mRNA and apoptotic markers.
Quantitative transfection efficiency and downstream apoptosis marker evaluation
In vivo gene transfection efficiency was evaluated by quantitative polymerase chain reaction (qPCR). For mRNA extraction, tumors were excised and stored in RNAlater® (Invitrogen, Carlsbad, CA) at 4°C overnight and then -20°C for long-term storage. mRNA of tumor samples was extracted using Powergen 125 tissue homogenizer (Fisher Scientific, Waltham, MA) and TRIzol® Reagents, PureLink® RNA Mini Kit (Invitrogen, Carlsbad, CA). Extracted RNA was measured with Nano-Drop® 2000 (Thermo-Scientific, Wilmington, DE). cDNA was synthesized from 2 μg of extracted mRNA with SuperScript® III First-Strand Synthesis SuperMix Kit (Invitrogen, Carlsbad, CA). 2 μL of synthesized cDNA and LightCycler® 480 SYBR Green kit (Roche, Indianapolis, IN) were used for qPCR in LightCycler® 480 and analyzing mRNA levels of Flag-p53 and corresponding downstream transcription factors. Primer sequences (Additional file 1: Table S1) for p53, Bax, Bcl-2, β-actin, DR5, Apaf-1, PUMA, caspase 3 and caspase 9 were synthesized in Eurofins MWG Operon (Huntsville, AL). All the results for gene expression have been calculated and reported relative to the control group.
Qualitative transfection efficiency and downstream apoptosis marker evaluation
Proteins were extracted from tumors using Total Protein Extraction Kit (Millipore, Billerica, MA) and Powergen 125 tissue homogenizer (Fisher Scientific, Waltham, MA). Tissue lysate samples were analyzed for total protein concentration using BCA assay (Pierce, Rockford, IL, USA). 50 μg of total protein extract was run on pre-cast 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system at 200 V for 30 minutes. Subsequently, protein bands on gel were transferred onto PVDF membrane by iBlot® Dry Blotting System (Invitrogen, Carlsbad, CA). Membrane was blocked with 5% milk in Tween®-containing Tris buffer saline (TBS-t) for 1 hour at room temperature. Membrane was cut and incubated with 1:1,000 dilution of primary rabbit β-actin antibody, 1:500 primary rabbit cleaved PARP antibody, 1:500 primary rabbit cleaved caspase 3 (Cell Signaling Technology Inc., Danvers, MA) or 1:1000 dilution of primary mouse monoclonal anti-FLAG®M2 antibody (Sigma-Aldrich, St. Louis, MO) separately overnight at 4°C. Membranes were then washed three times with TBS-t and incubated with 1:2,000 dilutions of secondary anti-rabbit or anti mouse horse-radish peroxidase-conjugated IgG(Cell Signaling Technology Inc., Danvers, MA) in TBS-t for 1 hour at room temperature. After rinsing excess antibody with TBS-t and water, 4 ml ECL substrate (Pierce, Rockford, IL, USA) was added and mixed with membranes for 5 minutes, which is cleaved by peroxidase to give a chemiluminescent product. The membranes were visualized using Kodak Digital X-ray Specimen (DXS) System. β-actin was used as protein loading control.
Terminal deoxynucelotidyl transferase dUTP nick end labeling (TUNEL) analysis
TUNEL analysis was performed on the tumor sections to confirm the DNA fragmentation as a result of activation of apoptotic signaling cascade. Excised tumors were embedded in frozen section medium (Richard-Allan Neg 50, Thermo Scientific, Waltham, MA), flash frozen in liquid nitrogen, and stored at -80°C until use. Embedded tumors were thawed to -20°C, cryo-sectioned into 10 μm thick sections using the Microm® HM550 cryostat (MICROM International GmbH, Germany), and mounted onto glass slides (SuperFrost Plus®, Thermo Scientific, Waltham, MA). Sections were air dried at room temperature and then stored at -20°C. DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI) was used for tissue staining. After staining, tissues were mounted with Fluoromount-G (Southern Biotech, AL), covered with a coverslip, sealed with nail polish and imaged by Olympus BX61 microscope.
All the statistical analysis was performed using Prism 5.0 software (Graph Pad Software Inc., San Diego, CA). Results were expressed as mean ± SD of the at least three independent experiments. Data was analyzed by Student’s t-test or one way ANOVA followed by Bonferroni’s post hoc analysis for multiple comparisons. Differences were considered statistically significant at p < 0.05.