Unless otherwise stated, reagents used were from Sigma (Dorset, UK).
PC-3 prostate cancer cells were purchased from ATCC, and maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA). PC-3M-luc-2 were purchased from Caliper Life Sciences (Buckinghamshire, UK) and maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA). Cells were cultivated in 175 cm2 flasks in a humidified incubator; once 80–90% confluency was reached, cells were passed to maintain exponential growth. Mycoplasma absence was confirmed monthly, using Plasmotest (Invivogen, France).
Plasmid DNA preparation
MAX Efficiency® DH5α™ Competent Cells transformed with pEGFP-N1 or CMV-iNOS plasmids were cultured in a shaking incubator overnight at 37 °C in Luria broth containing 50 μg/ml ampicillin. Plasmid DNA was isolated and purified using PureLink® HiPure Plasmid Maxiprep Kits (Life Technologies, Paisley, UK), as recommended by the manufacturer. Plasmid DNA, dissolved in ultrapure water, was stored at − 20 °C.
Nanoparticle complexation and characterization
RALA, supplied as a desalted lyophilized powder was reconstituted in ultrapure water to a stock concentration of 5.8 mg/ml. Aliquots were stored at − 20 °C until use.
Plasmid DNA (pDNA)/RALA nanocomplexes were prepared as described previously (McCarthy et al. 2014); electrostatic interaction between cationic RALA and anionic pDNA (30 min at room temperature) facilitates the formation of particles with size and charge characteristics suitable for gene delivery (McCarthy et al. 2014; Bennett et al. 2015; McCaffrey et al. 2016). Nanoparticles were complexed at N:P10 (the N:P ratio is the molar ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the pDNA backbone—at N:P10, 14.5 μg of RALA is complexed with 1 μg of DNA); nanoparticle size and charge can be altered by modifying the N:P ratio. For analysis of intracellular nanoparticle behavior, nanoparticles were complexed with RALA conjugated to fluorescein isothiocyanate (FITC) (Biomatik) and pDNA labeled with Cy3 using a Mirus Bio LabelIt® kit (Cambridge Bioscience, Cambridge, UK)). Nanoparticle physicochemical properties were analyzed using a Nano ZS Zetasizer and DTS software (Malvern Instruments, UK).
Transmission electron microscopy
RALA/DNA complexes were prepared at N:P 10 with 1 µg pCMV-iNOS in a total volume of 30 µl. Nanoparticles were loaded onto a carbon-coated copper 400 mesh grid (TAAB Laboratories, UK) and allowed to dry. Following drying, the samples were stained with 5% uranyl acetate in methanol at room temperature for 1 min, washed with 50% ethanol then molecular grade water and allowed to dry again. Nanoparticles were imaged using a JEM-1400Plus Transmission Electron Microscope (Joel, USA) at an accelerating voltage of 120 kV. Settings were as follows: pinhole (m) 95.5 µm, pinhole (airy) 999.4 µm, laser (Argon, visible) On (29%), Laser (DPSS 561, visible) On, laser (HeNe 633, visible) On, optical magnification with 10× and a 63× oil immersion objective, whole section depth was 19.13 µm, with 39 Z sections. Each section was 0.5 µm thick. The Z position of the XY image was 24, meaning 12 µm from the top of the cell.
Cellular uptake of FITC-RALA/Cy3-pDNA NPs
PC-3M-luc2 were seeded in 24-well plates at 104 cells per well, and incubated overnight. Cells were conditioned for 2 h in Opti-MEM (Life Technologies) before addition of nanoparticle complexes (NPs complexed at N:P 10), and cells were transfected with NPs equivalent to 0.5 µg DNA per well. Cellular FITC/Cy3 content was assessed over the following 120 h by flow cytometry using a CytoFLEX instrument (Beckman Coulter, Labplan, Dublin, Ireland). FITC and Cy3 contents were assessed using manufacturer settings for FITC and PE-A. For the 120-h timepoint, cells were transferred from the wells of 24-well plates to those of 6-well plates to allow for proliferation.
Intracellular nanoparticle tracking
PC-3s were seeded in 24-well plates on round coverslips at 104 cells per coverslip, and incubated overnight. Cells were conditioned for 2 h in Opti-MEM (Life Technologies) before addition of nanoparticle complexes, and cells were transfected for 240 min. Cells were fixed using 4% paraformaldehyde in PBS, and coverslips were mounted onto microscope slides using Diamond Antifade with DAPI (Life Technologies). Nanoparticle localization was analyzed by confocal fluorescence microscopy using a Leica SP5 microscope and LAS-AF software.
PC-3M-luc2 were seeded in T25 culture flasks at a density of 106 cells per flask, and incubated overnight. Following a 2-h starvation in Opti-MEM, cells were transfected with RALA/CMV-iNOS nanoparticle formulations, equivalent to 6 μg DNA per flask; following a 6-h transfection, transfection media were replaced with normal growth medium, and cells were incubated overnight. Following 24 h, cells were trypsinized, counted and plated in triplicate in 6-well plates at 500/1000 cells per well. Plates were incubated at 37 °C for 12 days, following which, colonies were fixed and stained using 0.4% crystal violet (Sigma) in 70% methanol; excess stain was removed by gentle washing in water, and once dry, colonies were manually counted.
In vivo immunological response to RALA/pDNA nanoparticles
In order to determine whether nanoparticles complexed of RALA and pDNA provoked an immune response, a range of ex vivo assays in C57BL/6 mice were performed. Mice were subjected to single or repeated administrations of PBS, RALA/pEGFP-N1 nanoparticles or polyethylenimine (PEI)/pEGFP-N1 nanoparticles. Each injection delivered nanoparticles (at N:P 10) equivalent to 10 µg pDNA, and injections were weekly for 3 weeks. 48 h after each injection, three mice were sacrificed; blood was collected by cardiac puncture, and the serum was extracted for analysis of total IgG, IgM, IL-1β, and IL-6 using assay kits (Enzo Life Sciences, Exeter, UK). The significance of the impact on these mediators was assessed using two-way ANOVA with Dunnett’s multiple comparisons test.
To determine whether repeated administrations of nanoparticles provoke neutralizing antibody responses, similar administrations were performed. Following sacrifice, blood was collected by cardiac puncture, serum was isolated, and sera from triplicate mice were pooled, heat inactivated and stored at − 20 °C. 5 × 103 PC-3 were seeded in triplicate wells of 96-well plates and allowed to adhere overnight. Cells were starved in Opti-MEM for 2 h prior to transfection. Freshly prepared RALA/pEGFP-N1 nanoparticles were incubated for 30 min in sera from mice that had received one of the indicated treatments. Sera/nanoparticle mixtures were diluted in Opti-MEM, and used to transfect PC3s. Transfections were for 6 h, following which, Opti-MEM was replaced with RPMI-1640. After 48 h, cells were analyzed for eGFP expression by flow cytometry using a BD FACSCalibur.
iNOS transgene expression
PC-3M-luc2 were plated (104 cells per well of a 24-well plate) and allowed to adhere overnight, and were transfected with RALA/CMV-iNOS for 6 h, following which Opti-MEM was replaced with phenol red-free MEM/10% fetal bovine serum. (RPMI-1640 is nitrite-rich, which would interfere with the nitrite content assay.) Medium nitrite content was assayed 48 h later using Greiss test for nitrites (Active Motif, Belgium), following the manufacturer’s instructions. Cellular iNOS expression was measured by western blot as previously described (Ning et al. 2012).
Establishment of metastatic disease
All animal experiments were carried out in accordance with the Animal (Scientific Procedures) Act 1986 and conformed to the current UKCCCR guidelines. Mice were bred in-house and maintained using the highest possible standard of care, and priority was given to their welfare.
Mice (6–8 weeks old) were anesthetized using isoflurane (3% in O2) and restrained using surgical adhesive tape in a supine position. Thoracic fur was removed using Veet hair removal cream. Using a 1-ml syringe/26G needle, mice were inoculated with 105 PC-3M-luc2 in 100 μl PBS via the left cardiac ventricle (Lim et al. 2011)—the cell suspension was gently injected into the ventricle, following which the needle was held in place for 10 s to minimize leakage from the ventricle. Mice were imaged using an IVIS200 (Xenogen) instrument to confirm appropriate ventricular delivery. Mice were injected intraperitoneally with 150 mg/kg d-luciferin; following a 15-min incubation, mice were anesthetized using isoflurane and imaged. Appropriate left ventricular delivery is characterized by luminescence throughout the body, while inappropriate delivery is characterized by luminescence that is limited to the thoracic cavity.
iNOS gene therapy
Gene therapy treatment began 48-h post-inoculation, with mice receiving treatments twice weekly, totaling five treatments. RALA/CMV-iNOS nanocomplexes (corresponding to 5 × 10 μg DNA per mouse) at N:P 10 were delivered via the tail vein. Solvent (PBS) and vehicle (RALA equivalent to the mass of RALA used in the gene therapy regimen) controls were also performed.
Mice were monitored for micrometastases development using routine IVIS imaging, as well as body mass measurement. A loss of 20% original body mass was deemed sufficient to necessitate sacrifice of the mouse. The degree of whole body luminescence in mice was determined using Living Image software (Perkin Elmer).
All statistics were performed using GraphPad Prism, version 6.0 g for Mac OS X. The tests used are described throughout.