N-isopropylacrylamide (NIPAM, 97%, Aldrich) was recrystallized from 70:30 hexane:toluene mixtures and dried in vacuo at room temperature. 2-(dimethylamino) ethyl methacrylate (DMAEM, Aldrich) was distilled under reduced pressure (45 ºC, 5 mmHg). 2,2′-azobi-sisobuyronitrile (AIBN, Aldrich) was recrystallized from methanol and dried in vacuo. 1,4-dioxane (ACS grade, Fisher), 2-mercaptoethanol (Aldrich) and ethidium bromide (Sigma) were used as received. Distilled deionized (DDI) water was prepared with a Millipore system and used for dilution purposes throughout.
The human nasopharyngeal cancer epithelial cell lines, CNE-1 and C666-1, used in this study have been described elsewhere (Cheung et al 1999
; Li et al 2002
). The CNE-1 cells were cultured in alpha minimum essential medium (α-MEM, Sigma) and C666-1 in RPMI-1640 (Sigma), respectively at 37 ºC in a humidified incubator containing 5% CO2
. The media were supplemented with L-glutamine (200 mM), penicillin (10,000 units), streptomycin (10 mg in aqueous sodium chloride 0.9%, Sigma) and 10% Fetal Calf Serum (FCS, Wisent Inc.). The cells were passaged by trypsinizing nearly confluent cells in T-75 flasks at 1:6 dilution for CNE-1 cells, and 1:3 dilution for C666-1 cells.
Synthesis and characterization of PNIPAM/DMAEM copolymers
To a Schlenk flask, DMAEM and NIPAM were added in the desired feed proportions to 100 mL of 1,4-dioxane (10%w/v monomers) and heated to 60 °C. For synthesis of polymers of low molecular weight, 2-mercaptoethanol was added as a chain transfer agent (CTA) at a 10:1 monomer/CTA molar ratio. The monomer mixture was allowed to thermally equilibrate and degassed under nitrogen for 10 minutes, followed by rapid injection of 2 mL of free-radical initiator, AIBN, degassed at 60 °C, at a 100:1 monomer/initiator molar ratio. After polymerization for 12 h under nitrogen, the reaction mixture was cooled and the resultant polymers were collected by precipitation into hexane and dried in vacuo. The polymers were dissolved in DDI water and exhaustively dialyzed for 72 h using a SpectraPor 3 membrane with molecular weight cut-off 3500.
The copolymer composition was determined by 1H NMR spectroscopy using Gemini 300 (Varian, Palo Alto, CA) in D2O at 300 MHz. The relative abundance of NIPAM and DMAEM in the copolymer samples was calculated from the ratio of the integrated area under the peaks corresponding to proton resonances unique to the monomers, that is, δ = 3.9 ppm for NIPAM, 1H, s, CONHCH(CH3)2, and δ = 4.4 ppm for DMAEM, 2H, t, COOCH2CH2N(CH3)2.
The intrinsic viscosity of polymers was determined from polymer solutions of concentrations ranging from 1 mg/mL to 5 mg/mL in 270 mmol/L KCl using a Cannon Ubbelohde #75 dilution type viscometer. The monovalent electrolyte was added to minimize electrostatic effects on the viscosity of polymer solutions. The average molecular weight of the polymers was determined using the Mark-Houwink (M-H) equation, η = K
, where K
values for the copolymers were computed from previously published data for NIPAM, KNIPAM
= 5.75 × 10–5
= 0.78, and for DMAEM, K
= 9.13 × 10–4
, respectively (Egoyan 1985
; Ganachaud et al 2000
), under the assumption of linear additivity of the parameters with respect to the copolymer composition determined by 1
The pKa of the copolymers was determined by potentiometric and conductometric coupled titration of 10 mg/ mL polymer solutions with 0.05 N NaOH at 25 °C using a Radiometer Copenhagen ABU-93 triburette autotitrator mated to a Radiometer Copenhagen Model CDM92 conductivity meter. The equivalent points in the titrations of polymer solutions we found from the inflection points of the titration curves. The pKa values were derived from the potentiometric data and represented the pH at one half the volume of base required to fully titrate tertiary amino groups on DMAEM. There was good agreement between pKa values derived potentiometrically and those obtained conductomerically.
Preparation and purification of plasmid PDC312/oriP.luc
Double stranded plasmid DNA (pDNA) PDC312/oriP.luc
(6 kb) expressing firefly luciferase (luc
) under control of the oriP promoter was prepared by ligating the SalI/BamHI fragment, containing the oriP.luc
cassette, isolated from pΔ E1sp1A/oriP.luc
as described by Li et al (2002)
with PDC312 cut with the same enzymes. The plasmid was transformed into Escherichia coli
DH5-a competent cells and purified using a QIAGEN plasmid Mega kit. The DNA concentration and purity of the pDNA was assessed by measurement of the UV absorption at 260 and 280 nm.
Preparation and purification of Adenovirus
Adenovirus vectors expressing luc
under the control of a cytomegalovirus (CMV) promoter were amplified in 293 cells using established methods (Graham and Eb 1973
), and purified from cell lysates by banding twice on CsCl gradients. Purified virus was then desalted overnight in 1:1000 parts by volume virus solution to Tris-HCl pH 8.0 buffer. Viral concentrations and purity were determined by the absorbance at 260 and 280 nm. The concentration of viral particles was calculated from the optical density at 260 nm (OD260
), using the formula 1 OD260
= 1.1 × 1012
particles/ml as derived by Maizel et al (1968)
Preparation and characterization of pDNA polyelectrolyte complexes (PECs) and polymer-modified adenovirus
PECs were prepared by the addition of aliquots of polymer stock solution (200 μg/mL in PBS) to pDNA (2 μg) to give the desired molar ratio of nitrogen atoms in the polymers to phosphorous atoms in the DNA (N:P). The N:P ratio was calculated from the equation:
is the weight fraction of DMAEM in the copolymer, wpol
is the weight of the copolymer, wDNA
is the weight of pDNA, and the constants 157 and 325 represent the weight of DMAEM and DNA per nitrogen and phosphorous atom, respectively.
The total volume of solution was adjusted to 400 μl with PBS and vortexed for several seconds. The quantity of reagents was scaled up when necessary. Complex formation was carried out for 30 min at room temperature with gentle mixing using a hematological mixer. PECs used in transfection experiments and for complexation with Ad5 were prepared in the same manner except that either α-MEM or RPMI-1640 was used in place of PBS.
Type I complexes between cationic polymers and Ad5 particles were prepared by mixing stock polymer solutions in either α-MEM (CNE-1 cell infection) or RPMI-1640 (C666-1 cell infection, with Ad5 dilutions at the desired ratio of polymer to virus (typically 100 to 1000 polymer molecules/Ad5 particle) and MOI (see Figure legends). Type II complexes were prepared by mixing PECs as described above with Ad5 dilutions.
Dynamic light scattering and zeta-potential analysis of PECs
The volume–average particle size and size distribution of freshly prepared PECs of different N:P ratios in deionized water at 25 °C was measured using a NICOMP 380ZLS dual zeta/dynamic light scattering instrument equipped with a 10 mW, 632.5 nm laser in particle sizing mode. For zeta-potential measurements, the NICOMP 380ZLS was switched to zeta-mode. All particle size and zeta-measurements were run in triplicate using 5 iterative cycles per sample. Prior to measurements, the NICOMP 380ZLS was calibrated using polystyrene latex particles (Polysicences Inc.) of known hydrodynamic size and surface charge.
Circular dichroism spectroscopy
Complexation of polymers with Ad5 and pDNA was examined by circular dichroism (CD) spectroscopy. PECs and polymer-modified Ad5 were prepared as described earlier. CD spectra of pDNA, Ad5 and PEC and Ad5 complexes were recorded at 20 ºC in a 1 mm path length cuvette using an AVIV model 62A DS spectropolarimeter (Lakewood, NJ). The integration time was 1s and the slit width 2 nm.
Ethidium bromide displacement assay
The ethidium bromide assay was carried out to probe the association of polymers with pDNA. A 200 μL aliquot of 50 μg/mL pDNA in PBS solution was diluted to 2 mL volume in a 1 cm cuvette. A 1 μL aliquot of a 400 μg/mL EtBr solution was introduced to the pDNA and mixed by gentle inversion. The fluorescence emission was recorded (λex
= 512 nm, λem
= 600 nm, slit width ex/em = 5 nm/5 nm) on a Spex FluoroMax-3 fluorometer. A 5 mg/mL polymer solution was titrated into the pDNA/EtBr solution and the fluorescence emission monitored. The relative fluorescence was calculated from the ratio of the observed fluorescence in presence of polymer relative to that in its absence, correcting for EtBr fluorescence according to the relation:
is the fluorescence emission intensity
Gel retardation assay
The gel retardation assay was used to evaluate that binding of the polycation to pDNA was the result of condensation. PECs incorporating 1 μg of pDNA were formed by mixing pDNA stock solution with aliquots of polymer stock solutions (100 μg/ml in PBS). The total volume was adjusted to 200 μl with PBS and complexation was carried out for 1 h at 25 °C. The PEC solution (25 μl) was run on a 0.7 wt.% agarose gel (100 V) in 1 × TAE (40 mM Tris-acetate and 1 mM EDTA, pH 8.3) buffer. DNA bands were visualized by ethidium bromide staining.
In vitro transfection of CNE-1 and C666-1 NPC cells by PECs and polymer-modified Ad5
CNE-1 and C666-1 cells were seeded in a 24-well culture plate at 1 × 105 and 2 × 105 cells/well, respectively. For infection tests, the growth media were removed and the cells rinsed with PBS. A 200 μL aliquot of the Ad5 or polymer-modified Ad5 vector in 2% heat-inactivated FBS media was introduced to the cells, typically at a multiplicity of infection [MOI] = 50 and incubated for 1 h at 37 °C (5% CO2). The cells were washed 3 times with 200 μL of PBS, followed by addition of a fresh medium containing 10% FBS. The cells were incubated for 24 h to allow for luciferase expression. The medium was then removed and the expressed luciferase was isolated according to the protocol supplied with the Dual-Light® Reporter Gene Assay System (Tropix, Applied Biosystems, Foster City, CA). All samples were run in triplicate using a ThermoLabsystems Luminoskan Ascent luminometer (Thermo Electron Corp., Waltham, MA, USA) for chemiluminescent detection.
Assay of cellular toxicity of NIPAM/ DMAEM polymers
CNE-1 cells were seeded in 96-well culture plates at 2 × 104 cells/well in α-MEM. The medium was removed from plated cells and replaced with various concentrations of polymer (0.25–10 mg/ml) in serum-free α-MEM for 1 h at 37 °C under 5% CO2. The polymer solutions were then removed and replaced with α-MEM plus 10% FBS. The cells were allowed to proliferate for various time points (ie, 8–48 h) prior to the addition of 100 μL of MTT reagent (α-MEM, 2% FBS). After 3 h incubation in presence of MTT reagent, 175 μL of 0.1% HCl in isopropyl alcohol was added to each well and pipette tip aspirated. The absorbance of the solution was read at 570 nm using a BioRad 3550 microplate reader (BioRad, Hercules, CA). The cytotoxicity was calculated as the percentage of viable cells.
The experiment was repeated in triplicate using three different sets of cultured cells. Within each experiment, the values of six independent measurements (6 wells) at each concentration were obtained. The results are expressed as the mean ± S.D. from data obtained from the three separate measurements.
Transmission electron microscopy
A 50 μL aliquot containing 9 × 109 Ad5 particles in α-MEM was combined with a 50 μL aliquot of polymer solution at a polymer/Ad5 ratio of 100 polymer molecules/particle. The solution was gently aspirated and incubated at 37 °C for 15 min. 10 μL aliquots of polymer-modified Ad5 fixed with OsO4 and dispersed onto a Formvar-coated TEM grid, stained with lead citrate, and counterstained with 1% aqueous uranyl acetate for 30s. The solution was then removed and the grids dried in air. TEM images were captured using a Hitachi H-7000 (Tokyo, Japan) transmission electron microscope.