Polyethylenimine (branched PEI, average molecular weight of 25 kDa), was purchased from Sigma-Aldrich (St Louis, MO, USA). PEI was purified by dialysis against water (dialysis tubing with MWCO 3500; Pierce, Rockford, IL, USA) for 3 days and lyophilization. Chitosan C390 (MW 390 kDa, deacetylation degree 83.5%) was kindly provided by Vanson Halosource (Redmond, WA, USA). Chitosan solution was prepared in 5 mM sodium acetate (NaAc) buffer at a concentration of 0.02 % (w/v). The pH of chitosan solution was adjusted to 5.5 and the solution was sterile-filtered through a 0.22 μm filter (Millipore, Billerica, MA, USA).
Amplification of plasmid DNA
Plasmid pVR1255 is a 6,413b pcDNA encoding luciferase driven by human CMV promoter (a gift from Dr Carl J Wheeler, Vical Inc., San Diego, CA, USA). The plasmid was amplified in Escherichia coli DH5α and purified by Qiagen Giga plasmid purification kit (endotoxin free, Qiagen, Hilden, Germany). Purified plasmids were dissolved in saline and kept in aliquots at a concentration of 1 to 2 mg/mL. Terrific Broth and ampicillin were purchased from Gibco BRL (Grand Island, NY, USA).
Preparation of chitosan-DNA and PEI-DNA nanoparticles
A chitosan solution (0.02% in 5 mM sodium acetate buffer, pH 5.5) and a DNA solution (100 μg/mL in 5 mM of NaAc solution) at an N/P ratio of 3 (N/P = molar ratio of chitosan nitrogen to DNA phosphate) were preheated to 50–55°C separately. An equal volume of both solutions were quickly mixed while vortexing. PEI-DNA nanoparticles were prepared by mixing PEI solution (133 μg/ml in 5% glucose) with equal volume of DNA solution (100 μg/ml in 5% glucose) at an N/P ratio of 10. Both chitosan-DNA and PEI-DNA nanoparticles were incubated at room temperature for 30 min before characterization and transfection. The particle size and zeta potential of chitosan-DNA and PEI-DNA nanoparticles were measured on a Zetasizer® 3000 (Malvern Instruments, UK).
Bile was isolated from rat common bile ducts over 30- to 60-min periods and frozen at −80°C within a few hours. It was added to chitosan-DNA nanoparticles, PEI-DNA nanoparticles, and naked DNA solutions to reach the final concentrations of 10% and 50% (v/v), respectively. The solutions were then incubated at 37°C. At different time points, aliquots of samples were taken, quenched at 4°C before analysis by electrophoresis on a 1% agarose gel and ethidium bromide staining for visualization.
To investigate the degradation mechanism of bile, an aliquot of rat bile sample was pretreated by heating in a boiling water bath for 15 min and cooled on ice. It was added to chitosan-DNA nanoparticles and naked DNA solutions at a final concentration of 10%. In parallel, aliquots of chitosan-DNA nanoparticles and naked DNA were incubated with 10% untreated bile, 10% untreated bile with 50 mM of NAC, or 10 mM of hydrogen peroxide. The samples were incubated at 37°C. At 3-hour and 5-day time points, aliquots of samples were quenched at 4°C before analyzed by electrophoresis on a 1% agarose gel and ethidium bromide staining for visualization.
Six- to eight-week-old male Wistar rats were obtained and housed in National University of Singapore Animal Holding Unit. Rats were maintained on ad libitum rodent feed and water at room temperature, 40% humidity. All animal procedures were approved by the National University of Singapore Faculty of Medicine Animal Care and Use Committee.
Isolation and culture of hepatocytes, Kupffer cells, and normal rat cholangiocytes (NRC)
Hepatocytes were harvested from male Wistar rats weighting from 200 to 250 g by a two-step in situ collagenase perfusion as described previously (Zhang et al 2001
). Hepatocytes were collected by centrifugation twice at 50×g for 3 min. Hepatocytes (pellet) were seeded on collagen coated substrates, and maintained in William’s E medium supplemented with 1 mg/mL BSA, 10 ng/mL of EGF, 0.5 μg/mL of insulin, 5 nM dexamethasone, 50 ng/mL linoleic acid, 100 U/mL penicillin, and 100 μg/mL streptomycin. The non-parenchymal cell fraction in the supernatant was washed with buffer and centrifuged at 650×g for 7 min at 4°C. Cell pellets were centrifuged on a density cushion of Percoll (25% and 50%) at 2500×g for 15 min at 4°C. The KCs fraction was then collected and seeded in a tissue culture flask, and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cell viability was determined using the Trypan Blue exclusion method. All cell culture media and supplements were from Gibco BRL (Grand Island, NY, USA) unless specified. The NRC cell line was kindly provided by Dr Nicholas F LaRusso (Mayo Clinic, Rochester, MN, USA) and were maintained on collagen-coated tissue culture inserts with polyester membrane (6.5 mm, Nalge Nunc, Rochester, NY, USA) in DMEM/Ham’s F-12 medium with supplements as described by Vroman and LaRusso (Vroman and LaRusso 1996
Transfection of primary rat hepatocytes, KCs, and NRCs
Isolated hepatocytes and KCs were seeded 24 hours prior to transfection into a 24-well plates at an initial density of 3 × 105 cells per well. Before transfection, the medium in each well was replaced with 0.5 ml of fresh William’s E medium or RPMI 1640 for hepatocytes or KCs, respectively, with or without 10% bile. Nanoparticles containing 3 μg of DNA or 3 μg naked DNA was added to each well. The medium was refreshed after 4 h of incubation with cells. After 48 hours, cells were lysed with cell lysis buffer (Promega). Transfection of NRCs was conducted in transwell inserts (Nalge Nunc) in a 24-well tissue culture plate. When the NRCs reached 70% confluency, nanoparticles containing 3 μg of DNA or 3 μg naked DNA was added to each well. The medium was refreshed after 4 hours of incubation with cells. The analysis of luciferase expression was conducted according to the same procedure described above.
Luciferase activity in the cell extract was measured on a luminometer (LUMAT LB9507, Berthold, Germany) using a luciferase assay system from Promega (Madison, WI, USA). The relative light units (RLU) are normalized against protein concentration in the cell extracts using a micro-BCA protein assay kit (Pierce, Rockford, IL, USA).
Luciferase expression following RII, intraportal, and intravenous injections of nanoparticles and plasmid DNA
Wistar rats (male, 200–250 g) were randomly assigned to groups of 15–17 rats. Animals were laparotomized under general anesthesia and the liver was isolated from the surrounding tissue. A 33G needle was inserted into the common bile duct and a tie was used to secure the needle. Nanoparticles and naked DNA were administered at the dose equivalent to 200 μg of plasmid (~0.8 mg/kg of body weight) in 4 mL of medium into the common bile duct over 20 min (0.2 ml/min) using a syringe pump. A tie was then placed around the bile duct between the liver and the point of infusion to prevent back flow, and the needle was withdrawn. After 30 min, all ties were removed. The needle hole in the bile duct might require stitches with 10-O nylon suture (Ethicon, Somerville, NJ, USA) to prevent bile leakage, whenever necessary. Rats were kept on normal diet. For portal vein injection using a syringe pump, surgical operation was performed as previously reported (Zhang et al 2001
). Nanoparticles or naked DNA was infused at 0.2 mL/min using the same DNA concentration as described above. Tail vein injection was also performed using a syringe pump through a 30 G needle over 2 min.
On days 3, 7, and 14, 5 rats from each group were sacrificed. Major organs (liver, heart, lung, spleen, and kidney) were harvested and stored at −80°C for analysis. Each liver was divided into 4 sections composed of median, left, right, and caudate lobes. Two mL of lysis buffer (0.1% Triton X-100, 2 mM EDTA, and 0.1 M Tris-HCl, pH 7.8) per g of tissue was used for each sample, and the tissue was homogenized and subjected to 2 freeze-thaw cycles. The homogenates were centrifuged at 14000 rpm for 10 min. Luciferase activity in the homogenate was measured for 10 sec on a luminometer and converted to the mass of luciferase expressed per gram of tissue using a standard curve generated in parallel on the same luminometer. Student’s t-test was used to compare the difference between selected groups. Differences were considered statistically significant when p<0.05.
DNA distribution after RII of nanoparticles and naked DNA
To characterize DNA distribution, animals were injected with DNA nanoparticles containing 200 μg of Cy5-labeled plasmid DNA (pGeneGrip, Gene Therapy Systems, Inc.) through RII. Four hours after injection, major organs including liver, heart, spleen, lung, and kidney were resected, embedded in OCT medium, and frozen in liquid nitrogen as described above. For immunofluorescence staining, cryosections (μ8 μm) on microslides were fixed in cold acetone, and incubated with Hoechst 33258 to stain cell nuclei. Staining of endothelial cells was performed with a mouse anti-rat mAb against rat endothelial cell antigen (5 μg/mL, RECA-1, ab9774, Abcam, Cambridge, MA, USA) and a goat anti-mouse IgG2a HRP conjugate (1:100; Zymed, San Francisco, CA, USA), using a Tyramide Signal Amplification Fluorescein System (NEN, PerkinElmer, Wellesley, MA, USA). For staining of KCs, slides were incubated with mouse anti-rat macrophage F-6-J mAb [ab8173; Abcam; 10 μg/mL in I-Block (Tropix, Applied Biosystems, Foster City, CA, USA) and subsequently with a F(ab)2 goat anti-mouse IgG-FITC Ab (Abcam; 7 μg/mL in I-Block). Slides were mounted with Gel/Mounting medium (Abcam) and evaluated under a Nikon confocal microscope equipped with an imaging system (Nikon Singapore, Singapore).
Damage/toxicity to liver and biliary tree following RII
Blood samples were drawn on days 1, 2, 3, 7, and 14 from the tested rats. ALT, AST, ALP, and bilirubin levels in serum samples were analyzed using a multiparametric automatic analyzer in the Clinical Chemistry Laboratory at the National University Hospital in Singapore. Histopathological examination was performed on liver tissues collected at same time points. For collection of tissue, rats were anesthetized, perfused with 4% paraformaldehyde (PFA) in PBS, and euthanized. Extracted tissue will be further fixed in 4% PFA, and routinely processed for paraffin-section. Sections will be stained with hematoxylin-eosin.