Polyethylenimine (PEI, branched, MW 25 kDa), methoxy polyethylene glycol (PEG, Mw 2 KDa), sodium, carbon tetrachloride (CCl4
), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), triethylamine, triisobutylaluminum, linoleic acid, glutamine, ZnSO4
, dexamethasone, CuSO4
, Dulbecco's Modified Eagle's Medium (DMEM), Williams' E medium and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO), if not specified. Fetal calf serum and fetal bovine serum were purchased from HyClone (Logan, UT). Epidermal growth factor, penicillin–streptomycin, insulin and HEPES buffer were purchased from Invitrogen (Carlsbad, CA). N1
-bis(trifluoroacetyl) dipropyltriamine was synthesized following the procedure reported by O'Sullivan et al [20
]. The cyclic monomer 4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane was prepared as reported previously [21
2.2. Amplification and purification of plasmid DNA
VR1255C is a 6.4 Kb plasmid DNA encoding firefly luciferase driven by the cytomegalovirus (CMV) promoter (a gift from Carl Wheeler, Vical, San Diego, CA). The plasmid was amplified in Escherichia coli DH5α and purified by an endotoxin free QIAGEN Giga plasmid purification kit (QIAGEN, Valencia, CA). Purified plasmid DNA was dissolved in distilled water.
2.3. Synthesis and characterization of PEG-b-PPA copolymer
The synthesis of PEG-b
-PPA is summarized in . PEG-O−
) was prepared by reacting 1.0 g of methoxy PEG with potassium granules (over stoichiometry) in 50 mL of anhydrous THF for 8 h under refluxing [22
]. The concentration of PEG-O−
was determined by titration using 50 mM HCl. The polymerization of 4-alkyl-2-oxo-2-hydro-1,3,2-dioxaphospholane was initiated by adding PEG-O−
solution into the reaction vessel at a molar ratio of 1:500. The mixture was stirred at room temperature for 48 h. The precursor polymer (2
) was obtained by precipitation into anhydrous toluene followed by vacuum drying. The precursor polymer (2
) (3.0 g) was then dissolved in 20 mL of anhydrous DMF under argon. To this solution was added 27.2 g of N1
-bis(trifluoroacetyl)dipropyltriamine, followed by addition of 10 mL of anhydrous triethylamine and 10 mL of anhydrous CCl4
. The mixture was stirred at 0°C for 30 min then at room temperature for 24 h. The reaction mixture was then precipitated into ether and dried under vacuum to yield polymer (3
). The residue was suspended in 25% ammonia solution and stirred at 60°C for 16 h. The solution was concentrated and dialyzed in dialysis tubing (MWCO 3500, Spectrapor, Spectrum Labs, CA) against distilled water for 2 days with frequent water change. The PEG-b
) was obtained after lyophilization (0.6 g, yield 12%). PPA control (Mn = 24.8 KDa, polydispersity = 1.64) was prepared by a method we reported previously [14
]. The molecular weight of PEG-b
-PPA and PPA control was determined using a Waters 2690 HPLC module equipped with a Phenomenex PolySep-GFCP 4000 HPLC column (Torrance, CA), which was connected to a multiangle light scattering detector (MiniDawn, Wyatt Technology, Santa Barbara, CA) and a differential refractive index detector (Optilab DSP interferometric refractometer, Wyatt Technology). Phosphate buffer (0.1M, pH 7.4) with 0.15 M NaCl was used as the mobile phase (flow rate = 0.5 mL/min).
2.4. Compaction ability of PEG-b-PPA polymer
VR1255 DNA solution (25 μL) in DI water containing at a concentration of 20 μg/mL was added to an equal volume of PEG-b-PPA solution in DI water at various concentrations. The mixture was vortexed for 20 sec, incubated for 30 min at room temperature, and then electrophoresed on a 0.8% (w/v) agarose gel for 40 min at 80 V. The gel was stained with ethidium bromide and visualized on an UV illuminator (Eagle Eye II, Stratagene, La Jolla, CA).
2.5. Size and zeta potential of PEG-b-PPA/DNA micelles
Nanoparticle size and zeta potential were measured by photon correlation spectroscopy and laser Doppler anemometry, respectively, using a Zetasizers 3000 (Malvern Instruments, Southborough, MA, USA). Size measurement was performed at 25°C at a 90° scattering angle. The mean hydrodynamic diameter was determined by cumulative analysis. The zeta potential measurements were performed using an aqueous dip cell in the automatic mode.
2.6. Transmission electron microscopy
Ten μL of PEG-b-PPA/DNA micelle solution (N/P = 8, containing 25 μg/mL of DNA) and PPA/DNA complex solution (N/P = 8, containing 25 μg/mL of DNA) were added to a holey carbon TEM grid (SPI, West Chester, PA) and incubated for 5 min at 25°C, followed by washing with deionized water. The grid was further stained with uranyl acetate (1% solution) and washed with deionized water twice.
2.7. In vitro gene transfection
gene transfection was performed in HepG2 (human hepatocellular carcinoma) cells and primary rat hepatocytes. HepG2 cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum at 37°C and 5% CO2
. Cells were seeded in 24-well plates at a density of 6 × 104
cells per well and incubated for one day. Primary rat hepatocytes were harvested from male Wistar rats weighing from 250 to 300 g by a two-step in situ
collagenase perfusion method [23
]. The concentration and viability of hepatocytes were determined by Trypan blue exclusion test. Rat hepatocytes were plated in Type I collagen-coated 24-well plates (Collaborative Biomedical Products, Becton Dickinson, Bedford, MA) at a density of 3 × 105
cells per well, and allowed to incubate overnight before washing off the unattached cells. Hepatocytes were cultured in Williams' E medium supplemented with 10% fetal bovine serum, 10 ng/mL epidermal growth factor, 100 μg/mL penicillin–streptomycin, 5 nM dexamethasone, 0.5 μg/mL insulin, 15 mM HEPES, 50 ng/mL linoleic acid, 2 mM glutamine, 0.5 mg/mL bovine serum albumin, 50 pM ZnSO4
, 0.1 μM CuSO4
and 3 nM Na2
-PPA/DNA micelles or PPA/DNA complexes were added to each well at a dose of 2 μg of plasmid DNA. After 4 h of incubation, the culture media were refreshed. Two days later, the culture media were removed, and cells were washed with 0.5 mL of phosphate buffered saline (pH 7.4). Cells were then lysed with a reporter lysis buffer (0.2 ml/well, Promega, Madison, WI), and subjected to two freeze-thaw cycles. The suspensions were centrifuged at 14,000 rpm for 5 min. Twenty μL of cell lysate supernatant was mixed with 100 μL of luciferase substrate (Promega), and the light units were measured on a luminometer (Lumat LB9507, Berthold, Germany). The luciferase activity was converted to the amount of luciferase using recombinant luciferase (Promega) as the standard, and normalized against protein content using the BCA protein assay (Bio-Rad Laboratories, Hercules, CA).
2.8. Gene delivery in rats though intrabiliary infusion
The animal protocols were approved by the Animal Care and Use Committee at Johns Hopkins University School of Medicine. Male Wistar rats aged 6−8 weeks (200−300 g) were grouped randomly). The rat was first anesthetized with a mixture of Ketamine (60 mg/mL) and Xylazine (8 mg/mL), and the liver was separated from the surrounding tissue. A 33 gauge needle was inserted into the common bile duct and a tie was used to secure the needle. Four mL of PEG-b-PPA/DNA micelles (N/P = 8) or PPA/DNA complexes containing 20 μg VR1255 DNA in 5% glucose solution were administered through the needle 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 before the needle was withdrawn. After 30 min, all ties were removed. Stitches with 10-O nylon (Ethicon, Somerville, NJ) were used to repair the needle hole in the bile duct to prevent bile leakage, whenever necessary.
Three days after administration, three rats from each group were sacrificed, and major organs (liver, heart, lung, spleen and kidney) were harvested, weighed and homogenized with a tissue homogenizer (Heidolph, Schwabach, Germany). The homogenate was subjected to two freeze-thaw cycles and centrifuged at 14,000 rpm for 5 min at 4°C. Luciferase activity was analyzed as described above and normalized against the weight of whole tissue.
2.9. Evaluation of liver function and toxicity
Blood samples were collected from rat tail vein on Days 1, 2 and 3 after bile duct infusion, incubated at 4°C for 4 h and centrifuged at 2,000 rpm for 15 min. Serum was isolated and stored at −80°C. Liver functions including alanine aminotranferease (ALT) and aspartate aminotransferase (AST) activities were tested at the Clinical Chemistry Lab at the Department of Pathology, Johns Hopkins School of Medicine.
For histochemical analysis, liver tissue was isolated three days after injection, fixed in phosphate buffered formalin (4%), washed, and embedded in paraffin. H&E staining of sectioned tissues was performed by the Pathology Lab at Department of Comparative Medicine, Johns Hopkins School of Medicine.