**2. Materials and Methods**

#### **2.1. Reagents**

properties and lacking the disadvantageous aspects would emerge as the most attractive one

A major barrier to the non-viral delivery is low uptake of DNA across the plasma mem‐ brane of a cell owing to the inappropriate and ineffective interactions of the DNA delivery vehicle with the cell membrane. Negatively charged DNA molecules are usually condensed with cationic reagents to allow formation of the complexes carrying net positive charges. The resulting complexes can interact electrostatically with anionic heparan sulfate proteoglycans (syndecans) on cell surface and reach the cytoplasmic side in the form of endosomes through endocytosis [1]. The extremely low pH and enzymes within the late endosomes usually bring about degradation of entrapped DNA and associated complexes. Finally, DNA that survives both endocytic processing and cytoplasmic nucleases must dissociate from the condensed complexes either before or after nuclear translocation through nuclear pore or during cell

Many therapeutic applications demand a vehicle with capability of delivering transgene(s) to a selective cell type in order to increase the expression efficacy and alleviate any side effect. A common strategy in non-viral case involves attachment of a targeting moiety onto a poly‐ cation (lipid or polymer) backbone which finally condenses the DNA through ionic interac‐ tions. Targeting moiety can enable the resulting DNA carrier to bind to a receptor, lectin, antigen or cell-adhesion molecule on plasma membrane prior to internalization via endocy‐ tosis or phagocytosis. Polylysine, the first backbone used for gene delivery has been conjugat‐ ed to a diverse set of cell-specific ligands, such as asialoorosomucoid [2], transferrin [3], epidermal growth factor (EGF) [4], mannose [5], fibroblast growth factor (FGF) [6] and anti‐ bodies [7] for targeting, respectively, hepatocytes via asialoglycoprotein receptors, transfer‐ rin receptor-positive cells, EGF receptor-carrying cells, macrophages through membrane lectins, FGF receptor-bearing cells and lymphocytes via surface-bound antigens. In the similar fash‐ ions, polymers like polyethylenimine and liposomes have been coupled to other cell surface receptor-specific ligands in addition to those described above, such as integrin-binding pep‐ tide conjugated onto PEI to target integrins on cell surfaces [8] and vitamin folate conjugat‐ ed onto liposomes through a polyethylene spacer to target folate receptor-bearing cells [9]. Cell adhesion molecules (integrin, syndecan, cadherin, selectin) which are a diverse group of cell surface proteins mediating interactions between cells, and between cells and the ex‐ tracellular matrix, are valuable targets for precise gene delivery to haematopoietic cells, air‐ way epithelial cells, tumor cells and vascular endothelial cells using synthetically designed

Recently, we have reported on the development of a safe, efficient nano-carrier system of carbonate apatite which can assist both intracellular delivery and release of DNA leading to very high level of trans-gene expression in cancer and primary cells [11-13]. We have also revealed a new approach of organic-inorganic hybrid carrier devised by complexing fibronec‐ tin and E-cadherin-Fc chimera electrostatically with nano-particles of carbonate apatite [14, 15]. Specific recognition to cell surface integrin and E-cadherin molecules through double ligand-coated nano-particles, resulted in synergistic acceleration of transgene delivery and consequential expression into embryonic stem cells. Instead of simultaneous mixing of DNA and cell-adhesive molecules in particle-preparation medium and subsequent incubation, step-

for implementation in research laboratories and gene therapy.

266 Advances in Biomaterials Science and Biomedical Applications

division.

non-viral vectors [10].

Plasmids, pGL3 (Promega) containing a luciferase gene under SV40 promoter and pEGFP-N2 (CLONTECH Laboratories, Inc.) having a green fluorescence protein gene under CMV promoter were propagated in the bacterial strain XL-1 Blue and purified by QIAGEN plas‐ mid kits. Lipofectamine 2000 and DMEM were purchased from Invitrogen and Gibco BRL, respectively. Fibronectin was bought from Sigma and expression as well as purification of Ecad-Fc fusion proteins was done according to the previously described report [16].

#### **2.2. Cell Culture**

HeLa cells were cultured in 75-cm2 flasks in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 50 μg penicillin ml-1, 50μg streptomycin ml-1 and 100μg neomycin ml-1 at 37°C in a humidified 5% CO2-containing atmos‐ phere. F9, a mouse teratocarcinoma stem cell line and EB3, a mouse embryonic stem cell line were cultured in gelatin-coated 25-cm2 flasks. F9 cells were maintained in Dulbecco's modi‐ fied Eagle's medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2-containing atmosphere. Feeder-free murine ES cells were maintained in KNOCKOUT-DMEM (Invitrogen), supplemented with 1 mM L-glutamine, 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma Chemical), 10% FBS and 1,000 units/ml leukemia inhibitory factor (LIF) (Chemicon). All media contained 50 μg/ml penicillin, 50 μg/ml streptomycin, and 100 μg/ml neomycin.

### **2.3. Transfection of cells**

Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. 3 to 6 μl of 1 M CaCl2 was mixed with 2 μg of plasmid DNA in 1 ml of fresh serum-free HCO3- - buffered (pH 7.5) medium (DMEM) and incubated for 30 min at 370 c for complete generation of DNA/carbonate apatite particles. For generation of ECM protein-embedded carbonate apatite particles, fibronectin and E-cad-Fc proteins were added either alone or together to a final concentration of 5 μg/ml, to Ca2+ and DNA-containing DMEM followed by incubation at 370 c for 30 min. Medium with generated ECM protein-associated or non-associated, DNA-containing particles was added with 10% FBS to the rinsed cells. After 4 hr incubation, the medium was replaced with serum supplemented medium and the cells were cultured for 1 day. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, USA). Each transfection experi‐ ment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein. For lipofectamine-mediated transfection, protocol provided by Invitro‐ gen was followed in a 24-well plate. Cells were incubated with DNA/lipofactamine com‐ plexes in serum-free media for 4 hr and like above, grown for 1 day after replacement with fresh serum media.

For transfection with calcium phosphate-DNA co-precipitation, briefly, 12 μg of plasmid DNA was added to 300 μl of a solution containing 250 mM CaCl2. This solution was added to 300μl of a 2×HBS (50 mM Hepes, 140 mM NaCl, 1.5 mM Na2HPO4.2H2O, pH 7.05) and mixed rapidly by gentle pipetting twice. The DNA/CaPi mixture was incubated at room temperature for the period of time indicated. After addition of 100 μl of the incubated mixture drop-wise to 1 ml serum supplemented media of each well, cells were incubated for 4 hr and like above, after replacement with fresh serum media, grown for 1 day.

#### **2.4. MTT assay**

HeLa cells were transfected and cultured for 1 day as described above. 30 μl of MTT solu‐ tion (5mg/ml) was added to each well and incubated for 4 hrs. 0.5 ml of DMSO was added after removal of media. After dissolving crystals and incubating for 5 min at 370 C, absorb‐ ance was measured in a microplate reader at 570 nm with a reference wavelength of 630 nm.

#### **2.5. Chemical analysis**

Following generation of carbonate apatite as described above, using 6 mM Ca2+ and no DNA, precipitated particles were lyophilized after centrifugation and washing with distilled deion‐ ized water. Other apatite particles generated as described above, were also similarly lyphi‐ lized. Calcium and phosphorus contents were determined using SPS 1500 VR Atomic Absorption Spectrophotometer. Carbon and fluorine were estimated by CHNS-932 (Leco, USA) and SXelements micro analyser, YS-10 (Yanaco, Japan), respectively.

#### **2.6. Infrared spectroscopy**

Fourier transform-infrared spectroscopy of apatite particles prepared as described above, was performed using FT/IR-230, JASCO. The samples were ground in a mortar and approximate‐ ly 1 mg was thoroughly mixed with 300 mg of ground spectroscopic grade KBr. Transpar‐ ent pellets were prepared in a KBr die with an applied load of 8000 kg, under a vacuum of 0.5 torr.

#### **2.7. X-ray diffraction**

The x-ray diffraction powder reflections of the particles prepared as described above, were recorded using M18XHF-SRA diffractometer system.

#### **2.8. Particle size measurements**

For visualization by a scanning electron microscope (SEM), a drop of DNA-carbonate apa‐ tite suspension prepared according to the instructions in transfection protocol, was added to a carbon-coated SEM stage and dried, followed by observation by a high resolution SEM (S-800, Hitachi, Japan). Dynamic light scattering (DLS) measurement for particle suspension was carried out with a Super-dynamic Light Scattering Spectrophotometer, 'Photal' (Otsuka Elec‐ tronics) at 75 mW Ar laser.

#### **2.9. Confocal laser scanning microscopy**

gen was followed in a 24-well plate. Cells were incubated with DNA/lipofactamine com‐ plexes in serum-free media for 4 hr and like above, grown for 1 day after replacement with

For transfection with calcium phosphate-DNA co-precipitation, briefly, 12 μg of plasmid DNA was added to 300 μl of a solution containing 250 mM CaCl2. This solution was added to 300μl of a 2×HBS (50 mM Hepes, 140 mM NaCl, 1.5 mM Na2HPO4.2H2O, pH 7.05) and mixed rapidly by gentle pipetting twice. The DNA/CaPi mixture was incubated at room temperature for the period of time indicated. After addition of 100 μl of the incubated mixture drop-wise to 1 ml serum supplemented media of each well, cells were incubated for 4 hr and like above, after

HeLa cells were transfected and cultured for 1 day as described above. 30 μl of MTT solu‐ tion (5mg/ml) was added to each well and incubated for 4 hrs. 0.5 ml of DMSO was added

ance was measured in a microplate reader at 570 nm with a reference wavelength of 630 nm.

Following generation of carbonate apatite as described above, using 6 mM Ca2+ and no DNA, precipitated particles were lyophilized after centrifugation and washing with distilled deion‐ ized water. Other apatite particles generated as described above, were also similarly lyphi‐ lized. Calcium and phosphorus contents were determined using SPS 1500 VR Atomic Absorption Spectrophotometer. Carbon and fluorine were estimated by CHNS-932 (Leco, USA) and SX-

Fourier transform-infrared spectroscopy of apatite particles prepared as described above, was performed using FT/IR-230, JASCO. The samples were ground in a mortar and approximate‐ ly 1 mg was thoroughly mixed with 300 mg of ground spectroscopic grade KBr. Transpar‐ ent pellets were prepared in a KBr die with an applied load of 8000 kg, under a vacuum of 0.5

The x-ray diffraction powder reflections of the particles prepared as described above, were

For visualization by a scanning electron microscope (SEM), a drop of DNA-carbonate apa‐ tite suspension prepared according to the instructions in transfection protocol, was added to a carbon-coated SEM stage and dried, followed by observation by a high resolution SEM (S-800, Hitachi, Japan). Dynamic light scattering (DLS) measurement for particle suspension was

C, absorb‐

after removal of media. After dissolving crystals and incubating for 5 min at 370

fresh serum media.

**2.4. MTT assay**

**2.5. Chemical analysis**

**2.6. Infrared spectroscopy**

**2.7. X-ray diffraction**

**2.8. Particle size measurements**

torr.

replacement with fresh serum media, grown for 1 day.

268 Advances in Biomaterials Science and Biomedical Applications

elements micro analyser, YS-10 (Yanaco, Japan), respectively.

recorded using M18XHF-SRA diffractometer system.

pGL3 vector was labeled with PI at a PI/DNA ratio of 1:1 and particles generated with this labeled plasmid (described in transfection protocol), were incubated with HeLa cells for 6 hours. Acidic compartments were labeled with 5μM LysoSensor, according to the instruc‐ tions provided by Molecular Probes, and membrane-bound precipitates were removed by 5 mM EDTA in PBS before observation by LEICA TCS-NT.

#### **2.10. SDS-PAGE and Western blotting**

Following generation of carbonate apatite as described above using 3 mM Ca2+ and required amount of fibronectin or E-cad-Fc chimera and no DNA and centrifugation at 15000 rpm for 5 min at 40 C, precipitated particles were washed with water with several centrifugation steps to remove unbound proteins and dissolved with 50 mM EDTA in PBS for subsequent analy‐ sis by 7.5% SDS-PAGE in reducing condition. In order to see particle-bound fibronectin, after SDS-PAGE, the gel was stained with Coumassie blue, washed and dried. For detection of particle-associated E-cad-Fc, proteins after being run by SDS-PAGE were transferred to PVDF membrane (Immobilon, Millipore) and 80 mA current was applied for 90 min to complete transfer of the proteins. The PVDF membrane was washed with PBS (-)-containing 0.1% Tween 20 and then blocked for 1 hr at room temperature by "Blocking One" (Nacalai Tesque, Ja‐ pan). The membrane was incubated with horseradish peroxidase (HRP)-conjugated antimouse IgG for 1 hr and washed with PBS-T three to four times to completely remove nonspecific interactions. Enhanced chemiluminescence system (Amersham Bioscience) was used for visualization.

#### **2.11. DNA labelling, fluorescence microscopy and flow cytometry**

Plasmid DNA was labelled non-covalently with propidium iodide (PI) using 1:1 weight ratio of DNA to PI in the particle preparation medium. Labelled DNA inside the cells was ob‐ served by a fluorescence microscope (Olympus-IX71), following 4 hr incubation of differen‐ tially formulated particle suspensions with F9 cells and removing extracellularly bound particles by 5 mM EDTA in PBS. For flow cytometric analysis using FACS Calibur (Becton, Dickinson and Company), 1 day after transfection with pEGFP plasmid DNA, F9 cells were collected in a sorter buffer following treatment with trypsin-EDTA and repeated centrifugation and wash‐ ing of the resulting cell pellet with PBS (-) (2 times).
