Methods

2.2.1 Preparation of Lipo-PEI-PEG Complex (LPPC)

DOPC and DLPC (50mg/ml) were added into the round bottom flask, and 1 ml methanol was as well. Then the container of lipid mixture was placed to the rotary evaporator (37℃, without vacuum treatment, minimum rotary speed) until dry. After about 2 days, the dry lipid film was hydrated by steam (about 37℃) for 2~3 hours. And 5ml aqueous medium (containing 0.675g PEI and 0.22g PEG) was added gently to the container of lipid. Then the container was kept agitating violently for 10 minutes. After vortex, place the LPPC at RT overnight. And the turbid medium of LPPC was extruded through 200 nm mesh nine times. Then the product could be used for the following experiment.

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2.2.2 Nanoparticle characterization by Scanning Electron Microscopy

The morphology of nanoparticles was observed by scanning electron microscopy (SEM). The nanoparticle formulations were prepared as described above and diluted in demineralized water. Then the sample was directly transferred and dried onto a silicon chip by vacuum freeze-drying system. The SEM picture was taken at ×10,000, 10 kV.

2.2.3 DNA retarding assay

The degree of complexation between different amount of LPPC with constant amount of DNA was observed by the DNA retarding assay. 1μg DNA was pre-incubated with SYBR Green I nucleic acid gel stain (Molecular Probes, Invitrogen), and the stained DNA was then incubated with different amount of LPPC to form liposome-DNA complex (lipoplex) for 30 minutes at room temperature. The liposome-DNA complexes were electrophoresed in a 0.8%

agarose gel; the gel-running time was 30 minutes at 100V and 500 mA. The liposome-DNA complexes were treated with heparin (1 μg) as the control groups to see if heparin could release DNA from the liposome-DNA complexes.

The gel was photographed over a UV box.

2.2.4 Evaluation of particle size and charge

The particle size of the LPPC-DNA complexes was measured by the dynamic light scattering. Complexes used in the measurement were of 18 μg DNA content and formed in 1 ml ddH2O that was pre-filtered with a 0.22 μm filter. Measurements were made in automatic mode.

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The surface charge of the LPPC-DNA complexes was measured as zeta potential by a ZetaPlus Z-potential analyzer.

2.2.5 Plasmids DNA extraction

Plasmids pAsRed2-N1 and pLEGFP were two reporter genes who encoded red fluorescent protein and green fluorescent protein, respectively. Besides, the pIL-6 plasmid was created by inserting a human IL-6 gene into a pAAV-MCS vector. And these plasmids were purified from transformed Escherichia coli using a NucleoBond PC 100 kit (Macherey-Nagel, Duran, Germany) according to the manufacturer’s instructions. At first, a single colony of E. coli was inoculated in 100 ml of LB broth contained antibiotics and at 37℃ at agitation for 16 hours. The bacteria were recovered by centrifugation at 8000 rpm for 15 minutes at 4℃. The pellet was collected, and 4 ml buffer S1 with RNase (Macherey-Nagel, Duran, Germany) was added to dispense the pellet. Then 4 ml buffer S2 (Macherey-Nagel, Duran, Germany) was added to the suspension. The lysate was mixed gently and incubated at room temperature for 3 minutes (no more than 5 minutes). The pre-cooled 4 ml buffer S3 (Macherey-Nagel, Duran, Germany) was then added to the solution and mixed gently until a homogeneous suspension containing and off-white flocculate as formed. The mixture was incubated on ice for 5 minutes and then spun at 13000 rpm for 25 minutes at 4℃.

The supernatant was loaded onto the NucleoBond AX 100 Midi column which was equilibrated with 2.5 ml buffer N2 (Macherey-Nagel, Duran, Germany).

The flow-through was emptied by gravity flow and discarded. 10 ml buffer N3 (Macherey-Nagel, Duran, Germany) was added to wash the column twice. The DNA was eluted by 5 ml buffer N5 (Macherey-Nagel, Duran, Germany). Then 3.5 ml isopropanol was added to precipitate the DNA. The mixture was

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incubated on ice for 10 minutes and recovered by centrifugation at 13000 rpm for 30 minutes at 4℃. 6 ml 70% ethanol was added to the pellet and the solution was spun at 13000 rpm for 5 minutes. Finally, the pellet was dissolved in appropriate amount of ddH₂O and stored at -20 .℃

2.2.6 In vitro transfection

Cells were seeded in the 6-well plate at a density of 2.5×10⁵ cells/well and cultured with 2 ml growth medium for 24 hours respectively. Cells were transfected plasmid DNA encoding GFP with different amounts of LPPC.

Briefly, 3 μg plasmid DNA and 50μg LPPC were each diluted into 250μl Opti-MEM I medium (Gibco, Grand Island, NY) and vortexed. The DNA solution was added into LPPC solution after 5 minutes (Notice: do not reverse the order), and then vortexed. After 25 minutes, the cells were rinsed and supplemented with 200μl Opti-MEM I medium. The LPPC/DNA mixture was gently added to each well. Finally, 600μl Opti-MEM I medium were added into each well. After 12 hours incubation, 2 ml fresh growth medium were added into each well. After 48 hours the gene expressions were measured by FACScan flow cytometry (Becton Dickinson, San Jose, CA).

2.2.7 Generation of red fluorescent protein-secreting BALB/3T3 and green fluorescent protein-secreting B16-F10 to build up fusion model

BALB/3T3 was transfected with pAsRed2-N1 (BD Biosciences Clontech ) and B16-F10 was transfected adenovirusly with plasmids DNA encoding GFP green fluorescent protein, respectively. To select the fluorescent-permanent cell lines, limit dilution and usage of antibiotics, G418 (500 μg/ml), were performed to BALB/3T3_pAsRed cells. Besides, only G418 selection (1.5 mg/ml) was

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used to B16-F10_pLEGFP cells. The intensities of fluorescence expression were analysis by FACS analysis. These two fluorescent proteins-expressed cell lines were used to measure the effect of LPPC addition on fusion efficiencies as the model.

2.2.8 Generation of bone marrow-derived DCs and phenotype of cell surface

Erythrocyte-depleted murine bone marrow cells obtained from BALB/c mouse femurs and tibias were cultured in CM supplemented with 200U/ml recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (Lutz, Kukutsch et al. 1999). At day 0 bone marrow precursors were seeded at 2.5 × 10⁶ per 100mm bacteriological dish in 10 ml CM containing rmGM-CSF. At day 3 another 10 ml CM containing 200U/ml rmGM-CSF were added to the plates. At day 6 half of the culture supernatant was collected, centrifuged, and the cell pellet resuspended in 10 ml fresh CM containing 200 U/ml rmGM-CSF, and given back into the original plate. At day 8 non-adherent cells were collected by gentle pipetting, centrifuged at 300g for 5 min at RT, and resuspended in 10 ml CM into a fresh 100 mm tissue culture plastic dish containing 200U/ml rmGM-CSF and 0.5 ϻg/ml LPS for 2 days for complete maturation. Then the non-adherent cells with the typical morphologic features of DCs were used for the experiment. For phenotypic analysis, immature DCs at day 8 and mature DCs at day 10 were stained with a panel of antibodies, including MHC class I and II, CD86 and CD11c (BioLegend, San Diego, CA) and quantified by flow cytometry (Becton Dickinson, San Jose, CA).

2.2.9 Co-transfection-fusion protocol

Bone marrow-derived DCs were transfected and fused with BALB/3T3 at a

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2:1 ratio using LPPC and polyethylene glycol (PEG; m.w. 1450) solution (Sigma-Aldrich), as described (Wang, Saffold et al. 1998). In brief, BALB/3T3 and DCs were mixed and washed twice with serum-free medium prewarmed to 37℃. LPPC/IL-6 complex was then added to the mixed cells for 2 hr at 37℃,

5% CO2. The cells were centrifuged at 300 × g for 5 min. After removing the medium, 1 ml of PEG was added to the cell pellet over 1 min and the suspension was stirred gently for 2 min. An additional 10 ml of serum-free medium was added to the cell suspension over the next 3 min with continued stirring. The resultant cell mixture was pelleted at 400 × g, 5 min and grown for 48hr in CM with GM-CSF. Then the fusion cells were prepared for further experiment, and the supernatant of fusion cells was collected to detect the production of IL-6 protein or TGF-β1 protein used ELISA kits (Human IL-6 and Mouse TGF-β1, R&D Systems). The fusion model of BALB/3T3_pAsRed and B16-F10_pLEGFP were transfected and fused at 1:1 ratio using the same protocol.

2.2.10 TGF-β secretion of different cell lines detected by ELISA

Supernatants of different cell lines were collected at 12 hours, 24 hours and 36 hours and stored immediately at -20℃ until further use respectively.

Bioactive and total TGF-β secretions were determined by ELISA (R&D Systems). The supernatants were acidified according to the manufacturer’s instructions.

2.2.11 TGF-β derived from immortalized cells on BMDC’s phenotypes

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Bone marrow cells derived from BALB/c mouse femurs and tibias were cultured in CM supplemented with 200 U/ml rmGM-CSF as described above.

On day 6 of culture, DCs were collected and cultured at 10⁶ cells/ml in CM supplemented with rmGM-CSF and with or without the addition of conditioned medium from BALB/3T3 cells for 6 days. Cytokine and conditioned medium were replenished every 2 days. DCs were matured with 0.5 g/ml LPS (SIGMA-ALDRICH) for 2 days. The expressions of MHC II and CD86 molecules were analyzed by FACScan flow cytometry (Becton Dickinson, San Jose, CA).

2.2.12 Splenocyte isolation

Mice were sacrificed by dislocation and their spleens were quickly harvested in a laminar flow hood. Spleens were placed in a 280 μm-pored mesh and chopped by scissors. 10 ml of RPMI 1640 (Invitrogen Co., USA) supplemented with 10% FBS, 0.2% NaHCO₃ and 1% PSA were slowly added onto the mesh while spleens were being ground until the spleen tissue became white. Single cell suspension was collected in a Petri dish and recovered by centrifugation at 1200 rpm at 4℃ for 5 min. Supernatant was discarded and 10 ml 1× ACK lysis buffer was added for 5 min at room temperature. 1× ACK lysis buffer can lyse the red blood cells while leaving the rest of the lymphocytes and leucocytes. The mixture was then diluted by 10 ml of RPMI 1640 and cells were recovered by centrifugation at 1200 rpm at 4℃ for 5 min. After the supernatant was discarded, the cells were rinsed by 10 ml PBS once again. Finally, cells were resuspended in RPMI 1640 and underwent cell calculation by trypan blue exclusion. For the cell proliferation of splenocytes, cells were seeded in a

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96-well at 2.5 × 10 ⁵ per well. For the cytokine profiles for splenocytes, cells were seeded in a 24-well plate at 1 × 10 ⁶ per well.

2.2.13 Animal immunization

BALB/c mice were vaccinated once (i.p.) on day 0 with 1 × 10⁶ DC fusion vaccine made by LPPC with IL-6 gene and PEG treatments (LPPC/IL-6/PEG DC vaccine) or DC fusion vaccine made by PEG treatment alone (PEG DC vaccine). To test the efficacy of the vaccines, each group of the animals were sacrificed and the splenocytes of the immunized mice were seeded in 96-well plates (2.5 × 10⁵ cells/well) or 24-well plates (1 × 10⁶ cells/well) with Ags to measure the proliferation or cytokines expressions. For cell proliferation, MTT assay was used to estimate at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of splenocytes alone. For cytokines expressions, supernatants of each sample were collected at 48 hrs and frozen at -80℃. Supernatants concentrations of TNF-α, IL-2, IFN-γ, IL-10 and IL-4 were measured by ELISA.

2.2.14 Statistical analysis

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All figures are expressed as mean ± SD. All data were computed by student-test. All statistical significant was set up at p < 0.05.

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