1. Volume 7, Issue 3, Pages 409-418 (March 2003)
Increased uptake of liposomal–dna complexes by lung metastases following intravenous administration 
Isao Ito, Gopalan Began, Imran Mohiuddin, Tomoyuki Saeki, Yuji Saito, Cynthia D Branch, Ara Vaporciyan, L Clifton Stephens, Nancy Yen, Jack A Roth, Rajagopal Ramesh 
Molecular Therapy 
Volume 7, Issue 3, Pages (March 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Effect of liposome–DNA complex transfection on cell viability and luc expression in tumor cells and normal cells. Cells analyzed were human lung tumor cells (H1299, H358, H460, A549), normal lung fibroblast cells (WI38, CCD-16, MRC-9), normal bronchial epithelial cells (NHBE), human umbilical vein endothelial cells (HUVEC), murine fibroblasts (3T3, 10T1/2), and murine fibrosarcoma (UV2237m, K1735) cells. Cells (5 × 105) were either not transfected (control) or transfected with DOTAP:Chol–luc DNA complex and harvested at 24 and 48 h after transfection and analyzed for cell viability by trypan exclusion assay (A) and for luc activity using a luciferase assay kit (B). No significant toxicity was observed in transfected tumor and normal cells of both human and murine origin compared to untransfected control cells (A). luc expression was higher in both human tumor cells (P = 0.01) and murine tumor cells (P = 0.04) compared to normal cells (B). luc activity was expressed as nanograms per milligram of total protein. Each time point represents the mean of triplicate wells. Error bars represent standard errors.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Increased uptake of liposome–DNA complex by tumor cells. Tumor cells (H1299, A549, H460), normal cells (WI38, MRC-9, CCD-16), and endothelial cells (HUVEC) were transfected with fluorescently labeled DOTAP:Chol–DNA complex. Cells were analyzed for liposome–DNA complex uptake by fluorescence microscopy and flow cytometry and for luciferase expression by luminometer. Increased uptake was observed in tumor cells and in endothelial cells compared to normal cells. Increased uptake by tumor cells and HUVEC correlated with increased luciferase expression. Luciferase expression is expressed as nanograms per milligram of protein.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Increased uptake of liposome–DNA complex by tumor cells is due to phagocytosis. Tumor cells (H1299) were transfected with 2 μm fluorescent microspheres (A) or fluorescently labeled DOTAP:Chol–luc DNA complex (B) in the presence or absence of cytochalasin B. Cytochalasin B, an inhibitor of phagocytosis, inhibited uptake of fluorescently labeled DOTAP:Chol–luc DNA complex by tumor cells (original magnification ×200). Hoechst staining of cells indicates intact nucleus (C).
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Increased luciferase expression by lung tumors in vivo is due to increased uptake of liposome–DNA complex. UV2237m lung-tumor-bearing mice were injected with fluorescently labeled DOTAP:Chol–luc DNA complex via tail vein (C, D). Animals receiving no treatment served as controls (A, B). The animals were euthanized by CO2 inhalation at 24 h after injection, and their lungs were isolated. Bright-field microscopy of hematoxylin-stained lung tissue sections revealed the presence of tumors in the lungs (A, C). Fluorescence microscopy of an unstained lung tissue section demonstrated greater fluorescence by tumor cells than by surrounding normal cells (D). Minimal fluorescence was observed in control tissue sections (B). Immunohistochemical analysis of lung tumor sections for luciferase protein demonstrated increased luciferase expression by tumors as indicated by the brown staining (E) compared to their surrounding normal tissue (H) and non-tumor-bearing lung tissue (G). Tumor-bearing lungs receiving no treatment (F) and tissue sections stained with no secondary antibody (I) served as controls (original magnification ×200). Arrows indicate cells staining positive for luciferase protein. Semiquantitative analysis of the areas staining positive indicated tumors staining more positively than surrounding normal tissues (P = 0.01) (J).
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Increased uptake of liposome–DNA complex by A549 lung tumors in vivo. Nude mice were injected with A549 lung tumor cells via tail vein. Animals were injected with DOTAP:Chol–luc DNA complex 2 weeks (small tumors) and 3 weeks (large tumors) after tumor cell inoculation. Tumor-bearing animals receiving no treatment served as controls. Immunohistochemical analysis of lungs harvested 24 h after treatment demonstrated higher luc expression (P = 0.01) in the tumors compared to surrounding normal tissues as indicated by the brown staining (A, B). Increased luciferase expression was observed in tumors that were small and in tumors that were large. Arrows indicate positive staining.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Comparison of luc expression in alveolar macrophages isolated from tumor-bearing and non-tumor-bearing animals. Alveolar macrophages isolated from UV2237m lung-tumor-bearing and non-tumor-bearing animals were transfected with DOTAP:Chol–luc DNA complex and analyzed for luc expression 24 h after transfection. luc expression was significantly (P = 0.001) higher in macrophages isolated from non-tumor-bearing animals compared to those from tumor-bearing animals (A). Cell viability assay indicated no significant difference in proliferation between macrophages isolated from tumor-bearing animals and those isolated from non-tumor-bearing animals (B). Analysis of lungs for luc expression demonstrated no significant difference between tumor-bearing and non-tumor-bearing animals (C). luc activity was expressed as nanograms per milligram of total protein. Experiments were done in triplicate and bars denote standard errors.
Molecular Therapy 2003 7, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions