Tissue-dependent factors affect gene delivery to tumors in vivo - Nature

Gene Therapy (2003) 10, 1079–1088 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt

RESEARCH ARTICLE

Tissue-dependent factors affect gene delivery to tumors in vivo B Smrekar1, L Wightman1, MF Wolschek2,3, C Lichtenberger3, R Ruzicka1, M Ogris4, W Ro¨dl4, M Kursa1, E Wagner1,4 and R Kircheis1,5 1

Boehringer Ingelheim Austria, Dr Boehringer Gasse 5-11, Vienna, Austria; 2Department of Clinical Pharmacology, Section of Experimental Oncology, University of Vienna, Vienna, Austria; 3Department of Internal Medicine IV, Division of Gastroenterology and Hepatology, University of Vienna, Vienna, Austria; 4Pharmaceutical Biology-Biotechnology, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Germany; and 5IGENEON Immuno Therapy of Cancer, Brunner Strae 69/3, Vienna, Austria

Systemic application of surface-shielded transferrin-polyethylenimine/DNA complexes leads to predominant DNA uptake and gene expression in Neuro2a tumors in syngeneic A/J mice. Similarly, high expression levels were found in Huh-7 and HepG2 human tumor xenografts in SCID mice after systemic application of surface-shielded EGF-PEGPEI/DNA complexes. Significant DNA uptake but low gene expression were found in the M-3 melanoma while no DNA uptake and no gene expression were found in KB, 518A2, A549, and SW480 xenograft tumor models. To elucidate the reasons for these differences, the tumors were analyzed for vascularization and infiltration of macrophages. Neuro2a, Huh-7, and HepG2 tumors are well vascularized, with a high

density of partially immature blood vessels and low numbers of infiltrating macrophages. The M-3 melanoma is well vascularized correlating with significant DNA uptake, however, necrosis and intensive infiltration by macrophages lead to rapid degradation of DNA. In contrast, the KB, 518A2, A549, and SW480 tumors are poorly vascularized, correlating with undetectable DNA uptake and gene expression. Using two different vector systems the data indicate that gene delivery to tumors in vivo is affected by tissuedependent factors. Uptake of DNA into the tumor depends on vascularization of the tumor, while necrosis and macrophage infiltration may facilitate degradation of the DNA. Gene Therapy (2003) 10, 1079–1088. doi:10.1038/sj.gt.3301965

Keywords: polyethylenimine,PEI; in vivo gene transfer; tumor targeting,tumor vasculature; transferrin; EGF,epithelial growth factor

Introduction Gene therapy is receiving increasing attention as a promising strategy for cancer treatment. In particular for cancer where widespread metastases are not amenable to standard therapies, gene therapy may present a potential modality for treatment. However, gene therapeutic strategies are critically dependent on the development of vectors that enable efficient and specific delivery of therapeutic genes to the desired target tissue. The polycation polyethylenimine (PEI) has been shown to be a promising backbone for the design of nonviral gene delivery vectors,1,2 combining DNA condensation activity with an intrinsic endosomolytic activity.1,3 In particular, low molecular weight PEI molecules (22, 25 kDa) have shown high efficacy and good biocompatibility for applications in vivo.4–8 In order to further increase transfection efficiency and incorporate target specificity, the DNA delivering activity of PEI has been combined with specific mechanisms for receptormediated cellular uptake.9,10 Targeting moieties, such as transferrin, have been coupled to the PEI backbone, generating transferrin-PEI/DNA complexes which have Correspondence: Dr R Kircheis, IGENEON Immuno Therapy of Cancer, Brunner Strae 69/3, A-1230 Vienna, Austria. E-mail: [email protected] Received 30 August 2002; accepted 23 November 2002

demonstrated efficient gene delivery in a broad variety of applications in vitro and promising results in topical application in vivo.10,11 A more sophisticated approach is the development of vector systems that are able to deliver therapeutic genes efficiently and specifically to the target cells after systemic application into the systemic blood circulation. Studies comparing different types of vectors for systemic application have demonstrated that the physical parameters of the transfection complexes such as particle size, stability and surface charge determine complex biodistribution, toxicity and gene transfer efficacy in vivo.12–17 While positively charged ligand-PEI/DNA complexes show high transfection efficacy in cell culture applications, additional modifications have to be introduced into the transfection particles to overcome barriers encountered after systemic application in vivo. These barriers include circulation in the blood, passage through various biological barriers, and diffusion through tissues, which are dependent on the particle size and colloidal stability of the complexes. Furthermore, for targeted delivery to the desired target tissue particles have to be protected from unspecific interactions with blood components, extracellular matrix, and nontarget cells.14,18 We have developed two surface-shielded targeted gene delivery systems, based on transferrin-PEI or PEGylated EGF-PEI, respectively, that are able to target

Factors for gene delivery to tumors B Smrekar et al

gene expression to distant tumors following systemic application.19–23 In the present study, using these two delivery systems for systemic application we compared DNA biodistribution and gene expression in a variety of tumor models. Major differences in the efficacy of gene delivery between different tumor models were found. The histological characteristics of the tumor, such as blood vessel formation and infiltration of macrophages, can influence DNA uptake into the tumor and DNA degradation, explaining the differences in DNA accumulation and DNA expression observed in the different tumor models.

Neuro 2a

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Transfection of tumor cell lines with transferrin-PEI/ DNA complexes in vitro Transfection efficacy was estimated by luciferase reporter gene expression using the pCMVL plasmid condensed with PEI (25 kDa). PEI25/DNA complexes or Tf-PEI/ PEI25/DNA complexes, which additionally have the cell-binding ligand transferrin linked to PEI25 (Tf-PEI), were compared for their ability to transfect the murine neuroblastoma cell line Neuro2a, the murine melanoma cell line M-3, and the human adenocarcinoma cell line KB (Figure 1). PEI25/DNA complexes resulted in significant luciferase reporter gene expression with 105 relative light units (RLUs) for the Neuro2a cells and 106 RLU for the M-3 and KB cells. Incorporation of transferrin ligand into the complex (Tf-PEI:PEI25 at a ratio of 1:10) resulted in a 30–300-fold increase in transfection efficacy, depending on the cell line, compared to the ligand-free complexes, reaching 107 to nearly 108 RLU per million cells (*Po0.01 Tf-PEI/PEI/ DNA versus PEI/DNA, two-tailed T-test). Viability of the cells after transfection was higher than 90%. Therefore, as a prerequisite for our in vivo studies, the data demonstrate that all three tumor cell lines can be transfected efficiently in vitro by transferrin-PEI/PEI25/DNA complexes.

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Gene delivery in vivo after systemic application After demonstrating that Tf-PEI complexes are able to efficiently transfect the tumor cell lines in vitro, transfection complexes were tested for their ability to transfect these tumors in vivo following systemic application. One million Neuro2a or M-3 cells were injected subcutaneously in syngeneic A/J mice or DBA/2 mice, respectively, KB cells were injected subcutaneously in NMRI nu/nu mice. All tumors had comparable growth rates. Two weeks after tumor implantation, when tumors had reached 10–13 mm in size, DNA complexes containing the pCMVL plasmid were injected systemically via the tail vein. Efficacy of gene delivery was assessed by measuring luciferase reporter gene expression in the tumor and the major organs, including lung, heart, liver, spleen, and kidney. Previously, we demonstrated that for targeting gene delivery to distant tumors following systemic in vivo application a higher ratio of transferrin in the complexes (eg Tf-PEI:PEI25 at a ratio of 1:4) is necessary to efficiently shield the positive surface charge of the complexes and to reduce unspecific interactions.19 Surface-shielded Tf-PEI/PEI/DNA complexes (zetapotential: +7 mV) and, as a control, positively charged PEI/ Gene Therapy

PEI25

Tf-PEI/PEI25

Figure 1 Transfection of tumor cell lines in vitro using PEI/DNA or TfPEI/PEI/DNA complexes. Murine Neuro2a neuroblastoma cells (a) or M3 melanoma cells (b), and human KB adenocarcinoma cells (c) were transfected by PEI25/DNA or Tf-PEI/PEI25/DNA complexes. For transfection 200 000 cells per well were seeded 24 h prior to transfection. Plasmid DNA (pCMVL; 2 mg per well) was complexed with PEI or TfPEI/PEI at a molar ratio of PEI nitrogen to DNA phosphate N/P¼4.8, rigorously mixed, incubated 20 min, and added to the cells growing in 1.5 ml culture medium. After 4 h incubation medium was replaced. Cells were harvested 24 h after transfection. Luciferase activity was recorded using a Lumat LB9501/16 instrument, and is expressed as RLUs per 106 cells. One million RLUs correspond to 2 ng luciferase. Values after transfection with PEI/DNA or Tf-PEI/PEI/DNA complexes were compared by two-tailed T-test, *Po0.01.

DNA complexes (zetapotential: +30 mV) were injected systemically. Figure 2 shows luciferase reporter gene expression 24 h after systemic application of Tf-PEI/ PEI/DNA or PEI/DNA complexes into Neuro2a (a), M-3 (b), and KB (c) tumor-bearing mice. Different reporter gene expression patterns were found in the three tumor models, and with shielded and unshielded complexes. In


the three tumor models. Significant gene expression in the tumor was found in the Neuro2a model. In contrast, only low expression in the M-3 and near background expression levels in the KB models were found. Application of surface-shielded Tf-PEI/PEI/ DNA complexes resulted in a decrease in lung expression in all three models and high luciferase expression in the Neuro2a tumor (Po0.05 tumor versus all major organs, two-tailed T-test). In contrast, very low expression levels were found in the M-3 melanoma and no significant expression was found the KB tumor model.

Figure 2 Gene expression after systemic gene delivery into tumor-bearing mice. Tf-PEI/PEI/DNA complexes (N/P¼4.8; containing 50 mg pCMVL/ 250 ml) were injected into the tail vein of Neuro2a tumor-bearing A/J mice (a), or M-3 tumor-bearing DBA/2 mice (b), or KB tumor-bearing NMRI nu/nu mice (c). For comparison PEI/DNA complexes (N/P¼4.8; containing 50 mg pCMVL/250 ml) were applied (insets). Gene expression 24 h after application was measured by luciferase assay. Luciferase background (100–200 light units) was subtracted from each value. Luciferase activity represents mean7SEM, nX6. Luciferase background levels of untreated animals are below 200 RLU/organ (not shown). Statistical significance was calculated using two-tailed T-test. * Po0.05, tumor versus all major organs.

accordance with previous reports,6–8,19 positively charged PEI/DNA complexes (Figure 2a–c insets) resulted in significant lung expression in all three models, while the expression in the other organs varied, and particularly in the tumor differed greatly between

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DNA biodistribution after systemic application The differences in reporter gene expression between the three tumor models could be caused by differences in the DNA uptake, for example, the ability of the DNA complexes to extravasate from the blood system and penetrate the tumor mass. To test this hypothesis the biodistribution of DNA 4 h after tail vein injection of TfPEI/PEI/DNA complexes and the uptake of the DNA into the Neuro2a, M-3, and KB tumors was studied by Southern blot analysis using a pCMVL-specific probe as described in Materials and methods. By this technique intact DNA and partially degraded DNA can be distinguished. In accordance with the gene expression data (Figure 2) the DNA biodistribution pattern showed high amounts of DNA, up to 2% of the total injected DNA, in the Neuro2a tumor (Figure 3a). High amounts of total DNA were also found in the liver reaching nearly 5% of the total injected DNA. However, most of the DNA was at least partially degraded 4 h after injection, with 96% of the DNA in the liver being degraded (Figure 3a, b). This high level of DNA uptake and degradation has been attributed to the Kupffer cells in this organ,19,24,25 indicated by rapid uptake of rhodamine-labeled DNA (GeneGrip) by Kupffer cells (Figure 3g). In the M-3 model, similarly highest amounts of total DNA were found in the liver followed by the tumor, however, the overall amounts detected were slightly lower than those observed in the Neuro2a model. Again the highest ratio of degraded DNA was found in the liver (Figure 3c), and most of the intact DNA was found in the tumor, followed by liver and kidney (Figure 3d). In contrast to the Neuro2a and M-3 tumor models, no DNA uptake was found in the KB tumors (Po0.05 Neuro2a versus KB tumor, T-test, onetailed, correlating with the gene expression data Figure 2), while the levels of DNA accumulation found in the liver were comparable with those found in the other models (Figure 3e, f). The DNA biodistribution data demonstrate pronounced differences in DNA uptake into the tumor in the different tumor models. The high transgene expression found in Neuro2a tumors in contrast to the KB tumors (see Figure 2) correlates with significant DNA accumulation in the Neuro2a tumors whereas no DNA was found in KB tumors following systemic application of transfection complexes (Figure 3). Furthermore, there is a high uptake of DNA by macrophages following application in vivo irrespective to the tumor model, illustrated by the rapid uptake of DNA particles by Kupffer cells in the liver, removing DNA from the blood circulation. Gene Therapy


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Figure 3 Biodistribution of plasmid DNA after systemic gene delivery. TfPEI/PEI25/DNA complexes (N/P¼4.8; containing 50 mg pCMVL) were injected into the tail vein of Neuro2a tumor-bearing A/J mice (a, b), or M-3 tumor-bearing DBA/2 mice (c, d), or KB tumor-bearing NMRI nu/nu mice (e, f). (a–f) DNA biodistribution 4 h after injection was studied by Southern blot hybridization with a probe specific for pCMVL as described in Materials and methods. The amounts of total DNA (a, c, e) and intact DNA (b, d, f) were determined. DNA accumulation in Neuro2a, M-3, and KB tumors was compared: *Po0.05, Neuro2 versus KB tumor (one-tailed T-test). Black bars: intact DNA, grey bars: partially degraded DNA. (g) Uptake of plasmid DNA by Kupffer cells in the liver. Tf-PEI/PEI25/DNA transfection complexes containing rhodamine-labeled plasmid DNA (pGeneGripTM) (N/P¼4.8; 50 mgDNA/mouse) were injected into the tail vein of Neuro2a tumor-bearing A/J mice. DNA distribution in the tissue is visualized by the red fluorescence of pGeneGripTM (left picture). Kupffer cells were stained with the macrophage specific F4/80 antibody (FITC labeled, middle picture). Cell nuclei are visualized by DAPI staining. The superimposition of all three stainings is shown in the right picture. Slides were evaluated under a Zeiss Axioskop II fluorescence microscope equipped with a Hamamatsu CCD camera. 400 magnification.

Gene delivery is influenced by tissue-dependent factors of the tumor To investigate whether morphological differences between the tumors could explain the differences in DNA biodistribution and DNA expression in the three models, Neuro2a, M-3, and KB tumors were analyzed concerning their vascularization status and infiltration by macrophages (Figures 4 and 5). Major morphological differences were found between the different tumor models Gene Therapy

Figure 4 Morphology of tumor models. Dissections from Neuro2a neuroblastoma (a), M-3 melanoma (b) and KB adenocarcinoma (c) are shown. Representative tumors out of more than 30 tumor samples analyzed for each tumor type are shown.

already when compared on the macroscopic level. Figure 4 shows representative tumor samples out of more than 30 tumors analyzed for each tumor type. The Neuro2a tumor showed well-developed vascularization (Figure 4a). The M-3 tumors (Figure 4b) appeared black in color because of the melanin produced by the melanoma cells, in some areas fine red blood vessels could be seen. In contrast, the macroscopic view of dissected KB tumor (Figure 4c) revealed a very poor vascularization of this tumor. The tissue of the KB tumors appeared as a uniformly pale, white solid mass with only a few fine red hairlines indicating a very low density of blood vessels.


areas of compact tumor tissue FITC staining of endothelial cells highlighted well-established vessel structures (Figure 5d). Furthermore, only few rhodamine-labeled macrophages were found in these areas. In contrast, necrotic areas were generally negative for endothelial cells and highly infiltrated by macrophages (Figure 5e). In KB tumors (Figure 5f, g), areas with tumor cells forming compact tissue but also numerous large necrotic regions were found. Endothelial cell staining was largely negative (Figure 5f) confirming a poor vascularization of this tumor. In contrast, large infiltration of macrophages was observed throughout the KB tumor (Figure 5g).

Figure 5 Vascularization and macrophage infiltration in the different tumor models. Neuro2a (a–c), M-3 (d, e) and KB tumors (f, g) were dissected, and microslides were stained for macrophages using F4/80 antibody (rhodamine labeled), and for endothelial cells using antiCD31 antibody (FITC labeled). Cell nuclei are visualized by DAPI staining. Slides were evaluated under a Zeiss Axioskop II fluorescence microscope equipped with a Hamamatsu CCD camera.

Furthermore, in both M-3 and KB model internal tumor necrosis and multiple areas with loss of the regular tissue structure, and in some cases liquid-filled cysts were found. Microscopic examination and staining of vascular endothelium and infiltrating macrophages were performed on the three different tumor types. In all, 20 to 30 microslides from different parts of the tumor, and from at least three tumor samples of each tumor type, Neuro2a, M-3, and KB, respectively, were analyzed. Microslides were stained with DAPI for visualization of the cell nuclei. Macrophage-specific staining was performed using a rhodamine-labeled F4/80 antibody. Endothelial cell-specific staining was performed using a FITClabeled anti-CD31 antibody. Figures 5a–c show structures typically found in the Neuro2a tumor. The DAPI-stained nuclei of the tumor cells are closely packed, indicating histologically compact tumor tissue without major necrotic areas. Beside established mature vessel structures (Figure 5b, c), also immature and discontinuous type blood vessels (Figure 5a), and in some cases lacunalike vessels (Figure 5b) were found. Rhodamine-F4/80 staining for macrophage infiltration (Figure 5c) was generally infrequent in the Neuro2a tumor. Microscopic examination showed the M-3 tumor tissue histologically to be more heterogeneous compared to the Neuro2a tumor (Figure 5d, e). DAPI staining of cell nuclei showed closely packed tumor cells in some areas of the tumor (Figure 5d), while other areas were characterized by loose connections and gaps between the tumor cells, indicating areas of necrosis (Figure 5e), which could already be seen macroscopically. In the

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Tissue-dependence of gene delivery efficacy: confirmation with other DNA formulations To further prove the general applicability of the correlation found between the efficacy of gene delivery and tissue-dependent characteristics of the target tumor a panel of tumor xenografts in SCID mice were compared concerning efficacy of gene delivery. In these experiments, the luciferase reporter gene was formulated in a second transfection formulation, which was also developed specifically for systemic gene delivery.22,23 PEGylated PEI/DNA complexes based on the linear PEI (22 kDa) and containing EGF as a cell binding ligand, EGF-PEG-PEI/DNA complexes, were systemically applied into SCID mice bearing a variety of subcutaneously growing human tumor xenografts: Huh-7 or HepG2 hepatoma, 518A2 melanoma, A549 lung carcinoma or SW480 colon carcinoma. Figure 6 shows the gene delivery efficacy in comparison to macroscopic morphology and histology of the tumors. High expression levels of the luciferase reporter gene of 2 106 RLUs were found with Huh-7 or HepG2 hepatoma xenografts, while low to near background levels were found in the 518A2 melanoma, A549 lung carcinoma and SW480 colon carcinoma models (Po0.01 HepG2 versus 518A2, A549, and SW480; Po0.05 Huh-7 versus 518A2, A549, and SW480, two-tailed T-test) (Figure 6a). In contrast to the pronounced differences in gene expression after systemic application in vivo, all tumor types showed highly efficient transfection in vitro (Figure 6b). Comparing the macroscopic morphology the Huh-7 and HepG2 hepatoma showed intense red color and blood-filled areas indicating a well-developed vascularization whereas the other tumor models showed a white or pale color indicating very poor vascularization. Endothelial cellspecific staining of microsections using a FITC-labeled anti-CD31 antibody showed high numbers of wellestablished large vessel structures as well as discontinuous blood vessels throughout the Huh-7 and HepG2 hepatomas, in some cases lacuna-like structures were found. In contrast, almost no large blood vessels were found in A549, 518A2, and SW480 xenografts confirming a much poorer vascularization of these tumors (Figure 6c). Furthermore, the fine vessel structures found in these tumor models were generally continuous as compared to the multiple types of blood vessels (including discontinuous and lacuna-like vessels) found in the Huh-7 and HepG2 hepatomas. In contrast to the pronounced differences in tumor vascularization a moderate infiltration of macrophages – mostly in necrotic areas – was found in all xenografts Gene Therapy


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Figure 6 Tissue-dependence of gene delivery efficacy in various tumor xenografts. Human tumor xenografts: Huh-7 hepatoma, HepG2 hepatoma, 518A2 melanoma, A549 lung carcinoma, and SW480 colon carcinoma were grown in SCID mice. EGF-PEG-PEI/DNA complexes (N/P¼6; containing 50 mg pCMVL) were injected into the tail vein of tumor-bearing mice. Luciferase reporter gene expression (RLU) in the tumor following systemic application in vivo is shown (n¼5). Expression levels were statistically compared by two-tailed T-test, *Po0.05, **Po0.01 (a). For comparison transfection efficacy of the same tumor cell lines in vitro is shown (b). Microslides of the tumors were analyzed for vascularization by endothelial cell-specific staining using FITClabeled anti-CD31 antibody (c). Magnification: 100 (c, middle), 400 (c, right). Dissections of the tumors showing the macroscopic appearance of the tumors are shown for comparison (c, left).

with no major differences between the different tumor models.

Discussion Nonviral gene delivery vectors that can efficiently and specifically trigger transgene expression in the desired target cells would be valuable tools for the treatment of numerous human illnesses. While a variety of nonviral vectors, including lipoplexes and polyplexes, have been shown to be able to deliver DNA efficiently in vitro, only a minority of them are capable of delivering DNA efficiently in vivo. One of the most promising nonviral vectors being developed is the polycation PEI, which can efficiently condense DNA and intrinsically initiate release of the DNA complexes from the endosomal compartment into the cytoplasm.1,3 Goula et al and others using the linear low molecular weight PEI (22 kDa)/ DNA complexes for systemic application have shown efficient gene delivery with high gene expression in the lung and lower expression in a variety of organs including heart, liver, spleen, and kidney.6–8,26 While nonviral vectors such as linear PEI22 give sufficiently high transgene expression in first-pass organs such as the lungs, targeting gene expression to distant sites, such as distant tumors seems to be more difficult. We have modified PEI-based vectors by covalent attachment of transferrin or EGF, developing surface-shielded transferGene Therapy

rin-polyethylenimine (Tf-PEI)-based10,12,19 or PEGylated EGF-PEI-based gene delivery systems,22,23 respectively. Shielding of surface charge was shown to protect the transfection particles from interaction with blood components,14,19 and accordingly no aggregation of transfection particles was found after incubation of stabilized transfection particles with murine plasma (see Materials and methods). Using surface-shielded transfection systems, we could demonstrate targeted gene expression in distant tumors after systemic application in several murine tumor models, such as the Neuro2a neuroblastoma growing in syngeneic A/J mice, as well as Huh-7 and HepG2 hepatoma xenograft tumor models in SCID mice. However, when testing surface-shielded transfection complexes more broadly in different tumor models major differences in the efficacy of gene delivery were observed. The aim of this study was to elucidate tumor-specific factors, which could play a role in affecting gene delivery efficacy observed in the different tumor models. The understanding of these factors could be most helpful for the prediction of the expression pattern of transgenes in certain tumor situation, as well as for further optimization of gene delivery vectors. Possible differences observed in gene delivery to the different tumor models could be through a number of anatomical and biological reasons: (i) the ability of different types of tumor cells to take up and express complexes after binding to the cell surface, (ii) differ-


ences in maturation and leakiness of blood vessels, thus in vivo affecting DNA complex extravasation from the systemic blood circulation into the tumor, (iii) once extravasated, the complexes could be taken up and inactivated by innate immune cells, such as macrophages, or bind nonspecifically to extracellular matrix or necrotic tissue. The ability of different tumor cells to take-up and express genes was initially tested in vitro, all cell lines used in this study were efficiently transfected by the TfPEI/DNA or EGF-PEG-PEI/DNA complexes, respectively, with no major differences observed between the different tumor cell lines. These results indicate that if complexes will reach the tumor cells potentially the complexes can be internalized and the DNA expressed. Targeted gene delivery to distant tumors after systemic application is hypothesized to be dependent on passive targeting, with the complexes being shielded from nonspecific interactions leading to prolonged blood circulation time, and allowing the complexes to reach the tumor.14,18,19 Furthermore, within the tumor, there is a higher permeability of the vasculature compared to normal vasculature.27–29 This higher permeability will favor the passive accumulation of particles at these sites of leakiness, a process which is also used by stealth liposomes.30 Hence, for passive targeting of transfection complexes to the tumor, sufficient vascularization of the tumor, and a higher leakiness of the tumor vascular bed compared to normal vasculature are required. When transfection complexes were applied systemically in vivo, major differences were found between the different tumor models. High luciferase reporter gene expression was found in Neuro2a tumors, while M-3 tumors and KB tumors expressed low or undetectable levels of luciferase activity. We hypothesized that the differences in gene expression observed between the different tumor models could result from different conditions for the complexes to extravasate from the blood system into the tumor. Analyzing the three tumor models for vascularization major differences were observed. The Neuro2a tumor appeared to be well vascularized with a high density of partially immature blood vessels correlating with efficient DNA uptake and significant gene expression in the tumor. Similarly, a well-developed vascularization was found in the M-3 melanoma model correlating with significant DNA uptake. Still the more mature type of blood vessels found in the M-3 tumors may be less suitable for extravasation of the complexes into the tumor tissue because of lower leakiness compared to the more immature types of blood vessels in Neuro2a tumors. In contrast, in the KB tumor model blood vessel formation was generally very poor, which correlated with almost undetectable DNA levels in this tumor. These data indicate that a sufficient vascularization of the tumor is necessary for the uptake of the circulating DNA complexes after systemic application. Besides the level of vascularization obviously the permeability of the vascular bed is a prerequisite for efficient extravasation of particles from the systemic circulation into the tumor. In this context a more immature vessel structure, for example, as frequently observed in Neuro2a tumor specimens can favor a passive extravasation of transfection complexes into the tumor tissue. A great variety of blood vessel types in tumors has been found, including immature, discontinuous, and mosaic vessel types,

which are thought to contribute to the high leakiness of tumor vasculature compared to normal vasculature.27 The high variation in the vessel structures in different tumors may also account for the differences in vascular permeability and the different cutoff sizes observed in different tumors.28,29 Employing transfection complexes of larger particle size (340–770 nm, Tf-PEI/PEI/DNA) and small particle size (160–190 nm, EGF-PEG-PEI/ DNA), respectively, the present studies give some indication about the differences in the cutoff size of the vasculature which can be found in different tumor models. However, even within a given tumor a certain heterogeneity concerning vascular leakiness seems to exist, as previous data have shown that uptake of plasmid DNA as well as gene expression were not equally distributed within the tumor, but rather were found predominantly at distinct sites within the tumor. These foci were often located close to the most immature blood vessels.12,19,23 The dependency of gene delivery efficacy on the vascularization of the target tumor was reconfirmed in a panel of human tumor xenografts in SCID mice using a second gene delivery system designed for systemic in vivo application. In contrast to the Tf-PEI/ DNA system used in the first part of this study where high amounts of transferrin in the complex serve for both, cell binding ligand and shielding of the surface charge,19 in the EGF-PEG-PEI/DNA system EGF serves as the cell binding ligand and the surface charge of the complex is shielded by a PEG coat. Furthermore, the more powerful linear PEI226–8 was used in this system.2,22,23 Using the EGF-PEG-PEI/DNA system a similar correlation between efficacy of gene delivery/ gene expression and vascularization of the tumor xenografts was found as shown with the Tf-PEI/PEI/ DNA system. Another factor that will affect DNA delivery and finally the uptake of DNA into the tumor cells, is the presence of necrotic tissue and infiltration by macrophages. Fluorescence staining with the macrophagespecific F4/80 antibody illustrated the rapid uptake of DNA complexes by macrophages, that is, Kupffer cells in the liver, removing the DNA from the circulation, this way preventing DNA delivery to other cells and tissues, and decreasing the efficacy of gene delivery in vivo in general. Analyzing the tumor models, only very limited areas of tissue necrosis and low numbers of infiltrating macrophages were found in the Neuro2a, Huh-7, and HepG2 tumors correlating with significant gene expression in the tumor. In contrast, large areas of necrosis and intensive infiltration by macrophages were found in the M-3 melanoma model correlating with only low gene expression in spite of significant DNA uptake into this tumor. Necrotic tissue and heavy infiltration of macrophages will cause rapid degradation of the DNA similarly as observed in the liver. A rapid attraction of macrophages by luciferase reporter gene delivery and rapid degradation of the transgene has been demonstrated in previous studies.12 Finally, in the KB tumor model, large areas of necrosis were found, probably being also a result of the insufficient vascularization of this tumor. The necrotic areas were highly infiltrated by macrophages. The aim of this study was to elucidate factors that affect the efficacy of gene delivery and gene expression to distant tumors after systemic gene delivery. The data

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indicate that uptake of DNA into the tumor is critically dependent on the vascularization of the tumor with the number and size, but also the type of blood vessels affecting efficacy of gene delivery. Furthermore, necroses and infiltration by macrophages are major factors affecting particle distribution within the tumor, facilitating rapid degradation and removal of the DNA. These data will have implications for the design and applicability of nonviral vectors that will be suitable in systemic gene therapy protocols.

Materials and methods Plasmids and polycations Endotoxin-free plasmid pCMVL coding for the Photinus pyralis luciferase gene was obtained from Elim Biopharmaceuticals, Inc. (CA, USA). PEI, molecular weight 25 kDa (branched) and 22 kDa (linear), were obtained from Sigma, Milwaukee, WI and MBI Fermentas, St Leon-Rot, Germany. The synthesis of Tf-PEI, and PEGPEI or EGF-PEG-PEI conjugates has been described previously (19, and 22, 23, respectively). Cells Murine Neuro2a neuroblastoma cells (ATCC CCL-131) were cultured in RPMI 1640 medium (Gibco BRL)/10% fetal calf serum (Gibco BRL)/1% L-glutamine (Bio Whittaker Europe). Murine M-3 melanoma cells (ATCC CCL53.1) were cultured in HAM’s F10 medium (Bio Whittaker Europe)/15% Horse serum (Gibco BRL)/5% FCS/ 1% L-glutamine. For cultivation of M-3 cells the flasks and six-well plates (used for transfections) were precoated with 0.1% gelatine. KB cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL)/10% FCS/1% L-glutamine. Human Huh-7 and HepG2 hepatoma, 518A2 melanoma, A549 lung carcinoma and SW480 colon carcinoma cells were cultured in DMEM/F12 Medium (Gibco BRL)/10% FCS. Formulation of transfection complexes and transfection in vitro In vitro transfections were performed in six-well plates. At 24 h prior to transfection 200 000 cells per well were seeded. Tf-PEI/PEI/DNA complexes. For transfection indicated amounts of plasmid DNA (eg 2 mg DNA per well) and PEI (25 kDa) or a mixture of PEI (25 kDa) and Tf-PEI (25 kDa) were each prediluted in 250 ml 0.5 HBS, rigorously mixed and incubated at room temperature. PEI/DNA or Tf-PEI/PEI/DNA complexes were formed at various molar ratios of PEI nitrogen to DNA phosphate (N/P; for example N/P¼4.8: eg 6 mg PEI per 10 mg DNA).19 After 20 min incubation, glucose (from a 50% stock solution) was added to a final concentration of 2.5% (w/v) for iso-osmolaritiy, and 0.5 ml of the complexes (containing eg 2 mg DNA) were added to the cells growing in 1.5 ml culture medium. After 4 h incubation, transfection medium was replaced with fresh medium. Cells were harvested 24 h after transfection, lysed in lysis buffer (25 mM Tris phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100) and snap frozen in liquid nitrogen. For luciferase reporter gene expression assay cell lysates Gene Therapy

were thawed and centrifuged for 10 min at 14 000 g and 41C to pellet debris. Luciferase activity was recorded using a Lumat LB9501/16 instrument (Berthold, Bad Wildbad, Germany). Luciferase background (100–200 light units) was subtracted from each value. Transfection efficiency was expressed as RLUs per 106 cells. Transfection complexes for in vivo application were prepared similarly, but at a higher DNA concentration (200 mg/ml).19

PEGylated EGF-PEI/DNA complexes (EGF-PEG-PEI/ DNA). These were prepared by flash-mixing of indicated amounts of plasmid DNA with PEI (linear, 22 kDa) and PEG20-PEI22 and EGF-PEG20-PEI25 derivatives as described.22,23 Briefly, EGF-PEG-PEI/ DNA were prepared at a ratio of: PEI22/EGF-PEG20PEI25/PEG20-PEI22¼6.5:1:2.5, and an overall N/P ratio of 6 (N/P¼6: 40 mg PEI per 50 mg DNA). EGF-PEG-PEI/ DNA complexes were mixed in 20 mM Hepes, pH¼7.1 at a final DNA concentration of 200 mg/ml. Different from the previously described transfection complexes23 in this study glucose was added directly after complex formation (before freezing the complexes) to a final concentration of 5%.31 Transfection complexes were snap-frozen in liquid nitrogen and subsequently stored at 201C or 801C. Complexes were kept at room temperature for 15–20 min after thawing before being used in physical characterization or transfection experiments. Measurement of particle size and zeta-potential of the Polycation/DNA complexes Particle size of transfection complexes was analyzed before and after incubation with murine plasma. Transfection complexes (50 ml) were incubated with 50 ml murine plasma (50%, Sigma, P-9275) for 30 min at 371C. After incubation transfection complexes were diluted with water to 700 ml and particle size was measured by laser-light scattering using a Malvern Zetasizer 3000 (Malvern Instruments, Spring Lane South, Malvern, Worcs., WR 14 1XZ) as described previously.13 The average particle size was 770750 and 340720 nm (Tf-PEI/PEI/DNA complexes), and 190710 and 16072 nm (EGF-PEG-PEI/DNA complexes) before and after incubation with murine plasma, respectively. In murine plasma alone particles with an average size of 150 nm could be measured. The presented data are means of several measurements (nX3). For estimation of the surface charge DNA complexes were diluted in 10 mM NaCl and the zeta-potential was measured using a Malvern Zetasizer 3000 as described.14 Tf-PEI/PEI/DNA complexes and EGF-PEG-PEI/DNA complexes showed a zetapotential of +4 to +7 and +0.5 to +3 mV, respectively. The data represent the means of at least three measurements. Animals A/J mice, DBA/2 mice, and NMRI nude/nude mice (7–8 weeks, female) were purchased from Harlan (Bicester, UK). C.B-17-scid/scid (SCID) mice (7–9 weeks, female) were obtained from Harlan Winkelmann (Borchen, Germany).


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Tumor models and systemic application in tumorbearing mice A/J mice were injected subcutaneously with 1 106 Neuro2a cells. DBA/2 mice were injected subcutaneously with 1 106 M-3 cells. NMRI nude/nude mice were injected subcutaneously with 1 106 KB cells. C.B17-scid/scid (SCID) mice were injected subcutaneously with human tumor cells (5 106, Huh-7; 10 106, HepG2, A549, 518A2, SW480) suspended in 100 ml PBS. After 2–5 weeks, when tumors had reached approximately 10–13 mm in size, transfection complexes (containing 50 mg DNA/250 ml per mouse) were injected intravenously. Luciferase reporter gene expression assay Animals were killed and the organs were resected and homogenized in 250 mM TRIS-buffer, pH 7.5, using an IKA-Homogenizer, snap frozen in liquid nitrogen and stored at 801C. The tissue lysates were centrifuged at 14 000 g for 10 min at 41C to pellet debris. Luciferase background (100–200 light units) was subtracted from each value and transfection efficacy is expressed as RLU per organ. Immunofluorescence staining For immunofluorescence staining microslides (cryosections 6–7 mm) were blocked with PBS/1% BSA for 10 min, and 10% goat serum for 15 min. For specific staining of macrophages slides were incubated for 1 h with the primary antibody: anti-mouse macrophage F4/ 80 [rat], Serotec, Oxford, UK, clone: Cl:A3-1 (F4/80), diluted 1:50. After washing slides were incubated for 30 min with the secondary antibody: F(ab0 )2 goat anti-rat IgG, FITC labeled, Serotec, Oxford, UK, diluted 1:50, or anti-rat F(ab0 )2 Texas red [goat], Cedarlane, Ontario, Canada, diluted 1:200. For specific staining of endothelial cells slides were incubated for 1 h with an anti-murine CD31 [rat] mAb-FITC labeled, Pharmingen, diluted 1:50. Slides were mounted with DAKOs Fluorescent mounting medium, and evaluated under a Zeiss Axioskop II fluorescence microscope equipped with a Hamamatsu CCD camera. For visualization of cell nuclei slides were stained with DAPI (Boehringer Mannheim, 1 mg/ml in methanol). For each tumor type 20–30 microslides representing different parts of the tumor, obtained from 3 to 6 tumor samples were analyzed. Determination of DNA biodistribution after systemic application At 4 h after injection animals were killed and organs were resected and homogenized in 250 mM TRIS-buffer, pH 7.5, using an IKA-Homogenizer, frozen in liquid nitrogen, and stored at 801C. Isolation of DNA was performed according to the QIAamps Tissue Kit protocol (Qiagen Cat. No. 29304). Southern blots were hybridized with a probe generated from plasmid pCMVL and washed as described in the DIG High Prime DNA Labeling and Detection Starter Kit II (Boehringer Mannheim, Cat. No. 1585614). The hybridized probes were immunodetected with anti-digoxigenin-AP Fab fragments, visualized with the chemiluminescence substrate CSPD, and recorded on an X-ray film (Boehringer Mannheim).

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