Effective gene transfer to melanoma cells using bacterial ghosts

Available online at www.sciencedirect.com

Cancer Letters 262 (2008) 54–63 www.elsevier.com/locate/canlet

Effective gene transfer to melanoma cells using bacterial ghosts Pavol Kudela a,c,*, Susanne Paukner b,c, Ulrike Beate Mayr b,c, Dana Cholujova a, Gudrun Kohl c, Zuzana Schwarczova a, Jozef Bizik a, Jan Sedlak a, Werner Lubitz b,c b

a Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, SK-833 91 Bratislava, Slovakia Department of Medicinal Chemistry, Faculty of Life Science, University of Vienna, A-1090 Vienna, Austria c BIRD-C GmbH & Co. KEG, Hauptstrasse 88, A-3420 Kritzendorf, Austria

Received 12 October 2007; received in revised form 21 November 2007; accepted 23 November 2007

Abstract Bacterial ghosts (BG) are cell envelopes preparations of Gram-negative bacteria devoid of cytoplasmic content produced by controlled expression of PhiX174 plasmid-encoded lysis gene E. Eight melanoma cell lines were investigated for their capacity to bind and phagocyte BG derived from Escherichia coli NM522 and Mannheimia haemolytica A23. High capability to bind BG was observed in almost all of the analyzed cell lines, furthermore cells were able to take up BG independently of the used bacterial species. Further, transfection efficiency of BG loaded with DNA in vitro was measured. The Bowes cells exhibited a high expression level of GFP and the incubation of cells with plasmid loaded BG led up to 82% transfection efficiency.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Melanoma; Bacterial ghost; DNA carrier; Gene expression

1. Introduction The rate of incidence and mortality of patients with malignant melanoma is increasing worldwide and has doubled over the past decade. Melanoma represents the most malignant tumor of the skin and therefore needs to be detected and excised early because of its high metastatic potential. Successful treatment of melanoma patients is possible only after detection in early stages and no significant *

Corresponding author. Address: Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, SK-833 91 Bratislava, Slovakia. Tel.: +421 2 5932 7402; fax: +421 2 5932 7250. E-mail address: [email protected] (P. Kudela).

treatment is known once tumor metastases are detected. Because of its ‘‘fatal’’ character, new approaches in the treatment of malignant melanoma need to be developed. Melanoma belongs to the group of immunogenic and very intensively studied tumors [1,2]. This type of tumor is one of a few cancers with known multiple tumor-associated antigens capable of eliciting specific immune response including mutated antigens (CDKN2A); differentiation antigens (melanA/MART-1, gp100) or the shared tumor-specific antigens of the cancer/testis family (MAGE-1, MAGE-3, NY-ESO-1) [3–6]. Recently a number of different types of vaccines have been tested including the melanoma derived proteins as immu-

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.031

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notherapeutic agents; vaccines against the melanoma and tumor-associated antigens; the modified melanoma cell vaccine-modification of cytokines genes, the costimulatory molecules, the modification of immune effector cells – APCs and the tumor immunization or passive immunotherapy with specific monoclonal antibodies [7–16]. Gene therapy is a promising technology in the development of potential effective vaccines. Successful delivery of antigens or DNA to the target cells requires the adequate form and suitable compartments of the selected delivery system. DNA vaccines consist of bacterial plasmids encoding the antigen under the control of strong eukaryotic promoters. The expression of the delivered gene should induce strong immune response or change the behavior of the targeted cells. There are several systems allowing delivery of such genetic material into the target cell including viral vectors, attenuated bacteria, polycation/DNA complexes or cationic liposomes [17–19]. The use of attenuated viral or bacterial systems still bears potential danger due to their pathogenicity. The BG system, developed during recent years, represents a new platform in vaccine development. Cell envelopes from Gram-negative bacteria are produced by controlled expression of lysis gene E. All cell surface structures of bacteria remain in native form while the cytoplasmic content is expelled [20,21]. Therefore the BG could replace the use of live or attenuated bacteria in vaccine development. BG combine the properties of adjuvant because of the presence of all the immune stimulating compounds on the surface such as LPS, peptidoglycans and lipid A and their high inner space capacity which can be loaded with antigens, plasmids or chemical agents [22–28]. Previous studies have shown that BG are able to deliver heterologous genes to professional antigen presenting cells (APC) including monocyte-derived DCs and macrophages [23,24,26]. Melanoma cells have many of the same functions as APCs, including the ability to phagocyte, process and present antigens [29–32]. In the present study we have investigated the capacity of two BG prepared from Escherichia coli NM522 and Mannheimia haemolytica A23 to target melanoma cells and determined adherence rates, as well as the capability of melanoma cells to phagocyte BG. Moreover, we studied the ability of E. coli NM522 and M. haemolytica A23 ghosts loaded with plasmid encoding the green fluorescent protein (pEGFP) to deliver DNA to the target cells and to mediate transgene

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expression at varying amounts of BG per cell and DNA loads. We demonstrated that BG loaded with pEGFP are able to deliver plasmids to melanoma cells. 2. Materials and methods 2.1. Cell lines and reagents Human melanoma cell lines: Bowes, SK-Mel-28 and A-375 were obtained from American Type Culture Collection (ATCC, Rockville, MD); 1F6 and 1F6m were kindly provided by T. de Vries, Department of Pathology, University Medical Centrum, Nijmegen, NL; WM-164, WM-239 and WM-373 were the kind gifts of M. Herlyn, the Wistar Institute, Philadelphia, PA. All cell lines were maintained in RPMI 1640 medium (Gibco BRL, Paisley, Scotland) supplemented with 2 mM glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 lg/ml streptomycin (Gibco), 10% FCS (Gibco), in a 5% CO2 humidified incubator at +37 C. Dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), paraformaldehyde (PFA), fluorescein diacetate (FDA) and propidium iodide (PI) were obtained from Sigma–Aldrich Co. (St. Louis, MO). 2.2. Production of BG BG from E. coli NM522, M. (Pasteurella) haemolytica A23 were produced by the controlled expression of the phage derived lysis protein E as described previously [20,21,33]. The non-lysed bacteria were inactivated with gentamycin (Gibco) (50 lg/ml) and streptomycin (100 lg/ml). Subsequently the BG were washed three times with PBS (phosphate buffered saline, pH  7.4), resuspended in distilled water and were lyophilized. Lyophilized BG were stored at +4 C. 2.3. Preparation of plasmid DNA and of FITC-labeled linear dsDNA for loading of BG The plasmid pEGFP-N1 (4.7 kb, Clontech, Palo Alto, CA, USA) was prepared from 4 L overnight culture of E. coli by a large scale protocol using the standard alkaline method described by Horn et al. [34]. LPS, proteins and RNA were removed by ammonium acetate precipitation and RNaseI digestion. The DNA, after the final precipitation with ethanol, was dissolved in HPLC water (Sigma–Aldrich Co.) at a concentration of approximately 25 mg/ml and stored frozen at 20 C. The concentration of plasmid DNA was determined by measurement of the absorbance at 260 nm and 280 nm, and the absence of RNA and the chromosomal bacterial DNA was confirmed by agarose gel electrophoresis. The fluorescence labeling of the DNA was performed by PCR. A randomly chosen 400 bp DNA fragment of the archaebacterial phage /CH1 was amplified with the 5-FITC-labeled

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primers 5 0 -CGGCAGGTTTCATCCAGGAG-3 0 and 5 0 -TAACAGCACGCCGGAACTGA-3 0 (VBC Genomics, Vienna, Austria). The 50 ll reactions containing 1.75 nM dNTPs, 0.5 lM of both oligonucleotide primers, 1 lg / CH1 DNA and 1-U Taq DNA polymerase in polymerase buffer were subjected to the following conditions: 4 min predenaturation at +94 C; 35 cycles of 30 s denaturation at +94 C, 30 s annealing at +60 C and 1 min extension at +68 C; 5 min at +4 C final temperature. The amplified PCR products (linear, dsDNA) were stored frozen at 20 C protected from light. 2.4. Loading of BG with DNA Loading of BG with pEGFP-N1 (Clontech; Qiagen maxi kit preparation) was performed by diffusion of plasmid DNA into the BG through the lysis holes. The lyophilized BG (10 mg) were resuspended in 100 ll HEPES buffered saline (HBS, 100 mM NaCl, 10 mM sodium acetate, 10 mM HEPES) containing pEGFP-N1 (1 lg/ll) and incubated with agitation at a temperature between +28 C and +37 C for 10 min. Prior to incubation, the DNA-ghosts suspension was supplemented with CaCl2 (25 mM final concentration). For the determination of the optimal Ca2+ concentration, the Ca2+ was adjusted to final concentrations of 25 mM. The BG were then spun down, washed once with ml of HBS supplemented with the appropriate Ca2+ concentration and resuspended in HBS. The loaded BG were then aliquoted and were stored frozen as pellets at 80 C for more than 2 months without any significant loss of the loaded DNA. Furthermore, the sterility was confirmed by determination of the colony forming units in 1 mg BG on LB at +28 C. 2.5. Isolation of the loaded plasmids and quantitation by real time PCR The BG-associated pEGFP-N1 was quantified by real time PCR after plasmid isolation with the mini preparation kit (Peqlab, Erlangen, Germany). The plasmid DNA was eluted from the columns with twice 50 ll Tris– HCl (10 mM, pH 8). The evaluation of the mini preparation kit was done by six parallel extractions followed by quantitation in duplicate. This revealed that on average 97.2% of the loaded DNA was extracted. The amplification of the egfp gene (700 bp) was performed in the Corbett Research Rotor-Gene 2000 (Corbett Research, Sydney, Australia) and monitored with intercalating dye SYBRGreen I (Roche, Basel, Switzerland). The oligonucleotide 5 0 -GGT GAG CAA GGG CGA GGA G-3 0 and 5 0 -TTA CTT GTA CAG CTC GTC CAT G-3 0 (Oligo Sequencing Service, Vienna, Austria) were used as forward and reverse primers. A pEGFP-N1 preparation purified with the mini preparation kit (peqlab, Erlangen, Germany) was used for calibration. It was shown to be free of RNA and chromosomal DNA by agarose gel electrophoresis.

The DNA concentration of the calibration pEGFP-N1 was quantified by the measurement of absorption at 260/ 280 nm. Aliquots of the calibration DNA were stored at 20 C. To obtain a standard curve at least five serial dilutions (102–106) of the pEGFP-N1 were prepared. Each sample and standard was quantified in duplicate by real time PCR. Two reactions with water instead of template served as blank control. Each 25 ll reaction included DyNAzymeTM II DNA polymerase (Finnzymes, Finland; 1 U), the DyNAzymeTM reaction buffer, dNTPs (0.4 mM each), oligonucleotide primers (2 pmol) and SybrGreenI (final dilution of 1:105). The thermal cycling conditions were as follows: the initial denaturation of +94 C (3 min), followed by +94 C (45 s), +60 C (1 min), +72 C (1 min) for 26 cycles. Fluorescent signal data were acquired at the end of each extension phase. This was followed by a 3 min hold at +72 C and the melt curve analysis from +72 C to +99 C. The final melt curve analysis and agarose gel electrophoresis ascertained that there were no artifacts like primer dimers present in the amplification mixture. For the quantitative analysis, only the standard curves with a correlation coefficient of at least 0.9998 were used. As the pDNA isolation and the real time PCR were each done in duplicate, the mean value and the standard deviation for each sample were calculated from four values. 2.6. FITC-labeled BG uptake The efficiency of the endocytic activity of the analyzed cell lines was measured as the uptake of FITClabeled BG. Five milligrams of BG were resuspended in 500 ll 0.1 M Na2CO3; pH  9.0. Stock solution volume of 25 ll FITC (1 mg FITC in 1 ml DMSO) was added to the BG suspension and shaken for 2 h at room temperature. Afterward the BG were carefully washed with PBS and stored at +4 C protected from light. The cells cultured in 24-well plates (2 · 105 cells/ well) were incubated with FITC-BG (500 per cell) for 2 h at both +37 C and +4 C. After the incubation cells were washed three times with PBS to remove the excess BG. Finally the cells were trypsinized, washed with PBS, fixed in cold 1% PFA in PBS and analyzed on an EPICS ALTRATM Flow Cytometer (Beckman Coulter, Miami, FL). 2.7. Toxicity assay for BG The cells cultured in 24-well plates (2 · 105 cells/well) were incubated with different amounts of BG (0; 10; 50; 100; and 500 per cell) for 2 h at +37 C. After incubation the cells were carefully washed three times with PBS to remove the excess of BG and incubated in culture medium (2 ml/well) for the next 48 h at +37 C. Finally the cells were carefully trypsinized, washed twice with cold PBS and resuspended in 400 ll of PBS/0.2%

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BSA containing 10 nM of FDA (from a 5 mM stock in DMSO) followed by incubation for 30 min at room temperature. The cells were then cooled and 4 ll of PI (1 mg/ml) was added. Cells were analyzed on an EPICS ALTRATM Flow Cytometer (Beckman Coulter). 2.8. Gene transfection in vitro

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secondary Abs (DAKO, Glostrup, Denmark) at +4 C for 30 min and washed afterwards three times with FACS staining/washing buffer. The cells were fixed with 1% PFA in PBS at +4 C for 30 min before the fluorescence was analyzed with EPICS ALTRATM Flow Cytometer (Beckman Coulter). 2.9. Statistical analysis

Cells were plated in 24-well plates at 1 · 105 cells/ well 12 h prior to transfection. The transfections were performed in 200 ll/wells of complete media containing the BG (500 per cell) loaded with pEGFP (1 and 2 lg per sample). After 2 h the cells were washed to remove the excess BG and incubated with culture medium for 48 h. The Effectene-mediated transfection (Qiagen, Hilden, D) was performed according to the manufacturer’s instructions. The cells were incubated for 10 min with transfection complex at room temperature. Next, the transfection complex was aspired, the cells were washed and complete culture medium was added to the cells. Transfection efficiency was analyzed 48 h after transfection by FACS analysis and defined as the percentage of cells with increased fluorescence confirmed by specific monoclonal antibodies over the untransfected controls. The cells were trypsinized, washed with PBS and incubated with 0.5 ml of 1· FACS Permeabilizing Solution 2 (Becton Dickinson Immunocytometry Systems, San Jose, CA), vortexed, incubated for 10 min at room temperature and washed with FACS staining/washing buffer (0.5% FCS and 0.1% NaN3 in PBS), followed by incubation with mAb anti-GFP (BD Living Colors GFP Monoclonal Antibody, BD Biosciences Clontech, Palo Alto, CA) at +4 C for 30 min and washed with FACS staining/washing buffer. In the next step the cells were incubated with the appropriate R-PE-conjugated

The statistical significance of the difference between two groups was evaluated by Student’s t-test and between more than two groups by the one-way ANOVA. Differences were considered to be significant with p < 0.05. 3. Results 3.1. Phagocytosis of BG by melanoma cells To evaluate phagocytic activity of human melanoma cells, we investigated the capability of 8 human melanoma cell lines to bind and uptake BG, by adding FITC-labeled BG (500 per cell) derived from E. coli NM522 and M. haemolytica A23 (Fig. 1). The uptake rates were found to be similar for both types of BG for each melanoma cell line. We detected a high capacity (more than 75%) to uptake BG in 6 out of 8 tested human melanoma cell lines (Bowes, SK-Mel-28, 1F6, 1F6m, WM-164, WM-239) without restriction to bacterial species. Lower uptake capacities were detected for A-375 (at least 62%) and for WM-373 (30%) cells. 4 out of the 6 cell lines with increased uptake of BG exhibited preferential uptake (less than 10%) of E. coli NM 522 ghosts- (1F6, 1F6m, WM-164, WM-239) and 2 with preferential uptake (15%) of M. haemolytica A23 ghosts (Bowes, SK-Mel-28). Slightly higher uptake

Fig. 1. Comparative analysis of FITC-labeled BG uptake by 8 different melanoma cell lines. 2 · 105 cells were incubated with FITC-BG (500 per cell) – E. coli NM522 (empty bar) and M. haemolytica A23 (full bar) for 2 h at +37 C and at +4 C (negative control). The endocytic activity of melanoma cell lines varies slightly depending on the bacterial strain from which the BG were prepared. Values were calculated as the percentage of cells with increased fluorescence incubated at +4 C subtracted from the percentage of positive cells incubated at +37 C. Endocytic capacity of melanoma cells was measured by EPICS ALTRATM Flow Cytometer (Beckman Coulter). Representative of four independent experiments is shown.

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rates for E. coli NM522 ghosts were also detected for A-375 cells while for WM-373 cells no difference in uptake of BG was observed between the BG derived from two different bacterial strains. The highest uptake of both used types of BG (more than 90%) was observed by cells of the WM164 and WM-239 cell lines. Both, the poorly metastatic cell

line 1F6 and its metastatic subline 1F6m displayed almost the same preferential uptake of BG derived from E. coli NM522 compared to BG derived from M. haemolytica A23. A subtle increase in distinct uptake of BG derived from two different bacterial strains was detected by 1F6m cells compared to 1F6 cells.

Fig. 2. Effect of BG on viability of melanoma cells. Melanoma cells were incubated for 2 h with different amounts of BG (E. coli NM522full squares with dotted lines; M. haemolytica A23-full circles with full lines) followed by cultivation in complete medium without BG for the next 48 h and stained with PI and FDA, and analyzed by flow cytometry. Data represent means of three independent experiments performed in triplicates ±SD.

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3.2. Effect of BG on the viability of melanoma cells Potential toxic effect of BG on the viability of melanoma cells was investigated by flow cytometry. The changes in the number of viable cells after incubation with BG were calculated using the following formula: A [difference in viability (%)] = B [% of viable cells incubated with BG]  C [% of viable cells incubated without BG] (Fig. 2). No significant changes in cell viability were observed after incubation of cell lines Bowes, SK-Mel-28 and A-375 with both analyzed types of BG. A slight decrease of cell viability was detected in 1F6 cells but the opposite effect was observed in its metastatic subline 1F6m. However taking the standard deviation into account there was no significant effect of BG on the viability of these cell lines. BG prepared from E. coli NM522 affected the viability of WM-164 and WM-239 more than the BG prepared from M. haemolytica A23. In general, BG prepared from E. coli NM522 tended to result in a more decreased viability than BG prepared from M. haemolytica A23, except for cell line 1F6m. The strongest effect on the viability of melanoma cells by BG was detected after incubation of WM-373 cells with both types of BG. Interestingly, these cells showed the lowest binding capacity and uptake rates of BG; furthermore their viability was significantly reduced in correlation with increased amounts of BG used during 2 h of incubation (Figs. 1 and 2). 3.3. Gene transfer to melanoma cells using BG The capability of BG to mediate heterologous gene transfer to the melanoma cell line Bowes was investigated. Bowes cells were selected according to two criteria: (1) they had one of the highest capacities to phagocyte BG and (2) no effect of BG was detected on the viability of Bowes cells. Melanoma cells were co-incubated with BG (500 per cell) loaded with either 1 or 2 lg of the pEGFP plasmid DNA. In parallel, Bowes cells were transfected with 1 lg and 2 lg of the pEGFP with the Effectene reagent. Transfection efficiency was evaluated as the percentage of cells expressing GFP. Cells incubated with and without the empty BG were used as negative control. The obtained results showed that the BG loaded with DNA have an even greater potential to deliver heterologous genes to the Bowes cells than Effectene reagent (Fig. 3A). We have observed a greater than 20% increase of GFP positive cells after incubation with the plasmid DNA loaded BG compared to the cells transfected with Effectene. Use of higher amounts of DNA did not result in a significant increase of transfection efficiency for either method (less than 10%). Furthermore, our results showed that preferential uptake of M. haemolytica A23 ghosts compared to E. coli NM522 ghosts by Bowes cells did not significantly affect the gene delivery by BG to melanoma cells. There were only 5% more GFP expressing cells after incubation with M. haemolytica A23

Fig. 3. Comparison of transfection efficiencies in the Bowes cells using the Effectene transfection reagent and BG (500 per cell) derived from E. coli NM522 and M. haemolytica A23 loaded with pEGFP with different DNA concentrations (1 lg and 2 lg of DNA per sample) (A). Each bar represents means of three independent experiments ±SD performed in duplicates. No meaningful increase in transfection efficiency was detected after the co-cultivation of Bowes cells with different amounts of BG derived from M. haemolytica A23; total concentration of DNA was 1 lg throughout (B). Data represent means ± SD of three independent experiments performed in duplicates.

ghosts compared to those incubated with E. coli NM522 ghosts (Fig. 3A). Next, we decided to test varying amounts of BG per cell, using the same amount of total DNA (1 lg) per sample. No significant difference in transfection efficiency was detected after the co-cultivation of Bowes cells with varying amount of M. haemolytica A23 ghosts containing a constant amount of DNA; the number of cells expressing the delivered gene varied from 68% to 82%. The most efficient amount of BG for heterologous gene transfer to Bowes cells by bacterial ghosts was 100 BG per cell (82% cells expressing GFP). Furthermore, very high transfection efficiency (more than 70%) was detected even after incubation of Bowes cells with the lowest amount of BG (1 per cell) (Fig. 3B). Altogether, obtained data demonstrate the high capacity and efficiency of BG to deliver heterologous genes to melanoma cells.

4. Discussion Efficient gene delivery to melanoma cells would allow a new approach in the therapy of this disease.

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In this study, we investigated the transfection efficiency of gene delivery using the non-viral BG system. The panel of melanoma cell lines was tested for their capacity to phagocyte BG derived from Gram-negative bacteria. We have shown that these melanoma cells have a high capability to bind and internalize BG, and these phenomena were associated with the ability of BG to successfully deliver heterologous DNA to melanoma cells. Quantitative analysis by flow cytometry showed high expression levels of the marker protein GFP (up to 82%) in Bowes cells after incubation with BG loaded with plasmids encoding GFP. Moreover, we demonstrated that BG seem to be a better delivery system compared to the non-liposomal lipid transfection reagent Effectene for the delivery of transgenes to malignant cells derived from solid tumors. It has been shown previously that melanoma cells have many functions in common with APCs, including their phagocytic activity [29,30] . Melanoma cells are capable of phagocyting both apoptotic and live cells as well as plastic beads [31,32]. Our data demonstrate that BG belong to the group of attractive targets phagocyted by melanoma cells. This fact might be related to the presence of the LPS on the surface of BG which may lead to an activation of melanoma cells. Melanoma cells respond to LPS through the TLR-4 receptor that is constitutively expressed by this type of tumor cell. Activation of melanoma cells by LPS results in enhanced production of IL-8 and cell adhesion [35]. Although it was reported that TLR-4 is not involved in cellular LPS uptake by monocyte or endothelial cells [36] the connection between TLR-4-mediated melanoma cell activation by LPS and their phagocytic activity remains to be defined. Many tumor cells are beyond the reach of the immune system. This immune evasion might be related to the loss of relevant tumor antigens, and/ or defects in the antigen processing and presentation machinery [37]. Delivery of gene(s) encoding appropriate protein(s) to tumor cells with known defects might lead to the recovery of the cell’s native functions and the potential induction of the immune response. Another important possibility for increasing the immunogenicity of tumor cells is their transfection with genes encoding cytokines attracting the professional antigen presenting cells and other crucial factors in the activation and development of the immune responses, including the response against the tumor cells [38–40]. As shown in our

results, BG represent a promising and efficient vehicle for transfer of DNA to melanoma cells. Melanoma cells revealed high capabilities to internalize BG, and this phenomenon was also observed for other types of tumor cells including colon carcinoma [27] and leukemia cells (Kudela and Lubitz, unpublished data). Uptake of BG loaded with Doxorubicin (Dox), antineoplastic drug commonly used in cancer therapy, by CaCo2 cells led to its release from lysoendosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of colon carcinoma cells were significantly decreased after internalization of Dox loaded BG compare to cells incubated with free Dox [27]. The same effect was observed after incubation of leukemia cells with Dox loaded BG (Kudela and Lubitz, unpublished data). These results indicate high capacity of BG to target various histological types of cancer. Previously published studies have shown that BG are also efficiently internalized and phagocyted by professional antigen-presenting cells, and are able to deliver the heterologous genes to both non-dividing cells (monocyte-derived DCs) and dividing cells (macrophages) [23,24]. Furthermore, intradermal and intramuscular immunization of Balb/c mice with BG loaded with pCMVp encoding b-galactosidase demonstrated efficient humoral and cellular responses against the delivered antigen. Moreover, b-galactosidase-specific immune response was detected after intravenous immunization of mice with autologous DCs transfected ex vivo with pCMVb-loaded BG [26]. In addition, BG can be loaded with a combination of peptides, drugs or plasmids which gives us an opportunity to design new types of polyvalent vaccines [41,42]. Combined intratumoral vaccination using BG loaded with both antineoplastic drug agent and plasmids encoding genes for tumor-specific antigen, together with adoptive transfer of autologous DC ex vivo transfected by BG loaded with plasmid encoding the same tumor antigen and/or ex vivo expanded autologous tumor antigen-specific T cells might lead to increased and effective immune response against tumor cells, but using of this strategy in prospective cancer immunotherapy should be further extensively investigated. The present study revealed that despite the high loading capacity of BG, relatively low concentrations of DNA are sufficient for effective gene delivery and its expression by target cells. This suggests that BG could be loaded by multiple plasmids and

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used to deliver heterologous genes to different types of cells in particular tumor types (i.e., proliferating and non-proliferating). Furthermore, it was shown that loading of BG with DNA could be simplified by using self-immobilizing plasmid (SIP) that is retained by the carrier envelope due to a specific interaction between cytoplasmic membrane anchored proteins with minicircle DNA during and after protein E-mediated lysis. This technique allows removal of plasmid sequences dispensable for vaccination, e.g., the origin of replication and the antibiotic resistance marker, resulting in production of BG containing minicircle DNA with an increased safety profile [43,44]. Further, the cytoplasmic space of BG can be filled with various substances, e.g., water-soluble proteins, plasmids, drugs, further extending their utility as a potential therapeutic vehicle [42]. Several delivery systems including viral and nonviral systems have been investigated for the delivery of transgenes to tumor cells [8,17,19,45,46]. Despite the fact that viral vectors provide efficient gene transfer, safety concerns are still present due to the immunogenicity of most viruses that limits the duration of gene expression and the ability to re-administer the genes. Similarly, live bacterial vaccines have been successfully used as carriers for vaccine antigens but their use bears a risk of reversion to their original pathogenic forms [47]. Therefore, the development of delivery systems with low cytotoxicity and high efficiency of gene delivery means considerable improvement compared to current viral and bacterial methods. BG are produced by protein E-mediated lysis of Gram-negative bacteria, which is able to fuse inner and outer membrane forming an specific tunnel through which all the cytoplasmic content of bacteria is expelled. Empty body of BG is devoid of nucleic acids, ribosomes and other constituents, whereas inner and outer membrane structures remain intact [20]. Moreover, using a nuclease together with the protein E-mediated lysis system insure degradation of total bacterial DNA including antibiotic resistance genes [48]. Therefore, there is no risk of reversal to pathogenic form in contrast to attenuated bacteria used as bacterial delivery system. The main advantage of BG is their non-living character, while still retaining all of the surface morphological, structural and antigenic components of their living counterparts [41]. A potential problem in using BG as carriers and/or adjuvant for future therapeutic applications is the LPS (i.e., endotoxin) content of the Gram-negative

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bacteria cell envelopes from which they are derived. However, previous immunological studies have shown that BG induce dose-dependent antibody responses against bacterial cell components and LPS without inducing fever. Safe profile of BG was confirmed using a Limulus-assay, where purified LPS (E. coli, O26:B6) expressed endotoxic activity values 100-times higher than the BG. Therefore, endotoxicity does not limit the use of BG as a candidate vaccine [49]. In conclusion, our results demonstrate ideal use of BG as a non-viral gene delivery system, exhibiting both a high transfection efficiency and low rate of endotoxicity, with study results showing that up to 82% of melanoma cells expressed the plasmidencoded reporter gene delivered by BG. As an alternative to the current viral systems, these findings merit further studies for the optimization of this non-viral gene delivery system and the development of this system for future use in clinical trials for melanoma and other types of cancer. Acknowledgements This work has been supported by BIRD-C GmbH & Co. KEG, Kritzendorf, Austria. We thank Jan Markus for his helpful discussion and suggestions for improvement of this manuscript. References [1] T. Boon, P.G. Coulie, B. VandenEynde, Tumor antigens recognized by T cells, Immunology Today 18 (1997) 267– 268. [2] F.O. Nestle, G. Tonel, A. Farkas, Cancer vaccines: the next generation of tools to monitor the anticancer immune response, PLoS Medicine 2 (2005) 951–952. [3] T. Saida, Recent advances in melanoma research, Journal of Dermatological Science 26 (2001) 1–13. [4] Y. Kawakami, S. Eliyahu, C. Jennings, K. Sakaguchi, X.Q. Kang, S. Southwood, P.F. Robbins, A. Sette, E. Appella, S.A. Rosenberg, Recognition of multiple epitopes in the human-melanoma antigen Gp100 by tumor-infiltrating Tlymphocytes associated with in-vivo tumor-regression, Journal of Immunology 154 (1995) 3961–3968. [5] R.F. Wang, S.L. Johnston, G. Zeng, S.L. Topalian, D.J. Schwartzentruber, S.A. Rosenberg, A breast and melanomashared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames, Journal of Immunology 161 (1998) 3596–3606. [6] T. Boon, P.G. Coulie, B.J. Van den Eynde, P. van der Bruggen, Human T cell responses against melanoma, Annual Review of Immunology 24 (2006) 175–208. [7] M. Tanaka, Y. Kaneda, S. Fujii, T. Yamano, K. Hashimoto, S.K.S. Huang, D.S.B. Hoon, Induction of a systemic immune response by a polyvalent melanoma-associated

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