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Department of Drug Delivery Research1 , Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto; The Japan Society for the Promotion of Science (JSPS)2 , Tokyo; Institute for Integrated Cell-Material Sciences (iCeMS)3 , Kyoto University, Kyoto, Japan
Enhancement of gene expression by transcriptional activation using doxorubicin-loaded liposome/pDNA complexes K. Un 1,2 , Y. Kono 1 , M. Yoshida 1 , F. Yamashita 1 , S. Kawakami 1 , M. Hashida 1,3
Received September 12, 2011, accepted October 31, 2011 Mitsuru Hashida, Ph.D. and Shigeru Kawakami, Ph.D., Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
[email protected],
[email protected], Pharmazie 67: 400–405 (2012)
doi: 10.1691/ph.2012.1702
Gene therapy is a promising treatment option for cancers generated by mutation of oncogenes or tumor suppressor genes. The transcriptional process is activated by doxorubicin (DXR), and gene expression efficiency followed by gene transfection can be enhanced by the combination-use of DXR. Therefore, coencapsulation of plasmid DNA (pDNA) and DXR into non-viral gene carriers can enhance gene expression. Here, we prepared DXR-loaded liposome/pDNA complexes (DXR-loaded PEGylated lipoplexes) by coencapsulating pDNA and DXR into liposomes. Gene expression was enhanced by DXR encapsulation into lipoplexes in colon-26 cells and cultured mouse macrophages, and this gene expression level was significantly higher than that obtained by the combination of PEGylated lipoplexes and free DXR. Moreover, the activation profiles of transcriptional factors induced by DXR-loaded lipoplexes were different from those induced by free DXR; therefore, co-encapsulation of pDNA and DXR into gene carriers might be contributed to effective enhancement of gene expression. These findings provide a new approach for achieving effective gene transfection using PEGylated lipoplexes. 1. Introduction Gene therapy is a promising treatment option for cancer and congenital diseases based on gene mutation. In particular, cancer is generated by mutation of oncogenes and tumor suppressor genes (Vogelstein and Kinzler 2004); therefore, anti-cancer effects have been observed by delivery of pDNA encoding tumor suppressor genes or short hairpin RNA against oncogene into the tumor (Seth et al. 1996; Deng et al. 2007; Jiang et al. 2009). Many solid tumors show extensive angiogenesis, defective vascular architecture, and impaired lymphatic drainage/recovery systems (Maeda et al. 2000; Maeda 2001). Long-lived nanoparticles circulating in the blood, such as polyethylene-glycol (PEGylated) modified carriers, accumulate in tumor tissues via enhanced permeability and retention (EPR) properties (Maeda et al. 2000; Maeda 2001). Many researchers have been developed the transfer methods of nucleic acids into tumors by PEGylated carriers (Kaul and Amiji 2005; Lu et al. 2006; Sonoke et al. 2008; Noh et al. 2010; Gjetting et al. 2010; Morille et al. 2011); however, effective gene transfer into tumors is difficult due to pulmonary accumulation by cationic properties in the gene transfection. To obtain sufficient gene expression following gene transfection, not only high level of gene transfer efficiency into the targeted organs/cells but also activation of intracellular transcriptional/translational process is important factors (Thomas and Klibanov 2003). Various physical stimulations, such as electric pulse, physical pressure, water pressure, and US exposure, activates transcriptional processes (Pazmany et al. 1995; Mukai et al. 2010; Nishikawa et al. 2008; Chiu et al. 2008), and this transcriptional activation is involved in enhanced gene expression in hydrodynamics method, physical pressuremediated method, and sonoporation method (Ochiai et al. 2007; 400
Nishikawa et al. 2008; Mukai et al. 2010; Un et al. 2011a). Recently, it was reported that transcriptional processes are activated by anticancer agents, including DXR and vitamins (Brantley-Finley et al. 2003; Griesenbach et al. 2009; Crispen et al. 2007; Charoensit et al. 2008; Zanotto-Filho et al. 2009; Charoensit et al. 2010), and moreover, in vivo gene expression efficiency obtained by non-viral gene carriers is enhanced by co-administration of free DXR (Griesenbach et al. 2009). Since the in vivo distributing characteristics of gene carriers and DXR are different, co-encapsulation of pDNA and DXR into gene carriers might be more effective at enhancement of gene expression. In the present study, we prepared DXR-loaded liposome/pDNA complexes (DXR-loaded PEGylated lipoplexes) co-encapsulated pDNA and DXR into liposomes by remoteloading method (Haran et al. 1993; Fritze et al. 2006). First, the transfection efficiency and cytotoxicity followed by gene transfection with DXR-loaded PEGylated lipoplexes were investigated in colon-26 cells and cultured mouse macrophages. Moreover, the effect on transcriptional factors, such as AP-1 and NF-B, followed by gene transfection using DXR-loaded PEGylated lipoplexes were investigated, and compared with that of free DXR. Finally, the transfection efficiency in colon-26 cellderived tumors by intratumor administration of DXR-loaded PEGylated lipoplexes was evaluated. 2. Investigations, results and discussion First, we prepared DXR-loaded PEGylated lipoplexes encapsulated by remote-loading method and complexed with pDNA. The particle sizes of PEGylated liposomes/lipoplexes with or without DXR were approximately 100 nm (Fig. 1A). Following Pharmazie 67 (2012)
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Fig. 1: Physicochemical properties of DXR-loaded PEGylated lipoplexes. (A) Particle sizes and ζ-potentials of PEGylated lipoplexes with or without DXR. Each value represents the mean ± S.D. (n = 3). (B) Confirmation of pDNA complex by gel-retardation assay. Naked pDNA and PEGylated lipoplexes were run on a 1% agarose gel and detected by ethidium bromide-staining. (C) Release of DXR from PEGylated liposomes/lipoplexes in the presence of mouse serum (37 ◦ C).
gel electrophoresis of pDNA and PEGylated lipoplexes, pDNA complexes were formed with or without DXR (Fig. 1B). Moreover, the -potentials of PEGylated lipoplexes with or without DXR were approximately + 40 mV, and lower than that of PEGylated liposomes (Fig. 1A); suggesting that pDNA is attached to the surface of PEGylated liposomes. These results are consistent with other reports on PEG-modified liposomes/pDNA complexes (Sonoke et al. 2008; Ogawara et al. 2009; Un et al. 2010a). The loading efficiency of DXR into PEGylated liposomes was 95.8 ± 1.4 % (n = 5). Following evaluation of the release of DXR from PEGylated liposomes/lipoplexes under in vivo conditions (Emanuel et al. 1996; Maurer-Spurej et al. 1999), the release of DXR from PEGylated liposomes/lipoplexes was approximately 4% at 180 min after incubation in the presence of mouse serum (Fig. 1C). These findings are consistent with other reports about DXR-loaded PEG-modified liposomes (Emanuel et al. 1996; Fritze et al. 2006), and suggest that DXR is stably sustained into PEG-modified lipoplexes in extracellular situations. The release of DXR encapsulated into PEG-modified liposomes by remote-loading method is dependent on extra-liposomal pH, and increased release of DXR from PEGylated liposomes is achieved under low pH-conditions in endosomes (Maurer-Spurej et al. 1999; Fritze et al. 2006). To activate transcriptional processes, DXR needs to transfer into the cytoplasm/nucleus effectively. Therefore, the pH-triggered release of DXR in endosomes based on remote-loading method is reasonable for enhancing gene expression. Then, we investigated the enhancing efficiency of gene expression followed by gene transfection using DXR-loaded PEGylated lipoplexes in vitro and in vivo. Following preliminary experiments, DXR was loaded into PEGylated liposomes/lipoplexes at a final concentration of 0.1 M in cultured medium to minimize in-vitro cytotoxicity. As shown in Figs. 2A and 3, the level of gene expression obtained by DXR-loaded PEGylated lipoplexes was significantly enhanced compared with that by PEGylated lipoplexes in colon-26 cells, mouse cultured macrophages, and colon-26 cell-derived tumor Pharmazie 67 (2012)
tissues. Moreover, the level of gene expression obtained by DXR-loaded PEGylated lipoplexes was higher than that by combination-use of free DXR (Fig. 2A). As shown in Fig. 2B, the cytotoxicity induced by DXR was not observed in this experimental condition, and moreover, the cytotoxicity followed by gene transfection using DXR-loaded PEGylated lipoplexes was not observed in colon-26 cells and cultured mouse macrophages (Fig. 2B). It was reported that combination-use of free DXR was enhanced the gene expression followed by gene transfection using non-viral carriers (Griesenbach et al. 2009). In this study, the levels of gene expression obtained by DXR-loaded lipoplexes were higher than that by combination-use of PEGylated lipoplexes and free DXR (Fig. 2A). These observations suggest that the enhancing effects of gene expression are higher because of same intracellular transport by co-encapsulation of pDNA and DXR. Then, we evaluated the gene expression and intranuclear transport of transcriptional factors, such as AP-1 and NF-B, followed by gene transfection using DXR-loaded PEGylated lipoplexes in vitro. Following examination of the expression properties for c-fos and c-jun, which are the components of AP-1 (Eferl and Wagner 2003), c-fos and c-jun mRNA expression was enhanced transiently in colon-26 cells and cultured mouse macrophages in the gene transfection using DXR-loaded PEGylated lipoplexes (Fig. 4). Following evaluation of expressing properties and intranuclear transporting properties of NF-B (Perkins 2007; Pereira and Oakley 2008) followed by gene transfection using DXR-loaded PEGylated lipoplexes, p105 (precursor of p50) and p65 mRNA expression was not enhanced by gene transfection using DXR-loaded PEGylated lipoplexes (data not shown). On the other hand, the amount of intranuclear p50 and p65 increased transiently in colon-26 cells and cultured mouse macrophages in the gene transfection using DXR-loaded PEGylated lipoplexes (Fig. 5). Although these phenomena were also observed in the addition of free DXR, mRNA expression and intranuclear transport of transcriptional factors followed by free DXR were enhanced earlier and more transiently com401
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Fig. 2: The level of luciferase expression (A) and cell viability (B) followed by PEGylated lipoplexes, PEGylated lipoplexes with free DXR and DXR-loaded PEGylated lipoplexes (5 g pCMV-Luc, 0.1 M DXR) at 24 hr after transfection in colon-26 cells and primary mouse peritoneal macrophages. Each value represents the mean + S.D. (n = 4). ** p < 0.01, compared with the corresponding group of PEGylated lipoplexes. † p < 0.05; †† p < 0.01, compared with the corresponding group of PEGylated lipoplexes + DXR.
pared with that by DXR-loaded PEGylated lipoplexes. It has been reported that transcriptional activation followed by DXR stimulation is involved in enhanced expression of AP-1 and intranuclear transport of NF-B via phosphorylation of ERK, p38. and JNK (Brantley-Finley et al. 2003), and our results are corresponding to these reports. However, although free DXR is transport into the cells via passive diffusion, pDNA delivered by PEGylated lipoplexes is transport via endocytosis (Un et al. 2011b). Therefore, it is considered that activating characteristics of transcriptional factors, such as enhanced expression of AP-1 and intranuclear transport of NF-B, was different in the gene transfer using PEGylated lipoplexes with free DXR or DXR-loaded PEGylated lipoplexes. Moreover, the difference of enhancing level of gene expression might be contributed by the correlation with intracellular transport of pDNA and transcriptional activation by DXR. These considerations suggest that the gene carriers co-encapsulation of pDNA and DXR, such as DXR-loaded PEGylated lipoplexes, can be achieved the enhancement of gene expression via DXR-mediated transcriptional activation. In conclusion, we constructed DXR-loaded PEGylated lipoplexes, and achieved the enhanced gene expression in the gene transfection using DXR-loaded PEGylated lipoplexes in vitro. These levels of gene expression obtained by DXRloaded PEGylated lipoplexes were higher than that by combination-use of PEGylated lipoplexes and free DXR. Moreover, the activating profiles of transcriptional factors induced by DXR-loaded PEGylated lipoplexes were different from that by free DXR, and co-encapsulation of pDNA and DXR into the gene carriers might be contributed to the effective enhancement of gene expression. These findings may help in the development of an effective gene transfection method using PEGylated lipoplexes.
Polar Lipids (Alabaster, AL) and NOF (Tokyo, Japan), respectively. Cholesterol (DSPC) and doxorubicin (DXR) were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. RPMI-1640 was purchased from Nissui Pharmaceutical (Tokyo, Japan) and fetal bovine serum (FBS) was purchased from Japan Bioserum (Hiroshima, Japan). Opti-MEM I was obtained from Invitrogen (Carlsbad, CA). All other chemicals purchased were of the highest purity available. 3.2. pDNA and cells The pCMV-Luc was constructed in our previous reports (Takagi et al. 1998). Briefly, pCMV-Luc was constructed by subcloning the HindIII/Xba I firefly luciferase cDNA fragment obtained from the pGL3-control vector (Promega, Madison, WI) into the polylinker of pcDNA3 vector (Invitrogen). The pDNA was amplified in the E. coli strain DH5␣, isolated and purified using a QIAGEN Endofree Plasmid Giga Kit (QIAGEN, Hilden,
3. Experimental 3.1. Materials 1,2-Distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (CH3 O-PEG2000 -DSPE) were purchased from Avanti
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Fig. 3: The level of luciferase expression in the tumor followed by PEGylated lipoplexes with or without DXR (5 g pCMV-Luc, 0.1 g DXR) at 24 hr after intratumor administration into colon-26 cell-derived tumors. Each value represents the mean + S.D. (n = 4). *** p < 0.001, compared with PEGylated lipoplexes.
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Relative c-jun mRNA copy numbers (c-jun mRNA/gapdh mRNA)
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Fig. 4: Time-course of c-fos (A,C) and c-jun (B,D) mRNA expression levels followed by PEGylated lipoplexes (5 g of pCMV-Luc) with or without DXR (0.1 M) at predetermined times after transfection in colon-26 cells and primary mouse peritoneal macrophages. Each value represents the mean ± S.D. (n = 4). * p < 0.05; ** p < 0.01, compared with the corresponding group of N.T.. N.T., no treatment.
Germany). Colon-26 adenocarcinoma cells were obtained from American Type Culture Collection (ATCC; Manassas, VA), and cultured in RPMI1640 at 37 ◦ C in 5% CO2 . The media were supplemented with 10% FBS, 100 IU/ml penicillin, 100 g/ml streptomycin, and 2 mM l-glutamine. Mouse peritoneal macrophages were harvested and cultured according to our previous report (Un et al. 2010b). Briefly, the macrophages were harvested from female ICR mice (4-week-old; Japan SLC, Shizuoka, Japan) at 4 days after intraperitoneal injection of 2.9% thioglycolate medium (1 mL). The collected macrophages were washed and suspended in RPMI-1640 medium supplemented with 10% FBS, 100 IU/mL penicillin, 100 g/mL streptomycin and 2 mM l-glutamine, and plated on culture plates. After incubation for 2 h at 37 ◦ C in 5% CO2 , non-adherent cells were washed off with culture medium, and the macrophages were incubated for another 72 h.
lipoplexes were determined by a Zetasizer Nano ZS instrument (Malvern Instrument, Worcestershire, UK). 3.4. DXR release experiments The release of DXR from liposomes was evaluated by equilibrium dialysis method. In brief, liposomal suspension was mixed with mouse serum, and the mixture was loaded into the membrane tube (Spectra/Por Membrane, MWCO: 12,000–14,000; Spectrum Laboratories, Breda, Netherlands). The dialysis tubes were placed into PBS, and were incubated at 37 ◦ C for 12 h. The released DXR was detected at excitation; 480 nm and emission; 590 nm wavelengths. 3.5. In-vitro gene transfection
3.3. Preparation of DXR-loaded lipoplexes Liposomes were constructed according to the procedures described previously (Un et al. 2010b). Briefly, DSTAP, cholesterol. and CH3 OPEG2000 -DSPE were mixed in chloroform at a molar ratio of 56:39:5, and the mixture was dried by evaporation, vacuum desiccated and the resultant lipid film was resuspended in 250 mM ammonium sulfate (pH 5.4). After hydration for 30 min at 65 ◦ C, the dispersion was sonicated for 10 min in a bath-type sonicator and for 3 min in a tip-type sonicator to produce liposomes. Liposomes were sterilized by passage through a 0.45 m membrane filter (Nihon-Millipore, Tokyo, Japan). DXR was loaded into liposomes by remote-loading method (Haran et al. 1993; Fritze et al. 2006). Briefly, the external phase of liposomes was replaced with PBS (pH 8.0) by gel filtration using a Sephadex G-25 column (PD-10; GE Healthcare, Buckinghamshire, UK). DXR in PBS (pH 8.0) was added to liposomes at a drug-to-lipid molar ratio of 1:10 and incubated at 60 ◦ C for 1 h. Our preliminary experiments showed that the loading efficiency of DXR into liposomes was more than 96% reproducibly. The DXR-loaded lipoplexes were prepared by gently mixing with equal volumes of pDNA and DXR-loaded liposome solution at a charge ratio of 1.0:2.3 (−: + ). The particle sizes and -potentials of
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The culture medium was replaced with Opti-MEM I, and added the DXRloaded lipoplexes (DXR: 0.1 M) complexed with 5 g pCMV-Luc. Then, 6 h after the addition of lipoplexes, the incubation medium was replaced with RPMI-1640 and incubated for an additional 18 h. After incubation, the cells were scraped from the plates and suspended in lysis buffer (0.05% Triton X-100, 2 mM EDTA, 0.1 M Tris; pH 7.8). Cell suspensions were mixed and then centrifuged at 10,000 × g, 4 ◦ C for 10 min. The supernatant was mixed with luciferase assay buffer (Picagene; Toyo Ink, Tokyo, Japan) and the luciferase activity was measured by a luminometer (Lumat LB 9507; EG&G Berthold, Bad Wildbad, Germany). Luciferase activity was normalized to the protein content of cells. Protein concentration was determined using a Protein Quantification Kit (Dojindo Molecular Technologies, Tokyo, Japan). 3.6. MTT assay The cytotoxicity was evaluated by MTT assay (Mosmann 1983). Briefly, at 24 h after in-vitro gene transfection using DXR-loaded lipoplexes (DXR: 0.1 M), 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide (MTT; Nacalai Tesque) solution was added to each well and incubated for 4 h. The resultant formazan crystals were dissolved in 0.04 M HCl-
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Intranuclear p50 (pg/mg protein)
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Fig. 5: Time-course of intranuclear p50 and p65 levels followed by PEGylated lipoplexes (5 g of pCMV-Luc) with or without DXR (0.1 M) at predetermined times after transfection in colon-26 cells and primary mouse peritoneal macrophages. Each value represents the mean ± S.D. (n = 3). ** p < 0.01, compared with the corresponding group of N.T.. N.T., no treatment.
isopropanol and sonicated for 10 min in a bath-type sonicator. Absorbance values at 550 nm (test wavelength) and 655 nm (reference wavelength) were measured and the results were expressed as viability (%).
were calculated for each sample from the standard curve using the instrument software (‘Arithmetic Fit Point analysis’ for the Lightcycler). Results were expressed as relative copy numbers calculated relative to gapdh mRNA (copy numbers of c-fos and c-jun mRNA/copy numbers of gapdh mRNA).
3.7. In-vivo gene transfection Six-week-old BALB/c female mice were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were carried out in accordance with the Principles of Laboratory Animal Care, as adopted and propagated by the U.S. National Institutes of Health and the Kyoto University Guidelines for Animal Experiments. To prepare colon-26 cell-bearing mice, colon-26 cells (1 × 106 cells/mouse) were subcutaneously administrated in the back of mice. DXR-loaded lipoplexes was administered into the tumor tissues when the tumor grew up to 300∼500 mm3 in volume. Then, 24 h after the administration of lipoplexes, mice were sacrificed, and tumor tissues collected, washed twice with cold saline, and homogenized with lysis buffer (0.05% Triton X-100, 2 mM EDTA, 0.1 M Tris, pH 7.8). After three cycles of freezing and thawing, the homogenates were centrifuged at 10,000 × g, 4 ◦ C for 10 min. The luciferase activity of resultant supernatant was determined by luciferase assay, and normalized to the protein content of tissues. 3.8. Quantitative RT-PCR Total RNA was isolated from the cells and organs using GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO). Reverse transcription of mRNA was carried out using PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan). The detection of c-fos, c-jun and gapdh cDNA was carried out by real-time PCR using SYBR® Premix Ex Taq (Takara Bio) and Lightcycler Quick System 350S (Roche diagnostics, Indianapolis, IN, USA) with primers. The primers for c-fos, c-jun and gapdh cDNA were constructed as follows: primer for c-fos, 5’-CCA GTC AAG AGC ATC AGC AA-3’ (forward) and 5’-AAG TAG TGC AGC CCG GAG TA-3’ (reverse); primer for c-jun, 5’-TCC CCT ATC GAC ATG GAG TC-3’ (forward) and 5’-TGA GTT GGC ACC CAC TGT TA-3’ (reverse); primer for gapdh, 5’-TCT CCT GCG ACT TCA ACA-3’ (forward) and 5’-GCT GTA GCC GTA TTC ATT GT-3’ (reverse) (Sigma-Aldrich). The mRNA copy numbers
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3.9. Measurement of intranuclear NF-B Cells were collected at predetermined times after gene transfection, and the nuclear extracts from cells were prepared using a Nuclear Extract Kit (Active Motif, Carlsbad, CA). Nuclear protein was divided into aliquots and stored at -80 ◦ C for later use. The protein concentration was measured with a Protein Quantification Kit (Dojindo Molecular Technologies). The amounts of p50 and p65, which are the components of NF-B in the cellular nuclear extract was measured using a NF-B (p50) Transcription Factor Kit (Thermo Fisher Scientific, Waltham, MA) and a NF-B (p65) transcription Factor Assay Kit (Cayman Chemical, Ann Arbor, MI), respectively, according to the manufacturer’s protocols.
3.10. Statistical analyses Results were presented as the mean ± S.D. of greater than three experiments. Analysis of variance (ANOVA) was used to test the statistical significance of differences among groups. Two-group comparisons were performed by Student’s t-test. Multiple comparisons between control and test groups were performed by Dunnett’s test, and multiple comparisons between all groups were performed using the Tukey-Kramer test.
Acknowledgement: This work was supported in part by a Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This research paper was presented during the 8th Conference on Retrometabolism Based Drug Design and Targeting, June 2–4, 2011, Graz. Austria.
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