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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer

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Soo-Yeon Kim a , Sang-Jin Lee b, *, Soo-Jeong Lim a, **

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a b

Department of Bioscience and Bioengineering, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea Genitourinary Cancer Branch, Research Institute, National Cancer Center, Goyang 410-769, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 March 2014 Received in revised form 28 July 2014 Accepted 14 August 2014 Available online xxx

Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus–emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus–emulsion complex may act as a lung-targeting adenoviral gene delivery system. ã 2014 Published by Elsevier B.V.

Keywords: Cationic emulsion Adenovirus Gene therapy Caveolin Endocytosis

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1. Introduction

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Designing gene transfer vehicles that efficiently introduce foreign genes into target cells at high expression levels remains a major hurdle for utilizing gene transfer therapies. Gene transfer vehicles can be classified into viral and non-viral vectors. Despite safety concerns, viral vectors are more efficient transfer vehicles than non-viral vectors. Recombinant adenoviruses are particularly promising viral vectors because they are capable of infecting a wide range of cell types at all stages of cell division (Han et al., 2008). Adenovirus (adenovirus type 5) entry into cells requires attachment to a primary receptor, coxsackievirus and adenovirus receptor (CAR) on the target cell membrane via the adenovirus fiber knob. The CAR-docked adenovirus particles activate the secondary integrin receptor and the activated receptor triggers

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* Corresponding author. Tel.: +82 31 920 2460; fax: +82 31 920 2139. ** Corresponding author. Tel.: +82 2 3408 3767; fax: +82 2 3408 4334. E-mail addresses: [email protected] (S.-J. Lee), [email protected] (S.-J. Lim).

endocytosis, particularly clathrin-dependent ones (Meier and Greber, 2003; Zhang and Bergelson, 2005). Once adenoviruses are internalized, they rapidly escape the endosome and enter the nucleus directed by proteins inherent to the adenovirus (Fasbender et al., 1997). Therefore, gene transfer efficiencies of adenovirusderived vectors are not limited by endosomal escape as in the case of non-viral vectors. Rather, they are limited by CAR expression levels on the surface of the target cell. Several studies have shown that CAR receptor expression is frequently down-regulated in cancer cells, hampering adenovirus binding (Lee et al., 2004). In addition to the limited entry of adenoviral vectors on CAR-deficient target cells, several factors present major obstacles for the clinical application of adenovirus-mediated gene therapy, including elevated immune response due to a high prevalence of neutralizing antibodies, liver toxicity caused by adenovirus sequestration. Chemical modification of the adenovirus surface with polymers that shield the adenoviral vectors has shown promise in overcoming the current limitations of adenovirus-mediated gene transfer (Kim et al., 2012). Surface conjugation with polyethyleneglycol reduced liver accumulation and prevented antibody

http://dx.doi.org/10.1016/j.ijpharm.2014.08.024 0378-5173/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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neutralization (O’Riordan et al., 1999). However, chemical modification of viral proteins can cause decreased production efficiency, impaired intracellular trafficking and difficulties in purification (Rogee et al., 2007). Electrostatic complexation of cationic lipidderived vectors with negatively charged adenovirus particles is an alternative strategy that has been shown to facilitate viral attachment to negatively charged cell membranes and improve adenoviral transgene expression (Han et al., 2008; Lee et al., 2004). In our previous studies, complexation of adenoviral vectors with cationic liposomes allowed increased gene expression particularly in CAR-deficient cells in vitro as well as in vivo (Han et al., 2008; Lee et al., 2004). Furthermore, cationic liposome complexation also resulted in reduced immunogenicity of adenovirus vectors (Singh et al., 2008). Such a hybrid system is advantageous because of its simple preparation method and because it capitalizes on the unique features associated with non-viral- and viral-vector systems. Cationic lipids can form liposomes, solid lipid nanoparticles and emulsions depending on the preparation conditions (Liu and Yu, 2010; Vighi et al., 2010; Davaa et al., 2013). Several studies on nonviral gene transfer utilizing cationic lipid-based formulations have shown that oil-in-water type emulsions are more efficient gene transfer vehicles than liposomes formed without an oil core (Chung et al., 2001; Kim et al., 2003; Liu and Yu, 2010). The success of emulsions as gene transfer vehicles stems from the increased stability of the plasmid–emulsion complex as compared to plasmid–liposome complexes. This is particularly true of formulations in the presence of destabilizing components such as serum. In an effort to further improve adenoviral gene transfer efficiency, cationic emulsions and adenoviruses were complexed by electrostatic interactions similar to those in emulsion–plasmid complexes. To our knowledge, this is the first reported utilization of cationic emulsions to enhance carrier efficiency in adenoviral gene transfer. Optimized cationic emulsions containing Lipiodol as the oil core effectively enhanced adenoviral gene transfer in vitro, in a manner related to the increased stability of emulsion–virus complexes, leading to the improved cellular uptake through activation of caveolae-dependent endocytosis. Furthermore, systemic administration in vivo demonstrates that the emulsion– adenovirus complexes reduce transgene expression in liver tissue while significantly increasing expression in lung tissue.

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2. Materials and methods

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2.1. Cell lines and reagents

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1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 (PEG-PE), and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-lissamine rhodamine B sulfonyl (RhoPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (CHOL), chlorpromazine, methyl-b-cyclodextrin, amiloride and wortmannin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Tetramethylrhodamine-70,000 MW neutral Dextran (TMR-dextran) was purchased from Molecular Probes (Eugene, OR, USA). Lipiodol, an iodinated ethyl ester of poppy seed oil (Kweon et al., 2010) was purchased from Guerbet Antre (Aulnay-sous-Bois, France). Soybean oil, safflower oil, sesame oil, corn oil, squalene and cottonseed oil were purchased from Sigma– Aldrich (St. Louis, MO, USA). Poppy seed oil was purchased from Jean-Marc Montegottero (Beaujeu, France). Mouse B16-F10 melanoma cells and human HL-60 promyelocytic leukemia cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM (B16-F10) or RPMI1640 (HL-60) medium (Welgene, Daegu, Korea)

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supplemented with 10% penicillin/streptomycin and 10% heatinactivated fetal bovine serum (Gibco, NY, USA) and kept in humid incubator at 5% CO2 and 37  C. The recombinant adenoviral Ad-GFP (replication-defective adenovirus type 5) viruses and Ad-Luc carrying green fluorescence protein (GFP) and luciferase (Luc) reporter genes respectively under the control of cytomegalovirus promoter were constructed in E1/E3-deleted RightZap1.2 vector (OD260, Boise, ID, USA) and propagated in a permissive 293 cell line. Produced adenovirus particles were purified by ultra-centrifugation through cesium chloride gradients. Viral titers were determined by absorbance at 260 nm. Plaque-forming unit (PFU) was calculated by determining the maximal dilution factor to be able to lyze 293 cells in 96-well plate. The viral stock was kept frozen at 80  C until use. All other chemicals were of reagent grade and used without further purification.

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2.2. Preparation of liposomes and emulsions

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Liposomes and emulsions were prepared by sonication method (Choi et al., 2004). DOTAP and DOPE (or CHOL), with or without addition of PEG-PE, were mixed at a molar ratio of 1:1:0.01 in tertbutyl alcohol (Junsei, Saitama, Japan). After rapid freezing at 80  C, mixtures were subjected to freeze-drying by freeze dryer (EYELA FDU-1200, Japan). After overnight freeze drying, the obtained lipid cakes were hydrated with 5% dextrose dissolved in distilled water. For preparing emulsions, oil was first added to the dried lipid cakes and the 5% dextrose was added dropwise to the oil–lipid mixture. The hydrated lipid dispersion was briefly vortexed and sonicated in bath type sonicator for 2 h at 37  C. When required, liposomes were extruded through a 200 nm polycarbonate membrane using an extruder (Lipofase-Basic, Avestin, Canada). The prepared liposomes and emulsions were stored at 4  C until use.

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2.3. Characterization by dynamic light scattering

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Mean particle sizes of liposomes, emulsions or their complexes with adenovirus were determined using a fiber-optic particle analyzer (FPAR-1000, Otsuka Electronics, Japan). Prior to measurement, samples were diluted with filtered 5% dextrose solution. The system was used in the auto-measuring mode. Particle size analysis data were evaluated using volume distribution mode to detect even a few large particles. Zeta potential was determined using a Zen 600 zetasizer (Malvern, England). Prior to measurement, samples were diluted to 1 ml with deionized water. Default instrument settings and automatic analysis were used for all measurements. Each measurement was carried out in triplicate. In case of samples containing adenoviruses, adenovirus at a concentration of 2  109 pfu was included in samples. To evaluate the serum and salt effect on the stability of adenovirus-cationic lipid carrier complexes, samples containing complexes (350 nmol lipid/2  109 pfu) were diluted to 1 ml with 4 mM phosphate buffered saline (pH 7.4) containing 0.5% fetal bovine serum and the size changes of complexes were monitored by dynamic light scattering at designated time points during incubation at 37  C.

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2.4. In vitro adenoviral gene transfer

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Adenoviral infection was preformed as described previously (Lee et al., 2004). Briefly, cells were seeded in 12-well tissue culture plates at a density of 105 cells/well and were used when 70–80% confluent. Complexes of Ad-GFP and emulsions were made by mixing adenoviral vectors and emulsions using gentle pipette tip

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aspiration, followed by incubation for 20 min at room temperature. Cells were washed with phosphate-buffered saline (PBS), and virus –liposome complexes were added together with 400 ml medium with or without serum supplementation. The amount of adenovirus was fixed at 800 pfu/cell. After 4-h incubation in a CO2 incubator at 37  C, cells were washed with PBS to remove the complexes, and 1 ml fresh 10% serum-containing medium was added. Cells were then incubated for an additional 30-h before assessing GFP expression. To examine the effect of endocytosis inhibitors on the adenovirus-mediated gene transfer, cells were preincubated with specific inhibitors for 1-h and further incubated with adenovirus– emulsion complexes for 3 h.

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2.5. Evaluation of transgene expression

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After infection, cells were harvested by using a scraper. Harvested cells were washed twice with PBS, resuspended in fresh medium supplemented with 10% serum. Cells were fixed with 1 ml 1% paraformaldehyde for 30 min at 4  C. After brief centrifugation, the supernatant was discarded and fixed cells were resuspended with PBS. The expression of GFP in suspended cells was determined by using Beckman coulter flow cytometer (Beckman Coulter korea, Seoul, Korea) using Cell Quest program (BD Bioscience). Ten thousand fluorescent events per sample were acquired using a 530/15 band pass filter for the green fluorescence protein signal obtained with fluorescence emission centered at 530 nm. To evaluate the transgene expression by confocal microscopy, cells were seeded on a cover glass placed in 12 well plates and then infected with adenovirus complexes by using the similar protocol as described above. After removing the culture media, cells were washed three times with cold PBS and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then washed three times with cold PBS. Mounting solution (Dako korea, Seoul, Korea) was dropped on a slide glass and the cover glass was put on slide glass to contact the mounting solution. The fixed cells were observed by Leica TCS SP5Confocal Laser Scanning Microscope (Wetzlar, Germany). Fluorescence imaging of each sample was obtained using 488 nm excitation line and a band-pass BP495555 emission filter.

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2.6. Cell uptake study For measuring cell binding and uptake of liposomes or emulsions, liposomes and emulsions labeled with Rho-PE (0.5 mol% of total lipids) were prepared as described above. Cells were seeded in 12 well plates at a density of 105 cells/well. After overnight incubation, the culture medium was changed to serumfree media before adding aliquots of Rho-PE labeled liposomes or emulsions at 37  C. At designated time, cells were washed twice with PBS, harvested, fixed and then resuspended in PBS. Cell fluorescence by rhodamine attachment and uptake was analyzed by flow cytomtery using Beckman coulter flow cytometer (Beckman Coulter Korea, Seoul, Korea). The cellular uptake of liposome or emulsion was also evaluated by confocal microscopy, after preparing cell samples by using the similar uptake protocol as described above. Fluorescence imaging of each sample was obtained using 543 nm excitation lined and a band-pass BP550-620 emission filter. When pretreatment with endocytosis inhibitors was required, prior to adding emulsions, cells were preincubated with a genistein (200 mM), chlorpromazine (60 mM), wortmannin (10 mM), or amiloride (5 mM), for 3 h. Inhibitor concentrations were chosen according to previous works (Cardarelli et al., 2012; Rejman et al., 2004).

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2.7. In vivo study

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The protocols for animal studies were reviewed and approved by the Experimental Animal Ethical Committee of Sejong University (Seoul, Korea) according to the Guide for the Care and Use of Laboratory Animals. Six-week-old male C57BL6 mice were purchased from Doo Yeol Biotech. (Seoul, Korea). To determine the transgene expression in various organs of mice by administration of viruses and virus–emulsion complexes, random groups of mice (n = 5 for each group) were administered with PBS, 1 109 pfu/mouse Ad-luc or emulsion–Ad-luc complex (50 ml emulsion (175 nmol as DOTAP)/109 pfu) via tail vein in a final volume of 100 ml. At 24-h post-injection, mice were sacrificed and organs (heart, liver, lung, spleen and kidney) were collected. The collected organs were chopped into several pieces and minced to more fine pieces with liquid nitrogen. Each sample was put into round-bottomed tubes and homogenized in ice-cold Luciferase Cell Culture Lysis 2X Reagent (Promega Corp., Madison, WI) with a homogenizer (IKA-Werke, Stufen, Germany), 1 ml volume for liver and 500 ml for other organs. After centrifugation, the separated lysate supernatant was analyzed for the luciferase activity using a Luciferase Assay Reagent (Promega Corp., Madison, WI) according to manufacturer’s instruction. The luminescence intensity was measured using a luminometer (Lumat LB 9507, Berthold). The protein content of the same lysate supernatant was determined using Bradford assay method with Bio-Rad protein Assay kit (Hercules, CA, USA).

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2.8. Statistical analysis

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Statistically significant differences between values obtained in different types of formulations or under different experimental conditions were determined using two-tailed unpaired Student’s t-tests.

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3. Results and discussion

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3.1. Effect of liposome composition on adenoviral gene transfer

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In our previous studies (Han et al., 2008; Lee et al., 2004), cationic liposomes were prepared using DOTAP as the cationic lipid and DOPE as a helper lipid for complexation with the adenovirus. DOPE is frequently used as a helper lipid to enhance gene transfection by liposome–plasmid complexes. However, the mechanism by which DOPE assists gene transfection is known to be fusogenic, thereby providing endosome-disrupting properties that are not required for adenoviruses that efficiently escape the endosome and enter the nucleus. In other studies, CHOL has been suggested as an effective helper lipid in vitro and in vivo, by improving liposome stability and by reducing serum protein interactions with liposomes (Crook et al., 1998; da Cruz et al., 2004). To improve adenoviral gene transfer efficiency, CHOL was explored as a helper lipid in place of DOPE. As shown in Fig. 1, GFP expression in B16-F10 cells following incubation with liposome– Ad-GFP complexes was dependent on liposome dose regardless of which helper lipid was incorporated. Cells treated with DOTAP/ CHOL liposome complexes showed significant up-regulation of adenovirus-mediated GFP expression as compared to DOTAP/DOPE liposome complexes. This demonstrates that the inclusion of CHOL in DOTAP liposomes was more advantageous in enhancing adenovirus-mediated transgene expression than DOPEcontaining liposomes. Dynamic light scattering analysis was performed to compare the physicochemical characteristics of adenoviral complexes with DOPE- or CHOL-containing liposomes. The surface charge of naked adenovirus was found to be negative as reported in numerous

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Fig. 1. Effect of helper lipid on efficacy of liposome-complexed adenoviral gene transfer. Liposomes were prepared with DOTAP:DOPE or DOTAP:CHOL at 1:1 molar ratio. B16-F10 cells were incubated with a fixed amount of Ad-GFP pre-complexed with liposomes at a ratio of 3.5 or 7 nmol/107 pfu (total lipid/Ad-GFP). Infection was performed in the presence of 25% serum. After infection, the percentage of GFPexpressing cells was determined by FACS analysis. The data shown are mean  SD (n = 3).

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studies (Singh et al., 2008; Zeng et al., 2012). The incubation of adenovirus with liposomes produced larger particles, whose surface charge turned more positive in a liposome concentration-dependent manner (Table 1), indicating that cationic liposomes were associated with adenovirus particles through electrostatic interaction to form complexes. In comparing DOPE- and CHOL-containing liposomes, adenovirus incubation with 3.5 nmol of DOTAP/CHOL liposomes produced complexes smaller in mean size with more positive surface charge, compared with that of DOTAP/DOPE liposomes. It indicates that the negative surface of viral particles was more effectively shielded by CHOLcontaining liposomes. Strong affinity between adenovirus and CHOL (Worgall et al., 2000) may be responsible for more tight association, resulting in enhanced adenoviral gene transfer. With this regard, it is of interest that Van den Bossche et al. (Van den Bossche et al., 2011) recently have shown that DOPE-containing liposomes were superior to CHOL-containing ones in adenoviral gene transfer, contrary to our observation. Adenovirus particles were fully enveloped by lipid layers in their studies by encapsulating adenovirus particles during liposome preparation process and the reduced gene expression by DOTAP/CHOL liposomes was because those fully enveloped adenovirus required fusogenic lipid to escape from endosomes. In the present study, adenovirus particles were mixed with pre-formed liposomes, thus not being fully surrounded by liposomes and it seems likely that they retained their ability to escapse from endosomal compartment.

3.2. Effect of oil component constituting emulsion on adenoviral gene transfer

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Since cationic emulsion complexes with plasmid DNA have shown superior effectiveness in non-viral gene transfer, it was of interest whether this result could potentially be translated to adenoviral gene transfer. In addition, the oil component in cationic emulsions has been recognized as an important factor affecting non-viral gene transfer efficiency (Chung et al., 2001). To investigate whether emulsion-complexed viral gene transfer is affected by a similar mechanism, Ad-GFP–emulsion formulations containing various oils were tested. Complexing Ad-GFP with varying emulsion formulations containing varying oils tended to exhibit superior gene transfer efficiency as compared to complexing with liposomes where identical lipid components (DOTAP/ CHOL) were used, the only difference being the inner oil core (Fig. 2A). Squalene- and poppyseed oil-containing emulsions showed slightly less GFP expression than liposome vehicles; however, the difference was not statistically significant (p > 0.01). Soybean, cottonseed, sesame, corn, safflower and Lipiodolcontaining emulsions all increased GFP expression in B16-F10 cells compared to liposome controls. Among them, Lipiodolcontaining emulsions displayed the highest transgene expression efficiency, followed by sesame oil, cottonseed oil, corn oil and finally safflower oil-containing emulsions. Specifically, GFP expression increased by 1.9-fold in cells that were administered with Lipiodol emulsion vehicles compared to comparable liposome vehicles and increased by 16.7-fold compared to cells that were administered with virus alone (83.3%, 43.6% and 5.0% increase in GFP expression in cells after incubation with emulsion complex, liposome complex and virus alone, respectively). These data indicate that oil/water emulsions using cationic lipids as a stabilizer were more efficient than cationic liposomes in enhancing the adenoviral transgene expression. The success of these emulsions may be attributed to the higher physical stability of adenovirus–emulsion hybrid vehicles compared to adenovirus–liposome hybrid vehicles. In addition, differences in gene transfer efficiency among different emulsion formulations may be due to the differences in physical stability of emulsions, which are known to be greatly affected by the physicochemical properties (e.g., surface tension and viscosity) of oil (Chung et al., 2001). To investigate this, five different emulsion (liposome) formulations selected. Since the emulsion stability is generally dependent on the initial particle size, the initial mean sizes of formulations were first checked. The initial mean sizes followed the order of squalene emulsion < liposome < safflower emulsion < poppyseed emulsion < Lipiodol emulsion. Lipiodol emulsions was 2.8-fold larger than the smallest squalene emulsion (Fig. 2B, inset). After complexation, the mean sizes followed the order of liposome < squalene emulsion < safflower emulsion < poppyseed emulsion < Lipiodol emulsion. Therefore, Lipiodol emulsions formed the largest complex with adenovirus despite of their effectiveness in gene transfer. When the

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Table 1 Mean particle size, polydispersity (PI) index and zeta potential of liposomes before or after complexation with adenovirus determined by dynamic light scattering. Each value represents the mean  SD (n = 3). Size (nm)

Adenovirus DOTAP/DOPE DOTAP/Cholesterol a b

z (mV)

PI

Virus ( )

Virus (+)

Virus ( )

Virus (+)a

Virus ( )

Virus (+)

– 168  23 163  29

105  1 365  49a 173  3a

– 0.207  0.026 0.178  0.020

0.020  0.019 0.385  0.049 0.198  0.022

– +55.7  0.9 +59.0  1.2

18.5  0.6 +39.1  1.8a +44.7  2.3a

+47.8  2.6b +54.5  1.2b

3.5 nmol of liposomes were complexed with 107 pfu adenovirus. 7 nmol of liposomes were complexed with 107 pfu adenovirus.

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Fig. 2. (A) Effect of oil type on efficacy of emulsion-complexed adenoviral gene transfer. The o/w emulsions containing 11 ml/ml of varying oil as an inner phase was stabilized by 1:1 DOTAP:CHOL mixture at a concentration of 7 mmol/ml. B16-F10 cells were incubated with Ad-GFP pre-complexed with each emulsion at a ratio of 3.5 nmol/107 pfu (total lipid/Ad-GFP). Infection was performed as described in the text in the presence of 25% serum and evaluated by FACS analysis. For comparison, liposomes prepared with DOTAP/CHOL mixture at the same content were also used. The data shown are mean  SD (n = 3). Significant differences are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001, compared to DOTAP/CHOL liposomes. (B) Stability of adenovirus–emusion complexes as assessed by seruminduced changes of mean size as a function of time. Inset: the initial particle size of each formulation without adenovirus complexation. The data shown are mean  SD (n = 3).

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destabilization of these complexes were induced by a salt/serum condition modified from an earlier study (Kim et al., 2003), the mean size of complexes increased in a time-dependent manner regardless of formulations (Fig. 2B). However, the extent of increase varied among formulations: at 60-min postincubation, the sizes of adenovirus complexes with liposomes increased by 3.9-fold while those with Lipiodol emulsions increased by 1.7fold. As a result, the mean sizes of complexes at 60-min postincubation followed the order of Lipiodol emulsion < liposome < squalene < poppyseed oil. In case of adenovirus–safflower emulsion complexes, large aggregate was formed at 60-min and their sizes could not be determined. Since the proteins and counterpart ions present in PBS supplemented with serum would bind to the adenovirus-cationic lipid carrier complexes, our data imply that adenovirus particles complexed with Lipiodol emulsions are more resistant to protein/ion bindings compared to other emulsions and liposomes.

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3.3. Effect of PEG-PE inclusion and oil content of emulsions on adenoviral gene transfer

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Lipiodol-containing emulsions were further studied, since they exhibited the most potent enhancement in adenoviral gene transfer. Previous work demonstrated that the small amount of inclusion of PEG-PE in cationic liposomes further improved adenoviral gene transfer in the presence of serum proteins (Lee et al., 2004). DOTAP/CHOL emulsions were prepared with or without PEG-PE to examine whether this phenomenon translated to emulsion gene transfer vehicles. Incorporation of PEG-PE significantly reduced the mean droplet size of DOTAP/ CHOL emulsions from 489  44 nm to 182  37 nm, indicating that PEG-PE contributed to the formation of a stable fine-particle emulsion. Mixing surfactants of high and low hydrophilic lipophilic balance (HLB) is known to reduce the droplet size of emulsions (Kang et al., 2012; Perrier et al., 2010); therefore, inclusion of the higher HLB PEG-PE with the lower HLB DOTAP/CHOL mixture effectively stabilized the emulsion by reducing particle size. Ad-GFP complexation with DOTAP/CHOL/ PEG-PE emulsions slightly increased GFP expression in B16-F10 cells compared to that with DOTAP/CHOL emulsions (Fig. 3A) but the extent of improvement was not significant (p > 0.01). DOTAP/CHOL/PEG-PE emulsions containing varying Lipiodol content were prepared to determine whether adenoviral gene transfer enhancement was dependent on Lipiodol content. Increasing the Lipiodol content in emulsions from 0 ml/ml to 6 ml/ml then to 11 ml/ml enhanced the adenoviral transgene expression in a linear fashion (Fig. 3B). Lipiodol content beyond 11 ml/ml and as high as 23 ml/ml was no more effective in increasing GFP expression in cells, suggesting that the effect was saturated. B16-F10 cells treated with Ad-GFP via DOTAP/CHOL/ PEG-PE emulsion hybrid vehicles containing 11 ml/ml of Lipiodol resulted in much higher GFP expression compared to cells treated with Ad-GFP alone as well as those treated with DOTAP/CHOL/PEGPE liposomes (Fig. 3C). These results indicate that emulsions, particularly those containing Lipiodol, are highly effective in enhancing adenoviral gene transfer and transgene expression of GFP in B16-F10 cells.

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3.4. Effect of serum on the adenoviral gene transfer mediated by emulsion or liposome complexes

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Due to the electrostatic binding of serum proteins to gene transfer vehicles, serum is known to interfere with gene transfer in both non-viral and viral systems (Lee et al., 2004; Uchida et al., 2002) and previous studies have demonstrated that liposome– adenovirus complexation improves gene transfer efficiency under serum conditions ranging from 0 to 80% (Lee et al., 2004). To ascertain the effectiveness of emulsions as a hybrid carrier with adenovirus in the presence of serum, in vitro studies comparing the complex stability and the gene transfer efficiency in the presence of varying serum content were performed. Lipiodol emulsions stabilized by DOTAP/CHOL/PEG-PE were slightly smaller than corresponding liposomes and Ad-GFP complexation slightly increased the mean size of both of liposomes and emulsions (Table 2). The positivity of zeta potential of emulsion was higher than that of liposomes (+64.2 vs. +54.7 mV). Incorporation of Lipiodol in emulsions might affect the arrangement of lipid components, increasing the positivity of surface charge. Ad-GFP complexation decreased the zeta potential of liposomes from +54.7 to +39.8 mV, whereas the decrease was less in case of emulsions (+64.2 to +60.5 mV). These data suggest that Lipiodol emulsions were more effective in shielding the negative charge of adenoviral surface compared to liposomes.

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Fig. 3. (A) Effect of PEG-PE inclusion in emulsions on efficacy of adenoviral gene transfer. Emulsions containing Lipiodol as an oil phase (11 ml/ml) were prepared with 1:1 DOTAP:CHOL mixture with or without addition of 0.5 mol% of PEG-PE. B16-F10 cells were infected with Ad-GFP–emulsion complexes at a ratio of 3.5 nmol/107 pfu (lipid/ Ad-GFP) in the presence of 25% serum and subjected to FACS analysis. (B) Effect of Lipiodol content of emulsions on efficacy of gene transfer by virus–emulsion complexes. Emulsions containing indicated amount of Lipiodol was prepared by using 1:1:0.01 mixture of DOTAP:CHOL:PEG-PE. After infection in the presence of 25% serum, the percentage of GFP-expressing cells was determined by FACS analysis. Each point represents the mean  SD (n = 3). (C) Representative fluorescent images of cells after GFP gene transfer using adenovirus alone, virus–liposome complexes or virus–emulsion complexes. At 30-h post incubation, the photographs of cells were taken using a confocal microscope (20 magnification). For preparing liposomes and emulsions, 1:1:0.01 mixture of DOTAP:CHOL:PEG-PE were used. Lipiodol was incorporated in emulsions at 11 ml/ml. 444 445 446 447 448 449 450 451

When the stability of adenovirus–emulsion complex (E complex) was compared with that of adenovirus–liposome complex (L complex) by incubating each complex in the presence of PBS containing 0.5% serum, salt and serum increased the size of both L complex and E complex in a time-dependent manner (Fig. 4A). At 60-min postincubation, the size of L complex was already larger than 1 mm, reaching 1.7 mm following 240-min incubation. In contrast, the size of E complex remained below 1 mm up to 240-min

incubation (870 nm). The polydispersity index increased from 0.209 to 0.596 (L complex) whereas it did from 0.308 to 0.334 (E complex). These data demonstrate the higher stability of E complex compared to L complex. Serum content higher than 0.5% could not be tested due to the interference by serum components in measuring the particle size by dynamic light scattering method. The addition of 50% serum had little effect on the expression of GFP in B16-F10 cells after treatment with adenovirus–liposome

Table 2 Mean particle size, polydispersity index and zeta potential of cationic lipid formulations before or after complexation with adenovirus. Liposomes or emulsions were prepared with identical lipids (1:1:0.01 mixture of DOTAP/CHOL/PEG-PE) except the inner oil core (11 ml Lipiodol/ml) included in emulsions. Liposomes or emulsions corresponding to 3.5 nmol of lipids were complexed with 107 pfu Ad-GFP. Each value represents the mean  SD (n = 3). Size (nm)

Liposomes Emulsions

z (mV)

PI

Virus ( )

Virus (+)

Virus ( )

Virus (+)

Virus ( )

Virus (+)

168  22 182  37

175  22 197  19

0.198  0.043 0.207  0.026

0.209  0.067 0.308  0.039

+54.7  2.1 +64.2  1.7

+39.8  6.6 +60.5  1.5

Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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Fig. 4. (A) Comparison of stability of L complex and E complex as assessed by serum-induced changes in mean size and PI as a function of incubation time. Transgene expression following complex treatment of (B) B16-F10 and (C) HL-60 cells. Infection was performed with L complex) or E complex at a ratio of 3.5 nmol/107 pfu in the presence of indicated serum content. The percentage of GFP- expressing cells was determined by FACS analysis. Each point represents the mean  SD (n = 2). Significant differences are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

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complexes (48.3  5.9% and 46.2  6.2% with 0% and 50% serum). GFP expression in cells treated with adenovirus–emulsion complexes rather increased from 63.4  4.7% to 83.2  4.6% when 50% serum was used (Fig. 4B). In the presence of 90% serum, GFP expression was greatly reduced regardless of liposome or emulsion complexation, although the percentage of GFP expressing cells was still higher in samples treated with the emulsion vehicle. Adenovirus treatment with no vehicle barely introduced GFP in B16-F10 cells in the presence of 90% serum (2.8  0.5%). These results indicate that Lipiodol emulsion vehicles allow more effective adenoviral gene transfer in the presence of high content of serum. The decrease in the presence of 90% serum may be due to the severe binding of serum components not only to adenoviral complexes but also to cellular membranes, thereby interfering the attachment/uptake of adenoviral particles/complexes into cells regardless of formulations. Considering that in vivo microenvironments are dynamic while in vitro experimental conditions are

static, 50% serum condition is regarded as a condition most closely mimicking the physiological condition (Zhang and Anchordoquy, 2004; Zhang et al., 2014) and our data imply that E complex may be the most effective in in vivo gene transfer. In view of our previous results demonstrating the reduced adenoviral gene transfer by DOTAP/DOPE/PEG-PE liposome vehicles in the presence of 10% serum (Lee et al., 2004), our data indicate that CHOL inclusion and the emulsion formulation itself contribute to increased gene transfer in the presence of high content of serum. Yoo et al. (Yoo et al., 2004) observed that addition of Lipiodol in squalene emulsions improved non-viral gene transfer efficiency in adherent cells, but did not improve gene transfer in non-adherent cells. They reasoned that adding high density Lipiodol (1.3 g/ml) increases the density of the resulting adenovirus–emulsion complexes, thereby increasing the contact time between adherent cells and the emulsion–plasmid complex. To check whether the enhancement in adenoviral gene transfer observed by

Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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Lipiodol-containing emulsion vehicles is limited by cell type, adenoviral transgene expression was measured in nonadherent HL-60 cells. In the absence of serum, cellular GFP expression increased 4.8-fold and 15.3-fold by liposome- and emulsioncomplexation, respectively, compared to cells treated with Ad-GFP alone. GFP expression by E complex in the presence of 0 and 90% serum was 3.2-fold and 1.6-fold higher than L complex indicating that Lipiodol-containing emulsions were more effective than L-complex in HL-60 cells, albeit the difference at serum 90% was not statistically significant (Fig. 4C). The present data indicate that Lipiodol-containing DOTAP/CHOL/PEG-PE emulsions enhance the adenoviral gene transfer in both a nonadherent cell type, HL-60, and an adherent cell type, B16-F10, making the enhancement unlikely to be due to a simple density increase.

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3.5. Evaluation of emulsion uptake into cells

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Based on our earlier works, the enhancement of adenoviral gene transfer by liposome vehicles is mediated by an increase in cellular uptake due to an electrostatic attraction between the positively charged liposomes and the negatively charged cellular membrane (Lee et al., 2004). To elucidate the cause of the observed enhancement of adenoviral transgene expression by emulsion vehicles, the rate and extent of liposome and emulsion uptake were compared using rhodamine-labeled PE. The extent of both liposome and emulsion uptake into B16-F10 cells increased in a time-dependent manner. The extent of emulsion uptake was 2.0-, 1.6- and 1.6-fold higher than liposome uptake after incubation for 10 min, 1 h and 4 h, respectively (Fig. 5A). The extent of liposomeor emulsion uptake was also concentration-dependent (Fig. 5B). Cells incubated with the emulsion vehicle at 3.5, 7 and 14 nmol DOTAP dose for 4 h showed an increase in the percent of rhodamine-positive cells that was 1.7-, 1.5- and 1.6-fold greater than observed after liposome incubation. In Fig. 5C, representative fluorescent and phase-contrast images of cells demonstrate that emulsion treatment yielded higher rhodamine uptake in cells than liposome treatment suggesting that enhanced adenoviral transgene expression by emulsion vehicles is associated with an increase in the attachment and uptake of the emulsion-complexed adenoviruses. The increased positivity of zeta potential of emulsion complex (Table 2), together with increased stability of E complex (Fig. 4A), may provide stronger electrostatic attachment to anionic components of cellular membranes such as glycosaminoglycan (Vercauteren et al., 2012), leading to higher rates of internalization.

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3.6. Effect of endocytosis inhibitors on emulsion uptake and transgene expression To express the transgene at the cellular level, adenoviral vectors must undergo cellular binding, endocytosis, endosomal escape and nuclear import. Previous findings indicate that adenoviral entry into cells occurs mainly via the clathrin-dependent pathway (Meier and Greber, 2003), while most cationic nanoparticles are internalized through various endocytosis pathways, including clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis, although the contribution of each pathway varies depending on cell type and the characteristics of the nanoparticle themselves (Billiet et al., 2012; Goncalves et al., 2004). Since the intracellular trafficking of adenovirus complexes initiated from endosomal escape would be dictated by the properties of adenovirus rather than the nanoparticles, it was of interest whether emulsion complexation affects the entry routes of adenovirus. To clarify the mechanisms involved in the cellular internalization of emulsion complexes, experiments were performed to

Fig. 5. (A) Time-dependent and (B) dose-dependent uptake of liposome/emulsions to cells. Liposome/emulsions prepared with DOTAP/cholesterol/PEG-PE/Rho-PE mixture were used. (A) Time-dependent uptake was determined after incubating cells with liposomes or emulsions at a concentration of 14 nmol as DOTAP. (B) Dosedependent uptake was determined by FACS analysis after incubating cells with liposomes or emulsions at indicated DOTAP concentration for 4 h. Each bar represents the mean  SD (n = 3). (C) Representative fluorescent images of cells incubated with 4 ml liposomes or emulsions for 2 h. Images were taken by confocal microscopy at 20 magnification.

assess the extent of uptake and transgene expression in the presence of various endocytosis inhibitors. When B16-F10 cells were incubated with emulsion vehicles in the presence of chlorpromazine, a pharmacological inhibitor of clathrindependent endocytosis, or genistein, an inhibitor of caveolaedependent endocytosis (Cardarelli et al., 2012; Kanda et al., 2013; Rejman et al., 2004), emulsion uptake significantly decreased (Fig. 6A). In contrast, treatment with either wortmannin or amiloride, chemicals commonly employed to inhibit macropinocytosis (Cardarelli et al., 2012), did not affect the cellular uptake of emulsion vehicles. To ensure that these macropinocytosis markers are working, wortmannin and amiloride were also tested on the entry of 70-kD TMR-dextran, a marker of macropinocytosis (Faille et al., 2012) and the entry of dextran was inhibited by these inhibitors (Fig. 6A, inset). Combined, these data indicate that the entry of emulsions is mediated mainly via the clathrin-dependent and caveolae-dependent endocytosis

Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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Fig. 6. Effect of endocytosis inhibitors on emulsion uptake (A) and transgene expression by adenovirus alone or as an emulsion complex (B). (A) After 1-h pretreatment with specific inhibitors, B16-F10 cells were incubated with 4 ml of emulsions (14 nmol as DOTAP) labeled with rhodamine-PE. Extent of emulsion uptake was determined as percentage of rhodamine-positive cells by FACS analysis and the percentage of rhodamine-positive cells without pretreatment was used as an index for a 100%. Inset: the percentage of TMR-positive cells pretreated with amiloride or wortmannin. Cells were 1-h pretreated with specific inhibitor prior to incubating with 0.5 mg/ml TMR-Dextran. At 3-h postincubation, the percentage of TMR-positive cells was determined by FACS analysis. (B) After 1-h pretreatment with specific inhibitors, B16-F10 cells were infected with Ad-GFP alone or E complex at fixed adenovirus content (6400 and 800 pfu/cell, respectively). The percentage of GFP-expressing cells infected with Ad-GFP alone or E complex without pretreatment was used as an index for a 100% (control), respectively and after 30-h post incubation, this value was used to obtain the remaining percentage of GFPexpressing cells in cells pretreated with specific inhibitors, respectively. Each bar represents the mean  SD (n = 3). Significant differences are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001, compared to control cells without pretreatment. 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589

pathways, while macropinocytosis minimally contributes to internalization of the emulsion vehicles. Adding chlorpromazine, decreased the GFP expression in cells treated with naked Ad-GFP by 34% (p < 0.001), confirming that naked Ad-GFP entry is by the clathrin-dependent endocytic pathway, as reported in the literature (Meier and Greber, 2003). Genistein, however, did not affect GFP expression in cells, indicating that naked Ad-GFP does not enter the cells via caveolae-dependent endocytosis in B16-F10 cells (Fig. 6B). By contrast, genistein treatment effectively inhibited the emulsion– adenovirus complex (E complex) internalization, significantly decreasing GFP expression (p < 0.0001). Chlorpromazine treatment was also effective at inhibiting transgene GFP expression (p < 0.01), suggesting that adenovirus–emulsion complexes are internalized via both clathrin- and caveolae-dependent endocytic mechanisms. It is therefore likely that emulsion complexation of adenoviral vectors provides an additional entry route through

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caveolae-dependent endocytosis, thereby enhancing transgenic expression. Adenoviral docking on CAR mainly uses clathrincoated pit endocytosis route for cell entry, followed by release from endosomes into the cytoplasm and translocation into the nucleus. In contrast, intracellular trafficking of adenovirus that entered cells through caveolin-dependent endocytosis appears to be different, including the formation of mega-caveosome (Rogee et al., 2007). It would be therefore of interest to explore the gene therapy potential of emulsion as a hybrid with adenoviral vector with impaired intracellular trafficking as a result of surface modification.

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3.7. Effect of emulsion complexation on in vivo adenoviral gene transfer

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It is well known that the in vivo fate of drugs or genes encapsulated in or complexed with nanoparticles is quite different from that of freely soluble drugs or genes and is highly dependent on the properties of encapsulating nanoparticles. Following intravenous administration, nanoparticles composed of cationic lipids are known to become entrapped in the pulmonary capillary bed after binding to erythrocytes and serum proteins, resulting in increased accumulation of encapsulated drug in the lung (Schroeder et al., 2012). Earlier works have shown that systemic injection of cationic liposomes as plasmid vehicles led to accumulation and gene expression in lung tissue (Chollet et al., 2002; Herber-Jonat et al., 2011). To investigate how emulsion complexation of adenovirus affects gene expression in various organs, mice were injected with Ad-luc or Ad-luc–emulsion complex via tail veins and luciferase activity was measured in the liver, spleen, lung, heart and kidneys. Twenty four hours after injection with naked adenovirus, the highest levels of luciferase activity were detected in the liver and spleen, followed by lungs and kidneys with the lowest activity measured in the heart. Luciferase activity in the liver and spleen was 4800- and 2967-fold higher than was measured in control mice (PBS injection), but activity was only 209-, 37- and 10-fold higher in the lungs, kidneys and heart (Fig. 7). Increased adenovirus gene expression in the liver and spleen has been consistently reported by other groups (Kim et al., 2012; Singh et al., 2008). Twenty four hours after injection with adenovirus–emulsion complex, the highest luciferase activity was found in the lungs, followed by the spleen and liver with the lowest activity measured in the heart and kidneys. Luciferase activity detected in the lungs of mice receiving the adenovirus–emulsion complex was 12,933-fold higher than in

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Fig. 7. Comparison of transgene expression in various organs of mice following systemic administration of naked adenovirus or adenovirus–emulsion complex. At 24-h postinjection of Ad-luc or Ad-luc–emulsion complex, liver, lung, heart, kidney and spleen were obtained, homogenized and luciferase activity was measured as described in the text. Luciferase activity in each tissue was represented by relative light units (RLU) per milligram of tissue proteins. The data represents the mean  SD (n = 5). Significant differences are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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control mice (PBS injection), whereas activity only increased by 873-, 304-, 4- and 10-fold in the spleen, liver, kidneys and heart. In comparing luciferase activity between mice injected with naked adenovirus and mice injected with the emulsion-complexed adenovirus, the emulsion group had 62-fold greater activity in the lungs (p < 0.0001) and 16-fold and 3-fold lower activity in the liver (p < 0.01) and spleen (p > 0.01), respectively. This indicates that emulsion complexation significantly alters the biodistribution of adenovirus in vivo, greatly enhancing transgene expression in the lung while reducing expression in the liver. Adenovirus accumulation in the liver, primarily mediated by binding to blood coagulation factors during systemic circulation (Yilmazer et al., 2013), causes liver toxicity, limiting the clinical usefulness of these adenoviral vectors (Kim et al., 2012; Singh et al., 2008). This study shows that adenovirus–emulsion complexes decreased transgene expression in the liver, suggesting that this complexation may be a promising strategy for reducing adenoviral accumulation and toxicity in liver tissue. Other studies have also shown reduced adenoviral accumulation in the liver with the use of cationic nanoparticle vehicles (Han et al., 2008; Ma et al., 2002; Singh et al., 2008). Increased transgene expression in the lungs, coupled with reduced expression in the liver, makes the adenovirus–emulsion complex a potential lung-targeted adenoviral gene delivery system. In earlier studies, systemic administration of the adenovirus administered via a liposomal vehicle led to higher adenovirus accumulation in the lungs with no complementary increase in transgene expression in lung tissue (Singh et al., 2008). Singh et al. speculated that the lack of transgene expression was due to short-term or transient residence of the adenovirus– liposome complex in the pulmonary vasculature. The adenovirus– laden emulsion vehicles studied here may offer longer residence time in lung capillary beds by increasing direct cellular-membrane binding in pulmonary tissue. Caveolae, flask-shaped invaginations of the cell membrane composed of membrane microdomains rich in cholesterol and glycosphingolipids, are particularly abundant in endothelial cells (Conner and Schmid, 2003). Increased complex binding and uptake to pulmonary endothelial cells through caveolin-dependent endocytosis may contribute to increased adenoviral gene expression in the lungs following emulsion complex administration.

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4. Conclusion

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The present study explored the potential of a cationic lipid (DOTAP)-based emulsion as a vehicle for improved adenoviralmediated gene transfer. Formulation optimization identified DOTAP/CHOL/PEG-PE emulsions containing a Lipiodol oil core as a gene transfer vehicle that significantly enhanced adenovirus transfer efficiency from among a variety of potential candidates. The data suggest that Lipiodol-containing DOTAP/CHOL/PEG-PE emulsions form complexes with adenovirus particles through more stronger electrostatic interaction, providing increased cellular uptake through an additional cellular entry mechanism, namely caveolae-dependent endocytosis. Furthermore, systemic administration of adenovirus–emulsion complexation enhanced transgene expression in the lungs while reducing expression in the liver, implying that emulsion complexation may be a promising strategy for overcoming the hurdles that limit safe and efficient application of recombinant adenoviruses in gene therapy. This emulsion formulation represents a cost-effective method to enhance transgene expression without expensive adenovirus modifications, which is practical for a variety of different adenoviruses and may also have other applications in addition to gene therapy.

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Acknowledgements

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This work was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2012R1A1A2042768) and the Global Core Research Center (GCRC) grant (No. 20110030678).

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Please cite this article in press as: Kim, S.-Y., et al., Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.024

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