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Published online before print July 20, 2006, 10.1148/radiol.2403051261
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(Radiology 2006;240:771-777.)
© RSNA, 2006

Molecular Imaging

Intraarterial Gene Delivery in Rabbit Hepatic Tumors: Transfection with Nonviral Vector by Using Iodized Oil Emulsion1

Young Il Kim, MD, Jin Wook Chung, MD, Jae Hyung Park, MD, Joon Koo Han, MD, Jung Wan Hong, MA and Hesson Chung, PhD

1 From the Department of Radiology and Center for Liver Cancer, Research Institute and Hospital, National Cancer Center, Gyeonggi-do, Korea (Y.I.K.); Department of Radiology and Institute of Radiation Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea (J.W.C., J.H.P., J.K.H.); and Biomedical Research Center, Korea Institute of Science and Technology, Seoul, Korea (J.W.H., H.C.). From the 2002 RSNA Annual Meeting. Received July 27, 2005; revision requested September 30; revision received November 25; final version accepted January 2, 2006. Address correspondence to J.W.C. (e-mail: chungjw{at}radcom.snu.ac.kr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To evaluate the feasibility of an iodized oil emulsion that is used for the chemoembolization of hepatocellular carcinoma as a modifier of a nonviral gene transfer system for intraarterial gene delivery in experimentally induced hepatic tumors.

Materials and Methods: Experiments were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the animal research committee at Seoul National University Hospital. VX2 carcinoma was implanted into the liver of 26 rabbits. Four nonviral gene transfer systems were prepared by using pCMV-luc+ as a reporter gene. The first system consisted of a DNA and polyethylenimine (PEI) complex (n = 7); the second, of a DNA and PEI complex mixed with iopamidol and iodized oil (n = 7); the third, of a DNA and PEI complex mixed with iopamidol (n = 7); and the fourth, of a DNA and PEI complex mixed with iodized oil (n = 5). For the DNA and PEI complex that was mixed with iopamidol and iodized oil, iopamidol was used to stabilize the emulsion. Twenty days after tumor implantation, intraarterial gene delivery was performed by selective catheterization of the hepatic artery. Rabbits were euthanized 24 hours after gene delivery. Luciferase activity was assayed in the tumor, left hepatic lobe, right hepatic lobe, and other organs and was statistically analyzed for comparison between complexes by using the Kruskal-Wallis test.

Results: Luciferase activity in the tumor was significantly higher for the group that received DNA, PEI, iopamidol, and iodized oil than for any other group (Kruskal-Wallis test, P < .05). Luciferase activity in the left hepatic lobe, right hepatic lobe, and other organs was not significantly different between complexes. Selective gene expression in tumor cells was confirmed by means of immunohistochemical analysis for luciferase.

Conclusion: It is feasible to use an iodized oil emulsion system for the intratumoral transfection of nonviral vectors in experimentally induced hypervascular hepatic tumors.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
The treatment of liver cancer, especially advanced hepatocellular carcinoma (HCC), remains difficult. Curative surgical resection is possible in only one-third of patients because of advanced tumor extension and associated liver cirrhosis at initial presentation (1,2). Currently, other available treatment options include systemic or intraarterial administration of chemotherapeutic agents, transcatheter arterial chemoembolization (TACE), local ablation therapy, and radiation therapy. The overall clinical outcome, however, is not promising.

The transcatheter approach for the treatment of HCC is based on the physiologic background that the blood supply for HCC is obtained from the hepatic artery, while that for the normal liver parenchyma is obtained mainly from the portal vein. TACE was reported by Doyon et al (3). In the early 1980s, iodized oil (Lipiodol; Andre Guerbet, Aulnay-sous-Bois, France), a lymphangiographic dye, was found to remain selectively in the neovasculature and extravascular spaces of HCC when injected into the hepatic artery (4,5). Thereafter, TACE with iodized oil emulsion and various anticancer drugs has been used as a standard treatment for unresectable or postoperatively recurrent HCC and as an alternative to surgery, even for resectable tumors (6,7).

Multiplicity of tumor foci and associated advanced liver cirrhosis, however, still make HCC difficult to treat. Thus, investigators have evaluated other therapeutic approaches, one of which is gene therapy (8). For gene therapy, viral and nonviral vectors are the two main strategies for gene delivery. The main advantage of viral vectors is that they are generally efficient in terms of the number of transfected cells. Viral vectors, however, can provoke immune and/or toxic reactions. Because nonviral vectors induce minimal host immune responses, they have been increasingly proposed as a safer alternative to viral vectors (9,10). Nonviral vectors, however, have much lower transfection efficiency than do viral vectors. To overcome this problem, local delivery is suggested as a way to increase the direct uptake of DNA and vector complexes into the target tissue. For hepatic tumors, local delivery would be performed via the hepatic artery.

For TACE, the iodized oil functions as a contrast agent, as a vehicle for chemotherapeutic agents, and as an embolic agent. For this experiment, we hypothesized that an iodized oil emulsion might improve the transfection efficiency of nonviral vectors in hypervascular hepatic tumors, as is similarly achieved during chemoembolization. Thus, the purpose of our study was to evaluate the feasibility of using an iodized oil emulsion that is used for the chemoembolization of HCC as a modifier of a nonviral gene transfer system for intraarterial gene delivery in experimentally induced hepatic tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Components of Gene Delivery Systems
The reporter gene, pCMV-luc+, consisted of a cytosolic form of firefly (Phontinus pyralis) luciferase complementary DNA that was derived from pGL3 (Promega, Madison, Wis) by using XbaI and HindIII restriction and was subcloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, Calif). These plasmids were amplified in the Escherichia coli DH5-{alpha} strain and were purified by using a mega kit (Plasmid Mega Kit; Qiagen, Chatsworth, Calif) according to the manufacturer's instruction.

Polyethylenimine (PEI) 25 kDa (Aldrich, St Louis, Mo) was used as a nonviral vector. PEI stock solution was prepared by mixing 1 mg PEI in 1 mL water and was stored at 4°C.

In preparing the iodized oil emulsion, iodized oil (Lipiodol; Laboratories Guebet) was used as the oil phase. The iodized oil that was used in our experiments was composed of an iodinated ethyl ester of poppyseed oil consisting of mono-, di-, and triiodinated ethyl esters of linoleic (73%), oleic (14%), palmitic (9%) and stearic (3%) acids, with an iodine content of 38% by weight.

To adjust the specific gravity of the inner aqueous phase so that it was equivalent to that of the iodized oil, a water-soluble nonionic contrast medium (iopamidol, Iopamiro 300; Bracco Industria Chimica, Milano, Italy) was used as the aqueous phase for the iodized oil emulsion.

In Vitro Experiment to Determine Stabilizing Effect of Iopamidol
The state of the DNA, PEI, and iodized oil complex was compared with that of the DNA, PEI, iopamidol, and iodized oil complex to investigate the effect of iopamidol as a stabilizing agent (Y.I.K., J.W.H., H.C.). Both complexes were made according to the conventional pumping method (11) and were put into a slide glass bottle separately. The appearance of each complex was examined immediately after mixing and 30 minutes later; phase characteristics of the complexes were determined by using an optical microscope (Olympus, Tokyo, Japan).

Transarterial Gene Delivery in Rabbits
The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, as approved by the National Research Council of the National Institutes of Health and by the committee on the care and use of animals in research at Seoul National University Hospital.

A total of 28 adult New Zealand white rabbits weighing 2.5–3.0 kg were used (Fig 1). The animals were divided into four groups according to delivery complex (Fig 2), and seven rabbits were allocated to each group. Group 1 received the DNA and PEI complex alone; group 2 received the DNA and PEI complex mixed with iopamidol and iodized oil; group 3 received the DNA and PEI complex mixed with iopamidol; and group 4 received the DNA and PEI complex mixed with iodized oil. Two rabbits in group 4 died during the experiment; one died of hepatic artery dissection during catheterization, and the other died during induction of general anesthesia.


Figure 1
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  		Figure 1: Flow chart of the experiment.

 

Figure 2
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  		Figure 2: Diagram shows experimental groups according to DNA vector complexes that were used for intraarterial gene delivery.

 
To identify the role of iopamidol or iodized oil when used separately, the groups that received the DNA and PEI complex mixed with either iopamidol or iodized oil (groups 3 and 4, respectively) were included in the experiment. When making the DNA, PEI, iopamidol, and iodized oil complex, iopamidol was first added to the DNA and PEI complex, which was then mixed with iodized oil by using the conventional pumping method. The VX2 strain had been maintained by successive transplantation into the hind limb of a carrier rabbit. The animals were anesthetized with an intramuscular injection of a mixed solution of ketamine hydrochloride (Ketalar; Yuhan Yanghang, Seoul, Korea) and 2% xylazine hydrochloride (Rompun; Bayer, Seoul, Korea), and 0.5 mL/kg of the mixed solution was administered to each rabbit. Tumor implantation was performed by using an aseptic technique. The rabbits underwent laparotomy through a subxyphoid abdominal incision, and a total of 0.1 mL of minced tumor suspension was implanted with a 20-gauge needle at the lateral segment of the left lobe of the liver (Y.I.K., J.W.C.).

Follow-up spiral computed tomography (CT) (Somatom Plus 4; Siemens, Erlangen, Germany) was performed to document the formation of hepatic tumor 20 days after implantation (Fig 3a). CT was performed with animals in the supine position and covered the entire liver (3-mm collimation, pitch of 1.5, and 1-mm reconstruction interval). For contrast material–enhanced CT, 13 mL of contrast medium was injected at a rate of 0.5 mL/sec through the auricular vein. By using a bolus tracking technique, arterial and portal phase scans were obtained with a 16-second interval. Spiral CT was also used to measure the volume (V) of each tumor, which was calculated according to the equation V = L x S2/2, where L is the longest diameter of the tumor and S the shortest diameter of the tumor (12).


Figure 3
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  		Figure 3a: CT and microangiographic images of implanted VX2 carcinoma before and after delivery of DNA, PEI, iopamidol, and iodized oil complex. (a) Transverse CT scan obtained during hepatic arterial phase shows round enhancing mass (arrow) in left lobe of liver. (b) Common hepatic arteriograph with microcatheter reveals hypervascular mass (arrow) in left lobe of liver. (c) Postdelivery spot arteriograph shows iodized oil retention around tumor (arrow).

 
Because the effectiveness of gene delivery can be influenced by tumor volume, rabbits with similar-sized tumors were evenly distributed between groups. The mean tumor volume was 3332.6 mm3 ± 331.3(± standard deviation) in group 1, 3201.6 mm3 ± 458.09 in group 2, 3270.4 mm3 ± 361.6 in group 3, and 3406.0 mm3 ± 522.2 in group 4. No statistically significant difference was noted between groups by using the Kruskal-Wallis test (P = .759).

The gene delivery complexes were prepared by mixing 100 µL of pCMV-luc+ that contained firefly luciferase complementary DNA driven by the cytomegalovirus promoter with an appropriate amount of carrier, PEI (100 µL in 1 mg/mL of PEI stock solution), and iopamidol (100 µL). The water-in-oil emulsion of iodized oil (300 µL) and the DNA, PEI, and iopamidol complex (300 µL) were made by using the conventional pumping method (11).

One day after CT, intraarterial gene delivery was performed by using fluoroscopic guidance (Y.I.K., J.W.C., J.H.P.). While the animals were anesthetized as previously described, an incision was made to expose the right femoral artery, and a 5-F dilator was inserted into the artery as a substitute for an arterial sheath. To evaluate arterial anatomy and tumor vascularity, celiac angiography was performed by using a 3-F microcatheter (Microferret; Cook, Bloomington, Ind) and 3 mL of contrast medium (Pamiray, Dongkook Pharmaceutical, Seoul, Korea), which was injected at a rate of 0.5 mL/sec. The left hepatic artery that fed the tumor was selectively catheterized with a 3-F microcatheter. When the catheter was advanced to an adequate position that was as close to the implanted tumor as was possible within the left hepatic artery, the gene delivery complex was carefully injected, avoiding efflux out of the artery (Fig 3b, 3c). The catheter was then removed, and the femoral artery was ligated. The surgical wound was closed to complete gene delivery. All the procedures were performed aseptically.


Figure 3
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  		Figure 3b: CT and microangiographic images of implanted VX2 carcinoma before and after delivery of DNA, PEI, iopamidol, and iodized oil complex. (a) Transverse CT scan obtained during hepatic arterial phase shows round enhancing mass (arrow) in left lobe of liver. (b) Common hepatic arteriograph with microcatheter reveals hypervascular mass (arrow) in left lobe of liver. (c) Postdelivery spot arteriograph shows iodized oil retention around tumor (arrow).

 

Figure 3
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  		Figure 3c: CT and microangiographic images of implanted VX2 carcinoma before and after delivery of DNA, PEI, iopamidol, and iodized oil complex. (a) Transverse CT scan obtained during hepatic arterial phase shows round enhancing mass (arrow) in left lobe of liver. (b) Common hepatic arteriograph with microcatheter reveals hypervascular mass (arrow) in left lobe of liver. (c) Postdelivery spot arteriograph shows iodized oil retention around tumor (arrow).

 
Transgene Expression
One day after gene delivery, all rabbits were euthanized by using a rapid intravenous injection of pentothal sodium (5 mL). The tumor, left hepatic lobe, right hepatic lobe, and other organs (spleen, kidney, heart, and lung) were harvested, and each was homogenized in a high-speed homogenizer (T-25-Ultra-Turrax; Janke & Kunkel, Staufen, Germany) with lysis buffer (0.1-mol/L Tris-HCL, 2-mmol/L ethylenediaminetetraacetic acid, and 0.1% Triton X-100 [Sigma, St Louis, Mo]). The lysis buffer had a pH of 7.8, and the entire amount of lysis buffer for each collected organ was 5 µL/mg. The amount of lysis buffer was 5 mL per milligram of tissue. The homogenized organ lysates were centrifuged at 4°C for 15 minutes at 14 000 rpm in an Eppendorf centrifuge (micro-17R; Hanil Science Industrial, Incheon, Korea). A portion of the supernatant was assayed for protein concentration by using a protein assay kit (Bio-Rad Laboratories, Hercules, Calif). The amount of luciferase activity in the lysates was quantified by using a kit (Promega, Madison, Wis) and a luminometer (Model TD-20/20; Turner Designs, Sunnyvale, Calif). Peak light emission was measured for 10 seconds at room temperature. Conversion from relative light units to luciferase protein mass was calculated from a standard curve by using the following equation: picograms of luciferase = –0.267 + 236.993 x relative light unit. For this equation, purified firefly luciferase (Calbiochem, San Diego, Calif) was used as the reference standard.

To localize transgene expression in tissues, pCMV-luc+ genes were delivered to the hepatic tumor (n = 2) by using the DNA, PEI, iopamidol, and iodized oil complex in the same way as mentioned above (Y.I.K.). One day after gene delivery, tumors were dissected, frozen in optimal cutting temperature embedding compound (Tissue-Tek; IMEB, San Marcos, Calif), and stored at –70°C. Cryosections measuring 4 µm in thickness were sampled and placed on silane-coated glass slides, which were fixed with cold acetone for 5 minutes at –20°C. After being treated with a mixed solution of methanol and H2O2 (7:3) for 5 minutes, the slides were washed with Tris-buffered saline and Tween 20 (Lab Vision, Fremont, Calif). The samples were incubated for 1 hour with a mouse monoclonal antiluciferase antibody (Calbiochem), which was used at a 1:25 dilution. Next, the samples were incubated by using a secondary goat antimouse antibody (Dako, Carpinteria, Calif) for 40 minutes. The luciferase protein was detected during immunoperoxidase staining with 3,3'-diaminobenzidine tetrahydrochloride (Dako), and the slides were counterstained with Mayer hematoxylin. Tissue sections were observed to determine the location of luciferase expression by using light microscopy (Olympus, Tokyo, Japan) (Y.I.K., J.K.H., J.W.H.). Negative controls were obtained by omitting the primary antibody.

Statistical Analysis
Statistical analysis for the comparison of luciferase activity among experimental groups was performed by using the Kruskal-Wallis test (SPSS, version 11.0 for Windows; SPSS, Chicago, Ill). A P value of less than .05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
When the appearance of DNA, PEI, and iodized oil was compared with that of DNA, PEI, iopamidol, and iodized oil, the former was separated into two phases immediately after the pumping preparation. The complex that was composed of DNA, PEI, and iodized oil was separated into two phase immediately after the pumping preparation. However, the complex that was composed of DNA, PEI, iopamidol, and iodized oil showed a milky appearance, without phase separation. Optical microscopy (Fig 4) demonstrated no emulsion formation in the DNA, PEI, and iodized oil complex, but fragmented drops of various sizes, which represented emulsion formation, were scattered throughout the solution that contained DNA, PEI, iopamidol, and iodized oil.


Figure 4
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  		Figure 4a: Optical microscopic images of (a) DNA, PEI, and iodized oil and (b) DNA, PEI, iopamidol, and iodized oil 30 minutes after pumping. No emulsion formation is seen in a, whereas b shows various sized droplets, which represent a water-in-oil–type emulsion. (Original magnification, x100.)

 

Figure 4
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  		Figure 4b: Optical microscopic images of (a) DNA, PEI, and iodized oil and (b) DNA, PEI, iopamidol, and iodized oil 30 minutes after pumping. No emulsion formation is seen in a, whereas b shows various sized droplets, which represent a water-in-oil–type emulsion. (Original magnification, x100.)

 
The amount of luciferase activity (Fig 5) in the tumors was 1.57 pg per milligram total protein ± 2.17 for the group that received DNA and PEI; 129 pg per milligram total protein ± 74.9 for the group that received DNA, PEI, iopamidol, and iodized oil; 4.84 pg per milligram total protein ± 4.58 for the group that received DNA, PEI, and iopamidol; and 2.60 pg per milligram total protein ± 1.31 for the group that received DNA, PEI, and iodized oil.


Figure 5
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  		Figure 5: Luciferase expression is shown for each complex. Note that tumoral luciferase activity is highest in the group that was administered DNA, PEI, iopamidol, and iodized oil. DP = DNA and PEI; DPI = DNA, PEI, and iopamidol; DPIIO = DNA, PEI, iopamidol, and iodized oil; DPIO = DNA, PEI, and iodized oil; error bars = standard deviation.

 
Tumoral luciferase expression was higher in the group that received DNA, PEI, iopamidol, and iodized oil than in any other group, and the increase in luciferase activity was determined to be significant by using the Kruskal-Wallis test (P = .001). In the group that received DNA, PEI, iopamidol, and iodized oil, luciferase expression was higher in the tumor than in other organs, which suggests that the gene stayed mostly in the tumor and was not transferred to other organs. In the group that received DNA, PEI, and iopamidol, the highest level of gene expression was not observed in the tumor, and the difference between the tumor and other organs were not significant. The group that received DNA, PEI, and iodized oil showed very low transfection efficiency in all organs and tumor tissues, which suggests that iodized oil alone has no advantage in improving transfection efficiency. Therefore, iodized oil should be emulsified by using iopamidol, as is usual for TACE.

The amount of luciferase activity in left hepatic lobe that surrounded the tumor was 4.3 pg per milligram total protein ± 4.48 in the group that received DNA and PEI; 16.9 pg per milligram total protein ± 14.6 in the group that received DNA, PEI, iopamidol, and iodized oil; 15.1 pg per milligram total protein ± 15.9 in the group that received DNA, PEI, and iopamidol; and 2.44 pg per milligram total protein ± 1.5 in the group that received DNA, PEI, and iodized oil. Thus, the groups that received the DNA and PEI complex mixed with either iopamidol and iodized oil or iopamidol alone showed slightly higher luciferase activity in the left hepatic lobe than did those that received the other complexes, but there was no statistical significance. Luciferase activity in the right hepatic lobe and in other organs was very low and showed no significant difference between complexes.

Because luciferase activity in the tumor was higher for the group that received DNA, PEI, iopamidol, and iodized oil than for any other group, we repeated intraarterial delivery by using DNA, PEI, iopamidol, and iodized oil in two additional rabbits and performed luciferase immunohistochemical analysis to localize transgene expression. In both animals, the immunohistochemical activity of luciferase was concentrated in an area of the tumor cells rather than in the normal hepatocytes (Fig 6).


Figure 6
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  		Figure 6a: Immunohistochemical stains of VX2 carcinoma show reporter gene expression after gene transfection with DNA, PEI, iopamidol, and iodized oil. Brown color indicates active luciferase expression. Images obtained at (a) x12.5 and (b) x100 magnification show that luciferase activity is concentrated in tumor area. (c) Image obtained by using a high-powered light microscope (x400) reveals strong activity of luciferase in tumor area, whereas little activity is seen in (d) the surrounding normal parenchyma.

 

Figure 6
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  		Figure 6b: Immunohistochemical stains of VX2 carcinoma show reporter gene expression after gene transfection with DNA, PEI, iopamidol, and iodized oil. Brown color indicates active luciferase expression. Images obtained at (a) x12.5 and (b) x100 magnification show that luciferase activity is concentrated in tumor area. (c) Image obtained by using a high-powered light microscope (x400) reveals strong activity of luciferase in tumor area, whereas little activity is seen in (d) the surrounding normal parenchyma.

 

Figure 6
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  		Figure 6c: Immunohistochemical stains of VX2 carcinoma show reporter gene expression after gene transfection with DNA, PEI, iopamidol, and iodized oil. Brown color indicates active luciferase expression. Images obtained at (a) x12.5 and (b) x100 magnification show that luciferase activity is concentrated in tumor area. (c) Image obtained by using a high-powered light microscope (x400) reveals strong activity of luciferase in tumor area, whereas little activity is seen in (d) the surrounding normal parenchyma.

 

Figure 6
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  		Figure 6d: Immunohistochemical stains of VX2 carcinoma show reporter gene expression after gene transfection with DNA, PEI, iopamidol, and iodized oil. Brown color indicates active luciferase expression. Images obtained at (a) x12.5 and (b) x100 magnification show that luciferase activity is concentrated in tumor area. (c) Image obtained by using a high-powered light microscope (x400) reveals strong activity of luciferase in tumor area, whereas little activity is seen in (d) the surrounding normal parenchyma.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
An ideal gene delivery system has to fulfill several properties. First, because of biophysical requirements and anatomic constraints (such as migration through blood capillaries, extravasation through small vascular fenestrations, and diffusion into tissue and cellular uptake), the particles carrying the DNA should be small and should not have a tendency to aggregate during in vivo application.

Second, the nonspecific environment that the DNA carrier has to pass through should be minimal. For intravenous administrations, undesirable interactions between vectors and blood components or nontarget cells, such as those in the reticuloendothelial system, are important obstacles for targeting genes in vivo (13). Therefore, it would be desirable to administer the vector in a way that avoids the obstacles that can be expected with systemic delivery, and intraarterial delivery might help to overcome these problems.

Local delivery increases the direct uptake of the DNA carrier complex into the target tissue compared with systemic delivery, which induces more loss of the DNA carrier complex into the systemic circulation. Furthermore, though the intravenously infused genes enter the liver efficiently, it has been shown that most of them are completely trapped by Kupffer cells, thereby making it impossible to direct a gene to hepatocytes or tumor cells (14).

To meet the requirement of a nonviral gene carrier, the carrier must be stable and have a high positive surface charge to bind with DNA, which has a negative net charge. Compared with other nonviral gene delivery systems (especially liposomal or cationic lipid systems), cationic polymers have been shown to be promising carriers because of their better chemical stability (15). PEI, one of the most successful polymers used in gene delivery research today, can form small complexes with DNA to enhance transfection efficiency (16); this is because PEI exhibits the highest positive charge density when fully protonated to neutralize the negative charge of the DNA and condense its structure (17–19). Thus, PEI protects DNA from nuclease degradation (20), and the DNA and PEI complex interacts with negatively charged plasma membranes fast enough to be found in endosomes only 2 hours after exposure (16).

Several water-soluble contrast agents have been used to dissolve chemotherapeutic drugs and to adjust the specific gravity of the inner aqueous phase so that it is equivalent to that of iodized oil during TACE. Iopamidol, a nonionic water-soluble contrast medium, was used to adjust the specific gravity of the inner aqueous phase in our study, as is similarly achieved during TACE. The DNA, PEI, and iodized oil complex showed immediate phase separation after pumping preparation but had no gain in transfection efficiency compared with the DNA and PEI complex. These results are the same as those that can be expected from the typical clinical use of water-soluble contrast agents to adjust the specific gravity of the inner aqueous phase so that it is equivalent to that of iodized oil. Nakamura et al (21) reported that an emulsion of water in iodized oil can be stabilized by adding water-soluble contrast agents to chemotherapeutic drugs.

For more than a decade, much attention has been given to iodized oil–based TACE for the treatment of HCC. When injected into the hepatic artery by using a vascular catheter, iodized oil is retained in HCC for several weeks to more than a year but is cleared from normal liver parenchyma within 7 days (22). Iodized oil has three functions. First, it acts as a contrast medium, thereby facilitating the visualization of the tumors during TACE. Second, it slows arterial circulation by acting as an embolic agent, thereby blocking the blood supply to the HCC effectively. Third, it shows preferential uptake into tumor tissue and can be used as a vehicle for the targeted delivery of cytotoxic or radiotherapeutic drugs (23).

In this study, we attempted to develop an iodized oil emulsion that remained in the tumor for a long period; released the DNA, PEI, and iopamidol complex in a sustained release pattern; and minimized the environment that could destabilize the vector. The DNA, PEI, and iopamidol complex was transferred successfully to the hepatic VX2 carcinoma when the complex resided in the water phase of the water-in-oil emulsion that was formed by the iodized oil. Adding iodized oil to the DNA, PEI, and iopamidol complex improved the effective transfection in the target tumor and its neighboring cells, and the highest level of gene expression was observed in the VX2 carcinoma compared with other organs.

Slow arterial flows that are induced by the iodized oil might increase the contact time between the infusate and the tumor. Localized ischemia that results from the embolizing effect of the iodized oil might also induce tumor vessels to be more permeable to gene vector complexes, which can easily access the tumor cells. An emulsion system made from iodized oil could help to localize the tumor inside the liver and to release DNA or a DNA carrier complex in a sustained fashion to the target tumors.

Our study had some limitations. First, we could not show what percentage of cancer cells were transfected. To assay luciferase activity, we harvested and homogenized the whole tissues containing tumor cells that were transfected or not transfected by genes. It is also true that parts of a tumor supplied by a different arterial branch can be missed. The rate of tranfection is very important in realizing the clinical efficacy of the gene delivery system. To overcome these problems, nonviral vectors like the gene delivery systems used in our study might be administered repeatedly, with minimal host immune responses which have a concern with viral vectors. Second, we could not predict changes in luciferase activity with the lapse of time. Considering the fact that the iodized oil emulsion preferentially resides in tumor tissues and releases infusates slowly, we should have studied the changes in luciferase activity over time. To do so, however, would have required many more animals. Other methods for assaying luciferase activity over time are needed. Optical in vivo imaging systems that can acquire and quantify bioluminescence data may be a solution.

There are a variety of vectors that differ in their composition, tissue specificity, and transfection efficiency. This variety suggests that there is not a single ideal vector that can be applied to all tissues and all diseases. On the basis of the tumor-specific physiologic background and established therapeutic methods, we developed DNA carrier complexes, by using iodized oil and iopamidol, that may be used as modifiers for intraarterial gene delivery.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: HCC = hepatocellular carcinoma • PEI = polyethylenimine • TACE = transcatheter arterial chemoembolization

Authors stated no financial relationship to disclose.

See also Science to Practice in this issue.

Author contributions: Guarantors of integrity of entire study, all authors; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, Y.I.K.; experimental studies, Y.I.K., J.W.C., J.H.P., J.W.H., H.C.; statistical analysis, Y.I.K., J.W.C., J.K.H., J.W.H.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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