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Clinical Cancer Research Vol. 7, 3606-3612, November 2001
© 2001 American Association for Cancer Research

Regular Articles

Tumor Targeting and Imaging of Intraperitoneal Tumors by Use of Antisense Oligo-DNA Complexed with Dendrimers and/or Avidin in Mice1

Noriko Sato, Hisataka Kobayashi2,3, Tsuneo Saga, Yuji Nakamoto, Takayoshi Ishimori, Kaori Togashi, Yasuhisa Fujibayashi, Junji Konishi and Martin W. Brechbiel

Department of Nuclear Medicine and Diagnostic Imaging [N. S., T. S., Y. N., T. I., J. K.], and Hitachi Medical Co. chaired Department of Diagnostic and Interventional Imagiology [H. K., K. T.], Kyoto University, Kyoto 606-8507, Japan; Molecular Imaging Division, Biomedical Imaging Research Center, Fukui Medical University, Fukui 910-1193, Japan [Y. F.]; and Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, MD 20892 [M. W. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To establish an effective nonviral gene delivery and a corresponding imaging method for i.p.-disseminated tumors, various oligonucleotide-carrier complexes were synthesized, and their in vitro and in vivo properties were examined.

The 20-mer multiamino-linked oligonucleotide (oligo), synthesized as antisense against the c-erbB-2 sequence, and the 3'-biotinylated form of the same oligonucleotide (oligo-Bt) were 111In labeled through a diethylenetriaminepentaacetic acid chelate. 111In-oligo was mixed with generation 4 polyamidoamine dendrimer (G4) or with biotinylated G4 (G4-Bt), which are positively charged to form electrostatic complexes. 111In-oligo/G4-Bt and 111In-oligo-Bt were conjugated to avidin (111In-oligo/G4-Av and 111In-oligo-Av, respectively). 111In-oligo/G4, 111In-oligo/G4-Av, 111In-oligo-Av, and carrier-free 111In-oligo (2.96 kBq/22.4–45.9 ng of oligo) were examined for internalization in vitro in human ovarian cancer cells (SHIN3). Biodistribution of 111In-oligo-carrier complexes or 111In-oligo was examined in normal (n = 4–7) or i.p. SHIN3 tumor-bearing (n = 6–10) mice 2–24 h after i.p. injection (74 kBq/125–300 ng). Scintigraphy of i.p. tumor-bearing and normal mice was performed at various times postinjection of 111In-oligo-carrier complex or 111In-oligo (1.85 MBq/2.2 ng).

111In-oligo-carrier complexes bound to the tumor cells were internalized at a rate of 34–56% at 24 h. In vivo, G4, G4-Av, and Av significantly enhanced tumor delivery of 111In-oligo [9.1, 14.5, and 24.4% of injected dose per g of tissue (ID/g) at 24 h; P < 0.05, < 0.01, and < 0.0001, respectively] compared with delivery without carrier (0.8% ID/g). Scintigrams of 111In-oligo delivered to the i.p.-disseminated tumors by the carriers were successfully obtained.

In conclusion, G4, G4-Av, and Av can effectively deliver 111In-oligo to i.p.-disseminated tumors. 111In-oligo-carrier complexes also have potential as tracers for imaging and monitoring of gene delivery.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonviral gene transfection systems, which can carry both oligonucleotides and plasmid DNA (1) , have an advantage of lower immunogenicity when compared with viral systems (2) . A number of nonviral carriers for gene transfer have been synthesized, including polylysines (3) , cationic liposomes (4) , polypeptides (5 , 6) , recombinant histones (7) , and other reagents (8, 9, 10) . However, the transfection/expression efficiency of these nonviral systems remains insufficient, especially in the presence of serum (11) .

Recently, starburst polyamidoamine dendrimers have emerged as a novel synthetic gene carrier (12 , 13) . Dendrimers are highly branched spherical polymers that have a unique surface of primary amino groups with high solubility in water (13) . Because of the high number of positive charges on their surfaces, dendrimers form stable electrostatic complexes with negatively charged nucleic acids in buffer at physiological pH. Dendrimers facilitate gene transfer to various cell lines with a higher efficiency than polylysines and are also less cytotoxic (12 , 14 , 15) . Moreover, dendrimers are efficient in delivering oligonucleotides even in the presence of high concentrations of serum proteins by protecting the oligonucleotides from the degradation by exonuclease (15) . Studies of gene/antisense therapy using dendrimers as a vector have been actively carried out, and expression of reporter genes was reported (2 , 12 , 14, 15, 16, 17, 18, 19, 20) . However, the behavior of the dendrimer-nucleotide complexes in a biological environment and the mechanism of dendrimer-mediated cellular uptake of oligonucleotides have not been clarified. A mechanism similar to that proposed for cationic lipid complexes (2 , 21) has been suggested. The DNA-dendrimer complex that possesses a net positive charge (17) binds to negatively charged receptors such as sialylated glycoproteins on the cell surface. After internalization, dendrimers buffer the endosome and inhibit pH-dependent lysosomal nucleases; at the same time, the electrostatic bonding between oligonucleotide and dendrimer is weakened by the low pH environment in the lysosome, which allows migration of intact DNA to the nucleus (2) .

Av,4 a natural polycationic protein of egg white, has been used as a nonviral carrier of DNA through the biotin-Av system and was reported to introduce a high internalization (transfection) rate in vitro by absorption-mediated endocytosis (22) . Endocytosis of Av into the liver and tumor cells was accelerated by its highly glycosylated side chains (23 , 24) . Although the mechanism of this Av-mediated cellular uptake of oligonucleotides is also not fully understood, Av is known to show a high accumulation in tumors when injected i.p. into mice bearing i.p.-disseminated adenocarcinoma tumors (25) .

Peritonitis carcinomatosa is a late manifestation of gastrointestinal and gynecological malignancies, and therapeutic methods for the widely spread tumors in the peritoneal cavity are limited to i.v. or i.p. chemotherapy, which produce unsatisfactory results. Gene/antisense therapy is one of the anticipated therapies for peritonitis carcinomatosa, and experimental studies have been carried out by several investigators (26, 27, 28) .

In the present study, to establish effective nonviral gene transfer carriers for i.p.-disseminated tumors, three antisense oligonucleotide-carrier complexes were synthesized, using a dendrimer and/or the biotin-Av system, and their tumor targeting abilities were examined in vitro and in vivo. The word "targeting" has been used with two different meanings among researchers in different fields: gene targeting in molecular biology and organ/tissue targeting in pharmacology. In the present study, we focused on developing new gene carriers that deliver the gene to the tumor in vivo. The properties of these synthesized oligonucleotide-carrier complexes were examined in terms of tumor targeting, not gene targeting. Furthermore, to examine the potential of the carriers themselves, we used an antisense oligonucleotide and tumor cells that are not specific to each other in our experiments. Thus, we chose an antisense oligonucleotide for the primer of c-erbB-2, which is well known to be related to carcinogenesis of various cancers, and for tumor cells, we chose an ovarian adenocarcinoma cell line that often causes peritonitis carcinomatosa and is also c-erbB-2 negative. In addition, scintigraphic imaging of the i.p. tumor-bearing mice that received injections of the synthesized complexes was also performed. Imaging of the gene/antisense delivery to the targeted tumors or organs may be a promising method for monitoring a gene/antisense therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antisense Oligonucleotides.
The 20-mer antisense oligonucleotide 5'-CCAGGUCCAUGGUGGUCACT-3' [where U indicates the substitution of dT with amino-linked dU (Mr 5418); hereafter designated "oligo"] synthesized based on the c-erbB-2 sequence, and the 3'-biotinylated version of the same internally amino-linked oligonucleotide (oligo-Bt) were purchased from JENSET (Paris, France). c-erbB-2 was used because it is a well-known oncogene that is related to many kinds of cancers.

Conjugation to Chelate and 111In Labeling of Oligonucleotides.
The diethylenetriaminepentaacetic acid chelate, 1B4M, was used to label oligonucleotides with 111In, according to the method published previously (29) . Briefly, for the conjugation of chelate with oligonucleotides, 78 nmol of oligo or oligo-Bt was dissolved in H2O (1.0 mM) and mixed with 11.8 mg of Na3PO4. The mixture was then incubated overnight with 3.14 µmol of 1B4M at 37°C. The conjugation ratio of 1B4M to both oligo and oligo-Bt was calculated to be 2.0, based on incubation of 4 µl of the mixture with 10 µl of 111InCl3 (74 MBq/ml in 0.01 M HCl) and 50 µl of a 0.1 M ammonium citrate solution (pH 5.0) for 1 h at room temperature, followed by high-performance liquid chromatography or Sephadex G-25 gel filtration (Pharmacia, Piscataway, NJ). The reaction mixture was diluted to 1 ml with H2O and applied to a Sep-Pak C18 cartridge (Waters, Milford, MA) pretreated with 5 ml of acetonitrile, 10 ml of H2O, and 10 ml of 0.15 M NaCl. The eluate was reapplied to the cartridge twice. After the cartridge was washed with 20 ml of H2O, the oligo fraction was eluted with 3 ml of 30% acetonitrile (v/v) in H2O.

The oligo or oligo-Bt solution (150 µl) was mixed with 450 µl of a 0.1 M ammonium citrate solution (pH 5.0) and then incubated with 11.1 MBq (300 µCi) of 111InCl3 (74 MBq/ml in 0.01 M HCl) for 1 h at room temperature. Labeling yields, determined by high-performance liquid chromatography or Sep-Pak C18 cartridge were >90% for 111In-oligo and >70% for 111In-oligo-Bt. The specific activities of 111In-oligo and 111In-oligo-Bt were 443–804 and 297–804 kBq/µg, respectively.

G4 and Biotinylation.
G4 (Aldrich Chemical Co., Milwaukee, WI) has an ethylenediamine core and 64 reactive amino groups on its surface, with a molecular weight of 14,215 (13) . Ten mg of G4 were dissolved in 900 µl of 0.05 M Na2HPO4-0.05 M NaH2PO4 (pH 7.4) and mixed with 1.17 mg of Bt (Pierce Chemical Co., Rockford, IL), at a molar ratio of 1:3, at room temperature for 30 min. The mixture was applied to a diafiltration membrane (Centricon 10; Amicon, Inc., Beverly, MA) to remove unbound Bt and to concentrate the solution to 50 µg/µl.

111In-Oligo-Carrier Complexes.
Acetonitrile in the 111In-oligo or 111In-oligo-Bt solutions was evaporated by exposing the solutions to a stream of nitrogen gas. The 111In-oligo was mixed with a 100-fold molar excess of G4 or G4-Bt, and the pH of the solution was adjusted to 7 with 0.5 M Na2HPO4-0.5 M NaH2PO4 (pH 9) to form the electrostatic complex of 111In-oligo/G4 or 111In-oligo/G4-Bt. Av (Pierce) was added to 111In-oligo/G4-Bt solution at a G4-Bt-to-Av molar ratio of 4:1 to form 111In-oligo/G4-Av complex. In parallel, 111In-oligo-Bt was mixed with Av at a 1:1 molar ratio after adjustment of the pH of the 111In-oligo-Bt solution to 7 with 0.5 M Na2HPO4-0.5 M NaH2PO4 (pH 9) and incubated at room temperature for 10 min. 111In-oligo-Av was formed.

All of the oligonucleotide-carrier complexes were purified by Sephadex G-50 gel (Pharmacia) filtration. The schemes for all three preparations are shown in Fig. 1Citation .



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  		Fig. 1. Schemes for preparing the three oligo-carrier complexes used in this study.

 
Internalization Assay.
The human ovarian cancer cell line SHIN3 (3 x 105), preseeded in 35-mm dishes and cultured overnight in RPMI 1640 (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% FCS (Life Technologies, Inc., Grand Island, NY) and 0.03% L-glutamine in a 5% CO2 environment, were used. Subconfluent cells were incubated with 2.96 kBq (0.08 µCi)/22.4 ng (as oligo) of 111In-oligo/G4, 111In-oligo/G4-Av, or carrier-free 111In-oligo or with 2.96 kBq/45.9 ng (as oligo) of 111In-oligo-Av in 1 ml of RPMI 1640 with 10% FCS for 1 h at 37°C or 4°C as a noninternalized control. The medium containing the tracers was then replaced with 1 ml of standard RPMI 1640. The cells were further cultured at 37°C or 4°C to examine internalization of radioactivity. At 0, 1, 3, 10, and 24 h (n = 4), the medium was removed, and the cells were washed with 0.5 M Na2HPO4-0.5 M NaH2PO4-0.1 M NaCl (pH 7.4). Membrane-bound radioactivity was removed by washing the cells with acidic solution [0.05 M glycine-HCl-0.1 M NaCl (pH 2.8)], which eliminated the charge-related binding of the complexes to the cell surface. The radioactivity in the medium and membrane-bound and acid-resistant internalized radioactivity was assessed by gamma counter (Aloka, Tokyo, Japan). The percentages of membrane-bound fraction and internalized fraction in the total bound radioactivity were calculated. The assay at 4°C could be performed only up to 3 h because cells died and detached thereafter.

Biodistribution Study.
To develop an i.p. tumor model, 3–4 x 106 SHIN3 cancer cells were injected i.p. into 5-week-old female BALB/c nu/nu mice. Twenty days later, numerous small i.p.-disseminated tumors had formed, especially around the stomach, subphrenic region, at the hepatic and splenic hila, and on the mesentery. Seventy-four kBq (2 µCi) of 111In-oligo/G4, 111In-oligo/G4-Av, 111In-oligo-Bt-Av, or carrier-free 111In-oligo were injected i.p. into groups of mice bearing i.p. tumors (n = 6–10; 125–167 ng each for 111In-oligo/G4, 111In-oligo/G4-Av, and 111In-oligo; 240–300 ng for 111In-oligo-Av, by oligo weight), and then biodistribution was examined. As a control, biodistribution in normal mice was compared for 111In-oligo/G4, 111In-oligo-Av, and 111In-oligo (n = 4–7). The mice were sacrificed by ether inhalation at 2, 10, or 24 h after i.p. injection of radioactive oligo. Blood, i.p.-disseminated tumors, and various organs were removed and weighed, and their radioactivity counts were measured by gamma counting. The % ID/g for these data were determined, and the results were normalized to a 20-g mouse, using following formula:

.

All animal experiments were carried out in accordance with the regulations of the Kyoto University animal facility regarding animal care and handling.

Scintigraphy by 111In-Oligo-Carrier Complexes.
A total of 1.85 MBq (50 µCi)/2.2 µg of 111In-oligo-carrier complexes or carrier-free 111In-oligo were injected i.p. into i.p. tumor (SHIN3)-bearing mice or normal mice. Scintigrams were obtained at 0, 2, and 10 h and 1, 2, and 3 days after injection. Four days after injection of the oligo-carrier complexes, the mice were sacrificed, and the locations of the i.p.-disseminated tumors were examined and correlated with the scintigrams. In addition, to further confirm that the foci of radioactivity shown on the images actually represented delivery of 111In-oligo to the organs and/or tumors, the organs and i.p. tumors in tumor-bearing mice were taken out, washed with H2O to remove unbound 111In-oligo (-carrier complexes), and placed side by side. Scintigrams were then obtained of these samples and compared with corresponding mouse scintigrams obtained from the live mice.

Statistical Analyses.
A one-way ANOVA with Fisher’s protected least significant difference test was used for statistical analyses of the internalization assay and the biodistribution study. All tests were two-sided, and a probability (P) < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Internalization Assay.
The tumor cells incubated at 37°C showed an internalization of radioactivity (Fig. 2)Citation , whereas those incubated at 4°C did not (data not shown). The percentages of 111In-oligo/G4, 111In-oligo/G4-Av, and 111In-oligo-Av internalized at 24 h were 34, 51, and 56% of the total bound fraction, respectively, all of which were significantly higher than that of 111In-oligo (23%; P < 0.01 for 111In-oligo/G4 and P < 0.0001 for 111In-oligo/G4-Av and 111In-oligo-Av). Significantly more 111In-oligo/G4-Av and 111In-oligo-Av was internalized than 111In-oligo/G4 (P < 0.01).



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  		Fig. 2. Internalization of 111In-oligo complexes or 111In-oligo bound to SHIN3 ovarian cancer cells at 37°C. The percentage of the internalized radioactivity in the total bound radioactive is indicated. The left panel is a magnification of the lower left-hand corner of the right panel. 111In-oligo-carrier complexes internalized significantly higher than 111In-oligo at 24 h (P < 0.01 for 111In-oligo/G4; P < 0.0001 for 111In-oligo/G4-Av and 111In-oligo-Av). •, 111In-oligo/G4; {circ}, 111In-oligo/G4-Av; {blacktriangleup}, 111In-oligo-Av; {triangleup}, 111In-oligo.

 
111In-oligo/G4 or 111In-oligo/G4-Av bound to the cells (both membrane-bound and internalized radioactivities) at 37°C was 12–13% of the total radioactivity at 1 h. The total bound radioactivity decreased thereafter to 9 and 5% for 111In-oligo/G4 and 111In-oligo/G4-Av, respectively, at 24 h. In contrast, the amount of 111In-oligo-Av bound to the tumor cells did not decrease over time and was 11% at 24 h (data not shown).

Biodistribution Study.
Biodistribution data in normal and tumor-bearing mice at 2 and 24 h postinjection are shown in Figs. 3Citation and 4Citation , respectively. In normal mice, 111In-oligo without carrier accumulated highly in the kidneys at 2 h (7.5% ID/g), representing a rapid renal excretion of 111In-oligo. When G4 was used as a carrier, the complex accumulated in the spleen (7.3% ID/g) and stomach (6.6% ID/g) at 2 h in addition to the kidney, all of which quickly decreased with time. In contrast, the high accumulation of 111In-oligo-Av in the liver (6.1% ID/g), spleen (9.6% ID/g), and stomach (7.0% ID/g) at 2 h further increased after 24 h had elapsed. The liver uptake of 111In-oligo-Av was significantly higher than that of 111In-oligo/G4 or 111In-oligo (P < 0.0001).



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  		Fig. 3. Biodistribution of 111In-oligo complexes or 111In-oligo in normal mice 2 h (A) and 24 h (B) post-i.p. injection. {square}, In-oligo/G4; , 111In-oligo-Av; {blacksquare}, 111In-oligo.

 


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  		Fig. 4. Biodistribution of 111In-oligo complexes or 111In-oligo in i.p. tumor-bearing mice 2 h (A) and 24 h (B) post-i.p. injection. Conjugation of 111In-oligo with G4, G4-Av, or Av significantly increased the delivery of 111In-oligo to the tumors compared with carrier-free 111In-oligo at 24 h (P < 0.05, < 0.01, and < 0.0001 for 111In-oligo/G4, 111In-oligo/G4-Av, and 111In- oligo-Av, respectively). {square}, In-oligo/G4; , 111In-oligo/G4-Av; , 111In-oligo-Av; {blacksquare}, 111In-oligo).

 
In the tumor-bearing mice, G4, G4-Av, or Av delivered significantly larger amounts of 111In-oligo to the i.p.-disseminated tumors than did 111In-oligo without carrier (Fig. 4Citation ; P < 0.05, < 0.01, and < 0.0001 for G4, G4-Av, and Av, respectively, at 24 h). Radioactivity uptake in the liver (for 111In-oligo-Av), stomach, and spleen, which was high in normal mice, was low in tumor-bearing mice. The tumor accumulation of 111In-oligo/G4 was 12.4 and 9.1% ID/g at 2 and 24 h post-i.p. injection, respectively. 111In-oligo/G4-Av exhibited tumor uptake as high as 41.7% ID/g at 2 h, which decreased to 14.5% ID/g at 24 h. 111In-oligo-Av exhibited very high tumor accumulation at 2 h (21.3% ID/g), which persisted up to 24 h (24.4% ID/g; P < 0.01 and < 0.05 compared with 111In-oligo/G4 and 111In-oligo/G4-Av, respectively). The tumor uptake of carrier-free 111In-oligo was as low as 0.8% ID/g at 24 h. The high tumor radioactivity uptake resulted in very high tumor-to-nontumor radioactivity ratios for 111In-oligo/G4, 111In-oligo/G4-Av, and 111In-oligo-Av (data not shown).

Scintigraphy of 111In-Oligo-Carrier Complexes.
Tumor targeting of the three 111In-oligo-carrier complexes also correlated with scintigraphy. The tumors shown by scintigraphy were confirmed when mice were sacrificed and the locations of the tumors were compared with the scintigraphic findings. Small tumors that accumulated and agglutinated around the stomach and subphrenic region, at the hepatic and splenic hila, and on the mesentery were visualized as if they were large tumors. Because of splenomegaly of the tumor-bearing mice and accumulated tumors at the splenic hilum and around the stomach, the intestine was slightly shifted to the right in the peritoneal cavity. Consequently, the mesenteric tumors were shown on the right side in the scintigrams. Scintigrams of the organs and tumors taken out from the mice also showed a high visualization of tumors when 111In-oligo-carrier complexes were used (images not shown). Fig. 5Citation shows the scintigrams of normal and i.p. tumor-bearing mice at 24 h after i.p. injection of 111In-oligo/G4, 111In-oligo-Av, and free 111In-oligo. 111In-oligo/G4 faintly showed the stomach, and 111In-oligo-Av accumulated in the liver in addition to the bladder in normal mice, whereas in the tumor-bearing mice, the i.p.-disseminated tumors were clearly visualized with sufficient contrast to the normal organs. The scintigram of free 111In-oligo showed only renal and bladder uptake, and no other organ or tumor was identified. The scintigram at 0 h revealed immediate renal excretion of 111In-oligo after i.p. injection in both tumor-bearing and normal mice (images not shown).



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  		Fig. 5. Scintigrams of i.p. tumor-bearing (A, C, and E) and normal (B, D, and F) mice 24 h after i.p. injection of 111In-oligo/G4 (A and B), 111In-oligo-Av (C and D), and 111In-oligo (E and F). i.p.-disseminated tumors were visualized with 111In-oligo/G4 (A) and 111In-oligo-Av (C; arrows).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The three oligo-carrier complexes synthesized for this study by the G4 and/or the Bt-Av system showed a high tumor accumulation for both the in vitro and the in vivo i.p. tumor models. For synthesis of 111In-oligo-Av, 3'-biotinylated oligo was used because biotinylation of the 3' end of an oligonucleotide, followed by binding to Av, has been reported to provide protection against serum 3'-exonuclease degradation (30 , 31) .

The internalization study showed a gradual internalization of the complexes to the tumor cells in the presence of serum. However, the level of the total bound 111In-oligo/G4 and 111In-oligo/G4-Av decreased with time. This decrease was possibly caused by some release of 111In-oligo from the complexes at the cell surface prior to internalization. Contrary to this possibility, the total bound 111In-oligo-Av did not decrease, suggesting that the binding of oligo-Bt to Av was stronger than that of oligo to either G4 or G4-Av.

In both normal and tumor-bearing mice, carrier-free 111In-oligo demonstrated only renal and urinary bladder uptake as a result of rapid excretion through the kidney. By complexing or conjugating 111In-oligo with G4, G4-Av, or Av, we significantly increased the delivery of 111In-oligo to the tumors. The hepatic or splenic accumulation of the complexes may be attributable to the uptake by reticuloendothelial systems; however, the high gastric uptake demonstrated was unexpected, and the reason is unclear. 111In-oligo/G4 and 111In-oligo/G4-Av showed decreasing tumor uptake over time, whereas 111In-oligo-Av maintained high tumor accumulation up to 24 h. These findings seem to be comparable to the in vitro study results, which also found a decrease of the total tumor cell binding with the 111In-oligo/G4 and 111In-oligo/G4-Av complexes. The markedly high uptake of 111In-oligo/G4-Av to the tumor at 2 h postinjection is not surprising because 111In-oligo/G4-Av may accumulate in the tumor by virtue of both dendrimer and Av mechanisms. Although the tumor uptake of 111In-oligo/G4-Av rapidly decreased to a level close to that of 111In-oligo/G4 at 24 h postinjection, it was still as high as 14.5% ID/g at 24 h. An extremely high i.p. tumor uptake of i.p.-injected Av has been reported (25) , in addition to the findings that the i.p. injection can provide higher tumor uptake in i.p. tumor models because of greater accessibility compared with the i.v. route (32) . Another potential reason for the high tumor uptake shown in this study is that after i.p. inoculation, SHIN3 cells form small tumors with a broader surface, which may lead to higher tumor uptake compared with that of other larger tumors (33) .

At the mixing ratio of 1:100 for 111In-oligo to G4 (or G4-Av) used in this study, the complexes formed were positively charged and readily bound to the negatively charged cell membrane. The complexes are considered to be in a clustered form, similar to that of DNA-polylysine complexes (34) , and oligo are in a condensed form, which facilitates cellular uptake of the oligo (19) . In previous ultrafiltration experiments using complexes with 1:100 or 1:1 DNA:generation 3 dendrimer (Mr 6909) molar ratios, 80–90% of the complexes were in the range of 10–1000 kDa range, and 30% of the complexes were <30 kDa. Some fractions of the complexes that are large (~1000 nm in diameter; Ref. 17 ) are suspected to internalize inefficiently (12) , which allows some 111In-DNA to be released from the complexes at the cell surface.

To determine the complexation ratio of G4 with oligo, a preliminary study using Sephadex G-50 gel filtration was performed. At a molar mixing ratio of 1:1 for 111In-oligo and G4, >70% of 111In-oligo was not complexed with G4. At higher G4 molar ratios, the ratio of 111In-oligo bound to G4 increased, and at 1:100, all of the 111In-oligo was complexed with G4; we consequently selected the mixing ratio of 1:100 for in the present study.

Recent studies have indicated that the concentration of the gene, the DNA-to-dendrimer charge ratio, and the size (generation) of the dendrimer, which are the determining factors for the size and charge of the entire complex (4) , directly affect the transfection efficiency (16) . The optimal charge ratio of DNA:dendrimer examined in a serum-free in vitro environment (12 , 19) might not be directly comparable with experiments carried out in the presence of serum or in vivo. A higher dendrimer:oligonucleotide ratio increased transfection efficiency, especially at a higher serum concentration (16) . Further detailed studies to determine adequate DNA:dendrimer charge ratio are required. Many investigators have compared transfection and transcription efficiencies between different dendrimer generations (12 , 16 , 19 , 20 , 35) ; however, the appropriate size to be used as a carrier has not been confirmed yet.

Imaging in conjunction with gene therapy is now in the developmental stage. A variety of imaging techniques, including positron emission tomography, gamma scintigraphy and magnetic resonance imaging, have been investigated (36) . Imaging with radioisotopes has an advantage because of the high sensitivity compared with magnetic resonance imaging, which enables visualization of even a smaller amount of gene delivered to the target organ/tissue or a smaller amount of gene expressed in the target. The delivery of 111In-oligo to the i.p.-disseminated tumors by G4, G4-Av, or Av as a carrier could be successfully visualized. The scintigraphy of 111In-oligo-Av in normal mice illuminated the liver, which is in good agreement with the high liver accumulation of this complex shown in our biodistribution studies in normal mice. In the i.p. tumor-bearing mice, 111In-oligo-Av was delivered to the tumors, giving low liver accumulation. The scintigrams of the 111In-oligo/G4, 111In-oligo/G4-Av, and 111In-oligo-Av complexes successfully visualized tumors with sufficient contrast to the normal tissues, in accordance with the high tumor-to-nontumor radioactivity ratios obtained in the biodistribution studies. Thus, these 111In-oligo-carrier complexes have potential as tracers to monitor gene delivery. Neither the scintigrams nor the biodistribution data provide explicit evidence of internalization of 111In-oligo into the i.p. tumor cells; however, this condition is strongly suggested from the results of the in vitro internalization study.

One potential obstacle to using radiolabeled oligonucleotides as a tracer to monitor gene delivery is evidence that oligonucleotides undergo exocytosis in vitro in some cell lines (37 , 38) . If a radiolabeled oligonucleotide underwent cytosolic metabolism after internalization and the free oligonucleotide exocytosed, leaving radioactive metal chelate in the cytosolic component, the information obtained from the radioactivity would not accurately be related to the localization of oligonucleotide (39) . The combination of a very high tumor uptake of the 111In-oligo-carrier complexes presented in this study combined with the results of previous reports of increased transfection/transcription efficiency of DNA complexed with dendrimer or Av compared with free DNA (12 , 16 , 17 , 22) indicate that the amount of exocytosed radiolabeled DNA may be negligible. However, the fate of DNA complexed with dendrimer and/or Av after internalization is still unclear.

In this study, we used an antisense oligonucleotide as the primer for c-erbB-2 and SHIN3 cancer cells, which do not show high expression of c-erbB-2. 111In-oligo-carrier complexes were delivered to the tumor cells by non-sequence-specific targeting both in vitro and in vivo. The high tumor accumulation of 111In-oligo complexes indicates that the oligonucleotide carriers used in the present studies, i.e., G4, G4-Av, and Av, have a possibility of being vectors in a variety of i.p. tumor-targeting gene therapies, irrespective of the sequence. Double-stranded DNAs or phosphorothioate oligonucleotides, which would be more stable in vivo (38) , can be conjugated to these vectors. Conjugation with a plasmid containing reporter genes would also enable detection of expression of the delivered gene. Additionally, dendrimers have their own potential for modification through their surface amino groups, so as to form conjugate with a tumor-specific antibody and to create a tumor-specific vector (40 , 41) .

In conclusion, G4, G4-Av, and Av complexes had been shown to effectively deliver 111In-oligo to i.p.-disseminated tumors. These complexes have a great potential for modification and can be a vector for a variety of oligonucleotides. These 111In-oligo-carrier complexes can also be tracers for imaging of correlation of gene delivery and have enabled the first tumor images with 111In-labeled oligo to be obtained.


   ACKNOWLEDGMENTS
 
We thank Nihon Mediphysics, Japan, for kindly providing InCl3.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Konica Imaging Science Foundation, Tokyo, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Diagnostic and Interventional Imagiology, Graduate School of Medicine, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-4955; Fax: 81-75-751-4954. Back

4 The abbreviations used are: Av, avidin; Bt, sulfosuccinimidyl-6-(biotinamide) hexanoate; 1B4M, 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid; G4, generation 4 starburst polyamine dendrimer; % ID/g, percentage of injected dose per g of tissue. Back

3 Present address: Metabolism Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4N109, 10 Center Drive, MSC 1374, Bethesda, MD 20892. Phone: (301) 435-8344; Fax: (301) 496-9956; E-mail: hisataka{at}kuhp.kyoto-u.ac.jp Back

Received 5/21/01; revised 8/ 3/01; accepted 8/ 6/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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