intraperitoneal delivery of liposomal sirna for therapy of advanced ovarian cancer

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[Cancer Biology & Therapy 5:12, 1708-1713, December 2006]; ©2006 Landes Bioscience

Research Paper

Intraperitoneal Delivery of Liposomal siRNA for Therapy of Advanced Ovarian Cancer

Charles N. Landen1

William M. Merritt1 Lingegowda S. Mangala1 Angela M. Sanguino2

Corazon Bucana3

Chunhua Lu1 Yvonne G. Lin1 Liz Y. Han1 Aparna A. Kamat1

Rosemarie Schmandt1

Robert L. Coleman1

David M. Gershenson1 Gabriel Lopez-Berestein2

Anil K. Sood1,3,*1Department of Gynecologic Oncology; 2Department of Experimental Therapeutics; and 3Department of Cancer Biology; MD Anderson Cancer Center; Houston, Texas USA

*Correspondence to: Anil K. Sood; Departments of Gynecologic Oncology and Cancer Biology; The University of Texas MD Anderson Cancer Center; 1155 Holcombe Boulevard, Unit 1362; Houston, Texas 77030 USA; Tel.: 713.745.5266; Fax: 713.792.7586; Email: [email protected]

Original manuscript submitted: 07/04/06Manuscript accepted: 09/30/06

Previously published online as a Cancer Biology & Therapy E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3468

KeY WoRDS

siRNA, liposomes, intraperitoneal, DOPC, EphA2, ovarian cancer

ACKNoWLeDGeMeNtS

See page 1713.

ABStRACtPurpose: Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows

efficient delivery to tumor tissue, and has therapeutic efficacy in preclinical proof‑of‑concept studies using EphA2‑targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as IV delivery.

Experimental design: SiRNA was incorporated into the neutral liposome 1,2‑dioleoyl‑ sn‑glycero‑3‑phosphatidylcholine (DOPC). Alexa555‑siRNA‑DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared.

Results: Alexa555‑siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2‑targeting siRNA‑DOPC reduced tumor growth by 48–81% compared to paclitaxel/control siRNA‑DOPC IP (HeyA8: 0.34 g v 0.66 g; SKOV3ip1: 0.04 v 0.21, p < 0.01). This reduction was comparable to concurrently‑treated mice with paclitaxel and EphA2 siRNA‑DOPC injected IV, which showed a reduction in growth by 45–69% compared to paclitaxel/con‑trol siRNA‑DOPC injected IV (HeyA8: 0.23g v. 0.42g; SKOV3ip1: 0.04 v. 0.13 g).

Conclusions: IP injection of siRNA incorporated in DOPC allows intra‑tumoral delivery and has therapeutic efficacy in orthotopic ovarian tumors. These findings may have thera‑peutic implications for siRNA‑based strategies.

INtRoDuCtIoNRNA interference (RNAi) has become a powerful tool in examining protein function,

gene discovery, drug development.1-3 Through these methods, specific RNA sequences can be targeted for destruction, preventing translation of the proteins under investigation. The use of small interfering RNA (siRNA), one mechanism of RNAi, has recently been examined as a therapeutic modality.4,5 Several methods of siRNA delivery into an in vivo system have been described.6-15 However, most of these delivery systems have impractical or limited clinical use, such as delivery under high pressure and volume, injections directly into the tumor or contained cavities, or use of viral vectors. Development of a modality that can be administered systemically and safely would have the potential to specifically target a single gene for destruction, introducing an exciting novel method of therapy for cancer or other diseases in which protein inhibition would be desirable.

We have examined siRNA as a therapeutic modality in an orthotopic mouse model of advanced ovarian cancer.5,16 We have previously demonstrated that by incorporating siRNA into a neutral liposome (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine-DOPC), siRNA in DOPC injected intravenously is delivered to tumor cells in vivo, the targeted protein is downregulated, and tumor growth can be modulated. Ovarian cancer is an excellent model for novel therapeutics, as it is the leading cause of death among gyneco-logic malignancies, fifth overall, with an estimated 20,180 cases and 15,310 deaths in the United States in 2006.17 The majority of ovarian cancer patients respond to initial therapy of tumor cytoreductive surgery and platinum-based chemotherapy, but about 70% of patients with an initial positive response will recur and succumb to disease.18 Thus, novel therapeutic strategies are urgently needed to improve the outcome of women with ovarian

cancer.

1708 Cancer Biology & Therapy 2006; Vol. 5 Issue 12

www.landesbioscience.com Cancer Biology & Therapy 1709

Intraperitoneal In Vivo siRNA-Mediated Anti-EphA2 Therapy

Many protein targets would be candidates for downregulation by siRNA therapy in hopes of decreasing tumor growth. We have examined EphA2 as a target, given its expression by a variety of cancer types, limited expression in normal tissues, and the efficacy demonstrated as a tumor target in ovarian cancer.19 EphA2 is a tyrosine kinase receptor in the ephrin family that plays a key role in neuronal development,20,21 but has also been shown to function as an oncoprotein. Induced overexpression transforms nonmalig-nant breast cells into a malignant phenotype,22 and treatment with EphA2-downregulating agents was shown to reduce tumor growth in breast, pancreatic and ovarian cancer models.5,23-27 EphA2 is overexpressed in many types of human tumors, including lung, breast, colon, prostate and melanoma.28-35 About 70% of human ovarian tumors overexpress EphA2, which is associated with a poor outcome.35 EphA2 is an attractive target in tumor therapy, not only because of the high expression across multiple lines and the demon-strated efficacy of EphA2-targeting methods, but also because of the anticipated low toxicity rate, given low EphA2 expression in normal human tissues.19,36,37

In a mouse model of advanced ovarian cancer, intravenous admin-istration of liposome-incorporated siRNA allowed delivery to the tumor and reduced tumor growth when EphA2 was targeted.5 It is not known whether intraperitoneal delivery of siRNA-DOPC would result in either local or systemic delivery. Therefore, we examined whether delivery by intraperitoneal injection of liposomal siRNA would also allow adequate tumoral delivery and therapeutic efficacy when targeting EphA2 in an ovarian cancer model. This would reduce the need for labor-intensive use of intravenous delivery in animals. In addition, it is a clinically-appropriate question, as the use of intraperitoneal chemotherapy is increasing in treatment of human ovarian cancer patients.38

MAteRIALS AND MetHoDSCell lines and culture. The ovarian cancer cell lines HeyA8 and

SKOV3ip139 were maintained in RPMI 1640 supplemented with 15% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). For in vivo injection, cells were trypsinized and centrifuged at 1,000 rpm for 7 minutes at 4˚C, washed twice, reconstituted in serum-free HBSS (Life Technologies, Carlsbad, CA) at a concentration of 5 x 106 cells/mL (SKOV3ip1) or 1.25 x 106 cells/mL (HeyA8) for 200 mL i.p. injections.

Small interfering RNA constructs. SiRNA was obtained from Qiagen (Valencia, CA) in three formulations. A nonsilencing siRNA sequence, which does not share homology with any known human mRNA (target sequence 5'-AATTCTCCGAACGTGTCACGT-3') and tagged with Alexa 555, was used to determine uptake and distri-bution in various tissues when given in vivo. EphA2-targeting siRNA, with the target sequence 5'-AATGACATGCCGATCTACATG-3', has been shown to reduce EphA2 expression in vitro and in vivo.5,25 A nonsilencing siRNA construct (sequence as above without an Alexa 555 tag) was used as control for EphA2-targeting experiments.

Liposomal preparation. For in vivo delivery, siRNA was incor-porated into 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as previously described.5 Briefly, DOPC and siRNA were mixed in the presence of excess tertiary butanol (1:10 w/w siRNA-DOPC), followed by Tween 20 (1:19 Tween 20:siRNA-DOPC), subsequently lyophilized and stored at -80˚C until use. Immediately prior to in vivo administration, the lyophilized preparation was hydrated with 0.9% saline at a concentration of 15 mg/mL to achieve the desired dose in 150 to 200 mL per injection.

Orthotopic in vivo model of advanced ovarian cancer and tissue processing. Female athymic nude mice (NCr-nu) were obtained from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD), housed in specific pathogen- free conditions, and cared for in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care, the USPHS “Policy on Human Care and Use of Laboratory Animals,” and The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee. Tumors were estab-lished by IP injection of cells prepared as above. Studies to determine uptake and tissue distribution of single-dose fluorescent siRNA in tissue or silencing potential of siRNA against EphA2 were initiated once IP tumors reached a size of 0.5–1.0 cm3 as assessed by palpation (about 17 days after injection). The HeyA8 cell line used for these experiments typically produce 1-2 dominant nodules at this stage of growth. Although exact size cannot be determined by palpation, it does allow identification of presence of a clinically significant tumor in which siRNA uptake can be assessed. In these mice, liposomal siRNA (5 mg) was given as a 200 mL IP bolus, and tumor and other tissues were harvested 6 or 24 hours after the last dose (6 hours for fluorescent experiments, 24 hours for EphA2 downregulation experiments). Tissue specimens were divided, placed in formalin for preparation of paraffin clines, snap frozen for lysate preparation, or frozen in OCT medium for frozen slide preparation.

In an attempt to assess the extent of direct diffusion of siRNA into the tumor, an experiment was conducted where a 0.5 cm3 tumor was harvested from the intraperitoneal cavity and bathed in 1 mL RPMI-1640 media containing 10 mg/mL Alexa-555 siRNA for 16 hours at 37˚C. It was then frozen in OCT media as described above and cut slides were examined for fluorescence.

For separately-conducted long-term treatment experiments to assess tumor growth, therapy began 1 week after tumor cell injection. Paclitaxel (100 mg) or vehicle was injected IP once weekly; liposomal siRNAs (nonspecific or EphA2 targeting, 150 mg/kg) were injected twice weekly IP in 150 to 200 mL volume (depending on mouse weight). Mice (n = 10 per group) were monitored for adverse effects, and tumors were harvested after 3 weeks of therapy. Mouse weight, tumor weight and distribution of tumor were recorded.

Immunofluorescence. Tissue for immunofluorescence examina-tion was collected from sacrificed mice, immediately placed in OCT

medium, and rapidly frozen near liquid nitrogen. Frozen sections were cut at 8 mm sections, fixed with acetone, exposed to 1.0 mg/mL

Hoescht (Molecular Probes, in PBS) for 10 minutes to stain nuclei, washed, and covered with propylgallate and cover slips for micro-scopic evaluation. Conventional microscopy was done with a Zeiss AxioPlan 2 microscope (Carl Zeiss, Inc, Germany), Hamamatsu ORCA-ER Digital camera (Hamamatsu Corp, Japan), ImagePro software (Media Cybernetics, Silver Spring, MD).

Immunohistochemistry. Immunohistochemistry for EphA2 was performed as previously described5 on paraffin-embedded sections. After deparaffinization, antigen retrieval was done by heating in steam cooker in 0.2 M Tris-HCl (pH 9.0) for 10 minutes. Nonspecific proteins and exposed endogenous mouse IgG antibodies were blocked with 0.13 mg/mL mouse IgG Fc blocker (The Jackson Laboratory, Bar Harbor, ME) in 0.5% blocking agent (TSA Biotin System kit, Perkin-Elmer, Boston, MA) overnight at 4˚C. Slides were incubated in primary antibody (5 mg/mL mouse anti-EphA2 clone EA5, a kind gift of Dr. Michael Kinch, MedImmune, Inc, Gaithersburg, MD) for 4 hours at 4˚C followed by 1.5 mg/mL biotinylated horse anti-mouse (Vector Labs, Burlingame, CA) for



10 minutes at room temperature. Signal was amplified with strep-tavidin-HRP, detected with 3,3’-diaminobenzidine (DAB; Phoenix Biotechnologies, Huntsville, AL), counterstained with Gill no. 3 hematoxylin (Sigma).

Statistical analysis. For in vivo therapy experiments, 10 mice in each group were used as directed by a power analysis to detect a 50% reduction in tumor weight (b error = 0.2). Continuous variables (i.e., tumor weight) were analyzed for statistical significance (achieved if p < 0.05) with Student’s t test for two-group comparisons and ANOVA for multiple-group comparisons. The Mann-Whitney rank sum test was used if values were not normally distributed. Stata 8 software (College Station, TX) was used for data analysis.

ReSuLtSDelivery of small interfering RNA to orthotopic tumors by

intraperitoneal injection. We have previously shown that intrave-nous injection of siRNA incorporated in DOPC results in highly efficient delivery into intraperitoneal tumors. To determine the extent of delivery by intraperitoneal injection, 5 mg of fluores-cently-tagged siRNA in DOPC was injected intraperitoneally into mice bearing HeyA8 intraperitoneal tumors, which were harvested six hours later. Sections from the tumor exhibited fluorescence within the tumor parenchyma, whereas untreated tumors have no

endogenous fluorescent emissions (Fig. 1). To demonstrate the wide distribution of siRNA within the tumor, low-power photographs were taken in adjacent segments of the tissue and compiled in a panoramic view (Fig. 1A). However, the small liposomes are difficult to appreciate at this magnification. Therefore, high-power photographs within each segment were taken to more accurately depict the perinuclear distribution of siRNA within tumor cells (Fig. 1B). Following a single dose of siRNA-DOPC, 37–68% (mean 56.9%, S.D. 13.9%) of cells incorpo-rated siRNA into the cytoplasm. Regarding distribution over the entire tumor, 98% of fields examined contained some degree of siRNA delivery via the IP route. Therefore, tumor delivery of liposomal siRNA with IP administration appears to be at least, if not more, effective compared to IV administration.

In an attempt to assess the degree that siRNA uptake into tissues might be due to direct diffusion, rather than uptake from the intraperitoneal cavity into the vascu-lature then redelivery to the tumor, an ex vivo experiment was conducted. A 0.5 cm3 tumor was excised from the intraperitoneal cavity of a sacrificed mouse, and bathed in 1mL of RPMI-1640 containing 10 mg/mL of Alexa-555 siRNA for 16 hours. The tumor was then processed identically to freshly-collected tumors and examined for fluorescence. A great deal of the siRNA is noted to collect at the periphery of the tumor, as examined by low power (Fig. 1C),

which is not seen in tumors collected after in vivo IP administration (Fig. 1A). When examined at high power, minimal uptake of siRNA is seen in the deeper parenchyma of the tumor (Fig. 1D). However, uptake by this method is lower than that of in vivo-delivered tumors, with 3.5% of cells showing fluorescence (S.D. 2.2%, p = 0.004 compared to 56.9 ± 13.9% for in vivo delivery). Although this ex vivo experiment is limited in its comparability to in vivo tumors, in that observations may be altered by factors such as metabolic changes in the tumor after excision, and higher pressures in the intraperito-neal cavity, it does suggest that some degree of direct diffusion is possible.

In vivo therapy experiments with liposomal anti‑EphA2 small interfering RNA. Based on encouraging results regarding tumor penetration following intraperitoneal injection of siRNA incorpo-rated into DOPC, we next examined the therapeutic efficacy of this approach. Female nude mice (n = 50 per cell line, 10 per group) were injected with SKOV3ip1 or HeyA8 cells into the peritoneal cavity. One week after tumor cell injection, animals were randomly allocated to five treatment groups: (a) empty liposomes injected IP, (b) pacli-taxel IP plus a nonspecific siRNA-DOPC injected IP, (c) paclitaxel IP plus EphA2-targeted siRNA-DOPC IP, (d) paclitaxel IP plus nonspecific siRNA-DOPC IV, (e) paclitaxel IP and EphA2-targeting siRNA-DOPC IV. EphA2-targeting siRNA alone was previously shown to be inferior to combination therapy with paclitaxel, there-

Figure 1. Uptake if Liposomal siRNA into Tumor Parenchyma after IP Administration. Mice bearing intraperitoneal HeyA8 tumors were injected intraperitoneally with a single dose of 5 mg liposomal nonsilencing Alexa555‑tagged siRNA‑DOPC. After six hours, tumors were harvested and examined for fluorescence. Multiple adjacent images captured from the edge of the tumor inward were examined at low (A, 10x) and high (B, 40x) power. Close examination shows that the siRNA is taken up by the tumor cells (nuclei in blue) deep into the tumor parenchyma. The scale of each power is shown in millimeters or microns. In a separate ex vivo experiment, tumor was harvested and bathed with 10 mg/mL siRNA in RPMI‑1640 for 16 hours, examined for fluorescence within the tumor. While most of the fluorescence is noted to collect at the peripheral tumor capsule (C), which was not seen in in vivo IP delivery, rare uptake deep into the tissue is noted when examined at higher power (D).



fore was not repeated.5 After three weeks of therapy, the animals were sacrificed and necropsies were done. Tumors were excised and weighed, with mean and individual weights reported in Figure 2. As previously shown, paclitaxel and control siRNA-DOPC led to reduced tumor growth compared to empty liposomes in each model. When compared to paclitaxel and control siRNA-DOPC, treatment with paclitaxel and anti-EphA2 siRNA-DOPC injected IP led to a reduction in tumor growth by 81% in the SKOV3ip1 model (from 0.21 to 0.04g, p < 0.01) and 48% in the HeyA8 model (from 0.66 to 0.34g, p < 0.05). This reduction in tumor growth was comparable to that achieved with IV injection of siRNA-DOPC, with reductions of 69% and 45% in the SKOV3ip1 and HeyA8 models, respectively. Although the overall mean tumor growth in IV-treated mice was generally less than that in comparably-treated mice by the IP route, the differences were not statistically significant (p = 0.27–0.79).

No obvious clinical toxicities were noted in treated mice, including behavior, eating habits and mobility. Mouse weights were not significantly different among the five groups of animals, suggesting that eating and drinking habits were not affected, and no diarrhea was noted.

EphA2 downregulation with intraperitoneal siRNA delivery. To confirm that IP delivery of siRNA-DOPC in conjunction with paclitaxel was downregulating EphA2 expression, separate experi-ments were conducted in which mice bearing intraperitoneal tumors were treated with two doses (three days apart) of EphA2-targeting or control siRNA-DOPC, in conjunction with one dose of paclitaxel. Tumors were excised, subjected to IHC for EphA2, and examined by an observer blinded to treatment group. Twenty-four hours after the second dose of EphA2-targeting siRNA-DOPC, EphA2 expression was noted to be significantly reduced compared to paclitaxel and control siRNA-DOPC (Fig. 3).

siRNA distribution in other organs. We expect that there is some direct delivery of siRNA-DOPC into intraperitoneal tumors following IP injection. However, given the extensive tumor penetra-tion, deeper than that explained by direct diffusion, we next asked whether there is also systemic uptake and organ delivery. To deter-mine if intraperitoneal administration of siRNA-DOPC resulted in delivery of siRNA to other organs, the liver, kidney, heart, spleen, pancreas, and brain were collected six hours after a single dose of fluorescent siRNA-DOPC. As previously noted after intravenous injection, uptake in the liver and kidney after IP injection was prominent (Fig. 4). The sections shown are from deep within the organ tissue, not the surface, therefore most likely represent uptake of siRNA-DOPC into the circulation with subsequent deposition through the vasculature, rather than direct diffusion from the intra-peritoneal cavity. Uptake was not noted in the heart. Definitive conclusions regarding uptake in the brain, pancreas and spleen cannot be made due to autofluorescence present in these organs (pictures not shown).

DISCuSSIoNWe have shown that siRNA is taken up by the tumor parenchyma

when incorporated into DOPC liposomes and injected intraperi-toneally into mice. Moreover, the EphA2 protein can be effectively targeted by siRNA treatment, such treatment leads to significantly reduced tumor growth when combined with paclitaxel in an orthotopic mouse model of advanced ovarian cancer.

We have previously demonstrated that in a preclinical mouse model of ovarian cancer, therapy with EphA2- or FAK-targeting siRNA

leads to reduced tumor growth when injected intravenously.5,16 This report extends our prior work where intravenous administration of siRNA-DOPC was effective, showing an equivalency when injected by the intraperitoneal route. There are two primary advantages to use of the intraperitoneal route. First, with regard to preclinical studies, this method of administration is much easier than intravenous administration via the tail vein in mice. IV therapy is labor inten-sive, time consuming, requires more training than IP injections. If access is lost during injection, the volume of injection is less reliably known. Repeated injections make access more difficult as veins are damaged. Furthermore, such injections are more acutely stressful to mice, which increasing evidence suggests may adversely affect tumor growth.40

Secondly, the intraperitoneal route may have a therapeutic advan-tage, especially in ovarian cancer. Three randomized prospective trials have demonstrated a survival advantage when compared to intravenous administration.41-43 Based on those trials, the National Cancer Institute has released a statement that intraperitoneal chemo-therapy should be encouraged as the standard of care for women with advanced ovarian cancer.38 Future studies are ongoing to delineate the optimal dose and treatment schedule for combined intraperi-toneal and intravenous chemotherapy, and as targeted therapies are considered for additional therapy, those with efficacy via the intra-peritoneal route may confer an advantage.

Traditionally it has been felt that uptake of chemotherapy when delivered by the intraperitoneal route is both by direct diffusion into the cells,44 as well as by systemic uptake into the vasculature and subsequent delivery to the tumor. In fact, intraperitoneal chemo-therapy is only recommended in patients with less than 1 cm of bulky disease remaining after tumor reductive surgery.45 However, with delivery of siRNA in DOPC by IP injection, we have observed uptake in the kidney, a retroperitoneal organ, and deep into the liver

Figure 2. In vivo Therapy of Ovarian Cancer in Mice with Anti‑EphA2 Liposomal siRNA. Mice were injected intraperitoneally with either SKOV3ip1 (A) or HeyA8 (B) tumor cells. One week later, treatment was initiated, consisting of control siRNA‑DOPC IP alone, or the combination of paclitaxel (Pac) given IP and control siRNA‑DOPC or EphA2 siRNA‑DOPC, with siRNA given either IV or IP. After three weeks of therapy, tumors were harvested and total tumor weight recorded. Weights in each group were compared with student’s t‑test if data set was normally distributed, or the Mann‑Whitney test if not.



parenchyma, suggesting that delivery via uptake into the vasculature followed by redistribution of siRNA is significant and rapid (less than six hours). Therefore, such siRNA therapy would not be limited to ovarian or other cancer patients with minimal residual disease.

While siRNA use has yielded tremendous value in experimental studies of gene function and drug discovery,3 its use as a therapeutic agent is in its infancy. The primary hurdle lies in developing an effective method of clinically appropriate delivery.4 Initial methods of in vivo administration included high-pressure, high-volume intra-venous injection that are not clinically feasible in humans.6 Other reported methods of administration include direct intratumoral injection,11 and injection of naked siRNA into directed sites such as joint cavities,46 the thecal space,14 and the vitreous humor of the eye.12 Reports of efficacy with intravenous injection of naked siRNA are sparse. Use of chemically-modified siRNA has been described,47 but the effects of such modifications are not fully elucidated. We have previously described that when incorporated into neutral DOPC liposomes, intravenous tail vein injection of small volumes under normal pressure led to tumor uptake of the siRNA, downregula-tion of the desired target, decreased tumor growth.5 Testing of this method in other cancer models, utilizing other targets, by other investigators is underway. Liposomes have long been used in patients. For example, liposomal doxorubicin is well-tolerated, effective, FDA approved for use in recurrent ovarian cancer patients.48 Therefore delivery of siRNA within liposomes is a realistic and clinically- feasible modality.

The relative reduction in tumor growth with EphA2-directed therapy was similar between the intravenous and intraperitoneal routes (50–80% in each). However, it is reasonable to speculate whether the systemic response to IV versus IP therapy would be different. It has been suggested that siRNA sequences that have no known homology to murine mRNA may still yield a response.49,50 We have observed this in the HeyA8 line, as described previously.5 Others have provided data suggesting that siRNA administration may induce production of interferon, or other nonspecific immune responses.51,52 Theoretically, the induction of these systems may be different with different routes of administration. Whether the efficacy by the IP route would be different for other tumors is yet to be seen. However, in the ovarian cancer model, our data suggest that use of IP administration is valid in development of siRNA-directed therapy. Further characterization of effects via the different routes is warranted before one particular route is declared superior regarding therapeutic efficacy.

The use of siRNA as a therapeutic agent is indeed an exciting concept. Much remains to be learned and validated regarding safety and specificity of this modality. Importantly, because the administra-tion by either the intravenous or intraperitoneal route is systemically distributed, appropriate choice of a target will be essential to mini-mize toxicity to normal tissues. EphA2 is minimally expressed in normal tissues, and may provide such a target. As further data are gathered, siRNA may develop into a feasible therapeutic modality in ovarian and other cancer patients.

Figure 3. Downregulation of EphA2 with IP‑Delivered Liposomal siRNA. Mice bearing 0.5–1.0 cm3 sized tumors were treated on day 1 with paclitaxel and either control siRNA‑DOPC or EphA2 siRNA‑DOPC, all administered IP, a repeat dose of siRNA on day 4, with tumor collection on day 5. Tissue subjected to immunohistochemistry for EphA2 expression demonstrates reduced expression of EphA2 in treated tissue (C) compared to tissue treated with IgG (B). The negative control for IHC with no primary antibody is depicted in (A).

Figure 4. Uptake of Liposomal siRNA into Distant Organs. After IP injection of siRNA‑Alexa‑555/DOPC, significant siRNA signal is seen in the liver (B) and kidney (E), compared to the absence of fluorescence in organs after injection of nonfluorescent control siRNA‑DOPC (C and E). H&E sections of each organ are shown in (A and D).



AcknowledgementsThis research was funded in part by grants from the

Bettyann Asche-Murray Fellowship Award at the University of Texas MD Anderson Cancer Center awarded to CNL, the Department of Defense (#W81XWH-04-1-0227), a Program Project Development Grant from the Ovarian Cancer Research Fund, the Zarrow Foundation and the UT MD Anderson Cancer Center ovarian cancer SPORE grant (#2P50CA083639).

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intraperitoneal delivery of liposomal sirna for therapy of advanced ovarian cancer

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