Nanocarriers for ovarian cancer active drug targeting

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

Nanocarriers for ovarian cancer active drug targeting F. Delie1*, E. Allémann1, M. Cohen2 1

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, 1211 Geneva, Switzerland 2 Department of Obstetrics and Gynecology, Faculty of Medicine, 30, boulevard de la Cluse, 1211 Geneva, Switzerland *Correspondence: [email protected]

Ovarian cancer ranks fifth amongst the most fatal form of female cancers in Europe and the United States. It is characterized by the silent development of a tumor in the ovarian/fallopian area followed by the dissemination of micrometastases in the peritoneal space. Currently, the treatment consists in surgical debulking in combination with chemotherapy. Unfortunately, most women will undergo a relapse, and the chances of a cure are slim. Nanocarriers have been proposed to improve the efficacy of chemotherapy. They offer the advantage of passive biodistribution at the tumor site due to enhanced permeation and retention effect. Functionalizing drug delivery systems with chemical moieties that are able to recognize molecular elements expressed specifically by cancer cells or structures involved in tumor development may enhance biodistribution to cancer sites, further improving therapeutic efficiency. In this review, the characteristics of ovarian cancer and current treatments are described. The potential targets identified to inhibit ovarian cancer development and the main advances obtained with targeted nanocarriers are also presented. Several strategies were tested using either versatile ligands or very specific targets. Promising data are presented; most of them demonstrate the in vitro proof of concept, while some confirm the potential of this approach with in vivo evidence. Key words: Ovarian cancer – Active targeting – Nanocarriers – Liposomes – Nanoparticles – Folate – EGFR – GRP78 – Anticancer therapy.

ovarian malignancies. EOC is characterized by a high degree of heterogeneity. A complex classification based on the morphology and molecular characteristics has been developed. Four main histological subtypes of EOC have been defined: serous, clear cell, endometrioid and mucinous [9]. Serous adenocarcinoma is the most frequently diagnosed EOC. Among serous tumors, low- versus high-grade tumors have different molecular mechanisms of pathogenesis, which suggests that the origin of invasive EOC could be different and remains to be defined [10]. More generally, EOC develops in the fallopian-ovarian area and spreads out into the peritoneal cavity as micrometastases. Depending on the progression of the disease, ovarian cancers are ranked in four categories (stages I, II, III, IV) as presented in Table I. EOC survival rate is dependent on the disease stage at the time of diagnosis (Figure 1). Due to the lack of specific symptoms and reliable ovarian cancer biomarkers, 75 % of EOC patients present with advanced disease when first diagnosed, which makes EOC the most lethal gynecologic malignancy. The excellent survival rates for women diagnosed at early stages provide a strong impetus to support efforts in developing strategies to identify the disease before it spreads outside the pelvis. Unfortunately, no routine screening test is yet available for the early detection of ovarian cancer. Tests such as the search for tumor markers, cancer antigen 125 (CA125), transvaginal ultrasonography and pelvic examination are not suitably specific or sensitive. The early detection of ovarian cancer remains an enormous challenge. Maximal cytoreductive surgery followed by platinum and/or taxane-based chemotherapy is the standard therapy for advanced EOC. Seventy-five percent of patients will show a complete clinical and biological response after the initial treatment. However, the majority

Cancer remains the second leading cause of death after cardiovascular disease. With surgery and/or radiotherapy, chemotherapy is the first-line treatment for cancer management. The main limitation of conventional chemotherapy is the non-specific distribution of drugs to both cancerous and healthy tissues, which leads to high toxicity and serious adverse effects. Furthermore, resistance to treatment is frequently encountered upon repeated exposure to the drug. Nanocarriers have emerged as a promising strategy for improving cancer therapy; for review, refer to [1, 2]. Nanocarriers or colloidal drug delivery systems include micelles, liposomes, organic or inorganic nanoparticles, solid lipid nanoparticles, nanogels and dendrimers in which drugs may be encapsulated; for review, refer to [3, 4]. After intravenous (i.v.) administration, colloidal carriers distribute rapidly and preferentially in organs of the reticuloendothelial system (RES). Surface modifications with the development of so-called stealth carriers with long circulating properties allow partial or complete avoidance of the RES. Subsequently, smart carriers able to specifically target the tumor by carrying recognition elements at the surface were designed to enhance the specificity of biodistribution of the carrier and thus of the drug. Ovarian cancer ranks fifth amongst the most fatal form of female cancers in Europe and the United States. It is the most lethal of gynecological malignancies. It is characterized by the silent development of the tumor in the ovarian/fallopian area followed by the dissemination of micrometastases in the peritoneal space. The development of new diagnostic and therapeutic approaches is mandatory to improve the outcome of the disease. Recent advances in our understanding of the molecular biology of cancer have been exploited to achieve more selective treatments. Actively targeted strategies seem best suited for this type of multifocal tumor. This review article proposes first to briefly present the characteristics and current treatment of ovarian cancer. Then, the strategy used to identify targets and the main advances obtained with targeted nanocarriers are presented.

Table I - Ovarian cancer staging according to the International Federation of Gynecology and Obstetrics staging system.

I. Ovarian cancer physiopathology

Ovarian cancer is the most common gynecologic cancer. Despite a high sensitivity to chemotherapeutic agents during fist-line treatment, it remains the fifth cause of cancer mortality in women [5-8]. Epithelial ovarian cancer (EOC) accounts for the vast majority of

Grading

Clinical symptoms

Stage I Stage II Stage III

limited to one or both ovaries pelvic extension or implants microscopic peritoneal implants outside of the pelvis; or limited to the pelvis with extension to the small bowel or omentum distant metastases to the liver or outside the peritoneal cavity

Stage IV

421

Nanocarriers for ovarian cancer active drug targeting F. Delie, E. Alléman, M. Cohen

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

5-year survival rate (%)

100

tage of the EPR effect to accumulate in tumor tissues and locally release chemotherapeutic agents. Active targeting takes advantage of the understanding of cancer cell biology and molecular signature by combining classical chemotherapeutic agents to specific ligands towards molecules expressed exclusively by tumor cells or endothelial cells in neovessels. This strategy is particularly attractive to reduce the high toxicity of current chemotherapeutic treatments, which exhibit unfavorable biodistribution to both cancerous and healthy tissues. Furthermore, this targeted approach is of greatest value when the cancer has spread as micrometastases, as encountered in ovarian cancer. Several strategies have been proposed including the direct combination of a drug with the targeting entity. Although attractive, this methodology implies direct and sometimes complex chemistry between both parts. Therefore, the use of a carrier is very appealing. The concept is based on the encapsulation of a chemotherapeutic agent in colloidal carriers that act as cargo, offering the advantage of a high drug payload capacity. The surface of the nanocarrier may be functionalized with specific ligands such as antibodies, aptamers, glycoproteins, lectins or peptides to promote the interaction with a defined target cell. Selection of the targeting moiety is driven by the cancer physiopathology. It may be versatile, targeting a wide variety of cancers by being directed, for example, towards a molecule involved in neovascular development or “nutrient-related”, or it may be very specific to a particular cancer type. Efforts have been made to identify specific ovarian tumor targets [20-22]. Active targeting of nanocarriers may be mediated via a direct approach when the ligand, also called the element of recognition, is directly linked to the surface of the carrier [23-25]. Sometimes, the conjugation chemistry may be difficult to perform or the ligand-to-target affinity is weak. Indirect targeting implying a multi-step process is, then, preferred. The multi-step approach generally takes advantage of the strong, although not covalent, affinity between biotin and avidin. This approach implies a “pretargeting” step where the recognition moiety associated to biotin is incubated or injected beforehand, and the drug associated with avidin is subsequently administered. A threestep strategy has also been proposed. A biotin-conjugated ligand is first administered followed by free avidin and finally, a biotinylated drug-loaded carrier is delivered. The different approaches developed for active targeting are schematically depicted in Figure 2. Only a few articles report the development of active targeting with nanocarriers for ovarian cancer. They are summarized in Table II. Several studies have focused on the use of folate as a targeting moiety. Folate receptors are overexpressed in a large variety of cancers, including ovarian, and are rarely found at the surface of healthy cells, making this a good recognition target for chemotherapeutic agents [26]. pH-sensitive doxorubicin (dox)-loaded mixed micelles were prepared with folate ligands at the surface to simultaneously target folate receptors

93

75

69

50

45 30

25

0

Localised

Regional

Distant

All stages

Stage of diagnosis

Figure 1 - Evolution of the five-year survival rate in patients diagnosed at different stages of ovarian cancer, adapted from [7, 47].

will relapse within 18 to 24 months. Maintenance treatments are less effective with the progressive resistance to drugs, which contributes to the poor five-year survival rate. The physiopathology and molecular biology of ovarian cancer development have been thoroughly studied in an effort to develop a tailored cancer therapy. New strategies that take advantage of typical biological or unique molecular features at the development site of the tumors have emerged. In general, cancer tissues are characterized by uncontrolled cell division and tissue growth. Therefore, cells in need of nutrients overexpress receptors such as transferrin receptors, folate receptors and LDL receptors. Rapidly growing tumors will also trigger angiogenesis, the formation of new blood vessels. Due to the urgent demand for blood supply, the architecture of the neovasculature is disorganized and leaky. Indeed, endothelial cells display numerous fenestrations with intercellular pores as wide as 300 nm and up to 1 µm in certain cases. As the lymphatic drainage of the tumor is usually impaired, and thus less efficient than in healthy tissue, it causes the retention of large molecules and small particles such as macromolecules or nanocarriers. This phenomenon is termed the enhanced permeability and retention (EPR) effect. In association with neovascularization, endothelial cells at the site of tumor development overexpress specific cellular biomarkers, such as epidermal growth factor receptor (EGFR) and integrins (e.g. αvβ3, αvβ5), which represent potential targets for cancer therapy [11, 12]. Depending on the nature and the origin of the tumor, cells will express specific elements at their membranes, such as the HER2/ neu (also known as erbB-2, cd340) receptors in prostate, breast and ovarian cancers; CA125 in ovarian, endometrial, fallopian tube, lung, breast and gastrointestinal cancers; prostate specific membrane antigen (PSMA) in prostate cancer [13]; and GRP-78 in many type of cancers [14]. Efforts to improve the poor long-term outcome of patients with advanced EOC have generated a variety of approaches with the aim of increasing the response to first-line treatment or maintenance therapy. New strategies in cancer therapy have moved away from non-specific cytotoxic drugs to innovative targeted therapies. Targeted therapy is a novel approach aimed at blocking cancer development by interfering with specific target molecules involved in carcinogenesis and tumor growth. Several reviews have recently been published on this topic [6, 15-19].

II. Active targeting strategies for ovarian cancer

Several strategies based on the accepted knowledge of cancer physiopathology have been designed to improve tumor management. Passive targeting based on the mere physico-chemical properties (mainly the size) of the molecules or nanocarriers will take advan-

Figure 2 - Schematic representations of direct and indirect (via two or three steps protocol) active targeting strategies. 422

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

Nanocarriers for ovarian cancer active drug targeting F. Delie, E. Alléman, M. Cohen

Table II - Targeted nanocarriers for the treatment or diagnosis of ovarian cancer. Targeting ligand

Active compound(s)

Nanocarrier Mean size (nm)

Methods Cell lines

Ref.

Folate

Doxorubicin

pH sensitive mixed micelles 150

In vitro/in vivo A2780/DOXr and KB cells

[27]

Folate

Gene therapy, pDNA

Trimethylchitosan NP 130 to 480 depending on N/P ratio

In vitro SKOV-3, KB, A549, NIH3T3

[28]

Folate

90

ChemoRads 75

In vitro/in vivo SKOV-3, OVCAR-3

[29]

RGD

SiRNA

Chitosan NP 200

In vitro/in vivo SKOV-3 ip1, HeyA8, A2780

[32]

EGFR specific peptide

Paclitaxel, lonidamine

PLGA-PEG/PCL NP 140

In vitro 9 cell lines among which: SKOV-3, OVCAR-5

[33]

OV-TL-3

14

C-sucrose

Liposomes ND

In vitro/in vivo OVCAR-3

[35]

OV-TL-3

Doxorubicin

Liposomes ND

In vitro/in vivo OVCAR-3

[36]

Herceptin

Paclitaxel

PLA-NP 240

In vitro/in vivo SKOV-3

[38]

Herceptin

Doxorubicin

PLGA-NP 210

In vitro SKOV-3, MES-SA, Dx5

[39]

Anti-Her2 antibody

Imaging and photothermal agents

Gold nanocomplex 185

In vitro OVCAR-3, MDA-MB-231

[40]

Biotin

Fluorescent tag

Dendrimers ND

In vitro OVCAR-3, HEK2938

[34]

Biotin/CA 125

FITC, 125I

Liposomes 110

In vitro SKOV-3, OVCAR-3

[44]

Biotin/CA 125

Paclitaxel

Poloxamer-PLA NP 190

In vitro SKOV-3, OVCAR-3

[45]

Y, paclitaxel

pDNA: plasmid DNA, NP: nanoparticle, PLA: poly(lactic acid), PLGA: Poly(lactic-co-glycolic), RGD: arginine-glycine-aspartic acid peptide sequence, siRNA : small interfering RNA, FITC: fluorescein isothiocyanate, N/P ratio: ratio between the DNA and polymer used in the preparation, ND: not documented.

drug delivery is the possibility of co-encapsulating drug molecules that may act synergistically in the same carrier. Folate-targeted NPs were designed to deliver paclitaxel (Tx) as a direct chemotherapeutic agent and 90Y as a radiotherapeutic compound [29]. Cytotoxicity was always higher with folate-coated particles compared to uncoated carriers; however, it was significantly better when particles were loaded with both therapeutic entities. This was confirmed in vivo in a peritoneal metastasis model after i.v. administration of the different formulations. In a survival study, the targeted particles loaded with both agents were the most effective treatment. Unfortunately, no significant difference between the functionalized and the control particles was observed in vivo, most likely due to the rather small sample size. Developing new strategies against folate is a valuable tool. Indeed, folate receptors are expressed on different types of cancer cells. Therefore, carriers designed to target this receptor would be versatile and could be used for different patients. Ovarian cancer is characterized by the presence of peritoneal metastases, and no evidence of efficacy has been provided in a relevant in vivo model with use of the folate receptor approach. The αvβ3 integrin is overexpressed in a number of different tumors as well as in activated endothelial cells but absent in normal tissue. The amino acid sequence Arginine-Glycine-Aspartic acid (RGD peptide) binds with high affinity to integrins. Therefore, it has been used as a recognition element in numerous drug delivery systems [30] and therapeutic approaches for ovarian cancer [31]. For example, it was linked to chitosan (Ch) to deliver siRNA to ovarian cancer cells (SKOV-3 ip1, HeyA8, A2780, A2780ip2 and MOEC) with different levels of αvβ3 integrin expression [32]. In the αvβ3-deficient cell

and favor the intracellular release of the drug in multi-drug-resistant (MDR) cells [27]. These constructs were designed to target cancer cells and circumvent the efflux pump by inducing endosomal release. pH-sensitive micelles proved to be more efficient than pH-insensitive micelles and free dox. To assess biodistribution and the extent of extravasation of the drug into the tumor, ovarian cancer cells were grown in a skin-fold window chamber model in mice. Although it was not quantitative, this method showed that dox-loaded micelles extravasated later than free dox. The intensity of the effect lasted longer with the targeted formulation. The direct measurement of dox distribution in tissues showed that the targeted formulations significantly increased tumoral concentration by 10-fold for the pH-sensitive formulation and 4-fold for the pH-insensitive formulation, as compared to free dox. Furthermore, dox accumulation in the heart was reduced by either of the two micelle formulations. In another study, folate-conjugated Ntrimethyl chitosan was synthesized to obtain DNA delivery colloidal systems after cross-linking with sodium alginate [28]. The efficiency of these particles was assessed in SKOV-3 ovarian cancer cells and KB cells, an oral squamous cell carcinoma cell line. Both cell lines overexpress the folate receptor. A549 and NIH 3T3, folate receptordeficient cell lines of human lung carcinoma and mouse embryo fibroblast origin, respectively, were used as controls. The presence of folate at the surface of the particles significantly increased the cellular uptake of the complexes. The specific involvement of folate receptors was demonstrated by an inhibition assay in the presence of an excess of free folate in the medium. Transfection efficiency was increased with folate-modified carriers in KB and SKOV-3 cells, whereas it remained low in control cells. Another advantage of using carriers for 423

Nanocarriers for ovarian cancer active drug targeting F. Delie, E. Alléman, M. Cohen

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

lines, no binding of the particles was observed, whereas dose-related binding was observed in SKOV-3 cells expressing the integrin. The periostin silencing activity of the system was assessed after the encapsulation of anti-periostin siRNA. Periostin is a protein involved in cell invasion, survival and angiogenesis, which plays a role in the metastatic potential of cancer cells. SKOV-3 ip1 and A2780 cells were injected by intraperitoneal (i.p.) administration in mice. Then, 24 h after a single administration of either Ch-NP or RGD-Ch-NP, the animals were sacrificed. The expression of periostin in the tumor was reduced by 51 % in the targeted-treated mice and by 20 % in the Ch-NP-treated animals. Therapeutic efficacy was further tested using different delivery systems associated or not with the chemotherapeutic agent docetaxel. Anti-periostin siRNA in Ch-NP-RGD was more efficient than the irrelevant siRNA counterpart and the non-targeted carrier. The most effective treatment associated the relevant targeted carrier with free docetaxel. In the A2780 model, the targeted system was efficient; however, no significant inhibition was observed when compared to the control siRNA-Ch-NP RGD carrier. Other biomarkers overexpressed in stressed cells, such as cancer cells, are the growth factors (particularly EGFR) often associated with aggressive cancers. The poly-lactide-co-glycolide-PEG polymer was conjugated with a peptide-recognizing EGFR. Then, it was introduced in the formulation of active targeted NP prepared in the presence of poly(lactic-co-glycolic acid)-PEG (PLGA-PEG) and poly(epsiloncaprolactone) in the respective mass ratio of 20/10/70 [33]. Tx and lonidamine were used as chemotherapeutic agents. EGFR-targeted NPs were more efficiently taken up by the cells overexpressing EGFR than were non-targeted systems. The most effective cytotoxic effect was obtained when both drugs were associated in EGFR-targeted nanocarriers. In vivo confirmation is required to appreciate the value of this targeting approach. Biotin is an essential nutrient for cell growth and development. As it is not synthesized in vivo, organisms depend on exogenous sources. Cancer cells with a higher rate of division have a high avidity for biotin. Therefore, it is a good candidate as a ligand that would be ideal for reaching tumors. Biotinylated dendrimers were prepared and loaded with fluorescent dyes to follow their uptake and cellular fate after incubation with OVCAR-3 cells, an ovarian cancer cell line, and HEK 293T cells, a human embryonic kidney cell line [34]. Dendrimers are synthetic, three-dimensional constructs with a treelike structure. Dendrimers are formed using a nano-scale, multi-step synthesis process. Each synthesis step results in a new “generation” that has twice the complexity of the previous generation. With their multiple branches, dendrimers provide versatile conjugation opportunities. With each synthesis of a new generation, the number of biotin molecules bound to the dendrimers increased, and the cellular uptake was related to the amount of biotin. The presence of free biotin in the medium inhibited uptake of the targeted carriers at low concentrations; however, at higher concentrations, it did not. Therefore, the involvement of both adsorptive and biotin-receptor dependent mechanisms was suggested. These carriers are also presented as interesting for a pretargeting strategy for further in vivo study. Nassander et al. demonstrated the specific targeting of immunoliposomes in vitro and in vivo using OV-TL 3 antibodies directed against the antigen OA3, which is present at the cell surface of more than 90 % of all human ovarian carcinomas [35]. After i.p. administration, targeted radiolabeled liposomes localized to OVCAR-3 cells more rapidly and in a greater number. More than 80 % of the injected dose was still associated with the tumor after 24 h, whereas only 10 % of the dose remained associated when the carrier was not targeted. The same liposomes loaded with dox were tested in a follow-up study to assess their antitumor efficiency [36]. Targeted liposomes were more toxic to OVCAR-3 cells in vitro than the control untargeted formulation. In vivo, after i.p. administration, dose-dependent efficiency was reported for both types of liposomes; however, no difference was observed

between antibody-coated and uncoated liposomes. The absence of a difference was most probably due to rapid and premature release of the drug from the liposome before reaching the target. More rigid and more solid liposomes were formulated and tested, but they were less efficient in targeting the tumor than the liposomes investigated previously. Her-2, Human Epidermal Growth Factor Receptor-2, is overexpressed in some patients diagnosed with breast or ovarian cancers. Herceptin comprises anti-Her-2 monoclonal antibodies and is a potent drug approved for the treatment of breast and gastric cancer patients. Hapca-Cirstoiu et al. have demonstrated the efficacy of a Tx-loaded NP surface functionalized with Herceptin (NPs Tx HER). These particles were internalized in SKOV-3 cells expressing Her-2 but not in Daudi cells, a human lymphoma cell line, which did not express Her-2 [37]. The efficacy of these immunoparticles was evaluated by bioluminescence imaging and survival rate in a disseminated xenograft ovarian cancer model induced by the i.p. inoculation of SKOV-3 cells [38]. The bioluminescence study clearly showed the superior anti tumor activity of NPs Tx HER as compared to free Tx. As a confirmation, a significantly longer survival rate was observed for mice treated with NPs Tx-HER compared to free Tx, Herceptin alone or Tx-loaded nanoparticles functionalized with an irrelevant mAb (Mabthera, rituximab). The biodistribution pattern of Tx was assessed on healthy and tumor-bearing mice after i.v. or i.p. administration. An equivalent biodistribution profile was observed in healthy mice for Tx encapsulated in either non-coated nanoparticles (NPs-Tx) or in NPs Tx HER after i.v. or i.p. injections, except for a reduced drug accumulation in lungs when formulations were administered i.p. Encapsulated Tx accumulated preferentially in the RES related organs, such as liver and spleen, probably due to uptake of the nanoparticles by phagocytic cells, whereas free Tx had a non-specific distribution in all tested organs. Compared to free Tx, a single injection of encapsulated Tx in tumor-bearing mice increased tumor accumulation. However, no difference in overall tumor accumulation between NPs Tx HER and NPs-Tx was observed. This work and the interest of targeting Her-2 were confirmed with dox-loaded PLGA immunoparticles, which induced higher toxicity compared to free dox or uncoated particles [39]. Ovarian cancer is frequently called the silent killer because the disease is already at advanced stages when it is usually detected and the chances of a successful treatment are low. Therefore substantial effort is invested in the attempt to develop tools for early diagnosis. Chen et al. have engineered gold nanoparticles for infrared imaging coupled with a photothermal agent [40]. This strategy is known as the theranostic approach due to the combination of diagnostic and therapeutic approaches. In this study, the targeting ligand was a rabbit-specific antibody against the HER2(c-erbB-2)/HER-2/neu epitope. Proof of concept that the particles targeted Her-2 expressing cells and did not interact with non-expressing cells was obtained in vitro. Therefore, only the targeted cells may be visualized and further killed by thermal ablation when activated by a near-infrared laser. However, these encouraging results still need to be confirmed in vivo. These studies using an anti-Her-2 approach have demonstrated the value of targeting for the diagnosis and treatment of ovarian tumors. Nevertheless, Her2 is also expressed in certain healthy cells and is rather erratically expressed in ovarian cancer cells; 10 to 60 % of cases, depending on the study [5, 6]. Although these studies provided irrefutable in vitro and in vivo proof of concept, Her-2 might not be the most suitable target for ovarian cancer. This is also supported by the fact that ovarian cancer treatments based on Herceptin were not successful [41]. Specific cell surface ovarian cancer proteins are particularly interesting for combining targeting and targeted therapy. Glucose regulated protein 78 (GRP78) has been abundantly found at the cell surface in ovarian cancer cells [20]. The interaction of cell surface GRP78 with antibodies directed against the COOH-terminal domain of GRP78 was shown to suppress Ras/MAPK and PI3-kinase/AKT signaling 424

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

Nanocarriers for ovarian cancer active drug targeting F. Delie, E. Alléman, M. Cohen

while promoting caspase activation in human prostate cancer cells [42]. Moreover, GRP78 autoantibodies purified from ovarian cancer patients’ sera were shown to increase the response to the drug and decrease the invasiveness of ovarian cancer cells [43]. Thus, the use of antibodies against this protein on nanocarriers could both target cancer cells and inhibit tumor development. As proof of concept, we recently developed Tx-loaded NP coated with commercial antibodies against the C-terminus portion of glucose-regulated proteins (GRPs) [44]. These NP exhibited increased binding to cells compared to uncoated nanoparticles with a limited internalization rate. Nevertheless, they significantly increased the sensitivity of Bg-1, an ovarian cancer cell line, to the drug compared to other treatments (free paclitaxel, unloaded carrier or uncoated NP). These observations confirm the value of targeting GRP78 to increase drug accumulation and the magnitude of the response in ovarian cancer cells. Xiao et al. used a three-step indirect targeting strategy to improve the efficacy of anti-cancer agents in radioimmunotherapy [45]. As a first step, biotinylated anti-CA125 antibodies were incubated with cells. Avidin was then added and the excess washed away before incubation with radionuclide-loaded biotinylated liposomes. A two step strategy was also proposed involving BsMAb, antibodies presenting both anti-CA125 and anti-biotin paratopes and thereby avoiding the step of avidin recognition. This study demonstrated that biotinylated liposomes could specifically bind to pre-labeled NIH-OVCAR3 cells expressing CA125. Non-pre-labeled cells did not interact with the liposomes. A three-step approach with biotinylated poloxamer/PLA nanoparticles loaded with paclitaxel and targeted against CA125 was also developed [46]. The higher toxicity of the targeted system observed in OVCAR3 cells and not in SKOV-3 cells confirmed the interest of this approach. Although promising, the indirect targeting procedure has not been demonstrated yet in vivo for ovarian cancer therapy. 

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

12.

*

13.

Epithelial ovarian cancer is characterized by asymptomatic growth and the subsequent presence of multiple micrometastases in the peritoneum, which leads to high mortality. It appears, therefore, as a motivating pathology to develop targeted drug delivery systems to bring the right drug to the right place. The ultimate goals of this strategy are to reduce the adverse effects of chemotherapeutic agents and to improve drug distribution at the tumor site. Better understanding of the molecular, cellular and clinical biology of ovarian cancer has allowed researchers to define relevant targets either specific to EOC or less specific, related to cancer development in general. The second approach is appealing because it would provide more versatile tools that would be useful for the management of other cancers. Nanocarriers offering a high loading rate with the possibility of co-encapsulating synergistic drugs and a large surface area available for ligand coupling may be designed as a combinatorial approach associating active targeting and targeted therapy. The available data confirms the efficacy of active targeting via nanocarriers. However, further research will be required before these tools can be applied in a clinical setting. Many issues have to be considered. The first is the safety of nanosystems. Despite some encouraging data obtained from animal models, caution is needed when translating data to a clinical setting. The second issue is the transfer of technology from a bench to a manufacturing scale. Despite the advantages expected with the use of NPs for active targeting, no active targeted nanosystem has been commercialized. No data are available regarding the comparison of versatile and specific targets. Therefore, whether the trend should evolve towards versatile systems applicable to different types of cancers or more to specific targets remains to be elucidated.

14.

15. 16. 17. 18. 19. 20.

21.

22.

23.

425

Brigger I., Dubernet C., Couvreur P. - Nanoparticles in cancer therapy and diagnosis. - Adv Drug Del Rev., 54, 631-651, 2002. Heidel J.D., Davis M.E. - Clinical developments in nanotechnology for cancer therapy. - Pharm Res., 28, 187-199, 2011. Sunderland C.J., Steiert M., Talmadge J.E., Derfus A.M., Barry S.E. - Targeted nanoparticles for detecting and treating cancer. - Drug Develop.Res., 67, 70-93, 2007. Emerich D.F., Thanos C.G. - Targeted nanoparticle-based drug delivery and diagnosis. - J. Drug Target., 15, 163-183, 2007. Markman M. - The promise and perils of 'targeted therapy' of advanced ovarian cancer. - Oncology, 74, 1-6, 2008. Tagawa T., Morgan R., Yen Y., Mortimer J. - Ovarian cancer: opportunity for targeted therapy. - J. Oncol., doi:10.1155/2012/682480, 2012. Jemal A., Siegel R., Xu J., Ward E. - Cancer statistics, 2010. CA Cancer J. Clin., 60, 277-300, 2010. Pignata S., Cannella L., Leopardo D., Pisano C., Bruni G.S., Facchini G. - Chemotherapy in epithelial ovarian cancer. - Cancer Lett., 303, 73-83, 2011. Kurman R.J., Shih Ie M. - Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer--shifting the paradigm. - Hum Pathol., 42, 918-931, 2011. Bonome T., Lee J.Y., Park D.C., Radonovich M., Pise-Masison C., Brady J., Gardner G.J., Hao K., Wong W.H., Barrett J.C., Lu K.H., Sood A.K., Gershenson D.M., Mok S.C., Birrer M.J. - Expression profiling of serous low malignant potential, lowgrade, and high-grade tumors of the ovary. - Cancer Res., 65, 10602-10612, 2005. Altintas I., Kok R.J., Schiffelers R.M. - Targeting epidermal growth factor receptor in tumors: from conventional monoclonal antibodies via heavy chain-only antibodies to nanobodies. - Eur. J. Pharm. Sci., 45, 399-407, 2012. Yoncheva K., Momekov G. - Antiangiogenic anticancer strategy based on nanoparticulate systems. - Expert Opin. Drug Deliv., 8, 1041-1056, 2011. Sanna V., Sechi M. - Nanoparticle therapeutics for prostate cancer treatment. - Maturitas, 2012. Sato N., Matsuda K., Sakuma C., Foster D.N., Oppenheim R.W., Yaginuma H. - Regulated gene expression in the chicken embryo by using replication-competent retroviral vectors. - J. Virol., 76, 1980-1985, 2002. Bovicelli A., D'Andrilli G., Giordano A. - New players in ovarian cancer. - J. Cell Physiol., 226, 2500-2504, 2011. Kalachand R., Hennessy B.T., Markman M. - Molecular targeted therapy in ovarian cancer: what is on the horizon? Drugs, 71, 947-967, 2011. Suh D.H., Kim J.W., Kim K., Kang S.B. - Major clinical research advances in gynecologic cancer in 2010. - J. Gynecol. Oncol., 21, 209-218, 2010. Banerjee S., Kaye S. - The role of targeted therapy in ovarian cancer. - Eur. J. Cancer, 47, Suppl. 3, S116-130, 2011. Campos S.M., Ghosh S. - A current review of targeted therapeutics for ovarian cancer. - J. Oncol., doi:10.1155/2010/149362, 2010. Shin B.K., Wang H., Yim A.M., Le Naour F., Brichory F., Jang J.H., Zhao R., Puravs E., Tra J., Michael C.W., Misek D.E., Hanash S.M. - Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. - J. Biol. Chem., 278, 7607-7616, 2003. Springer D.L., Auberry D.L., Ahram M., Adkins J.N., Feldhaus J.M., Wahl J.H., Wunschel D.S., Rodland K.D. - Characterization of plasma membrane proteins from ovarian cancer cells using mass spectrometry. - Dis. Markers, 19, 219-228, 2003. Sasaroli D., Gimotty P.A., Pathak H.B., Hammond R., Kougioumtzidou E., Katsaros D., Buckanovich R., Devarajan K., Sandaltzopoulos R., Godwin A.K., Scholler N., Coukos G. - Novel surface targets and serum biomarkers from the ovarian cancer vasculature. - Cancer Biol. Ther., 12, 169-180, 2011. Nobs L., Bucheger F., Gurny R., Allemann E. - Current methods for attaching targeting ligands to liposomes and nanoparticles. - J. Pharm. Sci., 93, 1980-1992, 2004.

Nanocarriers for ovarian cancer active drug targeting F. Delie, E. Alléman, M. Cohen

J. DRUG DEL. SCI. TECH., 22 (5) 421-426 2012

24. 25. 26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Nobs L., Buchegger F., Gurny R., Allemann E. - Biodegradable nanoparticles for direct or two-step tumor immunotargeting. Bioconjug. Chem., 17, 139-145, 2006. Trapani G., Denora N., Trapani A., Laquintana V. - Recent advances in ligand targeted therapy. - J. Drug Target., 20, 1-22, 2012. Sudimack J., Lee R.J. - Targeted drug delivery via the folate receptor. - Adv. Drug Deliv. Rev., 41, 147-162, 2000. Kim D., Gao Z.G., Lee E.S., Bae Y.H. - In vivo evaluation of doxorubicin-loaded polymeric micelles targeting folate receptors and early endosomal pH in drug-resistant ovarian cancer. - Mol. Pharm., 2009. Zheng Y., Cai Z., Song X., Yu B., Bi Y., Chen Q., Zhao D., Xu J., Hou S. - Receptor mediated gene delivery by folate conjugated N-trimethyl chitosan in vitro. - Int. J. Pharm., 382, 262-269, 2009. Werner M.E., Karve S., Sukumar R., Cummings N.D., Copp J.A., Chen R.C., Zhang T., Wang A.Z. - Folate-targeted nanoparticle delivery of chemo- and radiotherapeutics for the treatment of ovarian cancer peritoneal metastasis. - Biomaterials, 32, 85488554, 2011. Temming K., Schiffelers R.M., Molema G., Kok R.J. - RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. - Drug Resist. Updat., 8, 381-402, 2005. Gamble L.J., Borovjagin A.V., Matthews Q.L. - Role of RGDcontaining ligands in targeting cellular integrins: Applications for ovarian cancer virotherapy (Review). - Exp. Ther. Med., 1, 233-240, 2010. Han H.D., Mangala L.S., Lee J.W., Shahzad M.M., Kim H.S., Shen D., Nam E.J., Mora E.M., Stone R.L., Lu C., Lee S.J., Roh J.W., Nick A.M., Lopez-Berestein G., Sood A.K. - Targeted gene silencing using RGD-labeled chitosan nanoparticles. - Clin. Cancer Res., 16, 3910-3922, 2010. Milane L., Duan Z., Amiji M. - Development of EGFR-targeted polymer blend nanocarriers for combination paclitaxel/lonidamine delivery to treat multi-drug resistance in human breast and ovarian tumor cells. - Mol. Pharm., 8, 185-203, 2011. Yellepeddi V.K., Kumar A., Palakurthi S. - Biotinylated poly(amido) amine (PAMAM) dendrimers as carriers for drug delivery to ovarian cancer cells in vitro. - Anticancer Res., 29, 2933-2943, 2009. Nassander U.K., Steerenberg P.A., Poppe H., Storm G., Poels L.G., De Jong W.H., Crommelin D.J. - In vivo targeting of OV-TL 3 immunoliposomes to ascitic ovarian carcinoma cells (OVCAR-3) in athymic nude mice. - Cancer Res., 52, 646-653, 1992. Vingerhoeds M.H., Steerenberg P.A., Hendriks J.J., Dekker L.C., Van Hoesel Q.G., Crommelin D.J., Storm G. - Immunoliposomemediated targeting of doxorubicin to human ovarian carcinoma in vitro and in vivo. - Br. J. Cancer, 74, 1023-1029, 1996. Cirstoiu-Hapca A., Bossy-Nobs L., Buchegger F., Gurny R., Delie F. - Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles. - Int. J. Pharm., 331, 190-196, 2007. Cirstoiu-Hapca A., Buchegger F., Lange N., Bossy L., Gurny R., Delie F. - Benefit of anti-HER2-coated paclitaxel-loaded immuno-nanoparticles in the treatment of disseminated ovarian cancer: therapeutic efficacy and biodistribution in mice. - J. Control. Release, 144, 324-331, 2010. Lei T., Srinivasan S., Tang Y., Manchanda R., Nagesetti A., Fernandez-Fernandez A., McGoron A.J. - Comparing cellular uptake and cytotoxicity of targeted drug carriers in cancer cell lines with different drug resistance mechanisms. - Nanomedicine, 7, 324-332, 2011.

40. 41. 42.

43.

44.

45.

46.

47.

Chen W., Bardhan R., Bartels M., Perez-Torres C., Pautler R.G., Halas N.J., Joshi A. - A molecularly targeted theranostic probe for ovarian cancer. - Mol. Cancer Ther., 9, 1028-1038, 2010. Wilken J.A., Webster K.T., Maihle N.J. - Trastuzumab sensitizes ovarian cancer cells to EGFR-targeted therapeutics. - J. Ovarian Res., 3, 7, 2010. Misra U.K., Pizzo S.V. - Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells. - Cancer Biol. Ther., 9, 142-152, 2010. Cohen M., Petignat P. - Purified autoantibodies against glucoseregulated protein 78 (GRP78) promote apoptosis and decrease invasiveness of ovarian cancer cells. - Cancer Lett., 309, 104109, 2011. Delie F., Ribaux P., Petignat P., Cohen M. - Anti-KDEL-coated nanoparticles: a promising tumor targeting approach for ovarian cancer? - Biochimie, in press, 2012 June 17 [Epub ahead of print]. Xiao Z., McQuarrie S.A., Suresh M.R., Mercer J.R., Gupta S., Miller G.G. - A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro. - J. Biotechnol., 94, 171-184, 2002. Xiong X.Y., Gong Y.C., Li Z.L., Li Y.P., Guo L. - Active targeting behaviors of biotinylated pluronic/poly(lactic acid) nanoparticles in vitro through three-step biotin-avidin interaction. - J. Biomater. Sci. Polym. Ed., 22, 1607-1619, 2011. Cannistra S.A. - Cancer of the ovary. - N. Engl. J. Med., 351, 2519-2529, 2004.

Abbreviations CA125: cancer antigen 125 Ch: chitosan Dox: doxorubicin EOC: epithelial ovarian cancer EPR: enhanced permeability and retention EGFR: epidermal growth factor receptor i.p.: intraperitoneal i.v.: intravenous MDR: multidrug resistance NP: nanoparticle PEG: poly(ethylene glycol) PLA: poly(D,L-lactic acid) PLGA: poly(lactic-co-glycolic acid) RES: reticulo-endothelial system Tx: paclitaxel VEGF: vascular endothelial growth factor

Acknowledgments The authors are very grateful to Dr. Magali Zeisser-Labouèbe for critical reading of the manuscript and her pertinent suggestions.

Manuscript Received 12 March 2012, accepted for publication 11 May 2012.

 

 

426