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Therapeutics, Targets, and Chemical Biology

“OA02” Peptide Facilitates the Precise Targeting of Paclitaxel-Loaded Micellar Nanoparticles to Ovarian Cancer In Vivo

Kai Xiao, Yuanpei Li, Joyce S. Lee, Abby M. Gonik, Tiffany Dong, Gabriel Fung, Eduardo Sanchez, Li Xing, Holland R. Cheng, Juntao Luo and Kit S. Lam
DOI: 10.1158/0008-5472.CAN-11-3883 Published April 2012

Abstract

Micellar nanoparticles based on linear polyethylene glycol (PEG) block dendritic cholic acids (CA) copolymers (telodendrimers), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers, are reported. The micellar nanoparticles have been decorated with a high-affinity “OA02” peptide against α-3 integrin receptor to improve the tumor-targeting specificity which is overexpressed on the surface of ovarian cancer cells. “Click chemistry” was used to conjugate alkyne-containing OA02 peptide to the azide group at the distal terminus of the PEG chain in a representative PEG5k-CA8 telodendrimer (micelle-forming unit). The conjugation of OA02 peptide had negligible influence on the physicochemical properties of PEG5k-CA8 nanoparticles and as hypothesized, OA02 peptide dramatically enhanced the uptake efficiency of PEG5k-CA8 nanoparticles (NP) in SKOV-3 and ES-2 ovarian cancer cells via receptor-mediated endocytosis, but not in α-3 integrin-negative K562 leukemia cells. When loaded with paclitaxel, OA02-NPs had significantly higher in vitro cytotoxicity against both SKOV-3 and ES-2 ovarian cancer cells as compared with nontargeted nanoparticles. Furthermore, the in vivo biodistribution study showed OA02 peptide greatly facilitated tumor localization and the intracellular uptake of PEG5k-CA8 nanoparticles into ovarian cancer cells as validated in SKOV3-luc tumor–bearing mice. Finally, paclitaxel (PTX)-loaded OA02-NPs exhibited superior antitumor efficacy and lower systemic toxicity profile in nude mice bearing SKOV-3 tumor xenografts, when compared with equivalent doses of nontargeted PTX-NPs as well as clinical paclitaxel formulation (Taxol). Therefore, OA02-targeted telodendrimers loaded with paclitaxel have great potential as a new therapeutic approach for patients with ovarian cancer. Cancer Res; 72(8); 2100–10. ©2012 AACR.

Introduction

Ovarian cancer is the ninth most common cancer, with an estimated 22,280 new cases in 2012, but is the fifth most deadly, with an estimated 15,500 deaths in 2012 (1). The standard treatment for patients with advanced-stage disease usually involves surgical staging and debulking followed by adjuvant chemotherapy, typically with platinum and paclitaxel (PTX). However, the more extensive use of chemotherapeutic drugs such as paclitaxel is often limited by its severe side effects, including hypersensitivity reactions, myelosuppression, and neurotoxicity, which may be attributed to their nonspecific systemic organ distribution and inadequate intratumor concentrations, resulting in suboptimal efficacy (2, 3). Despite the intensive chemotherapy, more than 70% of patients with ovarian cancer will suffer from disease relapse or recurrence, and ultimately die of this disease. Therefore, there is a tremendous incentive to refine existing treatment modalities to avoid or delay the recurrence and to treat recurrent ovarian cancer more effectively. Optimization of chemotherapeutic drug delivery is among the critical approaches to improve the therapeutic index of cytotoxic agents.

Nanotechnology is an emerging field that has shown great promise in the development of novel diagnostic and therapeutic agents for a variety of diseases, including cancers (4). As the vasculature in tumors is known to be leaky, and the tumor lymphatic system is also deficient, nanoparticles can preferentially accumulate in the tumor site via the enhanced permeability and retention (EPR) effects (5). Polymeric micelles represent one of the most promising nanocarriers due to their unique core-shell structure formed by amphiphilic block copolymers, which could facilitate the solublization of poorly soluble drugs and protect the drugs from degradation and metabolism. We have recently developed a series of novel linear dendritic block copolymers (telodendrimers) comprising polyethylene glycol (PEG) and dendritic cholic acids (CA), which can encapsulate high concentrations of hydrophobic drugs such as paclitaxel and self-assemble to form stable core-shell micelles under aqueous condition (6–12). The representative PEG5k-CA8 micellar nanoparticles possess the ideal properties for drug delivery, including high drug loading capacity, optimal particle size (20–60 nm), outstanding stability (more than 6 months at 4ºC), and sustainable drug release profile. Paclitaxel-loaded PEG5k-CA8 nanoparticles have been shown to exhibit superior antitumor efficacy and toxicity profile than free drug (Taxol) and paclitaxel/human serum albumin nanoaggregate (Abraxane) at equivalent paclitaxel doses, in nude mice bearing human ovarian cancer (SKOV-3) xenografts (6).

To further facilitate the residence, penetration, and cancer cell uptake of delivered drugs within the tumor sites for more efficient cancer treatment, an attractive approach is to decorate the nanoparticles surface with targeting ligands that specifically recognize receptors on cancer cells (active targeting). Active targeting might result in higher retention of nanoparticles drugs at tumor sites (i.e., by reducing passive transport away from tumor) and enhanced uptake of the drugs by cancer cells via receptor-mediated endocytosis (13–15). Furthermore, actively targeted nanoparticles have also shown the potential to overcome multidrug resistance via bypassing of P-glycoprotein–mediated drug efflux (16). Combining passive and active targeting in a single platform will further improve the therapeutic index of nanocarrier delivered drugs (17, 18). A wide variety of targeting ligands, including antibodies and single-chain Fv fragment (19, 20), peptides (21, 22), small molecules (23), and aptamers (24, 25) have been used with varying degrees of success to functionalize nanoparticles for their potential application in targeted cancer therapy. Although antibodies or antibody fragments are effective as targeting agents, there are some innate problems such as decreased receptor affinity as a result of conjugation methods, potential immunogenicity, nonspecific uptake by reticuloendothelial system, and relative poor stability (17). In contrast, peptides or peptidomimetics with high binding affinity and specificity to cancer cells may have many favorable characteristics, including deep tumor penetration due to the smaller size, lack of immunogenicity, easy synthesis and scale-up, and good stability especially if d-configuration and unnatural amino acids are used (26).

Integrins are a family of heterodimeric transmembrane glycoproteins involved in a wide range of cell-to-extracellular matrix (ECM) and cell-to-cell interactions (27, 28). It has been found that integrins are overexpressed on various cell types such as angiogenic endothelial cells and certain cancer cells. For example, α-3 integrin is overexpressed in several types of cancers, especially ovarian cancer, breast cancer, and melanoma (29). The overexpression of α-3 integrin on these cancer cells has been exploited as a promising pharmacologic target for the selective drug delivery in the treatment of these cancers. In addition, during the cell locomotion and migration, integrins can undergo endocytosis after the activation with anchoring ligands, which may facilitate the intracellular delivery of nanoparticles drugs into cancer cells, when these nanoparticles are decorated with integrin-targeting ligands. A high-affinity α-3 integrin-targeting peptide “OA02” has been identified in our laboratory through screening one-bead one-compound (OBOC) combinatorial peptide libraries (30). This “OA02” peptide has been shown to bind strongly to α-3 integrin–overexpressing ovarian cancer cells and specifically target ovarian cancer xenografts (ES-2) in nude mice when conjugated to near-infrared fluorescence (NIRF) dyes (30).

In the present study, we hypothesize that the incorporation of “OA02” peptide ligand onto our newly developed micellar nanoparticles (NP) will facilitate the precise homing of drug payload to α-3 integrin–overexpressing ovarian cancer cells. First, the alkyne-modified “OA02” peptide was synthesized and conjugated to the azide-functionalized PEG5k-CA8 telodendrimer via copper-catalyzed cyloaddition (“click chemistry”). Then, the binding specificity, uptake efficiency, and in vivo tumor–targeting property of fluorescence-labeled OA02-NPs were evaluated in human ovarian cancer cells and xenograft mouse model, respectively. Finally, the antitumor effect of paclitaxel-loaded OA02-NPs against ovarian cancer was studied both in vitro and in vivo.

Materials and Methods

Materials

Diamino PEG was purchased from Rapp Polymere. Cy5.5 Mono NHS ester was purchased from Amersham Biosciences. Hydrophobic fluorescence dye DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate, D-307), 4′,6-diamidino-2-phenylindole (DAPI), and LysoTracker Red were purchased from Invitrogen. Paclitaxel was purchased from AK Scientific Inc. Taxol (Mayne Pharma) was obtained from the UC Davis Cancer Center Pharmacy. Cholic acid, MTT, and fluorescein isothiocyanate (FITC) and all other chemicals were purchased from Sigma-Aldrich.

Synthesis of OA02-conjugated PEG5k-CA8 telodendrimer

Boc-NH-PEG5k-CA8 telodendrimer was first synthesized as described previously (6). N3-PEG5k-CA8 was obtained by the coupling of 4-azidobutyric acid NHS ester to the terminus of PEG after deprotecting Boc group of Boc-NH-PEG5k-CA8 with 50% (v/v) trifluoroacetic acid (TFA) in dichloromethane. The telodendrimer was then dialyzed and finally lyophilized.

Alkyne-modified OA02 peptide (cdG-HoCit-GPQc-Ebes-K-alkyne) was synthesized via solid-phase synthesis on Fmoc-Rink Amide MBHA Resins using the standard Fmoc chemistry as described previously (30). 5-Hexynoic acid was coupled onto the ε-amino group of lysine on the peptide. Alkyne-modified OA02 peptide was conjugated to the N3-PEG5k-CA8 telodendrimer via CuI-catalyzed cyloaddition (21). The conjugation was confirmed by the amino acid analysis (AAA). The molecular structure and molecular weight of OA02-PEG5k-CA8 telodendrimer were measured by 1H-NMR (nuclear magnetic resonance) and matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry (MS), respectively.

FITC- or Cy5.5-labeled telodendrimers were synthesized by coupling FITC or Cy5.5 NHS ester to the amino group of the proximal lysine between PEG and cholic acid after the removal of Dde protecting group by 2% (v/v) hydrazine in dimethylformamide.

Preparation and characterization of paclitaxel-loaded OA02-NPs

Paclitaxel-loaded OA02-NPs (PTX-OA02-NPs) were prepared using the mixture (1:1) of blank PEG5k-CA8 and OA02-PEG5k-CA8 telodendrimers via a dry-down (evaporation) method as described previously (6). To determine the amount of paclitaxel loaded in the nanoparticles, paclitaxel-loaded nanoparticles were dissolved in dimethyl sulfoxide (1:9, v/v) and measured by high-performance liquid chromatography (HPLC). The encapsulation efficiency (EE) was calculated according to the following formula:

EE (%) = (mass of paclitaxel encapsulated in nanoparticles/mass of paclitaxel added) × 100%

The morphology, particle size distribution, and zeta potential of PTX-OA02-NPs were characterized by cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS, Microtrac), respectively. The in vitro drug release kinetics from PTX-OA02-NPs was measured by the dialysis method. Briefly, aliquots of PTX-OA02-NPs solution were injected into dialysis cartridges with the molecular weight cutoff value of 3.5 kDa. The cartridges were dialyzed against 1 L PBS and shaken at 37°C at 100 rpm with activated charcoal to create an ideal sink condition. The concentration of paclitaxel remained in the dialysis cartridge at different time points were measured by HPLC.

Cell culture and animals

SKOV-3, ES-2, and K562 cells were purchased from American Type Culture Collection. SKOV3-luc cells were obtained from Caliper Life Sciences. All these cancer cell lines were authenticated by the suppliers and passaged in the laboratory for fewer than 6 months after resuscitation. Cells were maintained in a 37°C/5% CO2 humidified chamber in McCoy's 5A (SKOV-3, ES-2, and SKOV3-luc) or RPMI-1640 (K562) media supplemented with 10% FBS.

Female nude mice, 6 to 8 weeks age, were purchased from Harlan Laboratories. All animal protocols were approved by the Institutional Animal Care and Use Committee. Ovarian cancer xenograft mouse model was established by subcutaneously injecting 5 × 106 SKOV-3/SKOV3-luc cells in a 100 μL of mixture of PBS and Matrigel (1:1, v/v) at the right flank in female nude mice.

Confocal microscopy

SKOV-3 and ES-2 cells were seeded in 8-well chamber slides. When the cells were almost confluent, cells were incubated with 2 μmol/L FITC fluorescent-labeled nanoparticles and OA02-NPs for 2 hours at 37°C with 5% CO2, respectively. Then, cells were washed 3 times with cold PBS, fixed with 4% paraformaldehyde for 10 minutes, and the nuclei were counterstained by DAPI. The slides were mounted with coverslips and observed by Olympus FV1000 confocal microscopy. In another set of experiment, excess amount of α-3 integrin antibody or free OA02 peptide (200 μmol/L) were added into the medium 30 minutes before the incubation of 2 μmol/L FITC-labeled OA02-NPs with cells, followed by the same procedure as earlier.

Flow cytometry

To show the overexpression of α-3 integrin, SKOV-3 and ES-2 cells were incubated with Alex Fluor 488–conjugated α-3 integrin antibody (Chemicon International, 1:500) for 30 minutes at 4°C, followed by PBS wash twice, and then resuspended in PBS for the flow cytometric analysis.

SKOV-3, ES-2, and K562 (α-3 integrin negative) cells were incubated with 2 μmol/L FITC-labeled nanoparticles or OA02-NPs for 2 hours at 37°C, respectively. Then the cells were washed with PBS 3 times and resuspended in PBS for the flow cytometric analysis. A total of 10, 000 events were collected for each sample. For peptide inhibition experiments, free OA02 peptides with the final concentration from 2 to 200 μmol/L were added into the medium 30 minutes before the incubation of 2 μmol/L FITC-labeled nanoparticles or OA02-NPs with cells.

Intracellular tracking of OA02-NPs in live ovarian cancer cells

To simultaneously track the payload and carrier of OA02-NPs, DiD dyes were encapsulated as drug surrogates into FITC-conjugated OA02-NPs. SKOV-3 ovarian cancer cells were seeded in the coverglass chamber slides. After reaching 80% confluence, cells were incubated with DiD/FITC dual-labeled OA02-NPs. After 1.5 hours, LysoTracker Red (50 nmol/L) was added in the medium and the cells were further incubated for another 30 minutes (31). Then, the live cells were observed under the Olympus FV1000 confocal microscopy.

MTT assay

MTT assay was used to evaluate the in vitro cytotoxicity of blank/paclitaxel-loaded nontargeted nanoparticles and OA02-NPs against ovarian cancer cells (32). Cells were treated with blank/paclitaxel-loaded nanoparticles and OA02-NPs, respectively. After 2 hours of treatment, cells were washed with PBS 3 times, and fresh media were replaced in the plates. At 72 hours, MTT was added to each well and further incubated for another 4 hours. The absorbance at 570 nm with a reference wavelength of 660 nm was detected with a microplate reader. Untreated cells served as a control. Results were shown as the average cell viability [(ODtreat − ODblank)/(ODcontrol − ODblank) × 100%] of triplicate wells.

In vivo and ex vivo NIRF optical imaging

Nude mice bearing subcutaneous SKOV3-luc tumors were intravenously injected with 4 nmol/L Cy5.5 fluorescent-labeled nanoparticles and OA02-NPs, respectively. At different time point (0.5, 2, 4, 8, and 24 hours) postinjection, mice were scanned with Kodak imaging system IS2000MM. At 24 hours, tumors and major organs were excised for ex vivo imaging. For the microscopic analysis, excised tumors were frozen in optimum cutting temperature (OCT) medium at 80°C. The corresponding slices (10 μm) were prepared, air dried for 10 minutes, and fixed with 4% paraformaldehyde for 10 minutes. The α-3 integrin expression in the tumor section was stained by Alex Fluor 488–conjugated α-3 integrin antibody (1:500) for 1 hour at room temperature. The blood vessel was stained by rat anti-mouse CD31 primary antibody (Millipore, 1:100) for 1 hour and Cy3-conjugated goat anti-rat IgG secondary antibody (Millipore, 1:1,500) for 1 hour at room temperature.

Therapeutic study

The antitumor efficacy and toxicity profiles of different paclitaxel formulations were evaluated in the subcutaneous xenograft mouse model of SKOV-3 ovarian cancer. The treatment was initiated when tumor volume reached 100 to 200 mm3 and this day was designated as day 0. The maximum tolerated dose (MTD) of Taxol in mice is approximately 10 mg/kg (6, 33), and the micellar formulations of paclitaxel were expected to be better tolerated than Taxol according to our previous report (6). Mice were administrated intravenously with PBS, Taxol (10 mg/kg), PTX-NPs (10, 30 mg/kg), and PTX-OA02-NPs (10, 30 mg/kg), respectively (n = 8–10). The dosage was given every 3 days for a total of 6 doses. Tumor sizes were measured with a digital caliper twice per week. Tumor volume was calculated by the formula (L × W2)/2, where L is the longest and W is the shortest in tumor diameters (mm). Relative tumor volume (RTV) equals the tumor volume at given time point divided by the tumor volume before initial treatment. For humane reasons, animals were sacrificed when the implanted tumor volume reached 1,500 mm3, which was considered as the end point of survival data. Survival rate was analyzed using a Kaplan–Meier plot. The potential toxicities after treatment were monitored by the animal behavior observation and the body weight measurement twice per week.

Statistical analysis

Statistical analysis was conducted by the Student t test for comparison of 2 groups, and one-way ANOVA for multiple groups, followed by Newman–Keuls test if overall P < 0.05.

Results and Discussion

Synthesis of OA02-PEG5k-CA8 telodendrimer

N3-PEG5k-CA8 telodendrimer was first synthesized via stepwise solution-phase condensation reactions as reported previously (6). “Click chemistry” was used to covalently conjugate alkyne-containing OA02 peptide onto the azide group at the PEG terminus of N3-PEG5k-CA8 telodendrimer, resulting in OA02-PEG5k-CA8 telodendrimer (Fig. 1A). The conjugation of OA02 peptide onto PEG5k-CA8 telodendrimer was confirmed by AAA (Supplementary Fig. S1). Each amino acid of OA02-PEG5k-CA8 telodendrimer was hydrolyzed and quantitatively measured by HPLC. As summarized in Supplementary Table S1, the determined numbers of each amino acid by AAA were almost identical to their corresponding theoretical values in the molecular formula of OA02-PEG5k-CA8 telodendrimer, indicating the successful conjugation of OA02 peptide to the telodendrimer. The molar ratio of OA02 peptide to telodendrimer was almost 1:1, which meant that there was approximately one targeting peptide molecule per telodendrimer monomer. The molecular weight of OA02-PEG5k-CA8 telodendrimer was measured with MALDI-TOF MS. The monodispersed mass trace was detected, and its molecular weight from MALDI-TOF MS was almost identical to the theoretical value (Supplementary Fig. S2A). The chemical structure of OA02-PEG5k-CA8 telodendrimer was also determined by 1H-NMR spectrometry. As shown in Supplementary Fig. S2B, the signals at 0.6 to 1.3 and 3.5 to 3.7 ppm could be assigned to cholic acids and PEG chains, respectively. The 1H-NMR signals of OA02 peptide were overlapped with the signals from the telodendrimers, and no distinguishable signal was observed.

Figure 1.
Figure 1.

Chemical structure of OA02 peptide functionalized PEG5K-CA8 telodendrimer (A) and the preparation of stealth-targeted nanoparticles by the self-assembly of blank/OA02-functionalized telodendrimers (B). Alkyne-containing OA02 peptide was conjugated to the azide group on the distal terminus of PEG chain in PEG5K-CA8 telodendrimer via “click chemistry”. The Dde-protected amino group on the proximal lysine in OA02-PEG5K-CA8 telodendrimer can be removed and used for the conjugation of fluorescence dyes such as FITC or Cy5.5 for cell and animal imaging. The OA02 peptides presented on the surface of targeted nanoparticles are able to specifically recognize and bind the α-3 integrin receptors, which are overexpressed on the cell membrane of ovarian cancer cells.

Preparation and characterization of paclitaxel-loaded OA02-PEG5k-CA8 nanoparticles

The critical micelle concentration (CMC) of OA02-PEG5k-CA8 telodendrimer was found to be comparable with that of blank PEG5k-CA8 telodendrimer, with the range of 6 to 12 μmol/L (Table 1 and Supplementary Fig. S3), indicating its excellent micelle-forming property. Using the dry-down method, hydrophobic drugs such as paclitaxel can be readily encapsulated into the core of micellar nanoparticles. When the feeding ratio of drug paclitaxel/telodendrimer (w/w) was 1:4, the encapsulation efficiency of PEG5k-CA8 nanoparticles and OA02-PEG5k-CA8 nanoparticles were approximately 92% and 96%, respectively. To enable the developed nanoparticles to possess both antibiofouling (“stealth”) and cell-specific–targeting properties (34), OA02-PEG5k-CA8 telodendrimer was mixed with blank PEG5k-CA8 telodendrimer (1:1, w/w) to prepare PTX-OA02-NPs for the subsequent cell and animal studies (Fig. 1B). The cryo-TEM image (Fig. 2A) showed that PTX-OA02-NPs were spherical, with uniform particle sizes of around 50 nm in diameter, which was similar with the result obtained from DLS measurement (Fig. 2B). The zeta potential of PTX-OA02-NPs in PBS was almost neutral (−0.64 mV). The stability of PTX-OA02-NPs was evaluated by measuring the changes of particle sizes over time at different conditions. PTX-OA02-NPs were found to be very stable at 4°C for more than 3 months, and also stable at 37°C for at least 72 hours when incubated with 50% FBS (data not shown), which indicated that they will likely be able to maintain their stability and integrity during their in vivo applications. The drug release pattern from PTX-OA02-NPs was similar with that from PTX-NPs, which both were biphasic, with the initial rapid release of paclitaxel during the first 4 hours, followed by the slow linear release over the subsequent few days (Fig. 2C). In summary, the decoration of OA02 peptide had negligible impact on the physicochemical properties of PEG5k-CA8 nanoparticles, including CMC, morphology, particle size, drug-loading capacity, encapsulation efficiency, stability, and drug release profile. This is probably because the OA02 peptide conjugation occurs at the distal end of PEG chain which is located on the shell of self-assembled micellar nanoparticles without interfering with the drug-holding hydrophobic core unit.

Figure 2.
Figure 2.

The morphology (A) and particle size distribution (B) of PTX-OA02-NPs (5 mg paclitaxel in 20 mg/mL telodendrimer) measured by cryo-TEM and DLS, respectively. A, white arrows point to nanoparticles, while tobacco mosaic virus (TMV) was used as calibration standard (18 nm in width). The ice crystals usually have clear edge contrast. C, in vitro paclitaxel release kinetics from PTX-NPs or PTX-OA02-NPs in PBS at 37°C. The concentration of paclitaxel remained in the dialysis cartridge at various time points was measured by HPLC. Error bars were obtained from triplicate samples.

View this table:
Table 1.

The physicochemical characteristics of PEG5K-CA8 and OA02-PEG5K-CA8 telodendrimers

Cellular uptake studies

α-3 Integrin was showed to be overexpressed on both SKOV-3 and ES-2 cells, as measured by the flow cytometric analysis (Supplementary Fig. S4). The uptake profiles of FITC-labeled OA02-NPs in ovarian cancer cells were first qualitatively observed by the confocal microscopy. Nontargeted nanoparticles had minimal nonspecific cellular uptake after 2-hour incubation, whereas the decoration of OA02 peptide greatly increased the extent of nanoparticles uptake in both SKOV-3 (Fig. 3A) and ES-2 cells (Supplementary Fig. S5A). Most FTIC-labeled nanoparticles (green) distributed around the perinuclear region, which meant that these nanoparticles were internalized into the cytoplasm. More importantly, the uptake of OA02-NPs in both ovarian cancer cells was able to be remarkably inhibited by excess amount of α-3 integrin antibody or free OA02 peptide, suggesting the α-3 integrin receptor-dependent internalization of OA02-NPs.

Figure 3.
Figure 3.

The uptake of FITC-labeled OA02-NPs in SKOV-3 ovarian cancer cells and K562 leukemia cells (α-3 integrin negative). A, confocal microscopic images of SKOV-3 cell incubated with FITC-labeled nanoparticles and OA02-NPs (green) for 2 hours. To show the α-3 integrin-dependent uptake, excess amount of α-3 integrin antibody, or free OA02 peptide was added before the incubation of OA02-NPs with SKOV-3 cells. The flow cytometric analysis of OA02-NPs uptake in K562 cells (B) and SKOV-3 cells (C). The MFI of cells incubated with nanoparticles and OA02-NPs are shown as insets. *, P < 0.05. D, the addition of free OA02 peptides at concentrations from 2 to 200 μmol/L inhibited cellular uptake of OA02-NPs in SKOV-3 cells in a dose-dependent manner.

The uptake efficiencies of FITC-labeled OA02-NPs in ovarian cancer cells and K562 leukemia cells (α-3 integrin negative) were further quantitatively measured by the flow cytometric analysis. Both nontargeted nanoparticles and OA02-NPs had similar low nonspecific uptake in K562 cells (Fig. 3B). However, OA02-NPs exhibited significantly higher uptake than the nontargeted nanoparticles in both ovarian cancer cells (P < 0.05), with almost 6-fold higher uptake in SKOV-3 cells (Fig. 3C) and 4-fold higher uptake in ES-2 cells (Supplementary Fig. S5B), respectively. The addition of free OA02 peptide was able to inhibit the uptake of OA02-NPs in SKOV-3 cells (Fig. 3D) and ES-2 cells (Supplementary Fig. S5C) in a dose-dependent manner, further confirming the α-3 integrin–targeting specificity of OA02-NPs. It should be noted that significantly higher concentration of free peptide (10- to 100-folds) was required to inhibit the cellular uptake of OA02-NPs, whereas the equal concentration of free peptide did not produce obvious inhibition effect. This could be possibly explained by the increased binding affinity of OA02 peptides presented on the nanoparticles surface due to the multivalency effects (35).

Intracellular tracking of dual fluorescent-labeled OA02-NPs in live cells

After 2-hour incubation, both FITC-labeled telodendrimer carrier (green) and DiD dye payload (red) were simultaneously internalized into the SKOV-3 cells with colocalized dot-shape fluorescent foci in the perinuclear region of the cytoplasm (Fig. 4A, bottom left), indicating that the intact OA02-NPs were taken up into the cytoplasm. These fluorescent foci were generated as a result of the accumulation of OA02-NPs in the endocytic vesicles (i.e., endosomes and lysosomes). This was evidenced by the partial colocalization of internalized FITC-labeled OA02-NPs (green) with lysosomal compartment (red), producing yellow fluorescence in the merge images (Fig. 4A, bottom right). Similar observation was also reported by Gu and colleagues in the LNCaP prostate cancer cells incubated with A10 aptamer–targeted PLGA-b-PEG-b-Apt nanoparticles (34).

Figure 4.
Figure 4.

A, intracellular tracking of dual fluorescent-labeled OA02-NPs upon their uptake in live SKOV-3 cells. DiD dye (red) was encapsulated as drug surrogates into FITC (green) covalently labeled OA02-NPs. SKOV-3 cells were incubated with DiD/FITC dual-labeled OA02-NPs for 2 hours, and the cells were stained with lysosome tracker (red) before the live-cell imaging by confocal microscopy. B, the differential cytotoxicities of 0.5 μg/mL paclitaxel-loaded nanoparticles (PTX-NPs), paclitaxel-loaded OA02-NPs (PTX-OA02-NPs), and the equivalent dose of blank nanoparticles, OA02-NPs against SKOV-3 ovarian cancer cells measured by MTT assay. Nanoparticles were incubated with cells for 2 hours, and the cells were subsequently washed and incubated in fresh media for a total of 72 hours before assessing cell viability in each group. *, P < 0.05.

In vitro cytotoxicity study

PTX-OA02-NPs were found to be significantly more cytotoxic against both SKOV-3 (Fig. 4B) and ES-2 cells (Supplementary Fig. S6), when compared with nontargeted PTX-NPs at the equivalent paclitaxel concentration (P < 0.05). The enhanced cytotoxicity of PTX-OA02-NPs is likely related to the capability of OA02 peptide to facilitate the uptake of PTX-OA02-NPs into ovarian cancer cells, thus increasing the intracellular paclitaxel concentration. Furthermore, there was no observable cytotoxic effect associated with both blank nontargeted nanoparticles and OA02-NPs at the equivalent nanoparticles concentration, which eliminated the possibility of OA02 peptides or nanoparticle-induced cytotoxic activity.

Biodistribution and tumor targeting specificity in vivo

NIRF optical imaging is an important tool for visualizing molecular processes in vivo, as NIRF dyes with deep penetration, low autofluorescence, and low tissue absorption and scattering enable the high-resolution tissue imaging (26). Both Cy5.5 fluorescent-labeled nanoparticles and OA02-NPs distributed throughout the body of the mice immediately after the intravenous injection and gradually accumulated into the SKOV3-luc tumor. However, the uptake rate of OA02-NPs in the tumor site was faster than that of nontargeted nanoparticles. Substantial contrast between tumors and background in the mice injected with OA02-NPs was observed at around 4-hour postinjection, and these nanoparticles were able to be retained in the tumor throughout the 24-hour period, whereas the accumulation of nontargeted nanoparticles in the tumor was not apparent until 8-hour postinjection (Fig. 5A). Ex vivo images at 24 hours showed that both nontargeted nanoparticles and OA02-NPs exhibited relatively high uptake in the SKOV3-luc tumor than in normal organs except the liver (macrophage uptake), as the result of EPR effect (Fig. 5B). However, the mean fluorescence intensity (MFI) of tumors for OA02-NPs was approximately 1.7-fold higher than that for nontargeted nanoparticles (Fig. 5C, P < 0.05). The histologic distribution of nanoparticles in the tumor tissue was further observed under the confocal microscopy. As shown in Fig. 5D, the majority of Cy5.5-labeled nontargeted nanoparticles (red) were mainly distributed in the perivascular region, which is in concordance with previous histologic observations of passive accumulation of liposomes and micelles in tumor tissues (14). In contrast, OA02-NPs were able to extravasate from the tumor vasculature, penetrate deep into the interstitial space of the tumor, bind to α-3 integrin–overexpressing tumor cells, and eventually become internalized. Similar observations were also reported by Kirpotin and colleagues using PEGylated liposome targeted by an anti-HER-2 antibody (36). The enhanced tumor localization and intracellular uptake of OA02-targeted nanoparticles into ovarian cancer cells is probably attributed to the specific interaction between the OA02 peptide and α-3 integrin, thus facilitating the homing of targeted nanoparticles to ovarian cancer, and the receptor-mediated endocytosis.

Figure 5.
Figure 5.

In vivo and ex vivo NIRF optical imaging of Cy5.5-labeled nanoparticles or OA02-NPs biodistribution after i.v. injection in subcutaneous SKOV3-luc tumor–bearing mice. A, in vivo optical images of real-time tumor-targeting characteristics of nanoparticles or OA02-NPs. Tumor-bearing mice were injected via tail vain with 4 nmol/L Cy5.5-labeled nanoparticles or OA02-NPs and were scanned with Kodak multimodal imaging system IS2000MM at different time point. B, representative ex vivo optical images of tumors and organs of SKOV3-luc tumor–bearing mice sacrificed at 24 hours. C, quantitative fluorescence intensities of tumors and organs from ex vivo images (n = 3). D, histologic analysis of nanoparticles or OA02-NPs distribution (Cy5.5, red) in tumor cryosections. The nuclei were stained by DAPI (blue), α-3 integrin expression on SKOV-3 tumor cells and vascular endothelial cells were visualized by anti-α-3 (green) and anti-CD31 (orange) staining, respectively. AU, arbitrary units.

Therapeutic study

The antitumor effect of PTX-OA02-NPs was evaluated in the subcutaneous SKOV-3 ovarian cancer xenograft mouse model when compared with nontargeted PTX-NPs and paclitaxel clinical formation (Taxol). As shown in Fig. 6A, all the paclitaxel formulations significantly inhibited the tumor growth, when compared with the control group (P < 0.05). However, both PTX-NPs and PTX-OA02-NPs exhibited better tumor growth inhibition than Taxol, at equivalent paclitaxel dose of 10 mg/kg. The superior tumor growth inhibition of paclitaxel micellar formulations might be attributed to the larger amount of paclitaxel delivered to the tumor site via the EPR effect. More importantly, mice treated with PTX-OA02-NPs had significantly slower tumor growth rate than those treated with nontargeted PTX-NPs at the equivalent paclitaxel doses (P < 0.05). The survival rate of mice in each group is presented by the Kaplan–Meier survival curve, respectively (Fig. 6B). In general, compared with PBS control, all the paclitaxel formulations significantly prolonged the survival rates of tumor-bearing mice. However, mice treated with 30 mg/kg PTX-OA02-NPs exhibited the longest survival time among these treatment groups. The median survival time of mice in the group of PBS control, Taxol, PTX-NPs (10, 30 mg/kg), and PTX-OA02-NPs (10, 30 mg/kg) were 20, 27, 29, 69, 32, and 95 days, respectively. It was also noted that complete response (CR), defined as a complete disappearance of palpable tumor nodule, was achieved in 5 of 10 mice (50%) in the 30 mg/kg PTX-OA02-NPs group, and 2 of 8 mice (25%) in the 30 mg/kg PTX-NPs group. The better tumor growth inhibition, prolonged survival time, and higher complete tumor response rate observed in the PTX-OA02-NPs group are likely due to the more efficient and specific delivery of paclitaxel to the ovarian cancer cells by the unique ligand-targeted nanoformulation, as mentioned earlier. Several independent investigations have also showed that some other targeted nanoparticles such as antibody-targeted liposomes (37), transferrin-targeted polymeric nanoparticles (38), and folate-targeted polyamidoamine (PAMAM) polymers (39) can significantly enhance the antitumor effects as compared with their nontargeted counterparts.

Figure 6.
Figure 6.

In vivo tumor growth inhibition (A), Kaplan–Meier survival curves (B), and body weight changes (C) of SKOV-3 tumor–bearing mice after the intravenous treatment of various paclitaxel formulations. Tumor-bearing mice were administered intravenously with PBS (control), Taxol (10 mg/kg), PTX-NPs (10 and 30 mg/kg), and OA02-PTX-NPs (10 and 30 mg/kg), respectively, every 3 days on days 0, 3, 6, 9, 12, and 15 for a total of 6 doses. Data represent mean ± SEM (n = 8–10).

Toxicities were assessed by direct observation of animal behavior and body weight monitoring. Mice treated with 10 mg/kg Taxol showed a decline of overall activity during the first 30-minute postinjection, which is likely a sign of hypersensitivity reaction related to the diluent (Cremophor EL and ethanol; ref. 33). In addition, this group of mice was found to have significant amount of body weight loss (with a nadir of 6.7%) after receiving the first few doses, when compared with the control group (P < 0.05). In contrast, the mice in both PTX-NPs and PTX-OA02-NPs groups tolerated the regimens well. The treatments did not seem to have obvious adverse impact on their activity level and body weight (Fig. 6C). Overall, the paclitaxel nanoformulations, both PTX-NPs and PTX-OA02-NPs, showed with an improved systemic toxicity profile, which might be attributed to their Cremophor-free composition, prolonged blood retention time, and sustained drug release features (40).

Conclusion

We have successfully developed OA02 peptide–targeted polymeric micelles system to effectively deliver chemotherapeutic drugs to ovarian cancer. High-affinity and high-specificity “OA02” peptide against α-3 integrin was successfully conjugated to the distal PEG terminus of PEG5k-CA8 telodendrimer via “click chemistry” and displayed on the surface of self-assembled micellar nanoparticles. OA02 peptide decoration dramatically enhanced the intracellular delivery of nanoparticle drugs into α-3 integrin–overexpressing ovarian cancer cells via receptor-mediated endocytosis, resulting in higher in vitro cytotoxicity of PTX-OA02-NPs against those cancer cells than nontargeted PTX-NPs. OA02 peptide also significantly facilitated the distribution of targeted nanoparticles into tumor tissues and cells in SKOV3-luc tumor–bearing mice. Furthermore, PTX-OA02-NPs were found to be more efficacious and less toxic than the equivalent doses of nontargeted PTX-NPs and Taxol in ovarian cancer xenograft mouse model. Therefore, OA02 peptide–targeted nanoparticles drug delivery system has great translational potential in the treatment of patients with ovarian cancer.

Disclosure of Potential Conflicts of Interest

K.S. Lam is the founding scientist of LamnoTherapeutics which plan to develop the nanotherapeutics described in the manuscript. J. Luo and K. S. Lam are the inventors of pending patent on telodendrimers. J.S. Lee has received Commercial Research Grant from Hyundai Hope on Wheel Pediatric Cancer Research Grant.

Authors' Contributions

Conception and design: K. Xiao, Y. Li, R.H. Cheng, J. Luo, K.S. Lam

Development of methodology: K. Xiao, Y. Li, J. Luo

Acquisition of data: K. Xiao, Y. Li, A.M. Gonik, E. Sanchez, L. Xing, R.H. Cheng

Analysis and interpretation of data: K. Xiao, Y. Li, J.S. Lee, A.M. Gonik, R.H. Cheng, J. Luo, K.S. Lam

Writing, review, and/or revision of the manuscript: K. Xiao, Y. Li, J.S. Lee, A.M. Gonik, J. Luo, K.S. Lam

Administrative, technical, or material support: J.S. Lee, T. Dong

Study supervision: K.S. Lam

Grant Support

This study was supported by NIH/NCI R01CA140449 (to J. Luo), R01CA115483 (to K.S. Lam), R01EB012569 (to K.S. Lam), and DoD Postdoctoral Fellowship Award (W81XWH-10-1-0817; to K. Xiao).

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.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

    K. Xiao and Y. Li contributed equally to this work.

  • Received November 28, 2011.
  • Revision received February 3, 2012.
  • Accepted February 19, 2012.

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Cancer Research: 72 (8)
April 2012
Volume 72, Issue 8

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“OA02” Peptide Facilitates the Precise Targeting of Paclitaxel-Loaded Micellar Nanoparticles to Ovarian Cancer In Vivo
Kai Xiao, Yuanpei Li, Joyce S. Lee, Abby M. Gonik, Tiffany Dong, Gabriel Fung, Eduardo Sanchez, Li Xing, Holland R. Cheng, Juntao Luo and Kit S. Lam
Cancer Res April 15 2012 (72) (8) 2100-2110; DOI: 10.1158/0008-5472.CAN-11-3883
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