Design and Function of Engineered Protein Nanocages as a Drug

Laboratory of Tumor Biology, Takasaki University of Health and Welfare, Takasaki, Gunma 370-0033, Japan. Mol. Pharmaceutics , 2015, 12 (5), pp 1422–...
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Design and Function of Engineered Protein Nanocages as a Drug Delivery System for Targeting Pancreatic Cancer Cells via Neuropilin‑1 Masaharu Murata,*,†,‡ Sayoko Narahara,†,‡ Takahito Kawano,†,‡ Nobuhito Hamano,† Jing Shu Piao,† Jeong-Hun Kang,§ Kenoki Ohuchida,†,∥ Takashi Murakami,⊥ and Makoto Hashizume†,‡ †

Department of Advanced Medical Initiatives, Faculty of Medical Science, ‡Innovation Center for Medical Redox Navigation, and Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan § Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Osaka 565-8565, Japan ⊥ Laboratory of Tumor Biology, Takasaki University of Health and Welfare, Takasaki, Gunma 370-0033, Japan ∥

S Supporting Information *

ABSTRACT: We describe the development of neuropilin 1binding peptide (iRGD)−nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD−nanocages for their target cells. An engineered L30iRGD−nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD−nanocage by heating the mixture. The OSU03012-loaded L30-iRGD−nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD− nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery. KEYWORDS: drug delivery system, protein nanocages, pancreatic cancer, neuropilin-1

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INTRODUCTION

issue for improving their efficacies in the treatment of pancreatic cancer. Nanoparticles are promising materials for enhancing the delivery of therapeutic drugs to their intended targets in the human body. To enhance the performance of nanomaterials, it is essential that we develop precise sizes, shapes, and surface chemistries of nanoscale molecules.5−8 In particular, nanoparticles ranging in size from 10 to 200 nm show a prolonged circulation in the blood by avoiding the host’s defense systems, including the reticuloendothelial system (RES).9 Thus, such nanoparticles display an enhanced permeability and retention (EPR) effect in solid tumors, which improves tumor targeting.10−12 Protein-based nanoparticles are particularly interesting because of their inherent advantages, including their well-defined structures based on multiple copies of one or a few biomaterial-based building blocks formed through self-

Pancreatic cancer is one of the most lethal human cancers and continues to be a major unsolved health problem in industrialized countries. Annually, >17000 patients in Japan and ∼30000 in the United States die from pancreatic cancer.1 Pancreatic cancer is a particularly aggressive malignancy with a 5-year mortality rate of 97−98%, and it is usually associated with widespread metastatic disease.2 Because of the absence of early specific symptoms and the lack of early detection methods, pancreatic cancer patients are often not diagnosed until an advanced stage, at which time the tumors are often unresectable and are best treated with chemotherapy.3 However, most cases of pancreatic cancer show poor or no response to chemotherapy. In the last 10 years, several novel chemotherapies that target specific markers in pancreatic cancer have been developed using molecular approaches and may offer promising strategies in the diagnosis and treatment of pancreatic cancer.4 Many of those molecular approaches, including gene therapy, small molecule inhibitors, and antibody therapy, have been tested in preclinical models. Enhancing the delivery of chemotherapies and adjuvant therapies is a major © XXXX American Chemical Society

Received: October 24, 2014 Revised: March 9, 2015 Accepted: March 26, 2015

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DOI: 10.1021/mp5007129 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Technologies (Gaithersburg, MD, USA) unless otherwise specified. DNA Cloning and Expression. The structures of recombinant plasmids encoding four protein nanocages linked to the iRGD peptide via flexible linkers (−(GGS)n−) (HSPG41C-L3-iRGD [L3-iRGD], HSPG41C-L11-iRGD [L11-iRGD], HSPG41C-L21-iRGD [L21-iRGD], and HSPG41C-L30-iRGD [L30-iRGD]) are shown in Figure 1B and Figure S1 in the Supporting Information. The expression vectors encoding the protein nanocages were transfected into Escherichia coli BL21 CodonPlus (DE3) (Agilent Technologies, Phoenix, AZ, USA) using a T7 expression system. The transfected cells containing an expression vector were grown in 2× YT medium (Sigma-Aldrich, St. Louis, MO, USA) with 100 mg/mL ampicillin at 37 °C. Recombinant protein production was initiated by the addition of isopropyl thiogalactopyranoside (IPTG; Wako Pure Chemical Industries, Ltd., Osaka, Japan) to a final concentration of 1 mM with incubation at 37 °C for 4 h. The cells were pelleted by centrifugation at 4 °C; the pellet was resuspended in 8 mL of sample buffer (25 mM KH2PO4-KOH, pH 7.0, 2 mM DTT, and 1 mM EDTA), and the resulting suspension was ultrasonicated (Bioruptor UCD-300; Tosho Denki, Yokohama, Japan) on ice for 4 min (H-amplitude, 20 s sonication at 20 s intervals). DNase I and RNase A were then added to final concentrations of 5 and 1 mg/mL, respectively, and the cell lysate mixture was incubated at 4 °C for 30 min. Insoluble material was removed by centrifugation (20000g, 20 min) at 4 °C. The recombinant protein was purified by ion-exchange chromatography in which the supernatant was loaded on a HiLoad 16/10 Q Sepharose HP anion-exchange column (GE Healthcare, Little Chalfont, UK) and the target protein was eluted in sample buffer containing 1 M NaCl. The fractions containing the nanocages, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were loaded onto a silica-based size exclusion chromatography (SEC) column (TSKgel G3000SW; Tosoh Corp., Tokyo, Japan), and the nanocages were eluted in storage buffer (25 mM NaH2PO4/Na2HPO4, pH 7.0, and 0.1 M NaCl). Characterization of the Nanocages. SEC confirmed that monodisperse nanocages were successfully obtained with sufficient purity. The purified nanocage fractions were analyzed by SDS-PAGE using 15% gels according to a standard protocol. The sizes of the nanocages in physiological conditions were measured by dynamic light scattering (DLS) (Malvern Nanosizer ZS; Malvern Instruments Ltd., Malvern, UK). The molecular weight of the purified nanocages was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (Bruker Daltonics, Autoflex speed; Bruker Corp., Billerica, MA, USA) with a sinapinic acid matrix. Fluorescence Microscopy. To link the nanocage with a fluorescent dye, we subsituted Gly41 located inside the native HSP16.5 nanocages with Cys. These nanocages were incubated with a 5-fold molar excess of Alexa488-maleimide (Invitrogen/ Life Technologies, Carlsbad, CA, USA) in phosphate buffer (pH 7.0) at 50 °C for 1 h. Excess dye was removed by Zeba desalt spin column (Thermo Scientific, Rockford, USA). To determine the cellular localization of nanocages, we seeded AsPC-1/CMV-Luc and MIA-PaCa2 cells at 1 × 104 cells/well in 6-well μ-Slides (Ibidi, Martinsried, Germany) and incubated them with colorless DMEM containing 1% antibiotic/ antimycotic mix and 10% FBS overnight. Then, dye-labeled

assembly, allowing for the reproducible production of highly symmetric and uniformly sized architectures.13−17 In the present study, we describe the development of nanoparticles that specifically target pancreatic cancer cells. The nanoparticles were developed using a genetic engineering approach involving the addition of iRGD peptides to the Cterminal regions exposed on the outer surface of the nanoparticle via several linker moieties. The iRGD peptide, which was discovered using a phage display library, can interact with integrin and neuropilin-1 receptors on pancreatic cancer cells to mediate cellular internalization and extravasation, and facilitate deep tissue penetration, to improve imaging sensitivity and therapeutic efficacy.18−21 Neuropilin-1 expression is upregulated not only in pancreatic cancer, but also in other cancer types, including prostate cancer, lung cancer, colorectal cancer, and glioma.22,23 Although the role of neuropilin-1 in cancer progression is not fully elucidated, neuropilin-1 plays a critical role in tumorigenesis, cancer invasion, metastasis, and angiogenesis through the VEGF, PI3K, and Akt pathways.24 As a model nanoparticle, we focused on small heat shock protein (HSP) 16.5, a naturally occurring protein in Methanococcus jannaschii that forms a cage-like structure by the self-assembly of 24 subunits. The outer and inner diameters of the cage are 12 and 6.5 nm, respectively.25−28 To examine the therapeutic potential of these nanocages, we loaded OSU03012,29−31 a novel celecoxib derivative, into the interior of the iRGD− nanocages. Using pancreatic cancer cells, the cytotoxic effects of OSU03012-loaded iRGD−nanocages were compared to OSU03012-loaded control nanocages and free OSU03012 coadministered with the synthetic iRGD peptide. The results suggest that the iRGD−nanocages are a useful nanocarrier for specifically delivering therapeutic drugs to pancreatic cancer cells (Figure 1).

Figure 1. Schematic illustration of the engineered nanocages for targeted therapy of pancreatic cancer.

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EXPERIMENTAL SECTION Cell Culture. All cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). AsPC-1, Suit-2, and MIA-PaCa2 were cultured in Dulbecco modified Eagle’s medium (DMEM; Wako, Tokyo, Japan), and HT-29 and MCF-7 were cultured in RPMI 1640 medium (Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic mix. Cells were maintained in a humidified incubator containing 5% CO2 at 37 °C. All chemical reagents were purchased from Life B

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Molecular Pharmaceutics nanocages were added to each well to a final concentration of 1 μM. After incubation for 6 h, nonincorporated nanocages were removed by washing the cells twice with phosphate-buffered saline (PBS) and adding fresh medium. In addition, the cells were stained with Hoechst 33342 (Invitrogen/Life Technologies, Carlsbad, CA, USA), a fluorescent DNA-binding dye. Fluorescent images were captured with a fluorescence microscope BZ-9000 (Keyence, Osaka, Japan) using Hoechst 33342 (Ex 360/40, DM 400, Em 460/50) and Alexa Fluor 488 (Ex 470/50, DM 495, Em 535/50) filter sets. Phase-contrast images were recorded before fluorescence recording. All fluorescence images were acquired under the same conditions, including the objective lens (Nikon Plan Fluor ELWD 20 × /0.45 NA), exposure time (1/12 s for Hoechst 33342 and 1/5.5 s for Alexa Fluor 488), and gain (+12 dB). Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Total RNA was isolated with QIA shredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RT-qPCR was performed on an iQ5 (Bio-Rad, Hercules, CA, USA). Each 20 μL reaction mixture contained 10 μL of 2× iTaq Universal SYBR Green One-Step Kit (Bio-Rad), 0.25 μL of iScript Reverse Transcriptase (Bio-Rad), 0.3 μM of neuropilin-1 forward primer (5′CTC AGA ATG GAG CTG CTG GG-3′), 0.3 μM of neuropilin-1 reverse primer (5′-GTT CCC GTT GGG AGT GGT C-3′), 8.55 μL of RNA- and nuclease-free water. These primers were designed according to previous reports.32,33 The RT-qPCR amplification profile was as follows: cDNA synthesis for 10 min at 50 °C, followed by reverse transcriptase inactivation for 5 min at 95 °C, and 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. Once amplification was complete, melting curves were obtained across a temperature gradient of 65−95 °C at 0.1 °C/s. Fluorescence was continually measured during the heating period, and the melting peaks were visualized by plotting the fluorescence versus the temperature. The data were normalized to ribosomal protein 18S mRNA as a control housekeeping gene. iCycle iQ real-time PCR detection system software version 3.1 (Bio-Rad) was used for data analyses. Intracellular Fluorescence Intensity Assay. Transfection was assessed by seeding 1 × 104 cells in 100 μL of complete growth medium per well in 96-well microtiter plates 12 h before nanocage addition to allow for cell attachment. After replacing the cell culture medium, the cells were incubated for the specified times with dye-labeled nanocages at a final concentration of 1 μM. Cells were counted after Hoechst 33342 staining, and the intracellular fluorescence intensity of the dye-labeled nanocages was determined using an ArrayScan VTI HCS reader (Thermo Fisher Scientific, Waltham, MA, USA). Cellular uptake was assessed by seeding 2 × 105 cells in 2 mL of complete growth medium (Dulbecco’s modified Eagle’s medium with 10% fetal calf serum or RPMI1640 with 10% fetal calf serum) per well in 6-well microtiter plates 24 h before adding the nanocages to allow for cell attachment. After the cell culture medium was replaced, the cells were incubated for the specified times with dye-labeled nanocages at a final concentration of 0.5 μM. After incubation for 3 h, cultured cells were harvested by exposure to Accutase (Innovative Cell Technologies, San Diego, CA, USA) for 5 min and washed twice with PBS buffer. The resulting single cell preparations were suspended in ice-cold PBS and analyzed using an EC800 flow cytometer (Sony, Tokyo, Japan). The cells incorporated

with the dye-labeled nanocages were excited at 488 nm, and emission spectra were collected using a 525/50 band-pass filter. Data were collected for 10000 gated events and analyzed using EC800 Software version 1.3.5. In inhibition experiments, AsPC-1 cells were seeded at 2 × 105 cells/well in 6-well plates and incubated in DMEM containing 1% antibiotic/antimycotic mix and 10% FBS overnight. Then, 0.5 μM of dye-labeled nanocages was added to each well with the appropriate concentrations of a synthetic iRGD peptide [c(CRGDKGPDC)] as a competitive inhibitor or a synthetic iRGE peptide [c(CRGEKGPDC)] as a negative control. These circular peptides were purchased from Operon Biotechnologies/Eurofins Genomics (Luxembourg, Luxembourg). After incubation for 3 h, cultured cells were harvested by exposure to Accutase for 5 min and washed twice with PBS. The resulting single cell preparations were suspended in icecold PBS and analyzed using an EC800 flow cytometer. The cells incorporated with the dye-labeled nanocages were excited with an argon laser (488 nm). Data were collected for 10000 gated events and analyzed using EC800 Software version 1.3.5. Evaluation of in Vitro Cytotoxic Activity. Drug-loaded nanocages were prepared as follows. First, a solution of OSU03012 (Cayman Chemical, Ann Arbor, MI, USA) in water/dimethyl sulfoxide (50:50, v/v) was mixed with a PBS solution of nanocages in a final volume of 100 μL followed by incubation at 55 °C for 30 min. The final concentrations (μM) of OSU03012 and nanocages in the solution were 400 and 4.2 (100 μM of subunit protein), respectively. Unloaded OSU03012 was removed using a Zeba desalt spin column (Thermo Scientific, Rockford, USA). The concentration of entrapped OSU03012 within the nanocages was determined by reverse-phase HPLC. Chromatographic measurements were performed on a Jasco gradient HPLC system (Jasco, Tokyo, Japan) consisting of two PU-2080i pumps and an MD-2015 multiwavelength detector operating at 260 nm. Runs were carried out on a WP300 C4 column (150 × 4.6 mm i.d., 5 μm, GL sciences, Inc., Tokyo, Japan). Mobile phase A was 0.05% trifluoroacetic acid (TFA) in water, and B was acetonitrile. Separations were performed at room temperature using a linear gradient of 20−70% B for 20 min followed by an isocratic step of 5 min at an eluent flow rate of 1 mL/min. Prior to HPLC analysis, OSU03012-loaded nanocages were treated with 0.05% TFA in water to collapse the cage structure under acidic conditions. AsPC-1 cells were seeded at 0.5 × 104 cells/well in 96-well microtiter plates in complete growth medium in the presence of drug-loaded nanocages containing 5 μM of OSU03012. After incubation for 72 h, the number of viable cells was determined using a CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Apoptosis was analyzed by measuring caspase3/-7 activities using a CellPlayer 96-Well Kinetic Caspase-3/7 Apoptosis Assay Kit (Essen BioScience, Ann Arbor, MI, USA). During the assay, the luminescent and fluorescent intensities of each sample were quantified using an ARVO plate reader (PerkinElmer, Waltham MA, USA). The integrated objectcounting algorithm was used to isolate the fluorescent nuclear signal from the background signal, segment the signal into individual objects, and count objects on a per area basis for each time point. Results were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Results of three experiments were compared using Student’s t test. C

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Figure 2. Design and function of engineered nanocages. A, Domain boundaries of individual subunits of the recombinant nanocage. The light brown rod indicates the helix domains in the N-terminal region (residues 1−33) of HSP16.5. The green dots and lines indicate the iRGD peptide and hydrophilic linker (−(GGS)n−), respectively. The blue line indicates a Gly residue at position 41, which was substituted with a Cys residue for conjugation with AlexaFluor 488-maleimide. B, SDS-PAGE analysis of the recombinant nanocages. Lane 1, molecular weight standards; lane 2, HSPG41C−nanocages; lane 3, L3-iRGD−nanocages; 4, L11-iRGD−nanocages; 5, L21-iRGD−nanocages; 6, L30-iRGD−nanocages. The protein was stained with Coomassie brilliant blue. C, Fluorescence microscopic observation of the uptake of the nanocages in the human pancreatic cancer cell lines AsPC-1 and MIA-PaCa2. Fluorescent image after internalization of each nanocage following incubation for 6 h at 37 °C. Green indicates AlexaFluor 488-labeled nanocages; blue indicates cell nuclei. Experiments were performed at least three times. D, Quantitative analysis of the fluorescence intensity of pancreatic cancer cells incubated with the indicated nanocages at 37 °C for 12 h. Data are expressed as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control (HSPG41C) nanocages (Student’s t test).

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RESULTS AND DISCUSSION Characterization of the iRGD−Nanocages. Nanocages linked to iRGD peptides using linkers of different lengths and control nanocages (HSPG41C) (Figure 2A) were prepared using an E. coli protein expression system. The proteins were purified by sequential anion-exchange chromatography followed by native SEC. The purified proteins, separated by SDSPAGE, appeared as a single band by Coomassie blue staining. As shown in Figure 2B, proteins with the expected size of the expressed nanocages were present in each lane (lanes 2−6). The concentration of the purified fusion protein was determined using Bradford’s method and ranged from 2.5 and 4.0 mg/L of cell culture medium. The observed molecular weight of the purified iRGD−nanocages, as determined by MALDI-ToF with a sinapinic acid matrix, agreed with calculations (Figure S2, Supporting Information). The sizes of the nanocages under physiological conditions were measured by DLS analysis. The mean outer diameters of

the HSPG41C−, L3-iRGD−, L11-iRGD−, L21-iRGD−, and L30-iRGD−nanocages were 12.0, 12.6, 12.9, 13.6, and 14.2 nm, respectively (Figure S3, Supporting Information). The sizedistribution peaks of the nanocages shifted toward larger sizes with repeated additions to the C-terminal region. All of the nanocages were highly monodispersed, and the nanocage structure was maintained in aqueous medium without the formation of large aggregates. Cellular Uptake of the iRGD−Nanocages. For cellular uptake experiments, Alexa488-labeled nanocages were prepared by the Michael addition reaction of AlexaFluor488 C5 maleimide to Cys41 located on the interior wall of the HSP16.5 cages. SDS-PAGE confirmed that the nanocages were successfully labeled with the fluorophore (data not shown). The close spatial proximity of the AlexaFluor 488 molecules in the nanocages resulted in autoquenching of the fluorescence (Figure S4, Supporting Information). The fluorescence intensity of AlexaFluor 488-labeled nanocages decreased with an increase in the labeling ratio (fluorophore per subunit D

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Molecular Pharmaceutics protein) of AlexaFluor 488 into the nanocages up to ∼0.5. Further increases in the labeling ratio did not result in any noticeable change in fluorescence intensity. For quantitative fluorescence measurements, highly modified nanocages (labeling ratio >0.8) were used for cellular uptake experiments. To determine whether the fusion of iRGD to the HSP16.5 protein nanocage enhanced the binding affinity of the nanocage toward human pancreatic cancer cell lines (AsPC-1, a metastatic cell line established from the ascitic fluid of a female patient; MIAPaCa2, a primary cell line established from a male patient),34 we performed transfection assays using cells expressing the specific target of the iRGD peptide. As shown in Figure 2C, the fluorophore-labeled iRGD−nanocages were efficiently taken up by AsPC-1 cells but not by MIA-PaCa2 cells. Incubation with these nanocages resulted in specific accumulation of these proteins in the cytoplasm but not in the nucleus of pancreatic cancer cells. In contrast, the nonspecific HSPG41C nanocages were slightly taken up by both cell lines under the same conditions. This phenomenon was explained by a difference in the expression level of neuropilin-1the target receptor of the iRGD peptidebetween the two human cancer cell lines. RTqPCR, performed with RNA isolated from both cell lines revealed that the mRNA expression of neuropilin-1 was higher in AsPC-1 cells than in MIA-PaCa2 cells (Figure 3). Similar

length was adjusted by incorporating 1−10 GGS repeats. To quantify the contributions of the linker moiety to the cellular uptake of the engineered nanocages by pancreatic cancer cells, we determined the intracellular fluorescence intensity of iRGD−nanocages using cell-based assays. As shown in Figure 2D, the intracellular fluorescence intensity of the iRGD− nanocages taken up by AsPC-1 cells increased with increasing linker length. In contrast, the cellular uptake of the iRGD− nanocages by MIA-PaCa2 cells was not significantly affected by the linker length. These results suggest that longer flexible linkers have greater advantages in terms of the functional properties of the ligand domain than shorter, rigid linkers. To determine the cellular specificity of L30-iRGD nanocages, the nanocages were transfected into five different human tumor cells, including colon (HT-29), breast (MCF-7), and pancreatic tumor cell lines (AsPC-1, MIA-PaCa2, and Suit-2). Cellular uptake behavior was evaluated comparatively by flow cytometry. As shown in Figure 4, the L30-iRGD−nanocages were taken up by two pancreatic tumor cell lines (AsPC-1 and Suit-2) in an efficient manner compared to uptake of nonspecific HSPG41C−nanocages. However, there was a slight difference in cellular uptake between L30-iRGD−nanocages and HSPG41C−nanocages for other cell lines (HT-29, MCF-7, and MIA-PaCa2). This cell-specificity and efficiency of transfection of iRGD−nanocages was in agreement with the expression level of neuropilin-1 on the cell surface as expected (Figure S5, Supporting Information). Moreover, in a preliminary in vivo experiment, iRGD− nanocages also efficiently accumulated in tumors (Figure S5, Supporting Information). The intratumoral microdistribution of nanocages with or without the iRGD moiety along the surfaces of tumor sections was assessed to investigate the iRGD-dependent penetration of nanocages in AsPC-1 tumorbearing mice. In this tumor model, the immunofluorescence signals from the L30-iRGD−nanocages were considerably stronger than the fluorescence of HSPG41C control nanocages. The immunofluorescence was uniformly distributed throughout the tumor section at 6 h postinjection. This phenomenon could not be explained by blood circulation or EPR effects because the prepared nanocages were of similar sizes and had similar surface charge properties. Therefore, it seems reasonable to suggest that the iRGD moiety enhanced the tumor-homing properties of the nanocages in vivo. We will evaluate the in vivo application of the nanocages in more detail in future articles. Mechanisms Involved in Cellular Uptake of iRGD− Nanocages. To evaluate the mechanism involved in the internalization of iRGD−nanocages into AsPC-1 cells, we performed competition assays using synthetic iRGD peptide. The incorporation of fluorescently labeled L30-iRGD− nanocages in the presence of various concentrations of synthetic iRGD peptides was intensity normalized to 100 for untreated AsPC-1 cells (Figure 5). The synthetic iRGD peptides dose-dependently inhibited the incorporation of labeled L30-iRGD−nanocages. In contrast, synthetic iRGE peptides and the glutamic acid-substituted negative control peptide did not significantly affect the uptake of the iRGD− nanocages into AsPC-1 cells. These findings suggest that the binding and uptake of the iRGD−nanocages is specifically due to the presence of the iRGD domain, and that internalization was achieved by interactions between the iRGD domain with neuropilin-1 expressed on the surface of these cells. Ruoslahti et al. proposed that homing peptide-induced activation of an endocytic trans-tissue transport pathway, termed the CendR-

Figure 3. RT-qPCR expression analysis of neuropilin-1 mRNA expression in human pancreatic cancer cell lines. The calculated expression of the target gene was normalized to the expression of the endogenous housekeeping gene ribosomal protein 18S. All data are presented as the ratio of the target gene/18S in three independent experiments. **p < 0.01 (Student’s t test).

observations were obtained from flow cytometric analysis of both cell lines using antihuman neuropilin-1 antibody (Figure S5, Supporting Information) as reported by other research groups.32,33 Interestingly, the uptake of the iRGD−nanocages into AsPC1 cells was influenced by the length of the linker between the nanocage and the iRGD domain. An important architectural factor that influences the function of proteins is the flexibility of the linkers between each domain in multidomain proteins that are involved in many biological processes.35 In this study, we constructed a series of fusion proteins of the protein nanocage and the peptide ligand domain for the target cell-surface receptors (i.e., nanocage−linker−iRGD) in which the linker E

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Figure 4. Representative histograms of the fluorescence intensities of various cells incubated with fluorophore-labeled nanocages in the presence of 10% fetal bovine serum. Cells were treated with 0.5 μM of Alexa Fluor 488-labeled L30-iRGD− nanocages (magenta line) or Alexa Fluor 488-labeled HSPG41 control nanocages (green line) in the presence of 10% fetal bovine serum. Untreated control cells are displayed by a black line..

nanocages to the specific delivery of drugs to pancreatic cancer cells. Cytotoxic and Apoptotic Effects of OSU03012-Loaded iRGD−Nanocages on AsPC-1 Cells. We next investigated the use of iRGD−nanocages for delivering drugs to pancreatic cancer cells. In this study, we used OSU03012 as an anticancer drug and prepared the drug-containing iRGD−nanocages as described in the Experimental Section. OSU-03012 is a bioavailable, third-generation celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity. OSU-03012 induces cell death in a variety of tumor types, including pancreatic, colon, and breast cancer and glioblastoma.37 Previous studies have demonstrated that OSU-03012 specifically inhibits PDK-1mediated phosphorylation of Akt with median inhibitory concentrations (IC50) in the low micromolar range38 and induces cell death through multiple mechanisms in pancreatic cancer cells.29 The amount of OSU03012 entrapped within the L30-iRGD−nanocages was determined by HPLC analysis. It was estimated that each iRGD−nanocage entrapped ∼40−50 OSU03012 molecules under experimental conditions. It is wellknown that the hydrophobic region of soluble HSPs (sHSP) plays important roles in the self-association of dynamic oligomeric structures and their chaperone-like activity on substrate proteins.39 This α-crystallin domain and the Nterminal regions of sHSP can incorporate hydrophobic reporter molecules, such as bis-4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid.40,41 Thus, we believe that hydrophobic drugs, such as OSU03012, are physically entrapped in the hydrophobic region of the nanocages.

Figure 5. Inhibitory effects of the synthetic peptide following transfection of L30-iRGD−nanocages into AsPC-1 cells. Data are representative of three independent experiments per group. ***p < 0.001 vs the control group (Student’s t test).

pathway, occurs in the target tissues of this peptide.18,36 This prototypic peptide contains the integrin-binding RGD motif. RGD mediates tumor targeting by binding to αv integrins, which are selectively expressed on various endothelial tumor cells. This is followed by proteolytic cleavage to expose the CendR element (RGDR or RGDK), which is required for cellular uptake. Our results suggest that the iRGD domain might be recognized by the target receptor in a similar way. These characteristics are important for applying the iRGD− F

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Molecular Pharmaceutics Because cell cytotoxicity is an important factor in selecting the materials used for developing drug carriers, we characterized the effects of these nanocages on cell viability under the same conditions that were used to determine cellular uptake. This experiment revealed that the HSPG41C− and L30iRGD−nanocages did not have appreciable cytotoxic effects on AsPC-1 cells under these experimental conditions (Figure S7, Supporting Information). The cytotoxicity of OSU03012loaded iRGD−nanocages against AsPC-1 cells was measured after a 48 h incubation using the CellTiter-Glo luminescent cell viability assay, which detects ATP bioluminescence as a marker of cell viability. The IC50 value was 10.5 and 9.2 μM for free OSU03012 and OSU03012-loaded HSPG41C−nanocages as a control, respectively (Figure 6). The cytotoxicity of the

Figure 7. Effects of free OSU03012 and OSU03012-loaded nanocages on caspase-3/-7 activation in AsPC-1 cells. AsPC-1 cells were seeded at 0.5 × 104 cells/well in 96-well microtiter plates in complete growth medium in the presence of nanocages loaded with 5 μM of OSU03012 or with 5 μM of free OSU03012. Caspase-3/-7 activation was measured using Cellplayer kinetic caspase-3/-7 reagent at each time point. Data are representative of three independent experiments per group. ***p < 0.001 vs the control group (Student’s t test).

Interestingly, the OSU03012-loaded iRGD−nanocages induced cell death more efficiently than the same concentrations of free OSU03012 coadministered with synthetic iRGD peptide in vitro (Figure S8, Supporting Information). Ruoslahti et al. proposed a novel mechanism involving homing peptideinduced activation of an endocytic trans-tissue transport pathway. In this sophisticated mechanism, called the bystander effect, the iRGD peptide can transport coadministered drugs into the target tissue without chemically coupling the compound with the peptide.20,43 Although the reason why the OSU03012-loaded iRGD−nanocages showed greater cytotoxic effects than the synthetic iRGD peptide coadministered with OSU03012 is unclear, iRGD−nanocages containing 24 iRGD moieties per nanocage may be effectively taken up into AsPC-1 cells together with OSU03012 via allosteric effects of the iRGD peptide.

Figure 6. Cytotoxicity of free OSU03012 and OSU03012-loaded nanocages against AsPC-1 cells. AsPC-1 cells were seeded at 0.5 × 104 cells/well in 96-well microtiter plates in complete growth medium in the presence of nanocages containing various concentrations of OSU03012 or free OSU03012. After incubation for 72 h, the number of viable cells was determined using the CellTiter-Glo luminescent cell viability assay according to the manufacturer’s instructions. Data are representative of five independent experiments per group. The inhibitory concentration 50% (IC50) was calculated from a nonlinear regression curve using Prism4 software (GraphPad Software, San Diego, CA, USA).

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OSU03012-loaded iRGD−nanocages against AsPC-1 cells was greater than that of free OSU03012 or OSU03012-loaded HSPG41C−nanocages (IC50 = 4.7 μM). This is because the neuropilin-1-mediated endocytic uptake of the nanocages was more specific, leading to greater cellular uptake of OSU03012, which enhanced the cytotoxicity of OSU03012. Although the drug release mechanism of OSU03012 from nanocages is unclear, we believe that small molecules such as OSU03012 can gradually pass through the pores of nanocages by diffusion. To assess the mechanism involved in the cytotoxicity of the OSU03012-loaded iRGD−nanocages, we next examined the time-course of changes in caspase-3/-7 activities using a fluorogenic substrate peptide containing a caspase-3 and -7 DEVD recognition motif (Asp-Glu-Val-Asp). This profluorescent peptide releases a DNA dye upon cleavage by caspase-3/-7, resulting in green fluorescent staining of nuclear DNA. Caspase-3 and -7 are critical mediators of apoptosis.42 Figure 7 shows caspase-3/-7 activation in AsPC-1 cells in the presence of nanocages loaded with 5 μM of OSU03012 or with 5 μM of free OSU03012. Both caspases were only activated in AsPC-1 cells incubated with OSU03012-loaded iRGD− nanocages for 72 h. These results imply that OSU03012 induces the apoptotic death of AsPC-1 cells by activating the caspase cascade under experimental conditions.

CONCLUSIONS In conclusion, we have developed a protein nanocarrier expressing the neuropilin-1 binding peptide iRGD on the exterior surface of a naturally occurring HSP cage that specifically targets pancreatic cancer cells. The iRGD− nanocages were selectively taken up by AsPC-1 cells with high neuropilin-1 expression but not by MIA-PaCa2 cells with low neuropilin-1 expression. The uptake of fluorophore-labeled iRGD−nanocages by AsPC-1 cells was inhibited by excess iRGD peptide, indicating that the iRGD−nanocages are taken up by specific receptors on AsPC-1 cells. The specificity of the iRGD−nanocages for their target cells was improved by the inclusion of longer, flexible linkers between the iRGD domain and the nanocage. Furthermore, exposure to OSU03012-loaded iRGD−nanocages induced cell death in AsPC-1 cells by activating the caspase cascade. The OSU03012-loaded iRGD−nanocages induced cell death more effectively than that of the same concentrations of free OSU03012. The genetic engineering strategy used here is effective for producing nanocages that are directed to and are taken up by specific cell types, target organs, and cancer cells. This represents an important development for a variety of applications. Ligandmediated active binding to sites and cellular uptake are G

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Molecular Pharmaceutics

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particularly valuable for the delivery of therapeutic and imaging agents that are not easily taken up by cells.

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ASSOCIATED CONTENT

* Supporting Information S

Additional experimental details as noted in the text. This material is available free of charge via the Internet at http:// pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Health Labour Sciences Research Grant (Research on Publicly Essential Drugs and Medical Devices) from the Ministry of Health, Labour and Welfare of Japan; Special Coordination Funds for Promoting Science and Technology (SCF funding program “Innovation Center for Medical Redox Navigation”); and a Grant-in Aid for Scientific Research (No. 24300172) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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DOI: 10.1021/mp5007129 Mol. Pharmaceutics XXXX, XXX, XXX−XXX