Remote Control of Light-Triggered Virotherapy - ACS Nano (ACS

Nov 7, 2016 - Clinical virotherapy has been successfully approved for use in cancer treatment by the U.S. Food and Drug Administration; however, a num...
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Remote Control of Light-Triggered Virotherapy S.-Ja Tseng,†,‡ Kuo-Yen Huang,§ Ivan M. Kempson,⊥ Shih-Han Kao,# Meng-Chia Liu,¶ Shuenn-Chen Yang,§ Zi-Xian Liao,*,¶ and Pan-Chyr Yang*,†,∥,§ †

Graduate Institute of Oncology and ∥Department of Internal Medicine, National Taiwan University College of Medicine, Taipei 10051, Taiwan ‡ National Taiwan University Cancer Center (YongLin Scholar), Taipei 10051, Taiwan § Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan ⊥ Future Industries Institute, University of South Australia, Mawson Lakes, S.A. 5095, Australia # Research Center for Tumor Medical Science, China Medical University, Taichung 40402, Taiwan ¶ Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan S Supporting Information *

ABSTRACT: Clinical virotherapy has been successfully approved for use in cancer treatment by the U.S. Food and Drug Administration; however, a number of improvements are still sought to more broadly develop virotherapy. A particular challenge is to administer viral therapy systemically and overcome limitations in intratumoral injection, especially for complex tumors within sensitive organs. To achieve this, however, a technique is required that delivers the virus to the tumor before the body’s natural self-defense eradicates the virus prematurely. Here we show that recombinant adeno-associated virus serotype 2 (AAV2) chemically conjugated with iron oxide nanoparticles (∼5 nm) has a remarkable ability to be remotely guided under a magnetic field. Transduction is achieved with microscale precision. Furthermore, a gene for production of the photosensitive protein KillerRed was introduced into the AAV2 genome to enable photodynamic therapy (PDT), or light-triggered virotherapy. In vivo experiments revealed that magnetic guidance of “ironized” AAV2-KillerRed injected by tail vein in conjunction with PDT significantly decreases the tumor growth via apoptosis. This proof-of-principle demonstrates guided and highly localized microscale, light-triggered virotherapy. KEYWORDS: virotherapy, adeno-associated virus, nanoparticle, microtransduction, photodynamic therapy

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Additionally, clinical trials involving adeno-associated virus (AAV)-mediated gene delivery have enabled successful treatment of a number of monogenic disorders5,7 and developments in tissue engineering.8 Directed localization reduces therapeutic dose and consequently lowers risks of AAV-directed immune response, ectopic expression, and oncogene activation that leads to mutagenesis. Furthermore, improved approaches to engineer AAV capsids9 and eliminate CpG motifs from the AAV genome10 have reduced the AAV’s immunogenicity by avoiding binding to neutralizing antibodies produced from natural exposure of humans to AAV. Interestingly, AAV capsids

he U.S. Food and Drug Administration (FDA) has approved a genetically engineered virus to treat patients with advanced melanoma.1 Among innovative treatments for cancer therapy, virotherapy represents a class of promising cancer therapeutics, with viruses from several families currently being evaluated in clinical trials.2−4 Most clinical trials of virotherapy have treated patients via intratumoral injection.4 However, one of the most important technical solutions needed for clinical virotherapy is enhanced systemic delivery.1−5 Achieving efficacious and accurate systemic delivery will greatly broaden opportunities in virotherapy. Significant developments in technological solutions improving delivery, potency, and purity for virotherapy have given rise to recent success.2,4,6 Specificity in viral delivery however will greatly enhance therapeutic gains. © 2016 American Chemical Society

Received: September 7, 2016 Accepted: November 2, 2016 Published: November 7, 2016 10339

DOI: 10.1021/acsnano.6b06051 ACS Nano 2016, 10, 10339−10346

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Figure 1. Remote-controlled “ironized” virus. (A) Concept of a remotely directed ironized virus by a single tail vein injection for microvirotherapy. Ironized AAV2 rapidly accumulates in the target tumor site when directed with magnetic-field-enforced delivery. Here, KillerRed is expressed by tumor cells infected by AAV2-KillerRed. Light triggers virotherapy. Illumination of KillerRed protein generates reactive oxygen species (ROS) and subsequent intracellular damage, promoting cell death. (B) Schematic conjugation of ironized AAV2. Twostage conjugation of iron oxide nanoparticles with AAV2 via EDC/sulfo-NHS. Photograph shows the transparent yellow solution of Ironized AAV2. (C) TEM image of iron oxide nanoparticles with carboxylic acid. Bar = 50 nm. (D) TEM image of ironized AAV2 prepared at a molar ratio (1/20) of nanoparticle/EDC showing iron oxide nanoparticles associated with virus. Bar = 200 nm. (E) Percentages of GFP-expressing cells 6 days post-transduction by ironized AAV2 for varying molar ratios of nanoparticle/EDC, analyzed by flow cytometry (#, P > 0.25; ##, P < 0.005; based on a two-tailed t test, assuming unequal variances). Data show the mean of measurements conducted in sextuplicate ± SD. (F) Viability of HEK293 cells after exposure to ironized AAV2 at various mole ratios of nanoparticle/EDC. Cell viability is given as the percentage of viable cells remaining after treatment for 24 h, compared against the unexposed cells. Cell numbers were determined by the standard MTS assay (*, P > 0.2; **, P > 0.5; based on a two-tailed t test, assuming unequal variances). Data show the mean of measurements conducted in sextuplicate ± SD.

and tissue level down to the microlevel. Notably, AAV serotype 2 (AAV2) shows significant promise at both the preclinical and clinical level as a delivery agent for human gene transfer.5,7,9 Taken together, this provides strong motivation for the design of a remotely directed “ironized” virus for microvirotherapy. An established concept would provide well-defined microtransduction (Figure 1A). The validity of this concept was tested with a genetic approach to photodynamic therapy (PDT), circumventing PDT sensitizer-based side effects and providing highly specific light-triggered virotherapy in AAV2-infected cells. Sensitization is achieved intracellularly with expression of the photosensitive KillerRed protein.18

engineered to express light-dependent factor motifs bound to a light-switchable protein tagged with a nuclear localization sequence, upon exposure to light, display a significant increase in gene delivery efficiency.11 However, accurate and specific delivery of genetic material with an appropriate dosage has been a major challenge. For systemically administered viruses, the liver is often the default destination5 and represents a barrier when other organs/tissues are the intended targets. Magnetic nanoparticles provide accelerated vector accumulation in target sites when directed with magnetic-field-enforced delivery.12 Effective magnetic-mediated delivery technologies have significant potential in biomedical applications13 and have inspired various approaches to promote delivery to specific sites.14−17 Interestingly, magnetic nanoparticle coated-virus delivery can improve the activity of viral infection16,17 and stabilize modified virus against the inhibitory effects of serum.17 An appropriate magnetic field strength can be operated with a microscale “spot”, shifting the remote guidance from the organ

RESULTS AND DISCUSSION Preparation of Iron Coating for Virus. To validate this concept rigorously, adeno-associated virus serotype 2 was chemically conjugated with iron oxide nanoparticles with carboxylic acid (size: 5 nm) at various molar ratios of 10340

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Figure 2. Remote-controlled “ironized” virus for microtransduction. (A) Representative confocal images of AAV2 distribution over the period of magnetic field exposure (5, 10, or 30 min), immunostained by using anti-AAV2 antibody and the secondary antibody conjugated to Alexa Fluor 488. (B) Unmodified AAV2 at magnetization for 30 min was used as a control. Bars = 1000 μm. Profile curves of fluorescence intensity of GFP-expressing cells infected by ironized AAV2 (C, D) or AAV2 (E) incubated with magnetic field exposure (diameter: 1500 μm) for 30 min and a subsequent 6 days’ transduction. Images showing the GFP-positive cells infected by ironized AAV2 or AAV2 were observed by confocal microscopy. All fluorescence intensities from images were assayed by confocal microscopy. The cells were stained by DAPI to label the cell nuclei (adjusted to select red fluorescence). Data show means of measurements conducted in sextuplicate. Bars = 1000 μm.

AAV2 incubated with human embryonic kidney 293 (HEK293) cells (Figure 1F) or human non-small-cell lung cancer (H1975) cells (Figure S2). Overall, ironized AAV2 at the optimized molar ratio of 1/20 was suited to efficiently infect cells by AAV2 with low toxicity for magnetically guided transduction and photosensitization. We further evaluated the stability of ironized AAV2 by way of its transduction efficiency over 1-day and 7-day periods of storage in phosphate-buffered saline (PBS) solution or complete culture medium at 4 or 37 °C. At 4 °C, activity was maintained for 7 days in either medium (Figure S3). Significant deterioration in transduction was observed by way of GFP expression in cells after storage at 37 °C for 7 days (Figure S3). This temperature-dependent degradation is likely to be due to thermal deterioration, serum protein coating, and aggregation or neutralization.5,9,10 Remote Magnetic Control of Ironized AAV2 Distributions. To further evaluate the capability of remote magnetic control of ironized AAV2, we utilized the immunostain assay using anti-AAV2 antibody and the secondary antibody conjugated to Alexa Fluor 488 for the observation of AAV2 distribution in cell cultures. Appreciable fluorescence accumulated with 5, 10, or 30 min exposure to a magnetic field (2000− 2200 G) for producing a localized control of AAV2 distribution (Figure 2A). In contrast, unmodified AAV2 had a homogeneously random distribution with magnetic field exposure for 30 min (Figure 2B). Likewise, we examined the GFP expression of cells infected by ironized AAV2 when treated cells were incubated at 6 days post-transduction after exposure to the

nanoparticle/1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) through EDC/N-hydroxysulfosuccinimide (sulfo-NHS) conjugation via the AAV surface proteins’ amino groups (Figure 1B). In various formulations of ironized AAV2 molar ratios, ironized AAV2 had a hydrodynamic diameter of ca. 30−90 nm (Figure S1A). Transmission electron microscopy (TEM) morphology of iron oxide nanoparticles or ironized AAV2 showed diameters of ca. 5 nm (Figures 1C and S1B) and 30−40 nm, respectively (Figures 1D and S1C). In vitro characterization and assays for viral infection were undertaken in complete culture medium (10% fetal bovine serum, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin). To evaluate the effect of chemical conjugation on AAV2 transduction efficiency, we used an AAV2-GFP (green fluorescent protein) assay detected with flow cytometry (Figure 1E). At 6 days post-transduction without a magnetic field, cells treated with ironized AAV2 maintained a constant green fluorescent protein (GFP) expression for molar ratios of 1/1 to 1/20 (P > 0.25) relative to the control treatment of AAV2. Transduction efficiency dropped to 55.6% (P < 0.005) with a molar ratio of 1/25 and to 38.7% (P < 0.005) with a molar ratio of 1/100. These data clearly indicate that the nature of the chemical bond used to covalently couple the iron oxide nanoparticles with carboxylic acid to the protein of the AAV2 surface influenced the efficiency of viral transduction due to competition of surface ligands19 and corresponds with the hydrodynamic diameter of ironized AAV2 (Figure S1A). No cytotoxicity was observed for any of the molar ratios of ironized 10341

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Figure 3. In vitro light-triggered virotherapy. (A) Sequence map of pAAV-KillerRed. AAV2-KillerRed was produced by the expression plasmid of pAAV-KillerRed and the packaging plasmids (pHelper and pAAV-RC2). Profiles of fluorescence intensity of death and nuclei distribution of cells infected by ironized AAV2 (B, C) or AAV2 (D) after illumination of KillerRed protein. Cells were incubated with a magnetic field (diameter: 1500 μm) for 30 min subsequent to 6 days when treated with ironized AAV2 or AAV2 before irradiation. After irradiation for 20 min, the infected cells were observed using live/dead fixable far red dead cell stain kit. Right panel: Representative confocal images show the red fluorescence (cell death). Furthermore, the treated cells were stained by DAPI to reveal the cell nuclei, and the confocal images were merged with red fluorescence. All fluorescence intensities from images were assayed by confocal microscopy. Data show the mean of measurements conducted in sextuplicate. Bars = 1000 μm.

same magnetic field with a cylindrical magnet of 1500 μm diameter for a maximum duration of 30 min. The distribution of GFP-expressing cells is represented by the fluorescence intensity in Figure 2C and D, indicating a “microtransduction” profile 2000 μm in diameter. In contrast, unmodified AAV2treated cells expressing GFP were distributed randomly (Figure 2E). Light-Triggered Virotherapy Using Ironized AAV2KillerRed. With data confirming successful microtransduction of cells, we performed light-triggered virotherapy utilizing AAV2-KillerRed (Figure 3A) with a corresponding wavelength of 561 nm for 20 min irradiation.13 Consistent with the observed GFP-expressing cells infected by ironized AAV2, the KillerRed expression resulted in a circular area only (Figure S4A). Also consistent with the GFP expression, the AAV2KillerRed control had no preferential spatial transduction (Figure S4B). As KillerRed possesses photoinstigated toxicity, we observed cell death after irradiation with yellow light in the cells expressing the KillerRed protein. Distribution of cell death was effectively accumulated in the magnetic field microspot and

produced no phototoxicity when not infected by AAV2KillerRed (Figure 3B and C), demonstrating remotely controlled ironized AAV2 for light-triggered virotherapy as compared to unmodified AAV2 (Figure 3D). Antitumor Activity and Biodistribution in Preclinical Studies through the Bloodstream. To translate the proofof-principle results (Figures 1−3) to preclinical application, we performed light-triggered virotherapy treatment using remotely ironized AAV2-KillerRed in athymic BALB/c nude mice with EGFR-TKI-resistant H1975 (EGFRL858R/T790M) xenograft tumors (Figure 4A). Notably, treatment with ironized AAV2KillerRed was associated with strong suppression of tumor growth (Figures 4B and S5), contrasted by a large area of tumor necrosis indicated by H&E (hematoxylin and eosin) staining (Figure 4C), extensive positive staining by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay (Figure 4D), and the nucleic acid labeled by DAPI (4′,6diamidino-2-phenylindole) staining (Figure 4E) compared with other treatments. Also, the light blue colored area stained with Prussian Blue indicated the distribution and increased presence 10342

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Figure 4. In vivo systemic remotely controlled virotherapy. (A) Treatment protocols assessing remotely controlled delivery, light-triggered virotherapy using various conditions. (B) Tumor growth of various virus-treated EGFR-TKI-resistant H1975 (EGFRL858R/T790M) xenograft tumors via tail vein injection under magnetization (M) and/or light irradiation (L). Tumor sizes were measured by a caliper on the described days (*, P < 0.015; **, P < 0.001; based on a two-tailed t test, assuming unequal variances). Data show means of measurements conducted in sextuplicate ± SEM. Representative images of tumor sections from mice per group (n = 6) after various treatments on day 15 were stained with hematoxylin and eosin (H&E) (C), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (D), DAPI (E), and Prussian Blue (F). Bars = 500 μm. (G) Biochemical analysis of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic (GPT), total bilirubin (TBIL), and creatinine (CRE) levels was performed in serum obtained from blood after administration of ironized AAV2-KillerRed in athymic BALB/c nude mice at day 0, day 2, day 7, and day 14. Data show means of measurements conducted in triplicate ± SD. (H) Body weight of mice after various treatments. Body weight of mice were measured at described days in response to the treatments of various formulations by tail-vein injection with and without exposure to M or L. Results show means of measurements conducted in sextuplicate ± SD. (I) Representative IVIS images of mice taken at day 14 after tail vein injection of various formulations using AAV2 encoded luciferase as a detection signal. (J) Representative IVIS images of organs from mice injected i.v. with various treatments at day 14.

of iron in the samples exposed to the magnetic field and ironized AAV2 (Figure 4F). Single administration of ironized AAV2-KillerRed injected by tail vein resulted in significantly

suppressed tumor outgrowth; however it lacked long-term suppression. Impressively, when we further injected ironized AAV2-KillerRed at day 8, a complete cessation of volume 10343

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(Milwaukee, WI, USA). Plasmid DNA of pKillerRed-dMito was purchased from Evrogen JSC (Moscow, Russia). Virus (AAV2luciferase) and plasmid DNAs of pHelper, pAAV-RC2, pAAV-GFP, and pAAV-MCS were purchased from Cell Biolabs (San Diego, CA, USA). Plasmid pAAV-KillerRed was constructed as follows. First, we added the EcoRI and the Sall sites into a KillerRed fragment from pKillerRed-dMito by using polymerase chain reaction (PCR) with the following sequences of primers: 5′-GGCGAATTCGCCACCATGGGTTCAGAGGGCGGCCCCGCCC-3′ and 5′-ACGCGTCGACTTAATCCTCGTCGCTACCGATGGCGCTGGT-3′. The PCRgenerated KillerRed cDNA (0.71 kb) was then cloned into the EcoRISall site of pAAV-MCS to yield pAAV-KillerRed (Figures 3A and S7). The human embryonic kidney 293 (HEK293, CRL-1573, ATCC) and 293T (CRL-3216, ATCC) cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin. Cells were cultured in a 37 °C incubator with 5% CO2. The human nonsmall-cell lung cancer cell line (H1975, CRL-5908, ATCC) was cultured in RPMI (Roswell Park Memorial Institute) 1640 medium with 10% FBS. Cells were cultured in a 37 °C incubator with 5% CO2. Production, Purification, and Titration of Virus. AAV2-GFP or AAV2-KillerRed production was performed using the AAV-2 helper free packaging system (Cell Biolabs). Briefly, AAV2-reporter was produced by PEI-mediated cotransfection of plasmid DNAs (pHelper, pAAV-RC2, and pAAV-transgene) in 293T cells. For each 100 mm dish, 293T cells were transfected with 20 μg of pHelper, 10 μg of pAAV-RC2, and pAAV-GFP (or pAAV-KillerRed). The three plasmid DNAs were mixed with 40 μg of PEI in serum-free culture medium and then thoroughly mixed for 30−60 s by vortex mixing and left for at least 20−30 min. Transfection time was performed for only 30 min. Transfected cells were harvested 3 days after transfection. Purification and titration of AAV2-GFP or AAV2-KillerRed were performed according to the protocols of the ViraBind AAV purification kit (Cell Biolabs) and QuickTiter AAV quantitation kit (Cell Biolabs) for viral transduction. The number of genome copies (GC) per milliliter of AAV2-GFP or AAV2-KillerRed stock for each batch (8 × 100 mm dishes) of virus production ranged from 1011 to 1012. Purified viruses were stored at −80 °C until use. Preparation and Characterization of Ironized Virus. Ironized AAV2 was prepared according to the procedures of chemical conjugation (Figure 1B). Reaction mixtures were prepared, which contained the iron oxide nanoparticles with carboxylic acid (25 μg, 0.1725 nmol), and EDC (0.1725, 0.865, 1.73, 3.46, 4.325, 8.65, or 17.3 nmol) in MES-buffered saline solution, and to the mixtures was gently added sulfo-NHS with stirring at 25 °C for 15 min to achieve a homogeneous solution of iron oxide nanoparticles with amine-reactive sulfo-NHS ester. The AAV2 stock (0.5 μL, 1 × 1012 GC mL−1) in PBS was added dropwise to the mixtures and then reacted at constant temperature of 25 °C for 120 min. After the chemical conjugation process, the yellow solution was purified by using a size desalting column (molecular weight cutoffs: 100 K) that was equilibrated with PBS and solvent-exchanged to PBS (Figure S8). After the process of purification, a recycle yield of ∼90% was achieved by using PCR with the following sequences of primers for AAV2-KillerRed: 5′G CCCA TGA GCT GGAA GCC- 3′ and 5′-C GAT GGCG CTGGTGATGC-3′; Figure S9). The obtained PCR fragments of AAV2-KillerRed corresponded with the expected size of 539 bp. The amount of iron associated with the ironized AAV2 after chemical conjugation was determined with inductively coupled plasma atomic emission spectroscopy (ICP-AES).22 Samples were digested by heating for 24 h to completely evaporate medium. Subsequently, 1 mL of 37% HCl was added and vortexed to allow complete dissolution of the iron oxide nanoparticles into an ionic state. Samples were reheated again to 70 °C for 12 h to evaporate HCl. Finally, the sample was added into 3 mL of 2% nitric acid and filtered using a 0.2 μm surfactant-free cellulose acetate membrane filter (Thermo Scientific Inc.) to remove organic matter and impurities. ICP-AES analysis of the ironized AAV2 indicated a yield of 24 ± 2.1, 38 ± 2.5, 45 ± 1.7, 53 ± 2.3, 61 ± 3.2, 66 ± 3.5, or 75 ± 2.6% iron oxide nanoparticles (total amount 25 μg) for molar ratios of nanoparticle/EDC of 1/1, 1/5, 1/

growth was achieved for a further 5 days and growth was significantly inhibited beyond this (P < 0.015). The roles of magnetization and light irradiation on the efficacy of the ironized AAV2-KillerRed were independently tested with regard to suppression of tumor growth. Neither condition contributed to altering tumor growth independently. Furthermore, delivery of AAV2-KillerRed in the presence of a magnetic field and light irradiation also did not result in any statistically relevant antitumor effect due to the most intense expression in the liver tissue indicating rapid clearance after systemic injection.20 Taken together, these results imply that ironized AAV2 without the magnetic guidance accumulates in the liver and is consistent with systemic clearance13 and AAV2’s natural fate.20 Concurrent delivery is consistent with other studies to assist in overcoming the inherently difficult challenge in achieving systemic delivery.1−5 Animals treated with ironized AAV2-KillerRed were also evaluated for levels of glutamic oxallotransaminase (GOT), pyruvic oxallotransaminase (GPT), total bilirubin (TBIL), and creatinine (CRE) to monitor liver and kidney function. These biochemical analyses did not show any significant liver or kidney toxicity (Figure 4G). In all experimental groups, no significant loss of body weight was detected, representing a lack of any serious ironized virus, magnetic field exposure, or light irradiation-related toxicity (Figure 4H). Biodistribution was considered at day 7 (Figure S6) and day 14 (Figure 4I) with bioluminescence for animals treated comparably to the in vivo studies but with ironized AAV2-luciferase. Consistent with those findings, we observed significant bioluminescence in the tumor at day 7 and day 14 when magnetic guidance was utilized, and this coincides with the tumor suppression shown in Figure 4B. This reinforces the dynamic dependence upon remotely controlling specificity in delivery. As expected, bioluminescence was also observed in the liver, consistent with the clearance pathway of viruses and nanoparticle (Figure 4J).18

CONCLUSIONS In summary, we have demonstrated specificity in antitumor effects with light-triggered virotherapy achieved with remotely guided “ironized” virus delivery. Such a technological concept could be harnessed to improve therapeutic efficacy and accuracy with systemic delivery via the bloodstream. There are several distinguishing features of our ironized AAV2, such as targeted delivery, light-triggered activation of virotherapy, lack of recombination and genomic integration,5 and strong preclinical safety record,5 that define potential advantages of this concept. Furthermore, magnetic resonance imaging (MRI) instruments can be applied to create pulsed magnetic field gradients in the desired direction,21 and it may provide the prospect of shaping the accumulation within an internal 3D volume. EXPERIMENTAL SECTION Materials and Cell Culture. Iron oxide nanoparticles with carboxylic acid (lot number: 051413A; size: 5 nm; zeta potential: −30 to −50 mV) were purchased from Ocean NanoTech (San Diego, CA, USA). (1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride), N-hydroxysulfosuccinimide, and 2-(N-morpholino)ethanesulfonic acid (MES)-buffered saline were purchased from Thermo Scientific Inc. (Rockford, IL, USA). Phosphate-buffered saline was purchased from Sigma Co. (St. Louis, MO, USA). Branched polyethylenimine (PEI, Mw = 25 000) was purchased from Aldrich 10344

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As per the previous study of KillerRed activation,18 we further chose the irradiation time of 20 min with a 561 nm argon laser for optimized reactive oxygen species (ROS) generation and KillerRed phototoxicity for our studies. After irradiation of KillerRed, the infected cells were observed using a live/dead fixable far red dead cell stain kit (Thermo Fisher Scientific Inc.) as described by the manufacturer. The treated cells were stained by DAPI to label the cell nuclei. Mice Studies. All procedures involving animals were permitted by Academia Sinica Institutional Animal Care and Utilization Committee. Athymic BALB/c nude mice (6-week-old males) were provided by National Laboratory Animal Center (Taiwan). Mice were maintained in a controlled environment with a 12 h/12 h light/dark cycle, housed in groups of 5 maximum, and allowed food and water ad libitum. In Vivo Light-Triggered Virotherapy Efficacy. To assess the light-triggered virotherapy effect of ironized AAV2 or unmodified AAV2, with or without an external magnetic field on the tumor site, EGFR-TKI-resistant H1975 cells that carry the L858R and T790M xenograft tumors were established by injecting 2 × 106 cells subcutaneously into abdomens of 6-week-old male athymic nude mice as prepared according to the trials in Figure 4A. Once tumors reached ∼200 mm3 volume, mice were randomized into five groups and tail vein injected with 100 μL of PBS with ironized AAV2 (5 × 109 GC mouse−1) or AAV2 (5 × 109 GC mouse−1). The treatment of PBS injection was used in control mice. In the treatments consisting of applying a magnetic field to the targeted tumor, the H1975 (EGFRL858R/T790M) xenograft tumors were exposed to magnetic fields at 1.5 T Gauss for 2 h. At day 3, animals were treated with 1.5 mW mm−2 total irradiance for KillerRed activation. The tip of the laser fiber was mounted above the tumor, perpendicular to the animal. This regimen was determined following initial optimization experiments.18 The laser treatment was administered to the tumor for 20 min every day for 5 days starting from the third day after injection as described in Figure 4A. Tumor growth was measured every day following treatment using calipers. The length (L) and width (W) of the tumor were measured, and the tumor volume was calculated according to the following formula: tumor volume = (0.5L2)W. Tumor size examination was conducted 24 h after the last treatment. Histological and Immunohistochemical Analysis. H1975 (EGFRL858R/T790M) xenograft tumors were harvested 15 days after innoculation. Harvested xenograft tumors were fixed in 10% formalin and paraffin-embedded, and 5 mm sections were stained with H&E and examined by microscopy. Xenograft tumor sections were also stained with Prussian Blue or Click-iT Plus TUNEL assay with Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) to detect the iron oxide nanoparticle of ironized AAV2 within tumors or observe them in in situ apoptosis detection. Blood Analysis. After administration of ironized AAV2-KillerRed at day 0, day 2, day 7, and day 14, the blood serum obtained from athymic BALB/c nude mice was collected by using orbital sinus blood sampling. Biochemical analysis of GOT, GPT, TBIL, and CRE levels was evaluated. For determination of GOT and GPT levels, the enzymatic method by measuring relative aspartate aminotransferase and alanine aminotransferase activity was used. Also, the level of TBIL, which is an indicator of hepatic cellular damage including hepatoma and hepatitis, was determined by using Randox diagnostic test kits according to the manufacturer’s instructions. As the indicator for kidney function, CRE level was tested by using Randox diagnostic test kits. In Vivo Bioluminescence Imaging. Ironized AAV2-luciferase or AAV2-luciferase in sterile-filtered PBS solution (100 μL total volume containing 5 × 109 GC AAV2) was injected via tail vein in mice with or without an external magnetic field on xenograft tumor. Bioluminescence imaging was achieved at day 7 and day 14 after treatments. Mice were anesthetized in a chamber filled with 2% isoflurane in oxygen. The luminescent images after intraperitoneal (i.p.) injection of luciferin (∼240 μL, Caliper Life Sciences Inc., Hopkinton, MA, USA) were captured at 5−10 min postincubation by an IVIS imaging system (Xenogen IVIS-50 with Living Image software), with a constant image acquisition time of 5 min (Bin:

10, 1/20, 1/25, 1/50, or 1/100, respectively. All data have been presented as a percentage of the initial iron concentration. Data are shown as the mean ± the standard deviation for experiments performed in triplicate. For TEM (JEOL JEM-1400) analysis, a drop of ironized AAV2 solution was allowed to air-dry onto a Formvar-carbon-coated 200 mesh copper grid. TEM images were then acquired on a JEOL-1400 microscope operating at an accelerating voltage of 100 kV. The measurements of hydrodynamic diameter of ironized AAV2 at different molar ratios of nanoparticle/EDC were carried out using a Zetasizer Nano ZS (Malvern Instruments). The temperature for the measurement was kept at 25 °C. Transduction. All experiments of viral infection and transduction used culture medium with 10% FBS, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin. To measure the transduction ability of ironized virus or unironized virus without any magnetic fields, we used the AAV2-GFP as the signal indicator. HEK293 cells were seeded in 24well plates at 1 × 105 cells well−1 and infected the next day. The test samples of ironized AAV2 at various molar ratios of nanoparticle/EDC and the AAV2-GFP only were added to cells in DMEM with 10% FBS for 6 days’ transduction, respectively. The GFP-expressed cells by viral transduction were quantitatively assessed by flow cytometry (Beckman Coulter, Fullerton, CA, USA). Cells were detached by 0.025% trypsin, and suspensions were transferred to microtubes and fixed by 4% paraformaldehyde. Cells were appropriately gated by forward and side scatter, and 10 000 events per sample were collected. The untreated cells were used as the negative control. Toxicity of Ironized Virus. A total of 7 × 104 HEK293 or H1975 cells were seeded in each of the wells of a 24-well plate and fed with culture medium for 12 h. The cells were then exposed to test ironized AAV2 at different molar ratios of nanoparticle/EDC and incubated at pH 7.4 for 24 h. After 24 h of incubation, the transfection media containing test samples were removed. Additionally, the iron oxide nanoparticles or AAV2 was only incubated at pH 7.4 for 24 h. The CellTiter 96AQueous One solution cell proliferation assay system (Promega, Madison, WI, USA) was used to determine the cell proliferation as per previous studies.18,23 The optical density of formazan at 490 nm quantified the cell viability. The reagent contained the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and inner salt (MTS), the reduction of MTS achieved by untreated cells was set at 100%, and that of test cells was expressed as a percentage of untreated cells. Stability of Ironized Virus. From the results shown in Figure 1E and F, we subsequently chose a molar ratio of nanoparticle/EDC of 1/ 20 for the ironized AAV2 in subsequent in vitro and in vivo studies. To determine the stability of ironized virus, 100 μL of the ironized AAV2 (5 × 108 GC) solution using AAV2-GFP was added to 1 mL of PBS solution (pH 7.4) or complete culture medium (pH 7.4) and stored at 4 or 37 °C for 1 day or 7 days. Measurement of the transduction ability of ironized virus was then conducted as described in the section on Transduction. Micro-transduction by an External Magnetic Field. HEK293 cells were seeded in 35 mm dishes at 2.5 × 105 cells well−1 and infected the next day. The test samples of ironized AAV2 or AAV2 only were incubated with cells in DMEM with 10% FBS followed by analysis at different time courses (0, 5, 10, or 30 min) of external magnetic field (2000−2200 G) with a diameter of 1500 μm. Subsequently, samples were fixed by 4% paraformaldehyde, and the immunostained virus was performed using anti-AAV antibody (Abcam, Cambridge, MA, USA) specific to the amino acid 75−86 of major coat protein VP3 of AAV2 for the observation of AAV2 distribution. The signal amplification was developed with goat anti-rabbit IgG H&L conjugated to Alexa Fluor 488 (Abcam) and observed with the confocal microscope. Alternatively, infected cells were observed and analyzed for GFP expression by a confocal microscope after 6 days of transduction. The infected cells were stained by DAPI to label the cell nuclei. 10345

DOI: 10.1021/acsnano.6b06051 ACS Nano 2016, 10, 10339−10346

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06051. In vitro and in vivo data of ironized AAV2-KillerRed or ironized AAV2-luciferase (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] (Z.-X. Liao). *E-mail: [email protected] (P.-C. Yang). Author Contributions

S.J.T., Z.X.L., and P.C.Y. designed the experiments. S.J.T., Z.X.L, and I.M.K. performed the synthesis and characterizations of ironized virus. S.H.K., Z.X.L., and M.J.C. constructed and amplified all viral plasmids. S.J.T. and Z.X.L. prepared and produced all viral particles. S.J.T. carried out the in vitro assays. Z.X.L., S.J.T., I.M.K., and K.Y.H. conceived and planned the photodynamic therapy component of the research. K.Y.H., S.J.T., Z.X.L., and S.C.Y. carried out the in vivo mice studies. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported through Academia Sinica Research Program on Nanoscience and Nanotechnology (2399-1050200) and the Taiwan Ministry of Science and Technology Grant (MOST105-2628-E-110-001-MY3, MOST105-2628-B110-004-MY3, and MOST105-2218-E-002-031). The authors thank Ms. Yun-Shan Chen for technical support in the Molecular and Genetic Imaging Core, Taiwan Mouse Clinic and Pathology Core Laboratory in the Institute of Biomedical Sciences, Academia Sinica. REFERENCES (1) Ledford, H. Cancer-Fighting Viruses Win Approval. Nature 2015, 526, 622−623. (2) Bell, J.; McFadden, G. Viruses for Tumor Therapy. Cell Host Microbe 2014, 15, 260−265. (3) Russell, S. J.; Peng, K. W.; Bell, J. C. Oncolytic Virotherapy. Nat. Biotechnol. 2012, 30, 658−670. (4) Miest, T. S.; Cattaneo, R. New Viruses for Cancer Therapy: Meeting Clinical Needs. Nat. Rev. Microbiol. 2014, 12, 23−34. (5) Kotterman, M. A.; Schaffer, D. V. Engineering Adeno-Associated Viruses for Clinical Gene Therapy. Nat. Rev. Genet. 2014, 15, 445− 451. (6) Zamarin, D.; Holmgaard, R. B.; Subudhi, S. K.; Park, J. S.; Mansour, M.; Palese, P.; Merghoub, T.; Wolchok, J. D.; Allison, J. P. Localized Oncolytic Virotherapy Overcomes Systemic Tumor Resistance to Immune Checkpoint Blockade Immunotherapy. Sci. Transl. Med. 2014, 6, 226ra32. (7) Naldini, L. Gene Therapy Returns to Centre Stage. Nature 2015, 526, 351−360. 10346

DOI: 10.1021/acsnano.6b06051 ACS Nano 2016, 10, 10339−10346