Mild Hyperthermia Induced by Gold Nanorod-Mediated Plasmonic

Nov 2, 2016 - Oncolytic adenovirus (Ad) is a promising candidate for cancer gene therapy. However, as a monotherapy, it has shown insufficient therape...
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Mild Hyperthermia Induced by Gold NanorodMediated Plasmonic Photothermal Therapy Enhances Transduction and Replication of Oncolytic Adenoviral Gene Delivery Bo-Kyeong Jung,†,⊥ Yeon Kyung Lee,‡,⊥ JinWoo Hong,† Hamidreza Ghandehari,*,‡,§ and Chae-Ok Yun*,† †

Department of Bioengineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, Korea ‡ Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Korea § Departments of Pharmaceutics and Pharmaceutical Chemistry and of Bioengineering, Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah 84112, United States S Supporting Information *

ABSTRACT: Oncolytic adenovirus (Ad) is a promising candidate for cancer gene therapy. However, as a monotherapy, it has shown insufficient therapeutic efficacy in clinical trials. In this work, we demonstrate that gold nanorod (GNR)-mediated mild hyperthermia enhances the cellular uptake and consequent gene expression of oncolytic Ad to head and neck tumor cells. We examined the combination of oncolytic Ad expressing vascular endothelial growth factor promoter-targeted artificial transcriptional repressor zinc-finger protein and GNR-mediated mild hyperthermia to improve antitumor effects. The in vitro mechanisms of increased transduction in the presence and absence of hyperthermia were explored followed by evaluation of efficacy of this combination strategy in an animal model. Exposure to optimized hyperthermia conditions improved endocytosis of oncolytic Ad, transgene expression, viral replication, and subsequent cytolysis of head and neck cancer cells. GNR-mediated plasmonic photothermal therapy resulted in precise control of tumor temperature and induction of mild hyperthermia. A combination of oncolytic Ad and GNRs resulted in potent tumor growth inhibition of head and neck tumors. KEYWORDS: oncolytic adenovirus, hyperthermia, endocytosis, gold nanorods, head and neck cancer

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example, in a clinical study of ONYX-015, which is an E1B 55 kDa-deleted Ad, feasible therapeutic efficacy was observed in patients with pancreatic tumors, but viral replication was not detectable.10 Successful oncolytic Ad-mediated cancer gene therapy requires a strategy for combination therapy to increase viral replication and/or cellular uptake. Additionally, synergistic therapeutic efficacy of combination treatment can lead to dose reduction of oncolytic Ad, resulting in attenuated risk of shedding of viral vector to nontarget tissues and any adverse inflammatory response.

ncolytic adenovirus (Ad) is a promising candidate for cancer gene therapy. Oncolytic Ad has several beneficial characteristics such as no risk of insertional mutagenesis, facile production in high-titer, high transgene expression level, selective replication in cancer cells, and subsequent replication-mediated oncolysis.1 Replicating viral agents in tumors leads to improved efficacy over nonreplicating Ads because of their inherent ability to multiply, lyse infected cancer cells, and spread to surrounding cancer cells.2 Importantly, replication of oncolytic Ad and subsequent secondary infection of neighboring cells increases expression level of therapeutic gene in cancer-specific manner, leading to potent antitumor effect with minimal potential side effects.3−9 Despite these attributes, oncolytic Ad as a monotherapy induces insufficient therapeutic efficacy in clinical trials. For © 2016 American Chemical Society

Received: September 28, 2016 Accepted: November 2, 2016 Published: November 2, 2016 10533

DOI: 10.1021/acsnano.6b06530 ACS Nano 2016, 10, 10533−10543

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ACS Nano Reports in the literature suggest that heat shock proteins (HSPs) mitigate the oncolytic effect of replication-competent human Ad.11 The oncolytic and burst kinetics of a replicationcompetent Ad has been tested after exposure to heat shock, resulting in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA.12 Other studies have further examined heat shock promoter activity in replicationdeficient Ads and on the effects of temperature on adenoviral infection and replication.13,14 These in vitro studies suggest that mild hyperthermia can enhance the therapeutic efficacy of oncolytic Ads. The influence of mild hyperthermia on enhancing the endocytosis of oncolytic Ads however is largely unexplored. One way to generate heat for cancer therapy is laser light source. Laser light is noninvasive, tunable, allows for multiple pulsed regimens, and can create heat energy leading to the destruction of the cancer tissue.15 However, high intensity laser can damage adjacent normal tissues due to nonspecific absorption. To reach the tumor site for therapeutic dose, more energy is required, which could cause burning, blistering, and pain.16 Therefore, further improvements are necessary to enhance the delivery of laser-induced heat to the tumor region for attenuated adverse effects.17 Gold nanoparticles (GNPs) absorb near-infrared (NIR) wavelengths and exhibit plasmonic properties. Plasmon-resonant gold nanorods (GNRs), which have large absorption cross sections at near-infrared (NIR) frequencies, have been utilized for localized hyperthermia.18 We have shown that moderate hyperthermia with GNR-mediated plasmonic photothermal therapy (PPTT) can be used to guide the delivery of macromolecular therapeutics to solid tumors and improve efficacy.19−21 However, utility of GNR-mediated mild hyperthermia for the delivery of viral gene carriers has not been explored. Accessible head and neck tumors are candidates for a combination of GNR-mediated mild hyperthermia and intratumoral oncolytic therapy. Herein we demonstrate that GNR-mediated mild hyperthermia enhances the cellular uptake and consequent gene expression of oncolytic Ad to head and neck tumor cells. We have examined the combination of oncolytic Ad expressing vascular endothelial growth factor (VEGF) promoter-targeted artificial transcriptional repressor zinc-finger protein22 and GNR-mediated mild hyperthermia to improve antitumor effect against head and neck cancer. The in vitro mechanisms of increased transduction in the presence of hyperthermia are explored, followed by evaluation of therapeutic efficacy of this combination strategy in an animal model of head and neck cancer.

Figure 1. Enhanced cell killing effect of oncolytic Ad combined with mild hyperthermia. JHU-022 cells were treated with various backbones of oncolytic Ad at 5 MOI with heat shock at 42 °C or northermia post-infection. Cell viability was assessed by MTT assay. The serum-free media-treated group (cell only) was normalized at 100%. Data are presented as mean ± SD ***P < 0.001.

exposed to mild hyperthermia compared to those treated under normal physiological temperature. Hyperthermia-mediated enhancement in cancer cell killing efficacy of oncolytic Ad was not dependent on the status of Ad fiber, E1A gene composition, promoter to drive E1A expression, or insertion of therapeutic gene. These results demonstrate that mild hyperthermia can enhance the efficacy of oncolytic Ads regardless of their genetic structures, implying that such enhancement is a general phenomenon and can be used for a variety of viral constructs. Enhancement of Viral Replication with Mild Hyperthermia. Cytolytic effect of oncolytic Ad is dependent on its viral replication capacity, as intracellular accumulation of oncolytic Ad progenies induces cell lysis and ultimately causes secondary infection of neighboring cancer cells.27 We investigated whether the enhanced cytolytic effect of oncolytic Ads under mild hyperthermia was mediated by active viral replication. As shown in Figure 2a, viral replication significantly increased in cancer cells at 48 h post-infection under hyperthermia compared to northermia (P < 0.01). Furthermore, expression level of Ad E1A, an integral component of Ad replication, was markedly higher under mild hyperthermia compared to physiological temperature at 12 and 24 h following Ad treatment (Figure 2b). This is in good agreement with previous reports, showing that HSPs 70 and 72 enhance the replication of Ad.25,26 These results indicate that hyperthermia-mediated augmentation of oncolytic Ad replication leads to induction of a potent cancer cell killing effect. It is noteworthy that at later time points, e.g., 48 h post-heat treatment, this augmentation was reduced (Data not shown). The reason for this reduction in difference between hyperthermia and northermia condition could be due to either limited cell pool in vitro and/or the normalization of cell

RESULTS AND DISCUSSION Enhanced Tumoricidal Effect of Oncolytic Ads with Mild Hyperthermia. Our previous work demonstrated that E1B 55 kDa-deleted oncolytic Ad can preferentially replicate in cancer cells, however, its cytopathic effect was attenuated in several cancer cells due to a partial loss of viral replication function related to E1B 55 kDa.23,24 It has been reported that the activation of heat shock response is essential for replication of Ad and that induction of HSP is mediated by E1 gene products.25 Furthermore, HSP 72 expression enabled viral replication of clinically approved E1B 55 kDa-deleted oncolytic Ad, ONYX-015, in nonpermissive cells.26 Based on these previous studies, we evaluated whether hyperthermia can affect the cancer cell killing effect of several oncolytic Ads with different genome structures. As shown in Figure 1, cell killing efficacy of oncolytic Ads was significantly increased in cells 10534

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The large GTPase dynamin (100 kDa) is known as one of the host cell proteins that regulate clathrin-mediated endocytosis.32 Dynamin is related to clathrin-coated membrane invaginations and mediates the constriction of coated pits and the budding of coated vesicles from the plasma membrane.33,34 The expression level of dynamin under hyperthermia conditions was assessed by Western blot. As shown in Figure 3b, the expression level of dynamin under mild hyperthermia was higher than that measured at 37 °C. Pretreatment of cancer cells with dynasore, a cell-permeable dynamin inhibitor, attenuated Ad-mediated GFP expression level in comparison, same as clathrin, and transduction efficiency decreased in the presence of dynasore, a cell-permeable inhibitor of dynamin. Dynamin regulates clathrin-mediated endocytosis, a major pathway for uptake into eukaryotic cells,35 and significantly influences the uptake of Ad. It must be noted that the normalized GFP expression at 100 μM concentration of dynasore did not change compared to northermia, suggesting that Dynasore-mediated inhibition of dynamin is a saturable process at this concentration. Caveolins are major structural proteins of caveolar membranes.36 We investigated whether mild hyperthermia can influence the expression of caveolin-1. Results demonstrate that the caveolin-1 expression level increases under mild hyperthermia (Figure 3c). Mild hyperthermia-induced higher GFP expression in cells treated with naked Ad compared to northermia. Presence of Genistein, a caveolin inhibitor, did not affect the transduction efficiency of Ad under both hyperthermia and northermia conditions, suggesting that enhanced transduction of Ad under hyperthermia was not regulated by caveolin-mediated endocytosis. These findings are in good agreement with previous reports demonstrating that endocytosis of Ad is usually not dependent on caveolin-mediated cellular uptake pathways.37 Together, these results demonstrate that hyperthermia-induced enhancement in transduction of Ad was mediated by increased cellular expression level of dynamin and clathrin, which facilitated cellular internalization of Ad. To further illustrate the relative entry of Ad with hyperthermia, inhibition of Ad transduction experiment was performed with amiloride as a known micropinocytosismediated endocytosis inhibitor.38 These results demonstrate that Ad-mediated GFP expression is significantly blocked to a higher extent under mild hyperthermia compared to northermia (Figure 3d). The relative fluorescence intensity of GFP expression under mild hyperthermia showed about an 80% decline at 500 μM concentration of amiloride. These data support that the cellular uptake of the Ad under mild hyperthermia is partially mediated by macropinocytosis. Time Dependence of Transduction Efficiency. Sequential order in which hyperthermia and viral transduction occurs, as well as time of exposure to hyperthermia, can influence the transduction efficiency of Ad. To investigate this phenomenon, transduction efficiency of Ad was examined at various time points over an 18 h period before and after exposure to hyperthermia. As shown in Figure 4, transduction efficiency of Ad was highest when cells were exposed to mild hyperthermia 2 h prior to transduction. Based on this finding, all subsequent experiments had cells exposed to mild hyperthermia 2 h prior to infection. In Vitro Viral Replication and Cellular Uptake of Oncolytic Ad under Pretreated Mild Hyperthermia. In order to assess whether enhanced transduction efficiency of Ad under hyperthermia was due to increased cellular uptake,

Figure 2. Enhanced viral replication of oncolytic Ad in the presence of mild hyperthermia. JHU-022 cells were treated with oncolytic Ad with heat shock at 42 °C or northermia post-infection. (a) At 4, 24, or 48 h post-infection, supernatant and cell pellets were harvested, and the copy number of viral genome in each sample was quantified by Q-PCR. (b) Representative Western blot analysis of E1A from the cell lysate at 6, 12, or 24 h post-infection. Data are presented as mean ± SD, **P < 0.01.

temperature back to 37 °C over time. Future studies to further sustain this augmentation may involve repeated administration of heat. Such repeated administration can be accomplished by noninvasive exposure to laser beam using GNR mediated PPTT. Endocytosis Mechanisms of Ad Uptake. To investigate possible reasons for the observed improved efficacy, we evaluated the mechanisms of endocytosis of Ad under hyperthermia and northermia conditions. It is known that mild hyperthermia improves the rate of cellular uptake.28,29 Ads have been reported to be internalized into cells via clathrinmediated endocytosis.30 In order to verify cellular uptake efficiency of Ad related to proficiency of Ad replication, JHU022 cells were treated with mild hyperthermia for 2 h, and the cells were harvested to measure the expression of clathrin at various time points. As shown in Figure 3a, the cellular expression level of clathrin markedly increased under mild hyperthermia compared to northermia. Furthermore, the cells treated with only naked Ad showed that replicationincompetent Ad-mediated GFP expression level was markedly higher under hyperthermia compared to northermia. This indicates that increased cellular expression level of clathrin under mild hyperthermia resulted in increased cellular uptake of Ad. Of note, pretreatment with chlorpromazine, a clathrin inhibitor,31 resulted in significantly greater attenuation of GFP expression under mild hyperthermia compared to northermia. These results suggest that internalization of Ad under mild hyperthermia is more pronounced and dependent on clathrinmediated endocytosis compared to northermia. This is due to the increased cellular expression level of clathrin under hyperthermia compared to northermia. 10535

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Figure 3. Mechanism of cellular uptake of oncolytic Ad with mild hyperthermia. JHU-022 cells were incubated at 42 °C for 2 h. The expression levels of (a) clathrin, (b) dynamin, or (c) caveolin were verified at 4 h. To assess the endocytosis mechanism of Ad with mild hyperthermia, JHU-022 cells were pretreated for 30 min with inhibitors of (a) clathrin-, (b) dynamin- (c) caveolin-, or (d) macropinocytosismediated endocytosis diluted in serum-free media at indicated concentrations. Ad was subsequently added in the presence of inhibitors for an additional 2 h with 42 °C incubation or northermia. After 48 h, GFP expression was observed by fluorescence microscopy and flow cytometry. Original magnification: × 200. Data presented as mean ± SD ***P < 0.001. 10536

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Figure 4. Transduction efficiency of oncolytic Ad at various time points with mild hyperthermia or northermia. JHU-022 cells were seeded and infected with 5 MOI of GFP-expressing Ad and incubated with various time points at 42 °C for 2 h. The transduced cells were incubated for an additional 48 h. Cells were imaged using fluorescence microscopy and analyzed for flow cytometry. Original magnification: × 200. Data presented as mean ± SD ***P < 0.001 versus no heat.

Figure 5. Enhanced viral replication of oncolytic Ad delivery in the presence of mild hyperthermia. (a) The cellular uptake of Ad in the presence or absence of mild hyperthermia. At 5, 30, or 120 min post-infection, the cellular uptake was quantified by assessment of FITC intensity through FACS analysis. Data are presented as mean ± SD, ***P < 0.001. (b) Quantification of viral replication by Q-PCR. (c) VEGF ELISA.

cellular uptake of fluorescein isothiocyanate (FITC)-labeled oncolytic Ad with or without heat shock treatment was analyzed by FACS. As shown in Figure 5a, the cellular uptake of FITC-labeled oncolytic Ad increased by 1.3-fold under mild hyperthermia compared to those incubated without heat shock at 120 min, indicating that heat shock can enhance the cellular internalization of oncolytic Ad (P < 0.001). Furthermore, viral production of oncolytic Ad was significantly increased by 5.9fold following pretreatment with heat shock in comparison to

northermia, implying that increased cellular uptake of oncolytic Ad, which positively correlated with elevated expression level of dynamin and clathrin, can augment viral production (Figure 5b). Further, VEGF expression was significantly attenuated under hyperthermia compared to northermia (Figure 5c). These results suggest that a combination of heat shock treatment and oncolytic Ad might induce potent antitumor effect, as tumor cell lytic effect of oncolytic Ad is mediated by active viral replication. 10537

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Figure 6. NIR laser-induced heating with PEG-GNRs in vitro and in vivo. (a) Changes of temperature in PEG-GNRs suspension depending on concentration of GNRs and NIR laser powers in vitro. Error bar presents the mean ± SD (n = 3). (b) Mild hyperthermia induced by various laser powers at fixed PEG-GNR concentration (48 ug/mL) and (c) the confirmation of GNRs (48 ug/mL) leading to mild hyperthermia at 1.0 W/cm2 laser power in JHU-022 tumor models. *** Indicates a statistically significant difference (P < 0.001) by one-way analysis of variance. Each error bar is represented as ± SD (n = 3).

IR SPR peaks have greater electric field intensity and absorption cross sections and are considered as more promising than other spherical nanoparticles for in vivo biomedical applications.46 Before use, GNRs were modified by PEGylation to reduce serum protein adsorption and improve suspension stability.47 Zeta potential of PEGylated GNRs was −10.4 mV to ascertain a stable colloidal suspension (Figure S2c). The aspect ratio (∼4.4), size (59.7 × 13.5 ± 5.3 × 1.3 nm) and SPR peak of the PEG-GNRs (810 nm) as observed by UV−vis absorption spectrophotometer were measured (Figure S2a,b). Mild hyperthermia was investigated and monitored as a function of various concentrations of GNRs and NIR-laser powers. GNRinduced hyperthermia was conducted in 96-well plates with NIR laser irradiation. PBS buffer and GNRs with PBS buffer were placed in the 96-well plates, and NIR laser irradiation was applied for inducing hyperthermia. Final temperature was measured for 10 min post-laser irradiation using a 33-gauge needle thermocouple. At 0.06 W/cm2 laser power, the temperature did not reach hyperthermia regardless of GNR concentration. At 0.4 W/cm2 laser power, the temperature increased from 23.5 to 41.2 °C at the 24 μg/mL of GNRs. PEGylated GNR concentration of 48 μg/mL resulted in the desired temperature for the in vivo studies at 42 °C (Figure 6a). PEGylated GNR at 48 μg/mL was injected intratumorally to optimize the laser power for inducing mild hyperthermia in JHU-022 tumor xenografts. When tumors were treated with 0.9, 1.0, and 1.1 W/cm2 laser powers at a fixed GNR concentration (48 μg/mL), the average equilibrium temperature inside the tumors was achieved at 37, 42, and 47 °C, respectively (Figure 6b). To verify that PEGylated GNR and NIR laser-induced mild hyperthermia, treatment with laser alone (1.0 W/cm2 laser power) and GNRs (48 μg/mL) plus laser (1.0 W/cm2) was conducted in tumor bearing animals.

Transduction Efficiency of Fiber Knob-Modified Adenovirus. The coxsackie-adenovirus receptor (CAR) expression is highly variable in cancer cells, especially malignant tumor cells which have low CAR expression, resulting in poor Ad infectivity.39 We investigated the transduction efficiency of two different kinds of fiber-modified Ads; RGD modified and k35,40 along with Ad harboring wild-type fiber as a control. As shown in Figure S1, control Ad and RGD-modified Ad showed very limited transduction efficiency in JHU-022 cells, while transduction of k35-modified Ad showed the most efficient transduction. This result demonstrates that CAR expression level of JHU-022 is low, thus fiber-modified oncolytic Ad harboring k35 fiber was utilized for subsequent experiments to facilitate Ad uptake into JHU-022 xenograft tumor models. Optimization of GNR-Mediated Mild Hyperthermia in Vivo. Based on the promising in vitro results, we hypothesized that the combination of oncolytic viral delivery and mild hyperthermia will enhance efficacy in vivo. The generation of localized mild hyperthermia in the tumor site should be limited to transferring sufficient heat without destroying normal tissue.41 Induction of hyperthermia in solid tumors by plasmonic gold nanostructures has been reported.42,43 GNRs were applied in this study because they have a well-defined structure and exhibit a localized surface plasmon resonance (SPR) peak in the near-infrared wavelength.44 For in vivo animal studies, GNRs with SPR peak in the near-infrared region (700−900 nm) are preferred because light within this spectrum window can penetrate tissue more deeply than the visible light and also is not substantially absorbed by the aqueous environment. GNRs exhibit two SPR peaks, the transverse SPR around 520−600 nm and the longitudinal surface plasmon resonance in the near IR region, with the exact wavelength tunable by controlling the aspect ratio of the nanorods.45 Near 10538

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Figure 7. In vivo antitumor efficacy of Ad with GNRs. (a) PBS, GNR + laser, Ad, Ad + GNR + laser were treated on JHU-022 xenografts established in nude mice. Tumor growth was assessed every other day by measuring tumor volume using the formula: ab2 × 0.523, where a is the largest and b is the smallest diameter. Data represent the mean ± SE (n = 6). **P < 0.01 versus virus only. ***P < 0.001 versus control and laser + GNR treatment. (b) Histological analysis in tumor tissue. Histological analysis in tumor tissues treated with PBS, GNR + laser, Ad, Ad + GNR + laser. Light micrographs depicting tumor tissues stained with H&E, TUNEL, Ad E1A-specific antibody, VEGF-specific antibody, or CD31-specific antibody. Original magnification: H&E × 100; TUNEL × 100; E1A × 100, × 400; VEGF × 400; CD31 × 400.

Tumors were subsequently treated with laser for GNR or GNR plus RdB-k35/KOX group. The concentrations of GNR and laser power were chosen, as optimized above (48 μg/mL and 1.0 W/cm2). Tumor growth was evaluated for 43 days after intratumoral injection. As shown in Figure 7a, the tumor volume treated with PBS grew rapidly, whereas the inhibition rate of tumor growth in the GNR plus laser and oncolytic Ad only treated groups was 39% and 46%, respectively, compared to the PBS group (P < 0.001). In marked contrast, oncolytic Ad in the presence of GNR-mediated mild hyperthermia showed 78% tumor growth inhibition, demonstrating superior antitumor efficacy of combination strategy with respect to the two

The presence of GNRs significantly increased the tumor temperature and induced mild hyperthermia (Figure 6c). According to these results, intratumoral administration of 48 ug/mL of GNRs and 1.0 W/cm2 laser power was used for further in vivo efficacy studies. Evaluation of Antitumor Efficacy in Vivo. The therapeutic efficacy of k35-modified oncolytic Ad under GNR-mediated mild hyperthermia was evaluated in a xenograft model bearing a JHU-022 head and neck cancer tumor. When tumors grew to about 100 mm3 in volume, GNRs and VEGFspecific zinc finger protein-expressing oncolytic Ad (RdB-k35/ KOX) were administered intratumorally at days 0 and 3, along with PBS, GNR alone, or RdB-k35/KOX alone as controls. 10539

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that GNR-mediated enhancement of oncolytic viral therapy can be used in the treatment of a variety of cancers.

groups treated with mild hyperthermia or oncolytic Ad alone (P < 0.01). Histological examinations of the tumor tissue sections were performed to evaluate the antitumor activity of each treatment. In the hematoxylin and eosin (H&E) staining and TUNEL assays, oncolytic lesions and apoptotic tumor cells were localized in the tumors treated with oncolytic Ad alone or oncolytic Ad with GNR plus laser groups (Figure 7b). Of note, the mice treated with oncolytic Ad in the presence of mild hyperthermia exhibited greater oncolytic and apoptotic areas compared to oncolytic Ad alone group. Furthermore, a higher quantity of E1A-positive spots was detected in GNR-mediated mild hyperthermia plus Ad combination group compared to those treated with Ad alone. Both VEGF expression and CD31positive microvessel density were markedly decreased in the tumor tissues treated with oncolytic Ad alone or oncolytic Ad with GNR plus laser groups compared to PBS treated group, suggesting that RdB-k35.Kox under either hyperthermia or northermia can induce potent antiangiogenic effect. Together, these results clearly suggest that combination therapy with Ad plus GNR laser irradiation augmented antitumor efficacy of oncolytic tumor therapy. Techniques for inducing tumor hyperthermia using radiofrequency or hyperthermic intraperitoneal perfusion are limited in their capacity to selectively deliver heat to solid tumors.48 Plasmonic photothermal therapy provides the opportunity for localized delivery of heat in a selective and tunable fashion. In addition, it provides the opportunity for repeated administration of hyperthermia noninvasively. Though gene therapy based on delivery of oncolytic viruses shows promise, specific and sufficient gene delivery to cancer cells remains a hurdle. To avoid these problems such as slow diffusion rate into tumor cells from systemic circulation and rapid clearance by liver, intratumoral injection of viral particles into cancer cells has been the preferred method of administration.49 When combined with oncolytic viral gene therapy, controlled viral replication can be achieved to improve therapeutic efficacy. While the studies described above relate to intratumoral delivery of viral gene carriers and GNRs, a similar concept can be used for systemic delivery of viral particles decorated with polymeric constructs and targeting moieties where GNRmediated hyperthermia can enhance the EPR effect, as observed before,19 and improve cellular uptake and efficacy while minimizing adverse effects of viral gene therapy. Some of the drawbacks of using GNRs to induce mild hyperthermia include limited depth of penetration of laser light, heterogeneous delivery of GNRs to cancer cells, and nonspecific uptake by nontarget organs. For GNR-mediated enhanced delivery of oncolytic viruses to reach clinical practice, these shortcomings need to be addressed.

MATERIALS AND METHODS Materials. Hexadecyltrimethylammonium bromide (CTAB, ≥99.0%), L-ascorbic acid (BioXtra, ≥99.0%), silver nitrate (AgNO3, >99%), sodium borohydride (NaBH4, 99%), and gold(III) chloride solution (HAuCl4, 99%) were obtained from Sigma (St. Louis, MO). Cell Lines and Cell Culture. JHU-022 oral cavity cancer cell line was a kind gift from Professor David Sidransky of Johns Hopkins University (Baltimore, MD, U.S.A.). JHU-022 cells were in Advanced Roswell Park Memorial Institute (RPMI 1640) medium supplemented with 2 mM L-glutamine and 10% fetal calf serum (Gibco, Carlsbad, CA, U.S.A.). Cells were maintained at 37 °C in a humidified atmosphere at 5% CO2. MTT Assay. JHU-022 cells were seeded at 96-well plates and infected with various backbones of oncolytic adenovirus (0.5−50 MOI). The infected cells were incubated at 42 °C at the same time or 4 h post-infection for 2 h. The infected cells were then incubated at 37 °C, and at 48 or 72 h post-infection, 200 μL of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma Chemical, St. Louis, MO, U.S.A.) in phosphate-buffered saline (PBS; 2 mg/mL) was added to each well. After 4 h of incubation at 37 °C, the supernatant was discarded, and the formazan was solubilized with 150 μL of dimethyl sulfoxide. Absorbance was measured on a microplate reader at 540 nm. Synthesis and Characterization of Gold Nanorods. GNRs were prepared by the seed-mediated growth method as previously described.50 Briefly, 5 mL of 0.5 mM HAuCl4 was added to 5 mL of 0.2 M CTAB solution. Immediately after, 600 μL of fresh NaBH4 was added to HAuCl4/CTAB solution. This seed solution was placed in a 28 °C water bath before further manipulation. To prepare the growth solution, 100 mL of 0.2 M CTAB and 6 mL of 4 mM AgNO3 were mixed in a glass bottle, and the bottle was placed in 28 °C water bath. 100 mL of 1 M HAuCl4 solution was added to the growth solution and gently mixed. 1.4 mL of 0.078 M ascorbic acid was introduced, and the solution was vigorously stirred until clear. 267 μL of seed solution was injected to the growth solution, and the bottle was placed in a 28 °C water bath overnight. CTAB-GNRs were separated by centrifugation at 13,000 rpm for 20 min and washed by deionized water three times to remove excess CTAB. For PEGylation of GNRs, methoxy-PEGthiol (5 kDa, Creative PEGWorks, Winston Salem, NC, U.S.A.) was added to the CTAB-GNR suspension (OD: 8) at a 100 μM PEG concentration and stirred overnight. The mPEG-GNRs were centrifuged and washed in deionized water to remove unreacted PEG. The shape, size, zeta potential, and SPR peak of GNRs were obtained by transmission electron microscopy (TEM), DLS Malvern Zetasizer and UV-spectrophotometer. The TEM specimens were prepared by drop-casting the GNRs onto a carbon coated 300 mesh copper grid. The UV−vis absorption profile of GNRs was measured by a spectrophotometer. Quantification of Replicated Viral Genomes. To quantify replicated Ad, JHU-022 cells were seeded onto 12-well plates at a density of 5 × 104 cells per well. The next day, the cells were incubated at 42 °C for pretreatment with heat or northermia. After 2 h, all cells were infected with 1 MOI of Ad. Among those not treated with heat shock, some were incubated at 42 °C for 2 h, and after 4 h of Ad infection, the media was changed. At 48 h after viral infections, supernatants and cell lysates were collected. For real-time Q-PCR analysis of viral genomes, 5 μL of each sample was analyzed by Q-PCR (TaqMan PCR detection; Applied Biosystems, Foster City, CA, U.S.A.) as previously reported.51 Western Blot. To assess clathrin, dynamin, or caveolin expression levels, JHU-022 cells were treated at 42 °C for 2 h. At 4 h, the cells were lysed in lysis buffer (50 mM HEPES containing 0.15 M NaCl, 0.5% Nonidet P40, and proteinase inhibitors phenylmethylsulfonyl fluoride, tosyl-L-lysine chloromethyl ketone, and N-tosyl-L-phenylalanine chloromethyl ketone). The precleared lysates and cell supernatants were separated by 10% sodium dodecyl sulfate-

CONCLUSION In summary, the combination of oncolytic adenovirus delivery with mild hyperthermia produced by GNR-mediated plasmonic photothermal therapy shows promise for improved treatment of head and neck tumors. Exposure to optimized hyperthermia conditions improved endocytosis of oncolytic Ad, transgene expression, viral replication, and subsequent cytolysis of head and neck cancer cells. GNR-mediated PPTT resulted in precise control of tumor temperature and induction of mild hyperthermia. Treatment with combination of oncolytic Ad and GNRs resulted in significant reduction in tumor growth in JHU-022 xenograft tumor model. Together these results show 10540

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U.S.A.) and antirabbit CD31 (Abcam, Cambridge, MA, U.S.A.) primary antibodies to assess the antiangiogenic effects of oAd. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out using a TdT-FragEL DNA fragmentation detection kit (Merck, Darmstadt, Germany) for presence of apoptotic cell death areas according to the manufacturer’s instructions.

polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membranes were incubated with primary antibodies against clathrin heavy chain (P1663) antibody (Cell Signaling Technology, Beverly, MA, U.S.A.), purified mouse anti-dynamin II (BD Biosciences PharMingen, San Diego, CA, U.S.A.), caveolin-1 (N20) rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), and β-actin (Cell Signaling Technology). Bound antibodies were detected by a horseradish peroxidase-conjugated antimouse secondary antibody (Cell Signaling Technology) or a horseradish peroxidase-conjugated antirabbit secondary antibody (Cell Signaling Technology) and developed using the enhanced chemiluminescence system (Pierce, Rockford, IL, U.S.A.). FACS Analysis. JHU-022 cells were seeded onto a 12-well plate and cultured to 60% confluence 1 day prior to the transduction assay. Cells were infected with 5 MOI of GFP-expressing Ad and incubated for various time points (12, 6, 4, or 2 h prior to infection or same time, 45 min, 2, 4, 6 h post-infection) at 42 °C for 2 h. The transduced cells were incubated for an additional 48 h. Cells were imaged using fluorescence microscopy (Olympus IX81; Olympus Optical, Tokyo, Japan). Subsequently, cells were detached and washed with PBS three times. After PBS washing, 1% paraformaldehyde in PBS solution was added to perform flow cytometry analysis using BD FACScan analyzer (Becton-Dickinson, San Jose, CA, U.S.A.) and the CellQuest software (Becton-Dickinson). Human VEGF ELISA. Human VEGF was quantified in cell supernatants using the human VEGF quantikine immunoassay kit (R&D Systems, Minneapolis, MN, U.S.A.), according to the manufacturer’s recommendations. Serial dilutions of a known concentration of purified recombinant human VEGF-A were used to establish a standard curve. In Vitro Optimization of NIR Laser-Induced Mild Hyperthermia. PBS buffer and various concentrations of free GNRs with PBS buffer such as 24, 48, and 72 μg/mL were placed into the different wells of a 96-well plate (0.1 mL/well). The samples were irradiated by 808 nm NIR laser diode (Oclaro Inc., San Jose, CA, U.S.A.) for 10 min. The changes in intratumoral temperature were measured by 33gauge needle thermocouple (Omega, Stamford, CT, U.S.A.). NIR Laser-Induced Mild Hyperthermia with GNRs in Vivo. Animals and Tumor Model. Male BALB/c nude mice (6 weeks old) were purchased from Orient Bio Inc. (Seongnam, Korea). All animal studies were performed according to the institutional guidelines of the Korea Institute of Science and Technology (KIST). 3 ×106 JHU-022 cells were injected subcutaneously into the left flank of BABL/c nude mice. Tumor volume was measured and calculated by V = L(w)2 × 0.523, where L is the length and w is the width of tumor. In Vivo Optimization of NIR Laser-Induced Mild Hyperthermia with GNRs. In vivo animal model (3 per group) bearing JHU-022 tumor cells, 0.1 mL of GNRs (48 μg/mL), and various 808 nm NIR laser powers (0.9, 1.0, and 1.1 W/cm2) were used to irradiate the tumors for 10 min. The changes in intratumoral temperature were measured by a 33-gauge needle thermocouple (Omega, Stamford, CT). The mice injected with PBS were used as control. In Vivo Therapeutic Efficacy. The antitumor effect of oncolytic adenovirus was investigated in head and neck cancer xenografts bearing JHU-022 cells. When the tumor volumes reached about 100 mm3, mice were randomly split into 4 groups (6 per group). The mice were anesthetized and administered intratumorally with PBS, GNRs plus laser, oncolytic adenovirus (oAd) only, and oAd plus GNR plus laser, respectively. The concentrations of GNRs and Ad were normalized as 48 μg/mL and 2 × 109 viral particles in 0.1 mL PBS solution, respectively. The tumors were treated with 808 nm laser diode (1.0 W/cm2) for 10 min after injection. Adenoviruses were injected intratumorally two times on days 0 and 3. Tumor growth was recorded for 42 days. To further analyze antitumor effect, the tumors at 28 days after treatment were excised, and samples were stained with H&E. To visualize viral replication in tumor tissues, immunohistochemistry with Ad-2/5 E1A-specific antibody (SC-430; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) was performed as described previously.49 Further, the tumor sections were also immunostained with monoclonal antirabbit VEGF (Laboratory Vision, Fremont, CA,

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06530. Transduction efficiency of modified Ad and complete details of GNR characterization using TEM, DLS and UV−vis spectra are provided (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Global Innovative Research Center program of the National Research Foundation of Korea (2012K1A1A2A01055811) and by the Intramural Research Program (Global RNAi Carrier Initiative) of Korean Institute of Science and Technology, a grant from the U.S. National Institutes of Health (R01CA107621, Dr. Ghandehari), and the National Research Foundation of Korea (2010-0029220, Dr. C.-O. Yun). REFERENCES (1) Walther, W.; Stein, U. Viral Vectors for Gene Transfer: A Review of Their Use in The Treatment of Human Diseases. Drugs 2000, 60, 249−271. (2) Choi, J.-W.; Lee, J.-S.; Kim, S. W.; Yun, C.-O. Evolution of Oncolytic Adenovirus for Cancer Treatment. Adv. Drug Delivery Rev. 2012, 64, 720−729. (3) Graham, F. L.; Prevec, L. Methods for Construction of Adenovirus Vectors. Mol. Biotechnol. 1995, 3, 207−220. (4) Russell, S. J.; Peng, K.-W.; Bell, J. C. Oncolytic Virotherapy. Nat. Biotechnol. 2012, 30, 658−670. (5) Green, N. K.; Hale, A.; Cawood, R.; Illingworth, S.; Herbert, C.; Hermiston, T.; Subr, V.; Ulbrich, K.; Van Rooijen, N.; Seymour, L. W. Tropism Ablation and Stealthing of Oncolytic Adenovirus Enhances Systemic Delivery to Tumors and Improves Virotherapy of Cancer. Nanomedicine 2012, 7, 1683−1695. (6) Bachtarzi, H.; Stevenson, M.; Fisher, K. Cancer Gene Therapy with Targeted Adenoviruses. Expert Opin. Drug Delivery 2008, 5, 1231−1240. (7) Kim, J.; Cho, J. Y.; Kim, J.-H.; Jung, K. C.; Yun, C.-O. Evaluation of E1B Gene-Attenuated Replicating Adenoviruses for Cancer Gene Therapy. Cancer Gene Ther. 2002, 9, 725−736. (8) Kim, J.; Kim, J.-H.; Choi, K.-J.; Kim, P.-H.; Yun, C.-O. E1A-and E1B-Double Mutant Replicating Adenovirus Elicits Enhanced Oncolytic and Antitumor Effects. Hum. Gene Ther. 2007, 18, 773−786. (9) Bischoff, J. R.; Kirn, D. H.; Williams, A.; Heise, C.; Horn, S.; Muna, M.; Ng, L.; Nye, J. A.; Sampson-Johannes, A.; Fattaey, A. An Adenovirus Mutant That Replicates Selectively in p53-Deficient Human Tumor Cells. Science 1996, 274, 373−376. 10541

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