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Magnetically Guided Viral Transduction of Gene-Based Sensitization for Localized Photodynamic Therapy to Overcome Multi Drug Resistance in Breast Cancer Cells Zi-Xian Liao, Ivan M Kempson, Yu-Chen Fa, Meng-Chia Liu, Li-Chen Hsieh, Kuo-Yen Huang, and Li-Fang Wang Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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Bioconjugate Chemistry

Magnetically

Guided

Viral

Transduction

of

Gene-Based

Sensitization for Localized Photodynamic Therapy to Overcome Multi Drug Resistance in Breast Cancer Cells Zi-Xian Liao,*a Ivan M. Kempson,*b Yu-Chen Fa,a Meng-Chia Liu,a Li-Chen Hsieh,a Kuo-Yen Huang,c,d and Li-Feng Wang,*e

a

Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan.

b

Future Industries Institute, University of South Australia, Mawson Lakes, S.A. 5095, Australia.

c

Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.

d

Graduate Institute of Health Industry Technology and Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan

e

Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan.

Corresponding Author *E-mail: [email protected] (Z.-X.L.) *E-mail: [email protected] (I.M. Kempson) *E-mail: [email protected] (L.-F.W.)

ACS Paragon Plus Environment

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ABSTRACT Chemotherapy represents a conventional treatment for many cancers at different stages either solely prescribed or concomitant to surgery and/or radiotherapy. However, treatment is tempered in instances of acquired drug resistance in response to either chemo- or targeted-therapy, leading to therapeutic failure. To overcome this challenge, many studies focus on how cancer cells manipulate their genomes and metabolism to prevent drug influx and/or facilitate efflux of accumulated chemo drugs. Herein we demonstrate magnetic adeno-associated virus serotype 2 (Ironized AAV2) has an ability to be magnetically guided, and transduce the photosensitive KillerRed protein to enable photo-dynamic therapy (PDT) irrespective of drug resistance.

INTRODUCTION Chemotherapy represents a conventional treatment for many cancers at different stages either solely prescribed or concomitant to surgery and/or radiotherapy. However, some cancers develop resistance to traditional chemotherapies, leading to therapeutic failure. Resistance is correlated with upregulated membrane transport mechanisms that ‘pump’ chemotherapeutic agents out of the cells. To overcome this challenge, many studies focus on how cancer cells manipulate their genomes and metabolism to prevent drug influx and/or facilitate efflux of accumulated chemo drugs.1-3 In this work, we obtained a chemo drug (doxorubicin, DOX) resistant human breast adenocarcinoma MCF-7 (CDRMCF-7) cell line to mimic this clinical challenge. As a function of DOX concentration

CDR

MCF-7 cells are significantly less challenged compared to MCF-7 cells

(Figure S1). At a dose of 50 µg mL-1, 78 % of CDRMCF-7 cells remain viable compared to 2.5 % of MCF-7 cells. An alternative to chemotherapy is therapy targeting the epidermal growth factor receptor (EGFR) family.4 However, the increased response rates to EGFR inhibitors in certain cancers with EGFR tyrosine kinase domain mutations are also reported to acquire resistance within one year.5 Despite the efforts to overcome drug resistance, it remains as a major cause of cancer chemotherapy and targeted therapy failure. A variety of delivery concepts have emerged for innovative treatment in cancer therapy largely attempting to improve specificity for drugs and proteins but also in gene-based concepts. Specificity can be opportunistically achieved via exploiting tumour microenvironment physico-chemical properties such as pH, for example.10-12 However, these are dependent on these properties being exhibited by a solid tumour volume. To


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guide delivery independent of tumour characteristics magnetic nanoparticles (MNP) conjugated to a carrier/vector provide accelerated accumulation in target cells when directed with magnetic fields.8 Over the past several years, effective magnetically directed delivery technologies have demonstrated significant potential in biomedical engineering and has inspired various approaches to promote delivery to specific sites.8,9,13-15 This includes conjugation of magnetic nanoparticles to viral vectors. Virotherapy in itself represents a class of promising cancer therapeutics6 and has successfully advanced to clinical use for cancer treatment with approval by the US Food and Drug Administration (FDA).7 We previously developed ‘controllable’ virus’ for either recombinant adeno-associated virus serotype 2 (AAV2)8 or lentivirus9 via chemical conjugation with iron oxide nanoparticles that could be directed with magnetic fields. Furthermore, a gene of the photosensitive protein or short hairpin RNA (shRNA) was introduced into the AAV2 or lentivirus genome to enable photo-dynamic therapy (PDT).8 or RNA interference (RNAi).9 In the case of PDT, irradiation with a specific light bandwidth generates reactive oxygen species leading to irreversible DNA damage and cell killing via an apoptotic pathway8,10; offering additional options in cancer treatment. Here, we present an improvement to this previous work that promotes viral transduction in chemo drug resistant-MCF-7 (CDRMCF-7) cells under magnetic field, and express the KillerRed (KR) protein in transduced cells for light-triggered virotherapy (AAV2-KR) (Figure 1).


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Figure 1. Light-triggered virotherapy. Schematic representation of light-triggered virotherapy to a chemo drug resistance-cancer cell. a) AAV2 carrying the KillerRed gene and conjugated with magnetic iron oxide nanoparticles can be specifically directed with magnetic fields. b) Viral delivery transduces the KillerRed gene inside cells leading to expression of the KillerRed protein. c) Irradiation of KillerRed with laser light produces reactive oxygen species (ROS). d) ROS lead to DNA double strand breaks and apoptosis. RESULTS AND DISCUSSION Effect of Ironized Modified Virus on Transduction and Cytotoxicity. All in vitro experiments were undertaken in medium (10% fetal bovine serum, 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin) for mimicking in vivo environment. To validate this concept in CDR

MCF-7 or MCF-7 cells, Ironized AAV2 were chemically conjugated with iron oxide

nanoparticles (~ 5nm, Figure S2A) as described previously.8 To estimate the influence of molar ratios of nanoparticle/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) on viral transduction and cell viability in

CDR

MCF-7 cells, we used an AAV2-GFP (green

fluorescent protein) assay detected with flow cytometry when treated cells were incubated for 6 days post-transduction (Figure 2). Interestingly, magnetic nanoparticle coated-virus delivery can improve the activity of viral infection and is consistent with previous observations.8,16 Overall, Ironized AAV2 transfection efficiency in

CDR

MCF-7 cells was optimal at a molar ratio of 1/20.

This ratio also maintained the maximum efficiency for MCF-7 cells. As such we chose a molar


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ratio of 1/20 for the Ironized AAV2 in subsequent in vitro studies. Furthermore, transmission electron microscopy (TEM) morphology of Ironized AAV2 at an optimized ratio of 1/20 showed diameters of 30 – 40 nm (Figures S2B).

Figure 2. Effect of ironized virus. Percentages of GFP-expressing cells 6 days post-transduction by Ironized AAV2 for varying molar ratios of nanoparticle/EDC, analyzed by flow cytometry. Data shows mean of measurements conducted in sextuplicate ± s.d.. Viability of 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. Data shows mean of measurements conducted in sextuplicate ± s.d.. Effect of magnetic field. To assess the role of magnetic field on viral infection in

CDR

MCF-7

cells, treated cells exposed to a magnetic field for 120 min of varying strength were incubated for 6 days post-transduction (Figure 3). A constant magnetic field of 1,500 or 2,000 Gauss did not significantly alter viral transduction (34.5 and 33.9 % respectively). An increase in transduction was recorded for increasing the magnetic field to 3,500 Gauss (45.8%), however the benefit of increasing field strength was lost when the field was further increased to 5,000 Gauss (39.5 %), at which point the field appeared to cause precipitation and aggregation of Ironized AAV2. Alternatively, shorter periodic exposures were tested. Exposure to the external magnetic field was repeated for four 1-hour cycles consisting of 30 min exposure to a 5,000 Gauss field and 30 min of 0 Gauss. Under this condition there was approximately a 2-fold (68.4 % expression) increase in viral expression of GFP in the CDRMCF-7 cells compared with negative control (33.6


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%). Microscopic observation indicated that cyclic exposure reduced precipitation and aggregation of Ironized AAV2 and promoted accumulation along with uptake within the magnetic field. Thus the stronger field could be utilised for increasing the localized transfection while avoiding detrimental aggregation.

Figure 3. Effect of magnetic field exposure. Percentages of GFP-expressing cells 6 days posttransduction by Ironized AAV2 after 120 min of varying strengths of magnetic field, analyzed by flow cytometry. Cycling of the magnetic field for 4 cycles over 4 hours, but equivalent total time of exposure, achieved significant increase in transfection. Data shows mean of measurements conducted in sextuplicate ± s.d.. KillerRed expression. To evaluate the effect of the ‘ironized’ coating or magnetic field (5,000 Gauss) on the viral transduction or cytotoxicity using Ironized AAV2-KR in CDRMCF-7 or MCF7 cells, efficiency of KillerRed-expressed cells were quantified by flow cytometry when treated cells were incubated for 6 days post-transduction. Magnetic field exposure was applied for 30 mins, then removed for 30 mins, and repeated for a total of 4 exposures to the magnetic field. CDR

MCF-7 cells treated with Ironized AAV2-KR under a magnetic field (M+) considerably

improved KillerRed-expression to 48.8 % compared to the control treatment of Ironized AAV2 (M-) or AAV2 (23.5 % or 12.1 % respectively) (Figure 4A).


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Figure 4. Light-triggered virotherapy. (A) Percentages of KillerRed-expressed cells 6 days posttransduction by Ironized AVV2 or virus, with or without magnetic fields (M), analyzed by flow cytometry. Data shows mean of measurements conducted in sextuplicate ± s.d.. (B) Toxicity of Ironized AAV2-KR or virus only under different conditions determined by MTS assay against MCF-7 cells or CDRMCF-7 cells. Data shows mean of measurements conducted in sextuplicate ± s.d.. Representative images of KillerRed distribution in cell cultures from fluorescence microscopy are given in Figure S3. The main pathway for AAV2 infection is via endocytosis17, however CDR

MCF-7 cells can avoid particle influx or facilitate efflux of accumulated particles. These data

imply that Ironized AAV2 can improve the viral transduction in CDRMCF-7 cells under magnetic field due to enhancement of Ironized AAV2 influx and/or prevention of accumulated AAV2 efflux. Furthermore, significant cytotoxicity was observed in both

CDR

MCF-7 and MCF-7 cells

treated with Ironized AAV2-KR after light-exposure of 20 min (561 nm, 100 mW cm-2) due to reactive oxygen species (ROS) generation triggered by KR protein activation (Figure 4B). Reactive oxygen species generation by KillerRed activation. With data confirming improved viral transduction of

CDR

MCF-7 cells, we performed light-triggered virotherapy

utilizing AAV2-KR with a corresponding wavelength of 561 nm. We further chose the irradiation time of 20 min for optimized ROS generation.8,10,18 The generation of ROS was visualized in the photoactivated KillerRed-expressed cells in

CDR

MCF-7 or MCF-7 cell cultures

with CellROXTM staining. In the cells treated with the Ironized AAV2-KR ± magnetic field, the ironized coating had a positive effect on the ROS generation with a pronounced improvement in CDR

MCF-7 compared to virus only (Figure 5A) due to enhanced cellular uptake of viral particles.


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Consistent with the observed CellROS-positive

CDR

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MCF-7 cells, the CellROS-positive MCF-7

cells also displayed an increased in fluorescence intensity under magnetic field (Figure S4).

Figure 5. ROS generation triggered by KillerRed. (A) Images show ROS generation mediated by KillerRed photoactivation observed in infected cells and irradiated for 20 min, in which the CellROXTM and DAPI stains identify ROS and nuclei. Cells co-cultured with ironized AAV2 or AAV2 were applied a magnetic field (M) (30 min cycle-1 for 4 cycles with 120 mins between each exposure) and incubated for 6 days post-transduction prior to irradiation. Bar = 500 µm. (B) Flow cytometry quantified percentage of CellROX-positive cells. Data shows mean of measurements conducted in sextuplicate ± s.d.. Additionally, flow cytometry quantified the efficiency of achieving CellROX-positive CDR

MCF-7 or MCF-7 cells (Figure 5B). The Ironized AAV2 showed high percentage (93.8 %) of

CellROX-positive MCF-7 cells under magnetic field. In the CDRMCF-7 cells infected by Ironized AAV2-KR, the percentage of CellROX-positive cells exhibited significantly different percentages (89.1 % or 55.6 %) of CellROX-positive cells with or w/o magnetic field respectively. A characteristic of the drug resistant cell line is the reduced influx and/or enhanced efflux of particles. These data indicate the magnetic field prevents the efflux of accumulated


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Ironized AAV2 in

CDR

MCF-7 cells and consequently enhances expression of the KillerRed

protein.

Figure 6. Apoptosis via light-triggered virotherapy. Images of CDRMCF-7 (A) or MCF-7 cells (B) showing KillerRed-mediated apoptotic cells stained by TUNEL assay incubated after lightexposure of 20 min. Cells were cultured with Ironized AAV2-KR, AAV2-KR only, iron oxide nanoparticles only, or DOX only, exposed to a magnetic field and incubated for a further 6 days before irradiation. The treated cells were stained by DAPI to reveal the cell nuclei. Bars = 500 µm. Light-triggered virotherapy. We further desired to identify if the ROS generation led to cell death and, if so, if it were due to apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was used to identify apoptotic cells.8-10 Consistent with the observed CellROX-positive

CDR

MCF-7 cells infected by Ironized AAV2 (Figure 5), a small

amount of TUNEL fluorescence in the cells treated with AAV2 or Ironized AAV2 was observed (Figure 6A). After addition of the magnetic field, the TUNEL staining resulted in a significant increase in fluorescence from

CDR

MCF-7 cells. Conversely, we observed no evidence of cell


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death when treated with DOX (1 µg mL-1). Evidently, this result shows the

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CDR

MCF-7 cells

prevent DOX accumulation or facilitate efflux of accumulated DOX.1-3 In contrast to chemo drug-resistant cancer cells, distribution of TUNEL fluorescence in MCF-7 cells was evident for both treatment with Ironized AAV2-KR with magnetic field or with DOX only (Figure 6B). Live (green fluorescent) & Dead (red fluorescent) assay in

CDR

MCF-7 (Figure 7A) and MCF-7 cells

(Figure 7B) for Ironized AAV2-KR with magnetic field treatment concurred with the data on apoptosis.

Figure 7. Anti-proliferation by photo-dynamic therapy (PDT). Images of Live/Dead and nuclei distribution of CDRMCF-7 (A) or MCF-7 cells (B) infected by Ironized AAV2-KR, AAV2-KR only, iron oxide nanoparticles only, or DOX only under a magnetic field and incubated for a further 6 days before irradiation. After irradiation for 20 min, the infected cells were observed using Live/Dead® Fixable Far Red Dead Cell Stain Kit. Bars = 500 µm. CONCLUSIONS We have demonstrated highly efficient photosensitization with the gene-based KillerRed photosensitizer. Loco-regional specificity was achieved for viral transduction via utility of a


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magnetic field and conjugation of magnetic nanoparticles to the virus. This demonstrated successful anti-proliferation of chemotherapy drug resistant cancer cells. Such a technological model with improved virotherapy loco-regional specificity could be harnessed to overcome drug resistant cancer cells.

EXPERIMENT SECTION Materials. Plasmid DNA of pKillerRed-dMito was purchased from Evrogen JSC (Moscow, Russia). Plasmid DNAs (pAAV-GFP and pAAV-MCS) and virus (AAV2-GFP-control virus) were purchased from Cell Biolabs (San Diego, CA). Furthermore, plasmid pAAV-KillerRed was constructed as described previously.8 Phosphate buffered saline (PBS) and doxorubicin hydrochloride (DOX) were purchased from Sigma Co. (St. Louis, MO). Branched polyethyleneimine (PEI, Mw = 25,000) was purchased from Aldrich (Milwaukee, MI). (1-ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), and 2-(N-morpholino)ethanesulfonic acid (MES) buffered saline were purchased from Thermo Scientific Inc. (Rockford, IL). Iron oxide nanoparticles with carboxylic acid (Lot number: 051413A; size: 5 nm; zeta potential: -30 mV to -50 mV) were purchased from Ocean NanoTech (San Diego, CA). Cell Culture. The Human breast adenocarcinoma MCF-7 cells (HTB-22, 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 doxorubicin (DOX) resistant human breast adenocarcinoma MCF-7 (CDRMCF-7) cell line was obtained from the Biomedical Technology and Device Research Laboratories (Hsinchu, Taiwan). Cytotoxicity of Doxorubicin. 104

CDR

MCF-7 or MCF-7 cells were seeded in each of the

wells of a 96-well plate and cultured with complete medium for 12 hours. The cells were then exposed to DOX at various concentrations (0.5, 1,5, 10, 25, 50, or 100 µg mL-1) and incubated for 24 hours. The CellTiter 96® AQueous one solution cell proliferation assay system with the tetrazolium

compound

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium, inner salt; MTS) was used to measure the survival rate and proliferation of mammalian cells.8,10 The optical density (OD) of formazan at 490 nm quantified


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the cell viability. 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. Viral Production, Purification, and Titration. Production of adeno-associated virus serotype 2 (AAV2)-KillerRed was performed using the AAV-2 Helper Free Packaging System (Cell Biolabs). Briefly, AAV2-KillerRed was produced by PEI-mediated co-transfection of plasmid DNAs (pHelper, pAAV-RC2, and pAAV-KillerRed) in 293T cells.8 Transfected cells were harvested 3 days after transfection. Purification and titration of AAV2-KillerRed were performed according to the protocols of the ViraBind™ AAV Purification Kit (Cell Biolabs) and QuickTiter™ AAV Quantitation Kit (Cell Biolabs). The number of genome copy (GC) per milliliter of 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.8 Reaction mixtures contained the iron oxide nanoparticles with carboxylic acid (25 µg, 0.1725 nmol), EDC (0.1725, 0.865, 1.73, 3.46, 4.325, 8.65, or 17.3 nmol) in MES buffered saline solution, and the mixtures had gently added sulfo-NHS for 15 min to achieve a homogeneous solution of iron oxide nanoparticles with amine-reactive sulfo-NHS ester groups. The AAV2 stock (0.5 µL, 1 × 1012 GC mL-1) in PBS was added dropwise to the mixtures and then reacted for 120 min. After chemical conjugation, the solution was purified by using a size desalting column (molecular weight cut-offs: 100K) that was equilibrated with PBS and solvent-exchanged to PBS. After purification, a recycle yield of ~90%

was

achieved

by

PCR

(sequences

of

primers

for

AAV2-KillerRed:

5’-

GCCCATGAGCTGGAAGCC-3’ and 5’-CGATGGCGCTGGTGATGC-3’). A drop of the sample solution was allowed to air-dry onto a Formvar-carbon-coated 200 mesh copper grid for transmission electron microscopy (TEM, JEOL JEM-2100F) analysis. TEM images were then acquired on a JEOL-1010 microscope operating at an accelerating voltage of 100 kV. In Vitro Viral Transduction and Cytotoxicity of Ironized Virus. All experiments of transduction used culture medium (10% FBS, 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin). To measure the transduction ability of ironized virus or un-ironized virus without any magnetic fields, we used the AAV2-GFP (green fluorescent protein) as the signal indicator. CDR

MCF-7 or MCF-7 cells were seeded in 24-well plates at 1 × 105 cells well-1 and infected the


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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. 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. Suspensions were then 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. CDR

MCF-7 or MCF-7 cells (7 ×104 cells) were seeded in each of the wells of a 24-well

plate and fed with culture medium for 12 hours. The cells were exposed to test Ironized AAV2 at different molar ratios of nanoparticle/EDC and incubated at pH 7.4 for 24 hr. After 24 hr incubation, the media containing test samples were removed. Additionally, the iron oxide nanoparticles or AAV2 only was incubated at pH 7.4 for 24 hr. The CellTiter 96® AQueous one solution cell proliferation assay system (Promega, Madison, WI, USA) was used to determine the cell proliferation. The optical density (OD) of formazan at 490 nm quantified the cell viability. 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. Viral Transduction under Magnetic Field. From the results shown in Figure 2, Ironized AAV2 at the optimized molar ratio of 1/20 was suited to efficiently infect

CDR

MCF-7 or MCF-7

cells with low toxicity, and we chose a molar ratio of 1/20 for the Ironized AAV2 in subsequent in vitro studies. The test samples of Ironized AAV2 or AAV2 only were incubated with cells in DMEM with 10% FBS followed by analysis at 120 min of external magnetic field (1,500, 2,000, 3,500, or 5,000 Gauss). Alternatively, the introduction of external magnetic field was repeatable for four cycles (30 min cycle-1) of magnetic field changes between 0 and 5,000 Gauss. Transduced cells were observed or analyzed for KillerRed expression by a fluorescence microscope or flow cytometry (BD Bioscience, San Jose, CA, USA) equipped with a 561 nm argon laser for quantification of the percentage of KillerRed-expressed cells after 6 days transduction. As per the previous study of KillerRed activation,1,3 we further chose the irradiation time of 20 min with 561 nm argon laser (~100 mW cm-2 as measured by an optical-power meter (Thorlabs, Inc., Newton, New Jersey, USA)) for optimized reactive oxygen species (ROS) generation and KillerRed phototoxicity for our studies.KillerRed-expressed cells with or without irradiation were quantified for cell viability by MTS assay. The reduction of MTS achieved by


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untreated cells was set at 100% and that of test cells was expressed as a percentage of untreated cells. In Vitro Assays. After irradiation of KillerRed, the KillerRed-expressed cells were observed using CellROX® Green Reagent (Invitrogen, Camarillo, CA), In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), or 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. The percentage of CellROX-positive cells through KillerRed activation were further analyzed using flow cytometry. Additionally,

CDR

MCF-7 or

MCF-7 cells was incubated at 1 µg mL-1 DOX as a control. Statistical Analysis. Data are shown as the mean ± the standard deviation for experiments performed in sextuplicate. In statistical significance testing, P values were calculated using a two-tailed t test, assuming unequal variances.

ASSOCIATED CONTENT Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Z.-X. Liao) *E-mail: [email protected] (I. M. Kempson) *E-mail: [email protected] (L.F. Wang) ORCID Zi-Xian Liao: 0000-0002-3051-0728


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Funding This work was supported through the Taiwan Ministry of Science and Technology Grant (MOST105-2628-E-110-001-MY3 and MOST105-2628-B-110-004-MY3) and NSYSU-KUM Joint research Project (104-P026 and 105-P018). Notes The authors declare no competing financial interest.


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(16) Tresilwised, N., Pithayanukul, P., Holm, P. S., Schillinger, U., Plank, C., and Mykhaylyk, O. (2012) Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes. Biomaterials 33, 256-69. (17) Nonnenmacher, M., and Weber, T. (2011) Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 10, 563-76. (18) Liao, Z. X., Li, Y. C., Lu, H. M., and Sung, H. W. (2014) A genetically-encoded KillerRed protein as an intrinsically generated photosensitizer for photodynamic therapy. Biomaterials 35, 500-8.


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