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Gold Nanoparticle-incorporated Molecularly Imprinted Microgels as Radiation Sensitizers in Pancreatic Cancer Aoi Yoshida, Yukiya Kitayama, Kentaro Kiguchi, Takuya Yamada, Hiroaki Akasaka, Ryohei Sasaki, and Toshifumi Takeuchi ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00766 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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ACS Applied Bio Materials

Gold Nanoparticle-incorporated Molecularly Imprinted Microgels as Radiation Sensitizers in Pancreatic Cancer

Aoi Yoshida1, Yukiya Kitayama1,3*, Kentaro Kiguchi1, Takuya Yamada1, Hiroaki Akasaka2, Ryohei Sasaki,2,3, Toshifumi Takeuchi1,3*

1

Graduate School of Engineering, Kobe University, 1-1, Rokkodai-cho, Nada-ku, Kobe

657-8501, Japan 2

Division of Radiation Oncology, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku,

Kobe 650-0017, Japan

3

Medical Device Fabrication Engineering Center, Graduate School of Engineering, Kobe

University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

Corresponding Author: Toshifumi Takeuchi, E-mail: [email protected]

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ABSTRACT

Radiation therapy is a powerful approach for treating pancreatic cancer, a representative refractory cancer with a high fatality rate, and efforts have been made to decrease the radiation dose and suppress the side effects related to damage to normal tissues during radiation therapy. Gold nanoparticles (Au NPs) are known to possess radiosensitizing activity and low biotoxicity; however, Au NP-incorporated biomaterials have not been investigated as feasible radiosensitizers for use in vivo. Accordingly, in this study, Au NP-incorporated molecularly imprinted polymer microgels (Au-MIP microgels) were created as radiation sensitizers using a newly

developed

one-pot

seeded

precipitation

polymerization

method,

and

the

radiation-sensitizing effects of the Au-MIP microgels were investigated in mice bearing pancreatic tumors. In mice injected with the Au-MIP microgels, tumor sizes were smaller than those in control mice injected with buffer solution when X-ray irradiation was performed. Furthermore, biotoxicity was not observed in mice injected with the Au-MIP microgels because of negligible body weight loss in these mice. Based on these findings, Au-MIP microgels may have applications as novel radiation sensitizers in radiation therapy.

Keywords: gold nanoparticles, pancreatic cancer, radiation therapy, radiosensitizer, microgel


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Introduction Cancer is a major public health problem that causes one in four deaths in the United States of America (USA). Pancreatic cancer is a representative refractory cancer with a high fatality rate that is difficult to diagnose and is typically identified at a late stage.1-2 Patients with pancreatic cancer often have a poor prognosis, and the 5-year survival rate is as low as 6% in the USA.3-7 Therefore, improved approaches for the treatment of pancreatic cancer are urgently required. Radiation therapy is an effective and minimally invasive approach for cancer therapy that allows patients to be treated on an outpatient basis.8-9 In radiation therapy, the tumor tissue is irradiated with X-rays, and reactive oxygen species (ROS), such as superoxide radicals and hydrogen radicals, are generated via radiation reacted with water and oxygen molecules.10 The generation of ROS leads to cellular damage by interacting with biological molecules, such as DNA, lipids, and proteins, directly or by exerting oxidative stress indirectly, thereby causing cell death. However, a large radiation dose leads to damage to normal tissues during X-ray irradiation, thereby limiting the dose of radiation that can be applied. The use of radiation sensitizers may be an effective approach for low-risk radiation cancer therapy with decreased radiation doses.11-15 Gold nanoparticles (Au NPs) are one type of radiation sensitizer that can absorb approximately three times the radiation of iodine, another



known radiation sensitizer.16-20 In addition, Au NPs are inert and biocompatible materials. In the presence of a radiation sensitizer under X-ray irradiation, secondary radiation and/or secondary electrons are generated at the surface of the radiation sensitizer in addition to the primary radiation, resulting in accelerated generation of ROS. Drug delivery may be an excellent approach to realizing efficient cancer treatment. For drug delivery, the development of nanocarriers is important for the targeted delivery of therapeutic agents selectively to the tumor tissue.21-27 In our previous study, polymer nanogels capable of albumin recognition (molecularly imprinted nanogels [MIP-NGs]) were successfully created by molecular imprinting,28 which is a template polymerization technique for obtaining artificial polymer-based molecular recognition materials.29-37 In molecular imprinting, a template molecule is complexed with functional monomers bearing functional groups for the template molecule, and the complex is copolymerized with comonomers and a crosslinker to form the template molecule-incorporated polymer matrix. The removal of the template molecule leaves cavities complementary in size and shape to the template molecules. A long circulation time was achieved in the blood stream by acquiring stealth capability due to the albumin-rich protein corona formation on the surface of MIP-NGs due to the specific adsorption of albumin in situ. In this study, we attempted to incorporate cancer treatment agents, such as radiation sensitizers, into MIP-NGs to create novel stealth nanomaterials for radiation therapy, in which 4 ACS Paragon Plus Environment

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high biocompatibility and low agglomeration property can be expected due to the possible formation of albumin-rich protein corona. To date, various studies have reported the radiation sensitizing effects of Au NPs, and Au NPs have been stabilized using citric acid, glucose, polyethylene glycol (PEG), proteins, and PEG-containing block copolymers.38 The radiation-sensitizing effects of PEGylated nanogels containing Au NPs at cellular levels have been reported by Yasui et al.39 However, so far, radiation sensitizing effects of Au NP-incorporated polymer nano/microgels have not been evaluated in vivo, and the effectiveness of these materials in radiation therapy remains unclear. Herein, we prepared Au NP-incorporated MIP microgels (Au-MIP microgels) via seeded precipitation polymerization, and the radiation-sensitizing effects of these Au-MIP microgels were investigated using a mouse model of pancreatic cancer (Scheme 1).



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Scheme 1. Radiation therapy model of pancreatic cancer using Au-MIP microgels as radiation sensitizers prepared by seeded precipitation polymerization

Experimental Section Materials Na2CO3, citric acid, NaCl, MgSO4, diethyl ether, N-isopropylacrylamide (NIPAm), and N,N’-methylenebisacrylamide (MBAA) were purchased from Nacalai Tesque Co. (Kyoto, Japan). Na2HPO4, dichloromethane, N-Boc-3-hydroxypyrrolidine, ethyl acetate, hexane, acetone, HSA,

transferrin

(Tf),

cell-counting

assay

kit

(CCK-8),

and

2,2’-azobis

(2-methylpropionamidine)dihydrochloride (V-50) were purchased from Wako Co. Ltd. (Osaka, Japan). Immunoglobulin G (IgG) was purchased from SIGMA-ALDRICH (MO, USA). NaH2PO4 was purchased from Katayama Chemical Industries Co. Ltd. (Osaka, Japan). N,N-Diisopropylethylamine, acryloyl chloride, and 5-amino fluorescein were purchased from 6 ACS Paragon Plus Environment

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Tokyo Chemical Industries (Tokyo, Japan). 2-Methacryloyloxyethyl phosphorylcholine (MPC) was purchased from NOF Corporation (Tokyo, Japan). Pyrrolidyl acrylate (PyA) was prepared as previously reported.40 The Au NP (24 nm) aqueous dispersion was from Tanaka Holdings Co. Ltd. (Tokyo, Japan). The stock solution of APF (5 mM in DMF) was purchased from SEKISUI MEDICAL CO., LTD. (Tokyo, Japan), and deionized water was obtained from a Millipore Milli-Q purification system.

Characterization UV-Vis spectral measurements were performed using a V-560 spectrophotometer (JASCO Ltd., Tokyo, Japan). The particle size distribution and zeta potential were obtained using a DLS system (Zetasizer Nano ZS; Malvern Instruments Ltd., Malvern, UK). Transmission electron microscopy (TEM) images of the purified Au-MIP microgels were obtained using a JEM-1230 (JEOL, Tokyo, Japan).

Preparation of Au-MIP microgels NIPAm (204 mg, 1.8 mmol), MBAA (15.4 mg, 0.1 mmol), MPC (29.5 mg, 0.1 mmol), PyA (21 mg, 0.15 mmol), HSA (6.6 mg, 0.2 μmol), and V-50 (108.5 mg, 0.4 mmol) were mixed in 10 mM phosphate buffer (pH 7.4, 45 mL). The Au NP dispersion (69 μg/mL, 5 mL, 15 nm) was 7 ACS Paragon Plus Environment


then added to the prepolymerization mixture. Precipitation polymerization was carried out at 70°C for 12 h. The Au-MIP microgels were purified by size-exclusion chromatography (Sephadex G-100) using 10 mM phosphate buffer (pH 7.4) as an eluent. The lower critical solution temperature (LCST) of polyNIPAm-based gels is approximately 60°C;28 therefore, Au-MIP microgels were swollen and stably dispersed in 10 mM phosphate buffer (pH 7.4) at 37°C. After the purification by size-exclusion chromatography, solid contents of Au-MIP microgels

were measured by gravimetry, where the gels were dried for 24 h at 80°C, which is

higher than the LCST of poly(NIPAm)-based microgels, and under these conditions, hydration in the gels may not occur, facilitating the drying. From the gravimetry measurements, the yield of Au-MIP microgels was estimated to be approximately 63%.

Quantitative analysis of Au contents in Au-MIP microgels Au-MIP microgels, purified by size-exclusion chromatography (0.725 mg/mL, 2 mL) and dispersed in 10 mM phosphate-buffer (pH 7.4), were dried, and the nitric acid/sulfuric acid (1:1 v/v) mixture (1.5 mL) was added and evaporated on a hot plate. The aqua regia (concentrated hydrochloric acid/concentrated nitric acid = 3/1 v/v, 1.0 mL) was added to the residue and reacted at room temperature for 2 h to ionize Au NPs. The aqueous solution was diluted with 8 ACS Paragon Plus Environment

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pure water (4.0 mL). Au contents were determined by ICP-AES (ULTIMA 2000, Jobin Yvon Horiba).

Radiation therapy All animal experiments were performed according to Kobe University Animal Experimentation Regulations. MIAPaCa-2 human pancreatic cancer cells, obtained from American Type Culture Collection (Rockville, MD, USA), were cultured in RPMI-1640 medium. BALB/c nude mice (male, 4 weeks) were purchased from Charles River Laboratories Japan Inc. (Yokohama, Japan). The mice were maintained in specific pathogen-free animal care facilities under isothermal conditions with regulated photoperiods. Twenty mice received subcutaneous injections of 2 × 106 MIAPaCa-2 cells suspended in Matrigel (BD Biosciences) and were then randomly divided into 5 groups (4 mice per group). Au-MIP microgels were concentrated by ultrafiltration (molecular weight cutoff = 30 kDa). The solid content of Au-MIP microgels was calculated by gravimetry. Au-MIP microgels (29.1 mg/mL, 100 μL) dispersed in 10 mM PBS (pH 7.4, 140 mM NaCl) were locally injected into the tumor tissue of BALB/c nude mice bearing pancreatic cancer tumors; 10 mM PBS (pH 7.4, 140 mM NaCl) was injected as a control group with X-ray radiation. The mice were anesthetized by intraperitoneal administration of somnopentyl (0.1 mg/g body weight), and the bodies of mice, excluding tumor tissues, were protected from 9 ACS Paragon Plus Environment


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irradiation damage using a lead plate. X-ray irradiation (0.5 Gy/min at the target; total dose: 5 Gy) was carried out using an MBR-1505R2 (Hitachi Medical Co., Tokyo, Japan) at a current of 5 mA and a voltage of 150 kV with a 1-mm-thick aluminum filter. The data of mice body weights and therapeutic efficacy of radiation were expressed as mean ± standard deviation. The tumor size was measured by calculating the volume using the formula L ×W2 × (π/6), where L and W are the longest and shortest diameters of the tumor, respectively.41-43 The L and W values were measured using calipers. Data of outlier tumor sizes were removed from the datasets, where the outlier was defined as |(tumor volume measured – tumor volume mean)|/standard deviation > 2. The

statistical significance of differences in therapeutic efficacy between two groups possessing unequal variance was calculated using the Student’s t-test with two-sided test. Differences were considered significant at a 95% confidence level (p < 0.05).

Results and Discussion Au NP-incorporated polymer nano/microgels have been prepared using various approaches,44 including in-situ synthesis of Au NPs in polymer microgels,45-50 assembly of Au NPs on polymer microgels,51-52 seeded polymerization with prefunctionalized Au NPs,50,

51

multistep seeded

polymerization,53-54 and liquid-liquid dispersion with three immiscible liquids.55 In multistep seeded polymerization, the Au NPs were first coated with a poly(styrene-divinylbenzene) inner 10 ACS Paragon Plus Environment

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shell, and a poly(NIPAm-MBAA) outer shell was then formed.54 These approaches were necessary for multistep preparation of Au NP-incorporated polymer nano/microgels. Herein, a one-step simple method for synthesis of Au-MIP microgels was developed, and the obtained nanocomposite microgels were used as radiation sensitizers. Au-MIP microgels were prepared by seeded precipitation polymerization with Au NPs (24 nm, Figure 1a) as seed particles in 10 mM phosphate buffer (pH 7.4) at 70°C for 12 h. NIPAm and MBAA were used as the main monomer and crosslinking agent for microgels, respectively. MPC was used as a highly hydrophilic biocompatible monomer for improving particle stability. PyA was also used because this monomer was applied as a functional monomer for creation of stealth nanogels in our previous study.28 The stability of seed Au NPs in the prepolymerization mixture was investigated by UV-Vis measurements because the coagulation state of Au NPs was estimated using their localized surface plasmon resonance properties.56-57 When the Au NPs were contained in the prepolymerization mixture, the maximum absorption wavelength was clearly shifted to 526 nm from 626 nm (Figure S1), and the size of Au NPs was increased to 845 nm from 24 nm in number-average particle size (dn; Figure S2). In contrast, in the presence of albumin, the spectral shift derived from Au NPs was negligible (528 nm), and the size of Au NPs was maintained at a similar value (dn = 24 nm), indicating that the albumin worked as a stabilizer of Au NPs in seeded precipitation polymerization. 11 ACS Paragon Plus Environment


After seeded precipitation polymerization, the particle size of the obtained Au-MIP microgels was 190 nm as dn and was clearly larger than that of seed Au NPs (dn = 24 nm; Figure 1b). The spectral shift was not observed in UV-Vis measurements (527 nm), indicating that the Au NPs were not coagulated during the polymerization in 10 mM phosphate buffer (pH 7.4) (Figure S3). In addition, the time-course of particle size during polymerization was also investigated, and the size was maintained at a similar value after 6 h of polymerization, indicating that the particle size was saturated at around 6 h (Figures S4 and S5). The particle morphology observed by TEM indicated that the Au NPs were incorporated into the polymer microgels (Figure 1c). To quantify the Au content in the Au incorporated polymer nanogels, ICP atomic emission spectroscopy measurements were performed. The amount of A incorporated into Au-MIP microgels was approximately 5.0 μg/mg particles. The HSA recognition property of Au-MIP microgels was evaluated by microBCA assay using IgG and Tf as reference proteins. The HSA bound amount was greater than those for IgG and Tf, indicating that the molecular recognition capability of Au-MIP microgels was confirmed (Figure S6), and as was the case of previously reported MIP-NGs [28], the stealth capability in body fluids can be expected. From these results, the Au-MIP microgels were successfully prepared by a one-step seeded precipitation polymerization method. In the CCK-8 assay using a


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standard fibroblast cell line NIH3T3, almost no cytotoxicity was observed up to 2910 μg/mL of Au-MIP microgels.

(c)

Figure 1. Particle size distributions measured by DLS of Au NPs (a), Au-MIP microgels (b), and TEM image of Au-MIP microgels.



In the next step, the biocompatibility of the Au-MIP microgels was checked in vivo using BALB/c nude mice bearing pancreatic cancer tumors (Figure 2). The body weights of mice injected with Au-MIP microgels with and without X-ray irradiation were similar. In addition, the body weights of the X-ray-irradiated mice injected with Au-MIP microgels were also similar to those of X-ray-irradiated control mice injected with phosphate buffer, indicating that the Au-MIP microgels had high biocompatibility (low biotoxicity).

Figure 2. Body weight changes in pancreatic cancer model mice treated with (triangles) and without (squares) Au-MIP microgels with X-ray irradiation, and with (circles) and without (diamonds) Au-MIP microgels without X-ray irradiation. 14 ACS Paragon Plus Environment

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The radiation sensitizing effect of Au-MIP microgels was investigated in vitro. Because Au possesses a high atomic number, it can absorb X-ray, generating secondary electrons as well as characteristic x-ray or Auger electrons (photoelectric effect).18 These radiations and electrons further react with water and oxygen molecules, accelerating the generation of ROS such as hydroxyl radials. To examine the radiation-based ROS generation in Au-MIP microgels, a ROS-responsive fluorescent dye, aminophenyl fluorescein (APF), was employed to detect ROS generation.58 As expected, the fluorescence increased with X-ray irradiation (5 and 10 Gy), confirming that Au-MIP microgels possess radiation-based ROS generation capability (i.e. radiation sensitizing effect). Finally, to investigate the radiation-sensitizing effects of Au-MIP microgels, the tumor volumes of treated mice were measured. Au-MIP microgels were locally injected into the pancreatic tumor, and X-ray irradiation was performed just after the injection, in which Au-MIP microgels were retained in the tumor during the X-ray irradiation. When Au-MIP microgels enhanced ROS generation, the ROS generated from the locally injected Au-MIP microgels attacked the DNA even more than several μm far from the AuNP.59



Figure 3. Tumor volume changes in X-ray irradiated pancreatic cancer model mice injected with and without Au-MIP microgels. The relative tumor volume is the ratio of a tumor volume divided by an initial tumor volume.

As shown in Figure 3, the tumor sizes of mice injected with Au-MIP microgels were gradually increased when X-ray irradiation was not performed. The trend of tumor growth was similar with that for the control mice without addition of Au-MIP microgels, indicating the Au-MIP microgels did not show the tumor growth inhibition property without X-ray irradiation 16 ACS Paragon Plus Environment

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(Figure 3). In contrast, tumor growth was effectively suppressed in mice injected with Au-MIP microgels (29.1 mg/mL) when X-ray irradiation was performed (p < 0.05, Figure 3). Furthermore, the tumor sizes in mice injected with nanocomposite microgels treated with X-ray irradiation were clearly smaller than those of phosphate buffer-injected control mice (Figure 4). These results indicated that Au-MIP microgels represent effective radiation sensitizers as previously reported for Au NPs, stabilized by various molecules such as PEG, citric acid, and proteins,60-64 and could have applications in radiation therapy for pancreatic cancer treatment.



(-) Au-MIP microgels (-) X-ray irradiation

(+) Au-MIP microgels (-) X-ray irradiation

(+) Au-MIP microgels (+) X-ray irradiation

(-) Au-MIP microgels (+) X-ray irradiation

Figure 4. Photographs of pancreatic cancer model mice treated with (right, top) and without (left, top) Au-MIP microgels with X-ray irradiation, and with (right, bottom) and without (left, bottom) Au-MIP microgels without X-ray irradiation


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Conclusions Au-MIP microgels were prepared using a newly developed one-pot seeded precipitation polymerization method in the presence of seed Au NPs. The Au-MIP microgels were further used as radiation sensitizers for radiation therapy for mice bearing pancreatic tumors, resulting in effective suppression of tumor growth in mice injected with Au-MIP microgels compared with that in control mice injected with phosphate buffer when X-ray irradiation was performed. Our findings confirmed that Au-MIP microgels could function as radiation sensitizers for radiation therapy. Further studies are needed to develop innovative radiation sensitizers based on composites of polymer nanogels and Au NPs in the near future.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:xxx

UV-Vis spectra and size distribution of Au NPs in prepolymerization mixture and Au-MIP microgels, size distributions at different polymerization times, HSA recognition property of



Au-MIP microgels, cytotoxicity, ROS generation, and effect of Au-MIP microgel concentration on radiation therapy are available free of charge via the Internet.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (T.T.)

*E-mail: [email protected] (Y.K)

ORCID Toshifumi Takeuchi: 0000-0002-5641-2333

Yukiya Kitayama: 0000-0002-7418-301X Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENT

This study was supported by JSPS KAKENHI Grant Number JP18H05398. The authors thank the Foundation for Biomedical Research and Innovation at Kobe for supporting this work. 20 ACS Paragon Plus Environment

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REFERENCES

(1)

Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.;

Thun, M. J. Cancer Statistics, 2004. CA Cancer J. Clin. 2004, 54 (1), 8-29. (2)

Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 2017,

67 (1), 7-30. (3)

Gillen, S.; Schuster, T.; zum Buschenfelde, C. M.; Friess, H.; Kleeff, J.

Preoperative/neoadjuvant therapy in pancreatic cancer: A Systematic Review and Meta-Analysis of Response and Resection Percentages. PLoS Med. 2010, 7 (4), e1000267. (4)

Kamisawa, T.; Wood, L. D.; Itoi, T.; Takaori, K. Pancreatic Cancer. Lancet 2016, 388

(10039), 73-85. (5)

Zhou, B.; Xu, J. W.; Cheng, Y. G.; Gao, J. Y.; Hu, S. Y.; Wang, L.; Zhan, H. X. Early

Detection of Pancreatic Cancer: Where Are We Now and Where Are We Going? Int. J. Cancer 2017, 141 (2), 231-241. (6)

Makohon-Moore, A.; Iacobuzio-Donahue, C. A. Pancreatic Cancer Biology and

Genetics from An Evolutionary Perspective. Nat. Rev. Cancer 2016, 16 (9), 553-565.



(7)

Kleeff, J.; Korc, M.; Apte, M.; La Vecchia, C.; Johnson, C. D.; Biankin, A. V.; Neale,

R. E.; Tempero, M.; Tuveson, D. A.; Hruban, R. H.; Neoptolemos, J. P. Pancreatic Cancer. Nat. Rev. Dis. Primers 2016, 2, 16022. (8)

Sultana, A.; Smith, C. T.; Cunningham, D.; Starling, N.; Tait, D.; Neoptolemos, J. P.;

Ghaneh, P. Systematic Review, Including Meta-Analyses, On the Management of Locally Advanced Pancreatic Cancer Using Radiation/Combined Modality Therapy. Br. J. Cancer 2007, 96 (8), 1183-1190. (9)

Sulaiman, N. S.; Fujii, O.; Demizu, Y.; Terashima, K.; Niwa, Y.; Akagi, T.; Daimon, T.;

Murakami, M.; Sasaki, R.; Fuwa, N. Particle Beam Radiation Therapy Using Carbon Ions and Protons for Oligometastatic Lung Tumors. Radiat. Oncol. 2014, 9, 183. (10)

Babaei, M.; Ganjalikhani, M. The Potential Effectiveness of Nanoparticles as Radio

Sensitizers for Radiotherapy. BioImpacts 2014, 4 (1), 15-20. (11)

Hainfeld, J. F.; Dilmanian, F. A.; Slatkin, D. N.; Smilowitz, H. M. Radiotherapy

Enhancement with Gold Nanoparticles. J. Pharm. Pharmacol. 2008, 60 (8), 977-985. (12)

Loehrer, P. J.; Feng, Y.; Cardenes, H.; Wagner, L.; Brell, J. M.; Cella, D.; Flynn, P.;

Ramanathan, R. K.; Crane, C. H.; Alberts, S. R.; Benson, A. Gemcitabine Alone Versus Gemcitabine Plus Radiotherapy in Patients with Locally Advanced Pancreatic Cancer: An Eastern Cooperative Oncology Group Trial. J. Clin. Oncol. 2011, 29 (31), 4105-4112. 22 ACS Paragon Plus Environment

Page 22 of 32

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13)

Kuncic, Z.; Lacombe, S. Nanoparticle Radio-Enhancement: Principles, Progress and

Application to Cancer Treatment. Phys. Med. Biol. 2018, 63 (2), 02TR01. (14)

Nakayama, M.; Sasaki, R.; Ogino, C.; Tanaka, T.; Morita, K.; Umetsu, M.; Ohara, S.;

Tan, Z. Q.; Nishimura, Y.; Akasaka, H.; Sato, K.; Numako, C.; Takami, S.; Kondo, A. Titanium Peroxide Nanoparticles Enhanced Cytotoxic Effects of X-Ray Irradiation Against Pancreatic Cancer Model Through Reactive Oxygen Species Generation In Vitro and In Vivo. Radiat. Oncol. 2016, 11, 91. (15)

Young, S. W.; Qing, F.; Harriman, A.; Sessler, J. L.; Dow, W. C.; Mody, T. D.; Hemmi,

G. W.; Hao, Y. P.; Miller, R. A. Gadolinium(III) Texaphyrin: A Tumor Selective Radiation Sensitizer That Is Detectable by MRI. Proc. Natl. Acad. Sci. USA 1996, 93 (13), 6610-6615. (16)

Zygmanski, P.; Sajo, E. Nanoscale Radiation Transport and Clinical Beam Modeling for

Gold Nanoparticle Dose Enhanced Radiotherapy (GNPT) Using X-rays. Br. J. Radiol. 2016, 89 20150200. (17)

Dongre, P. Gold Nanoparticle-Assisted Radiation Therapy 2017. p 145-158.

(18)

Ashton, J. R.; Castle, K. D.; Qi, Y.; Kirsch, D. G.; West, J. L.; Badea, C. T.

Dual-Energy CT Imaging of Tumor Liposome Delivery after Gold Nanoparticle-Augmented Radiation Therapy. Theranostics 2018, 8 (7), 1782-1797.



(19)

Chithrani, D. B.; Jelveh, S.; Jalali, F.; van Prooijen, M.; Allen, C.; Bristow, R. G.; Hill,

R. P.; Jaffray, D. A. Gold Nanoparticles as Radiation Sensitizers in Cancer Therapy. Radiat. Res. 2010, 173 (6), 719-728. (20)

Laprise-Pelletier, M.; Simao, T.; Fortin, M. A. Gold Nanoparticles in Radiotherapy and

Recent Progress in Nanobrachytherapy. Adv. Healthc. Mater. 2018, 7 (16), e1701460. (21)

Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique Features of Tumor Blood

Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug. Deliver. Rev. 2011, 63 (3), 136-151. (22)

Cho, K. J.; Wang, X.; Nie, S. M.; Chen, Z.; Shin, D. M. Therapeutic Nanoparticles for

Drug Delivery in Cancer. Clin. Cancer Res. 2008, 14 (5), 1310-1316. (23)

Sill, T. J.; von Recum, H. A. Electro spinning: Applications in Drug Delivery and

Tissue Engineering. Biomaterials 2008, 29 (13), 1989-2006. (24)

Sun, C.; Lee, J. S. H.; Zhang, M. Q. Magnetic Nanoparticles in MR Imaging and Drug

Delivery. Adv. Drug. Deliver. Rev. 2008, 60 (11), 1252-1265. (25)

Kumari, A.; Yadav, S. K.; Yadav, S. C. Biodegradable Polymeric Nanoparticles Based

Drug Delivery Systems. Colloids Surf B Biointerfaces 2010, 75 (1), 1-18. (26)

Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery.

Nat. Mat. 2013, 12 (11), 991-1003. 24 ACS Paragon Plus Environment

Page 24 of 32

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27)

Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery:

Design, Characterization and Biological Significance. Adv. Drug. Deliver. Rev. 2012, 64, 37-48. (28)

Takeuchi, T.; Kitayama, Y.; Sasao, R.; Yamada, T.; Toh, K.; Matsumoto, Y.; Kataoka,

K. Molecularly Imprinted Nanogels Acquire Stealth In Situ by Cloaking Themselves with Native Dysopsonic Proteins. Angew. Chem. Int. Ed. 2017, 56 (25), 7088-7092. (29)

Komiyama, M.; Mori, T.; Ariga, K. Molecular Imprinting: Materials Nanoarchitectonics

with Molecular Information. Bull. Chem. Soc. Jpn. 2018, 91 (7), 1075-1111. (30)

Pan, J. M.; Chen, W.; Ma, Y.; Pan, G. Q. Molecularly Imprinted Polymers as Receptor

Mimics for Selective Cell Recognition. Chem. Soc. Rev. 2018, 47 (15), 5574-5587. (31)

Xing, R. R.; Wang, S. S.; Bie, Z. J.; He, H.; Liu, Z. Preparation of Molecularly

Imprinted Polymers Specific to Glycoproteins, Glycans and Monosaccharides via Boronate Affinity Controllable-Oriented Surface Imprinting. Nat. Protoc. 2017, 12 (5), 964-987. (32)

Horikawa, R.; Sunayama, H.; Kitayama, Y.; Takano, E.; Takeuchi, T. A Programmable

Signaling Molecular Recognition Nanocavity Prepared by Molecular Imprinting and Post-Imprinting Modifications. Angew. Chem. Int. Ed. 2016, 55 (42), 13023-13027. (33)

Chen, L. X.; Wang, X. Y.; Lu, W. H.; Wu, X. Q.; Li, J. H. Molecular Imprinting:

Perspectives and Applications. Chem. Soc. Rev. 2016, 45 (8), 2137-2211.



(34)

Takeuchi, T.; Sunayama, H.; Takano, E.; Kitayama, Y. Post-Imprinting and In-Cavity

Functionalization. In Molecularly Imprinted Polymers in Biotechnology, Mattiasson, B.; Ye, L., Eds., 2015; Vol. 150, pp 95-106. (35)

Whitcombe, M. J.; Chianella, I.; Larcombe, L.; Piletsky, S. A.; Noble, J.; Porter, R.;

Horgan, A. The Rational Development of Molecularly Imprinted Polymer-Based Sensors for Protein Detection. Chem. Soc. Rev. 2011, 40 (3), 1547-1571. (36)

Chen, L. X.; Xu, S. F.; Li, J. H. Recent Advances in Molecular Imprinting Technology:

Current Status, Challenges and Highlighted Applications. Chem. Soc. Rev. 2011, 40 (5), 2922-2942. (37)

Takeuchi, T.; Hayashi, T.; Ichikawa, S.; Kaji, A.; Masui, M.; Matsumoto, H.; Sasao, R.

Molecularly Imprinted Tailor-Made Functional Polymer Receptors for Highly Sensitive and Selective Separation and Detection of Target Molecules. Chromatography 2016, 37, 43-64. (38)

Her, S.; Jaffray, D. A.; Allen, C. Gold Nanoparticles for Applications in Cancer

Radiotherapy: Mechanisms and Recent Advancements. Adv. Drug. Deliver. Rev. 2017, 109, 84-101. (39)

Yasui, H.; Takeuchi, R.; Nagane, M.; Meike, S.; Nakamura, Y.; Yamamori, T.; Ikenaka,

Y.; Kon, Y.; Murotani, H.; Oishi, M.; Nagasaki, Y.; Inanami, O. Radiosensitization of Tumor


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cells Through Endoplasmic Reticulum Stress Induced by PEGylated Nanogel Containing Gold Nanoparticles. Cancer Lett. 2014, 347 (1), 151-158. (40)

Inoue, Y.; Kuwahara, A.; Ohmori, K.; Sunayama, H.; Ooya, T.; Takeuchi, T.

Fluorescent Molecularly Imprinted Polymer Thin Films for Specific Protein Detection Prepared with Dansyl Ethylenediamine-Conjugated O-Acryloyl L-Hydroxyproline. Biosens. Bioelectron. 2013, 48, 113-119. (41)

Akasaka, H.; Mizushina, Y.; Yoshida, K.; Ejima, Y.; Mukumoto, N.; Wang, T. Y.;

Inubushi, S.; Nakayama, M.; Wakahara, Y.; Sasaki, R. MGDG Extracted from Spinach Enhances the Cytotoxicity of Radiation in Pancreatic Cancer Cells. Radiat. Oncol. 2016, 11, 153. (42)

Tailor, T. D.; Hanna, G.; Yarmolenko, P. S.; Dreher, M. R.; Betof, A. S.; Nixon, A. B.;

Spasojevic, I.; Dewhirst, M. W. Effect of Pazopanib on Tumor Microenvironment and Liposome Delivery. Mol. Cancer Ther. 2010, 9 (6), 1798-1808. (43)

Shim, W. S. N.; Teh, M.; Mack, P. O. P.; Ge, R. W. Inhibition of Angiopoietin-1

Expression in Tumor Cells by an Antisense RNA Approach Inhibited Xenograft Tumor Growth in Immunodeficient Mice. Int. J. Cancer 2001, 94 (1), 6-15. (44)

Karg, M.; Hellweg, T. New "Amart" Poly(NIPAM) Microgels and Nanoparticle

Microgel Hybrids: Properties and Advances in Characterisation. Curr. Opin. Colloid Interface Sci. 2009, 14 (6), 438-450. 27 ACS Paragon Plus Environment


(45)

Kim, J. H.; Lee, T. R. Hydrogel-Templated Growth of Large Gold Nanoparticles:

Synthesis of Thermally Responsive Hydrogel-Nanoparticle Composites. Langmuir 2007, 23 (12), 6504-6509. (46)

Akamatsu, K.; Shimada, M.; Tsuruoka, T.; Nawafune, H.; Fujii, S.; Nakamura, Y.

Synthesis of pH-Responsive Nanocomposite Microgels with Size-Controlled Gold Nanoparticles from Ion-Doped, Lightly Cross-Linked Poly(Vinylpyridine). Langmuir 2010, 26 (2), 1254-1259. (47)

Zhang, J. G.; Xu, S. Q.; Kumacheva, E. Polymer Microgels: Reactors for

Semiconductor, Metal, and Magnetic Nanoparticles. J. Am. Chem. Soc. 2004, 126 (25), 7908-7914. (48)

Suzuki, D.; Kawaguchi, H. Modification of Gold Nanoparticle Composite

Nanostructures Using Thermosensitive Core-Shell Particles as a Template. Langmuir 2005, 21 (18), 8175-8179. (49)

Suzuki, D.; Kawaguchi, H. Gold Nanoparticle Localization at the Core Surface by

Using Thermosensitive Core-Shell Particles as a Template. Langmuir 2005, 21 (25), 12016-12024. (50)

Suzuki, D.; Kawaguchi, H. Hybrid Microgels with Reversibly Changeable Multiple

Brilliant Color. Langmuir 2006, 22 (8), 3818-3822.


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(51)

Karg, M.; Pastoriza-Santos, I.; Perez-Juste, J.; Hellweg, T.; Liz-Marzan, L. M.

Nanorod-Coated PNIPAM Microgels: Thermoresponsive Optical Properties. Small 2007, 3 (7), 1222-1229. (52)

Das, M.; Sanson, N.; Fava, D.; Kumacheva, E. Microgels Loaded with Gold Nanorods:

Photothermally Triggered Volume Transitions Under Physiological Conditions. Langmuir 2007, 23 (1), 196-201. (53)

Alvarez-Puebla, R. A.; Contreras-Caceres, R.; Pastoriza-Santos, I.; Perez-Juste, J.;

Liz-Marzan, L. M. Au@pNIPAM Colloids as Molecular Traps for Surface-Enhanced, Spectroscopic, Ultra-Sensitive Analysis. Angew. Chem. Int. Ed. 2009, 48 (1), 138-143. (54)

Contreras-Caceres, R.; Sanchez-Iglesias, A.; Karg, M.; Pastoriza-Santos, I.; Perez-Juste,

J.; Pacifico, J.; Hellweg, T.; Fernandez-Barbero, A.; Liz-Marzan, L. M. Encapsulation and Growth of Gold Nanoparticles in Thermoresponsive Microgels. Adv. Mat. 2008, 20 (9), 1666-1670. (55)

Budhlall, B. M.; Marquez, M.; Velev, O. D. Microwave, Photo- and Thermally

Responsive Pnipam-Gold Nanoparticle Microgels. Langmuir 2008, 24 (20), 11959-11966. (56)

Sepulveda, B.; Angelome, P. C.; Lechuga, L. M.; Liz-Marzan, L. M. LSPR-Based

Nanobiosensors. Nano Today 2009, 4 (3), 244-251.



(57)

Mayer, K. M.; Hafner, J. H. Localized Surface Plasmon Resonance Sensors. Chem. Rev.

2011, 111 (6), 3828-3857. (58)

Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. Development of

Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. J. Biol. Chem. 2003, 278 (5), 3170-3175. (59)

Tran, H. N.; Karamitros, M.; Ivanchenko, V. N.; Guatelli, S.; McKinnon, S.; Murakami,

K.; Sasaki, T.; Okada, S.; Bordage, M. C.; Francis, Z.; El Bitar, Z.; Bernal, M. A.; Shin, J. I.; Lee, S. B.; Barberet, P.; Tran, T. T.; Brown, J. M. C.; Hao, T. V. N.; Incerti, S. Geant4 Monte Carlo Simulation of Absorbed Dose and Radiolysis Yields Enhancement from a Gold Nanoparticle under Mev Proton Irradiation. Nucl. Instrum. Meth. B 2016, 373, 126-139. (60)

Wolfe, T.; Chatterjee, D.; Lee, J.; Grant, J. D.; Bhattarai, S.; Tailor, R.; Goodrich, G.;

Nicolucci, P.; Krishnan, S. Targeted Gold Nanoparticles Enhance Sensitization of Prostate Tumors to Megavoltage Radiation Therapy In Vivo. Nanomed-Nanotechnol 2015, 11 (5), 1277-1283. (61)

Zhang, X. D.; Wu, D.; Shen, X.; Chen, J.; Sun, Y. M.; Liu, P. X.; Liang, X. J.

Size-Dependent Radiosensitization of PEG-Coated Gold Nanoparticles for Cancer Radiation Therapy. Biomaterials 2012, 33 (27), 6408-6419.


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(62)

Chang, M. Y.; Shiau, A. L.; Chen, Y. H.; Chang, C. J.; Chen, H. H. W.; Wu, C. L.

Increased Apoptotic Potential and Dose-Enhancing Effect of Gold Nanoparticles in Combination with Single-Dose Clinical Electron Beams on Tumor-Bearing Mice. Cancer Sci. 2008, 99 (7), 1479-1484. (63)

Zhang, X. D.; Chen, J.; Luo, Z. T.; Wu, D.; Shen, X.; Song, S. S.; Sun, Y. M.; Liu, P.

X.; Zhao, J.; Huo, S. D.; Fan, S. J.; Fan, F. Y.; Liang, X. J.; Xie, J. P. Enhanced Tumor Accumulation of Sub-2 nm Gold Nanoclusters for Cancer Radiation Therapy. Adv. Healthc. Mater. 2014, 3 (1), 133-141. (64)

Chen, N.; Yang, W. T.; Bao, Y.; Xu, H. L.; Qin, S. B.; Tu, Y. BSA Capped Au

Nanoparticle as an Efficient Sensitizer for Glioblastoma Tumor Radiation Therapy. RSC Adv. 2015, 5 (51), 40514-40520.



Graphical Abstract


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Gold Nanoparticle-Incorporated Molecularly Imprinted Microgels as

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