Implantable chemothermal brachytherapy seeds_ A

European Journal of Pharmaceutics and Biopharmaceutics 129 (2018) 191–203

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Implantable chemothermal brachytherapy seeds: A synergistic approach to brachytherapy using polymeric dual drug delivery and hyperthermia for malignant solid tumor ablation

T

Ludwig Erik Aguilara, Reju George Thomasb, Myeong Ju Moonb, Yong Yeon Jeongb, ⁎ ⁎ Chan Hee Parka, , Cheol Sang Kima, a b

Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Republic of Korea Department of Radiology, Chonnam National University Hwasun Hospital, Hwasun 58128, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Chemothermal seeds Cancer treatment Hyperthermia therapy Polymeric chemotherapy Thermal brachytherapy

Chemothermal brachytherapy seeds have been developed using a combination of polymeric dual drug chemotherapy and alternating magnetic field induced hyperthermia. The synergistic effect of chemotherapy and hyperthermia brachytherapy has been investigated in a way that has never been performed before, with an indepth analysis of the cancer cell inhibition property of the new system. A comprehensive in vivo study on athymic mice model with SCC7 tumor has been conducted to determine optimal arrays and specifications of the chemothermal seeds. Dual drug chemotherapy has been achieved via surface deposition of polydopamine that carries bortezomib, and also via loading an acidic pH soluble hydrogel that contains 5-Fluorouracil inside the chemothermal seed; this increases the drug loading capacity of the chemothermal seed, and creates dual drug synergism. An external alternating magnetic field has been utilized to induce hyperthermia conditions, using the inherent ferromagnetic property of the nitinol alloy used as the seed casing. The materials used in this study were fully characterized using FESEM, H1 NMR, FT-IR, and XPS to validate their properties. This new approach to experimental cancer treatment is a pilot study that exhibits the potential of thermal brachytherapy and chemotherapy as a combined treatment modality.

1. Introduction Several types of cancer (CA) that exhibit as solid tumor malignancies have always been a challenge to treat, especially due to their rapid and aggressive growth rate. Examples of these are mammary, prostate, and hepatic tumors. These types of solid malignancies are treated with aggressive Radiation Therapy (RT) and an adjuvant of Chemotherapy (CT). In the United States alone, breast and prostate CA constitute (27 and 29)% of all solid malignancy cases, respectively [1]. In the clinical setting, multimodal therapies are required to enhance the prognosis of the patient. Radio- and Chemo-therapies are staple treatments, and surgery is also known to be an effective form of intervention. A multimodal non-surgical approach has been the mode of treatment for patients with advanced stages of CA, to reduce the size of the tumor, and halt the spread of malignant cells. RT and CT are also known to be used as adjuvant therapies to enhance the effect of one another [2–4]. Another modality that has been known to effectively treat solid

⁎

Corresponding authors. E-mail addresses: [email protected] (C.H. Park), [email protected] (C.S. Kim).

https://doi.org/10.1016/j.ejpb.2018.06.003 Received 2 October 2017; Received in revised form 1 June 2018; Accepted 2 June 2018 Available online 04 June 2018 0939-6411/ © 2018 Published by Elsevier B.V.

malignancies is brachytherapy (BT), where radioactive seeds are implanted in and around the tumor, and give off high and low doses of radiation over time [5–8]. This is known to improve the prognosis of prostate and breast CA patients, and other forms of solid malignancies [9–11]. We borrow the idea of implanting seeds, but use nitinol and its inherent ferromagnetic property to induce heat around and inside the tumor site, while also releasing chemotherapeutics over time to treat cancer. The combined effects of hyperthermia therapy (HT) via alternating magnetic field induced heating and dual drug delivery via pH responsive polymers were studied in this work to assess the effectiveness of these innovative chemothermal seeds (CTS) as a new form of CA treatment. However, one setback of HT is the inappropriate dosage of thermal energy transfer into the tumor tissue. Overheating and under heating is still a major issue of HT; in order to maximize this treatment, we needed an exact analysis of the thermal capability of the CTS. The exact amount and array of CTS can be determined via analysis of the isotherm using in vivo tumor model. In the treatment modality of HT, where the surrounding site of the tumor and the tumor itself are heated


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efficiency was measured via High-performance liquid chromatography (HPLC) (retention time of 6.633; wavelength 263 nm) (JASCO, USA), by measuring the amount of BTZ left in the supernatant after the loading process, and comparing it to the amount of BTZ present before the drug loading procedure. The entrapment efficiency was then calculated using the following equation:

to (43–45) °C to create a cytotoxic environment [12–14], HT is known to enhance the effect of RT and CT by delaying or impeding DNA recombination of damaged cells, leading to higher cell death [15–18]. There are multiple approaches to creating hyperthermia conditions in the body; local and systemic HT are utilized in clinical settings, and these are conducted using microwave implants and heat baths [19,20]. In contemporary research, magnetic nanoparticles are introduced and utilized for HT, examples of which are Iron II magnetite and III maghemite nanoparticles functionalized with ligands and chemotherapeutic drugs for the active targeting and treatment of cancer, and promising results have been reported by such studies [21–28]. However, recent studies have drawn attention to the side effects of magnetic nanoparticles (SPIONS), like liver and kidney toxicities, fluctuations in anti-oxidant and tissue nitrite levels, and phenotypic gene alteration, leading us to rethink our strategy of creating hyperthermia conditions in the body [29–31]. In this research, we utilized polymeric vehicles for drug delivery, especially stimuli responsive polymers. We exploited the catecholboronic link that is pH cleavable to deliver the drug payload to malignant tissue. We covered the surface of the nitinol tubes with electropolymerized dopamine to act as a pH sensitive drug delivery platform, and conjugated it with a boron-containing drug bortezomib (BTZ) via catechol-boronic bonding. In order to enhance the effect of the chemotherapy, we partnered it with 5-Fluorouracil (5-FU), and loaded it into an acidic pH soluble poly (dopamine-co-acrylamide)-compl-poly (boronic acid-co-acrylamide) hydrogel. The hydrogel is injected into the nitinol tube, and can deliver its drug payload under low pH. This method can provide a dual drug delivery platform in adjunct to hyperthermia therapy, using stimuli responsive macromolecules and bulk ferrogmagnetic material.

Entrapment Efficiency (%) =

amount of drug entrapped × 100 amount of drug used

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2.4. Synthesis of poly (dopamine-co-acrylamide)-compl-poly(boronic acidco-acrylamide) hydrogel loaded with 5-fluorouracil The polymerization followed a simple radical process. First, 30 ml DMSO was purged with N2 gas for 30 min, and 2.2 Mol at 99.5:0.5 ratio of acrylamide and 4-vinylphenylboronic acid was prepared, and subsequently added to the solvent under constant stirring and N2 gas blanket. The temperature was raised to 65 °C, and maintained for 24 h. The resulting turbid white solution was then washed using diethyl ether to isolate the solid copolymer. The solid copolymer was next dissolved in pure ethanol, and further washed using diethyl ether for a second time, to removed unreacted monomers and residual solvent. The same process was followed for the polymerization of poly(dopamine-co-acrylamide) copolymer, but with DMF as a solvent, and the molar ratio was changed to 2.3 Mol at 99.5:0.5 ratio. The resulting copolymers were then dried for 24 h using a vacuum pump. Once the copolymers were synthesized, they were dissolved in ultrapure distilled water at 5 wt/vol%. The dissolved copolymers were then added at 1:1 ratio in a vial, and 10 wt% of 5-FU in respect to the copolymer weight was added to the final solution. The pH of the solution was raised to 9 by adding 10 µl of 1.0 M NaOH solution. After the solution was stirred, the gelation process followed. The viscous gel was then loaded into 12 ml syringe with a 27 gauge needle, and the hydrogel was injected into the CTS.

2. Experimental 2.1. Materials Nitinol tubes of 0.7 mm OD and 0.58 ID were purchased from Alfa Aesar (South Korea); bortezomib from LC Laboratories (USA); Tris, NaCl, and KCl were all obtained from Samchun Chemicals (South, Korea); and dopamine hydrochloride, 5-Fluorouracil, 4-vinylphenylboronic acid, dimethyl formamide, and dimethyl sulfoxide were all purchased from Sigma-Aldrich. All aqueous solutions were prepared with ultrapure water purified with a Milli-Q UV-Plus water purification system (Millipore, Bedford, MA). The water had a resistivity of > 1018 MΩ cm−1.

2.5. Characterization of the chemothermal seed The morphology of the coating and hydrogel were observed using Field Emission Scanning Electron Microscopy (Carl Zeiss Supra 40VP). The samples were sputter coated under argon, in order to make them electrically conductive. The excitation voltage used to capture the images was set at 2 kV. Fourier transform Infrared spectroscopy data was obtained using the Spectrum-GX FTIR spectrometer (PerkinElmer Co., USA). The scanning range was set at (500–4000) cm−1 with a resolution of 1 cm−1. The elemental composition and surface state of the samples were checked using X-ray photoelectron spectroscopy (XPS, AXIS-NOVA, Kratos, Inc.) with an Al Kα irradiation source. Thermogravimetric analysis was obtained using TMK 0017 universal analysis (TA Instruments Co., USA) and (Q50, TA Instruments), respectively. The heating rate was set at 10 °C/min, and ranged from (10 to 400) °C. The gas flow rate of nitrogen was set at 40 ml/min for both analyses, which were to determine the thermal stability of the polymers and drugs used in this study. Rheological studies of the hydrogel were conducted using HR-1 Discovery Rheometer (TA Instruments, USA) to determine the injectability of the hydrogel into the narrow nitinol tubes, as seen in Fig. S2 of the SI. Thermal analysis of the synthesized copolymers was performed using Thermal Analyzer (TAQ20, TA Instruments, USA) (Fig. S3 of the SI). Gel permission chromatography multi-angle light scattering (MALS) was conducted to evaluate the degree of polymerization of the synthesized copolymers; the solid copolymers were dissolved in ultra pure water, and analyzed (Fig. S4 and Table S1 of the SI). H1 NMR spectroscopy (400 MHz/FT NMR spectrometry, JNM-AL400, JEOL,

2.2. Electropolymerization process Multiple scanning cyclic voltammetry was used for the electrochemical polymerization of dopamine. Fig. S1 of the Supplementary Information (SI) shows the typical CV of dopamine polymerized at a concentration of 1 mg/mL Tris buffer saline solution (TBS, pH 7.4) on a nitinol tube at (1.5 to −1.5) A, with the scanning rate set at 50 mV/s. After each cycle, the area under the curve in the voltammogram decreases as a result of the deposition of polydopamine onto the working electrode. After 10 cycles (20 segments), the amount of polymer that had been deposited led to almost complete insulation of the working electrode. 2.3. Loading of bortezomib to the electropolymerized dopamine coated nitinol tubes First, the drug was weighed at 3.0 mg, and dissolved in a 10 ml DW/ DMSO mixture (V/V = 10:1), with a resulting drug concentration of 300 µg/ml. The pH of the drug solution was then adjusted to 9.0 with drops of 1.0 M NaOH. The coated tubes were then fully submerged in the BTZ solution for 24 h at room temperature. The drug loading 192


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in using squamous cell carcinoma, mouse-derived (SCC7) cancer cell line (Korean Cell Line Bank, KCLB, Korea). CCK8 assay, live/dead staining using resazurin-red and sytoxgreen dyes, and morphological studies using DAPI and Rhodamine dyes were obtained, to demonstrate the anti-cancer properties of the CTS. The hyperthermia conditions were set at 43 °C. The alternating magnetic field used was set at 270 kHz, with 3 s exposure time and the temperature therapeutic range was maintained for 2 min. The cell proliferation index was calculated using the following equation:

USA) was conducted to determine the chemical structure of the yielded copolymers, using DMSO‑d6 and D2O as solvents for poly(4-vinylphenilboronic acid-co-acrylamide) and poly(dopamine-co-acrylamide) copolymers, respectively. 2.6. In vitro analysis drug release study The in vitro drug release test was carried out to test the pH-dependent release of bortezomib from the electropolymerized dopamine and the solubility of the poly (DOPAMINE-co-ACRYLAMIDE)-compl-poly (BORONIC ACID-co-ACRYLAMIDE) hydrogel. Two PBS solutions were used with different pH levels that mimic the environmental pH of a necrotic tumor. The pH of the PBS was adjusted by directly tittering hydrochloric acid to the PBS solutions. PBS with pH of (4 and 6) was maintained throughout the drug release test, to avoid result fluctuations. Three pieces of 5 mm CTS were then submerged in the PBS solutions, and 1 ml aliquots were taken at specific time intervals. The PBS was then replaced with pre-warmed fresh PBS solutions to maintain the media concentration. The aliquots were analyzed using HighPerformance Liquid Chromatography (JASCO, USA). The experiment was done in triplicates, and the reported values were the averaged values obtained.

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Cell Proliferation Index = ND/ND1 = 1

where ND = Cell number on day D, and ND1=1 = Cell number on day 1. Apoptosis detection studies were also conducted using in vitro Fluorescein staining; the cells were harvested, and stained using In Situ Cell Death Detection Kit, Fluorescein (Roche, Germany), according to the manufacturer’s instructions. The green fluorescence was viewed under a ZOEY™ fluorescent cell imager (Bio-Rad, USA). CASPASE3 activity was measured using ApoTarget® Caspase-3 colorimetric protease assay (Invitrogen, USA). The cells were harvested, and lysed after treatment, to detect the caspase 3 protein. The samples were then read using a Spark® microplate reader (TECAN, Switzerland) at 400 nm wavelength.

2.7. In vitro biocompatibility study 2.10. In vivo pilot study to determine optimal parameters for implantation and alternating magnetic field (AMF)/hyperthermia duration

For the biocompatibility test, the material used was e-pdopa-nitinol foil to effectively seed the utilized cells. The fibroblast (NIH-3T3) cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin-streptomycin. The cells were grown on the foils for (1, 3, and 5) days, and the proliferation was investigated using Dojindo’s cell counting kit-8, according to the manufacturer’s instructions. The e-pdopa-nitinol foil was sterilized using Ultraviolet irradiation overnight, and placed on 48-well plates. Then, 100 µl of NIH-3T3 (fibroblast) cell suspension (5000 cells/well for samples without drug content and 5000 cells/well for samples with drug content, DMEM/high glucose (supplemented with 10% FBS and 1% penicillin-streptomycin) was dispensed in a pre-incubated 96-well plate containing mats, and allowed to incubate in a humidified atmosphere of 5% CO2 at 37 °C for the designated time. The morphological study of the cells was observed using DAPI and actin-green staining, and was subsequently viewed under confocal microscopy LSM-510 (Carl-Zeiss, Germany). Also, for the biocompatibility testing of the hydrogel, solubilized form of the gel was used to evaluate the effects of the polymeric component on normal cells. We co-cultured the cells with the extracts of the hydrogel following the same protocol as described above.

An initial study was conducted to determine the optimal amount of implants needed, and the length of exposure to hyperthermia treatment. The implants were made up of bare nitinol tubes without any modification. This was to determine the effect of hyperthermia on the tumor growth. The CTS implantation protocol was carried out aseptically by creating pilot holes in the tumor at approximately 5 mm distance using a 27 gauge needle, Once the holes were made, the CTS were manually inserted by hand using a McIndoe forcep. The implantation was done under full mice sedation, using a steady flow rate of isoflurane mixed with oxygen gas. The experiments were performed in accordance with the guidelines of the Ethics Committee of the Chonnam National University Medical School (CNUIACUC-H-2011-5). Athymic mice (nu/ nu-ncr, Balb/c mice, (5–6) weeks old, (20–25) g) were obtained from Jungang Lab Animal, Inc., Korea. In a typical procedure, the mice were subcutaneously injected with SCC7 cancer cells (1 × 106 cells), and we waited until the tumor size grew to (200 ± 15) mm3. The following treatment plans were studied as described in Table 1: 2.11. In vivo evaluation of the chemothermal seeds After determining the optimal treatment parameter, we proceeded to conduct the main in vivo experiments. Athymic mice model with induced SCC7 cancer cell line tumor at their hind right leg region was made to evaluate the tumoricidal effects of the CTS. The tumor was induced first via culturing of (SCC7) in vitro. The mice were randomly allocated into four experimental groups of 4 mice each (n = 4). The first group was used as the control, i.e., without any treatment. The second, third and fourth groups were implanted with CTS and unmodified nitinol tube, and they were divided into treated (AMF-on) and (AMF-off) groups. The groups were named accordingly as CTS (no

2.8. Combination index study of bortezomib and 5-fluorouracil on SCC7 cancer cell line SCC7 cancer cells were cultured using RPMI culture media supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillinstreptomycin. The cells were seeded using a 48-well polystyrene plate with a cell density of 5000 cells per well. The doses used for the combination index studies are fully described in the supplementary material. After adding the set doses, we measured the number of remaining living cells using CCK8 assay, and the results were evaluated using Compusyn® software to determine the combination index score of the drugs used in the study (Figs. S6 and S7 and Table S2 of the supplementary material)

Table 1 In vivo Pilot Study Treatment Plans.

2.9. In vitro study of the synergism of hyperthermia brachytherapy and chemotherapy for cancer treatment A cytotoxicity test was done to evaluate the effectiveness of the CTS 193

Group (n = 4)

No. of Implants

Length of hyperthermia (s)

1 2 3 4

6 × 2.5 mm 6 × 2.5 mm 3 × 5.0 mm 3 × 5.0 mm

800 400 800 400

implants implants implants implants


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making it analogous to the traditional brachytherapy treatment. However, this is the only similar aspect that our therapy shares with radio brachytherapy, as we have designed a system that releases two types of chemotherapeutics, while being able to induce hyperthermia at the same time. Fig. 1C explains how each chemothermal can release two types of chemotherapeutics by virtue of pH-dependent release, and deliver thermal dosage via eddy current heating, when exposed to an external alternating magnetic field source. Each seed is coated with an electropolymerized polydopamine, which coating is capable of bonding to a boronic acid containing drug, such as BTZ. The link is pH cleavable under acidic pH of lower than 5.0, while on the other hand, the hydrogel component contains 5-FU, and the release mechanism is slightly different from the coating, because the drug is not directly conjugated to the polymer, but is contained inside the hydrogel network, Once the hydrogel is solubilized under low pH, 5-FU can also be released into the tumor environment. The dual drug therapy is just one component of the chemothermal seed, because the seed is also made up of nitinol alloy with 49% nickel that is ferromagnetic, so is also capable of inducing hyperthermia inside the tumor environment. This creates a synergistic effect that makes the tumor sensitive to chemotherapy drugs.

Table 2 Main In vivo Experiment Treatment Plans. Group (n = 4)

No. of Implants

Length of hyperthermia (s)

Control (no treatment) CTS (Chemotherapy only)

0 3 × 5.0 mm implants 3 × 5.0 mm implants 3 × 5.0 mm implants

0 400

Control/AMF (Hyperthermia only) CTS/AMF (Combination)

400 400

hyperthermia/with chemotherapy), CTS/AMF (hyperthermia/chemotherapy), Control/AMF (with hyperthermia/no chemotherapy), and Control (no treatment). Table 2 describes the details of the main in vivo experiment. The Implantation protocol was designed to implant 3 pieces of 5 mm CTS in parallel at approximately 5 mm distance from one other. The placement of the CTS was verified using micro CT-scan. After implantation, the mice were allowed to rest for 24 h, before the first hyperthermia treatment. The hyperthermia treatment was carried out using an alternating magnetic field generator (OSH-120-B, OSUNG HITECH, Republic of Korea) with a power output of 12.5 kA m−1 and frequency of 270 kHz; the system has a copper solenoid coil with a diameter of 45 mm and 3 loops, and is cooled using a water circulator. The hyperthermia treatment was carried out for 400 s, and the superficial temperature was monitored real time using NEC thermal camera TS9230. The temperature points were plotted accordingly. The hyperthermia treatment was repeated for the next 4 days, and the tumor size of the mice was recorded using a digital Vernier caliper, and was calculated using the following equation:

Volume = length × width2/2

3.2. Characterization of the fabricated chemothermal seeds The components of the CTS have been characterized. First, we want to probe the surface morphology of both bare and coated nitinol tubes and the hydrogel. Fig. 2A–F show the FESEM photographs that demonstrate the electropolymerized surface of the nitinol tube, and the porosity of the hydrogel. Electropolymerization of electrically conductive mononer dopamine has been carried out using cyclic voltammetry [37–39]. The bare nitinol shown changes in surface morphology after the deposition of the polydopamine thin film. It has been established that polymerization of natural phenolics, such as dopamine, can create a thin film that ranges from nano to micro in thickness, depending on the number of cycles that was run during the electropolymerization process. A subtle change to the surface morphology of the nitinol tube can be seen, as the micro pits and crevices were smoothed out by the polymer coating; however, on zooming out, it is apparent that that the coating is not as uniform as expected, due to variation of the dopamine nucleation sites. The nitinol tube is utilized as the working electrode in the electrochemical cell to deposit dopamine in the solution. Fig. S1A of the SI shows the cyclic voltammogram of the process. The observed voltammetric responses could be attributed to the redox couples of dopamine/dopaminequinone and leucodopaminechrome/dopaminechrome, and eventual formation of the polydopamine thin film structure. In consequence, the conductivity of the working electrode decreases as the cycle progresses, further indicating the deposition of polydopamine. In order to validate the coating, we further conducted ATR-FT-IR spectroscopy and XPS, as shown in Fig. 2G–H. The characteristic peaks of polydopamine as shown by the (1610 and 1292) cm−1 correspond to the stretching vibration of the C]C and the phenolic CeOH of polydopamine, while the 820 cm−1 peak can be attributed to the amine group of polydopamine, and bortezomib’s CeN and OeH in the (1080 and 3351) cm−1 peaks, respectively, were also observed. The XPS spectra also indicate the presence of BTZ in the coating, as evidenced by the B1s peak with an atomic weight of 1%. The tall C1s and N1s peaks on the coated samples indicate the phenolic carbon and amine groups, which corroborate the ATR-FT-IR results, as those peaks are not seen in the pure nitinol sample. The hydrogel component of the CTS was made up of poly(dopamine-co-acrylamide) and poly(4-vinylphenylboronic acid-co-acrylamide) copolymers. The synthesized copolymers had undergone complexation to form a hydrogel that was designed to be soluble at acidic pH, which constitutes the environmental pH of solid necrotic tumors, which was capable of being injected into the CTS’s narrow diameter. Fig. 2I and J show the H1 NMR spectra, suggesting the structure of the

(3)

The tumor was then excised and weighed (Kent Scientific, Connecticut USA), and the weight in grams was plotted against the control. It was then further evaluated using Haemotoxylin and Eosin (H &E) staining for necrosis evaluation, and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for apoptosis determination. 2.12. Statistical analysis All relevant data were compared using one-way ANOVA, (OriginLab® software). Data are expressed as mean ± SD of measurements (*p < 0.05). 3. Results 3.1. Fabrication and general idea of treatment using chemothermal seeds Brachytherapy as a modality for cancer treatment has been around for decades, and the implantation of radioactive seeds has been deemed less invasive, compared to other implantation protocol, and can make a patient fully functional immediately after the procedure [7,32–36]. In our study, the idea of implanting metallic seeds that have the same size and diameter as the conventional brachytherapy seeds has been adapted and modified to release dual chemotherapeutic drugs using pHsensitive polymeric carriers (hydrogel and coating), and partnering it with induced hyperthermia using an external alternating magnetic field. As seen in Fig. 1A and B, the design of the chemothermal seeds (CTS) has been specified and defined. The drug containing hydrogel and coating serves as the chemotherapy part of the system, while the inherent ferromagnetic property of Nitinol was used as the heat source for hyperthermia therapy. The dimensions of the chemothermal seeds are similar to commercially available brachytherapy seeds in the market, 194


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Fig. 1. Photograph of (A) Chemothermal Seed, and (B) Specifications of the dimensions and materials used for chemothermal seed fabrication. (C) Schematic of chemothermal seed capability to release dual chemotherapeutics under acidic pH conditions via the electropolymerized polydopamine coating that contains bortezomib and the dissolution of the poly(dopamine-co-acrylamide)-compl-poly(4-vinylphenilboronic acid-co-acrylamide) hydrogel that contains 5-Fluorouracil. The nitinol alloy casing of the chemothermal seed can create hyperthermia conditions when exposed to an external alternating magnetic field, creating a synergistic dual modal brachytherapy.

3.3. In vitro pH responsive drug release study

yielded copolymer, having the acrylamide as the main chain, and dopamine and 4-vinylphenylboronic acid showing on the side chains. All representative peaks were identified for the macromolecules that were yielded. The proposed gelation/complexation process can be seen in Fig. S1C of the SI, where the mechanisms for crosslinking and solubility under different pH conditions are demonstrated. The side chain cathecol and boronic acid moeities of the poly(dopamine-co-acrylamide) and the poly(4-vinylphenylboronic acid-co-acrylamide) can be crosslinked together when the pH of the system is increased to a weak alkali condition (pH (8–9)), and can be dissociated when the pH of the system becomes acidic (pH < 5) [40,41]. The formed hydrogel has an injection window of 20 min, as shown in Fig. S2 of the SI. The hydrogel’s G′ modulus increases from (180 to 720) Pa over time, possibly due to the increasing degree of crosslinking of the poly(dopamine-co-acrylamide) and poly(4-vinylphenylboronic acid-co-acrylamide) copolymers in the hydrogel network. After 20 min, the G′ of the hydrogel stabilizes at 900 Pa, making it capable of remaining inside the nitinol tube cavity after the injection process. This ensures that the hydrogel will not leak out before the implantation, and will remain intact inside the chemothermal seeds.

The drug release of the CTS has been demonstrated to be pH responsive, with both drugs having higher release profile at acidic pH level of 4, compared to pH 6. The CTS drug loading for both BTZ and 5FU were determined using high performance liquid chromatography and each CTS can load up to 40% of BTZ or 10 µg BTZ/CTS, and 10 wt/ vol% of 5-FU or 20 µg 5-FU/CTS. The main drug payload 5-FU shows higher drug release profile at both pH conditions compared to the BTZ, as seen in Fig. 3. The complexation links between the boronic-catechol moieties of the copolymers in the hydrogel were weakened by the acidic pH, making it soluble in the media, consequently releasing its drug payload. Also, the mechanism of drug release from the coating follows the same principle, where the boronic-catechol moiety complexation dissociates, and releases the BTZ payload. It is important to note the pH responsiveness of the drug release, since the microenvironment of the tumor has been known to be acidic, compared to normal physiologic tissue environment. This is due to less oxygenation (hypoxia) that causes lactic acid formation from anaerobic glycolysis, and faulty and leaky vasculature formation of malignant tumors [42]. We can exploit this inherent property of cancer as a drug release 195


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Fig. 2. Complete characterization of the chemothermal seed components; FESEM images of nitinol tube surface: (A) and (D) uncoated nitinol, (B) and (E) electropolymerized dopamine coating, (C) and (F) poly(dopamine-co-acrylamide)-compl-poly(4-vinylphenilboronic acid-co-acrylamide) Hydrogel Network, (G) ATR-FTIR spectra of electropolymerized dopamine coating, (H) X-ray photon spectroscopy of the polydopamine coating, drug loaded coating, and pure nitinol. (I) and (J) H1 NMR spectra of poly(dopamine-co-acrylamide) and poly(4-Vinylphenilboronic acid-co-acrylamide), respectively.

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(7.35); this would mean that it could spare normal cells, and would limit its toxicity. 3.5. In vitro study of the synergism of hyperthermia brachytherapy and chemotherapy for cancer treatment To evaluate the effects of hyperthermia and chemotherapy on SCC7 cancer cell line, in vitro studies were conducted to demonstrate that the components of the CTS can indeed cause synergistic anti-cancer effects. As demonstrated by the morphological studies using DAPI and Rhodamine staining in Fig. 5A, we observed cells that are undergoing possible apoptotic stages, as evidenced by the stunted cytoskeleton of the cells, and blebbing of the nucleus. The chemotherapy group shows the same morphological effect as the hyperthermia group. The highest effect was observed in the combination treatment, where almost all of the cells can be found in a damaged state. We also conducted a live and dead staining, where we harvested the cells after treatment, and Fig. 5B shows that the highest ratio of dead/injured cells is obtained in combination treatment, as evidenced by the dominant sytox green fluorescence. The CPI scores were also obtained using CCK8 assay to monitor the day-by-day reduction for all treatment groups. The CPI scores after 3 days of incubation were (0.75, 0.5, and 0.05) for Chemotherapy, Hyperthermia, and Combination groups, respectively, indicating that the combination of hyperthermia and dual drug chemotherapy has a synergistic effect on SCC7 cancer cell line inhibition. Apoptotic response of the cells in the treatment groups was then investigated, and is shown in Fig. 5D. Fluorescein stain imaging showed a higher amount of cells that underwent apoptosis in the combination therapy, compared to in CT or HT alone. This was then corroborated by the CASPASE3 assay, which shows that the combination therapy has higher caspase3 activity with an OD score of 0.16, with Chemotherapy and Hyperthermia therapy both having 0.12 OD scores. Caspase 3 is an indicator protein that leads to the caspase-activated apoptotic pathway through interaction with Caspase 8 and Caspase 9 protein, also known as the caspase cascade. The in vitro study shows that cancer inhibition treatment is more effective when partnered with another modality. This is apparent in this study, where the anti-cancer effects are increased by combining chemotherapy and hyperthermia therapy. The cancer cells become more sensitive to chemotherapeutics when exposed to elevated temperatures, due to the denaturing effects of heat on repair enzymes, and impairing DNA and RNA synthesis inside the cell body. Cancer cells tend to create a salvaging effort in their damaged DNA, making chemotherapeutics less effective in inducing apoptosis. But when the CA cells are exposed to hypethermia temperatures after being exposed to chemotherapeutics, the salvaging mechanism can be halted by the unfolding of protiens and enzymes responsible for such efforts.

Fig. 3. In vitro Drug Release Study of the chemothermal seeds under two pH conditions, where it is demonstrated that BTZ and 5-FU can be released in bulk under pH 4.0 conditions, and has a lesser release profile under pH 6.0 (n = 3, mean +/− SD).

triggering mechanism to be selective only in a malignant environment. We also conducted a combination index study for the drug of choice, to determine whether they have a synergistic, additive, or antagonistic effect. The combination index study shows that at all combination doses used, the technique can lead to a synergistic effect in killing SCC7 cancer cells. Also, BTZ and 5-FU are known to be used clinically in solid tumor chemotherapy, so it is only logical to use both of them in this study [16,43–45]. The use of dual drug therapy shows increased anticancer property compared to single drug therapy alone; also, in the clinical setting, multiple chemotherapy drugs are being used to enhance and improve the prognosis of patients [12,46,47].

3.4. In vitro biocompatibility study of the components used in fabricating the chemothermal seeds We needed to evaluate normal cellular response in the presence of the CTS components, since we are dealing with metallic implants that could potentially stay inside the body of the patient for a long duration of time [48–51]. Fig. 4A and B show that the NIH-3T3 fibroblast cells can proliferate in the presence of the hydrogel, the coating, and the pure nitinol substrate, as evidenced by the increasing CPI score after day 5 of incubation on all samples. Interestingly, there is no significant difference in the CPI of all nitinol substrates, even with the drug-loaded coating; DAPI and actin-green staining indicate the cellular morphology of the incubated cells showing normal actin growth in all samples (Fig. 4C–G). The BTZ payload that is conjugated in the polydopamine coating cannot cause any severe cytotoxic effect on the NIH-3T3 cells due to the normal pH of the media, therefore maintaining the catecholboronic link between the drug and polydopamine. Only during the presence of acidic environment can the link be dissociated, and lead to cytotoxic effects [52,53]. The hydrogel component itself doesn’t cause any cytotoxic effects on fibroblast cells, as evidenced by the elongated actin growth and normal nucleus size. The hydrogel was made with ultra pure water, and no harmful solvents were used during complexation. Also, we make sure that there are no residual solvents left after the radical polymerization process, by washing the solid copolymers twice in Diethyl ether, and vacuum drying them for 24 h. The stability of the complexation in the hydrogel network also depends on the pH of the system, and only during the presence of acidic conditions (pH < 5.0) can the drug payload be delivered. The hydrogel will maintain its water content when exposed to normal physiological pH

3.6. In vivo pilot study to determine optimal parameters for implantation and AMF/Hyperthermia duration In order to determine the optimal amount and final specifications of CTS needed for the in vivo experiment, a pilot in vivo study was conducted. Table 1 describes the different treatment plans. After evaluating the results, the treatment plan for group 4 shows the most favorable outcome, and therefore we inferred that three 5 mm CTS and hyperthermia exposure time of 400 s were the optimal parameters for the main in vivo experiment. The effect of in vivo hyperthermia can be seen in Fig. 6. The results show that the number of implants and exposure time to hyperthermia can affect the therapeutic outcome of the treatment. The optimal parameter for hyperthermia exposure using the CTS implant is at 400 s, indicating that prolonged exposure doesn’t translate to higher therapeutic effect. The size of the chemothermal seed also plays an important role, as 5 mm seeds can give out a more uniform isotherm, compared to CTS with 2.5 mm length. Due to the crude implantation protocol, we could not ensure the correct alignment of the 197


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Fig. 4. Biocompatibility Studies of the chemothermal seed components (A) and (B) CPI index of NIH-3T3 fibroblast cells exposed in the Hydrogel and polydopamine coating with and without BTZ and pure nitinol. Morphological Studies using DAPI and Actin Green Staining (C) control, (D) Hydrogel, (E) pure nitinol, (F) E-pdopa coated nitinol, and (G) E-pdopa-BTZ nitinol (n = 3, mean +/− SD).

resilient and harder to inhibit, even with succeeding treatments. Thermotolerance is due to the production of heat shock proteins that can help in inhibiting apoptotic factors in the cell [2,15,54]. Another important aspect of hyperthermia in the CTS system is that it can lead to higher intratumoral acidosis than is required as the triggering mechanism for the chemotherapy component of the CTS [55,56]. The tumor was based on SCC7 cell line that is known to be highly aggressive [57]. It was then let to grow to a significant volume before starting treatment, making it more challenging to reduce in size; interestingly, the CTS/AMF group shows a reduction in tumor growth after 4 treatments of hyperthermia (Fig. 8A). The control group without any treatment shows a fast growth rate that significantly outpaces the other treatment groups (Fig. 8B). Using separate treatment only leads to retardation of growth for the tumor, while the combination of dual drug chemotherapy and hyperthermia therapy could cause the tumor to reduce in size by up to 70%. This is in agreement with the in vitro results of this work, and proves that the concept of using dual chemotherapeutic drugs and hyperthermia can lead to a higher expression in malignant tumor ablation. Fig. 8C shows that the histological assessment of the tumor reveals the malignant tissue underwent ablation for both groups that were exposed to hyperthermia treatment, and the TUNEL staining exhibited positive fluorescence for apoptosis on all treatment groups, except for the control. CTS group (chemotherapy only) shows apoptotic cells due to the release of chemotherapeutic drugs, and Control/AMF group (hyperthermia only) also exhibited the same results, but the CTS/AMF group shows that the combined effects of HT and CT have higher amount of cells that underwent apoptosis, proving that hyperthermia can indeed increase the sensitivity of cancer cells to chemotherapeutics, and vice versa. Hyperthermia can create synergistic effects to chemotherapy, due to the denaturing property of heat to proteins involved in fixing damaged DNA [15,16,58,59]. This is the first time in the literature where a chemothermal brachytherapy treatment has been evaluated for tumor inhibiting property in vivo. Although there have

implants inside the tumor. With lesser but longer implants, we could achieve uniform tumor heating and ablation, regardless of the alignment of the seeds. In future studies, we would like to design a sophisticated implantation device, to ensure that proper alignment and spacing of the CTS is performed. 3.7. Chemothermal seed capability assessment on destroying solid tumor malignancy Following the pilot study, implantation of the CTS was followed and planned accordingly as seen in Fig. 7A. First, pilot holes were made using a solid needle to mimic the actual implantation of brachytherapy seeds. Once the holes were made, the CTS were inserted into the pilot holes, and the implants were confirmed by micro-CT scan. The radioopacity of the CTS was verified, as seen in Fig. 7B. It can be noted that nitinol alloys have dense metallic structure; therefore, X-rays can be absorbed readily, and can be easily seen using any imaging technique, such as that we used in this study. CTS placement is crucial, as the isotherms are important to achieve uniform heating on the whole tumor structure. After the implantation procedure, the mice were then rested for 24 h before the first hyperthermia treatment, which is to start the chemotherapy phase of the treatment, and make the tumor respond better to hyperthermia. As seen in the thermal photographs and graph in Fig. 7C and D, the CTS hyperthermia induction of the tumor was efficient enough to achieve therapeutic temperature, and the bioheat transfer was observed to be uniform all throughout the tumor. The hyperthermia temperature of 43 °C was reached on both AMF groups. To demonstrate also that the AMF cannot induce eddy current heating in the tissues of the mice, a control group was checked, and demonstrated that there was no increase of temperature on the body or tumor of the mice in the presence of alternating magnetic field. Reaching the therapeutic temperature is indeed crucial in making in vivo hyperthermia feasible to treat cancer tumors. If the temperature is lower than 43 °C, it can only lead to thermotolerance, making the tumor more 198


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Fig. 5. In vitro cancer cell inhibition properties of the chemothermal seeds and study of individual therapies and combined therapy used in this study. (A) Morphological study of SCC7 cancer cell line using DAPI and Rhodamine stains, (B) Live and Dead staining using Resazurin Red (live) and Sytox green (Dead) stains, (C) Cell proliferation index of SCC7 cancer cell line under different treatment groups. (Broken red line is when the AMF is turned on). Data are expressed as mean ± SD, n = 3. *p < 0.05) (one way ANOVA post hoc Tukey test). Apoptosis detection using (D) CASPASE 3 activity assay. inset; in situ TUNEL (fluorescein staining) (n = 3, mean +/− SD). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

may be quite difficult. One factor that motivated us to perfom another method of hyperthermia therapy is the noted hepatic and renal toxicities created by magnetic nanoparticles. In order to increase the thermal capacity of these materials, high doses of magnetic nanoparticles are usually needed, and that comes with increasing the risks for the said toxicities. The idea of bypassing the hepatic and renal system comes to mind by directly implanting the chemothermal seeds into the tumor site [62]. This reduces the need for the introduction of magnetic nanoparticles to the vasculature, and therefore could create a non-toxic treatment. The heating capability of the the chemothermal seed is also seen as sufficient to create a uniform and correct thermal isotherm and dose. Also, nitinol as a casing is beneficial, due to the known biocompatibility and mechanical stability of the alloy. It has been used in multiple biomedical applications, such as in vascular and non-vascular stents

been computer simulation studies and in vitro studies with thermobrachytherapy partnered with radiation, they lack further evaluation on actual living systems [5,60,61]. With this new concept of chemothermo brachytherapy, we can now definitively prove the effectiveness of the technology even further. 4. Discussion The idea of using heat as an adjuvant therapy for chemotherapy and radiotherapy has already been established by contemporary works. However, there are still shortcomings that need to be addressed, such as the correct thermal dose, and the uniform isotherm that must be created in the tumor environment in order for the treatment to be successful. Thermotolerance is still one of the limiting factors for hyperthermia therapy, as reaching the needed thermal range using other materials 199


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Fig. 6. (A) Tumor volume graph. (see Table 1 for legends) (B) Ablated tumor after day 4. (C) Alternating magnetic field induced hyperthermia thermal image.

Fig. 7. (A) Illustration of the implantation protocol of the chemothermal seeds, 200


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Fig. 8. In vivo evaluation of the chemothermal seeds (A) photographs of day-by-day tumor reduction of all treatment groups, showing the visible ablation of the tumor on the groups exposed to alternating magnetic field hyperthermia, (B) Tumor volume over time graphical representation (n = 4, mean +/− SD), (C) H&E and TUNEL Staining of the excised tumor after final treatment of hyperthermia. Visible ablation is evidenced by the necrotic tissue of the groups exposed to alternating magnetic field hyperthermia, Apoptotic cells are also visible in groups that received treatment with higher incidence in the CTS/AMF group.

compl-poly(BORONIC ACID-co-ACRYLAMIDE) hydrogel used in this study, we have exploited the pH responsive complexation of boronic acid and catechol functionality of the said polymers to be used as a smart drug delivery platform. In this case, electropolymerization was employed to create a thin film on the surface of the chemothermal seed. The polydopamine thin film serves a dual purpose for this design. (1) It serves as a drug carrier, due to the catechol moiety of polydopamine, and (2) it also serves as another passivation layer, adding to the natural oxide layer of the nitinol casing, ensuring biocompatibility. Also, the synthesized hydrogel itself doesn’t cause any toxicity to normal fibroblast cells, as demonstrated in the biocompatibility experiment. The cells can grow and thrive in the presence of the hydrogel, and don’t show any abnormalities in growth and proliferation. Another important thing to consider is the selectivity of the material to deliver the drug payload only in the conditions where it is needed. In an environment such as in a cancer tumor, the pH of the microenvironment is acidic, compared to the neutral pH environment of normal tissue. The design of the drug release can only happen in the presence of acidic pH; therefore, this could spare the normal tissue and cells in the body. In clinical setting, the seed is not only implanted in the tumor site, but also in the surrounding normal tissue. The chemothemal seeds exhibit no toxicity when exposed to normal physiological pH, except when heat is

and orthopedic implants. However, it should be noted that when using ferromagnetic materials, artifact formation during magnetic resonance imaging (MRI) can form and obscure the imaging signal. Usually, nitinol is used for its shape memory effect, but in this case it is exploited for its inherent ferromagnetic property, due to its equiatomic intermetallic composition ((49–60)% Nickel, the rest Titanium). Compared to magnetic nanoparticles, the bulk nitinol alloy casing can heat up faster and longer in the presence of alternating magnetic field, due the capability of bulk materials to maintain heat. Energy conversion and bioheat transfer is much more efficient when materials are used in bulk [63]. This can be translated to anticancer activity, as shown in the in vivo experiment. The tumor growth is retarded using separate treatment, but when with an adjuvant therapy, the hyperthermia treatment has better outcome when partnered with dual drug chemotherapy. The design of the chemothermal seed must also be optimized to create a favorable treatment outcome. The use of polymers as drug delivery platform has been studied and proven on numerous accounts [64]. Polymers have the capability of being stable, biocompatible, and functional when it comes to delivering their drug payload [65,66]. In particular with the use of stimuli responsive polymers, such as electropolymerized dopamine and the poly (DOPAMINE-co-ACRYLAMIDE)201


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introduced during hyperthermia treatment. Thermal brachytherapy has been a technology that is still currently in evaluation for the potential application to treating solid malignancies. However, it lacks in vivo evaluation to know whether the technique can translate to clinical applications. The current study still has a long way to go; with better implantation protocol using a better implantion device, we could create a much more uniform isotherm, and could therefore enhance the effect of hyperthermia. Nevertheless, we have proven that the use of chemotherapeutics and hyperthermia in a single treatment method can be achieved without using precarious nanomaterials, and that the chemothermal brachytherapy seeds can be used in animal models, and has the potential to be translated to clinical applications. This study is a proof of concept that the technology of using implantable seeds can be translated to cancer therapy on a level that surpasses current evaluations.

[8] P. de la Puente, A.K. Azab, Delivery systems for brachytherapy, J. Control. Release 192 (2014) 19–28. [9] C.F. Njeh, M.W. Saunders, C.M. Langton, Accelerated partial breast irradiation (APBI): a review of available techniques, Radiat. Oncol. (Lond., Engl.) 5 (2010) p. 90–90. [10] J. Skowronek, M. Wawrzyniak-Hojczyk, K. Ambrochowicz, Brachytherapy in accelerated partial breast irradiation (APBI) – review of treatment methods, J. Contemp. Brachyther. 4 (3) (2012) 152–164. [11] A. Challapalli, et al., High dose rate prostate brachytherapy: an overview of the rationale, experience and emerging applications in the treatment of prostate cancer, Brit. J. Radiol. 85 (Spec. Iss. 1) (2012) S18–S27. [12] L.E. Aguilar, et al., On-demand drug release and hyperthermia therapy applications of thermoresponsive poly-(NIPAAm-co-HMAAm)/polyurethane core-shell nanofiber mat on non-vascular nitinol stents, Nanomed. Nanotechnol. Biol. Med. 13 (2) (2017) 527–538. [13] Z. Teng, et al., A magnetic droplet vaporization approach using perfluorohexaneencapsulated magnetic mesoporous particles for ultrasound imaging and tumor ablation, Biomaterials 134 (2017) 43–50. [14] R. Das, et al., Boosted hyperthermia therapy by combined AC magnetic and photothermal exposures in Ag/Fe3O4 nanoflowers, ACS Appl. Mater. Interfaces 8 (38) (2016) 25162–25169. [15] B. Hildebrandt, et al., The cellular and molecular basis of hyperthermia, Crit. Rev. Oncol. Hematol. 43 (1) (2002) 33–56. [16] R.D. Issels, Hyperthermia adds to chemotherapy, Eur. J. Cancer 44 (17) (2008) 2546–2554. [17] S. Patra, et al., Dual-responsive polymer coated superparamagnetic nanoparticle for targeted drug delivery and hyperthermia treatment, ACS Appl. Mater. Interfaces 7 (17) (2015) 9235–9246. [18] L. Tan, et al., Biocompatible hollow polydopamine nanoparticles loaded ionic liquid enhanced tumor microwave thermal ablation in vivo, ACS Appl. Mater. Interfaces 8 (18) (2016) 11237–11245. [19] P. Wust, et al., Hyperthermia in combined treatment of cancer, Lancet Oncol. 3 (8) (2002) 487–497. [20] W.K. Park, et al., Evaluation of a novel thermal accelerant for augmentation of microwave energy during image-guided tumor ablation, Theranostics 7 (4) (2017) 1026–1035. [21] Y.J. Kim, M. Ebara, T. Aoyagi, A smart hyperthermia nanofiber with switchable drug release for inducing cancer apoptosis, Adv. Funct. Mater. 23 (46) (2013) 5753–5761. [22] D.H. Kim, et al., Temperature-sensitive magnetic drug carriers for concurrent gemcitabine chemohyperthermia, Adv. Healthcare Mater. 3 (5) (2014) 714–724. [23] H. Wang, et al., Chitosan-gated magnetic-responsive nanocarrier for dual-modal optical imaging, switchable drug release, and synergistic Therapy, Adv. Healthc. Mater. (2017) 6(6). [24] W. Gao, et al., A smart, phase transitional and injectable DOX/PLGA-Fe implant for magnetic-hyperthermia-induced synergistic tumor eradication, Acta Biomater. 29 (2016) 298–306. [25] A.R.K. Sasikala, et al., An implantable smart magnetic nanofiber device for endoscopic hyperthermia treatment and tumor-triggered controlled drug release, Acta Biomater. 31 (2016) 122–133. [26] K. Hayashi, et al., Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging and hyperthermia treatment, Theranostics 3 (6) (2013) 366–376. [27] C.S.S.R. Kumar, F. Mohammad, Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery, Adv. Drug Deliv. Rev. 63 (9) (2011) 789–808. [28] W.J. Lokerse, et al., Investigation of particle accumulation, chemosensitivity and thermosensitivity for effective solid tumor therapy using thermosensitive liposomes and hyperthermia, Theranostics 6 (10) (2016) 1717–1731. [29] J.M. Rojas, et al., Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion, Nanomed. Nanotechnol. Biol. Med. 12 (4) (2016) 1127–1138. [30] A. Sabareeswaran, et al., Effect of surface-modified superparamagnetic iron oxide nanoparticles (SPIONS) on mast cell infiltration: An acute in vivo study, Nanomed. Nanotechnol. Biol. Med. 12 (6) (2016) 1523–1533. [31] A.H. Silva, et al., Superparamagnetic iron-oxide nanoparticles mPEG350–and mPEG2000-coated: cell uptake and biocompatibility evaluation, Nanomed. Nanotechnol. Biol. Med. 12 (4) (2016) 909–919. [32] J. Nohra, et al., Sexuality after brachytherapy for localized prostate cancer: longterm results, J. Urol. 179 (4) (2008) 299–300. [33] B.R. Pieters, et al., Comparison of biologically equivalent dose-volume parameters for the treatment of prostate cancer with concomitant boost IMRT versus IMRT combined with brachytherapy, Radiother. Oncol. 88 (1) (2008) 46–52. [34] A.T. Porter, et al., Brachytherapy for prostate cancer, CA Cancer J. Clin. 45 (3) (1995) 165–178. [35] H. Ragde, et al., Modern prostate brachytherapy, CA Cancer J. Clin. 50 (6) (2000) 380–393. [36] G.S. Merrick, et al., Short-term sexual function after prostate brachytherapy, Int. J. Cancer 96 (5) (2001) 313–319. [37] J.L. Wang, et al., Electropolymerization of dopamine for surface modification of complex-shaped cardiovascular stents, Biomaterials 35 (27) (2014) 7679–7689. [38] J.G. Manjunatha, et al., Sensitive Voltammetric Determination of Dopamine at Salicylic Acid and TX-100, SDS, CTAB Modified Carbon Paste Electrode, Int. J. Electrochem. Sci. 5 (5) (2010) 682–695. [39] X.L. Luo, X.Y.T. Cui, Electrochemical deposition of conducting polymer coatings on magnesium surfaces in ionic liquid, Acta Biomater. 7 (1) (2011) 441–446. [40] S. Wu, et al., pH-responsive drug delivery by amphiphilic copolymer through

5. Conclusion This new way of performing dual modal therapy for cancer is a proof of concept that an implantable device can lead to a synergistic tumoricidal effect. The chemothermal seed treatment concept has shown biocompatibility, pH-responsive drug release, and efficient heating capability that is crucial in administering effective treatment, compared to single therapy alone. This work can be a basis for the future design of better chemothermal seeds that can be used in chemotherapy and thermal brachytherapy, as it has shown the synergistic effect in inhibiting cancer both in vitro and in vivo. Acknowledgement This paper was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (Project no. 2016R1A2A2A07005160), (Project no NRF2015R1C1A1A02036404) and (Project no. 2018R1D1A1B07048782). The paper was also partially supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2015H1C1A1035635) Author Declaration The Authors involved in this research declare that they have no conflicts of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ejpb.2018.06.003. References [1] L. Butcher, Solid tumors: prevalence, economics, and implications for payers and purchasers, Biotechnol. Healthc. 5 (1) (2008) 20–21. [2] N.R. Datta, et al., Local hyperthermia combined with radiotherapy and-/or chemotherapy: recent advances and promises for the future, Cancer Treat. Rev. 41 (9) (2015) 742–753. [3] R.D. Issels, et al., Neo-adjuvant chemotherapy alone or with regional hyperthermia for localised high-risk soft-tissue sarcoma: a randomised phase 3 multicentre study, Lancet Oncol. 11 (6) (2010) 561–570. [4] Y. Chen, et al., Drug-loaded mesoporous tantalum oxide nanoparticles for enhanced synergetic chemoradiotherapy with reduced systemic toxicity, Small 13 (8) (2017) p. 1602869–n/a. [5] G. Warrell, D. Shvydka, E.I. Parsai, Use of novel thermobrachytherapy seeds for realistic prostate seed implant treatments, Med. Phys. 43 (11) (2016) 6033–6048. [6] S.M. Glaser, et al., Brachytherapy boost for prostate cancer: trends in care and survival outcomes, Brachytherapy 16 (2) (2017) 330–341. [7] V. Srougi, et al., Early outcomes of focal brachytherapy for localized prostate cancer: comparison with whole gland brachytherapy, J. Urol. 197 (4) (2017) E1364–E1365.

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L.E. Aguilar et al.

[54] S.K. Calderwood, D.R. Ciocca, Heat shock proteins: stress proteins with Janus-like properties in cancer, Int. J. Hyperth. 24 (1) (2008) 31–39. [55] H.I. Bicher, et al., Effects of hyperthermia on normal and tumor microenvironment, Radiology 137 (2) (1980) 523–530. [56] M.W. Dewhirst, et al., Will hyperthermia conquer the elusive hypoxic cell? implications of heat effects on tumor and normal-tissue microcirculation, Radiology 137 (3) (1980) 811–817. [57] G.Y. Lee, et al., Anti-tumor and anti-metastatic effects of gelatin-doxorubicin and PEGylated gelatin-doxorubicin nanoparticles in SCC7 bearing mice, J. Drug Target. 14 (10) (2006) 707–716. [58] H. Dahring, et al., Improved hyperthermia treatment of tumors under consideration of magnetic nanoparticle distribution using micro-CT imaging, Mol. Imag. Biol. 17 (6) (2015) 763–769. [59] R.D. Issels, et al., Improved overall survival by adding regional hyperthermia to neo-adjuvant chemotherapy in patients with localized high-risk soft tissue sarcoma (HR-STS): long-term outcomes of the EORTC 62961/ESHO randomized phase III study, Eur. J. Cancer 51 (2015) p. S716–S716. [60] B. Gautam, et al., Dosimetric and thermal properties of a newly developed thermobrachytherapy seed with ferromagnetic core for treatment of solid tumors, Med. Phys. 39 (4) (2012) 1980–1990. [61] B. Gautam, et al., Practical considerations for maximizing heat production in a novel thermobrachytherapy seed prototype, Med. Phys. 41 (2) (2014). [62] J.L. Schaal, et al., Injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy, J. Control. Release 228 (2016) 58–66. [63] R. Pfeifer, et al., Noninvasive induction implant heating: an approach for contactless altering of mechanical properties of shape memory implants, Med. Eng. Phys. 35 (1) (2013) 54–62. [64] P. Kallinteri, et al., Novel Functionalized Biodegradable Polymers for Nanoparticle Drug Delivery Systems, Biomacromolecules 6 (4) (2005) 1885–1894. [65] A. Mickova, et al., Core/shell nanofibers with embedded liposomes as a drug delivery system, Biomacromolecules 13 (4) (2012) 952–962. [66] D. Sivakumaran, D. Maitland, T. Hoare, Injectable microgel-hydrogel composites for prolonged small-molecule drug delivery, Biomacromolecules 12 (11) (2011) 4112–4120.

boronate-catechol complexation, ChemPlusChem 78 (2) (2013) 175–184. [41] J. Park, et al., Polydopamine-based simple and versatile surface modification of polymeric nano drug carriers, ACS Nano 8 (4) (2014) 3347–3356. [42] Y. Kato, et al., Acidic extracellular microenvironment and cancer, Cancer Cell Int. 13 (1) (2013) 89. [43] P. DiFazio, et al., Radiofrequency ablation of solid tumors in combination with bortezomib causes ER-stress, Gastroenterology 150 (4) (2016) p. S5–S5. [44] C.Y. Huang, et al., Bortezomib enhances radiation-induced apoptosis in solid tumors by inhibiting CIP2A, Cancer Lett. 317 (1) (2012) 9–15. [45] C.A. Coltman, et al., Further clinical studies of combination chemotherapy using cyclophosphamide, vincristine, methotrexate and 5-flourouracil in solid tumors, Am. J. Med. Sci. 261 (2) (1971) 73. [46] M.R. King, Z.J. Mohamed, Dual nanoparticle drug delivery: the future of anticancer therapies? Nanomedicine 12 (2) (2017) 95–98. [47] N. Schleich, et al., Dual anticancer drug/superparamagnetic iron oxide-loaded PLGA-based nanoparticles for cancer therapy and magnetic resonance imaging, Int. J. Pharm. 447 (1–2) (2013) 94–101. [48] J. Li, et al., Zr61Ti2CU25Al12 metallic glass for potential use in dental implants: Biocompatibility assessment by in vitro cellular responses, Mater. Sci. Eng. C-Mater. Biol. Appl. 33 (4) (2013) 2113–2121. [49] R. Okner, et al., Electropolymerized tricopolymer based on N-pyrrole derivatives as a primer coating for improving the performance of a drug-eluting stent, ACS Appl. Mater. Interfaces 1 (4) (2009) 758–767. [50] A. Parsapour, S.N. Khorasani, M.H. Fathi, Effect of Surface Treatment and Metallic Coating on Corrosion Behavior and Biocompatibility of Surgical 316L Stainless Steel Implant, J. Mater. Sci. Technol. 28 (2) (2012) 125–131. [51] B. Thierry, M. Tabrizian, Biocompatibility and biostability of metallic endovascular implants: state of the art and perspectives, J. Endovasc. Ther. 10 (4) (2003) 807–824. [52] A.R.K. Sasikala, et al., A smart magnetic nanoplatform for synergistic anticancer therapy: manoeuvring mussel-inspired functional magnetic nanoparticles for pH responsive anticancer drug delivery and hyperthermia, Nanoscale 7 (43) (2015) 18119–18128. [53] J. Su, et al., Catechol polymers for pH-responsive, targeted drug delivery to cancer cells, J. Am. Chem. Soc. 133 (31) (2011) 11850–11853.

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