Encapsulation of superparamagnetic iron oxide nanoparticles with polyaspartamide biopolymer for hyperthermia therapy

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Encapsulation of superparamagnetic iron oxide nanoparticles with polyaspartamide biopolymer for hyperthermia therapy Minh Phuong Nguyen, Minh Hoang Nguyen, Jaeyun Kim, Dukjoon Kim

⁎

School of Chemical Engineering, Sungkyunkwan University, Suwon, Kyunggi 16419, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Hyperthermia therapy Biopolymers Polysuccinimide (PSI) Polyaspartamide Super-paramagnetic iron oxide nanoparticles Biomedical applications

We report a delicate synthesis process of polyaspartamide-encapsulated superparamagnetic iron oxide nanoparticles (PA-encapsulated SPIONs) with their sufficiently obtained grain-size below 100 nm for hyperthermia application. Iron oxide nanoparticles with a high magnetization have been applied as nano-heaters while polyaspartamide (PA) is a biocompatible and biodegradable polymer with a polysuccinimide (PSI) backbone. Multi-functional polymer PA could be conjugated with other groups such as biotin to enhance the uptake capability by receptors of cancer cells. Consequently, encapsulating SPIONs nano-heaters with PA biopolymer is an attractive roadmap for hyperthermia therapy application. Our results revealed that PA-encapsulated SPIONs showed excellent biocompatible behavior based on cell viability test. With Prussian blue staining of cancer cells (4T1), cellular uptake of PA-encapsulated SPIONs was significantly increased in the presence of biotin conjugated on the outer shell. Furthermore, PA-encapsulated SPIONs exhibited effective cancer killing activities in both in vitro and in vivo hyperthermia experiments. Therefore, PA-encapsulated SPIONs might have potential for hyperthermia therapy.

1. Introduction Persistent development of polymer technology has promoted scientists to explore new polymeric types with novel properties for a series of diverse applications, especially for biomedical applications. For these bio-applications, biopolymers must fulfil some important prerequisites. For example, they need to be non-toxic, biocompatible, and biodegradable. Using biopolymers in medical applications is emerging as potential roadmap for many institutions and medical centers. Many efforts have been made to investigate various biopolymer-types such as polyethylene glycol [1], polyglutamic acid [2], polyvinylpyrrolidone [3], polyaminoacid hydrogel [4], polypeptides [5], and polylactic-coglycolic acid based hydrogel [6] for clinical applications. Polyaspartamide (PA) has attracted significant attention in the biomedical field due to its substantially advanced features such as non-toxic, high biodegradable and biocompatible, easy conjugating or grafting with other organic groups, and possible encapsulation of diverse bioactive inorganic agents for combination therapy [7–13]. As the cancer is a mortal disease, the number of patients diagnosed with cancer is increasing every year. To treat this disease, many advanced therapies have been suggested [14–16]. However, an efficient method for treating cancer remains a challenge [17]. In recent years, hyperthermia therapy has been proposed as a promising method for ⁎

combating cancer [18–21]. This method destroys cancer tissues by heating local tissues without affecting nearby tissues. It is essential to select an appropriate way to deliver heat to cancer cells [22]. With nanotechnology advancements, using magnetic nanoparticle hyperthermia is an appropriate solution because magnetic nanoparticles have excellent hyperthermia effect. In addition, they can penetrate deeply into small regions of cancer tumors [22]. Strategies to produce biocompatible, biodegradable, super-sized, and superparamagnetic nano-heaters for hyperthermia therapies have been attractive topics. They are continuously investigated by scientists. In this research, we report a delicate synthesis process of encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) with PA biopolymers to combat cancer cells by hyperthermia therapy. PA plays an important role in encapsulating SPIONs to form a hydrophilic, biocompatible, and biodegradable system while SPIONs with superparamagnetic feature are utilized as nano-heaters for destroying thermal sensitive cancer cells. Encapsulation was implemented by hydrophobic interaction between oleic acid on the surface of SPIONs and hydrophobic tails on polysuccinimide (PSI) – octadecylamine (C18). The hydrophilic surface of SPIONs is formed by grafting o-(2-aminoethyl) polyethylene glycol (PEG) on PSI. As a multi-functional drug carrier, it can be also conjugated with biotin to effectively enhance targeting and uptake capability of SPIONs to cancer cells. The synthesis

Corresponding author. E-mail address: [email protected] (D. Kim).

https://doi.org/10.1016/j.eurpolymj.2019.109396 Received 29 July 2019; Received in revised form 20 November 2019; Accepted 29 November 2019 0014-3057/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Minh Phuong Nguyen, et al., European Polymer Journal, https://doi.org/10.1016/j.eurpolymj.2019.109396

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2.4. Encapsulating iron oxide nanoparticles with PA

process, characteristics, in vitro and in vivo experiments of PA-encapsulated SPIONs system are discussed.

Encapsulation was implemented by hydrophobic interaction. PA (0.03 g) was dissolved in 5 mL DI water. SPIONs (0.005 g) were dispersed in 5 mL chloroform. SPIONs sol were then dropped into PA solution followed by stirring at room temperature for 24 h. After that, solvents were evaporated in a vacuum oven at 60 °C. Unreacted polymer was removed after centrifuging at 7000 rpm for 10 min. PAencapsulated SPIONs were then obtained after washing several times with distilled water and centrifuging at 15000 rpm for 1 h followed by drying.

2. Experimental In this section, we address a series of material preparations including PSI, PA, and SPION preparations. Materials used for these preparations are tabulated in supplementary information (Table S1). These materials were purchased from Sigma-Aldrich (Milwaukee, WI, USA). After material preparations, encapsulating of SPIONs with PA, an important step for in vitro and in vivo testing, was conducted.

3. Characteristics and methods 2.1. Synthesis of polysuccinimide (PSI)

3.1. Size and morphology

PSI plays a role as the backbone for PA. Thus, PSI preparation was one of the essential initial steps for subsequent PA preparation. In this step, a mixture of 25 g L-aspartic acid, 70 g mesitylene, and 30 g sulfolane was firstly prepared. The mixture was polymerized under a nitrogen inlet at 70 °C with catalysis of 0.643 mL phosphoric acid. A Dean-Stark trap was used to remove water, the by-product, from the reaction. After 8 h, the residual product was precipitated in methanol, filtered, and washed several times with distilled water. The final product, PSI, was dried in vacuum for 24 h.

Particle size, morphology, and size distribution of nanoparticles were examined by high-resolution transmission electron microscopy (HR-TEM, JEM-2100F, JEOL, Japan). For measurement, samples were prepared by dropping the nanoparticle solution onto carbon-coated copper grids and drying them in air. Hydrodynamic diameters, Zeta potential of nanoparticles were measured using a dynamic light scattering analyzer (DLS, ELS-Z model, Otsuka, Japan). 3.2. Molecular weight and chemical structure of the polymer

2.2. Synthesis of polyaspartamide (PA)

The molecular weight of PSI was identified using a Ubbelohde viscometer. PSI was dissolved in DMF and measured at 25 °C in a circulating water bath. The degree of polymerization was calculated according to Eq. (1) [23]:

Firstly, the obtained PSI (10 g) and C18 (1.3882 g) were mixed in 25 mL DMF. The obtained solution was then heated in a nitrogen gas environment at 70 °C for 24 h. After reaction, product was precipitated in methanol. The filtered precipitation, PSI-C18, was washed several times with distilled water and dried at 70 °C in vacuum for 24 h. Secondly, to graft PEG on PSI, the synthesized PSI-C18 (0.1553 g) and PEG (0.2 g) were dissolved in 20 mL of DMF. This solution mixture was reacted under nitrogen gas at 70 °C for 48 h. Product (PSI-C18-PEG) was formed after removing solvent using dialysis membrane for 3 days followed by freeze-drying. Finally, biotin was conjugated to PEG by stirring a solution consisting of 1 mol of biotin, 2 mol of PSI-C18-PEG, 1.1 mol of DCC, and 3 mol% of DMAP in 25 mL of DMF at 0 °C for 5 min and room temperature (25 °C) for 3 h. After that, the mixture was dialyzed and freeze-dried for 3 days to obtain PSI-C18/PEG-biotin.

1.56 n = 3.52 × ηred

(1)

where ηred was reduced viscosity of the polymer solution. Chemical structure of the synthesized polymer was analyzed by hydrogen-1 nuclear magnetic resonance (1H NMR) spectroscopy (Unity Inova 500 MHz, Varian, Palo Alto, CA, USA). Samples were dissolved in dimethyl sulfoxide-d₆ (DMSO‑d6) and measured at room temperature. 3.3. Magnetic property and magnetic heating measurements Magnetic property and magnetic strength of SPIONs and PA-encapsulated SPIONs were determined using a superconducting quantum interference device (SQUID, Quantum Design MPMS XL, San Diego, CA, USA) at 300 K. An automation magnetic hyperthermia system manufactured by MSI (7.0 KW, Wichita, KS, USA) was used for magnetic heating measurements. SPION and PA-encapsulated SPION sols at different concentrations were placed in glass vials for measurement. All samples were exposed to an alternating magnetic field at fixed frequency (f) of 325 kHz and room temperature (25 °C). Change in temperature of the sample upon heating excitation with a continuous alternating magnetic field was recorded with a fiber optic thermometer system (Neoptix, USA) at one-minute intervals.

2.3. Synthesis of iron oxide nanoparticles Thermal decomposition method was used to synthesize superparamagnetic iron oxide nanoparticles (SPIONs). As the first step, an iron oleate complex precursor was prepared. Briefly, 2.7 g of iron (III) chloride hexahydrate and 9.125 g of sodium oleate were mixed with 35 mL hexane, 20 mL ethanol, and 15 mL distilled water. The mixture was then stirred at 70 °C. After 4 h of reaction, the solution was separated into two layers. Byproduct NaCl and unreacted components crowded in the lower layer were removed using a separatory funnel. The iron oleate complex obtained in the upper layer was washed three times with distilled water. Residual solvents were then removed by evaporation. The final form of the iron oleate complex was a dark brown wax. In second step, SPIONs were synthesized. Briefly, 9 g of synthesized iron oleate precursor and 1.425 g of oleic acid were dissolved in 50 g of octadecene. The mixture was heated from room temperature (25 °C) up to 320 °C at a rate of 3.3 °C min -1 and then maintained at 320 °C for 30 min. After the reaction, the solution turned black due to formation of SPIONs. The black solution was cooled to room temperature and precipitated in 125 mL of ethanol. Finally, SPIONs were obtained after washing many times with ethanol, followed by drying.

3.4. Cellular uptake Cellular uptake of PA-encapsulated SPIONs was evaluated by Prussian blue staining as described previously [24]. Briefly, 20% HCl solution and 10% potassium hexacyanoferrate (II) trihydrate solution were mixed at a volume ratio of 1:1 to obtain a working solution. Then 1 × 104 4T1 breast cancer cells and 3T3 murine cells were seeded into 6-well microplates. After 24 h of incubation, PA-encapsulated SPION with and without biotin were added into wells and incubated for another 24 h. Subsequently, cells were washed with PBS solution and fixed with 2% neutral buffered formalin. The working solution was then added to each well, followed by incubation for 30 min. The solution 2

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Fig. 1. 1H NMR spectrum of (a) polysuccinimide (PSI) and (b) PA.

(control group) was injected only with the PBS solution without hyperthermia heating process. Group 2 was injected only with PA-encapsulated SPIONs without hyperthermia heating process. Group 3 was injected with PA-encapsulated SPIONs and then treated under hyperthermia heating process; however, these PA was intentionally exceptional absence of biotin groups. Finally, Group 4 was injected with PA-encapsulated SPIONs and then treated under hyperthermia heating process. The mice were injected with the nanoparticles (2.5 mg Fe/kg) by intravenous injection and heated every two days for 10 days. For the heating, the mice were placed in the heating coil of the MSI automation magnetic hyperthermia system (7.0 KW, Wichita, KS, USA) at a fixed frequency (f) of 325 kHz. The average size of tumors was measured every two days using a digital caliper. Tumor volume (V) was calculated using the following formula: V = 0.523 × (length × width2) [25].

was then removed. Subsequently, cells were washed again with PBS solution and observed under a microscope. 3.5. Cell viability To determine cell viability after treatment with PA-encapsulated SPION solution at different concentrations, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was performed. Briefly, 1 × 104 of 4T1 breast cancer cells and 3 T3 murine cells were seeded into 96-well microplates and incubated for 24 h. PAencapsulated SPION was then added to wells and further incubated for 24 h. Subsequently, 20 µL MTT solution (5 mg mL−1) was added to each well. After 4 h of incubation, the culture medium was removed and 200 µL DMSO was added into each well to dissolve formazan crystals. After incubation for 30 min, the UV absorbance intensity of cells was measured at wavelength of 570 nm.

4. Result and discussion

3.6. In vitro hyperthermia experiment

4.1. Characteristic of PA derivatives

Cell proliferation assay was performed to evaluate in vitro effect of the hyperthermia treatment. Briefly, 1 × 105 4T1 and 3T3 cells were seeded into 6-well microplates and incubated for 24 h. Six wells were divided into three groups (two wells for each group). The first group was the control group without nanoparticles. For the remaining two groups, PA-encapsulated SPIONs were added at two different concentrations (0.6 mg mL−1 and 1 mg mL−1) and then cultured for 24 h. Cells were then trypsinized and added into centrifuge tubes. Fresh culture medium was added to the centrifuge tube to reach a final volume of 0.4 mL. Subsequently, one centrifuge tube of each group was placed in the copper coil of the MSI automation magnetic hyperthermia system (7.0 KW, Wichita, Kansas, USA) while being heated for 30 min. After heating, 100 µL of each sample was drawn from the centrifuge tube and added to 96 well-microplates. The cells continued to incubate at 37 °C for 24 h. The cells were then examined in the same way as the cell viability measurements.

The average molecular weight of PSI demonstrates roughly the formation of polymer chain. From Equation (1), the average molecular weight of PSI was calculated to be 60,000 g mol−1 corresponding to an intrinsic viscosity of 27. This indicated that the PSI chain formed completely to conjugate with other functional groups. Conjugation of PSI with various functional groups was detected efficiently based on 1H NMR spectra shown in Fig. 1. The 1H NMR spectrum (Fig. 1a) showed one peak at 5.3 ppm and two peaks at 3.2 and 2.7 ppm. These peaks were characteristic of methine and methylene protons on the main chain of the PSI chemical structure, respectively [26,27]. Fig. 1b shows appearance of C18-groups with peaks at 0.85 and 1.23 ppm [28]. In addition, proton signals at 3.58 and 4.5 ppm indicating the presence of PEG [28] were observed. Furthermore, conjugation of biotin to PEG was confirmed based on two peaks at 4.2 and 4.3 ppm that were characteristic of methane protons and another two peaks at 6.3 and 6.4 ppm corresponding to urea protons [29]. The degree of substitution (DS) known to indicate the integration of proton signal intensity was calculated from the 1H NMR spectrum. DS values of C18, PEG, and biotin were 9%, 2%, and 0.5%, respectively, when C18, PEG, and biotin were fed at ratios of 10%, 2.5%, and 1%, respectively. DS values nearly corresponded to feed ratios of functional groups, indicating that the conjugation of functional groups on PSI was possibly well controlled by the initial feed ratio.

3.7. In vivo hyperthermia experiment The in vivo effect of the hyperthermia treatment was evaluated in BALB/c mice. Six-week old female-mice (BALB/c) were purchased from the Hanlim Experimental Animal Laboratory (Seoul, Korea) and tumor models were created by subcutaneous injection of 2 × 106 4T1 cells into the back of the mice. When the tumors reached the average size in range of ~100–150 mm3, we separated the mice into 4 groups (5 mice/ group) and treated them on different ways. For examples, group 1 3

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Fig. 2. Particle size, distribution, and hydrodynamic diameter of SPIONs and PA-encapsulated SPIONs measured by TEM, and DLS: (a) TEM image of SPIONs; (b) DLS result of SPIONs; (c) TEM image of PA-encapsulated SPIONs; (d) DLS result of PA-encapsulated SPIONs.

7.4 (physiological pH). Zeta potential value characterizes for electrostatic force between nanoparticles in a solvent, therefore, the stability of nanoparticles in a solvent increases with increasing of Zeta potential (or surface charge) [33]. The obtained Zeta potential value demonstrates the probably high stability of the PA-encapsulated SPIONs in physiological environment and thus great potential in hyperthermia therapy [34,35].

4.2. Characterization of PA-encapsulated IONs Fig. 2 shows size, morphology, and distribution of nanoparticles confirmed by TEM and DLS measurements. TEM images (Fig. 2a) showed the appearance of uniformly distributed homogeneous as-synthesized SPIONs with spherical shape and average size of 10 nm. DLS results (Fig. 2b) corroborated the average diameter of around 10 nm for nanoparticles. As mentioned earlier, the surface of as-synthesized SPIONs needs to be modified to be hydrophilic, biocompatible, and biodegradable for bio-applications. Therefore, SPIONs were encapsulated with PA by hydrophobic interaction between oleic acid and C18. Fig. 2c shows TEM images of spherical PA-encapsulated SPIONs with average size below 100 nm. Inside these particles, it appeared that many tiny nanoparticles were gathered together into a group which was separated completely with other groups. These results indicated that PA probably encapsulated a group of numerous 10 nm-SPIONs. It should be noticed that the average diameter of encapsulated SPIONs from DLS measurement (Fig. 2d) was around 150 nm which was higher than that of TEM result (Fig. 2c). Such differences of obtained average diameter size from these two methods were probably due to encapsulation of PA on the surface of SPIONs. It is probably difficult to detect a highly transparent “outer-shell” of PA with TEM measurement. Nanoparticle diameter remains the most essential parameter in almost all applications because physical and chemical properties of nanoparticles depend strongly on this parameter. The diameter of around 100 nm is believed to be an appropriate nanoscale of PA-encapsulated SPIONs for bio-applications, especially hyperthermia therapy [30–32]. Moreover, Zeta potential value of PA-encapsulated SPIONs is −13.8 ± 0.1 mV at pH

4.3. Magnetic property and magnetic heating measurements Fig. 3 shows hysteresis loops of SPIONs and PA-encapsulated SPIONs. It was found that SPIONs and PA-encapsulated SPIONs exhibited superparamagnetic feature due to the absence of hysteresis, meaning that magnetization could decline to zero in case of the absence of an external magnetic field. It is a preferred behavior for hyperthermia therapy applications because it allows for easy observation of heating variation of particles under an alternating magnetic field [36]. In addition, a high saturation magnetization to maintain efficient heating of nanoheaters under alternating magnetic field is an important parameter for such applications [18]. Calculated saturation magnetization values (Ms) of SPIONs and PA-encapsulated SPIONs were 45.03 emu g−1 and 10.46 emu g−1, respectively. It could be easily inferred that encapsulation of SPIONs with PA can cause decrease in saturation magnetization feature due to nonmagnetic characteristic of PA. To demonstrate the heating ability of PA-encapsulated SPIONs, we tested nano-magnetic hyperthermia effect at room condition (25 °C) by applying an AC magnetic field at a frequency of 325 kHz. Fig. 4 shows heating ability of PA-encapsulated SPIONs at various concentrations 4

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that SPIONs encapsulated with PA still well maintained its heating ability in hyperthermia treatment. However, it should be noticed that the heating ability of PA-encapsulated SPIONs in this experiment was tested in laboratory room-condition. It might be completely different when it is tested under in vivo testing condition. This will be discussed more clearly with in vivo experiments shown in the next section. 4.4. Cell viability As aforementioned, non-toxicity is one of essential prerequisites for biomedical applications. It is known that cells can degrade and utilize iron oxide compound thanks to the metabolism process. Thus, iron oxide seems to be intrinsically non-toxic to cells [37,38]. However, cytotoxicity can be caused by high concentrations of SPIONs [39]. To demonstrate clearly the nano-magnetic hyperthermia effect of PA-encapsulated SPION in in vitro and in vivo experiments, it is necessary to first confirm that using a certain SPION concentration is almost nontoxic to cells. Consequently, we used MTT viability assay to test cell viability after treatment with PA-encapsulated SPIONs at 0.1–3 mg mL−1. Fig. 5 shows cell viability results of the normal cells (3T3) and cancer cells (4T1) after 24 h incubation with various concentrations of PA-encapsulated SPIONs. For both cell-lines, cell viability was stable over 90% after treatment with PA-encapsulated SPIONs at concentration ranging from 0.1 to 0.8 mg mL−1 compared to the control group. It was decreased slightly to about 85% after treatment with PA-encapsulated SPIONs at 1 mg mL−1. It was significantly decreased to less than 70% after treatment with PA-encapsulated SPIONs at 2 mg mL−1 or higher concentrations. This indicated that PA-encapsulated SPIONs at concentration over 1 mg mL−1 was highly toxic to cells. Thus, such concentration would be inappropriate for hyperthermia therapy. Since PA-encapsulated SPIONs at concentration of 1 mg mL−1 resulted in cell viability of more than 85%, 1 mg mL−1 was chosen as the standard concentration of PA-encapsulated SPIONs for subsequent in vitro and in vivo hyperthermia testing.

Fig. 3. SQUID measurements of the SPIONs and the PA-encapsulated SPIONs.

4.5. Cellular uptake In this part, the role of biotin functional group in enhancing targeting and uptake ability to cancer cells is discussed. It is well-known that biotin (vitamin H or B7) is one of important growth promoters for cells. Cells normally use receptor on its surface to uptake biotin. Fastgrowing cells such as cancer cells generally require more biotin than normal cells for their violent growth. As a result, some cancer cell lines such as 4T1 need to overexpress biotin-receptors on their surface as much as possible to have high biotin uptake [40,41]. Based on this feature, biotin has been used as tumor-targeting molecular in biopolymers [41–43]. We compared biotin-uptake efficiency between normal cells (3T3) and cancer cells (4T1). Fig. 6 shows Prussian blue staining microscope images of cells after incubation with PA-encapsulated

Fig. 4. The temperature increase of the PA-encapsulated SPIONs at different concentrations versus time (f = 325 kHz, 4.9 kW).

from 1 to 100 mg mL−1. An increase in temperature by at least 5 °C from an initial room temperature to 29 °C could be achieved in 30 min even at a low concentration of 1 mg mL−1. The temperature tended to strongly increase with increasing concentration. These results showed

Fig. 5. Cell viability of PA-encapsulated SPIONs on (a) 3T3 cells and (b) 4T1 cells. 5

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Fig. 6. Microscope images for Prussian blue staining of 3T3 and 4T1 cells after incubated with the PA-encapsulated SPIONs: (a) Control of 4T1 cells, (b) PAencapsulated SPIONs without biotin on 4T1 cells, (c) PA-encapsulated SPIONs with biotin on 4T1 cells, (d) control of 3T3 cells, (e) PA-encapsulated SPIONs without biotin on 3T3 cells, and (f) PA-encapsulated SPIONs with biotin on 3T3 cells.

treatment.

SPIONs. In these images, the appearance of blue pigments indicates cell’s ability to uptake nanoparticle. These blue pigments are caused by interaction between SPIONs and ferrocyanide of working-solution. It was apparent that images of control groups without SPIONs (Fig. 6a and d) had no blue pigments. However, blue pigments appeared densely and clearly in images of 4T1 cells (Fig. 6b and c), while they were not so dense or clear in images of 3T3 cells (Fig. 6e and f). This demonstrated that SPIONs were transported to cancer 4T1 cells better than those to 3T3 cells. This means that PA-encapsulated SPIONs seem to be mainly uptaken by cancer 4T1 cells. This could really benefit hyperthermia treatment. In particular, it should be noticed that blue pigments in Fig. 6c with biotin were significant larger and more crowded than those in Fig. 6b without biotin. This comparison clearly demonstrated the essential role of biotin functional group in enhancing targeting and uptake of PA-encapsulated SPIONs into cancer cells for hyperthermia

4.6. In vitro hyperthermia Fig. 7 shows hyperthermia effect on cell viability of both 3T3 and 4T1 cell lines as a function of PA-encapsulated SPION concentration. For exact comparison, control groups without PA-encapsulated SPIONs of both cell-lines were tested in hyperthermia treatment. As shown in Fig. 7, cell viability of the control group with hyperthermia treatment was about 85% which was slightly lower than that of the control group without hyperthermia treatment (100%). This showed that hyperthermia treatment insignificantly affected cell viability of both cell lines. As seen in Fig. 7a, cell viability of 3T3 cells treated with PAencapsulated SPIONs after hyperthermia treatment was nearly stable compared to that of the control group. This demonstrated that the 6

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Fig. 7. Effects of (a) in vitro hyperthermia experiments on 3T3 cells, and (b) in vitro hyperthermia experiments on 4T1 cells.

magnetic-nanoparticle hyperthermia effect on normal cells (3T3) was insignificant. However, as seen in Fig. 7b, cell viability of 4T1 cancer cells treated with PA-encapsulated SPIONs was decreased significantly compared to that of the control group after hyperthermia treatment. In particular, it should be noticed that cell viability of 4T1 with biotin was decreased considerably compared to that without biotin. In addition, in vitro hyperthermia treatment on cancer cells seemed to be more effective with PA-encapsulated SPIONs. However, as demonstrated in cytotoxicity experiment (part 4.4), a further increase in PA-encapsulated SPIONs concentration seemed to be inappropriate for hyperthermia treatment. Based on cellular uptake experiment (part 4.5), it was apparent that biotin functional group played an important role in enhancing the targeting and uptake of PA-encapsulated SPIONs to cancer cells, resulting in more effective hyperthermia treatment.

the same as group 1, although its volume seemed to be slightly lower. Especially, groups 3 and 4 showed significantly slow growths of tumor volume as a function of days. After 28 days, the volume of group 3 reached around 1500 mm3 while that of group 4 gained around 300 mm3 which was significantly smaller than that of the control group. These results demonstrated that hyperthermia treatment with PA-encapsulated SPIONs possibly inhibited the growth of tumor cells considerably. In addition, it should be particularly noticed that the tumor volume of group 4 with biotin was more effectively inhibited than that of group 3 without biotin. Thus, we can conclude that conjugation with biotin can help PA bio-polymer enhance targeting and uptake to cancer cells, thus leading to more effective hyperthermia treatment. This role of the biotin group was discussed clearly in previous sections.

4.7. In vivo hyperthermia

5. Conclusion

We determined the hyperthermia effect of PA-encapsulated SPIONs by performing in vivo anti-tumor experiments. We implemented tumor xenograft models of 4T1 cells in BALB/c mice and separated them into four treatment groups as mentioned in Section 3.7 (in vivo hyperthermia experiment). Tumor volume of each group as a function of days is shown in Fig. 8. Results showed that at 7 days after injection of 4T1 cells, tumors of all groups reached nearly the same volume of around 100–150 mm3. From this standard volume, we began to test hyperthermia effect and observe volume-growth of tumor as a function of days. Fig. 8 indicated that tumor volumes had considerable difference after 28 days. In the control group, the volume increased intensely within days, reaching a large volume of around 3000 mm3 after 28 days. The volume growth of the second group tended to be nearly

SPIONs were synthesized by thermal decomposition method. The diameter of SPIONs was around 10 nm as confirmed by TEM and DLS. These SPIONs were encapsulated by PA to enhance their biocompatible and biodegradable capabilities necessary for clinical applications. After encapsulation, the diameter of PA-encapsulated SPIONs was below 100 nm with saturation magnetization value of around 10.46 emu g−1. With such magnetization, PA-encapsulated SPIONs exhibited good heating ability even low concentration of 1 mg mL−1 in room-condition via hyperthermia experiment. Cytotoxicity testing using normal and cancer cells indicated that the cell viability was over 85% after cells were treated with SPION at concentration below 1 mg mL−1 which could be effectively utilized for in vivo and in vitro hyperthermia experiments. Cellular uptake results indicated that conjugation of biotin to PA on the external surface increased cancer cellular uptake capability of synthesized particles, resulting in excellent heating ability of in vitro hyperthermia treatment. Finally, after in vivo hyperthermia treatment with PA-encapsulated SPIONs, cancer tumor growth was inhibited significantly compared to the control group. These results will open further potential for cancer treatment in the future.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments This research was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean Government (MEST) (NRF-2017R1A5A1070259).

Fig. 8. Tumor volume of the mice at different groups as a function of days after tumor inoculation. 7

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Appendix A. Supplementary material [21]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.109396.

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