Magnetic Prussian blue nanoparticles for combined enzyme-responsive drug release and photothermal therapy

Abstract

Multifunctional nanoparticles are attracting increasing attention as novel agents for efficient tumor therapy. In this study, a core–shell nanoparticle (NP) system is synthesized by growing gelatin–doxorubicin (Gel–DOX)-stabilized Prussian blue (PB) nanoshells on Fe₃O₄ nanocores (Fe₃O₄@PB@Gel–DOX NP), for combined photothermal therapy and enzyme-responsive chemotherapy under magnetic field enhancement. The composite nanoparticles exhibit excellent superparamagnetism (31.6 emu g⁻¹), contributing to their enhanced therapeutic effect under the magnetic field. Drug release from this nanocomplex is triggered in the presence of enzyme in solutions or in live cells. A photothermal effect is evident under near infrared (NIR) laser irradiation, owing to the high photothermal conversion efficiency of the PB nanoshell, resulting in more than 80% cell death of Hela cells treated with 40 μg mL⁻¹ of the nanoparticles. The effects of combining photothermal and drug-induced tumor ablation with magnetic field enhancement are evaluated using a tumor cell viability assay in vitro. It is expected that this nanosystem integrating superparamagnetism, photothermal therapy and chemotherapy will foster new avenues for developing the next generation of multifunctional platforms towards effective cancer treatment.

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Introduction

Despite advances in modern healthcare and medical technologies, cancer remains one of the main diseases responsible for increasing yearly mortality rate both in the developed and developing worlds.¹ In treatment of cancer, various nanoparticle (NP) platforms are used to meet the challenges associated with nonspecific drug distribution, rapid clearance, insufficient drug concentration within the tumor, and nonspecific toxicity to normal cells.^2,3 Multifunctional NPs have been developed by combining various functional molecules into a single NP carrier for synergistic tumor therapy.^4–6 However, the intrinsic toxicity, level of complexity, and the cost of time and resources to obtain approval from authority have prevented most recently reported nano-agents from being applied on real patients. Thus, there exists a pressing demand to develop multifunctional therapeutic nano-agents for clinical applications based on materials already approved by the US Food and Drug Administration (FDA).

Interest has grown in photothermal therapy (PTT)-associated multifunctional therapeutic agents, which ablate tumor cells through heat produced by photothermal conversion under near infrared (NIR) irradiation,^7–9 because of their specificity and non-invasiveness. The NIR (wavelength 700–1100 nm) light has a greater tissue penetration depth and is acceptable to normal tissues. There are many well-designed therapeutic agents which have been developed for photothermal therapy, such as carbon-based nanostructures,^10–12 gold-based nanomaterials^13–15 and copper chalcogenide semiconductors.^16–18 These demonstrate high efficacy based on strong optical absorbance in the NIR tissue transparency window. However, some issues have arisen, such as potential long-time toxicity and complicated synthesis procedure, which have limited their further applications. Prussian blue (PB), used for clinical treatment of internal radioactive exposure, is FDA-approved because of its excellent biosafety in the human body.¹⁹ Recently, PB-based NPs have been explored as potential ablation agents for PTT because of their strong photothermal conversion efficiency, broad optical absorbance in the NIR region, as well as outstanding photothermal stability.^20–23 The PB-based nanostructure features a high molar extinction coefficient of 10⁹ M⁻¹ cm⁻¹ at 808 nm, which is comparable with that of Au nanorods (5.24 × 10⁹ M⁻¹ cm⁻¹ at 808 nm). Additionally, a broad optical absorbance band was observed in PB-based nanostructures from 500 nm to 900 nm with a typical absorption peak at 710 nm in the NIR region, making them desirable as NIR laser-driven photothermal agents. Moreover, PB NPs are cost-effective materials and can be mass-produced using a facile method by simply mixing FeCl₃ and K₄[Fe(CN)₆] under acidic conditions.²⁴ Gelatin (Gel) derived from partially hydrolyzed collagen has been used successfully for stabilizing various types of NPs, such as PB NPs,^22,25 Fe₃O₄ NPs²⁶ and CdTe quantum dots.²⁷ Additionally, gelatin can be easily hydrolyzed into its sub-compounds (peptides, amino acids) by gelatinase, which is a type of endogenous proteolytic enzyme usually overexpressed in tumor tissues in comparison with normal tissues.²⁸ The significantly elevated level of gelatinase is suggested to assist tumor cells during metastasis to distant tissues or organs far from the primary tumor.²⁹ Therefore, covalent conjugation of gelatin with nanocarriers can cause enzyme-responsive drug release in tumors.^22,30 Doxorubicin (DOX), a typical anti-cancer chemotherapy drug for treatment of a wide range of tumor types, can be conjugated with functionalized polymers for controlled and sustained drug release.^31,32

Meanwhile, magnetic NPs, particularly those made of superparamagnetic Fe₃O₄, have attracted great interest for guiding drug to a target region under localized magnetic field gradients.^33–35 This treatment causes a large dose of drug to accumulate in the tumor region via magnetic drug carrier, and minimizes potential adverse side effects to normal tissues. More importantly, Fe₃O₄ NPs have been approved for clinical application in magnetic resonance imaging (MRI), indicating their acceptable biosafety for patients.^19,36 Thus, composite NP synthesized from PB, Fe₃O₄, and gelatin offers a promising system as a drug carrier for clinical usage because of the outstanding biosafety of these materials. In this paper, we propose a multifunctional Fe₃O₄@PB@Gel–DOX agent for combined chemo- and photothermal therapy enhanced by a magnetic field (Fig. 1). The gelatin–DOX complex is prepared using a traditional periodate oxidation method.^30,37 Multifunctional Fe₃O₄@PB@Gel–DOX NPs are synthesized by growing PB nanoshells on superparamagnetic Fe₃O₄ nanocores following a shell-growing procedure,^38,39 while Gel–DOX serves as the colloid to stabilize the magnetic NPs. Thus, magnetic field enhanced photothermal and chemotherapy can be achieved using this core–shell complex. The synergistic effect of photothermal and chemotherapy with Fe₃O₄@PB@Gel–DOX NPs is demonstrated in vitro on Hela cells derived from cervical cancer which overexpress gelatinase.⁴⁰

Fig. 1 Schematic illustration of Fe₃O₄@PB@Gel–DOX NPs for combined chemo- and photothermal destruction of tumor cells enhanced by a magnetic field.

Experimental

Materials

Potassium hexacyanoferrate(ii) trihydrate (K₄[Fe(CN)₆]·3H₂O), iron(III) chloride hexahydrate (FeCl₃·6H₂O), iron(II) sulfate heptahydrate (FeSO₄·7H₂O), citric acid, sodium hydroxide (NaOH), sodium periodate (NaIO₄), sodium cyanoborohydride (NaCNBH₃) and gelatin type A (∼300 bloom, powder form derived from porcine skin) were purchased from Sigma-Aldrich, Singapore. Gelatinase was provided by ITS Science & Medical, Singapore. Doxorubicin hydrochloride (DOX) was purchased from Dalian Meilun Biotech, China. DMEM culture medium (supplemented with 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin and 10% fetal bovine serum), calcein AM and PrestoBlue cell viability assay kits were obtained from Life Technologies, Singapore. Deionized water with a resistivity of 18.2 MΩ cm was obtained through Millipore Synthesis A10 (Molsheim, France).

Synthesis of Gel–DOX conjugates

The principle of conjugating doxorubicin onto gelatin based on a traditional periodate oxidation method, is shown in Scheme S1 (ESI†). Soluble Gel–DOX conjugates were synthesized with a feeding ratio of 1 : 20 (DOX : gelatin) by weight according to a previous report.³⁰ 10 mg of DOX was dissolved in 4 mL of PBS (10 mM) in a 25 mL flask. Then, 0.2 mL of NaIO₄ (0.1 M) was added to the DOX solution followed by 1 h incubation at 40 °C in the dark. Afterwards, 15 μL of glycerol (0.1 M) was added under stirring to neutralize excess periodate, and stirring was continued for 30 min. Then, 4 mL of gelatin (50 mg mL⁻¹) dispersed in carbonate buffer solution (pH = 9.5) was added to the solution followed by 1 h incubation at 40 °C in the dark. 0.5 mL of NaCHBH₃ (0.1 M) was added to the reacting system and maintained for 2 h in the dark. The final solution was dialyzed at 40 °C for one week and subsequent lyophilization was allowed to proceed for three days.

Synthesis of Fe₃O₄ NPs

Superparamagnetic Fe₃O₄ NPs were prepared based on in situ co-precipitation of ferrous (Fe²⁺) and ferric (Fe³⁺) ions under base conditions.⁴¹ Briefly, 5 mL of FeCl₃·6H₂O (10 mM) and FeSO₄·7H₂O (5 mM) aqueous solution was added dropwise to 50 mL of NaOH (2 M) solution under vigorous stirring at 80 °C. After 30 min of stirring, the superparamagnetic Fe₃O₄ was harvested by magnetic separation and washed with deionized water.

Synthesis of Fe₃O₄@PB@Gel–DOX NPs

An improved shell-growing method was implemented to synthesize Fe₃O₄@PB@Gel–DOX NPs.³⁸ Briefly, 10 mL of Fe₃O₄ NPs (0.2 mg mL⁻¹) was re-dispersed into 20 mL of aqueous K₄[Fe(CN)₆] solution (10 mM). The solution was adjusted to pH = 2 by dropwise addition of hydrochloric acid (0.1 M) under stirring. The color of the solution gradually changed to green during the addition process. 10 mg of Gel–DOX complex was then added dropwise to the solution, and this was stirred continuously for 30 min, followed by Fe₃O₄@PB@Gel–DOX NP separation and purification with deionized water based on magnetic decantation. For reference, pure PB NPs were prepared by adding citric acid (0.5 mmol) to FeCl₃ aqueous solution (20 mL, 1 mM) at 60 °C under stirring. Products were harvested after dropwise addition of K₄[Fe(CN)₆] aqueous solution (1 mM) containing citric acid (0.5 mmol) to the experimental solution at 60 °C under stirring.

Characterization of Fe₃O₄@PB@Gel–DOX NPs

The UV-vis absorbance spectra of the synthesized PB NPs, Fe₃O₄ NPs, Gel–DOX and Fe₃O₄@PB@Gel–DOX NPs were measured using a UV-vis spectrophotometer (UV-2450, Shimadzu Scientific Instruments, Japan) in the range of 400–900 nm with a quartz cuvette of 1 cm optical path length. The size distribution of NPs was determined using Zetasizer Nano ZS (Malvern Instruments Ltd, UK). Morphology of the NPs was examined by transmission electron microscopy (TEM, Libra 120 Plus, Carl Zeiss, US) at an accelerating voltage of 120 kV. The magnetic response of the NPs was tested using a magnet adjoining a cuvette containing an aqueous dispersion of the NPs, to observe their movements under a magnetic field. Field-dependent magnetization of NPs was measured at 25 °C using a vibrating sample magnetometer (VSM, Lakeshore 7400).

In vitro drug release

In vitro drug release was studied using a conventional dialysis method.⁴² Briefly, 1 mL of Fe₃O₄@PB@Gel–DOX NPs with a concentration of 1 mg mL⁻¹ was placed in a dialysis bag, which was immersed in a vial containing 25 mL of PBS (10 mM, pH = 7.4, with or without 1 μg mL⁻¹ gelatinase) and incubated at 37 °C for 48 h under stirring. At predetermined time points, 2 mL of release medium was withdrawn from the vial, which was replenished with 2 mL of fresh PBS. The released DOX was analyzed using a calibration curve derived from fluorescence spectra (excitation at 480 nm, emission at 590 nm) using a fluorescent spectrometer (PerkinElmer, Waltham, MA, USA).

Measurement of photothermal effect

3 mL aqueous dispersions of Fe₃O₄@PB@Gel–DOX NPs with various concentrations in cuvettes (4 mL) were irradiated by a continuous-wave diode NIR laser (output power = 2 W, wavelength = 808 nm, Sintec Optronics Technology, Singapore) for 10 min. The solution temperature was monitored by a digital thermometer at intervals of 10 s during the irradiation process. The photothermal stability of Fe₃O₄@PB@Gel–DOX NPs was characterized by continuous laser irradiation for four cycles. Specifically, the NP suspension was first irradiated for 10 min, followed by cooling to room temperature before the next irradiation.

Magnetic field enhanced photothermal effect

The magnetic field enhanced photothermal cell ablation effect of Fe₃O₄@PB@Gel–DOX NPs was tested using Hela cells, a human cervical carcinoma cell line. The cells were seeded into a 12-well plate with fresh culture medium at a density of 10⁵ cells per well. After overnight incubation in a humidified atmosphere containing 5% CO₂ at 37 °C, the culture medium was replaced by fresh medium containing Fe₃O₄@PB@Gel–DOX NPs (1 mL, 25 μg mL⁻¹). To apply the magnetic field, a permanent magnet was placed beneath the cell culture plate for 2 h. For the control group, cells were cultured without any magnet for 2 h. Afterwards, the cells were irradiated by a NIR laser (output power = 2 W, wavelength = 808 nm) for 10 min. After further incubation for 1 h, cells were stained with calcein AM for live cell imaging (green fluorescence), resulting from hydrolyzation of calcein AM by intracellular esterase in live cells.

The cell viability was evaluated after application of the magnetic field, laser or/and drug treatment using a PrestoBlue assay. Hela cells were seeded onto 96-well plates and incubated at 37 °C for 12 h. Then, the culture medium was replaced with 200 μL fresh medium containing Fe₃O₄@PB@Gel–DOX NPs or Fe₃O₄@PB@Gel NPs with gradient concentrations from 0 to 80 μg mL⁻¹. After 4 h incubation, the cells were exposed to NIR laser irradiation (2 W, 808 nm) for 10 min. For the group subjected to the magnetic field, a permanent magnet was placed beneath the 96-well plates for 2 h, followed by 2 h incubation without magnet. After further incubation for 24 h, cell viability was determined using the PrestoBlue cell viability assay.

Flow cytometry analysis

1 × 10⁵ Hela cells were seeded into a 12-well cell culture plate and incubated at 37 °C with 5% CO₂ for 12 h. Then, the old medium was replaced by 100 μg mL⁻¹ of Fe₃O₄@PB@Gel–DOX NPs dispersed in fresh culture medium. The cultures incubated with NPs were divided into two groups. The first group was incubated for 24 h without a magnetic field. The second group was subjected to a magnetic field by placing a permanent magnet beneath the 12-well plates for 2 h, followed by further incubation for 22 h. Subsequently, excess NPs were thoroughly washed away using 1× PBS three times and attached cells were resuspended in 500 μL of 1× PBS for flow cytometry (LSRII, BD Biosciences). The fluorescence of Hela cells was detected using a FITC channel (excitation = 488 nm, emission = 500–560 nm) with 1 × 10⁴ gated cells. The cells without treatment of NPs were used as blank controls. The data were further analyzed using FlowJo software.

Statistical analysis

Statistical significance was analyzed using one-way analysis of variance (ANOVA) with OriginPro 8.5.1 (OriginLab, MA, USA). A p-value of less than 0.05 (*p < 0.05, n = 4) was considered to be statistically significant between two groups.

Results and discussion

NP preparation and characterization

The conjugated gelatin–DOX complex easily dispersed in aqueous conditions (Fig. 2a), and showed a strong UV-vis absorption peak at 490 nm (Fig. 2b) attributed to the conjugated doxorubicin.^30,37 To coat the PB onto Fe₃O₄ NPs, the magnetic nanocore functioned as a nucleation site for PB precipitation. The core–shell structure was formed under acidic conditions with Fe³⁺ ions preferentially created on the nanocore surface, which further conjugated with hexacyanoferrate ions to produce a PB coating layer on the Fe₃O₄ nanocore.^38,39 Simultaneously, gelatin–DOX was introduced as a protective colloid to stabilize the core–shell structure and form monodispersed Fe₃O₄@PB@Gel–DOX NPs.²⁵ The purified NPs were harvested by magnetic separation to remove impurities.

Fig. 2 (a) Images of aqueous dispersion of Fe₃O₄ NPs, PB NPs, Gel–DOX conjugates and Fe₃O₄@PB@Gel–DOX NPs; (b) UV-vis-NIR absorbance spectra of Fe₃O₄ NPs, PB NPs, Gel–DOX conjugates and Fe₃O₄@PB@Gel–DOX NPs dispersed in DI water; (c) TEM image of Fe₃O₄@PB@Gel–DOX NPs; (d) the size distribution of Fe₃O₄@PB@Gel–DOX NPs measured by dynamic light scattering; (e) UV-vis-NIR absorbance spectra of Fe₃O₄@PB@Gel–DOX NPs dispersed in DI water under various concentrations; (f) Fe₃O₄@PB@Gel–DOX NPs dispersed in DMEM medium with gradient concentrations (from left to right: 100, 80, 60, 40, 20 and 0 μg mL⁻¹).

Transmission electron microscopy (TEM) was used to characterize the morphology of the Fe₃O₄@PB@Gel–DOX NPs, which have diameter of 20–25 nm (Fig. 2c). This is consistent with the hydrodynamic diameter of 22.7 ± 3.1 nm measured by the dynamic light scattering (DLS) method (Fig. 2d). The diameter of pure Fe₃O₄ NPs was measured as 12–15 nm (Fig. S1 and S2, ESI†). Therefore, the thickness of the nanoshell comprising PB and Gel–DOX can be estimated as 8–10 nm. The NPs were able to uniformly disperse in a colloidal form as a result of the presence of gelatin–DOX. The thickness of the PB nanoshell could be controlled by varying the amount of hexacyanoferrate ions added during the reaction. However, the magnetic properties of NPs were reduced when the nanoshell was thick. The aqueous dispersion of Fe₃O₄@PB@Gel–DOX NPs exhibited a dark green color, which could be attributed to color overlap among blue PB nanoshells, the brown Fe₃O₄ nanocore and the red gelatin–DOX complex (Fig. 2a). Compared with the pure Fe₃O₄ NPs, the complex Fe₃O₄@PB@Gel–DOX NPs exhibited peak absorbance in the spectrum of 700–800 nm (Fig. 2b), indicating good potential as a NIR-induced photothermal agent. The major absorbance at 600–900 nm is mainly attributed to the Prussian blue component in PB NPs and Fe₃O₄@PB@Gel–DOX NPs, or more specifically, the charge transfer between Fe²⁺ and Fe³⁺.²⁰ The absorbance intensity is dependent on the concentration of Prussian blue in the sample (Fig. 2e). Fig. 2b shows that Fe₃O₄@PB@Gel–DOX NPs (100 μg mL⁻¹) exhibited lower absorbance intensity compared with PB NPs (100 μg mL⁻¹). This is because the Fe₃O₄ nanocore and the conjugated Gel–DOX complex reduced the effective proportion of Prussian blue component in the synthesized complex NPs. The absorbance intensity increased almost linearly with the NP concentration, indicating their excellent dispersity under aqueous conditions (Fig. 2e). Furthermore, Fe₃O₄@PB@Gel–DOX NPs also could be uniformly dispersed in DMEM culture medium without aggregation for at least 3 days (Fig. 2f).

Magnetic property

Superparamagnetism is a critical property of NPs for drug delivery guided or enhanced by a magnetic field. A permanent magnet was placed against cuvettes containing aqueous dispersions of NPs to demonstrate their magnetic responses (Fig. 3a). Both Fe₃O₄ NPs and Fe₃O₄@PB@Gel–DOX NPs exhibited fast aggregation toward the magnet within 5 min, and redispersed quickly with a gentle shake after removal of the magnet. Further characterization using field-dependent magnetization at 25 °C (Fig. 3b) showed no hysteresis loop or remnant magnetization, indicating the superparamagnetism of Fe₃O₄ NPs, Fe₃O₄@PB NPs and Fe₃O₄@PB@Gel–DOX NPs. Compared with Fe₃O₄ NPs (63.6 emu g⁻¹) and Fe₃O₄@PB NPs (47.5 emu g⁻¹), Fe₃O₄@PB@Gel–DOX NPs exhibited a lower saturation magnetization value (31.6 emu g⁻¹), which implies that the nanoshells surrounding the Fe₃O₄ nanocores can reduce its magnetism. On the other hand, the Fe₃O₄ nanocores contribute to the outstanding superparamagnetic property of Fe₃O₄@PB@Gel–DOX NPs.

Fig. 3 Characterization of the magnetic properties of Fe₃O₄ NPs, Fe₃O₄@PB NPs and Fe₃O₄@PB@Gel–DOX NPs: (a) particle aggregation toward a permanent magnet; (b) magnetic hysteresis loops at 25 °C.

In vitro drug release

Enzyme triggered drug release from Fe₃O₄@PB@Gel–DOX NPs was investigated in vitro in PBS solution with or without gelatinase (Fig. 4). In the absence of gelatinase, 18.6% of drug was released during the first 6 h, which could be because of slight thermolysis of gelatin–DOX conjugates during NP synthesis. Moreover, only 21.2% cumulative drug release was observed in 48 h, suggesting a relatively high drug retaining efficiency in physiological conditions. In contrast, in the presence of gelatinase, 54.3% of drug release was detected within the first 6 h, which is ∼2-fold higher than the release rate in the system without enzyme, indicating efficient enzyme-triggered hydrolyzation of gelatin–DOX complex. Cumulatively, 72.2% of drug was released during 48 h. It is speculated that the incomplete drug release could be a result of residual gelatin on the surface of NPs. The results demonstrate that enzyme-mediated drug release from Fe₃O₄@PB@Gel–DOX NPs is good.

Fig. 4 In vitro characterization of enzyme-responsive drug release from Fe₃O₄@PB@Gel–DOX NPs.

Photothermal effect

The aqueous solution containing Fe₃O₄@PB@Gel–DOX NP exhibits strong absorbance in the spectrum of 700–800 nm (peak at 710 nm), indicating its good potential as a photothermal agent for NIR-induced PTT. The photothermal property of Fe₃O₄@PB@Gel–DOX was characterized by monitoring temperature elevation of 3 mL of NP aqueous dispersion (0, 10, 50, 100 and 200 μg mL⁻¹) under NIR laser irradiation for 10 min. The Fe₃O₄@PB@Gel–DOX NP dispersion exhibited significant temperature increase during laser irradiation, whereas no obvious temperature change was observed in the DI water (Fig. 5a). Moreover, the temperature elevation rate was highly dependent on the concentration of Fe₃O₄@PB@Gel–DOX NPs. At an NP concentration of 100 μg mL⁻¹, the temperature elevation can reach up to 45.6 °C within 10 min, exceeding the critical temperature (43 °C) required for tumor cell ablation.

Fig. 5 (a) Temperature elevation of Fe₃O₄@PB@Gel–DOX NP dispersions at various concentrations under NIR laser irradiation; (b) temperature variations of Fe₃O₄@PB@Gel–DOX NP dispersions (50 μg mL⁻¹) under repeated NIR irradiation for four cycles.

Photothermal stability is related to the capability of the NP agent to maintain photothermal conversion efficiency and structural integrity after repeated laser irradiation. Excellent photothermal stability is an essential feature of an NIR laser-driven photothermal agent for continuous use in clinical applications. There are many indirect methods for evaluation of photothermal stability, such as using temperature changes, color changes or variation in the UV-vis-NIR spectrum after repeated irradiation. In this study, the photothermal stability of Fe₃O₄@PB@Gel–DOX NPs was investigated by periodically exposing the NP dispersion to NIR laser irradiation. The temperature variation of the NP dispersion during four consecutive irradiation cycles (∼42 min for each cycle including 10 min irradiation) is shown in Fig. 5b. The peak temperature elevation decayed less than 1% after four irradiation cycles, indicating the excellent photothermal stability of Fe₃O₄@PB@Gel–DOX NPs as an efficient PTT agent.

Magnetic field enhanced photothermal and chemo-toxicity

To evaluate the photohyperthermic effect on tumor cell ablation under a magnetic field, Hela cells were incubated with Fe₃O₄@PB@Gel–DOX NPs with or without external magnetic field prior to NIR laser irradiation. Fluorescence microscopy images of Hela cells stained with calcein AM after various treatments (Fig. 6) show the viable cells. Compared with the negative control (Fig. 6a), no obvious change in cell viability was observed in the presence of either laser irradiation or NPs alone (Fig. 6b–d). However, a partially ablated (dark green) region was observed in Hela cells treated by both NPs (25 μg mL⁻¹) and NIR laser irradiation but without magnetic field, indicating a certain level of hyperthermic effect on Hela cells, although this was insufficient for complete cell ablation (Fig. 6e). With the aid of an external magnetic field, a completely ablated (dark) region was observed under the same bulk concentration of NPs and NIR laser irradiation (Fig. 6f), indicating significant cell toxicity caused by NIR-induced hyperthermic destruction. The dark region corresponded well with the laser irradiation spot, while the cells outside the laser irradiation spot remained viable and exhibited vivid green fluorescence. These results suggest that the aggregated NPs under a localized magnetic field can significantly enhance the photothermal ablation of tumor cells.

Fig. 6 Photothermal cell destruction: fluorescence images of Hela cells with various treatments with Fe₃O₄@PB@Gel–DOX NP dispersion (25 μg mL⁻¹) after calcium AM staining: (a) agent−, irradiation−; (b) agent−, irradiation+; (c) agent+, irradiation−; (d) agent+, irradiation−, magnetic field on; (e) agent+, irradiation+; (f) agent+, irradiation+, magnetic field on. NIR laser irradiation was performed at a wavelength of 808 nm with power of 2 W for 10 min. A magnetic field was applied by placing a permanent magnet beneath the cell culture plate for 2 h.

The chemo-cytotoxicity of Fe₃O₄@PB@Gel–DOX NPs was investigated using a PrestoBlue cell viability assay (Fig. 7a). In the absence of an external magnetic field and laser irradiation, the cell viability generally decreased with increasing NP concentration from 0 to 80 μg mL⁻¹. The results showed that Fe₃O₄@PB@Gel–DOX NPs had some inhibitory effect on growth of Hela cells, which could be caused by leached doxorubicin. After NPs were taken up by Hela cells through endocytosis, gelatin–DOX conjugates were hydrolyzed by endogenous gelatinase overexpressed in Hela cells.⁴⁰ When the magnetic field was applied, the cell viability was further reduced, particularly at high NP concentration (80 μg mL⁻¹). For combined photothermal- and chemo-therapy (Fig. 7b), a significant reduction was observed in cell viability after Hela cells were treated with NPs at concentrations higher than 10 μg mL⁻¹. Most importantly, there were prominent differences between the groups subjected to a magnetic field and the control groups at NP concentrations from 10 to 40 μg mL⁻¹.

Fig. 7 Viability assay of Hela cells treated with different dosages of Fe₃O₄@PB@Gel–DOX NPs: (a) without NIR laser irradiation; (b) under NIR laser irradiation (808 nm, 2 W) for 10 min. Data are shown as mean ± SD (n = 4; *p < 0.05).

Previous studies reported that inorganic NPs with similar diameters such as Fe₃O₄ NPs⁴³ and PB NPs⁴⁴ could be efficiently taken up by viable cells through endocytosis. Moreover, there is evidence that an external magnetic field can increase the number of magnetic NPs attaching to the cell surface because of local concentration, and thus enhance the cellular uptake.^45,46 Flow cytometry analysis further confirmed that application of a magnetic field can enhance the internalization of Fe₃O₄@PB@Gel–DOX NPs by individual Hela cells (Fig. 8).† The fluorescence intensity of the Hela cells treated with NPs was much higher than that of the unlabeled cells. The fluorescence became even stronger in cells subjected to a magnetic field, indicating a higher level of NP intake. The major findings in the present study are all consistent with the previously reported phenomena, indicating that application of a magnetic field can dramatically enhance therapeutic efficiency of Fe₃O₄@PB@Gel–DOX NPs through combined photothermal- and chemo-ablation of tumor cells.

Fig. 8 Flow cytometry analysis on cellular uptake of Fe₃O₄@PB@Gel–DOX NPs by Hela cells after 24 h incubation with and without magnetic field: (a) histograms; (b) dot plots. The control represents the unlabeled cells.

Conclusion

Magnetic Prussian blue nanoparticles loaded with doxorubicin (Fe₃O₄@PB@Gel–DOX) were designed successfully and synthesized for combined chemo- and photothermal therapy enhanced by a magnetic field, which can efficiently eradicate tumor cells under appropriate dosages and NIR irradiation through a synergistic therapeutic effect. The key components, PB, DOX, Fe₃O₄ and gelatin, are all typical FDA-approved materials, which ensures the biosafety of the multifunctional NPs. Moreover, multimodality therapeutics require a much smaller amount of drug, and thus could potentially reduce any side effects related to overdosage of anti-cancer drugs in conventional chemotherapy. Magnetic NPs based on Fe₃O₄ have been explored extensively as contrast agents for enhanced magnetic resonance imaging (MRI).^47,48 The Fe₃O₄@PB@Gel–DOX NP developed in this study contains a nanocore made of Fe₃O₄. Therefore, it could be used also as a MRI contrast agent with multiple functions, such as MRI-guided photothermal therapy. Further studies in vivo are necessary to systematically characterize the performance of this therapeutic nanosystem, which are beyond the scope of the present study because of limited time and resources. Such a versatile therapeutic system could foster innovations in future multifunctional nanoplatforms for cancer treatment.

Acknowledgements

Y.K. gratefully acknowledges the Tier 2 Academic Research Fund (ARC 22/13) and Tier 1 Academic Research Fund (RG 37/14) from the Ministry of Education of Singapore. Y.Z. acknowledges the start-up research grant from the Nanyang Technological University and the Tier-1 Academic Research Funds from the Singapore Ministry of Education (RGT 30/13 and RGC 6/13). P.X. is grateful for the assistance by Cangjie Yang for TEM and Shiying Liu for flow cytometry.

Notes and references

1. A. Jemal, R. Siegel, J. Q. Xu and E. Ward, Ca-Cancer J. Clin., 2010, 60, 277–300.
2. M. K. Yu, J. Park and S. Jon, Theranostics, 2012, 2, 3–44.
3. C. E. Ashley, E. C. Carnes, G. K. Phillips, D. Padilla, P. N. Durfee, P. A. Brown, T. N. Hanna, J. W. Liu, B. Phillips, M. B. Carter, N. J. Carroll, X. M. Jiang, D. R. Dunphy, C. L. Willman, D. N. Petsev, D. G. Evans, A. N. Parikh, B. Chackerian, W. Wharton, D. S. Peabody and C. J. Brinker, Nat. Mater., 2011, 10, 389–397.
4. M. E. Gindy and R. K. Prud'homme, Expert Opin. Drug Delivery, 2009, 6, 865–878.
5. C. X. Guo, Y. S. Jin and Z. F. Dai, Bioconjugate Chem., 2014, 25, 840–854.
6. V. P. Torchilin, Adv. Drug Delivery Rev., 2006, 58, 1532–1555.
7. J. Yang, J. Choi, D. Bang, E. Kim, E. K. Lim, H. Park, J. S. Suh, K. Lee, K. H. Yoo, E. K. Kim, Y. M. Huh and S. Haam, Angew. Chem., Int. Ed., 2011, 50, 441–444.
8. D. Jaque, L. M. Maestro, B. del Rosal, P. Haro-Gonzalez, A. Benayas, J. L. Plaza, E. M. Rodriguez and J. G. Sole, Nanoscale, 2014, 6, 9494–9530.
9. X. H. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. Chem. Soc., 2006, 128, 2115–2120.
10. H. K. Moon, S. H. Lee and H. C. Choi, ACS Nano, 2009, 3, 3707–3713.
11. L. F. F. Neves, J. J. Krais, B. D. Van Rite, R. Ramesh, D. E. Resasco and R. G. Harrison, Nanotechnology, 2013, 24, 375104.
12. Y. Hashida, H. Tanaka, S. W. Zhou, S. Kawakami, F. Yamashita, T. Murakami, T. Umeyama, H. Imahori and M. Hashida, J. Controlled Release, 2014, 173, 59–66.
13. H. T. Ke, J. R. Wang, Z. F. Dai, Y. S. Jin, E. Z. Qu, Z. W. Xing, C. X. Guo, X. L. Yue and J. B. Liu, Angew. Chem., Int. Ed., 2011, 50, 3017–3021.
14. J. Lin, S. J. Wang, P. Huang, Z. Wang, S. H. Chen, G. Niu, W. W. Li, J. He, D. X. Cui, G. M. Lu, X. Y. Chen and Z. H. Nie, ACS Nano, 2013, 7, 5320–5329.
15. B. Jang, J. Y. Park, C. H. Tung, I. H. Kim and Y. Choi, ACS Nano, 2011, 5, 1086–1094.
16. M. Zhou, R. Zhang, M. A. Huang, W. Lu, S. L. Song, M. P. Melancon, M. Tian, D. Liang and C. Li, J. Am. Chem. Soc., 2010, 132, 15351–15358.
17. Y. B. Li, W. Lu, Q. A. Huang, M. A. Huang, C. Li and W. Chen, Nanomedicine, 2010, 5, 1161–1171.
18. Y. Huang, Y. Lai, S. Shi, S. Hao, J. Wei and X. Chen, Chem.–Asian. J., 2015, 10, 370–376.
19. U.S. Food and Drug Administration, www.fda.gov.
20. G. L. Fu, W. Liu, S. S. Feng and X. L. Yue, Chem. Commun., 2012, 48, 11567–11569.
21. H. A. Hoffman, L. Chakrabarti, M. F. Dumont, A. D. Sandler and R. Fernandes, RSC Adv., 2014, 4, 29729–29734.
22. P. Xue, K. K. Y. Cheong, Y. F. Wu and Y. Kang, Colloids Surf., B, 2015, 125, 277–283.
23. G. L. Fu, W. Liu, Y. Y. Li, Y. S. Jin, L. D. Jiang, X. L. Liang, S. S. Feng and Z. F. Dai, Bioconjugate Chem., 2014, 25, 1655–1663.
24. M. Shokouhimehr, E. S. Soehnlen, A. Khitrin, S. Basu and S. P. D. Huang, Inorg. Chem. Commun., 2010, 13, 58–61.
25. F. Shiba, Colloids Surf., A, 2010, 366, 178–182.
26. B. Gaihre, S. Aryal, N. A. M. Barakat and H. Y. Kim, Mater. Sci. Eng., C, 2008, 28, 1297–1303.
27. S. J. Byrne, Y. Williams, A. Davies, S. A. Corr, A. Rakovich, Y. K. Gun'ko, Y. P. Rakovich, J. F. Donegan and Y. Volkov, Small, 2007, 3, 1152–1156.
28. R. E. Sawaya, M. Yamamoto, Z. L. Gokaslan, S. W. Wang, S. Mohanam, G. N. Fuller, I. E. McCutcheon, W. G. StetlerStevenson, G. L. Nicolson and J. S. Rao, Clin. Exp. Metastasis, 1996, 14, 35–42.
29. W. G. Stetlerstevenson, S. Aznavoorian and L. A. Liotta, Annu. Rev. Cell Biol., 1993, 9, 541–573.
30. Z. B. Zha, S. H. Zhang, Z. J. Deng, Y. Y. Li, C. H. Li and Z. F. Dai, Chem. Commun., 2013, 49, 3455–3457.
31. H. S. Yoo and T. G. Park, J. Controlled Release, 2001, 70, 63–70.
32. A. Kakinoki, Y. Kaneo, Y. Ikeda, T. Tanaka and K. Fujita, Biol. Pharm. Bull., 2008, 31, 103–110.
33. X. X. Ma, H. Q. Tao, K. Yang, L. Z. Feng, L. Cheng, X. Z. Shi, Y. G. Li, L. Guo and Z. Liu, Nano Res., 2012, 5, 199–212.
34. J. Zhang, M. C. Shin and V. C. Yang, Pharm. Res., 2014, 31, 579–592.
35. B. Polyak and G. Friedman, Expert Opin. Drug Delivery, 2009, 6, 53–70.
36. Y. X. Wang, Quantitative Imaging in Medicine and Surgery, 2011, 1, 35–40.
37. D. C. Wu, C. R. Cammarata, H. J. Park, B. T. Rhodes and C. M. Ofner, Pharm. Res., 2013, 30, 2087–2096.
38. C. Thammawong, P. Opaprakasit, P. Tangboriboonrat and P. Sreearunothai, J. Nanopart. Res., 2013, 15, 1689.
39. X. Q. Zhang, S. W. Gong, Y. Zhang, T. Yang, C. Y. Wang and N. Gu, J. Mater. Chem., 2010, 20, 5110–5116.
40. M. W. Roomi, J. C. Monterrey, T. Kalinovsky, M. Rath and A. Niedzwiecki, Oncol. Rep., 2010, 23, 605–614.
41. J. Salado, M. Insausti, L. Lezama, I. G. de Muro, E. Goikolea and T. Rojo, Chem. Mater., 2011, 23, 2879–2885.
42. N. Chidambaram and D. J. Burgess, AAPS PharmSciTech, 1999, 1, 32–40.
43. Y. J. Ma and H. C. Gu, J. Mater. Sci.: Mater. Med., 2007, 18, 2145–2149.
44. M. Shokouhimehr, E. S. Soehnlen, J. H. Hao, M. Griswold, C. Flask, X. D. Fan, J. P. Basilion, S. Basu and S. P. D. Huang, J. Mater. Chem., 2010, 20, 5251–5259.
45. Z. Cao, X. L. Yue, X. D. Li and Z. F. Dai, Langmuir, 2013, 29, 14976–14983.
46. C. Plank, O. Zelphati and O. Mykhaylyk, Adv. Drug Delivery Rev., 2011, 63, 1300–1331.
47. S. Santra, C. Kaittanis, J. Grimm and J. M. Perez, Small, 2009, 5, 1862–1868.
48. F. Yang, Y. X. Li, Z. P. Chen, Y. Zhang, J. R. Wu and N. Gu, Biomaterials, 2009, 30, 3882–3890.

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Footnotes

† Electronic supplementary information (ESI) available: Additional scheme and figures. See DOI: 10.1039/c5ra01616a

‡ These authors contributed equally to this work.

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