Doxorubicin-loaded ionic liquid–polydopamine nanoparticles for combined chemotherapy and microwave thermal therapy of cancer

Abstract

Recently, combinational therapy has been increasingly employed in the treatment of cancer. In this study, hollow polydopamine nanoparticles were developed as favorable biocompatible delivery nanoplatforms for chemotherapy and microwave thermal therapy. By loading polydopamine nanoparticles with ionic liquid, the functionalized polydopamine nanoparticles became chemotherapeutic drug nanocarriers for combinational therapy. The obvious antitumor efficacy of doxorubicin-loaded ionic liquid–polydopamine nanocomposites was demonstrated in in vitro and in vivo experiments for combined chemotherapy and microwave thermal therapy. Moreover, the combination of chemotherapy with microwave thermal therapy applied to cancer therapy based on drug-loaded ionic liquid–polydopamine nanocomposites is a promising therapy for future cancer treatment in clinical applications.

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Introduction

The common methods of tumor therapy in clinical applications, such as chemotherapy, radiotherapy and surgery, are not able to completely cure cancer because of their many disadvantages.^1–3 Chemotherapy, a traditional treatment, is disadvantaged by the nonspecific biodistribution, lack of water solubility, less effective concentration, and toxic side effects.^4–6 Combinational therapy based on two or more therapies has been receiving remarkable attention in recent years.^7–12 Bao and his co-workers synthesized PEGylated plasmonic MoO_3−x hollow nanospheres for the photoacoustic imaging guided chemo-photothermal combinational therapy of cancer.¹³ Liu and his co-workers developed PEGylated MoS₂ nanosheets for combined photothermal and photodynamic therapy.¹⁴ Zhong's group produced a biocompatible multifunctional nanocarrier for combined radioisotope therapy and chemotherapy of cancer.¹⁵ These study efforts have provided certain guiding significance to cancer therapy.

Currently, microwave thermal therapy (MWTT) has attracted tremendous interest for its excellent properties such as non-intrusive, deep penetration, less damage to normal tissues and splendid antitumor effects.¹⁶ MWTT has been used in some parenchymal organ tumors such as liver cancer, with excellent efficiency.¹⁷ However, as a potential treatment method, combinational therapy involving chemotherapy and MWTT with desired therapeutic effects has not yet been achieved because chemotherapy drugs cannot effectively reach the tumor site. An advanced multifunctional delivery carrier that can deliver chemotherapy drugs to tumor sites and enhance MWTT efficiency is urgently needed.

To improve the efficiency of the delivery of the drug to the tumor sites, nanoparticles have been used as delivery carriers due to their special structures and functions such as tunable size, passive tumor targeting based on enhanced permeability and retention (EPR) effect, high loading rate and low cytotoxicity.^18–29 Various nanocarriers, including polymers,^30–33 carbon nanostructures^34,35 and metallic compounds,^36–38 are being explored. Among these drug delivery systems, polydopamine (PDA) nanoparticles have been developed for tumor therapy. PDA can form coatings on multifarious substrates to shape nanostructures with excellent biocompatibility.^39–43 These nanoparticles are an ideal candidate for double-effect delivery of chemotherapy and MWTT. However, there have been no reports about PDA as the drug delivery carrier for combined chemotherapy and MWTT.

In this study, PDA nanoparticles with hollow structures were synthesized as multifunctional nanocarriers (Scheme 1). Ionic liquid (IL) with high susceptibility to microwave irradiation⁴⁴ was loaded into the hollow PDA nanoparticles as a microwave sensitization agent (IL–PDA) and doxorubicin (DOX) was loaded on the IL–PDA nanoparticles for chemotherapy and MWTT. In vitro cytotoxicity tests and tumor-burdened animal experimental results demonstrated the high antitumor efficiency of the combinational therapy based on the special nanostructures. Furthermore, the good biocompatibility of the nano-composites was also studied in vitro and in vivo. As a result, the DOX-loaded IL–PDA nanocomposites (IL–PDA–DOX) were proved to be promising nanoagents for coordinating chemotherapy with MWTT to treat cancer.

Scheme 1 Schematic of the synthetic process of IL–PDA–DOX nanocomposites for combined chemotherapy with MWTT in the treatment of cancer.

Experimental

Materials

Dopamine hydrochloride (DA) was purchased from Sigma-Aldrich Co. Ltd. (Shanghai, China). 1-Butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF₆) was purchased from Chengjie Chemical Co. Ltd. (Shanghai, China). 1,4-Dioxane was purchased from Beijing Chemical Reagents Company (China). DOX was purchased from Beijing Huafeng United Technology Co. Ltd. All the chemicals in this study were used without further purification.

Characterization

Transmission electron microscopy (TEM, JEM-2100, JEOL) and scanning electron microscopy (SEM, Models 4300, Hitachi) were used to characterize the morphology and size distribution of IL–PDA–DOX nanocomposites. Fourier transform infrared spectrometry (FT-IR, Varian, Model 3100 Excalibur) was used to examine the composition and surface functional groups. UV-Visible spectra were obtained using a JASCO V-570 spectrophotometer. Infra-red thermal mapping in this study was monitored via a FLIR SC620 apparatus. The images of ICR mice bearing H22 tumors were taken with a Canon DS126231 digital camera. Photomicrographs of the tissue samples were captured with an Olympus X71 microscope.

Synthesis of IL–PDA–DOX nanocomposites

IL–PDA–DOX nanocomposites were synthesized via a three-step method. First, hollow PDA nanoparticles were synthesized by a template method. In brief, 20 mL/4 mL/55.04 mg/50 μL of deionized water/alcohol/SiO₂ nanoparticles/ammonia were mixed and stirred for 30 min. Then, 60 mg of DA were added and stirred for another 16 h, and 0.9 mL of HF solution was added to the mixture. The as-prepared hollow PDA nanoparticles were collected by centrifugation and washed with deionized water three times.

Second, 0.5 mL/0.5 mL/8 mL of IL/1,4-dioxane/alcohol were mixed equably and 1 mL of hollow PDA nanoparticles (10 mg mL⁻¹) was added and then the reaction system was retained for 30 min under vacuum conditions using a water pump. The as-prepared IL–PDA nanoparticles were obtained.

Finally, DOX was loaded on the IL–PDA nanoparticles. In brief, 1 mL DOX (10 mg mL⁻¹) was added to 10 mL of IL–PDA nanoparticles (1 mg mL⁻¹) dispersed in deionized water and stirred for 16 h. The obtained DOX-loaded IL–PDA (IL–PDA–DOX) nanocomposites were collected by centrifugation and washed with PBS buffer three times. The supernatants were collected for DOX concentration measurement.

Microwave sensitization of IL–PDA–DOX in vitro

The IL–PDA–DOX nanocomposites (20 mg) were dispersed in saline (1 mL) in the microwave plate, then irradiated by a microwave device (Beijing Muheyu Electronics Co. Ltd) for 5 min. The power of the microwave was 1.5 W. The temperature of the solution was monitored with an optical fiber temperature sensor (Beijing Dongfang Ruizhe Technology Co. Ltd). Saline was used as the control group. The heating process was monitored using a FLIR SC620 apparatus. The infra-red images were taken at one minute intervals.

Cytotoxicity of PDA and IL–PDA nanoparticles toward cancer cells in vitro

Cytotoxicity was evaluated by MTT assay. HepG2 cells were seeded on 96-well plates at a density of 7000 cells per well and incubated at 37 °C in 5% CO₂ for 24 h. Different concentrations of nanomaterials were added into the wells and cultured for another 24 h. The relative cell viabilities were then determined by MTT assay.

Chemotherapy effects of IL–PDA–DOX without MW irradiation on cancer cells

HepG2 and Hela cells were seeded in a 96-well plate at a density of 7000 cells per well and incubated at 37 °C in 5% CO₂ for 24 h. Different concentrations of IL–PDA–DOX nanocomposites were added to the wells and cultured for another 24 h. The relative cell viabilities were then determined by MTT assay.

Study of the combination anticancer effects of IL–PDA–DOX in vitro under MW irradiation

IL–PDA–DOX nanocomposites diluted in DMEM medium at different concentrations (50, 25, 12.5, 6.25, 3.125, 0 μg mL⁻¹) were added to a plate and mixed uniformly with HepG2 cells. The mixture was then irradiated by MW for 5 min. After 4 h incubation, the relative cell viabilities were determined by MTT assay and LDH assay.

Cancer combination therapy experiment in vivo

All animal experiments in this study were performed in accordance with the Institutional Animal Care and Use Committee of Technical Institute of Physics and Chemistry. ICR mice bearing H22 tumors were divided into 5 groups randomly (n = 4 per group) and treated with (1) PBS, (2) free DOX, (3) IL–PDA–DOX, (4) IL–PDA + MW, (5) IL–PDA–DOX + MW. All the agents were intravenously injected. The power of the microwave was 1.5 W. The tumor volume and body weight of the mice were monitored every other day. After sixteen days of normal feeding, all the mice were sacrificed and the major organs (heart, liver, spleen, lungs and kidneys) were obtained for hematoxylin and eosin (H&E) staining. The tumor volume was calculated using the formula as follows: length × width × width/2.

Biocompatibility in vivo

Female ICR mice of similar quality were randomly divided into five groups (n = 5 per group) and injected with PBS, free DOX (8 mg kg⁻¹), IL–PDA–DOX nanocomposites (8 mg kg⁻¹, 5 mg kg⁻¹, 3 mg kg⁻¹ of DOX) via the tail vein. During the 14 days of normal feeding, the weight of the mice was monitored every other day. Then, all the mice were sacrificed and the major organs, such as heart, liver, spleen, lungs and kidneys, were obtained for H&E staining. The blood was collected for routine blood testing.

Statistical analysis

All data were collected as mean + standard deviation (SD). The level of statistical significance was taken at a value of p < 0.05.

Results and discussion

Synthesis and characterization of IL–PDA–DOX nanocomposites

The typical SEM and TEM images of SiO₂–PDA nanoparticles and IL–PDA–DOX nanocomposites are shown in Fig. 1a and b, respectively. The spherical morphology of SiO₂–PDA and hollow structures of IL–PDA–DOX are demonstrated. The size distributions of the synthetic IL–PDA nanoparticles and the IL–PDA–DOX nanocomposites were further characterized, and it was indicated that the as-prepared IL–PDA nanoparticles and IL–PDA–DOX nanocomposites were spherical and well proportioned, with an average diameter of 140 nm (ESI Fig. S1 and S2†). TEM images further revealed that the IL–PDA–DOX nanocomposites were obviously hollow. In addition, the thickness of PDA in the IL–PDA–DOX nanocomposites was about 25 nm (Fig. 1c). Zeta potentials of the as-synthesized PDA, IL–PDA and IL–PDA–DOX were −17.1, −15.1 and −19.9 mV, respectively (ESI Fig. S3†). The difference in surface charge proved that IL and DOX were successfully loaded. To investigate the DOX loading efficiency of IL–PDA–DOX nanocomposites, FTIR analyses were performed, as shown in Fig. 1d. The strong peaks at 2920 (C–H) and 1574 (N–H) cm⁻¹ on IL–PDA–DOX nanocomposites were attributed to DOX.⁴⁵ The predominant peak at 850 cm⁻¹ was attributed to P–F.⁴⁶ These results demonstrated that DOX and [Bmim]PF₆ were successfully connected on the PDA nanoparticles. TGA was carried out to further confirm the composition of the nanocomposites (ESI Fig. S4†). Three main weight loss rates are shown in the IL–PDA–DOX curve. Moreover, IL, PDA and DOX curves show that when the temperature was below 200 °C, only PDA showed weight loss. At 200 °C, DOX started to decompose. When the temperature reached 400 °C, IL began to decompose quickly. Therefore, the different weight loss rates from room temperature to 500 °C in IL–PDA–DOX curves were caused by PDA, DOX and IL, respectively. These results revealed that DOX and IL were successfully loaded. The loading ratio of IL could be measured by the IL, PDA, and IL–PDA TGA curves. Weight loss of IL, PDA, and IL–PDA were 100%, 36.1% and 50.6%, respectively. Therefore, according to calculation, the loading ratio of IL was 22.8%. The DOX loading was calculated according to the results of UV-Vis absorption spectra. Fig. 1e shows the UV-Vis absorption spectra of free DOX with different concentrations, with the peak located at 480 nm. Fig. 1f shows the standard curve of DOX obtained by curve fitting a series of UV-Vis absorption characteristic peaks. The loading rate of DOX was calculated to be 10.79% by following the UV-Vis absorption spectra of the supernatant in IL–PDA–DOX nanocomposites (the inset of Fig. 1f). To the best of our knowledge, tumor cells could be killed at about 42–45 °C.⁴⁷ The microwave heating properties of IL–PDA–DOX nanocomposites were determined using a microwave device and FLIR apparatus. The temperature of IL–PDA–DOX nanocomposites increased from 30.4 to 53.5 °C in less than 5 min of MW irradiation, whereas the saline solution only increased from 30 to 44.7 °C (Fig. 1g). The corresponding MW heating effects were visually demonstrated by infra-red images (Fig. 1h). Therefore, suitable MW heating effects were achieved for IL–PDA–DOX nanocomposites under MW irradiation. Based on these promising results, the excellent MW heating effects of IL–PDA–DOX made it possible to kill cancer cells by combining chemotherapy with MWTT.

Fig. 1 Fundamental characterization. (a) SEM and TEM images of SiO₂–PDA nanoparticles. (b) SEM and TEM images of IL–PDA–DOX nanocomposites. (c) Magnified TEM images of IL–PDA–DOX nanocomposites. (d) FTIR spectra of the IL–PDA–DOX nanocomposites, DOX, PDA nanoparticles and IL. (e) UV-Vis spectra of free DOX with different concentrations. (f) The standard curves of free DOX; the inset shows UV-Vis absorption spectra of supernatants from IL–PDA–DOX nanocomposites. (g) Heating curves of IL–PDA–DOX nanocomposites dispersed in saline solution (1 mL, 10 mg mL⁻¹) that were irradiated with MW for 5 min. (h) The FLIR images corresponding to (g); images were taken at one minute intervals.

Evaluation of cells in vitro

The premise for the biological applications of nanomaterials is their biocompatibility; therefore, the cytotoxicity of PDA and IL–PDA was measured. Fig. 2a shows that the PDA and IL–PDA nanoparticles were biocompatible with HepG2 cells. The low cytotoxicity demonstrated that IL–PDA exhibited good biocompatibility, which inspired us to further explore them as a promising nanoplatform for cancer treatment.

Fig. 2 The results of cell experiments (a) cytotoxicity of PDA and IL–PDA for HepG2 cells by MTT assay. (b) The inhibition rate in vitro of IL–PDA–DOX nanocomposites for different cells by MTT assay. (c) Evaluation of the influence of IL–PDA–DOX on HepG2 cells under microwave irradiation, by MTT assay. Data are shown as mean ± SD (n = 4).

To evaluate the therapeutic effect in vitro, the potential ability of IL–PDA–DOX nanocomposites to kill HepG2 and Hela cells without MW irradiation was carried out by MTT assay. For HepG2 cells, after being cultured together for 24 h, the inhibition rates of the IL–PDA–DOX nanocomposites were 26.9%, 42.8%, 50.5%, 56.3%, 59.5%, 62.0% and 66.9%, for 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 μg mL⁻¹, respectively (Fig. 2b); the inhibition rates of the IL–PDA–DOX nanocomposites against HepG2 cells were enhanced with the increase in concentration from 0.78 to 50 μg mL⁻¹. For Hela cells, a similar result was obtained. As a result, the cell-killing ability of IL–PDA–DOX was demonstrated.

To further demonstrate the in vitro therapeutic efficacy of the combined chemotherapy and MWTT, the relative cell viabilities of HepG2 cells treated with IL–PDA–DOX with or without MW irradiation were determined by MTT assay (Fig. 2c). Under MW irradiation, the viability of the cells decreased drastically as the concentration of IL–PDA–DOX nanocomposites increased, exhibiting higher cytotoxicity than the group without MW irradiation (Fig. 2c). To further explore the therapeutic efficacy, the relative cell viabilities of HepG2 cells treated with or without IL–PDA–DOX under MW irradiation were determined by LDH assay (ESI Fig. S5†). It is well known that the more serious the cell damage, the higher the LDH activity. The LDH activity of cells in the MW group was higher than in the control group. After incubating with IL–PDA–DOX nanocomposites and irradiating under MW, the LDH activities of the cells were all higher than that of the MW only group. Moreover, with the increase in concentrations of IL–PDA–DOX nanocomposites, the LDH activity of the cells was improved gradually. Both LDH and MTT assay results suggested that IL–PDA–DOX nanocomposites combined with MW irradiation greatly increased the possibility of killing tumor cells. It demonstrated that IL–PDA–DOX was a prospective agent for combined chemotherapy and MWTT for cancer treatment.

Combined chemotherapy and MWTT on tumor bearing mice

To monitor the MW thermal effect of IL–PDA–DOX nanocomposites in vivo, the temperature change in the tumor region was recorded with an IR thermal camera. Mice bearing H22 tumors were anesthetized and exposed to MW irradiation for 5 min (Fig. 3a). During the irradiation, the temperature in the region of the tumor injected with IL–PDA–DOX nanocomposites rapidly increased from 30.8 °C to 52.6 °C, which was a higher temperature than that required for tumor cell damage. When injected with saline, the temperature only increased from 30.5 °C to 41.5 °C under the same MW irradiation conditions. The antitumor experiment was designed to evaluate the combination effect of chemotherapy and MWTT in vivo. Mice bearing H22 tumors were randomly divided into five groups and received treatments with PBS, free DOX, IL–PDA + MW, IL–PDA–DOX, IL–PDA–DOX + MW, respectively. From the representative images of mice at day 0 and day 16 (Fig. 3b), the tumors treated with IL–PDA–DOX combined with MW disappeared, which implied that they were completely healed. The tumor volumes and body weights of all mice were recorded at day 16 (Fig. 3d and e). As shown in Fig. 3d, compared with the control group, the tumors in the mice treated with IL–PDA + MW were diminished two days after the treatment, but there was a relapse after six days. In the IL–PDA–DOX group, the tumors were decreased and completely ablated without regrowth, which might be attributed to DOX release from IL–PDA–DOX nanocomposites and cooperation with MWTT. The body weights increased gradually and there was no difference among these groups. After 16 days of normal feeding, all mice were sacrificed and the tumors were extracted and weighed. The tumors were photographed and mean tumor weights are shown in Fig. 3c and f. The tumors in the IL–PDA–DOX + MW group disappeared and the tumors in the IL–PDA + MW group were smaller than in the three other groups. The representative H&E staining images of main organs (heart, liver, spleen, lung and kidney) are shown in Fig. S6,† (ESI). There were no significant pathological changes in these tissues. In addition, H&E staining images of tumor tissue slices (Fig. 3g) illustrated that in the IL–PDA + MW group, the tissues showed large areas of necrosis when compared with the control group. An obvious antitumor efficacy of IL–PDA–DOX nanocomposites combined with MWTT was demonstrated via the above data.

Fig. 3 In vivo studies of combined chemotherapy and MWTT in ICR mice bearing H22 tumors (n = 4, per group). (a) Infrared thermal images of ICR mice in the MW and IL–PDA–DOX + MW groups; images were taken at 1 min intervals. (b) Images at days 0 and 16 of representative mice from each group. (c) Images of tumors in each group after excision. (d) Change in tumor volume in each group. (e) Change in mean body weights in each group. (f) Mean tumor weights in each group. (g) H&E staining images of tumor tissue in different groups.

The biocompatibility of IL–PDA–DOX nanocomposites in vivo

Biocompatibility evaluation was performed to examine the potential toxicity of IL–PDA–DOX nanocomposites in vivo. Mice with similar weight were divided into five groups and injected with PBS, free DOX (8 mg kg⁻¹) and IL–PDA–DOX nanocomposites by intravenous injection via the tail vein. No obvious adverse manifestations were observed during the experiments. The weights of the mice in the five groups were recorded every other day and the relative weights were calculated (ESI Fig. S7†). There were no significant differences among all the treated groups. Two weeks later, the mice were sacrificed and tissue sections of the heart, liver, spleen, lungs, and kidneys were retained for histology analysis. After staining with H&E, the tissue slices were observed under a microscope. As shown in Fig. 4, compared with the control group, no inflammation or histopathological abnormalities were observed in all organs except some black materials (red arrow in Fig. 4) in lung, kidney, liver and spleen tissues of I-8. The qualitative biodistribution of materials in the main organs was also revealed and the decreasing order of concentration was spleen, lung, liver, kidney and heart was observed. The blood of the mice was collected for routine blood testing (ESI Fig. S8†). No distinct diversifications were found statistically among all groups, which indicated that no palpable toxicity was caused by the IL–PDA–DOX nanocomposites when they were injected into the blood.

Fig. 4 Representative H&E staining images of organs (heart, liver, spleen, lung and kidney) for the biocompatibility of IL–PDA–DOX nanocomposites via tail vein injection in vivo in different groups. The dose of IL–PDA–DOX nanocomposites was (8 mg kg⁻¹, 5 mg kg⁻¹, 3 mg kg⁻¹) abbreviated as I-8, I-5 and I-3, respectively (scale bar: 100 μm).

Conclusions

Multifunctional IL–PDA–DOX nanocomposites were successfully developed for the combination of chemotherapy and MWTT to treat cancers. The loading rate of DOX reached 10.79% in the IL–PDA–DOX nanocomposites. IL–PDA–DOX nanocomposites possessed admirable microwave sensitization effects under lower microwave power. They also exhibited very high inhibition effects when combined with MW irradiation, and acted as an effective chemotherapy and MWTT agent for tumor ablation under MW irradiation without inducing any appreciable tissue toxicity. The drug loaded IL–PDA nanocomposites could be promising for cancer theranostics via the combination of chemotherapy and MWTT. To achieve more convenient and effective tumor theranostics, enhancement of the drug loading rate and incorporation of some materials with special properties, such as nuclear magnetic materials, fluorescent materials, and CT imaging materials, will be carried out in the future.

Acknowledgements

The financial of this work was supported by the National Hi-Technology Research and Development Program (863 Program) (no. 2013AA032201 and 2012AA022701) and the National Natural Science Foundation of China (NSFC) (61171049, 30800258, 31271075, 51202260 and 81201814).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental data. See DOI: 10.1039/c6ra02434c

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