Facile Fabrication of Magnetic Microrobots Based on Spirulina

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Biological and Medical Applications of Materials and Interfaces

Facile Fabrication of Magnetic Microrobots Based on Spirulina Templates for Targeted Delivery and Synergistic Chemo-Photothermal Therapy Xu Wang, Jun Cai, Lili Sun, Shuo Zhang, De Gong, Xinghao Li, Shuhua Yue, Lin Feng, and Deyuan Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15586 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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ACS Applied Materials & Interfaces

Facile Fabrication of Magnetic Microrobots Based on Spirulina Templates for Targeted Delivery and Synergistic Chemo-Photothermal Therapy Xu Wang†, Jun Cai*, †, ‡, Lili Sun†, Shuo Zhang‡, §, De Gong†, Xinghao Li†, Shuhua Yue‡, §, Lin Feng†, ‡, Deyuan Zhang†, ‡. †School

of Mechanical Engineering and Automation, Beihang University, No. 37 Xueyuan Road,

Haidian District, Beijing 100191, China ‡Beijing

Advanced Innovation Center for Biomedical Engineering, Beihang University, No. 37

Xueyuan Road, Haidian District, Beijing 100191, China §School

of Biological Science and Medical Engineering, Beihang University, No. 37 Xueyuan

Road, Haidian District, Beijing 100191, China KEYWORDS: magnetic microrobots, Spirulina, drug loading, targeted delivery, chemophotothermal therapy

ACS Paragon Plus Environment

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ABSTRACT

Magnetic microrobots can be actuated in fuel-free conditions and are envisioned for biomedical applications related to targeted delivery and therapy in a minimally invasive manner. However, mass fabrication of microrobots with precise propulsion performance and excellent therapeutic efficacy is still challenging, especially in a predictable and controllable manner. Herein, we propose a facile technique for mass-production of magnetic microrobots with multiple functions using Spirulina (Sp.) as biotemplate. The core-shell structured Pd@Au nanoparticles (NPs) were synthesized in Sp. cells by electroless deposition, working as photothermal conversion agents. Subsequently, the Fe3O4 NPs were deposited onto the surface of the obtained (Pd@Au)@Sp particles via sol-gel process, enabling them to be magnetically actuated. Moreover, the anticancer drug doxorubicin (DOX) was loaded on the (Pd@Au)/Fe3O4@Sp microrobots, which endows them with additional chemotherapeutic efficacy. The as-prepared biohybrid (Pd@Au)/Fe3O4@Sp-DOX microrobots not only possess efficient propulsion performance with the highest speed of 526.2 μm/s under a rotating magnetic field, but also have enhanced synergistic chemo-photothermal therapeutic efficacy. Furthermore, they can be structurally disassembled into individual particles under near-infrared (NIR) laser irradiation and exhibit pHand NIR-triggered drug release. These intriguing properties enable the microrobots to be a very promising and efficient platform for drug loading, targeted delivery and chemo-photothermal therapy. INTRODUCTION Recent advances in magnetically steered microrobots have exhibited considerable potential for targeted delivery.1-6 Compared with fuel-driven motors, magnetic microrobots exempt the


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demand of either harmful chemical conditions or power system, thus causing negligible interference to the physiological environments they confront.7-9 Therein, helical magnetic microrobots, inspired by bacterial flagellum propulsion, have attracted particular attention owing to their capability of transforming the rotation around their helical axis into a translational motion along the helical axis to provide an efficient and precise three-dimensional locomotion behavior.10-11 To date, various feasible methods have been proposed for fabricating helical microrobots, such as self-scrolling,12 3D direct laser writing (DLW),13-15 glancing angle deposition (GLAD)16 and biotemplated synthesis.17-18 Among these methods, using biological templates can be cost-effective and noncytotoxic, which is highly demanded for a range of biomedical applications.17 Nature has created abundant helical structures over millions of years of evolution, which can be used for fabricating biohybrid magnetic microrobots.19-21 Taking advantage of their intrinsic helical morphology can elide the process of fabricating spiral configuration compared to other manufacturing methods of microrobots. Spirulina (Sp.) is a microorganism with naturally intact three-dimensional helical structure, which can be cultivated in great quantities and already commercialized as nutritional supplements, demonstrating its desirable feasibility and security in biomedical applications. All these fascinating characteristics render it an ideal biotemplate for fabricating biohybrid magnetic microrobots for drug loading and targeted delivery. Magnetic microrobots can deliver anti-cancer drug to specific spots to treat various tumors such as lung cancer, breast cancer, brain cancer, etc. Though the drug-based chemotherapy is restricted by several undesirable effects such as its safe dosage and drug resistance of some cancer cells, which may impact the therapeutic efficacy, it’s still the main route for cancer treatment nowadays. Meanwhile, other therapeutic approach has been developed such as


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photothermal therapy (PTT), which can convert absorbed light energy to heat by photothermal agents, thus causing thermal ablation to target cancer cells.22-23 With the development of therapeutic technology, combining multiple methods such as chemotherapy and PTT has been proposed with distinct advantages since it can significantly boost therapeutic efficacy as compared to sole treatment.24-27 To date, a lot of noble metal nanomaterials such as Pt,28 Au,29-30 Pd31 and Ag32 have been extensively taken as effective photothermal therapeutic agents for PTT due to their prominent near-infrared (NIR) absorption capacity. Therein, bimetallic nanomaterials such as Pd@Au nanoplates and Pd@Ag core-shell nanooctapod are highlighted due to their higher photothermal conversion efficiency than that of monometal nanomaterials.3335

The nanoparticles (NPs) usually take effect via vascular injection or transdermal delivery for

PTT, which has the shortcoming of particle aggregation. Some researchers use biocompatible scaffolds such as polymer molecules29 or silicate hollow microspheres36 to solve this problem. Taking all the above-mentioned factors into account, in this study, we propose the strategy of using biomaterial Sp. as a scaffold to fabricate magnetic microrobots, which can load both anticancer drug and photothermal agents to achieve synergistic and enhanced therapeutic efficacy, meanwhile to avoid NP aggregation. To the best of our knowledge, although some relative researches of magnetic microrobots for targeted delivery have been carried out,7, 17, 19 there are few reports focused on multifunctional microrobots combining targeted delivery and chemophotothermal therapy so far. Herein, we propose a facile route to fabricate biohybrid magnetic microrobots (BMMRs) for targeted delivery and synergistic chemo-photothermal therapy (Scheme 1). First, the Pd@Au core-shell NPs were embedded both in the Sp. cells and on the cell walls, which exhibit excellent photothermal conversion ability. Subsequently, Fe3O4 NPs are deposited on the as-prepared


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(Pd@Au)@Sp particles, enabling the obtained (Pd@Au)/Fe3O4@Sp microrobots to be actuated in an external magnetic field. Moreover, chemotherapeutic doxorubicin (DOX) is further loaded, thus the (Pd@Au)/Fe3O4@Sp-DOX platform is successfully established, which can be used as drug delivery system and perform synergistic chemo-photothermal therapeutic capacity for EC109 and 769-P cancer cells upon an 808 NIR laser irradiation. In addition, the biohybrid microrobots can be degraded under NIR irradiation and exhibit pH- and NIR-triggered drug release. This work provides an integrated route for fabricating multifunctional microrobots with highly swimming speed, precisely directional guidance, ultrahigh drug loading efficiency as well as efficient photothermal conversion ability towards applications of targeted delivery and synergistic chemo-photothermal therapy in a single platform. EXPERIMENTAL SECTION Reagents and Materials Sp. cells were offered by Kang Sheng Algae Co., Ltd. Stannous chloride (SnCl2), palladium chloride (PdCl2), hydrochloric acid (HCl, 37%), sodium stannate trihydrate (Na2SnO3·3H2O), sodium hypophosphite monohydrate (NaH2PO2·H2O), chloroauric acid (HAuCl4·4H2O, >99%), trisodium citrate dihydrate (Na3C6H5O7·2H2O, 99%), potassium hydroxide (KOH), potassium nitrate (KNO3), and ferrous sulfate heptahydrate (FeSO4·7H2O) were purchased from Lan Yi Chemical Reagent Co., Ltd (Beijing, China). Methyl thiazolyl tetrazolium (MTT), RPMI-1640 and phosphate buffer solution (PBS) were obtained from Life Technologies Corporation (USA). Dimethyl sulfoxide (DMSO) was purchased from Amresco (USA). Calcein-AM (CA) and propidium iodide (PI) were purchased from YEASEN (Shanghai, China). Human renal cancer


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cell line 769-P was available in the cell store of Peking University First Hospital. Esophageal carcinoma cell line EC109 was retrievable from Peking Union Medical College Hospital. Synthesis of (Pd@Au)@Sp Composites Sp. cells were activated with colloidal palladium and the details could be found in our previous literature.37 The Pd seeds were then exposed via peptization process and functioned as catalytic core to deposit Pd@Au NPs on the Sp. cells through electroless plating.38-40 The activated and peptized Sp. cells (1 g) were washed repeatedly to remove the unbound Pd particles from the solution, and then dispersed in deionized (DI) water (100 mL) in a beaker heated by a water bath (90°C), in which HAuCl4 aqueous solution (2 mL, 1 wt%) was subsequently added. Afterwards, Na3C6H5O7 solution (10 mL, 1 wt%) was added dropwise to the mixture and stirred for 20 min. Finally, the products were washed thoroughly with DI water, and accumulated for the following Fe3O4 deposition. Fabrication of (Pd@Au)/Fe3O4@Sp Microrobots The Fe3O4 depositing process was based on the sol-gel method.41 Firstly, KOH solution (20 mL, 0.25 M) was added in the beaker, and then KNO3 solution (20 mL, 1 M) was added and mixed adequately, followed by the addition of FeSO4·7H2O solution (10 mL, 0.25 M) containing (Pd@Au)@Sp cells. After the gel solution was formed, the beaker was kept in water bath for 30 min at 90°C. Subsequently, the mixture was rinsed with DI water and the obtained (Pd@Au)/Fe3O4@Sp microrobots were collected. Characterization


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The morphology and main elements of the obtained biohybrid magnetic microrobots (namely BMMRs) were characterized by field emission scanning electron microscope (FESEM, SU8010LA, Hitachi) equipped with energy dispersive spectrometer (EDS). The cross sections of samples were observed by transmission electron microscope (TEM, JEM-1400, JEOL). The XRay Diffraction (XRD-6000, Shimadzu) patterns were collected to analyze the crystallization of the as-prepared BMMRs. The content of the photothermal agents loaded on the BMMRs was measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Thermo, Waltham, USA). Magnetic properties of the BMMRs were tested using a vibrating sample magnetometer (VSM, 730T, LAKESHORE). The swimming capabilities of the BMMRs through wireless magnetic actuation were tested by a self-built triaxial Helmholtz coil system that provides homogeneous rotational magnetic fields. Photothermal Effect Measurements To investigate the heat conversion induced by NIR laser, BMMRs dispersed in water with a series of concentrations (0, 2, 4, 8, 16 and 32 mg/mL) were irradiated with an 808 nm NIR fiber laser (MDL-H-808 nm) at a power density of 2.5 W/cm2 for 10 min. Besides, 16 mg/mL of BMMRs dispersed in water were exposed to laser at 1, 1.5, 2, 2.5 and 3 W/cm2 for 10 min respectively to investigate the effect of power density. In addition, temperature variation of the solution containing BMMRs was monitored over 4 cycles of the heating/cooling process to evaluate the photothermal stability, which was recorded by a thermocouple (TES Co., Ltd., Taiwan, China). Measurements of DOX Loading Efficiency and Release Profile


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To assess the potential of the BMMRs as targeted drug delivery carriers, DOX loading efficiency and release mode were investigated. The UV–visible (UV-Vis) spectra of the DOX solution before and after interacting with the BMMRs were recorded on a spectrometer (UV-3600, Shimadzu). We incubated BMMRs with phosphate buffered saline (PBS) solution (pH=7.4, 0.01 M) containing DOX at various concentrations (5, 10, 20, 40, 80 and 160 μg/mL). The mixture was stirred at room temperature for 24 h and then separated by a magnet. The amount of DOX in the supernatant was quantified with the assistance of the standard concentration curve of DOX (at the absorption of 480 nm) generated by a series of DOX solution of various concentrations. The drug loading efficiency of BMMRs was calculated by Equation 1. The drug release behavior of the BMMRs were determined in different environment (pH=7.4, pH=5.4). And the NIR laser was turned on to heat the solution and maintained for 30 min at a time interval of 30 min without irradiation. The released DOX was calculated by UV-vis analysis using the Equation 2. Drug Loading Efficiency (%) = (𝑀0 ― 𝑀𝑡)/𝑀0 × 100%

(1)

where M0 and Mt are the masses of DOX in the initial solution and the supernatant, respectively. Accumulative Release (%) = 𝐷𝑟/𝐷𝑡 × 100%

(2)

where Dr is the accumulative amounts of released DOX and Dt is the total amounts of DOX incorporated in the BMMRs. In Vitro Targeted Drug Delivery The targeted drug delivery of BMMRs was carried out by magnetically actuating the DOXloaded BMMRs towards cancer cells. 769-P cells were first seeded in a Petri dish (35 mm in diameter) and cultured for 24 h at 37°C in a 5% CO2-95% air humidified atmosphere to allow for


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cell adhesion. Subsequently, 1 mg/mL of DOX-loaded BMMRs was added and magnetically propelled to specific region of 769-P cells. And then the laser irradiation (2.5 W, 5 min) was employed to trigger the release of DOX, followed by the live/dead staining using Calcein-AM/PI kit. According to the manufacturer’s instructions, after 20 min of staining, the cell viability was assessed with a fluorescence microscope at wavelength of 490 nm (Calcein-AM, green fluorescent signal for viable cells) and 545 nm (PI, red fluorescent signal for dead cells). In Vitro Chemo-Photothermal Therapy The cell viability of chemo-photothermal therapy was further evaluated by MTT assay and live/dead staining. We adopted the widely-used method of mixing the BMMRs or DOX-loaded BMMRs with cancer cells. 769-P and EC109 cells were seeded into 96-well plates (4×103 cells per well) and cultured at 37°C in a 5% CO2-95% air humidified atmosphere for 24 h. And then the culture medium in each well was withdrawn and replaced separately with 200 μL of medium containing BMMRs or DOX-loaded BMMRs. After incubated for 24 h, the BMMRs were treated with NIR laser irradiation (2.5 W/cm2) for 3 min and 5 min, respectively. Yet the DOXloaded BMMRs were treated with or without laser irradiation. Subsequently, 200 μL of MTT was added into each well, and incubated for another 4 h (37°C). Afterwards, the culture medium was removed and 200 μL of DMSO was added into each well, followed by shaking on a horizontal shaker for 10 min. The absorbance was measured at 560 nm using enzyme-labeling analysis equipment (Thermo Scientific Varioskan Flash). Moreover, the live/dead staining using Calcein-AM/PI was also conducted. Firstly, 769-P cells were incubated with BMMRs and DOXloaded BMMRs respectively, followed by irradiation with laser (2.5 W/cm2) for 3 min. After that, they were stained by live/dead kit and observed under a fluorescence microscope with green fluorescent signal for viable cells and red fluorescent signal for dead cells.


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RESULTS AND DISCUSSION Characterization of (Pd@Au)/Fe3O4@Sp Microrobots The biohybrid microrobots were fabricated by several steps based on Sp. templates. The Pd@Au NPs were synthesized both in the cytoplasm and on the cell walls of Sp. cells, and then Fe3O4 NPs were deposited on the obtained (Pd@Au)@Sp composites. Optical microscope, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to study the morphology of the Sp. cells at different stages. From the optical images, we can clearly see that the standard helical shape of Sp. template was perfectly remained during the whole processes. Compared with the original Sp. cell with a green appearance (Figure 1-a1), the (Pd@Au)@Sp (Figure 1-b1) and (Pd@Au)/Fe3O4@Sp (Figure 1-c1) composites with the metal luster appearance changed to golden yellow and yellow green. Corresponding SEM images show that the original Sp. cell have smooth surface (Figure 1-a2), yet the (Pd@Au)@Sp (Figure 1-b2) and (Pd@Au)/Fe3O4@Sp (Figure 1-c2) have relatively rough surface. To gain insight into the depositing activities of NPs on the Sp. cells, the samples at various stages were sliced up and analyzed by TEM. The interior space of the original Sp. cell appears to have sponge structure (Figure 1-a3, a4). After deposition process, spherical Pd@Au NPs were homogeneously distributed inside the sponge cytoplasm as well as on the cell walls (Figure 1-b3, b4), and the rodlike Fe3O4 NPs were deposited on the surface of (Pd@Au)@Sp particles successively (Figure 1-c3, c4). The Pd@Au NPs on the Sp. cells were characterized via cross-sectional TEM image, and then the average particle size was measured to be 14.79 ± 4.05 nm for the shown sample (Figure 2a). We also identified the core-shell nanostructure of Pd@Au NP via the high resolution


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transmission electron microscopy (HRTEM) (Figure 2b). The lattice fringes with d = 0.225 nm and d = 0.235 nm are distinct, which could be ascribed to the planes of Pd (111) and Au (111), respectively. The corresponding fast Fourier transformation pattern (FFT) taken from Pd and Au confirms their lattice-matched interfaces (Figure 2c). Furthermore, the element mapping of (Pd@Au)/Fe3O4@Sp particles indicates the presence of Pd, Au and Fe elements (Figure 2d). The XRD patterns were used to further identify the crystalline nature and composition of the products. As shown in Figure 3a, the original Sp. cell is of amorphous state. The (Pd@Au)@Sp composites have peaks at 2θ of 38.2°, 44.4°, 64.5° and 77.5°, corresponding to diffraction from the (111), (200), (220) and (311) planes of the face centered cubic (fcc) crystal structure of Au, and the (Pd@Au)/Fe3O4@Sp composites demonstrate two sets of diffraction peaks assigned to Au and Fe3O4. The (220), (311), (400), (422), (511) and (440) lattice planes of Fe3O4 can be easily observed excluding the planes of Au described above. Peaks for the Pd atoms are invisible, which may due to the minor content of Pd. The statistical XRD analysis results reveal that Au NPs and Fe3O4 NPs are successfully deposited on the Sp. cells, which are corresponding to element mapping (Figure 2d) and the EDS results (Figure S5). The deposition mechanism of Pd@Au NPs on the Sp. cells is very similar to that of Pd@Ag deposition, which has been described in detail in our previous literature.37 In brief, activation process is required in order to deposit metallic NPs on the non-catalytic Sp. template. Herein, we applied colloidal palladium activation to fulfill this procedure, of which the composition and working conditions are shown in Table S1. After that, the acceleration process exposed catalytic Pd NPs both intracellularly and extracellularly (Figure S1). Finally, metallic Au was deposited on the Pd core by way of electroless deposition. Notably, in this study, we omitted extra permeability treatment of Sp. cells, yet only used the hydrochloric acid (HCl) contained in the


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colloidal palladium to enhance permeability, which makes the whole process simpler compared to the Pd@Ag deposition. A comparison of Pd@Au NPs synthesized in the solution and loaded on Sp. cells was shown in Figure S2, the prepared Pd@Au NPs in the solution aggregated into irregular NPs. In contrast, the Pd@Au NPs loading on Sp. cells were dispersed homogeneously, demonstrating that the cell substrates endow abundant porous structure and deposition sites, which allowed the Pd@Au NPs to form in confined space and avoid aggregation. Moreover, similar to previous research,37 the size and distribution of Pd@Au NPs can be controllably adjusted by changing the electroless deposition composition (Figure S3). The magnetic property of Fe3O4 NPs contributes to the controlled propulsion of the BMMRs. Hence, we first evaluated the magnetization of BMMRs before magnetic propulsion experiments. The M-F curve shows that the saturation magnetization of the BMMRs was 28.79 emu/g (Figure 3b), which is enough for magnetic-responsive targeted delivery.23 Inset images are the magnified image within 500 Gs and photographs demonstrating the oriented motion of BMMRs under an external magnetic field. The magnified image exhibited that the coercivity was 79.86 Gs, indicating their ferromagnetic property. Previous reports have demonstrated that different magnetic properties are essentially related to the formation of different phases, morphologies and crystallite sizes of the Fe3O4 nanostructures. Domain wall movement and shape anisotropy of the nanorods are expected to be generated and contribute to the high value of coercivity.42 Consequently, in this study, the coercivity and ferromagnetic property may be ascribed to the shape anisotropy of the rodlike Fe3O4 NPs. Propulsion Performance of Magnetic Microrobots


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The magnetic propulsion performance of the obtained BMMRs was tested using a self-built triaxial Helmholtz coil system that could generate a uniform rotating magnetic field. Schematics of the triaxial Helmholtz coil system and magnetic actuation mechanism of BMMRs was shown in Figure 3c, where B denotes the strength of the magnetic field and V represents the translational velocity. In order to quantitatively investigate the factors influencing the propulsion performance of the BMMRs, we tested the motion of the BMMRs with different geometric parameters (Table S2, BMMR-1, BMMR-2 and BMMR-3 namely) in the magnetic field (80 Gs) at different frequencies ranging from 5 to 60 Hz. According to previously reported results, various parameters such as chirality, body length, helical diameter, helical angle and the applied frequency would affect the propulsion speed of the helical microrobots.19,

43-44

As shown in

Figure 3d, the BMMR-3 with turn number of 4.5 and pitch of 36 μm exhibits a higher speed compared to the BMMR-1 and the BMMR-2, and its highest value can reach 526.2 μm/s, which corresponds to a relative speed of ~3 body length/s. The speed of the BMMR-3 increases linearly from 62.6 to 526.2 μm/s as the applied frequency changed from 5 to 40 Hz, yet it slowed down greatly when the frequency further increased, which suggests that 40 Hz is the step-out frequency for the BMMR-3. In general, the fluidic drag will exceed the maximal available magnetic torque when the applied frequency is higher than the step-out frequency, thus leading to a decrease in velocity as the BMMRs rotate out of step with the magnetic field.10, 17, 45 The real-time propulsion performance of the BMMRs in DI water under rotating magnetic field (100 Gs, 20 Hz) was shown in Video S1, and the time lapse images over 2 s time intervals delineate the motion trajectories of BMMRs (Figure 3e). The results confirm that highly efficient and


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precisely controllable propulsion of the BMMRs can be achieved, which is crucial for precise manipulation of targeted delivery. Drug-loading Performance of the BMMRs The BMMRs has been successfully fabricated. In order to achieve targeted chemo-photothermal therapy of cancer, DOX should be further loaded on the BMMRs. To investigate the drug loading performance of the BMMRs, the absorption spectra of the DOX solution before and after incubating with BMMRs was acquired (Figure S6a, b). The drug loading efficiency of BMMRs at different concentrations of DOX (Figure S6d) can be calculated using the corresponding standard calibration curve (Figure S6c). From the results we can find that the drug loading efficiency of the BMMRs increased as the initial DOX concentration increased ranging from 5 to 80 μg/mL, whereas it decreased when the DOX concentration continually increased to 160 μg/mL. Hence, the highest amount of drug loading efficiency was determined to be 92.1%. Compared to previous studies of micro/nanocarrier,24, 36 the DOX loading efficiency of BMMRs is much higher, which may be partially due to the good permeability and hydrophilicity of the Sp. template. In addition, as shown in Figure S7, the obtained (Pd@Au)Fe3O4@Sp microrobots exhibited negative charge through zeta potential measurements, contrary to the positive charge of DOX. Accordingly, DOX could be loaded intracellularly and extracellularly via electrostatic interaction onto the BMMRs, indicating that the as-prepared BMMRs can be used as an effective carrier for drug-loading. Moreover, the loaded DOX could be protected against the harsh environment they may confront, which is intensely required for targeted drug delivery. Evaluation of Photothermal Properties


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In order to evaluate the photothermal performance of the obtained BMMRs, NIR laser is chosen as the light source. NIR is commonly used in PTT induced by AuNPs due to the optical window in the near-infrared, where the light absorption by tissue chromophores is reduced, thus deeper light penetration was allowed. Yet visible light has only been applied to superficial epithelial cells or transparent organs due to its low transmittance to human body.46-47 Herein, we prepared Pd@Au NPs in the solution without Sp. templates and then tested their UV-vis-NIR absorption spectra. As shown in Figure S8a, the peak of absorption spectra is 750-900 nm, exactly in the NIR spectral region (780-2526 nm). Compared with that, the BMMRs exhibit relatively stable light absorption at a broad range of wavelength including 808 nm NIR laser (Figure S8b). Taking all the factors into consideration, 808 nm NIR laser was chosen as light source for the photothermal performance evaluation of the BMMRs. Regarding the photothermal conversion efficiency of NPs with different sizes, some studies have been reported previously.29, 48 In general, the absorption peaks of NPs could be shifted along with the change of grain sizes, which may provide a direct guidance for laser selection. However, the photothermal performances of metal NPs are primarily related to their concentrations in solution instead of the sizes.24, 49 Therefore, the concentrations of BMMRs for photothermal conversion were taken into key considerations in this work. Different concentrations (0, 2, 4, 8, 16 and 32 mg/mL) of the BMMRs were irradiated with an 808 nm NIR laser (2.5 W/cm2) for 10 min, and the temperature variation along with the irradiation time was shown in Figure 4a. The results show that the change of temperature in pure water is almost negligible, yet the temperature increases rapidly with increasing the concentration of BMMRs as well as the irradiation time, especially within 6 min, whereas the temperature increment is unconspicuous when we further increase the irradiation time. For the


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BMMRs at a concentration of 16 mg/mL, the temperature increased from 29 to 54.4oC within 10 min, indicating a rapid and efficient conversion of the NIR laser energy into thermal energy. As the concentration further increased, no distinct improvement of the photothermal performance was found, thus we determined 16 mg/mL as a proper concentration for further evaluation. To investigate the effect of power density on the temperature change, 16 mg/mL of BMMRs dispersed in water were exposed to the laser at power density of 1, 1.5, 2, 2.5 and 3 W/cm2 for 10 min, respectively. The photothermal heating curves display a distinct laser-power-dependent photothermal effect for the BMMRs, and the temperature increased to 54.4°C and 57.3°C at 2.5 W/cm2 and 3 W/cm2, respectively (Figure 4b). Since the temperature elevation at the power density of 2.5 W/cm2 is enough for photothermal therapy (Generally, 42°C is sufficient to induce target cell death),24 it was chosen as a proper power density for further experiments. The excellent photothermal performance of BMMRs can be ascribed to the well-dispersed photothermal agent Pd@Au NPs on BMMRs. The contents of Au and Pd in the BMMRs were measured to be 1.113 wt% and 0.14 wt% by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Previous reports have demonstrated that the bimetallic materials present better NIR absorption ability and photothermal conversion effect than that of monometallic materials.24, 49 Particularly, the Pd@Au core-shell NPs have an excellent photothermal effect due to the plasma resonance of the Au nanoshell and Pd core, which is capable of converting light energy into heat that results in a rise of temperature. The temperature elevation of the BMMRs solution is compared with previously reported NPs. As shown in Table S3, the photothermal conversion capacity of BMMRs is comparable or even better to previously reported NPs, such as AuNRs@C,49 AuNRs@PPy,50 Au@HSN-PGEA51 and Pd@Au nanoplates,52 which suggests that the BMMRs are promising materials for PTT.


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In addition to the photothermal conversion capability, photothermal stability is also significant during PTT. The temperature profiles were tested for 4 successive cycles of heating/cooling processes (Figure 4c). The results show that the temperature elevation was perfectly maintained during 4 cycles of testing, exhibiting highly stable photothermal conversion ability of BMMRs. Both the remarkable photothermal conversion efficiency and stability support the BMMRs as an excellent candidate for PTT applications. During the photothermal conversion experiments, it was observed that the structure of both BMMRs and (Pd@Au)@Sp composites tended to break down gradually when irradiated under NIR laser for a long time (Figure 5a-d and Figure S10), and most of them became segments after 20 min. To reveal the mechanism of BMMRs degradation, we conducted the experiment of laser irradiation on pure Sp. templates with power density of 2.5 W/cm2. As shown in Figure S11, the original Sp. templates were of intact spiral shape. After irradiated for 40 min under NIR, morphological changes on Sp. templates appeared, yet structure degradation was not observed. Consequently, the degradation was not due to the NIR irradiation on the composition of Sp. templates. Moreover, it was neither ascribed to the use of chemical agents, the photothermal performance of BMMRs was tested in DI water, and chemical regents were not employed. Excluding the possible factors discussed above, we consider that the mechanism of BMMRs degradation can be attributed to local hyperthermia of Pd@Au nanosites inside the Sp. cells under laser irradiation. During the photothermal conversion experiments, NIR worked with the photothermal agent Pd@Au NPs, thus energy highly centered and high heat was produced, which destroyed the structure of BMMRs. Notably, the NIR-triggered degradation capability can simultaneously facilitate drug release. For biomedical applications, an ideal drug delivery system requires controlled drug release according to the physiological response and clinical


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requirements. As depicted in Figure 4d, DOX release from the BMMRs exhibited pH and laser stimuli dependence. In all cases the release rate can be significantly varied via switching the laser on or off. The laser-on state induced a steep increase in drug release, whereas the laser-off state allowed only a minimal amount of drug release. Besides, the released amount of DOX from BMMRs significantly accelerated at pH 5.4 compared to physiological conditions of pH 7.4, thus BMMRs show great potential for targeted delivery to weak acidic environment where cancer cells resided. The maximum amplitude of DOX release triggered by NIR irradiation increased by 20% as pH decreased. The results suggest that with the assistance of pH and NIR laser, large doses of drug release required by some therapies can be achieved,23 which may dramatically improve the synergistic chemo-photothermal therapeutic efficacy for further clinical applications. In Vitro Targeted Drug Delivery The targeted delivery of DOX to 769-P cells via BMMRs was studied in the magnetic field and the time-lapse images were shown in Figure 6a, the DOX-loaded BMMRs can be magnetically propelled to specific region of the Petri dish, reaching and adhering to the targeted 769-P cells quickly. In order to investigate the effect of magnetic propulsion on the cancer therapy, the viabilities of the NIR-treated cancer cells with and without DOX-loaded BMMRs were tested by live/dead staining. The cells without DOX-loaded BMMRs maintained high viability with green color (Figure 6b-i). In contrast, the cells with DOX-loaded BMMRs presented low viability with red fluorescence signal due to the function of released DOX and NIR laser irradiation (Figure 6b-ii). The results show the feasibility of using the guidable BMMRs to achieve targeted drug delivery and synergistic chemo-photothermal therapy of cancer cells. In Vitro Chemo-Photothermal Therapy


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We have already demonstrated the in vitro targeted drug delivery ability of BMMRs. To assess the therapeutic capacity of the BMMRs towards 769-P and EC109 cancer cells, MTT assay and live/dead cell staining were further carried out. The biocompatibility of BMMRs with different concentrations ranging from 0 to 2000 μg/mL was first investigated. Previous studies have demonstrated that the content of Sp-templated magnetic microrobots (containing Sp. templates and Fe3O4 NPs) within 800 μg/mL had very low cytotoxicity to normal 3T3 cell line.21 Besides, Au NPs are often used in biomedicine due to their good biocompatibility, which make the BMMRs biocompatible. Furthermore, in this study, 769P cells were incubated with BMMRs of different concentrations ranging from 0 to 2000 μg/mL, cell viabilities are above 80% for all the samples even at high concentrations (Figure S12), demonstrating that the cytotoxicity was very low. Consequently, BMMRs with different concentrations ranging from 0 to 2000 μg/mL were suitable for further biomedical application due to the good biocompatibility. Herein, we investigated the effects of PTT, chemotherapy and synergistic chemo-photothermal therapy on cancer cells, respectively. For PTT, different concentrations (0, 2, 4, 8, 16 and 32 mg/mL) of the BMMRs were incubated with 769-P cells for 24 h and then irradiated under an 808 nm NIR laser (Figure S13a). As mentioned above, 16 mg/mL of BMMRs was proper for photothermal evaluation. Yet when it was employed in the PTT, the cancer cells were killed completely, which can be verified by Calcein-AM/PI staining (Figure S14), showing a red fluorescence of dead cells. In order to figure out how the concentration of BMMRs affected PTT, we further optimized the concentrations of BMMRs ranging from 0 to 2000 μg/mL (Figure S13b). Different concentrations of BMMRs (0~2000 μg/mL) were incubated with 769-P cells, and then irradiated with NIR laser (2.5 W/cm2). As shown in Figure 7a, cell viability decreased


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remarkably with increasing the concentration of BMMRs. About 62.5% and 79.4% of 769-P cells were killed after the treatment of BMMRs at 2000 μg/mL under laser irradiation for 3 min and 5 min, respectively. The results indicate that BMMRs could trigger time- and dosedependent cytotoxicity to cancer cells and higher concentration of them could more effectively convert NIR light energy into heat, which is likely to cause coagulative necrosis, mitochondrial dysfunction and protein denaturation of cancer cells.53-54 Although irradiation for 3 min or 5 min would not totally disassemble the BMMRs and may result in incomplete drug release, the chemo-photothermal therapeutic efficacy for cancer cells is still high enough. This is because that the drug loading efficiency of BMMRs is higher compared to previous studies of micro/nanocarrier. Furthermore, we do not expect the drug to be released totally at a time, so as to maintain longer treatment periods with good effect. We then evaluated the chemotherapeutic effects of DOX-loaded BMMRs on 769-P cells. As shown in Figure 7b, the viabilities of cancer cells decreased with increasing DOX concentrations from 0 to 80 μg/mL. Notably, only ~20% of 769-P cells was alive when the DOX concentration reached to 80 μg/mL, suggesting that DOX-loaded BMMRs are highly cytotoxic and have a dose-dependent chemotherapeutic effect towards 769-P cells. To further explore synergistic chemo-photothermal therapeutic effects on cancer cells, two groups of MTT assays using 769-P and EC109 were independently carried out. As shown in Figure 7c, compared to the control group (no drug and no irradiation), the cell viabilities of 769P and EC109 cells were up to 92.6% and 82.2% respectively when they were irradiated with laser (2.5 W/cm2, 3 min) yet without addition of BMMRs, showing that separate laser irradiation would not affect cell viability on a large scale. By contrast, for the BMMRs+Laser group, 2 mg/mL of BMMRs leads to ~ 40% loss of cell viability under laser irradiation, suggesting an


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effective inhibition of the photothermal effect of BMMRs. We also compared the cell viability of BMMRs with DOX-loaded BMMRs groups to further study the chemotherapeutic efficacy. As can be seen, the cell viabilities were above 80% when BMMRs were incubated with 769-P cells. The decrease of living cells would be attributed to the selective toxicity towards tumor cells of biocompound C-phycocyanin contained in Sp. cells, which can arrest the cell cycle and induce apoptosis.55 In contrast, for the DOX-loaded BMMRs group, the cell viability of 769-P cancer cells decreased to 37.3%, which suggest an excellent chemotherapeutic efficacy of DOX-loaded BMMRs. Compared to cancer treatment with either BMMRs-DOX group or BMMRs+laser group, the cell viability of DOX-loaded BMMRs irradiated with NIR laser dropped dramatically to 10% for 769-P cells. The results indicate that the combination of hyperthermia effects and NIR-triggered drug release of the DOX-loaded BMMRs leads to the highest cell mortality rate among all the groups. The synergistic chemo-photothermal method could indeed enhance the therapeutic efficacy for cancer cells as compared to sole treatment.25, 56-58 In addition, the synergistic effect of chemo-photothermal therapy was also verified by CalceinAM/PI staining. Calcein-AM can permeate into the cell membrane and be hydrolyzed by intracellular esterases to a green flurescent dye of calcein in living cells, whereas the cell nucleus dye of propidium iodide (PI) can only cross the damaged cell membranes and embed into double stranded DNA to show a red fluorescence in dead cells. They were selected to indicate the viability of cells intuitively. As shown in Figure 7d, the control group of 769-P cells without any treatment showed high viability with green color. In comparison, the BMMRs+Laser group and BMMRs-DOX group display blended green and red color, whereas the cells incubated with DOX-loaded BMMRs under NIR laser irradiation showed substantial cell death with red color, leaving a minimal number of live cells. The results demonstrate that synergistic chemo-


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photothermal therapy could induce a higher therapeutic efficacy than that of single chemotherapy or PTT, which is corresponding to the MTT assay. Overall, the superior synergistic chemo-photothermal therapeutic efficacy of the BMMRs can be attributed to the following reasons: 1) The bimetallic core-shell configuration of Pd@Au NPs exhibits better NIR absorption ability and photothermal conversion effect than that of monometallic materials; 2) The ultrahigh payload of anticancer drug DOX on the BMMRs can contribute to excellent chemotherapeutic efficacy; 3) The capability of NIR-triggered structural disassembly would enhance drug release. CONCLUSIONS In summary, we developed a systematic technique for mass-production of (Pd@Au)/Fe3O4@SpDOX magnetic microrobots and explored their synergistic chemo-photothermal therapeutic efficacy for cancer cells. The obtained BMMRs can be precisely steered under a rotating magnetic field and perform ultrahigh drug loading efficiency, as well as remarkable photothermal conversion capacity. Furthermore, the BMMRs can be structurally disassembled under NIR laser irradiation and exhibited controlled pH- and NIR-triggered drug release mode. All these advantages make the BMMRs a very promising and efficient platform for targeted delivery and enhanced synergistic chemo-photothermal therapy compared with single treatment. Considering the scale of our microrobots, oral delivery might be an effective and secure route for biomedical applications, such as gastrointestinal cancer therapy. In addition, Au and Fe3O4 NPs are also CT and MR imaging contrast agents, demonstrating that the real-time monitoring with these BMMRs during anti-cancer therapy may be feasible, which is worth to be explored in the future work.


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Scheme 1. (A) Fabrication processes of the (Pd@Au)/Fe3O4@Sp-DOX microrobots. (B) Schematic illustration of propulsion, NIR-triggered degradation and DOX release of magnetic microrobots, and their synergistic chemo-photothermal therapy towards cancer cells.


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Figure 1. The optical, SEM, TEM images and corresponding enlarged view of the original Sp (a1-a4), (Pd@Au)@Sp (b1-b4) and (Pd@Au)/Fe3O4@Sp (c1-c4).


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Figure 2. (a) TEM image of Pd@Au NPs on the Sp. cell, inset image is the particle size distribution. (b) HRTEM images of core-shell Pd@Au NPs. (c) The corresponding fast Fourier transformation pattern from the area outlined by red squares in (b). (d) Element mapping of (Pd@Au)/Fe3O4@Sp, the yellow, purple and green points represent Pd, Au and Fe, respectively.


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Figure 3. (a) The XRD pattern of original Sp, (Pd@Au)@Sp and (Pd@Au)/Fe3O4@Sp, respectively. (b) VSM magnetization curves of (Pd@Au)/Fe3O4@Sp. Inset images are the magnified image within 500 Gs and photographs demonstrating the oriented motion of (Pd@Au)/Fe3O4@Sp under an external magnetic field. (c) Schematics of the triaxial Helmholtz coils and magnetic actuation of BMMRs, where V represents the translational velocity and B denotes the strength of the magnetic field. (d) Velocity-frequency profiles for different samples


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of biohybrid magnetic microrobots (BMMR-1, BMMR-2 and BMMR-3) in the magnetic field with the intensity of 80 Gs. (e) Time lapse images of BMMRs under a rotating magnetic field (100 Gs, 20 Hz) over 2 s time intervals. Scale bar: 200 μm.

Figure 4. (a) Photothermal curves of different concentrations of BMMRs under NIR irradiation (808 nm, 2.5 W/cm2) for 10 min. (b) Photothermal curves of BMMRs under NIR irradiation with power density of 1, 1.5, 2, 2.5 and 3 W/cm2 for 10 min, respectively. (c) Temperature changes of the BMMRs over 4 cycles of irradiation/cooling. (d) Drug release profiles from DOX-loaded BMMRs at different pH environment (with or without NIR irradiation).


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Figure 5. Degradation of BMMRs under NIR irradiation with power density of 2.5 W/cm2. (a) 0 min, (b) 4 min, (c) 12min and (d) 20 min. Scale bar: 50 μm.


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Figure 6. (a) Time-lapse images of the magnetic manipulation of DOX-loaded BMMRs towards the 769-P cells. (b) Schematic illustration of in vitro targeted drug delivery. DOX-loaded BMMRs were added to one side of the medium of 769-P cells and propelled to specific region, the laser irradiation was employed and the cells were stained with the Calcein-AM/PI staining kit to test the cell viability. Results of the live/dead staining of 769-P cells in the region without (i) and with (ii) DOX-loaded BMMRs.


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Figure 7. Cell viabilities of 769-P cells (a) Incubated with various concentrations of BMMRs and irradiated with laser (2.5 W) for 3 min and 5 min, respectively. (b) Incubated with different concentrations of DOX-loaded BMMRs. (c) Cell viabilities of 769-P cells and EC109 cells treated with RPMI-1640, laser only, BMMRs only, BMMRs+Laser, BMMRs-DOX and BMMRs-DOX+Laser, respectively. (d) Fluorescence microscopy images of 769-P cells stained with the live/dead kit after treated with PBS, BMMRs+Laser, BMMRs-DOX and BMMRsDOX+Laser, respectively. Green and red colors represent live and dead cells. Scale bar: 200 μm.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website.


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Supporting figures and tables (PDF) The propulsion of magnetic microrobots in DI water using a rotating magnetic field (100 Gs, 20 Hz) (AVI) AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected] (J.C.)

ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (Grant No.51775022) and the Fundamental Research Funds for the Central Universities.

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with

in-Situ-Formed

Reil

Gold

Nanorod

Cores

for

Complementary

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BRIEFS The fabricated (Pd@Au)/Fe3O4@Sp microrobots endow efficient propulsion performance, ultrahigh drug loading efficiency and excellent photothermal conversion ability, which make them a very promising and efficient platform for targeted delivery and synergistic chemophotothermal therapy for cancer. SYNOPSIS


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Facile Fabrication of Magnetic Microrobots Based on Spirulina

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