Red Blood Cell-Mimicking Micromotor for Active Photodynamic

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23392−23400

Red Blood Cell-Mimicking Micromotor for Active Photodynamic Cancer Therapy Changyong Gao, Zhihua Lin, Daolin Wang, Zhiguang Wu,* Hui Xie, and Qiang He* Key Laboratory of Microsystems and Microstructures Manufacturing (Ministry of Education), State Laboratory of Robotics and System (HIT), Harbin Institute of Technology, Yikuangjie 2, Harbin 150080, China

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ABSTRACT: Photodynamic therapy (PDT) is a promising cancer therapeutic strategy, which typically kills cancer cells through converting nontoxic oxygen into reactive oxygen species using photosensitizers (PSs). However, the existing PDTs are still limited by the tumor hypoxia and poor targeted accumulation of PSs. To address these challenges, we here report an acoustically powered and magnetically navigated red blood cell-mimicking (RBCM) micromotor capable of actively transporting oxygen and PS for enhanced PDT. The RBCM micromotors consist of biconcave RBC-shaped magnetic hemoglobin cores encapsulating PSs and natural RBC membrane shells. Upon exposure to an acoustic field, they are able to move in biological media at a speed of up to 56.5 μm s−1 (28.2 body lengths s−1). The direction of these RBCM micromotors can be navigated using an external magnetic field. Moreover, RBCM micromotors can not only avoid the serum fouling during the movement toward the targeted cancer cells but also possess considerable oxygen- and PS-carrying capacity. Such fuel-free RBCM micromotors provide a new approach for efficient and rapid active delivery of oxygen and PSs in a biofriendly manner for future PDT. KEYWORDS: micromotor, acoustically-driven, cell membrane camouflaging, active delivery, photodynamic therapy

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INTRODUCTION Photodynamic therapy (PDT) is emerging as a promising approach for cancer therapy, offering advantages such as being highly localized, sparing the normal tissues, being minimally invasive, and being inexpensive.1 There are three essential components in PDT: photosensitizer (PS), oxygen, and light.2 Under the irradiation of light, the excited PS transfers energy to the surrounding oxygen and then produces reactive oxygen species that can cause irreversible cellular damage to cancer cells.3 Despite the progress that has been achieved in using the PDT for tumor treatments, critical issues that limit the application of PDT remain that need to be solved.4 One of the limitations is the hypoxia in tumors.5 In most solid tumors, because of the poor vascular architecture from preexisting venues, oxygen supply is insufficient, which leads to hypoxia.6 The inadequate oxygen supply can minimize the photodynamic effect of PS and thus reduce the PDT efficacy. Another challenge is the low tumor-targeted accumulation capability of PSs.7 The poor selectivity of conventional PSs results in lower PDT efficacy and higher phototoxicity to normal tissues.8 To enhance the PDT efficiency for tumor treatment, it is urgent to target transport of oxygen and PSs efficiently to predefined cancer cells. To overcome these limitations, various kinds of approaches have been proposed. For example, hemoglobin has been widely studied as a type of blood substitute because of its oxygencarrying capability. 9 Previous reports have shown that hemoglobin-based oxygen carriers exhibit high oxygen affinity.10 To improve the selectivity and accumulation efficacy of PSs in © 2019 American Chemical Society

tumors, various delivery systems have been developed. However, it is challenging to determine how to escape the immune clearance in intravascular circulation and actively deliver oxygen and PSs to the targeted tissues.11 A promising strategy to address these questions is to develop self-propelled delivery systems that could overcome the clearance by the reticuloendothelial system and targeted transport of oxygen and PSs to the sites of diseases and thus enhance the tumor PDT efficiency. Recently, synthetic micro-/nanomotors have received considerable interest because of their potential applications including drug delivery, object separation, nanosurgery, nanolithography, and environmental remediation.12−19 Self-propulsion of these synthetic micro-/nanomotors could be realized by converting chemical energy or other external energy resources such as light, magnetic, or electronic fields.20−23 Despite the advance of these motors, especially catalytic motors, there are still many inherent limitations, such as the toxic fuels (e.g., H2O2) and poor biodegradability or biocompatibility.24−28 Because low power ultrasonic waves are biocompatible and have been widely applied in clinical applications, considerable attention has been recently paid to enable ultrasonic waves as a propelling force.29,30 The acoustically actuated motors are not only in no need of fuel supply but also allow their speed to be Received: May 7, 2019 Accepted: June 5, 2019 Published: June 5, 2019 23392

DOI: 10.1021/acsami.9b07979 ACS Appl. Mater. Interfaces 2019, 11, 23392−23400

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ACS Applied Materials & Interfaces

Figure 1. Characterization of RBCM micromotors. (A) Schematic fabrication procedure of RBCM micromotors. (B) CLSM images, (C) SEM, (D) zeta potential, and (E) hysteresis loop of the RBCM micromotors. Scale bar: 5 (B), 2 μm (C).

acoustically propelled RBCM micromotors denote a multifunctional platform with capabilities of resistance to immune clearance and active targeting transport for precision therapy in future.

changed by adjusting the applied power, which has become one of the most important physical stimuli for the fuel-free propulsion of micro-/nanomotors. It is well known that blood cells possess long circulation capability. Inspired by the natural cells, based on the method of hypotonic dilution/encapsulation, a red blood cell motor was fabricated by loading iron oxide nanoparticles into normal red blood cells and then propelled by an ultrasound field.31 However, because of the hypotonic treatment, the hemoglobin in the red blood cells was leaked, and the resulting red blood cell motor no longer has the oxygencarrying capacity like natural red blood cells. Herein, we developed an acoustic-powered, magnetic navigable red blood cell-mimicking (RBCM) micromotor, acting as an oxygen- and PS-active delivery platform to improve the photodynamic cancer therapy efficacy. The new RBCM micromotors are prepared by wrapping RBC-shaped hemoglobin particles encapsulating Fe3O4 nanoparticles and PS (indocyanine green, ICG) with red blood cell membranes because natural cell membrane-camouflaged particles have displayed long blood circulation time in living organisms.32,33 The obtained RBCM micromotors can move autonomously in fresh whole blood by converting ultrasonic energy into movement because of their RBC-shaped biconcave discoidal structure and higher density. The orientation of motion of RBCM micromotors can be conveniently modulated by applying an external magnetic field. Significantly, the RBC membrane camouflaging protects the RBCM micromotors against biofouling and immune clearance, thus enhancing their capability of motion in whole blood. Because of the excellent oxygen- and PS-carrying functionality and admirable directional motion capacity of the RBCM micromotors, the anticancer efficacy of the PDT could be greatly enhanced. These

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RESULTS AND DISCUSSION Figure 1A demonstrates the fabrication process of the PS (ICG)loaded RBCM micromotors. RBC-shaped magnetic Hb microparticles encapsulating ICG were first fabricated by chemical coprecipitation according to the previously reported procedures with some modifications.34 Briefly, 100 mL of CaCl2 (0.05 M) solution containing 5 g Hb, 500 mg dextran sulfate, and 10 mg ICG was mixed with 20 mL of NaOH (0.5 M) solution containing 500 mg of Fe3O4 nanoparticles. After encapsulating more Hb via a layer-by-layer method, the resulting RBC-shaped magnetic microparticles were camouflaged by using RBC membrane vesicles according to our published paper.35 After the removal of the Ca(OH)2 templates, RBCM micromotors were obtained. The as-prepared RBCM micromotors were characterized by confocal laser scanning microscopy (CLSM). Prior to the cell membrane cloaking, RBC membrane vesicles and Hb particles were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD, excitation/ emission = 644/665 nm), and fluorescein isothiocyanate (FITC, excitation/emission = 495/525 nm). The CLSM images in Figure 1B show a green (FITC channel) or red (DiD channel) biconcave discoidal morphology, indicating the successful camouflage of Hb particles (green color) by RBC cell membranes (red color). The structure of RBCM micromotors was further confirmed by using scanning electron microscopy (SEM). The SEM image in Figure 1C shows a biconcave 23393


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Figure 2. Motion of acoustically powered RBCM micromotors. (A) Schematic illustration of ultrasound-powered and magnetically guided motion of RBCM micromotors. (B) Time-lapse image and (C) MSD vs time of RBCM micromotors and RBCs in blood. (D) Acoustic power-dependent average velocity of RBCM micromotors in blood. (E) “On/off” motion of RBCM micromotors in serum propelled by an external ultrasound field. (F) Multimagnetic guidance of RBCM micromotors in serum by applying an external magnetic field under ultrasound field. (G) Time-lapse motion images of RBCM micromotors in PBS, serum, and blood. (H) Corresponding quantitative velocities of RBCM micromotors in different solutions. (I) Quantitative velocity of the RBCM micromotors in different viscosity HA solution; scale bar: 50 μm. Acoustic field condition: 0.8 W, 2.15 MHz.

2C). These results suggest that the RBCM micromotors possess active motion capacity under the propulsion of ultrasound field. It has been reported that both the asymmetric structure and high density are important to acoustic-propelled directional motion.36,37 In order to confirm the effect of density on the acoustically powered motion of the RBCM micromotors, the movement behaviors of RBCM micromotors with different magnetic nanoparticle loads were studied. As shown in Figure S1 and Video S2, upon propulsion with 0.8 W ultrasound field, the RBCM micromotors with magnetic nanoparticles loading percentage of 1, 5, 20, and 30% traveled in water at average velocities of 4.21, 8.03, 15.06, and 20.07 μm s−1, respectively, indicating that the material density effectively affects the motion velocity of RBCM micromotors. Compared with the density of normal RBCs varying between 1.085 and 1.115 g cm−3, the obtained RBCM micromotors have higher density because of a nearly pure hemoglobin component and the loading of magnetic nanoparticles. Therefore, the discoidal structure and higher density of RBCM micromotors result in pressure gradients and thus induce the movement of RBCM micromotors toward lower pressure areas between nodes and antinodes, as shown in the Figure S2 and Video S3. Moreover, an effective motion of RBCM micromotors with high concentration was observed under the propulsion of acoustic field (Figure S3 and Video S4). Figure 2D shows the dependence of the speed of RBCM micromotors in the blood on the applied acoustic power. It is shown that the velocity of RBCM micromotors can be easily modulated by changing the ultrasound power. Also, the controllable “on/off” motion of these RBCM micromotors

discoidal structure of the resulting RBCM micromotors with diameters of 2.1 ± 0.3 μm, like RBC morphology. As a result of red blood cell membrane camouflage, the surface zeta potential switched from −23.71 ± 1.62 mV (bare Hb particles) to a more negative value (−30.71 ± 1.28 mV) close to that of original RBCs (−34.57 ± 1.83 mV) (Figure 1D). This result not only further confirms the successful RBC membrane coating but also suggests that the RBC membrane maintained its original orientation. To evaluate the content of each component in the obtained RBCM micromotors, their concentration before and after coprecipitation was determined, respectively. It was found that the content of Hb, Fe3O4 nanoparticle, RBC membrane, and ICG in the as-prepared RBCM micromotors was 71.1, 23.6, 5.1, and 0.2%, respectively. Moreover, the amount of each constituent in the final RBCM micromotors could be regulated by controlling their initial concentrations. Figure 1E shows that the RBCM micromotors exhibited a super-paramagnetic property and high saturation magnetization value, which allows a remote guidance of RBCM micromotors using an external magnetic field. Next, we evaluated the capacities of RBCM micromotors for acoustically powered and magnetically guided motion (Figure 2A). The time-lapse image shows that as-synthesized RBCM micromotors and normal RBCs, respectively, traveled 113.22 μm (red line) and 34.40 μm (green line) in 20 s, indicating a higher velocity of RBCM micromotors (Figure 2B and Video S1). The mean square displacement (MSD) of RBCM micromotors versus time intervals shows a steeply parabolic curve, compared to a gentle increase of normal RBCs (Figure 23394


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Figure 3. Prolonged propulsion of RBCM micromotors in blood. Actual movement time-lapse images of RBCM micromotors (A) and bare Hb particles (B) after being incubated with blood for 0, 24, and 48 h. Scale bar: 20 μm. Ultrasound field conditions: 0.8 W, 2.15 MHz.

S5). These data indicate that the red blood cell membrane as the surface of biomimetic motors effectively evades the absorption of human blood proteins, and thus, the obtained RBCM micromotors possess high antipollution capability and longterm motion capacity. Next, we evaluated the cell cytotoxicity and the antiphagocytosis ability of RBCM micromotors. To examine the potential cytotoxicity of RBCM micromotors, suspensions containing 0, 5, 10, and 25 μg mL−1 RBCM micromotors were incubated with normal hepatocyte (L02 cell line) for 24 h, and then, the viability of L02 cells was measured through the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 4A, the RBCM micromotors have no evident toxic impact on hepatocyte cells, suggesting the good cell compatibility of RBCM micromotors. For hemolysis analysis, RBCM micro-

could be realized by switching on/off the propulsion of external acoustic field as displayed by the tracking line in Figure 2E. To navigate the motion of RBCM micromotors, an external magnetic field of 1 mT was employed. Figure S4 and Video S5 show that this weak magnetic field only enables the rotation of RBCM micromotors at the same position and cannot propel them. Under the propulsion of the ultrasound field and navigation of the magnetic field, the movement direction of RBCM micromotors could be continually changed and the motion velocities did not obviously decrease, indicating that the orientation of motion of RBCM micromotors could be guided by using a weak magnetic field (Figure 2F). The motion capability of RBCM micromotors was further evaluated by measuring their motion speed. As shown in Figure 2G and Video S6, RBCM micromotors could move in phosphate-buffered saline (PBS), serum, and blood. The velocities of RBCM micromotors in PBS, serum, and blood were 15.39, 10.13, and 7.15 μm s−1, respectively, at an ultrasound power of 0.8 W and a frequency of 2.15 MHz (Figure 2H). These differences in velocity arise from the solution viscosity. To better assess the influence of the viscosity on the velocity of RBCM micromotors, hyaluronan (HA) was used to tune the solution viscosity. Figure 2I shows that the speed of acoustically propelled RBCM micromotors dramatically decreased with the increase of viscosity under a low ultrasound power of 0.8 W; however, they could still move at a speed of 2.34 μm s−1 at a high viscosity of 15.5 mPa s, suggesting the practicability of RBCM micromotors for various biomedical applications. We further investigated the anti-biofouling and long-term motion capability of RBCM micromotors in whole blood after the RBC membrane surface camouflaging. Figure 3, taken from the Videos S7 and S8, illustrates the movements of RBCM micromotors and bare Hb particles in whole blood after incubation for 0, 24, and 48 h at an acoustic power of 0.8 W and a frequency of 2.15 MHz, respectively. One can see that the velocity of RBCM micromotors before and after a long period of incubation in blood (7.86 μm s−1 at 0 h, 7.75 μm s−1 at 24 h, and 6.87 μm s−1 at 48 h) has no obvious decrease, as shown by the tracking trajectories in Figure 3A and the calculated velocities in Figure S5. In contrast, the bare Hb particles showed notably hindered propulsion after the same treatments (Figures 3B and

Figure 4. Biocompatibility of RBCM micromotors. (A) Cell cytotoxicity of RBCM micromotors. (B) Hemolysis assay of RBCM micromotors. (C) Macrophage uptake of Hb particles, RBCM micromotors, and RBCs by J774 cells. (D) Absorption spectrum of RBCs before and after the ultrasonic treatment at different ultrasound power values from 0 to 5 W. 23395


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Figure 5. Targeted motion of RBCM micromotors toward HeLa cells in a microfluidic channel. (A) Schematic illustration of the targeted movement of RBCM micromotors in a PDMS microchannel with the guidance of a magnetic field. CLSM images of the distribution of RBCM micromotors at different positions in the microchannel before (B) and after (C) treatment. RBCM micromotors are shown in green (FITC channel), and HeLa cells are stained blue (DAPI channel). Scale bar: 100 μm.

Figure 6. In vitro PDT by the RBCM micromotors. (A) Schematic mechanism for the PDT using RBCM micromotors. (B) UV−vis spectra of RBCM micromotors at different binding states. (C) ODCs of the RBCM micromotors. (D) CLSM images of HeLa cells after different treatments. Living cells are displayed in green (calcein AM) and apoptotic cells are shown in red propidium iodide (PI). Scale bar: 250 μm.

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the affinity of hemoglobin for oxygen.38 Oxygen dissociation curves (ODCs) in Figure S7 show that the p50 value of RBCM micromotors (10.3 mmHg) is smaller than that of free Hb (24.4 mmHg), suggesting that the resulting RBCM micromotors have good oxygen affinity. The high oxygen affinity of the RBCM micromotors is caused by the quaternary state of Hb. It has been documented that when the quaternary state of Hb is in the Rstate, the polymerized Hb will possess low p50 (high oxygen affinity); otherwise, if the state is T-state, the polymerized Hb will have high p50 (low oxygen affinity).39 Research studies also have shown that a low p50 value is an essential factor for blood substitutes, and the suitable p50 value is 5−10 mmHg.40 Once the p50 value of oxygen carriers is high, the oxygen will be prematurely released in the precapillary arterioles and cause vasoconstriction. More importantly, the ultrasonic treatment has no significant influence on the p50 value of RBCM micromotors. The generation of cytotoxic singlet oxygen from RBCM micromotors was monitored by using a reactive oxygen species probe [9,10-anthracenediyl-bis(methylene)dimalonic acid, ABDA]. It is well known that the absorbance intensity of ABDA will decrease with the increasing concentration of singlet oxygen.41 As shown in Figure 6C, notable decreases of the ABDA absorption peaks in RBCM micromotors dispersion were observed with the increasing exposure time under 808 nm light, indicating that the RBCM micromotors possess good singlet oxygen generation capacity. Finally, we assessed the PDT efficacy of RBCM micromotors. HeLa cells were stained with PI or calcein-AM to identify apoptotic or live cells. As shown in Figure 6D, compared with the PBS-treated group, only an 808 laser or RBCM micromotors alone caused a few cell deaths. In the absence of the PS, ICG, the bare oxygen-loaded RBCM micromotors plus NIR laser had little effect on HeLa cells. Because of the presence of oxygen in the solution, the HeLa cells showed more death in the only ICG-encapsulated RBCM micromotors plus NIR laser-treated group. Under the combined treatment of dual-oxygen-ICG-loaded RBCM micromotors and the 808 nm laser, a noteworthy stronger PDT effect on HeLa cells was observed. The PDT effect was quantified by evaluating the viability of HeLa cells. The flow cytometry results in Figure S8 show that, upon exposure with 0.35 W cm−2 808 nm laser for 15 min, more than 75% HeLa cells were killed by the dualoxygen-ICG-loaded RBCM micromotors. The MTT assay measurement further confirms that the anticancer efficacy of dual-oxygen-ICG-loaded RBCM micromotors was significantly higher than that of other control groups (Figure S9). Overall, these results indicate that an enhanced photodynamic anticancer efficacy could be achieved by using RBCM micromotors as the active delivery platform.

motors were incubated with normal blood. After incubation for 3 h, the blood compatibility of RBCM micromotors was quantified by measuring the content of hemoglobin in the supernatant. Figure 4B shows that no obvious hemolytic effects were observed, indicating that the RBCM micromotors have good hemocompatibility. These are mainly attributed to the natural resources of hemoglobin and red blood cell membranes. To investigate the antiphagocytosis capability of RBCM micromotors, FITC-labeled Hb particles, FITC-labeled RBCM micromotors, and DiD-labeled red blood cells were incubated with equivalent numbers of J774 macrophage cells for 6 h. The flow cytometry measurement shows that the RBC coating was found to significantly decrease the uptake of RBCM micromotors by macrophages, compared to bare Hb particles (Figure 4C). This suggests that the RBCM micromotors could indeed inhibit phagocytosis like natural RBCs. Furthermore, we evaluated the stability of the natural RBCs upon external acoustic fields for a long period. As shown in Figure 4D, the UV−vis absorption spectrum of RBCs showed minor changes after treatment at different ultrasound powers from 0 to 5 W for 1 h. This result indicates that the used ultrasound conditions had no adverse effect on the blood cells. Taken together, these results demonstrate that RBCM micromotors possess good biological compatibility, which allows it to be used in future biomedical applications. Then, we tested the targeted motion capability of RBCM micromotors to predefined cancer areas by using a microfluidic chip (Figure 5A). Note that a green fluorescent FITC was used to label RBCM micromotors and a blue-fluorescent probe 4′,6diamidino-2-phenylindole (DAPI) was chosen to indicate HeLa cells. RBCM micromotors were initially injected in the left-side reservoir, and magnetic field was placed near the right-side reservoir. As shown in Figure 5B, the RBCM micromotors displayed no directional motion without the propulsion of an acoustic field. Under the propulsion of the ultrasound field and navigation of the external magnetic field, it was found that the RBCM micromotors rapidly moved into the right-side reservoir along the microfluidic channels (Figure 5C). These results suggest that the RBCM micromotors have the potential to be used for targeted delivery of oxygen and PSs to cancer cells. We further explored whether the RBCM micromotors could enhance the efficacy of photodynamic cancer therapy, as schematically illustrated in Figure 6A. Abundant oxygen and PSs are necessary for PDT. First, the oxygen- and ICG-carrying capabilities of RBCM micromotors were assessed by using an UV−vis spectrum. The UV−vis−NIR spectra of both the oxygenated and deoxygenated RBCM micromotors in Figure 6B show pronounced absorption peaks at 808 nm, indicating the successful encapsulation of ICG with high packing capability. The fluorescence image in Figure S6 further demonstrates that ICG was truly encapsulated into the RBCM micromotors. We also found that the oxygenated RBCM micromotors showed a characteristic peak at 414, nm and the deoxygenated RBCM micromotors exhibited a characteristic peak at 430 nm. The change of the characteristic peaks of RBCM micromotors before and after the deoxygenation demonstrates the preservation of the bioactivity of Hb proteins in RBCM micromotors, indicating that the as-prepared RBCM micromotors could bind and release oxygen. Next, the oxygen-carrying capacity of RBCM micromotors was evaluated using a parameter p50. Here, p50 is defined as the oxygen partial pressure when oxygen saturation reached 50%, representing the capacity of the blood to transport oxygen and

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CONCLUSIONS We have successfully developed a fuel-free, acoustically propelled, and magnetically navigated RBCM micromotor as an active oxygen and PS transporter for enhanced photodynamic cancer therapy. The biconcave RBC-like structure and higher density of RBCM micromotors allow for precise directional control and high-speed propulsion. We can not only conveniently regulate the speed of the RBCM micromotors but also control their motion orientation through the external ultrasonic and magnetic fields. More importantly, owing to the red blood cell membrane coating, the resulting RBCM micromotors possess anti-biofouling capability and immune clearance escape capacity and thus possess prolonged motion 23397


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After the co-precipitation, the microparticles were isolated from the solution through centrifugation at 2000g for 2 min. Then, the free ICG in the supernatant was collected and measured using a UV−vis spectrometer at the wavelength of 784 nm. The residual magnetic nanoparticles in the supernatant were collected by using a magnet. The collected magnetic nanoparticles were fried and weighed after repeated washing. To calculate the amount of the RBC membrane in the RBCM micromotors, the residual RBC membrane in the supernatant was collected by ultracentrifugation (50 000g, 30 min) and then weighed. The weight percentage of each component in the RBCM micromotors was calculated according to the equation: weight percent (%) = [weight(total) − weight(free))]/weight(RBCM micromotors) × 100%. Acoustic Propulsion. The acoustic-propelled motion of the RBCM micromotors was carried out using a method as reported previously.31 RBCM micromotors were added dropwise into an acoustic cell and covered with a thin glass slide. A 5 mm inner diameter, 10 mm outside diameter, and thickness of 0.5 mm piezoelectric transducer, which was connected with a waveform generator and a power amplifier, was used to provide ultrasound field. This piezoelectric transducer was attached to the bottom center of the acoustic cell. Then, a frequency of 2.15 MHz and varying power between 0 and 20 W were applied to the piezoelectric transducer. In this experiment, the RBCM micromotors were powered by the scattering acoustic waves which were produced at that 2.15 MHz frequency. Owing to the biconcave discoidal structure and asymmetrical distribution of the magnetic nanoparticles within the RBCM micromotors, these micromotors could be able to convert the acoustic energy into movement. In the whole movement guidance experiments, the ultrasound-propelled RBCM micromotors were oriented using an external magnetic field of 1 mT. Biocompatibility Assay. To evaluate the cytotoxicity of RBCM micromotors, normal hepatic (L02) cells were cultured in 96-well plates at a density of 1 × 104 cells per well. When L02 cells reached a concentration of 90%, different amounts of RBCM micromotors were added and co-cultured with L02 cells for 24 h. Then, L02 cells were washed with PBS, and the cell viability was tested using cell proliferation kit I under manufacturer’s instruction. All these test experiments were repeated three times. The blood biocompatibility of RBCM micromotors was tested via antihemolytic experiments. Bare Hb particles and RBCM micromotors were suspended in PBS solution and then co-cultured with red blood cells at a concentration of 50 μg mL−1, respectively. As the positive control, 0.5 mL of water was added into 0.5 mL of blood. In addition, 0.5 mL of PBS was incubated with 0.5 mL blood for the negative control. After incubation for 3 h, all the samples were centrifuged, and the supernatant parts were tested at 540 nm. The hemolysis rates were calculated according to the equation: hemolysis rate % = [(Asample absorbance − Anegative control)/(Apositive control − Anegative control)] × 100%. The antiphagocytosis ability of RBCM micromotors was evaluated using flow cytometry. J774 macrophages were cultured in 25 cm2 flasks at the density of 1 × 105 cells per flask and then individually coincubated with equivalent numbers of FITC-labeled Hb particles and FITClabeled RBCM micromotors. DiD-labeled leukocytes were incubated with J774 macrophages and were used as a control experiment. After 6 h of incubation, the noninternalized particles were removed using PBS. The antiphagocytosis capacity of RBCM micromotors was quantified using flow cytometry. Hemoglobin Carry and Release of Oxygen. The purified RBCM micromotors were added dropwise into a cell for flow-through measurements (Hellma Gmbh & Co. KG, Germany). Then, CO2 gas was injected into the cell at a low flow rate. After 2 h, a small amount of sodium dithionite was added. Moreover, the light absorption of RBCM micromotors was measured using an UV−vis spectrometer. Following that, O2 gas was flowed for 2 h, and the light absorption of RBCM micromotors was recorded. To minimize light scattering, a low concentration of RBCM micromotors (0.05 mg mL−1) was used for UV−vis measurement. Oxygen Dissociation Curve. The ODC of RBCM micromotors was measured using a described method with modification.38 An

time in the blood. Upon irradiation of 808 nm light, the generated photodynamic effect of RBCM micromotors could rapidly kill cancer cells with an enhanced photodynamic cancer therapy efficacy. Such acoustically propelled RBCM micromotors with excellent drug carrying capacity, as a new class of fuel-free propulsion and controllable platform, hold great potential in practical cancer therapy.

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EXPERIMENTAL SECTION

Materials. FeCl3·6H2O, FeCl2·4H2O, dextran sulfate, HA, 1,1′dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD), ICG, PI, calcein-AM, FITC, DAPI, ABDA, and cell proliferation kit I (MTT) were bought from Sigma-Aldrich Co., Ltd. Hemoglobin from Swine was obtained from Tokyo Chemical Industry CO., Ltd. HeLa cells, HTP-1 cells, J774 macrophage cells, and normal hepatocyte (L02 cell line) were purchased from the American Type Culture Collection. Blood was obtained from Kunming mice. Preparation of Magnetic Nanoparticles. The citrate-stabilized iron oxide nanoparticles were prepared via the method as reported in ref 42. Briefly, 1.17 g of FeCl3·6H2O and 0.43 g of FeCl2·4H2O were mixed with 20 mL of N2-treated water, followed by heating to 80 °C under the protection of N2. Then, 2.5 mL of NH4OH was added during vigorous stirring, and heating was continued for 30 min. After that, the nanoparticles were collected to the reaction flask by using a magnet, and the supernatant was decanted. Fresh water (20 mL) and citric acid solution (2 mL, 0.5 g mL−1) were added into the reaction flask, and the reaction mixture was heated to 95 °C for 90 min. Under the protection of N2, the reaction mixture was cooled to 25 °C. The obtained magnetic nanoparticles were collected and washed with deionized water three times. Preparation of RBC Membrane Nanovesicles. The preparation of RBC membrane nanovesicles consists of two steps: isolation of the RBC membrane and preparation of RBC membrane nanovesicles. To isolate the RBC membrane, the fresh whole blood was first withdrawn from the female imprinting control region mice (8−10 weeks) purchased from Harbin Medical University and then centrifuged at 800g for 5 min at 4 °C. After being washed with ice-cold 1 × PBS three times, the resulting RBCs were suspended into ice-cold 0.25 × PBS for 25 min. The RBC ghosts were obtained by centrifugation at 800g for 5 min. To prepare the RBC membrane nanovesicles, the resultant RBC ghosts were sonicated using a bath sonicator at a power of 100 W and frequency of 53 kHz for 10 min. Then, the suspension was extruded through 400, 200, and then 50 nm pore-sized carbonate porous membranes by using an Avanti mini extruder for 15 passes, separately. The prepared RBC membrane nanovesicles were stored in PBS at 4 °C. Preparation of RBCM Micromotors. The RBCM micromotors were synthesized according a previously reported protocol with slight modification.34,35 Briefly, 100 mL of mixture solution of CaCl2 (0.05 M), Hb (0.05 g mL−1), ICG (0.1 mg mL−1), and dextran sulfate (0.005 g mL−1) were quickly mixed with 20 mL of solution of NaOH (0.5 M) and Fe3O4 nanoparticles (25 mg mL−1). After 30 s, the mixture was stopped. The products were collected by centrifugation and washed with PBS three times. Then, the obtained microparticles were crosslinked by glutaraldehyde (0.05%) for 2 h, followed by centrifugation and washing. To encapsulate much more Hb, five bilayers of Hb/ glutaraldehyde were deposited on these Hb−Ca(OH)2 particles via a layer-by-layer method. After that, to fully camouflage Hb microparticles with the RBC membrane, 1 mg of these above Hb- and ICG-loaded microparticles was cosonicated with RBC membrane nanovesicles obtained from 107 RBCs for 2 h at 4 °C using a bath sonicator at a power of 100 W and a frequency of 53 kHz. Then, RBC membrane-coated microparticles were collected by centrifugation at 5000g for 1 min. Finally, the core of Ca(OH)2 was removed using Na2EDTA solution (0.1 M, pH 7.4). The obtained RBCM micromotors were stored at 4 °C. Component Analysis of RBCM Micromotors. The content of each component (Hb, Fe3O4 nanoparticle, ICG, and RBC membrane) in the as-prepared RBCM micromotors was determined by measuring the concentrations of each component before and after coprecipitation. 23398


Research Article

ACS Applied Materials & Interfaces ORCID

oxygen electrode (GMH 3630, Greisinger electronic GmbH, Germany) was used to detect the partial oxygen pressure (pO2). Hb or RBCM micromotors were suspended in PBS solution (pH 7.4). Then, at the pO2 values between 0 mmHg (Adeoxy) and 160 mmHg (Aoxy), the absorption (A) at 576, 580, and 588 nm was measured, respectively. The value of oxygen saturation (Y) was calculated at a given pO2 according to the equation: Y = (A − Adeoxy)/(Aoxy − Adeoxy). Microfluidic Experiment. Polydimethylsiloxane (PDMS) microfluidic channels were prepared using conventional soft lithography techniques. Then, PDMS microchannels were filled with diluent serum solution, the left-side reservoir was injected with RBCM micromotors, and the right-side reservoir was filled by HeLa cells. The movement of RBCM micromotors was propelled by acoustic field, and the movement orientation was guided by a magnetic field, which was placed near the right-side reservoir. PDT Experiments. To evaluate the PDT efficacy of RBCM micromotors, HeLa cells were treated differently and different indicators marked their growths. In the experiments, the PBS-treated group was chosen as the blank group. The only 808 nm laser-irradiated group, RBCM micromotors alone cocultured group, the bare oxygenloaded RBCM micromotors plus 808 nm laser-irradiation treated group, and the only ICG-loaded RBCM micromotors plus 808 nm laser-irradiation treated group were used as negative groups. The dualoxygen-ICG-loaded RBCM micromotor-treated group was chosen as the experimental group. The power of the 808 nm laser was 0.35 W cm−2, and the exposure time was 15 min. After treatments, PI and calcein AM were added to each group. The PDT effects were observed using CLSM and quantified via the MTT assay and flow cytometry.

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Changyong Gao: 0000-0001-8137-7894 Zhiguang Wu: 0000-0002-0570-0757 Hui Xie: 0000-0003-4299-2776 Qiang He: 0000-0002-3557-6865 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the National Natural Science Foundation of China (nos. 21573053, 21603047) and National Postdoctoral Program for Innovative Talents (BX201700065).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b07979. Movement of the RBCM micromotors with different magnetic nanoparticles loading percentage and in high concentration; propulsion performance of the RBCM micromotors; corresponding quantitative velocities of RBCM micromotors and bare Hb particles; fluorescence image of ICG-loaded RBCM micromotors; oxygen dissociation curves of the RBCM micromotors; quantitative analysis of apoptotic HeLa cells by flow cytometry; and PDT efficacy calculated by determining the HeLa cell viability (PDF) Movement of RBCM micromotors under an ultrasound field (AVI) Acoustic propelled motion of RBCM micromotors in various media (AVI) Movement of RBCM micromotors in serum under various external stimulus conditions (AVI) Movement of the RBCM micromotors in the whole blood initially and following 24, 48 h incubation (AVI) Motion of the Hb particles in the whole blood initially and following 24 and 48 h incubation under an acoustic field (AVI) Motion of RBCM micromotors in various media including PBS, serum, and blood (AVI) Movement of the RBCM micromotors in the whole blood initially and following 24 and 48 h incubation (AVI) Movement of the Hb particles in the whole blood initially and following 24 and 48 h incubation (AVI)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.W.). *E-mail: [email protected] (Q.H.). 23399


Research Article

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Red Blood Cell-Mimicking Micromotor for Active Photodynamic

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