Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte

Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte Membrane-Coated Magnetic Nanoparticles for Enhanced Imaging-Guided Cancer Therapy Lang Rao,† Bo Cai,† Lin-Lin Bu,‡ Qing-Quan Liao,† Shi-Shang Guo,† Xing-Zhong Zhao,† Wen-Fei Dong,*,§ and Wei Liu*,† †

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China ‡ Department of Oral Maxillofacial Head Neck Oncology, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, China § Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163, China S Supporting Information *

ABSTRACT: Biomimetic cell membrane-coated nanoparticles (CM-NPs) with superior biochemical properties have been broadly utilized for various biomedical applications. Currently, researchers primarily focus on using ultrasonic treatment and mechanical extrusion to improve the synthesis of CM-NPs. In this work, we demonstrate that microfluidic electroporation can effectively facilitate the synthesis of CM-NPs. To test it, Fe3O4 magnetic nanoparticles (MNs) and red blood cell membrane-derived vesicles (RBCvesicles) are infused into a microfluidic device. When the mixture of MNs and RBC-vesicles flow through the electroporation zone, the electric pulses can effectively promote the entry of MNs into RBC-vesicles. After that, the resulting RBC membrane-capped MNs (RBC-MNs) are collected from the chip and injected into experimental animals to test the in vivo performance. Owing to the superior magnetic and photothermal properties of the MN cores and the long blood circulation characteristic of the RBC membrane shells, core−shell RBCMNs were used for enhanced tumor magnetic resonance imaging (MRI) and photothermal therapy (PTT). Due to the completer cell membrane coating, RBC-MNs prepared by microfluidic electroporation strategy exhibit significantly better treatment effect than the one fabricated by conventional extrusion. We believe the combination of microfluidic electroporation and CM-NPs provides an insight into the synthesis of bioinpired nanoparticles to improve cancer diagnosis and therapy. KEYWORDS: microfluidic electroporation, iron oxide magnetic nanoparticles, red blood cell membrane, tumor magnetic resonance imaging, cancer photothermal therapy ancer is a leading cause of mortality among humans.1 Current cancer therapy strategy mainly includes surgery, radiotherapy, and chemotherapy.2 However, surgery is often not able to completely remove all cancer cells in the human body, and radiotherapy and chemotherapy elicit severe side effects in normal tissues and have limited specificity for cancer cells.3,4 Photothermal therapy (PTT), which utilizes photosensitive nanoparticles to convert light to heat, can effectively kill cancer cells under light irradiation without causing substantial damage to normal tissues in the dark.5 Various nanomediators with superior photothermal conversion properties, such as gold nanoparticles,6 carbon nanotubes,7

graphene oxide nanosheets,8 and black phosphorus-based nanospheres,9 have been employed for PTT applications. More recently, clustered Fe3O4 magnetic nanoparticles (MNs) have been reported to possess broad photoabsorption in the near-infrared (NIR) range and have further been used as a class of photosensitizers for tumor PTT.10,11 Compared with other photosensitive nanomaterials, MNs possess various unique

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© 2017 American Chemical Society

Received: January 7, 2017 Accepted: March 8, 2017 Published: March 8, 2017 3496

DOI: 10.1021/acsnano.7b00133 ACS Nano 2017, 11, 3496−3505

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Figure 1. Microfluidic electroporation-facilitated synthesis of RBC-MNs for enhanced imaging-guided cancer therapy. (a) Microfluidic electroporation facilitates the synthesis of RBC-MNs. (b) Subsequently, the RBC-MNs, which are collected from the microfluidic chip, enrich in the tumor site after the blood circulation. (c) Biomimetic RBC-MNs are further used for enhanced in vivo tumor MRI and PTT.

poration.31−35 Meanwhile, microfluidic devices, with the advantages of high throughput, quantitative format, and parallel dependence, have emerged as a promising platform for synthesis of versatile nanomaterials.36−40 Thus, it is conceivable to use microfluidic electroporation to promote the entry of nanoparticles into cell membranes and facilitate the synthesis of RBC membrane-capped MNs (RBC-MNs). In this work, as shown in Figure 1, we fabricated a microfluidic chip used for electroporation in which MNs and RBC membrane-derived vesicles (RBC-vesicles) were completely merged and then flowed through the electroporation zone. The electric pulses between two electrodes could effectively promote the entry of MNs into RBC-vesicles and further facilitate the synthesis of RBC-MNs. Subsequently, the resulting RBC-MNs were collected from the chip and injected into experimental animals. After systematic circulation, RBC-MNs enriched in the tumor site via the enhanced permeability and retention (EPR) effect. Finally, through the classical endocytosis process including phagocytosis and pinocytosis with clathrin-mediated and clathrin-independent modes, RBC-MNs entered the cytoplasm of cancer cells.41 With the superior magnetic and optical absorption properties of the MNs and the long blood circulation time of the RBC membranes, core−shell RBCMNs were used for enhanced MRI-guided cancer PTT, demonstrating the potential of microfluidic electroporationfacilitated RBC-MNs in cancer diagnosis and therapy.

advantages, such as superior biocompatibility and biodegradability (MNs can be degraded into iron ions in vivo and excess iron ions can be transferred into ferritin proteins for iron storage and detoxification),12 and most importantly good superparamagnetic properties and utility as contrast agents for magnetic resonance imaging (MRI).13−15 However, most Fe3O4 nanomaterials, as with other exogenetic biomaterials, are easily recognized as intruders by the immunity system and cleared out from systematic circulation by the mononuclear phagocyte system (MPS).16 The current gold standard method to reduce the MPS uptake is surface modification of nanoparticles with a layer of hydrophilic poly(ethylene glycol) (PEG).17 However, accumulating reports have demonstrated that PEG triggers an “accelerated blood clearance (ABC)” phenomenon in experimental animals: a second dose of PEGylated nanoparticles given several days after the first injection is rapidly cleared out from blood circulation.18 Recent advances in surface chemistry have revealed that superhydrophilic poly(carboxybetaine) (PCB) can be used as an alternative to PEG to effectively alleviate the ABC effect.19 Moreover, in our previous report, we have demonstrated that red blood cell (RBC) membranes could serve as a biomimetic nanocoating to prolong the blood circulation time, reduce the ABC phenomenon, and preserve the cancer targeting capability in biological fluidics.20,21 To date, bioinspired cell membrane-coated nanoparticles are generally prepared by two steps: preparing cell membranederived vesicles and coating nanoparticles with the obtained vesicles.22 Researchers have mostly used ultrasonic treatment and mechanical extrusion to prepare these cell-mimicking nanoparticles.23−28 However, although the two methods can effectively derive cell membrane vesicles from source cells, ultrasonic treatment may destroy the nanoparticle cores, and mechanical extrusion requires prohibitively large forces in the process of extruding hard particles through a porous membrane. Thus, more facile and effective membrane coating strategies need to be developed. Electroporation, which has been widely employed to facilitate cell transfection, relies on applied electric fields to break down the dielectric layer over cell membranes and create multiple transient pores for biomolecules and nanoparticles to enter.29 Since the proof-of-concept of microfluidic electroporation was reported in 2001,30 a variety of microfluidic chips have been developed to improve the transfection performance and to decrease the applied voltage needed for efficient electro-

RESULTS AND DISCUSSION Microfluidic Electroporation-Facilitated Synthesis of RBC-MNs. A microfluidic chip used for electroporation was developed (Figure 2a) that consisted of five primary parts: two inlets, a Y-shaped merging channel, an S-shaped mixing channel, an electroporation zone and one outlet. To test the reliability of our chip, we measured the relative optical density (OD) at five detection points (C1−C5) in the polydimethylsiloxane (PDMS) channel to estimate whether two reagents were adequately mixed (Figure 2b).42 As shown in Figure 2c, the distribution of the OD is relatively uneven at point C1. From C1 to C5, the OD becomes increasingly smooth and steady. At C5, the OD along the line perpendicular to the channel is nearly uniform, demonstrating that the two reagents have been completely mixed. Then, we infused MNs and RBC-vesicles into the microfluidic chip. MNs of ∼80 nm in diameter were synthesized by using a modified solvothermal method (Figure S1, Supporting 3497


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24 h in FBS, while MNs exhibited significant size increases caused by the nanoparticle aggregation (Figure S4). Then, we attempted to optimize the pulse voltage, duration, and flow velocity. RBC-MNs were collected from the microchip outlet, and the size was individually measured after being stored in FBS for 24 h. As shown in Figure 2e, when the pulse voltage, duration, and flow velocity were set at 50 V, 200 μs, and 20 μL min−1, respectively, the resulting nanoparticles exhibited the smallest diameter, indicating a complete cell membrane coating onto the nanoparticles. It should be noted that, Kinosita et al. used a single square-wave electric pulse of intensity 3.7 kV cm−1 to electrodes to format of pores on human RBCs,50 and Teissie et al. and Neumann et al. used a single square-wave electric pulse of intensity 20−80 kV cm−1 to induce pores on vesicles.51,52 However, the duration of the pulse they used is 5−20 μs. In our work, although the intensity was ∼3 kV cm−1, the duration was 200 μs, which is around 10 times larger than the one they used. We speculate it was the larger duration that led to the effective permeabilization of 200 nm RBC-vesicles. Structure and Performance Characterization of RBCMNs. Physicochemical characterizations were induced to verify the core−shell structure of RBC-MNs. Dynamic light scattering (DLS) demonstrated that after the fusion of MNs and RBCvesicles, the hydrodynamic diameter and zeta potential of the obtained RBC-MNs increased to approximately 100 nm and −10 mV (Figure 3a), respectively, indicating successful RBC

Figure 2. On-chip manipulation and optimization. (a) Photo of the electroporation-integrated microfluidic chip. (b) DI water and blue dye merged in the Y-shaped PDMS microchannel and thoroughly mixed in the S-shaped channel. After the intensive mixing, the mixture flowed through the electroporation zone. (c) The normalized OD of the merging solution at various locations. (d) The mixture of MNs and RBC-vesicles flowed between two electrodes at the electroporation zone. (e) The influences of pulse voltage, duration, and flow velocity on the diameter of RBCMNs after being stored in 100% FBS for 1 d. Data represented as mean ± SD (n = 3).

Information).43,44 RBC-vesicles were prepared according to the previous reports.22,45 RBCs purified from fresh mouse blood were treated hypotonically to obtain empty RBCs (Figure S2). The resulting empty RBCs were subjected with ultrasonic treatment and extruded through 400 and 200 nm porous membranes, yielding the RBC-vesicles of ∼200 nm in diameter and ∼−6.4 mV in zeta potential (Figure S3). After being adequately mixed in the S-shaped channel, the mixture of MNs and RBC-vesicles was flowed through the electroporation zone (Figure 2d). Advances in bioscience and material science have demonstrated that various nanoparticles (e.g., gold nanoparticles and quantum dots) could be delivered into cells by electroporation.46,47 Both experimental results and theoretical calculations have confirmed that electroporation could induce the multiple transient pores on the cell membrane and thus enhance the transport of nanoparticles into cells.48,49 Thus, we hypothesize the effects of electric pulses could promote the entry of MNs into RBC-vesicles and facilitate the synthesis of the RBC-MNs. We first explored the influences of pulse voltage, duration, and flow velocity on the synthesis of RBC-MNs. In our previous reports, we demonstrated that after complete cell membrane coating was achieved, the nanoparticles were stable in size.20 In contrast, incomplete coverage would expose the surface of nanoparticles to ionic buffers, resulting in significant nanoparticle aggregation. Thus, we hypothesize the diameter of RBC-MNs can be used as an index to indicate the degree of integrity of RBC membrane coating on MNs. We first used the conventional extrusion method to coat MNs with excess RBCvesicles and tested the stability of the resulting RBC-MNs and original MNs in 100% fetal bovine serum (FBS). We found that, as desired, RBC-MNs exhibited a relatively stable size over

Figure 3. Structure characterization of RBC-MNs. (a) Mean diameter and zeta potential of MNs and RBC-MNs. (b) Representative TEM image of RBC-MNs. The inset shows the TEM image of single RBC-MN. (c) SDS-PAGE image of MNs and RBC-MNs. All data represented as mean ± SD (n = 3).

membrane coating onto MNs. Also, transmission electron microscopy (TEM) visualization revealed a Fe3O4 core of ∼80 nm in diameter and a lipid shell of ∼9.4 nm in thickness (Figure 3b and Figure S5), which was consistent with the reported RBC membranes of 5−10 nm in thickness.53 Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands suggested that relative to basic no-protein adsorption on MNs, RBC-MNs copied the protein contents from RBCs (Figure 3c). To head-to-head compare the performances of RBC-MNs fabricated by the conventional extrusion and the electroporation-facilitated 3498


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Figure 4. Performance characterization of RBC-MNs. (a) Photos of PBS containing RBC-MNs-E without (left) and with (right) an external magnet. (b) Field-dependent magnetization curves of nanoparticles. (c) T2 relaxation rate (1/T2) of nanoparticles at different Fe concentrations. The inset shows the T2-weighted MRI image of RBC-MNs-E at various concentrations. (d) UV−vis absorption spectra of nanoparticles. (e) Temperature curves of PBS and PBS containing nanoparticles after being exposed to an 808 nm laser. (f) Hydrodynamic size change curves of nanoparticles in 100% FBS over 15 d. Data represented as mean ± SD (n = 3). Compared with the RBC-MNs-E group, * and *** individually represent P < 0.1 and P < 0.001, respectively.

method, two kinds of nanoparticles are designated RBC-MNsC and RBC-MNs-E, respectively, in the upcoming description of experiments. MNs have been widely adopted in biomedical applications due to their superior magnetic and photosensitive properties and their use as MRI contrast agents and PTT photosensitizers.54,55 After confirming the coating of RBC membranes onto MNs, we tested the magnetic properties of nanoparticles using a physical property measurement system (PPMS) and found that both of the RBC-MNs possessed good water solubility and superparamagnetic properties (Figure 4a,b). Furthermore, a 6.0 T imaging system was employed for MRI with various nanoparticles in dispersion (Figure 4c). Additionally, the T2-weighted relaxation rate (R2) of RBCMNs-E was calculated to be 88.95 mM−1 S−1, which was close to that of MNs and indicated that the RBC membrane shell did not compromise the MRI capability of the MNs core. Meanwhile, the UV−vis absorption spectrum demonstrated that RBC-MNs-C and RBC-MNs-E obtained an additional absorption band at ∼400 nm (Figure 4d), which may be due to the influence of RBC-vesicles. Two kinds of RBC-MNs inherited an applicable absorption at ∼808 nm from the MNs core, suggesting the potential of using them for PTT applications. After that, phosphate buffer solution (PBS) or PBS containing MNs or RBC-MNs were exposed to an 808 nm NIR laser device, and the temperature was measured using an infrared (IR) thermal imaging system. As shown in Figure 4e, three suspensions containing nanoparticles exhibited a remarkable temperature increase; in contrast, the PBS control only showed a negligible increase, indicating that two kinds of RBC-MNs had similar photothermal conversion efficiency to MNs and could be used for highly efficient tumor PTT. Notably, relative to bare MNs and RBC-MNs-C, RBC-MNs-E exhibited improved colloidal stability over 15 d (Figure 4f),

which demonstrated that hydrophilic surface glycans of RBC membranes played a key role in the stabilizing effect and indicated that the nanoparticles possessed a more complete cell membrane coating after the microfluidic electroporation effect. To confirm it, TEM was employed to observe the morphology of RBC-MNs-C, and the image demonstrated that not all RBCMNs-C were completely coated by RBC membranes (Figure S6). It should be pointed out that, in our previous report,20 we found RBC-MNs-C could maintain a stable size at the lowest membrane-to-core ratio of around 0.5 mL of blood per 1 mg of MN. In this work, we demonstrated that, by using the electroporation, the membrane-to-core ratio could further lower to 0.2 mL of blood per 1 mL of MN, which is crucial in the clinical transformation of this kind biomimetic nanoparticles. In Vitro Characterization of Immune Escape and PTT Effect of RBC-MNs. Accumulating evidence suggests that the capability of RBCs to escape the macrophage uptake is due to the synthetic functions of the cell membrane components.56 Specifically, CD47, which embedded in RBC membranes as a biomarker, is able to effectively reduce the phagocytosis by macrophages through interactions with its receptor.57 After ensuring the RBC membranes on MNs, the antiphagocytosis capability of RBC-MNs was investigated by incubating RAW 264.7 murine macrophage-like cells with nanoparticles. We first performed a cell viability assay and found that the effects of nanoparticles on RAW 264.7 cells were negligible (Figure 5a), demonstrating the favorable biocompatibility of RBC-MNs. Subsequently, MNs, RBC-MNs-C, and RBC-MNs-E were individually incubated with RAW 264.7 cells over various durations, and inductively coupled plasma-atomic emission spectrometry (ICP-AES) was employed to measure the Fe content to quantitatively analyze the nanoparticle uptake by macrophages. As shown in Figure 5b, the uptake rate 3499


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immune escape capability of nanoparticles after being capped with RBC membranes. Subsequently, MCF-7 human breast cancer cells were used to test the in vitro PTT effect of nanoparticles. The nanoparticle uptake by MCF-7 cells was first analyzed, and three kinds of nanoparticles exhibited similar uptakes (Figure S8), ensuring the feasibility of the following PTT experiments. After that, the cells were incubated with MNs, RBC-MNs-C, or RBC -MNs-E and then exposed to NIR laser irradiation. As shown in Figure 5c, uncoated and RBC membrane-coated nanoparticles exhibited obvious cytotoxicity to cancer cells under laser irradiation. In contrast, even after 5 min of irradiation, the laser alone without nanoparticles caused negligible cell death. Furthermore, the confocal laser scanning microscopy (CLSM) image revealed that RBC-MNs-E possessed a superior effect on selectively ablating cancer cells at the irradiation site (Figure 5d). Along with the in vitro toxicity results (Figure S9), it has been demonstrated that after the surface modification of RBC membranes, MNs possessed better biocompatibility without compromising the capability of photothermally killing cancer cells. In Vivo MRI-Guided Tumor PTT with RBC-MNs. After confirming the magnetic and photothermal properties of MNs and the immune escape capability of RBC membranes, BALB/c nude mice bearing MCF-7 human breast tumor xenografts received an intravenous (i.v.) injection of MNs, RBC-MNs-C, or RBC-MNs-E to test the in vivo performance of nanoparticles. At various time points after the injection, fresh blood was harvested to measure the Fe content with ICP-AES. It can be seen in Figure 6a that the RBC-MNs-E group exhibited improved blood retention over 48 h, which can be attributed to the cell membrane coating on RBC-MNs-E. Before and at 24 h after the injection, the mice were also used for in vivo MRI. For RBC-MNs-E, an unambiguous tumor darkening was observed in the tumor site of the mice after the injection; in contrast, the tumor site was not obviously changed after the injection of

Figure 5. In vitro characterization of the immune escape and PTT capabilities of RBC-MNs. (a) RAW 264.7 macrophage-like cell viability after the incubation with nanoparticles at different concentrations. The cells without the addition of nanoparticles were used as a control. (b) Nanoparticle uptake by RAW 264.7 cells with various incubation durations. (c) MCF-7 cancer cell viability after various treatments for different durations. (d) Representative CLSM image of MCF-7 cells after NIR laser irradiation. The cells were incubated with RBC-MNs-E, treated with laser irradiation, and then stained with FDA and PI. Green and red fluorescence indicates live and dead cells, respectively. White line represents the boundary of area with and without irradiation. All data represented as mean ± SD (n = 4).

significantly increased at first and then gradually slowed. Also, we investigated the influence of nanoparticle concentration on the cell uptake and found that compared with uncoated MNs, two kinds of RBC membrane-coated nanoparticles exhibited similarly lower uptakes (Figure S7), suggesting the favorable

Figure 6. In vivo tumor MRI with RBC-MNs. (a) Pharmacokinetic curves of nanoparticles. (b) Representative in vivo T2-weighted MRI images of tumor-bearing mice before and after the injection of PBS or PBS containing nanoparticles. Red arrows indicate the tumor sites. (c) Biodistribution of nanoparticles in mice at 48 h after the injection. All data represented as mean ± SD (n = 6). Compared with the MNs group, ** and *** individually represent P < 0.01 and P < 0.001, respectively, and # represents P < 0.05. 3500


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Figure 7. In vivo tumor PTT with RBC-MNs. (a) Representative in vivo IR thermal images of tumor-bearing mice before and after the treatment. Black arrows indicate the tumor sites. (b) Tumor volume curves of mice after the treatment. (c) Average tumor weight of various treatment groups. The inset shows ex vivo tumor photo of mice after the treatment. (d) Representative H&E- and TUNEL-stained tumor slice images of mice after the treatment. All data represented as mean ± SEM (n = 6). Compared with the PBS group, *, **, and *** individually represent P < 0.05, P < 0.01, and P < 0.001, respectively, and ## represents P < 0.01.

uncoated MNs (Figure 6b), confirming that RBC-MNs-E possess better enrichment in tumors than MNs and RBC-MNsC. At 48 h after the injection, all mice were euthanized, and their tumors and major organs were harvested and quantified with ICP-AES. As shown in Figure 6c, RBC-MNs-E showed higher tumor accumulation and lower enrichment in the spleen and liver (the two main parts of the MPS), further confirming that RBC-MNs-E inherited immune evasion ability from the source cells and gained a more complete cell membrane surface coating. Next, another group of tumor-bearing mice received an i.v. injection of PBS or PBS containing MNs, RBC-MNs-C, or RBC-MNs-E and were treated with laser irradiation 1 h after the injection. Then, the temperature of the tumor surface was measured using the IR camera. As shown in Figure 7a, the mice treated with RBC-MNs-E + laser exhibited a temperature increase in the tumor site from 34.5 to 55.2 °C within 5 min, even better than the RBC-MNs-C + laser group, which met with our expectations on highly efficient photothermal conversion. To evaluate the antitumor effects of core−shell RBC-MNs, the mice tumor volumes were further measured every other day after the laser treatment. As shown in Figure 7b, the mice treated with RBC membrane-coated MNs and laser irradiation showed superior tumor ablation. Specifically, the RBC-MNs-E + laser group exhibited almost complete tumor inhibition, demonstrating that microfluidic elelctroporation could facilitate MNs to enter into RBC-vesicles and thus RBC-MNs-E possess a completer cell membrane coating. In contrast, the mice with the treatment of PBS or PBS + laser or MNs + laser showed flourishing tumor growth, which may due to the unfavorable tumor accumulation of MNs. It should be noted that the treatments of i.v. injection and laser irradiation did not affect the mouse body weight obviously (Figure S10). Finally, all mice were euthanized after 16 d of therapy, and their tumors were harvested and weighed. As expected, the RBCMNs-E + laser group exhibited the best outcome in tumor

inhibition (Figure 7c). Furthermore, histological examination was employed to evaluate the photothermal damage to tumor cells. As shown in Figure 7d, the hematoxylin and eosin (H&E)- and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labeling (TUNEL)-stained slice images demonstrated that after the PTT effect induced by the treatment of RBC-MNs-E + laser, the structure of the tumor tissue was lost, and many cells in the tissue were killed or apoptotic. It also should be noted that Zimmermann et al. have demonstrated drugs could be electroloaded into RBCs with MNs, and this carrier system could specifically target other organs by using an external magnetic guide.58,59 Thus, we believe the antitumor effect of our biomimetic RBC-MNs-E could be further enhanced after the use of an external magnetic field. Systematic toxicity has been a considerable misgiving for biomedical materials all along.60 Although we have demonstrated that RBC membrane-coated nanoparticles possess good biocompatibility in our previous reports,20 microfluidic chip and electroporation treatment may induce unexpected changes to nanoparticles. Thus, in this work, ICR mice received an i.v. injection of PBS or PBS containing RBC-MNs-E to test the systematic toxicity. Neither death nor a distinct weight difference was observed between the control and the treated group over 30 d (Figure S11), which suggested that no overall side effects were induced by the injection of RBC-MNs-E. Finally, all mice were euthanized, and their blood and organ samples were individually harvested for histology analysis and blood examination. No distinct differences were detected in tissue slices and blood parameters (Figures S12 and S13), further confirming the superior in vivo compatibility of RBCMNs-E.

CONCLUSIONS In summary, we have demonstrated that microfluidic electroporation can facilitate MNs to enter into RBC-vesicles. After 3501


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On-Chip Operation and Observation. Five mL of PBS containing 1 mg of MNs and RBC-vesicles obtained from 0.2 mL of mouse blood was introduced into the chip through polyethylene tubes by syringe pumps (TS2−80, Longer Precision Pump, China). A homemade high-voltage control system was employed,69 and electric pulses were applied to the solution passing between the electrodes. The voltage and duration of the electric pulses (the pulse frequency was fixed at 100 Hz) and the flow velocity of the solution were optimized for efficient electroporation. When the duration and flow velocity was set at 200 μs and 20 μL min−1, the voltage was adjusted from 20 to 70 V; when the voltage and flow velocity was set at 50 V and 20 μL min−1, the duration was adjusted from 50 to 300 μs; and when the voltage and duration was set at 50 V and 200 μs, the voltage was adjusted from 10 to 50 μL min−1. After electroporation, the obtained mixture was collected from the outlet. The process of solution in the microchip was observed, recorded, and further analyzed with Image-Pro Plus 6.0 software.42 Structure Characterization of RBC-MNs. One mL of PBS containing 50 μg of MNs or RBC-MNs was used to measure the mean diameter and zeta potential of nanoparticles with a DLS (Nano-Zen 3600, Malvern Instruments, UK). The morphology of nanoparticles was characterized using a TEM (JEM-2010HT, Japan). To prepare the TEM samples, the droplet containing MNs or RBC-MNs was contacted with copper grids and then negatively stained with uranyl acetate. SDS-PAGE was further employed to analyze the proteins on nanoparticles. MNs and RBC-MNs were individually prepared in SDS buffer and heated at 95 °C for 5 min. Then 20 μg of solution was added into each well at a 10% gel and run at 120 V for 2 h. The resulting gel was stained with Coomassie blue, washed, and observed. In addition, RBC-MNs, prepared by the conventional extrusion method at a membrane-to-core ratio of around 0.2 mL of blood per 1 mg of MNs, were introduced as a control.20,22 Performance Characterization of RBC-MNs. The magnetic property of nanoparticles was assessed at room temperature using a PPMS-9 system (Quantum Design, USA). The MRI capability of nanoparticles was measured using a small animal MRI system (6.0 T, Bruker, Germany). T2-weighted images of RBC-MNs-E at different Fe concentrations (i.e., 0.05, 0.1, 0.2, and 0.4 mM) were captured to determine the R2. The photoabsorption properties of nanoparticles were measured at room temperature using an UV−vis spectrophotometer (UV-2550, Shimadzu, Japan). The photothermal conversion efficiency of nanoparticles was characterized by measuring the temperature increase under the laser irradiation. Various dispersions containing 50 μg Fe mL−1 different nanoparticles were treated with an 808 nm NIR laser device (MDL-N-808, Changchun New Industries Optoelectronics Technology, China) at 5 W cm−2 for different durations (i.e., 0, 1, 2, 3, 4, and 5 min). The temperature was measured using an IR thermal imaging system (HBT-2A, Haobo Technology, China). The stability of nanoparticles was evaluated by measuring the diameter of various nanoparticles in 100% FBS for 15 d with DLS. Cell Viability Assay. First, RAW 264.7 and MCF-7 cells were individually cultured in 96-well culture plates for 12 h. Then different concentrations of MNs, RBC-MNs-C, and RBC-MNs-E (i.e., 12.5, 25, 50, 100, 200, 400, and 800 μg mL−1) were added to the culture medium, and the cells grown without the addition of nanoparticles were used as a control. Then the cells were further incubated for 24 h and washed with PBS for three times. At the end of the incubation, 100 μL of PBS containing 5 mg mL−1 of CCK-8 was added, and the cells were incubated for another 4 h. Finally, the cell viability was assessed using a microplate reader (Emax Precision, USA). The cytotoxicity was calculated by dividing the OD values of the treated groups (T) by the OD values of the control (C) (T/C × 100%). In Vitro Immune Escape Evaluation. First, RAW 264.7 cells were cultured in 12-well culture plates for 12 h. Then different concentrations of MNs, RBC-MNs-C, and RBC-MNs-E (i.e., 25, 50, and 100 μg Fe mL−1) were added to the culture medium, and the cells grown without the addition of nanoparticles were used as a control. The cells were further incubated for 4 h and washed. To quantify Fe uptake by cells, the cells were lysed by the addition of 0.5 mL 1% Tween 80 to each well. The cell lysate was then treated with

being collected from the microchip, RBC-MNs were injected into the experimental mice to test the in vivo performance. Due to the immune evasion capabilities of RBCs and the magnetic and optical absorption properties of MNs, RBC-MNs were successfully used for enhanced MRI-guided cancer PTT. In addition, compared to RBC-MNs-C fabricated by the conventional extrusion, microfluidic electroporation-facilitated RBCMNs-E exhibited better colloidal stability and improved in vivo MRI and PTT performance, further demonstrating that microfluidic electroporation treatment endowed nanoparticles with a completer cell membrane coating. Scalability and storing capacity are two key indicators to evaluate the possibility of translation of biomaterial platforms to the clinical. Relative to other cell-based theranostic systems (e.g., RBC- and macrophage-based platforms),61,62 the convenience of synthesis and storage of our core−shell nanoparticles is a promising indicator of future clinical transformation.63 Meanwhile, considering the advantages of microfluidic techniques in the synthesis of nanoparticles,37 we have reason to believe that the combination of microfluidic electroporation and bioinspired cell membrane-coated nanoparticles will offer strong industrialization prospects. By autologous extraction of RBC-vesicles and administration of RBC membrane-coated nanoparticles, the biomimetic membrane coating endowed nanoparticles with an immune compatible invisibility cloak. From this finding, we believe that it is possible to derive RBCvesicles from patients to prepare RBC membrane-capped nanoparticles for personalized diagnosis and therapy. It should be noted that “exosomes” are a kind of 50−100 nm extracellular vesicle that is secreted by most, if not all, cells.64 Due to its physiologic function, exosomes have been widely employed for drug delivery and also for surface functionalization of nanoparticles.65,66 Although there are similarities between cell membrane-derived vesicles and exosomes, the size and function of exosomes cannot be effectively controlled. In contrast, our method could accurately control the size and function of vesicles to coat the final nanoparticles for various biomedical applications.

MATERIALS AND METHODS Materials and Reagents. SU-8 2050 negative photoresist, AZ5214 positive photoresist, and PDMS were purchased from MicroChem (USA), Shipley (USA), and GE Toshiba Silicone (USA), respectively. PBS and FBS were obtained from Thermo-Fisher (USA). Uranyl acetate, cell counting kit-8 (CCK-8), fluorescein diacetate (FDA), and propidium iodide (PI) were purchased from SigmaAldrich (USA). SDS gel and buffer were obtained from Beyotime (China) and Invitrogen (USA), respectively. H&E and TUNEL were purchased from Roche (Switzerland). The other used solvents were gained from Aladdin-Reagent (China) and Sinopharm Chemical Reagent (China). Microfluidic Chip Design and Fabrication. All channels in the microfluidic chip were 50 μm in height and 200 μm in width. A pair of 1 mm long Au electrode was symmetrically placed at the electroporation zone under the PDMS channel, ensuring the electroporation effect on fluids in the channel. A silicon wafer mold with designed patterns was fabricated using SU-8 2050 photoresist by standard soft lithography.67 Currently, Au electrodes were patterned on a glass substrate using AZ-5214 photoresist by soft lithography and thermal evaporation.68 Then, a PDMS (A:B = 10:1) layer was cast from a silicon mold, punched, and finally bonded to the electrode-patterned glass substrate using an oxygen plasma system (Harrick Scientific, USA). Finally, the obtained microfluidic device was heated at 120 °C for 2 d to ensure soundness and regain hydrophobicity. 3502


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concentrated nitric acid and 30% H2O2 (1:2), and the resulting solution was left in an airing chamber for 12 h at room temperature and then kept in oil bath for 6 h at 80 °C to remove acids, yielding the samples for Fe content quantification using an ICP-AES (Iris Intrepid II XSP, Thermo Elemental, USA). Also, the nanoparticle concentration was fixed at 100 μg Fe mL−1, the incubation duration (i.e., 1, 2, 4, 8, 16, and 24 h) was changed, and the followed steps were processed as described above to investigate the influence of cell incubation time on the nanoparticle uptake. In Vitro PTT Assay. First, MCF-7 cells were cultured in 96-well culture plates for 12 h. Then 100 μg Fe mL−1 MNs, RBC-MNs-C, or RBC-MNs-E was added into the culture medium, and the cells grown without the addition of nanoparticles were used as a control. The cells were further incubated and washed. Subsequently, an 808 nm laser at 5 W cm−2 was employed to plates for various durations (i.e., 0, 1, 2, and 3 min). After the laser treatment, all cells were further incubated for 12 h, and the cell viability was assessed as mentioned above. To observe the capability of RBC-MNs-E to kill cells by the PTT effect, MCF-7 cells after the NIR irradiation treatment were costained with 5 μg mL−1 FDA and PI,70 and then observed using a CLSM (IX81, Olympus, Japan). In Vivo MRI, Pharmacokinetics, and Distribution. Twenty-four BALB/c nude mice bearing MCF-7 human breast tumor xenografts (n = 6) received an i.v. injection of 100 μL of PBS or PBS containing MNs, RBC-MNs-C, or RBC-MNs-E at a dose of 2.5 mg Fe kg−1. Before and 24 h after the injection, all mice were anesthetized by intraperitoneal (i.p.) injection of 80 μL 10% chloral hydrate, and T2weighted transversal cross section images were captured using the MRI system. At different time points after the injection (i.e., 0.5, 1, 2, 4, 8, 24, and 48 h), 20 μL of blood was harvested from the tail veins, and ICP-AES was employed for Fe content analysis as mentioned above. Total weight of blood was estimated to be 6% of the mouse body weight, and the Fe content in the mice injected PBS only was subtracted. To investigate the biodistribution of nanoparticles, all mice were euthanized 48 h after the injection, and their tumors and organs (i.e., livers, spleens, hearts, lungs, and kidneys) were harvested for ICPAES analysis. In Vivo PTT Assay. Twenty-four tumor-bearing mice (n = 6) received an i.v. injection of 100 μL of PBS or PBS containing MNs, RBC-MNs-C, or RBC-MNs-E at a dose of 2.5 mg Fe kg−1 every other day. One h after the injection, the mice were anesthetized, and their tumor sites were treated with the laser irradiation at 5 W cm−2 for 5 min. Another 6 mice injected with PBS only were used as a control. After each PTT treatment, the temperatures of the tumor site, the mice body weights, and tumor volumes were individually measured. On the 16th d after the first therapy, all mice were euthanized, and their tumors were harvested, weighed, and fixed in 4% neutral formalin, processed into paraffin, and sectioned at 4 μm. Finally, the sections were stained with H&E and TUNEL and examined. Statistical Analysis. All data were analyzed using one-way analysis of variance (ANOVA) following post-Tukey comparison tests with GraphPad Prism 5.0 software. P < 0.05 indicates a significantly statistical difference.

Wei Liu: 0000-0003-4789-362X Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors would like to thank Jinwen Yang and Dr. Yaoyao Ren (Center for Electron Microscopy, Wuhan University) for their kind help in TEM characterization. This work was supported by National Natural Science Foundation of China (Grant No. 61474084) and National Research and Development Program for Major Research Instruments (Grant No. 81527801). REFERENCES (1) Miller, K. D.; Siegel, R. L.; Lin, C. C.; Mariotto, A. B.; Kramer, J. L.; Rowland, J. H.; Stein, K. D.; Alteri, R.; Jemal, A. Cancer Treatment and Survivorship Statistics, 2016. Ca-Cancer J. Clin. 2016, 66, 271− 289. (2) Breugom, A. J.; Swets, M.; Bosset, J.-F.; Collette, L.; Sainato, A.; Cionini, L.; Glynne-Jones, R.; Counsell, N.; Bastiaannet, E.; van den Broek, C. B. M.; Liefers, G.-J.; Putter, H.; van de Velde, C. J. H. Adjuvant Chemotherapy after Preoperative Radiotherapy and Surgery for Patients with Rectal Cancer: A Systematic Review and MetaAnalysis of Individual Patient Data. Lancet Oncol. 2015, 16, 200−207. (3) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (4) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161−171. (5) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869−10939. (6) Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Gold Nanoparticles in Chemical and Biological Sensing. Chem. Rev. 2012, 112, 2739−2779. (7) Moon, H. K.; Lee, S. H.; Choi, H. C. In Vivo Near-InfraredMediated Tumor Destruction by Photothermal Effect of Carbon Nanotubes. ACS Nano 2009, 3, 3707−3713. (8) Yang, K.; Zhang, S.; Zhang, G.; Sun, X.; Lee, S.-T.; Liu, Z. Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient Photothermal Therapy. Nano Lett. 2010, 10, 3318−3323. (9) Shao, J.; Xie, H.; Huang, H.; Li, Z.; Sun, Z.; Xu, Y.; Xiao, Q.; Yu, X.-F.; Zhao, Y.; Zhang, H.; Wang, H.; Chu, P. K. Biodegradable Black Phosphorus-Based Nanospheres for In Vivo Photothermal Cancer Therapy. Nat. Commun. 2016, 7, 12967. (10) Chu, M.; Shao, Y.; Peng, J.; Dai, X.; Li, H.; Wu, Q.; Shi, D. Near-Infrared Laser Light-Mediated Cancer Therapy by Photothermal Effect of Fe3O4 Magnetic Nanoparticles. Biomaterials 2013, 34, 4078− 4088. (11) Ren, X.; Zheng, R.; Fang, X.; Wang, X.; Zhang, X.; Yang, W.; Sha, X. Red Blood Cell Membrane-Camouflaged Magnetic Nanoclusters for Imaging-Guided Photothermal Therapy. Biomaterials 2016, 92, 13−24. (12) Lee, N.; Yoo, D.; Ling, D.; Cho, M. H.; Hyeon, T.; Cheon, J. Iron Oxide-Based Nanoparticles for Multimodal Imaging and Magnetoresponsive Therapy. Chem. Rev. 2015, 115, 10637−10689. (13) Gao, J.; Gu, H.; Xu, B. Multifunctional Magnetic Nanoparticles: Design, Synthesis, and Biomedical Applications. Acc. Chem. Res. 2009, 42, 1097−1107. (14) Ho, D.; Sun, X.; Sun, S. Monodisperse Magnetic Nanoparticles for Theranostic Applications. Acc. Chem. Res. 2011, 44, 875−882. (15) Yang, P.; Gai, S.; Lin, J. Functionalized Mesoporous Silica Materials for Controlled Drug Delivery. Chem. Soc. Rev. 2012, 41, 3679−3698.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00133. Additional experimental details; nanoparticle synthesis and characterization; cytotoxicity and cell uptake; in vivo toxicity (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 3503


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Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte

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