Photosensitizer Decorated Red Blood Cells as an Ultrasensitive Light

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Photosensitizer Decorated Red Blood Cells as an UltraSensitive Light-Responsive Drug Delivery System Min Gao, Aiyan Hu, Xiaoqi Sun, Chao Wang, Ziliang Dong, Liangzhu Feng, and Zhuang Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15444 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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ACS Applied Materials & Interfaces

Photosensitizer Decorated Red Blood Cells as an Ultra-Sensitive Light-Responsive Drug Delivery System Min Gao, Aiyan Hu, Xiaoqi Sun, Chao Wang, Ziliang Dong, Liangzhu Feng, Zhuang Liu* Institute of Functional Nano & Soft Materials (FUNSOM), the Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, P.R. China KEYWORDS: drug delivery system, red blood cells, chlorin e6, photodynamic, light-responsive release ABSTRACT: Red blood cells (RBCs) have been widely explored as a natural drug delivery system (DDS) owing to their inherent biocompatibility and large internal cavities to load various types of functional molecules. Herein, we uncover that a photosensitizer, chlorin e6 (Ce6), could be decorated into the membrane of RBCs upon simple mixing, without affecting the membrane integrity and stability in dark. Upon light irradiation with a rather low power density, the singlet oxygen generated by Ce6 would lead to rather efficient disruption of RBC membrane. With doxorubicin (DOX), a typical chemotherapy drug, as the model, we engineer a unique type of light-responsive RBC-based DDS by decorating Ce6 on the cell membrane and loading DOX inside cells. The light triggered cell membrane break down would thus trigger instant release of 1 ACS Paragon Plus Environment


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DOX, enabling light-controlled chemotherapy with great specificity. Beyond that, our RBC system could also be utilized for loading of larger biomolecules such as enzymes, whose release as well as catalytic function is also controlled by light. Our work thus presents a unique type of biocompatible cell-based DDS that can be precisely controlled by mild external stimuli, promising not only for cancer therapy but also for other potential applications in biotechnologies.

Introduction In the past several decades, drug delivery systems (DDSs) have received tremendous attention for their excellent performance in improving the bioavailabilities of many drugs for disease treatment, especially for those anticancer drugs.1-6 DDSs, especially nanoscale DDSs, have found to be able to greatly improve therapeutic outcomes while obviously diminish side effects of those loaded drugs owning to their improved in vivo pharmacokinetic behaviors.7-10 Moreover, many pioneering studies have uncovered that precise control on the cellular uptake and drug release profiles of those nano-DDSs could further enhance the treatment efficacy and selectivity. Apart from tumor-specific internal stimuli such as reduced pH, tumor-associated enzymes, and reactive oxygen species (ROS), many external stimuli including light, heat, ultrasonication, magnetic field and some others have also been demonstrated to be efficient in modulating the cellular uptake and / or release profiles of those nano-DDSs, contributing to more efficient cancer treatment.11-19 As the most abundant type of blood cells, red blood cells (RBCs),20 have been widely explored as a natural DDS with great biocompatibility, high drug loading capacity, long blood circulation half-life, and lack of immunogenicity.21-23 With large inside cavities, RBCs have found to be efficient for loading of drugs, enzymes and even nanoparticles for diverse bio-

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applications.24-26 Recently, several different groups including ours have attempted to fabricate novel RBC-based DDSs responsive to external stimuli such as light and magnetic field to realize tumor-specific drug delivery.27-28 By disruption of cell membrane under physical stimuli, drug cargoes inside RBCs would then be effectively released.29 In 2012, Delcea and coworkers demonstrated that gold nanoparticles anchored on the surface of RBCs with increased absorbance in the near infrared (NIR) region could dramatically improve the drug release from the inner cavities under the irradiation of a NIR laser.30 In a later work by our group, we engineered another type of photothermal responsive RBC-based drug carrier by loading indocyanine green (ICG) attached bovine serum albumin (BSA) inside RBCs and then explored their potential for light-controlled cancer combination therapy.26 However, the photothermal effect to destroy RBC membrane and release loaded drugs would require a relatively high laser power density and long irradiation time to render sufficient heating. It would thus be interesting to develop light-responsive RBC-based drug delivery carriers with greater sensitivity to light via a different mechanism. It is known that the photodynamic effect of photosensitizers could induce severe hemolysis under laser irradiation.31-32 Therefore, in this work, we report a unique type of RBC-based DDS that is responsive to light-triggered photodynamic effect so as to effectively release molecules loaded inside cells (Scheme 1). In our system, it is found that chlorin e6 (Ce6), a photosensitizer, could be easily inserted into the membrane of RBCs upon mixing without obviously disturbing the structure of RBC membrane in dark. Upon exposure to 660-nm light with a rather low power density in a short period of time, such Ce6-decorated RBCs (RBC-Ce6) could be rapidly disrupted owing to the photodynamic destruction of RBC membrane. After loading with doxorubicin (DOX), a model chemotherapeutic agent for cancer therapy, into the inner aqueous



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cavities of RBCs, the generated DOX@RBC-Ce6 shows a rapid release profile of DOX upon exposure to the 660-nm light. As the result, light-controllable drug release and cancer cell killing could be realized with such DOX@RBC-Ce6 system. In addition to loading of small molecules, those Ce6 decorated RBCs could also be loaded with larger biomolecules such as horse radish peroxidases (HRP), a model enzyme, whose release and catalytic activity towards extracellular substrates is also precisely controlled by external light irradiation. Our results thus demonstrate a new method to engineer RBC-based DDS that is highly sensitive to external light irradiation, enabling remotely controllable delivery of therapeutic molecules or functional biomolecules with great specificity and selectivity.

Experimental methods 1. Materials: RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc.. Penicillin and streptomycin were purchased from Beyotime Institute of Biotechnology Co., Jiangsu, China. DOX was purchased from Dalian Meilun Biotech Co, Ltd. Chlorin e6 (Ce6) was purchased from Frontier Scientific, inc. Horse radish peroxidase (HRP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Chemiluminescence substrate reagents were purchased from BEIJING KEYBIOTECH CO. LTD. 2. Preparation of RBC-Ce6: To obtain RBCs, the orbital sinus of healthy ICR mice was punctured to extract blood, in which RBCs were then separated by centrifugation (3000 rpm, 5 min) and washed with phosphate buffered saline (PBS, pH=7.4) for twice. Then, we suspended 300 µL of the packed 4 ACS Paragon Plus Environment

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RBCs in 700 µL of PBS for further use. Later, the loading of Ce6 onto RBCs was accomplished by mixing different concentrations of Ce6 (0, 1.5, 3, 6, 12 µg/mL) with RBCs (66 µL, 30% hematocrit) in 2 mL PBS buffer for 0.5 h at room temperature. Free Ce6 was removed by washing with PBS for three times, incubation in PBS containing 10% fetal bovine serum (FBS) for 4 h to remove any loosely adsorbed Ce6, and washing with PBS once again. Subsequently, those obtained RBC-Ce6 were re-suspended in 2 mL fresh PBS and stored at 4 °C. Then at different time points (0, 6, 24, 48 h), 100 µL of above solutions were taken out and centrifuged to obtain the supernatants. UV-Vis-NIR spectra was used to measure the amounts of hemoglobin released from those RBC-Ce6 groups. Moreover, for qualitatively measuring the loading of Ce6, RBC-Ce6 were firstly washed with PBS for three times and then analyzed by a Calibur flow cytometer (BD biosciences, USA). Confocal fluorescence images of cell samples were taken by a laser scanning confocal microscope (Lecia SP5II). The amounts of Ce6 inserted in the membrane of RBCs were measured by its characteristic absorption peak at 660 nm in the RBCCe6 sample after subtracting the background absorption of blank RBCs, and this experiment was conducted based on our previous report.28 Meanwhile, a singlet oxygen sensor green (SOSG) dye was used to test the generation of singlet oxygen based on the previously protocol reported.33 In brief, 10 µL SOSG (5 mM in methanol) was mixed with 1990 µL RBC-Ce6 (10 µL/mL) obtained by RBCs incubated with different concentrations of Ce6 (0, 1.5, 3, 6, 12 µg/mL). The fluorescence intensity of SOSG was detected (λex = 494 nm, λem = 534 nm) before and after irradiation by a 660-nm light source (5 mW/cm2). 3. Characterization of RBC-Ce6: To investigate the drug releasing manner of the engineered RBC-Ce6, Ce6 released from 5 ACS Paragon Plus Environment


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RBC-Ce6 under incubation in PBS was collected by centrifugation (8000 rpm, 5 min) at different time points (1, 2, 3, 4, 5 day). UV-Vis-NIR absorbance spectra were recorded to measure the released Ce6 in the supernatant solutions. The anti-hemolysis ability of RBC-Ce6 comparing with pure RBCs was also investigated. Firstly, RBC-Ce6 (33 µL, 30% hematocrit) were re-suspended in PBS with different mOsm (30, 60, 120, 150, 180, 210, 240, 270, 300 mOsm) for 10 min. Then, those suspensions were centrifuged (8000 rpm, 5 min) and the obtained supernatants were measured by UV-Vis-NIR spectra. Subsequently, the rest of RBC-Ce6 (33 µL, 30% hematocrit) were disrupted to determine the total amount of hemoglobin. As the control, blank RBCs were treated with the same operations. Light-triggered drug release behavior of RBC-Ce6 was carried out by 660-nm light irradiation. Briefly, 1 mL suspensions with 83 µL 30% hematocrit RBC-Ce6 were exposed to 660-nm light with 5 mW/cm2 power densities for different periods of time (0, 2, 5, 10 min). 1 mL suspension with 83 µL 30% hematocrit RBCs which were incubated with Ce6 at different concentration (0, 1.5, 3, 6, 12 µg/mL) were exposed to the 660-nm light with 5 mW/cm2 power density for 10 min. Finally, those suspensions were centrifuged and the obtained supernatants were measured by UV-Vis-NIR spectra. As the controls, the supernatants of RBC-Ce6 without light irradiation were also detected. After the RBC-Ce6 sample was irradiated by 660-nm light (5 mW/cm2, 10 min), the fluorescence imaging was carried out with a laser scanning confocal microscope by Ce6 fluorescence. RBC-Ce6 without light treatment was chosen as control group. Besides, after the samples were exposed to 660-nm light, the size change of DOX@RBC-Ce6 was also measured by dynamic light scattering (DLS). 6 ACS Paragon Plus Environment

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4. Preparation of DOX@RBC-Ce6: DOX@RBC was produced based on the “hypotonic dialysis encapsulation” method reported previously.24, 26, 34, 35 The loading efficiency of DOX into RBCs was determined to be ~17% based on the fluorescence of DOX after lysis of DOX@RBC and extraction of free DOX by HCl / isopropanol. Subsequently, DOX@RBC was treated with Ce6 as preparations of RBCCe6 to get DOX@RBC-Ce6. The sample was stored in 4 °C for further use. 5. Characterization of DOX@RBC-Ce6: For flow cytometry measurement, RBCs were washed with PBS buffer for three times and then analyzed by flow cytometer. Confocal fluorescence images of DOX@RBC-Ce6 were performed by laser scanning confocal microscope. Due to the overlap of hemoglobin absorption and DOX spectra,35-36 DOX@RBC was treated with a lysis buffer with free DOX extracted from the cell lyate by an HCl / isopropanol solvent to quantify the cellular uptake of DOX.26, 37 As the controls, blank RBCs without DOX loading were also treated by the same method. 6. Cell culture experiments The 4T1 murine breast cancer cell line was originally obtained from American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C under 5% CO2. To study the cellular internalization of DOX, six groups were used in our experiments: (1) DOX@RBC-Ce6 plus light, (2) DOX@RBC-Ce6, (3) DOX@RBC plus light, (4) DOX@RBC, (5) Free DOX, (6) PBS. In each group, 4T1 cells were seeded in a 24-well plate containing 15-mm circle glass cover slides at a density of 5*10^4 cells per well. After 24 h, for group (1) - (5), RBCs (50 µL, 30% hematocrit) containing 1% penicillin/streptomycin were added and further incubated for 0.5 h. Subsequently, groups (1) and (3) were exposed to the 660-nm light irradiation with a power density of 5 mW/cm2 for 10 min.



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After additional incubation for 6 h, RBCs on culture dish were fully lysed with RBCs lysis solution (NH4Cl) for 5 min. Then those cancer cells were washed for three times with ice-cold PBS buffer. Confocal fluorescence microscope and flow cytometer were applied to examined the above samples which had been stained with 4′,6-diamidino-2-phenylindole (DAPI). For in vitro MTT experiments, 4T1 cells (5*10^4 cells) were cultured in a 24-well plate containing 1 mL RMPI-1640 medium and eight groups were designed in this experiments: (1) DOX@RBC-Ce6 plus light, (2) DOX@RBC-Ce6, (3) DOX@RBC plus light, (4) DOX@RBC, (5) RBC-Ce6 plus light, (6) RBC-Ce6, (7) free DOX, (8) control group. 50 µL of 30% hematocrit DOX@RBC-Ce6 (1*10^8) containing 1% penicillin/streptomycin were added into the plate after 24 h. Afterwards, 660-nm light (5 mW/cm2) irradiation was employed to treat groups (1), (3), (5) for 10 min, with the other control groups kept in dark. After further incubation for another 24 h, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was carried out to determine relative cell viabilities of various samples. 7. Enzyme loading and release HRP were per-modified with fluorescent dye FITC to get HRP-FITC. After that, HRP were loaded into RBC like DOX by the “hypotonic dialysis encapsulation” method and then mixed with Ce6 to obtain HRP@RBC-Ce6. Confocal images of HRP@RBC-Ce6 and light-triggered HRP release behaviors were conducted following the same protocol as that used for DOX@RBC-Ce6. HRP@RBC-Ce6 and RBC-Ce6 were centrifuged before and after LED light irradiation and then the supernatants were incubated with HRP substrate in the same volume. Chemiluminescece phenomena were observed using an in vivo imaging instruments (IVIS) spectrum system with 60 s exposure time after incubated for 3 min. Meanwhile, the in vitro MTT assay of HRP@RBC-Ce6 system also was conducted like DOX@RBC-Ce6 system. 4T1 cells


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(5*10^4 cells) were cultured in a 24-well plate containing 1 mL RMPI-1640 medium and five groups were designed in this experiments: (1) HRP@RBC-Ce6 plus light, (2) HRP@RBC-Ce6, (3) RBC-Ce6 plus light, (4) RBC-Ce6, (5) control group. 50 µL of 30% hematocrit HRP@RBCCe6 (1*10^8) containing 1% penicillin/streptomycin were added into the plate after 24 h. Afterwards, groups (1), (3) were exposed to 660-nm light (5 mW/cm2 for 10 min), while the control groups were kept in dark. Subsequently, all samples were incubated for another 24 h before the MTT assay.

Results and discussion In our experiments, we accidentally found that Ce6, a porphyrin-based photosensitizer, could be directly adsorbed into the lipid bilayer structure of RBC membrane upon simple mixing. RBCs were incubated with Ce6 at various concentrations in phosphate buffered saline (PBS) for 0.5 h, and then purified by washing with PBS containing 10% FBS to remove excess Ce6 and loosely adsorbed Ce6 on RBCs (see method section for details). Although a high Ce6 concentration would lead to hemolysis of RBC, RBCs incubated with Ce6 at concentrations below 6 µg/mL appeared to be rather stable in dark without significant hemolysis within 48 h (Figure 1A). After removal of excess free Ce6, UV-VIS-NIR spectra of those cells showed an obvious peak at 660 nm (Figure 1B), which increased as the rise of Ce6 incubating concentrations, suggesting the successful loading of Ce6 onto RBCs. Meanwhile, flow cytometry datas also uncovered strong Ce6 fluorescent signals from RBCs after being mixed with Ce6 (Figure 1C). We then investigated the distribution of Ce6 on RBCs by confocal fluorescence microscope. Intense Ce6 fluorescence signals were observed around the periphery of RBCs (Figure 1D), revealing that most of those Ce6 molecules were attached on the membrane of



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RBCs instead of inside the cavities of those cells. Notably, Ce6 molecules absorbed on RBC membrane appeared to be quite stable, with ~97% Ce6 molecules remained on RBC membrane after incubation in PBS for 5 days (Figure S1A). We speculate that such a behavior may be attributed to the possible insertion of hydrophobic Ce6 molecules into the lipid bilayer structure of RBC membrane, instead of loose adsorption of Ce6 molecules on the surface of RBCs.38 Moreover, loading of Ce6 on RBCs would not affect the stability of those cells under reduced osmatic pressures (Figure S1B&C). Photodynamic agents could kill cancer cells under light irradiation by producing singlet oxygen and reactive oxygen species.28,

39

We thus determined the hemolysis results and the

singlet oxygen generation ability of RBC-Ce6 prepared under different concentrations of Ce6 loading (Figure 2A&B). When Ce6 incubating concentration was 1.5 µg/mL or above, over 95% RBCs were broken up and a great deal of singlet oxygen could be produced by Ce6 under the 660-nm light emitting diode (LED) irradiation at the power density of 5 mW/cm2 for 10 min. In our following experiments, we chose 3 µg/mL of Ce6 as the incubation concentration to prepare the RBC-Ce6 sample, in which ~6*10^6 Ce6 molecules were loaded onto each RBC as estimated by the characteristic Ce6 absorption peak (see method section for experimental details). In fact, light irradiation for as short as 2 min appeared to be sufficient to trigger hemolysis for those Ce6-decorated RBCs (Figure 2C&D). As revealed by confocal fluorescence microscope, the vast majority of those Ce6-decorated RBCs could be completely destructed after light irradiation (Figure 2E). The sizes of the broken-up RBC-Ce6 after light exposure were determined to be mostly in ~150 nm, which was much smaller than intact RBCs, suggesting that Ce6 decorated RBCs had been broken up into nanosacle fragments (Figure 2F).


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The hypotonic dialysis encapsulation method that had been commonly used to load small molecules inside RBCs was employed in this experiment.26, 40-41 As shown in Scheme 1, by using the hypotonic dialysis encapsulation method, model drug DOX molecules were encapsulated inside RBCs, which were then loaded with Ce6 by insertion into RBC membrane upon mixing, obtaining Ce6-decorated, DOX-loaded RBCs (DOX@RBC-Ce6). As revealed by confocal fluorescence microscope, while Ce6 remained on the cell membrane of RBCs, DOX was loaded inside the cavities of those cells (Figure 3A). Flow cytometry data further confirmed that both DOX and Ce6 were successfully loaded on RBCs in our DOX@RBC-Ce6 system (Figure 3B&C). We then studied the photo-induced drug release behavior of DOX@RBC-Ce6. When those cells were exposed to 660-nm LED light at 5 mW/cm2 for 10 min, RBCs were effectively disrupted (Figure 3D), leading to instant release of DOX from RBCs in the DOX@RBC-Ce6 system (Figure 3E). Therefore, the photodynamic effect of Ce6 could be utilized to trigger highly efficient disruption of RBC membrane structures under light exposure, to allow rapid release of the payload molecules loaded inside cells. Afterwards, we would like to apply such DOX@RBC-Ce6 for light-controllable drug release and chemotherapy. 4T1 murine breast cancer cells were incubated with DOX@RBC-Ce6 and then exposed to 660-nm light irradiation for 10 min (5 mW/cm2). Several other control groups included 4T1 cells incubated with DOX@RBC-Ce6 in dark, cells incubated with the equivalent concentration of free DOX, as well as cells incubated with DOX@RBC in the presence or absence of light exposure. After incubation for 6 h, RBCs were removed from 4T1 cells by washing and then treatment with a red blood cell lysis solution. Those 4T1 cells were imaged by confocal fluorescence imaging and also examined by the flow cytometry analysis. Compared to cells treated with DOX@RBC (no Ce6) regardless of light exposure, or



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DOX@RBC-Ce6 in dark, 4T1 cells treated with DOX@RBC-Ce6 with light irradiation showed much stronger intracellular DOX fluorescence, to a level similar to those cells treated with free DOX (Figure 4A, Figure S2A). Those results suggested that the encapsulated DOX could be released from DOX@RBC-Ce6 as triggered by the 660-nm LED light and then get into cancer cells. The potential of our engineered drug-loaded RBCs for in vitro light-controlled killing of cancer cell killing was then investigated. 4T1 cells were incubated with DOX@RBC-Ce6 and then exposed to the 660-nm light for 10 min (5 mW/cm2). After further incubation for 24 h, the MTT assay (Figure 4B) was then carried out. Compared to DOX@RBC-Ce6 in dark, DOX@RBC-Ce6 plus light irradiation showed obviously enhanced therapeutic effect in killing cancer cells, owing to the light triggered DOX release from RBCs to subsequently kill cancer cells. Meanwhile, the improved efficiency compared with free DOX group could be due to DOX has potentiate PDT responses, which means the cytotoxic effect of DOX could damage the tumor cells which is further damaged by PDT.42 On the other hand, cells treated with DOX@RBC regardless of light irradiation showed much weaker cancer cell killing effects due to the limited drug release from DOX loaded RBCs without the photodynamic-induced RBC destruction mechanism. All those results collectively suggested that our DOX@RBC-Ce6 system could act as a smart DDS for light-controllable drug release and cancer cell killing. In addition to loading of small molecules such as DOX, we lastly wondered whether larger protein molecules such as functional enzymes could also be loaded inside those RBCs for lightcontrollable release in this system.43 Horse radish peroxidase (HRP) was chosen as a model enzyme in this work to demonstrate this concept (Figure 5A). Using the hypotonic dialysis encapsulation method, HRP was encapsulated in RBCs, which were then loaded with Ce6 by


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simple mixing. Confocal fluorescence microscope images confirmed that HRP (fluorescently labeled) could be effective loaded inside RBCs, with Ce6 attached on the RBC membrane (Figure 5B). Irradiation by the 660-nm LED light would lead to 97% of HRP release from HRP@RBC-Ce6 within 10 min, while the same sample showed no appreciable release of HRP in dark without light exposure (Figure 5C). We then added the substrates of HRP into the supernatants of HRP@RBC-Ce6 and RBC-Ce6 samples, with or without light irradiation. The emitted chemiluminescence was detected by an in vivo bioluminescence imaging system with an exposure time of 60 s. As expected, for HRP@RBC-Ce6 samples in dark without light-triggered release, as well as RBC-Ce6 without HRP loading, no significant chemiluminescence signals were observed (Figure 5D). In contrast, strong chemiluminescence signals showed up for HRP@RBC-Ce6 samples after the 660-nm LED light irradiation (Figure 5D), suggesting that the light-triggered disruption of RBC membrane would lead to release of HRP to react with its extracellular substrates. Meanwhile, this HRP@RBC-Ce6 system showed no obvious cytotoxicity compared with other groups (Figure S3A). Therefore, such RBC-Ce6 system could also serve as a light-controllable DDS for loading and controlled release of functional enzymes. Conclusions In summary, we have successfully engineered a light-controllable RBC-based drug delivery system for loading and remotely controlled release of molecular payloads. Upon simple mixing, photosensitizer Ce6 could be directly adsorbed into the membrane of RBC, which while being stable in dark would be disrupted under light irradiation as the result of photodynamic effect. As the result, therapeutic small drug molecules or larger biomolecules such as enzymes loaded inside those RBCs could be instantly release as triggered by light with a rather low power density and short exposure time. Such light-controllable RBC-based drug release systems show a



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number of unique advantages: 1) RBCs are natural carriers with inherent biocompatibility and ultra-long blood half-life. 2) The system is rather easy to be fabricated upon simple mixing of RBCs and Ce6. 3) The photodynamic effect triggered release much more sensitive than previously reported light-response NDDSs based on systematic nanoparticles, or RBC-based systems replying on the photothermal effect, which require irradiation by light with much higher power densities or longer time. Such RBC-based light-controllable DDS may be promising not only for remotely controlled cancer therapy, but also for other smart nano-biotechnology applications.

ASSOCIATED CONTENT Supporting Information. The release profile of Ce6 from RBC-Ce6 over time in dark, hemolytic test of RBC-Ce6 and pure RBCs at different osmotic pressures and flow cytometric analysis of intracellular DOX fluorescence intensity of 4T1 cells with various treatments were supported as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Prof. Z. Liu* Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu 215123, China. E-mail: [email protected] ACKNOWLEDGMENT


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This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203), a Jiangsu Natural Science Fund for Distinguished Young Scholars (BK20130005), Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Scheme 1. The preparation of DOX@RBC-Ce6, in which DOX was loaded inside RBCs while Ce6 was inserted into RBC membrane by hydrophobic interaction. With 660 nm light irradiation, the RBC membrane structure would be disrupted in this system and the loaded DOX would be instantly released.


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Figure 1. Ce6 loading on RBCs. (A) Stability test of RBC-Ce6 prepared by incubating RBCs with Ce6 at different concentrations (0, 1.5, 3, 6, 12 µg/mL). No significant hemolysis of RBCs was observed for RBC-Ce6 prepared at Ce6 concentrations below 6 µg/mL. (B) UV–Vis–NIR absorbance spectra of RBC-Ce6 prepared by incubation with Ce6 at different concentrations (0, 1.5, 3, 6, 12 µg/mL). The peak at ~660 nm evidenced the successful inserting of Ce6 into RBC membrane. (C) Flow cytometry data of native RBC and RBC-Ce6 prepared by incubating RBCs with Ce6 at different concentrations. (D) Confocal images of RBC-Ce6 revealing that Ce6 molecules were mostly attached on the membrane of RBCs instead of inside cells. Red color represents Ce6 molecules and the scale bar is 25 µm.



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Figure 2. Light triggered disruption of RBC-Ce6. (A) Quantitative detection of light-triggered release of hemoglobin from RBC-Ce6 prepared by incubating RBCs different concentrations of Ce6. (B) The changes of SOSG relative fluorescence intensity for RBC-Ce6 samples in (A). (C) UV–Vis–NIR absorbance spectra of the supernatants from RBC-Ce6 irradiated by 660 nm LED light at 5 mW/cm2. Inset is a photo of centrifuged RBC-Ce6 samples before and after light irradiation. (D) Quantitative detection of light-triggered release of hemoglobin from RBC-Ce6 samples in (C). (E) Confocal images of RBC-Ce6 before and after 660 nm LED light irradiation at 5 mW/cm2 for 10 min. The scale bar is 25 µm. (F) DLS data of RBC-Ce6 before and after light irradiation. Error bars were based on standard deviation (SD) of triplicate samples in this figure. 20 ACS Paragon Plus Environment

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Figure 3. DOX loading and release for DOX@RBC-Ce6. (A) Confocal images of DOX@RBCCe6. Red and green colors represent DOX and Ce6 fluorescence, respectively. The scale bar is 25 µm. (B&C) Flow cytometry data of native RBC (B) and DOX@RBC-Ce6 (C). (D) Photographs of DOX@RBC-Ce6 before and after irradiation by the 660 nm LED light for 10 min. (E) Kinetics of light-triggered release. The samples were irradiated with 660 nm LED light at the power density of 5 mW/cm2 for 10 min. Error bars were based on SD of triplicate samples.



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Figure 4. In vitro light-controllable killing of cancer cells with DOX@RBC-Ce6. (A) Confocal fluorescence images of 4T1 cells incubated with DOX@RBC-Ce6, DOX@RBC, or free DOX (20 µg/mL) for 30 min, followed by being exposed to the 660 nm LED light irradiation at 5 mW/cm2 for 10 min (or without light irradiation), and then further incubated for 6 h. Red and blue colors represent DOX fluorescence and DAPI-stained cell nuclei, respectively. Scale bar = 25 µm in all images. RBCs were removed by adding red blood cell lysis solution before imaging and analysis. (B) Relative viabilities of 4T1 cells incubated with RBC-Ce6， DOX@RBC, DOX@RBC-Ce6, or free DOX (20 µg/mL) for 30 min, followed by being exposed to 660 nm LED light irradiation (or without light irradiation), then changed the culture medium and further incubated for 24 h. Error bars are based on at least quadruplicate measurement.


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Figure 5. Enzyme loading and light-triggered release. (A) A scheme showing light-triggered release of enzyme (e.g. HRP) from Ce6-decorated RBCs. (B) Confocal images of HRP@RBCCe6. HRP were pre-modified with fluorescent dye FITC. Red and green colors represent FITC and Ce6 fluorescence, respectively. The scale bar is 15 µm. (C) Kinetics of light-triggered release of HRP without or with irradiation by the 660-nm LED light at the power density of 5 mW/cm2 for 10 min. Error bars were based on SD of triplicate samples. (D) Light-triggered chemiluminescence from HRP@RBC-Ce6. HRP@RBC-Ce6 and RBC-Ce6 were treated or untreated with the LED light. The supernatant of those samples were incubated with HRP substrates for 3 min, and then imaged by an in vivo bioluminescence imaging system (IVIS) with 60 s exposure time.



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TOC:


Photosensitizer Decorated Red Blood Cells as an Ultrasensitive Light

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