Letter Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Red Blood Cell-Shaped Microparticles with a Red Blood Cell Membrane Demonstrate Prolonged Circulation Time in Blood Koichiro Hayashi,*,†,‡ Shota Yamada,† Wataru Sakamoto,† Eri Usugi,§ Masatoshi Watanabe,§ and Toshinobu Yogo† †
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Division of Materials Research, Institute of Materials and Systems for Sustainability, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ‡ Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0044, Japan § Oncologic Pathology Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu 514-8507, Japan S Supporting Information *
ABSTRACT: Prolonging the circulation time (CT) of microparticles (MPs) in the blood is key for a successful microparticlebased medicinal approach to serve as drug delivery systems (DDSs). Previously, we reported that MPs that mimic the shape of red blood cells (RBCs) avoid accumulation in the spleen and lungs. We now describe the effectiveness of mimicking not only the shape of RBCs but also their surface structure for the prolongation of CT. RBC-shaped MPs (RBC-MPs) were electrosprayed with cellulose and covered with a native RBC membrane (RBCM) collected from mouse blood. Seven hours after intravenous injection, approximately twice as many RBCM-covered RBC-MPs (RBC-MPs@RBCM) were present in the blood of mice compared to unmodified RBC-MPs. Twenty-four hours postinjection, the concentration of RBC-MPs@RBCM in the blood was 4 times higher. These findings suggest that an RBCM covering the MPs contributed to significant CT prolongation, which may positively impact their applications as DDSs. KEYWORDS: biomimetics, biodistribution, drug delivery, smart materials, nanomedicine
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the induction of anti-PEG antibodies.7 This immune response results in the accelerated blood clearance (ABC) of subsequently injected PEGylated MPs, causing them to undergo immediate clearance from the blood because of the anti-PEG immunoglobulin M specifically produced by the first injection of the PEGylated MPs.7 Mimicking red blood cells (RBCs) may prove to be a promising strategy because RBCs by nature circulate in the blood for extended periods of time. The long CT of RBCs in blood is a result of their surface characteristics, shape, and mechanical properties.8−10 With regard to their surface characteristics, RBCs have a negatively charged surface due to sialic acid residing on the RBC membrane (RBCM), producing electrostatic repulsion between RBCs and other cells, especially other RBCs.9 The electrostatic repulsion helps to prevent the aggregation of RBCs. Furthermore, cluster of differentiation (CD) 47 residing on the RBCM has been identified as a self-marker that inhibits macrophage phagocytosis through interactions with the receptor for signalregulatory protein alpha.10 With respect to shape and mechanical properties, RBCs acquire deformability owing to their intricate shape (i.e., concave discoidal shape) and
icroparticles (MPs) are promising in the diagnosis and treatment of several diseases, with their ability to deliver substances.1 Controlling their biodistribution is a key for success in using MPs for the diagnosis and treatment of medical conditions.1 For example, prolonging the circulation time (CT) of MPs in the blood enhances their therapeutic efficacy in the treatment of arteriosclerosis and ischemic diseases.2 Micrometer-sized MPs (carriers) can deliver multiple substances or collections of cargo simultaneously in relatively abundant amounts, even if the cargo is nanoscale in size.3 In contrast, nanometer-sized carriers have difficulty delivering abundant and multiple nanoscale collections of cargo because of the small difference in size between the carrier and cargo.3 For example, a carrier diameter of 100 nm can carry a maximum of 455 molecules of protein, such as antihuman immunoglobulin G (IgG) with a molecular weight of 150 kDa (about 13 nm in diameter).4 Furthermore, it is difficult to load in the carriers genes such as plasmid DNA (pDNA) that are several micrometers in length. In contrast, a micrometer-sized carrier can carry 4.2 × 105 particles of IgG and can even deliver pDNA.5 Previously, the modification of MPs with poly(ethylene glycol) (PEG) in a process called PEGylation has been used to prolong their CT in the blood.6 Unfortunately, the PEG moiety is immunogenic and elicits an immune response with © XXXX American Chemical Society
Received: February 19, 2018 Accepted: July 10, 2018
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DOI: 10.1021/acsbiomaterials.8b00197 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering
Figure 1. Scheme for coating the red blood cell microparticles (RBC-MPs) with an RBC membrane (RBCM). Blood was collected from male ddY mice and used to generate the RBCM. The RBCM and the synthesized RBC-MPs were combined and sonicated to produce membrane-covered microparticles (RBC-MPs).
elasticity and flexibility, providing them with desirable biodistribution.8 Hence, RBCs are able to flow through blood capillaries smaller than the size of the actual RBC due to their shape being easily deformed.8 Hu et al. recently coated spherical MPs with RBCM.11 The coating of the spherical MPs with RBCM was effective for prolonging CT in the blood compared to PEGylation. In addition, Rao et al. achieved a reduction of the ABC by coating spherical MPs with RBCM.12 However, these studies used spherical MPs, not MPs with an RBC-like shape and mechanical properties. Thus, other RBC advantages such as deformability were not exploited. Considering that the extended CT of RBCs is a result of not only surface characteristics but also the shape and mechanical properties, coating RBC-like MPs (RBC-MPs) with RBCM has the potential to produce MPs that even further resemble natural RBCs. We previously reported an original technique for generating RBC-MPs by electrospraying cellulose under precisely adjusted conditions.13 Owing to their shape and mechanical properties, RBC-MPs acquire deformability and can flow through pulmonary blood capillaries and splenic interendothelial slits that are narrower than their diameter.13 As a result, the RBCMPs successfully avoid accumulation in these organs.13 In contrast, spherical MPs produced by the electrospraying of cellulose accumulate heavily in the spleen and lungs.13 Furthermore, the RBC-MPs carried heavily functional substances such as superparamagnetic nanoparticles and fluorescent dyes while retaining their RBC-like shape.14,15 In addition, we could endow the RBC-MPs with the ability to selectively trap substances, waste, and toxins in the blood, in excess.14 When the RBC-MPs were composed of superparamagnetic nanoparticles, we could collect the RBC-MPs after trapping these substances using a permanent magnet.14 Thus, prolonging the CT of RBC-MPs in the blood probably contributes to the enhancement of the trapping efficiency. It has been rtheeported that discoidal particles accumulated in tumors, independent of the enhanced permeability and retention (EPR) effect, even if their size was on the micrometer scale.16 According to this report, RBC-MPs have the potential to target tumors by prolonging their CT in the blood because they also have a discoidal shape. In the current study, we prepared RBC-MPs covered with native RBCM (RBC-MPs@RBCM) in an effort to prolong the CT of RBC-MPs in the blood. The RBC-MPs were prepared by electrospraying cellulose as previously described. To generate the RBC-MPs@RBCM, the RBCM was prepared from mouse blood, suspended in phosphate-buffered saline (PBS), and mixed with the RBC-MPs. The mixed suspension was ultrasonicated for several seconds (Figure 1). The RBCMPs@RBCMs produced were collected by centrifugation. The
effects of the RBCM coating of RBC-MPs on CT in blood were analyzed in vivo using a murine animal model. As shown by scanning electron microscopy (SEM), the RBC-MPs@RBCM maintained the shape of RBC-MPs before RBCM modification (Figure S1) and had a shape similar to that of natural RBCs (Figure 2A). The hydrodynamic
Figure 2. (A) SEM image of an RBC-MPs@RBCM. (B) Bright-field and fluorescent images of an RBC-MPs@RBCM stained with lipophilic fluorescent dye CellVue to label the cell membrane. (C) SDS-PAGE (10%) analysis of membrane proteins from (a) RBCMPs@RBCM and (b) RBCM. The gel was stained with Coomassie Brilliant Blue. Molecular-weight protein markers are indicated. The corresponding protein band for CD47 is indicated by an arrow.
diameters of RBC-MPs before and after RBCM modification were 2180 and 2530 nm, respectively (Figure S2). Furthermore, the zeta potentials of RBC-MPs before and after RBCM modification were −41 and −10 mV, respectively. According to the literature, the zeta potential of an RBCM is −6.4 mV.12 Thus, the zeta potential of RBC-MPs after RBCM modification moved on to that of the RBCM. The changes in the hydrodynamic diameter and zeta potential before and after RBCM modification suggested that the RBCM was linked to RBC-MPs. For visual confirmation that the RBC-MPs@ RBCMs were covered with a membrane, they were stained with CellVue, a fluorescent lipophilic agent used for cell membrane labeling. As shown in the confocal microscopic image, green fluorescence was emitted along the margins of the RBC-MPs (Figure 2B), demonstrating that the RBC-MPs were bound to some RBC components. Next, preservation of the native RBCM structure was confirmed by protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The RBC-MPs@RBCM had the same protein electrophoretic pattern as did native RBCM (Figure 2C). It is reported that the band for CD47 appears at 47−52 kDa.17 In the image of RBC-MPs@RBCM, the band for CD47 was observed at 52 kDa. Furthermore, CD47 was detected by Western blot (WB) analysis, as shown in Figure S3. These results demonstrated that the RBC-MPs had been successfully covered with an RBCM to generate the desired RBC-MPs@RBCM particles. B
DOI: 10.1021/acsbiomaterials.8b00197 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering Immediately, a week, and a month after dispersing the RBCMPs@RBCM in PBS, the hydrodynamic diameter was measured by dynamic light scattering analyseis. Although the size distribution somewhat broadened with time, there is little change in the average hydrodynamic diameter (Figure S4). Thus, the RBC-MPs@RBCMs were stably dispersed in PBS. The cytotoxicity of the RBC-MPs@RBCM was evaluated by the water-soluble tetrazolium salt (WST)-1 assay. The cell viability was nearly 100% at concentrations below 100 μg mL−1 (Figure S5), demonstrating that the RBC-MPs@RBCM had no cytotoxicity at these concentrations. To investigate the effects of the RBCM coating on the prolonging of CT in blood, we prepared RBC-MPs containing lipophilic near-infrared fluorescent dye XenoLight DiR (DiR) by electrospraying DiR-containing cellulose solution and coating the DiR-containing RBC-MPs (DiR-RBC-MPs) with RBCM. The DiR allowed for in vivo tracking of the MPs with fluorescence imaging. We have already confirmed little leakage of the dye from the RBC-MPs.13 The fluorescence intensity of purified DiR-RBC-MPs@RBCM was nearly equal to that of non-membrane-covered particle DiR-RBC-MPs (Figure S6). The DiR-RBC-MPs@RBCM and DiR-RBC-MPs were injected intravenously into ddY mice at a dose of 10 μg/g. At 1, 3, 7, and 24 h after injection, the fluorescent images of caudal areas, which were located well away from organs and unaffected by fluorescence from the organs, were acquired. Furthermore, the blood samples (500 μL) were collected from the mice and the fluorescent images were captured using a multispectral fluorescence imager. At all time points, the caudal area and blood of mice injected with DiR-RBC-MPs@RBCM emitted significantly higher fluorescence than did those of mice injected with DiR-RBC-MPs (Figure 3A,B). The fluorescence intensities per square meter of blood components were quantified from the fluorescent images using the region of interest (ROI) tool installed in the fluorescence imager. We confirmed that no fluorescence was observed in the fluorescent image of blood of nontreated mice (Figure S7A) and RBCM alone (Figure S7B). At 1, 3, and 7 h after injection, the fluorescence intensities of blood samples from mice injected with DiR-RBC-MPs@RBCM were approximately 2 times higher than those from mice injected with DiR-RBC-MPs (Figure 3C). Notably, at 24 h, the former was approximately 4.5 times higher than the latter. The concentrations of RBCMPs@RBCM and RBC-MPs in the blood samples were estimated using a calibration curve method (Figure S8). The concentrations of RBC-MPs@RBCM in the blood samples 1, 3, 7, and 24 h after injection were 12.3, 13.0, 11.6, and 7.4 μg/ mL, respectively. Those of RBC-MPs were 7.7, 6.0, 6.7, and 1.9 μg/mL, respectively. These results demonstrated that the RBCM coating created approximately a 2-fold increase in the concentration of RBC-MPs in the blood by 7 h after injection and approximately a 4-fold increase at 24 h. Thus, the RBCM coating was effective at significantly prolonging the CT of MPs in the blood. Perhaps natural RBCs may act as drug carriers. However, to use natural RBCs as drug carriers, we probably need to encapsulate the aimed substances in RBCs after the destruction of RBCM. In that case, the loading efficiency of substances in RBCs would be too low. Furthermore, the substances potentially interact with RBCM, which may prevent the restoration of RBCM to its original state. In contrast, in the case of the use of RBC-MPs, we can heavily load aimed substances in RBC-MPs by electrospraying the solution
Figure 3. (A) Fluorescent images of mouse caudal areas 1, 3, 7, and 24 h after the intravenous injection of (a) DiR-RBC-MPs@RBCM and (b) DiR-RBC-MPs. (B) Bright-field and fluorescent images of blood samples collected from mice 1, 3, 7, and 24 h after intravenous injection of (a) DiR-RBC-MPs@RBCM and (b) DiR-RBC-MPs. (C) Fluorescence intensities per square meter of blood samples collected from mice injected with (a) DiR-RBC-MPs@RBCM and (b) DiRRBC-MPs. Data were expressed as the mean ± standard deviation. The differences in the means of the fluorescence intensity of blood samples per square meter between groups injected with DiR-RBCMPs@RBCM and DiR-RBC-MPs were compared with Student’s t tests. P ≤ 0.05 was considered to be a significant difference.
containing the substances. Our previous studies revealed that RBC-MPs contained almost all of the added substances while maintaining the RBC-like shape.14,15 In addition, these MPs can be endowed with the ability to selectively trap excess intravital substances such as cortisol.14 Moreover, we can collect and remove the excess substances using a magnet with RBC-MPs containing magnetic nanoparticles.14 These applications may not be achieved using natural RBCs because it is difficult to endow natural RBCs with the above advantages and functions. Thus, because RBC-MPs have various functions, prolonged CT in the blood could provide beneficial effects. Such multifunctional RBC-MPs with prolonged blood CT may serve as agents for the dialysis and treatment of arteriosclerosis, ischemic diseases, depression, cancers, and so forth. The present study focused on the effectiveness of the RBCM coating of RBC-MPs and their CT in the blood. In the future, we plan to investigate the kinetics and distribution of RBCMPs@RBCM in various tissues in order to determine the influence of mimicking both the shape and surface structure of RBCs regarding the biodistribution of these MPs throughout the body. C
DOI: 10.1021/acsbiomaterials.8b00197 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
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(12) Rao, L.; Bu, L.-L.; Xu, J.-H.; Cai, B.; Yu, G.-T.; Yu, X.; He, Z.; Huang, Q.; Li, A.; Guo, S.-S.; Zhang, W.-F.; Liu, W.; Sun, Z.-J.; Wang, H.; Wang, T.-H.; Zhao, X.-Z. Red blood cell membrane as a biomimetic nanocoating for prolonged circulation time and reduced accelerated blood clearance. Small 2015, 11, 6225−6236. (13) Hayashi, K.; Yamada, S.; Hayashi, H.; Sakamoto, W.; Yogo, T. Red blood cell-like particles with the ability to avoid lung and spleen accumulation for the treatment of liver fibrosis. Biomaterials 2018, 156, 45−55. (14) Hayashi, K.; Hayashi, H.; Yamada, S.; Sakamoto, W.; Yogo, T. Cellulose-based molecularly imprinted red-blood-cell-like microparticles for the selective capture of cortisol. Carbohydr. Polym. 2018, 193, 173−178. (15) Hayashi, K.; Ono, K.; Suzuki, H.; Sawada, M.; Moriya, M.; Sakamoto, W.; Yogo, T. Electrosprayed synthesis of red-blood-celllike particles with dual modality for magnetic resonance and fluorescence imaging. Small 2010, 6, 2384−2391. (16) van de Ven, A. L.; Kim, P.; Haley, O.; Fakhoury, J. R.; Adriani, G.; Schmulen, J.; Moloney, P.; Hussain, F.; Ferrari, M.; Liu, X.; Yun, S. H.; Decuzzi, P. Rapid tumoritropic accumulation of systemically injected plateloid particles and their biodistribution. J. Controlled Release 2012, 158, 148−155. (17) Mouro-Chanteloup, I.; Delaunay, J.; Gane, P.; Nicolas, V.; Johansen, M.; Brown, E. J.; Peters, L. L.; Kim, C. L. V.; Cartron, J. P.; Colin, Y. Evidence that the red cell skeleton protein 4.2 interacts with the Rh membranecomplex member CD47. Blood 2003, 101, 338− 344.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00197. Experimental methods, SEM image, size distributions, WB analysis, fluorescent image, and calibration curve (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Koichiro Hayashi: 0000-0002-3147-5784 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI (JP15K14146, JP17H03403, and JP15H02296). REFERENCES
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DOI: 10.1021/acsbiomaterials.8b00197 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
