Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A

Letter pubs.acs.org/journal/abseba

Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A Bioengineered CT-Contrast Agent Santosh Aryal,*,†,‡ Tuyen Duong Thanh Nguyen,†,‡,§ Arunkumar Pitchaimani,†,‡,⊥,§ Tej B. Shrestha,⊥ David Biller,∥ and Deryl Troyer⊥ †

Department of Chemistry, Kansas State University, 1212 Mid-Campus Drive North, Manhattan, Kansas 66506, United States Nanotechnology Innovation Center of Kansas State (NICKS), Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, United States ⊥ Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, 228 Coles Hall, Manhattan, Kansas 66506, United States ∥ Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, A-111 Mosier Hall, Manhattan, Kansas 66506, United States ‡

S Supporting Information *

ABSTRACT: Red blood cells (RBCs) are the natural resident of the vascular lumen, therefore delivery of any agents within the vascular lumen could benefit by unique natural transporting features of RBCs. RBCs continuously circulate for ∼100 days before being sequestered in the spleen, they only extravasate at sites of vascular hemorrhage. Taking advantages of these features, we engineered RBC as a carrier in order to design a unique delivery system capable of delivering X-ray computed tomography (CT) contrast agents, gold nanoparticles (AuNPs), thereby acting as CT-contrast agent. A strategic membrane fusion technique was used to engineer the surface of RBC with gold nanoparticles in this in vitro study without altering its shape, size, and surface properties. KEYWORDS: gold nanoparticle, erythrocytes, fusion, drug delivery, CT-contrast

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erythrocytes normally have a lifespan of 100−120 days, travel ∼250 km through the cardiovascular system, and function as a natural carrier for oxygen.6 Hydrodynamic forces in the circulation and the endothelial glycocalyx minimize RBC interactions with vascular walls.6 Therefore, RBCs are ideal candidates to use as a delivery system targeting vascular lumen. To date, RBCs have been used as circulating intravenous slow-release carriers for the delivery of various therapeutic agents including antibiotics and cardiovascular drugs.7−10 These drugs were loaded into the carrier RBC either by encapsulation or by ligand−receptor binding. Examples include chemical coupling of agents to the RBC surface (either covalent, or noncovalent),11,12 partial rupturing RBC and resealing,8,13,14 coupling to RBC membrane of a receptor that binds to a therapeutic agent,15 and conjugation of therapeutics with affinity ligands (e.g., antibodies or their fragments) that bind to RBC thereby anchoring cargoes on RBC.6,16,17 One such effort has been presented by Muzykantov et al., in 1992, where the authors discussed the avidin-induced lysis of biotinylated

ptimization of vascular delivery of several classes of therapeutic and diagnostic agents is an important biomedical problem because of a multifarious biological environment. This problem is especially acute in the case of delivery of potent and specific agents, which in most cases require precise localization in the target site. It is even more problematic when the goal is to target the circulatory system in order to find vascular defects. Rapid elimination via reticuloendothelial system (RES), physical fenestration, and extravasation to the tissues are major barriers for agents to remain for prolonged periods in the vascular lumen. One way to overcome these problems is by coupling agents to carriers, such as polymers, phospholipids, albumin, antibodies, or other biological molecules, and physically encapsulating into the nano/microstructured delivery platforms.1−5 Use of carrier provides optimal blood circulation half-life, restricts unintended cellular uptake, minimizes untoward effects in nontarget sites by targeting to the intended therapeutic site, and provides favorable timing of the action. Among other carriers, red blood cells (RBCs, erythrocytes, non-nuclear biconcave discs with a diameter of ∼7 μm, thickness of ∼2 μm, and plasma membrane surface area of ∼160 μm2) represent a potentially attractive unique carrier for therapeutic agents. Human © XXXX American Chemical Society

Received: September 21, 2016 Accepted: December 21, 2016 Published: December 21, 2016 A

DOI: 10.1021/acsbiomaterials.6b00573 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 1. Schematic illustration of strategic gold nanoplating of erythrocytes using fusogenic liposomes. (A) Strategic fusion mechanism showing gold nanoplating. (B) Microscopic demonstration of RhB-labeled fusogenic liposome fused RBC, phase contrast, fluorescent, and merged images. (C) Quantitative fusion study of RhB-labeled fusogenic liposome onto the RBC. (D) Fusion study conducted under the principle of FRET mechanism in which a FRET pair (fluorescent dye NBD (donor fluorophore, λem 525 nm) and RhB-labeled (acceptor fluorophore, λem 595) fusogenic liposomes were fused with RBC. Upon fusion FRET effect would be reduced once the FRET-pair-labeled liposome fuses with the RBC that do not contain fluorophore, upon which the larger spacing between the donor and acceptor fluorophore would lower the FRET efficiency and result in fluorescence recovery of the donor fluorophore at λem 525 nm.

have adapted liposome-mediated surface modification of RBCs to develop magnetic resonance contrast agent.26 Here, we report complementary engineering strategies for the gold nanoplating of RBCs to design a CT-contrast agent for the first time with optimized biocompatibilities, which could also open a door for the surface functionalization of RBC for different clinical applications including diagnosis and therapy. The cell membrane fusion strategy presented herein is based on the fusion of two different structures in which nanosized thiol-functionalized fusogenic liposomes were fused with the RBCs without altering the shape, size, and surface properties of RBCs.26 When the nanosized thiol-functionalized fusogenic liposome comes in contact with the substrate (for example RBC), having a larger surface area in phospholipids, it tends to fuse and assemble along with the phospholipids of the larger substrate as demonstrated in schematic representation (Figure 1A). To prove the concept of a fusion between two different structures, we prepared the red fluorescent (L-α-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (PE-RhB)-labeled liposome and incubated with RBCs for 15 min. As can be seen in Figure 1B in the merged micrographs of bright-field and fluorescent PE-RhB field, most of the fluorescent signals due to PE-RhB were localized on the surface of the RBC. This broadly distributed fluorescence was painted over the entire RBC surface area, which can be explained by the fusion of nanoscale fusogenic liposomes in the manner similar to that of our earlier work where fusogenic gadolinium liposomes were fused onto the surface of RBC.26 This broadly distributed RhB fluorescence onto the RBC was also confirmed from the experiment where increasing numbers of RhB labeled liposomes when fused with RBCs, increases the

erythrocytes. The stability of erythrocytes has been increased either by reducing the surface density of avidin or by blocking the biotin binding sites. This study illustrated the bioconjugation techniques for the membrane stability and mechanism of erythrocytes lysis.18 Similarly, various techniques to engineer drug-loaded RBC for the drug delivery purposes have been reviewed and its roadmap for possible marketing approval have been discussed.19 In addition to RBC, similar approaches have been studied as cell-based drug delivery systems where nanoparticles loaded stem cells were used as a “Trojan horse” for targeted drug delivery.20−23 Given that macrophages usually capture foreign materials, they could be easily loaded with therapeutic agents in vitro. RNA-loaded liposomes, magnetic NPs, Au nanoshells, imaging-agent-loaded NPs (zirconium-89 or quantum dot), and drug-loaded NPs were able to be phagocytosed by macrophages and accumulate in tumor tissue or even migrate through the blood−brain barrier into brain tumors.20,23−25 It is clear from the aforementioned improvements in different cell-based delivery systems that there is a huge interest in the use of endogenous materials in the field of medical application and this could be the promising way to move forward as a personalized medicine in near future. In the meantime, the engineering challenge to engineer such systems cannot be avoided as protocols have adapted covalent conjugation or hypotonic treatment of the cells to load agents of interest, which utilizes the pretreatment with a number of chemicals. Such chemical exposure could change the nature of RBCs and cells, like shape, size, chemistry, and mechanical properties, which could alter the in vivo fate of the system. More specifically, the change in shape, size, and deformability of RBC can significantly vary its pharmacokinetics. Recently, we B


Letter


Figure 2. Characterization of RBC. (A) Confocal laser scanning microscopic image showing z-stack to ensure RhB labeled liposome and RBC surface fusion. (B) Determination of the concentration of AuNPs onto the Au nanoplated RBC. (C) Stability of Au-RBC at various AuNPs input and comparison with pure RBC measured by measuring the released hemoglobin during nanoplating. The experiment was conducted at a fixed number of RBC for 2 h. (D) Retention of RBC surface protein in Au-RBC analyzed using gel electrophoresis.

energy transfer mechanism between the fluorophores.27−29 First, a fusogenic FRET liposome was fabricated using fluorescence donor (L-α-phosphatidylethanolamine-N-(4-nitrobenzo-2-oxa-1,3-diazole) (ammonium salt) (egg-transphosphatidylated, chicken), (PE-NBD) and a fluorescence acceptor PERhB as liposome building blocks along with other phospholipids. When donor and acceptor are in close proximity, the donor can transfer energy to the acceptor, thereby diminishing its own emission energy.27 However, when the liposome fuses with RBC, the distance between the donor and acceptor increases. As can be seen in Figure 1D, it is clear that the fluorescent intensity of the donor increases with fusion, which is only possible when FRET-liposome fuses with RBC with subsequent increases in the distance between the two fluorophores, resulting in the fluorescent recovery of the donor. With the confirmation in molecular level (Figure 1C, D), we imaged RBC under a confocal laser scanning microscope (CLSM) to proof our claim of surface fusion. As shown in Figure 2A, the z-stack image clearly revealed the RhB fluorescent signal from liposome is broadly distributed onto the surface of RBC. Z-stack records images at different focal planes to visualize entire sample volume; therefore, with this measurement and along with FRET study we can confirm that the liposome was fused to distribute throughout the surface of RBCs. Next, RBCs modified with thiol liposomes were incubated with AuNPs 5 ± 0.5 nm at 4 °C in PBS at pH 7.4. In a typical experiment 8, 16, 40 μg/mL of AuNPs were treated with liposome infused RBCs (fixed number of RBC = 1 × 107) for the period of 2h at 4 °C. The resulting Au nanoplated RBCs were purified by repeated centrifugation and collected at suspension in PBS. The Au content was

Figure 3. Transmission electron microscopic images of RBC, AuNP, and Au-RBC. Inset of Au-RBC electron micrographs of Au-RBC shows the plating of RBC surface with clusters of 5 nm AuNPs.

fluorescent signal of RhB at 595 nm (Figure 1C). To further understand the fusion behavior at a molecular level, we conducted a fluorescence resonance energy transfer (FRET) experiment.26 FRET is a widely used technique that measures the distance of molecules at the molecular level based on the C


Letter


Figure 4. Hyperspectral images showing structural integrity of pure RBC and AuNP plated RBC with distinctive surface plasmon resonance spectrum of AuNPs.

measuring the hemoglobin release during the nano plating process (Figure 2C) in the presence and absence of surface infused thiolated liposome. Over the range of input concentration of AuNPs, RBC infused with 1016 number of liposomes showed the higher stability when the input was 16 μg/mL (Figure 2C). Prior to Au nanoplating, a challenge experiment was also done with a various number of thiolated liposomes (Figure S1) in order to optimize the proper concentration of liposome with higher RBC stability. It was found that the maximum percentage of liposomes that we can infuse onto the RBC was ∼60% with a minimal release of hemoglobin (less than 10%). Therefore, this sample was used in our plating experiment. In all challenged samples, the hemoglobin release is less than 10%, which also suggests that the liposome fusion process is smooth and compatible with RBC. At this stage, the size of the RBC and Au-RBC was also evaluated using Image-J software and calculated the overall size distribution before and after Au nanoplating to ensure the intact structure of RBCs (Figure S2). Figure S2 revealed no significant change in the size distribution of RBCs before and after Au nanoplating. The size of the RBCs before and after Au nanoplating ranges between 7 and 8 μm. RBC surface intactness was further analyzed and confirmed by zeta measurement indicating that there is not a significant change in surface charge before (−26 ± 4 mV) and after Au nanoplating (−28 ± 3 mV). Furthermore, we conducted gel electrophoresis of both RBC and Au-RBC and found that all the surface markers and proteins present in RBCs were intact Figure 2D. More specifically, a major characteristic band of RBCs such as spectrin and protein 4.1 (Band 4.1) which interact with the inner leaflet of the lipid bilayer is well resolved in Au-RBC,30 which is the indicative of structural preservation of RBC after fusion and Au nanoplating. Next, we analyzed the electron microscopic features of RBC to understand how these AuNPs distributed onto the surface of RBC. Figure 3 confirms that the AuNPs were localized onto the

Figure 5. Measurement of CT-contrast enhancement in engineered Au-RBC. (A) CT images of Pure RBC, Au-RBC, and AuNPs embedded in agarose gel. (B) X-ray attenuation (Housefield Unit, HU) of AuNPs, Au-RBC, RBC (*P < 0.05, Student’s t test).

determined by inductively coupled plasma-mass spectroscopy (ICP-MS). As shown in Figure 2B, with various initial inputs of AuNPs, it has been observed that 20% of AuNPs were successfully plated onto the RBCs. In addition to AuNPs loading, we further analyzed the stability of Au-RBC by D



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surface of RBC and the evidence of the formation of larger clusters of AuNPs. Moreover, the surface properties before and after Au nanoplating were studied using Hyperspectral Imaging System (HIS) with enhanced dark field microscopy for probing and characterizing changes in RBCs. HIS is a rapidly growing modality for biomedical applications.31−33 The technique provides both spectral and spatial information in one measurement and does not require contact between the object and the sensor. The HIS integrated-dark field-based microscope technology is specifically designed for mapping of surfaces and surface material identification.31,34,35 The HIS spectrometer allows analysis of scattered light at pixel-by-pixel level; thus, samples can be imaged by acquiring hundreds of contiguous wavelengths or bands producing extensive spatial and spectral data for each pixel.34 Figure 4 shows the hyperspectral image and corresponding spectrum of unplated and Au nanoplated RBC. A distinct light scattering signature due to surface plasmonic material in Au-RBC (compared to unplated RBC) confirms the surface modification of RBC with AuNPs. Light scattering from ordinary RBC gave rise to three visible and near-infrared (VNIR) spectra, all characterized by the presence of three peaks at about 510, 555, and 590 nm; among them, the latter had a slightly higher intensity. With Au nanoplating, a significant change in the VNIR spectra was observed. Peaks at 510 nm in unplated RBCs disappeared or possibly might have coupled with a peak at 555 nm in the case of Au-RBC. The intensity of the peak at 555 nm is significantly higher, which is only possible due to the presence of surface AuNPs clusters. AuNPs have characteristic surface plasmon band (SPB) ranges from visible to NIR range depending on the size and shape. AuNPs used for plating of RBC are of 5 nm and shows SPB at 520 nm (Figure S3). Here, after Au nanoplating onto the surface of RBCs, the formation of clusters of Au shifted SPB to 555 nm. The formation of Au nanoclusters is also supported by the TEM micrographs (Figure 3) in which the presence of AuNPs on the surface of RBC was clearly visible. Finally, the X-ray attenuation properties as an application of engineered Au-RBC was investigated in comparison with a free RBC. CT-contrast phantom images (Figure 5A) of the aqueous suspensions of AuNPs, Au-RBC, and RBC embedded in agarose gel shows that the Au-RBC gives stronger darker contrast than that of RBC. As shown in Figure 5B, the change in the attenuation coefficient of the Au-RBC is lower than that of RBC because of the darker contrast. Further, by improving the loading of AuNPs onto the RBC the contrast efficiency of Au-RBC can be improved. This demonstrates that the AuNPs were attached to the RBC, yielding a distinguishable CT attenuation number that is lower than that of typical RBC, thus making the Au-RBC detectable in the pool of blood. In conclusion, we have demonstrated a preliminary, yet informative contribution, to the engineering of RBCs. RBCs are naturally designed intravascular carriers characterized by unique longevity in the bloodstream, biocompatibility, and safe physiological mechanisms for metabolism. Because of these unique natural and transporting features, vascular delivery of CT-contrast agents may benefit from the carriage by RBCs, because it naturally stays within the vascular lumen, in order to visualize vascular abnormalities. Considering the RBC’s longer plasma circulation half-life, the double structural fusion approach presented here has a potential to design a drug delivery system where drug longevity in plasma is crucial.

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00573. Detailed materials and methods sections, size distribution analysis of RBC and Au-RBC, RBC stability, UV−vis of AuNPs, biocompatibility of AuNPs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Santosh Aryal: 0000-0002-7807-6342 Author Contributions §

T.D.T.N. and A.P. contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the support from Nanotechnology Innovation Center of Kansas State (NICKS) and Johnson Cancer Research Center (JCRC), Kansas State University, Manhattan, Kansas. Confocal core supported by CVM-KSU.

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REFERENCES

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Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A

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