Morphological Characteristics, Hemoglobin Content, and Membrane Mechanical Properties of Red Blood Cell Delivery Systems

Abstract

Red blood cell (RBC)-based systems are under extensive development as platforms for the delivery of various biomedical agents. While the importance of the membrane biochemical characteristics in relation to circulation kinetics of RBC delivery systems has been recognized, the membrane mechanical properties of such carriers have not been extensively studied. Using optical methods in conjunction with image analysis and mechanical modeling, we have quantified the morphological and membrane mechanical characteristics of RBC-derived microparticles containing the near-infrared cargo indocyanine green (ICG). We find that these particles have a significantly lower surface area, volume, and deformability as compared to normal RBCs. The residual hemoglobin has a spatially distorted distribution in the particles. The membrane bending modulus of the particles is about twofold higher as compared to normal RBCs and exhibits greater resistance to flow. The induced increase in the viscous characteristics of the membrane is dominant over the elastic and entropic effects of ICG. Our results suggest that changes to the membrane mechanical properties are a result of impaired membrane−cytoskeleton attachment in these particles. We provide a mechanistic explanation to suggest that the compromised membrane−cytoskeleton attachment and altered membrane compositional and structural asymmetry induce curvature changes to the membrane, resulting in mechanical remodeling of the membrane. These findings highlight the importance of membrane mechanical properties as an important criterion in the design and engineering of future generations of RBC-based delivery systems to achieve prolonged circulation.

Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells

“…Hypotonic treatment did not result in complete depletion of hemoglobin in μEGs and μEGs +ICG . These results are consistent with the absorption spectrum (Figure ) showing spectral peaks at 412, 542, and 575 nm, respectively, corresponding to the Soret, fundamental ( Q 0 ), and its vibronic tone ( Q v ) associated with oxyhemoglobin, as well as quantitative optical phase images, demonstrating the presence of residual oxyhemoglobin in μEGs and μEGs +ICG …”

“…Combined with reductions in ankyrin, band 3, and protein 4.2, these changes may be sufficient to alter the membrane mechanical properties of μEGs +ICG and weaken the adhesion between the membrane and the cytoskeleton. 51 Consistent with our SDS-PAGE analysis (Figure 4), the proteomic profiles obtained from TMT-MS showed relatively lower abundance levels of dematin (69.2%), tropomodulin (43.5%), and tropomyosin (33.1−64.8%) in nEGs +ICG as compared to normal RBCs. In addition, the actin junctional complex-related proteins αand β-adducin in nEGs +ICG also showed lower relative abundance levels of about 44.8 and 60.5%, respectively, as compared to RBCs.…”

“…These proteins included actin with increased expression of 10%, tropomodulin (36.8%), α-/β-adducin (96.8 and 56.6%, respectively), tropomyosin (76.8%), and dematin (112.4%). Combined with reductions in ankyrin, band 3, and protein 4.2, these changes may be sufficient to alter the membrane mechanical properties of μEGs +ICG and weaken the adhesion between the membrane and the cytoskeleton …”

“…Our findings suggest that hypotonic treatment and ICG loading had little to no marked effect on the presence of these proteins in μEGs and μEGs +ICG (Figure ). Nevertheless, it should be recognized that the linkages between the membrane and cytoskeleton may be disrupted, resulting in changes to the membrane mechanical properties and deformability of these RBC-like particles . Weakened vertical linkages between the membrane and cytoskeleton can lead to dissociation of the spectrin tetramers into dimers. , Furthermore, hypotonic treatment is associated with clustering and rearrangement of band 3. , …”

“…These results are consistent with the absorption spectrum (Figure 3) showing spectral peaks at 412, 542, and 575 nm, respectively, corresponding to the Soret, fundamental (Q 0 ), and its vibronic tone (Q v ) associated with oxyhemoglobin, 55 as well as quantitative optical phase images, demonstrating the presence of residual oxyhemoglobin in μEGs and μEGs +ICG . 51 The nanosized particles (nEGs and nEGs +ICG ), fabricated by mechanical extrusion of the micro-sized particles, had several faint and missing protein bands as compared to nonextruded micro-sized particles (μEGs and μEGs +ICG ) and RBCs (Figure 2). During the extrusion process, the cells are passed through cylindrical pores with nanosized diameters, resulting in cell lysis and reassembly of membrane lipids to form nanosized vesicles (e.g., nEGs).…”