Mechanical characterization of erythrocyte-derived optical microparticles by quantitative phase imaging and optical tweezers

Abstract: Erythrocyte-based carriers can serve as theranostic platforms for delivery of imaging and therapeutic payloads. Engineering these carriers at micro- or nanoscales makes them potentially useful for broad clinical applications ranging from vascular diseases to tumor theranostics. Longevity of these carriers in circulation is important in delivering a sufficient amount of their payloads to the target. We have investigated the circulation dynamics of micro (∼4.95 μm diameter) and nano (∼91 nm diameter) erythrocyte-derived carriers in real time using near-infrared fluorescence imaging, and evaluated the effectiveness of such carrier systems in mediating photothermolysis of cutaneous vasculature in mice. Fluorescence emission half-lives of micro- and nanosized carriers in response to a single intravenous injection were ∼49 and ∼15 min, respectively. A single injection of microsized carriers resulted in a 3-fold increase in signal-to-noise ratio that remained nearly persistent over 1 h of imaging time. Our results also suggest that a second injection of the carriers 7 days later can induce a transient inflammatory response, as manifested by the apparent leakage of the carriers into the perivascular tissue. The administration of the carriers into the mice vasculature reduced the threshold laser fluence to induce photothermolysis of blood vessels from >65 to 20 J/cm2. We discuss the importance of membrane physicochemical and mechanical characteristics in engineering erythrocyte-derived carriers and considerations for their clinical translation.

Abstract: Nanoparticles with longer blood circulation, high loading capacity, controlled release at the targeted site, and preservation of camptothecin (CPT) in its lactone form are the key characteristics for the effective delivery of CPT. In this regard, natural membrane-derived nanovesicles, particularly those derived from RBC membrane, are important. RBC membrane can be engineered to form vesicles or can be coated over synthetic nanoparticles, without losing their basic structural features and can have prolonged circulation time. Here, we developed a hybrid system to encapsulate CPT inside the amphiphilic micelle and coat it with RBC membrane. Thus, it uses the dual ability of polymeric micelles to preserve CPT in its active form, while maintaining its "stealth" effect due to conserved RBC membrane coating. The hybrid system stabilized 60% of the drug in its active form even after 30 h of incubation in serum, in contrast to 15% active form present in free drug formulation after 1 h of incubation. It showed strong retention inside the Ehrlich Ascites Carcinoma (EAC) mice models for at least 72 h, suggesting camouflaging ability conferred by RBC membrane. Additionally, the nano formulation retarded the tumor growth rate more efficiently than free drug, with no evident signs of necrotic skin lesions. Histopathological analysis showed a significant reduction in cardiac atrophy, hepato-renal degeneration, and lung metastasis, which resulted in the increased overall survival of mice treated with the nano formulation. Hence, CPT-loaded polymeric micelles when coated with RBC membrane can prove to be a better system for the delivery of poorly soluble drug camptothecin.

Abstract: Parasitization by malaria-inducing Plasmodium falciparum leads to structural, biochemical, and mechanical modifications to the host red blood cells (RBCs). To study these modifications, we investigate two intrinsic indicators: the refractive index and membrane fluctuations in P. falciparum-invaded human RBCs (Pf-RBCs). We report experimental connections between these intrinsic indicators and pathological states. By employing two noninvasive optical techniques, tomographic phase microscopy and diffraction phase microscopy, we extract three-dimensional maps of refractive index and nanoscale cell membrane fluctuations in isolated RBCs. Our systematic experiments cover all intraerythrocytic stages of parasite development under physiological and febrile temperatures. These findings offer potential, and sufficiently general, avenues for identifying, through cell membrane dynamics, pathological states that cause or accompany human diseases.

Abstract: Using a novel noncontact technique based on optical interferometry, we quantify the nanoscale thermal fluctuations of red blood cells (RBCs) and giant unilamellar vesicles (GUVs). The measurements reveal a nonvanishing tension coefficient for RBCs, which increases as cells transition from a discocytic shape to a spherical shape. The tension coefficient measured for GUVs is, however, a factor of 4-24 smaller. By contrast, the bending moduli for cells and vesicles have similar values. This is consistent with the cytoskeleton confinement model, in which the cytoskeleton inhibits membrane fluctuations [Gov et al., Phys. Rev. Lett. 90, 228101, (2003).

Abstract: Development of theranostic nano-constructs may enable diagnosis and treatment of diseases at high spatial resolution Some key requirements for clinical translation of such constructs are that they must be non-toxic, non-immunogenic, biodegradable, with extended circulating lifetime Cell-based structures, particularly those derived from erythrocytes, are promising candidate carrier systems to satisfy these requirements One particular type of theranostic materials utilize light-sensitive agents that once photo-activated can provide diagnostic imaging capability, and elicit therapeutic effects Here we demonstrate the first successful engineering of hybrid nano-scale constructs derived from membranes of hemoglobin-depleted erythrocytes that encapsulate the near infrared chromophore, indocyanine green We show the utility of the constructs as photo-theranostic agents in fluorescence imaging and photothermal destruction of human cells These erythrocyte-mimicking nano-structures can be derived autologously, and may have broad applications in personal nanomedicine ranging from imaging and photo-destruction of cancerous tissues to vascular abnormalities, and longitudinal evaluations of therapeutic interventions

Abstract: In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane-cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in membrane cholesterol content.

Abstract: Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k0 and k1 (with k0>k1), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k0 and k1 in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k0 stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k0 stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation.