Erythrocyte abundance, mobility, and carrying capacity make them attractive as a platform for blood analyte sensing as well as for drug delivery. Sensor-loaded erythrocytes, dubbed erythrosensors, could be reinfused into the bloodstream, excited noninvasively through the skin, and used to provide measurement of analyte levels in the bloodstream. Several techniques to load erythrocytes, thus creating carrier erythrocytes, exist. However, their cellular characteristics remain largely unstudied. Changes in cellular characteristics lead to removal from the bloodstream. We hypothesize that erythrosensors need to maintain native erythrocytes’ (NEs) characteristics to serve as a long-term sensing platform. Here, we investigate two loading techniques and the properties of the resulting erythrosensors. For loading, hypotonic dilution requires a hypotonic solution while electroporation relies on electrical pulses to perforate the erythrocyte membrane. We analyze the resulting erythrosensor signal, size, morphology, and hemoglobin content. Although the resulting erythrosensors exhibit morphological changes, their size was comparable with NEs. The hypotonic dilution technique was found to load erythrosensors much more efficiently than electroporation, and the sensors were loaded throughout the volume of the erythrosensors. Finally, both techniques resulted in significant loss of hemoglobin. This study points to the need for continued development of loading techniques that better preserve NE characteristics.
Keys to successful treatment of disease include early diagnosis and timely treatment. It is hypothesized that early clotting events may contribute to a pro-thrombotic state that exacerbates atherothrombotic vascular disease. Brillouin spectroscopy involves inelastic coupling of light with phonons and enables viscoelastic characterization of samples at the microscale. In this work, we apply Brillouin spectroscopy to a model fibrinogen-thrombin clotting system with the goal of measuring clotting dynamics at the microscale and providing characterization that is not possible with standard rheometric techniques. Here, the clotting dynamics of the model clotting system are measured at various fibrinogen and thrombin concentrations.
Erythrocytes, or red blood cells, transport oxygen to and carbon dioxide from the body's tissues and organs. Red blood cell mechanical properties are altered in a number of diseases such as sickle cell anaemia and malaria. Additionally, mechanically modified red blood cell ghosts are being considered as a long-term, biocompatible carrier for drug delivery and for blood analyte sensing. Brillouin spectroscopy enables viscoelastic characterization of samples at the microscale. In this report, Brillouin spectroscopy is applied to characterize the mechanical properties of red blood cells and red blood cell ghosts.
For diabetics, continuous glucose monitoring and the resulting tighter control of glucose levels ameliorate serious complications from hypoglycemia and hyperglycemia. Diabetics measure their blood glucose levels multiple times a day by finger pricks, or use implantable monitoring devices. Still, glucose and other analytes in the blood fluctuate throughout the day and the current monitoring methods are invasive, immunogenic, and/or present biodegradation problems. Using carrier erythrocytes loaded with a fluorescent sensor, we seek to develop a biodegradable, efficient, and potentially cost effective method to continuously sense blood analytes. We aim to reintroduce sensor-loaded erythrocytes to the bloodstream and conserve the erythrocytes lifetime of 120 days in the circulatory system. Here, we compare the efficiency of two loading techniques: hypotonic dilution and electroporation. Hypotonic dilution employs hypotonic buffer to create transient pores in the erythrocyte membrane, allowing dye entrance and a hypertonic buffer to restore tonicity. Electroporation relies on controlled electrical pulses that results in reversible pores formation to allow cargo entrance, follow by incubation at 37°C to reseal. As part of the cellular characterization of loaded erythrocytes, we focus on cell size, shape, and hemoglobin content. Cell recovery, loading efficiency and cargo release measurements render optimal loading conditions. The detected fluorescent signal from sensor-loaded erythrocytes can be translated into a direct measurement of analyte levels in the blood stream. The development of a suitable protocol to engineer carrier erythrocytes has profound and lasting implications in the erythrosensor’s lifespan and sensing capabilities.