The Molecular Size of Bioengineered Oxygen Carriers Determines Tissue Oxygenation in a Hypercholesterolemia Guinea Pig Model of Hemorrhagic Shock and Resuscitation.

Polymerized human hemoglobin (PolyhHb) has shown promise in preclinical hemorrhagic shock settings. Different synthetic and purification schemes can control the size of PolyhHbs, yet research is lacking on the impact of polymerized hemoglobin size on tissue oxygenation following hemorrhage and resuscitation in specialized animal models that challenge their resuscitative capabilities. Pre-existing conditions that compromise the vasculature and end organs, such as the liver, may limit the effectiveness of resuscitation and exacerbate the toxicity of these molecules, which is an important but minimally explored therapeutic dimension. In this study, we compared the effective oxygen delivery of intermediate molecular weight PolyhHb (PolyhHb-B3; 500-750 kDa) to high molecular weight PolyhHb (PolyhHb-B4; 750 kDa-0.2 µm) for resuscitative effectiveness in guinea pig models subjected to hemorrhagic shock. We evaluated how the size of PolyhHb impacts hemodynamics and tissue oxygenation in normal guinea pigs and guinea pigs on an atherogenic diet. We observed that while PolyhHb-B3 and -B4 equivalently restore hemodynamic parameters of normal-dieted guinea pigs, high-fat-dieted guinea pigs resuscitated with PolyhHb-B4 have lower mean arterial pressures, impaired tissue oxygenation, and higher plasma lactate levels than those receiving PolyhHb-B3. We characterized the plasma of these animals following resuscitation and found that despite similar oxygen delivery kinetics, circulating PolyhHb-B3 and -B4 demonstrated a size-dependent increase in the plasma viscosity, consistent with impaired perfusion in the PolyhHb-B4 transfusion group. We conclude that intermediate-sized PolyhHbs (such as -B3) are ideal for further research given the effective resuscitation of hemorrhagic shock based on tissue oxygenation in hypercholesterolemic guinea pigs.

Biophysical Analysis and Preclinical Pharmacokinetics-Pharmacodynamics of Tangential Flow Filtration Fractionated Polymerized Human Hemoglobin as a Red Blood Cell Substitute.

Red blood cell (RBC) substitutes tested in late-phase clinical trials contained low-molecular-weight hemoglobin species (<500 kDa), resulting in vasoconstriction, hypertension, and oxidative tissue injury; therefore, contributing to poor clinical outcomes. This work aims to improve the safety profile of the RBC substitute, polymerized human hemoglobin (PolyhHb), via in vitro and in vivo screening of PolyhHb fractionated into four molecular weight brackets (50-300 kDa [PolyhHb-B1]; 100-500 kDa [PolyhHb-B2]; 500-750 kDa [PolyhHb-B3]; and 750 kDa to 0.2 µm [PolyhHb-B4]) using a two-stage tangential flow filtration purification process. Analysis showed that PolyhHb's oxygen affinity, and haptoglobin binding kinetics decreased with increasing bracket size. A 25% blood-for-PolyhHb exchange transfusion guinea pig model suggests that hypertension and tissue extravasation decreased with increasing bracket size. PolyhHb-B3 demonstrated extended circulatory pharmacokinetics, no renal tissue distribution, no aberrant blood pressure, or cardiac conduction effects, and may therefore be appropriate material for further evaluation.

Lipid Chemical Structure Modulates the Disruptive Effects of Nanomaterials on Membrane Models.

Understanding the mechanisms by which engineered nanomaterials disrupt the cell plasma membrane is crucial in advancing the industrial and biomedical applications of nanotechnology. While the role of nanoparticle properties in inducing membrane damage has received significant attention, the role of the lipid chemical structure in regulating such interactions is less explored. Here, we investigated the role of the lipid chemical structure in the disruption of lipid vesicles by unmodified silica, carboxyl-modified silica, and unmodified polystyrene nanoparticles (50 nm). The role of the lipid headgroup was examined by comparing nanoparticle effects on vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vs an inverse phosphocholine (PC) with the same acyl chain structure. The role of acyl chain saturation was examined by comparing nanoparticle effects on saturated vs unsaturated PCs and sphingomyelins. Nanoparticle effects on PCs (glycerol backbone) vs sphingomyelins (sphingosine backbone) were also examined. Results showed that the lipid headgroup, backbone, and acyl chain saturation affect nanoparticle binding to and disruption of the membranes. A low headgroup tilt angle and the presence of a trimethylammonium moiety at the vesicle surface are required for unmodified nanoparticles to induce membrane disruption. Lipid backbone structure significantly affects nanoparticle-membrane interactions, with carboxyl-modified particles only disrupting lipids containing cis unsaturation and a sphingosine backbone. Acyl chain saturation makes vesicles more resistant to particles by increasing lipid packing in vesicles, impeding molecular interactions. Finally, nanoparticles were capable of changing the lipid packing, resulting in pore formation in the process. These observations are important in interpreting nanoparticle toxicity to biological membranes.

Novel Experimental Modules to Introduce Students to Nanoparticle Characterization in a Chemical Engineering Course.

The increasing industrial and biomedical applications of nanomaterials have enhanced the need to educate a well-trained nanotechnology workforce. This need has led to efforts to introduce hands-on, nanotechnology-based, experimental modules into high school or college-level courses in science or engineering. However, the majority of such efforts have focused on nanoparticle synthesis techniques, and an equally important aspect of working with nanomaterials, i.e. nanoparticle characterization, has received less attention. Herein, we report a series of nanoparticle characterization experiments, as part of a newly developed "Nano and Biointerfaces" course, to familiarize upper undergraduate students as well as graduate students in chemical engineering with nanoparticle characterization techniques. An inquiry-based approach was used in that the composition and properties of nanoparticles were not revealed to the students beforehand and students were asked to perform experiments to characterize nanoparticle composition, size, morphology, and surface area. The results of these experiments were compared with certificates of analysis for particles, provided by the vendor, and the differences in measured properties were discussed. Assessment was performed through evaluation of laboratory memos and presentations, a question in the end of semester final exam, and a student survey. The modular nature of these experiments allows for them to be implemented, with modifications as needed, in other higher education institutions, or in high schools, to familiarize students with nanoparticle characterization.

Quantitative analysis of red blood cell membrane phospholipids and modulation of cell-macrophage interactions using cyclodextrins.

The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell-cell interactions.

Loss of membrane asymmetry alters the interactions of erythrocytes with engineered silica nanoparticles.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.