Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (O2 ) carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration purification process to remove almost all undesirable Hb species (>0.2 µm and <500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Scalable manufacturing platform for the production of methemoglobin as a non-oxygen carrying control material in studies of cell-free hemoglobin solutions.

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-[Formula: see text], and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.

Magnetophoretic and spectral characterization of oxyhemoglobin and deoxyhemoglobin: Chemical versus enzymatic processes.

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids.

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5-100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

Purification of Lumbricus terrestris Mega-Hemoglobin for Diverse Oxygen Therapeutic Applications.

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.

Synthesis of Hemoglobin-Based Oxygen Carrier Nanoparticles By Desolvation Precipitation.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) present an alternative to red blood cells (RBCs) when blood is not available. However, the most widely used synthesis techniques have fundamental flaws, which may have contributed toward disappointing clinical application. Polymerized Hb contains a heterogeneous distribution of particle size and shape, while Hb encapsulation inside liposomes results in high lipid burden and low Hb content. Meanwhile, there are a variety of other nanoparticle synthetic techniques which, having found success as drug delivery vehicles, may be well suited to function as an HBOC. We synthesized desolvated Hb nanoparticles (Hb-dNPs) with diameters of approximately 250 nm by the controlled precipitation of Hb with ethanol. Oxidized dextran was found to be an effective surface stabilizing agent that maintained particle integrity. In vitro biophysical characterization showed a high-affinity oxygen delivery profile (P50 = 7.72 mm Hg), suggesting a potential for therapeutic use and opening a new avenue for HBOC research.

Novel manufacturing method for producing apohemoglobin and its biophysical properties.

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and ß subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.

TRAIL stabilization and cancer cell sensitization to its pro-apoptotic activity achieved through genetic fusion with arginine deiminase.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) binds to death receptors and induces apoptosis in various cancer cell lines while sparing normal cells. Recombinant TRAIL has shown good safety and efficacy profiles in preclinical cancer models. However, clinical success has been limited due to poor PK and development of resistance to death receptor-induced apoptosis. We have addressed these issues by creating a fusion protein of TRAIL and arginine deiminase (ADI). The fusion protein benefits from structural and functional synergies between its two components and has an extended half-life in vivo. ADI downregulates survivin, upregulates DR5 receptor and sensitizes cancer cells to TRAIL induced apoptosis. ADI-TRAIL fusion protein was efficacious in a number of cell lines and synergized with some standard of care drugs. In an HCT116 xenograft model ADI-TRAIL localized to the tumor and induced dose-dependent tumor regression, the fusion protein was superior to rhTRAIL administered at the same molar amounts.

Wearable wireless User Interface Cursor-Controller (UIC-C).

Controlling a computer or a smartphone's cursor allows the user to access a world full of information. For millions of people with limited upper extremities motor function, controlling the cursor becomes profoundly difficult. Our team has developed the User Interface Cursor-Controller (UIC-C) to assist the impaired individuals in regaining control over the cursor. The UIC-C is a hands-free device that utilizes the tongue muscle to control the cursor movements. The entire device is housed inside a subject specific retainer. The user maneuvers the cursor by manipulating a joystick imbedded inside the retainer via their tongue. The joystick movement commands are sent to an electronic device via a Bluetooth connection. The device is readily recognizable as a cursor controller by any Bluetooth enabled electronic device. The device testing results have shown that the time it takes the user to control the cursor accurately via the UIC-C is about three times longer than a standard computer mouse controlled via the hand. The device does not require any permanent modifications to the body; therefore, it could be used during the period of acute rehabilitation of the hands. With the development of modern smart homes, and enhancement electronics controlled by the computer, UIC-C could be integrated into a system that enables individuals with permanent impairment, the ability to control the cursor. In conclusion, the UIC-C device is designed with the goal of allowing the user to accurately control a cursor during the periods of either acute or permanent upper extremities impairment.

Mesenchymal Stem Cells Synergize with 635, 532, and 405 nm Laser Wavelengths in Renal Fibrosis: A Pilot Study.

OBJECTIVE: To address whether a single treatment of one of three visible light wavelengths, 635, 532, and 405 nm (constant wave, energy density 2.9 J/m2), could affect the hallmarks of established renal fibrosis and whether these wavelengths could facilitate mesenchymal stem cell (MSC) beneficence. BACKGROUND DATA: Chronic kidney disease is a global health problem with only 20% receiving care worldwide. Kidneys with compromised function have ongoing inflammation, including increased oxidative stress and apoptosis, peritubular capillary loss, tubular atrophy, and tubulointerstitial fibrosis. Promising studies have highlighted the significant potential of MSC-based strategies to mitigate fibrosis; however, reversal of established fibrosis has been problematic, suggesting that methods to potentiate MSC effects require further development. Laser treatments at visible wavelengths have been reported to enhance mitochondrial potential and available cellular ATP, facilitate proliferation, and inhibit apoptosis. We hypothesized that laser-delivered energy might provide wavelength-specific effects in the fibrotic kidney and enhance MSC responses. MATERIALS AND METHODS: Renal fibrosis, established in C57BL6 mice following 21 days of unilateral ureter obstruction (UUO), was treated with one of three wavelengths alone or with autologous MSC. Mitochondrial activity, cell proliferation, apoptosis, and cytokines were measured 24 h later. RESULTS: Wavelengths 405, 532, and 635 nm all significantly synergized with MSC to enhance mitochondrial activity and reduce apoptosis. Proliferative activity was observed in the renal cortices following combined treatment with the 532 nm laser and MSC; endothelial proliferation increased in response to the 635 nm laser alone and to the combined effects of MSC and the 405 nm wavelength. Reductions of transforming growth factor-ß were observed with 532 nm alone and when combined with MSC. CONCLUSIONS: Specific wavelengths of laser energy appear to induce different responses in renal fibrotic tissue. These findings support further study in the development of a customized laser therapy program of combined wavelengths to optimize MSC effects in the treatment of renal fibrosis.