Andre F. Palmer

Biocompatible and Biodegradable Polymersome Encapsulated Hemoglobin : A Potential Oxygen Carrier

This work describes the development of polymersome-encapsulated hemoglobin (PEH) self-assembled from biodegradable and biocompatible amphiphilic diblock copolymers composed of poly(ethylene oxide) (PEO), poly(caprolactone) (PCL), and poly(lactide) (PLA). In the amphiphilic diblock, PEO functions as the hydrophilic block, while either PCL or PLA can function as the hydrophobic block. PEO, PCL, and PLA are biocompatible polymers, while the last two polymers are biodegradable. PEH dispersions were prepared by extrusion through 100 nm pore radii polycarbonate membranes. In this work, the encapsulation efficiency of human and bovine hemoglobin (hHb and bHb) in polymersomes was adjusted by varying the initial concentration of Hb. This approach yielded Hb loading capacities that were comparable to values in the literature that supported the successful resuscitation of hamsters experiencing hemorrhagic shock. Moreover, the Hb loading capacities of PEHs in this study can also be tailored simply by controlling the diblock copolymer concentration. In this study, typical Hb/diblock copolymer weight ratios ranged 1.2-1.5, with initial Hb concentrations less than 100 mg/mL. The size distribution, Hb encapsulation efficiency, oxygen affinity (P 50), cooperativity coefficient (n), and methemoglobin (metHb) level of these novel PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. Taken together, our results demonstrate the development of novel PEH dispersions that are both biocompatible and biodegradable. These novel dispersions show very good promise as therapeutic oxygen carriers.

Self-Assembled Poly(butadiene)-b-Poly(ethylene oxide) Polymersomes as Paclitaxel Carriers

In this work, self‐assembled poly(butadiene)‐ b‐poly(ethylene oxide) (PB‐PEO) polymersomes (polymer vesicles) and worm micelles were evaluated as paclitaxel carriers. Paclitaxel was successfully incorporated into PB‐PEO polymersomes and worm micelles. The loading capacity of paclitaxel inside PB‐PEO colloids ranged from 6.7% to 13.7% w/w, depending on the morphology of copolymer colloids and the molecular weight of diblock copolymer. Paclitaxel loaded OB4 (PB219‐PEO121) polymersome formulations were colloidally stable for 4 months at 4 °C and exhibited slow steady release of paclitaxel over a 5 week period at 37 °C. Evaluation of the in vitro cytotoxicity of paclitaxel‐polymersome formulations showed that the ability of paclitaxel‐loaded polymersomes to inhibit proliferation of MCF‐7 human breast cancer cells was less compared to paclitaxel alone. By increasing the concentration of paclitaxel in polymersomes from 0.02 to 0.2 μg/mL, paclitaxel‐polymersome formulations showed comparable activity in inhibiting the growth of MCF‐7 cells. Taken together, these results demonstrate that paclitaxel‐polymersomes have desirable restrained release profile and exhibit long‐term stability.

Determination of Size Distribution and Encapsulation Efficiency of Liposome-Encapsulated Hemoglobin Blood Substitutes Using Asymmetric Flow Field-Flow Fractionation Coupled with Multi-Angle Static Light Scattering

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome‐encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field‐flow fractionation (AFFF) in series with multi‐angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF‐MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl‐phosphatidylcholine, cholesterol, and dimyristoyl‐phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400‐nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200‐nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100‐, 80‐, and 50‐nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen‐carrying properties as indicated by their P50 and cooperativity coefficients. LEHbs extruded in PB had an average P50 of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen‐binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB‐LEHb dispersions are significantly low even in the absence of antioxidants such as N‐acetyl‐l‐cysteine.

High-efficiency matrix modulus-induced cardiac differentiation of human mesenchymal stem cells inside a thermosensitive hydrogel

Mesenchymal stem cells (MSCs) experience an extremely low rate of cardiac differentiation after transplantation into infarcted hearts, in part due to the inability of stiff scar tissue to support differentiation. We hypothesized that delivering MSCs in a hydrogel with a modulus matched to that of native heart tissue should stimulate MSC differentiation into cardiac cells. We have developed a thermosensitive and injectable hydrogel suitable for the delivery of cells into the heart, and found that the appropriate gel modulus can differentiate MSCs into cardiac cells with high efficiency. The hydrogel was based on N-isopropylacrylamide, N-acryloxysuccinimide, acrylic acid and poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel solution can be readily injected through needles commonly used for heart injection, and is capable of gelling within 7s at 37°C. The formed gels were highly flexible, with breaking strains (>300%) higher than that of native heart tissue and moduli within the range of native heart tissue (1-140 kPa). Controlling the concentration of the hydrogel solution resulted in hydrogels with three different moduli: 16, 45 and 65 kPa. The moduli were decoupled from the gel water content and oxygen diffusion, parameters that can also influence cell differentiation. MSCs survived in the hydrogels throughout the entire culture period, and it was observed that gel stiffness did not affect cell survival. After 14 days of culture, more than 76% of MSCs had differentiated into cardiac cells in the 45 and 65 kPa gels, as confirmed by the expression of cardiac markers at both the gene and protein levels. MSCs in the hydrogel with the 65 kPa modulus had the highest differentiation efficiency. The differentiated cells also developed calcium channels that imparted an electrophysiological property, and gap junctions for cell-cell communication. The efficiency of differentiation reported in this study was much higher than for the differentiation approaches described in the literature, such as chemical induction and co-culture of MSCs and cardiomyocytes. These results indicate that the novel hydrogel holds great promise for delivering MSCs into an infarcted heart for the generation of new heart tissue.

Tangential flow filtration of hemoglobin.

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P50) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers.

Effect of Glutaraldehyde Concentration on the Physical Properties of Polymerized Hemoglobin-Based Oxygen Carriers

Artificial blood substitutes based on glutaraldehyde cross‐linked hemoglobin (PolyHb) are currently being developed for use in human subjects needing blood transfusions. Despite the commercial development of PolyHb dispersions, a systematic study of the effect of varying the glutaraldehyde to hemoglobin (G‐Hb) molar ratio on the resulting PolyHb physical properties (molecular weight distribution and oxygen binding parameters) has not been conducted to date. The results of this study show that increasing the G‐Hb molar ratio elicits a general decrease in the P50 (partial pressure of oxygen at which Hb is half saturated with oxygen) and cooperativity and a simultaneous increase in the weight averaged molecular weight (Mw) of the PolyHb dispersion and methemoglobin (MetHb) level. Three PolyHb dispersions (20:1, 30:1, and 40:1 G‐Hb molar ratios) displayed potential as artificial blood substitutes. The 20:1 PolyHb dispersion resulted in the presence of more intramolecularly cross‐linked and non‐cross‐linked tetramers versus cross‐linked species that were larger than a tetramer (∼75% tetrameric and ∼25% higher‐order species), lower MetHb level (8%), and P50 (20.1 mmHg) similar in magnitude to that of non‐cross‐linked Hb. The 30:1 PolyHb dispersion consisted of more higher‐order species (∼76%), higher MetHb level (28%), and lower P50 (13.3 mmHg). The 40:1 PolyHb dispersion resulted in a similar P50 of 13.0 mmHg and similar MetHb level (30%); however, this PolyHb dispersion only consisted of species larger than a tetramer. The molecular weight distribution of PolyHb dispersions was determined using asymmetric flow field‐flow fractionator (AFFF) coupled with multiangle static light scattering (MASLS). This is the first time that AFFF‐MASLS has been used to characterize the molecular weight distribution of PolyHb dispersions.

Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction.

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

Basic science focus on blood substitutes: a summary of the NHLBI Division of Blood Diseases and Resources Working Group Workshop, March 1, 2006.

In March 2006, a workshop sponsored by the National Heart, Lung, and Blood Institute was convened to identify the role of basic science research in clarifying issues that are impeding progress in the development of hemoglobin‐based oxygen carrying (HBOC) solutions. These discussions resulted in a consensus that, although HBOCs have shown clinical promise, various side effects have inhibited further development and regulatory approval, with cardiovascular events being of particular concern. As a consequence, workshop participants focused on formulating research recommendations to better understand and mitigate these side effects. In addition, several important corollary issues were identified, including better understanding of the impact of HBOC infusion on human physiology; the need for rapid, noninvasive methods for the measurement of tissue oxygenation in human patients to better inform transfusion decisions; further investigation of routes and consequences of hemoglobin metabolism; optimization of clinical protocols for HBOC use; and assessment of the impact of HBOC formulation excipients. Also discussed was the possibility and desirability of developing new HBOCs with improved characteristics, such as prolonged functional intravascular persistence, greater stability, and a decreased propensity to generate reactive oxygen species. One practical limitation in this area is the consistent availability of pure, well‐characterized HBOC solutions for the research community. This communication summarizes the opinion of workshop participants on these issues and concludes with a list of specific recommended areas of research that could positively impact the development of blood substitutes.

Addition of ascorbic acid solution to stored murine red blood cells increases posttransfusion recovery and decreases microparticles and alloimmunization.

The storage of red blood cells (RBCs) results in numerous changes, which over time result in decreased recovery of transfused RBCs. In addition (at least in animal models), stored RBCs can be more immunogenic and also stimulate the systemic release of inflammatory cytokines in transfusion recipients. One component of the RBC storage lesion is the accumulation of oxidative damage. We tested the hypothesis that adding a chemical antioxidant (ascorbic acid) to stored RBCs would improve the quality of the stored RBCs.

Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers.

The production of a fully functional bioartificial liver assist device (BLAD) would greatly enhance available treatment options for patients suffering from acute liver failure. Currently, inadequate oxygen provision to hepatocytes seeded within hollow fiber bioreactors hampers development of a viable hollow fiber-based BLAD. Experimentally, oxygen provision to primary rat hepatocytes cultured within hollow fiber bioreactors was measured, it was observed that supplementation with an oxygen carrier (bovine red blood cells at ∼ 2% human hematocrit) did not significantly improve oxygenation compared to the absence of an oxygen carrier. Therefore, an oxygen transport model of an individual hollow fiber within the bioreactor was developed and simulated (up to ∼ 10% human hematocrit) to more fully examine the effect of oxygen carrier supplementation on oxygenation within the bioreactor. The modeling analysis, supported via the experimental results, was utilized to predict optimal bioreactor operating conditions for the delivery of in vivo-like oxygen gradients to cultured hepatocytes in clinically relevant settings.