Incorporation of reversibly cross-linked polyplexes into LPDII vectors for gene delivery

LPDII vectors are synthetic vehicles for gene delivery composed of polycation-condensed DNA complexed with anionic liposomes. In this study, we evaluated the stability and transfection properties of polyethylenimine (PEI, 25 kDa)/DNA polyplexes before and after covalent cross-linking with dithiobis(succinimidylpropionate) (DSP) or dimethyl x 3,3'-dithiobispropionimidate x 2HCl (DTBP), either alone or as a component of LPDII vectors. We found that cross-linking PEI/DNA polyplexes at molar ratios > or =10:1 (DSP or DTBP:PEI) stabilized these complexes against polyanion disruption, and that this effect was reversible by reduction with 20 mM dithioerythritol (DTE). Transfection studies with polyplexes cross-linked at molar ratios of 10:1-100:1 in KB cells, a folate receptor-positive oral carcinoma cell line, showed decreasing luciferase gene expression with increasing cross-linking ratio. Subsequently, polyplexes, cross-linked with DSP at a molar ratio of 10:1, were combined with anionic liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol), diolein/CHEMS/poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) (6:4:0.05 mol/mol), or diolein/CHEMS/folate-PEG-cholesterol (folate-PEG-Chol) (6:4:0.05 mol/mol) for LPDII formation. Transfection studies in KB cells showed that LPDII vectors containing cross-linked polyplexes mediated approximately 2-15-fold lower gene expression than LPDII prepared with un-cross-linked polyplexes, depending on the lipid:DNA ratio. Inclusion of PEG-DSPE at 0.5 mol % appeared to further decrease transfection levels approximately 2-5-fold. Compared with LPDII formulated with PEG-DSPE, LPDII incorporating 0.5 mol % folate-PEG-Chol exhibited higher luciferase activities at all lipid:DNA ratios tested, achieving an approximately 10-fold increase at a lipid:DNA ratio of 5. Compared with cross-linked LPDII vectors without PEG-DSPE, inclusion of folate-PEG-Chol increased luciferase activities 3-4-fold between lipid:DNA ratios of 1 and 5. Interestingly, inclusion of 1 mM free folate in the growth media during transfection increased transfection activity approximately 3-4-fold for cross-linked LPDII vectors and LPDII containing folate-PEG-Chol, but had no effect on the transfection activity of LPDII formulated with PEG-DSPE. However, in the presence of 5 mM free folate, the luciferase activity mediated by LPDII vectors containing folate-PEG-Chol was reduced approximately 6-fold. Transmission electron micrographs were also obtained to provide evidence of LPDII complex formation. Results showed that cross-linked LPDII vectors appear as roughly spherical aggregated complexes with a rather broad size distribution ranging between 300 and 800 nm.     

Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine.

Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl.3,3'-dithiobispropionimidate*2HCl (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M(w)) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the N/P ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intra-cross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.     

Cation channels trigger apoptotic death of erythrocytes

Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca(2+)-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca(2+) and mimicked by ionomycin (1 microM). Osmotic shrinkage and oxidative stress activate Ca(2+)-permeable cation channels and increase cytosolic Ca(2+) concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca(2+)-permeable cation channels in erythrocytes, thus increasing cytosolic Ca(2+) activity and triggering erythrocyte apoptosis.     

Near-neighbor relationships of the subunits of cytochrome c oxidase.

Cytochrome c oxidase in detergent dispersion has been cross-linked with two reversible cross-linking agents, dithiobissuccinimidylpropionate (DSP) and dimethyl-3,3'-dithiobispropionimidate (DTBP), and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, several subunit pairs were seen including II and V, V and VII, IV and VI. With higher levels of DSP, larger aggregates were seen until a cross-linked product with an apparent molecular weight of 140 000 was the predominant band on gels. This is the smallest molecular weight aggregate to contain all seven subunits of the enzyme and most likely represents the "unit" or two heme and two copper containing complex of cytochrome c oxidase.     

Membrane permeability of bifunctional,amino site-specific,cross-linking reagents

The membrane permeability of a series of reversible cross-linking reagents which are diazide tartarate derivatives has been compared with that of dimethyl-3,3′-dithiobispropionimidate (DTBP). The diazide tartarate derivatives tested include tartryl-diazide (TDA), tartryl-di(glycylazide) (TDGA), tartryl-di(β-alanylazide) (TDAA), tartryl-di-(γ-aminobutyrylazide) (TDBA), tartryl-di (?-aminocaproylazide) (TDCA). TDA, which has the shortest chain length of the diazide tartarate derivatives tested, proved to be readily permeable through the erythrocyte membrane. When added at equal concentration to unsealed ghosts, TDGA was at least as reactive as DTBP in its ability to cross link the internally displayed proteins 1, 2, 4.1, 4.2, and 6. Treatment of resealed ghosts by DTBP produced oligomeric complexes of these proteins plus apparent homooligomeric complexes of hemoglobin. TDGA at the same concentrations did not cross-link any of these components, indicating its membrane-impermeable nature. As the chain length of the homologous series increased from TDGA to TDCA, the cross-linkers became increasingly permeable through the erythrocyte membrane.     

Stabilization of vasopressin-induced membrane events by bifunctional imidoesters

Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross- linking is maximal at pH 10, and is reduced as pH is lowered. Freeze- fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta- mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events.     

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.     

Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.     

Steady-state volumes and metabolism-independent osmotic adaptation in mammalian erythrocytes

Erythrocytes of various mammalian species -- including human -- maintain osmotic balance with the blood plasma (osmotic activity 270-310 mosmol). However, their intracellular levels of osmotically active ions (potassium, sodium, chloride, and hydrogencarbonate), water content and osmotic resistance deviate significantly. In the present report we study the relationship among intracellular water, potassium and sodium levels of the erythrocytes of various mammalian species and in the developing calf. In addition, the osmotic resistance, K(+) (Rb(+)) uptake and the DPH fluorescence anisotropy of various erythrocytes and erythrocyte ghost membranes were correlated. The results show no statistically significant relationship between erythrocyte water content and [K(+)+Na(+)] levels or K(+)/Na(+) ratios. The reversal of erythrocyte K(+)/Na(+) ratios coincides with the decrease of steady-state ATP levels in the developing calf. The mobility of lipids within the hydrophobic inner layer of the plasma membrane relates closely to passive K(+) (Rb(+)) uptake, and plays a significant role in regulatory volume changes.     

The in vivo cross-linking of proteins and DNA by heavy metals

Cross-linking of proteins to DNA in live, intact Novikoff ascites hepatoma cells exposed in vitro to different concentrations of CuSO4, Pb(NO3)2, HgCl2, and AlCl3 was studied. Protein-DNA complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by electrophoretic separation of proteins associated with the DNA-containing pellets. Concentration dependence experiments showed that the optimal cross-linking occurred at metal concentration of 0.5 mM for CuSO4, HgCl2, and AlCl3 while the optimal cross-linking for Pb(NO3)2 was at 5 mM. For some metals at concentrations higher than optimal, the amounts of cross-linked proteins decreased significantly. Immunochemical analysis of the cross-linked proteins using antibodies to matrix, chromatin, lamins, and cytokeratin fractions demonstrated that some, but not all, members of these protein families became cross-linked to the DNA. Each metal exhibited a cross-linking pattern of its own, different from those of the other metals. Radioactive labeling experiments showed that all the metals tested became associated with the DNA-protein pellets within 1 h after their addition to the incubation medium. However, hexavalent chromium required more than 2 h before appearing in the DNA-protein pellets in significant amounts.     

The effect of copper on erythrocyte deformability. A possible mechanism of hemolysis in acute copper intoxication

Although the development of hemolytic anemia as a complication of acute copper intoxication is well documented, the precise mechanism by which copper produces accelerated erythrocyte destruction is unknown. Normal erythrocyte survival depends in part on the ability of the cell to deform and pass through narrow areas of microcirculation in the liver and especially in the spleen. In the present study, it is demonstrated that toxic concentrations of copper rapidly and markedly reduce erythrocyte deformability. This reduction in cell deformability is associated with a marked increase in membrane permeability and osmotic fragility of copper-treated cells. Further, the decrease in deformability occurs despite normal levels of cell ATP and the apparent absence of oxidative damage to the cell. These observations indicate that copper-mediated changes in the erythrocyte membrane may be responsible for reducing the flexibility of the cell. The loss of deformability could act to reduce erythrocyte survival and thus explain the hemolysis associated with copper intoxication in vivo.     

Chemical cross-linking of Ia alloantigen alpha and beta chains with dimethyl 3,3'-dithiobispropionimidate.

We have examined the polypeptide chain composition of membrane-bound and detergent-solubilized Ia antigens using the chemical cross-linking reagent dimethyl 3,3'-dithiobispropionimidate (DTBP). Products of the I-E/C subregion of the major histocompatibility complex, which were solubilized from spleen cells with the detergent NP-40 and partially purified by affinity chromatography on lentil lectin-agarose, could be almost completely cross-linked by DTBP. Thus, the characteristic 33,000 m.v. (alpha) and 28,000 (beta) polypeptide chains seen on sodium dodecylsulfate polyacrylamide gels disappeared and a major new species of 60,000 m.w. appeared after cross-linking. When isolated and reduced with 2-mercaptoethanol, the 60,000 m.w. peak was found to be comprised to alpha and beta chains. Similar results were obtained when I-E/C, as well as I-A, alpha and beta chains were crosslinked on the cell surface. These data demonstrate that the alpha and beta chains of the Ia antigens exist primarily in the form of a dimer both in detergent solution and in situ.     

Cross-linking of cytokeratins to DNA in vivo by chromium salt and cis-diamminedichloroplatinum(II)

The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.     

Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.     

Cross-linking of phospholipids to proteins in the erythrocyte membrane

Erythrocytes treated with the cross-linking agents difluorodinitrobenzene and suberimidate are rendered refractory to lysis. When ghosts are treated with these reagents 8.4% and 2.3% of the total lipid phosphate is cross-linked to protein by difluorodinitrobenzene and suberimidate respectively. This represents 20 and 5.8% of the amino-phospholipids. The lipids extracted from treated ghosts do not react with ninhydrin as do lipids extracted from control ghosts. Thus essentially all the amino-phospholipids of the ghosts react with these cross-linking agents and up to 20% becomes cross-linked to proteins.     

pH effects on red cell deformability

The effect of pH on erythrocyte deformability was determined in the ektacytometer. In isotonic solutions, cell deformability was essentially unaltered at pH values from 6.44 to 7.7, despite the known cell volume changes that occur over this pH range. Analysis by osmotic scan ektacytometry showed that erythrocyte deformability is homeostatically maintained constant during acidosis and alkalosis because of a balance between the countervailing effects of changes in intracellular viscosity and in the surface to volume ratio. This balance is perturbed in the sickle erythrocyte, so that acidosis improves deformability.     

Evaluation of Integrin Molecules Involved in Substrate Adhesion

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the pi integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both α5β1 and α5β1 integrins. α6 was cross-linked only in cells plated on laminin whereas α5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either α5 or β1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.     

Use of spin label and the flow-induced ESR spectral difference for studying erythrocyte deformation

ESR spin-labeling method is expanded to measure the macroscopic visco-elastic properties of erythrocytes. A suspension of erythrocytes with an incorporated fatty acid spin label was forced to flow through a flat ESR sample cells, and the ESR spectral change caused by the shear flow was utilized to assess the cell deformability. Chemical cross-linking or heat denaturation of membrane proteins to make the cells less deformable without any morphological change was found to reduce the relative spectral difference (delta h/h). This result indicates that the spectral difference is related to the cell deformation that accompanies the orientation of the cells in the shear flow. In addition, the average decay time (tau av) for the spectral difference observed when the flow was abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation at 49 degrees C. Since the observed tau av is much shorter than the expected rotational correlation time for the erythrocyte, the decay is attributed to the deformation recovery process. It is demonstrated that the measurements of both delta h/h and tau av by ESR spectroscopy give qualitative information on the viscosity and the elasticity of the cell membrane system.     

The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.     

Engineering select physical properties of cross-linked red blood cells and a simple a priori estimation of their efficacy as an oxygen delivery vehicle within the context of a hepatic hollow fiber bioreactor

Bovine red blood cells (bRBCs) can potentially provide a simplistic and economic means of improving oxygenation within hollow fiber (HF) bioreactor cell cultures. Bovine RBCs are also interesting since many of their physical properties can be altered as a result of glutaraldehyde (G) cross-linking. Cross-linking bRBCs produces an oxygen carrier that is expected to be beneficial under specific circumstances (i.e., delivery of oxygen to cells that are sensitive to free hemoglobin (Hb) and cells that require low inlet oxygen tensions). We have examined the osmotic stability and electrophoretic mobility of cross-linked bRBCs and observed that cross-linking improves osmotic stability while minimally impacting electrophoretic mobility. The oxygen binding/dissociation properties (P(50) and n) of cross-linked bRBCs were also measured, and under the reported reaction conditions, cross-linking increased the oxygen affinity and reduced the cooperativity of bRBCs. A basic Krogh tissue cylinder model was then utilized to provide a quick a priori estimate of oxygen delivery and release to hepatocytes housed within a HF bioreactor in order to demonstrate potential oxygenation benefits arising with both normal and cross-linked bRBC media supplementation. This model showed that bRBCs generally improved oxygen delivery and release to HF cell cultures and that cross-linked bRBCs are particularly beneficial in specifically targeting oxygen delivery to cells maintained at low inlet oxygen tensions. Additionally, the model showed that bRBC supplementation can significantly improve oxygen delivery without requiring extreme bRBC concentrations.