3 Results and Discussion

After synthesis and purification, the biophysical properties of PolybHb were characterized via various techniques. The resulting O­2 affinity, cooperativity coefficient, hydrodynamic diameter, ligand binding/release kinetics, autoxidation kinetics, and average MW are listed in Table 1 .

3.1 Oxygen Affinity and Offloading.

In this study, the effect of the quaternary state of bHb during polymerization on oxygen affinity (P50) and Hill cooperativity coefficient (n) were explored. Figure 3A displays the oxygen equilibrium curves of bHb, T- and R-state PolybHb. Fitting the OECs to the Hill equation yields a P50 of 41.3 ± 3.3 mm Hg, 43.4 ± 7.0 mm Hg and 38.9 ± 2.5 mm Hg for T-state PolybHb 25:1, 30:1, and 35:1, respectively. These P50 values are comparable to the commercial PolybHb HBOC-201® ( ~38 mm Hg) (Biopure, Cambridge, MA) (J.S., M., & J.C., 2008; Pearce, Gawryl, Rentko, Moon-Massat, & Rausch, 2006). The P50s of the T-state PolybHbs prepared in this study are, on average, higher than the commercial human PolyHbs, PolyHeme® (28-30 mm Hg) (Northfield Laboratories, Evanston, IL) and Hemolink® (33.5 mm Hg) (Hemosol Inc., Toronto, Canada) (Lou Carmichael et al., 2000; Rice et al., 2008). The right-shifted O2 equilibrium curve of T-state PolybHb can be ascribed to deoxygenation of bHb during PolybHb synthesis. Conversely, the left-shifted O2 equilibrium curve of R-state PolybHb was due to oxygenation of bHb during PolybHb synthesis. Interestingly, the molar ratio of glutaraldehyde to bHb during polymerization did not have a significant effect on the P50 and n (p < 0.05). In comparison to T-state PolyhHbs and unmodified bHb, R-state PolybHbs exhibit much lower P50s. In Figure 3C , we found significant differences (p < 0.05) between the P50 of bHb and PolybHb in both quaternary states. In Figure 3D , the Hill cooperativity coefficient showed no significant difference (p < 0.05) between T- and R-state PolybHbs. Both T- and R-state PolybHbs displayed no cooperativity (n~1.0), which is lower than PolyHeme® (1.7) and HBOC-201® (1.4) (Day, 2003; Lou Carmichael et al., 2000; Napolitano, 2009a). The absence of cooperativity implies that the α and β globin subunits can no longer go through conformational changes which is indicative of the higher cross-link density of PolybHb in this study. This can be explained by the “locking effect” that the protein cross-linker glutaraldehyde has on the quaternary structure of Hb (Buehler et al., 2005).
Figure 3B shows the O2 offloading kinetics for bHb and PolybHbs at various glutaraldehyde:bHb molar ratios. T-state PolybHbs released O2 faster than R-state PolybHbs. Both T-state and R-state PolybHbs released O2 slower than native bHb. Figure 3E displays the effect of the glutaraldehyde:bHb molar ratio on \(k_{off,\ \text{\ O}_{2}}\). R-state PolybHb 25:1 exhibited a significantly higher\(k_{off,\ \text{\ O}_{2}}\) than R-state PolybHb 30:1 and 35:1, indicating that the \(k_{off,\ \text{\ O}_{2}}\) of R-state PolybHb can be engineered by varying the glutaraldehyde:bHb molar ratio. The\(k_{off,\ \text{\ O}_{2}}\) of T-state PolybHb was not sensitive to the glutaraldehyde:bHb molar ratio.
Figure 3F displays the effect of quaternary state on\(k_{off,\ \text{\ O}_{2}}\). T-state PolybHb possessed significantly higher \(k_{off,\ \text{\ O}_{2}}\) than R-state PolybHb (p < 0.05), suggesting that the \(k_{off,\ \text{\ O}_{2}}\) of PolybHb can be modified by maintaining the quaternary state of bHb before and during polymerization. The \(k_{off,\ \text{\ O}_{2}}\) of bHb was significantly higher than PolybHb (p < 0.05), suggesting that polymerization regulates O2 offloading kinetics. As a result, PolybHbs will not inherit the cooperative O2offloading feature of native bHb. In comparison to Hemolink® (130 ± 3.5 s-1) and Oxyglobin® (61.8 ± 1.6 s-1) (Chen, Chen, Liou, & Chao, 2015; Jia, Ramasamy, Wood, Alayash, & Rifkind, 2004), both T-state and R-state PolybHbs synthesized at higher cross-link density with negligible fraction of free bHb resulted in a reduced \(k_{off,\ \text{\ O}_{2}}\). R-state PolybHb exhibited increasing \(k_{off,\ \text{\ O}_{2}}\) with decreasing glutaraldehyde:bHb molar ratio.

3.2 Size and Molecular Weight.

Figure 4A displays the size distribution of bHb, T-state PolybHb, and R-state PolybHb at glutaraldehyde:bHb molar ratios of 25:1, 30:1, and 35:1. All PolybHb solutions contained negligible free Hb tetramers (0 to 1.2%, 0th order species, i.e. free Hb), which is considerably less than previous PolybHbs synthesized from our lab (Zhou et al., 2011). A right-shifted distribution of PolybHb MW was observed with increasing glutaraldehyde:bHb molar ratio, suggesting that the size of PolybHb is tunable. Both 35:1 T-state and 35:1 R-state PolybHb possessed the highest average MW (1194 ± 106 kDa, 1316 ± 81 kDa respectively) compared to the other PolybHbs synthesized in this study. However, the increased size of 35:1 R-state PolybHb resulted in negligible passage through 0.2 μm HF filter modules. Zhang et al . synthesized 40:1 and 50:1 T-state PolyhHb which had a weight averaged MW of 3.7 and 18 MDa, respectively (Zhang et al., 2011). Since the PolyhHbs in that study were synthesized at higher glutaraldehyde:Hb molar ratios than the 30:1 ratio in this study, it should have contained a larger fraction of polymers > 0.2 μm in size than the PolybHbs produced in this study. Previously, Liu et al . found that nanoparticles with the diameter > 300 nm tended to trigger macrophage uptake in the reticuloendothelial system (Liu, Mori, & Huang, 1992). Thus, the PolybHbs synthesized in this study may be less likely to be quickly cleared versus PolyHbs that were synthesized in previous work (Zhang et al., 2011). In Figure 4B , analysis of the polymer order of the majority of T-state and R-state PolybHbs yielded less than 1% free bHb in solution (0th order, 1.31 ± 0.87%). In comparision, the commercial HBOCs, PolyHeme® and Hemopure® contained less than 5% of free Hb (Levy et al., 2002; Marret et al., 2004). The average MW of PolyHeme® and Hemopure® were 250 kDa and 150 kDa, respectively. Unfortunately, both commercial HBOCs induced severe hypertension when transfused in vivo . Fortunately, 35:1 T-state and R-state PolybHbs possessed MWs 4-fold higher than PolyHeme®and Hemopure® (Gould et al., 1998; Moore et al., 2009).
All PolybHbs have less than 0.9% of 128 kDa 1st order polymers (∼0.14 ± 0.32%). T-state PolybHb at a 25:1 cross-link density contained a significantly higher fraction of 3rd and 4th order PolybHb (27.6 ± 8.7%; 51.7 ± 2.0%) than those at a 30:1 cross-link density (8.7 ± 3.7%; 33.1 ± 2.1%) and 35:1 cross-link density (5.0 ± 3.4%; 22.2 ± 2.9%). All PolybHbs possessed similar amounts of 5th order polymers (25.8 ± 6.9%). The fraction of 5th order polymers in both the T- or R-state PolybHb increased with increasing glutaraldehyde:bHb molar ratio. The fraction of lower polymer orders (2nd and 3rd) exhibited the opposite trend, since smaller polymers were crosslinked to form larger clusters with increasing glutaraldehyde:bHb molar ratio. Previously, it was observed that the presence of free Hb was responsible for the renal toxicity, vasoconstriction, and hypertension observed when PolyHb was transfusedin vivo [26, 37–39]. Thus, both T-state and R- state PolybHb synthesized in this study are unlikely to induce those side-effects, because of the extremely low levels of stroma-free bHb in the final products. It is interesting to note that the 35:1 T-state PolybHb and 30:1 R-state PolybHb have a similar MW distribution even though they were syntheszied at different glutaraldehyde:bHb molar ratios. The shift in MW distribution observed via HPLC-SEC is consistent with the hydrodynamic diameter measured via DLS and is shown in Figure 4D . In contrast, Figure 4B shows that there was a minor difference between the polymer-order composition of T-state PolybHb 35:1 and R-state PolybHb 30:1. T-state PolybHb 35:1 contained a higher fraction of 2nd and 5th order polymers compared to R-state PolybHb 30:1.
Figure 4C shows the SDS-PAGE of bHb, T-state PolybHb, and R-state PolybHb synthesized in this study. All lanes show a band at 32 kDa, representing αβ dimers. For bHb (control) in lane 1, a clear band at approximately 16 kDa was observed, which indicates the presence of α (15.043 kDa) and β (15.946 kDa) subunits that dissociated from tetrameric native bHb (α2β2). Lanes 2 and 3 display both T-state and R-state PolybHb at a 25:1 glutaraldehyde:bHb molar ratio, where T-state PolybHb migrated as a broader band compared to R-state PolybHb. This pattern is also observed at other glutaraldehyde:bHb molar ratios (30:1 and 35:1). This finding aligns with the polymer order distribution in Figure 4B as in general, T-state PolybHb had a smaller fraction of 4thand 5th order of polymers compared to R-state PolybHb at the same cross-link density. Within the same quaternary state in lanes 3, 6 and 9 (or 2, 5 and 8), the MW of T-state (or R-state) PolybHb increased with increasing glutaraldehyde:bHb molar ratio.
Figure 4D displays the hydrodynamic diameter of bHb and PolybHb. The diameter of PolybHb grew with increasing glutaraldehyde:bHb molar ratio, which is consistent with Figures 4A, 4B and 4C . Both 35:1 T-state and 35:1 R-state PolybHb had the largest effective hydrodynamic diameters (64.1 ± 9.3 and 166.9 ± 4.1 nm, respectively) compared to 25:1 and 30:1 PolybHbs. PolybHb is 5-10 times on average larger in size compared to Oxyglobin® (5.85–10.49 nm), Hemolink® (5.28–11.13 nm), HBOC-201® (5.73–11.10 nm) and PolyHeme® (5.59–10.31 nm) (Day, 2003; R. M. J. Palmer, Ferrige, & Moncada, 1987). InTable 1 , the polydispersity index (PDI) of PolybHb is shown. All PolybHbs possess a PDI lower than 0.4, which corresponds to moderate polydispersity.

3.3 Viscosity.

The viscosity of T-state and R-state PolybHbs synthesized in this study is listed in Table 1 . Increasing the glutaraldehyde:bHb molar ratio increased the viscosity of PolybHbs. Both T-state PolybHb 35:1 and R-state PolybHb 30:1 possesed a higher viscosity at 5 g/dL compared to PolyHeme® (~2.1 cP) and Hemopure® (~1.3 cP) at approximately a protein concentration of 10 g/dL (Napolitano, 2009b). PolybHbs with high viscosity are desirable for intravenous transfusion, since they induce the release of endothelial-derived relaxing factors (EDRFs) via mechanotransduction, which might further mitigate the vasoconstrictive effects due to PolyHb NO scavenging or O2 oversupply (R. M. J. Palmer et al., 1987). However, the ultra-high viscosity of R-state PolybHb 35:1 might induce retinal dilation and conjunctival hemorrhage if transfused in vivo (Gertz & Kyle, 1995). Thus, R-state PolybHb 35:1 may not serve as an ideal RBC substitute.

3.4 PolybHb-Haptoglobin Binding Kinetics.

Free Hb in plasma tends to dissociate into two pairs of αβ dimers, which are scavenged via the Hb binding protein haptoglobin (Hp). The resulting Hb-Hp complex can then bind to the CD163+ macrophages and monocytes. The CD163 receptor scavenges both Hb and Hb-Hp and essentially cleares Hb from the systemic circulation by receptor endocytosis into macrophages and monocytes (Etzerodt & Moestrup, 2013). To determine the potential for PolybHb to be cleared via CD163 mediated endocytosis, the ligand-binding kinetics of Hp with bHb/PolybHb was monitored by rapidly mixing Hp with bHb/PolybHbs at various concentrations.
The kinetics of Hp-bHb/PolybHb binding is shown in Figure 5A . It was evident that both T-state and R-state PolybHbs quenched a lower number of Hb binding sites in Hp compared to bHb. By fitting the kinetic traces in Figure 5A to a mono-exponential equation, the pseudo first order binding rate constants at different concentrations of bHb and PolybHbs were plotted in Figure 5B . To calculate the 2nd order (bHb/PolybHb)-Hp binding rate constant (k Hp-Hb), we performed a linear fit to the data in Figure 5B to determine the slope of the pseudo first order reaction rate constant as a function of PolybHb/bHb concentration. Overall, the k Hp-Hb of T-state (0.0136 – 0.0228 μM−1 s−1) and R-state PolybHbs (0.0069 – 0.0097 μM−1 s−1) were significantly reduced compared to unmodified bHb (0.145 μM−1 s−1). Thek Hp-Hb of Hp-bHb binding was consistent with a previously reported value of 0.15 μM−1s−1 (Meng et al., 2018a). Thek Hp-Hb of T-state PolybHb was significantly (p < 0.05) greater than that of R-state PolybHb at the same glutaraldehyde:bHb molar ratio. The k Hp-Hb for 25:1 and 30:1 R-state PolybHb were lower than Oxyglobin® (0.011 μM−1 s−1) as reported previously in the literature (Meng et al., 2018a). The k Hp-Hbfor 25:1 T-state PolybHb is similar to Oxyglobin® (0.011 μM−1 s−1) (Meng et al., 2018a). Thek Hp-Hb of the rest of PolybHbs was similar to Oxyglobin® (0.011 μM−1 s−1) (Meng et al., 2018a).

3.5 Autoxidation Rate.

Autoxidation of bHb, T-state PolybHb, and R-state PolybHb is shown inFigure 5C and 5D . We found that both T- and R-state PolybHbs displayed two-phase autoxidation kinetics which was not observed for native bHb. The fast-step autoxidation rate constant of all PolybHbs except 25:1 T-state PolybHbs was higher than that of bHb (kox = 0.021 ± 0.003 h−1). This finding aligns with previous studies which demonstrated that HBOCs with chemical modifications exhibit higher autoxidation rate constants compared to unmodified Hb (Gertz & Kyle, 1995; Meng et al., 2018a). For the slow-step autoxidation kinetics, all PolybHbs yielded a decreased rate of autoxidation compared to native bHb. R-state PolybHbs had autoxidation rates ∼2-fold lower than that of bHb. These results might be attributed to the different autoxidation rate between α and β subunits (Tsuruga, Matsuoka, Hachimori, Sugawara, & Shikama, 1998). The slow-step autoxidation rate constant of T-state PolybHbs (<0.0162 h-1) was higher than that of R-state PolybHbs (<0.0069 h-1). The higher autoxidation rate constants of T-state PolybHbs compared to R-state PolybHb are consistent with that of previously reported HBOCs (Zhang et al., 2011). For instance, it was reported that 50:1 T-state PolybHb had an increased kox in comparison to 40:1 R-state PolybHb (Zhang et al., 2011). For T-state PolybHb, the duration of slow-step phase was found to increase when decreasing the glutaraldehyde:bHb molar ratios.

3.6 CD Spectra Analysis.

Figure 6A displays the CD spectra of both T-state and R-state PolybHb in the far-UV region (190 - 260 nm). No significant differences (p < 0.05) between PolybHb and bHb were observed, suggesting that the secondary structure of bHb remained unchanged in both T-state and R-state PolybHbs.

3.7 MALDI-TOF MS Analysis.

The results of MALDI-TOF mass spectral analysis is shown inFigure 6B, 6C and 6D . In Figure 6B , the spectrum in the 0-3000 Da m/z mass range for bHb exhibited a peak around 616 Da that corresponded to the heme group (Bonaventura, Henkens, Alayash, & Crumbliss, 2007; Etzerodt & Moestrup, 2013). A heme peak around 616 Da was also observed for both T- and R-state PolybHb along with other unidentified low abundance peaks in the 0-3000 Da m/z range (data not shown).
Figure 6C displays the mass spectra of bHb and R-state PolybHb in the in the 10,000-80,000 Da m/z mass range. The mass spectrum of bHb exhibited two distinct peaks at 15,043 and 15,946 m/z that correspond to the α and β subunits of bHb (Elmer et al., 2011). The spectra for all PolybHb showed a flat noisy line with low intensity in the mass range 20-80 kDa signifying the virtual absence of smaller species (αβ dimers or α2β2 tetramers) in the polymerized samples. These results align with the findings from HPLC-SEC and SDS-PAGE analysis which showed that <1% free bHb was present in most of T- and R-state PolybHb preparations. Furthermore, the sample preparation conditions (acetonitrile and TFA) did not induce substantial Hb monomer formation as observed by the low-intensity peaks in the monomer region for PolybHb samples. This further confirmed that the crosslinked bHb was extremely stable and did not dissociate upon ionization in the mass spectormeter. In the inset of Figure 6C , R-state PolybHbs exhibited a low-intensity peak at 15,043 m/z, representing uncrosslinked α subunits (<1%). While for T-state PolybHb, two distinct low-intensity peaks were observed at both 15,043 and 15,946 m/z which correspond to the α and β subunits. This difference indicated that R-state PolybHb contains more β-β crosslinked polymers compared to T-state PolybHb.

3.8 Meta-Data Analysis.

In Figure 7A , for both T-state and R-state PolybHbs, the gas-liquid exchange contact time did not correlate with the metHb level. Figure 7B shows that increasing the temperature in the reactor during dithionite injection lead to higher metHb levels. In Figure 7C, while the PolybHb yield seemed to decrease linearly with the number of diafiltration cycles, no significant effect of diafiltration cycles on yield was observed (p < 0.05). Hence, we can enhance the purity of PolybHb without being concerned about lowering the PolybHb yield. Figure 7D displays the effect between the number of diafiltration cycles and metHb level, where a significant variance (p < 0.05) was found between the group with 14 diafiltraion cycles compared to others. 14 diafiltration cycles yielded the lowest metHb for both T-state and R-state PolybHb. Figure 7E and 7F show the correlation between glutaraldehyde:bHb molar ratio and yield of PolybHbs. R-state PolybHb 35:1 and T-state PolybHb 35:1 both exhibit significantly higher yields than the other molar ratios (p<0.05). Unfortunately, 35:1 R-state PolybHb was diafiltered on a 0.2 μm HF filter instead of a 500 kDa HF filter compared to the other PolybHbs. Thus, only 30:1 R-state PolybHb and 35:1 T-state PolybHb are desirable PolybHbs with clearly higher yield. In Figure 7G and 7H, neither the quaternary state nor glutaraldehyde:bHb molar ratio influenced the PolybHb metbHb level (3.7 ± 1.5 %).