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 %).