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Artificial Cells, Blood Substitutes, and Biotechnology, 35: 6979, 2007 Copyright Q Informa Healthcare ISSN: 1073-1199 print/1532-4184

online DOI: 10.1080/10731190600974541

Trapping Hemoglobin in Rigid Matrices: Fine Tuning of Oxygen Binding Properties by Modulation of Encapsulation Protocols
Stefano Bruno, Luca Ronda, Stefano Bettati, and Andrea Mozzarelli
Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy

Abstract: Encapsulation of hemoglobin in a biocompatible matrix is a potential strategy for obtaining blood substitutes. Such a system would retain most of the immunogenic and functional properties of the physiologically relevant oxygen carrier but would prevent protein extravasation and dimer=dimer dissociation. We applied this approach by entrapping hemoglobin in wet nanoporous silica gel, in the presence and absence of allosteric effectors. Silica gels, although not suitable for intravenous perfusion, are inert and optically transparent, thus allowing a full characterization of the functional and structural properties of encapsulated hemoglobin by spectroscopic techniques. Results indicate that hemoglobin molecules, entrapped using different protocols, exhibit an oxygen affinity that can be modulated between 12 and 140 torr. This tunability could be exploited to meet distinct clinical needs.
Keywords: Bioencapsulation; Blood substitutes; Hemoglobin; Silica gel

INTRODUCTION The risks associated with blood transfusions and possible shortages of blood supplies have prompted research into alternative oxygen carriers, particularly intended for the treatment of traumatic or surgical blood losses, ischemic states and, in combination with chemotherapy, cancer.
This work was supported by a grant of the European Union LSHB.CT-2004S03023 to A.M. Address correspondence to Andrea Mozzarelli, Department of Biochemistry and Molecular Biology, University of Parma, Via GP Usberti 23/A, 43100 Parma, Italy. E-mail: andrea.mozzarelli@unipr.it 69

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Most of the potential oxygen therapeutics formulated so far are based on hemoglobin either from recombinant molecules, transgenic animals, or outdated banked human blood. Cell-free hemoglobin as such proved unsuitable as a blood substitute, due to its renal toxicity caused by tetramer dissociation in a-b dimers and to a severe vasoconstriction due to nitric oxide scavenging [1-3]. In order to overcome these side effects, several chemically modified human or bovine hemoglobins were tested. The problem of tetramer dissociation was first tackled by introducing covalent intramolecular linkers, but diaspirin-cross-linked hemoglobin (DCLHb) failed in two clinical trials [4] and experiments on animals revealed myocardial lesions as a side effect [5]. Intermolecular polymerization proved more successful, with two products currently in phase III clinical trials. Particularly, PolyHeme1, a pyridoxylated glutaraldehyde-polymerized human hemoglobin derivative, showed encouraging results as potential therapeutic in the early treatment of severely injured trauma patients [6]. The South African government recently approved the use of Hemopure1, a glutaraldehyde-polymerized bovine hemoglobin, for the treatment of acute anemia [6,7]. A third strategy of hemoglobin modification consists in the surface conjugation of the protein with bulky substituents, particularly polyethylene glycol (PEG). MP4, a low-p50, non-vasoactive maleimide-activated polyethylene glycol derivative, is due to enter advanced clinical trials [8]. A different approach consists in the preparation of liposomes or nanoparticles [23] containing hemoglobin [9]. This formulation shows only a limited circulation half-life. An increase of intravascular persistence was achieved either by introducing an actin matrix into the liposome aqueous core [6,7,10] or by coating the surface of liposomes with PEG groups [11,12]. The level of PEG conjugation was further increased by using amphiphilic diblock copolymers as building blocks of the liposome membrane (polymerisomes) [13]. Hemoglobin-loaded liposome has the advantage of avoiding the need for covalent modifications of hemoglobin. Moreover, the vesicles size can be partially modulated by regulating the extrusion process, thus giving flexibility to the end product. Neither chemical modifications of hemoglobin nor loading of hemoglobin in liposomes yield a full control over oxygen binding properties. A system in which hemoglobin affinity could be finely regulated without changes in the overall chemical and biophysical properties of the product would be greatly needed for both clinical and experimental purposes. Historically, it was taken for granted that an effective blood substitute should possess oxygen binding properties resembling those of hemoglobin inside the red blood cells, where endogenous allosteric effectors are present. Recently, this view was challenged by the socalled autoregulatory vasoconstriction theory, according to which the severe vasoconstriction

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observed upon administration of most hemoglobin-based blood substitutes is caused by the response of the vessels to an excessive supply of oxygen, rather than by the nitrogen monoxide scavenging activity of hemoglobin [14]. This principle led to high affinity hemoglobin derivatives, of which MP4 is the one at the most advanced stage of clinical experimentation. Our goal was to obtain a system with a tunable p50. This was achieved by encapsulating hemoglobin in wet nanoporous silica gels, using different protocols, with and without allosteric effectors. Silica gel is not fully biocompatible per se, but it is a good model for rigid, hydrated, inert matrices, the surface polarity of which depends on the precursor. Small molecules can freely diffuse inside the matrix. The silica gel approach has been exploited for the development of bioreactors entrapping enzymes, biomedical devices and biocompatible coating [15]. The most striking property of silica gel encapsulation is the retention of protein bioactivity with respect to solution. The polymer network cages the protein, preventing or dramatically slowing down tertiary and quaternary conformational changes. It was shown that hemoglobin encapsulated under anaerobic conditions retains T-like binding properties, even if exposed to saturating concentrations of either carbon monoxide or oxygen. At 15C, the T to R quaternary transition takes place on a timescale of days [16]. It was also possible to selectively block different tertiary states within the T quaternary conformation exhibiting a 10-fold difference in oxygen affinity and no cooperativity [17]. Here, we report the protocols used for obtaining hemoglobin-doped gels endowed with a tunable p50 ranging from 12 to 140 torr at 15C, and the functional and spectroscopic characterization of these hemoglobin gels. MATERIALS AND METHODS Materials CM-sephadex, DEAE-sephadex, tetramethyl orthosilicate (TMOS), potassium phosphate, sodium dithionite, HEPES, sodium chloride, inositol hexafosphate (IHP), and bezafibrate (Bzf) were of the best commercially available quality and were used without further purification. All gases were research grade. Hemoglobin Purification Human hemoglobin from a nonsmoker was purified according to procedures described previously [18,19]. In order to obtain a higher degree of purity, the DEAE-sepharose column was followed by a CM-sephadex column, developed with a pH gradient from 6.9 to 8.6 in 10 mM HEPES

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buffer. The purest fractions were collected, concentrated to a final concentration of 100 g=l and flash-frozen in liquid nitrogen. Encapsulation of Hemoglobin in Silica Gel Encapsulation in silica gel was carried out by promoting the polymerization of hydrolyzed TMOS in a solution containing 1020 g=l hemoglobin. The hydrolysis of TMOS is triggered by mixing 750 ml of TMOS, 169 ml of water and 11 ml of hydrochloric acid (protocol 1). The reaction between the two phases comes to completion by sonicating the mixture for 20 minutes at 4C. The resulting solution, adjusted to pH 6.0 by adding a concentrated stock of a buffer solution, was deoxygenated by bubbling nitrogen for 3040 minutes. Finally, 1.5 parts of a hemoglobin solution, buffered at pH 7.2, were added. Gelification occurs within a few minutes at room temperature. The gels were stored at 4C, covered by a buffered solution at neutral pH. In an alternative protocol (protocol 2), one part of TMOS is mixed with one part of a buffer at pH 6.2 and vortexed for 2 minutes. Nitrogen is bubbled in the emulsion for a time ranging from 30 to 90 minutes. The bubbling time affects the degree of hydrolysis of TMOS, thus changing the tightness of the polymer network around the protein. Finally, an equal volume of hemoglobin solution is added. The gelification occurs within 1 hour. As for protocol 1, the gels are kept hydrated by layering a buffered solution on them. Both protocols were used to encapsulate hemoglobin equilibrated in nitrogen (T state gels) or in carbon monoxide or oxygen atmosphere (R state gels). Deoxyhemoglobin was also encapsulated in the presence of allosteric effectors at different final concentrations: . 10 mM IHP 2 mM Bzf 200 mM sodium chloride . 100 mM phosphate . 200 mM sodium chloride When present, allosteric effectors were added to both hemoglobin solutions and storing solutions. T state gels were stored under anaerobic conditions, in the presence of 30 mM sodium dithionite. All gels were kept at 4C before use. Determination of Oxygen Binding Properties Absorbance spectra of hemoglobin gels were recorded by a Zeiss MPM03 microspectrophotometer. Before measurements, silica gels were washed several times under anaerobic conditions with the deoxygenated storing

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buffer. Gels were anaerobically loaded in a Dvorak-Stotler flow cell [20] covered with a gas-permeable silicon copolymer membrane. The flow cell was mounted on the thermostated stage of the microspectrophotometer. Spectra were recorded in the wavelength range 450 to 700 nm, at 1-nm intervals, using gels that absorb usually less than one optical density unit. Oxygen pressures between 0 and 760 torr were prepared by mixing oxygen and helium with either an ENVIRONICS 200 or an ENVIRONICS 4000 gas mixer. The humidified gas mixture was flowed through the sample cell. The fractional saturation at each oxygen pressure was determined by fitting absorption spectra to a linear combination of the spectra of the fully deoxy-, oxy- and met-hemoglobin species (reference spectra), plus a baseline and a slope to take into account the non perfect optical quality of the gel surface [16]. The estimation of K1, the affinity constant of the first oxygen for hemoglobin in solution, was carried out by using a home-made tonometer, consisting of a cuvette fused to a small glass reservoir and connected to the gas mixture generators ENVIRONICS 200 and ENVIRONICS 4000. The use of two gas mixers in sequence allows us to reliably prepare gas mixtures at very low oxygen partial pressure. As a consequence, the quality of the data is better with respect to those collected using a traditional tonometer. The humidified mixture was continuously flown into the modified tonometer through gas-tight connections. Equilibration at each oxygen pressure occurred within 20 minutes, at controlled temperature. Spectra were collected using a CARY 400 spectrophotometer and analyzed as described above. Only fractional saturations below 1% were used for the estimation of the K1.

RESULTS AND DISCUSSION A representative example of a spectrum of hemoglobin gels, collected using the microspectrophotometer, and the spectral components determined by linear fitting with the reference spectra are reported in Figure 1. The fitting yields the fractional saturation with oxygen of reduced hemes and the fraction of met-hemoglobin, which is lower than 10% in all the experiments. By collecting spectra at different oxygen pressures, a complete binding curve can be obtained. Similar measurements were carried out on hemoglobin in solution using a spectrophotometer and modified tonometer. Representative examples of Hill plots determined for hemoglobin gels obtained under different encapsulation conditions are presented in Figure 2 and compared to those measured for hemoglobin in solution in the absence of allosteric effectors. The fitting of the Hill plot in the

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Figure 1. Representative absorption spectrum of hemoglobin encapsulated in silica gel (solid line). The contributions of deoxy- (dashed line), oxy- (dash-dot line) and met-hemoglobin (dash-dot-dot line) spectra resulting from the linear fitting are reported. The calculated fractional saturation is 0.45 and the fractional met-hemoglobin content is 0.1.

saturation range 20% to 80% yields the p50 and the Hill coefficient n. The calculated values are reported in Figure 3 and Table 1, where, for comparison, the p50 values of T states hemoglobin, under different solution conditions, estimated from the K1, are also reported. The Hill coefficient is very close to 1 for the highest and lowest affinity hemoglobin gels, obtained in the absence of any allosteric effectors or in the presence of saturating concentrations of strong allosteric effectors, respectively (conditions 1, 5 and 6 in Table 1). This suggests that these hemoglobin gels contain homogeneous, noncooperative binding forms. The p50s for these two forms are very close to those estimated for T state hemoglobin in solution under the same conditions (conditions 8 and 9 in Table 1), indicating that the silica matrix entraps conformations present in solution

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Figure 2. Hill plots of hemoglobin encapsulated in the absence of allosteric effectors (condition 1 in Table 1) (open inverted triangles), in the presence of strong allosteric effectors (condition 5 in Table 1) (open triangles), in the presence of a saturating concentration of chloride ions (condition 4 in Table 1) (closed squares). For comparison, the Hill plot of hemoglobin in solution in the absence of allosteric effectors (condition 7 in Table 1) (closed circles) is also reported. Solid lines through data points are the fit to the Hill equation.

without perturbing their functional properties. Furthermore, the p50 of hemoglobin gels prepared in the presence of weak allosteric effectors (Fig. 2, conditions 3 and 4 in Table 1) is intermediate between those of the two extreme forms. Chloride ions at saturating concentrations affect the p50 of encapsulated hemoglobin more than phosphate ions. The Hill coefficient lower than unity observed for these hemoglobin gels is unlikely to be due to negative cooperativity. It could be explained by functional heterogeneity. In particular, these intermediate gels seem to be constituted by a mixture of the two populations of T state hemoglobin endowed with highest and lowest affinity. In order to test this hypothesis, the oxygen binding curves were fitted to the sum of two hyperbole equations. The p50s and the Hill coefficients were fixed to the values experimentally determined for the lowest and highest affinity T states, allowing to change their relative contribution. According to this fitting, the hemoglobin gels prepared in the presence of chloride ions (condition

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Figure 3. p50 values (white bars) and Hill coefficients (black bars) of hemoglobin encapsulated in silica gels prepared according to conditions 16 in Table 1.

4 in Table 1) would consist of 55% of the lowest affinity state and 45% of the highest affinity state. In hemoglobin gels prepared in the presence of phosphate ions (condition 3 in Table 1), the two populations are 36% and 64%, respectively. Further modulation of the p50 was obtained by modifying the encapsulation protocol for the highest affinity state. Protocol 2 allowed us to obtain hemoglobin gels with a p50 of 12.4 or 26 torr by changing the time allowed for hydrolysis of TMOS from 30 to 90 minutes (conditions 1 and 2 in Table 1). Titrations of the methanol produced during the hydrolysis of TMOS demonstrate that the reaction does not occur immediately after the aqueous emulsion is formed and takes several hours to be completed (data not shown). The time allowed for hydrolysis could affect the degree of branching of the silica network and, therefore, the tightness of the gel matrix around the protein. This might affect the conformational state distribution. This would explain the different p50 of deoxyhemoglobin depending on variation of the hydrolysis=polymerization steps. However, protocol variations do not change the p50 as much as the presence of allosteric effectors. When high concentrations of strong allosteric effectors are present, the variation of the encapsulation protocols seems to have a limited effect. For instance, encapsulation with protocol 1 produces hemoglobin gels exhibiting a p50 only slightly lower than those

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Table 1. Protocols, p50s and Hill coefficients of hemoglobin encapsulated in silica gel and in solution Sample 1 2 3 4 5 6 7 8 9 Deoxyhemoglobin, no effectors Deoxyhemoglobin, no effectors Deoxyhemoglobin, 100 mM phosphate Deoxyhemoglobin, 200 mM Cl Deoxyhemoglobin, 10 mM IHP, 2 mM Bzf, 200 mM Cl Deoxyhemoglobin, 10 mM IHP, 2 mM Bzf, 200 mM Cl Solution, no effectors Estimated p50 (from K1) in solution, no effectors Estimated p50 (from K1) in solution, 10 mM IHP, 2 mM Bzf, 200 mM Cl Protocol 2 (300 ) 2 (900 ) 1 2 1 2 p50 (torr) 12.4 0.2 26 1 33.6 0.5 71.1 3.8 139 4 134 5 2.61 0.05 14.1 5 124 4 n 0.94 0.02 0.95 0.03 0.80 0.01 0.84 0.04 0.93 0.03 0.93 0.03 2.36 0.06 1.0 0.3 1.07 0.07

prepared using protocol 2 (Table 1). In this case, the effect of IHP and Bzf seems to prevail over the degree of tightness of the gel matrix in determining the functional properties of hemoglobin. In order to rule out a perturbation of the protein conformation by the gel matrix, we have carried out an extensive spectroscopic characterization of encapsulated hemoglobin. Flash photolysis experiments show that hemoglobin encapsulated in the T and R state exhibits CO rebinding properties very close to those observed in solution, take into account that large conformational relaxations are frozen in the gel in the experimental time window [21]. As a matter of fact, deoxyhemoglobin gels, exposed to CO immediately before the experiment, behave as predicted for T state hemoglobin, i.e. absence of geminate rebinding and slow bimolecular phase. On the other hand, gels encapsulated in the R state show a typically high fraction of geminate rebinding and a fast bimolecular phase. These rebinding kinetics are simple if compared to hemoglobin solutions, where the conformational transition from R to T state takes place in the timescale of the bimolecular rebinding. The results of the analysis of rebinding kinetics of gels encapsulated under different conditions showed a more complex behavior, though in keeping with the recently proposed Tertiary Two State model of hemoglobin allostery [21]. T state and R state gels were also shown to possess the same resonance Raman spectroscopic markers of the liganded and unliganded states in solution [22], thus suggesting that encapsulation does not perturb the individual conformations, but only shows down their interconversion. Similar results were obtained with circular dichroism spectroscopy (data not

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shown), according to which T state gels maintain typical T state structural markers even when bound to carbon monoxide or oxygen. CONCLUSIONS Encapsulation in silica gel entraps protein conformations without altering their functional properties. By modulating the encapsulation protocols, different quaternary and tertiary states of hemoglobin can be selectively stabilized in the gel matrix, as pure species or defined mixtures. The overall functional properties of the hemoglobin gel change accordingly. In particular, the oxygen affinity can be tuned in the range between 12 and 140 torr for deoxyhemoglobin, thus satisfying different clinical needs. The challenge is now to design a fully biocompatible polymeric matrix with comparable effects on protocol-dependent hemoglobin functional properties. REFERENCES
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