Polymerization of modified diaspirin cross-linked hemoglobin (DCLHb) with 1,6-bismaleimic-hexane

Abstract

Increasing the size of hemoglobin (Hb) by polymerization offers the benefits of reduced renal clearance and increased duration in the vascular circulation. With this goal, diaspirin cross-linked hemoglobin (DCLHb) was modified in order to keep one thiol group on the surface and then polymerized with 1,6-bismaleimic-hexane (1,6-BMH) to increase the molecular weight. The HPLC results indicated that approximate 20% dimers to tetramers of DCLHb desired were achieved after the polymerization. It was also demonstrated that the oxygen-carrying capacity of the products was similar to natural heme. The present study is expected to improve the efficacy of the DCLHb as an oxygen therapeutic agent.

Keywords:

• Cross-linking agent
• DCLHb
• oxygen affinity
• thiol group

Introduction

The primary biological function of natural red blood cells (RBCs) is to transport hemoglobin-bound oxygen and carbon dioxide to and from tissues, respectively. Hemoglobin, by which in the RBCs oxygen is carried, is a tetramer of two α- and two β-polypeptide chains, each bound to an iron-containing heme group capable of binding one oxygen molecule. The molecular weight (MW) of hemoglobin is about 64,500, whose subunits are noncovalently associated through van der Waals forces, hydrogen bonds, and salt bridges.

Administering a cell-free suspension of hemoglobin as oxygen carrier has numerous advantages such as a reduced need for compatibility testing, and the ability to sterilize by ultrafiltration or low heat (Squires 2002). However, unmodified cell-free human hemoglobin is not suitable for use as an oxygen-delivering therapeutic agent. First, due to the lack of 2,3-diphosphoglycerate (2,3-DPG), the oxygen affinity of extracellular human hemoglobin is too high to deliver oxygen to tissues effectively (Christa et al. 2013). Then, extracellular hemoglobin is prone to dissociate into α–β dimers in the circulation; high concentrations of these dimers overwhelm the haptoglobin scavenging system and accumulate in the tubules of the kidney, where they are nephrotoxic (Buehler et al. 2010, Chang 2006, Haney et al. 2005, Pimenova et al. 2009). Furthermore, extracellular hemoglobin results in vasoconstriction and hypertension by NO, inhibiting endothelial cell-relaxing factors (Rioux et al. 1995). Finally, extracellular hemoglobin can alter the blood volume and change the colloidal osmotic pressure of the blood.

In order to improve the safety and efficacy of hemoglobin, the tetrameric hemoglobin has been intramolecular cross-linked between the dimeric subunits of the native hemoglobin (Gao et al. 2013). Hence the duration time in the vascular circulation increases and the renal toxicity reduces (Bolin and DeVenuto 1983, Labrude et al. 1986). For example, bis-(3,5-dibromosalicyl)fumarate (DBBF; Chatterjee et al. 1986), a cross-linking reagent, which creates cross-links between the α and β chains to form diaspirin cross-linked hemoglobin (DCLHb), showed an increase in circulation time up to 12 h compared to <6 h for untreated hemoglobin (Squires 2002). In addition, bifunctional cross-linking reagent such as glutaraldehyde has also been used to create polyhemoglobin molecules (Bian et al. 2013, Sehgal et al. 1983). However, it is hard to control polymer MW and consistent quality of the products. In other words, the polymerized products were mixtures of hemoglobin, oligomers of hemoglobin, and polymers of hemoglobin having a spectrum of sizes and MW. If a product in a specific size range was desired, it had to be separated from the bulk product mixture by chromatographic or ultrafiltration methods. The separation of such product mixtures into the various size ranges on a commercial scale is technically difficult, time consuming, and costly.

Although DCLHb and other cross-linked hemoglobins were successful in overcoming most of the shortcomings of cell-free hemoglobin, the full potential of extracellular hemoglobin solutions as an oxygen-delivering therapeutic agent could not be met by tetrameric hemoglobin solutions. A primary drawback of chemically modified tetrameric hemoglobins, such as DCLHb, was the limited duration of the hemoglobin in the circulatory system (Buehler et al. 2010, Haney et al. 2005, Pimenova et al. 2009). In general, larger hemoglobins are retained longer in the vascular system, but those polyhemoglobin molecules that are too large are prone to aggregation and/or precipitation. Therefore, polymerization of DCLHb was directed to obtain a polymerized DCLHb (polyDCLHb) product having hemoglobin molecules with molecular mass in the range of 130–260 kDa.

With this in mind, it is necessary to increase the molecular size of the resulting hemoglobin composition. Further modifications to the DCLHb have been developed to extend the circulatory duration of the hemoglobin. Moreover, whether the molecular mass could be controlled in a proper range is also of great importance for DCLHb to improve the efficiency. In the present study, DCLHb reacted first with bromoacetic acid to block the residual thiol groups of protein chains, and then an amino group on the surface of one DCLHb tetramer was converted into thiol group with 2-iminothiolane hydrochloride (2-IT); therefore, a thiol group was located on the DCLHb, which was called “DCLHb-SH” and could react with cross-linking reagent (Figure 1). Next, the “DCHLb-SH” reacted with 1,6-bismaleimic-hexane (1,6-BMH, Figure 2) so that a specific intermolecular cross-linking could be formed between two converted thiol groups (Hai et al. 1998), aiming at an appropriate increase of molecular mass of hemoglobin (Figure 2). The properties of the cross-linked DCLHb were characterized, and P₅₀ (the O₂ pressure at which Hb is half saturated) of the product was also evaluated. This research explores a novel approach to produce oligomers of DCLHb within a proper MW range by specific cross-linking, hence preventing the formation of high molecular weight (HMW) polymers.

Figure 1. Conversion of amino group of DCLHb to thiol group with 2-IT.

Figure 2. Structure of 1,6-BMH.

Experimental

Reagents and instruments

Human umbilical cord blood was obtained from Tianjin Stem Cell Gene Engineering Company. Maleic anhydride, 1,6-diaminohexane, fumaryl chloride, and 2-IT were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China) and used without further purification. Other chemicals used were of analytical grade, and the solutions were prepared with deionized water.

Spectrophotometry was conducted using SHIMADZU UV-2550, and analytical RP-HPLC was carried out with SHIMADZU CBM-10AVP. Multiangle laser light scattering (MALLS) was performed using Wyatt DAWN HELEOSII. The NMR test was performed by NanoBay 400 MHz (Avance™III HD, Bruker), and oxygen equilibrium measurements of samples were carried out using Hemox analyzer (TCS Scientific, PA)

Methods

1. Hemoglobin – Purified Hb from human placentas was prepared following the methods described by Walder et al. (1994). The final product in the retentates was collected and stored at −80 °C.

2. Synthesis of 1,6-BMH – maleic anhydride (19.6 g, 0.2 mol) was combined with 100 mL of DMF. The mixture was stirred at RT until all the maleic anhydride dissolved in the solvent, then 1,6-diaminohexane (12 g, 0.1 mol) was added in one portion to the solution. The mixture was heated to 90 °C for 0.5 h under N₂ and cooled. To this solution, 3 mg of nickel diacetate, 1 mL of triethylamine, and 40 mL of acetic hydride were added. The mixture was reacted for another 0.5 h at 90 °C under N₂. After cooling, the resulted red–brown solution was poured into 800-mL ice water. The mixture was allowed to stand overnight and a dark brown solid was formed. The solid was collected by filtration and recrystallized twice with ethyl acetate. The final product was dried under vacuum to give 11.3 g (41%) of 1,6-BMH, brown crystals. mp: 139–140 °C. ¹H NMR (CDCl₃, ppm) δ 6.70 (s, 4H), 3.50 (t, 4H), 1.57 (m, 4H), 1.31 (m, 4H).

3. Synthesis of DCLHb – Purified Hb was intramolecularly modified by DBBF to form DCLHb, this preparation was carried out as described earlier (Walder et al. 1979).

4. Preparation of DCLHb-SH and polymerization of DCLHb-SH with 1,6-BMH – 16 mL of unpurified DCLHb solution (15.6 μmol, total content of Hb) was deoxygenated by successive vacuum/nitrogen cycles for 2 h, then cooled to 0 °C. A solution of bromoacetic acid (31.2 μmol) in 0.2-M phosphate-buffered saline (PBS) buffer (2 mL, pH 7.4) was introduced rapidly to the deoxy-DCLHb solution. The reaction mixture was maintained at 0 °C and stirred under nitrogen for 2 h to block the thiol groups of Hb. Then, 5-mg 2-IT was added to the solution and the reaction continued at 4 °C for 3 h. This procedure was carried out in order to convert amino groups into corresponding thiol groups. Next, 1,6-BMH (0.6 molar equivalent of Hb) was added. The solution was stirred at 5 °C under nitrogen for 3 h and the final polymerized product was concentrated to 6.4  ±  0.2 g per dL into sterile glass bottles, and then stored frozen at –20 °C for further characterization.

5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed based on the method of Laemmli (1970).

6. UV-Vis wavelength scanning – UV-Vis full wavelength scanning of the Hb solution; DCLHb-SH solution and the polyDCLHb solution were recorded in 200–700 nm range.

7. Size exclusion chromatography-MALLS (SEC-MALLS).

   Molecular size and polydispersity were determined for each Hb fraction by SEC coupled to MALLS. SEC-MALLS data were acquired and analyzed with Astra version 5.1 software (Wyatt Technology Corp., Santa Barbara, CA, USA). Samples were injected in 25-μL volume and separated on a Superdex^TM 200 Increase 10/300 GL column (300 × 10 mm), eluted in 0.1-M PBS, pH 7.4, at 0.5 mL/min. The sample passed through a Wyatt DAWN HELEOSII laser photometer for MALLS. The detector was operated at a wavelength of 658 nm. All SEC-MALLS measurements were made at ambient temperature.

8. HPLC analysis

   The samples were loaded on a Superdex^TM 200 Increase 10/300 GL column (300 × 10 mm). The columns were equilibrated and eluted with 1M MgCl₂ at the flow rate of 0.5 mL/min and an excitation wavelength of 280 nm. All volumes of the samples were 20 μL.

9. Oxygen affinity

   Oxygen binding curve of native Hb was measured using a Hemox analyzer at 37 °C in PBS, pH 7.4. PolyDCLHb was measured on identical conditions, with 2-IT (2 mmol/L) and cross-linking agent (1 mmol/L) added. Triplicate measurements for each sample were performed to obtain the average value of P₅₀, which was obtained from the oxygen equilibrium curves.

Results and discussion

SDS-PAGE

The SDS-PAGE analysis of purified Hb, DCLHb, DCLHb-SH, and polyDCLHb is shown in Figure 3. Purified Hb migrated as a single band at 16 kDa, while unpurified DCLHb migrated as two primary bands at 16 (non-cross-linked Hb chains) and 32 kDa (Buehler et al. 2006). Finally, polyDCLHb migrated as a more complex mixture of species; there was a new band which indicated that a further intermolecular cross-linking was successful.

Figure 3. SDS-PAGE. (A–D) represent the elution of MW standards, purified Hb, DCLHb, and DCLHb-SH, respectively. E and F are polyDCLHb (cross-linked by 1,6-BMH).

UV-Vis spectrum

Native Hb was used as a contrast. It is evident from Figure 4 that the absorption intensity of Hb before and after modification is not changed obviously. As we know, the absorption peak of heme is in the 415 nm. This demonstrated that there is no change in the spatial conformation around heme. Meanwhile, the absorption peaks of DCLHb-SH and polyDCLHb, which are at 540 nm and 577 nm, respectively (Hirsch et al. 1999), showed a good oxygenation state. This means that the modification of DCLHb has no obvious effects on the function of carrying oxygen.

Figure 4. UV-Vis wavelength scanning of Hb, DCLHb-SH, and polyDCLHb.

Light scattering of studies

The SEC-MALLS spectrum of the solution of polyDCLHb is shown in Figure 5. The MWs of main components and their corresponding peak of time are summarized in Table 1. Among these peaks, Peak 3 (from 20 min to 21.5 min) represents the tetramers of modified Hb. It was notable that there appeared a peak from18 min to 20 min (Peak 2), whose MW was ∼130.7 kDa. This peak indicates that there is a formation of polyDCLHb, which is cross-linked by two modified DCLHbs and one 1,6-BMH, so that the specific polymerization of DCLHb is feasible. Meanwhile, Peak 4 (from 21.5 min to 22.5 min) suggests the dissociation of unintramolecular Hb into dimers.

Figure 5. SEC-MALLS of polyDCLHb (ratio of 2-IT to Hb).

Table 1. MW distribution of polyDCLHb by SEC-MALLS.

Entry	Retain time (min)	MW (g/mol)
Peak 1	15.744–18.004	2.649 × 10^5
Peak 2	18.051–19.913	1.307 × 10^5
Peak 3	20.007–21.483	6.894 × 10^4
Peak 4	21.530–22.536	3.763 × 10^4

HPLC analysis of polymerization

The HPLC spectra of purified Hb, DCLHb, and polyDCLHb are shown in Figure 6. Due to the dissociation of unintramolecular Hb, there appeared only a single peak at about 21.7 min, which indicated the dimers of Hb (Figure 6A). After the cross-linking with DBBF, the MWs of DCLHb were increased, as a result, the corresponding peak at 20.7 min in HPLC demonstrated the existence of DCLHb (Figure 6B). When 1,6-BMH was used to further cross-link the DCLHb, the polymerizaiton gave a product mixture having a HPLC profile consisting of three peaks (Figure 6C). These peaks were identified as “tetrimers” (DCLHb), polyDCLHb, and “HMWs” of DCLHb that eluted in the excluded volume of the column. Among these peaks, the one which was in the 18.7 min corresponded to the polyDCLHb, and from the area of the peak, it could be concluded that polyDCLHb was up to about 20% of the total products (Table 2). This result confirmed that it was feasible to increase and control the MWs of polyDCLHb within a specific range by intermolecular cross-linking.

Figure 6. HPLC of the products. (a) purified Hb, (b) DCLHb, and (c) polyDCLHb mobile phase 1 mol/L MgCl₂.

Table 2. The constituents and corresponding proportion of the chemical modified Hb.

Entry	Retain time (min)	Peak area	Peak area (%)
HMWs	17.484	154,341	7.14
PolyDCLHbs	18.660	416,739	19.29
Tetrimers	20.726	13,203,13	61.11
Dimers	21.704	269,332	12.46

Considering that the dosages of bromoacetic acid and 2-IT had influence on the control of MWs, we carried out a series of experiments in which the quantity of both reagents was varied to optimize the experimental condition. The results are listed in Table 3.

Table 3. The proportion of polyDCLHb in the polymerization of DCLHb with 1,6-BMH with varial quantity of bromoacetic acid and 2-IT.

Entry	Molar ratio of bramoacetic acid to Hb	Molar ratio of 2-IT to Hb	Retain time (min)	Peak area	Peak area (%)
A1	3	1.5	18.708	268,012	14.33
A2	3	2	18.659	298,208	15.15
A3	3	3	18.704	289,441	14.95
B1	2	1.5	18.715	290,138	17.09
B2	2	2	18.713	385,831	18.16
B3	2	3	18.708	374,684	17.92

When 3 molar equivalents of bromoacetic acid were used, the percentage of polyDCLHb in the total products was in the range of 14–15% (Table 3; Entry A1–A3). Nevertheless, using 2 molar equivalents of bromoacetic acid resulted in the increase of polyDCLHb up to the percentage of 17–18% (Table 3; Entry B1–B3). The increase of the desired polyDCLHb with less bromoacetic acid indicated that using more bromoacetic acid was adverse to the formation of polyDCLHb, which might hinder the conversion of amino group to thiol group. Hence the percentage of polyDCLHb decreased. In addition, the results of 3 molar equivalents of 2-IT were not as good as those of 2 molar equivalents of 2-IT. Excess of 2-IT might be expected to convert a small amount of polyDCLHb to “HMWs”, which consequently led to the light percentage decrease of polyDCLHb.

Oxygen affinity

The oxygen affinity (P₅₀) (Benesch et al. 1982) of purified Hb and polyDCLHb are shown in Figures 7 and 8, respectively. Due to the lack of 2,3-DPG, purified Hb showed a high affinity of O₂, whose P₅₀ reduced to a lower value (10.88  mmHg) than native Hb (26  mmHg). However, after the cross-linking, the polyDCLHb showed a P₅₀ value for 28.8 mmHg, which is similar to that of human blood (26 mmHg). The oxygen equilibrium curve indicated that polyDCLHb possessed favorable biological ability, which offers the benefits to carry and release O₂.

Figure 7. Oxygen binding curve of native hemoglobin was measured using a Hemox analyzer at 37 °C in PBS, pH 7.4. Vertical axis is the fraction of hemoglobin sites to which oxygen is bound. The partial oxygen pressure at 50% saturation is expressed in mmHg.

Figure 8. Oxygen binding curve of polyDCLHb was measured using a Hemox analyzer at 37 °C in PBS, pH 7.4 (2-IT 2 mmol/L, cross-linking agent 1 mmol/L).

Conclusion

The present study prepared a cross-linking agent, 1,6-BMH; then DCLHb was modified to produce DCLHb-SH and polymerized by 1,6-BMH to prepare polyDCLHb with proper MWs (∼130.7  kDa). Within this range of MWs, not only could polyDCLHb increase duration in the vascular circulation but also prevent aggregation/precipitation. Experimental data demonstrated the successful preparation of polyDCLHb and indicated its good oxygen affinity. This work put forward a new method to prepare chemical-modified polyDCLHb with proper MWs.

Disclosure statement

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Funding information

This work is supported by The National Science Foundation of China (No. 21202117/B020703) and The National High Technology Research and Development Program (“863” Program) of China (No. 2012AA021903).