Modulation of oxidative stability of haemoglobin inside liposome-encapsulated haemoglobin

Abstract

The major hurdle in the formulation of liposome-encapsulated haemoglobin (LEH) is the oxidation of haemoglobin (Hb) into methaemoglobin during storage and after administration. In order to reduce this oxidative degradation, we tested various reducing conditions in the presence of catalase. We found that at 37°C more than 50% of Hb oxidized to methaemoglobin within 24 h, whereas in presence of catalase, the oxidation was significantly reduced. The effect of catalase was further enhanced by a reduction mixture containing β-NAD, d-glucose, adenine, inosine, MgCl2, KCl, KH2PO4 and Na2HPO4; only 14% methaemoglobin was generated in the presence of catalase and reduction mixture. Contrary to the expectation, glutathione, deferoxamine and homocysteine enhanced Hb oxidation. The presence of CRM inside liposomes (250 nm) significantly decreased Hb oxidation. The results suggest that catalase and a well-defined mixture of co-factors may help control Hb oxidation for improvement in the functional life of LEH.

Figure 1

Effect of temperature and duration of incubation on oxidation of Hb to methaemoglobin (*p < 0.05).

Figure 2

Presence of catalase reduces the amount of methaemoglobin formation: (a) Hb was incubated for 24 h at 37°C with increasing concentrations of bovine catalase. Approximately, 25 000 U/mL was found to be optimum catalase concentration. The percent methaemoglobin is plotted as percent of control where the control is Hb incubated at 37°C for 24 h without any additions. Asterisk in the figure indicates that the difference from control incubation (0 U/mL) is significant (p < 0.05) and (b) temperature dependence of catalase activity in controlling Hb oxidation. In all incubations, 24 000 units of catalase was used per millilitre volume. Asterisk indicates significance (p < 0.05) with respect to respective control without catalase.

Figure 3

Effect of reductant and catalase (25 000 U/mL) combination on Hb oxidation for 24 h at 37°C: (a) glutathione (GSH); (b) deferoxamine (DFM); and (c) homocysteine (HC). The reduction in methaemoglobin formation by these reducing agents was because of the remarkable precipitation and denaturation of Hb found in the samples marked ρ. Asterisk indicates significance (p < 0.05) with respect to respective control without catalase.

Figure 4

Addition of RM to the Hb solution decreases methaemoglobin formation at 37°C for 24 h. Asterisk in the figure indicates that the difference from control incubation is significant (p < 0.05).

Figure 5

Catalase (Cat) and RM synergistically decrease Hb oxidation over 24 h at 37°C. Asterisk in the figure indicates that the difference from control incubation is significant (p < 0.05).

Figure 6

(a–f) Spectra of Hb extracted from LEH incubated at various temperatures for 24 h. The spectrum of sample without CRM kept at 4°C was similar to that shown for the sample with CRM at 4°C, implying no oxidative degradation. However, the spectrum at 37°C is markedly altered as compared to that at 4°C. The presence of CRM subdues Hb oxidation, as is demonstrated by the relatively unaltered spectrum of 37°C + CRM sample. The results are representative of two separate LEH preparations each run in duplicates.

Figure 7

(a) The absorbance values of variously treated Hb at isobestic point (563 nm from Figure 6) were used to calculate %MetHb in Hb extracted from LEH (*p < 0.05); (b) the protective effect of CRM on Hb oxidation is visualized by reduced darkening of Hb in CRM-stabilized LEH (+CRM) as compared to the LEH samples without CRM (−CRM). The LEH samples incubated at 37°C for 24 h are shown; (c) characteristics of LEH preparation carrying co-encapsulated CRM; (d) a transmission electron micrograph of LEH preparation is shown; and (e) A DiO-stained confocal image of an LEH particle. The DiO-stained LEH was prepared at a larger size because the resolution of confocal microscopy does not permit elucidation of structural details of nanometre-sized liposomes.