Synthesis of Nanoparticles Fully Made of Hemoglobin with Antioxidant Properties: A Step toward the Creation of Successful Oxygen Carriers

Abstract

Transfusion of donor red blood cells (RBCs) is a crucial and widely employed clinical procedure. However, important constraints of blood transfusions include the limited availability of blood, the need for typing and cross-matching due to the RBC membrane antigens, the limited storage lifetime, or the risk for disease transmission. Hence, a lot of effort has been devoted to develop RBC substitutes, which are free from the limitations of donor blood. Despite the potential, the creation of hemoglobin (Hb)-based oxygen carriers is still facing important challenges. To allow for proper tissue oxygenation, it is essential to develop carriers with high Hb loading since Hb comprises about 96% of the RBCs’ dry weight. In this work, nanoparticles (NPs) fully made of Hb are prepared by the desolvation precipitation method. Several parameters are screened (i.e., Hb concentration, desolvation ratio, time, and sonication intensity) to finally obtain Hb-NPs with a diameter of ∼568 nm and a polydispersity index (PDI) of 0.2. A polydopamine (PDA) coating is adsorbed to prevent the disintegration of the resulting Hb/PDA-NPs. Due to the antioxidant character of PDA, the Hb/PDA-NPs are able to deplete two harmful reactive oxygen species, namely, the superoxide radical anion and hydrogen peroxide. Such antioxidant protection also translates into minimizing the oxidation of the entrapped Hb to nonfunctional methemoglobin (metHb). This is a crucial aspect since metHb conversion also results in inflammatory reactions and dysregulated vascular tone. Finally, yet importantly, the reported Hb/PDA-NPs are also both hemo- and biocompatible and preserve the reversible oxygen-binding and releasing properties of Hb.

Metal-Phenolic Networks as Broad-Spectrum Antioxidant Coatings for Hemoglobin Nanoparticles Working as Oxygen Carriers

“…For this, the Palmer group employed glutaraldehyde and oxidized dextran to stabilize the NPs through protein cross-linking, while in our group we coated the resulting Hb-NPs with PDA through self-polymerization of dopamine. 24,36 While covalent modification of Hb with crosslinkers will inevitably alter Hb conformation that will, in turn, affect its ability to bind and release oxygen, PDA coatings also come with important limitations. These include an uncontrollable degradation behavior in physiological conditions or potential neurotoxicity issues.…”

“…Hb-NPs were first formed by desolvation precipitation following our previously reported protocol. 24 After addition of EtOH, which was used as the antisolvent to create Hb-NPs, the MPN coating was deposited by simply adding TA and a Fe 3+ solution to the suspension. The color of the suspension immediately turned dark red, indicating that the MPN coating to render MPN@Hb-NPs had been formed instantaneously (Figure 1A).…”

“…•− scavenging properties for trolox, MPN@Hb-NPs, and for free Hb that oxidizes into metHb in the presence of ROS. 24 However, upon increasing the concentration, the results were different and both MPN@ Hb-NPs and trolox promoted a more pronounced decrease in nAbs than free Hb. These results were more evident for the highest studied concentration (i.e., 1 mg mL −1 ) where MPN@ Hb-NPs and trolox promoted a ∼77 and ∼100% decrease in nAbs, respectively (as compared to only a ∼55% decrease for free Hb).…”

“…First, Hb-NPs were fabricated following our previous protocol. 24 In brief, EtOH absolute (3 mL, 4 °C) was added dropwise to a Hb solution in MQ (2 mL, 15.0 mg mL −1 , 4 °C), placed in an ice bath and under continuous stirring at 420 rpm using a magnetic stirring apparatus. The resulting turbid suspension (980 μL) was transferred to a 1.5 mL Eppendorf tube and a TA solution (10 μL, 40 mg mL −1 in MQ) was rapidly added and vortexed for 5 s. Next, FeCl 3 •6H 2 O (10 μL, 2.5 mg mL −1 in MQ) was rapidly added and the turbid suspension was vortexed by an additional 5 s. The resulting dark red turbid suspension was spun down (3000g, 5 min, 4 °C) and washed in MQ (3×, 3000g, 5 min, 4 °C) using a bench-top centrifuge (Eppendorf, centrifuge 5804R, Hamburg, Germany).…”

“…18,22,23 We recently reported a promising approach to create antioxidant HBOCs with a high Hb content by the fabrication of small solid nanoparticles (NPs) fully made of Hb (Hb-NPs). 24 We employed the desolvation precipitation method and screened several process parameters (i.e., the Hb concentration, ethanol (EtOH) to water phase ratio, sonication time, and amplitude) to obtain Hb-NPs with a size of ∼570 nm and a PDI of 0.2. Additionally, to stabilize the as-prepared Hb-NPs, a polydopamine (PDA) coating was deposited.…”