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Abstract
Oxygen (O2) is indispensable for our survival, as our cells and tissues need a constant O2 supply to carry out their basic metabolic activities. Hemoglobin (Hb), the main component of red blood cells (RBCs), is the molecule responsible for transporting ~98% of the O2 in our body, playing a crucial role in maintaining our homeostasis. Thus, any condition related to blood loss or the impairment of Hb’s function may result in devastating consequences. While there are multiple medical situations that require blood, blood transfusion is the only widely used and well-established clinical procedure that alleviates this necessity. Despite being a life-saving procedure, blood transfusions present important drawbacks such as limited availability, the need for typing and crossmatching, and short storage lifetime. In order to find alternatives that overcome these limitations, research efforts have been dedicated towards developing Hb-based O2 carriers (HBOCs). Although prominent advances have been achieved in the past decades, there are still shortcomings that need to be addressed, including the development of HBOCs encapsulating high concentrations of Hb while maintaining its functionality.
This PhD thesis addresses these challenges and presents HBOCs, not only with high Hb content, but also with antioxidant properties to minimize the oxidation of Hb into nonfunctional methemoglobin. In addition, two different strategies were explored to confer stealth properties on the HBOCs. In parallel, to solve inconsistencies experienced in the characterization of the different HBOCs and ensure the accurate quantification of Hb, multiple quantification methods were assessed and compared.
First, polymeric, and metal-organic framework-based nanoparticles (NPs) were investigated as encapsulation platforms. Optimizing the parameters for the polymeric poly(lactic-co-glycolic acid)-based NPs resulted in a loading capacity of 27% and an entrapment efficiency (EE) of 40%. In contrast, the optimization of the zeolitic imidazolate framework (ZIF)-8 NPs resulted in a DL as high as 99% and an EE of 85%. Specifically, a formulation containing poly(ethylene glycol) (PEG), named Hb40PEG200@ZIF-8 NPs, contained 34 mg mL-1 Hb, which was 7.3 times more than any previously reported Hb-loaded ZIF-8 (Hb@ZIF-8) NPs. Furthermore, the entrapped Hb showed outstanding retention of its O2 carrying ability, preserving its functionality in a 95%. Thus, to explore different antioxidant and stealth coatings, only the Hb40PEG200@ZIF-8 NPs were considered.
The surface modification of the Hb40PEG200@ZIF-8 NPs with a metal-phenolic network (MPN) coating clearly enhanced the stability of the NPs in the most common buffers (e.g., PBS) and cell media, expanding their potential biomedical applications. In addition, the MPN coating conferred antioxidant properties to the Hb40PEG200@ZIF-8 NPs, as demonstrated by their ability to scavenge a nitric oxide reactive specie (i.e., DPPH•).
While native RBCs have a lifespan of 120 days, the circulation half-life of free Hb is only a few hours. Therefore, when developing HBOCs, it is very important to consider strategies that extend their circulation time. Consequently, to provide the MPN-coated Hb@ZIF-8 NPs with stealth properties, PEGylation was performed, as PEG is considered the gold standard antifouling polymer. The PEGylated NPs presented improved resuspension but also reduced immunoglobulin G and increased bovine serum albumin adsorption, potentially leading to extended circulation times. Alternatively, a human serum albumin (HSA) coating was explored as a non-immunogenic option. Both strategies resulted in NPs in which the entrapped Hb was able to reversibly bind and release O2, showing promising biocompatibility as demonstrated by cell viability studies.
Finally, to further assess the functionality of the as-synthesized HBOCs in a microenvironment that better mimics the physiological conditions, the Hb40PEG200@ZIF-8 NPs with different surface coatings were assessed by perfusion in vessel-on-chip models. Their biocompatibility with human induced pluripotent stem cell-derived endothelial cells and their ability to release O2 in hypoxic conditions was evaluated, highlighting the potential use of HSA-coated Hb@ZIF-8 NPs as HBOCs.
All in all, this thesis presents various multifaceted HBOCs, contributing to moving forward a new generation of HBOCs that more closely mimic our RBCs and aim to restore tissue oxygenation in emergency situations.
This PhD thesis addresses these challenges and presents HBOCs, not only with high Hb content, but also with antioxidant properties to minimize the oxidation of Hb into nonfunctional methemoglobin. In addition, two different strategies were explored to confer stealth properties on the HBOCs. In parallel, to solve inconsistencies experienced in the characterization of the different HBOCs and ensure the accurate quantification of Hb, multiple quantification methods were assessed and compared.
First, polymeric, and metal-organic framework-based nanoparticles (NPs) were investigated as encapsulation platforms. Optimizing the parameters for the polymeric poly(lactic-co-glycolic acid)-based NPs resulted in a loading capacity of 27% and an entrapment efficiency (EE) of 40%. In contrast, the optimization of the zeolitic imidazolate framework (ZIF)-8 NPs resulted in a DL as high as 99% and an EE of 85%. Specifically, a formulation containing poly(ethylene glycol) (PEG), named Hb40PEG200@ZIF-8 NPs, contained 34 mg mL-1 Hb, which was 7.3 times more than any previously reported Hb-loaded ZIF-8 (Hb@ZIF-8) NPs. Furthermore, the entrapped Hb showed outstanding retention of its O2 carrying ability, preserving its functionality in a 95%. Thus, to explore different antioxidant and stealth coatings, only the Hb40PEG200@ZIF-8 NPs were considered.
The surface modification of the Hb40PEG200@ZIF-8 NPs with a metal-phenolic network (MPN) coating clearly enhanced the stability of the NPs in the most common buffers (e.g., PBS) and cell media, expanding their potential biomedical applications. In addition, the MPN coating conferred antioxidant properties to the Hb40PEG200@ZIF-8 NPs, as demonstrated by their ability to scavenge a nitric oxide reactive specie (i.e., DPPH•).
While native RBCs have a lifespan of 120 days, the circulation half-life of free Hb is only a few hours. Therefore, when developing HBOCs, it is very important to consider strategies that extend their circulation time. Consequently, to provide the MPN-coated Hb@ZIF-8 NPs with stealth properties, PEGylation was performed, as PEG is considered the gold standard antifouling polymer. The PEGylated NPs presented improved resuspension but also reduced immunoglobulin G and increased bovine serum albumin adsorption, potentially leading to extended circulation times. Alternatively, a human serum albumin (HSA) coating was explored as a non-immunogenic option. Both strategies resulted in NPs in which the entrapped Hb was able to reversibly bind and release O2, showing promising biocompatibility as demonstrated by cell viability studies.
Finally, to further assess the functionality of the as-synthesized HBOCs in a microenvironment that better mimics the physiological conditions, the Hb40PEG200@ZIF-8 NPs with different surface coatings were assessed by perfusion in vessel-on-chip models. Their biocompatibility with human induced pluripotent stem cell-derived endothelial cells and their ability to release O2 in hypoxic conditions was evaluated, highlighting the potential use of HSA-coated Hb@ZIF-8 NPs as HBOCs.
All in all, this thesis presents various multifaceted HBOCs, contributing to moving forward a new generation of HBOCs that more closely mimic our RBCs and aim to restore tissue oxygenation in emergency situations.
Original language | English |
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Publisher | DTU Health Technology |
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Number of pages | 277 |
Publication status | Published - 2024 |
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Dive into the research topics of 'Towards the development of multifaceted oxygen carriers encapsulating high concentrations of fully functional hemoglobin'. Together they form a unique fingerprint.Projects
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Biomaterials-based approach for the creation of artificial red blood cells
Satué, C. C. (PhD Student), Hosta Rigau, L. (Main Supervisor), Lind, J. U. (Supervisor), Bülow, L. (Examiner) & Jørgensen, L. (Examiner)
01/12/2020 → 15/07/2024
Project: PhD