Enzyme-loaded rod-like microgel shapes: a step towards the creation of shape-specific microreactors for blood detoxification purposes.

Rapid removal of toxic substances is crucial to restore the normal functions of our body and ensure survival. Due to their high substrate specificity and catalytic efficiency, enzymes are unique candidates to deplete toxic compounds. While enzymes display several limitations including low stability and high immunogenicity, these can be overcome by entrapping them in a diverse range of carriers. The resulting micro/nanoreactors shield the enzymes from their surroundings, preventing their misfolding or denaturation thus allowing them to conduct their function. The micro/nanoreactors must circulate in the blood stream for extended periods of time to ensure complete depletion of the toxic agents. Surprisingly, while it is widely acknowledged that non-spherical carriers exhibit longer residence time in the bloodstream than their spherical counterparts, so far, all the reported micro/nanoreactors have been assembled with a spherical architecture. Herein, we address this important issue by pioneering the first shape-specific microreactors. We use UV-assisted punching to create rod-like microgel shapes with dimensions of 8 µm × 1 µm × 2 µm and demonstrate their biocompatibility by conducting hemolysis and cell viability assays with a macrophage and an endothelial cell line. Upon encapsulation of the model enzyme ß-lactamase, the successful fabrication of rod-shaped microreactors is demonstrated by their ability to convert the yellow nitrocefin substrate into its hydrolyzed product.

Metal-organic framework-based oxygen carriers with antioxidant activity resulting from the incorporation of gold nanozymes.

Blood transfusions are a life-saving procedure since they can preserve the body's oxygen levels in patients suffering from acute trauma, undergoing surgery, receiving chemotherapy or affected by severe blood disorders. Due to the central role of hemoglobin (Hb) in oxygen transport, so-called Hb-based oxygen carriers (HBOCs) are currently being developed for situations where donor blood is not available. In this context, an important challenge that needs to be addressed is the oxidation of Hb into methemoglobin (metHb), which is unable to bind and release oxygen. While several research groups have considered the incorporation of antioxidant enzymes to create HBOCs with minimal metHb conversion, the use of biological enzymes has important limitations related to their high cost, potential immunogenicity or low stability in vivo. Thus, nanomaterials with enzyme-like properties (i.e., nanozymes (NZs)) have emerged as a promising alternative. Amongst the different NZs, gold (Au)-based metallic nanoparticles are widely used for biomedical applications due to their biocompatibility and multi-enzyme mimicking abilities. Thus, in this work, we incorporate Au-based NZs into a type of HBOC previously reported by our group (i.e., Hb-loaded metal-organic framework (MOF)-based nanocarriers (NCs)) and investigate their antioxidant properties. Specifically, we prepare MOF-NCs loaded with Au-based NZs and demonstrate their ability to catalytically deplete over multiple rounds of two prominent reactive oxygen species (ROS) that exacerbate Hb's autoxidation (i.e., hydrogen peroxide and the superoxide radical). Importantly, following loading with Hb, we show how these ROS-scavenging properties translate into a decrease in metHb content. All in all, these results highlight the potential of NZs to create novel HBOCs with antioxidant protection which may find applications as a blood substitute in the future.

Hemoglobin-based oxygen carriers camouflaged with membranes extracted from red blood cells: Optimization and assessment of functionality.

Despite being an indispensable clinical procedure, the transfusion of donor blood has important limitations including a short shelf-life, limited availability and specific storage requirements. Therefore, a lot of effort has been devoted to developing hemoglobin (Hb)-based oxygen carriers (HBOCs) that are able to replace or complement standard blood transfusions, especially in extreme life-threatening situations. Herein, we employed a Hb-loaded poly(lactide-co-glycolide) core which was subsequently coated with nanozymes to protect the encapsulated Hb from oxidation by reactive oxygen species. To render HBOCs with long circulation in the vasculature, which is a crucial requirement to achieve the high oxygen demands of our organism, the carrier was coated with a red blood cell-derived membrane. Three coating methods were explored and evaluated by their ability to repel the deposition of proteins and minimize their uptake by an endothelial cell line. Preservation of the oxygen carrying capacity of the membrane-coated carrier was demonstrated by an oxygen-binding and releasing assay and, the functionality resulting from the entrapped nanozymes, was shown by means of superoxide radical anion and hydrogen peroxide depletion assays. All in all, we have demonstrated the potential of the membrane-coated nanocarriers as novel oxygen carrying systems with both antioxidant and stealth properties.

Optimization of Hemoglobin Encapsulation within PLGA Nanoparticles and Their Investigation as Potential Oxygen Carriers.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) display the excellent oxygen-carrying properties of red blood cells, while overcoming some of the limitations of donor blood. Various encapsulation platforms have been explored to prepare HBOCs which aim to avoid or minimize the adverse effects caused by the administration of free Hb. Herein, we entrapped Hb within a poly(lactide-co-glycolide) (PLGA) core, prepared by the double emulsion solvent evaporation method. We study the effect of the concentrations of Hb, PLGA, and emulsifier on the size, polydispersity (PDI), loading capacity (LC), and entrapment efficiency (EE) of the resulting Hb-loaded PLGA nanoparticles (HbNPs). Next, the ability of the HbNPs to reversibly bind and release oxygen was thoroughly evaluated. When needed, trehalose, a well-known protein stabilizer that has never been explored for the fabrication of HBOCs, was incorporated to preserve Hb's functionality. The optimized formulation had a size of 344 nm, a PDI of 0.172, a LC of 26.9%, and an EE of 40.7%. The HbNPs were imaged by microscopy and were further characterized by FTIR and CD spectroscopy to assess their chemical composition and structure. Finally, the ability of the encapsulated Hb to bind and release oxygen over several rounds was demonstrated, showing the preservation of its functionality.

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

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.

Hemoglobin-Based Oxygen Carriers Incorporating Nanozymes for the Depletion of Reactive Oxygen Species.

While transfusion of donor blood is a reasonably safe and well-established procedure, artificial oxygen carriers offer several advantages over blood transfusions. These benefits include compatibility with all blood types, thus avoiding the need for cross matching, availability, lack of infection, and long-term storage. Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being explored as an "oxygen bridge" to replace or complement standard blood transfusions in extreme, life-threatening situations such as trauma in remote locations or austere battlefield or when blood is not an option due to compatibility issues or patient refusal due to religious objections. Herein, a novel HBOC was prepared using the layer-by-layer technique. A poly(lactide-co-glycolide) core was fabricated and subsequently decorated with Hb and nanozymes. The Hb was coated with poly(dopamine), and preservation of the protein structure and functionality was demonstrated. Next, cerium oxide nanoparticles were incorporated as nanozymes, and their ability to deplete reactive oxygen species (ROS) was shown. Finally, decorating the nanocarrier surface with poly(ethylene glycol) decreased protein adsorption and cell association/uptake. The as-prepared Hb-based oxygen nanocarriers were shown to be hemo- and bio-compatible. Their catalytic potential was furthermore demonstrated in terms of superoxide radical- and peroxide-scavenging abilities, which were retained over multiple cycles. Overall, these results demonstrate that the reported nanocarriers show potential as novel oxygen delivery systems with prolonged catalytic activity against ROS.

A dual-component carrier with both non-enzymatic and enzymatic antioxidant activity towards ROS depletion.

While ROS display crucial functions in many physiological processes, elevated ROS levels are also related to the initiation and progression of many severe diseases such as cancer, cardiovascular conditions or neurologic disorders. Research approaches to diminish ROS levels during disease progression are currently being focused on the therapeutic administration of antioxidant enzymes. However, enzyme administration suffers from several limitations including their fast elimination from blood upon administration, thus making crucial the development of enzyme encapsulating platforms. We have recently reported a multicompartment architecture constituted by two inherently different types of materials, i.e., polymeric microgels and liposomes. Poly(N-isopropylacrylamide-co-acrylic acid) microgels decorated with liposomes and subsequently coated by a protective poly(dopamine) shell (PDA) combine the benefits of both systems while minimizing some of their drawbacks. Herein, we exploit this dual-component platform as a microreactor for ROS depletion. We combine the intrinsic PDA's antioxidant properties with the encapsulation of the catalase enzyme within the liposomal compartments. The surface of the carrier is further functionalised with a poly(ethylene glycol) layer and the low fouling properties are demonstrated in terms of reduction of protein adsorption and cellular uptake. The potential of the carrier as an antioxidant microreactor is shown by its ability to deplete superoxide radicals and hydrogen peroxide, which can also take place in the presence of the two relevant cell lines.