Handbook of Immunological Properties of Engineered Nanomaterials

The following sections are included:

• Preface
• Contents
• List of Contributors

Whenever nanoparticles (NPs) come into contact with biological fluids, a layer of proteins (“protein corona”) may adsorb onto their surfaces. This corona enshrouds the NPs and largely defines their biological identity. The nature of the corona and the efficiency of its formation can be a decisive factor for the biological response of an organism to NP exposure. Here, we describe methods presently available for determining the composition of the protein corona under physiological conditions and recent findings in this developing field. Mechanistic aspects of protein adsorption to NP surfaces are reviewed in the context of the current literature. Additionally, the effects of protein corona formation on cellular uptake and biodistribution of NPs are discussed. Finally, we comment on the significant role of the protein corona for contemporary nanomedical applications.

Proteins bound to nanoparticle surfaces are known to affect particle clearance by influencing immune cell uptake and distribution to the organs of the mononuclear phagocytic system. The composition of the protein corona has been described for several types of nanomaterials, but the role of the corona in nanoparticle biocompatibility is not well established. In this study, we investigate the role of nanoparticle surface properties (PEGylation) and incubation times on the protein coronas of colloidal gold nanoparticles. While neither incubation time nor PEG molecular weight affected the specific proteins in the protein corona, the total amount of protein binding was governed by the molecular weight of PEG coating. Furthermore, the composition of the protein corona did not correlate with nanoparticle hematocompatibility. Specialized hematological tests should be used to deduce nanoparticle hematotoxicity.

This article discusses the role of the protein corona in delivery systems with tagged nanoparticles and how knowledge of the protein corona can help in optimizing delivery. The basic question is whether and how the binding of proteins and other biomolecules at the nanoparticle surface interfere with the interaction between a tag and its receptor. This is an interesting problem in many respects, but most intriguing are the observed differences in delivery efficiency in vivo compared with protein-free in vitro conditions. In order to understand possible situations that the nanoparticle will face in a protein-rich biological environment, we will first describe the formation of a protein corona and thereafter discuss potential perturbations of the delivery systems when moving from in vitro testing to in vivo applications. We emphasize the role of mathematical modeling in optimizing the design of functionalized nanoparticles to achieve high success of delivery.

Both engineered and naturally occurring nanomaterials (NMs) may come in contact with blood through different pathways, either directly (e.g., as nanoparticles carrying chemotherapy agents injected intravenously, imbedded in a coating on a vascular graft, or as wear debris released from an orthopedic device) or indirectly (e.g., through inhalation, oral, or topical exposure routes). Due to the ubiquitous importance of red blood cells in sustaining the different tissues of the body, in this chapter we review the possible adverse effects of NMs on erythrocytes and the experimental techniques which have been applied to evaluate and better understand their mechanisms of interaction. This chapter provides a comprehensive overview of current literature, outlines practical approaches to study nanoparticle interactions with erythrocytes, describes challenges and suggests solutions to overcome them.

The vascular endothelium forms the inner lining of the vasculature and is an essential system in multicellular organisms that delivers nutrients and oxygen, removes CO₂ and waste products, and facilitates interorgan communication. Endothelial dysfunctions may lead to life-threatening vascular disorders, including hypoperfusion and organ damage, and thrombotic and/or bleeding pathologies. As various engineered nanomaterials are being designed for biomedical applications for intravascular use and other nanomaterials may reach the vasculature as a result of occupational, environmental, or other types of exposure, it is necessary to evaluate the effects of engineered nanomaterials on the vascular endothelium. In the introduction of this chapter, the physiology of endothelial cells (ECs) and the pathophysiology of the possible adverse effects of nanomaterials on ECs are briefly described. Next, the methods that are used for the in vitro evaluation of the effects of nanomaterials on ECs are reviewed. Investigations of the effects of nanomaterials on ECs are primarily based on the observation of structural and functional changes in the different types of cultured ECs upon (usually) short-term exposure (up to 72 h) to various doses of nanomaterials. Numerous microscopic methods are employed to study morphological and structural changes of ECs and nanomaterial uptake. Different cell biology assays are used to assess the cytotoxicity of nanomaterials and their effects on proliferation and induction of apoptosis, necrosis, and autophagy. In addition, assays of various EC activation markers, nitric oxide (NO) and reactive oxygen species (ROS) production or functional EC mobility and morphogenesis, monolayer permeability, and vasoactivity assays also provide valuable information on nanomaterial effects on the vascular intima. Lastly, a summary of the published results regarding the in vitro effects of various nanomaterials on ECs, as well as selected assay protocols used in our laboratory are presented.

The plasma coagulation system (PCS) consists of plasma proteins and other factors that, together with platelets and vascular endothelial cells, maintain hemocoagulation balance. Under physiological conditions, these systems prevent blood clotting and, in cases of vascular injury, facilitate hemostasis to prevent blood loss. The dysregulation of hemocoagulation may lead to life-threatening thrombotic and/or bleeding pathologies. Since 1) various engineered nanomaterials are being designed for biomedical applications that will come into contact with the blood and 2) other nanomaterials may reach the circulation as a result of occupational, environmental, or other types of exposure, it is necessary to evaluate the effects of engineered nanomaterials on the PCS. In the introduction of this chapter, the components and mechanisms of the kallikrein–kinin system (KKS), the PCS, fibrinolysis, and the pathophysiology of the possible adverse effects of nanomaterials on these systems are briefly described. Next, the methods that are used for the in vitro evaluation of the effects of nanomaterials on the KKS, PCS, and fibrinolysis are reviewed. Screenings for the effects of nanomaterials are primarily based on their preincubation with citrated platelet-poor plasma (PPP). The PPP is subsequently subjected to clotting tests, including measurements of the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and recalcification time. In certain studies, plasmas that are deficient in specific plasma coagulation factors are used for comparison purposes. The activities of certain specific coagulation factors can be assayed photometrically using chromogenic or fluorogenic substrates, while antigens can be detected using ELISAs. Thromboelastography is another technique that is used for the complex analysis of the effects of nanomaterials on blood clotting and fibrinolysis. Gravimetric in vitro thrombolytic assays have also been used to this end. Lastly, a summary of the published results regarding the in vitro effects of various nanomaterials on the KKS, the PCS, and fibrinolysis is presented.

Platelets (PLTs) are small discoidal cells that are anucleated in mammals and circulate in the blood, where they play a critical role in hemostasis and thrombosis. PLT adhesion at the site of vascular injury and their activation and aggregation result in the formation of the primary hemostatic plug. This plug is enforced by a fibrin network structure as a result of the activation of the plasma coagulation system (PCS). Thus, PLTs, together with the PCS and vascular endothelial cells, maintain the hemocoagulation balance. Under physiological conditions, these systems prevent blood clotting, while in cases of vascular injury, they facilitate hemostasis to prevent blood loss. The dysregulation of hemocoagulation can lead to life-threatening thrombotic and/or bleeding pathologies. As various engineered nanomaterials that will come into contact with the blood are being designed for biomedical applications and other nanomaterials may reach the circulation as a result of occupational, environmental, or other routes of exposure, it is necessary to evaluate the effects of engineered nanomaterials on PLTs. In the introduction of this chapter, both the physiology of PLTs and the pathophysiology of the possible adverse effects of nanomaterials on PLTs are briefly described. Next, the methods that are used for the in vitro evaluation of the effects of nanomaterials on PLTs are reviewed. Screenings for the effects of nanomaterials on PLTs are generally based on PLT aggregation assays. These tests include cell counter-based assays, light transmission aggregometry, and impedance aggregometry. The adhesion of PLTs to nanomaterial surfaces is frequently monitored by scanning electron microscopy or light microscopy. Ultrastructural changes are examined using transmission electron microscopy. PLT membrane activation markers (CD62P, CD63, PAC-1) and the release of PLT membrane microparticles are analyzed by flow cytometry or via immunomicroscopic techniques. Soluble activation markers that are released by PLTs (e.g., βTG, PF4, and TSP-1) are analyzed using ELISA. PLT integrity is assayed using a colorimetric lactate dehydrogenase (LDH) assay in the PLT supernatant. Other techniques are used to investigate the effects of nanomaterials on specific signaling pathways. Lastly, a summary of the published results regarding the in vitro effects of various nanomaterials on PLTs is presented.

Nanoparticles are increasingly being used in industry, biology, and medicine, but the benefits of using nanotechnology for industrial and biomedical applications are tempered by concerns about the safety of these new materials. Nanoparticles can be immunotoxic, however, no novel (i.e., specific to nano) immunotoxicity has been described. Therefore, it is our working assumption that the standard panel of immunotoxicity methods and parameters used to understand the safety of drugs and medical devices is also applicable to engineered nanomaterials, but that these methods may require modification and optimization to account for differences in nanoparticle distribution, route of entry, and interference. While nanoparticle interaction with various constituents of the immune system has been reviewed before, little attention was given to nanoparticle effects on the blood coagulation system. Herein, we review recent advances in our understanding of nanoparticle interactions with plasma coagulation factors, platelets, endothelial cells and leukocytes. We will discuss studies demonstrating how nanoparticle physicochemical properties (e.g., size, charge, hydrophobicity) can be engineered to achieve desirable effects on the coagulation system and to avoid undesirable toxicities.

The complement system is the most important biochemical cascade in the blood for the recognition, opsonization, and elimination of foreign materials. To date, the leading causes of death in the United States include cancer, cardiovascular and neurodegenerative diseases, and diabetes. New treatments are urgently needed to treat these devastating diseases and nanotechnology potentially provides new avenues to fight such illnesses. These avenues include the development of novel nanocarriers that deliver drugs in a specific and controlled manner, while minimizing secondary effects. The success of bioengineering effective nanocarriers for drug delivery purposes requires a deep understanding of the interaction between the complement system and the nanocarriers. This review focuses on reporting the current state of complement activation by different nanomaterials. Here, we assess various important parameters that influence the activation of the complement system, which include the physicochemical characteristics of both nanocarriers and complement proteins. We next evaluate the most recent engineering approaches to prevent or reduce complement activation. Finally, we discuss different in vitro and in vivo procedures to assess complement activation.

PEGylated liposomal doxorubicin (Doxil^®) is the first FDA-approved nanomedicine (1995); it has been successfully used for cancer therapy for nearly 20 years. Despite its “stealth” properties, Doxil activates the complement system, which leads to a hypersensitivity reaction (HSR) known as Complement Activation Related Pseudoallergy (CARPA). Herein we provide a comprehensive overview of both in vitro and in vivo aspects of this phenomenon, and review recent clinical experience with Doxil and its generic formulations. We discuss the complexity of the mechanism of CARPA, which, in addition to the engagement of different complement activation pathways, may include triggering of mast cells, macrophages and other allergy-mediating secretory cells, leading to the liberation of multiple vasoactive mediators and a variety of effects on the cardiovascular and other organs. We describe furthermore the approaches of inhibiting Doxil-induced CARPA in vivo in experimental animals and humans. The case study of Doxil is likely applicable to many other reactogenic nanomedicines that share common toxicities related to complement activation and for which CARPA represents an immune barrier to successful clinical use.

Introduction: The lymphatic system has a critical role in the immune system's recognition and response to disease and it is an additional circulatory system throughout the entire body. Extensive multidisciplinary investigations have been carried out in the area of lymphatic delivery, and lymphatic targeting has attracted a lot of attention for providing preferential chemotherapy and improving bioavailability of drugs that undergo hepatic first-pass metabolism.

Areas covered: This review focuses on progress in the field of lymphatic therapeutics and diagnosis. Moreover, the anatomy and physiology of the lymphatic system, particulate drug carriers and different physicochemical parameters of both modified and unmodified particulate drug carriers and their effect on lymphatic targeting are addressed.

Expert opinion: Particulate drug carriers have encouraged lymphatic targeting, but there are still challenges in targeting drugs and bioactives to specific sites, maintaining desired action and crossing all the physiological barriers. Lymphatic therapy using drug-encapsulated lipid carriers, especially liposomes and solid lipid nanoparticles, emerges as a new technology to provide better penetration into the lymphatics where residual disease exists. Size is the most important criteria when designing nanocarriers for targeting lymphatic vessels as the transportation of these particles into lymphatic vessels is size dependent. By increasing our understanding of lymphatic transport and uptake, and the role of lymphatics in various diseases, we can design new therapeutics for effective disease control.

The following sections is included:

• Index