Handbook of Immunological Properties of Engineered Nanomaterials

The following sections are included:

• Contents

Clear benefits of using engineered nanoparticles for biomedical applications are often challenged by concerns about the safety of these materials. Since the main job of the immune system is to efficiently detect and eliminate foreign materials from the body, nanoparticle effects on and interaction with various components of the immune system are active areas of research in current bionanotechnology and nanomedicine. Nanoparticles can be engineered to either avoid immune recognition or to specifically interact with the immune system. Below, we will provide a top level overview of the current state of science in the area of nanoimmunotoxicology, highlight common challenges associated with research of immunological reactivity of engineered nanomaterials, identify current gaps in our understanding of nanoparticle interaction with components of the immune system, and introduce other chapters in this book.

Physicochemical characterization seeks to define the physical and chemical properties, composition, identification, quality, purity, and stability of the material. Since so many parameters influence nanoparticle immunological properties, thorough characterization is essential to draw meaningful conclusions. This chapter will discuss the parameters that need to be monitored and why, and the means of doing so with the appropriate instrumentation, with emphasis on dynamic light scattering and zeta potential measurements.

Nanotherapeutics (NTs) are complex systems with multiple components, each of which could be susceptible to the damaging effects of the sterilization procedure in a different way. Sterilization by autoclaving could result in heat-induced chemical and physical changes in NTs and sterilization by gamma irradiation could induce free radical formation, which could result in immediate chemical changes as well as impact the stability of the product. A suitable battery of tests needs to be utilized during developmental studies to understand the impact of sterilization. The panel of analytical methods must be appropriate for monitoring the physical and chemical changes to NTs. Due to the impact of sterilization, it is necessary to perform all characterization studies after sterilization procedures and to establish a stability program suitable for the detection of chemical degradants during the shelf-life of NTs.

Endotoxin is a common contaminant in engineered nanomaterial formulations and can confound the results of efficacy and toxicity studies due to its high immunostimulatory potential. There is a growing need for methods to reliably detect, quantify, and remove endotoxin from nanoformulations prior to biological studies. This chapter provides a general overview of the endotoxin structure and its mechanism of immune cell activation, review methods traditionally used for endotoxin detection and quantification, and the applicability of these methods to engineered nanomaterial formulations. We also highlight challenges associated with endotoxin detection and quantification in nanomaterial formulations, and provide practical suggestions for overcoming these challenges.

Surface characteristics of nanomaterials are well recognized to contribute prominently to their unique properties and applications. It is less appreciated that the same surfaces have high intrinsic reactivities, typically distinct from identical bulk material chemistries. These size-dependent properties facilitate rapid, ubiquitous surface adsorption and contamination from many sources: storage containers; nanosynthesis components, by-products, and stabilizers; ubiquitous and adventitious environmental pollutants in clean room air and water; and diverse biomolecules that nanomaterials might encounter throughout their life cycle in biological, biomedical, and environmental exposures. Given their extremely large specific surface areas and high surface reactivities, nanoparticle surfaces are dynamic and rarely (if ever) the pure nanomaterial. Instead, such surfaces comprise complex mixtures of poorly characterized and controlled adsorbates, depending on the nanomaterial exposure history. Many adsorbates bring their own biological and toxicological activities. Significantly, surface adsorption may alter otherwise benign or innocuous adsorbates to render certain antigenic or inflammatory responses on a nanomaterial surface. To understand if such a scenario truly exists and the possible risks involved, rigorous nanomaterial surface assessments combined with careful immune system activation studies are necessary. However, the appropriate analytical tools to readily ascertain such interactions and mechanisms remain lacking.

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.

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 the current literature, outlines practical approaches to study nanoparticle interaction with erythrocytes, describes challenges, and suggests solutions to overcome them.

Disclaimer:

The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. The conclusions and opinions expressed in this article are those of the authors and do not constitute FDA policy, and the mention of any products and/or manufacturers does not imply FDA endorsement.

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 briefl y 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.

Disclaimer:

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.

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.

Disclaimer:

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.

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 briefl y 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.

Disclaimer:

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.

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.

Nanoparticle (NP) uptake by the mononuclear phagocytic system (MPS) has been described through several in vitro and in vivo studies, and is a field of research that is constantly evolving and continuing to grow. While many NP agents contain traditional small molecules that have been used therapeutically for decades, the unique delivery system of NPs allows for greater exposure and efficacy of the active chemical entity in the body. Once an NP enters the body, they encounter a much different host response than that observed with small molecule administration. The network of opsonins and circulating and tissue phagocytes is very complex, and their interaction with NPs often depends on the biological atmosphere as well as the physicochemical properties of the NP. This chapter will discuss NP uptake by the MPS and the resulting clinical manifestations. More specifically, key concepts will include differences in NP physicochemical properties, cell lines and/or animal models used, and the bidirectional interaction between the MPS and NPs in patients.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that induce the primary immune response and are responsible for the activation of T and B cells. The main function of DCs is to serve as a link between the innate and adaptive immunity. Understanding the role of DCs in controlling the immune system can provide powerful tools for the successful manipulation of immune cells to optimize vaccine delivery and to expand possibilities of immunotherapeutic approaches. Nanoparticle (NP) applications for targeted delivery of antigens to the DCs is a promising strategy for vaccine development. Herein, I present an overview of different NPs and approaches of their delivery to DCs, review the interaction between NPs and DCs, NP effects on the maturation, activation, and function of DCs, and outline future directions for NPenhanced immune control to further foster the development of successful vaccination strategies and cancer immunotherapies.

This chapter reviews the observations made during the last decade in the area of nanoparticle effects on bone marrow cells such as nanoparticle biodistribution in the host including the bone marrow, nanoparticle-assisted imaging of the bone marrow, immunomodulatory effects, toxicity to bone marrow cells, and the use of nanoparticles for the radioprotection of the bone marrow. Depending on the nature of nanoparticles and their surface modifications, their biodistribution can be controlled to some extent, allowing the increase or decrease of their uptake into the bone marrow for the intended application. Magnetic resonance imaging with magnetic iron oxide nanoparticles has been gaining momentum with studies being conducted both in patients and animal models. Nanoparticles have proven to be useful in helping to induce the suppression or stimulation of the bone marrow as well as avoid the immunomodulatory effects of certain drugs altogether. The delivery of radioprotectors to the bone marrow by nanoparticles helps to enhance their radioprotective effect. Since the application of nanomaterials in biomedical research and medicine is a relatively new field, the most thorough evaluation of their potential toxicity, especially to the bone marrow, is paramount. While biodegradable nanoparticles in general lack bone marrow toxicity, metal and metal oxide nanoparticles vary in their effects on the bone marrow depending on the dose and route of administration, with TiO₂ particles having the highest cause for concern. Hence, research in this area should continue for any newly introduced nanomaterial.

Interest in nanoparticles and their use as vaccine carriers and adjuvants has greatly increased in recent times. However, despite current intense research in this field, the ways in which the immune system responds to nanoscale particulates are still being defined. This chapter will review the physical and chemical characteristics of nanoparticles 1–1000 nm in diameter, considering size, shape, surface charge and chemistry, and their effects on the immune system, including drainage to the lymph nodes (LNs), uptake by antigen-presenting cells (APCs) and the triggering of intracellular signalling pathways. We examine how particle size affects nanoparticle uptake by the key innate stimulators of the immune system, i.e., dendritic cells (DCs), and how nanoparticles modulate DCs and the induction of multiple arms of the immune response, including antibody production and CD4 and CD8 T cell responses via conventional and cross-priming pathways. We further discuss how inert nanoparticles, which by themselves may not necessarily promote the significant inflammation usually associated with adjuvants, can nevertheless induce powerful immunity, suggesting nanotechnology has outstanding potential to deliver safe synthetic vaccines against today's major diseases such as cancer and malaria. Biodegradable or biocompatible nanoparticles, such as polymeric particles, chitosan, polystyrene, gold/silver particles and magnetic/metallic particles, are discussed in relation to the induction of immune responses and vaccine formulations. An indepth understanding of how nanoparticles physicochemically modulate the immune system supports the rational development of nanoparticle-based vaccines, as well as safe nanoparticulate drug delivery systems.

The ever-exploding scientific advancements in the field of nanotechnology have enabled its integration into biosciences and medicine. Such interdisciplinary approaches allow researchers to engineer novel tools for diagnosis, therapy, and prognosis of different clinical complications. One of the most successful nano-tools that have emerged in recent years are nano-drug delivery devices. The use of such nano-sized carriers to load therapeutic agents (drugs, vaccines, biomolecules, and enzymes) aid in overcoming their pharmacological and toxicological hurdles through site-specific and controlled drug delivery. This chapter is a comprehensive account on the recent developments in the delivery of anti-inflammatory drugs using nanocarriers. Here, various kinds and types of nanocarriers and their countless inherent and engineered properties which make them ideal for delivering anti-inflammatory drugs, and the special characteristics of inflammation and inflammatory cells and tissues, which make them vulnerable to these nano-tools, will be discussed. The potential active and passive targeting approaches using drug-loaded nanocarriers for various inflammatory disorders are also elaborated.

Nanostructures can interact with biological systems, e.g., the immune system, inducing a response to these foreign elements in different ways, including the induction of phagocytosis and activation or inhibition of the immune cells. However, it can also respond by triggering hypersensitivity or allergy reactions. The process of allergy to nanostructures is still relatively unknown, but the process of pseudoallergy (which involves complement activation and infl ammatory responses) is the one most described. This chapter summarizes the types of hypersensitivity reactions, the most frequent symptoms, several examples of allergy induced by nanomaterials, and the most common methods of diagnosis.

The immune system is highly versatile at recognizing foreign substances and mounting a multi-stage response against them. Antigenicity is a subtype of the immunogenic response characterized by the formation of an antibody specific to the given type of foreign substance, called an antigen. Due to their small size, nanoparticles are not antigenic themselves. However, some of them act as haptens and become antigenic when conjugated to a protein carrier. Antibodies against several types of engineered nanomaterials have been produced by the conjugation of the nanomaterial to a protein carrier and immunization in the presence of strong adjuvants. Conversely, engineered nanomaterials not conjugated to a protein, and those specifically designed to carry therapeutics proteins, were shown to be non-antigenic and moreover, helped to reduce the antigenicity of therapeutic proteins attached to them. However, accidental non-engineered nanomaterials which contaminate clinical formulations of therapeutic proteins are believed to contribute to their antigenicity. This chapter will review the scientific literature regarding the antigenic response to engineered nanomaterials, describe nanoparticle physicochemical properties contributing to antigenicity, with focus on the role of nanoparticle surface coating in antibody generation, and discuss the role of accidental nanoparticles in the antigenicity of therapeutic proteins.

Preclinical immunotoxicity studies are usually conducted to identify potential causes for concern before a new drug or a medical device is used in clinical trials. Traditionally, these studies consist of in vivo standard toxicity studies and specialized in vivo immune function tests. In immunotoxicology, in vitro assays are used to support and further explore the findings of in vivo studies in cases where adverse effects were triggered by the test material. This strategy, established over multiple years of use in the pharmaceutical industry, is also applicable to engineered nanomaterials. However, because nanoparticles are complex and may be expensive and time-consuming to synthesize, they are often only available in extremely limited quantities during early preclinical development. There is a need to rapidly screen small amounts of nanomaterials to identify toxic candidates and understand the physicochemical properties that contribute to toxicity. This allows for the fine-tuning of these properties before the nanomaterial is produced at large scales. A battery of reliable and predictive in vitro tests would meet this need, but would require a firmly established correlation between in vitro and in vivo results. In this chapter, we summarize the current literature on this subject and also share our own experiences with in vitro immunological screening of engineered nanomaterials for toxicity to the immune system.

The last decade has seen an explosion in the use of nanomaterials (NMs). From their increasing use in improving and developing new technologies for industrial purposes, to their potential in medical applications, these materials offer exciting promise to a variety of research fields due in large part to their novel properties, including small size, increased specific surface area, physicochemical properties (such as morphology, surface charge, and chemical makeup), and surface modifications. There is much concern, however, that NM interactions with biological systems can lead to harmful or toxic effects as a result of these novel properties. In particular, the small size of NMs makes them a target for uptake by phagocytic cells of the immune system and subsequent biodistribution into lymphoid tissues such as the spleen, lymph nodes, and bone marrow. Current in vitro screening techniques typically do not correlate well with observed in vivo toxicity. Therefore, evaluating the immunotoxic effects of NMs in vivo is increasingly important, as the use of these materials for industrial, research, and medical applications continues to increase. This chapter aims to discuss how the adverse effects of NMs on the immune system can be evaluated in light of their unique characteristics, to consider various in vivo models by which NM-mediated immune effects can be detected, and to review the immune effects of three different types of NMs with differing primary routes of human exposure.

Nanoscale materials are being developed for use in a wide variety of products including pharmaceuticals that are regulated by the U.S. Food and Drug Administration (FDA). Some of these represent reformulations of existing drugs or slow release degradable systems. A different category are drugs conjugated to durable nanoparticles (NPs), which do not degrade in the body. In all cases, these novel materials are subject to the same rules and regulations concerning drug development that apply to small molecules and therapeutic proteins. All drugs, including NP–drug conjugates, should be evaluated for immunotoxicity during development. This evaluation is governed by the ICH S8 guidance for small molecule drugs or by the ICH S6 guidance for protein drugs. These guidances continue to apply for drugs conjugated to NPs. Under S8, the primary level of evaluation is a weight of evidence review of the data collected in standard toxicology studies. If data suggestive of immunotoxicity are found, further focused studies may be needed. For proteins, the primary issue is drug hypersensitivity and samples should be collected from non-clinical studies to fully evaluate the role of any anti-drug antibodies. Several studies from CDER/FDA laboratories are used to illustrate how the principles described in the guidance documents could be applied in a real-world situation.

Disclaimer:

This report is not an official U.S. FDA guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred.

The following sections are included:

• Index