Handbook of Immunological Properties of Engineered Nanomaterials

The following sections are included:

• Preface
• Contents
• List of Contributors

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.

Nanoparticles (NPs) can offer many advantages over traditional drug design and delivery, as well as toward medical diagnostics. As with any medical device or pharmaceutical drug intended to be used for in vivo biomedical applications, NPs must be sterile. However, very little is known regarding the effect of sterilization methods on the intrinsic properties and stability of NPs. Herein a detailed analysis of physicochemical properties of two types of AuNPs upon sterilization by means of five different techniques is reported. In addition, cell viability and production of reactive oxygen species are studied. The results indicate that sterilization by ethylene oxide seems to be the most appropriate technique for both types of NPs. It is concluded that it is crucial to test several methods in order to establish the specific type of sterilization to be performed for each particular NP.

Silver nanoparticles are commonly used in a variety of commercial and medical products. Here, we investigate the effects of standard sterilization methods, including heat/steam (autoclave) and gamma irradiation on the structural integrity and biocompatibility of citrate-stabilized silver nanoparticles with nominal sizes of 20, 40, 60 and 80 nm. Particle size, shape and in vitro biocompatibility were studied preand post-sterilization. Sterilization by gamma irradiation at dose levels commonly used in medical device industry (15, 25 and 50 kGy) resulted in dramatic changes in particle size and morphology, as monitored by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Exposing the particles to a chemical producer of hydroxyl radicals (N-hydroxy-2-pyridinethione) allowed us to duplicate the sterilization-based changes in size and morphology, implying a free radical mechanism of action. Compared to untreated controls, we also observed a three-to five-fold increase in tendency of sterilized silver nanoparticles to cause platelet aggregation, a sensitive in vitro indicator of thrombogenicity.

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.

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.

Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here we tested the traditional Limulus Amebocyte lysate (LAL) gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of or covered by the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement control (IEC) revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica-surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein–particle interactions alone are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution (MVD) was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.

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.

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 engineered nanomaterials (ENMs). From their increasing use in improving and developing new technologies for industrial use to their potential for use 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, physico-chemical properties (such as morphology, surface charge, and chemical make-up), and surface modifications. There is much concern, however, that ENM interactions with biological systems can result in harmful or toxic effects as a result of these novel properties. In particular, the small size of ENMs 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 ENMs in vivo is increasingly important, as the use of these materials for industrial, research, and medical applications continues to increase. This chapter aims to consider how the adverse effects of ENMs on the immune system can be evaluated in light of their unique characteristics, to consider various in vivo models by which ENM-mediated immune effects can be detected, and to review the immune effects of three different types of ENMs, each with differing primary routes of human exposure.

Preclinical characterization of novel nanotechnology-based formulations is often challenged by physicochemical characteristics, sterility/sterilization issues, safety and efficacy. Such challenges are not unique to nanomedicine, as they are common in the development of small and macromolecular drugs. However, due to the lack of a general consensus on critical characterization parameters, a shortage of harmonized protocols to support testing, and the vast variety of engineered nanomaterials, the translation of nanomedicines into clinic is particularly complex. Understanding the immune compatibility of nano formulations has been identified as one of the important factors in (pre)clinical development and requires reliable in vitro and in vivo immunotoxicity tests. The generally low sensitivity of standard in vivo toxicity tests to immunotoxicities, inter-species variability in the structure and function of the immune system, high costs and relatively low throughput of in vivo tests, and ethical concerns about animal use underscore the need for trustworthy in vitro assays. Here, we consider the correlations(or lack thereof) between in vitro and in vivo immunotoxicity tests as a means to identify useful in vitro assays. We review literature examples and case studies from the experience of the NCI Nanotechnology Characterization Lab, and highlight assays where predictability has been demonstrated for a variety of nanomaterials and assays with high potential for predictability in vivo.

The following sections is included:

• Index