Nano Today
Volume 10, Issue 4, August 2015, Pages 487-510
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Review
Nanoparticle uptake: The phagocyte problem

https://doi.org/10.1016/j.nantod.2015.06.006Get rights and content

Highlights

  • Diverse populations of different macrophages very effectively clear nanomaterials using specific and non-specific uptake mechanisms.

  • Nanomaterials are very efficiently scavenged from circulating blood and tissues by macrophages resident in tissue and filtration organs (MPS system), severely limiting particle targeting.

  • To avoid rapid uptake, clearance and to improve targeting, nanomaterials that avoid both specific and non-specific macrophage recognition must be developed to improve intra- and extra-cellular targeting.

  • Long-term residence of non-degradable systems within macrophages in clearance organs poses a unique challenge and could initiate inflammatory mechanisms.

Summary

Phagocytes are key cellular participants determining important aspects of host exposure to nanomaterials, initiating clearance, biodistribution and the tenuous balance between host tolerance and adverse nanotoxicity. Macrophages in particular are believed to be among the first and primary cell types that process nanoparticles, mediating host inflammatory and immunological biological responses. These processes occur ubiquitously throughout tissues where nanomaterials are present, including the host mononuclear phagocytic system (MPS) residents in dedicated host filtration organs (i.e., liver, kidney spleen and lung). Thus, to understand nanomaterials exposure risks it is critical to understand how nanomaterials are recognized, internalized, trafficked and distributed within diverse types of host macrophages and how possible cell-based reactions resulting from nanomaterial exposures further inflammatory host responses in vivo. This review focuses on describing macrophage-based initiation of downstream hallmark immunological and inflammatory processes resulting from phagocyte exposure to and internalization of nanomaterials.

Introduction

Nanoparticles in diverse forms and designs have substantial clinical potential; they may eventually be capable of directing specific cell recognition, internal trafficking and processing pathways. Additionally, nanoparticles may be able to overcome traditional drug delivery roadblocks that currently prevent many types of treatments from becoming viable therapies. For example, hydrophobic drugs, nucleic acids and proteins have been encapsulated within nanoparticles to reduce intracellular degradation, increase circulation times and improve therapeutic characteristics [1]. In 2013, 241 companies and institutions had a total of 789 ongoing clinical trials and 103 unique nanomedicine investigational products [2], seeking to exploit unique delivery properties.

However, despite promising potential and sharp increases in clinical trials and investigational products, only 38 products have received Food and Drug Administration (FDA) regulatory approval for patient use across 60 years of investigational research [2]. Nanomedicine translation faces significant challenges related to more efficient clinical development when compared to the 32 new but traditional small molecular weight therapeutics receiving FDA approval in 2012 alone. Nanomaterials, unlike small molecular weight, soluble therapeutics, approximate the same size scale as biologics, do not permeate epithelial membranes (gut, skin and eye) efficiently and often are decorated with diverse non-specifically adsorbed host proteins, increasing their non-specific cellular interactions. Both size and physical properties (e.g., materials chemistry, interfacial properties and particle transport dynamics) are often used to explain limitations in clinical translation, related to their observed increased cellular recognition that prompts host recognition, nanoparticle clearance and associated inflammatory effects [3], [4]. Issues plaguing nanomaterials circulation and targeting in humans can be attributed to rapid vascular filtration and clearance of therapeutics and diagnostics, and to induction of host inflammatory responses due to non-specific recognition and uptake of nanoconstructs by macrophages in vivo [5], [6], [7], [8], [9], [10], [11]. Rapid blood clearance limits nanomaterial accumulation at target delivery sites; nanoparticle accumulation in macrophages within clearance organs initiates inflammatory responses, inducing toxicity [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. A historical review of published literature indicates that approximately 95% of systemized nanomaterials doses are sequestered by filtration organs and never reach their intended targets [23], [24], [25]. This outcome is generally non-distinguishable from biodistributions of circulating microparticles studied in vivo for decades, and importantly, clinically insignificant for many drug classes in their therapeutic value [26].

Because mammals have been environmentally and continuously exposed to a dizzying array of nanomaterials for millennia (e.g., air-borne, water-borne, food-borne nanomaterials of many metallic siliceous and carbonaceous forms) without significant apparent overt toxicity, mammalian immunological surveillance systems must have evolved mechanisms to tolerate or eliminate adventitious, ambient assaults from daily particle burdens [27], [28], [29], [30]. Simple combinations of environmentally ubiquitous nanomaterials and abundant microbes would also suggest that nanoparticles carrying fragments of microbial organisms (e.g., antigens, nucleic acids and membrane chemistry) known to be highly immune-provocative to mammals (e.g., eDNA, dsRNA, endotoxins and exotoxins) would be subject to host immune processing and neutralization as a routine survival function. Therefore, host mechanisms for particle processing are, at some level, highly evolved and difficult to by-pass, despite the best efforts of materials engineering [27], [28], [29], [30].

Nanoparticle association with the host highly evolved mononuclear phagocytic system (MPS) is a function of particle opsonization upon contact with blood and rapid recognition of these opsonins via the MPS [31], [32]. This is particularly observed in structurally distinct fenestrated vasculature via liver Kupffer cells and splenic macrophages [33], [34]. If these macrophagic cells are indeed responsible for high particle clearance rates, disappointing imaging and therapeutic efficacy due to poor delivery efficiencies to specific targets and increased clearance organ accumulation are anticipated. Nanoparticle delivery vehicles designed to either avoid or specifically harness this host recognition system could improve payload delivery, reduce inflammatory effects and improve imaging and drug efficacy. However, to rationally design these improved systems, better understanding is needed of nanoparticle-macrophage interactions both at cellular and system-wide levels in physiological conditions.

Macrophages recognize opsonized proteins, specific surface chemistries, and other surface and biological characteristics that mark these nanoparticles, similar to analogous microparticle precedents, for clearance and/or toxicological fates [2], [29], [35], [36], [37], [38], [39]. Particle physicochemical characteristics can influence these interactions and may also potentiate toxicological mechanisms [2], [28], [38], [40], [41], [42], [43], [44], [45], [46], [47], [48]. What is not understood is how nanoparticle surfaces interact with the complex biological environment to influence phagocytic recognition, clearance, cellular processing and toxicological fates. Developing correlations between nanoparticle physicochemical characteristics and nanoparticle uptake, processing and clearance mechanisms in macrophages would provide a basis to overcome decades of frustration in particle systemic delivery and targeting, and facilitate design of new, more efficacious and safer nanomaterial platforms.

Mesothelioma, pneumoconiosis and silicosis are clinically relevant well known disease states that occur after post-environmental particulate exposure. These conditions share common features of morbidity, i.e., initiation of inflammation and presentation many years after initial exposure [49], [50], [51]. Development of inflammatory-mediated and damage from unresolved oxidative stress mechanisms is a chronic issue, distinct from acute effects in exposure, response and cumulative pathology. While particles and their associated disease etiologies might be very different from engineered nanomaterials introduced more recently, that the initial phases of these well-studied diseases follow similar patterns to what is reported for acute toxicity studies of engineered nanoparticles is concerning. Recent evidence suggests that long-term silica residence within MPS/RES clearance organs, including the lung, liver and spleen, initiate fibrotic-like lesions via infiltration and microgranulation of hepatocytes (in the liver) and long-term inflammatory responses and recruitment of macrophages/leukocytes [8], [51], [52]. Nanoparticles in circulation share many clearance mechanisms and fates of their microparticle analogs. Inhalation of nanoparticles has also initiated fibrotic-like lesions within lung tissue [50], [53]. Interestingly, fibrotic lesion production can be mitigated with particle surface modification (i.e., hydrophobicity and charge). For example, lung fibrosis was a hallmark for cationic silica nanoparticles, while those with polar or anionic surfaces tended to migrate to the mediastinal lymph nodes [54]. Nonetheless, chronic inhaled exposure to nanomaterials is shown to elicit deleterious lung effects from on-going oxidative stress, enhancing pro-inflammatory effects in airways of chronic obstructive pulmonary disease (COPD) patients [55]. Additionally, detrimental cardiovascular effects from inhaled nanomaterials exposure are observed in epidemiological studies, attributed to particle translocation across the respiratory epithelium into circulation and subsequent toxicity to vascular endothelium, disruption of normal blood coagulation and changes in autonomic nervous functions that eventually alter cardiac frequency and function [55]. This suggests that the body has developed a common local response to isolate these foreign particulate materials from host biological environments. Clearly, controlling biological fate for engineered nanomaterials requires focus on specific systemic processing mechanisms and their chronic consequences in either storage or elimination. Unanswered issues are how host local particle responses consequent to materials clearance, isolation, and elimination (i.e., oxidative stress, inflammatory and immune responses long term) are affected by nanomaterials chemistry, their morphologies or their biological conditioning, and how any of these downstream events initiate adverse chronic problems. Even more concerning are local particle clearance responses that might promote cancer and immunological disorders. Ultimately, rational design criteria for safe, effective nanomaterials with minimal human exposure risks, perhaps degradable inorganic or polymer systems, that reliably avoid adverse processing and long-term pathological responses are critical.

In this review, we argue that much nanoparticle drug delivery and imaging literature neglects defining the precise and defining relationships between host macrophages and in vivo nanoparticle processing, resulting in continued deficiencies in nanoparticle designs that target or circulate widely, and therefore also limit clinical reliability and translation. Additionally, the current lack of understanding of risks of chronic toxicity from on-going or repeated nanoparticle exposures to host filtration organs and macrophages could have serious consequences and impede clinical advancement. Key mechanistic details that initiate potentially toxic outcomes remain unknown. Long-term nanomaterial fates in clearance organs and possible risks for patients in both acute and chronic exposures are generally unexplored. In vitro and preclinical studies have not yet effectively recapitulated these exposures. This review identifies both nanomaterials failures and successes as a function of macrophage-nanomaterial interactions and resulting potential inflammatory effects, with the goal of improving therapeutic translational capacity.

Section snippets

What is a phagocyte/macrophage?

Macrophages and other phagocytes are leukocytic cells capable of phagocytizing or taking up bacteria, cellular debris and particles through energy-consuming membrane-engulfing as a characteristic phenotype [56], [57], [58]. Their primary role is early response to foreign material contamination and its clearance. Macrophages have been known to uptake foreign materials within a matter of minutes, increasing their rates of phagocytosis for positively charged and bacteria-specific proteins [59],

Recognition of nanomaterials by phagocytes

In vivo, the host particle surveillance and clearance systems (i.e., MPS or tissue-resident phagocytes) do not encounter bare nanomaterials. The immediate host biological conditioning produces protein adsorption to the biomaterial surface upon blood or tissue contact [83], [84].

Phagocytic internalization mechanisms

To limit, control or select nanomaterial uptake mechanisms and how they direct intracellular processes and fate, nanoparticle delivery could be directed with higher efficiency to specific cell populations (e.g., disease states) and cellular compartments. This capability would greatly improve drug delivery. Some drugs act only at specific disease sites and within cellular organelles and require internal trafficking efficiently to avoid off-site problems. For example, nucleic acid therapies act

Phagocytic intracellular fate

Control and manipulation of particle morphological and surface physicochemical properties to interact in predictable ways with physiological components (proteins, cells) would enable exploitation of rational particle engineering strategies to select specific cell types, transport routes, internal cell compartments and more control over dosing, biodistributions, therapeutic action and toxicity. Extracellular particle recognition and processing determines intra-cellular uptake and particle

In vitro macrophage model systems and their correlation in vivo

Little is known about mechanisms through which physicochemical characteristics of nanoparticles induce up-regulation of inflammatory genes or in which stage of nanoparticle-macrophage interaction inflammatory genes are upregulated (i.e., presentation to biological milieu, cellular contact, or after internalization). Better understanding of these mechanisms, activation stages and characteristics would be beneficial when designing nanoparticle systems. More importantly, inflammatory gene

Phagocyte-nanoparticle interactions in vivo

Particle biological conditioning, distributions and macrophage processing in vivo are significantly more complex than in vitro assays can duplicate, altering transport, physical states, tissue residence patterns and mechanisms of toxicity. Tools to dissect macrophages in organ-specific uptake, induction of toxicity and clearance in vivo are required to understand their current intrinsic lack of targeting, uncontrolled biodistributions, MPS accumulation, reduced circulation times and

Designing nanomaterials that reduce phagocytic recognition

Innovating the “bioinvisibility” of nanomaterials in the host to reduce macrophage recognition in vivo might yield new insights. Grafted and coated PEG and other hydrophilic polymer brush layers are commonly used in drug delivery to facilitate enhanced particle circulation times and reduced protein adsorption but this strategy has only produced incremental improvements after several decades of refinement. These systems are still recognized eventually by the innate immune system, taken up by

Drug delivery systems targeting macrophages or utilizing macrophages as a delivery platform

Nanoparticles residing in macrophages in clearance organs by virtue of abundant uptake mechanisms could be useful in certain disease states as a “targeted” therapeutic approach. Several labs have harnessed this power to target tumor-associated macrophages in cancer [263], [264], human immunodeficiency virus (HIV) [265], [266], [267], bacterial/fungal infections [220], [268], [269], among others. Additionally, more recently, utilization of macrophages themselves as mobile, disease-homing drug

Current clinically relevant nanoparticle-macrophage interactions

Therapeutic capacity and clearance mechanisms have been linked with macrophage activity in clinically relevant nanomaterial therapies. For example, liposomal chemotherapeutic intravenous formulations have been prescribed clinically on- and off-label for several years (e.g., Doxil™). These formulations exhibit increased plasma half-life as patients’ age increases or monocytic activity decreases (Figure 6) [3], [4]. Decreased particle clearance appears to be a direct result of reduced patient

Conclusions

Nanoparticle use in imaging, vaccines, drug delivery and theranostic systems has the potential to produce new breakthroughs for medical capabilities. However, long-standing limitations for particle-host interactions remain as formidable barriers for concept translation. Few nanoparticle systems have distinguished themselves in medical performance in full physiological test beds from decades of studies. Additionally, the enormous complexity of nanoparticle chemistries, surface modifications,

Acknowledgments

Support was provided by National Institutes of Health grants R01ES024681 (to HG) and R01EB000894 (to DWG) and a predoctoral fellowship (W81XWH-11-1-0057 to HH) from the Department of Defense Breast Cancer Research Program. DWG acknowledges support for the George S. and Dolores Eccles Foundation (USA).

Heather Herd Gustafson received her B.S.E. in Biomedical Engineering from Case Western Reserve University and Ph.D. in Bioengineering from The University of Utah. Her dissertation work focused on understanding macrophage nanoparticle interactions. During the course of her PhD she was awarded both a Whitaker Foundation Fellowship and Department of Defense Predoctoral Fellowship to elucidate the role that physicochemical characteristics played on nanoparticle intracellular uptake and fate within

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    Heather Herd Gustafson received her B.S.E. in Biomedical Engineering from Case Western Reserve University and Ph.D. in Bioengineering from The University of Utah. Her dissertation work focused on understanding macrophage nanoparticle interactions. During the course of her PhD she was awarded both a Whitaker Foundation Fellowship and Department of Defense Predoctoral Fellowship to elucidate the role that physicochemical characteristics played on nanoparticle intracellular uptake and fate within macrophages. She is currently a postdoctoral fellow at The Fred Hutchinson Cancer Research Institute working on the role that macrophages play in tumor development.

    Dolly Holt-Casper received her B.S. from Utah State University in Biological Engineering and M.S. and Ph.D. in Bioengineering at the University of Utah. She served as a fellow for both the American Heart Association and NSF IMSURE nanotechnology program. Her PhD focused on analyzing the foreign body response to various biomaterials including nanoparticles. She also completed a postdoctoral fellowship in Tissue Engineering from the University of Utah, working on creating 3D scaffolds for cardiac, cartilage, and adipose tissue regeneration. She received a Women Tech Award for academic excellence in the State of Utah, has filed 2 patents, and has won several idea and business plan competitions.

    David W. Grainger is a University Distinguished Professor and George S. and Dolores Doré Eccles Presidential Endowed Chair in Pharmaceutics and Pharmaceutical Chemistry, and Professor of Bioengineering at the University of Utah. Grainger's research focuses on improving drug delivery methods, implanted medical device and diagnostics performance, and nanomaterials toxicity. Grainger has published 176 research papers and 23 book chapters on biomaterials innovation in medicine and biotechnology, and novel surface chemistry. He has won research awards, including the 2013 Excellence in Surface Science Award (Surfaces in Biomaterials Foundation), the 2007 Clemson Award for Basic Research (Society for Biomaterials), and the 2005 American Pharmaceutical Research and Manufacturer's Association's award for “Excellence in Pharmaceutics”, and several teaching awards.

    Hamid Ghandehari is a Professor at the Departments of Bioengineering and Pharmaceutics and Pharmaceutical Chemistry, Director of Utah Center for Nanomedicine and Co-Founder and Co-Director of the Nano Institute of Utah at the University of Utah. His research focuses on the design of new polymers for gene therapy of head and neck cancer, targeted delivery of polymer therapeutics to solid tumors, oral delivery of chemotherapeutics, and assessing the biocompatibility of silica and dendritic nanoconstructs. Hamid is Editor in Chief of Advanced Drug Delivery Reviews, Fellow of the American Institute for Medical and Biological Engineering, the American Association of Pharmaceutical Scientists, and Controlled Release Society, Member of Center for Scientific Review College of Reviewers at the NIH, and serves on boards of several drug delivery journals and organizations. He has published nearly 150 peer reviewed articles. He received his BS in Pharmacy and PhD in Pharmaceutics and Pharmaceutical Chemistry from the University of Utah.

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