Toxicology and Applied Pharmacology: Marina A. Dobrovolskaia, Michael Shurin, Anna A. Shvedova

YTAAP-13553; No of Pages 12
Toxicology and Applied Pharmacology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Current understanding of interactions between nanoparticles and the

immune system
Marina A. Dobrovolskaia a,⁎, Michael Shurin b,c, Anna A. Shvedova d,e,⁎
a
Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, NCI at Frederick, Fred-
erick, MD 21702, USA
b
Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA
c
Department of Immunology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA
d
Health Effects Laboratory Division, National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV 26505, USA
e
Department of Physiology and Pharmacology, West Virginia University, Morgantown, WV 26506, USA
a r t i c l e i n f o a b s t r a c t
Article history: The delivery of drugs, antigens, and imaging agents benefits from using nanotechnology-based carriers. The suc-
Received 29 October 2015 cessful translation of nanoformulations to the clinic involves thorough assessment of their safety profiles, which,
Revised 24 December 2015 among other end-points, includes evaluation of immunotoxicity. The past decade of research focusing on nano-
Accepted 26 December 2015
particle interaction with the immune system has been fruitful in terms of understanding the basics of nanopar-
Available online xxxx
ticle immunocompatibility, developing a bioanalytical infrastructure to screen for nanoparticle-mediated
Keywords:
immune reactions, beginning to uncover the mechanisms of nanoparticle immunotoxicity, and utilizing current
Nanoparticles knowledge about the structure–activity relationship between nanoparticles' physicochemical properties and
Preclinical their effects on the immune system to guide safe drug delivery. In the present review, we focus on the most
Immunotoxicity prominent pieces of the nanoparticle–immune system puzzle and discuss the achievements, disappointments,
Immunology and lessons learned over the past 15 years of research on the immunotoxicity of engineered nanomaterials.
Drug delivery © 2015 Published by Elsevier Inc.
1. Introduction intended to stimulate or inhibit the immune system, as well as when

they are used for industrial and environmental applications. Specific
The immune system's function in the maintenance of tissue homeo- targeting of the immune system, on the other hand, provides an attrac-
stasis is to protect the host from environmental agents such as microbes tive option for vaccine delivery, as well as for improving the quality of
or chemicals, and thereby preserve the integrity of the body. This is anti-inflammatory, anticancer, and antiviral therapies (Mallipeddi and
done through effective surveillance and elimination of foreign and ab- Rohan, 2010; Gonzalez-Aramundiz et al., 2012; Zaman et al., 2013;
normal self cells and structures from the body. It is well known that cer- Tran and Amiji, 2015). Moreover, nanotechnology-based carriers can
tain environmental contaminants and xenobiotics, as well as other be used to reduce the immunotoxicity of traditional drugs (Libutti
drugs, may alter the immune system's normal function. Therefore, et al., 2010).
screening for immunotoxicity is a generally accepted step in toxicolog- Some nanomaterials, metal colloids and liposomes, for example,
ical research related to both environmental factors and pharmaceutical were in use more than a decade ago (Gregoriadis et al., 1974), yet
products (Luebke, 2012). most active research in this field began in early 2000, fueled by the at-
The interactions between nanoparticles and various components of tention paid by regulatory agencies, such as the United States Environ-
the immune system have become an active area of research in bio- mental Protection Agency (EPA) and the U.S. Food and Drug
and nanotechnology because the benefits of using nanotechnology in Administration (FDA), to the rapidly growing number of applications
industry and medicine are often questioned over concerns regarding containing various types of engineered nanomaterials. The increase in
the safety of these novel materials. The past decade of research has submissions was expected since innovative research in this area had
shown that, while nanoparticles can be toxic, nanotechnology engineer- been progressing for years, culminated by the establishment of several
ing can modify these materials to either avoid or specifically target the breakthrough technologies that led to the discovery of fullerenes
immune system. Avoiding interaction with the immune system is desir- (Benning et al., 1992), carbon nanotubes (Ramirez et al., 1994), den-
able when the nanoparticles are being used for medical applications not dritic polymers (Tomalia, 1991; Newkome et al., 2002), and quantum
dots (Takagahara, 1987). In 2005–2006, many worldwide initiatives
⁎ Corresponding authors.
were launched to improve the understanding of nanoparticle safety
E-mail addresses: marina@mail.nih.gov (M.A. Dobrovolskaia), ats1@cdc.gov and included, among others, the establishment of the Nanotechnology
(A.A. Shvedova). Task Force by the FDA (http://www.fda.gov/ScienceResearch/
http://dx.doi.org/10.1016/j.taap.2015.12.022
0041-008X/© 2015 Published by Elsevier Inc.
Please cite this article as: Dobrovolskaia, M.A., et al., Current understanding of interactions between nanoparticles and the immune system,
Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.12.022
2 M.A. Dobrovolskaia et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx
SpecialTopics/Nanotechnology/ucm2006658.htm), several nanotech- The immunotoxicity of environmental materials has also been
nology research programs by the EPA (http://www2.epa.gov/ reviewed elsewhere (Kagan et al., 2010b).
chemical-research/research-evaluating-nanomaterials-chemical- Herein, we focus on the most prominent pieces of the nanoparticle–
safety), the E56 committee by the American Society for Testing and Ma- immune system puzzle, discussing what worked, what didn't, and what
terials (ASTM) International (http://www.astm.org/COMMITTEE/E56. has been learned over the past 15 years of research on nanomaterials
htm), and the TC229 Nanotechnologies technical committee by the In- engineered for biomedical applications. A summary of achievements,
ternational Organization for Standardization (ISO) (http://www.iso. disappointments, and lessons learned is presented in Fig. 2, and is fur-
org/iso/iso_technical_committee?commid=381983). In addition to ther discussed below.
these efforts, the U.S. National Cancer Institute established the Nano-
technology Characterization Laboratory (NCL) to accelerate the transla- 2. Achievements
tion of nanotechnology-based concepts intended for medical
applications in the area of cancer diagnosis and therapy from bench to 2.1. Structure–activity relationship
bedside (http://ncl.cancer.gov/). One of the initial goals of the NCL was
to support the nanotechnology community by developing a so-called The physicochemical properties of nanoparticles determine their in-
assay cascade that would include, among other tests, a battery of immu- teractions with proteins in biological matrices (e.g. blood plasma and al-
nological assays. This assay cascade contributed to the initial under- veolar fluid) and with the immune cells. The structure–activity
standing of the interactions between nanoparticles and the immune relationships between the most prominent physicochemical properties
system and created a framework for stimulating discussions in the of nanoparticles and their effects on the immune system that lead to the
area of nano-immunotoxicology (Dobrovolskaia and McNeil, 2007; most common types of immunotoxicity are summarized in Fig. 3.
Marx, 2008; Dobrovolskaia et al., 2009a; Pantic, 2011; Smith et al., Below, we review several examples.
2013). Recently, the European Commission has established the Nanoparticles with cationic surfaces, or those that carry cationic li-
European Nanomedicine Characterization Laboratory (EU-NCL), which gands, interact with biological membranes electrostatically. This leads
shares several objectives with those of the NCL (https://ec.europa.eu/ to cellular damage, which triggers hemolysis, platelet activation, and ag-
jrc/en/news/eu-ncl-launched). gregation, and to the induction of leukocyte procoagulant activity (PCA)
The rapid growth of this field becomes obvious when one compares and disseminated intravascular coagulation (DIC) (Greish et al., 2012;
the number of publications searchable in PubMed using the key words Jones et al., 2012a; Jones et al., 2012b; Ziemba et al., 2012). For example,
“nanoparticles” and “immune system” between years 2000 and 2015 cationic dendrimers of different architecture and size (generation five
(Fig. 1). Reviewing these data reveals many advances, as well as disap- [G5] and generation four [G4] poly (propylene imine) [PPI] dendrimers
pointments. Moreover, delving into the mechanisms of nanoparticle [Bhadra et al., 2005; Agashe et al., 2006], G4 polyamidoamine [PAMAM]
immunotoxicity uncovered many challenges in material characteriza- dendrimers [Bhadra et al., 2003; Asthana et al., 2005], generation three
tion. Due to the wide variety of nanomaterials available, the characteri- [G3] PAMAM and G3 PPI dendrimers [Malik et al., 2000], as well as G4
zation of their physicochemical properties is directed toward poly-L-lysine [PLL] dendrimers [Agrawal et al., 2007]) were shown to
addressing parameters specific to certain type of particles (e.g. porosity be hemolytic both in vitro and in vivo. The in vitro percent hemolysis
is applicable to silicon nanoparticles, but is not informative for lipo- varied from 14 to 86% in whole blood from human donors and various
somes and dendrimers). The grand challenge in the particle characteri- animal species, and was dependent on the density of the surface groups.
zation that precedes immunotoxicity studies relates to the estimation of Likewise, cationic PAMAM dendrimers, but not their anionic and neutral
immunoreactive contaminants, such as synthesis byproducts (e.g. iron counterparts, altered key platelet functions and perturbed plasma coag-
catalysts in carbon nanotubes, cetyltrimethylammonium bromide ulation, which culminated with DIC (Greish et al., 2012; Jones et al.,
[CTAB] in gold nanorods), and bacterial endotoxins, as well as excipients 2012a; Jones et al., 2012b). The particle size, surface charge, and confor-
(e.g. Cremophor EL, polysorbate 80), and linkers (e.g. certain linkers mation of the polymer coating are important determinants of particle
used to attach poly(ethylene glycol) [PEG] to the nanoparticle surface) clearance by the mononuclear phagocytic system (MPS) in that smaller
(Crist et al., 2013). particles (100–200 nm) with unprotected surfaces and surfaces coated
The challenges related to the physicochemical characterization with a hydrophilic polymer in a “mushroom” configuration are primar-
(Clogston and Patri, 2013) and estimation of endotoxin contamination ily cleared by Kupffer cells in the liver; larger particles are eliminated by
have been recently reviewed elsewhere (Crist et al., 2013; red pulp macrophages in the spleen. The addition of a hydrophilic
Dobrovolskaia, 2015). polymer coating in a “brush” configuration protects particles from
immune recognition, while increasing the particle size above 300 nm
provides no protection, regardless of the polymer conformation
(Gbadamosi et al., 2002). Exposure to high aspect ratio particles (e.g.
carbon nanotubes, titanium nanobelts, cellulose nanofibers), as well as
certain metallic particles (e.g. Si), results in inflammasome activation
and the induction of proinflammatory cytokine interleukin (IL)-1β.
These particles, as well as certain cationic and carbon-based
particles, can exaggerate endotoxin-mediated inflammation (Baron
et al., 2015).
The immunotoxicity of a nanoparticle is also influenced by the
therapeutic payload it carries. For example, the induction of
cytokines and type I interferons, the inflammatory reaction, the
prolongation of plasma coagulation time, and complement activa-
tion are common dose-limiting toxicities of therapeutic nucleic
acids (Levin, 1999). These toxicities are also commonly observed
with nanoformulated nucleic acids, and this limits their translation
into clinical use (Dobrovolskaia and McNeil, 2015a; Dobrovolskaia
Fig. 1. Publications statistics. The PubMed data base was searched using the keywords
“nanoparticles” and “immune system” for the years 2000–2015. The data for 2015 were
and McNeil, 2015b). Cytotoxic DNA-intercalating drugs used to
excluded from the analysis because the publication year was incomplete at the time of treat cancer (e.g. doxorubicin, daunorubicin, and vincristine) are
the search. Each bar shows the total publication number per year. known to induce PCA and DIC (Wheeler and Geczy, 1990; Swystun
M.A. Dobrovolskaia et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx 3
Fig. 2. Achievements, disappointments, and lessons learned from the characterization of engineered nanomaterials over the past decade. This diagram outlines achievements (left circle)
and disappointments (right circle) based on the studies dedicated to investigating nanoparticle immunotoxicity over the past decade. The overlapping area shows the lessons learned from
these studies. API — active pharmaceutical ingredient; NP — nanoparticles; PCP — physicochemical properties, CARPA — complement activation-related pseudoallergy, ICH — International
Conference on Harmonization.
et al., 2009; Kim et al., 2011). Formulating these drugs using Depending on their size, particles are internalized by APCs via differ-
nanotechnology carriers may help avoiding the toxicity. However, ent pathways, including both pino- and phagocytosis (O'Hagan et al.,
if overcoming these toxicities is not considered during the design 2001; Fifis et al., 2004a). Moreover, macrophages utilize multiple routes
and optimization of nanoformulated versions of these drugs, both to take up the same types of nanoparticles (Franca et al., 2011). Several
PCA and DIC may not be resolved. studies reported that smaller particles (20–200 nm) elicit stronger im-
mune responses than their larger counterparts (O'Hagan et al., 2001;
Fifis et al., 2004a; Fifis et al., 2004b; Minigo et al., 2007; Mottram et al.,
2.2. Application of nanoparticles to improve vaccine efficacy 2007; Manolova et al., 2008). For example, Plebanski and her group con-
ducted a series of studies to demonstrate that 40–50 nm polystyrene
The efficacy of nanoparticle-based vaccines depends on the interac- particles induce potent CD4 + and CD8 + T-cell responses and do so
tions between the particles and the target cells, and is determined by more efficiently than their larger (N 500 nm) counterparts. In contrast,
the physicochemical properties of the particles (size, shape, and surface particles N 500 nm in size were more active in inducing interferon
functionalities) because these properties play a key role in particle rec- (IFN)-γ and antibody responses (Fifis et al., 2004a; Fifis et al., 2004b;
ognition by the antigen-presenting cells (APCs). The type of nanocarrier Minigo et al., 2007; Mottram et al., 2007). Several studies demonstrated
is generally selected based on the type of immune response desired that small (b 100 nm) nanoparticles quickly travel to the draining lymph
from the vaccine. Nanoparticles have been shown to provide a wide va- nodes (LNs) after intradermal injection and effectively target LN-
riety of advantages over conventional adjuvants. They can improve the resident dendritic cells (DCs), B-cells, and macrophages (Manolova
solubility of hydrophobic antigens; provide controlled and sustainable et al., 2008),(Reddy et al., 2007b). These data suggested that large parti-
release of antigens, therefore reducing the number of required immuni- cles depend on interaction with and uptake by tissue-resident APCs,
zations; target antigens to specific cells and tissues, thus reducing side while smaller particles utilize both cell-associated migration and lym-
effects; prevent antigen degradation and deliver multiple antigens con- phatic drainage, thus providing better antigen presentation (Manolova
currently (reviewed in [Xiang et al., 2008; Xiang et al., 2010]). As of et al., 2008). Manipulation of nanoparticle size, shape, surface chemis-
today, a wide variety of engineered nanomaterials from different classes try, and charge is generally employed to maximize antigen delivery to
(polymeric, chitosanic, magnetic, latex, gold, silica, and polystyrene) DCs. For example, small (b 100 nm) nanoparticles were shown to be
have been used successfully as antigen carriers and vaccine adjuvants taken up more efficiently by DCs, while large (1 μm) particles were pref-
(Tighe et al., 1998; Pavelic et al., 2001; Weiss et al., 2002; Walsh et al., erentially internalized by macrophages (Fifis et al., 2004a). Several
2003; Fifis et al., 2004a; Fifis et al., 2004b; Minigo et al., 2007; other studies have also reported that ~ 50 nm is the optimal
Mottram et al., 2007).
Fig. 3. Structure–activity relationship summary. Shown are the structure–activity relationships between nanoparticles and their effects on the immune system. Each block listed in the
bottom (structure) part of the figure is color-coded. To find what toxicity is related to the given structure block, please find the block in the top (activity) part of the figure marked
with the color matching that of the structure block. PCA — procoagulant activity, DIC — disseminated intravascular coagulation, CARPA — complement activation-related
pseudoallergy, MPS — mononuclear phagocytic system, IL — interleukin, PEG — polyethylene glycol, NP — nanoparticle, DXR — doxorubicin, API — active pharmaceutical ingredient,
DNA — deoxyribonucleic acid.
nanoparticle size for uptake by DCs (Aoyama et al., 2003; Nakai et al., determining the type of immunity induced. For example 40-nm nano-
2003; Wang et al., 2011). particles promoted Th1 and CD8+ T-cell responses, while 100-nm par-
Particle size was also reported as a primary factor in determining the ticles induced Th2 responses (Fifis et al., 2004a; Fifis et al., 2004b;
immuno-stimulatory profiles of vaccine formulations in that smaller Mottram et al., 2007).
particles (~ 220 nm) were more potent in inducing IFN-α responses, Tuning particle zeta potential is another approach that has been ex-
while their larger counterparts (~1200 nm) induced tumor necrosis fac- plored in vaccine design. For example, positively charged particles dem-
tor (TNF)-α (Rettig et al., 2010). This difference was attributed to the onstrate greater uptake by DCs (Thiele et al., 2003; Foged et al., 2005;
type of the cells that internalized these particles: plasmacytoid DCs Villanueva et al., 2009) and induction of DC maturation (Thiele et al.,
engulfed smaller particles, while macrophages preferred larger parti- 2001; Jilek et al., 2004; Little et al., 2004; Jilek et al., 2005; Reddy et al.,
cles. Moreover, particle size was also suggested to be a key factor in 2007a). Cationic poly(D,L-lactic-co-glycolic) (PLG) particles improved
the delivery of DNA adsorbed on the particle surface to APCs and in- 2005). Liposomes loaded with clodronate were used to specifically
duced greater cytotoxic T-lymphocyte responses compared to plain eliminate macrophages in a swine model to protect animals from
DNA antigen (Singh et al., 2000). More comprehensive coverage of endotoxin-mediated lung injury (Gaca et al., 2003). Liposomal and poly-
this subject is available elsewhere (Fesenkova, 2013; Xiang et al., 2013). meric nanoparticle reformulation of cyclosporine was reported to re-
duce off-target side effects (e.g. nephrotoxicity) (Freise et al., 1994;
2.3. Application of nanoparticles for delivery of antiretroviral, immunosup- Italia et al., 2007). Tacrolimus delivery using lipid nanoparticles resulted
pressive and anti-inflammatory drugs in improved skin penetration and tissue deposition, as well as a reduc-
tion in side effects (Pople and Singh, 2012). Polylactide nanoparticles
2.3.1. Antiretroviral. Antiretroviral drug delivery has many assorted were used for ex vivo delivery of cyclosporine A into DCc (Azzi et al.,
challenges, some of which are being effectively overcome using 2010). Reinjection of these drug-loaded DCs into the footpads of mice
nanotechnology-based carriers. In addition to improving the solubility improved drug delivery to the lymph nodes, where released cyclospor-
of antiretroviral drugs, nanoparticles are considered as means to imine suppressed T-cell proliferation (Azzi et al., 2010). Delivery of
prove drug delivery to tissues and cells serving as viral reservoirs. rapamycin by elastin-like polymeric nanoparticles resulted in reduced
When antiretroviral drugs are administered using conventional routes nephrotoxicity and injection site reactions, while demonstrating com-
and formulations, the concentrations of these drugs in the plasma are parable efficacy (Shah et al., 2013).
usually higher than the concentrations found in the lymphoid tissue, The activity of liposomal formulations of glucocorticoids provides
which serves as a major depot for the virus (Fletcher et al., 2014). HIV another example of how nanoparticles can alter a drug's tissue distribu-
can replicate in the lymphatic tissue even when the viral load in the pe- tion so that it provides additional beneficial effects. In this example, the
ripheral blood is low; therefore, the need to enhance drug delivery into free drug affects T-lymphocytes, while its liposomal counterpart targets
lymphoid tissue is recognized by many as an effective way of targeting macrophages and induces an alternatively activated M2 phenotype,
HIV both in systemic circulation and at its depot sites (Fletcher et al., leading to the expression of anti-inflammatory cytokines and, conse-
2014). quently, reduced inflammation (Schweingruber et al., 2011). Nanotech-
The physicochemical properties of nanoparticles that influence lym- nology is used not only to deliver single drugs, but also to co-deliver
phatic delivery are: size, charge, molecular weight, lipophilicity, and anti-inflammatory agents with different mechanisms of action. For ex-
surface ligands (surfactants, PEG, hyaluronic acid, biotin, peptides, anti- ample, dexamethasone-loaded PLGA nanoparticles can be combined
gens, and lectins) (Cho and Lee, 2014; Singh et al., 2014). Several antire- with siRNA-targeting COX-2 to suppress inflammatory responses
troviral drugs have been formulated using a wet-bead milling process (Park et al., 2012). More examples illustrating the benefits of delivering
and showed good stability and the desired tissue distribution. These ex- immunosuppressive and anti-inflammatory drugs using nanoparticles
amples include rilpivirine solid drug nanoparticles (Verloes et al., 2008; have been recently reviewed elsewhere (Ilinskaya and Dobrovolskaia,
Baert et al., 2009) and a cabotegravir (S/GSK1265744) nanocrystal for- 2014).
mulation (Spreen et al., 2013). Other approaches focusing on the use
of nanoformulations for oral delivery of these drugs are in progress in 2.4. Application of nanoparticles for cancer immunotherapy
order to address another problem related to poor patient adherence to
antiretroviral therapy (Prinapori and Di Biagio, 2015). A more compre- Cancer immunotherapy is another rapidly growing branch of
hensive review of this subject is available in a recent review by Liptrott nanomedicine. Drugs used for cancer immunotherapy vary both in
et al. (2016). structure and by mechanism of action. For example, Iipilimumab
(anti-CTLA4) directly interacts with and activates immune cells by re-
2.3.2. Immunosuppressive and anti-inflammatory. Nanoparticles can moving co-inhibitory signal, while GVAX (GM-CSF tumor vaccine) im-
be immunosuppressive per se or used to deliver immunosuppressive proves tumor recognition by making the tumor more
drugs. For example, inhaling carbon nanotubes was shown to suppress immunostimulatory (Ali and Lee, 2015; Lipson et al., 2015). A recent
humoral immune response via a mechanism involving the production study by Fiering et al. demonstrated the use of iron oxide nanoparticles
of transforming growth factor beta (TGF-β) by alveolar macrophages and an alternating magnetic field to induce local hyperthermia in mela-
and subsequent prostaglandin production by spleenocytes leading to noma. Interestingly, besides heat-mediated tumor ablation, this treat-
the systemic immunosuppression (Mitchell et al., 2009). Other exam- ment resulted in a potent CD8 + T-cell–dependent response against
ples of nanoparticles displaying immunosuppressive activities without the tumor, preventing the recurrence of tumor growth (Toraya-Brown
bearing a therapeutic payload include the imaging agent Resovist, a sin- et al., 2014). Another study demonstrated that tumor-associated
gle intravenous administration of which resulted in suppression of the myeloid-derived suppressor cells (MDSCs) characterized by high levels
antibody response to the model antigen (Shen et al., 2011). The of oxidative reactions may be responsible for the degradation of chemo-
water-soluble fullerene derivative polyhydroxy C60 was shown to in- therapeutic drugs in the tumor environment, and that this degradation
hibit type I hypersensitivity reactions to allergens, both in vitro and could be significantly reduced by drug loading onto functionalized car-
in vivo (Ryan et al., 2007). Likewise, allergen-loaded poly(D,L-lactic-co- bon nanotubes (Seo et al., 2015). This example offers an effective way to
glycolic) acid (PLGA) particles, chitosan, poly(lactic acid) (PLA), poly prolong drug function in the tumor environment by using a
[methyl vinyl ether-co-maleic anhydride) nanoparticles, and nanodelivery approach. Other recent examples include using poly
dendrosomes were used to suppress type I and type II reactions to envi- (ethyleneimine) nanoparticles for the delivery of antiPD-1 siRNA (Teo
ronmental and food allergens (Roy et al., 1999; Scholl et al., 2004; et al., 2015), branched polyethyleneimine-superparamagnetic iron
Balenga et al., 2006; Gomez et al., 2007; Gomez et al., 2008), while syn- oxide nanoparticles for the enhancement of Th1 cell polarization of
thetic peptide dendrimers were reported to block experimental allergic DCs (Hoang et al., 2015), 6-thioguanine-loaded polymeric micelles for
encephalomyelitis (Wegmann et al., 2008). the depletion of MDSCs (Jeanbart et al., 2015), and a CD4-targeted, oil-
Other studies have shown the benefits of using nanoparticles for the in-water emulsion for the co-delivery of IL-2 and TGF-β (McHugh
targeted delivery of immunosuppressive and anti-inflammatory drugs, et al., 2015).
and to prevent the undesirable immunosuppression of small-molecule
drugs (Stinchcombe et al., 2007). For example, using PLGA nanoparti- 2.5. Understanding the role of the immune system in nanoparticle
cles formulated to deliver glucocorticoids to inflamed joints in the biodegradation
mouse model of arthritis resulted in complete remission of the inflam-
matory response. The improved efficacy was due to the targeted and The therapeutic use of biodegradable nanoparticles is accompanied
controlled release of steroids from the nanocarrier (Higaki et al., by fewer safety concerns than those of durable nanomaterials. Not
surprisingly, the majority of currently marketed nanomedicines are rep- Other success stories demonstrating the reduction of immunotoxicity
resented by biodegradable, lipid-based materials (Etheridge et al., by reformulation of a traditional drug using nanotechnology-based car-
2013). Macrophages are known to play an active role in internalizing riers include the encapsulation of therapeutic antisense oligonucleo-
and degrading these nanocarriers (Song et al., 2012). tides into liposomes to prevent activation of the complement system
The use of nonbiodegradable nanoparticles is often associated with (Klimuk et al., 2000) and to reduce cytokine-mediated toxicities (Yu
concern regarding their bioaccumulation and long-term toxicity. In et al., 2013), as well as the reformulation of the small-molecule oncol-
this context, recent studies demonstrating the unique role of activated ogy drug 5-fluorouracil, using chitosan nanoparticles to decrease its
neutrophils in the enzymatic digestion of durable nanoparticles are hematotoxicity (Giacalone et al., 2013; Cheng et al., 2014).
very encouraging. Specifically, activated neutrophils were reported to
participate in the biodegradation of carbon nanotubes. 2.7. Understanding the applicability of the existing regulatory framework
Myeloperoxidase (MPO)-reactive intermediates and hypochlorous for immunotoxicity analysis of nanoformulations
acid (HClO) generated by MPO are believed to contribute to the biodeg-
radation process (Kagan et al., 2010a; Bianco et al., 2011; Shvedova Several nanotechnology-formulated drugs have already reached the
et al., 2012), and the existence of this mechanism was confirmed market. Examples include solid lipid nanoparticles (e.g. Leunesse®), li-
in vivo using MPO-deficient mice (Shvedova et al., 2012). MPO was posomes (e.g. Doxil®), protein-based nanoparticles (e.g. Abraxane), as
also found to have a role in the oxidative biodegradation of single-wall well as nanocrystals (e.g. Emend®). Regulatory experience gained
carbon nanotubes activated human neutrophils (Kagan et al., 2010a). from working with these formulations, along with data from preclinical
Moreover, additional research suggests that peroxynitrite (ONOO− reports, has helped to develop a better understanding of how to apply
)-driven oxidation, resulting from the enzymatic activities of NADPH- the regulatory framework established and used for small-molecule
oxidase/inducible nitric oxide synthase (iNOS), functions as another and macromolecular therapeutics to complex nanotechnology formula-
carbon nanotube biodegradation pathway in activated macrophages tions (Tyner and Sadrieh, 2011; Bancos et al., 2013; Cruz et al., 2013;
(Vlasova et al., 2012; Bhattacharya et al., 2014; Farrera et al., 2014; Tyner et al., 2015). The properties shared by nanotechnology-based car-
Kagan et al., 2014). Recent data demonstrated that tumor-associated riers that have already reached the market are biodegradability (e.g.
MDSCs expressing high levels of arginase, iNOS, NADPH oxidase, and emulsions and liposomes) and the ability to quickly dissolve in the
MPO were also able to biodegrade carbon nanotubes via oxidative path- body (e.g. nanocrystals). Durable nanoparticles (e.g. metals and metal
ways. These MDSC properties were recently utilized for an interesting colloids) are expected to accumulate in the body and, as such, raise im-
nanodelivery approach in which nitrogen-doped carbon nanotube munological safety concerns. These concerns stem from the notion that
cups (NCNCs) were loaded with therapeutic cargo (paclitaxel), sealed durable materials tend to distribute to the MPS (Sadauskas et al., 2009;
via conjugation with gold nanoparticles (GNPs), and opened by enzy- Di Gioacchino et al., 2011; Moon et al., 2011; Umbreit et al., 2012). Accu-
matic oxidative processes within, or in the immediate proximity of, mulation of these materials in the MPS may affect the normal function
MDSCs. This mechanism was proposed to enhance antitumor immune of this system, and therefore this concern should not be ignored.
responses by targeting and inactivating MDSCs using their own highly The FDA applies the same regulatory framework established for all
expressed oxidative enzymatic machinery (Zhao et al., 2015). small-molecule and macromolecular therapeutics to the regulation of
complex nanotechnology products (Bancos et al., 2013). The manufac-
2.6. Application of nanoparticles to reducing the immunotoxicity of tradi- ture, characterization, and non-clinical safety evaluation of combination
tionally formulated drugs products containing nanotechnology platforms have also been covered
in several recent regulatory documents, which recommend conducting
In addition to their use as carriers of novel drugs, engineered studies relevant to each component of the complex formulation (CDER,
nanomaterials are also increasingly being used to reformulate tradi- 2006; CDER, 2008). For example, the International Conference on Har-
tional drugs (low-molecular-weight drugs, therapeutic proteins, thera- monization (ICH) S8 and S6 guidelines are consulted to estimate the
peutic antibodies, and nucleic acids). Reformulation of traditional drugs immunotoxicity of small-molecule and macromolecular components,
using nanotechnology has been shown to improve drug solubility and respectively, of any nanotechnology-formulated combination product
pharmacokinetics, as well as to reduce undesirable side effects. Below, (CDER, 2001; Bancos et al., 2013).
we review several examples demonstrating how reformulation of tradi-
tional drugs using nanotechnology resulted in reduced immunotoxicity. 3. Disappointments
The traditional formulation of the cytotoxic oncology drug paclitaxel
relies on the polyethoxylated castor oil excipient Cremophor-EL ®, 3.1. Harmonization of testing is still in progress
which is known to induce anaphylaxis in sensitive individuals. The ana-
phylactic reaction to Cremophor-EL is mediated by its ability to activate Ten years ago, international standards development organizations
the complement system (Weiszhar et al., 2012). Due to this side effect, ASTM International and ISO established the E56 and TC229 committees,
the Cremophor-EL formulation of paclitaxel (Taxol ®) has to be admin- respectively, to lead the development of standard test methods for
istered via slow infusion, and after patient premedication with immu- nanomaterials. This effort resulted in the development of three stan-
nosuppressive drugs. In contrast to Taxol, the nanoalbumin dardized test methods — ASTM E56-2524-08(2013), ASTM E56-2525-
formulation of paclitaxel (Abraxane ®) is administered via push injec- 08(2013), and ISO 29701:210 — for the analysis of the hemolytic prop-
tion, and does not require premedication (Gradishar et al., 2005). Like- erties of nanoparticles, the effects of nanoparticulate materials on the
wise, TNF-α failed in clinical trials because it induces systemic formation of mouse colony-forming unit granulocyte/macrophage colo-
immunostimulation, resulting in fever and hypotension. However, nies, and of potential endotoxin contamination in nanomaterials, re-
TNF-α immobilized on the surface of PEGylated colloidal gold nanopar- spectively (http://www.astm.org/COMMIT/SUBCOMMIT/E5603.htm;
ticles (Cyt6091) has successfully passed a Phase I trial (Libutti et al., http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_tc_
2010). Therapeutic protein immunogenicity, which leads to the forma- browse.htm?commid=381983). While these harmonized methods are
tion of antibodies that neutralize the drug and, in some cases, endoge- important to support immunotoxicity studies, they are obviously insuf-
nous proteins, is a common reason for the discontinuation of such ficient to address the broad spectrum of end-points that are indicative of
drugs (Rosenberg et al., 2012). Liposomes were shown to reduce the nanoparticle immunotoxicity. Despite global efforts, there is no harmo-
immunogenicityof recombinant coagulation factor FVIII and to protect nization in the following areas: dose selection, dose metrics, assay for-
encapsulated protein from the antibodies formed in response to the tra- mat, cell species, and matrices. Recently, an integrated approach was
ditional formulation of this therapeutic protein (Ramani et al., 2008). proposed to estimate relevant in vitro doses of tested nanomaterials in
terms of total mass, surface area, and particle number (Cohen et al., (CLinDMA) and PEG-dimyristoylglycerol (Abrams et al., 2010), prot-
2014). While the results of this study are encouraging, adaptation of amine (Li et al., 1999), trimethyl ammonium propane-cholesterol
this approach by other laboratories and harmonization efforts are (Kim et al., 2007), and 1,3-dioleoyl-3-trimethylammonium propane
needed. (DOTAP) (Li et al., 1999; Vasievich et al., 2011). Clinical studies investi-
gating the safety of such formulations are often designed to prevent ad-
3.2. Lack of nanoparticle reference standards for immunotoxicity studies verse reactions by premedicating patients with immunosuppressive
cocktails containing immunosuppressive agents (e.g dexamethasone),
Reference standards are well-characterized materials with known antipyretic agents (e.g acetaminophen), histamine H1 receptor blockers
properties. These materials can be used to validate toxicology protocols (e.g. diphenhydramine), and histamine H2 receptor blockers (e.g. ranit-
and ensure the quality of measurements specific to a given protocol idine) (Coelho et al., 2013). It is clear that the advantages of tuning the
measuring given end points. It is generally acknowledged that the lack physicochemical properties of nanoparticles for the purpose of reducing
of nanoparticle reference standards limits the validation of instruments, drug immunotoxicity have not yet been fully explored. The continuing
protocols, and materials used to assess exposure to nanomaterials and evolution of the framework for evaluating nanoparticle immunotoxicity
understand their biocompatibility. While many types of nanomaterials is the likely reason for this observation.
have been linked to certain types of immunotoxicity (e.g. cationic
dendrimers are thrombogenic; PEGylated liposomes induce anaphy-
laxis), there are no standard reference materials to use for these and 3.4. The mechanisms of nanoparticle immunotoxicity are largely unknown
other types of immunotoxicity studies. Stefaniak et al. conducted a liter-
ature review and identified 25 nanomaterials that were considered to The majority of studies reported in the past decade were focused on
be candidate nanoparticle reference materials by standards develop- understanding the structure–activity relationship between the physico-
ment organizations worldwide (Stefaniak et al., 2013). Interestingly, chemical properties nanoparticles and immunotoxicity. Uncovering the
this study found a limited consensus regarding the types of candidate mechanisms of nanoparticle immunotoxicity is still a work in progress.
nanoparticles between various organizations involved in the develop- Several interesting mechanisms have been recently described. For ex-
ment of reference materials: the U.S. National Metrology Institute, the ample, the inhibition of phosphoinositol-3-kinase (PI3K) by cationic
U.S. National Institute of Standards and Technology, the REFNANO pro- PAMAM dendrimers has been suggested to contribute to the exaggera-
ject funded by the UK government, the Organization for Economic Co- tion of lipopolysaccharide (LPS)-induced leukocyte PCA in human pe-
operation and Development, and several NanoImpactNet projects ripheral blood mononuclear cells by these particles (Ilinskaya et al.,
(NanoImpactNet, NanoSustain, and NanoValid) funded by the 2014). The activation of mitogen-activated protein kinase (MAPK) p38
European Commission (Stefaniak et al., 2013). by gold nanoparticles functionalized with α-lipoyl-ω-methoxy poly
In the absence of consensus on and availability of relevant, well- (ethylene glycol) was proposed as a mechanism for exaggeration of
characterized reference materials, immunotoxicity studies are con- LPS-triggered nitric oxide and IL-6 secretion in murine macrophages
ducted using traditional positive controls, such as lipopolysaccharide (Liu et al., 2012). The binding of colloidal gold nanoparticles to high-
for cytokine induction, phytohemagglutinin for leukocyte proliferation, mobility group B-1 was implicated in the attenuation of nuclear
Triton X-100 for hemolysis, and cobra venom factor for complement ac- factor-kappaB (NF-kB) signaling, the phosphorylation of JNK, and the
tivation (Dobrovolskaia, 2015). Preclinical studies often rely on secretion of TNF-α triggered by TLR9 activation by CpG oligonucleotides
nanomedicines with known clinical immunotoxicities (e.g. Doxil or (Tsai et al., 2012).
Taxol for complement activation and anaphylaxis tests) The induction of oxidative and nitrosative stress by zinc oxide
(Dobrovolskaia, 2015). Despite the thorough characterization of their nanoparticles was described as a mechanism through which these
physicochemical properties, the main limitation in using these products particles activate redox-sensitive NF-κB and MAPK signaling path-
as reference materials is their expense and limited accessibility to the ways, leading to an inflammatory response in human monocytes
nanotechnology research community. (Senapati et al., 2015). The oxidative stress induced by the
nanoemulsion Cremophor-EL was also suggested to trigger IL-8 pro-
3.3. Examples of nanoformulations demonstrating reduced immunotoxicity duction by human monocytes via a mechanism that bypasses gene
of traditionally immunotoxic APIs are fewer than expected expression (Ilinskaya et al., 2015).
It is also important to note that the effects of nanomaterials on im-
Despite clear examples demonstrating the potential of nanotechnol- mune cells may result in both suppression of the immune effector
ogy for reducing the immunotoxicity of traditional active pharmaceuti- cells and activation of the immune regulatory (immunosuppressive)
cal ingredients (APIs), the systemic administration of many cells. Therefore, any discussion of nanoparticle immunotoxicity should
nanoformulations to patients still requires immunosuppressive and/or consider specific immune cell subsets. For instance, airborne carbon
antipyretic premedication. For example, the complexing of therapeutic nanotubes have recently been reported to induce rapid accumulation
nucleic acids with a nanocarrier is commonly based on electrostatic in- of pulmonary MDSCs in mice, which was associated with the acceler-
teractions. As such, the lipid and polymeric carriers used to formulate ated growth of lung carcinomas in vivo (Shvedova et al., 2013). Further
therapeutic nucleic acids tend to be cationic. As discussed earlier, cat- analysis of the mechanism revealed that carbon nanotubes may
ionic particles are known to be cytotoxic to a variety of immune cells, in- presensitize MDSCs to produce the immunosuppressive cytokine TGF-
duce cytokine secretion, exaggerate endotoxin-mediated toxicities, β, which contributes to the observed immunosuppression and, as a con-
activate complement, bind plasma proteins, trigger pro-coagulant activ- sequence, tumor growth (Shvedova et al., 2013). Thus, both uncovering
ity, and may also affect protein conformation and function (Pantic, the immunomodulatory properties of nanomaterials and understand-
2011; Boraschi et al., 2012; Dobrovolskaia and McNeil, 2013a). These ing the molecular and cellular mechanisms of their activities are impor-
data raise safety concerns. Indeed, several studies have demonstrated tant for the clinical translation of these materials and potential
that base modifications were insufficient to reduce the minimization of any undesirable immunoreactivity.
immunostimulatory activity of certain nucleic acids (e.g. siRNA). Fur- Further research is clearly needed to uncover additional mecha-
thermore, lipid nanocarriers were shown to contribute to the drug's nisms and to link them to the physicochemical properties of
immunostimulation (Abrams et al., 2010). Examples of nanoparticles.
immunostimulatory lipids used to prepare nanocarriers for therapeutic
nucleic acids include 2-(4-[(3β)-cholest-5-en-3-yloxy]butoxy)-N,N-di-
methyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine
3.5. Accidental nanoparticulate contaminants contribute to the immunoge- (Madani et al., 2013). Bacterial endotoxin is a common biological impu-
nicity of therapeutic proteins rity affecting over 30% of preclinical-grade nanomaterials (Crist et al.,
2013; Dobrovolskaia and McNeil, 2013b). If not properly identified in
The observation that particulate materials between 0.1 and 10 μm in and eliminated from nanoformulations, endotoxin can confound the re-
size can contaminate recombinant protein therapeutics and contribute sults of both nanoparticle immunotoxicity and efficacy studies. The
to their immunogenicity has generated increasing levels of concern elimination of endotoxin from nanoparticles was shown to reduce
(Carpenter et al., 2010). The mechanism by which this occurs has not their immunotoxicity (Vallhov et al., 2006). Moreover, some
been fully investigated; however, several factors, such as nanoparticle- nanomaterials, while not inflammatory themselves, were able to poten-
triggered protein aggregation (Mire-Sluis et al., 2011; Van Beers et al., tiate endotoxin-mediated inflammation. Silica- and carbon-based
2012); the adsorption of proteins on the particle surface, leading to nanomaterials, as well as some metal oxides, have been shown to exag-
the formation of highly immunogenic repeated-protein structures gerate endotoxin-mediated inflammation in the lungs (Inoue et al.,
(Mire-Sluis et al., 2011; Van Beers et al., 2012); and the exaggeration 2006; Inoue et al., 2007; Shi et al., 2010; Inoue, 2011; Inoue and
of inflammatory responses triggered by trace amounts of endotoxin Takano, 2011), while cationic PAMAM dendrimers were reported to ex-
(Dobrovolskaia et al., 2016) may play a role. For example, tungsten aggerate endotoxin-induced leukocyte PCA (Dobrovolskaia et al., 2012;
microparticles originating from the tungsten pins used in the Ilinskaya et al., 2014). Strategies for endotoxin detection have been
manufacturing process were shown to induce protein aggregation and discussed elsewhere (Dobrovolskaia, 2015).
increase the immunogenicity of a recombinant protein product (Liu
et al., 2010). 4.3. Thorough physicochemical characterization is needed prior to nano-
Another study suggested that hydrophobic metal, glass, and polysty- particle analysis in immunological assays
rene particulates adsorb protein on the particle surface and contribute
to protein aggregation and immunogenicity (Van Beers et al., 2012). Since the physicochemical properties of nanoparticles are the keys
Several other materials were named among common particulates to determining particle interaction with the immune system, thorough
found to contaminate therapeutic proteins, including cellulose and particle characterization is needed prior to immunotoxicity studies
glass fibers, silicon oil, rubber, stainless steel, fluoropolymers, and plas- (Clogston and Patri, 2013). Several examples have shown that instabil-
tics (Carpenter et al., 2010; Liu et al., 2010; Van Beers et al., 2012). Cel- ity of particle surface coatings results in inflammatory reactions, yet
lulose fibers were shown to exaggerate the production of endotoxin- partial loss of surface coating is undetectable by dynamic light scattering
induced pro-inflammatory cytokines (Dobrovolskaia et al., 2016). and zeta potential analysis, traditionally used for particle characteriza-
Gowns and other materials used in clean rooms during the manufactur- tion (Clogston and Patri, 2013; Crist et al., 2013). Certain processes
ing of therapeutic proteins — closures, filling pumps, containers, vial and reagents commonly involved in nanoparticle research (e.g. sterili-
stoppers, etc. — serve as sources of these particulate contaminants zation procedures and inhibitors used for signal transduction studies)
(Carpenter et al., 2010). These data led regulatory agencies to require may also affect nanoparticle integrity (Zheng et al., 2011;
the detection and characterization of particulate materials that can con- Dobrovolskaia et al., 2016). Missing such details may lead to misinter-
taminate therapeutic proteins; however, there is no general agreement pretation of the study results and faulty conclusions. Another important
as to what methods should be used (Carpenter et al., 2010). The major lesson has been learned from using nanoparticles for drug delivery:
challenge in understanding the mechanism of therapeutic protein im- drug conjugation to a nanotechnology-based carrier may change the
munogenicity in the presence of contaminating particles is the limited drug's original properties. For example, celastrol conjugated to a dendri-
quantities of these particulate materials. Even when a particulate con- mer carrier retained its ability to suppress LPS-induced nitric oxide re-
taminant is detected and isolated from the protein product, the quantity lease, but lost its ability to inhibit production of pro-inflammatory
of the isolate is usually insufficient to conduct a follow-up mechanistic cytokines (Boridy et al., 2012).
study (Carpenter et al., 2010). It is obvious that more research is needed
to address this important issue. 4.4. Total protein binding serves as good indicator of nanoparticle stealthi-
ness and its distribution to the MPS
4. Lessons learned
Proteins bind to a nanoparticle surface instantaneously upon entry
4.1. Nanoparticles' physicochemical properties are the keys to determining of the particle into the bloodstream. Some of these proteins stay on
particle interaction with the immune system the surface as long as the particles circulate in the bloodstream, while
others dissociate from the particle surface or get replaced by proteins
It is now well established that nanoparticles can be engineered to ei- with a higher binding affinity. Protein binding was shown to affect
ther avoid or specifically interact with the immune system. The tuning nanoparticle hydrodynamic size and charge (Dobrovolskaia et al.,
of nanoparticles to attain desirable attributes can be achieved by manip- 2009b), and was also suggested to influence the way cells and tissues in-
ulating the physicochemical properties (size, charge, hydrophobicity, teract with and process the particles, ultimately guiding cellular uptake,
shape) of the particle that determine its interaction with plasma pro- clearance route, and tissue distribution (Goppert and Muller, 2005;
teins and immune cells. This subject has been extensively reviewed Michaelis et al., 2006; Nagayama et al., 2007; Zensi et al., 2009). It is
elsewhere (Smith et al., 2013). We have also discussed many examples now well established that total protein binding can serve as an indicator
underlining this point in the achievement section of this paper. of particle “stealthiness.” Stealthy particles tend to stay in circulation
longer. In contrast, particles with proteins bound to their surface are
4.2. Chemical (e.g. synthesis byproducts) and biological (e.g. endotoxin) cleared by the cells of the MPS.
impurities can contribute to nanoparticle toxicity
4.5. Composition of protein corona is insufficient to predict nanoparticle
It is very important to distinguish nanoparticle-mediated immunocompatibility
immunotoxicities from those triggered by chemical and biological im-
purities. The presence of traces of CTAB used as a stabilizing agent in In contrast to total protein binding, the composition of the protein
the synthesis of gold nanorods was implicated in the cytotoxicity of corona has less predictive value. Complement and fibrinogen are abun-
these particles (Leonov et al., 2008). Iron and nickel used to catalyze re- dant plasma proteins, and they have been reported as part of the so-
actions involved in the synthesis of carbon nanotubes were shown to called “protein corona” for many engineered nanomaterials. However,
trigger inflammatory reactions in response to nanotube exposure the presence of these proteins on the particle surface per se does not
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J. Nanosci. Nanotechnol. 14, 868–880.
The Transparency document associated with this article can be Clogston, J.D., Patri, A.K., 2013. Importance of physicochemical characterization prior to
immunological studies. In: Dobrovolskaia, M.A., McNeil, S.E. (Eds.), Handbook of Im-
found, in the online version.
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Ltd., Singapore.
Acknowledgments Coelho, T., Adams, D., Silva, A., Lozeron, P., Hawkins, P.N., Mant, T., Perez, J., Chiesa, J.,
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M.D.'s work has been funded with federal funds from the National T., Hutabarat, R.M., Clausen, V.A., Alvarez, R., Fitzgerald, K., Gamba-Vitalo, C.,
Cancer Institute, National Institutes of Health, under contract Nochur, S.V., Vaishnaw, A.K., Sah, D.W., Gollob, J.A., Suhr, O.B., 2013. Safety and effi-
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YTAAP-13553; No of Pages 12

Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/ytaap

Current understanding of interactions between nanoparticles and the

1. Introduction intended to stimulate or inhibit the immune system, as well as when

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