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Overview
Bridge over troubled waters:understanding the synthetic
andbiological identities of engineerednanomaterialsBengt Fadeel,1∗
Neus Feliu,1 Carmen Vogt,1 Abuelmagd M.Abdelmonem2 and Wolfgang J.
Parak2
Engineered nanomaterials offer exciting opportunities for
‘smart’ drug deliveryand in vivo imaging of disease processes, as
well as in regenerative medicine. Theability to manipulate matter
at the nanoscale enables many new properties thatare both desirable
and exploitable, but the same properties could also give riseto
unexpected toxicities that may adversely affect human health.
Understandingthe physicochemical properties that drive
toxicological outcomes is a formidablechallenge as it is not
trivial to separate and, hence, to pinpoint individualmaterial
characteristics of nanomaterials. In addition, nanomaterials that
interactwith biological systems are likely to acquire a surface
corona of biomoleculesthat may dictate their biological behavior.
Indeed, we propose that it is thecombination of material-intrinsic
properties (the ‘synthetic identity’) and context-dependent
properties determined, in part, by the bio-corona of a given
biologicalcompartment (the ‘biological identity’) that will
determine the interactions ofengineered nanomaterials with cells
and tissues and subsequent outcomes. Thedelineation of these
entwined ‘identities’ of engineered nanomaterials constitutesthe
bridge between nanotoxicological research and nanomedicine. © 2013
WileyPeriodicals, Inc.
How to cite this article:WIREs Nanomed Nanobiotechnol 2013,
5:111–129. doi: 10.1002/wnan.1206
INTRODUCTION
Nanomaterials are in the same size range asbiomolecules and
cellular structures; this factlies at the very heart of
nanomedicine, a field in whichmany applications rely on nanoscale
interactions.However, this is also the reason for the
currentconcern surrounding nanomaterials: the interferenceof
man-made nanomaterials with biological systemscould also lead to
hazardous effects on humanhealth.1 While the interest in nanoscale
materials hasincreased tremendously in recent years,
importantobservations on their interactions with biological
∗Correspondence to: [email protected] of Molecular
Toxicology, Institute of EnvironmentalMedicine, Karolinska
Institutet, Stockholm, Sweden2Fachbereich Physik and
Wissenschaftlichen Zentrum für Material-wissenschaften, Philipps
Universität Marburg, Marburg, Germany
systems were reported much earlier. For instance,the fact that
nanoparticles are typically incorporatedby cells via endocytosis
was known for decades.2
In addition, colloidal nanoparticles were shown toinduce
alterations in the blood–air barrier in themouse lung more than
half a century ago.3 Numerousstudies have been published more
recently in whichexposure to engineered nanoparticles has been
linkedto toxicity.4–6 However, understanding which ofthe
physicochemical properties of nanomaterials thatare driving
toxicity remains a challenge; if onecould connect material
properties (size, shape, surfacecharge, porosity, colloidal
stability, purity/degree ofcontamination, etc.) with toxicity, then
this wouldenable prediction of potential hazards and could alsolead
to the design of nanomaterials with minimaltoxicity.7 In addition,
a thorough understanding of theproperties of nanomaterials that
determine biological
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responses would also facilitate the design of
betternanomedicines for the treatment of human disease.
In this review, we discuss the bridgingof nanotoxicological
research and nanomedicine.We suggest that a careful understanding
ofnanomaterial physicochemical properties, i.e., the‘synthetic
identity’, constitutes the bridge betweenthese two disciplines.
Moreover, we propose that the‘biological identity’ of nanomaterials
is determined,in part, by the adsorption of biomolecules onto
thenanomaterial surface upon introduction into a livingsystem. In
fact, as pointed out by Walkey and Chan8
in their excellent review on the protein corona asit applies to
nanomaterials, ‘once fully mapped, therelationships between
synthetic identity, biologicalidentity, and physiological response
will enableresearchers to predict the physiological responseof a
nanomaterial by characterizing its syntheticidentity’. This
statement points toward a predictivenanotoxicology, the ultimate
goal of which is todecode nanomaterial properties to enable
redesignof materials that are both useful and safe.9
NANOMATERIALS IN MEDICINE
Engineered nanomaterials offer great potential inmedical
applications.10 The variety of possibleapplications is very broad.
Here, we providesome highlights, and we attempt to emphasize
thephysicochemical properties that make nanomaterialsso favorable,
in particular for medical imaging anddrug or gene delivery.
Medical ImagingMedical imaging is typically based on the useof
contrast agents, which facilitate visualizationof tissues and
organs. For imaging, two basicproperties are required. First, the
contrast agentshould provide contrast compared to the
localenvironment, and second, the contrast agent shouldbe
specifically localized at the region of interest. Whatmay
nanoparticles contribute in this direction? Thefirst answer to this
question is relatively obvious:nanoparticles can provide higher
contrast because oftheir larger size compared to individual
molecules.Furthermore, instead of having only one fluorophorefor
fluorescence imaging, or just one chelated ion suchas Gd2+ to
provide contrast for magnetic resonanceimaging (MRI), several
fluorophores or Gd2+ ions canbe combined in one nanoparticle, and
this multivalentdisplay may provide higher contrast. In fact,
thecombination of different contrast agents in a singlenanoparticle
allows for multimodal imaging.11 The
combination of diagnostic and therapeutic functionsin a single
‘theranostic’ platform has also beenattempted. Yang et al.12
functionalized a reducedgraphene oxide–iron oxide nanoparticle
(RGO-IONP)complex with poly(ethylene glycol) (PEG), obtaininga
RGO-IONP-PEG theranostic nanoprobe that wasused for in vivo
trimodal fluorescence, photoacoustic,and MR imaging, uncovering
high passive tumortargeting, which was further exploited for
thermalablation of tumors in mice.
The second answer to the aforementioned ques-tion is less
obvious. Nonetheless, because of theirlarger size compared to
molecules, nanoparticlesare passively trapped in tumors; this
phenomenonis known as the enhanced permeability and reten-tion
(EPR) effect. Nanoparticles are small enoughto leak out from the
bloodstream into tumors, yetbig enough to be trapped in the tumor
vascula-ture. Hence, nanoparticles with many fluorophorescan
passively accumulate in tumors more efficientlythan the same
individual fluorophores.13 However,subsequent penetration into the
tumor itself is notreadily achieved. Wong et al.14 generated a
mul-tistage nanoparticle delivery system for deep pen-etration into
tumors. Hence, the gelatin core of100-nm nanoparticles was degraded
by proteasespresent in the tumor microenvironment thereby
releas-ing 10-nm quantum dots (QDs) after extravasation.Chauhan et
al.15 investigated how vascular normaliza-tion affects nanoparticle
delivery by studying whethera vascular endothelial growth factor
receptor-2-blocking antibody modulates nanoparticle
penetrationrates in mammary tumors in vivo. The authors
demon-strated that 12-nm particles penetrate tumors betterthan
larger particles (125 nm) once abnormal vesselsare repaired,
suggesting that small nanoparticles lessthan 12 nm are superior
because of higher tumorpenetration.
Kim et al.16 provided a particularly relevantexample of
nanoparticle-based imaging involving QD-based fluorescence
labeling, allowing for sentinellymph node mapping in large animals
under imageguidance. This approach could have significant impacton
such surgical procedures in cancer patients,provided that toxicity
of QDs is controlled. Thisis a nontrivial question, as QDs are
typically madefrom inherently toxic components such as cadmium,a
heavy metal with known adverse effects onhuman health. A recent
study in nonhuman primatessuggested that phospholipid
micelle-encapsulatedCdSe/CdS/ZnS QDs do not induce major signsof
toxicity up to 90 days postexposure; however,chemical analysis
revealed that most of the initial
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dose of cadmium remained in the liver, spleen, andkidneys.17
Finally, it is noteworthy that nanoparticles alsoafford
label-free detection. Hence, carbon nanotubeshave been shown to be
useful for photoacousticimaging, an approach that offers higher
spatialresolution and allows deeper tissues to be imagedcompared
with most optical imaging techniques.18
In a more recent study, Tong et al.19 reported thattransient
absorption microscopy offers an alternative,label-free method to
image both semiconducting andmetallic single-walled carbon
nanotubes (SWCNTs)in vitro and in vivo, in real time, with
submicrometerresolution.
Drug DeliveryNanoparticles clearly offer novel features for
‘smart’drug delivery.20 First of all, nanoparticles offerpotential
as passive carrier systems for delivery. Thisis due to the fact
that drug-loaded nanoparticlesinteract differently with cells than
the correspondingdrug alone.13 Nanoparticles can also be loaded
withdrugs in a way that allows for their slow release.Fine tuning
of the surface of nanoparticles allowsfor regulation of
nanoparticle interactions with cellsand thus the mode of
delivery.21,22 Nanoparticles canbe used to increase the local
concentration of drugsin, for instance, cancer cells. Ashley et
al.23 designedporous nanoparticle-supported lipid bilayers
termedprotocells that synergistically combined properties
ofliposomes and nanoporous particles. The protocellscan be loaded
with combinations of therapeuticagents, e.g., drugs or small
interfering RNAs. The veryhigh capacity of the high-surface area
nanoporouscore combined with the enhanced targeting efficacytoward
cancer cells enabled by the fluid-supportedlipid bilayer enabled a
single protocell loaded witha drug cocktail to kill a
drug-resistant humanhepatocellular carcinoma cell, representing a
million-fold improvement over comparable liposomes. Furtherin vivo
studies are certainly warranted. Davis et al.24
administered nanoparticles functionalized with atargeting ligand
(transferrin) systemically to a smallnumber of cancer patients and
were able todemonstrate successful RNA interference, i.e.,
specificinhibition of gene expression.
Notably, in a recent landmark study, the gapbetween preclinical
development and clinical transla-tion was bridged using targeted
doxorubicin-loadednanoparticles.25 The nanoparticles were
developedfrom a combinatorial library of more than 100 tar-geted
nanoparticle formulations varying with respectto particle size,
targeting ligand density, surface
hydrophilicity, drug loading, and drug release prop-erties. In
tumor-bearing mice, rats, and nonhumanprimates, doxorubicin-loaded
nanoparticles displayedpharmacokinetic characteristics consistent
with pro-longed circulation of nanoparticles in the
vascularcompartment and controlled release of the drug. Inaddition,
clinical data in patients with advanced solidtumors indicated a
pharmacokinetic profile consis-tent with the preclinical data as
well as some casesof tumor shrinkage at doses below the
solvent-baseddoxorubicin formulation dose typically used in
theclinic.25 This study shows that the ‘valley of death’between
preclinical research and clinical applicationscan be bridged
through a rational design approach(Figure 1).
Moreover, inorganic nanoparticles can be usedfor introducing new
functionalities. Here, twofascinating examples are given. First,
magneticnanoparticles can be used for locally trappingdrugs (which
are attached to the nanoparticles) byapplication of magnetic field
gradients.26 Magnetictargeting has been applied both in vitro27 and
invivo.28 Although clinical applications so far arelimited to pets,
this technology has the potentialfor being applied to humans in the
future, inparticular in cases of tumors close to the skin,as
sufficiently high magnetic field gradients can bedirected to the
body surface.28 Second, plasmonicnanoparticles, in particular those
based on gold,can be used for light-controlled release of
drugs.29
Upon optical excitation at the plasmon resonancefrequency,
collective motion of electrons ultimatelyleads to dissipation and
thus local heating of theenvironment of the nanoparticle surface.30
Initially,gold nanoparticles directed to tumor tissue have beenused
for local tissue destruction by light-inducedheating, also referred
to as hyperthermal ablation.31
Hence, nanoparticles may not only deliver drugs butcan also act
as therapeutic agents per se (furtherexamples of such
nanomaterial-intrinsic effects arediscussed below). The same
phenomenon, however,can be employed for controlled delivery. Upon
heatformation on the nanoparticle surface, molecularbonds can be
broken and attached molecules canthus be released based on light
triggers. Double-stranded DNA is a good linker, as it can be
moltenat temperatures well below the boiling point of
water.Light-controlled heating can also be used for theregulated
opening of the nanoscale containers.32 Thismay thus enable release
of drugs by local illumination.Because of absorption of light by
tissue [even inthe near-infrared (NIR)], the most likely in
vivoapplications will be for tumors located close to theskin.
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phys-chem
characterization
Nanomedicine
Nanotoxicology
Synthetic andbiological identity
Clinical trials
Pre-clinical studies
(a)
(b)
FIGURE 1 | Bridging nanotoxicology and nanomedicine. We
positthat careful assessment of the physicochemical properties of
engineerednanomaterials constitutes the ‘bridge’ between
nanotoxicologicalresearch and nanomedicine insofar as a detailed
understanding ofmaterial properties, i.e., the ‘synthetic identity’
is critical both fortoxicological assessment of nanomaterials and
for the development ofnovel nanomedicines (a). Furthermore,
understanding the ‘synthetic’and ‘biological’ identities of
nanomaterials will facilitate the bridging ofpreclinical studies
and the use of nanomaterials in medical imaging,drug delivery, and
regenerative medicine (b). The ‘biological’ identity ofa
nanomaterial is largely determined by the ‘corona’ of
biomoleculesthat forms in a biological environment; see text for
details.
Regenerative MedicineNanomaterials may also have considerable
impacton regenerative medicine, i.e., the replacement
orregeneration of human cells, tissues, or organs.33 Forinstance,
magnetic nanoparticles can be used to imageand guide stem cells to
their target in stem cell-basedtherapies.34,35 Cells interact with
the surroundingenvironment by making nanoscale interactions
withextracellular signals and nanomaterials can beemployed as
biomimetic scaffolds to stimulate tissuegrowth. Intriguingly,
supramolecular nanostructuresthat mimic, for instance, a growth
factor canbe used as a strategy for tissue regeneration
andrepair.36,37 Furthermore, in a recent clinical study,Jungebluth
et al.38 reported the first transplantationof an artificial trachea
in a cancer patient. Aftercomplete tumor resection, the patient’s
airway wasreplaced with a tailored bioartificial
nanocompositepreviously seeded with autologous bone
marrowmononuclear cells in a bioreactor. The cellsdifferentiated
into appropriate cell types. There areseveral advantages to this
approach. For instance, byusing the patient’s own stem cells to
populate the
scaffold, there are no concerns over rejection of
thetransplant.39
SAFETY ASSESSMENTOF NANOMATERIALS
Rational design of ‘nanomedicines’ began almost halfa century
ago, and several products including lipo-somes (i.e., passive
nanoscale carriers) have enteredinto routine clinical use (see
Duncan and Gaspar40 foran excellent historical perspective).
However, count-less other, more sophisticated nanomedicines are
inthe pipeline and the potential risks to human healthof these
novel entities need to be seriously considered.The latter is
certainly true for all pharmaceutical prod-ucts. Nanotoxicology
attempts to investigate the inter-actions of nanomaterials with
biological systems.41
However, there are several important and complicat-ing aspects
to address in nanotoxicological studiesincluding not only the need
for standardized assaysand reference materials9 but also the issue
of themost appropriate dose metric to use (surprisingly,
thisremains largely unresolved), and it may as yet be tooearly to
draw general conclusions regarding toxicityof nanomaterials; the
prevailing view today is thatnanomaterials should be studied on a
case-by-casebasis.42 Nevertheless, some lessons can be garneredfrom
studies conducted over the past several years. Inthe following
sections, we will discuss why engineerednanoparticles are
potentially hazardous, with the aimto elucidate physicochemical
properties that have beenlinked to toxicity. We also provide an
overview ofemerging trends in nanotoxicology including
high-throughput screening (HTS) and in silico
modelingapproaches.
High-Throughput ScreeningAs pointed out recently,43 results of
toxicological stud-ies using extraordinarily high doses of
nanomaterialshave to be interpreted with caution. Indeed, while
invitro tests may prove useful for hazard identification,in vivo
studies are needed to bridge the gap betweencell culture model
systems and the human exposuresituation, in order to understand
whether nanomate-rials pose any risk to human health. At the same
time,it is not ethically, economically, or practically feasi-ble or
reasonable to screen all nanomaterials usinganimal models.
Moreover, a model is only a model(and ‘essentially, all models are
wrong, but some areuseful’, as the statistician George Box famously
wrote)and we would be amiss to assume that results com-ing from
animal studies are always relevant.44 How,then, do we move forward?
Lai45 has proposed a
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nanotoxicity testing strategy based on short-term invivo animal
studies (i.e., shorter than a conventional90-day study) in
conjunction with HTS and mecha-nistic in vitro studies, and
comparing the data withthose of reference nanomaterials for the
specific sub-class in question—an approach in concordance withthe
‘Toxicity Testing in the 21st Century’ strategy forchemicals.46
To this end, more advanced in vitro modelsare needed, in
particular, assays that can be adaptedfor HTS. Huh et al.47
reported on a biomimeticmicrosystem that reconstitutes the critical
functionalalveolar-capillary interface of the human lung. This‘lung
mimic’ revealed that cyclic mechanical strainaccentuates toxic and
inflammatory responses of thelung to silica nanoparticles. The
authors concludedthat mechanically active ‘lung-on-a-chip’
microdevicesthat reconstitute tissue–tissue interfaces critical
toorgan function may provide low-cost alternatives toanimal studies
for toxicity testing. The ‘lung mimic’might also be amenable to
HTS.47
Naturally, it is important to validate in vitroassays. Han et
al.48 administered doses of titaniumdioxide nanoparticles of
different sizes (3–100 nm)to a rat alveolar epithelial cell line in
vitro andthe same nanoparticles by intratracheal instillationin
rats in vivo to examine the correlation betweenin vitro and in vivo
responses. The in vivo endpointwas the number of neutrophils in
bronchoalveolarlavage fluid following exposure to nanoparticles.The
correlations were based on toxicity rankingsof nanoparticles after
adopting surface area as dosemetric and response per unit surface
area as responsemetric. Slope analyses of the dose response
curvesshowed that in vitro and in vivo responses werewell
correlated. This study underlines the importanceof determining the
appropriate dose metric innanotoxicity studies. Shaw et al.49
applied a high-content approach, i.e., a battery of test for
multipleendpoints using multiple cell lines to test
nanoparticlesand derived detailed structure–activity
relationshipsfor the various nanomaterials tested.
Importantly,nanoparticles with similar activity profiles in
vitroexerted similar effects on monocyte numbers invivo.
HTS is a method for scientific experimentationthat comprises the
screening of large chemical librariesfor activity against
biological targets via the useof automation, miniaturized assays,
and large-scaledata analysis.50 HTS techniques have emerged asa
potentially useful tool to predict the possiblehazards of
nanomaterials.51,52 However, the fact thatnanomaterials may
interfere with commonly used invitro assays needs to be taken into
account.4 Indeed,
novel nanotoxicity assays based on label-free detectionof
cellular responses are needed.53
Mortimer et al.54 demonstrated that the so-called kinetic Vibrio
fischeri luminescence inhibitiontest is a potentially useful tool
for screening of thetoxicity of nanomaterials that can be adapted
forHTS of ecotoxicological effects of nanomaterials.Jan et al.55
reported on high-content screening for‘fingerprinting’ of
nanomaterials using cancer celllines of neuronal and hepatic
origin. George et al.56
provided evidence that an in vitro-based HTSapproach combined
with in silico data handling andzebrafish testing may constitute a
paradigm for rapidscreening of nanomaterials.
In Silico (Modeling) ApproachesToxicology assessment of
nanomaterials is expensiveand time-consuming. Therefore, in
addition toexperimental approaches for hazard assessment, thereis a
need for in silico methods in order to developstructure–activity
relationships that correlate toxicityendpoints. These
structure–activity relationships canbe quantitative or qualitative
in nature and theycan predict toxicological effects directly from
thephysicochemical properties of the entities, e.g.,nanoparticles
of interest.57
There are currently only a handful of nano-QSAR modeling
studies. In one recent study, theauthors developed a model to
describe the cytotoxicityof 17 different types of metal oxide
nanoparticles toEscherichia coli. The model was found to
reliablypredict the toxicity of metal oxide nanoparticles.58
Using a more extensive dataset of 109 nanoparticlespossessing
the same metal core but different organicmolecules on their
surface, Fourches et al.59 foundthat the cellular uptake of
nanoparticles can bepredicted by taking into account the
chemicalstructure of the coating molecules. The chemical
orstructural properties of nanomaterials are representedby
mathematical objects called descriptors, manyof which can be
calculated rather than measured.Examples of descriptors suitable
for nanomaterialsinclude particle size, shape, and surface
area,ionization potentials of metals, zeta potentials,
andphysicochemical properties of molecules covalentlybound to
nanoparticle surfaces.57 In a physiologicalenvironment,
nanoparticles selectively absorb proteinsto form a nanoparticle
‘corona’, a process governedby molecular interactions between
chemical groups onthe nanoparticle surfaces and the amino acid
residuesof the proteins (see below). Recently, a biologicalsurface
adsorption index (BSAI) was developed basedon the competitive
adsorption onto nanoparticles of
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a set of small-molecule probes that mimic aminoacid residues.60
By assuming that the adsorptionwas governed by five basic molecular
forces, themeasured adsorption coefficients were used to
developdescriptors, which, in turn, could be used to predict
theadsorption of small molecules to other nanomaterials.In a
subsequent study of a panel of 16 differentnanomaterials, the
nanomaterials were classified intodistinct clusters according to
their surface adsorptionproperties.61 It will be of interest to see
whether theBSAI could be used to predict the formation of aprotein
corona in a physiological setting.
Linking Toxicity to Material PropertiesTo systematically
investigate toxic effects of thenanoparticles, it would be highly
desirable tocorrelate their toxic effects with their
physicochemicalproperties.5,62 However, unfortunately, this
approachis not straightforward, as many physicochemicalproperties
are strongly entangled and are difficult tocontrol independently.7
Nevertheless, in the followingsection, we discuss selected studies
showing howmaterial properties may be linked to toxicity.
Carefulassessment of material properties serves as thebridge
between nanotoxicology and nanomedicine(Figure 1).
Size matters, in particular, for cellular uptakeof
nanoparticles. Moreover, in general, the greaterthe intracellular
dose of nanoparticles, the more thetoxic effects they generate.
Chan and coworkersdemonstrated in a series of experiments that
therecan be an optimal size for nanoparticle uptake.63,64
Similar claims have been made by several otherauthors, but the
latter work stands out, as sizewas controlled in an exclusive way.
Indeed, thenanoparticles were colloidally stable and thus werenot
agglomerated, i.e., they did not have the effectivelylarger
diameter of an agglomerate of nanoparticles,and surface chemistry
was the same for all sizes. In thisway, the size dependence of
nanoparticle uptake andcytotoxicity could be investigated. However,
cellularuptake is not mandatory for cytotoxicity to
occur:cobalt–chromium nanoparticles can damage humanfibroblasts
across an intact cellular barrier withouthaving to cross the
barrier. The outcome, whichincludes DNA damage without significant
cell death,is different from that observed in cells subjected
todirect exposure to nanoparticles.65
Also, shape can be important, though it isprobably overrated.
The classical example is carbonnanotubes, which are thought to
exert toxicity byvirtue of their ‘needle-like’ shape, i.e., an
extremelyhigh aspect ratio enabling these materials to piercecell
membranes. This may be relevant at least for
multiwalled carbon nanotubes (MWCNTs) with highwidth and,
therefore, high rigidity.66 Interestingly, inthe latter study, thin
and thick nanotubes similarlyaffected macrophages, while the
deleterious effects ofcarbon nanotubes on human mesothelial cells
werediameter-dependent. However, it is important to askwhen a fiber
is a fiber, and when is it, effectively, aparticle? Murray et al.67
have recently shown thatit is important to factor in agglomeration
whenassessing the in vivo toxicity of SWCNTs. Shape caninfluence
the mode of cellular uptake. Consider a rod-shaped nanoparticle and
a spherical nanoparticle ofthe same volume: the leading edge of the
rod-shapednanoparticle has a much smaller cross-section and
maytherefore penetrate cell membranes more effectively.However, in
many studies, in particular, in theoreticalsimulations, aspect
ratios are calculated for thenanoparticle cores, neglecting the
surface coating andthe adsorbed protein corona (discussed below),
whichreduces the effective aspect ratio and thus nullifiespotential
shape effects. Furthermore, agglomerationin physiological media may
rule out effects ofthe shape of individual nanoparticles.68
Schaeublinet al.69 investigated two gold nanoparticles
withdifferent aspect ratios using a keratinocyte cell line andfound
that gold nanospheres were nontoxic, whereasthe gold nanorods
induced apoptosis. Notably, bothnanoparticles formed agglomerates
in cell culturemedium, but the spherical particles had a large
fractaldimension (i.e., tightly bound and densely packed)while the
nanorod agglomerates had a small fractaldimension (i.e., loosely
bound).
Surface charge strongly influences uptakeof nanoparticles. In
general, positively chargednanoparticles are incorporated faster by
cells thannegatively charged ones, which is typically explainedby
the overall net negative charge of cellular surfaces.Although
studies exist that demonstrate that insome cases positively charged
nanoparticles interactwith cells differently when compared to
negativelycharged ones, resulting in different mechanisms
ofcytotoxicity, the higher toxicity of positively
chargednanoparticles is generally correlated to their
enhancedcellular uptake.70 To elucidate surface charge-dependent
toxicity, nanoparticles with differentsurface charge, but with
other physicochemicalparameters constant are required, which often
isexperimentally complicated to achieve.71,72 However,as pointed
out by Walkey and Chan,8 the proteincorona tends to give
nanomaterials a zeta potentialof about −10 to −20 mV irrespective
of thenanomaterial chemistry; this ‘normalization’ of
zetapotentials is related to the fact that most plasmaproteins
carry a net negative charge at physiological
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pH. In other words, the ‘biological’ identity mayoverride the
‘synthetic’ identity.
Perhaps, the most important physicochemicalparameter that
interferes with most others is colloidalstability. Obviously,
agglomerated nanoparticles donot have the size of the individual
nanoparticles butthe size of the agglomerate. This means,
therefore,that unless nanoparticles are very well dispersed,
anystatement about size- or shape-dependent uptake orcytotoxicity
is not sound, as the cell would interactwith the agglomerates and
not with the individualnanoparticles. Besides the fact that
agglomerationmasks effects of other physicochemical parameters,
itcan also directly affect interaction with cells.
‘Sticky’agglomerates of nanoparticles tend to precipitate ontop of
cells and thus can cause cytotoxic effects.73
Many metal and metal oxide nanoparticlescan undergo dissolution
within acidic compartments(lysosomes) in the cell which could drive
toxicity.This phenomenon, sometimes referred to as aTrojan
horse-type uptake mechanism because itcircumvents the plasma
membrane barrier and allowstoxic ions to ‘sneak’ into cells, has
been shown,for instance, for oxides of zinc, iron, manganese,and
cobalt.74 Cho et al.75 evaluated the pulmonaryinflammogenicity of
15 different metal/metal oxidenanoparticles and showed that
toxicity of thenanomaterials displayed a significant
correlationwith one of two physicochemical parameters:
zetapotential under acid conditions for low-solubilitynanoparticles
and solubility (degree of dissolution) forhigh-solubility
nanoparticles. The authors suggestedthat in the case of
high-solubility nanoparticles,inflammogenicity depends on the ions
that areproduced during dissolution of nanoparticles insidethe
acidic phagolysosomes of the cells.
Catalytic effects at the nanoparticle surfaceplay an important
role in the generation of reactiveoxygen species (ROS).76 Sayes et
al.77 studied theeffects of titanium dioxide nanoparticles in
cellculture and found that the extent to which nanoscaletitania
affected cellular behavior was not dependenton surface area; what
did correlate strongly tocytotoxicity, however, was the phase
composition ofthe nanoscale titania insofar as anatase TiO2 was
100times more toxic than rutile TiO2. The most
cytotoxicnanoparticle samples were also the most effective
atgenerating ROS.
In synopsis, it may seem disappointing thatone cannot pinpoint
how a certain physicochemi-cal parameter influences the toxicity of
(all) nano-materials. This is due, in part, to the fact thatmany
studies published to date are based on poorlydefined nanoparticles,
in which many physicochemical
parameters are entangled. In fact, it is nontrivial tochange
only one physicochemical parameter, withoutaffecting others. In
addition, not all nanomaterials arecreated equal. Thus, a
conclusive picture remains elu-sive. To be more conclusive,
toxicity studies should beperformed with well-defined model
nanoparticles, inwhich specific particle properties can be
independentlyvaried. Advanced synthesis approaches are pointingin
this direction, for instance by creating nanoparti-cles in which
surface charge can be tuned (almost)independently from other
particle properties.72 How-ever, most studies are performed with
nanoparticlesof poor definition and/or agglomerated
nanoparticlesystems. To remedy this situation, enhanced
commu-nication between material scientists and toxicologistsis
needed.
THE NANO-BIO-CORONA CONCEPT
To go one step further in terms of understandingthe interactions
of engineered nanomaterials withliving systems, we need to consider
the fact thatnanomaterials may adopt a ‘new’ identity throughthe
adsorption of biomolecules, a phenomenon that,in turn, is linked to
nanomaterial-intrinsic properties,e.g., size (surface curvature)
and hydrophobicity.Indeed, as stated recently by Mahon et al.,78
‘pristinenanoparticles in biological fluids act as a scaffoldfor
biomolecules, which adsorb rapidly to thenanoparticle surface,
conferring a new biologicalidentity’. Furthermore, the formation of
a ‘bio-corona’ on nanoparticles is an inherently
bilateralphenomenon, as proteins that adsorb to
nanoparticlesurfaces may also alter their behavior as a resultof
unfolding79 or fibrillation.80 The opsonization ofparticles with
serum proteins is, however, not really a‘new’ phenomenon81 even
though recent research hasprovided new insights into the parameters
that controlthis process.
It is generally believed that immediately aftercontact with
biological media, an initial corona isformed on nanoparticles by
loosely bound, low-affinity proteins. Prolonged incubation in
plasmaallows the formation of a denser, irreversibly attached‘hard’
corona with high-affinity proteins82,83 anda satellite ‘soft’
corona that undergoes intensiveexchange with the surrounding
media.84 The ‘hard’corona is formed because of the direct
interactionof proteins with the surface of the
nanoparticles,whereas protein–protein interactions dominate
theinteractions of the ‘soft’ corona with the ‘hard’corona.85 The
time scale of the process probably isvery short. The ‘hard’ protein
corona that is stronglyattached to the surface of nanoparticles is
likely
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the most relevant one for the in vivo fate of long-circulating
nanoparticles.86 Furthermore, changes inthe ‘hard’ corona may occur
when nanoparticlesare transferred to a new biological
compartment,e.g., upon translocation of nanoparticles across
theplasma membrane.86 Recent studies have shown thatthe corona of
biomolecules attached to nanoparticlesis degraded by the protease
cathepsin L within theendosomal compartment following endocytosis
ofnanoparticles.87 This needs to be taken into accountwhen
designing nanomaterials for intracellularapplications. Sund et
al.88 noted that the binding ofcytoplasmic proteins depends on the
surface chemistryof the nanoparticles. Hence, uncoated anatase
andrutile phases of TiO2 nanoparticles adsorbed proteinssimilarly,
whereas alumina and silicone-coated rutileforms of TiO2 bound only
a few proteins.
Walkey and Chan8 recently provided acompilation of 26 published
studies on the plasma-derived protein corona, and concluded that
‘theprotein corona is complex, that there is noone ‘universal’
plasma protein corona for allnanomaterials, and that the relative
densities ofthe adsorbed proteins do not, in general, correlatewith
their relative abundances in plasma’ (in otherwords, there is a
degree of specificity). Instead,it is suggested that the protein
corona dependson the ‘synthetic identity’ of each
nanomaterial.8
Indeed, the adsorption of biomolecules is drivenby surface
charge, hydrophobicity/hydrophilicity, andparticle size.83,89,90
Our recent studies show thatsuperparamagnetic iron oxide
nanoparticles (SPIONs)with different surface coating display
distinct plasmaprotein corona compositions (Vogt et al.,
manuscriptin preparation). Does the bio-corona cover
thenanoparticle surfaces completely or will targetingligands remain
accessible? Simberg et al.91 reportedthat both the dextran coat and
the iron oxide core ofdextran-coated SPIONs remained accessible to
specificprobes after incubation in plasma, suggesting that
thenanoparticle surface could be available for recognitionby cells
despite the bio-corona.
The majority of bio-corona studies have beenperformed with
plasma proteins,92 which is certainlyrelevant in cases when
nanoparticles are administeredinto the bloodstream. Nevertheless,
it is important toalso consider other portals of entry of
nanomaterialsinto the body, e.g., via inhalation or through theskin
or via the gastrointestinal tract as the coronacomposition is
likely to change as a function of theanatomical site and the
specific biofluids encounteredat each of these sites. Kapralov et
al.93 reported onthe in vivo formation of a lipid–protein corona
onthe surface of SWCNTs following administration
by pharyngeal aspiration in mice. The bio-coronawas identical to
lung surfactant and subsequent invitro studies demonstrated a role
for the surfactantcorona of lipids + proteins in macrophage uptake
ofcarbon nanotubes. Of note, plasma protein adsorptionto MWCNTs is
influenced by prior adsorption ofpulmonary surfactant lipids.94
There are, overall, few studies on long-termeffects of
nanomaterials and few, if any, of thesestudies have addressed the
potential role of the‘intrinsic’ versus the ‘biological’ identity
of thenanomaterials in question. Nevertheless, it may beuseful to
consider whether the bio-corona plays a roleunder such conditions.
In a recent study, Ruge et al.95
studied the impact of lung surfactant componentson macrophage
clearance of nanoparticles andthey concluded that because of the
interplay ofboth surfactant lipids and proteins, the
alveolarmacrophage clearance of nanoparticles is essentiallythe
same, regardless of different intrinsic surfaceproperties. The
latter study thus suggests that the‘biological’ identity may
override the ‘synthetic’identity of nanoparticles (at least in the
short term).However, we postulate that in the long term,
material-intrinsic properties (i.e., the ‘synthetic’ identity)
willcome into play and the long-term fate of nanoparticleswill
depend largely on whether the nanoparticlesundergo dissolution
and/or are susceptible tobiodegradation, or whether they escape
clearance bythe reticuloendothelial system and are
subsequentlycleared from the body. Indeed, in the chronic phase,at
which point the nanoparticles have left the systemiccirculation and
have been uptaken by cells, thebody’s own responses to the
nanoparticles maypredominate. For instance, inhalation of SWCNTsin
mice will trigger a cascade of pathological eventsrealized through
early inflammatory responses andthe induction of oxidative stress
culminating in thedevelopment of multifocal granulomatous
pneumoniaand interstitial fibrosis.96 Thus, while the
carbonnanotubes represent the initial offending trigger,
thelong-term effects (including potential carcinogeniceffects) are
manifested through subsequent cellularresponses to this trigger;
moreover, such organ andtissue responses may follow a common
pattern ofhost defense reactions (oxidative stress,
inflammation,etc.) toward foreign intrusion. In another
recentstudy, Mahler et al.97 showed that chronic oralexposure to
polystyrene nanoparticles can influenceiron uptake and iron
transport in an in vivo chickenintestinal loop model. Importantly,
chronic exposurecaused remodeling of the intestinal villi in
exposedanimals, which increased the surface area availablefor iron
absorption. In other words, the physiological
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responses triggered by the nanoparticles (in thiscase, tissue
remodeling) may determine long-termoutcomes, not the nanoparticles
per se and not thebio-corona.
Controlling the Bio-CoronaFrom a nanomedicine point of view, it
may bedesirable to avoid ‘nonspecific’ protein adsorption,i.e.,
bio-corona formation. This is commonly achievedby grafting PEG onto
nanoparticles; this may preventnonspecific uptake of nanoparticles
by cells of theimmune system, thereby prolonging their half-life
incirculation.10 Modifying the surface of nanoparticleswith an
antifouling polymer makes the proteinadsorption thermodynamically
unfavorable, whilethe high-molecular-weight polymeric chains
induceprotein ‘repulsion’ because of their
conformationalflexibility and induced steric hindrance.
PEGylationdoes not, however, prevent protein
adsorptionaltogether.78 Increased PEG grafting density on
thesurface of gold nanoparticles positively correlates witha
decrease in total protein adsorption and reduceduptake in J774A.1
murine macrophages.98
On the other hand, one may considerto exploit the bio-corona
phenomenon for tar-geting purposes.
PEG-polyhexadecylcyanoacrylate(PEG-PHDCA) nanoparticles have been
shown totranslocate into the brain after intravenous injectionin
rats, whereas PHDCA nanoparticles do not. Kimet al.99 found that,
after incubation with rat serum,apolipoprotein E (ApoE) adsorbed
more onto PEG-PHDCA than onto PHDCA nanoparticles. Moreover,ApoE or
ApoB-100 preadsorption onto PEG-PHDCAnanoparticles was required for
efficient penetrationinto rat brain endothelial cells. These data
suggestthe involvement of apolipoproteins in the transportof
PEG-PHDCA nanoparticles across the blood–brainbarrier, which could
be deployed for delivery of drugsinto the brain. Prapainop et
al.100 attempted cell-specific uptake of nanomaterials by
‘reprogramming’of the behavior of the protein corona on
nanomateri-als. Specifically, the surface of CdSe/ZnS QDs
possess-ing an amino-functionalized, PEGylated hydrophilicsurface
was decorated with the inflammatory metabo-lite, cholesterol
5,6-secosterol atheronal-B, and theresulting nanoparticles were
shown to bind to andinduce the misfolding of apolipoprotein B
leadingto uptake by RAW264.7 murine macrophages. Aspointed out by
the authors, the ability to programthe bio-corona on nanoparticles
with small moleculescould be developed to direct nanoparticles into
celltypes that they may not have been able to reachbefore.100
Impact of Bio-Corona on Cellular FunctionsThe protein corona has
been shown to play animportant role in modulating uptake and
toxicityof SWCNTs.101,102 However, it remains to be
firmlyestablished whether the biological identity of nanopar-ticles
is the result of a specific protein(s) in thenanoparticle corona or
a nonspecific effect relatedto the fact that proteins may alter the
agglomerationbehavior of nanoparticles leading to a difference
incellular uptake, which, in turn, has an impact on cyto-toxicity.
Ehrenberg et al.103 reported that the capacityof polystyrene
nanoparticle surfaces to adsorb pro-tein is indicative of their
tendency to associate withcells. However, removal of the most
abundant pro-teins from cell culture media did not affect the
levelof cell association, and the authors concluded thatcellular
association is not dependent on the identityof adsorbed proteins.
Lartigue et al.104 studied theadsorption of proteins on
biomedically relevant ironoxide nanoparticles by magneto-optical
birefringence;the effect of plasma at different concentrations
rang-ing from 1 to 100% on nanoparticle behavior wasassessed. It
was noted that at low plasma concen-trations (representative of
most in vitro conditions),the nanoparticles tended to form clusters
triggered byproteins such as fibrinogen, whereas at high
plasmaconcentrations (closer to the physiological situation)other
proteins such as apolipoproteins tended to coatand subsequently to
stabilize individual nanoparticles.This, in turn, affected in vitro
uptake by macrophages.Lesniak et al.105 reported that silica
nanoparticlesincubated with A549 cells in the absence of serumhave
a stronger adhesion to the cell membrane andhigher internalization
efficiency when compared withnanoparticles with a preformed surface
corona.
In a key study of the bio-corona phenomenon,Deng et al.79
demonstrated that negatively chargedpoly(acrylic acid)-conjugated
gold nanoparticles bindto and induce unfolding of fibrinogen,
whichpromotes interaction with integrin receptors onmacrophage-like
THP.1 cells, resulting in the releaseof inflammatory cytokines
(Figure 2). In a follow-up study, the authors showed that
fibrinogen boundwith high affinity to positively and negatively
chargedgold nanoparticles.106 However, only the negativelycharged
nanoparticles triggered cytokine release inTHP.1 cells, perhaps
because of a different orientationof the protein on the different
particles. Thus, whilecommon proteins may bind to different
nanoparticles,the physiological response may not be the same.
The complement system constitutes an impor-tant barrier to
infection or other foreign intru-sion. Nanoparticles may also
activate complement;
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00 2 4 6 8 10
50
100
150Size (nm)5 10 20
43557295
PAA-GNP (μg)
Unb
ound
fibr
inog
en (
%)
Mol
ecul
ar w
eigh
t (kD
a)
Unb
ound
fibr
inog
en (
%)
00 500Surface area (mm2)
1,000 1,500
50
100
150
D domain E domain D domain
~45 nm
C terminus ofα chain
C terminus ofα′ chain
Misfolded fibrinogen
NP NP THP.1 TNF-α
Bio-coronaformation
Mac-1
(a)
(c)
(d)
(b)
FIGURE 2 | Protein corona: role in proinflammatory
responses.Fibrinogen is the major human plasma protein bound by
poly(acrylicacid)-coated gold nanoparticles (PAA–GNP). (a) SDS–PAGE
of humanplasma proteins bound to PAA–GNP with diameters of 5, 10,
and20 nm. Three major protein bands were observed at 65, 55, and 45
kDa.(b) Unbound fibrinogen following pull-down with PAA–GNP
withdiameters of 5 nm (blue) or 20 nm (red). Purified fibrinogen
(0.6 mg)was incubated with increasing amounts of PAA–GNP. Inset:
unboundfibrinogen is plotted against total surface area for the
twonanoparticles. (c) Crystal structure of fibrinogen. The protein
was drawnusing Swiss-PdbViewer and coordinates for PDB entry 3GHG.
Commondomains are shown. Inset: the C-terminus of the g chain
(purple) thatinteracts with the Mac-1 receptor. (Reprinted with
permission from Ref79. Copyright 2011 Macmillan Publishers Ltd.)
(d) The schematicdiagram illustrates how unfolding of fibrinogen on
the surface ofPAA–GNP leads to interaction with the integrin
receptor, Mac-1, on thesurface of THP.1 monocytes, which in turn
increases NF-κB signalingleading to secretion of tumor necrosis
factor-α. It is pertinent to notethat fibrinogen, which has a
length of 45 nm and a diameter of5 nm, is much larger than the 5-nm
PAA–GNP. Deng et al.79 showedthat the maximum protein binding was 2
μg for the 5-nm PAA–GNP,which represents one to two nanoparticles
per fibrinogen molecule.
this may be viewed as a special case of bio-corona formation and
one that is of particularrelevance in nanomedicine. Nanomaterial
interac-tion with the complement system is complex andregulated by
interrelated physicochemical factorssuch as size, morphology, and
surface properties.107
Hamad et al.108 investigated polystyrene nanoparti-cles with
surface-projected polyethylene oxide chains
in ‘mushroom-brush’ and ‘brush’ configurations andfound that
distinct polymer architectures mediateswitching of complement
activation pathways. Aspointed out by the authors, these studies
suggest arational basis for the design of targetable nanosystemsfor
nanomedicine applications.
THE IN VIVO FATEOF NANOPARTICLES
In addition to understanding the synthetic and bio-logical
identities of nanomaterials, it is important totake into
consideration the context-dependent behav-ior of a nanomaterial. In
other words, to consider howthe biological identity of a
nanomaterial may changedepending on the specific biological
compartment (inthe body or within a cell). Indeed, as noted
previ-ously, ‘one of the key features of nanoscale materials,and
the one that may suggest novel and unantici-pated health risks, may
very well be the propensity ofsuch materials to cross biological
barriers in a man-ner not predicted from studies of larger
particles ofthe same chemical composition’.9 Here, we discusssome
studies illustrating how nanoparticles may crossbiological
barriers, and how material-intrinsic proper-ties may dictate such
interactions. We will also touchon factors that regulate
nanoparticle pharmacokinet-ics. Understanding the in vivo fate and
behavior ofnanomaterials is another area of common interest
innanotoxicology and nanomedicine.
Crossing Biological BarriersNanoparticles can cross biological
barriers and enterand distribute within cells by different
pathwaysand for this reason they are considered a primaryvehicle
for targeted therapies. In the body, we findcellular barriers that
include the cell membrane,and endosomal–lysosomal and nuclear
membranes,and physiological barriers that prevent extravasationof
foreign substances from the blood such as theblood–brain barrier.
The skin is the main barrier thatprotects our body from the
external environment.Understanding the barriers imposed by a
biologicalsystem is critical to the design of nanomaterials
forbiomedical applications (see Kievit and Zhang109
for an excellent review). It is also important toconsider
whether one should attempt to breachbiological barriers between
bodily compartments withnanoparticles as this may trigger
unexpected toxicitiesand disease processes.110
Yamashita et al.111 showed that silica andtitanium dioxide
nanoparticles with diameters of70 and 35 nm, respectively, can
cross the placenta
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and cause pregnancy complications when injectedintravenously
into pregnant mice. Larger (300 and1000 nm) silica particles did
not induce suchcomplications. It remains unclear if the
fetotoxicitywas caused by direct exposure to the nanoparticlesor by
the damage to the placenta. Nonetheless, thedetrimental effects
were abolished when the surfacesof the silica nanoparticles were
modified with carboxyland amine groups. Hence, size and surface
chargeboth may impact on the propensity of nanoparticlesto cause
damage to the unborn fetus. Similarly, Schlehet al.112 demonstrated
that size and surface chargeof gold nanoparticles determine
absorption acrossintestinal barriers and accumulation in
secondarytarget organs after oral administration in a ratmodel.
Choi et al.113 determined that nanoparticleswith hydrodynamic
diameter less than 34 nm withnoncationic surface charge translocate
rapidly fromthe lungs to regional lymph nodes in rats
followingintratracheal instillation. Furthermore, nanoparticleswith
a hydrodynamic diameter less than 6 nm werefound to traffic rapidly
from the lungs to lymph nodesand the bloodstream, ultimately being
cleared fromthe body through the kidneys. Moreover, as discussedin
further detail below, nanoparticle behavior wasfound to depend
strongly on surface coating. Thesefindings suggest strategies for
the rational design ofnanoparticles for drug delivery via lung
inhalation.Kannan et al.114 recently devised a prodrug approachto
treat cerebral palsy, a developmental disorderresulting from an
insult to a growing fetal or infantbrain. In this preclinical
study, N-acetyl-cysteine(NAC) was linked to polyamidoamine
dendrimersthat enabled NAC to cross the blood–brain barrier
andreach microglia and astrocytes. This nanoformulation(D-NAC) was
administered within 6 h of birth withimprovement in motor
performance and ameliorationof inflammation in newborn animals.
However, nanoparticles may disrupt or evenremodel biological
barriers. Mahler et al.97 reportedthat chickens acutely exposed to
carboxylatedpolystyrene nanoparticles had a lower iron
absorptionthan unexposed or chronically exposed birds.As mentioned
earlier, Chronic exposure causedremodeling of the intestinal villi,
which increased thesurface area available for iron absorption, and
thisincrease in intestinal surface area compensated forthe lowered
iron transport caused by nanoparticleexposure.
Biodistribution and Tumor TargetingPharmacokinetics is concerned
with quantifying theadsorption, distribution, metabolism, and
elimination(ADME) of chemicals and drugs in the body; the
aim is to relate drug dose or chemical exposure tobiological
effects.115 Evaluation of ADME propertiesof nanomaterials is
crucial for the medical imple-mentation of these materials. To this
end, in vivomodel systems are certainly needed. Riviere115
hasprovided a concise overview of studies on the invivo disposition
of fullerenes, carbon nanotubes, andQDs after parenteral
administration. Functionalized,water-soluble SWCNT and MWCNT may
negotiatethe glomerular filtration barrier and undergo
renalexcretion without extensive accumulation in the body,in a
manner dependent upon the degree of individu-alization of the
nanotubes.116,117 Notably, pristineSWCNTs may undergo enzymatic
biodegradation invitro118and in vivo119; biodegradation by
neutrophilsis promoted when the carbon nanotubes are coatedwith a
corona of immunoglobulins, which leads toenhanced cellular uptake
via Fc receptors expressed onneutrophils.118
How about the disposition of biomedically rel-evant
nanomaterials? Schädlich et al.120 investigatedthe influence of
the size of biodegradable PEG-PLAnanoparticles both in vivo and ex
vivo and foundthat nanoparticles of 111 and 141 nm accumulatedin
human xenograft tumor tissue while slightly biggernanoparticles
(166 nm) were rapidly eliminated by theliver. These studies
demonstrate how different biodis-tribution may occur because of
small nanoparticlesize differences. The importance of further
miniatur-izing nanocarrier size to optimize tumor accumula-tion and
penetration was recently shown121 (and seeabove, section on Medical
Imaging, for additionalexamples).
The EPR effect and/or targeting approaches mayenable nanoscale
carriers to reach a tumor, but thisdoes not necessarily mean that
the nanoparticles willalso penetrate into the tumor and deliver
their payloadof anticancer drugs. Cabral et al.122 compared
theaccumulation and effectiveness of different sizes(30, 50, 70,
and 100 nm) of long-circulating, drug-loaded polymeric micelles in
highly versus poorlypermeable tumors in a preclinical model, and
foundthat only the 30-nm micelles could penetrate poorlypermeable,
hypovascular pancreatic tumors to achievean antitumor effect.
Interestingly, the penetration andefficacy of the larger
nanoparticles could be enhancedby pharmacologically increasing the
permeability ofthe tumors.
Choi et al.113 followed the fate of intratra-cheally instilled
NIR fluorescent nanoparticles thatwere varied systematically in
size, surface modi-fication, and core composition and showed
thatnanoparticle behavior depends strongly on the sur-face coating,
which affects protein adsorption in body
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fluids; hence, for charged nanoparticles, nonspecificadsorption
of endogenous proteins, mostly albumins,resulted in a large
increase in hydrodynamic size ofthe nanoparticles, and this
affected the biodistributionof the nanoparticles following their
uptake in this ratmodel.
Finally, von Maltzahn et al.123 have provideda fascinating
example of ‘communicating’ nanopar-ticle systems based on
nanotechnological mimicry ofthe recruitment of immune cells to an
inflammatorylesion to improve in vivo tumor-targeting
efficiency.Hence, the authors designed multifunctional
systemswhereby the coagulation cascade in tumors is activatedby
photothermal heating of gold nanorods in order to‘broadcast’ tumor
location to clot-targeted nanopar-ticles, i.e., doxorubicin-loaded
liposomes coated withFactor XIII, a component of the coagulation
cas-cade. This approach, which thus takes advantage ofthe
endogenous coagulation cascade, yielded over 40times higher doses
of doxorubicin in tumors whenthe drug is loaded with Factor
XIII-covered liposomeswhen compared to plain liposomes.123
A CASE OF STOLEN IDENTITY
Nature may inspire the design of syntheticnanoparticles. Bertram
et al.124 developed ‘artificialplatelets’ based on Arg-Gly-Asp
(RGD)-functionalizednanoparticles, which halved the bleeding time
afterintravenous administration in a rat model ofmajor trauma. The
synthetic platelets consistingof poly(lactic-co-glycolic
acid)–poly-l-lysine blockcopolymer cores conjugated with PEG chains
termi-nated with RGD functionalities were cleared within24 h, and
no toxicity was seen up to 7 days postin-fusion. Hu et al.125
presented a novel approach inparticle functionalization by coating
biodegradablepolymeric nanoparticles with a corona of
naturalmembranes derived from red blood cells, includingboth
membrane lipids and associated membrane pro-teins, in order to
achieve ‘stealthy’, long-circulatingnanoparticles for drug
delivery. The latter study rep-resents an example of ‘borrowed
identity’ of nanoma-terials. Indeed, biomimetic design could be
exploitedfor drug delivery.126 Interestingly, the immune
systemutilizes its very own nanoparticles (exosomes) to trans-mit
information between cells. Exosomes may containboth mRNA and
microRNA, and the transferred exo-somal mRNA has been shown to be
translated inthe recipient cell.127 More recent studies
confirmedthat the transmitted microRNA is also functional.128
Hence, exosomes serve as a template for the delivery ofshort
RNAs for modulation of gene expression using
Physiological responses
Synthetic identity
NP NP
Biological identity
- tissue targeting
- cellular uptake
- cytotoxicity- cytokine secretion- immunogenicity- degradation,
excretion- etc
Bio-corona
Biomoleculeseg. protein, lipids
Material-intrinsicproperties
- size, shape, aspect-ratio- surface charge- colloidal
stability- stability / disssolution- catalytic properties- etc
Spatial determinants:- portal of entry- body fluid / organ-
subcellular compartmenttemporal determinants:- acute or long-term
effect
Context-dependentproperties
FIGURE 3 | Synthetic and biological identities of
nanomaterials.Schematic view of the ‘synthetic’ identity of
nanomaterials that isdetermined by material-intrinsic properties
and the ‘biological’ identitythat is manifested in a living system
and can be viewed as the sum ofthe context-dependent properties of
the nanomaterial. As discussed inthis review, the biological
identity is shaped, in part, by the adsorptionof biomolecules
(proteins and lipids) that form a ‘corona’ on the surfaceof
nanoparticles; the composition of the bio-corona depends on
theparticular biofluid (e.g., blood, lung fluid, and
gastrointestinal fluid) andmay exhibit dynamic changes as the
nanoparticle crosses from onebiological compartment to another. The
physiological responses tonanomaterials are dictated by the
synthetic and biological identities; apartial list of possible
biological/toxicological outcomes is shown in thisfigure.
nanoscale delivery vehicles that are, by
definition,biocompatible.
Stark has pointed out that nanoparticles dif-fer from molecules
in several respects; nevertheless, heconcludes that ‘from a
functional point of view, chem-ically well-defined nanoparticles
are an extension ofthe classical concept of the molecule’, as they
combinethe properties of solids with mobility (a property
ofmolecules).74 Indeed, certain nanoparticles appear tobridge the
gap between molecules and particles. Den-drimers are polymeric
nanoparticles with perfectlydefined structure and molecular
weight.129 Hayderet al.130 reported recently that
azabisphosphonate
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(ABP)-capped dendrimers selectively target monocytesand direct
them toward anti-inflammatory activation.The dendrimers also
exhibited antiosteoclastic activ-ity, thus preventing bone erosion.
Intravenous injec-tions of ABP-capped dendrimers inhibited the
devel-opment of inflammatory arthritis in two animalmodels. This
exciting study suggests that dendrimerscould function as novel
therapeutics for rheumatoidarthritis. Moreover, dendrimers
conjugated to glu-cosamine and glucosamine 6-sulfate were shown
topossess immunomodulatory and antiangiogenic prop-erties,
respectively, and when administered together,the nanoparticles
increased the long-term success ofglaucoma surgery in an animal
model by prevent-ing scar tissue formation.131 Thus, in some cases,
thesynthetic and biological identities of a nanomaterialappear to
blend into one: dendrimers may functionas drugs per se by virtue of
their unique physico-chemical properties, i.e., size and
multivalent surfacefunctionalities, which allow these nanoparticles
todirectly engage biological receptors and modulate
cellfunction.
CONCLUSIONS AND PERSPECTIVESThe Stone Age did not end because
they ran outof stones. New technologies inevitably replace oldones.
We are now at the dawn of a nanotech-nological revolution with
far-reaching implicationsfor society and it is crucial that we
ensure thesafety of these novel materials while not imped-ing their
implementation in important areas suchas in medicine. In this
review, we have attemptedto highlight the role of physicochemical
propertiesof engineered nanomaterials and their impact
onnanomaterial behavior in biological systems. Impor-tantly, a
growing body of evidence indicates that theadsorption of
biomolecules onto nanoparticle surfacesmay bestow a new ‘biological
identity’ onto thesematerials.8,78 This has considerable
ramifications notonly for nanotoxicological assessment of
syntheticnanoscale materials but also for their implementa-tion in
medicine. Of note, the bio-corona of serumproteins should not
necessarily be viewed as an unde-sirable biological phenomenon; the
bio-corona canbe controlled100 and may even be exploited for
drugdelivery.132 In addition, we would be amiss to
ignorefundamental physicochemical properties of nanoma-terials: the
cells may also ‘see’ what is beneath thecorona.
Careful assessment of material-intrinsic proper-ties and how
these properties are linked to physio-logical responses is thus
essential both in nanotoxi-cology and in nanomedicine. Notably, the
very sameproperty may be highly desirable for certain clini-cal
applications (for instance, the delivery of smallparticles to
exploit the EPR effect) but could alsoyield unwanted hazardous
effects. Taking into con-sideration not only the synthetic identity
but alsothe biological identity of nanomaterials, and howthese
identities may evolve over time and as afunction of different
biological compartments in thebody or at the subcellular level may
enable a betterunderstanding of nanomaterial-induced
physiologicalresponses (Figure 3). More studies are needed on
thelong-term effects of nanomaterials and on the rel-ative
importance of surface-adsorbed biomolecules(the bio-corona) versus
material-intrinsic propertiesof nanomaterials under such
conditions; understand-ing how common physiological reactions
(oxidativestress, inflammation, etc.) are triggered is also
ofimportance in order to mitigate adverse effects follow-ing
nanomaterial exposure. Furthermore, more refinedtechniques to study
the corona of adsorbed proteins,lipids, and other biomolecules on
nanomaterial sur-faces are warranted along with a greater emphasis
onthe potential impact of individual components of thecorona on
physiological responses. Bioinformatics-based approaches may prove
helpful when decipheringthe bio-corona data. New approaches
including HTSfor the rapid screening and ranking of the
hazardpotential of vast numbers of engineered nanomateri-als and
mathematical modeling of structure–activityrelationships of
nanomaterials may also facilitate thedevelopment of safe and useful
nanomaterials for invivo imaging, drug delivery, and other clinical
appli-cations.
ACKNOWLEDGMENTS
The authors are supported, in part, through grantsfrom the
Swedish Research Council for Environ-ment, Agricultural Sciences
and Spatial Planning(FORMAS), the Swedish Cancer and Allergy
Foun-dation, the Seventh Framework Programme of theEuropean
Commission (FP7-MARINA-263215 andFP7-NANOGNOSTICS-242264), and BMFM
Ger-many (Umsicht). BF holds a Senior Investigator Awardfrom the
Swedish Research Council.
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