Coatings 2014, 4, 139-159; doi:10.

3390/coatings4010139
OPEN ACCESS

coatings
ISSN 2079-6412
www.mdpi.com/journal/coatings
Review

Biomedical Nanoparticles: Overview of Their Surface

Immune-Compatibility
Olimpia Gamucci, Alice Bertero, Mariacristina Gagliardi and Giuseppe Bardi *

Center for MicroBioRobotics @SSSA, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34,
56025 Pontedera, Italy; E-Mails: [email protected] (O.G.); [email protected] (A.B.);
[email protected] (M.G.)

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +39-050-883-028; Fax: +39-050-883-101.

Received: 11 October 2013; in revised form: 8 January 2014 / Accepted: 30 January 2014 /
Published: 12 February 2014

Abstract: Diagnostic- and therapeutic release-aimed nanoparticles require the highest degree
of biocompatibility. Some physical and chemical characteristics of such nanomaterials are
often at odds with this requirement. For instance, metals with specific features used as
contrast agents in magnetic resonance imaging need particular coatings to improve their
blood solubility and increase their biocompatibility. Other examples come from the
development of nanocarriers exploiting the different characteristics of two or more
materials, i.e., the ability to encapsulate a certain drug by one core-material and the targeting
capability of a different coating surface. Furthermore, all these “human-non-self”
modifications necessitate proofs of compatibility with the immune system to avoid
inflammatory reactions and resultant adverse effects for the patient. In the present review we
discuss the molecular interactions and responses of the immune system to the principal
nanoparticle surface modifications used in nanomedicine.

Keywords: immune system; nanomaterials; immunogenicity; immunotoxicity; biodistribution;

mononuclear phagocytic cells; surface modifications
Coatings 2014, 4 140

1. Introduction

The focus of the present review is to describe the main molecular mechanisms of host defense
towards the principal surface modifications of nanoparticles (NPs) used in medical fields as diagnostics
or therapeutics.
A complex variety of organic materials (e.g., polymers, dendrimers, liposomes, proteins and
carbohydrates), allotropic forms of carbon (e.g., fullerenes, carbon nanotubes) and inorganic nanosized
particles (e.g., quantum dots, silica, gold or iron core NPs) are currently under investigation for drug
delivery and imaging purposes [1,2].
The nano size of drug delivery vectors is designed to improve solubility, pharmacokinetics and
biodistribution of small drugs, vaccines or other active molecules, otherwise less effective. Together
with these advantages, the introduction of such nano-systems may pose biocompatibility concerns that
go beyond the drug toxicological profile.
Complex living organisms have perfected their defense mechanisms towards exogenous pathogens
throughout the course of evolution. The immune system is able to distinguish between self and non-self
substances. The foreign molecules able to elicit immune responses by binding to specific host receptors
are called antigens (antibody generators). The immune system ensures a plethora of selective response
against several non-self molecules retaining immunological memory and preventing injury to host cells
during responses to microbes. Recognition and subsequent clearance of these alien elements are important
physiological mechanisms employed by the immune system to maintain the body homeostasis [3].

2. A Brief Description of Immune System Main Features

Different and subsequent lines of defense of increasing specificity are deployed by the immune
system to eradicate infections by exogenous organisms. Physical barriers, like the skin and mucosa, are
the first protection of the innate immunity to prevent the entrance of pathogens into the body. If infective
microorganisms overcome these barriers, another line of innate defense provides an immediate but
non-specific response. This line of control is carried out by different cellular and biochemical mediators.
Phagocytic cells, like neutrophils or macrophages, play a major role in wiping out the foreign pathogen
by encapsulating and destroying it with specific enzymatic reaction cascades. These cells recognize
unique molecular structures of microbial pathogens, called Pathogen-Associated Molecular Patterns
(PAMPs), by means of specific pattern-recognition receptors. Well known examples of PAMP-receptors
are the Toll-like receptors (TLRs) which recognize various microbial molecules including the
Gram-negative bacteria wall component lipopolysaccharide (LPS), the lipoteichoic acid (present on the
wall of Gram-positive bacteria) receptor or the receptors for mannose-rich oligosaccharides present on
the fungi. Innate immune cells are also able to respond to secondary effects like cell damages caused by
infections. Damage-Associated Molecular Patterns (DAMPs) indicate cell injuries induced by a myriad
of reasons, including toxins, traumas or decreased nutrient supply. Stress induced proteins, nuclear
proteins or crystal (i.e., monosodium urate) are recognized by DAMP receptors that initiate immune
responses. Furthermore, blood proteins can directly interact with the undesired invader and regulate its
fate. A heterogeneous group of cell derived proteins, named cytokines, synchronize all these immune
response mechanisms [3].
Coatings 2014, 4 141

By means of evolution, most of the vertebrates also possess a further level of protection, the adaptive
immune response, which can be activated by the innate immunity if pathogens have not been
successfully eliminated. The adaptive immune system improves its response during an infection and
“memorizes” pathogen features once it has been eliminated. The immunological memory allows a faster
and stronger reaction by subsequent exposure to an already recognized element. Indeed, this mechanism
is the basic principle of vaccination. The adaptive immunity is also called “acquired immunity”, as a
consequence of its responses against non-self elements “acquired” by experience. The peculiar cells of
adaptive immunity are the lymphocytes. These antigen-specific cells display receptors that are able to
recognize an incredible number of exogenous pathogens and to distinguish between very closely related
molecular structures.
We can describe two different types of adaptive responses, named humoral and cell-mediated
immunity, often working contemporaneously and in conjunction with the innate system. The humoral
immunity is mediated by proteins called antibodies. This family of proteins is secreted by B lymphocytes
(or B cells) once differentiated into antibody-secreting plasmacells. The process of recognition of
pathogens by B lymphocytes is also mediated by antibody receptors expressed on the cell surface that
trigger an intracellular signaling leading to cell differentiation upon foreign molecule binding. In
contrast, cell-mediated reactions begin with a coordinated interaction between a different type of
lymphocytes, the T lymphocytes (or T cells), and Antigen Presenting Cells (APCs). T cells are not
capable of producing antibodies and have a restricted specificity for antigens [4]. They recognize only
foreign peptides bound to host molecules, the major histocompatibility complex (MHC) exposed on the
presenting cells. One of the main tasks of adaptive cell-mediated immunity is to destroy microbes that
survive and proliferate inside the cells, like viruses. Functionally different subpopulations of
T lymphocytes play a role in the various kinds of infections. All these subsets are grouped in two main
families, known as T helper (THLs) and cytotoxic T cells (CTLs). The antigenic stimulation induces
T helper cells to secrete cytokines, which in turn foster proliferation and differentiation of T cells,
B cells, macrophages and other leukocytes. Cytotoxic T lymphocytes are equipped with specific set of
reactive proteins capable to kill cells that expose foreign antigens on their surface. A third group of
lymphocytes is represented by natural killer (NK) cells, which are principally involved in innate
response towards microbes and pathogens [5]. Lymphocytes and APCs circulate through the body and
home to the target sites of antigen exposure. Lymphoid organs, like spleen, liver or lymph nodes
represent the anatomical districts where these cells increase the possibility of antigen presentation and
start an immune response [6].
All the immune cells travel within the body in a strictly regulated manner. Differentiated cells from
all the tissues provide signals to drive the immune cell travelling, mainly releasing chemokines
(chemoattractant cytokines) [7]. This very special type of polypeptides has a conserved structure which
is maintained in the evolution, highlighting the importance of their role in cell migration. The tissue
modifications happening during inflammatory processes are intended for leukocytes recruitment from
the circulation to the injured tissue with the purpose of destroying pathogens. Indeed, the majority of the
cell movements implicated in the physiological tissue rearrangements and pathological conditions rely
on chemokine driven signaling [8].
Coatings 2014, 4 142

As well as microbes, most of the engineered nanomaterials that enter the body are recognized as
non-self by the immune system, which efficiently detects and tries to eliminate them through the
described mechanisms [9]. This biological event makes the immune system a crucial obstacle to deal
with, in order to apply nanotechnology to biomedicine.

3. Nanoparticle Interaction with Blood Components and Uptake by Phagocytic Cells

Critical physical and chemical features of NPs regulate the interaction with the immune system.
Surface electrostatic charges of engineered nanomaterials are fundamental to define immune
responses [10]. Positive-charged surfaces on the particles are often more cytotoxic than anionic and
neutral ones. Cationic NPs bind more efficiently than anionic charged or neutral molecules to the
negatively charged plasma membrane of target cells, which expose the negative charges of the
phospholipidic groups. Once NPs have entered the cell, NP surface positive charge slows down the
acidification of endosomes (responsible for the transport pathway from the plasma membrane to the
lysosome), thereby delaying the endosome–lysosome transition. Moreover, they can cause more
pronounced disruption of plasma-membrane integrity, stronger mitochondrial, lysosomal damage, and
increased number of autophagosomes [11,12]. Positively charged elements introduced into cells may
also form complexes with the negatively charged nucleic acids raising genotoxicity concerns. However,
a net positive surface charge helps the binding to plasma membranes. This physical effect can be
beneficial for drug delivery particles.
The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs
are important for the efficacy and selectivity of NPs. In general, neutral particles show lower interaction
with the cell membrane than charged (cationic or anionic) nanoparticles of the same size, due to the
lower number of electrostatic interactions between NP-surface and charged cell membranes. Designing
optimal coatings for drug delivery carriers requires a precise characterization of these physical interactions.
Drug or gene delivery nanocarriers for systemic injection in the bloodstream come across a biological
milieu composed of cells, proteins and solutes [13]. Injected NPs almost instantaneously adsorb and bind
different proteins, whose affinity kinetics depends on the nano-material surface, charge and size. The
protein layer created on the particle surface is called “corona” and it influences particle biodistribution
and circulation time. Proteins that increase the clearance of exogenous particles by specialized
mononuclear phagocytes are called opsonins. Blood released antibodies, for example, principally work
as opsonins enhancing phagocytosis. Molecules that activate the complement system (see below) are
also considered opsonins. Their binding to NPs injected in the bloodstream leads to the attachment of
macrophages followed by cell internalization. This type of internalization is highly regulated by cells
surface receptors and their capability of binding to the particular protein corona attached to the particle.
A detailed characterization of NP-specific opsonization process will help to design nanoparticle with
future clinical relevance.
The protein corona hiding the surface of NPs determines the effective size and the final
beneficial/dangerous effects of the material. Besides enhancing particle recognition by the host immune
cells, protein adsorption/opsonization increases the hydrodynamic diameter (HD) which contributes to
modify NP accumulation and tissue distribution. Generally HD is greatly larger than the NP diameter
measured after their synthesis. The HD of particles injected in the bloodstream is inversely related to
Coatings 2014, 4 143

glomerular filtration rate in the kidney, ultimately regulating the blood half-life of the solutes [14].
Molecules with an HD < 5 nm achieve a rapid equilibrium with the extravascular extracellular space
(EES), whereas larger particles have prolonged circulatory times due to slow transport across the
endothelium [15,16].
The opsonization process of NPs may involve the blood proteins of the complement system, which
refers to about 30 small blood molecules (serum proteins and cell membrane receptors) synthesized by
the liver [17]. This system helps and “complements” the action of innate and adaptive immunity and has
an important role in leukocytes chemotaxis and bacteria lysis. Upon stimulation, specific proteases
cleave downstream peptides to release cytokines, thus initiating an amplifying the proteolytic cascade to
further cleavages. The end-result is a massive amplification of the response and subsequent activation of
APCs, T and B cells. Complement can be activated through three different biochemical ways: the
classical, the alternative and the lectin pathway. The several types of NPs can differently activate the
complement system through one specific pathway or a combination of them [18]. The specificity of the
activated pathway by NPs depends on their surface properties, such as the conformation of a particular
polymer coating [19–21].
The activation of complement protein cascades can be responsible for some adverse effects
(i.e., hypersensitivity and anaphylaxis). To avoid such events, nano-formulations intended for systemic
administration of drug carriers are usually designed to prevent the complement activation.
As previously mentioned, NP physicochemical properties regulate their recognition and uptake by
phagocytic cells. In the past decades it was believed that phagocytosis was restricted to large matter
(>1 μm). However, like other cells, macrophages can also use different routes of internalization like
endocytosis and macropinocytosis to uptake nanosized particles. It has been observed that nanoparticles
>500 nm are primarily and more efficiently internalized via macropinocytosis or phagocytosis
mechanism than smaller ones with the same composition and surface properties [18,22]. NPs with
dissimilar morphologies interact with macrophages in a different way. As expected, the longest
dimensions exhibit the strongest attachment with macrophages membranes. Nonetheless, virus-sized
particles (20–200 nm) are efficiently taken up by macrophages or APCs, but mainly via clathrin-coated
mediated endocytosis [23].
Once internalized within immune cells, NPs can activate cytoplasmic multiprotein complexes called
inflammasomes, which are involved in the initiation of inflammatory responses [24]. These complexes
induce the proteolysis of inflammatory cytokine precursors and the following release of the bioactive
mediators. Endo/lysosome compartment damage and the release of hydrolytic enzymes, such as
Cathepsin B seem to play a major role in NP-induced inflammasome-dependent interleukin-1β (IL-1β)
release by macrophages and DCs [25].
Unfortunately, the information regarding the interaction of nanomaterials with the immune system is
still limited and fragmentary. Many results have been obtained in vitro using mouse or human
immortalized cell lines. The differences between mouse and human immune systems rise up some
concern on the potential clinic reliability. A second issue to be considered is the constitutive release of
inflammatory factors or growing hormones by immune cell lines, otherwise suppressed by primary cells
or temporary expressed after external stimuli. Furthermore, the several synthesis processes to produce
the same nanomaterials may introduce discrepancies in the resulting in vivo outcomes.
Coatings 2014, 4 144

In this review we report and comment on what is known in literature on the immunological
characterization of the principal inorganic and organic materials usually exploited in NP coating for
biomedical applications. To better focus on the surface material-related immune events, we will not
describe and discuss NP functionalization with peptides or other biological moieties intended for
specific cell targeting.

4. NP Surface Coatings

The employment of synthetic polymers to coat NPs increases their stability and solubility with
advantageous effects on cytotoxicity or inflammatory responses following administration [26]. Active
molecules can be encapsulated (adsorbed or bound) into the coated carriers to protected them from
metabolizing enzymes. Specific NP surfaces can confer protection to non-target tissues from a possible
unspecific toxic action of the drug payloads leading to side effects of the pharmaceutics. On the other
hand, they protect the drug from premature release or degradation in the biological environment of the
body, preserving their efficacy.
As well as delivery carriers, different type of shells cover NPs aimed at imaging and more in general
at diagnosis. For instance, contrast agents for Magnetic Resonance Imaging (MRI), like
super-paramagnetic iron oxide (SPIO) NPs or paramagnetic gadolinium-labeled NPs are often coated
with polymers to increase their solubility and biocompatibility [27].
Conversely, the coating can be chosen to produce immune-stimulant reactions, as demonstrated by
the application of NPs as adjuvant in vaccines production, which is becoming a field of fast development
in pharmaceutical industry [28]. In this case, reactive surfaces stimulate specific immune cells in order to
increase the response to antigens.
Therefore, NP surface features and materials must be considered as key factors that may significantly
contribute to adverse immune reactions and toxicity in current healthcare practice. In the following
paragraphs, representative observations will describe the type of interaction that the mainly used
NP-coating materials have with the immune system and their proved or potential effects.

5. Synthetic Polymeric Coatings

5.1. Polyethylene Glycol (PEG)

Hydrophobic surfaces generally tend to exclude water molecules causing aggregation and
precipitation of unstable colloids with limited biological applications. NP surface can be modified by
using highly hydrophilic polymers, such as poly(ethyleneglycol) (PEG) [26]. PEG is a polyether chain
able to absorb water and generate hydrogen bonds that allow solubilization in polar solvents and the
stabilization of the colloid either in acidic or basic pH environments. As we mentioned before, charged
nanoparticles bind more proteins than particles with neutral surfaces. Surface neutralization by coating
with PEG is one of the best approaches to protect nanoparticle surface from protein binding.
Two different approaches are mainly used to obtain PEG-coated nanoparticles: PEG adsorption or the
covalent attachment of the polyether chain on the surface, usually called PEGylation [29]. PEG
adsorption is the simpler approach, but it is barely useful in the case of NPs released in a biological
environment. Since this polymer is highly water-soluble, the coating might be immediately dissolved in
Coatings 2014, 4 145

salty fluids. PEGylation represents the best way to stably attach the polyether chain on the surface for
biomedical applications. A third way to obtain PEGylation is using amphiphilic di-block copolymers to
form NPs. In this case, self-assembling micellar structures can be produced and loaded with the
desired cargo [30].
As consequence of PEG coating NP internalization by phagocytes is reduced prolonging their
circulation time when released in the blood [13]. However, PEG covering of some materials like
liposomes does not protect from complement activation. The presence of specific chemical groups on
the polymer shell may modulate this effect. For instance, linkers like hydroxy and thiol-groups do not
activate the complement cascade, while methoxy groups potently trigger the cleavage reaction [21].
Different structural conformation and chemical group charges may determine the complement binding
with associated immune responses.
Most of the data present in literature describing antigenic responses to NPs surface coatings are
provided by experiments using PEGylated liposomes. Repeated systemic administrations of these
nano-systems induce an antibody-dependent rapid clearance of the second dose from the circulation [13].
This phenomenon is called accelerated blood clearance (ABC). The ABC effect seems to be
accompanied by the accumulation of both PEGylated and uncoated liposomes in the liver and spleen
immune resident cells. Phagocytic cells depletion attenuates this process, suggesting that macrophages
are involved in NPs accelerated clearance. The rate of the ABC phenomenon depends on many factors,
like the dose, the interval between the administered doses, the NP size, the surface charge, the liposomal
composition and the PEG density.
Nowadays, PEG coatings show many advantages to produce “stealth” NPs able to avoid a major
activation of immune system. This is proved by its abundant use as sole coating or in combination with
other polymers. Nevertheless, clinical studies employing PEGylated NPs for long time are not available
to assure its complete immune-safety.

5.2. PLGA

Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully developed polymers for
therapeutic devices, owing to its biodegradability and biocompatibility [31]. PLGA is synthesized by
co-polymerization of two different monomers, the cyclic dimers of glycolic acid and lactic acid.
Depending on the ratio of lactide to glycolide used for the polymerization, different forms of PLGA can
be obtained. Although the homopolymers of lactic acid (polylactide) and glycolic acid (polyglycolide)
show poor solubility, PLGA can be dissolved by a wide range of common solvents.
Under normal physiological conditions, PLGA undergoes hydrolysis in the body and produces its
original monomers. Since they are by-products of various metabolic pathways in the body, there is minimal
systemic toxicity associated with the use of PLGA for drug delivery or other biomedical applications.
PLGA is described in the literature as a promising carrier adjuvant for nasal subunit vaccines. Glycol
chitosan coated PLGA (GC-PLGA) NPs were found to elicit relatively stronger immune response as
compared to chitosan coated PLGA (C-PLGA) and PLGA NPs after nasal administration [32].
GC-PLGA NPs showed a better mucoadhesivity, with consequently prolonged nasal residence time.
Furthermore, GC-PLGA NPs resulted in a more efficient antigen uptake and transport across the nasal
Coatings 2014, 4 146

mucosa and into the circulatory system. The coating of NPs with chitosan and glycol chitosan did not
affect the integrity and release profile of the antigen.
Surface-modified PLGA microspheres (cationic PLGA microspheres) were also described as potent
nasal delivery systems for vaccines where mucosal, humoral and cellular responses after bacterial and
viral pathogens invasion are required [33].
It is worth noting that PLGA particulate vaccine adjuvants enhance LPS-induced NALP3
inflammasome and Caspase 1 activation leading to increased IL-1β secretion [34,35]. This observation
emphasizes the complexity of immune responses to NPs even for those usually considered non-toxic and
biodegradable. It can be hypothesized that the presence of PLGA coating helped to increase the
efficiency of the adjuvant delivery without a direct involvement in the immune response. The
enhancement of an inflammatory pathway born after particle internalization, that is, the inflammasome
activation, would suggest the hydrolyzation of the PLGA coating within the cell.
The advantage of using PLGA as a good and biodegradable protection for the desired cargo also
seems a good choice for the immune-compatibility of the polymer, based on the absence of detrimental
effects in the available literature.

5.3. Dendrimers

Dendrimers molecules are highly-branched symmetric structures which are synthesized in a

layer-by-layer fashion expressed in “generations” (G) around a core unit. This structure has repeated
dendrons with a single chemical linking group, called focal point. Their synthesis results in high level of
control over size, branching points and surface functionality. Several kinds of inorganic nanoparticles,
coated with dendrimers are reported in literature for drug and gene delivery [36–38] or imaging [39].
Many detailed results are available on dendrimer toxicity either in vitro or in vivo [40]. However,
information on the immune system reactions to dendrimer is still less well explored. Glycodendrimers
containing various surface modifications have been designed as anti-infective ligands with anti-viral or
antimicrobial effects. As examples, a polysulfonate G4 polyamidoamine (PAMAM) dendrimer show the
ability to block HIV-1 and HIV-2 activity in vitro [41]. Although the mechanism of infection prevention
is mainly related to structural entrapment of the virus by the branched structure, dendrimer-related
antimicrobial activity may also involve immune-modulation. For instance, N-acetyl-glucosamine-coated
G1 PAMAMs administered in mice bearing subcutaneous melanoma model, decrease tumor growth and
increase mice survival. These results were accompanied by an increase of CD69+ cells in the spleen and
the tumor tissue, associated to the up-regulation of IL-1h, IFN-g, TNF-a and IL-2 [42]. Another
indication of such phenomenon is provided by the over-expression of pro-inflammatory chemokines and
cytokines, namely MIP-1a, MIP-1h, IL-8, TNF-a, IL-1h and IL-6 induced in human dendritic cells and
macrophages by glucosamine-modified G3.5 PAMAMs [43].
Many different dendrimer compositions can be synthesized and each one should be studied using
primary immune cells and animal models in order to understand and eventually classify the different
features of immune responses. It is clear that cationic dendrimers can damage cell membranes and this
event can activate innate inflammatory reactions mediated by DAMP receptors, which could recognize
cell components escaping from their intracellular location.
Coatings 2014, 4 147

6. Natural Polymeric Coatings

Among the natural polymers, chitosan, sodium alginate and dextran are frequently used to cover NPs.
However, the information on immune effects is often restricted to the bulk material or to some
specific application.

6.1. Chitosan

Chitosan (Chi) is a linear polysaccharide, extensively diffused in biomedical applications for its
excellent properties including rapid blood clotting, biodegradability, non-toxicity, high charge density,
gelling and mucoadhesivity. These features give to chitosan an immense potential for various
pharmaceutical applications, such as the coating or entrapment of biochemicals, drugs and antigenic
molecules. An example is represented by chitosan microspheres, which have been studied as promising
carrier systems for mucosal vaccination (especially oral and nasal) to induce enhanced immune
responses [32].
This natural polymer can be employed both for inorganic and organic nano-structures. Literature
reports chitosan coatings of different metallic NPs [44,45]. Chi has been shown to enhance the function
of immune cells, such as neutrophils and macrophages stimulating the production of cytokines and
growth factors [46–49]. However, Chi is also controversially employed as a “fat binder” to reduce body
weight. The mechanism of interaction between chitosan and fat is not well understood and has not been
proved clinically. No indications of specific immune responses have been clearly shown following oral
administration of chitosan tablets or pills. On the other hand, due to its adjuvant properties that increase
inflammatory responses, caution should be used before choosing this polysaccharide as NP coating.

6.2. Sodium Alginate

Sodium alginate is a hydrophilic, water soluble and biocompatible polysaccharide obtained by marine
brown algae. Alginate consists of α-L-guluronate and β-D-mannuronate, arranged in a block structure as
homopolymer (polyguluronate or polymannuronate) or heteropolymer (a mixed sequence of these residues).
It is often employed as a coating for magnetic [50] and polymeric NPs [51,52]; alginate oligomers
obtained by enzymatic digestion of alginate polymer are able to induce the secretion of high levels of
inflammatory cytokine and chemokines, like MCP-1, RANTES in vitro and in vivo [53,54].
Combinations of alginate and poly-lactide-co-glycolide acid (PLGA) are developed for vaccination
purposes. Immunization studies in Balb/c mice by intradermal route demonstrated that incorporation of
alginate elicited humoral and cellular immune responses [33,55].
Enhancement of non-specific immune responses demonstrated in vivo suggests the employment of
alginate as coating for NPs aimed at immune stimulation. Either for vaccine adjuvant properties or for
diagnostic purposes to reveal deficient innate immune reaction, this brown algae-derived anionic
polysaccharide may represent a useful tool in biomedicine applications of NPs.
Coatings 2014, 4 148

6.3. Dextran

Dextran is a complex, branched polysaccharide composed of many glucose molecules. It is widely

used in medicine as an anti-thrombotic (anti-platelet action) and blood viscosity reducer. Larger
dextrans, which do not cross the vessels, are potent osmotic agents, and thus volume expanders in
hypovolemia.
Dextran is also diffused for the realization of metallic NP coatings to protect core oxidation. Dextran
coatings can be obtained by adsorption [56], chemical functionalization [27,57] or directly during
nanoparticle formation [58].
Even if extensively employed for NP shells, a few studies investigated the effects of such surfaces on
the immune system. Potential immune stimulatory effects on mouse splenocytes have been achieved
using drug loaded chitosan-carboxymethyl dextran NPs (CDNP) [59]. Unfortunately, it is difficult to
understand the specific contribution or role of the dextran in those kinds of immune responses.
The complex of two or more polymers can create surfaces with very different characteristics and
immune reactions.
Immune effects resulting from the release of free dextran molecules (not in nano-complexes) are not
useful to establish its immunogenicity as NP coating. Many inflammatory reactions depend on the
molecular weight of the chosen polysaccharide, its branching structure and the administered dose.

6.4. Starch

Starch is a different natural polymer produced by plants such as corn, potato, rise and cassava. It is
composed of two biomolecules, namely amylose and amylopectin. Amylose is a linear polymer of
glucose units mainly linked with alpha-1,4-bonds. Amylopectin, an extremely high molecular weight
polymer, has the same backbone structure of amylose, but with many alpha-1,6-linked branch points.
This polymer is highly biocompatible and biodegradable, with different physicochemical properties
according to the type of starch source [60].
Due to all these features, starch is widely used in many different biomedical applications ranging
from skin topical release [61] to degradable drug microsphere carrier [62]. Starch based coating have
also been employed for gold [63] or iron-core magnetic NPs [64,65].
Although commonly used, the literature regarding immune system interaction with starch coated NPs
is poor. It is unclear whether stereotyped immune reactions can be associated with the starch surface.
Due to its wide presence in food and its biodegradability, starch is considered a “safe” material. It is
expected, however, that potential allergic reaction will be experienced only in a restricted number of
sensitive human recipients, as well as for the other materials.

7. Inorganic NP Coatings

Inorganic covering of NPs is frequently used to produce NPs for applications involving specific
characteristics of the coating material. Due to the lower solubility of their coating properties, they are
less applied than the organic polymers for NP delivery in biomedicine. Data on the immune reactions
towards inorganic NPs are frequently associated to secondary effects following cell internalization of the
particles. NP size and charge play a major role in toxicity, intracellular localization and membrane
Coatings 2014, 4 149

damages that lead to DAMPs release by injured cells and inflammasome activation in the phagocytes.
Immune secondary events limit the possibility to find common responses to a certain coating material
and restrict the observations to the particles employed in a specific study.

7.1. Silica

Silicon dioxide NPs (SiO2 NPs) are exploited in a wide range of applications and in particular for
biomedical research. Dense or porous silica inorganic coatings are also used to shell different NP
cores [66]. SiO2 NPs have demonstrated low toxicity in vitro suggesting a positive role of silica shell
around different core materials, including CdSe/Zn QDs [67–69]. As described earlier, phagocytes such
as macrophages and monocytes react more efficiently to micro-size particles than to nano-size particles.
Moreover, increased cell damage has been shown for silica micro-particles than for nano-particles [18].
An interesting work by Fruijtier-Polloth and colleagues demonstrated that interaction between
amorphous silica nanoparticles and endothelial cells promotes the upregulation of endothelial adhesion
molecules expression. This effect enhances the adhesion of monocytes to endothelial cells, a typical
early event of inflammatory processes [70]. Moreover, many immunotoxic effects are due to the
interference with signaling pathways involved in activation of the immune response or the release of
pro-inflammatory cytokines or chemokines [71].
Morishige and colleagues demonstrated that chemical modification of silica NP surfaces can decrease
or suppress inflammasome activation and IL-1β release in THP-1 human monocyte/macrophage cell
line [25]. However, it is important to note that these inflammatory outcomes have been proved using
1 μm particles. Nano-scale silica particles, ranging from 30 to 300 nm, did not induce IL-1β release.
These results suggest that inflammatory responses of silica nanoparticles could depend on the
combination of several features, including size and surface functionalization.
It is worth mentioning that many indications come from cancer models where the immune system is
not in a physiological condition [72]. Further experiments specifically planned would help to better
decipher the silica coated NPs impact on the immune system.

7.2. Gold

Gold coatings are generally used for the preparation of bimetallic particles, containing a magnetic
core as platform for surface functionalization. Some strategies reported in literature describe different
core-shell NPs covered with this metal [73,74]. Gold-coated nanoparticles (Au-shelled NPs) have been
particularly designed for biomedicine, especially for drug delivery [75] and cancer applied hyperthermia
treatments [76,77].
There are controversial studies concerning the toxicological effects of engineering gold nanoparticles.
A study by Hashimoto et al. compared the exposure of cultured macrophages RAW264.7 to AuNPs
with AgNPs. Although an inflammatory response was observed for both the Au- and Ag-NPs, the
harmful cytotoxic effects of AuNPs were smaller than those of the Ag-NPs [78]. However, as described
for other kinds of nanomaterials, the interaction between cells and Au nanoparticles could be mediated
also by unspecific adsorption of serum proteins onto the gold surface [79].
Coatings 2014, 4 150

7.3. Titanium Dioxide (TiO2)

Titanium dioxide (TiO2) is another coating for inorganic nanoparticles, exploited to increase their
cytocompatibility. It was reported that a titanium oxide shell on Zn-based nanoparticles reduces zinc
ions release in human lung epithelial cell line [80]. More recent studies demonstrated that core/shell iron
oxide/titanium oxide nanoparticles can be used as doxorubicin vector for cancer cells [81]. There are
contentious data about TiO2 toxicity. TiO2 particles are considered to be inert and unable to pass
undamaged skin. For this reason, they are commonly used for cosmetics or sunscreens preparations as an
efficient filter of UV light [82]. In contrast, other studies reported cell toxicity and genotoxicity [83–85].
The discrepancy may be due to different TiO2 particle compositions, different cellular origin, or
variation in TiO2 sensitivity between the organisms. TiO2 acts as a modulator of neutrophil
degranulation, typically associated with inflammatory process. Actually, it enhances cell surface
expression of some granule markers, the secretion of some proteins in the supernatants and
metalloproteinases activity in human neutrophils [86].

8. Discussion and Conclusions

Nanoparticles designed for biomedical applications in vivo inevitably encounter the human Immune
System. Many aspects of nanoparticle-induced modulation of immune responses have been investigated
and comprehensive literature on the subject has been published [9,18]. In this review we have revised the
available literature focusing on the immune aspects specifically due to different surface coatings of NPs,
independently of their size, shape and core composition (summary in Table 1). We also excluded the
protein functionalization of the surfaces, which could inevitably induce a ligand-receptor driven
targeting of the particle or stimulate specific immune pathways on purpose.
It is important to have an overview on this subject; however, it is difficult to draw conclusions or even
compare the several ways to shell NPs. The choice of the NP coatings firstly relies on the chemical
feature of the employed materials. For example, not all the polymers can be covalently attached or
adsorbed onto different metal cores. The use of surfactants may be helpful for solubility improvement,
but can completely change the immune response. Experimental approaches that clearly avoid
false-positive or false-negative immunotoxicological results of nano-systems are not simple. Secondly,
the NPs end goal may limit the application of a specific NP coating. For instance, NPs aimed at clinical
applications could require high temperature sterilization processes. These particles cannot be coated
with polymers that liquefy at high temperatures. In this case the surface that the immune cells will face
would not be the original polymer but either a modified polymer or the core material.
Precise design of NP organic or inorganic coatings to avoid or specifically interact (e.g., coadjuvants)
with the Immune System could be done only if the binding sites were clearly known. Polymers differ by
chemical groups that present unique composition and structure. The different metallic or metal oxides
NPs show diverse reactivity on their surfaces depending on their atomic and crystal structure. So, several
features should be considered, like sizes of antibody binding sites vs. their potential interaction site on
the NP surface. Moreover, the biological conditions and opsonization by diverse molecules in vivo may
dramatically alter all these parameters.
Coatings 2014, 4 151

Table 1. Immune response summary of surface coatings.

Surface
Origin Immune response Cell mediator Applications References
coatings
Polyethylene Biomedical
Synthetic Complement activation Phagocytic cells [13,21,26,29,30]
glycol (PEG) applications
NALP3
Poly(lactic-co
inflammasome Drug delivery,
glycolic acid) Synthetic Antigen-mediated [31–35]
and Caspase 1 vaccine adjuvant
(PLGA)
activation
Dendritic cells
Innate DAMP Anti-infective
Dendrimers Synthetic and [36–43]
receptors-mediated ligands
macrophages
Innate immune Neutrophils and Coating of drugs,
Chitosan Natural [32,44–49]
response macrophages vaccine adjuvant
Coating for
Sodium Humoral and cellular Neutrophils and magnetic and
Natural [33,50,53–55]
alginate response macrophages polymeric NPs,
vaccines
Anti-platelet
Dextran Natural Not well defined Not well defined action, metallic [27,56–59]
NP coatings
Skin topical
Low immune and release, gold or
Starch Natural Not well defined [60–65]
allergic responses iron-core
magnetic NPs
Inflammasome Endothelial
Biomedical
Silica Inorganic activation cells, [18,25,66–72]
research
DAMPs release monocytes
Magnetic
Serum protein
core,cancer
Gold Inorganic mediated-inflammatory Not well defined [73–79]
applied
response
hyperthermia
Titanium Secretion of proteins,
Vector for cancer
dioxide Inorganic metalloproteinases Neutrophils [81–85]
cells, cosmetics
(TiO2) activation

In view of all this complexity, special attention must also be paid to in vitro and in vivo models for
immunology studies. Although currently used in the laboratory, these models provide limited
information regarding a potential immune response in human subjects. Immune cell lines are often used
as in vitro models. These cell types are proliferating clones that differ from primary immune cells which
are extracted from a donor in a physiological state. Primary cells are usually cultured and tested within a
couple of weeks. Although the window of time to perform experiments on each donor is shorter than
using a cell line, this represents the best in vitro model. Primary white blood cells represent a reliable
model to assess the cellular and molecular immune responses following cellular activation pathways by
the NPs. The possible release of inflammatory cytokines in the supernatant can also be carefully
Coatings 2014, 4 152

quantified in culture. The weak point of all the in vitro systems is the isolated and artificial environment
of the culture. Different sera, media and mutated cell lines of the same origin have produced
contradictory results. It is difficult to predict with reasonable certainty what will happen once NPs are
administered in vivo. Most of these experiments, however, are necessary to exclude some responses and
orientate the researches towards specific outcomes. If high doses of a certain NP do not induce
pico-molar doses of inflammatory cytokines in vitro it will be unlikely that the same particle will induce
cytokine release in relevant amounts in vivo. On the other hand, a different reaction cannot be excluded
in a living organism as a consequence of orchestrated reactions of different cell types.
In vivo experimental models are definitely required to understand systemic immune reactions. The
elected model for immunology is the mouse. Selected strains of mice are commercially available and
permit the investigation in fast reproducing and easy-to-handle mammals. Their immune system is not
exactly like the human one, but the cell types and the main features are very similar. Moreover, modern
biotechnology provides genetically modified animals missing genes involved in specific immune
reaction. Genetically modified mice for immunity genes provide the most straightforward evidence for
potential immune system activation induced by NPs. Model mice with a specific disease offer the
opportunity to study the specific immunological events induced by the nano-carrier potentially
employed as drug delivery system for the same disease. The latter consideration assumes that specific
disease states could present altered immune responses limiting the information collected from
experiments of NP release in healthy mice.
In vivo models are mandatory when the targets of delivery are protected organs like the Central
Nervous System (CNS). The optimal way to deliver NPs to the CNS would be through the blood
circulation. Nonetheless, the presence of the Blood-Brain-Barrier (BBB) increases the level of difficulty
for delivering drug carriers. The BBB acts as a rigorously regulated gate for the flow of ions,
macromolecules, and nutrients between blood and brain tissue, as well as potential hazards. It combines
physical and metabolic barriers to maintain the homeostasis of the CNS [87]. However, reaching the
CNS through blood flow is very difficult, although very attractive due to the reduced invasiveness of
intravenous administration compared to intracerebroventricular (ICV) or intraparenchymal (IP) injections.
The preparation of NPs for applications in the CNS implies a peculiar design of the surface and
specific particle properties. Polymeric NPs or coating with surfactants like polysorbates or PEG have
been traditionally used as a strategy to cross the BBB [88–90]. These approaches have been applied with
comprehensive chemical design of the nano-vectors and many observations of neural cell responses.
Fewer details are available on the immune responses happening in the CNS, once NPs have been
introduced in the neural parenchyma.
The CNS resident immune system is represented by microglial cells [91]. These cells are able to sense
pathological tissue alteration and perform immunological functions, besides many other emerging
functions in maintaining the homeostasis of the healthy CNS.
As already pointed out, PEGylation is one of the most used materials to reduce protein opsonization
on the NP surface, limiting the immune system response. It has been observed that uncoated lipid NPs,
which accumulate in the brain parenchyma after intravenous injection, cause considerable microglia
activation. PEG coating of those lipid NPs with PEG strongly reduce microglial activation [92].
We already mentioned the PEG or PLGA coating of magnetic NPs designed as contrast agents for
MRI imaging in the CNS. A novel approach is represented by magnetic core coated with lipid
Coatings 2014, 4 153

modified-PAMAM dendrimers [93]. No indications on immune reactions towards this type of NPs have
been presented yet. However, very recent results on G4 amine-PAMAMs and lipid-modified
amine-PAMAMs emphasize the importance of the dendrimer modification of the regulation of
inflammatory cytokine and chemokine receptors on microglia surface [94]. On the other hand, PAMAM
dendrimers alone have already been employed for CNS delivery in vivo demonstrating helpful features
for neuro-inflammatory disease models [95].
As described in this review, nanomaterials applied to biomedicine can be designed to avoid or target
the immune system. The advent of engineered NPs has highlighted the importance of immunosafety
tests to understand the behavior of immune cells in response to these formulations. Unfortunately,
nano-sized systems often interfere with the assays usually used by immunologists. Generally,
experimental results can be affected by chemical or biological contamination and by optical interference
related to the material density. Traditional in vitro tests do not usually contemplate the use of NPs and
their possible optical and catalytic interference with the employed reagents and cellular model.
Moreover, traces of impurities (solvents, carry-over molecules) and/or endotoxin within a
nano-formulation can also induce an inflammatory response, causing cellular- and immunotoxicity [96].
The development of new nanomaterial-aimed techniques and assays will help to validate the already
existing results and to unify the results obtained from different methods.

Acknowledgments

The activity presented in this work has been supported by Fondazione Istituto Italiano di Tecnologia.

Conflict of Interest

The authors declare no conflict of interest.

References

1. Labuzek, K.; Gorki, K.; Jaroszek, H.; Jarzabek, K.; Gabryel, B.; Okopien, B. Highly organized
nanostructures for brain drug delivery-new hope or just a fad? CNS Neurol Disord Drug Targets
2013, 8, 1271–1285.
2. Wilczewska, A.Z.; Niemirowicz, K.; Markiewicz, K.H.; Car, H. Nanoparticles as drug delivery
systems. Pharmacol. Rep. 2012, 64, 1020–1037.
3. Abbas, A.K.; Lichtman, A.H.; Pillai, S. Cellular and Molecular Immunology, 7th ed.; Elsevier:
Philadelphia, PA, USA, 2009.
4. Fu, H.; Wang, A.; Mauro, C.; Marelli-Berg, F. T lymphocyte trafficking: Molecules and
mechanisms. Front. Biosci. (Landmark Ed) 2013, 18, 422–440.
5. Campbell, K.S.; Hasegawa, J. Natural killer cell biology: An update and future directions.
J. Allergy Clin. Immunol. 2013, 132, 536–544.
6. Senovilla, L.; Galluzzi, L.; Zitvogel, L.; Kroemer, G. Immunosurveillance as a regulator of tissue
homeostasis. Trends Immunol. 2013, 10, 471–481.
7. Sallusto, F.; Baggiolini, M. Chemokines and leukocyte traffic. Nat. Immunol. 2008, 9, 949–952.
Coatings 2014, 4 154

8. Mithal, D.S.; Banisadr, G.; Miller, R.J. Cxcl12 signaling in the development of the nervous system.
J. Neuroimmune Pharmacol. 2012, 7, 820–834.
9. Dobrovolskaia, M.A.; Germolec, D.R.; Weaver, J.L. Evaluation of nanoparticle immunotoxicity.
Nat. Nanotechnol. 2009, 4, 411–414.
10. Hubbell, J.A.; Thomas, S.N.; Swartz, M.A. Materials engineering for immunomodulation. Nature
2009, 462, 449–460.
11. Frohlich, E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles.
Int. J. Nanomed. 2012, 7, 5577–5591.
12. Zahr, A.S.; Davis, C.A.; Pishko, M.V. Macrophage uptake of core-shell nanoparticles surface
modified with poly(ethylene glycol). Langmuir 2006, 22, 8178–8185.
13. Bertrand, N.; Leroux, J.C. The journey of a drug-carrier in the body: An anatomo-physiological
perspective. J. Control Release 2012, 161, 152–163.
14. Longmire, M.; Choyke, P.L.; Kobayashi, H. Clearance properties of nano-sized particles and
molecules as imaging agents: Considerations and caveats. Nanomedicine (Lond.) 2008, 3, 703–717.
15. Choi, H.S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J.P.; Itty Ipe, B.; Bawendi, M.G.;
Frangioni, J.V. Renal clearance of quantum dots. Nat. Biotechnol. 2007, 25, 1165–1170.
16. Lacerda, L.; Herrero, M.A.; Venner, K.; Bianco, A.; Prato, M.; Kostarelos, K. Carbon-nanotube
shape and individualization critical for renal excretion. Small 2008, 4, 1130–1132.
17. Ricklin, D.; Hajishengallis, G.; Yang, K.; Lambris, J.D. Complement: A key system for immune
surveillance and homeostasis. Nat. Immunol. 2010, 11, 785–797.
18. Dobrovolskaia, M.A.; McNeil, S.E. Handbook of Immunological Properties of Engineered
Nanomaterials; World Scientific Publishing: Singapore, 2013.
19. Al-Hanbali, O.; Rutt, K.J.; Sarker, D.K.; Hunter, A.C.; Moghimi, S.M. Concentration dependent
structural ordering of poloxamine 908 on polystyrene nanoparticles and their modulatory role on
complement consumption. J. Nanosci. Nanotechnol. 2006, 6, 3126–3133.
20. Andersen, A.J.; Hashemi, S.H.; Andresen, T.L.; Hunter, A.C.; Moghimi, S.M. Complement: Alive
and kicking nanomedicines. J. Biomed. Nanotechnol. 2009, 5, 364–372.
21. Hamad, I.; Al-Hanbali, O.; Hunter, A.C.; Rutt, K.J.; Andresen, T.L.; Moghimi, S.M. Distinct
polymer architecture mediates switching of complement activation pathways at the
nanosphere-serum interface: Implications for stealth nanoparticle engineering. ACS Nano 2010, 4,
6629–6638.
22. Hillaireau, H.; Couvreur, P. Nanocarriers’ entry into the cell: relevance to drug delivery. Cell Mol.
Life Sci. 2009, 17, 2873–2896.
23. Smith, P.J.; Giroud, M.; Wiggins, H.L.; Gower, F.; Thorley, J.A.; Stolpe, B.; Mazzolini, J.;
Dyson, R.J.; Rappoport, J.Z. Cellular entry of nanoparticles via serum sensitive clathrin-mediated
endocytosis, and plasma membrane permeabilization. Int. J. Nanomed. 2012, 7, 2045–2055.
24. Sun, B.; Wang, X.; Ji, Z.; Li, R.; Xia, T. NLRP3 inflammmasome activation induced by engineered
nanomaterials. Small 2013, 9, 1595–1607.
25. Morishige, T.; Yoshioka, Y.; Inakura, H.; Tanabe, A.; Yao, X.; Narimatsu, S.; Monobe, Y.;
Imazawa, T.; Tsunoda, S.; Tsutsumi, Y.; et al. The effect of surface modification of amorphous
silica particles on NLRP3 inflammasome mediated IL-1β production, ROS production and
endosomal rupture. Biomaterials 2010, 31, 6833–6842.
Coatings 2014, 4 155

26. Zhang, F.; Lees, E.; Amin, F.; Rivera Gil, P.; Yang, F.; Mulvaney, P.; Parak, W.J. Polymer-coated
nanoparticles: A universal tool for biolabelling experiments. Small 2011, 7, 3113–3127.
27. Tsuchiya, K.; Nitta, N.; Sonoda, A.; Nitta-Seko, A.; Ohta, S.; Takahashi, M.; Murata, K.;
Mukaisho, K.; Shiomi, M.; Tabata, Y.; et al. Evaluation of atherosclerotic lesions using
dextran- and mannan-dextran-coated uspio: Mri analysis and pathological findings. Int. J.
Nanomed. 2012, 7, 2271–2280.
28. Gregory, A.E.; Titball, R.; Williamson, D. Vaccine delivery using nanoparticles. Front. Cell. Infect.
Microbiol. 2013, 3, 13.
29. Jevsevar, S.; Kunstelj, M.; Porekar, V.G. Pegylation of therapeutic proteins. Biotechnol. J. 2010, 5,
113–128.
30. Rastogi, R.; Anand, S.; Koul, V. Flexible polymerosomes—An alternative vehicle for topical
delivery. Colloids Surf. B Biointerfaces 2009, 72, 161–166.
31. Danhier, F.; Ansorena, E.; Silva, J.M.; Coco, R.; Le Breton, A.; Preat, V. Plga-based nanoparticles:
An overview of biomedical applications. J. Control Release 2012, 161, 505–522.
32. Islam, M.A.; Firdous, J.; Choi, Y.J.; Yun, C.H.; Cho, C.S. Design and application of chitosan
microspheres as oral and nasal vaccine carriers: An updated review. Int. J. Nanomed. 2012, 7,
6077–6093.
33. Mata, E.; Igartua, M.; Patarroyo, M.E.; Pedraz, J.L.; Hernandez, R.M. Enhancing immunogenicity
to PLGA microparticulate systems by incorporation of alginate and rgd-modified alginate. Eur J.
Pharm. Sci. 2011, 44, 32–40.
34. Sharp, F.A.; Ruane, D.; Claass, B.; Creagh, E.; Harris, J.; Malyala, P.; Singh, M.; O’Hagan, D.T.;
Pétrilli, V.; Tschopp, J.; et al. Uptake of particulate vaccine adjuvants by dendritic cells activates
the NALP3 inflammasome. Proc. Natl. Acad. Sci. USA 2009, 106, 870–875.
35. Demento, S.L.; Eisembarth, S.C.; Foellmer, H.G.; Platt, C.; Caplan, M.J.; Saltzman, W.M.;
Mellman, I.; Ledizet, M.; Fikrig, E.; Flavell R.A.; Fahmy, T.M. Inflammasome-activating
nanoparticles as modular systems for optimizing vaccine efficacy. Vaccine 2009, 27, 3013–3021.
36. Wisher, A.C.; Bronstein, I.; Chechik, V. Thiolated pamam dendrimer-coated cdse/znse
nanoparticles as protein transfection agents. Chem. Commun. (Camb.) 2006, 15, 1637–1639.
37. Rouhollah, K.; Pelin, M.; Serap, Y.; Gozde, U.; Ufuk, G. Doxorubicin loading, release, and stability
of polyamidoamine dendrimer-coated magnetic nanoparticles. J. Pharm. Sci. 2013, 102,
1825–1835.
38. Pan, B.; Cui, D.; Sheng, Y.; Ozkan, C.; Gao, F.; He, R.; Li, Q.; Xu, P.; Huang, T.
Dendrimer-modified magnetic nanoparticles enhance efficiency of gene delivery system. Cancer
Res. 2007, 67, 8156–8163.
39. Lamanna, G.; Kueny-Stotz, M.; Mamlouk-Chaouachi, H.; Ghobril, C.; Basly, B.; Bertin, A.;
Miladi, I.; Billotey, C.; Pourroy, G.; Begin-Colin, S.; et al. Dendronized iron oxide nanoparticles
for multimodal imaging. Biomaterials 2011, 32, 8562–8573.
40. Duncan, R.; Izzo, L. Dendrimer biocompatibility and toxicity. Adv. Drug Deliv. Rev. 2005, 57,
2215–2237.
Coatings 2014, 4 156

41. Witvrouw, M.; Fikkert, V.; Pluymers, W.; Matthews, B.; Mardel, K.; Schols, D.; Raff, J.;
Debyser, Z.; De Clercq, E.; Holan, G.; et al. Polyanionic (i.e., polysulfonate) dendrimers can inhibit
the replication of human immunodeficiency virus by interfering with both virus adsorption and later
steps (reverse transcriptase/integrase) in the virus replicative cycle. Mol. Pharmacol. 2000, 58,
1100–1108.
42. Vannucci, L.; Fiserova, A.; Sadalapure, K.; Lindhorst, T.K.; Kuldova, M.; Rossmann, P.;
Horvath, O.; Kren, V.; Krist, P.; Bezouska, K.; et al. Effects of n-acetyl-glucosamine-coated
glycodendrimers as biological modulators in the b16f10 melanoma model in vivo. Int. J. Oncol.
2003, 23, 285–296.
43. Shaunak, S.; Thomas, S.; Gianasi, E.; Godwin, A.; Jones, E.; Teo, I.; Mireskandari, K.; Luthert, P.;
Duncan, R.; Patterson, S.; et al. Polyvalent dendrimer glucosamine conjugates prevent scar tissue
formation. Nat. Biotechnol. 2004, 22, 977–984.
44. Shi, S.F.; Jia, J.F.; Guo, X.K.; Zhao, Y.P.; Chen, D.S.; Guo, Y.Y.; Cheng, T.; Zhang, X.L.
Biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells. Int. J.
Nanomedicine 2012, 7, 5593–5602.
45. Sun, I.C.; Na, J.H.; Jeong, S.Y.; Kim, D.E.; Kwon, I.C.; Choi, K.; Ahn, C.H.; Kim, K.
Biocompatible glycol chitosan-coated gold nanoparticles for tumor-targeting ct imaging. Pharm.
Res. 2013, doi:10.1007/s11095-013-1142-0.
46. Mori, T.; Okumura, M.; Matsuura, M.; Ueno, K.; Tokura, S.; Okamoto, Y.; Minami, S.;
Fujinaga, T. Effects of chitin and its derivatives on the proliferation and cytokine production of
fibroblasts in vitro. Biomaterials 1997, 18, 947–951.
47. Peluso, G.; Petillo, O.; Ranieri, M.; Santin, M.; Ambrosio, L.; Calabro, D.; Avallone, B.; Balsamo,
G. Chitosan-mediated stimulation of macrophage function. Biomaterials 1994, 15, 1215–1220.
48. Porporatto, C.; Bianco, I.D.; Correa, S.G. Local and systemic activity of the polysaccharide
chitosan at lymphoid tissues after oral administration. J. Leukoc. Biol. 2005, 78, 62–69.
49. Porporatto, C.; Bianco, I.D.; Riera, C.M.; Correa, S.G. Chitosan induces different l-arginine
metabolic pathways in resting and inflammatory macrophages. Biochem. Biophys. Res. Commun.
2003, 304, 266–272.
50. Chen, K.L.; Mylon, S.E.; Elimelech, M. Enhanced aggregation of alginate-coated iron oxide
(hematite) nanoparticles in the presence of calcium, strontium, and barium cations. Langmuir 2007,
23, 5920–5928.
51. Demoulins, T.; Bassi, I.; Thomann-Harwood, L.; Jandus, C.; Kaeuper, P.; Simon, H.U.; von
Gunten, S.; McCullough, K.C. Alginate-coated chitosan nanogel capacity to modulate the effect of
tlr ligands on blood dendritic cells. Nanomedicine 2013, 9, 806–817.
52. Liu, Z.; Lv, D.; Liu, S.; Gong, J.; Wang, D.; Xiong, M.; Chen, X.; Xiang, R.; Tan, X. Alginic
acid-coated chitosan nanoparticles loaded with legumain DNA vaccine: Effect against breast cancer
in mice. PLoS One 2013, 8, e60190.
53. Yamamoto, Y.; Kurachi, M.; Yamaguchi, K.; Oda, T. Stimulation of multiple cytokine production
in mice by alginate oligosaccharides following intraperitoneal administration. Carbohydr. Res.
2007, 342, 1133–1137.
54. Yamamoto, Y.; Kurachi, M.; Yamaguchi, K.; Oda, T. Induction of multiple cytokine secretion from
raw264.7 cells by alginate oligosaccharides. Biosci. Biotechnol. Biochem. 2007, 71, 238–241.
Coatings 2014, 4 157

55. Salvador, A.; Igartua, M.; Hernandez, R.M.; Pedraz, J.L. Combination of immune stimulating
adjuvants with poly(lactide-co-glycolide) microspheres enhances the immune response of vaccines.
Vaccine 2012, 30, 589–596.
56. Yu, M.; Huang, S.; Yu, K.J.; Clyne, A.M. Dextran and polymer polyethylene glycol (peg) coating
reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2d and 3d cell culture. Int. J. Mol.
Sci. 2012, 13, 5554–5570.
57. Osborne, E.A.; Atkins, T.M.; Gilbert, D.A.; Kauzlarich, S.M.; Liu, K.; Louie, A.Y. Rapid
microwave-assisted synthesis of dextran-coated iron oxide nanoparticles for magnetic resonance
imaging. Nanotechnology 2012, 23, doi:10.1088/0957-4484/23/21/215602.
58. Jang, H.; Ryoo, S.R.; Kostarelos, K.; Han, S.W.; Min, D.H. The effective nuclear delivery of
doxorubicin from dextran-coated gold nanoparticles larger than nuclear pores. Biomaterials 2013,
34, 3503–3510.
59. Lin, Y.S.; Radzi, R.; Morimoto, M.; Saimoto, H.; Okamoto, Y.; Minami, S. Characterization of
chitosan-carboxymethyl dextran nanoparticles as a drug carrier and as a stimulator of mouse
splenocytes. J. Biomater. Sci. Polym. Ed. 2012, 23, 1401–1420.
60. Yuryev, V.P.; Tomasik, P.; Bertoft, E. Starch: Achievements in Understanding of Structure and
Functionality; Nova Science Publishers: Hauppauge, NY, USA, 2007.
61. Lane, M.E. Nanoparticles and the skin—applications and limitations. J. Microencapsul. 2011, 28,
709–716.
62. Hakansson, L.; Hakansson, A.; Morales, O.; Thorelius, L.; Warfving, T. Spherex (degradable starch
microspheres) chemo-occlusion—Enhancement of tumor drug concentration and therapeutic
efficacy: An overview. Semin. Oncol. 1997, 24, S6–100-S6–109.
63. Pender, D.S.; Vangala, L.M.; Badwaik, V.D.; Willis, C.B.; Aguilar, Z.P.; Sangoi, T.N.;
Paripelly, R.; Dakshinamurthy, R. Bactericidal activity of starch-encapsulated gold nanoparticles.
Front. Biosci. (Landmark Ed.) 2013, 18, 993–1002.
64. Cole, A.J.; David, A.E.; Wang, J.; Galban, C.J.; Yang, V.C. Magnetic brain tumor targeting and
biodistribution of long-circulating peg-modified, cross-linked starch-coated iron oxide nanoparticles.
Biomaterials 2011, 32, 6291–6301.
65. Cole, A.J.; David, A.E.; Wang, J.; Galban, C.J.; Hill, H.L.; Yang, V.C. Polyethylene glycol
modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor
targeting. Biomaterials 2011, 32, 2183–2193.
66. Vivero-Escoto, J.L.; Huxford-Phillips, R.C.; Lin, W. Silica-based nanoprobes for biomedical
imaging and theranostic applications. Chem. Soc. Rev. 2012, 41, 2673–2685.
67. Bardi, G.; Malvindi, M.A.; Gherardini, L.; Costa, M.; Pompa, P.P.; Cingolani, R.; Pizzorusso, T.
The biocompatibility of amino functionalized CdSe/Zn quantum-dot-doped SiO2 nanoparticles
with primary neural cells and their gene carrying performance. Biomaterials 2010, 31, 6555–6566.
68. Malvindi, M.A.; Brunetti, V.; Vecchio, G.; Galeone, A.; Cingolani, R.; Pompa, P.P. SiO2
nanoparticles biocompatibility and their potential for gene delivery and silencing. Nanoscale 2012,
4, 486–495.
69. Asefa, T.; Tao, Z. Biocompatibility of mesoporous silica nanoparticles. Chem. Res. Toxicol. 2012,
25, 2265–2284.
Coatings 2014, 4 158

70. Napierska, D.; Quarck, R.; Thomassen, L.C.; Lison, D.; Martens, J.A.; Delcroix, M.; Nemery, B.;
Hoet, P.H. Amorphous silica nanoparticles promote monocyte adhesion to human endothelial cells:
Size-dependent effect. Small 2013, 9, 430–438.
71. Fruijtier-Polloth, C. The toxicological mode of action and the safety of synthetic amorphous
silica—a nanostructured material. Toxicology 2012, 294, 61–79.
72. Rosenholm, J.M.; Mamaeva, V.; Sahlgren, C.; Linden, M. Nanoparticles in targeted cancer therapy:
Mesoporous silica nanoparticles entering preclinical development stage. Nanomedicine (Lond.)
2012, 7, 111–120.
73. Salgueirino-Maceira, V.; Correa-Duarte, M.A.; Farle, M.; Lopez-Quintela, M.A.; Sieradzki, K.;
Diaz, R. Synthesis and characterization of large colloidal cobalt particles. Langmuir 2006, 22,
1455–1458.
74. Robinson, I.; Tung le, D.; Maenosono, S.; Walti, C.; Thanh, N.T. Synthesis of core-shell gold
coated magnetic nanoparticles and their interaction with thiolated DNA. Nanoscale 2010, 2,
2624–2630.
75. Gangopadhyay, P.; Gallet, S.; Franz, E.; Persoons, A.; Verbiest, T. Novel superparamagnetic
core(shell) nanoparticles for magnetic targeted drug delivery and hyperthermia treatment. IEEE
Trans. Magn. 2005, 41, 4194–4196.
76. Elsherbini, A.A.; Saber, M.; Aggag, M.; El-Shahawy, A.; Shokier, H.A. Laser and
radiofrequency-induced hyperthermia treatment via gold-coated magnetic nanocomposites. Int. J.
Nanomedicine 2011, 6, 2155–2165.
77. Elsherbini, A.A.; Saber, M.; Aggag, M.; El-Shahawy, A.; Shokier, H.A. Magnetic
nanoparticle-induced hyperthermia treatment under magnetic resonance imaging. Magn. Reson.
Imaging 2011, 29, 272–280.
78. Hashimoto, M.; Toshima, H.; Yonezawa, T.; Kawai, K.; Narushima, T.; Kaga, M.; Endo, K.
Responses of raw264.7 macrophages to water-dispersible gold and silver nanoparticles stabilized
by metal-carbon sigma-bonds. J. Biomed. Mater. Res. A 2013, in press.
79. Chithrani, B.D.; Ghazani, A.A.; Chan, W.C. Determining the size and shape dependence of gold
nanoparticle uptake into mammalian cells. Nano Lett. 2006, 6, 662–668.
80. Hsiao, I.L.; Huang, Y.J. Titanium oxide shell coatings decrease the cytotoxicity of zno
nanoparticles. Chem. Res. Toxicol. 2011, 24, 303–313.
81. Arora, H.C.; Jensen, M.P.; Yuan, Y.; Wu, A.; Vogt, S.; Paunesku, T.; Woloschak, G.E.
Nanocarriers enhance doxorubicin uptake in drug-resistant ovarian cancer cells. Cancer Res. 2012,
72, 769–778.
82. Nohynek, G.J.; Dufour, E.K. Nano-sized cosmetic formulations or solid nanoparticles in
sunscreens: A risk to human health? Arch. Toxicol. 2012, 86, 1063–1075.
83. Zhang, R.; Bai, Y.; Zhang, B.; Chen, L.; Yan, B. The potential health risk of titania nanoparticles.
J. Hazard Mater. 2012, 211–212, 404–413.
84. Klien, K.; Godnic-Cvar, J. Genotoxicity of metal nanoparticles: Focus on in vivo studies. Arh. Hig.
Rada Toksikol. 2012, 63, 133–145.
85. Iavicoli, I.; Leso, V.; Fontana, L.; Bergamaschi, A. Toxicological effects of titanium dioxide
nanoparticles: A review of in vitro mammalian studies. Eur. Rev. Med. Pharmacol. Sci. 2011, 15,
481–508.
Coatings 2014, 4 159

86. Babin, K.; Antoine, F.; Goncalves, D.M.; Girard, D. TiO2, CeO2 and ZnO nanoparticles and
modulation of the degranulation process in human neutrophils. Toxicol. Lett. 2013, 221, 57–63.
87. Abbott, N.J.; Patabendige, A.A.; Dolman, D.E.; Yusof, S.R.; Begley, D.J. Structure and function of
the blood-brain barrier. Neurobiol. Dis. 2010, 37, 13–25.
88. Gagliardi, M.; Bardi, G.; Bifone, A. Polymeric nanocarriers for controlled and enhanced delivery of
therapeutic agents to the cns. Ther. Deliv. 2012, 3, 875–887.
89. Tosi, G.; Costantino, L.; Ruozi, B.; Forni, F.; Vandelli, M.A. Polymeric nanoparticles for the drug
delivery to the central nervous system. Expert Opin. Drug Deliv. 2008, 5, 155–174.
90. Bhaskar, S.; Tian, F.; Stoeger, T.; Kreyling, W.; de la Fuente, J.M.; Grazu, V.; Borm, P.;
Estrada, G.; Ntziachristos, V.; Razansky, D. Multifunctional nanocarriers for diagnostics, drug
delivery and targeted treatment across blood-brain barrier: Perspectives on tracking and
neuroimaging. Part. Fibre Toxicol. 2010, 7, 3.
91. Aguzzi, A.; Barres, B.A.; Bennett, M.L. Microglia: Scapegoat, saboteur, or something else?
Science 2013, 339, 156–161.
92. Huang, J.Y.; Lu, Y.M.; Wang, H.; Liu, J.; Liao, M.H.; Hong, L.J.; Tao, R.R.; Ahmed, M.M.;
Liu, P.; Liu, S.S.; et al. The effect of lipid nanoparticle pegylation on neuroinflammatory response
in mouse brain. Biomaterials 2013, 34, 7960–7970.
93. Boni, A.; Albertazzi, L.; Innocenti, C.; Gemmi, M.; Bifone, A. Water dispersal and
functionalization of hydrophobic iron oxide nanoparticles with lipid-modified poly(amidoamine)
dendrimers. Langmuir 2013, 29, 10973–10979.
94. Bertero, A.; Boni, A.; Gemmi, M.; Gagliardi, M.; Bifone, A.; Bardi, G. Surface functionalisation
regulates polyamidoamine dendrimer toxicity on blood-brain barrier cells and the modulation of
key inflammatory receptors on microglia. Nanotoxicology 2014, 2, 158–168.
95. Kannan, S.; Dai, H.; Navath, R.S.; Balakrishnan, B.; Jyoti, A.; Janisse, J.; Romero, R.;
Kannan, R.M. Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a
rabbit model. Sci. Transl. Med. 2012, 4, doi:10.1126/scitranslmed.3003162.
96. Oostingh, G.J.; Casals, E.; Italiani, P.; Colognato, R.; Stritzinger, R.; Ponti, J.; Pfaller, T.; Kohl, Y.;
Ooms, D.; Favilli, F.; et al. Problems and challenges in the development and validation of human
cell-based assays to determine nanoparticle-induced immunomodulatory effects. Part. Fibre
Toxicol. 2011, 8, doi:10.1186/1743-8977-8-8.

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).