hemolysis as expression of nanoparticles-induced...
TRANSCRIPT
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Abstract— Nanomedicine has a huge potential to bring
benefits in prophylaxis, diagnosis and treatment of various
diseases, with increased socio-economic benefits. Since
most of the future applications of therapeutic nanoparticles
(NPs) are based on intravenous/oral administration,
experiments on their interaction with human blood
components are of extreme importance. In this context, the
knowledge of the degree to which damage on red blood cells
following exposure to silver nanoparticles contributes to
the overall toxicity of various NPs is still highly needed. The
present paper unprecedentedly assesses the hemolytic
impact of biomedical nanoparticles on red blood cells
(RBCs).
Keywords— cytotoxicity, hemolysis, nanoparticles, erythrocyte,
therapy
I. INTRODUCTION
anotechnology implies the development of materials and
functional structures with at least one characteristic
nanometric dimension. Biomedical nanoparticles have
interesting electronic properties, based on the unique
size-dependent features thus exhibiting highly photothermal
effect with surface plasmon resonance and strong optical
absorption.[1-4] Their unique chemical stability make these
materials suitable for the attachment of biomolecules like
peptides, pharmacologically active substances and genes. So
far, these nanoparticles have proven several promising effects,
such as antitumor[5] gene therapy vector[6],
anti-inflammatory[7], antiviral[8] and antibacterial effects[9,
10]. Even though most literature data dedicated to therapeutic
nanoparticles refer to in vitro toxicological assessments[11, 12],
recent data brings evidences on their in vivo toxicity. Various
This work was funded by the Romanian Ministry of Education and Research
(PN-II-PT-PCCA-2011-3.1-1586, PN-II-RU-TE -2011-3-0251; PN-II-RU
-PD-2011-3-0287 PN-II-PT-PCCA-2011 -3.1-1551; PN-II-PT -PCCA- 2011-3.2-1289).
Department of Physiology, “Iuliu Hatieganu” University of Medicine and
Pharmacy, Victor Babes nr.8, 400012 Cluj-Napoca, Romania. *Correspondence to Teodora Mocan (e-mail: [email protected]).
animal models, including zebrafish[13], Sprague-Dawley
rats[14-17], mice[18] showed an increased level of toxicity of
these nanoparticles. Following administration, the transit of
these nanoparticles through the blood flow constantly occurs
independent of administration route.[14-19] Since most of the
future applications of therapeutic nanoparticles are based on
intravenous/oral administration, experiments on their
interaction with human blood components are of extreme
importance. In this context, the knowledge of the degree to
which damage on red blood cells following exposure to silver
nanoparticles contributes to the overall toxicity of various Nps
is still highly needed.
The present paper unprecedentedly assesses the hemolytic
impact of biomedical nanoparticles on red blood cells. (RBCs)
II. HEMOLYSIS
RBC lysis has been demonstrated for amorphous
nanomaterials such as silica NPs, thus limiting their rapid
application into human trials. The incriminated mechanisms is
centered on silanol groups existent on the outer surface of
NPs.[20] Also, tricalcium phosphate (TCP) and Hydroxyapatite
(HAP) nanoparticles (NPs), highly used in various medical
applications, have been proven to increase the hemolysis rate
with more than 5 % at 1000µg/ml concentration of RBC
exposure. The two types of nanoparticles revealed different
outcome, with higher hemolytic effect following TCP exposure.
The effect on RBC is accompanied by of reduction in
macrophages proliferation rate.[21] This is not the only report
on HAP nanoparticles to produce hemolysis. Another research
team selected HAP-NPs ranging from 30 to 80 nm in diameter.
Direct contact exposure as well as agar overlay techniques were
used to analyze cytotoxicity on rat macrophages. Also,
hemolysis test was used to assess effects of NPs exposure on
RBC.HSP70 identification by means of Western Blot was also
performed. Similar to other studies, an increase of over 5% in
hemolysis rate was found for concentrations of over 1000
µg/ml. HSP7 was up-regulated by concentrations of 100 µg/ml
HAP NPs and inhibition of macrophages’ proliferation could
be found at concentrations as low as 20 µg/ml. The study draws
a signal of concern regarding the opportunity of selecting HAP
NPs for medical applications.[22]
Another interesting study focused on the cytotoxic effects of
silver powders with different particulate diameter. Two
Hemolysis as Expression of
Nanoparticles-Induced Cytotoxicity in Red
Blood Cells
Teodora Mocan, MD, PhD,
N
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micron-sized and two nano-sized powders were prepared for
testing by water dispersion, followed by complete
characterization of obtained solution (SERS, TEM, and DLS).
Although two distinct methods were used for preparation of
solution needed for particle-RBC contact, nano-sized silver
particles revealed significantly higher hemolytic rates
compared to micro-sized silver particles are similar
concentration (220 μg/mL) regardless the method used.
According to presented data, this could be due to several factors,
including: increased silver ion release, increased surface area
or mechanical interaction with RBC. [23]. Similar data have
been reported by another group who showed a decreasing
hemolysis rate with increasing NP size for silver NPs exposure.
The authors incriminate, similar to other researchers, higher
effective surface with consecutive higher silver ions release in
smaller particulate size as compared to larger ones.[24] In all
this cases the hemolytic effect was considered to be induced by
the release of oxidative stress products following exposure.
(Figure 1)
Fig. 1. Schematic illustration of oxidative stress induced mechanisms of
hemolysis following administration of various nanoparticles.
Comparison between the observed effects following RBC
exposure to different types of nanoparticles has been proposed
by other studies. One of them focused on hemolysis,
erythrocyte sedimentation rate, membrane topography,
hemagglutination, and lipid peroxidation induces by three
distinct types of NPs: silver, gold and platinum following their
contact with RBC. Similar to the results presented, data
suggested silver NPs to be less hemocompatible as compared to
platinum and gold NPs. Following silver NPs exposure at
100 µg mL−1, evidences of hemolysis, membrane damage,
morphological alterations, and hemagglutination as well as
cytoskeleton changes were recorded. Also, RBC membrane
presented multiple depressions and pits following exposure.
None of these noxious effects were recorded following
macro-silver exposure. Interestingly, DNA alterations have
been recorded in nucleated cells as a result of their exposure to
hemolyzed erythrocyte fraction.[25] The hemolytic and
antimicrobial properties of silver nanoparticles and silver ions
(I) combined with various antimicrobial agents, including
polymimyxin B were tested. The antimicrobial activity against
Gram negative bacteria has demonstrated to act as an additive
effect of both materials and antimicrobial agents. Moreover, a
very low hemolytic activity of silver nanoparticles has been
reported as compared to AgNO3. The authors therefore
conclude that synergic treatment using the combined effect of
both polymyxin B and silver NPs represent a promising
antimicrobial treatment against Gram-negative bacteria.[26]
Other researchers have focused on nanomaterials like iron
oxide (Fe2O3) and zinc oxide (ZnO) as well as multi-walled
carbon nanotubes (MWCNTs) and their effect on RBC.
Possible effect modulation induced by their shape and
composition has also being studied. Results revealed extended
hemolysis for MWCNTs as well as absence of
hemagglutination following RBC exposure. This conclusion
was sustained by AFM images showing no signs of larger
particles formation due to RBC agglomeration. Based on the
already known biocompatibility of macro-carbon materials,
investigators draw the conclusion that the effects are due to the
shape alone and not the composition of the NPs. For iron oxide
NPs, results revealed an increased NP-RBC binding, and a
weak tendency of the NPs themselves to form large aggregates.
By contrast, zinc oxide NPs recorded high rate of clumping.
Such large, micrometer-sized aggregates formed by zinc oxide
NPs are very mobile within blood flow and become very toxic
to RBCs. Nanosilica NPs, following aggregate formation, has
been also said to promote hemolysis. The process follows
various steps including shape change from discocytes to
spherocytes, loss of membrane’s resiliency and flexibility,
swelling and volume increase and finally membrane break.[27]
Also, it has been demonstrated that silicon dioxide (SiO2) NPs
are able to induce a higher the 5% hemolysis rate following in
vivo continuous injection exposure.[28] Comparison between
different silica NPs has also been investigated. It has been
shown that amorphous silica is able to induce a significantly
higher hemolysis rate as compared to MCM-41-type
mesoporous silica NPs, thus making the latter a promising
candidate for in vivo applications.[29]
Research focusing on TiO2 NPs has brought evidences of
negative effects such as hemolysis (in a dose-dependent
manner), sedimentation rate, or hemagglutination. The process
is, most probably, passing, similar to other particles through
several starting with RBC attachment, TiO2 NPs
trans-membrane transport and oxidative stress and ending with
membrane breakage.[30]
Lipid-based nanoparticles with crystalline structure (LCNPs)
have also been intensely investigated. Different chemical
compositions have been tested for their safety as potential
intravenous drugs components. Results showed an intense
hemolytic effect for glycerol monooleate (GMO)-based LCNPs,
thus making such structures incompatible for human
intravenous administration. No hemolysis was observed in the
case of soy phosphatidylcholine (SPC)/GDO, making them an
appropriate candidate for various applications. Also,
intermediate hemolysis rate for as long chained molecules, such
as sponge phase nanoparticles based on
DGMO/GDO/polysorbate 80 (P80). Such molecules should be
used with caution in application heading in vivo intravenous
administration. All data, however, converge to the conclusion
that hemolytic properties of LCNPs are strongly connected to
the chemical structure of the monomers in the case [31]. Similar
data have been obtained in the case of partially quaternized
poly [thio-1-(N,N-diethyl-aminomethyl) ethylene]s, Q-P(T
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DAE) NPs. The nanostructures have been demonstrated to
induce RBC lipid bilayer disruption with hemolysis,
electrostatic attraction followed by hemagglutination. Also,
effects have demonstrated to be dependent on blood protein
content.[32] A group of researchers focused on synthesis and
characterization of surface-modified SLN for adsorptive
protein loading. Both the modulation of the emulsifier
concentration and the lipid matrix have been performed. As
demonstrated by complex techniques, such as X –ray
diffraction, thermoanalysis, electron microscopy demonstrated
the formation of crystalline and anisometrical particles.
Hemolysis test, performed as a measure of toxicity revealed
safe rates of RBC lysis of under 5 % (1.1 to 4.6%).[33]
Microemulsion-based nanoparticles (MEs) has been proposed
for in vivo applications. Various toxicological data have been
reported. At similar pharmacological concentrations sodium
caprylate induced significant hemolysis while MEs and
pluronic surfactants seem biocompatible.[34]
Other types of nanoparticles, like for example E78NPs were
found to absolutely harmless to RBC membrane integrity,
especially at low concentrations (1mg/mL), thus making the
suitable for blood-delivered applications regardless the surface
alteration (PEG-coated or non-pegylated E78 NPs).[35]
Similarly, de novo synthesized NPs, such as
heparin-immobilized polyethersulfone (PES) has passed
hemolysis test, demonstrating RBC lysis of less than 5%.[36]
Likewise, a study testing the effect of RBC exposure to
calix-arene based Solid Lipid Nanoparticles (SLNs)
demonstrated no hemolytic effect.[37] Crystalline carbon
nanoparticles(CNPs) with intense fluorescent properties
obtained by means of addition of different organic tags, such as
α-naphthylamine, fluorescein and rhodamine B. were
synthesized. Erythrocyte exposure to these type of
nanoparticles induced no signs of toxicity.[38] Minimal
cytotoxicity and RBC toxicity was observed for
polymethacrylate nanoparticles (NP), a type of NPs designed
for applications of oligodeoxynucleotides delivery.[39]
Cross-linked gelatin nanoparticles with the encapsulation of
cycloheximine, an already demonstrated inhibitor for protein
synthesis have also been designed. Data obtained from blood
compatibility testing experiments revealed minimal or no signs
of hemolysis.[40] An increasing interest has been generated by
layered double hydroxides (LDHs) for cell delivery of
bio-active molecules, drugs and genes. However, only limited
studies have focused on testing their biocompatibility testing.
One of these studies tested several chemical compositions
(MgAl-LDH as well as ZnAl-LDL). In vitro testing was
performed on carcinoma cells, normal human cells and RBCs.
Quantification of possible noxious effects was done by
measuring cell proliferation, membrane damage, cell
proliferation and hemolytic effect. None of the tested chemical
structures was able to induce significant hemolysis. A slightly
higher toxicity was recorded for ZnAl-LDH as compared to
MgAl-LDH, detectable as increased hemolysis and membrane
damage. The authors conclude that LDH could represent a
promising substrate for future in vivo drug delivery
applications.[41] By means of emulsion/solvent evaporation
technique, a study imagined the method to generate a distinct
type of NPs, made of a conjugate of
poly(D,L-lactide-co-glycolide) with alendronate (PLGA–ALE
NPs). Following administration, lysis of erythrocytes was
studied. No hemolysis was detected following PLGA–ALE
NPs exposure, the rate being similar to that recorded for PBS
exposure.[42] Another research group studied nanoparticles
based on complex polymeric structures, such as
poly(3-hydroxybutyrate)–poly(ethylene glycol)–poly(3-
hydroxyl butyrate) (PHB-PEG-PHB). They have been
proposed for drug delivery applications, therefore cytotoxicity
and hemolysis test were performed. No signs of cytotoxicity
and hemotoxicity were recorded, even for concentrations as
high as 120μg/mL. These results sustain the designed NPs’
potential for the intended applications.[43] Other types of NPs ,
like N-acyl chitosan nanoparticles, prepared by the
tripolyphosphate anions reactions, have been designed and
proposed for several applications. The testing experiments
revealed good hemocompatibility, as quantified by
thromboelastography and hemolysis.[44]
It has been already known that cytostatic therapies have
hematological toxicity as a common adverse effect. Various
methods have been proposed for diminishing these effects. One
of them was to encapsulate the drug into biocompatible
materials. One such treatment agent is Paclitaxel, a drug known
to present a low stability in aqueous media and to present
various adverse effects induced by the solubilizer used in
preparation. One group of researchers developed a new
formulation of amphiphilic cyclodextrin nanoparticles by
encapsulation of Paclitaxel. Hemolysis and cytotoxicity
experiments were performed to test the biocompatibility of the
newly synthesized NPs. Data were compared to a common
vehicle: cremophor: ethanol (50:50 v/v). Significantly reduced
hemolysis was obtained for the encapsulated nanoparticles, as it
could be inferred by SEM- imaging.[45] Another cytostatic
agent of interest was Doxorubicin. For the encapsulation
purpose, spherical self-assembled nanoparticles based on
oleoyl-chitosan (OCH) have been developed. The efficiency of
Doxorubicin loading was 52.6%. Drug releasing was complete
and rapid at PH =3.8. At PH 7.4 releasing process had two
distinct phases: a first burst release, followed by a sustained
release. Drug efficiency, quantified by the death rates following
in vitro exposure of various cancer cell lines, was significantly
higher than Doxorubicin solution alone. However, before
proposing any particular applications, the newly synthesized
NPs have been tested by means of MTT assay and hemolysis
test. No cytotoxicity signs were found on embryo fibroblasts.
All hemolysis experiments, performed under distinct
conditions resulted in acceptable hemolysis rates (under 5%).
All data suggest a good potential for the developed NPs
towards human anticancer applications.[46] Similarly,
self-assembled Fe3O4 magnetic nanoparticles (MNPs) were
developed for encapsulation of daunorubicin (DNR). By
contrast with DNR alone, the so-loaded capsule presented a
good hemocompatibility, with no hemolysis.[47] Similarly,
research has been developed for reducing the hemotoxicity of
camptothecin, a potent anticancer drug. Lipid nanoparticles
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based on different lipid cores were designed for its
encapsulation. The final NPs, holding camptothecin content
revealed a hemolysis rate of under 5%, thus placing the RBC
adverse reaction within acceptable limits.[48] Not only the
cytostatic agents raised the interest of researchers for
encapsulation. Anti-malarial drugs, such as arthemeter (ARM)
were loaded in lipid mass. For this purpose, a film of thin-film
hydration method was used. The lipid structures were glyceryl
soybean oil and trimyristate (solid lipid). Particles were further
loaded with the ARM 10%. Nanoparticulate surface was
optimized by means of non-ionic, cationic and anionic
surfactants. Testing of the newly developed NPs revealed a
level of hemolysis of under 7% and a significantly enhanced in
vivo anti-malarial activity as compared to the regular drug
solution, currently on the market.[49]
Interesting results were brought by experiments testing NPs
of mixed chemical structure. High biocompatibility, including
low hemolytic rates were found for monodisperse Au-silicate
nanoparticles (10.7 ± 1.6 nm in diameter).[50] Similar data
have been reported following RBC exposure to core–shell
Fe3O4@ Au composite magnetic nanoparticles (MNPs). All
concentrations used induces hemolysis rates of under 5%[51],
the already established cut-off for hemolytic effect .[52] Also, a
complex method has been imagined for superparamagnetic
Fe3O4nanoparticles synthesis using nickel-nitrilotriacetate
(Ni-NTA) structure modification. The opportunity of their
application for vivo imaging has been demonstrated by the lack
of hemolysis after RBC exposure.[53]
A very recent report demonstrates the modulation of
hemolytic properties of nanoparticles by the protein content of
plasma. Data shown demonstrate that RBCs exposure to
polystyrene nanoparticles (PS-NP) in protein-free medium
results in hemolysis (in a particle). It has also been reported
that PH can represent another modulator factor for the
hemolytic process. The authors focused on
protein-encapsulated hydrophobic γ-PGA (γ-hPGA)
nanoparticles. Due to a conformational change of γ-hPGA NPs,
hemolysis appears under PH conditions ranging from 7
downward to 5.5, but never at physiological PH.
Hydrophobicity, increased by the PH-induced conformational
change, might be the mechanism responsible for membrane
disruption. The authors conclude that the designed NPs could
find their utility into DNA, protein, drug delivery or other
systems based on endosome-disruptive NPs.[54]
These findings generated the idea to reduce the hemolysis
rate by different methods. One of them consists of using special
types of nanoparticles (such as magnet-controlled sorbent NPs:
MCS-B) for extracorporeal processing of blood. A group of
researchers performed complex experiments towards three
different aims. The first one was to detect the existence of a
relationship between the timing of hemolysis process debut and
the intensity of MCS-B exposure. The second objective was to
investigate specific metabolic pathways in exposed RBC: ATP
transport, Na-K and Ca-Mg ATPhases. Thirdly, the group
oriented their investigation towards standardizing and
optimizing the MCS-B protocol, so to obtain the best
anti-hemolytic effect. Results established that an optimal
desired effect can be best obtained by means of 1-2 sequences
of extracorporeal blood processing with MCS-B NPs. The
treatment decreased the Ca-Mg ATPhases without altering the
Na-K ATPhases.[55]
Another interesting research direction was to alter the
surface of NPs in order to reduce their hemolytic effects. One
such approach was that of gold NPs synthesized by a
biocompatible polymer (1900-DAPEG) which allowed surface
conformational alterations. Compatibility testing of the newly
synthesized NPs, performed by comparison with surfactant
stabilized gold-NPs (which served as control) revealed
decreased hemolytic rate. Also, other helpful properties were
observed, such as being significantly lower platelet activators
as their compared to their counterparts.[56] Significantly, it has
been also demonstrated that the rate of hemolysis is
significantly different between raw PLGA, PEG-ylated PLGA
and surfactant stabilized PLGA.[57] Observations and
experiments were carried out for obtaining an optimal
PEG-attachment at the surface of the nanoparticles. One such
study focused on mesoporous silica NPs (MCM-41) with
diameter of 150±20nm. PEGxk chains of various chain
densities and molecular weights were covalently linked to the
surface of the nanoparticles. PEG10k variant of newly
synthesized NPs showed a reduced hemolysis rate( 0.9%) as
compared to the rest of tested NPs.[58] It has also been
hypothesized that chitosan/pDNA-linked NPs could benefit
from a higher blood compatibility as compared to their raw
counterparts. Although an increased interaction was detected
between different particles, most probably coming from
positively charged surface or free polymer residues, the results
showed negative or neutral zeta potential. None of the so
designed NPs induced significant hemolysis.[59] The study
does not represent the only research focused on chitosan-based
NPs. Another report described the method for development of
Chitosan-N-trimethylaminoethylmethacrylate chloride–PEG
(CS-TM–PEG) copolymers, meant to reduce the toxicity of
chitosan. Results showed a hemolysis rate from10.3% to
13.6% for the non-PEGylated copolymer and from 4.76% to
7.05% for the PEGylated designed NP. The concentrations of
the copolymer used were 250-2000 μg/ml concentrated. The
study concludes that these 200-400nm wide nanoparticles,
showing an efficiency of 90% could be made more
hemocompatible by PEG-alteration of their surface.[60]
Another approach was described by a team of researchers
who performed de novo gold NPs synthesis using a natural
extract (Zingiber officinale). The extract used is known to
present double valence: both reducing and stabilizing. The
newly developed nanomaterials have demonstrated reduced
complement and platelet activating properties as well as low
hemolytic activity.[61] The auto fluorescence levels, represents
a strong indicator of erythrocytes health status. When we tested
the auto fluorescence changes following administration of
colloidal gold nanoparticles (20 nm) found no signs of toxicity
among the tested Au solutions. (Figure 2)
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Fig. 2. Autofluorescence image of human RBC incubated for 60 minutes with
gold nanoparticles at various concentrations. No noticeable changes were observed among the three tested solutions
None of the above aforementioned studies, however,
included NPs themselves as controls for the purpose of
identifying interferences with the assay they could come from
the specificity of the NPs.[62] The need for more complex
investigation techniques brought recent techniques into
consideration and usage into experimental studies, such as light
scattering by RBCs based on T-matrix formalism.[63] He-Ne
laser light scattering was also investigated by means of a
rouleau of n (2 ≤ n ≤ 8) RBCs of regular size. The rouleau
was formed by the regular agglomeration of RBC along the axis
of a vessel when low blood flow conditions are assured. The
RBC-RBC interactions were analyzed numerically by means of
fast Fourier transform methodology.[64] Development of
another method called tracking velocimetry (based on cell
magnetophoresis) , was also reported. The technique enables
researchers to quantify the migration of RBCs (both
deoxygenated and containing metHb). The unpaired electrons
contained in the four hem groups (deoxy and metHb) offers the
mentioned molecules paramagnetic properties, very different
from the diamagnetic properties of oxyhemoglobin. These
findings offer the theoretical backgrounds for differential
migration of the two classes of molecules when exposed to a
strong magnetic field. Such a method could be an useful tool for
biochemical analysis in cell characterization and separation
based on magnetic characteristics of self-molecules.[65]
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