hemolysis as expression of nanoparticles-induced ... · hemolysis rate with increasing np size for...

BIOTECHNOLOGY, MOLECULAR BIOLOGY AND NANOMEDICINE VOL.1 NO.1 OCTOBER 2013

ISSN: 2330-9318 (Print) ISSN: 2330-9326 (Online) http://www.researchpub.org/journal/bmbn/bmbn.html

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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



8

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



9

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



10

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)



11

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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hemolysis as expression of nanoparticles-induced ... · hemolysis rate with increasing np size for...

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