Emulsion-core and polyelectrolyte-shell nanocapsules: biocompatibility and neuroprotection against...

RESEARCH PAPER

Emulsion-core and polyelectrolyte-shell nanocapsules:biocompatibility and neuroprotection against SH-SY5Y cells

Marek Piotrowski • Krzysztof Szczepanowicz •

Danuta Jantas • Monika Leskiewicz •

Władysław Lason • Piotr Warszynski

Received: 23 May 2013 / Accepted: 30 September 2013 / Published online: 10 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The emulsion-core and polyelectrolyte-

coated nanocapsules, designed as water-insoluble neu-

roprotective drug delivery system, were synthesized

using layer-by-layer saturation method. The isopropyl

myristate was used as oil phase and docusate sodium

salt as emulsifier. For the polyelectrolyte shell prepa-

ration, synthetic polyelectrolytes, cationic (PDAD-

MAC, PAH, and PLL) and anionic (PGA) were used.

The particle size and zeta potential of nanocapsules

were characterized by the dynamic light scattering. The

average size of synthesized nanocapsules ranged from

*80 to *100 nm. Zeta potential values ranged from

less than approximately -30 mV for the polyanion

layers to greater than approximately ?30 mV for the

polycation layers. Biocompatibilities of the synthesized

nanocarriers were evaluated against SH-SY5Y human

neuroblastoma cells using various biochemical assays.

The results obtained show that synthesized nanocap-

sules coated with PLL and PGA were nontoxic to SH-

SY5Y cells, and they were used as nanocarriers for

model neuroprotective drug (a calpain inhibitor MDL

28170). The neuroprotective action of the encapsulated

MDL 28170 against hydrogen peroxide-induced oxi-

dative stress cytotoxicity was evaluated in the same cell

line. The results showed that nanoencapsulated form of

MDL 28170 were biocompatible and protected SH-

SY5Y cells against the H2O2 (0.5 mM/24 h)-induced

damage in 20–40 times lower concentrations than those

of the same drug added directly to the culture medium.

These data suggest that the nanoscale carriers of

neuroprotective drugs might serve as novel promising

therapeutic agents for oxidative stress-related neurode-

generative processes.

Keywords Nanoencapsulation � Layer-by-

layer � Polyelectrolytes � Cytotoxicity � Calpain

inhibitor-MDL 28170 � Oxidative stress

Introduction

Successful treatment of central nervous system (CNS)

disorders—such as Parkinson’s and Alzheimer’s dis-

eases, stroke, and multiple sclerosis, being serious

medical and social problems—still remains highly

challenging (Kabanov and Gendelman 2007). In the

recent decades, the significant progress in understand-

ing the biochemical mechanisms of neuronal cell

damage has been observed, and as consequence, new

approaches to the treatment and prevention of neuro-

degenerative diseases have been proposed. On the

M. Piotrowski (&) � K. Szczepanowicz � P. Warszynski

Jerzy Haber Institute of Catalysis and Surface Chemistry,

Polish Academy of Sciences, Niezapominajek 8,

30-239 Krakow, Poland

e-mail: [email protected]

D. Jantas � M. Leskiewicz � W. Lason

Institute of Pharmacology, Polish Academy of Sciences,

Krakow, Poland

123

J Nanopart Res (2013) 15:2035

DOI 10.1007/s11051-013-2035-1

molecular basis of the apoptosis, excitotoxicity, and

oxidative damage, some new potential neuroprotec-

tive drugs have been recently suggested, e.g., neuro-

peptides, proteases (calpains, caspases, etc.)

inhibitors, immunosuppressants, neurosteroids, anti-

oxidant enzymes, and nonenzymatic antioxidants. A

pharmacological classification of various neuropro-

tective agents is extensively described in The Hand-

book of Neuroprotection (Jain 2011). Unfortunately,

there are no clear indications as to how the vast

majority of these drugs should be practically used in

clinical trials, which may be related to their poor

absorption and biodistribution, and adverse reactions

of immune system after systemic applications. More-

over, numerous potential neuroprotectants do not

penetrate the blood–brain barrier (BBB) (Pardridge

2003). Thus, the effective progress of the treatment of

neurodegenerative diseases may depend not only on

the better understanding of the molecular mechanisms

of neuropathology but also on the favorable pharma-

ceutical properties of neuroprotective agents (Kanwar

et al. 2012). Nanotechnology creates new opportuni-

ties to challenge undesirable properties of drugs.

Nanometer size of the drug-loaded particles (diame-

ters of *100 nm) and use of biocompatible com-

pounds in their preparation are among the basic

principles governing nanocarriers design. Simulta-

neously, nanoencapsulation provides opportunities to

solubilize hydrophobic drugs for systematic delivery

(Wischke and Schwendeman 2008). Moreover,

numerous neuroprotective drugs in the nanoform are

capable of overcoming the limitations of CNS barriers

and reach therapeutic concentrations in brain tissue

(Begley 2004; Silva 2007). Further development of

nanocarrier systems is mainly focusing on targeted

drug delivery and sustained release of the drugs by

specific surface functionalization/modification (Pinto

Reis et al. 2006). In general, the nanostrategy may lead

to the improvement of in vivo bioavailability and

efficiency of neuroprotective agents that can contrib-

ute to their high effectiveness in therapeutic action

while reducing undesirable side effects.

There are many examples in the scientific literature

where research on nanoparticulate drug delivery

systems is directed toward neurological approaches.

Wilson et al. (2008a, b) described nanoparticles of

poly (n-butyl-cyano-acrylate) (PnBCA) coated with

polycarbonate 80 that significantly increased transport

of drugs (tacrine and rivastygmine) used to treat

Alzheimer’s disease into the brain tissue. Later reports

of encapsulation of these drugs confirmed the potential

of such nanopharmaceuticals for the modern molec-

ular neuroscience. Chitosan nanoparticles coated with

polysorbinate 80 optimize tacrine pharmacokinetic

parameters after intravenous administration in rats

(Wilson et al. 2010), while rivastigmine encapsulated

in poly(lactide-co-glycolide) (PLGA) and PnBCA

nanoparticles exhibits positive therapeutic outcome

in amnesic mice (Joshi et al. 2010). Z-DEVD-FMK

(N-benzyloxycarbonil-Asp(OMe)-Glu(OMe)-Val-

Asp(OMe)-fluoromethylketone)-loaded polyethylene

glycol (PEG)-overcoated chitosan nanospheres func-

tionalized with transferrin receptor monoclonal anti-

bodies (TfRMAb) (Karatas et al. 2009) are another

example of nanoscale carriers of neuroprotective

drugs. It was shown that this nanomedicine is rapidly

transported across the BBB and decreases neurolog-

ical deficit and ischemia-induced caspase-3 activity in

mice. Kumar et al. investigated encapsulation and

release of the anti-tuberculosis drug rifampicin using

hydrogen-bonded layer-by-layer (LbL) microshells of

poly(vinyl pyrrolidone) (PVP) and poly(methacrylic

acid) (PMAA). The rifampicin drug released from the

LbL microshells had the same efficacy as the free

drug, indicating that the drug properties are not

changed by the encapsulation and release processes

(Kumar et al. 2009). Mulik et al. described apolipo-

protein E3 (ApoE3)-mediated PnBCA nanoparticles

containing curcumin and tested their neuroprotective

action against beta amyloid-induced cytotoxicity in

SH-SY5Y cells. The results indicated a significant

reduction of cytotoxic effects in human neuroblas-

toma cells treated with this functionalized nano-

spheres as well as reduction of reactive oxygen

species (ROS) formation (Mulik et al. 2012). Reddy

et al. described PLGA nanoparticles loaded with

superoxide dismutase (SOD) that showed neuropro-

tective activity against oxidative stress-induced cyto-

toxicity in primary cultures of human neurons, which

appears to be because of the stability of the

encapsulated enzyme and its better neuronal uptake

after encapsulation (Reddy et al. 2008). Another

approach was proposed by Williams et al. who

synthesized PEG-glutathione (GSH) conjugates.

These nanoassemblies alleviate the oxidative stress-

induced cytotoxicity in SH-SY5Y cells (Williams

et al. 2009). Boridy et al. evaluated the ability of

cholesterol-bearing pullulan (CHP) nanogels to

of 12 J Nanopart Res (2013) 15:2035

123

interact with the amyloid beta 1–42 (Ab1–42)

oligomeric forms and to reduce their toxicity on

primary cortical and microglial cells (Boridy et al.

2009). More and more researches concerning neuro-

protective effects of drugs encapsulated in numerous

nanosystems, such as liposomes, polymer nanoparti-

cles, nanocrystals, dendrimers, nanoshells, nano-

tubes, etc., are currently in various stages of

advancement. There are a number of reviews that

summarize recent discoveries in the field of nano-

neuroscience (Brambilla et al. 2011; Kanwar et al.

2012; Wong et al. 2012).

In this article, we present a simple method of

synthesis of nanocapsules with emulsion cores and

polyelectrolyte shells, which can be promising deliv-

ery systems for neuroprotective drugs. The polyelec-

trolyte shell is formed by the LbL method. In general,

LbL assembly is performed by depositing subsequent

layers of oppositely charged polyelectrolytes on a

substrate until a multilayered structure of the desired

thickness is formed (Decher 1997). The important

innovation in the LbL method was realized through

the use a colloidal core as a substrate. This technique

was pioneered by (Sukhorukov et al. 1998) and was

further developed by other author groups (Caruso

et al. 2001; Szczepanowicz et al. 2010a; Bazylinska

et al. 2012) for various core types (Adamczak et al.

2012; Szczepanowicz et al. 2013). In the present

study, we synthesized nanocapsules on the emulsion

droplets using LbL saturation method as described

previously (Szczepanowicz et al. 2010b). Further-

more, we evaluated biocompatibility of the synthe-

sized nanocapsules against the model SH-SY5Y

human neuroblastoma cells, the main prerequisite

for their application as a neuroprotective drug deliv-

ery system. The SH-SY5Y cell line, a thrice-cloned

subline of SK-N-SH cells, originally established from

a bone marrow biopsy of a neuroblastoma patient,

possesses the capability of proliferating in culture for

long periods without contamination. Therefore,

despite some minor disadvantages, the SH-SY5Y cell

line has been widely used in experimental neurolog-

ical studies as in vitro cell model, including analysis

of neurodegenerative processes and neuroprotection

(Xie et al. 2010). Then, we investigated the neuro-

protective potential of nanoencapsulated model drug

against oxidative stress-induced cytotoxicity in the

same cell culture. On the basis of preliminary

experiments, hydrogen peroxide and calpain inhibitor

MDL 28170 were chosen as an oxidative stress-

inducing cytotoxic agent and a neuroprotective drug,

respectively. Connection of oxidative stress and

calpain (calcium-dependent cytosolic cysteine prote-

ase) activity is well documented. Oxidative stress

increases intracellular free Ca2? levels and activates

Ca2?-dependent enzymes which are key effectors in

neuronal cell death reaction cascades (Czogalla and

Sikorski 2005; Ray et al. 2000). Inhibition of calpains

by a number of developed chemicals (Calpastatin,

Calpeptin, MDL 28170, etc.) has been observed

(Johnson 2000). In particular, MDL 28170, shows

high selectivity for calpains (Thompson et al. 2010).

Materials and methods

Chemicals

Synthetic polyelectrolytes (Table 1): PLL (P2636),

PAH (283215), PDADMAC (409014), and PGA

(P4886); surfactant: docusate sodium salt (AOT)

(D4422); hydrophobic phase: isopropyl myristate

(IPM) (W355690); and model drug: MDL 28170

(M6690) were purchased from Sigma Aldrich. PGA-

g-PEG copolymer was synthesized according to

(Boulmedais et al. 2004). Dulbecco’s modified Eagle

medium (DMEM) (41966-029) and fetal bovine serum

(FBS) (10270-106) were from Gibco. The Cytotoxic-

ity Detection Kit (11644793001) was from Roche

Diagnostic. MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (M2128), dimethyl sulf-

oxide (DMSO) (D5879), Triton X-100 (T9284), and

penicillin/streptomycin mixture (P4458) were from

Sigma Aldrich. Hydrogen peroxide (H2O2) (30 %)

was from POCH. All chemicals were used without

further purification. Ultrapure water was obtained

using Direct Q UV (Millipore).

Nanocapsules’ synthesis

Nanocapsules were prepared according to the LbL

saturation method analogous to the previous method

(Szczepanowicz et al. 2010b). In brief, nanocapsules

were formed by addition of hydrophobic phase with

dissolved anionic surfactant to polycation solution

under gentle mixing with a magnetic stirrer. The

optimal surfactant and polycation concentrations

were determined by measuring zeta potential of

J Nanopart Res (2013) 15:2035 of 12

123

nanoemulsion drops and examining their stability.

Stable emulsion was obtained when zeta potential of

emulsion drops with adsorbed polyelectrolyte layer

reached the constant value. On such prepared emul-

sion droplets, polyelectrolyte multilayer shell was

constructed repeating the LbL saturation procedure.

Amount of polyelectrolyte used to form each layer was

determined by zeta potential measurements; addition

of polyelectrolyte was stopped when zeta potential of

measured droplets reached constant value close to the

value obtained for free polyelectrolyte in solution.

PEGylated polyanion was used to form external layer

that was adsorbed to improve biodistribution of

nanocarriers. This type of modification should prolong

their circulation time by elimination of opsonization

process and fast clearance (Jokerst et al. 2011). Drug-

loaded nanocapsules were synthesized as described

above except that before emulsification process, active

agent was dissolved in the hydrophobic phase. Nano-

capsules were prepared in sterile conditions. The

illustrative scheme of the synthesized nanocapsules is

presented in Fig. 1.

Nanocapsules’ zeta potential determination

The zeta potential was measured using Laser Doppler

Electrophoresis (LDE) by Zetasizer Nano Series (Mal-

vern Instruments). Each value was obtained as an

average from three runs of the instrument with at least 20

measurements. The zeta potential of capsules as well as

of polyelectrolytes in solution was measured in 0.015 M

NaCl. All measurements were performed at 25 �C.

Nanocapsules’ concentration measurements

Nanoparticle concentration was determined by NTA

(Nanoparticle Tracking Analysis) using NS500 instru-

ment (NanoSight). The concentration of nanocapsules

was measured in 0.015 M NaCl. All measurements

were performed at 25 �C.

Nanocapsules’ size analysis

The size distribution (hydrodynamic diameter) of

nanocapsules was evaluated by dynamic light scatter-

ing (DLS). Experiments were carried out using

Zetasizer Nano Series (Malvern Instruments). Each

value was obtained as average from three runs with at

least 10 measurements. The size of the nanocapsules

was also measured by NTA using NS500 (NanoSight).

All measurements were performed at 25 �C in

0.015 M NaCl.

Table 1 Characteristics of synthetic polyelectrolytes used in the synthesis of nanocapsules

Polyelectrolyte Abbrev. Formula MW (g/mol) Ionic

character

Zeta potential

(0.015 M NaCl)

(mV)

Poly(L-lysine hydrobromide) PLL (C6H12N2O2)n�xHBr 30,000–70,000 Cationic ?42.7

Poly(allylamine

hydrochloride)

PAH (C3H7N)n�xHCl *15,000 Cationic ?46.0

Poly(diallyl dimethyl

ammonium chloride)

PDADMAC (C8H16NCl)n 100,000–200,000 Cationic ?68.0

Poly(L-glutamic acid) sodium salt PGA (C5H9NNaO4)n 50,000–100,000 Anionic -48.0

Fig. 1 Scheme of I core of the nanocapsule, II multilayered

nanocapsule (n number of layers), III drug-loaded nanocapsule,

IV PEGylated nanocapsule


123

Nanocapsules’ stability studies

The stability test for nanocapsules was based on the

analysis of the time-dependent changes in their size

distribution and zeta potential. Nanocapsules suspen-

sions were stored at the room temperature. The

hydrodynamic diameter and zeta potential of nano-

capsules were periodically measured using Zetasizer

Nano Series (Malvern Instruments).

Nanocapsules’ visualization

Nanocapsules were visualized using scanning electron

microscopy (SEM) (JEOL JSM-7500F). Samples

were prepared by pouring nanocapsules suspension

in 0.015 M NaCl solution on the copper cylinders and

drying overnight.

SH-SY5Y cell culture

Human neuroblastoma SH-SY5Y (ATCC, passages

10–30) cells were grown in DMEM supplemented

with 10 % FBS and 0.1 % penicillin/streptomycin

mixture. Cells were maintained at 37 �C in a saturated

humidity atmosphere containing 95 % air and 5 %

CO2. Cells were seeded into 96-well plates with the

density of 4 9 105 per ml. 24 h before experiments,

culture medium was replaced by DMEM containing

1 % FBS.

Cell treatment

For determination of biocompatibility/cytotoxicity of

the synthesized cores/nanocapsules (AOT/PAH, AOT/

PDADMAC, AOT/PLL, AOT/PLL/PGA, and AOT/

PLL/PGA-g-PEG), SH-SY5Y cells were treated with

10 ll of particular solutions to achieve the final

concentration 5.7 9 109 particles/ml and 2.85 9 109

particles/ml. To establish the proper concentration of

hydrogen peroxide for neuroprotective studies, we

treated SH-SY5Y cells with series of dilutions of H2O2:

0.1, 0.25, 0.5, and 1 mM for 6 and 24 h. The H2O2

concentrations: 1 mM for 6 h and 0.5 mM for 24 h

were chosen for concomitant studies with calpain

inhibitor, MDL 28170 (1–10 lM). MDL 28170 stock

solution (10 mM) was prepared in DMSO and stored at

-20 �C. The final dilutions of MDL 28170 and H2O2

were prepared in distilled water. Nanocapsules were

stored and diluted in 0.015 M NaCl. The chemicals

were present in cultures at a final concentration of 1 %

(for H2O2 and MDL 2817) or 10 % (for nanocapsules).

The vehicle-treated cells were treated with relevant

solvents (10 % DMSO or/and 0.015 M NaCl).

Cell-viability quantification

Cell viability was quantified using 3-[4,5-dimethyl-

thiazol-2-yl]-2,5-diphenyltetrazolium bromide colori-

metric assay as described by (Jantas et al. 2011). In

brief, tested agents were added to each well, and cells

were incubated for 24 h at 37 �C. Next, 10 ll of MTT

solution (1.5 mg/ml) was added to each well and

incubated for 40 min at 37 �C. Subsequently, cells

were centrifuged at 1,000 rpm for 3 min, and after

removing culture medium and excess MTT, purple

formazan salt crystals were dissolved in 100 ll DMSO

(dimethyl-sulfoxide). After a brief agitation on a plate

shaker, the absorbance of each sample was measured

spectrophotometrically at a wavelength of 570 nm in a

96-well plate-reader (Multiscan, Labsystems). The

data were normalized to the absorbance in the control

group with vehicle-treated cells (control ? vehicle,

100 %). Results were expressed as a percent of the

control group (±SEM) established from n C 5 wells

from three separate experiments.

Cell death assessments

In order to estimate cell death, the level of lactate

dehydrogenase (LDH) released from damaged cells

into culture media was quantified as described by

(Jantas et al. 2011). A colorimetric assay was used, in

which the amount of red formazan salt, formed after

the conversion of lactate to pyruvate, proportional to

LDH activity and to the number of damaged cells in

the sample, was measured. After 24 h of cells’

incubation with tested agents at 37 �C, cell-free

culture supernatants (85 ll) were collected from each

well and incubated with the reagent mixture according

to the manufacturer’s instructions (Cytotoxicity

Detection Kit, Roche) for 20 min. The absorbance of

each sample was measured using 96 wells plate-reader

(Multiscan, Labsystems) at a wavelength of 490 nm.

Data were normalized to the activity of LDH released

from Triton X-100-treated cells (total, 100 %). Results

were expressed as a percent of the Triton X-100-

treated cells (±SEM) established from n C 5 wells

from three separate experiments.


123

Statistical analysis

Data after normalization to the control or total group

(±SEM) were analyzed using Statistica software

(StatSoft Inc.). One-way analysis of variance

(ANOVA) was used to determine overall significance.

Differences between control and experimental groups

were assessed using post-hoc Duncan test, with the

significant differences marked as follows: *p \ 0.05,

**p \ 0.01 and ***p \ 0.001 (vs control group) and

#p \ 0.05, ##p \ 0.01 and ###p \ 0.001 (vs H2O2-

treated cells).

Results and discussion

Preparation of nanocapsules

Nanocapsules were prepared using LbL saturation

method analogous to the method developed by other

authors (Szczepanowicz et al. 2010b). IPM with

dissolved AOT (330 g/l) was used as the hydrophobic

phase. It is worth noting that both AOT and IPM are

bearing GRAS (Generally Regarded as Safe) status

under FDA (Food and Drug Administration) regula-

tions. Polyelectrolytes were dissolved in 0.015 M

NaCl solutions. Emulsion cores of nanocapsules were

formed by adding a fixed 0.1 ml volume of AOT/IPM

to 200 ml of aqueous polycation solutions in the range

of concentrations: 5–50 ppm under mixing using a

magnetic stirrer at 300 rpm. The optimal surfactant/

polyelectrolyte ratio was determined by zeta potential

measurements (Fig. 2). Stable emulsion cores were

obtained when zeta potential of emulsion drops with

adsorbed polycation layer reached the constant value

(Table 2). By this way, the amount of free polyelec-

trolyte in the nanocapsules’ suspension was mini-

mized as most of it was consumed to reverse charge of

the emulsion drops.

Subsequent layers of the polyelectrolyte shell were

adsorbed on emulsion cores using similar procedure.

Fixed volume of positively charged nanoemulsion

core was added to the polyanion solutions, and proper

polyanion concentration was used in the synthesis

process. Amount of polyelectrolyte used to form each

layer was determined by zeta potential measurements.

Stable nanocapsules were obtained when zeta poten-

tial of emulsion core with adsorbed subsequent

polyelectrolyte layer reached the constant value close

to the value obtained for free polyelectrolyte in

solution. The observed layer-to-layer variations of

zeta potential provide evidence for the formation of

consecutive layers (Fig. 3). Zeta potential values

ranged from less than approximately -30 mV for

Fig. 2 Example of determining optimal surfactant/polyelec-

trolyte ratio: the dependence of zeta potential of AOT/PLL-

stabilized nanocapsules’ core on PLL concentration in the

water-phase of suspension before emulsification

Table 2 Zeta potential values of emulsion cores stabilized by

a complex of AOT and cationic polyelectrolyte

Emulsion core (IPM) Zeta potential (mV)

AOT/PDADMAC ?66.5

AOT/PAH ?78.5

AOT/PLL ?71.6

Fig. 3 Layer-to-layer variations of zeta potential of AOT(PLL/

PGA)n nanocapsules (n number of layers)


123

the polyanion layers to greater than approximately

?30 mV for the polycation layers. This value of zeta

potential (absolute value) indicates that the surface

charge of capsules is high enough to ensure electro-

static stabilization of their suspension, thereby pre-

venting aggregation and contributing to the long-term

stability.

PEGylated external shell layer, formed to improve

biodistribution of nanocapsules, was obtained by

coating positively charged AOT/PLL nanocapsules

with layer of PGA-g-PEG by means of the same LbL

procedure, i.e., by adding suspension of PLL-termi-

nated cores into filtered PGA-g-PEG solution. The

zeta potential of nanocapsules with PGA-g-PEG

external layer was -3 ± 4 mV, but they were stable

for up to 90 days because of steric stabilization by

PEG tails. Drug-loaded nanocapsules were synthe-

sized as described above except that before emulsifi-

cation process, model drug (MDL 28170) was

dissolved in AOT/IPM mixture.

Characterization of nanocapsules

The average size of synthesized AOT(PLL/PGA)1

nanocapsules measured by DLS and NTA was

*80 nm (Figs. 4, 5) and was increasing with the

number of adsorbed layers to *100 nm (Fig. 5).

Particle concentration determined by NTA was

5.7 9 1010 particles/ml. The example of SEM micro-

graph of the same AOT(PLL/PGA)1 capsules is shown

in Fig. 6. The size of the most of observed particles

were below 100 nm (*80 nm), which clearly con-

firmed the values obtained by DLS and NTA.

Drug encapsulation

The results of our previous study have indicated that

nanocapsules, formed by the LbL shell formation on

emulsion cores, can be successfully used as loaded

nanocarriers for some model drugs (e.g., beta-caro-

tene, vitamin A) (Szczepanowicz et al. 2010b).

Therefore, we decided to use this approach to directly

synthesize nanocapsules containing neuroprotective

Fig. 4 Size distribution of

AOT(PLL/PGA)n

nanocapsules by DLS:

n = 1 (left), n = 5 (right)

Fig. 5 Size distribution of AOT(PLL/PGA)1 nanocapsules by

NTA (inset of Fig. 5 nanocapsules visualization by the light

scattering)

Fig. 6 The SEM micrograph of AOT(PLL/PGA)1

nanocapsules


123

compounds. The model neuroprotective agent, calpain

inhibitor MDL 28170 (Fig. 7), was chosen as an active

component of the emulsion core of nanocapsules.

MDL of 25 mg was dissolved in 6 ml of AOT/IPM

solution to achieve a concentration 4.17 mg/ml and

was stored in 4 �C before emulsification process and

nanocapsules formation.

Biocompatibility of nanocapsules

In vitro biocompatibility testing is a common first step

in assessing nanoparticle-related health hazards. Since

our major driving force for synthesis of the nanocap-

sules is their potential use for practical neuropharma-

ceutical applications, SH-SY5Y human neuroblastoma

cells were used. Emulsion cores stabilized by various

AOT-polycation complexes were synthesized (AOT/

PAH, AOT/PDADMAC, and AOT/PLL). Subse-

quently, second layers were adsorbed onto AOT/PLL-

stabilized cores using polyanion PGA and PEGylated-

PGA (PGA-g-PEG). Cell-viability and cell death

assessments (by MTT and LDH assays, respectively)

were used to determine biocompatibility/cytotoxicity of

the synthesized nanocapsules. Both assays were done

24 h after treatment with the agents. The concentrations

of nanocapsules chosen for the biochemical tests were

5.7 9 109 and 2.85 9 109 particles/ml, which corre-

spond roughly to *1.5 9 105 and *0.75 9 105

particles/cell, respectively. The results of the biocom-

patibility testing are summarized in Figs. 8 and 9.

Statistically significant toxic effect is clearly

observed for nanocapsules stabilized by AOT/PAH

and AOT/PDADMAC complexes. By far, the least toxic

AOT/PLL-stabilized cores were chosen to be further

covered by a second polyanion layer. Polyanion PGA-

and PEGylated-PGA covered nanocapsules are non-

toxic to cells after 24 h of incubation. The lack of

increase of the cell viability in MTT tests with no LDH

change may suggest that nanocapsules exhibit no effect

on stimulation of cells proliferation, which is sometimes

observed when using unspecified, undifferentiated SH-

Fig. 7 Molecular formula of calpain inhibitor MDL 28170

Fig. 8 MTT reduction in SH-SY5Y culture after treatment with

IPM cores stabilized by AOT/PAH, AOT/PDADMAC, AOT/

PLL complexes; and AOT/PLL/PGA and AOT/PLL/PGA-g-

PEG nanocapsules. Each bar represents an average value and

SEM taken from n C 5 wells from three independent experi-

ments. Comparisons were made using analysis of variance

followed by Duncan’s test. ***p \ 0.001; *p \ 0.05 versus

control group

Fig. 9 LDH release in SH-SY5Y culture after treatment with

IPM core nanocapsules stabilized by AOT/PAH, AOT/PDAD-

MAC, AOT/PLL complexes; and AOT/PLL/PGA and AOT/

PLL/PGA-g-PEG nanocapsules. Each bar represents an average

value and SEM taken from n C 5 wells from three independent

experiments. Comparisons were made using analysis of

variance followed by Duncan’s test. ***p \ 0.001 versus

control group


123

SY5Y cells. Since nanoparticles, having large surface

areas and chemically active surfaces, may interfere with

viability assays, producing a false assessment of toxicity

and making it difficult to analyze, we have also

measured the extent of particle interference in MTT

and LDH assays. The obtained results suggested the lack

of nanocapsules’ interference on biochemical assays

(data not shown). We did not observe differences in

control and control ? vehicle groups. Consequently,

the impact of the vehicle (0.015 M NaCl) on the

cytotoxicity results can be excluded.

Cytotoxicity model

SH-SY5Y cells were treated with hydrogen peroxide

as an oxidative stress-inducing cytotoxic agent. Series

of dilutions of H2O2: 0.1, 0.25, 0.5, and 1 mM were

added to the culture medium. Afterward, MTT assay

was performed to quantify cells’ viability (Fig. 10).

Tests were performed after 6 and 24 h of incubation.

Investigations of cells’ viability under H2O2 condi-

tions showed time- and concentration-dependent

decrease in cell viability. The H2O2 concentrations

closest to the IC50 (the half maximal inhibitory

concentration) value were 1 mM after 6 h of incuba-

tion and 0.5 mM after 24 h of incubation. Conse-

quently, these concentrations were chosen as an

oxidative stress-inducing doses. The IC50 hydrogen

peroxide concentration of 0.5 mM (after 24 h) is in

good agreement with the finding of (Kwon et al.

2011a, b) who used H2O2 0.4 mM for 24 h and

reached the viability of incubated cells around 60 %.

The neuroprotective potential of model drug (MDL

28170) was evaluated against selected hydrogen

peroxide oxidative stress-induced cytotoxicity models

in the SH-SY5Y cell culture (Fig. 11). In particular,

the SH-SY5Y cells were treated with MDL 28170 in

the range of concentrations: 1–10 lM before the

exposure to 1 mM H2O2 for 6 h and 0.5 mM H2O2 for

24 h. The results indicated that the MDL 28170

concentration of 10 lM gave statistically significant

higher percentage of cell viability as compared to

H2O2-treated cells. The neuroprotective effect was not

observed after an incubation time of 6 h with a

concentration of hydrogen peroxide of 1 mM. In view

of the above, the neuroprotective potential of nanoen-

capsulated MDL 28170 was further evaluated against

0.5 mM H2O2-induced oxidative stress cytotoxicity in

SH-SY5Y human neuroblastoma cell culture for 24 h.

Neuroprotective action of encapsulated MDL

28170

MTT assay showed that nanoencapsulated MDL

28170 significantly attenuated the H2O2-induced

Fig. 10 MTT reduction in SH-SY5Y culture after H2O2-

induced oxidative stress. Each bar represents an average value

and SEM taken from n C 5 wells from three independent

experiments. Comparisons were made using analysis of

variance followed by Duncan’s test. ***p \ 0.001 versus

control group

Fig. 11 MTT reduction in SH-SY5Y culture after MDL 28170

treatment before cells exposure to the oxidative stress condi-

tions. Each bar represents an average value and SEM taken from

n C 5 wells from three independent experiments. Comparisons

were made using analysis of variance followed by Duncan’s test.

***p \ 0.001 versus control group; ##p \ 0.01 versus H2O2-

treated cells


123

cytotoxicity (Fig. 12). Similar to the results of MDL

28170 that was added directly to the culture medium,

encapsulated drug exerts neuroprotective effect on

SH-SY5Y cell lines as compared with untreated,

H2O2-exposed cells. It is worth noting that the

concentrations of the drug in nanoform in the culture

medium was 0.54 lM (5.7 9 109 particles/ml) and

0.27 lM (2.85 9 109 particles/ml) what is almost 20

and 40 times lower, respectively, than therapeutic

concentration of 10 lM of the same drug added

directly to the culture medium, having the same effect

on the cell viability (Fig. 11). This is very significant

in terms of pharmacology, since lower therapeutic

dose of the drug may reduce its undesired side effects.

Moreover, it is crucial from an economical point of

view, because the active agents are generally the most

expensive part of drug formulations. Although there is

a noticeable statistically significant difference

between nanoencapsulated drug-treated cells and

control group in MTT assay, these promising pre-

liminary results, however, indicate toward the neces-

sity of further studies of neuroprotective potential of

MDL 28170, such as microscopic analysis, flow

cytometry, etc. The results contributed to the further

plans for the extension of this research on primary

cultures of neurons and hippocampal slice cultures

using MDL 28170 in the form of nanocapsules.

Conclusion

We have described the preparation of nanocapsules

with emulsion cores stabilized by a complex of anionic

surfactant AOT and various cationic synthetic poly-

electrolytes. Nanocapsules’ shell consisting of anionic

and cationic polyelectrolytes was obtained using the

LbL method. The PEGylated external layer was

adsorbed to improve biodistribution of nanocarriers.

That type of modification should prolong circulation

time by elimination of the opsonization process and

fast clearance. Before the evaluation of neuroprotec-

tive action of nanoencapsulated model drug, MTT and

LDH biocompatibility tests of empty capsules were

performed. The results obtained show that synthesized

nanocapsules coated with PLL and PGA are nontoxic

to SH-SY5Y cells and they can be used as nanocarriers

for model neuroprotective drug—calpain inhibitor

MDL 28170. As a neurodegenerative model hydrogen

peroxide-induced cytotoxicity was chosen. The con-

centration of 0.5 mM H2O2 was able to induce

apoptotic cell death of SH-SY5Y cell culture within

24 h after incubation. The neuroprotective potential of

MDL 28170 was evaluated against oxidative stress

cytotoxicity in the same cell culture. The cells showed

the most significant viability at concentration of

10 lM. MTT assay showed that pretreatment with

encapsulated MDL 28170, similar to the same drug to

some extent, protected SH-SY5Y cells against H2O2-

induced cytotoxicity. The main advantage of nano-

capsulated MDL 28170 is that it exhibits neuropro-

tective action at c.a. 40 times lower dose than the drug

directly added to the assay.

The obtained results showed that the developed

nanocapsules are nontoxic to SH-SY5Y human neu-

roblastoma cells and could be the candidate for

nanocarriers for water insoluble neuroprotective

drugs, for instance, MDL 28170. This principal

prerequisite for nanomedicine applications provides

grounds for considering them for further experiments

that eventually may result in the more efficient

treatment of neurodegenerative disorders.

Acknowledgments This study was co-financed by the

Interdisciplinary PhD Studies: ‘‘Molecular sciences for

Fig. 12 MTT reduction in SH-SY5Y culture after H2O2-

induced oxidative stress incubated with encapsulated MDL

28170. Each bar represents an average value and SEM taken

from n C 5 wells from three independent experiments. Com-

parisons were made using analysis of variance followed by

Duncan’s test: ***p \ 0.001 versus control group; #p \ 0.05

versus H2O2-treated cells


123

medicine’’ (co-financed by the European Social Fund within the

Human Capital Operational Programme); the Polish National

Science Centre, grant no. DEC-2011/03/N/ST5/04808; and the

Marian Smoluchowski Krakow Research Consortium, a leading

National Research Centre KNOW, supported by the Ministry of

Science and Higher Education.

Conflict of interest The author reports no conflicts of interest

in this work.

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