RESEARCH PAPER

Development of curcumin-loaded poly(hydroxybutyrate-co-hydroxyvalerate) nanoparticles as anti-inflammatorycarriers to human-activated endothelial cells

Viorel Simion • Daniela Stan • Ana-Maria Gan • Monica Madalina Pirvulescu •

Elena Butoi • Ileana Manduteanu • Mariana Deleanu • Eugen Andrei •

Anamaria Durdureanu-Angheluta • Marian Bota • Marius Enachescu •

Manuela Calin • Maya Simionescu

Received: 24 July 2013 / Accepted: 29 October 2013 / Published online: 9 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Curcumin (Cm)-loaded poly(hydroxybu-

tyrate-co-hydroxyvalerate) (PHBV) nanoparticles

(CmPN) were obtained and characterized and their

effect on human endothelial cells (HEC) was assessed.

Different CmPN formulations have been prepared

using the emulsion solvent evaporation technique, and

characterized for size, structure, Zeta potential, Cm

entrapment efficiency, and in vitro Cm release. CmPN

cytotoxicity and cellular uptake have been followed

using HEC. Also, the effect of CmPN treatment on the

p38MAPK signaling pathway in endothelial cells was

investigated. The results obtained by electron and

atomic force microscopy revealed the spherical shape

of the CmPN formulation. Based on size and

encapsulation efficiency, the CmPN formulation with

the average diameter of 186 nm and with the highest

encapsulation efficiency (83 %) has been used in the

further studies. The release of Cm from CmPN was

*18 % after 8 h of incubation at 37 �C, followed by a

slow release until 144 h, when it reached 44 %,

indicating a controlled release. CmPN are taken up by

HEC and exhibited low cytotoxicity at concentrations

up to 10 lM. The pre-treatment of HEC with CmPN

before exposure to tumor necrosis factor-alpha (TNF-a)

determined a decrease of p38MAPK phosphorylation.

In conclusion, Cm encapsulated into PHBV nanopar-

ticles, at concentration up to 10 lM, has low cytotox-

icity and display anti-inflammatory activity on TNF-a-

activated HEC by suppressing the phosphorylation of

p38MAPK.

Keywords Curcumin � Endothelium �Inflammation � Polymeric nanoparticles �Poly(hydroxybutyrate-co-hydroxyvalerate)

Introduction

Curcumin (diferuloylmethane, Cm), having the chem-

ical structure 1,7-bis(4-hydroxy-3-methoxyphenyl)-

1,6-heptadiene-3,5-dione, is a polyphenol responsible

for the yellow color of Curcuma longa, a curry spice. It

has been reported that Cm has effective anti-cancer,

anti-inflammatory, and antioxidant properties (Menon

and Sudheer 2007; Jurenka 2009).

Electronic supplementary material The online version ofthis article (doi:10.1007/s11051-013-2108-1) contains supple-mentary material, which is available to authorized users.

V. Simion � D. Stan � A.-M. Gan � M. M. Pirvulescu �E. Butoi � I. Manduteanu � M. Deleanu �E. Andrei � M. Calin (&) � M. Simionescu

Institute of Cellular Biology and Pathology ‘‘Nicolae

Simionescu’’ of the Romanian Academy, 8, BP Hasdeu

Street, PO Box 35-14, 050568 Bucharest, Romania

e-mail: manuela.calin@icbp.ro

A. Durdureanu-Angheluta � M. Calin

Institute of Macromolecular Chemistry ‘‘Petru Poni’’

of the Romanian Academy, Iasi, Romania

M. Bota � M. Enachescu

Center for Surface Science and Nanotechnology,

Politehnica University, Bucharest, Romania

123

J Nanopart Res (2013) 15:2108

DOI 10.1007/s11051-013-2108-1

Curcumin mechanisms of action and intracellular

signaling pathways have been intensively studied for

its anti-cancer activity (Anand et al. 2008a; Singh and

Khar 2006). It was shown that in cultured cells,

curcumin induces apoptosis of tumor cells by inhib-

iting various intracellular transcription factors and

secondary messengers such as NF-kB, AP-1, c-Jun,

and the JAK-STAT pathway (Anand et al. 2008b;

Aggarwal et al. 2003).

The anti-inflammatory activity of Cm, as demon-

strated on human endothelial cells, involves down-

regulation of the nuclear factor-kappa B (NF-kB),

extracellular signal-regulated kinase (ERK1/2) and

p38 mitogen-activated protein kinase (p38MAPK)

signaling pathways and consequently, the decreased

production of interleukin-8 (IL-8), vascular cell

adhesion molecule-1 (VCAM-1), endothelial cell

leukocyte adhesion molecule-1 (ELAM-1), and inter-

cellular adhesion molecule-1 (ICAM-1) and the ensu-

ing decline of monocytes adhesion to endothelium.

(Wang and Dong 2012; Aggarwal and Harikumar

2009a; Pirvulescu et al. 2011).

Despite its extraordinary therapeutic effects, curcu-

min’s major drawback is its poor solubility and low

bioavailability after oral administration due to the rapid

elimination from the body. In phase I clinical studies,

Cm administration in cancer therapy produced minimal

side effects, and histological improvement of precan-

cerous lesions in one out of two patients with recently

resected bladder cancer and in two out of six patients

with Bowen’s disease (Cheng et al. 2001). However,

pharmacokinetic studies showed that only nanomolar

concentrations of Cm could be detected in serum, after

oral administration of several grams of curcumin per

day (Lao et al. 2006). Therefore, there was a need to

increase the circulation time and bioavailability of Cm.

To achieve these goals, many attempts have been made

to encapsulate Cm in different types of nanoparticles

(Bansal et al. 2011): liposomes (Li et al. 2005),

magnetic (Yallapu et al. 2012), gold (Manju and

Sreenivasan 2012), or polymeric nanoparticles employ-

ing polymers like poly(butyl)cyanoacrylate (Mulik

et al. 2012), poly(lactide-co-glycolide) (Anand et al.

2010), triblock poly(ethylene glycol)-poly(e-caprolac-

tone)-poly(ethylene glycol) (Feng et al. 2012), and

chitosan (Wan et al. 2012).

Since water solubility is one of the important

requirements for an ideal drug, in the present study we

devise a new approach by formulating curcumin into

PHBV (poly-hydroxybutyrate-co-hydroxyvalerate)

nanoparticles.

PHBV is a novel biodegradable and biocompatible

polymer which started to draw the attention of

scientists as a new candidate for particulate systems,

which offers the potential of releasing the encapsu-

lated drug by erosion of the tissue engineering

scaffolds (Sultana and Wang 2012) or nanoparticles

surface (Pignatello et al. 2009). PHBV is obtained by

copolymerization of polyhydroxybutyrate (PHB) and

hydroxyvalerate (HV), which are both polyhydrox-

yalkanoates. PHB is a rigid and highly crystalline

polymer with slow degradation rate, while PHBV has

a lower glass transition and melting temperatures and,

as a consequence, is more flexible and easier to

process than PHB (Masaeli et al. 2013). Different

molecular weights of PHB and PHBV polymers can be

obtained naturally, by varying the growing conditions

of bacteria (Verlinden et al. 2007). Because the main

degraded product of PHBV, R-3-hydroxybutyric acid,

is a normal constituent of the blood (Wiggam et al.

1997) and also found in the cell envelope of eukary-

otes (Reusch 2000), it was assumed that PHBV can be

well tolerated in vivo (Errico et al. 2009). The

hemocompatibility of PHBV polymer has been previ-

ously investigated by following the interaction of

blood components with PHBV films and the data

showed that the polymer did not exhibit hemolytic

properties on human red blood cells (Mendes et al.

2012), did not affect the platelets response and did not

activate the complement system (Sevastianov et al.

2003).

Most of the studies performed using Cm-entrapped

nanoparticles were focused on cancer therapy. Even

though it has been reported that Cm dissolved in

ethanol or DMSO protects from ischemia by prevent-

ing the blood–brain barrier damage (Jiang et al. 2007),

precludes homocysteine-induced dysfunction in endo-

thelial cells (Ramaswami et al. 2004), suppresses the

cholesterol accumulation in macrophage foam cells

and retards atherosclerosis in ApoE-/- mice (Zhao

et al. 2012), only few of the formulations of curcumin-

loaded nanoparticles have been introduced thus far for

cardiovascular disease therapy (Rogers et al. 2012;

Lobatto et al. 2010). We have chosen to employ, for

our experiments, PHBV nanoparticles because of the

beneficial properties of this polymer such as biode-

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gradability, biocompatibility, and low cost for its

production. Apart from forming a stable suspension in

water, these nanoparticles also exhibit fluorescent

properties due to the fluorescent characteristics of Cm.

The obtained curcumin-encapsulated nanoparticles

form a monodispersed population in water, which

also adds to its advantage. To our knowledge, no

attempt has been made to encapsulate curcumin in

PHBV nanoparticles and use it for anti-inflammatory

therapeutic purposes, thus far.

We report here that Cm encapsulated in PHBV

nanoparticles (CmPN), at concentration up to 10 lM,

has low cytotoxicity and displays anti-inflammatory

activity on tumor necrosis factor-alpha (TNF-a)-

activated human endothelial cells by suppressing the

phosphorylation of p38MAPK.

Materials and methods

Reagents

Reagents were obtained from the following sources:

curcumin, poly(3-hydroxybutyrate-co-3-hydroxyvaler-

ate (12 % hydroxyvalerate content), polyvinyl alcohol

(PVA), chloroform, Tween 80 and 3-[4,5-dimethyl-

thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

from Sigma-Aldrich Chemie (Germany). TNF-a, anti-

human pp38, anti-human p38, and anti-mouse second-

ary antibodies coupled with HRP from R&D Systems;

Dulbecco’s modified Eagle’s medium (DMEM), fetal

calf serum (FCS), penicillin G, streptomycin from

Gibco BRL (Gaithersburg, MD/USA), the cell culture

plates were supplied by Corning (New York, NY/

USA), acetonitrile (HPLC grade) was purchased from

Merck KGaA, Darmstadt, Germany and acetic acid

(HPLC grade) from Panreac Quimica, Spain. Deionised

(18.2 MX/cm) water was generated in-house using a

Milli-Q System from Millipore.

Preparation of curcumin-loaded PHBV

nanoparticles (CmPN)

Curcumin-containing polymer nanoparticles were pre-

pared using the emulsion solvent evaporation method

(Rosca et al. 2004). Briefly, the organic phase (com-

posed of PHBV and Cm dissolved in 2 ml chloroform)

and the aqueous phase (containing 30 ml of double

distilled water and different concentrations of Tween 80

and polyvinyl alcohol) were prepared separately. Then,

the aqueous phase was placed in a sonication bath and

the organic phase was added drop wise and homoge-

nized for 10 min at 21.000 rpm with a Heidolph Silent

Crusher M homogenizer. Further, the formed emulsion

was evaporated under vacuum for 60 min to eliminate

possible traces of the organic solvent.

Polymer nanoparticles characterization

Particle size and zeta-potential measurements

The mean diameter and size distribution of CmPN was

determined by dynamic light scattering (DLS) method

using a submicron particle analyzer (Nicomp Model

380 Particle Size Systems, USA) with a He–Ne Laser

of 5mW operated at 632.8 nm using a 900 angle

between incident and scattered beams. The data

obtained were subjected to Gaussian/Nicomp analysis

using Nicomp CW388 software with a viscosity of

0.933 cPoise and a refractive index of 1.331 at 230 C.

The CmPN size was evaluated in terms of volume–

weight distribution. The Zeta-potential was deter-

mined by electrophoretic light scattering (ELS), which

determines electrophoretic movement of charged

particles under an applied electric field, using a Delsa

Nano Submicron Particle Size Analyser (Beckman

Coulter).

Transmission electron microscopy (TEM)

The morphology of CmPN was assessed by TEM

using the negative staining technique. Briefly, equiv-

olumes of the sample and 1 % phosphotungstic acid

solution were mixed, and then a drop of the solution

was allowed to settle on a carbon-coated copper grid

for 1 min. After removing the excess sample with a

filter paper, the grids were air-dried in a desiccator,

and examined with a Philips EM 410 electron

microscope.

Atomic force microscopy (AFM) imaging

CmPN was diluted in water (1: 100) and deposited in

droplets onto a mica surface for 10 min. Upon

removing the excess with a filter paper, the sample

was dried under gentle N2 flux. The AFM images were

obtained with an intermittent contact (or tapping)

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mode with a scan speed of 1 Hz. The results were

analyzed with the XPMPro software.

Determination of Cm encapsulation efficiency

in CmPN (EE%)

Cm-entrapped nanoparticles were separated from the

non-entrapped free drug by centrifugation at

20,000 rpm, 4 �C, for 20 min, using Millipore Cen-

trifugal Filter Units 100 kDa (MW 100000 Amicon

Ultracentrifugal filter devices). The amount of free Cm

in the filtrate was mixed with ethanol (1:1 w/v) and

compared to a standard curve of Cm (1.5, 3, 6, 12, 25,

50, 100, 150, 200, 250, 300, and 400 lM) dissolved in

water/ethanol (1:1 v/v) and quantified using a TECAN

spectrophotometer (Infinite M200Pro, TECAN). The

EE% was calculated using the formula: EE %ð Þ ¼Drug½ �tot� Drug½ �free

� ��Drug½ �tot � 100:

In vitro release kinetics

The release of curcumin from nanoparticles was

carried out according to the method previously

described (Mohanty and Sahoo 2010; Rejinold et al.

2011). Briefly, 13.5 ml of CmPN suspension was

divided in 27 Eppendorf tubes (500 ll each), as the

experiment was performed in triplicates. The tubes

were kept in a Heidolph orbital shaker at 37 �C and

150 rpm. Free Cm is completely insoluble in water,

therefore, at predetermined intervals of time, the

solution was centrifuged at 3,000 rpm for 10 min

(Hettich Zentrifugen, Germany) to separate the

released (pelleted) curcumin from the curcumin

entrapped into nanoparticles. The released Cm was

quantified using a high performance liquid chroma-

tography (HPLC) method previously described (Li

et al. 2009). Cm was dissolved in 1 ml acetonitrile and

3 ll of this solution was injected in a ZORBAX

Eclipse Plus C18 column narrow bore RR

(150 mm 9 2.1 mm, 3.5 lm) maintained at 25 �C,

at a flow rate 0.25 ml/min. The mobile phase consisted

of a 5 % aqueous acetic acid and acetonitrile (25:75,

v/v). An Ultra HPLC (UHPLC) system (Agilent

Technologies 1290 Infinity) equipped with binary

solvent delivery pump (with four channels), autosam-

pler, thermostatted column compartment, a Diode

Array Detector and Agilent ChemStation software for

data acquisition and processing, was used. The

detector wavelength was set at 425 nm. The amount

of curcumin released at different time intervals was

determined using a calibration curve of curcumin

dissolved in acetonitrile (correlation coefficient was

r2 = 0.99984).

Cell culture

Human endothelial cells (HEC), EA.hy926 line, were

kindly donated by Dr. Cora Jean Edgell (Department

of Pathology, University of North Carolina and Chapel

Hill). The cells express the typical characteristics of

endothelial cells such as forming in culture a mono-

layer of closely apposed polygonal cells and express-

ing von Willebrand factor. The cells were grown to

confluence in Petri dishes (60 mm diameter) or in

tissue culture plates (6-, 24-, or 96 wells) in Dul-

becco’s modified Eagle’s medium (DMEM) supple-

mented with 10 % FCS, 100 U penicillin, 100 lg

streptomycin, 50 lg neomycin/ml, at 37 �C in a 5 %

CO2 incubator, as previously described (Edgell et al.

1983).

Cytotoxicity assay

The cytotoxicity of Cm, Cm-entrapped polymer

nanoparticles (CmPN), and control PN (identical

concentration of nanoparticles as CmPN, but without

curcumin) on HEC was evaluated by MTT assay

(Mosmann 1983). The method used for determining

the cell viability measures the reduction of tetrazolium

salt, MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphe-

nyltetrazolium bromide), by the action of dehydroge-

nase enzymes in metabolically active cells and the

generation of intracellular purple formazan, which can

be solubilized and quantified by spectrophotometry.

The MTT assay was performed in 96- and 24-well

plates following the standard procedure. Cells were

seeded at a density of 1 9 104 cells/well for 96-well

plates and 4 9 104 cells/well for 24-well plates and

allowed to attach. After 24 h, the culture medium was

replaced with fresh medium containing different

concentrations of free Cm, CmPN, and control PN

and incubated for 48 h. The media was then replaced

by the MTT solution (0.5 mg/ml in PBS) added to

each well. After 3 h incubation at 37 �C and 5 % CO2,

the media was removed and the formazan crystals

formed were solubilized with the lysis buffer contain-

ing 0.1 N HCl in isopropanol for 4 h at 37 �C. The

optical absorbance was measured at 570 nm with the

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reference wavelength at 720 nm on a microplate

reader (Tecan GENios). The results were expressed as

percentages relative to the results obtained with the

control cells (untreated cells). The differences in the

results obtained from different concentration of Cm,

CmPN, and control PN were statistically analyzed

using t test.

Cellular uptake of Cm-entrapped PHBV

nanoparticles

Flow cytometry analysis

To evaluate CmPN uptake efficiency, HEC were

seeded in 12-well plates for 24 h and then treated with

different concentrations of CmPN for 4 h. After

washing three times with PBS, the cultured HEC were

trypsinized and analyzed using a Gallios Flow

Cytometer (Beckman Coulter). Following flow

cytometry measurements, the samples were re-sus-

pended in a Trypan blue solution (Fluka, 200 lg/ml)

and measured again by flow cytometry. Trypan blue

was used to quench the extracellular fluorescence of

non-internalized CmPN. The difference between the

two measurements was considered as non-internalized

nanoparticles (Mo et al. 2012; Trewyn et al. 2008).

Confocal microscopy

The qualitative analysis of CmPN uptake by HEC was

performed using confocal microscopy (Das and Sahoo

2012). Human endothelial cells were seeded in a

Labtech Tissue Culture Dishes in 300 ll of complete

growth medium at a density of 80,000 cells/ml. After

24 h, the cells were incubated for 4 h with a suspen-

sion of 10 lM CmPN added to the cell culture

medium, washed for three times with PBS, fixed with

4 % paraformaldehyde, washed again with PBS for

three times and mounted with Roti-Mount FluorCare

DAPI from ROTH GmBH, Germany. Samples were

visualized with a Confocal laser scanning inverted

microscope (Leica TCS SP5) using 470 ± 20 nm

excitation and 525 ± 25 nm emission wavelengths

for free Cm and CmPN visualization, and

350 ± 20 nm excitation and 470 ± 20 nm emission

wavelengths for DAPI-stained nuclei. The images

were processed using LAS AF software (version 2.6).

Effect of CmPN treatment on p38MAPK signaling

pathway in endothelial cells

Western blot assay

HEC were seeded at a concentration of 60,000 cells/ml

and allowed to grow for 24 h then treated with 10 lM

Cm free or encapsulated into PN (CmPN) or with

control PN (without curcumin) for 6 h. Next, 10 ng/ml

TNFa was added to the incubation medium for another

12 h. Afterward, the cells were solubilized in 29

sodium dodecyl sulfate (SDS) gel sample buffer,

heated in a boiling water bath (5 min), sonicated and

diluted with SDS sample buffer (Pirvulescu et al.

2011). Then, 50 lg protein was subjected to 12 %

polyacrylamide gels electrophoresis and transferred

onto nitrocellulose membranes (using a Trans Blot

Semi-Dry system); blots were blocked in 5 % non-fat

dry milk in TBS and 0.05 % Tween (blocking buffer)

and subsequently incubated with anti-human pp38

(1:200 dilution) or anti-human p38 (1:200 dilution)

mouse monoclonal antibodies in blocking buffer,

overnight, at 4 �C. After washing, the membranes

were incubated with the secondary antibodies coupled

with HRP (1:2,000 dilution) in blocking buffer (1 h,

room temperature), washed in TBS containing 0.05 %

Tween 20 (wash buffer), incubated with enhanced

chemiluminescence reagents (ECL, 5 min), exposed

to an X-ray film and analyzed with a video system

(Image Master from Pharmacia); the optical density

was calculated using the Total Lab 1.11 software from

Pharmacia.

Statistical analysis

The results were expressed as mean ± SEM and

experiments were performed at least in triplicates.

Statistical differences were evaluated using Student’s

t test analysis. Differences were considered to be

statistically significant at a level of p \ 0.05.

Results

Preparation and characterization of CmPN

The schematic representation of the CmPN is depicted

in Fig. 1, where curcumin is entrapped in the poly-

mer matrix. First, we optimized the nanoparticles

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formulations by varying the concentration of PVA and

Tween 80. In general, the CmPN appeared as a clear

solution, suggesting the entrapment of Cm in the

polymer matrix of PHBV nanoparticles, which renders

the complete dispersion of Cm in water (d), as

compared to free Cm dissolved in water, where

macroscopic precipitates can be observed

(a) (Fig. 2). The fluorescence characteristic of Cm is

preserved by its encapsulation in nanoparticles as is

revealed by the emission spectrum of CmPN suspen-

sion in water compared to spectrum of free Cm

dissolved in ethanol. A spectral shift to the left when

Cm is entrapped in nanoparticles can be observed

(Fig. 2e).

Dynamic light scattering analysis revealed that the

formulated Cm-loaded PHBV nanoparticles had an

index of polydispersity in the range of 0.18–1.26, a

hydrodynamic diameter between 100 and 800 nm

and a negative zeta potential in the range of -12 to

-19 mV (Table 1). As expected, the average size,

polydispersity index, zeta potential, and encapsula-

tion efficiency were significantly affected by the

composition of formulated CmPN. Previous studies

showed that nanoparticles with slightly negative

Z-potential (approximately -5 to -20 mV) are the

most appropriate system to be used to mediate the

delivery of drugs at other sites than reticulo-endo-

thelial system because they are not opsonised as

rapid as nanoparticles with positive surface charge

Fig. 1 Schematic representation of curcumin loaded PHBV

nanoparticles (CmPN)

Fig. 2 Photographs

depicting curcumin (Cm)

dispersed in water (a),

ethanol (b), or entrapped

into polymeric PHBV

nanoparticles (CmPN)

(d) and empty polymeric

nanoparticles (PN) (c).

Emission spectra of free Cm

dissolved in water or ethanol

and empty (PN) or loaded

with curcumin PHBV

nanoparticles (CmPN)

(excitation wavelength

450 nm) (e)

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(Albanese et al. 2012) and do not stick non-specif-

ically to the cell’s surface (Jin et al. 2009). In our

study, it was observed a decrease of nanoparticles

size and a higher Z-potential value with the increase

of PVA concentration. Also, it can be observed that

the highest stability (indicated by the highest

Z-potential value) and highest encapsulation effi-

ciency are obtained in the case of nanoparticles with

the lowest polydispersity index (PI \ 0.2). These

results are in line with data reported in the literature

(Ranjan et al. 2012). The size and shape of

engineered nanoparticles were determined by trans-

mission electron microscopy (TEM) and atomic force

microscopy (AFM). Both methods revealed that

PHBV nanoparticles were spherical in shape, and

the size distribution was comparable to the values

determined by laser particle size analyzer (Fig. 3).

Upon evaluation of the six formulations under study,

we chose the CmPNf nanoparticles for further

experiments, because this formulation had the high-

est encapsulation efficiency and fulfilled the require-

ments of homogenous dispersed particles (Table 1).

In vitro release kinetics

The release kinetics of curcumin from the CmPN was

evaluated by HPLC. The initial release of curcumin

from nanoparticles (first hour of incubation) is

considered a burst effect of deposited or weekly

bounded drug molecules on the surface of nanoparti-

cles (Zhang et al. 2010). In our conditions,

18.05 ± 2.52 % of Cm was released after 8 h of

incubation, and then the concentration increased

steadily to 39.42 ± 1.23 % after 48 h of incubation,

followed by a slow release of Cm until 144 h, when it

reached 43.79 ± 0.68 %, indicating a controlled

release (Fig. 4).

Cytotoxicity of curcumin-loaded nanoparticles

By analyzing the cytotoxicity of CmPN on endothelial

cells, we aimed to establish the concentration of the

particles with the lowest possible cytotoxicity. The

experiments showed that the viability of HEC after

48 h incubation with concentrations of 0.5–5 lM

Table 1 Determination of the best formulation for curcumin (Cm) incorporation in poly(3 hydroxybutyrate-co-3-hydroxyvalerate

(PHBV) to construct curcumin-loaded polymeric nanoparticles (CmPN) by varying the concentration of polyvinyl alcohol (PVA) and

Tween-80

PN codes Cm (%) PHBV (%) PVA (%) Tween 80 (%) Size (nm) PI Zeta potential (mV) EE%

CmPNa 0.01 0.8 0.35 0.1 831 ± 8 0.53 ± 0.05 nd nd

CmPNb 0.03 0.8 0.35 0.35 308 ± 2 0.46 ± 0.001 -12 ± 0.04 62

CmPNc 0.01 0.8 1 0.35 230 ± 4 0.22 ± 0.002 -16 ± 0.7 80

CmPNd 0.01 0.8 1.3 1 113 ± 2 0.46 ± 0.002 -13 ± 0.03 nd

CmPNe 0.01 0.4 1.3 1.5 85 ± 8 1.265 ± 0.003 nd 37

CmPNf 0.01 0.8 1.3 0.8 186 ± 3 0.18 ± 0.001 -19 ± 0.8 83

PI polydispersity index, EE% encapsulation efficiency, nd not determined

Fig. 3 Engineered CmPN as seen by transmission electron microscopy (a), atomic force microscopy (b), and the nanosizer plot

diagram determined by laser particle size analyzer (c)

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CmPN was not affected, and only slightly diminished

at 10 lM CmPN concentration (*85 %). Concentra-

tions of 25 lM CmPN displayed a higher cytotoxicity

(*50 %). Thus, we established the 10 lM CmPN as

the maximum concentration to be employed to assess,

in further experiments, its effect on endothelial cells,

for a time period of maximum 48 h of incubation.

Empty nanoparticles (control PN) showed lower

cytotoxicity, which confirmed a good nanoparticles

formulation, suitable for further in vivo applications

(Fig. 5).

Uptake and internalization of CmPN

by endothelial cells

Nanocarriers, such as liposomes and polymeric nano-

particles, need to be internalized by the cells in order to

deliver the drug to the particular intracellular targets,

where its sites of action are located. To investigate the

internalization of CmPN by HEC, we employed

confocal microscopy and flow cytometry. Confocal

microscopy revealed that after 4 h incubation, the

cells take up CmPN, which appears as punctate

fluorescence dispersed within the cytosol (Fig. 6).

By comparison, the incubation of the cells with free

Cm (dissolved in ethanol) leads to a continuos

fluorescence in the cytosol, whereas in the case of

the incubation of the cells with control PN (empty,

without curcumin), no fluorescence could be observed.

In parallel experiments, flow cytometry analysis

demonstrated that the fluorescent signal increased as a

function of CmPN concentration (Fig. 7a). This

suggests that the amount of nanoparticles adhered

and/or internalized by the cells increased with the

increase of the Cm concentration entrapped into PN

(Fig. 7a).

To differentiate between the internalized nanopar-

ticles and that only adsorbed onto the cell membrane,

we exposed the cells to Trypan blue (0.4 %), which

was previously shown to quench the external fluores-

cence at the cell surface (Mo et al. 2012; Trewyn et al.

2008). Figure 7b depicted the decrease in the fluores-

cence of HEC incubated with 10 lM Cm entrapped

into PHBV nanoparticles (CmPN10) after adding of

Trypan blue solution (CmPN10 ? TB). Incubation of

HEC with different concentrations of CmPN, followed

by exposure to Trypan blue, showed that in the case of

25 and 50 lM CmPN, the internalization percent of

CmPN by endothelial cells is about 60–75 %, whereas

over 90 % of CmPN were internalized when 10 lM of

Cm entrapped PN was used (Fig. 7c).

CmPN suppress phosphorylation of p38MAPK

in HEC

Since it was demonstrated that curcumin is an inhibitor

of p38MAPK pathway in endothelial cells (Kim et al.

2007; Binion et al. 2008), we tested whether by

incorporating Cm into nanoparticles, the ability of Cm

to inhibit p38MAPK is still maintained. HEC were

seeded and allowed to grow overnight, then treated for

Fig. 4 In vitro release kinetics of curcumin from curcumin-

loaded polymeric PHBV nanoparticles (CmPN). Each point

represents the mean value ± SEM of a representative experi-

ment performed in triplicate

Fig. 5 Viability of cultured human endothelial cells subsequent

to incubation with free curcumin (Cm), curcumin-loaded

polymeric PHBV nanoparticles (CmPN) and empty polymeric

PHBV nanoparticles (PN) for 48 h. Each point represents the

mean value ± SEM (n = 3)

Page 8 of 15 J Nanopart Res (2013) 15:2108

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6 h with control PN (without curcumin), or with 1 and

10 lM Cm free or encapsulated into PN. Next, HEC

were activated with TNF-a for another 12 h and

further processed as described above (‘‘Effect of

CmPN treatment on p38MAPK signaling pathway in

endothelial cells’’ section).

The experiments showed that pre-treatment of

endothelial cells with CmPN before TNF-a-activation

reduced the activation of p38MAPK pathway to a

level similar to that obtained for free Cm at the same

concentration (Fig. 8). In the same experimental

conditions, empty PN had no effect on p38MAPK

Fig. 6 Confocal microscopy depicting human endothelial cells

incubated for 4 h at 37 �C with 10 lM free curcumin (free Cm),

10 lM curcumin-loaded polymeric nanoparticles (CmPN), and

empty polymeric nanoparticles (PN). The cells were visualized

for fluorescence of curcumin (green), DAPI-stained nuclei

(blue), and DIC (differential interference contrast) and the

merged images are presented. (Color figure online)

Fig. 7 Flow cytometry graph showing the uptake of CmPN by

endothelial cells as a function of concentration of the Cm

entrapped in polymeric nanoparticles, after 4 h incubation at

37 �C (a); pictograph of CmPN10 analyzed by flow-cytometry,

in the presence (CmPN10 ? TB) or absence (CmPN10) of

Trypan blue (b); percent of internalized CmPN determined by

Trypan blue method (c); CmPN10, CmPN25, CmPN50: PHBV

nanoparticles loaded with 10, 25, and 50 lM curcumin,

respectively; data are expressed as mean ± SEM of three

independent experiments

J Nanopart Res (2013) 15:2108 Page 9 of 15

123

pathway, which exhibited a similar level as in the cells

treated with TNF-a (Fig. 8). The experiments revealed

that the incorporation of curcumin in PN did not affect

the capability of Cm to inhibit the phosphorylation of

p38MAPK.

Discussions

Several investigators, including us, have documented

the potential effects of phytochemicals such as curcu-

min and other natural compounds as anti-inflamma-

tory therapeutics (Jurenka 2009; Wang and Dong

2012; Aggarwal and Harikumar 2009b; Pirvulescu

et al. 2011). In addition, the curcumin potential as an

anticancer agent is well established (Aggarwal et al.

2003; Anand et al. 2008a, b). However, some of its

drawbacks hamper the therapeutic efficacy of curcu-

min. First, its oral bioavailability is very low (Anand

et al. 2007) and hence, the oral administration of

curcumin is not feasible. Another drawback of curcu-

min is its photodecomposition (Bruzell et al. 2005).

The curative potential of curcumin and curcumi-

noids as anticancer agents were demonstrated using

cell culture studies, but in all the experiments, these

compounds were administered in a solution form

employing organic solvents like DMSO, ethanol, or

methanol (Aggarwal et al. 2007), which are not

recommended for in vivo use. Therefore, to exploit

the beneficial effects of Cm, there is a stringent need to

develop a drug delivery system capable to prevent

curcumin drawbacks and to achieve a maximum

therapeutic outcome. In this study, we aimed to

encapsulate curcumin into nanoparticles, in order to

outrun its hydrophobicity, protect it from degradation

and still reach its anti-inflammatory benefits.

Recently, the field of drug delivery progressed

especially with the evolution of nanotechnology,

wherein biocompatible nanoparticles have been devel-

oped as systemic carriers for therapeutic compounds

to target cells and tissues. Nanoparticles, as drug

delivery vehicles, enable passive targeting of tumors

and other inflamed tissues due to increased vascular

leakage as a result of increased production of

cytokines/chemokines and angiogenesis at these sites.

In the majority of solid tumors, the vascular cut-off

pore size range is between 380 and 780 nm (Yuan

et al. 1995), whereas normal vasculature is imperme-

able to particles larger than 2–4 nm (Gerlowski and

Jain 1986; Siflinger-Birnboim et al. 1987). At these

sites, the barrier function of the endothelium does not

function properly, and leakage of macromolecules and

exposure of the subendothelium to high concentrations

of plasma constituents, platelets, and red blood cells

lead to their accumulation in the interstitium. After

sustained inflammation, capillaries, and other small

vessels become leaky and angiogenesis may increase

(Joris et al. 1990). Injury of endothelial barrier

Fig. 8 Curcumin-loaded PHBV nanoparticles (CmPN)

decrease the phosphorylation of p38MAPK in TNF-a-activated

endothelial cells (HEC). Ratio of pp38MAPK/p38MAPK in

HEC (a) calculated from densitometric analysis of Western

Blots of pp38MAPK and p38 total MAPK (b). The data are

expressed as mean ± SEM of three independent experiments C:

non-activated HEC; TNF: HEC activated with TNF-a;

CmPN10: HEC exposed to CmPN loaded with 10 lM curcumin

before TNF-a activation; free Cm10: HEC exposed to 10 lM

free curcumin before TNF-a activation; Control PN 10: empty

nanoparticles used at the same concentration of nanoparticles as

CmPN 10; *p \ 0.05 significantly different, CmPN10 versus

TNF

Page 10 of 15 J Nanopart Res (2013) 15:2108

123

function occurs in arterioles after prolonged exposure

to histamine (Cuenoud et al. 1987; van Hinsbergh

1997), and also in arteries and veins in hypercholes-

terolemia or atherosclerosis conditions (Stemerman

1981).

Atherosclerotic lesions manifest an enhanced per-

meability of endothelium also due to endothelial cells

turnover in the large arteries. The death of endothelial

cells was found to be associated with massive albumin

leakage (63 %), comparing with normal aortic endo-

thelium (0.48 %) (Lin et al. 1990). Such changes in

vascular permeability enable the passive targeting of

inflamed tissues and tumors by NPs and their

accumulation generate an enhanced permeation and

retention (EPR) effect (Hobbs et al. 1998).

In this study, to overcome the drawbacks of Cm and

increase its bioavailability, we designed and prepared

curcumin-loaded PHBV nanoparticles (CmPN) and

tested their efficiency on activated HEC. We report

here that CmPN (1) exhibit high encapsulation

efficiency, (2) a controlled release of entrapped

curcumin, (3) low cytotoxicity on human endothelial

cells, (4) are efficiently internalized by the cells, and

(5) pre-incubation with CmPN induces a decrease of

p38MAPK activation in TNF-a-activated HEC. The

fate of any drug delivery system after in vivo admin-

istration mainly depends on its physicochemical

properties, and the size and structure of nanoparticles

affect their physical stability, biodistribution, and

cellular uptake. Thus, by encapsulating Cm inside of

PHBV nanoparticles, we aimed to prevent its degra-

dation and to obtain a sustained drug release.

The six different formulations of nanoparticles

developed by us (Table 1) were characterized for size,

structure, and encapsulation efficiency (by dynamic

light scattering, electron microscopy and atomic force

microscopy) and the formulation with the highest

encapsulation efficiency (CmPNf) has been chosen for

further investigations.

By copolymerization of PHB (which is a polymer

with very slow degradation rate) with 3-hydroxyval-

erate, a more flexible and easier to process copolymer,

PHBV is formed (Masaeli et al. 2013). Thus, previous

reports showed that progesterone was faster released

from microparticles obtained from PHBV copolymer

with 24 % HV content, than from microparticles of

PHBV copolymer with 9 % HV content, due to the

lower porosity of the matrix in the latter (Gangrade

and Price 1991; Gursel and Hasirci 1995). In our

studies, we used PHBV with 12 % HV content to

obtain polymeric nanoparticles, and reported a con-

trolled release of encapsulated curcumin from CmPN,

with *18 % release after 8 h of incubation at 37 �C,

followed by a slow release until 144 h, when 44 % of

the curcumin encapsulated in nanoparticles was

released.

Most of the curcumin-loaded polymeric nanopar-

ticles developed over time were employed in anti-

cancer studies, where the cytotoxic effects of curcu-

min at high concentrations was exploited (Anand et al.

2010) Although several studies proved the efficiency

of curcumin pre-treatment on inflammation, athero-

sclerosis and cardioprotection (Kumar et al. 1998;

Wang and Dong 2012), only few of the developed

nano-curcumin formulations are exploiting its anti-

inflammatory effects in cardiovascular diseases (Lob-

atto et al. 2011). When anti-inflammatory activity of

Cm is pursued, the low cytotoxicity of nanoparticles

formulations is a very important issue. In our study,

the experiments performed on cultured human endo-

thelial cells showed that the cell viability is not

considerably affected when concentrations of up to

10 lM Cm encapsulated into PHBV nanoparticles

were used (Fig. 5). The use of higher concentrations of

CmPN determined the significantly decrease of HEC

viability.

Based on the intrinsic fluorescence properties

exhibited by Cm, we found (by confocal microscopy)

that CmPN are internalized by HEC (Fig. 6). Since

confocal microscopy is only a qualitative analysis, the

uptake of CmPN by endothelial cells was quantified by

flow cytometry. The results showed that, as a function

of the concentration of Cm entrapped into nanoparti-

cles, the endothelial cells exhibited an enhanced

fluorescence, suggesting an increase in the adhered

and/or internalized nanoparticles by the cells

(Fig. 7a). To differentiate between the internalized

CmPN and those adsorbed on the cell membrane, the

external fluorescence of the cells was quenched by

employing Trypan blue, as previously reported (Mo

et al. 2012; Yallapu et al. 2010; Holpuch et al. 2010).

We detected the highest internalization efficiency of

CmPN by the endothelial cells (*90 %) at a concen-

tration of 10 lM Cm loaded into PNs. By increasing

the concentration of CmPN, a decreased internaliza-

tion efficiency was observed (Fig. 7b).

In a previous study, we reported that Cm (20 lM

concentration) display high anti-inflammatory effects

J Nanopart Res (2013) 15:2108 Page 11 of 15

123

by reducing the P-selectin and fractalkine expression

and the NADPH activation in human endothelial cells

(Pirvulescu et al. 2011). Also, it was reported that Cm

inhibits the p38MAPK pathway (Kim et al. 2007) and

reduces the expression of cell adhesion molecules in

activated endothelial cells through down regulation of

NF-kB transcription factor (Kumar et al. 1998;

Aggarwal et al. 2003). Thus, we tested whether Cm

activity and its effect on p38MAPK signaling pathway

is preserved after encapsulation in nanoparticles. We

found that the pre-incubation with CmPN decreases

the phosphorylation of p38MAPK in TNF-a-activated

HEC, similar to the effect of free curcumin (Fig. 8).

This indicates that encapsulated Cm maintains its

activity and suggests the anti-inflammatory effects of

CmPN on activated endothelial cells.

The advantages of the novel Cm-entrapped PHBV

nanoparticles introduced in this study over the existing

Cm nano-formulations are the low cost of PHBV

synthesis, which makes it attractive for large-scale

pharmaceutical production (Vilos and Velasquez

2012), the hemocompatibility (Mendes et al. 2012;

Sevastianov et al. 2003), and the excellent biocom-

patibility and biodegradability properties (Pignatello

et al. 2009; Sultana and Wang 2012; Wiggam et al.

1997; Errico et al. 2009). Moreover, PHBV is

naturally produced by bacteria, and this represent an

advantage when compared to synthetic polymers, like

poly(L-lactic acid) and poly(lactide-co-glycolide),

obtained in high temperature conditions, by employ-

ing catalysts and solvents, which should be completely

removed, in order to avoid problems with acceptance

of the polymer matrix formulation (Thomas and Lutz

2011).

The data provided in our study recommend Cm-

loaded PHBV nanoparticles as an appropriate system

for Cm delivery to endothelial cells and provide

support to continue the investigation of its pharmaco-

kinetic and toxicological evaluation in animal models.

Conclusion

Curcumin efficiently encapsulated into PHBV nano-

particles, exhibits very low cytotoxicity (at concen-

trations up to 10 lM), is efficiently internalized and

displays anti-inflammatory activity on activated

human endothelial cells by suppressing the phosphor-

ylation of p38MAPK. The engineered CmPN have the

advantage of maintaining the characteristic properties

of Cm, while reducing significantly its cytotoxicity,

thus being a novel and possible future drug-delivery

system and therapeutic tool. Our results indicate that

Cm has the potential to become a powerful therapeutic

agent in treating inflammatory diseases by encapsula-

tion into PHBV nanoparticles, which provides the

curcumin bioavailability and preserves its anti-inflam-

matory effects.

Acknowledgments This work was supported by UEFISCDI

(Executive Unit for Funding Education, Research, Development

and Innovation), Contract No. 4_001, Project NANODIATER

under the frame of EuroNanoMed, PNII-PCCE-ID-2011-2-

0028 Project; CARDIOPRO Project ID: 143, ERDF co-financed

investment in RTDI for Competitiveness and PN-II-ID-PCE-

2011-3-0928 Project, CNCS-UEFISCDI. The financial support

from European Social Fund ,,Cristofor I. Simionescu’’

Postdoctoral Fellowship Programme ID POSDRU/89/1.5/S/

55216 (M. Calin) and Doctoral Fellowship Programme

POSDRU/107/1.5/S/82839 (V. Simion) are gratefully

acknowledged.

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