Accepted Manuscript

Title: Assessment of sequential combination of

5-Fluorouracil-loaded-chitosan-nanoparticles and
ALA-Photodynamic therapy on HeLa cell line

Author: Marta Benito-Miguel Dolores Blanco Clara Gómez

PII: S1572-1000(15)00046-0
DOI: http://dx.doi.org/doi:10.1016/j.pdpdt.2015.05.001
Reference: PDPDT 650

To appear in: Photodiagnosis and Photodynamic Therapy

Received date: 24-3-2015

Revised date: 29-4-2015
Accepted date: 4-5-2015

Please cite this article as: Benito-Miguel M, Blanco D, Gómez C, Assessment of

sequential combination of 5-Fluorouracil-loaded-chitosan-nanoparticles and ALA-
Photodynamic therapy on HeLa cell line, Photodiagnosis and Photodynamic Therapy
(2015), http://dx.doi.org/10.1016/j.pdpdt.2015.05.001

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Full title: Assessment of sequential combination of 5-Fluorouracil-loaded-chitosan-

nanoparticles and ALA-Photodynamic therapy on HeLa cell line.

Running title: Combination of 5-Fluorouracil-loaded-chitosan-nanoparticles and ALA-

Photodynamic therapy.

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Authors: Marta Benito-Miguela,bPhD, Mª Dolores Blancob PhD, Clara Gómez*cPhD

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a Centro Universitario San Rafael-Nebrija, Madrid, Spain

b Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, UCM,

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Madrid, Spain.
c Departamento de Sistemas de Baja Dimensionalidad, Superficies y Materia
Condensada, Instituto de Química Física Rocasolano, CSIC, Madrid,
Spain.

(*) Corresponding author: an

M
Dr. C. Gómez
Departamento de Sistemas de Baja Dimensionalidad, Superficies y Materia Condensada
Instituto de Química Física Rocasolano
Consejo Superior de Investigaciones Científicas, CSIC
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C/Serrano 119, 28006 Madrid

Tel.: +34-91-561-9400 ext.961252
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Fax.: +34-91-564-2431
c.gomez@iqfr.csic.es
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Full title: Assessment of sequential combination of 5-Fluorouracil-loaded-chitosan-

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nanoparticles and ALA-Photodynamic therapy on HeLa cell line.

Running title: Combination of 5-Fluorouracil-loaded-chitosan-nanoparticles and ALA-

Photodynamic therapy.

Authors: Marta Benito-Miguela,bPhD, Mª Dolores Blancob PhD, Clara Gómez*cPhD

a Centro Universitario San Rafael-Nebrija, Madrid, Spain

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Page 1 of 31
b Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, UCM,
Madrid, Spain.
c Departamento de Sistemas de Baja Dimensionalidad, Superficies y Materia
Condensada, Instituto de Química Física Rocasolano, CSIC, Madrid,
Spain.

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(*) Corresponding author:
Dr. C. Gómez
Departamento de Sistemas de Baja Dimensionalidad, Superficies y Materia Condensada

cr
Instituto de Química Física Rocasolano
Consejo Superior de Investigaciones Científicas, CSIC
C/Serrano 119, 28006 Madrid

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Tel.: +34-91-561-9400 ext.961252
Fax.: +34-91-564-2431
c.gomez@iqfr.csic.es

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p te
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HIGHLIGHTS

CNPs were synthesized by the ionic crosslinking method via the TPP addition.

Sequential combination of 5Fu-CNPs and ALA-PDT exhibited the best photocytotoxic

efficiency.

Combination treatment (5Fu-CNPs and ALA-PDT) led to the highest ROS generation.

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Page 2 of 31
Combination treatment (5Fu-CNPs and ALA-PDT) led to the highest caspase-3
activation.

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Abstract

Background: Natural polymers are used as components of nanoparticles (NPs) for drug
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delivery, as they provide targeted, sustained release and biodegradability.

The purpose of this study was to increase the efficacy of the

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Photodynamic therapy (PDT) by the combination of 5-aminolevulinic

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acid (ALA) with 5-Fluorouracil-loaded-chitosan-nanoparticles (5-Fu-

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CNPs).
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Methods: Nanoparticles based on chitosan (CNPs) were synthesized by the ionic

crosslinking method via the TPP addition. 5-Fluorouracil (5-Fu), a first-

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line anticancer drug, was loaded into these 5Fu-CNPs, and they were

assayed as controlled delivery formulation. HeLa cells were incubated in

the presence of 5Fu-CNPs for 24h, next ALA was added to the culture

medium and 4 hours later, to complete the PDT, light irradiation took

place. Analysis of cell viability, reactive oxygen species (ROS)

production, observation of the apoptosis by fluorescence microscopy

followed by analysis of caspase-3 activity were carried out.

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Page 3 of 31
Results: Spherical 5Fu-CNPs with a mean diameter of 324±43nm, were successfully

synthesized and characterized by TEM and DLS. 5-Fu incorporation was

achieved successfully (12.3 g 5Fu/mg CNP) and the maximum 5-Fu

release took place at 2h. The combined administration of 5Fu-CNPs and

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PDT mediated by ALA (ALA-PDT) led to an improved efficacy of the

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antineoplastic treatment by generation of great cytotoxicity inducted

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through an increased ROS production. HeLa cells were destroyed by

apoptosis through activation of caspase pathway.

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Conclusions: This study proves that combination therapy (photodynamic “ALA” +

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chemical “5-Fu”+ immunoadjuvant ”chitosan”) may be an effective

approach for the treatment of cancer.

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Keywords: ALA; Photodynamic therapy; 5-Fu; chitosan; cell viability; ROS.

Introduction
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The use of NPs in biological applications is being widely explored e.g. polymeric NPs,
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metallic NPs and quantum dots have been found applicable in drug
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delivery, bioimaging and biosensing [1]. NPs are utilized for sustained
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release of drugs. NPs improve drug efficacy and reduce side effects as a

result of the lower doses that are administered.

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Drug delivery systems based on polymeric NPs, which use natural or synthetic polymer

material have received great attention due to their stability, ease of

surface modification, biocompatibility and biodegradable properties. The

natural polymer chitosan (N-deacetylated derivative of chitin), tends to

be internalized and degraded rapidly, thus enabling a moderate release of

the drug. Chitosan shows more advantages, such as its availability in

natural resources, low cost processing, stability and low toxicity. It has

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Page 4 of 31
 nonspecific antiviral and antitumor activities that have been already

described about three decades ago by Suzuki [2]. Strong systemic and

mucosal immune responses could be induced by chitosan [3]. In this

context, immune-stimulating activity such as increasing accumulation

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and activation of macrophages and polymorphonuclear cells, inducing

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cytokines after intravenous administration, has been reported after

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intravenous administration of chitosan [4]. Due to their bioadhesive,

biocompatibility, biodegradability and penetration-enhancement

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properties, chitosan nanoparticles (CNPs) enhance the immune response

by stimulating the uptake by phagocytotic cells and may also stimulate

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the immune system [3,5-7]. Therefore, the use of CNPs used as
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immunological adjuvants to induce both humoral and cell-mediated

immunity could be promising.

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5-Fluorouracil (5-Fu), is a first –line anticancer drug, which efficacy is affected by its
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low lipophilicity and low bioavailability and its several side effects [8].

In this manner, small-molecule drugs, as 5-Fu, carried by chitosan or its

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derivatives, result in extended release, improved bioavailability and

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reduced side effects [9]. Due to its pivotal role in the treatment of a
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variety of solid tumors, 5-Fu remains as one of the most commonly used

anticancer drugs and it has been included into a wide range of therapeutic

programs, either alone or in combination with other drugs.

Photodynamic therapy (PDT) is a promising therapeutic procedure for the management

of a variety of solid tumors and non-malignant lesions. It is a treatment

methodology dependent upon administration of a photosensitizer (PS)

and tissue penetrating light of a specific wavelength. When the PS is

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Page 5 of 31
 exposed to light, its molecules become excited and by decaying through

triplet state generate singlet oxygen (1O2) and reactive oxygen species

(ROS) toxic to the tissue cells [10]. The response to the treatment induces

intratumor cytotoxic reaction which involves inflammatory innate,

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adaptive immune reaction culminating in successful eradication of

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residual surviving cancer cells [11]. 5-aminolevulinic acid (ALA) is an

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endogenous cellular component and is metabolized within the heme

biosynthetic pathway to produce protoporphyrin IX (Pp IX), a potent

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endogenous PS. Because ALA is a precursor that bypasses the rate-

limiting enzyme of the heme synthesis pathway (ALA synthase),

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feedback inhibition of the pathway does not occur and PpIX rapidly
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accumulates in the cells. Dysplastic cells exhibit decreased ferrochelatase

activity and lower ferric ion concentration, which promotes further

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accumulation of PpIX than in normal cells. As a result, tumor cells

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become selectively photosensitized to the action of the light [12].

However, ALA appears to be less effective than other PDT agents

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(photofrin, etc.) in the case of deep seated tumors. To enhance the

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clinical efficacy of ALA-PDT, new methods have been proposed, such as

a combination with hyperthermia or ultrasound, chemotherapy [13,14].

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The efficacy of cancer treatment is not only being increased with the

combination therapy and the dose of photosensitizer and cytostatic drug

can also be reduced without compromising the therapeutic response

[14,15].

In this study, CNPs were prepared by the ionic crosslinking method via the TPP

addition, characterized and evaluated as delivery systems of 5-Fu. Next,

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Page 6 of 31
 5Fu-CNPs were incubated on HeLa cells followed by a photodynamic

treatment mediated by ALA (ALA-PDT), to evaluate the cytotoxic and

apoptotic effect of combined treatment.

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Materials and methods

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Preparation of the 5Fu-CNPs

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5Fu-CNPs were prepared by the ionic crosslinking method. A dissolution 0.5 % (w/v)

of chitosan in aqueous solution of acetic acid (1% v/v), was added

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dropwise into 100 mL of 1% (w/v) pH 3 sodium polyphosphate (TPP)

solution. The suspension was sonicated during the time of the addition of
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chitosan solution. The formation of CNPs started spontaneously via the

TPP initiated ionic gelation mechanism. Then, the suspension was stirred
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slowly and allowed to crosslink for 1 h at room temperature. Two hours

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later, the suspension was subject to repeated cycles of washing by

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deionized water and centrifugation (10,000 rpm for 5 min) to remove the
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unreacted chitosan and TPP, then lyophilized and stored at 4 °C.

Fifty milligrams of the above synthesized CNPs were suspended in 1 mL of 10mg/mL

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solution of 5-Fu in deionized water being under slowly magnetic stirring

during 24 h. After this time, the suspension was centrifuged (10,000 rpm

for 1 min), next the suspension was washed in deionized water and

centrifuged again (10,000 rpm for 1 min.). Finally, it was lyophilized and

stored.

Characterization of the 5Fu-CNPs

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Page 7 of 31
Particle size distribution was determined by dynamic light scattering (DLS) by the size

analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire,

UK) equipped with a 10 mW helium-neon laser working at a wavelength

of 633 nm, 173° scattering angle and temperature of 25 °C.

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Measurements were carried out with a suspension of CNPs in water

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(0.06% w/v), previously sonicated and incorporated into disposable

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cuvettes of polystyrene (Sarstedt AG&Co). The size and distribution of

CNPs were measured by putting the cuvette into the sample cell. Three

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measurements were made per sample. Morphology and size of the CNPs

was also observed by transmission electron microscopy (TEM) by a

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JEOL JEM-1010 microscope (JEOL, Korea LTD) at 80 Kv, with a
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resolution of 0.35 nm. CNPs were dispersed in distilled water and

deposited on 200 mesh Formvar/Carbon grids (Copper, Ted Pella, Inc.

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Canada). Photographs were analyzed by Megaview Imagine Systems

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Software (Olimpus Soft Imaging Megaview II Software).

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In Vitro Release of 5Fu-CNPs

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Thirty milligrams of 5Fu-CNPs were suspended in PBS (10 mL, 37ºC, pH = 7.4).

Release study was carried out at a constant temperature (37ºC) and orbital
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shaking (80 rpm) for 24 h by means of an orbital shaker incubator

(Ecotron INFORS HT). At different time intervals 150 L of suspension

were extracted and replaced with 150 L of PBS. The amount of 5-Fu

was measured by UV-vis spectrophotometry at the wavelength of

maximum absorption  max = 266 nm. The drug concentration in each

time is calculated from the calibration curve data to the UV absorption of

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Page 8 of 31
 5 known concentrations of 5-Fu in PBS, in the range from 0.01 to 0.005

mg/mL. Experiments were done in triplicate.

Cell culture

Human cervical cancer (HeLa) cells were maintained in DMEM (4.5 g/L Glucose)

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supplemented with 10% heat inactivated fetal bovine serum, L-Glutamine

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(2mM), penicillin (50 U/mL), streptomycin (50 μg/mL) (Invitrogen Life

Technologies, Grand Island, NY, USA) and gentamicin (50 μg/mL)

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(Lonza, Basel, Switzerland) at 37ºC under a humidified atmosphere of 5%

CO2 in air. Cells were plated in 75-cm2 flasks (Sarstedt Ag and Co.,

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Barcelona, Spain) and were passaged upon reaching 95% confluency, by

gentle trypsinization (0.05% trypsin/0.53 mM EDTA; Invitrogen Life

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Technologies).
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Photodynamic treatment
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Experiments were done in triplicate and each experimental condition performed on

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quintuplicate. HeLa cells were seeded in 96-well flat-bottom plates at

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10,000 cells/well. After 24 h, the culture medium was removed and in

some wells CNPs and 5Fu-CNPs were added in the amount needed for
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the release of 5-Fu in the two concentrations studied: 50 and 100 µM.

Two hours later, when 5-Fu release from 5Fu-CNPs was completed, other

wells were incubated with 5-Fu in solution (50 and 100 µM). As control

negative, in some other wells culture medium were added. All the wells

were incubated for 24 h, and next, the cells were washed twice with 100

L of serum-free DMEM. Afterward, ALA in solution in serum-free

DMEM (50 and 75 µM) was added and then cells were re-incubated for 4

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Page 9 of 31
 DMEM (50 and 75 µM) was added and then cells were re-incubated for 4

h before irradiation. Cells were irradiated using a LED Therapy Beauty

Equipment (L-35) (Guangzhou JIMY, China) (=640 nm, 40 mW cm-2)

applied at a distance of 8 cm from the cells during 7 min. After irradiation

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treatment, 10 L of FBS were added to each well.

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Cytotoxicity

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Cell viability was analyzed by MTT assay, method based on the activity of

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mitochondrial dehydrogenases. MTT assay was deemed a suitable test for

cell viability determination after PDT photoactivation. MTT (3-(4,5-

an
dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide; Sigma-Aldrich)

is reduced to a purple insoluble formazan derivative in living,

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metabolically active cells.
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After PDT cells were incubated for 20h before the MTT assay was performed. Fifty
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microliters of MTT solution (concentration 1 mg/mL) were added to each

well and the plate re-incubated at 37ºC for 2 h. MTT was then replaced
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with 100 μL DMSO (Sigma-Aldrich). Cell viability was determined by

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measuring the absorbance at 570 nm using a microplate reader

(Varioskan, Thermo Fisher Scientific, Barcelona, Spain). Results are

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presented as the percentage of surviving cells in relation to that of

untreated control cells.

ROS production

Using the fluorescent probe 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFDA)

(Molecular Probes/Invitrogen), the endogenous ROS production was

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Page 10 of 31
 quantified. Non-fluorescent H2DCFDA is converted to its fluorescent

2´,7´-dichlorofluorescein (DCF) by ROS species. The fluorescent

compound can be quantified using a Varioskan microplate reader. Cells

were pre-treated with H2DCFDA (Molecular Probes/Invitrogen) (100L,

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40 M) 10 min before ALA addition. Four hours later, immediately after

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PDT, the well plate was incubated for a further 1 h at 37ºC. Next, culture

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medium was removed and PBS was added (as culture medium interfered

the measurement). ROS fluorescence intensity (resulting from the

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oxidation of dye), was then measured (excitation wavelength at 488/12

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nm and emission wavelength at 520 nm). Then, MTT assay was carried

out to calculate ROS production per life cell.

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Observation of the apoptosis by fluorescence microscopy

Cells were seeded onto cover glasses, positioned in p24 at a concentration of 50,000
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cell/well in 1 mL of growth medium and then treated with the same

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protocol as described for PDT. Twenty minutes after irradiation, cells

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were washed with PBS and Annexin V and Propidium iodide (PI) were
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added according the manufacturer´s instructions (BD Pharmingen,

Annexin V-FITC Apoptosis Detection Kit I; BD Biosciences, San Diego,

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CA, USA). Fluorophores were incubated during 15 min. in dark and at

room temperature. Then the medium was discarded. The cells were fixed

for 10 minutes in 1% paraformaldehyde in PBS, and mounted onto glass

slides with Fluoromount G® (Souther Biotechnology Associates, Inc.).

To observe apoptosis and necrosis, images of stained cells were taken by

using a TSC SP2 confocal laser microscopy system (Leica, Wetzlar,

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Page 11 of 31
 Germany) equipped with an inverted DMIRE2 Leica microscope.

Fluorescence images were analyzed using LCS software from Leica.

Caspase-3 activity assay

HeLa cells were growth in p24 at a concentration of 100,000 cell/well in 1 mL of growth

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medium. Briefly, 24 h after PDT treatment, each cell conditions were

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collected in 150 L of lysis buffer and centrifuged at 13,000 rpm, 4ºC

during 15 min. Then the supernatant was stored at -80ºC. Estimation of

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total protein in the supernatant was performed by Bradford method before

running the caspase-3-activity assay with 40 g of the protein extract. The

fluorogenic caspase-3

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substrate (Caspase 3 Ac-DEVD-AMC,

Calbiochem; Alexis- Enzo Life Sciences) was added and incubated during
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1 h at 37ºC. Using the Varioskan microplaque reader, levels of cleaved

caspase substrate were monitorized at excitation/emission = 380 nm/460

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nm. Caspase-3 activity was normalized to total protein content and

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expressed as fluorescence intensity in arbitrary units per mg protein.

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Statistical analysis
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All data were expressed as the mean± SD of three independent experiments. Statistical
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analyses and calculations were performed with SPSS for Windows

software (version 15.0; SPSS, Chicago, III). Differences between groups

were analyzed using the analysis of variance (ANOVA). Previously, the

normality of the data was checked by using the Shapiro-Wilk test. Values

of p<0.05 and p<0.001 were considered statistically significant and very

statistically significant, respectively.

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Page 12 of 31
Results

Characterization of CNPs and 5-Fu release from CNPs

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TEM micrographs of 5Fu-CNPs showed individual and independent particles of very

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small size, most of them around ~ 18-20 nm with a very homogeneous

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morphology and spherical shape (Figure 1A), but usually CNPs tend to

form aggregates up to almost 200 nm (Figure 1B). DLS technique was

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also applied to investigate the average size of the particles, resulting in an

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average diameter of 324±43nm (Figure 1C). It should be pointed out that

the particle size determined by DLS technique is a hydrodynamic

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diameter. The larger diameter showed by DLS technique in contrast to

TEM measures might be a result of the aggregation of single CNPs while

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dispersed in water.
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Drug release studies were carried out at 37ºC. 5-Fu release from CNPs is shown in
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Figure 1D. Maximum 5-Fu release took place 2 hour after starting the
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experiment. The amount of drug incorporated was: 12.3 g 5-Fu/mg

CNP.
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Cell viability

To explore whether CNPs, 5Fu-CNPs, 5-Fu and ALA-PDT have positive interactions

with mortality of Hela cells when given in combination, it is necessary to

determine their own cytotoxicity. The 5-Fu doses examined were 50 and

100 M and 50 and 75 M for ALA. In the dark, all conditions without 5-

Fu demonstrated a consistent and low cytotoxicity (Figure 2A). 5-Fu

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Page 13 of 31
 exerts its cytotoxic action in a way similar both in solution and loaded

into the CNPs and higher at the highest dose (100M).

After 7 min of PDT, all conditions with ALA were affected to a greater extent with the

highest dose of ALA applied (75M). The combination treatment, either

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based on 5-Fu loaded into CNPs with ALA-PDT or 5-Fu solution with

ALA-PDT, enhances the final antitumorigenic effect, lowering decreasing

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doses of both 5-Fu and ALA and thus minimizing collateral effects

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(Figure 2B). Combination treatment significantly enhances the percentage

of cell death in comparison with the individual treatment even with the

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use of the lower doses (50 M for 5-Fu; 50 M for ALA) thereby

enabling reducing the doses and thus side effects. In addition, a slightly
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more effective effect was observed when 5-Fu was administered loaded

into CNPs than in solution into the combined treatment. To obtain this
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therapeutic reinforcement chemotherapy mediated by 5-Fu should be

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applied 24 h before ALA application and not concomitantly (see

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Supplementary Fig. 1, Supplemental Digital Content 1).

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Figure 2C shows how chitosan enhances (but not significantly) the effect of ALA. PDT

triggers an inflammatory response and enhances a specific antitumor

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response that could be further stimulated by the addition of adjuvants as

chitosan.

Reactive oxygen species levels

Figure 3 shows how ROS production can be correlated with the results shown in Figure

2B. At greater decrease in cell viability higher increased production of

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Page 14 of 31
 ROS was observed. Pretreatment with 5-Fu, augmented the expected

response of ROS production (which was higher when using 5Fu-CNPs

than with 5-Fu solution), due to ALA-PDT treatment. It is important to

point out that the individual treatment with 5-Fu either in solution or

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incorporated into CNPs was able to generate ROS.

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Observation of the apoptosis by fluorescence microscopy

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To prove that combination treatment reduced cell viability by inducing apoptosis, we

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investigated apoptotic cells through fluorescence microscopy by applying

Annexin V and Propidium iodine (AV/PI) double staining method.

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Results after light irradiation are shown in Figure 4, where late apoptotic

cells (AV+/PI+) were visualized, in increasing order of the number of

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apoptotic cells from CNPs (top), followed by the individual treatments

with 5-Fu, 5Fu-CNPs and ALA (middle), to the combination treatment

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(5-Fu solution plus ALA and 5-Fu loaded into CNPs plus ALA) (bottom).
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Caspase-3 activity assay

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Apoptosis can also be analyzed from a quantification of the enzymatic activity of

caspases. Therefore, the activity of the caspase-3 as apoptotic marker was

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determined (Figure 5). Results showed that the activity of caspase-3 was

higher after the combination treatment (although very similar for both

types of combination treatment: solution of 5-Fu with ALA-PDT and

5Fu-CNPs with ALA-PDT), than with the individual treatments applied.

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Page 15 of 31
Discussion

In the present study, CNPs were used to deliver 5-Fu. The ionic gelation method was

followed to prepare CNPs using medium molecular weight chitosan. The

size of the CNPs ranged from 281 to 367 nm. Previous studies revealed

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that CNPs could be used for delivery of various biological molecules and

drugs [16,17]. 5-Fu is a widely used anticancer drug that is toxic to

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normal cells and has a short plasma half-life of 15-20 min. In the present

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study, 5Fu-CNPs were synthesized with the aim of prolonging drug action

and thus improving therapeutic results. 5-Fu loaded into NPs based in

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chitosan could improve the bioavailability, the site-specific of delivery

and reduction of its side-effects providing obvious therapeutic advantages

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[9,18].

The effects of ALA-PDT and possible mechanisms involved in the treatment of cervical
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cancer in vitro and in vivo have been already explored [19,20]. PDT has
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several features that distinguish it among other antitumor therapies. Its

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unique mechanism of action include direct cytotoxic effects exerted

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towards tumor cells (lipid peroxidation, crosslinking of proteins and

damage to multiple cellular sites, including: membranes, DNA, and

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cytoskeleton), destruction of tumor and peritumoral vasculature and

induction of inflammatory innate, adaptive immune reaction culminating

in successful eradication of residual surviving cancer cells [21,22]. For

clinical applications, an ideal PDT should exhibit extensive killing of the

tumor cells with minimal damage to host tissues in the area of tumor. It is

still possible to damage neighboring healthy cells if the PS dose is too

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Page 16 of 31
 high. It would be ideal if a lower PS dose that is safe for healthy human

tissues can efficiently function as a tumoricide agent.

The potential benefits of combination therapies including PDT in association with other

pharmacological therapies over monotherapy for the treatment of several

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types of cancer, has been supported by some studies [13,15,23] PDT in

combination with chemotherapeutic agents has shown an increase of the

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direct damage to the targeted cells and thus an increase of the cytotoxicity

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by modulating signal pathways that may lead to apoptosis, alterations of

the tumor microvasculature and induction of a host immune response

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against cancer cells [23].

At least three different ways in which the 5-Fu can exert antitumor action have been
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described: inhibition of thymidylate synthase (enzyme essential for the

synthesis of the majority of thymidine nucleotides, precursor necessary in

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DNA synthesis); inhibition of RNA function and protein synthesis; and

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incorporation into DNA leading to their break [24,25]. Some studies also
p

have shown that pro-oxidant actions could enhance the antitumor efficacy
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of 5-Fu [26,27]. Therefore, it is expected that 5-Fu increases its

effectiveness when it is combined with therapies that induce oxidative

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effect as PDT.

Chitosan was applied to further enhance the immune response induced by PDT. By

stimulating inflammation and generating apoptotic and necrotic tissue,

PDT provides an ideal environment for initiation of an antitumor immune

response. Addition of adjuvants leads to PDT enhancement of the

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Page 17 of 31
 photodynamic effect [28,29].

In this work the efficacy of a combined treatment based on 5Fu-CNPs and ALA-PDT

was studied. HeLa cells were utilized as in vitro model of human cervical

cancer. From the MTT assay, 5-Fu had the same cytotoxic effect with and

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without light irradiation and higher at the highest dose. CNPs were not

cytotoxic with and without light irradiation, but enhanced the

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photodynamic effect of ALA. Combination of ALA-PDT with 5-Fu

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enhanced the percentage of cell death, and a slightly more when 5-Fu was

released from CNPs than by using 5-Fu in solution. Therefore, the

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administration of CNPs shows a slight added value. PDT leads to direct

tumor cell destruction and serves as the precursor for host immune
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responses by exposing tumor antigens [28]. Chitosan in combination with

PDT could stimulate and enhance the host immune system to mount a
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direct attack, with the help of the exposed antigens, against the remaining
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tumor cells [29]. CNPs could be outlined to be an interesting complement

of PDT.
p
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To maximize the effect of the combination treatment, chemotherapy mediated by 5-Fu

should be applied a day before ALA application (to ensure that the 5-Fu
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has exerted its action). However, simultaneous application of 5-Fu and

ALA did not result in a more effective therapy (see Supplementary Fig. 1,

Supplemental Digital Content 1).

ROS have been shown to destroy tumors by three distinct mechanisms: (1) direct

cytotoxicity towards tumor cells producing apoptosis and necrosis; (2)

destruction of tumor capillaries and microvasculature mediated by PDT

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Page 18 of 31
 damage towards endothelial cells; (3) an acute inflammatory response that

can activate host-defense mechanisms involving neutrophils and dendritic

cells [30]. In addition, it has been demonstrated that ROS generation is

higher in cells treated with PDT compared to the normal untreated cells

t
[31]. As PDT belongs to treatment modalities that generate ROS

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overproduction, generation of ROS was also analyzed in the present

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study. In addition, 5-Fu generates oxidative stress while its action is

favored in a pro-oxidant cellular environment as generated by PDT

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[26,27]. In our study, ROS production was higher in the cells treated with

ALA-PDT, (and higher at the highest concentration of ALA tested, 75

an
M) compared to the treatment without ALA application. The generation
M
of ROS in the HeLa cells treated with the combined treatment was higher

than the observed after the individual treatments (ALA, CNPs, 5-Fu in
d

solution, 5Fu-CNPs). This effect was more evident in the 5Fu-CNPs and
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ALA-PDT combined treatment.

p

Cells undergoing apoptosis display typical features. Dramatic nuclear changes are
ce

common during apoptotic death due to activation of endogenous

nuclease(s) which cleaves DNA into oligonucleosomal fragments.

Ac

Chromatin condensation, nuclear shrinkage and formation of apoptotic

bodies can easily be observed under fluorescence microscopy, after

appropriate staining of nuclei with DNA-specific fluorochromes. Annexin

and PI double staining were performed to determine if the combination

treatment had an enhanced apoptotic effect compared to the individual

treatments. It has been reported that PDT with mitochondrial localizing

PS, such as ALA, can induce rapid cell death apoptosis [32,33].

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Page 19 of 31
 PS, such as ALA, can induce rapid cell death via apoptosis [32,33].

Results of the present work showed that the number of late apoptotic cells

visualized by fluorescence microscopy increased in cells treated with

individual treatments and even more in cells treated with combination

t
treatment. These results were in agreement with those obtained from the

ip
cell viability analysis and ROS quantification.

cr
Insult or failure of the mitochondria could rapidly lead to the inhibition of cell survival

us
and proliferation, therefore the induction of mitochondrial impairment

may be a successful anticancer strategy. The opening of the mitochondrial

an
permeability transition pore can lead to a release of pro-apoptotic

molecules from the intermembrane space causing the activation of

M
caspase-9, which further activates caspase-3 [34]. Caspase-3, is a member

of the caspase family of 13 aspartate specific cysteine proteases that plays

d

a central role in the execution of the apoptotic program [35]. To find out
te

the possible pathway of the enhanced apoptosis observed after the

combination treatment, the expression level of caspase-3 was studied.

p

Results showed that enzyme activity of caspase-3 was higher in case of

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combination treatment compared to the control cells as well as the

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individual treatments. Increased caspase-3 activity leads to apoptosis and

finally cancer cell destruction. ROS are significant regulators of apoptosis

and activation of apoptosis is associated with generation of ROS. In this

study, combined treatment destroyed HeLa cells through an increased

ROS generation which mediates apoptosis by enhancing the activity of

caspase-3.

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Page 20 of 31
Conclusion

With this combination treatment, not only a higher efficacy can be achieved but also

both the effective dose of the chemotherapeutic drug and photosensitizer

t
ip
can be reduced, and hence, the adverse side effects of the drugs used can

cr
also be controlled. Immunoadjuvant contribution cooperates positively in

the final result. Further studies for assessing the efficacy and risk-benefit

us
of that PDT-combined treatment are needed to establish this combination

treatment as alternative for malignant disorders.

an
M
Acknowledgements/ Conflicts of Interest and Source of Funding: This research was
d

supported by a Grant from “UCM Group-Santander Bank (Group

te

920613)” and by the Spanish Research Project MICINN (MAT2010-

p

20646-C04-01). The authors have no other relevant affiliation or financial

ce

involvement with any organization or entity with a financial interest in or

financial conflict with the subject matter or materials discussed in the

Ac

manuscript apart from those disclosed.

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FIGURE LEGENDS

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Figure 1.- (A,B) TEM images of the CNPs. (C) Particle size distribution histogram of

CNPs obtained by DLS data. (D) Cumulative amount of 5-Fu released

from 5Fu-CNPs at 37ºC. Each value represents the mean±SD (n=3).

Figure 2.- (A) Viability of HeLa cells in percentage determined by the MTT assay in

t
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dark conditions. Each value represents the mean±SD of the three

independent experiments. (*) Statistically significant differences with a

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significant decrease from values of control cells (p<0.05). Combined

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treatment was performed at low doses (5-Fu 50 M and ALA 50 M) and

at high doses (5-Fu 100 M and ALA 75 M). (B) Viability of HeLa

an
cells after PDT treatment in which cells were irradiated for 7 min under

40mWcm-2 led lamp. Each value represents the mean±SD of the three
M
independent experiments. (*) p<0.05 and (**) p<0.001 Statistically and

very statistically significant differences, respectively, with a significant

d

decrease from the same treatment at dark conditions. (##) p<0.001 Very
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statistically significant differences with a significant decrease from ALA

p

group. Combined treatment was performed at low doses (5-Fu 50 M

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and ALA 50 M) and at high doses (5-Fu 100 M and ALA 75 M).

Figure 2C shows the effect of chitosan as adjunctive agent in the PDT

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mediated by ALA. Each value represents the mean±SD of the three

independent experiments. (*) p<0.05 and (**) p<0.001 Statistically and

very statistically significant differences, respectively, with a significant

ns
decrease from values of control cells. ( ) Not significant differences

between ALA+light and CNPs+ALA+light groups.

Figure 3.- ROS production after PDT treatment. Values have been normalized by values

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 of living cells. Data represent the mean±SD of the three independent

experiments. (*) p<0.05 and (**) p<0.001 Statistically and very

statistically significant differences, respectively, with a significant

increase from values of control cells.

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Figure 4.- Fluorescence microscope images of HeLa cells incubated with Annexin V

and PI 20 min after PDT for visualization by fluorescence microscopy.

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Experiment was done with the highest concentrations of ALA and 5-Fu

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used in the present study (75 M and 100 M, respectively). The green

fluorescence on the cells indicated apoptotic cells, the red fluorescence on

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the cells indicated late apoptotic cells. Scale bar: 100µm.

Figure 5.- Activity of caspase 3 in cell lysates. Experiment was done with the highest
M
concentrations of ALA and 5-Fu used in the present study (75 M and

100 M, respectively). Data represent the mean±SD of the three

d

independent experiments. (*) p<0.05 and (**) p<0.001 Statistically and

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very statistically significant differences, respectively, with a significant

p

increase from the same treatment at dark conditions.

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SUPPLEMENTAL DIGITAL CONTENT

Supplemental Digital Content 1.pdf

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