International Journal of Pharmaceutics 516 (2017) 6170

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Folic acid-decorated and PEGylated PLGA nanoparticles for improving

the antitumour activity of 5-uorouracil
Mazen M. El-Hammadia,b , ngel V. Delgadoc, Consolacin Melguizod,e, Jos C. Pradosd,e,
Jos L. Ariasa,d,e,*
a
Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, Spain
b
Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Damascus, Syria
c
Department of Applied Physics, Faculty of Sciences, University of Granada, Spain
d
Institute of Biopathology and Regenerative Medicine (IBIMER), University of Granada, Spain
e
Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS), University of Granada, Spain

A R T I C L E I N F O A B S T R A C T

Article history:
Received 28 August 2016 5-Fluorouracil (5-FU) is a broad spectrum cytotoxic agent being used in chemotherapy of malignancies.
Received in revised form 28 October 2016 However, 5-FU shows a number of limitations like short half-life, non-selective biodistribution, and the
Accepted 3 November 2016 development of drug resistances by tumour cells. It was investigated the potential use of folic acid-
Available online 5 November 2016 decorated and PEGylated poly(D,L-lactide-co-glycolide) nanoparticles (FOL-PEG-PLGA NPs) for the
targeted delivery of 5-FU to colon and breast cancers. PEG-PLGA and FOL-PEG-PLGA conjugates were
Keywords: synthesized and characterized. NPs of PLGA, PEG-PLGA, and FOL-PEG-PLGA were prepared by
Cancer chemotherapy nanoprecipitation under optimal formulation conditions. They were found to be haemocompatible,
5-Fluorouracil
and exhibited negligible cytotoxicity in normal (CCD-18 and MCF-10A) and tumour (HT-29 and MCF-7)
FOL-PEG-PLGA conjugate
human cell lines. 5-FU loading capabilities were also dened, and the NPs exhibited an initial burst drug
Haemocompatibility
Ligand-receptor mediated drug delivery release followed by a sustained 5-FU release. In vitro cytotoxicity studies in folate-overexpressed HT-29
Nanoprecipitation. colon cancer cells and MCF-7 breast cancer cells demonstrated that the half maximal inhibitory
concentration (IC50) of 5-FU-loaded FOL-PEG-PLGA NPs was approximately 4-fold less than that of the 5-
FU-loaded PLGA NPs (p < 0.05). Consequently, FOL-PEG-PLGA NPs could have great potential as a targeted
5-FU delivery system for colon and breast cancer treatment.
2016 Elsevier B.V. All rights reserved.

1. Introduction Current therapeutic approaches in the management of cancer

Cancer is one of the leading causes of morbidity and mortality
worldwide, with about 14 million new cases and 8.2 million deaths
in 2012 (Stewart and Wild, 2014). According to the World Health
Organization (WHO), the number of new cases is expected to
increase by around 70% rising from 14 million in 2012 to 22 million
within the next two decades. Among the most common causes of
cancer death, colorectal cancer (fourth) and breast cancer (fth)
accounted for 694,000 and 521,000 deaths by year, respectively, in
2012 (Stewart and Wild, 2014).
* Corresponding author at: Departamento de Farmacia y Tecnologa Farm-
acutica, Facultad de Farmacia, Universidad de Granada, 18071, Granada, Spain.
E-mail address: jlarias@ugr.es (J.L. Arias).
include chemotherapy, surgery, radiation, and biological therapies,
i.e. immune therapy and hormonal therapy. 5-uorouracil (5-FU), a
pyrimidine analogue, is among rst line cytotoxic agents employed
in the chemotherapy of malignancies. 5-FU is a broad spectrum
molecule that interferes with nucleoside metabolism and can be
incorporated into ribonucleic acid (RNA) and deoxyribonucleic
acid (DNA), mainly acting as a thymidylate synthase inhibitor and
ultimately activating apoptosis in cancer cells (Longley et al.,
2003). However, 5-FU shows a number of limitations including: i)
short in vivo half-life due to rapid metabolism, thus sustained
therapeutic effects cannot be obtained; ii) erratic oral bioavailabil-
ity owing to metabolism by dihydropyrimidine dehydrogenase; iii)
wide non-selective in vivo distribution leading to toxic effects, e.g.
myelosupression and gastrointestinal, haematological, neural and
dermatological adverse effects; and, iv) the development of drug
resistances by malignant cells.

http://dx.doi.org/10.1016/j.ijpharm.2016.11.012
0378-5173/ 2016 Elsevier B.V. All rights reserved.
62 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170
To address the above listed limitations, research has been
focused on developing targeted delivery systems for 5-FU using
polymeric nanoparticles (NPs), such as poly(D,L-lactide-co-glyco-
lide) (PLGA) NPs. PLGA, a Food and Drug Administration (FDA)-
approved polymer, has shown very promising applications in drug
delivery given its biocompatibility, biodegradability, and versatili-
ty (El-Hammadi and Arias, 2015; Holgado et al., 2011). Some
studies have reported the use of PLGA-based NPs to boost the
antitumour efciency of 5-FU against colon cancer (Shakeri-Zadeh
et al., 2015; Tang et al., 2014; Wang et al., 2015) and breast cancer
(Balasubramanian et al., 2014; Nair et al., 2011; Zhu et al., 2016).
While the properties of PLGA itself, such as the percentage of latic
acid and glycolic acid (Nair et al., 2011), can inuence the cytotoxic
effect of 5-FU, improved efciency and targeting characteristics
can be achieved by conjugation of PLGA with a hydrophilic polar
polymer, e.g. poly(ethylene glycol) (PEG) (Tang et al., 2014; Yadav
et al., 2010), and/or a targeting ligand, such as folic acid (FOL)
(Balasubramanian et al., 2014; Wang et al., 2015), hyaluronic acid
(Yadav et al., 2010), lactobionic acid (Dangi et al., 2014),
sulfadiazine (Guimares et al., 2015), transferrin (Balasubramanian
et al., 2014), and translocator protein 18 kDa (Laquintana et al.,
2014).
PEG is a non-ionic hydrophilic polyether that it is extremely
biocompatible. Conjugation of PEG to PLGA-based NPs (the so-
called PEGylation of the NPs) reports interesting advantages which
include enhanced aqueous solubility, suppressed opsonization,
reduced aggregation and improved stability, minimized immuno-
genicity, and prolonged biological half-life. For example, PEG-PLGA
NPs loaded with 5-FU have been shown to maintain drug release
over 5 days and to inhibit the peritoneal dissemination of colon
cancer in mice (Tang et al., 2014).
FOL is a water-soluble vitamin B that it is essential for the
production of new cells. A link has been established between low-
folate diet and increased risk of a number of malignancies,
including colon cancer (Fife et al., 2011) and breast cancer
(Manshadi et al., 2014). Folate receptors are overexpressed in
some tumour cells, such as in colorectal carcinomas (D'Angelica
et al., 2011) and breast cancer (Zhang et al., 2014), and have a high
afnity for FOL. Importantly, this afnity is maintained when FOL is
bound with other molecules which enables targeting of folate-
linked drugs or NPs to folate receptor-expressing tumour cells.
A targeting delivery carrier, such as the one that combines PLGA
with PEG and FOL, is expected not to only optimize the anticancer
efciency of encapsulated 5-FU, but it can also overcome the drugs
drawbacks through minimizing its toxicity, increasing its biological
(plasma) half-life, and overcoming the development of drug
resistances. Our group has recently shown that the incorporation
of 5-FU in poly(e-caprolactone) NPs can considerably enhance the
activity of this drug against colon cancer cells and reduce their
proliferation rate up to 40-times, as compared with the free
anticancer drug (Ortiz et al., 2012). We have also developed 5-FU-
loaded magnetoliposomes for combined hyperthermia and che-
motherapy against colon cancer (Clares et al., 2013).
In this work, we sought to synthesize a FOL-PEG-PLGA
conjugate and use it to fabricate targeted NPs loaded with 5-FU
for the treatment of colon and breast malignancies. 5-FU-loaded
PLGAS PEG-PLGA, and FOL-PEG-PLGA NPs, prepared by nano-
precipitation, were completely characterized and studied. The
PLGA-based NPs were examined for their haemocompatibility as
well as cytotoxicity against colon and breast cancer cells. Despite
FOL-PEG-PLGA nanostructures have been postulated for controlled
and targeted delivery of drug molecules to malignant cells, i.e.
capecitabine (Wei et al., 2014), paclitaxel (Liang et al., 2011),
saquinavir (Singh et al., 2015), to the best of our knowledge this is
the rst time that 5-FU-loaded FOL-PEG-PLGA NPs have shown
promising activity against colon and breast cancers.
2. Materials and methods
2.1. Materials
PLGA (50:50; molecular weight average, Mw: 15,300 Da),
hydroxyl-PEG-amine (Mw: 3000 Da), PEG bis(amine) (Mw:
3000 Da), FOL, N,N0 -dicyclohexylcarbodiimide (DCC), N-hydroxy-
succinimide (NHS), and triethylamine were purchased from
Sigma-Aldrich (Munich, Germany). Anhydrous dichloromethane
(DCM) and anhydrous dimethyl sulfoxide (DMSO) were purchased
from VWR (Barcelona, Spain). All other chemicals were of
analytical quality (Panreac, Barcelona, Spain). Finally, deionized
and ltered water was used in all the experiments (Milli-Q
Academic1, Millipore, Molsheim, France).
2.2. Methods
2.2.1. Synthesis of the FOL-PEG-PLGA conjugate
The conjugate was synthesized in a 4-step process as previously
described in the literature (Chen et al., 2012; Cheng et al., 2007;
Esmaeili et al., 2008; Saxena et al., 2012; Yoo and Park, 2004) with
some modications.
2.2.1.1. Activation of PLGA. PLGA (1836 mg; 0.12 mmol), DCC
(49.5 mg; 0.24 mmol) and NHS (27.6 mg; 0.24 mmol) were
dissolved in 4 mL of anhydrous DCM and left to stir at room
temperature under argon atmosphere for 24 h (PLGA/NHS/DCC
stoichiometric molar ratio: 1/2/2). The resultant solution was
ltered, to remove the by-product dicyclohexylurea, and
precipitated by dropping into ice-cold ether. The solid
precipitate was repeatedly washed in an ice-cold mixture of
ethyl ether and methanol to remove residual NHS. Ether was
decanted and the activated PLGA was completely dried under
vacuum.
2.2.1.2. PEGylation of PLGA. The activated PLGA (385 mg;
0.025 mmol) dissolved in 2 mL of anhydrous DCM was added
drop-wise to PEG bis(amine) (225 mg; 0.075 mmol) dissolved in
0.5 mL of anhydrous DCM. PEG bis(amine) was used in excess
[PLGA/PEG bis(amine) stoichiometric molar ratio: 1/3] to suppress
the formation of PLGA-PEG-PLGA triblock copolymers. The
reaction was carried out for 6 h with gentle stirring under argon
atmosphere. The reaction mixture was then precipitated with cold
methanol and washed with the same solvent to remove unreacted
PEG. The precipitated product, amine-terminated diblock
copolymer (PLGA-PEG-NH2), was ltered and dried under vacuum.
2.2.1.3. Activation of FOL (formation of NHS-folate). FOL (220.8 mg;
0.5 mmol) was activated with DCC (206.4 mg; 1 mmol) and NHS
(115 mg; 1 mmol) in 3 mL of DMSO in presence of 0.1 mL
triethylamine as a catalyst, under light protected argon
atmosphere overnight. The solution was ltered to remove the
dicyclohexylurea by-product, and then precipitated in ice-cold
anhydrous ether. The product was obtained in the dry state
following several steps of ether washing, decantation, and vacuum
drying.
2.2.1.4. Conjugation of FOL to PEG-PLGA. Activated FOL (15.9 mg;
0.036 mmol) and PEG-PLGA (329.4 mg; 0.018 mmol) were co-
dissolved in 3 mL of DMSO in light-protected conditions under
argon atmosphere at room temperature for 8 h. Excess amount of
activated FOL was used to get higher conjugation of FOL to PEG-
PLGA. The reaction mixture was then mixed with 100 mL cold
methanol and ltered through the paper lter. The precipitate on
the lter was dried under vacuum, and then dissolved in 50 mL
DCM; in this way, free FOL was precipitated in DCM but conjugated
 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170
FOL was dissolved, after centrifugation at 21,000g for 30 min
(Centrikon T-124 high-speed centrifuge; Kontron, Paris, France);
the supernatant was dried under vacuum (Esmaeili et al., 2008).
Folate content in the FOL-PEG-PLGA conjugate was computed
by dissolving a pre-weighed amount of FOL-PEG-PLGA in DMSO
and measuring the absorbance at 364 nm (UVvis Dinko spectro-
photometer, Dinko, Barcelona, Spain). A calibration curve was
constructed using serially diluted concentrations of free FOL in
DMSO (Yoo and Park, 2004).
2.2.2. Synthesis of the PEG-PLGA conjugate
The PEG-PLGA conjugate was synthesized following the rst 2
steps of FOL-PEG-PLGA synthesis (see Section 2.2.1.). Briey,
activated PLGA (704 mg; 0.046 mmol) dissolved in DCM was added
drop-wise to a hydroxyl-PEG-amine (138 mg; 0.046 mmol) solu-
tion in DCM. The reaction was continued for 6 h and the product
was precipitated with cold methanol, washed with methanol,
ltered, and dried.
The synthesis of the new conjugates, i.e. PEG-PLGA and FOL-
PEG-PLGA, was conrmed by proton nuclear magnetic resonance
(1H NMR) analysis (Bruker AM-300, Billerica, Massachusetts, USA),
using deuterated chloroform (CDCl3) as solvent.
2.2.3. Preparation of the PLGA-based nanoparticles
The NPs were fabricated by nanoprecipitation solvent evapora-
tion technique using a probe sonicator (Branson Sonier 450,
Emerson Electric Co., Danbury, Connecticut, USA). An organic
solution containing PLGA (50 mg; 3.268 mmol) (or an equivalent
amount of PEG-PLGA or FOL-PEG-PLGA, i.e. 59.8 mg or 60.4 mg,
respectively) in acetone was added at once to 10 mL of an aqueous
solution of polyvinyl alcohol (PVA), and sonication was started
immediately. Prior to addition, the microtip of the sonicator was
immersed in the PVA solution, leaving 1 cm distance from the
bottom of the vial without touching its walls. An aluminum foil
was placed over the opening of the vial to reduce the evaporative
loss of the liquid. Pulsed mode, with a cycle of 50%, was employed
because it helps to avoid foaming, retards the rate of temperature
increase in the medium, and enables better temperature control
than continuous mode operation. For further temperature control,
the sample vial was surrounded with ice during the sonication
process. 5-FU-loaded NPs were prepared by dissolving the
antitumour drug, at a given amount, in the organic phase prior
to addition to the aqueous phase. Finally, acetone was evaporated
using a Bchi Rotavapor1 rotary evaporator (Bchi, Flawil,
Switzerland) to obtain an aqueous suspension of pure NPs. The
dispersion was then centrifuged for 60 min at 10,000 rpm
(Centrikon T-124 high-speed centrifuge; Kontron, Paris, France),
the supernatant removed, and the NPs re-suspended in water.
To select the optimal formulation, several PVA concentrations
(0.5, 1, or 1.5%, w/v) and solvent ratios (3, 5, or 7 mL of the acetone
organic solution) were tested. Sonication conditions were also
studied by changing sonication time (0.5, 1, or 2 min) and sonicator
output (20, 40, or 50%). The criteria for evaluating the produced
NPs were the particle size and the polydispersity index (PdI).
2.2.4. Characterization of the PLGA-based nanoparticles
Mean particle size, size distribution (PdI), and zeta potential (z)
of the nanoformulations were determined using a Malvern
Zetasizer (Nano-ZS, Malvern Instruments Ltd., Worcestershire,
UK). Cell temperature was 25.0  0.5  C with a detection angle of
90 . All measurements were performed in triplicate after
appropriate dilution of the NP dispersions in water ( 0.1%, w/v).
High resolution transmission electron microscopy (HRTEM;
STEM CM20 microscope, Philips, Amsterdam, The Netherlands)
was employed to elucidate the shape, as well as to conrm the size
determinations of the NPs. Drops of dilute suspensions ( 0.1%, w/
63
v) were placed on copper grids with formvar lm, and the grids
were then dried at 25.0  0.5  C in a convection oven (J.P. Selecta, S.
A., Abrera, Barcelona, Spain).
Drug loading capacity was evaluated by determining the 5-FU
remained in the aqueous solution, collected after centrifugation of
the NPs dispersion, and deducting it from the total amount of the
used drug. Measurements were performed in triplicate using a
previously validated ultraviolet-visible (UVvis) spectrophotomet-
ric method at 266 nm (Ortiz et al., 2012). The incorporation of the
anticancer agent into the NPs was expressed in terms of drug
entrapment efciency (EE, %) [(amount of 5-FU loaded [mg]/initial
amount of 5-FU being used [mg])  100], and drug loading (DL, %)
[(amount of 5-FU loaded [mg]/amount of drug-loaded NPs being
obtained [mg])  100)].
2.2.5. Drug release studies
Drug release experiments using formulations prepared under
optimal conditions (5-FU: 15 mg; PLGA: 50 mg; PVA: 1%; solvent
ratio: 5:10; sonication time: 0.5 min; sonication output: 20%; see
Sections 3.2. and 3.3.) were performed in phosphate buffered
saline (PBS, pH 7.4  0.1) using the dialysis bag method. First, the
dialysis membrane tubing (cut-off: 2 kDa; Spectrum1 Spectra/
Por1 6 dialysis membrane tubing, New Brunswick, New Jersey,
USA) was soaked in water at 25.0  0.5  C for 12 h prior to use. NPs
dispersed in 2 mL of PBS (containing 5-FU at 0.5 mg/mL) were
transferred into a dialysis bag and the two ends were fastened with
clamps. The bag was placed in a beaker containing 200 mL of the
release medium, maintained at 37.0  0.5  C, and agitated at
200 rpm throughout the experiment. At selected time intervals
(0.25, 0.5, 1, 2, 3, 6, 9, 12, 24, 48, 72, 96, 120, and 144 h), 1 mL of the
medium was sampled and assayed at 266 nm 1 mL of PBS,
maintained at the same temperature, was added after each
sampling to maintain the volume of the release medium and
ensure sink conditions. The results were expressed as a drug
released percentage [(amount of 5-FU released in the medium
[mg]/amount of 5-FU loaded in the NPs [mg])  100]. The
experiments were performed in triplicate.
2.2.6. In vitro blood compatibility study
The interaction of the blank (drug unloaded) NPs (PLGA NPs,
PEG-PLGA NPs, and FOL-PEG-PLGA NPs) with blood components
was investigated to hypothesize their potential use for therapeutic
purposes, i.e. drug delivery against cancer. To that aim, blood
samples were obtained from three healthy female adults (24 to 41
years old), and poured into asks containing either ethylenedia-
minetetraacetic acid (EDTA, used in the haemolysis and platelet
activation experiments), or sodium citrate (used in the comple-
ment system activation and plasma clotting time experiments).
Following a previously established procedure (Arias et al., 2011;
Dash et al., 2010; Muoz de Escalona et al., 2016), the colloids were
placed in contact with blood aliquots to evaluate their effect on
erythrocytes, coagulation, and complement system. All the
determinations were performed in triplicate, and PBS was used
as a negative control. The UVvis spectrophotometric methodolo-
gies were successfully validated and veried for accuracy,
precision, and linearity.
2.2.7. In vitro proliferation studies
Normal human cell lines CCD-18 (colon broblast cells) and
MCF-10A (breast epithelial cells) were obtained from the Scientic
Instrumentation Centre (University of Granada, Granada, Spain)
and the American Type Culture Collection (ATCC, Manassas,
Virginia, USA), respectively. Human tumour cell lines HT-29
(colonic adenocarcinoma cells) and MCF-7 (breast adenocarcino-
ma cells) were obtained from ATCC (USA).
64 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170
These cell lines were grown in a Dulbeccos modied Eagles
medium (DMEM, Sigma-Aldrich Co., Saint Louis, Missouri, USA)
supplemented with 10% heat-inactivated fetal bovine serum (FBS,
Lonza Group AG, Basel, Switzerland), 14 mM sodium bicarbonate,
15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
(HEPES), 2 mM L-glutamine, 40 mg/mL gentamicin, and 500 mg/
mL ampicillin (Antibiticos de Len S.L.U., Len, Spain). They were
seeded in monolayer culture into 96-well plates (6  103 cells/
well), and maintained until use at 37.0  0.5  C in a humidied 5%
CO2 incubator.
Cytotoxicity of blank (5-FU unloaded) PLGA-based NPs, i.e.
PLGA NPs, PEG-PLGA NPs, and FOL-PEG-PLGA NPs, was investigat-
ed not only in normal human cells (CCD-18 and MCF-10A) but also
in the human tumour cell lines (HT-29 and MCF-7). To that aim,
quadruplicate cultures were evaluated by the 3-(4,5-dimethylth-
iazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT) proliferation
assay, determining mitochondrial dehydrogenase activity. NP
concentrations ranging from 0.1 to 200 mM (dilutions similar to
that of the drug-loaded particles used in these tests) were added to
the cells in the culture medium. After 72 h of incubation at
37.0  0.5  C, 20 mL of MTT solution were added to each well (5 mg/
mL in cell culture medium). The culture medium was removed
after incubation during 4 h at this temperature, and the resultant
formazan crystals were dissolved in 200 mL of DMSO. Finally, the
optical density (OD) of converted dye, which is proportional to the
number of viable cells, was measured at 570 and 690 nm (Titertek
multiscan colorimeter, Flow Laboratories, Irvine, California, USA).
Fig. 1. Synthesis procedures followed to prepare the PEG-PLGA and FOL-PEG-PLGA conjugates.
The relative cell viability (%) was calculated as [(OD treated cells/
OD control (untreated) cells)  100].
Similarly, the MTT assay was further used to dene the
cytotoxicity of the formulations involving the drug, i.e. free 5-
FU, 5-FU-loaded PLGA NPs, 5-FU-loaded PEG-PLGA NPs, and 5-FU-
loaded FOL-PEG-PLGA NPs, against HT-29 and MCF-7 cells. The
concentration of these preparations in contact with cells ranged
from 0.1 to 50 mM equivalent 5-FU concentration.
2.2.8. Statistical analysis
The IBM SPSS Statistics software package (version 17; IBM
Corporation, Chicago, Illinois, USA) was used to perform all
statistical analyses. Students t-test helped to compare results.
Data were expressed as means  standard deviations. Differences
were considered statistically signicant at a p value < 0.05.
3. Results and discussion
FOL-PEG-PLGA NPs were synthesized and investigated as a
potential carrier for 5-FU in the treatment of colon and breast
cancers.
3.1. Characterization of the synthesized PEG-PLGA and FOL-PEG-PLGA
conjugates
PEG-PLGA and FOL-PEG-PLGA were synthesized by following a
procedure previously described (Chen et al., 2012; Cheng et al.,
 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170 65

2007; Esmaeili et al., 2008; Saxena et al., 2012; Yoo and Park, 2004)
with some modications (Fig. 1). The synthesis of the two
compounds involves the formation of amide bonds. These bonds
can be formed by a well-established and a relatively simple
chemical reaction. A succinimide end group is rst produced from
a carboxyl group (COOH) in the presence of EDC, e.g. PLGA-COOH is
converted to PLGA-NHS. Next, the succinimide end group is paired
with an amine group (NH2). Upon conjugation of FOL to the
polymer, the succinimide derivative is obtained by conversion of
the a-COOH group of FOL to the more reactive NHS group, which is
then reacted with the free primary NH2 group of the PEG-PLGA
block copolymer. It is noteworthy that while a-COOH group
enables the preparation of the targeted conjugate, this conjugation
does not compromise the ability of FOL to bind to folate receptor
(Sudimack and Lee, 2000). The folate terminal moiety has been
found to reside on the surface when the NPs are formed (Low and
Kularatne, 2009).
Using 1H NMR analysis it was possible to identify the PLGA
moiety [-CH2- and CH3 peaks of poly(D,L-lactide), d = 5.2-5.3 and
1.5 ppm; CH2- peak of poly(glycolide), d = 4.8-4.9 ppm], the PEG
moiety (d = 3.6 ppm), and the characteristic peaks of the folate
moiety (d = 4.5, 6.6, 7.6, and 8.6 ppm) in the FOL-PEG-PLGA
conjugate (Fig. 2).
The conjugation percentage of FOL to PEG-PLGA was 42.3%
(molar ratio) measured by UV absorbance determinations at
364 nm.

Fig. 2. 1H NMR spectra of: (a) PEG-PLGA conjugate, and (b) FOL-PEG-PLGA conjugate.
66 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170

Table 1
Effects of formulation, sonication, and drug loading on the characteristics of the PLGA NPs. The values are the mean  standard deviation (SD) of triplicate experiments.

Factors Formulation Sonication Drug used Size (nm) PdI z (mV)

studied (mg)
PVA concentration (%, Solvent ratio (organic to aqueous phase ratio, Time Output
w/v) o:w) (min) (%)
Formulation 0.5 3:10 0.5 20 0 207.3  6.8 0.063  0.011 23.5  3.1
5:10 210.8  19.1 0.081  0.035 27.3  2.3
7:10 230.1  26.8 0.291  0.016 26.1  1.7
1 3:10 184.2  15.4 0.044  0.022 24.9  2.1
5:10 181.3  11.7 0.058  0.018 25.5  1.9
7:10 210.1  24.2 0.202  0.033 23.5  2.3
1.5 3:10 180.6  11.4 0.051  0.015 27.3  2.6
5:10 181.8  12.9 0.671  0.032 26.1  2.2
7:10 195.9  20.2 0.166  0.034 24.1  1.7
Sonication 1 5:10 0.5 40 0 180.3  8.4 0.081  0.024 26.2  2.1
50 211.1  10.4 0.086  0.064 27.9  2.4
1 20 183.2  9.1 0.053  0.021 26.3  1.8
40 188.5  10.4 0.075  0.042 28.9  2.8
50 217.4  12.3 0.092  0.079 23.1  1.9
5 20 197.5  14.2 0.036  0.016 24.5  1.3
40 210.9  16.8 0.112  0.021 27.9  2.6
50 239.5  17.1 0.244  0.134 26.2  1.5
5-FU loading 1 5:1 0.5 20 1 181.4  10.3 0.049  0.022 24.1  2.9
3 184.9  7.2 0.059  0.023 27.7  1.9
5 185.8  11.9 0.056  0.026 24.4  2.9
8 187.6  7.1 0.062  0.011 28.3  2.1
10 186.2  10.3 0.064  0.015 23.6  1.4
12 188.4  9.3 0.071  0.013 26.9  2.6
15 190.7  13.1 0.063  0.019 25.1  2.6

3.2. Characterization of the PLGA-based nanoparticles
The NPs were produced successfully by dissolving PLGA, PEG-
PLGA, or FOL-PEG-PLGA in acetone followed by nanoprecipitation
in water. Characterization of the NPs was performed using dynamic
light scattering (DLS) for particle size and z determinations, and
HRTEM for visualization of the NPs.
Effects of formulation, sonication, and drug loading on the
characteristics of the PLGA NPs (i.e. particle size, PdI, and z) were
investigated (Table 1). In these studies only the examined
parameter was varied while all the other parameters were kept
xed. When selecting formulation components, acetone was used
as the organic phase because it is a good solvent for 5-FU. In
addition, PVA was chosen as the stabilizer since it is commonly
employed in the preparation of PLGA NPs with a uniform size
distribution (Van de Ven et al., 2012).
Among formulation parameters, we rst investigated varying
the percentage of the stabilizer, i.e. PVA. Increasing PVA from 0.5%
to 1% (w/v) seemed to reduce the particle size and PdI, whereas a
higher percentage of 1.5% did not appear to add an additional effect
compared with the 1% PVA formulations. We also studied the effect
of altering the organic to aqueous phase ratio used. While particle
size showed a trend to grow with the increase of solvent/water
ratio, the effect of this ratio was more obvious on PdI. At acetone/
water ratio of 7:10, the signicant increase in PdI, between 3 and 6
folds as compared with the lower ratios, indicated a wider size
distribution. This effect may be due to a partial attenuation and
dissipation of the applied ultrasonic energy by the increased
volume of the medium.
Interestingly, short sonication time and lower output favoured
the production of NPs with a smaller size and lower PdI. For
instance, optimal particles with an average size of 181.3 nm and a
PdI of 0.058 were obtained by sonication for 0.5 min at 20% output,
whereas sonication for 5 min at 50% output signicantly increased
the average size to 239.5 nm (about 30%) and the PdI to 0.244
(about 400%). Although cavitational collapse produced during
sonication leads to particle size reduction, sonication-induced
aggregates can form under certain conditions (Taurozzi et al.,

Fig. 3. HRTEM images of PLGA NPs (a), PEG-PLGA NPs (b), and FOL-PEG-PLGA NPs (c). Bar lengths: 200 nm (a), and 150 nm (b and c).
 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170 67

2011). Increased collision frequency resulting from sonication and

augmented free energy owing to the increase in the liquid-solid
interface can enhance particleparticle interactions and favour
aggregate formation.
In general, drug loading into NPs is associated with an increase
in particle size. In this work, we observed a slight increase of up to
about 5% in the average particle size when increasing amounts of
5-FU, up to 15 mg, where used in NP preparation.
z values, ranging between 23 to 28 mV, did not seem to be
inuenced by formulation components, sonication conditions, nor
the amount of loaded drug.
Based on these ndings, the PVA concentration of 1%, the
organic to aqueous phase ratio of 5:10, sonication time of 0.5 min,
and sonication output of 20% were identied as parameters for the
optimal formulation of PLGA NPs. Under these parameters, the
mean particle size and PdI were found to be 181.3 nm and 0.058,
respectively. Furthermore, the volume of the organic solvent
(5 mL) was found to be sufcient to dissolve a suitable amount of 5-
FU.
The above dened optimal preparation conditions were also
used to obtain PEG-PLGA NPs and FOL-PEG-PLGA NPs character-
ized by a particle size of 145.3  10.4 nm and 149.2  7.0 nm,
respectively, with a PdI of 0.066  0.034 and 0.075  0.059,
correspondingly. These particle sizes were remarkably smaller
than those of the unmodied PLGA NPs. This result is in agreement
with previous ndings and it is thought to be due to the effect of
the hydrophilic PEG coating that improves the stability of the NPs
and results in a smaller particle size (Tao et al., 2012). Additionally,
while the negative surface charge of PEG-PLGA NPs, 22.3  2.1
mV, was similar to that of PLGA NPs (Table 1), FOL-PEG-PLGA NPs
had comparably a lower surface charge of 15.7  1.4 mV. This may
be owed to the effect of the protonated amino acid groups of folate
which diminish the negative charge of the PLGA carboxylic acid
groups (Liang et al., 2011).
HRTEM images conrmed the size determinations, and that
Fig. 4. Entrapment efciency (a) and loading (b) of 5-FU to the PLGA NPs (PLGA:
PLGA NPs, PEG-PLGA NPs, and FOL-PEG-PLGA NPs had similar 50 mg; PVA: 1%; solvent ratio: 5:10; sonication time: 0.5 min; sonication output:
morphologies (spherical shape, Fig. 3). 20%).

3.3. Drug loading studies

The EE (%) and DL (%) of 5-FU to the PLGA NPs as a function of

drug amount are collected in Fig. 4. As the drug amount used in NP
preparation was increased from 1 to 5 mg, the EE values increased
from 4.91  0.69 to 8.52  0.67 to reach a plateau at higher drug
amounts. Also, the loading percentage augmented linearly with
the amount of the drug and the maximum drug loading achieved
was 5.43  0.16 when 15 mg of drug were used. The relatively low
loading capacity may be due to the hydrophilic nature of 5-FU
which may encourage the immediate diffusion of the drug
molecules to the aqueous phase during preparation. In addition,
a relatively small amount of polymer (50 mg) was used in the
fabrication of the NPs which might also contribute to the resulting
loading capacity. Finally, similar drug incorporation results were
obtained when preparing 5-FU-loaded PEG-PLGA NPs and 5-FU-
loaded FOL-PEG-PLGA NPs. For instance, when 15 mg of 5-FU were
used, the 5-FU EE (%) values to the PEG-PLGA NPs and FOL-PEG-
PLGA NPs were 8.73  0.44 and 8.68  0.51, respectively, with a DL Fig. 5. In vitro release proles of 5-FU from: (^) PLGA NPs, (&) PEG-PLGA NPs, and
(%) of 5.52  0.14 and 5.49  0.15, correspondingly. (D) FOL-PEG-PLGA NPs, in PBS (pH 7.4  0.1) at 37.0  0.5  C. Data is presented as
mean value  standard deviation (SD) (n = 3).
3.4. In vitro drug release
approximately 2025% of the drug released after 1 h, followed
The in vitro release of 5-FU from the NPs was characterized in by a sustained, linear 5-FU release (Fig. 5). The initial burst release
PBS (pH 7.4  0.1) at 37.0  0.5  C, using a membrane dialysis of 5-FU could be due to the release of some weakly bound drug on
method. 5-FU-loaded PLGA NPs, 5-FU-loaded PEG-PLGA NPs, and the surface of the NPs which is rapidly released by diffusion.
5-FU-loaded FOL-PEG-PLGA NPs exhibited a similar drug release Whereas, the remained drug which is embedded in the NP
prole; characterized by an initial burst release, with structure was released slowly, likely due to polymer degradation
68 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170

by hydrolysis, in a more controlled manner over several days

(about 7580% was released over the 6-day experiment). Similar
release prole has been observed with PLGA NPs loaded with
different drugs (Menon et al., 2012; Park et al., 2009; Tewes et al.,
2007). However, PEG-PLGA NPs and FOL-PEG-PLGA NPs showed a
relatively higher 5-FU release compared with PLGA NPs; this
increased release was statically signicant (p value < 0.05) starting
from t = 2 h until the end of the experiment. The increase in drug
release may be attributed to variations in particle size, z values as
well as the greater hydrophilic surface of both PEG-PLGA NPs and
FOL-PEG-PLGA brought about by the PEG shell. The hydrophilic
surface can increase interaction with the aqueous medium, absorb
more water, and may subsequently induce faster degradation of
the NPs by hydrolysis which may lead to a faster 5-FU release. As a
result, 5-FU release from PEG-PLGA NPs and FOL-PEG-PLGA NPs
was completed in 4 days, while 6 days were needed by the PLGA
NPs to end the release of the loaded drug.

3.5. Haemocompatibility of the nanoparticles

Taking into consideration data in Table 2, the PLGA-based NPs

are expected to be characterized by a wide in vivo safety margin,
and suitability for parenteral administration (Arias et al., 2011;
Dash et al., 2010; Muoz de Escalona et al., 2016). In fact, a
negligible effect on haemolysis (even after 24 h), platelet activa-
tion, complement system activation, and plasma clotting time of
blood samples was observed (n = 3).

3.6. In vitro cytotoxicity experiments

As demonstrated in Fig. 6, blank (drug-unloaded) PLGA-based

NPs exhibited negligible cytotoxicity in normal (CCD-18, and MCF-
10A) and tumour (HT-29 and MCF-7) human cell lines at the
dilutions corresponding to that of 5-FU-loaded particles. Based on
these ndings, and also considering the data coming from blood
compatibility tests (see Section 3.5.), it can be assumed that these
polymeric nanosystems present an adequate biocompatibility and
safety for drug delivery purposes.
Fig. 7 displays the in vitro cytotoxic effect of free 5-FU, and 5-
FU-loaded NPs in HT-29 and MCF-7 cells. These formulations Fig. 6. In vitro cytotoxicity of PLGA NPs (a), PEG-PLGA NPs (b), and FOL-PEG-PLGA
inhibited cell proliferation in a dose-dependent manner. In both NPs (c) in CCD-18 human colon broblast cells, HT-29 human colon cancer cells,
MCF-10A human breast epithelial cells, and MCF-7 human breast cancer cells, after
cell lines, the half maximal inhibitory concentration (IC50) values of
72 h of exposure to a wide range of NP concentrations: 0.1 mM (grey column), 50 mM
5-FU-loaded PEG-PLGA NPs and 5-FU-loaded FOL-PEG-PLGA NPs (light grey column), 100 mM (black column), and 200 mM (white column). The
were approximately 2-fold and 4-fold, respectively, less than that values are the mean  standard deviation (SD) of quadruplicate cultures. Cells
of the 5-FU-loaded PLGA NPs (p < 0.05), whereas the IC50 of 5-FU- without treatment were used as control to calculate the relative cell viability (%).
loaded PLGA NPs was found to be similar to that of the free
antitumour agent (Fig. 7). The lower cytotoxicity of free 5-FU may (Pamujula et al., 2012). This effect was associated with the smaller
be attributed to the drug resistance resulting in the efux of 5-FU particle size of the PEGylated PLGA NPs, and similar ndings have
molecules by P-glycoprotein (P-gp) pumps (Fojo and Coley, 2007). been described in Section 3.2. on the effect of PEGylation on the
In addition, the higher cytotoxicity of 5-FU-loaded PEG-PLGA NPs size of the PLGA particles. The positive effect of PEGylation on NP
compared to 5-FU-loaded PLGA NPs is in agreement with a recent uptake by cancer cells overexpressing P-gp has been further
study in which PEGylation of PLGA NPs was found to signicantly described in liposomes (Kopecka et al., 2014) and poly(amido
increase cellular uptake by 4T1 murine breast cancer cells amine) (PAMAM) dendrimers (Liu and Chiu, 2013).

Table 2
Blood compatibility of blank (5-FU unloaded) PLGA-based NPs in terms of haemolysis (%), platelet activation (soluble P-selectin release, ng/mL), complement system
activation (C3a release: C3a desArg, ng/mL), and plasma recalcication time (T1/2max, min). Data is indicated as mean value  standard deviation (SD) (n = 4).

Sample Haemolysis (%) soluble P-selectin release (ng/mL) C3a desArg (ng/mL) T1/2max (min)

Incubation time

2h 4h 8h 24 h
PLGA NPs 0.8  0.1 1.1  0.2 1.9  0.2 1.8  0.4 103  4 296  13 12.8  1.6
PEG-PLGA NPs 1.1  0.3 1.2  0.4 0.9  0.3 1.1  0.1 105  3 301  14 11.3  1.4
FOL-PEG-PLGA NPs 1.2  0.2 0.9  0.3 1.7  0.4 1.9  0.3 101  5 299  11 13.1  1.5
Control (PBS solution) 0 0 0 0 98  7 290  11 10.9  2.1
 M.M. El-Hammadi et al. / International Journal of Pharmaceutics 516 (2017) 6170
Fig. 7. In vitro cytotoxicity of free 5-FU (grey column), 5-FU-loaded PLGA NPs (light
grey column), 5-FU-loaded PEG-PLGA NPs (black column), and 5-FU-loaded FOL-
PEG-PLGA NPs (white column) in HT-29 human colon cancer cells (a) and (b) MCF-7
human breast cancer cells, after 72 h of exposure to a wide range of NP
concentrations (up to 25 mM equivalent 5-FU concentration). The values are the
mean  standard deviation (SD) of quadruplicate cultures. Cells without treatment
were used as control to calculate the relative cell viability (%). Insets: IC50 values
(mM) of the 5-FU-based formulations.
Finally, the cytotoxic effect of 5-FU was found to be additionally
increased with the FOL-decorated and PEGylated (FOL-PEG-PLGA)
NPs (Fig. 7). These results may be associated to an enhanced
cellular uptake via folate receptor mediated endocytosis, thus
avoiding 5-FU efux by P-gp and leading to a signicantly
augmented effect against the folate receptor-overexpressed HT-
29 and MCF-7 cells, as previously reported in the literature
(Balasubramanian et al., 2014; Wang et al., 2015). The FOL-PEG-
PLGA nanosystem being described here has efcaciously improved
5-FU activity against MCF-7 cells as compared to alternative pure
PLGA NPs (Nair et al., 2011) and against HT-29 cells as compared to
alternative pure FOL-decorated (non-PEGylated) PLGA NPs (Wang
et al., 2015).
4. Conclusions
It has been reported for the rst time the use of FOL-PEG-PLGA
NPs as a targeted delivery system of 5-FU against colon and breast
cancers. To that aim, a PEG-PLGA copolymer was synthesized and
functionalized with FOL, which was then formulated as NPs by
nanoprecipitation. 5-FU loading conditions resulted in an adequate
drug vehiculization by the NPs and in sustained (biphasic) drug
release properties. FOL-PEG-PLGA NPs entrapping 5-FU showed a
superior cytotoxicity over non-targeted PLGA-based NPs in folate
receptor-overexpressed HT-29 colon and MCF-7 breast cancer cell
lines. On the other hand, blank (drug unloaded) NPs were
haemocompatible with negligible cytotoxicity, making them
promising candidates as drug carriers. Our ndings imply that
69
the FOL-decorated and PEGylated PLGA NPs may facilitate targeted
delivery of 5-FU to colon and breast tumours.
Acknowledgements and funding
Financial support of the Erasmus Mundus JOSYLEEM Program
is acknowledged as is the post-doctoral fellowship to Mazen M. El-
Hammadi. The authors gratefully acknowledge the assistance of Dr.
Ana Conejo Garca (Department of Organic Chemistry, Faculty of
Pharmacy, University of Granada, Spain) in the chemical syntheses
carried out in this work. Research leading to these results has
received funding from FEDER, Plan Nacional de Investigacin
Cientca, Desarrollo e Innovacin Tecnolgica (I + D + i), Instituto
de Salud Carlos III (FIS, Spain): projects PI11/01862 and PI11/02571;
and from Junta de Andaluca (Spain, project PE-2012-FQM-694).
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