Targeted poly (l-γ-glutamyl glutamine) nanoparticles of docetaxel against folate over-expressed breast cancer cells

Article abstract of DOI:10.1016/j.ijpharm.2014.03.033

A novel folate (FA) conjugated poly(l-γ-glutamyl glutamine) (PGG) nanoparticle loaded with docetaxel (DTX) was prepared to take advantage of both targeted drug delivery in breast cancer and reducing the overall side effects due to the adjuvant free formulation in comparison with Taxotere . Nanoprecipitation method was employed to prepare nanoparticles (NPs). The chemical structure of PGG synthesized polymers and PGG-FA conjugates and polymeric nanoparticles were characterized by H NMR, FTIR spectroscopy, field emission scanning electron microscopy, and laser scanning confocal microscopy. The average size of optimized nanoparticles with the aid of Box-Behnken experimental design was 131.96 ± 5.34 (nm) with polydispersity of 0.089 ± 0.019, zeta potential of -25.8 ± 2.21 (mV), and entrapment efficiency of 67.83 ± 3.29(%). In vitro cytotoxicity of the designed NPs was investigated by MTT assay against three chosen cell lines of MCF7, 4T1, and A549 based on their folate receptor expression capacity and was compared with Taxotere. Moreover, PGG-FOL NPs were loaded with 6-coumarin for cellular uptake investigation. In order to assess the antitumor efficacy and biodistribution of targeted NPs, 4T1 murine breast tumors were established on the balb/c mice and in vivo studies were performed. The obtained results showed that the novel designed system was highly effective against tumor cells and successfully localized in the tumor site.

Full text of DOI:10.1016/j.ijpharm.2014.03.033

International Journal of Pharmaceutics 467 (2014) 123–138
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Targeted poly ( -g-glutamyl glutamine) nanoparticles of docetaxel
L
against folate over-expressed breast cancer cells
Faranak Tavassolian a,b, Golnaz Kamalinia a,b, Hasti Rouhani , Mohsen Amini c,d,
a,b
e
f
a,b
Seyed Nasser Ostad , Mohammad Reza Khoshayand , Fatemeh Atyabi
,
a
a,b,
Morteza Rafiee Tehrani , Rassoul Dinarvand
*
a
Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Toxicology-Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b
c
d
e
f
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article history:
A novel folate (FA) conjugated poly(L-g-glutamyl glutamine) (PGG) nanoparticle loaded with docetaxel
Received 8 February 2014
Received in revised form 15 March 2014
Accepted 18 March 2014
(
DTX) was prepared to take advantage of both targeted drug delivery in breast cancer and reducing the
1
overall side effects due to the adjuvant free formulation in comparison with Taxotere . Nano-
precipitation method was employed to prepare nanoparticles (NPs). The chemical structure of PGG
synthesized polymers and PGG-FA conjugates and polymeric nanoparticles were characterized by H
NMR, FTIR spectroscopy, field emission scanning electron microscopy, and laser scanning confocal
microscopy. The average size of optimized nanoparticles with the aid of Box–Behnken experimental
design was 131.96 ꢀ 5.34 (nm) with polydispersity of 0.089 ꢀ 0.019, zeta potential of ꢁ25.8 ꢀ 2.21 (mV),
and entrapment efficiency of 67.83 ꢀ 3.29(%). In vitro cytotoxicity of the designed NPs was investigated by
MTTassay against three chosen cell lines of MCF7, 4T1, and A549 based on their folate receptor expression
Available online 28 March 2014
Keywords:
Nanoparticles
Folate targeting
Poly(L-g-glutamyl glutamine)
MTT assay
1
Docetaxel
capacity and was compared with Taxotere . Moreover, PGG-FOL NPs were loaded with 6-coumarin for
Antitumor efficacy
cellular uptake investigation. In order to assess the antitumor efficacy and biodistribution of targeted
NPs, 4T1 murine breast tumors were established on the balb/c mice and in vivo studies were performed.
The obtained results showed that the novel designed system was highly effective against tumor cells and
successfully localized in the tumor site.
ã 2014 Elsevier B.V. All rights reserved.
1. Introduction
effects with higher drug efficacy and desirable distribution, passive
targeting due to the leaky tumor vascular, enhanced permeability
It is difficult to disguise the fact that nanotechnology,
particularly nanoparticles, open a new route in drug administra-
tion and exclusively in cancer treatment strategies (Doane and
Burda, 2013). There are several types of nanoparticle systems such
as micelles, carbon based nanotubes, protein nanoparticles,
liposomes, dendrimers and polymeric nanoparticles (Couvreur,
and retention (EPR) effect and desirable biodistribution is amongst
NPs drug delivery systems benefits (Zhang et al., 2013). On the
other hand, targeting polymeric nanoparticles has received a great
deal of attention particularly in cancer treatment (Brannon-Peppas
and Blanchette, 2012; Byrne et al., 2008; Shapira et al., 2011;
Esmaeili et al., 2008; Taheri et al., 2011). Folate (FOL) receptors are
mostly noted due to their over-expression in tumors, e.g. breast
cancer (Liu et al., 2010; Wang et al., 2011a). Employing folic acid as
a targeting moiety is a promising strategy to enhance the
polymeric nanoparticles delivery to cancerous cells. Moreover,
reduced side effects and a lower damage to normal cells will occur
due to their specificity and high local concentration in tumor cells
2013; Hu and Zhang, 2012; Couvreur and Vauthier, 2006; Kope c9 ek,
2013; Tekade et al., 2009; Bangham et al.,1965). The advantages of
nano sized drug delivery systems are countless. Namely, less side
*
Corresponding author at: Tehran University of Medical Sciences, Faculty of
(
Werner et al., 2011; Zhao et al., 2011).
Poly- -glutamic acid has been widely used to prepare polymeric
nanoparticles during the recent years (Lee, 2006) and some of
Pharmacy, 16th Azar St., Tehran, Iran.
Tel.: +98 21 66959095/66959055; fax: +98 21 66959096/66959055.
E-mail address: dinarvand@tums.ac.ir (R. Dinarvand).
L
http://dx.doi.org/10.1016/j.ijpharm.2014.03.033
0
378-5173/ã 2014 Elsevier B.V. All rights reserved.

124
F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
these newly developed nanoparticulate formulations such as a
paclitaxel (PTX) conjugated poly- -glutamic acid formulation (CT-
103) have achieved great fortune in clinical trials (Wang et al.,
differentiated by their folate expression capacity (Mi et al.,
2011; Watanabe et al., 2012).
L
2
2
Determination of the in vivo performance of NPs is essential for
predicting their therapeutic response. In this study, 4T1 breast
tumor was established on balb/c mice as a tumor-bearing model.
Furthermore, the optimized NPs were evaluated in vivo by survival,
antitumor efficacy and biodistribution studies. Concisely, tissue
011b; Feng et al., 2010; Lee et al., 2013). There are only few studies
-glutamyl glutamine (PGG) polymers
properties and its characterization (Van et al., 2010; Yang et al.,
012a). PGG is prepared by a modification on poly( -glutamic acid)
PGA) polymer by addition of glutamic acid moieties on PGA side
available on poly
(L-g
2
L
1
(
distribution and tumor accumulation of Taxotere and targeted
chains (Yang et al., 2011a). PGG polymer in conjugation with
paclitaxel represented paramount therapeutic and pharmacoki-
netic results in comparison to Abraxane as reported by Van et al.
NPs were compared and analyzed.
2. Material and methods
2.1. Materials
1
(
Van et al., 2010). Additionally, desirable water solubility and
stability of PGG-PTX nano-conjugates and their appropriate
biocompatibility has been demonstrated by Yang et al. (2011a,b).
The anti-tumor activity and biodistribution of paclitaxel conjugat-
ed with PGG was also represented in Wang et al. study; indicating
that the prepared nanoparticles displayed both prolonged release
and favorable antitumor efficacy (Wang et al., 2010). Moreover, a
docetaxel (DTX) conjugated PGG has been also designed to act
against human non-small cell lung cancer cell line (Yang et al.,
Poly(
L
-glutamic acid) sodium salt (MW 20–30 kDa), N-(3-
0
dimethylamino propyl)-N -ethylcarbodiimide (EDC),
L-glutamic
acid di-tert-butyl ester hydrochloride, N-hydroxysuccinimide
(NHS), coumarin-6, MTT, fetal bovine serum (FBS), penicillin and
streptomycin were all obtained from Sigma–Aldrich (St. Louis, MO,
USA). Docetaxel (anhydrous) and folic acid were purchased from
Cipla (Mumbai, India). Roswell Park Memorial Institute 1640
(RPMI) was acquired from life technologies (Grand Island, NY,
USA). MCF7 human breast adenocarcinoma cells, 4T1 murine
breast cancer cells and A549 human pulmonary adenocarcinoma
cells were obtained from national cell bank of Iran (NCBI). Annexin
V-FITC/PI apoptosis kit was from Biovision (Germany, Lorrach). The
water used was pretreated with the TKA-GenPure water purifica-
2012b). The molecular weight is amongst the parameters that play
a major role in physicochemical properties of the polymer based
systems. Therefore, the effect of PGG-PTX molecular weight on
both toxicity and efficacy has been evaluated in a report by Yang
et al. (2012a).
DTX is a semisynthetic analogue of paclitaxel, a well-known
lipophilic anti-cancer agent that is obtained from the European
yew tree, Taxus baccata (Fite et al., 2007; Yousefi et al., 2009). DTX
seems to be a better choice in comparison with paclitaxel due to its
higher microtubules assembly promotion; however, neuro and
musculoskeletal toxicity due to the presence of tween 80 and
ethanol in Taxotere(r) formulation is still regarded as a matter of
concern, especially with considering the risk of extravastion and
hypersensitivity reactions and also the incompatibility of Taxotere
formulation with common administration sets (Liu et al., 2012).
The goal of the present study was to design a novel folate targeted
PGG polymeric nanoparticle free from tween 80 and ethanol that
could deliver DTX to breast cancerous cells not only with higher
specificity and efficacy but also with lower overall side effects.
Based on this knowledge we hypothesized that PGG nanoparticles
loaded by DTX and targeted by folic acid are more potent with a
higher efficacy on breast cancerous cell models in comparison with
Taxotere(r) especially with considering the overexpression of
folate receptors in tumor tissues and in cancerous cells (Esmaeili
et al., 2008). The objective of the current study was to prepare a
new surfactant and ethanol free formulation to prevent these two
excipients related side effects and in the same time develop a
targeted system (by targeting the over expressed folate receptors
in tumor tissues) and to specifically deliver the NPs to the targeted
tissue to enhance specificity and efficacy of drug formulation and
to reduce the overall side effect and to provide a better alternative
for the patients' chemotherapy regimen. Here, we report the
applied method for PGG synthesis and its conjugation with folate.
We employed the straight forward precipitation technology for
manufacturing the PGG-FA nanoparticle loaded with DTX, to
overcome the poor aqueous solubility of DTX (Bilati et al., 2005).
Additionally, the Box–Behnken experimental design was employed
and the obtained optimum formulation was further investigated
by various methods of nanoparticle characterization (Gazori et al.,
0
tion system. N,N -Dicylohexylcarbodiimide (DCC), ethylenedi-
amine, ethyl acetate, dichloromethane (DCM), acetonitrile
(ACN), dimethyl sulfoxide (DMSO) and methanol were purchased
from Merck (Darmstadt, Germany). All reagents used for HPLC
analysis were of HPLC grade.
2.2. Synthesis
2.2.1. Activation of PGA carboxyl group
Poly(L-glutamic acid) sodium salt was first dissolved in DCM,
and its carboxyl groups were activated by the addition of DCC and
NHS (1:9:10 molar ratio). The reaction was maintained under
nitrogen atmosphere and was stirred at room temperature for 24 h.
The reaction mixture was filtered and precipitated by adding
diethyl ether. The solvent was evaporated under reduced pressure
and was used for the subsequent step.
2.2.2. Synthesis of PGG
The activated PGA polymer was conjugated with L-glutamic acid
di-tert-butyl ester hydrochloride monomers (1:4 molar ratios) by
adding NHS, DCC, and DCM under constant stirring at room
temperature. Subsequently, a solution of HCl 10% was added to the
above solution and the mixture was well stirred for 2 h. The organic
phase was then separated and was dried by anhydrous sodium
sulfate. In the next step, the organic solvent was removed under the
vacuumandthestructureoffinalresultingconjugatedPGGpolymer
was confirmed using Fourier Transform Infrared Spectroscopy
(FTIR) by a Nicolet Magna IR-550, USA spectrophotometer.
2.2.3. Synthesis and purification of folate ethylenediamine
To prepare folate ethylenediamine, the carboxyl group of folate
structure was conjugated to ethylenediamine. As a result, NH
2
2
009). Moreover, in vitro cytotoxicity of the optimized PGG-FA
group on folic acid became ready for further reactions with the
PGG carboxylic groups.
1
nanoparticles loaded with DTX was compared with Taxotere
through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay on three different cell lines including
MCF7 (high folate receptors over expression), 4T1 (moderate
folate receptors over expression) and A549 (control cell line
without folate receptors over expression) which were
Folate carboxylic groups were activated by adding EDC and NHS
(1:10:9 molar ratios) in the presence of dried methanol as a
ꢂ
solvent. The mixture was stirred at 50 C for 24 h under light
protection and N
2
atmosphere. The final reaction was stirred
continuously for another 24 h in dark condition and in the presence

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
125
of dried pyridine with an equal molar ratio of N-BOC-ethylenedi-
amine. The solvent was then removed under vacuum.
from five optimized formulations were compared with the
suggested experimental responses.
To purify folate ethylenediamine from the above mixture,
distilled water was added to the reaction mixture and the pH was
adjusted to 12 by sodium hydroxide (5 M). Ethyl acetate was also
added to the mixture as an organic phase and the folate
ethylenediamine was extracted. The organic phase was then
separated and dried with anhydrous sodium sulfate. The solvent
was filtered and evaporated under reduced pressure. Methanol
was added to the residue and mixed for 20 min. The mixture was
filtered and the solid material was removed. The structure of folate
ethylenediamine was confirmed by proton nuclear magnetic
resonance (H NMR).
2.4. Preparation of DTX loaded PGG-FOL nanoparticles
DTX loaded PGG-FOL nanoparticles were prepared by a robust
and scalable nanoprecipitation method and according to the
optimized formulation achieved from employed Box–Behnken
experimental design (Bilati et al., 2005). Both DTX and FOL
conjugated PGG polymers were dissolved in a minute amount of
DMSO. Subsequently, the organic phase was injected to the
deionized water while gently stirred at 750 rpm at room
temperature. The nanoparticle suspension was finally centrifuged
thrice at 19,000 rpm for 30 min and was washed with deionized
water. After the final washing step, DTX loaded PGG-FOL nano-
2.2.4. Synthesis of PGG-folate conjugates
ꢂ
The synthesized folate ethylenediamine and the synthesized
particles were lyophilized and stored at ꢁ20 C until use and were
PGG polymer (1:1 molar ratio) were dissolved in dried methanol
subjected to dynamic light scattering (DLS) and scanning electron
microscopy (SEM) studies and prior to release and cytotoxicity
assays.
ꢂ
and were refluxed overnight for 24 h in 105 C while stirred
continuously. Subsequently, the solvent was removed and water
and DCM were added. The organic phase was separated and dried
with anhydrous sodium sulfate and was subsequently filtered. The
residue was then treated with water and was extensively dialyzed
2.5. Characterization of the nanoparticles
(
molecular weight cut off of 12,000 Da) against phosphate buffer
2.5.1. Particle size and size distribution
saline (PBS) with pH 7.4 for 48 h and then against distilled water for
The size and size distribution of DTX loaded PGG-FOL nano-
particles were measured by DLS method using a Malvern Zetasizer
Nano ZS (Malvern instrument, Worcestershire, UK). The nano-
particles were dispersed and diluted in deionized water and were
sonicated with ultrasonic Elmasonic bath (Elma HausSchmidbauer
GmbH & Co. KG) in advance to size determination and in order to
prevent their clumping.
2
4 h. The final solution was lyophilized (Christ, alpha 2-4 LD plus,
ꢂ
Germany) and stored at ꢁ20 C until use. The final resulting PGG-
FOL structure and its conjugated percentage were studied using H
NMR.
2.3. Experimental design studies
A response surface methodology design called Box–Behnken
2.5.2. Nanoparticles morphology study
was employed in the present study. Three quantitative indepen-
dent variables consisting of stirrer rate (rpm), polymer concentra-
tion (mg) and non-solvent volume (ml) were elucidated based on
the preliminary studies as shown in Table 1. Also, dependent
variables and responses, including particle size (nm), entrapment
efficiency (EE%) and drug loading (DL%) were evaluated with
The DTX loaded PGG-FOL nanoparticles were morphologically
investigated by Field Emission Electron Microscopy (FE-SEM,
S4160, and Hitachi, Japan) in which dried nanoparticles were
gently coated by a gold layer with a sputter coater and were
subjected to microscopy technique.
1
Design-Expert software (V. 7.0.0, Stat-Ease, Inc., Minneapolis,
2.5.3. Nanoparticles surface charge measurement
USA).
The NPs suspension was firstly diluted by deionized water while
the pH value (pH 7.0 ꢀ 0.1) and also the NPs concentration were
fixed before the surface charge was measured. The surface charge
of the drug-loaded NPs was evaluated by photon correlation
spectroscopy (Zeta Sizer, Nano-ZS Malvern, and UK).
As a result,17 runs were required for the optimized formulation
to be achieved and the second-order polynomial functions
explained the relationship between the dependent and the
independent variables as follows:
2
2
2
y1;2;3 ¼ b
0
þ b
1
A þ b
2
B þ b C þ b11A þ b22B þ b33C þ b12AB
3
2.5.4. Drug loading (DL) and encapsulation efficiency (EE)
measurements
þb13AC þ b23BC
HPLC was used for the quantitative measurement of DTX
entrapment in the PGG-FOL nanoparticles. The HPLC system
consisted of Knauer apparatus mode K-1001 (Berlin, Germany),
equipped with PDA K-2700 UV detector (Knauer, Germany).
A MZ-Analysentechnik C18 column (28 ꢃ 0.46 cm internal diam-
where A, B, and C are independent variables; and y is the predicted
dependent factor, b is the intercept, b , b , and b are linear
0
1
2
3
coefficients, b11, b22, and b33 are squared coefficients, and b12, b13
and b23 are the interaction coefficients of equation.
The goal of the design was to find the optimum formulation, not
only with minimum size but also with maximum entrapment
efficiency and drug loading. Consequently, to investigate the
adequacy and the level of model precision, obtained responses
,
eter, pore size 5 mm, Germany) was used. The mobile phase
consisted of acetonitrile/water (65:35 v/v). The flow rate was
ꢂ
1
ml/minute and the temperature was fixed on 25 C. Also the UV
detection wavelength was 228 nm. Three miligrams freeze dried
Table
1
Variable used in Box–Behnken experimental design.
Independent factors
Factor level
Dependent factors
Numeric factors
Stirring rate (rpm)
Polymeric concentration (mg/ml)
Non solvent volume (ml)
ꢁ1
500
5
0
750
10
1
1000
15
Particle size (nm): minimized
Entrapment efficiency (EE%): maximized
Drug loading (DL%): maximized
10
15
20

126
F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
NPs were firstly dissolved in the mobile phase and secondly
stirred for half an hour. Moreover, for the un-entrapped DTX
separation from the DTX loaded PGG-FOL nanoparticles, the
coumarin-6 fluorescence intensity which was detected in sample
wells versus thatof used in NPs suspensionwas determined and was
reported as the cellular uptake
solution was then filtered by 0.45
injecting to the HPLC. The following equation was used for
calculating the DL% and EE%:
m
m PVDF syringe filter prior to
To qualitatively evaluate the internalization of NPs by MCF7 and
A549 cells, confocal lasers scanning microscopy (CLSM) (Nikon Ti
Eclipse, Japan) was employed. Briefly, MCF7 and A549 cells were
cultivated in 6 well plates and the medium was removed after 48 h
and the cells were washed with PBS three times after incubation
with coumarin-6 loaded NPs for 4 h. The cells were then fixed by
ꢀW
ꢁ
actual amount of encapsulated drug in NPs
Wtotal drug
EE ¼
DL ¼
ꢃ 100
ꢃ 100
2
0 min incubation with 1.5 ml 75% ethanol and were evaluated
ꢀW
ꢁ
actual amount of encapsulateddrug in NPs
under CLSM by the help of an FITC filter.
W
total drugþpolymer
2
.9. MTT assay
The cytotoxicity of Taxotere , PGG-FOL nanoparticles (NPs),
2
.6. DTX in vitro release from nanoparticles
1
DTX release from drug loaded PGG-FOL nanoparticles was
and DTX loaded PGG-FOL NPs against MCF7, 4T1, and A549 cells
were measured using the MTT calorimetric assay. For this purpose,
each of the mentioned cell lines was seeded into 96-well plate
separately at 5000 cells per well. The plates were first incubated for
2
0
determined in two PBS medium with pH values of 5.2 and 7.4. The
acidic medium model was chosen due to its simulation to
endosomal and lysosomal compartments of the cancer cells. So,
the 10 mg DTX loaded NPs were dispersed in both media.
Subsequently, the dispersion was put in an orbital shaker and
4 h. Later, the various formulations at the concentration of 0.2, 0.4,
.6, and 0.8
m
m were used as a cell treatment, while the cells were
ꢂ
was introduced in 37 C. At predetermined time intervals, the
ꢂ
incubated for 24, 48, and 72 h at 37 C, and 5% CO
2
. At the
suspension was centrifuged at 17,000 rpm for 10 min. The
supernatant which contained the released amount of DTX from
PGG-FOL nanoparticles was separated. Moreover, the pellet was re-
suspended by adding adequate amount of fresh media in order to
continue the release profile with maintaining the sink condition.
Additionally, the supernatant DTX quantity was initially extracted
by DCM, and was later determined by HPLC procedure and based
on the calibration curve after its DCM was evaporated and filtered
through a 0.22 mm membrane filter. The in vitro release studies
were carried out in triplicate and all data were expressed as
mean ꢀ SD.
designated intervals the old medium was removed and 200 ml
fresh medium which contained 0.5 mg/ml MTT was added to wells.
ꢂ
The plates were incubated for 2–4 h at 37 C, and 5% CO
2
(
Memmert, Germany). The medium was then replaced with DMSO
solution in order to dissolve the water insoluble formazan crystals.
Finally, the absorbance of the solution was quantified by
measuring with a microplate reader (BioTek ELX800, USA) at
wavelength 570 nm and reference wavelength 620 nm. The final
concentration of DMSO was less than 1%.
1
Cell viability (%) of Taxotere and DTX loaded PGG-FOL NPs
against MCF7, 4T1, and A549 cells with 0.2 mM concentration of
DTX beside DTX free PGG-FOL NPs after 24, 48 and 72 h incubation
were also examined.
2.7. Cell culture
MTT assay results were obtained from six individual experi-
ments which all were expressed as the mean ꢀ SD. Cell viability
was measured according to the following equation:
MCF7 as a human breast cancer cell line with folate over
expression, 4T1 as a mouse metastatic breast cancer cell line with
lower folate over expression, and A549 cells which lack folate over
expression were used. RPMI 1640 supplemented with 10% FBS and
ꢀ
ꢁ
ODtest well
CV ¼
ꢃ 100
ODReference well
5
% penicillin–streptomycin was utilized as the cell culture
medium. Cells were cultivated in humidified environment at
Also the drug concentration that causes cell survival inhibition by
0% (IC50) was analyzed by Graph Pad Prism version 5.01. Each IC50
ꢂ
3
7 C with 5% CO
2
. Before experiment, the cells were pre-cultured
5
until desirable confluence was reached.
value was determined based on six cell viability measurements per
concentration.
2.8. In vitro cellular uptake studies
2
.10. Laser scanning confocal microscopy and apoptosis detection
For quantitatively investigating the cellular uptake of the
developed nanoparticles, MCF7 and A549 cells were cultivated in
6 well plates. Each well was surrounded by blank wells to avoid any
interferenceduetothefluorescenceemissionofadjacentwells.After
8 handby thetimethatthecultivatedcellsreached70% confluency,
the medium was replaced with fresh medium containing coumarin-
loadedNPswithacoumarin-6concentrationof1.25 g/mlandwas
incubated for 0.5,1, 2 and4 h. Coumarin-6loaded NPswere prepared
following the previously described method for preparing PGG-FOL
NPs loaded with DTX. Furthermore, untreated cells were used as
control groups. At mentioned periods, the NPs containing medium
Apoptosis detected by performing Annexin V-FITC/PI assay
9
based on the manufacturer’s protocol. After being treated with
targeted NPs containing DTX, MCF7 cells were harvested,
centrifuged, and washed twice with PBS. The cells were then
4
resuspended in 500
m
l of binding buffer, which contained 5
ml of
6
m
Annexin V-FITC and 10
m
l of PI (Annexin V-FITC Apoptosis Kit,
Biovision, Germany). The cells were subsequently incubated in a
ꢂ
dark condition for 15 min at 24 C. Finally, the stained cells were
washed and prepared and observed under a laser scanning
confocal microscopy (Nikon ECLIPSE Ti, Japan).
was removed and each well was washed with 100
remove the NPs which were not internalized. 100 l culture media
was then added to each well followed by 50 l Triton X-100 solution
ml cold PBS to
m
m
2.11. In vivo antitumor activity
in 0.2 N NaOH in order to lyse the cells. Subsequently, the
fluorescence intensity of each well was recorded by microplate
reader (BioTek, Synergy4, VT, USA) at an excitation wavelength of
The PGG-FOL NPs antitumor efficacy was assessed in tumor-
bearing mice. Mice were kept under specific pathogen-free (SPF)
conditions and had free access to food and water. All animal
470 nm and an emission wavelength of 520 nm. The percentage of

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
127
experiments were conducted in full compliance with guidelines
2.12. In vivo biodistribution
approved by the Animal Care Committee of Tehran University of
Medical Sciences.
Biodistribution study was performed in 3 groups of tumor-bearing
mice which were treated via tail vein injection by one of the following
formulations: (a) PGG-FOL NPs loaded with DTX (10 mg DTX/kg),
The shoulder of 6–8 weeks female balb/c mice (18–22 g) were
5
subcutaneously inoculated with a 0.1 ml suspension of 1 ꢃ10 4T1
1
murine breast cancerous cells in PBS. The tumor volume was
closely monitored by digital vernier calipers and calculated as
(b) Taxotere (10 mg/kg), (c) PBS as a control group. All animals were
fasted over night and had free access to water. Mice (n = 6 per
scheduled time) were sacrificed at 2, 4,12, and 24 h after dosing and
their tumor tissues beside other interest organs (spleen, kidneys,
heart, liver, lungs) were dissected for analyses. Tissues and tumors
2
a ꢃ b /2, where a was the longest and b was the shortest tumor
3
diameter. After the tumors became palpable (100–200 mm ), the
mice were randomly divided into 3 groups (n = 6), and weekly
1
ꢂ
treated with injection of Taxotere (10 mg/kg), or PGG-FOL NPs
were harvested and rinsed with buffer and stored at ꢁ80 C until
loaded with DTX (10 mg DTX/kg), and PBS as control via tail vein
injection. Animals body weight, and tumor volume along with the
overall morbidity was recorded daily for each of the tumor-bearing
mice.
Moreover, an additional group of 6 mice were treated weekly
with IV injection of different concentrations of optimized NPs (80,
analysis. The mentioned organs were weighed after washing with
NaCl 0.9%. The sectioned tissues or tumors were then homogenized
(Ultra-Turrax, IKA T-18, Germany) and centrifuged for 10 min while
methanol (1:1) was added for protein precipitation. At a prede-
termined point blood was collected from terminal cardiac puncture
with heparinized tube and the plasma separated by centrifugation
(1500 ꢃ g for 5 min). The DTX amount in the supernatants were
analyzed by HPLC as described above. The obtained data were
normalized with the control group and the mean values were
reported after the experiment was repeated three times.
120, 160, 200, 240, and 260 mg DTX/kg) in order to determine the
Maximum Tolerated Dose (MTD), which is defined as the
maximum dose without causing drug-related lethality and body
weight loss more than 20%.
Fig. 1. (a) Schematic view of PGG synthesis. (b) FTIR spectra of synthesized PGG polymer.

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F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
2.13. In vivo and ex vivo imaging assay
Secondly, after the purification and by the cleavage of bocilated
bond (Fig. 2b), amine group was attached covalently to PGG
carboxyl group as shown schematically in Fig. 3a. Therefore, the
PGG-FOL conjugates structure was confirmed by H NMR as
represented in Fig. 3b (Van et al., 2010).
The PGG-FOL conjugation percentage demonstrated from H
NMR area under curve statistics was 18%.
In order to investigate the in vivo tumor targeting of optimized
NPs, the precipitation method was employed to load the Cy7.5 dye
exactly the same way as that of DTX loaded NPs, but Cy7.5 was used
instead of DTX with similar loading content. In vivo imaging was
performed by FX PRO (Kodak, USA). The excitation and emission
wavelength were set 778 nm and 805 nm, respectively. Kodak
Molecular Imaging Software5.Xwas employed for image analyzing.
The PGG-FOL loaded with Cy7.5 were injected through tail vein of
tumor bearing mice at a dose of 5 mg/kg to examine both tumor
targeting efficiency and biodistribution of designed NPs. After 24 h
of dye loaded NPs administration, 5% chloral hydrate was used to
anesthetize the mice before taking in vivo whole body imaging.
Moreover, forexvivoimagingthe miceweresacrificed 6 hpostCy7.5
targeted NPs injection in order to excise both tumors and other
interest organs (kidneys, spleen, heart, and liver) from the mice.
3.2. Design of experiments and optimized formulation
A response surface methodology design, Box–Behnken design,
was employed in the present study. The obtained results from the 17
runs recommended by the Box–Behnken design, is shown inTable 2.
Based on the investigated results the range for particle size was
90–295 (nm), and for the entrapment efficacy was 7.1–69.1%, and
for drug loading was 1.3–6.3%, respectively. Briefly, it was
demonstrated that following modified quadratic second-order
polynomial equations were fit for modeling the particle size,
entrapment efficacy, and loading over the quantitative indepen-
dent variable in this project:
3
. Results
3
.1. Synthesis of PGG-folate conjugates
2
Size ¼ 132:20 ꢁ 12:25 A þ 52:00 B ꢁ 15:50 C þ 32:90 A
PGA carboxyl groups were activated by adding NHS, and DCC
2
2
þ78:40 B þ 4:40 C þ 16:00AB þ 2:00AC ꢁ 5:50BC
using a procedure that was described above. The activated carboxyl
group became ready for covalent attachment with amine group in
L-
Loading ¼ 0:058 ꢁ 6:625E ꢁ 003A þ 0:012 B ꢁ 2:000E ꢁ 003C
glutamic acid di-tert-butyl ester hydrochloride monomers (Fig. 1a).
Finally the synthesized PGG polymer structure was evaluated by
FTIR. Formation of the amide bond in PGG was confirmed by the
presence of the peak at 1659.79 in the FTIR spectrum (Fig. 1b).
As shown in schematic structure represented in Fig. 2a, the
carboxyl group of folate was activated in presence of NHS and DCC.
PGG-FOL conjugate was synthesized in two steps:
Firstly, the carboxylic group of folate, which was activated in
presence of NHS and DCC, was covalently attached to BOC-ethylene
diamine. The schematic structure of folate ethylenediamine is
represented in Fig. 2a.
2
2
2
ꢁ0:17A ꢁ 0:014B ꢁ 0:016C þ 1:000E ꢁ 003AB
þ2:750E ꢁ 003AC ꢁ 6:250E ꢁ 003BC
2
Entrapment Efficiency ¼ 0:64 ꢁ 0:088A þ 0:27B ꢁ 0:016C ꢁ 0:17A
2
2
ꢁ0:11B ꢁ 0:16C þ 2:500E ꢁ 004AB
0:011AC ꢁ 0:080BC
ꢁ
As represented in Fig. 4, the polymer concentration and the stirring
rate influenced on the final mean particle size, entrapment
Fig. 2. (a) Schematic mechanism of folate ethylene di amine synthesis. (b) H NMR spectra of synthesized folate ethylenediamine.

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
129
Fig. 3. PGG-FOL conjugates. (a) Schematic structure (b) H NMR spectra.
Table
2
Experimental run and NP's characterization suggested by Box–Behnken design in order to achieve optimum formulation.
Run
Independent factors
Stirrer rate Polymer (mg)
Dependent factors (responses)
Volume
(ml)
Size (nm)
Loading (L%) (mean ꢀ SD)
Entrapment efficiency (EE%) (mean ꢀ SD)
(
rpm)
(mean ꢀ SD)
1
2
3
4
5
6
7
8
9
750
5
15
10
5
20
15
20
15
20
10
15
15
10
15
20
10
15
15
15
10
15
145 ꢀ 7.21
289 ꢀ 12.87
162 ꢀ 7.32
164 ꢀ 10.48
249 ꢀ 21.64
192 ꢀ 11.58
125 ꢀ 9.65
132 ꢀ 7.24
173 ꢀ 10.28
291 ꢀ 24.49
151 ꢀ 13.45
296 ꢀ 27.47
139 ꢀ 14.29
131 ꢀ 9.52
134 ꢀ 12.38
170 ꢀ 15.93
230 ꢀ 20.41
2.0ꢀ 0.14
3.7 ꢀ 0.29
3.1 ꢀ 0.16
1.4ꢀ0.12
12.5 ꢀ 1.57
60.4 ꢀ 7.21
50.2 ꢀ 6.23
8.6 ꢀ 1.31
1000
500
1000
750
500
750
750
1000
500
1000
750
750
750
750
750
500
15
10
10
10
10
15
10
15
10
10
10
5
3.3 ꢀ 0.26
4.1 ꢀ 0.38
5.1 ꢀ 0.42
5.9 ꢀ 0.53
1.4 ꢀ 0.17
4.0 ꢀ 0.38
1.5 ꢀ 0.26
4.9 ꢀ 0.42
6.3 ꢀ 0.51
6.0 ꢀ 0.49
5.8 ꢀ 0.53
1.1 ꢀ 0.18
2.1 ꢀ 0.26
53.1 ꢀ 4.76
44.6 ꢀ 5.28
56.3 ꢀ 6.12
65.1 ꢀ 5.27
15.8 ꢀ 2.16
64.5 ꢀ 5.82
16.9 ꢀ 2.43
78.6 ꢀ 6.37
69.6 ꢀ 5.93
66.2 ꢀ 6.49
63.5 ꢀ 7.21
6.2 ꢀ 5.84
12.8 ꢀ 1.58
1
0
1
1
1
2
1
3
1
4
1
5
1
6
17
5

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F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
Fig. 4. 3-D response surface plots for size, loading, and entrapment of nanoparticles obtained from optimum formulation.
efficiency, and drug loading. Finally, the obtained optimized
independent variables for stirring rate was 718.29 rpm, for polymer
concentration was 10.58 mg, and for non-solvent volume was
(%) and ꢁ25.8 ꢀ 2.21 (mV). All data for particle size, zeta potential,
polydispersity index and encapsulation efficiency were repre-
sented in mean ꢀ SD, n = 6.
14.99 ml; the optimized predicted responses drawn from obtained
In order to image the surface morphology of NPs, field emission
scanning electron microscope (FESEM) was used. The FESEM
images proved that NPs displayed not only uniform spherical shape
but also narrow size distribution. Furthermore, the NPs size results
which obtained from FESEM and DLS were in agreement (Fig. 5).
data was 69.28% for encapsulation efficiency,145.08 nm for particle
size, and 6.03% for drug loading. The characteristics of fitted model
is demonstrated in Table 3.
The model predictability and its precision has been validated as
mentioned (n = 5). The mean entrapment efficiency substantiates
92.51% agreement and the mean drug loading verify 87.11%
3.4. In vitro drug release from nanoparticles
agreement with the predicted values, respectively.
Drug release experiments were carried out in the buffer
3.3. Characterization of the nanoparticles
medium with neutral and acidic pH to simulate NPs release profile
in cancerous cell medium via an HPLC method. A calibration curve
2
DLS was employed for the measurement of the particle size and
was developed in a range of 10–100
m
g/ml (R = 0.99) and accuracy
size distribution of nanoparticles. The obtained particle size and
the size distribution of the optimized DTX loaded PGG-FOL
nanoparticles were 131.96 ꢀ 5.34 nm and 0.089 ꢀ 0.019, respec-
tively. It should be noted that all of the NPs in this experiment
displayed unimodal size distribution as shown in Fig. 5, respec-
tively. Drug encapsulation efficiency is of the greatest importance
in the clinical application of NPs. DTX encapsulation efficiency of
optimized nanoparticles and their zeta potential were 67.83 ꢀ 3.29
and precision data were determined and the validated HPLC
method was utilized for determining the DTX concentration and to
evaluate drug release from the developed NPs. The DTX release
profile from drug loaded PGG-FOL optimized nanoparticles in pH
5.2 and 7.4 are shown in Fig. 6. Based on Noyes–Whitney equations,
by decreasing in particle size both solubility and dissolution rate
will increase (Hintz and Johnson, 1989). The obtained release data
over a 120 h period fitted with the Higuchi model (Sqaure
Table
3
Characteristics of fitted model to the responses.
Dependent variable
Best fitted model
R2
Adj. R2
Pred. R2
Adeq-precision
Lack of fit
Particle size (nm)
Entrapment efficiency (EE%)
Drug loading (L%)
Quadritic
Quadritic
Quadritic
0.9902
0.9575
0.9463
0.9776
0.9028
0.9272
0.8683
0.8339
0.8762
24.026
21.358
19.772
Insignificant (p value > 0.05)
Insignificant (p value > 0.01)
Insignificant (p value > 0.05)

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
131
Fig. 5. DTX loaded PGG-FOL nanoparticles characterization. Size and the polydispersity of the nanoparticles measured by (a) dynamic light scattering (DLS), and (b) field
emission scanning electron microscope (FESEM).
correlation coefficient or RSQ = 0.955). Obtained results show that
2
5–35% of DTX is released from nanoparticles in the first 12 h. In
addition a 65% cumulative release percentage was achieved after
20 h in acidic medium and was higher in comparison with neutral
medium.
1
3
.5. In vitro cellular uptake studies
1
The optimized NPs and Taxotere uptake in MCF7 and A549
cells after 4 h incubation were 67.2 ꢀ 6.1, 30.9 ꢀ 5.4, 48.9 ꢀ 4.6,
4
3.5 ꢀ 3.9, respectively. According to statistical evaluation, the
PGG-FOL NPs loaded with DTX cellular uptake (%) in MCF7 cells
was significantly higher when compared with A549 cells; also NPs
had significantly more entrance in both examined cell lines (p
1
value < 0.05) as shown in Fig. 7a. Moreover, Taxotere uptake
Fig. 6. The in vitro DTX release from DTX-loaded PGG-FOL optimized nanoparticles
in two pH one 7.4 the other 5.2. The cumulative amount of released DTX was
investigated by HPLC as described above.
shown no difference within different cell lines in the same period
of incubation time (p value > 0.05). The higher the incubation time

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F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
the higher the cellular internalization. This internalization was also
visualized by CLSM after 4 h incubation of MCF7 and A549 cells
with coumarin-6 loaded NPs as shown in Fig. 7b and c, respectively.
3
.6. Cytotoxicity assays
1
Taxotere and DTX loaded PGG-FOL optimized NPs formulation
cytotoxicity assay was performed against each cell line with the
same concentration 0.2, 0.4, 0.6, and 0.8 M as described below:
m
The in vitro cytotoxic activity of the optimized NPs was
determined by MTT assay in MCF7 cell line. The half maximal
1
inhibitory concentration (IC50
)
of Taxotere
and optimized
formulation of DTX loaded PGG-FOL NPs against MCF7 cell line
(
n = 6) after 24, 48 and 72 h incubation is shown in Table 4. The IC50
1
(m
M) for Taxotere in 24, 48 and 72 h was measured to be
.1468 ꢀ 0.016, 0.1206 ꢀ 0.024, 0.1003 ꢀ 0.033; and 0.2196 ꢀ 0.022,
.0852 ꢀ 0.013, 0.0652 ꢀ 0.027 for the DTX loaded PGG-FOL NPs,
0
0
respectively.
MTT assay was used to describe the suppressing effect of
1
Taxotere and the designed optimized NPs against murine breast
T1 cancerous cells which have a moderate folate over expression.
4
1
As depicted in Table 4, the IC50 results for Taxotere in 24, 48, and
7
0
0
2 h
.1056 ꢀ 0.014 (
.2734 ꢀ 0.027 (
The concentration (
were
0.1791 ꢀ0.014 (
M), and for NPs were found to be
M), 0.1287 ꢀ 0.016 ( M), 0.0855 ꢀ 0.022 ( M).
M) used for 50% inhibitory effect on A549
in 24, 48, 72 h was 0.1414 ꢀ 0.054,
m
M),
0.1473 ꢀ 0.027 (
mM),
m
m
m
m
m
1
cell for Taxotere
0
.1208 ꢀ 0.028, 0.0982 ꢀ 0.016, and for optimized folate targeted
NPs explained as 0.2246 ꢀ 0.091, 0.1315 ꢀ 0.041, 0.1007 ꢀ 0.029 as
shown in Table 4.
The compared figures for three cultured cell lines and the
achieved IC50 values in the predesigned incubation time is
represented in Fig. 8a–c.
Based on the results obtained from MTT assay, the drug free NPs
cell viability (%) were more than 90% which implies that the PGG-
FOL NPs have no cytotoxic effect on the mentioned cell lines
(Fig. 9).
3
.7. Apoptosis and laser scanning confocal microscopy
After treatment with targeted NPs containing DTX, MCF7 cells
were analyzed using a laser scanning confocal microscopy after
4 h and based on Annexin V-FITC apoptosis kit manufacturer
2
protocol. As shown in Fig. 10a Annexin V-FITC staining results in a
bright green fluorescence which determines the early apoptosis
stage in the treated cells, while the red fluorescence color observed
due to the late stage of apoptosis in the cell’s nuclei were stained by
propidium iodide (Fig. 10b). The dual-channel fluorescence view
and the corresponding view under the light microscopy are shown
in the Fig. 10c and d, respectively.
3
.8. Antitumor efficacy and survival study
The MTD of DTX loaded PGG-FOL conjugated NPs were
evaluated in the in vivo experiments. Based on the obtained
results, a single dose of 240 mg of DTX/kg of designed NPs was
administrated intravenously and was well tolerated in tumor
bearing mice with no significant side effects and remarkable
change in vital signs, whereas a single dose of 80 mg/kg of
1
Taxotere induced acute hypersensitivity reaction and various side
effects such as apathy, catatonia, respiratory distress, and severe
prostration. Additionally, no obvious toxicity was observed by the
DTX free PGG-FOL NPs (data not shown).
To compare the in vivo antitumor efficacy of conventional
1
formulation of Taxotere and novel targeted NPs, breast tumor
was established on the back of the mice by subcutaneous

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
133
Fig. 7. a) Cellular uptake of coumarin-6 loaded NPs after 0.5,1, 2, 4 h incubation by MCF7 and A549 cells. Confocal laser scanning microscopy (CLSM) of MCF7 (b) and A549 (c)
cells after 4 h incubation with coumarin-6 loaded NPs. * = significantly difference (p value < 0.05).
1
Fig. 8. Cytotoxicity of Taxotere and optimized formulation of DTX loaded PGG-FOL NPs with MTTassay (a) MCF7 human breast cancer cells with high folate over expression,
(
b) 4T1 murine breast cancer cells with moderate folate expression, (c) A549 as a cell line model with lack of folate over expression; obtained in 24, 48, and 72 h incubation
1
Fig. 9. In vitro viability of MCF7, 4T1 and A549 cell lines treated with drug free NPs, Taxotere and PGG-FOL NPs at 0.2 mM DTX equivalent concentration, after24, 48, and 72 h
incubation time.

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F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
Fig. 10. NP treated MCF7 cell apoptosis stages proved by laser scanning confocal microscope images. a) Membrane of apoptotic cells stained by Annexin v in green b) nuclei of
apoptotic cells stained by propidium iodide (PI) c) dual-channel fluorescence view d) corresponding view under the light microscopy.
1
Taxotere after 6 h; however, NPs showed 4.9 times higher
inoculation of 4T1 murine breast cancerous cell line. Mice were
randomly divided and treated by 4 weekly IV tail vein injection of
either Taxotere , DTX loaded PGG-FOL NPs, or PBS as control.
1
accumulation in tumor compared with Taxotere at 6 h after
1
injection (p < 0.05). DTX level detected in liver and spleen 1 h after
The animals were euthanized during the study period, either
designed NPs administration were 2.1 and 1.8 times higher than
1
due to the significant body loss (more than 20%) or due to the large
that of Taxotere , respectively. Moreover, NPs distributed to heart
3
1
tumor volume (more than 1500 mm ). The survival probability for
in much lower amount in comparison with Taxotere which
1
targeted NPs, and Taxotere group were 83.3 and 66.7(%),
mainly accumulated in heart 1 h after injection.
respectively. By day 13 the median survival time was reached
for the mice in the control group while it was not reached for those
receiving DTX either in Taxotere or designed NPs dosage form
3.10. In vivo and ex vivo imaging
1
until the end of the experiment (Fig. 11a).
The targeting efficacy of folate conjugated PGG NPs was
demonstrated by noninvasive in vivo whole body imaging and
followed by ex vivo imaging of heart, lung, kidneys, liver and tumor
tissues. The whole body image of tumor-bearing mice treated with
PGG-FOL loaded with fluorescent dye Cy7.5, shown in Fig. 13,
indicated the targeting efficiency of designed NPs with high tumor
localization after 12 h of injection. The ex vivo optical imaging also
confirmed the high accumulation of targeted NPs in tumor tissue
which is in consistency with biodistribution results (Fig. 13).
The results indicated that the tumor volume of mice treated
with designed targeted NPs showed stronger regression than those
1
treated with Taxotere in equivalent DTX concentration. Also, the
tumor size increased in the control group during the study
(
Fig. 11b). Notably, at the end of the experiment tumor were
dissected and depicted in Fig. 11d.
Moreover, body weight was also monitored during the in vivo
studies. The statistical analysis revealed that there was significant
difference in weight loss between the Taxotere and targeted NPs
group (p < 0.05) (Fig. 11c). Although Taxotere delayed the tumor
1
1
4. Discussion
growth, a significant loss in body weight was also observed in the
treated mice. It is worth noting that, on day 28, 4 out of 6 mice in
Taxotere group had body weight loss of more than 10% compared
to only 1 out of 6 in targeted NPs group.
Design of experiment (DoE) identifies the key factors and the
variable’s impact on the introduced responses. In other word,
design of experiment could be explained as a systematically
scientific study conductor that changes experimental variables
together in favor of their effect of a given response determination
with a minimum sample size. Four components that accompany
DoE are: designing the experiments, collecting the data, statistical
analyzing of the data and conclusion obtained from the experiment
results (Box and Behnken,1960; Ferreira et al., 2007). Based on the
DoE results, the higher polymer concentration the larger the
particle size will be. This seems to be due to the interfering effect of
the polymer solution viscosity on the solvent diffusion toward the
non-solvent. Increasing in the diffusion rate will alleviate the
alteration of polymer to nanoparticle structure. Moreover, by
strengthening the effective surface area, DTX binds to particles
more efficiently; hence, drug loading and entrapment efficiency
1
3.9. Biodistribution studies
For in vivo biodistribution studies, the breast cancer model balb/
c mice were sacrificed after single dose i.v. administration of
formulations at the prescheduled time. Both the selected organs
and tumors were analyzed for DTX concentration as described
above. The compared distribution profile is represented in Fig. 12.
As plotted in Fig. 12b, the DTX concentration could be measured in
plasma at 24 h which indicated the prolonged blood circulation
1
time for NPs compared to that of Taxotere . As a result, NPs have
adequate time to be presented more in the site of action. DTX
concentration increased in tumor tissue for both NPs and

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
135
1
Fig. 11. In vivo antitumor efficacy of PGG-FOL NPs loaded with DTX compared to that of Taxotere and control groups determined in Female balb/c mice as a breast cancer
1
model. Mice were treated weekly with Taxotere (10 mg/kg), DTX loaded PGG-FOL NPs (10 mg DTX/kg), blank NPs, and PBS. a) Kaplan–Meier survival curve, b) Mean tumor
3
volume (mm ), c) mean body weight of mice (g), and d) Photographs of tumor tissues after 28 days post treatment with mentioned dosage. Data presented as mean ꢀ SD
(n = 6).
get augmented. The NP size is reduced with increasing the stirring
rate. Despite the fact that the reduction in size was occurred by
higher stirring rate and higher transferred energy, there was a
great deal of bubble generation and solution splashing. As a result,
increased NPs hydrolyze rate and significant elevation in DTX
cumulative release percentage (p value < 0.05).
NPs internalization in cancerous cell has a direct relation with
their therapeutic effect. As a result, cumarin-6 was used as an
efficient compound for cellular uptake investigation not only due
to its high fluorescent intensity but also because of its small
leakage rate from NPs formulation. Based on the obtained results,
the cellular uptake was elevated as time went on; this could be
justified by an increasing amount of NPs cellular entrance.
Moreover, according to statistical evaluation the NPs cellular
uptake (%) in MCF7 cells was significantly higher when compared
with A549 cells (p value < 0.05) which could be explained by the
difference in the amount of folate receptor expression and the
targeting ability of the designed NPs formulation (Fig. 7a). Notably,
the receptor mediated endocytosis can be mentioned as one of the
major mechanisms involved in NPs internalization inside the cells.
This internalization was also visualized by CLSM after 4 h
incubation of MCF7 and A549 cells with coumarin-6 loaded NPs
as shown in the Fig. 7b and c, respectively. As it is demonstrated in
these figures, the fluorescence intensity of coumarin-6 is higher in
MCF7 cytoplasm when compared with A549 cells. This higher
amount of internalization indicates that targeted NPs can
effectively enter and distribute in the cellular cytoplasm with
specificity and selectivity. As a result, it can be proposed that the
750 rpm was considered to be the best stirring speed in this
process. By increasing the DMSO volume, particle size was also
decreased; but since the lipophil DTX has a very good solubility in
DMSO there was a decrease in the entrapment efficiency with an
increase in the DMSO fraction.
Additionally, surface charge plays a major role in nanoparticles
stability in the medium. The more repulsion represents the more
stable nanoparticle system. By holding this view, results showed a
negative charge on the NPs surface which is due to carboxyl and
folate groups (Table 4). Most importantly, appropriate obtained
results in size, zeta potential, and encapsulation efficiency, which
are shown in Table 4 indicate that the optimized DTX loaded PGG-
FOL NPs will have a great chance especially in targeted form of drug
delivery system.
The initial burst release obtained from the in vitro drug release
profile is due to the presence of DTX on the surface of
nanoparticles; however, it could be helpful in suppressing the
cancerous cell growth in the first 12 h of administration (Chen
et al., 2008; Kumari et al., 2010). Hence, the acidic pH leading to an

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F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
1
Fig. 12. Biodistribution of DTX (a) Taxotere , (b) PGG-FOL NPs loaded with DTX at 1, 6,12 and 24 h following i.v injection in breast cancer model of balb/c mice. Obtained data
were calculated as percentage of initial dose per g tissue and represented as mean ꢀ SD (n = 6).
currently developed these novel targeted NPs can achieve higher
cellular internalization and lower side effects.
assessed by MTT assay. MCF7 as a high folate over-expression cell
line, 4T1 with a moderate, and A549 with lack of folate receptor
expression were employed for the cytotoxicity assays (Liu et al.,
2011; Ernsting et al., 2012; Lu et al., 2009).
Development of a targeted drug delivery system is one of the
major goals of this study. The overexpression of folate receptors on
tumor tissues makes this receptor an attractive choice for targeting
practice. As a result, folate over expressed cell lines are usually
chosen to evaluate the cytotoxic effect of targeted drug delivery
systems. It goes without saying that the nanoparticle formulation
success in winning against cancerous cell is demonstrated by their
cytotoxicity. Three cell lines were chosen in this experiment based
on their folate expression capacity, and the NPs cytotoxicity was
The IC50 results against MCF7 cell line demonstrated that for
1
both Taxotere and NPs the elevation in their concentration and
increase in the incubation time will decrease the IC50 value. In the
1
first 24 h the optimized NPs IC50 was higher than Taxotere . This
can be described by NPs delayed release from the PGG-FOL
polymer. Instead, the IC50 results for both examined groups in 48
Fig. 13. (A) In vivo imaging after 24 h following the i.v. administration of Cy7.5 loaded PGG-FOL NPs and (B) ex vivo imaging after 6 h following the i.v. administration of Cy7.5
loaded PGG-FOL NPs in breast tumor bearing balb/c mice and excised tumor and interest organs (kidneys, spleen, heart and liver), respectively.

F. Tavassolian et al. / International Journal of Pharmaceutics 467 (2014) 123–138
137
and 72 h were lower for the optimized NPs formulation which
proof for efficient and specific tumor targeting ability of designed
verifies the potency of the designed NPs formulation.
NPs.
1
Based on the 4T1 MTT results, the Taxotere IC50 value was
lower after 24 h incubation but higher in 48 and 72 h in comparison
with DTX loaded PGG-FOL NPs. As mentioned above, this could be
explained by encapsulated NPs delayed release from the polymeric
system. Not surprisingly, the IC50 value for 4T1 stepped up for
optimized NPs which is due to the lower folate over expression of
5. Conclusion
A new folate targeted polymeric NP, loaded with DTX as a
potent anticancer drug and PGG as
a biodegradable and
biocompatible polymeric carrier, was successfully designed and
characterized. Sustained and continuous in vitro release profile and
higher cytotoxicity in folate over expressed cell line in comparison
4T1 cells in comparison with MCF7 culture cell line. It is clear that
the lower expression in folate, the lower drug uptake and the
higher IC50 value or equivalently lower mortality of the cells.
Moreover, the result demonstrated that not only due to the
sustained release of drug from the NPs targeted polymer core the
IC50 value for designed NPs was higher just in the first 24 h, but also
according to lack of folate over expression in A549 cultured cell line
there is no significant different among the data in 48 and 72 h (p
value > 0.05).
1
with Taxotere were proved by the obtained results. Here we focus
on how PGG-FOL NPs loaded with DTX differentiate from the
conventional DTX formulation of Taxotere . According to the
favorable results achieved in this study, the novel folate conjugated
DTX delivery NPs possess targeted therapy capability with lower
toxicity and higher anticancer therapeutic efficacy which signifi-
1
1
cantly inhibited tumor growth compared to that of Taxotere
To sum up, three cell lines have been chosen according to their
difference in folate expression and based on the logic of
investigating the efficiency and success of targeted designed
NPs formulation in agreement with the cancerous cell proliferation
suppressive effect. Inevitably, the cytotoxicity act is in accordance
with concentration-time dependent manner. Moreover, the higher
IC50 results for the first 24 h for DTX loaded PGG-FOL NPs indicate
its delayed release profile from the NP polymer core. More
appealing is the selectivity and enhancement of cancerous cell
uptake of the folate targeted NP delivery system in cell line with
greater folate over expression ability. In other word, IC50 as a
quantitative judgment for the in vitro efficiency of the NPs
formulation decreased 29.37% by treating MCF7 cancerous cells
after 48 h of incubation. Additionally, calculated from the
therapy that will hopefully be one step closer to the ideal goal in
cancer treatment regimens.
Declaration of interest
The authors report no declarations of interest.
Acknowledgments
This work is a part of first author’s PhD thesis and was
financially supported by a grant from Tehran University of Medical
Sciences. The authors also appreciate Ms. Salamian for her
technical assistance in cytotoxicity assays and Mr. Gholami for
his technical assistance in animal studies.
mentioned cell inhibitory effect data obtained from 48 h treatment
1
of MCF7 and 4T1 in the presence of Taxotere was 0.1206 mM,
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