DSpace at VNU: Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Article
Journal of
Biomedical
Nanotechnology

Copyright © 2013 American Scientific Publishers
All rights reserved
Printed in the United States of America

Vol. 9, 1416–1431, 2013
www.aspbs.com/jbn

Novel Multifunctional Biocompatible Gelatin-Oleic
Acid Conjugate: Self-Assembled Nanoparticles
for Drug Delivery
Phuong Ha-Lien Tran1 ∗ , Thao Truong-Dinh Tran1 , Toi Van Vo1 ,
Chau Le-Ngoc Vo2 , and Beom-Jin Lee2 ∗
1
2

International University, Vietnam National University–Ho Chi Minh City, 70000, Vietnam
College of Pharmacy, Ajou University, Suwon 443-749, Korea

In this work, a novel, biocompatible conjugates of gelatin and oleic acid (GOC) were synthesized by a novel aqueous
solvent-based method that overcame challenges of completely contrary solubility between gelatin and oleic acid (OA).
The GO nanoparticles (GONs) and Paclitaxel encapsulated nanoparticles (PTX-GON) were prepared by self-assembly
in water. These nanoparticles (NPs) were then conjugated with folic acid (FA) for targeting cervical cancer cells (Hela
cells) and were characterized for their various physicochemical and pharmaceutical properties. Fourier transform infrared
spectroscopy (FT-IR) and 1 H NMR studies indicated the successful synthesis of GOC which showed low critical aggreDelivered by Publishing Technology to: Rice University, Fondren Library
gation concentration in water (0.015
mg/ml). All NPs On:

wereSat,
stable
human
serum and their mean diameters were
IP: 63.142.225.118
10inOct
2015blood
07:05:21
below 300 nm suitable for passive targeting.
Powder
X-ray
diffraction
(PXRD)
diffractograms
showed the reduction in
Copyright: American Scientific Publishers
drug crystallinity and hence, leading to the solubility enhancement of PTX. The release of PTX from both PTX-GON
and FA conjugated PTX-GON (PTX-FA-GON) was controlled for a long time. The cytotoxicity results demonstrated great
advantages of PTX-FA-GON and PTX-GON over the conventional dosage form of pacliaxel (Taxol® . These results, therefore, indicate that GOC is a promising material to prepare drug encapsulated NP as a controlled delivery system and
PTX-FA-GON is a potential targeted delivery system for cancer therapy.

KEYWORDS: Gelatin-OA Conjugate (GOC), Self-Assembled Nanoparticles, Targeted Drug Delivery, Paclitaxel, Folic Acid (FA),
Protein Conjugation.

INTRODUCTION
Recent advancements in nanotechnology have had wide
applications in the pharmaceutical industry due to a number of advantages of placing nano-objects at the desired
position, enhancing sparingly soluble drugs and controlling the drug release rate. Since nanoparticles (NPs) are the
most widely used nano-objects,1 their generation is well
characterized and has been established by many chemical

scientists.2–5 However, their applications in pharmaceutical
formulations are still limited because of their incompatibility, low payload and a complicated preparation process.
For this reason, biocompatibility and self-assembled nature
are outstanding characteristics for a material to be applied
∗

Authors to whom correspondence should be addressed.
Emails: ,
Received: 27 October 2012
Accepted: 6 January 2013

1416

J. Biomed. Nanotechnol. 2013, Vol. 9, No. 8

in NP production. Thus, the authors have performed this
research for the investigation of a new self-assembled biomaterial which was synthesized by a simple method and
can be applied for encapsulating many types of drug with
the hope that this material will be widely used in the pharmaceutical industry.
An amphiphilic carrier which can form NPs in aqueous
medium by self-assembly is more preferable than others because it has been recognized as a promising nanosystem which can be applied to many biotechnological
and pharmaceutical fields with numerous types of drugs.6
These amphiphiles spontaneously form NPs by undergoing intra- and/or inter molecular associations between
hydrophobic moieties in an aqueous environment. The
hydrophobic segments make the inner core, which is a
host system for various hydrophobic drugs, whereas the
hydrophilic segments are oriented toward outer aqueous
environments to form the corona or outer shells. The shell
1550-7033/2013/9/1416/016

doi:10.1166/jbn.2013.1621


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

hinders the inactivation of the encapsulated drug by prepotential unwanted side effect.15 The nanoparticles in this
7
venting contact with inactivating species in blood. These
research have been expected to offer advantages over conNPs exhibit unique physicochemical characteristics such
ventional formulations including the ability of drug protecas special rheological features, a narrow size distribution, targeting the drug to the site of action, and reducing
tion, considerably lower critical aggregation concentrations
the side effects of chemotherapy. The multi-functional
and thermodynamic stability.8 9 However, new developnanoparticles, combining tumor targeting and tumor therment of amphiphilic carriers using generally recognized as
apy might be an ideal alternative carrier for controlled
safe (GRAS)-listed pharmaceutical excipients are of highly
delivery of anticancer drugs, which could not only reduce
motivated issue because of safety and diverse applications
the harmful side effects of chemotherapeutic agents but
in drug delivery and clinical therapy.
also maintain adequate drug levels in the body.
In this work, the gelatin-OA conjugate (GOC) as a
new biomaterial was originally designed to generate an
MATERIALS AND METHODS
amphiphilic structure comprised of two GRAS materiMaterials
als, gelatin and OA, using an activator, monoethanolamine
Gelatin was purchased from Kanto Chemical Co., Inc.
(MEA). Gelatin was chosen because it is a natural and
(Tokyo, Japan). OA was purchased from Shinyo Pure Chem10

biocompatible protein and a good wall material for
icals Co., Ltd. (Osaka, Japan). MEA was from Yakuri Pure
11
encapsulation. It possesses a preferably hydrophilic propChemicals Co., Ltd. Pyrene, TNBS (2,4,6-trinitrobenzene
erty, which facilitates gelatin solubility in water at body
sulfonic acid), FA, 1-ethyl-3-(3-dimethylamino-propyl)
temperature. Moreover, gelatin has multifunctional groups,
carbodiimide (EDC), N -hydroxysuccinimide (NHS),
including –COOH and –NH2 , which promote the capabil2-(N -morpholino)ethanesulfonic acid (MES), monoclonal
ity of gelatin to bind with other suitable agents. OA is
anti-FA clone VP-52, anti-mouse IgG gold conjugate
a biocompatible fatty acid which can not be dissolved in
(20 nm), Cremophor EL and 3-(4,5-dimethylthiazol-2-yl)water and also an agent that induces the stability of many
2,5-diphenyl tetrazolium bromide) (MTT) were purchased
12 13
NP systems.
The aim of our study was to develop
from Sigma (St. Louis, MO, USA). Hela cells were
an optimal preparation without the use of an organic solobtained from the American Type Culture Collection
vent for a wide application in manufacturing and for good
(ATCC,
USA). Dulbecco’s
Delivered
by Publishing
Technology
to: Rice University,
Fondren modified
Library Eagle’s medium
health and the environment.
Because

gelatin has
amine
IP:
63.142.225.118
On:
Sat,
10
Oct
2015
07:05:21
(DMEM),
fetal
bovine
serum
(FBS), trypsin-EDTA and
groups that cannot bind directly with OA in water, OA
Copyright: American Scientific
Publishers
penicillin-streptomycin mixtures were from Gibco BRL
must be activated for a reaction. Therefore, MEA was cho(Carlsbad, CA, USA). Paclitaxel (PTX) was obtained from
sen to activate OA for the conjugation of hydrophobic OA
Dae Woong Pharmaceutical Co. Ltd., (Seoul, Korea). The
and the hydrophilic parts of gelatin in water as an intersolvents were high performance liquid chromatography
mediate substance. MEA is a chemical intermediate that is
(HPLC) grade. All other chemicals were of analytical
soluble in water and is used in the manufacturing of cosgrade and were used without further purification.
metics and surface-active agents.14 Spray drying is selected
herein to collect GOC because it is a common method in
the pharmaceutical industry for rapidly drying solvents to
collect large quantities of samples, and hence, bringing a

further promising application in scalability. GOC was then
dispersed in water to obtain self-assembled GO nanoparticles (GON). The surface of the GON was further conjugated with folic acid (FA) as a ligand for actively targeting
to cancer cells since receptor-mediated drug targeting to
diseased sites is one of the most promising approaches
in chemotherapy to maximize drug efficacy and minimize
systemic toxicity.
The nanoparticles were applied to load paclitaxel (PTX),
an insoluble anticancer agent for controlling drug release.
Most of the anticancer drugs have limitations in clinical
administration because the drug delivery of these agents
often requires the use of adjuvants or excipients, which
often cause serious side effects. For example, Cremophor
EL (polyethoxylated castor oil) and ethanol are incorporating excipients in the pharmaceutical drug formulation of Taxol® for inducing drug solubility but its clinical
usefulness is often hampered by poor water solubility and
J. Biomed. Nanotechnol. 9, 1416–1431, 2013

Synthesis of NPs
Preparation of GOCs
Gelatin (1 g/100 ml) was dissolved in distilled water at
37 C to obtain an aqueous gelatin solution. OA was simultaneously mixed with MEA in distilled water to obtain
a uniform colloidal dispersion at two different concentrations (g/100 ml) of OA/MEA (GOC-1:0.1/0.04; GOC2:0.3/0.12). The gelatin solution was gradually poured into
this OA/MEA colloidal solution while stirring. The resulting clear solution was obtained after 1 h and delivered to
the nozzle of the spray dryer at a flow rate of 4 ml/min
using a peristaltic pump for spray-drying at 130 C inlet
and 80 C outlet temperatures. The pressure of the spray
air was 3 kg/cm2 , and the flow rate of the dry air was
approximately 3 mb. The diameter of the nozzle was
0.7 mm. The powder was washed three times with water
and ethanol by centrifugation at 10,000 rpm for 15 min
to remove OA and gelatin remaining. The supernatant was

discarded, and the powder was dried at room temperature
under vacuum.
1417


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Preparation of Self-Assembled GONs
The powder of GOCs was dispersed in aqueous media
(water, pH 1.2, pH 6.8 or pH 7.4) under gentle stirring
with a paddle at 50 rpm for different intervals of time
to prepare the self-assembled NPs. The resulting GONs
were collected via centrifugation at 40,000 rpm for 20 min
(Beckman Optima™ TL Ultracentrifuge, USA). The supernatant was discarded. The pellet was washed three times
with water and lyophilized via freeze-drying at −50 C for
2 days (Freeze Dryer, Ilshin Lab. Co., Ltd., Korea).

Tran et al.

Determination of the Degree of Substitution
The number of amino groups of gelatin that reacted with
OA was determined using the TNBS method. Samples
(2 mg) were dissolved in 5 ml of reaction buffer,
0.1 M NaHCO3 , and 2.5 ml of 0.01% TNBS (2,4,6trinitrobenzene sulfonic acid) was added and mixed well.
The samples were incubated at 37 C for 2 h. Finally,
1.25 ml of 1 N HCl was added to each sample and mixed,
and the absorbance of the solutions was measured at
335 nm. The free amino groups were determined for pure
gelatin, GOC-1 and GOC-2. The degree of substitution
(DS) was calculated as follows: DS = AG − AN /AG ×

100, where AG and AN are the absorbance of gelatin and
the NPs, respectively, at 335 nm. DS was defined as a percentage of the number of reacted amino groups relative to
the number of free amino groups in pure gelatin.
To specify how many pairs of GOCs are in one NP,
the amount of particles contained in 1 mg of NPs was
estimated based on the particle size and density of the
polymer. Based on the results of the above TNBS method
with the amount of reacted amine groups withdrawn from
the calibration of cystine, the number of gelatin-OA pairs
presented on one particle was determined.

Preparation of Drug-Loaded NPs
GOC-2 was selected in this experiment to prepare drugloaded NPs. GOC-2 and the drug (PTX) were dispersed
in dichloromethane; the loading amount of drug was 10%.
The solution (300 l) was emulsified in 10 ml of distilled water and sonicated for 20 min to form an oil/water
emulsion. Dichloromethane was evaporated under purge
nitrogen gas for 15 min. When present, large aggregates were removed by centrifugation at 1000 rpm for
5 min at 37 C. NPs were harvested and washed three
times with distilled water by centrifugation at 40,000 rpm
for 20 min at 37 C. The pellets were resuspended in
water, sonicated for 30 s, lyophilized and freeze-dried at
−50 C for 2 days. The obtained drug-loaded NPs were
Characterization of NPs
PTX-GONs.
Size Measurements
and Zeta Potential
Delivered by Publishing Technology to: Particle
Rice University,
Fondren Library
average

size of self-assembled NPs was meaIP: 63.142.225.118 On: Sat, The
10 Oct
2015particle
07:05:21
Preparation of Surface-Functionalized
Copyright: American Scientific
Publishers
sured using
a PAR-III Laser Particle Analyzer System
GONs with FA FA-GONs
(Otsuka Electronics, Japan). All measurements were perGON-2 and PTX-GON were selected in this experiment.
formed in triplicate with a He–Ne laser light source
GONs in MES buffer (1 mg/ml, pH 6.0) were activated by
(5 mW) at a 90 angle.
EDC/NHS for 30 min. A solution of FA (pH 7.5) made
The zeta potential of NPs was measured using an
by sodium phosphate (0.507 mg/ml) was mixed with the
Electrophoretic Light Scattering Spectrophotometer 8000
activated GON for 2 h to obtain FA-GON. The FA-GONs
(Otsuka Electronics, Japan) operated at −28.3 V/cm,
were collected using the same process mentioned above.
−0.1 mA and 28 C.
For PTX-FA-GON, the same process was carried out, but
The sample concentration was maintained at 1 mg/ml in
PTX-GON was used instead.
distilled water.
Characterization of GOCs
H NMR Spectroscopy
The synthesized conjugates were identified using 1 H
nuclear magnetic resonance (1 H NMR). The experiments

were performed on a NMR spectrometer type Bruker
Avance 600 MHz, and perdeutero DMSO-d6 was used as
a solvent.

1

Fourier Transform Infrared Spectroscopy FTIR
The spectra of the samples (gelatin, OA, conjugates, PTXGON, and PTX-FA-GON) were recorded using an IR
spectrophotometer (Excaliber Series UMA-500, Bio-Rad,
USA). KBr pellets were prepared by gently mixing 1 mg
of the sample with 200 mg KBr. Fourier transform infrared
spectra (400–4000 cm−1 were obtained with a resolution
of 2 cm−1 .
1418

Morphology of NPs
The solution of self-assembled NPs (1 mg/ml) was placed
on a copper grid to observe the morphology using transmission electron microscopy (TEM) (LEO 912AB-100,
Carl Zeiss, Korea Basic Science Institute-Chuncheon). The
samples of NPs were stained by 2% sodium phosphotungstate (PTA, pH 7.2) for 40 seconds and dried in a vacuum dryer at room temperature. Thereafter, the grid was
examined using a transmission electron microscope.
For TEM characterization of surface-functionalized NPs
with the FA ligand on the surface, further steps for sample preparation were required. The presence of the ligand
on the surface of NPs was detected through the presence of a gold NP probe. The surface-functionalized NPs
were incubated with a mouse anti-FA monoclonal antibody
followed by incubation with a 10 nm gold-labeled goat
J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

anti-mouse IgG. The NPs were initially incubated with a
10% BSA solution in PBS for 1 h and then with a folatespecific anti-FA antibody for 1 h. Unbound antibody was
removed by washing the particles with PBS. Particles were
incubated with gold-labeled IgG for 1 h, and unbound gold
particles were removed by washing twice with PBS.
High resolution transmission electron microscopy
(HRTEM) (Korea Advanced Institute of Science and Technology, Daejeon) was also applied to observe the morphology of NPs.

In Vitro Drug Release
Drug-loaded NPs (1 mg of PTX) were dispersed in 10 ml
at pH 7.4 in screw-capped tubes and placed in an orbital
shaker maintained at 37 C and shaken at 100 rpm. At predetermined time intervals, 0.5 ml samples were withdrawn
for analysis and the same amount of medium was replaced.
The samples were centrifuged at 40,000 rpm for 20 min
and the supernatant was taken for HPLC analysis to determine the drug release. The HPLC conditions were performed using the same process described in the above
section.

Measurement of Critical Aggregation
Concentration CAC
Biostability Study
The critical aggregation concentration (CAC) of GOC was
The stability of GONs with and without the targeting
determined using a probe fluorescence technique in which
ligand and the NPs loaded with PTX were evaluated by
pyrene was used as a hydrophobic probe.9 A series of vials
mixing in human blood serum. The particle size was conwere prepared as follows. Pyrene was dissolved in acetinuously monitored by DLS for 24 h. Tests in serum were
tone, and the concentration was controlled at 6 × 10−7 M.

conducted at 37 C to mimic physiological conditions.
After evaporation to remove the acetone at 50 C, 5 ml
of different concentrations of the GOC solution (0.00025,
Cancer Cell Killing Effect
0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25,
Hela (human cervical carcinoma) cells were cultured at
0.5 and 1 mg/ml) was added into pyrene. Sonication
37 C in a humidified 5% CO2 incubator. The culwas performed for 2 h to equilibrate the pyrene and the
ture medium was Dulbecco’s modified Eagle’s medium
NPs. The fluorescence spectrum was obtained with a flu(DMEM) supplemented with 10% (v/v) fetal bovine serum
orescence spectrophotometer (Perkin Elmer Asia LS-55B
(FBS), 100 IU/ml penicillin G sodium and 100 g/ml
M-2721, USA). The excitation wavelength was 336 nm,
streptomycin sulfate. A PTX stock solution (6 mg/ml) was
Delivered
by Publishing
Technology
to: Rice University, Fondren Library
and the emission spectra
of pyrene
were in the
range
prepared
dissolving
PTX in ethanol with an equal volIP:excitation
63.142.225.118
On: Sat,
10 Octby2015
07:05:21
of 35–450 nm. The slit opening for

and emisume
of
Cremophor
EL,
Copyright: American Scientific Publishers followed by sonication for 30 min,
sion was set at 10 nm and 5 nm, respectively. For the
which is the process used for the preparation of Taxol® 15
calculation of CAC, the intensity ratio measurement of
Hela cells were seeded in 96-well plates at a density of
the first energy band (374 nm, I1) to the third energy
1 × 104 cells/well in 200 L of culture media and incuband (385 nm, I3) in the emission spectra of pyrene was
bated for 24 h to allow the cells to attach to the dish. The
determined.
growth medium was removed, and the cells were washed
twice with PBS to remove the residual growth medium.
Powder X-Ray Diffraction PXRD
The cells were incubated with 200 L of fresh medium
Powder X-ray diffraction patterns of the samples (Gelatin,
containing drug-loaded NPs (e.g., PTX-GON and PTXOA, conjugates, PTX-GON, PTX- FA-GON) were anaFA-GON), blank NPs or Taxol® at different concentralyzed with a D5005 diffractometer (Bruker, Germany)
tions (concentration of PTX: 2.5, 25, 250, 2500, 12500,
using CuK radiation at a voltage of 40 kV and a curor 25000 ng/mL) for 24 h, 48 h and 72 h at 37 C. At
rent of 50 mA. The powder samples were scanned in steps
the determined time, the cells were washed twice with
of 0.02 from 5 to 60 (diffraction angle 2 with a rate
PBS to eliminate the remaining drug, and 200 L of the
of one second per step using a zero background sample
MTT solution (1 mg/ml in PBS) was added to each well.
holder.
Cells were incubated for a further 4 h at 37 C, and the
medium was carefully removed. Formazan was dissolved

Determination of Drug Loading
in isopropanol and incubated for 30 min at 37 C. The
Content and Encapsulation Efficiency
samples were covered with tinfoil and gently shaken on
After the supernatant was gathered and the washings were
an orbital shaker for 15 min. The absorbance was meacollected from the NP preparations, the drug loading consured on a VERSAmax tunable microplate reader (USA)
tent and encapsulation efficiency of the NPs were deterat a wavelength of 570 nm. Experiments were measured
mined indirectly by HPLC analysis (Waters™ , USA) with
in quadruplicate.
a reverse phase column (150 × 4 6 mm, Luna 5u C18
100 A) and a 20 l injection volume. For PTX analysis,
Cell viability = OD treated/OD control × 100%
the mobile phase consisted of a 55:45 (% v/v) mixture of
(OD treated: cells treated with NPs or Taxol® ; OD control:
acetonitrile and water; the flow rate was 1.0 ml/min, and
untreated cells).
the detection wavelength was 227 nm.
J. Biomed. Nanotechnol. 9, 1416–1431, 2013

1419


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Tran et al.

Regression analysis for dose-response curves was performed using SigmaPlot version 11.0 (Systat Software
Inc., Oint Richmond, CA, USA) to estimate the IC50 (half
maximal inhibitory concentration).

of gelatin, which was evident from an increased intensity of this peak. An N–H bending vibration of gelatin at
1535 cm−1 was also observed for GOC-1 and GOC-2, but
the intensity was increased, which implied an overlap. An
N–H stretching of gelatin at 3303 cm−1 was also observed
for GOC. Bands at 1338 and 1240 cm−1 , which indicated
RESULTS AND DISCUSSION
the in-phase combination of C–N stretching and C O
Synthesis and Identification of GO NPs
bending vibration, also appeared in the GOC-1 and GOC-2
When gelatin, OA and MEA were introduced into water
spectra. The NMR spectra also verified the OA binding
for conjugation, the resultant solution exhibited transparent
with gelatin through the presence of OA proton peaks in
at the ratio of OA to MEA 1:0.4 or lower. However, withthe GOC spectra, which was a prominently displayed peak
out gelatin, OA was not completely soluble regardless of
of the alkene bond –CH CH– of OA at 5.32 ppm. The
the amount of MEA, so the transparent solution indicated
disappearance of the proton peak of the –COOH group of
an interaction between gelatin and OA. Therefore, the
OA at 12 ppm in the GOC spectra indicated the reaction of
formulations of two conjugates (GOC-1 and GOC-2 for
–COOH groups with gelatin amine groups to form amides.
the low and high concentration of OA/MEA, respectively)
The number of amino groups of gelatin reacted with
were prepared with the aim of investigating the changes
OA was determined to reveal how many molecules of
in conjugates or NPs upon increasing amounts of OA.
OA and gelatin bind each other in GOC-1 or GOC-2.
The solution was spray-dried at various inlet temperatures,
Therefore, the formation of NPs composed of gelatin and

such as 80 C, 100 C and 130 C. The optimal temperOA can be observed. The principle of this calculation is
ature was 130 C because the excessive MEA evaporated
based on the free number of amino groups because their
completely above 100 C.16 Moreover, this temperature
absorbance can be measured by UV-spectroscopy using the
ensured the complete volatilization of water to produce dry
TNBS method.18 The reacted number of amino groups of
powder and allow amide bond formation from the dehyGOC can be determined indirectly from the determination
dration of the salts formed by gelatin’s amine groups and
of pure gelatin and the free amino groups remaining in
OA’s carboxylic groups. GOC was purified with ethanol
GOC-1 and GOC-2. The substitution percentage of GOCand water to eliminate free OA and gelatin. The purified
and University,
GOC-2 were
27 52 ±
0 63% and 60 07 ± 1 01%,
Delivered by Publishing Technology to: 1Rice
Fondren
Library
GOC was introduced to water IP:
for63.142.225.118
self-assembly. GONs
On: Sat, respectively.
10 Oct 2015Additionally,
07:05:21 gelatin possesses about 33 reacwere collected by a series of steps Copyright:
as follows:American
repeated Scientific
tive amino
groups per gelatin molecule of 1.000 amino
Publishers

ultracentrifugation, washing with water to eliminate excesacids.19 20 Consequently, the experiment exposed about 9
sive gelatin and finally, freeze-drying. Figure 1 (suppleor 20 amino groups per gelatin molecule of 1.000 amino
mentary data) shows the synthesis of the GOC and the
acids reacting with OA for GOC-1 or GOC-2, respecformation of GON. Furthermore, Table I shows that the
tively. In other words, 1 gelatin molecule of 1.000 amino
solubility of GOC was different from that of pure gelatin
acids was binding with 9 OA molecules for GOC-1 and 20
(the solubilities of GOC-1 and GOC-2 were quite simiOA molecules for GOC-2. To determine the formation of
GOCs into NPs and how many GOC molecules (1 GOC
lar), which indicates a property change of GOC compared
molecule composed of 1 gelatin molecule attached to 9 or
to pure gelatin. As observed after storage for 1 month,
20 OA molecule as mentioned above) could form one NP,
the properties of GOC-1 and GOC-2 were not changed.
1 mg of NPs was estimated to contain how many partiHerein, the solubility checked within 1 month is mentioned
cles. The conjugates under storage after freeze-drying were
as the first consideration of the system’s stability. Other
used, and the determination was based on the particle size
aspects will be mentioned below.
and density of the conjugates.21 The density of GOC-1
GOCs were characterized using FTIR and 1 H NMR
and GOC-2 measured in acetone was 0.37 and 1.62 g/cm3 ,
(Fig. 2). The peak changes of GOC-1 and GOC-2 showed
respectively. Considering that the average particle sizes of
a similar pattern. The frequency of the C O bonds of
GON-1 and GON-2 were 150 nm and 200 nm, respecOA and gelatin in FTIR spectra presented at 1709 and
tively, an average of 1 mg of GOC-1 and GOC-2 was
1638 cm−1 , respectively. GOC-1 and GOC-2 had a new
estimated to contain 1 5 × 1012 and 1 5 × 1011 particles,
peak at 1640 cm−1 and 1642 cm−1 , respectively, which

respectively. Based on the known average weight of GOCwas different from the C O peaks of gelatin and OA
1, GOC-2 and the average weight of one nanoparitcle, the
in both peak shape and position. This result indicated the
GON-1 and GON-2 was estimated to contain about 11,709
formation of an amide between the NH2 group of gelatin
and 77,684 GOCs, respectively.
and the C O of OA. If a molecule is conjugated, the
17
strong C O absorption is shifted to the right. Herein,
Characterization of GONs
the C O peak of OA at 1709 cm−1 was shifted to the
right by approximately 70 cm−1 . It may also be noted that
Morphology, particle size distribution and zeta potential of
the peak of GOC-1 and GOC-2 observed at 1640 cm−1 and
GONs under various conditions were evaluated. The histograms of the particle diameter (Fig. 3) revealed that most
1642 cm−1 , respectively, overlapped with the amide I band
1420

J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

oleic acid (OA)

MEA

O

C
O
N
H

H
C
CH 3

C

O
H
N

O
H
N

H
C

C

H
C

C

CH 2

N

H

O
H
N

H
C

C

H

Heat

C

N
H

CH 2
CH 2

CH 2

C

C

O

H
N

O

O-

NH

MEA

H
C

CH 2

C

OH

O
H
N

C
H

C

N

O
C
O

NH 2

NH 2

gelatin

Delivered by Publishing Technology to: Rice University, Fondren Library
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21
Copyright: American Scientific Publishers

GO conjugates

-NH2 : Gelatin

HOOCHOOC-

-NH2

Figure 1.

: Oleic acid
: Folic acid

Illustration of gelatin-OA conjugates’ synthesis and FA attached NPs.

of the GON-1 and GON-2 dispersed in water for 1 h were
152–182 nm and 196–236 nm in diameter, with an average
value of approximately 170 nm and 220 nm, respectively.
Figure 4(a) shows that the particles dispersed in water for
J. Biomed. Nanotechnol. 9, 1416–1431, 2013

1 h were spherical in shape for both of the NPs formed
from GOC-1 and GOC-2 (GON-1 and GON-2, respectively), with diameters in the range of 150–200 nm. The
fact that NP size in DLS was a little larger than the ones
1421


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery
Table I.

Tran et al.

Solubility property of GOCs in various solvents.
In organic solvent
(methanol, acetonitrile, etc.)

In water

In mixture of water
and organic solvent

Sample

Room temperature

37 C

Room temperature

37 C

Room temperature

Gelatin

Insoluble (2 phases:
gelatin solid/water)
Insoluble (turbid
solution)
Insoluble
(turbid solution)

Soluble

Insoluble (2 phases:
gelatin solid/solvent)
Sparingly soluble/Soluble

Insoluble (2 phases:
gelatin solid/solvent)
Sparingly soluble

Insoluble (2 phases:

gelatin solid/solvent)
Soluble

Sparingly soluble/Soluble

Sparingly soluble

Soluble

GOC-1
GOC-2

Insoluble (turbid
solution)
Insoluble
(turbid solution)

observed in the TEM images is reasonable because the
shows that the morphology of GON was not changed
within 1 h at 37 C in water or after storage of 1 month.
TEM images depicted the size in the dried state of the samThe pH of the medium had no apparent effect on the morple, but the laser light scattering method involved the meaphology of NPs (Fig. 4(c)). However, pH seemed to affect
surement of size in the hydrated state. The increased
the size of NPs because lower pH values resulted in NPs
amount of OA in the GON-2 induced larger particles comwith a little smaller size. Figure 4(d) shows that a tempared to GON-1. This result is reasonable because the
perature of 50 C did not affect the morphology of NPs
hydrophobic section of NPs from OA was increased, which
after 10 min in water. Body temperature (37 C) which
led to an enlargement of the NPs. Because the behaviors
renders the NPs eligible for any therapeutic treatment was
of GON-1 and GON-2 formation and the characterization

selected to examine the NPs over time. Figure 4(e) shows
in different temperatures and pH conditions were almost
the morphology of GON according to HRTEM, the difidentical, only the images of GON-2 are illustrated. GONs
ferent parts of OA and gelatin are clearly observed. This
in the self-assembling process with various temperatures
result affirmed the highly expressed core–shell structure of
(37 C and 50 C), times (intervals within 1 h and after
the GONs.
storage at room temperature for 1 month) or pH conditions
Delivered
Technology
University,Table
Fondren
Library
Furthermore,
II shows
the particle size distri(water, pH 1.2, pH 6.8
and pH by
7.4)Publishing
exhibited almost
iden- to: Rice
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21
bution
and
zeta
potential
of
GONs
using the conjugate
tical TEM images with round-shapedCopyright:

particles. Figure
4(b)
American Scientific Publishers
GOC-2

(a)

(b)
gelatin

1642
GOC-1

OA

1640

oleic acid

GOC-1

2839
2925
1709
gelatin

GOC-2
13381240
1638 1535

4000

3000

2000

1000

wavenumber (cm–1)
Figure 2.

1422

(a) FT-IR spectra of pure gelatin, OA, GOC-1 and GOC-2. (b) NMR spectra of pure gelatin, OA, GOC-1 and GOC-2.
J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

groups present was far greater than the number of remaining amine groups present on the surface. Therefore, the
zeta potential at pH 1.2 exhibited a low positive charge on
25
the surface of NPs, which was attributed to the remaining amine groups. The zeta potential of the GOs at other
20
pH values, such as pH 6.8 or 7.4, and at 50 C was
not significantly different from the zeta potential in water,
15
which indicated the stability of the conjugates. Table II

10
also shows the stability of the GONs after 1 month, and
the identical particle size distribution and zeta potential
5
were reserved under storage.
Stability of GONs was also exposed through criti0
cal
aggregation concentration (CAC) below which self60 80 100 120 140 160 180 200 220 240 260 280 300
assembled
systems found in disassemble and kinetic
Diameter (nm)
stability. Figure 5 shows I1 /I3 of pyrene emission spectra
as a function of the concentration of GOC-1 and GOC-2,
30
GON-2
respectively, in water. The CAC values of GOC-1 and
25
GOC-2 are 0.023 mg/ml and 0.015 mg/ml, respectively,
which is significantly lower than the critical micelle con20
centration (CMC) of sodium dodecyl sulfate (SDS) in water
(2.3 mg/ml)25 and OA in water (0.2033–0.9886 mg/ml).26
15
The low CAC value, expected to achieve the stable selfassembled NPs at a dilute condition such as body fluid,
10
is one of the important characteristics for polymeric
amphiphiles as a drug delivery carrier.9 18 27 It means that
5
GOC-1 and GOC-2 can form stable GONs under highly
Delivered by Publishing Technology diluted
to: Ricecondition.

University,
Fondren
0
Moreover,
theLibrary
lower CAC of GOC-2 was
60 80 100 120 140 160 180 200
240 260 280 300 On: Sat, 10 Oct 2015 07:05:21
IP: 220
63.142.225.118
considered herein as a parameter indicating the presence
Diameter (nm) Copyright: American Scientific Publishers
of the higher amount of OA in the NPs because the CAC
values are lowered with increasing hydrophobic moieties.28
Figure 3. Particle size distribution of GON-1 and GON-2 in
water after 1 h.
Taken together, these results show that the GONs possessed good stability.
GOC-1 and GOC-2 under various conditions. DLS analyCharacterization of the Functionalized
sis also determined that the polydispersity index (PDI) was
22
NP and Drug Loaded NPs
not above 0.15 reflecting a narrow dispersion of GONs.
Additionally, GONs had a tendency to slightly decrease in
Because GON-2 possessed more hydrophobic moiety,
size at lower pH values. This tendency was also observed
it was chosen in the further processes for attaching the ligin a previous study in which gelatin particle size was
and FA on the surface and loading the model anticancer
decreased at pH values lower than two.23 However, NP
drug, paclitaxel (PTX). Functionally modified NPs with
size was not affected by temperature and had a negligible

FA (FA-GON) containing PTX (PTX-FA-GON) were also
variation as a function of time, indicating the stability of
prepared to actively target cancer cells for the specific
GONs.
recognition of the soluble form of the folate receptor
NP stability was confirmed using a zeta potential evalexpressed on the surface of cancer cells. The effectiveness
uation of the GONs in aqueous media. The zeta potential
of a cancer therapy depends on the ability of the therapeuof the GONs was negatively charged at values greater than
tic to eradicate the tumor while affecting as few healthy
−30 mV and was not varied by the temperature and time
cells as possible. Interestingly, FA, a vitamin whose recepexcept pH, indicating a prediction of NP stability because
tor is frequently over-expressed on the surface of human
the surface charge resists the aggregation of the particles.24
cancer cells and is used as a tumor marker, is highly
This result also indicated that most of the amine groups of
restricted in most normal tissues.29 30 Therefore, the develgelatin took part in the reaction with OA, as demonstrated
opment of delivery systems that can preferentially localize
by the FTIR and 1 H NMR spectra. Consequently, the suragents to the tumor site has been a recent focus of research.
face of the GONs primarily stemmed from the COOH
Because most of the amine groups of gelatin were used,
groups of gelatin which is partly hydrolyzed into COO−
ligand-targeted NPs with an FA attachment were designed
and electrostatically stabilized the particle with an anionic
using EDC/NHS, a crosslinking agent soluble in water, to
activate the carboxylic groups of gelatin for the reaction of
surface charge. It is possible that the number of carboxylic
GON-1

Distribution (%)

Distribution (%)

30

J. Biomed. Nanotechnol. 9, 1416–1431, 2013

1423


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery
GON-1

(a)

(b)

8 min

(c)

GON-2

15 min

pH 1.2

Tran et al.

1 month

45 min

pH 6.8

pH 7.4

gelatin Fondren Library
(d) Technology to: Rice(e)
(f)
Delivered by Publishing
University,
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21
Copyright: American Scientific Publishers
50 ºC

OA

(g)

(h)

(i)

drug

OA

gelatin

Figure 4. TEM morphology of self-assembled NPs under various conditions (a) GON-1 and GON-2 at 37 C in water after 1 h;

(b) GON-2 at 37 o C in water as a function of time and after storage for 1 month; (c) GON-2 at various pH at 37 C after 10 min;
(d) GO-2 at 50 C after 10 min; (e) HR-TEM of GON-2; (f) TEM image of NPs with FA (FA-GON); (g) HRTEM of NPs with FA (FAGON) in which the arrow points out the black spots on the surface of NPs attributed to gold NPs, indicating the position of FA;
(h) HRTEM of gold NPs on the surface of NPs with FA (FA-GON); (i) HRTEM image of NPs without FA containing PTX (PTX-GON).

the amine functional groups of FA. As shown in Figure 1,
a superior characteristic of functionalized NPs with a
hydrophilic surface is the capability of transporting the
therapeutics to the target tissues or cells because they can
escape mononuclear phagocytes, macrophages and reticuloendothelial systems (RES) in the blood and organs.31
The dense surface concentration of hydrated polymer
1424

chains sterically hinders protein adsorption and opsonization to exhibit prolonged circulation times in in vivo.32
The attachment of FA is further elucidated through other
characterization techniques. Figure 4(f) shows a uniformly
spherical shape. To verify the presence of FA on the surface of NPs, GONs were incubated with an anti-FA antibody and with a gold-labeled secondary antibody (20 nm)
J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.
Table II.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Physicochemical properties of self-assembled GONs under various conditions.

pH

Temperature ( C)

1.2

37

10

6.8

37

10

7.4

37

10

water

37

8
10
15
30
45
60

50

Storage at room temperature/1 month,
then dispersing at 37 C/10 min/water

Particle
size (nm)

Polydispersity
index (PDI)

Zeta
potential (mV)

GON-1
GON-2
GON-1
GON-2
GON-1
GON-2
GON -1
GON-2
GON-1
GON-2

119 60 ± 1 34
175 69 ± 4 97
167 43 ± 5 93
235 04 ± 5 07
176 42 ± 6 02
220 54 ± 3 50
161 45 ± 2 70

205 72 ± 3 30
158 50 ± 4 42
200 06 ± 5 99

0 04 ± 0 01
0 03 ± 0 01
0 08 ± 0 04
0 06 ± 0 02
0 07 ± 0 07
0 08 ± 0 05
0 05 ± 0 01
0 11 ± 0 04
0 10 ± 0 04
0 09 ± 0 02

GON-1
GON-2
GON-1
GON-2
GON-1
GON-2
GON-1
GON-2
GON-1
GON-2

156 07 ± 6 68
215 45 ± 5 70
160 37 ± 5 54
218 22 ± 3 00

164 53 ± 5 18
213 66 ± 4 79
169 56 ± 5 43
219 65 ± 2 20
160 53 ± 3 41
220 00 ± 5 99

0 08 ± 0 02
0 02 ± 0 02
0 06 ± 0 03
0 09 ± 0 00
0 03 ± 0 04
0 05 ± 0 04
0 09 ± 0 02
0 05 ± 0 05
0 08 ± 0 05
0 04 ± 0 02

5 69 ± 1 46
4 16 ± 1 53
−35 70 ± 0 76
−37 45 ± 0 55
−36 73 ± 3 35
−38 19 ± 1 93
−32 30 ± 0 18
−30 48 ± 0 23
−38 78 ± 1 51
−40 25 ± 0 98
−36 59 ± 1 11
−35 45 ± 2 12

−43 07 ± 5 18
−38 89 ± 0 56
40 82 ± 0 19
−37 56 ± 0 80
−44 85 ± 5 66
−40 46 ± 2 79
−36 04 ± 1 41
−34 70 ± 0 48

GON -1
GON -2

168 44 ± 2 74
220 25 ± 4 16

0 07 ± 0 02
0 09 ± 0 01

−35 66 ± 0 95
−32 29 ± 0 74

Dispersion time (min)

10

Delivered
Publishing
to: Rice
University,
Fondren

Library rate, the biodisto recognize the position
where FAbyattached
to theTechnology
surface
affect
particle
size, the
biodegradation
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21
of the NPs. FA bound with the gold probe
is shown
in
tribution and
physicochemical properties.35 36 Therefore,
Copyright:
American
Scientific
Publishers
a surfactant-free nanoparticulate system has been sugFigure 4(g), in which the black spots (gold NPs) show the
gested for preparation. The GOCs are fortunately complace where FA attached to the surface of the NPs, conposed of components that possess surfactant properties for
firming the presence of this ligand on the surface of GONs.
stabilization, which leads to spontaneously stabilized NPs
HRTEM, which provided a closer view of FA-GONs on
without the necessity for surfactant. Therefore, the encapthe surface, showed the crystal lattice of the gold NPs
sulation of PTX in the GONs is performed in appropriate
(Fig. 4(h)). The FA-GON had a mean diameter of approxconditions with high efficiency (Table III). The entrapimately 200 nm.
ment of the drug involves the incorporation of the drug
PTX is one of the most effective anticancer agents.
into the hydrophobic cores of GONs due to hydrophoHowever, this drug has high incidences of adverse reacbic interactions, as previously shown in other research.37 38
tions, including neurotoxicity, myelosuppression and aller8

gic reactions. Cremophor EL (polyethoxylated castor oil),
Moreover, the solubility of PTX in the current drug-loaded
the excipient incorporated for increasing drug solubilnano systems was increased compared to the pure drug
ity, is also a cause to hamper the clinical usefulness of
(Table III).
PTX.15 Additionally, PTX is a hydrophobic drug with
The in vitro release profile of PTX-loaded NPs
with (PTX-FA-GON) or without FA (PTX-GON) was
a poor aqueous solubility of approximately 1 g/ml.33
investigated in PBS (pH 7.4) at 37 C (Fig. 6) and comFor this reason, the controlled release behavior of PTX
from polymeric NPs to suppress these adverse reactions
pared with the pure drug. The cumulative percentage
has been highly recommended. One common method to
release from PTX-FA-GON and PTX-GON was approxiprepare drug-encapsulated NPs is the solvent evaporamately 55% and 45%, respectively, for 6 days and reached
tion method in which the polymers containing the drug
100% for both conjugates after 1 month, which showed the
are solubilized in one of the phases of an emulsion, and
potential of the NPs as a controlled drug delivery system.
the polymer is precipitated to entrap the drug out of
The lower cumulative release of PTX from PTX-FA-GON
the solvent during solvent evaporation, which leaves the
was perhaps due to the presence of the ligand on the NP
drug-loaded particles suspended in the residual solvent.34
surface, which slightly increased the diffusion path. On the
other hand, drug release from Taxol® has been reported
Generally, a surfactant must be used to make smallsized NPs in this emulsion solvent evaporation system,
previously to be over 80% for PTX after 4–6 h.39 40 These
results indicated that the GONs in this study could be
but it can absorb to the NP surface to significantly
J. Biomed. Nanotechnol. 9, 1416–1431, 2013

1425


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Tran et al.

100

GOC-1
1.5

80

% drug released

1.4
1.3

I1/I3

1.2
1.1

60

40
PTX-GON
PTX-FA-GON

pure drug

20

1.0
0.9

0

0.8

0

5

10

15

20

25

30

Time (day)

0.7

Figure 6. Release profiles of PTX from NPs without and

with FA.

0.6
–4.0

–3.5

–3.0

–2.5

–2.0

–1.5

–1.0

–0.5

0.0

logC

FTIR analysis was used to investigate the molecular
interaction between excipient and drug. Figure 7 shows
the FTIR spectra of FA-GON, pure FA, pure PTX, PTX1.5
FA-GON and PTX-GON. The FTIR spectrum of FA-GON
1.4
was observed to contain the bands of GOC and another
peak at 1725 cm−1 , which indicated the presence of the

1.3
carboxylic group of FA. This spectrum affirmed the con1.2
jugation of FA with GON in the preparation in which the
carboxylic group of gelatin was activated by EDC/NHS
1.1
and reacted with the amine group of FA, leaving the carDelivered by Publishing Technology to: Rice University, Fondren Library
1.0
FA remaining. The amide I band and N–H
IP: 63.142.225.118 On: Sat, boxylic
10 Oct group
2015 of
07:05:21
bending
vibration
0.9
Copyright: American Scientific Publishersof FA-GON also exposed a changed peak
shape that moved slightly compared to the pure gelatin and
0.8
GOC. The 900–700 cm−1 region characteristic of the bending regions of the functional groups of FA also appeared
0.7
in the FA-GON spectrum. An N–H stretching of gelatin
0.6
at 3303 cm−1 was also observed for FA-GON. Bands at
1338 and 1240 cm−1 indicated the in-phase combination of
–4.0 –3.5 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5
0.0
C-N stretching, and a C O bending vibration appeared in
logC
the FA-GON spectrum but with a small changed position
Figure 5. Profile of intensity ratio (I1 /I3 from emission specfor FA-GON, implying a high net intensity of these bands

tra against versus logC of GOC-1 and GOC-2 to determine the
for FA-GON. The FTIR results also indicated that there
critical aggregation concentration.
was no interaction between the drug and GON or FA-GON,
employed as long-circulating drug carriers to retard PTX
as only drug peaks were observed. Therefore, the drug was
release over an extended period of time, especially for
only presented in the NPs by encapsulation.
PTX-FA-GON, which is helpful for decreasing drug leakPXRD analysis was performed to identify the crystalline
age from carriers before drug-loaded carriers arrive at the
state of PTX in NP. The disappearance of a sharp peak
target cells.39
of PTX in both PTX-FA-GO and PTX-GO diffractogram
I1/I3

GOC-2

Table III. Loading content, encapsulation efficiency and solubility of paclitaxel at pH 7.4 (loading amount of drug is 10% compared to polymer amount), particle size and zeta potential of targeted functionalized NPs (FA-GON) and PTX-loaded NPs (PTX-GON
and PTX-FA-GON).
Solubility at 48 h (37 C)
Samples
FA-GON
PTX-GON
PTX-FA-GON

1426

Loading
content (wt.%)

Encapsulation
efficiency (%)

Drug-loaded
nanoparticles ( g/mL)

Pure
drug ( g/mL)

Particle
size (nm)

Polydispersity
index (PI)

Zeta potential
(mV)

8 98 ± 0 22
9 13 ± 0 09

89 8 ± 2 2
91 3 ± 0 9

20 53 ± 1 15
16 08 ± 2 05

1 61 ± 0 07

253 69 ± 5 04

266 16 ± 4 89
285 31 ± 5 69

0 1 ± 0 03
0 08 ± 0 02
0 09 ± 0 04

−42 46 ± 1 19
−30 84 ± 2 78
−37 35 ± 1 87

J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

FA-GON
2840
2927
FA

1725

1262

1297
16461554

3002

PTX-FA-GON

1335
1689

PTX-FA-GON

PTX-GON
PTX-GON

PTX

pure PTX
4000

3000

2000

1000

Wavenumber (cm–1)
10

Figure 7. FT-IR spectra of FA, FA-GON, pure PTX, PTX-loaded
NPs without and with FA (PTX-GON and PTX-FA-GON).

20

30

40

50

60

2 theta
Figure 8. PXRD patterns of pure PTX, PTX-loaded NPs without and with FA (PTX-GON and PTX-FA-GON).

indicates the reduction of drug crystallinity (Fig. 8). This
result can explain the reason why the drug dissolution of
Delivered
Technology
to: Rice
University,
Library
tial
slightly
increasedFondren
the negative
charge due to the carPTX could be enhanced
comparedbytoPublishing
the pure drug
under
IP: 63.142.225.118 On: Sat,
10 Oct
2015of07:05:21

boxylic
groups
FA.
Therefore,
this
result also affirmed
the same conditions. The release of PTX
was controlled
Copyright:
American Scientific Publishers
the
aforementioned
characterization.
With the incorpofor a long time for both PTX-FA-GON and PTX-GON,
ration
of
PTX,
the
surface
charges
of
PTX-GON and
partly due to the enhanced solubility of PTX in these
PTX-FA-GON
were
slightly
decreased
but
still negatively
nanopartilces (Table III).

charged,
which
implied
a
good
stability
of
the systems.
The particle size from DLS measurements showed
The
localization
of
or
hydrophobic
drugs,
such
as tamoxthat most of the GON-FA, PTX-GON and PTX-FA-GON
ifen
paclitaxel,
within
the
micelle
corona
might
shield the
had an average diameter of approximately 250 nm, 260 nm
surface
charge
by
shifting

the
shear
plane
further
from the
and 280 nm, respectively (Table III), which is a some42
micelle
surface,
which
leads
to
a
reduced
zeta
potential.
what larger than the sizes observed in the TEM images.
These results are consistent with the results observed in the
Biostability Study
GONs and previous research.9 41 Drug-loaded NPs were
an effective drug carrier only if they did not lose their
In addition to checking the stability of the NPVs in aqueproperties at body temperature (37 C) and a physiologous systems as mentioned above, the stability of GON-2,
ical pH (7.4) until the release of drug at the target site
FA-GON, PTX-GON and PTX-FA-GON were tested in
was achieved. The particle size of the NPs after loading
100% human blood serum at 37 C to mimic physiological
with PTX was increased, because the hydrophobic drug
conditions. The stability of self-assembled nanoparticulate
entrapped in the hydrophobic core always creates a larger
systems as drug carriers is essential for in vivo applicaparticle size than drug-free micelles.39 The PDI of FAtions of these systems. Particle size values were monitored
GON, PTX-GON and PTX-FA-GON were not above 0.15,

as a function of the incubation time for 24 h to investigate
which indicated a narrow particle size distribution of the
the presence of particle aggregation (Fig. 9). Compared to
particles. This same observation was obtained after storage
the results observed in the aqueous conditions at pH 7.4,
of 1 month, which implied a stable nanoparticulate syswhich were to mimic the physiological condition (Tables II
tem. Table III also reveals the zeta potential of FA-GON,
and III), the size of these NPs changed negligibly over the
PTX-GON and PTX-FA-GON. The surface charge of
entire time-course of the test, which indicated an excellent
FA-GON was approximately −42 mV, also indicated that
stability and ultra-low fouling ability of the NPs. However,
the surface of the GONs stemmed primarily from the free
the plots of FA-GON and PTX-FA-GON showed good
carboxylic groups of gelatin and the presence of the FA
stability throughout the whole time compared to the NPs
without FA (GON-2 and PTX-GON). These results reinligand on the surface of the GONs because the zeta potenJ. Biomed. Nanotechnol. 9, 1416–1431, 2013

1427


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Tran et al.

24 hours

300
140
120

Cell viability (%)

Particle size (nm)

280

260

240

220

PTX-GON
PTX-FA-GON
Taxol

*/#
*/#

100

*/#

80
*/#
60

*/#

#

#

40
*
20

GON
FA-GON
PTX-GON
PTX-FA-GON

200

*/#

*

0
0

2.5

180

25

250

2500 12500 25000 drugfree

PTX concentration (ng/mL)
0

2

4

6

8

10

12

14

16

18

20

22

24

Incubation time (h)

force the further application of these systems in in vivo
experiments for the testing of their efficacy in the treatment
of cancer. However, the in vivo results would be expressed
in another report.

PTX-GON
PTX-FA-GON
Taxol

160
140

Cell viability (%)

Figure 9. Stability of NPs with and without targeting ligand
and those loaded with PTX in 100% human blood serum, continuously measured by DLS at 37 C (n = 3).

48 hours

180

120

*/#

*/#

100
80

60
40

*

**

*

*

20

Cell viability (%)

Delivered
Cancer Cell Killing
Effect by Publishing Technology to: Rice University, Fondren Library
IP:of63.142.225.118
On: Sat, 10 Oct0 2015 07:05:21
Instead, cancer cell killing effect
PTX-GON and PTX0
2.5
25
250 2500 12500 25000 drugCopyright: American
Scientific Publishers
free
FA-GON was evaluated herein in comparison with Taxol®
PTX concentration (ng/mL)
(PTX formulated in Cremophor EL). The MTT assay was

performed on PTX-GON and PTX-FA-GON for a compar72 hours
160
ison of their effects on the capability of anti-tumor treat*
ment using the Hela cell line. Cells were also incubated
140
PTX-GON
PTX-FA-GON
with empty NPs and the vehicle of Taxol® to ensure that the
Taxol
120
cytotoxicity was caused by PTX itself or the carrier-bearing
*
100
drug. The range of concentrations of PTX chosen for the
test corresponded to the plasma levels of the drug achiev80
able in humans.43 The vehicle of Taxol® and NPs chosen
60
for the testing of cytotoxic activity had the same concentration of Cremophor or blank NPs as the sample containing
40
**
the highest concentration of PTX (25,000 ng/ml). The cell
*/#
20
**
*/#
viability of Hela cells after treatment of PTX-GON, PTX0
FA-GON, Taxol® solution and vehicles after 24, 48 and 72
0
2.5
25

250 2500 12500 25000 drugh is shown in Figure 10. First, it should be emphasized
free
that no cytotoxicity activity was observed for the blank
PTX concentration (ng/mL)
NPs (without drug), even at a high concentration; the vehi*: Statistically significant difference, compared to Taxol®.
cle of Taxol® , Cremophor EL, which was absent in NPs,
#: Statistically significant difference between PTX-GON and PTX-FA-GON.
showed a significant effect. Therefore, the cytotoxicity of
Figure 10. Viability of Hela cells incubated for 24, 48 and
PTX-loaded NPs was only attributed to PTX, but the cyto®
72 hours with PTX-GON, PTX-FA-GON and Taxol® .
toxicity of Taxol was probably caused by Cremophor EL
rather than PTX. The cytotoxicity of Cremophor EL has
from an inhibition of cell growth or from cytotoxicity. The
been reported previously.8 44 45 These results also suggest
extent of cytotoxicity was influenced by both the concenthat GONs maintain the pharmacological activity of PTX
tration and the incubation time, in which higher concenand efficiently deliver PTX to the cells. The decrease in
trations of PTX inhibited a greater amount of cells and a
cell viability, as measured by the MTT test, can result
1428

J. Biomed. Nanotechnol. 9, 1416–1431, 2013


Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Delivered by Publishing Technology to: Rice University, Fondren Library
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21

Copyright: American Scientific Publishers

Figure 11.

Images of Hela cell viability upon the lowest concentration of PTX (2.5 ng/ml) at 72 hours under microscopy.

longer incubation period induced a larger number of cells
to enter the G2 and M cell cycle phases. Therefore, PTX
was more active45 and led to a higher reduction in cell
viability. PTX encapsulated in both of the researched NPs,
even the smallest concentration of PTX in the case of
PTX-FA-GON, had a higher killing effect on tumor cells
than Taxol® .
Figure 11 (supplementary data) indicates the more effective treatment of the NP through the appearance of cell
images with the lowest concentration of PTX (2.5 ng/ml)
at 72 h compared to the control image that contained culture media only. Cells with the lowest PTX concentration
of Taxol® showed almost the same viability as the control sample, but the cell viability in the images of PTXGON, PTX-FA-GON was very low. The solubilization of
PTX into NPs enhanced its in vitro anti-tumor activity
compared to Taxol® , showing a smaller IC50 than Taxol® .
PTX entrapped in PTX-GON or PTX-FA-GON was readily available to interact with cancer cells and retained its
cytotoxic activity. At 48 h, the IC50 of Taxol® as a control
was 30.07 ng/ml, but the cytotoxicity of PTX-FA-GON or
J. Biomed. Nanotechnol. 9, 1416–1431, 2013

PTX-GON was 0.54 ng/ml and 0.17 ng/ml, respectively.
Therefore, the results presented here are consistent with
other published data that showed that the incorporation of
PTX into NPs enhanced its anti-tumor activity compared
to Taxol® because it showed smaller IC50 than Taxol® 46 47

CONCLUSION
In summary, the current research investigated a novel synthetic method for the preparation of natural biocompatible GOCs. The pH, temperature and amount of OA and
gelatin were crucial for the formation of the self-assembled
GONs, which were thermodynamically formed via selfassembly of the inner hydrophobic part of OA and the
outer hydrophilic part of gelatin in an aqueous phase. The
NPs showed a uniform spherical shape and size and good
stability under storage for at least 1 month. The newly
synthesized GOC was also a good candidate for the encapsulation of a hydrophobic anti-cancer agent such as PTX,
which not only enhanced drug dissolution but also controlled drug delivery. This study also indicated that PTXloaded GO NPs, especially the NPs attaching the FA ligand
1429


Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

Tran et al.

on the surface, had a stronger cancer killing effect than
Taxol® , even at a low drug concentration. This result can
be attributed to the effect of PTX because no cytotoxicity was observed with the current NPs, but Cremophor
EL, the clinical vehicle of Taxol® , exhibited cytotoxicity.
Therefore, the NPs developed in this research could be
considered an effective anti-cancer drug delivery system
for cancer chemotherapy.

emulsion for lipophilic KW-3902, a newly synthesized adenosine
A1 -receptor antagonist. Chem. Pharm. Bull. 50, 87 (2002).
13. A. Ledo-Suárez, L. Rodríguez-Sánchez, M. C. Blanco, and M. A.
López-Quintela, Electrochemical synthesis and stabilization of
cobalt nanoparticles, Physica Status Solidi a 203, 1234 (2006).
14. G. Maxwell, Synthetic Nitrogen Products: A Practical Guide to the

Products and Processes, Springer, New York (2004).
15. A. K. Singla, A. Garg, and D. Aggarwal, Paclitaxel and its formulations. Int. J. Pharm. 235, 179 (2002).
16. National Code of Practice for the Preparation of Material Safety
Data Sheets, 2nd ed., [NOHSC:2011(2003)] Copyright ©Kilford &
Acknowledgments: This work was supported by the
Kilford Pty Ltd, October (2004).
MEST for Human Resource Development Center for Eco17. K. Alfred, G. Carola, and B. Simone, Molecular interactions in conjugates of dicarboxylic acids and amino acids. J. Mol. Struct. 661—
nomic Region Leading Industry Project and by a grant
662, 239 (2003).
from the Korean Health Technology R&D Project, Min18. Y.-Q. Ye, F.-L. Yang, F.-Q. Hu, Y.-Z. Du, H. Yuan, and H.-Y.
istry for Health and Welfare, Korea (A092018). We would
Yu, Core-modified chitosan-based polymeric micelles for controlled
like to thank the Central Research Laboratory, Kangwon
release of doxorubicin. Int. J. Pharm. 352, 294 (2008).
19. G. A. Digenis, T. B. Gold, and V. P. Shah, Cross-linking of gelatin
National University for the use of the FTIR. Finally, we
capsules and its relevance to their in vitro-in vivo performance.
acknowledge KBSI (Chuncheon, Korea) for the TEM and
J. Pharm. Sci. 83, 915 (1994).
particle size measurements.
20. C. Weber, C. Coester, J. Kreuter, and K. Langer, Desolvation process
and surface characterisation of protein nanoparticles. Int. J. Pharm.
194, 91 (2000).
REFERENCES
21. Y. B. Patil, U. S. Toti, A. Khdair, L. Ma, and J. Panyam, Single1. P. A. Maury, D. N. Reinhoudt, and J. Huskens, Assembly of nanoparstep surface functionalization of polymeric nanoparticles for targeted
ticles on patterned surfaces by noncovalent interactions, Current
drug delivery. Biomaterials 30, 859 (2009).
Opinion in Colloid and amp. Interface Science 13, 74 (2008).
22. V. Landry, B. Riedl, and P. Blanchet, Alumina and zirconia acry2. A. Dessey, A. M. Piras, M. Alderighi, S. S. Sandreschi, and F.
late nanocomposites coatings for wood flooring: Photocalorimetric

Chiellini, Doxorubicin loaded polyurethanes nanoparticles. Nano
characterization. Prog. Org. Coat. 61, 76 (2008).
Biomed. Eng. 4, 83 (2012).
23. L. Friedman and K. Klemm, Diffusion velocity and molecular
3. E. Anton, S. Kambalapally, T. Wilson, and N. Robert, Dextranweight, II, The effect of pH upon particle size in gelatin solutions.
Delivered
by Publishing
Technology
University, Fondren Library
containing nanocarriers
significantly
promote greater
anchorage to: Rice
J. Am. Chem. Soc. 61, 1747 (1939).
63.142.225.118
10 M.
OctJahanshahi
2015 07:05:21
dependent cell growth and density IP:
compared
to microcarriers.On:
NanoSat, 24.
and Z. Babaei, Protein nanoparticle: A unique
Copyright: American Scientific
Publishers
Biomed. Eng. 4, 29 (2012).
system as drug delivery vehicles. Afr. J. Biotechnol. 7, 4926
4. S. Hua, C. Xiufeng, R. Jing, P. Xia, W. Juan, W. Can, and
(2008).
B. Chenchen, Application of rotatable central composite design

25. K. H. Min, J.-H. Kim, S. M. Bae, H. Shin, M. S. Kim, S. Park,
in the preparation and optimization of poly(lactic-co-glycolic acid)
H. Lee, R.-W. Park, I.-S. Kim, K. Kim, I. C. Kwon, S. Y. Jeong,
nanoparticles for controlled delivery of HSA. Nano Biomed. Eng. 3,
and D. S. Lee, Tumoral acidic pH-responsive MPEG-poly( -amino
(2012).
ester) polymeric micelles for cancer targeting therapy. J. Control
5. H. Song, R. He, K. Wang, J. Ruan, C. Bao, N. Li, J. Ji, and
Release 144, 259 (2010).
D. Cui, Anti-HIF-1 antibody-conjugated pluronic triblock copoly26. K. Murakami, S. Y. Chan, and A. Routtenberg, Protein kinase C
mers encapsulated with Paclitaxel for tumor targeting therapy. Bioactivation by cis-fatty acid in the absence of Ca2+ and phospholipids.
materials 31, 2302 (2010).
J. Biol. Chem. 261, 15424 (1986).
6. K. Kataoka, A. Harada, and Y. Nagasaki, Block copolymer micelles
27. K. Y. Lee, I. C. Kwon, Y. H. Kim, W. H. Jo, and S. Y. Jeong,
for drug delivery: Design, characterization and biological signifiPreparation of chitosan self-aggregates as a gene delivery system.
cance. Adv. Drug Delivery. Rev. 47, 113 (2001).
J. Control Release 51, 213 (1998).
7. K. Akiyoshi and J. Sunamoto, Supramolecular assembly of
28. K. Na, D. H. Lee, D. J. Hwang, H. S. Park, K. H. Lee, and Y. H. Bae,
hydrophobized polysaccharides. Supramolecular Science 3, 157
pH-Sensitivity and pH-dependent structural change in polymeric
(1996).
nanoparticles of poly(vinyl sulfadimethoxine)–deoxycholic acid con8. J.-H. Kim, Y.-S. Kim, S. Kim, J.H. Park, K. Kim, K. Choi, H. Chung,
jugate. Eur. Polym. J. 42, 2581 (2006).
S. Y. Jeong, R.-W. Park, I.-S. Kim, and I. C. Kwon, Hydrophobi29. W. Franklin, M. Waintrub, D. Edwards, K. Christensen,
cally modified glycol chitosan nanoparticles as carriers for paclitaxel.
P. Prendegrast, J. Woods, P. Bunn, and J. Kolhouse, New anti-lung
J. Control Release 111, 228 (2006).
cancer antibody cluster 12 reacts with human folate receptors present

9. F.-P. Gao, H.-Z. Zhang, L.-R. Liu, Y.-S. Wang, Q. Jiang, X.-D.
on adenocarcinoma. Int. J. Cancer Suppl. 8, 89 (1994).
Yang, and Q.-Q. Zhang, Preparation and physicochemical character30. S. Weitman, R. Lark, L. Coney, D. Fort, V. Frasca, V. J. Zurawski,
istics of self-assembled nanoparticles of deoxycholic acid modifiedand B. Kamen, Distribution of the folate receptor GP38 in normal
carboxymethyl curdlan conjugates. Carbohydr. Polym. 71, 606
and malignat cell lines and tissues. Cancer Res. 52, 3396 (1992).
(2008).
31. Y.-Y. Yang, Y. Wang, R. Powell, and P. Chan, Polymeric core–
10. L. Silvio, R. G. Courteney-Harris, and S. Downes, The use of gelatin
shell nanoparticles for therapeutics. Clin. Exp. Pharmacol. Physiol.
as a vehicle for drug and peptide delivery. J. Mater. Sci.: Mater. Med.
33, 557 (2006).
5, 819 (1994).
32. D. D. Lasic, F. J. Martin, A. Gabizon, S. K. Huang, and
11. Y. L. Su, Z. Y. Fu, J. Y. Zhang, W. M. Wang, H. Wang, Y. C. Wang,
D. Papahadjopoulos, Sterically stabilized liposomes: A hypothesis
and Q. J. Zhang, Microencapsulation of radix salvia miltiorrhiza
on the molecular origin of the extended circulation times. Biochimica
nanoparticles by spray-drying. Powder Technol. 184, 114 (2008).
et Biophysica Acta BBA —Biomembranes 1070, 187 (1991).
12. T. Hosokawa, M. Yamauchi, Y. Yamamoto, K. Iwata, Y. Kato, and
33. V. Martin, Overview of paclitaxel (Taxol). Semin. Oncol. Nurs. 9, 2
(1993).
E. Hayakawa, Formulation development of a filter-sterilizable lipid

1430

J. Biomed. Nanotechnol. 9, 1416–1431, 2013

Tran et al.

Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

34. A. K. Bajpai and J. Choubey, In vitro release dynamics of an anticancer drug from swellable gelatin nanoparticles. J. Appl. Polym.
Sci. 101, 2320 (2006).
35. Y. Jeong, Y. Shim, K. Song, Y. Park, H. Ryu, and J. Nah,
Testosterone-encapsulated surfactant-free nanoparticles of poly(DLlactide-co-glycolide): preparation and release behavior. Bull. Korean
Chem. Soc. 23, 1579 (2002).
36. F.-Y. Cheng, S. P.-H. Wang, C.-H. Su, T.-L. Tsai, P.-C. Wu, D.-B.
Shieh, J.-H. Chen, P. C.-H. Hsieh, and C.-S. Yeh, Stabilizer-free
poly(lactide-co-glycolide) nanoparticles for multimodal biomedical
probes. Biomaterials 29, 2104 (2008).
37. Y. Zhang and R.-X. Zhuo, Synthesis and in vitro drug release
behavior of amphiphilic triblock copolymer nanoparticles based on
poly(ethylene glycol) and polycaprolactone. Biomaterials 26, 6736
(2005).
38. Y.-I. Jeong, J.-B. Cheon, S.-H. Kim, J.-W. Nah, Y.-M. Lee, Y.-K.
Sung, T. Akaike, and C.-S. Cho, Clonazepam release from core–
shell type nanoparticles in vitro. J. Control Release 51, 169
(1998).
39. J. You, X. Li, F. D. Cui, Y.-Z. Du, H. Yuan, and F. Q. Hu, Folateconjugated polymer micelles for active targeting to cancer cells:
Preparation, in vitro evaluation of targeting ability and cytotoxicity.
Nanotechnology 19, 1 (2008).
40. F. Danhier, N. Lecouturier, B. Vroman, C. Jérôme, J. MarchandBrynaert, O. Feron, and V. Préat, Paclitaxel-loaded PEGylated
PLGA-based nanoparticles: In vitro and in vivo evaluation. J. Control Release 133, 11 (2009).

41. S. Prabha, W.-Z. Zhou, J. Panyam, and V. Labhasetwar, Sizedependency of nanoparticle-mediated gene transfection: studies with
fractionated nanoparticles. Int. J. Pharm. 244, 105 (2002).
42. S. K. Sahoo, J. Panyam, S. Prabha, and V. Labhasetwar, Residual polyvinyl alcohol associated with poly(d,l-lactide-co-glycolide)

nanoparticles affects their physical properties and cellular uptake.
J. Control Release 82, 105 (2002).
43. E. Raymond, A. Hanauske, S. Faivre, E. Izbicka, G. Clark,
E. Rowinsky, and D. Von Hoff, Effects of prolonged versus shortterm exposure paclitaxel (taxol) on human tumor colony-forming
units. Anticancer Drugs 8, 379 (1997).
44. J. Liebmann, J. A. Cook, C. Lipschultz, D. Teague, J. Fisher, and
J. B. Mitchell, The influence of Cremophor EL on the cell cycle
effects of paclitaxel (taxol) in human tumor cell lines. Cancer
Chemother. Pharmacol. 33, 331 (1994).
45. F. Danhier, N. Magotteaux, B. Ucakar, N. Lecouturier, M. Brewster,
and V. Préat, Novel self-assembling PEG-p-(CL-co-TMC) polymeric
micelles as safe and effective delivery system for Paclitaxel. Eur. J.
Pharm. Biopharm. 73, 230 (2009).
46. U. Westedt, M. Kalinowski, M. Wittmar, T. Merdan, F. Unger,
J. Fuchs, S. Schäller, U. Bakowsky, and T. Kissel, Poly(vinyl
alcohol)-graft-poly(lactide-co-glycolide) nanoparticles for local
delivery of paclitaxel for restenosis treatment. J. Control Release
119, 41 (2007).
47. M. Breunig, S. Bauer, and A. Goepferich, Polymers and nanoparticles: Intelligent tools for intracellular targeting? Eur. J. Pharm.
Biopharm. 68, 112 (2008).

Delivered by Publishing Technology to: Rice University, Fondren Library
IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21
Copyright: American Scientific Publishers

J. Biomed. Nanotechnol. 9, 1416–1431, 2013

1431