Design and evaluation of clickable gelatin-oleic nanoparticles using fattigation-platform for cancer therapy

Article abstract of DOI:10.1016/j.ijpharm.2018.04.047

The principles of bioorthogonal click chemistry and metabolic glycoengineering were applied to produce targeted anti-cancer drug delivery via fattigation-platform-based gelatin-oleic nanoparticles. A sialic acid precursor (Ac₄ManNAz) was introduced to the cell surface. Gelatin and oleic acid were conjugated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry with the subsequent covalent attachment of dibenzocyclooctyne (DBCO) in a click reaction on the cell surface. The physicochemical properties, drug release, in vitro cytotoxicity, and cellular uptake of DBCO-conjugated gelatin oleic nanoparticles (GON-DBCO; particle size, ～240 nm; zeta potential, 6 mV) were evaluated. Doxorubicin (DOX) was used as a model drug and compared with the reference product, Caelyx. A549 and MCF-7 cell lines were used for the in vitro studies. GON-DBCO showed high DOX loading and encapsulation efficiencies. In A549 cells, the IC50 value for GON-DBCO-DOX (1.29 μg/ml) was six times lower than that of Caelyx (10.54 μg/ml); in MCF-7 cells, the IC50 values were 1.78 μg/ml and 2.84 μg/ml, respectively. Confocal microscopy confirmed the click reaction between GON-DBCO and Ac4ManNAz on the cell surface. Flow cytometry data revealed that the intracellular uptake of GON-DBCO-DOX was approximately two times greater than that of GON-DOX and Caelyx. Thus, the newly designed GON-DBCO-DOX provided a safe and efficient drug delivery system to actively target the anticancer agents.

Full text of DOI:10.1016/j.ijpharm.2018.04.047

InternationalJournalofPharmaceutics545(2018)101–112
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Design and evaluation of clickable gelatin-oleic nanoparticles using
fattigation-platform for cancer therapy
Nilesh M. Meghania, Hardik H. Amina, Chulhun Parka, Jun-Bom Parkc, Jing-Hao Cuid,
⁎
Qing-Ri Caod, Beom-Jin Leea,b,
a
College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea
Institute of Pharmaceutical Science and Technology, Ajou University, Suwon 16499, Republic of Korea
College of Pharmacy, Sahmyook University, Seoul 01795, Republic of Korea
College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
The principles of bioorthogonal click chemistry and metabolic glycoengineering were applied to produce tar-
geted anti-cancer drug delivery via fattigation-platform-based gelatin-oleic nanoparticles. A sialic acid precursor
(Ac4ManNAz) was introduced to the cell surface. Gelatin and oleic acid were conjugated by 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry with the sub-
sequent covalent attachment of dibenzocyclooctyne (DBCO) in a click reaction on the cell surface. The physi-
cochemical properties, drug release, in vitro cytotoxicity, and cellular uptake of DBCO-conjugated gelatin oleic
nanoparticles (GON-DBCO; particle size, ∼240 nm; zeta potential, 6 mV) were evaluated. Doxorubicin (DOX)
was used as a model drug and compared with the reference product, Caelyx®. A549 and MCF-7 cell lines were
used for the in vitro studies. GON-DBCO showed high DOX loading and encapsulation efficiencies. In A549 cells,
the IC50 value for GON-DBCO-DOX (1.29 µg/ml) was six times lower than that of Caelyx® (10.54 µg/ml); in
MCF-7 cells, the IC50 values were 1.78 µg/ml and 2.84 µg/ml, respectively. Confocal microscopy confirmed the
click reaction between GON-DBCO and Ac4ManNAz on the cell surface. Flow cytometry data revealed that the
intracellular uptake of GON-DBCO-DOX was approximately two times greater than that of GON-DOX and
Caelyx®. Thus, the newly designed GON-DBCO-DOX provided a safe and efficient drug delivery system to ac-
tively target the anticancer agents.
Bio-orthogonal click chemistry
Clickable nanoparticles
Doxorubicin
Fattigation-platform
Metabolic glycoengineering
Cancer therapy
1. Introduction
versatile and have already shown the potential to deliver poorly water-
soluble compounds and anticancer agents by increasing their water
Self-assembled nanoparticles composed of biodegradable and bio-
compatible polymers are promising drug delivery carriers owing to
their tunable physicochemical and biopharmaceutical properties. Nano
drug delivery systems can be tailored to target the disease site, improve
the bioavailability of poorly water-soluble drugs, and shield the drug
from the host immune system, thereby prolonging its circulation time
(Kamaly et al., 2016; Park et al., 2016). Several disease-related drugs
and/or biological molecules have been encapsulated in different types
of biodegradable systems (Cheng et al., 2015; Ma and Williams, 2018).
Recently, we designed a novel fattigation-platform to prepare self-
assembled gelatin-oleic nanoparticles (GON) with a combination of
naturally biocompatible protein, gelatin, and a fatty acid, oleic acid.
Owing to the biodegradable nature of the drug delivery system and the
abundance of functional groups such eCOOH and eNH2 for modifica-
tion, the fattigation-platform-based nanoparticles are extremely
solubility, enhancing their bioavailability, and targeting cancer cells
(Tran et al., 2014; Tran et al., 2013a; Tran et al., 2013b). Furthermore,
we demonstrated the advanced theranostic applications of this fatti-
gation-platform through a combination of GON with magnetic nano-
particles (MNP) (Nguyen and Lee, 2017; Tran et al., 2017).
In cancer treatment, the disease site-specificity via active targeting
is essential, as it leads to increased internalization and tumor cyto-
toxicity of the drug in addition to a reduction in side effects (Kudgus
et al., 2014; Tran et al., 2014; Xiong et al., 2005). Therefore, several
nanoparticles have been investigated over several decades with the use
of biological targeting moieties such as peptides, antibodies, and ap-
tamers. These “active targeting” nanoparticles bind to the target cell
surface receptors (Bazak et al., 2015; Choi et al., 2016). Other active
targeting moieties, such as the folate receptor, transferrin receptor,
human epidermal growth factor receptor 2, and G protein-coupled
⁎
Corresponding author at: College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea.
E-mail address: bjl@ajou.ac.kr (B.-J. Lee).
https://doi.org/10.1016/j.ijpharm.2018.04.047
Received 30 December 2017; Received in revised form 4 April 2018; Accepted 21 April 2018
Availableonline23April2018
0378-5173/©2018ElsevierB.V.Allrightsreserved.

N.M. Meghani et al.
InternationalJournalofPharmaceutics545(2018)101–112
receptor, are particularly advantageous for cancer treatment as they
reduce the distribution of potentially toxic drugs to healthy tissues,
which avoids undesirable side effects (Accardo et al., 2014; Liechty and
Peppas, 2012). However, for better cellular targeting and minimized
side effects, advanced functional biomaterials that allow precise control
and flexibility for successful drug delivery systems is highly encoura-
ging as a proof of concept. The adoption of bioorthogonal click chem-
istry (copper-free click chemistry) and metabolic glycoengineering for
the design of new biomaterial architecture and function to the fattiga-
tion-platform can provide an aggressive pathway for active targeting
anticancer agents.
tetramethylrhodamine (TAMRA)-DIBO-alkyne (Cat. No. C10410) were
purchased from Invitrogen (Carlsbad, CA, USA). DBCO-sulfo-NHS ester
was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). DOX
hydrochloride was received from Dong-A Pharmaceutical Co. Ltd.,
Korea. The reference compound, Caelyx® (liposomal DOX hydro-
chloride), was purchased from Janssen, Korea (Cat. No. EBBS700), and
4% paraformaldehyde was purchased from Electron Microscopy
Sciences – EMS (USA). Cell culture media and supplements were pur-
chased from Gibco (Grand Island, NY, USA). High-performance liquid
chromatography (HPLC)-grade solvents were obtained from Fisher
(Seoul, Korea). All other chemicals were of analytical grade and used
without further purification.
In the previous decade, click chemistry has paved the way for novel
synthesis reactions via more precise, more efficient, and faster ap-
proaches (Such et al., 2012). A typical click reaction is characterized by
high efficiency under mild conditions, limited side reactions, high yield,
and reduced byproduct formation (Bock et al., 2006). Several studies
have demonstrated the application of bioorthogonal click chemistry in
the field of biomedicine for various purposes, including the site-specific
tagging of proteins or nucleotides, analysis of metabolic pathways,
monitoring of zebrafish growth, and surface-modification of nano-
carriers (Laughlin et al., 2008; Ngo et al., 2009). Bioorthogonal
chemistry, which enables the specific, covalent attachment of a probe
molecule to the biological molecule of interest, has powerful applica-
tions in the biological field when used in combination with metabolic
glycoengineering. Metabolic engineering techniques have enabled the
introduction of unnatural glycans on the cell surface through the
feeding of specific precursors depending on their intrinsic metabolism.
This technique, which was mainly pioneered by the research group of
Bertozzi, has various applications, such as the exploitation of metabolic
pathways, the analysis of cellular glycans, and 3D cellular assembly (Du
et al., 2009; Prescher et al., 2004).
Previous attempts to target cancer by fattigation-platform-based
drug delivery systems have been mainly based on the leveraging of the
enhanced permeable and retentive vasculature of the disease, which
may result in undesirable side effects, rather than the active and site-
specific targeting of cancer. In this study, we reported the proof of
concept of a novel and facile approach for the preparation of clickable
GON, which can enhance the tumor targetability via an improvement in
the drug accumulation at tumor sites. This was partially achieved by the
enhanced permeability and retention (EPR) effect and mainly by en-
hanced internalization from the covalent binding of the nanoparticles
on the cell surface via metabolic glycoengineering (Hapuarachchige
et al., 2014; Lee et al., 2014b). Dibenzocyclooctyne (DBCO) was chosen
as the clickable material and doxorubicin (DOX) was used as the model
drug. We hypothesized that our dibenzocyclooctyne (DBCO)-functio-
nalized GON could effectively deliver DOX by targeting the azide-
modified sialic acid precursors on the cancer cell surface, which were
generated by bioorthogonal click chemistry and metabolic glycoengi-
neering. The physicochemical properties and cell targetability of
clickable GON were then comprehensively characterized.
2.2. Methods
2.2.1. Synthesis of DBCO-modified gelatin-oleic conjugates (GOC-DBCO)
2.2.1.1. Activation of OA. OA (200 µl) was dissolved in 10 ml of
absolute ethanol and 1 N NaOH (40 µl) was added to increase the pH
of the solution. EDC (310 mg) and NHS (200 mg) were added to the
above solution, which was stirred at 300 rpm for 15 min at room
temperature.
2.2.1.2. Preparation of gelatin-oleic conjugates (GOC). Gelatin (250 mg)
was dissolved completely in distilled water (20 ml) by heating at 37 °C
and stirring at 250 rpm in a water bath. This solution was slowly added
into the activated OA solution. The mixture was incubated in a shaker
(Biofree, Korea) at 200 rpm for 12 h at 37 °C. At the end of the reaction,
acetone (25 ml) was added to precipitate the GOC, which were then
collected after centrifugation at 15,000 rpm. The supernatant was
removed and the GOC were washed three times with distilled water
to remove excess alcohol, unconjugated OA, and gelatin. The final
product was dried in a vacuum drier (Jeio Tech, VDR-30G, Korea) for
2 days.
2.2.1.3. Synthesis of GOC-DBCO. GOC (15 mg) were dissolved in 50%
ethanol (3 ml) by stirring at 300 rpm for 2 h at 37 °C. DBCO-sulfo-NHS
(28 mg) was added to the solution, which was then stirred at 200 rpm
for 8 h at room temperature. After the reaction, the solution was
dialyzed through a cellulose membrane (molecular weight cut-off
[MWCO] 1000, Sigma-Aldrich Corporation, St. Louis, MO, USA)
against distilled water for 24 h at room temperature to remove excess
DBCO-sulfo-NHS and GOC; subsequently, the solution was vacuum-
dried for 12 h to obtain the GOC-DBCO.
2.2.2. Preparation of DBCO-modified GON (GON-DBCO)
GOC-DBCO (10 mg) was dissolved in 50% ethanol (3 ml) by stirring
at 300 rpm in a water bath at 37 °C. The solution was then stirred at
1500 rpm, with ethanol (4 ml) added dropwise at 1 ml/min by using a
peristaltic pump at 37 °C. As the solution turned into a colloidal solu-
tion, 100 µl of 25% glutaraldehyde was added for crosslinking, after
which the solution was stirred for 3 h. The colloidal dispersion was
centrifuged at 12,000 rpm for 25 min and the supernatant was dis-
carded to obtain the GON-DBCO, which were purified by three washes
with distilled water (Puris Expe-CB water system, Model: Expe-CB Ele
10, Korea) and centrifuged at 12,000 rpm for 15 min. The resulting
nanoparticles were vacuum dried for 2 days. The GON without DBCO
were prepared by a similar method.
2. Materials and methods
2.1. Materials
Gelatin (Type A; bloom strength, 300 gm), glutaraldehyde, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-ly-
sine, 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) and 4′,6-dia-
midino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich
Corporation (St. Louis, MO, USA). Oleic acid (OA) was purchased from
Samchun Pure Chemicals Co. Ltd., Korea. 1-(3-Dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC) was purchased from Alfa
Aesar (UK) and N-hydroxysuccinimide (NHS) was purchased from
Wako Pure Chemicals Industry Ltd., Japan. Click-IT® ManNAz
Metabolic Glycoprotein Labeling Reagent (tetraacetylated N-azidoa-
cetyl-D-mannosamine, Ac4ManNAz, Cat. No. C33366) and Click-IT®
2.2.3. Preparation of DOX-loaded GON-DBCO (GON-DBCO-DOX)
DOX was loaded into the GON-DBCO via the incubation method.
Briefly, GON-DBCO (10 mg) was dispersed in 50% ethanol (1 ml), and
water (9 ml) was added. DOX (2 mg) was added to the above solution
with stirring at 250 rpm for 12 h in a 37 °C water bath. After 12 h, the
solution was centrifuged at 12,000 rpm for 25 min and purified by three
washes in distilled water. The GON-DBCO-DOX were freeze-dried until
further use.
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InternationalJournalofPharmaceutics545(2018)101–112
2.2.4. Characterization of the drug delivery system
through the subtraction of the amount of unloaded drug from the total
amount of initial drug. The DL and EE were then calculated from the
following equation:
2.2.4.1. Attenuated
total reflectance-Fourier
transform
infrared
spectroscopy (ATR-FTIR) analysis. The IR spectra of gelatin, OA, GOC,
DBCO, and GOC-DBCO were measured by using ATR-FTIR spectroscopy
(UMA-500 microscope, Bio-Rad, USA). Except for OA, the dried samples
were used to measure the spectra, which were obtained at a resolution
weigh t of the drug in nanoparticles
total weight of the nanoparticles
DL(%) =
EE(%) =
× 100
× 100
of 4 cm−1
.
amount of the drug in nanoparticles
total amount of drug added initially
2.2.4.2. Proton nuclear magnetic resonance (1H NMR) analysis. The 1H
NMR spectra of gelatin, OA, GOC, and GOC-DBCO were measured
(Bruker AVANCE™ 600 MHz spectrometer, USA). OA, GOC, and GOC-
DBCO were dissolved in 2 ml of deuterated dimethyl sulfoxide (DMSO-
d6); gelatin was dissolved in deuterium oxide (D2O).
2.2.4.8. In vitro drug release. The GON-DBCO-DOX (1 mg of DOX) and
Caelyx® were dispersed in PBS (2 ml) at different pH conditions, i.e., pH
5.2 and 7.4, and then transferred to a dialysis membrane tube (MWCO
6000, Spectra/Por®, Spectrum Laboratories Inc., USA). Each membrane
tube was immersed in a tube containing 10 ml PBS solution at the same
pH as the solution in the membrane tube and shaken at 100 rpm and
37 °C. At predetermined time intervals, 1 ml of the sample was
withdrawn and fresh medium was added to maintain sink conditions.
The samples were centrifuged at 12,000 rpm for 20 min and the
supernatant was analyzed using a UV/visible spectrophotometer to
determine the drug release. Free DOX was used as a control; the DOX
calibration curve was produced from absorbance measurements at
480 nm.
2.2.4.3. Determination of the degree of substitution. The number of free
primary amino groups in gelatin that reacted with OA to form the GOC
was determined by the TNBS assay (Palmer and Peters, 1969). In this
method, 3 ml of gelatin, GOC, or GOC-DBCO solution (1 mg/ml) in
aqueous buffer (0.1 N NaHCO3, pH 8.3) was prepared by sonication for
1 h and 0.01% TNBS (2 ml) was added. The solutions were mixed well
and then incubated at 37 °C for 4 h. After incubation, 10% SDS (2 ml)
and 1 N HCl (1 ml) were added to each sample and the samples were
properly mixed by vortexing. The absorbance of each sample was
measured at 340 nm by using a UV/visible spectrophotometer (DU®
730, Beckman Coulter Inc., CA, USA). L-Lysine was used to construct a
calibration curve. The molar concentration of free primary amino
groups in gelatin, GOC, and GOC-DBCO was calculated from the
calibration curve, and the degree of substitution was calculated from
the following equation:
2.2.5. Cell culture and maintenance
A549, a human lung adenocarcinoma cell line, and MCF-7, a human
breast adenocarcinoma cell line, were purchased from the Korean Cell
Line Bank (KCLB, Seoul, Korea, KCLB No. 10185). The A549 cells were
grown in Roswell Park Memorial Institute (RPMI) 1640 medium and
the MCF-7 cells were grown in Dulbecco’s modified Eagle medium
(DMEM). Both cell culture media were supplemented with 10% FBS and
1% penicillin/streptomycin (PS) and the cells were allowed to attach
and stabilize at 37 °C in a humidified incubator with an atmosphere of
5% CO2.
DS = (Ca−Cb)/Ca × 100
where Ca and Cb represent the molar concentration of the free pri-
mary amino groups in gelatin and GOC, respectively.
2.2.4.4. Determination of the particle size and zeta potential. The particle
size and zeta potential of GON, GON-DBCO, and GON-DBCO-DOX were
determined by dynamic light scattering (DLS) using a zeta potential and
particle size analyzer (ELSZ-2000, Otsuka Electronics, Japan). The
samples were diluted in water (1:20) prior to measurement. The
particle size (intensity-weighed distribution) and zeta potential of
each sample were measured in triplicate by using a He-Ne laser light
source (5 mW) at a 90° angle, from which the average values were
determined.
2.2.5.1. Confirmation of the presence of azide groups by confocal
microscopy. A549 and MCF-7 cells (1 × 104 cells in 2 ml) were seeded
onto 35-mm glass-bottom in RPMI and DMEM medium with the sialic
acid precursor, Ac4ManNAz (5 and 50 µM), or without (control). After
incubation for 72 h, the cells were washed twice with Dulbecco’s (D)
PBS (pH 7.4). Click-IT® TAMRA-DIBO-alkyne (100 µM) was added to
the cells and incubated for 1 h at 37 °C. The cells were washed with
DPBS (pH 7.4) and fixed with 4% paraformaldehyde solution for
15 min. Triton X-100 (0.1% v/v) was added and the cells were
incubated at room temperature for 5 min. The nuclei were stained
with DAPI (5 µg/ml) and the cells were incubated in the dark for a
further 15 min. The cells were then washed with DPBS (pH 7.4) and
examined by using a confocal microscope (Nikon Eclipse Ti, Nikon,
Japan). The control cells were cultured without Ac4ManNAz and
treated with TAMRA-DIBO-alkyne.
2.2.4.5. Evaluation of the nanoparticle morphology. The morphological
evaluation of the GON, GON-DBCO, and GON-DBCO-DOX was
performed by field-emission scanning electron microscopy (FE-SEM,
JSM6700F, JEOL, Japan). The dried nanoparticle powder was placed on
a carbon tape, which was then coated with gold for 2 min under
vacuum. The samples were observed under an acceleration voltage of
5.0 kV and the images were taken at a resolution of 15000×.
2.2.5.2. Cell viability assay. The viability of cells after treatment with
free DOX, GON-DBCO, GON-DOX, GON-DBCO-DOX, and Caelyx® was
determined by using the MTT assay. A549 and MCF-7 cells
(1 × 104 cells/well) were seeded in 48-well plates with medium
containing Ac4ManNAz (50 µM) and incubated for 24 h. Subsequently,
Caelyx® and the nanoparticle formulations (DOX concentration
equivalent to 0.5, 1, 5, and 10 µg/ml in each group) were added to
the wells and the cells were incubated for a further 24 h. After a total
incubation time of 48 h, the cells were washed twice with PBS (pH 7.4)
and 25 µl of MTT (0.5 mg/ml in PBS) was added to each well. The plate
was incubated for 1 h at 37 °C. The culture medium was removed and
the formed formazan crystals were dissolved in DMSO (200 µl) after
incubation of the plate for 30 min in the dark at room temperature. The
absorbance of each well at 570 nm was measured by using a microplate
reader (BioTek, Vermont, USA). The results were compared with those
2.2.4.6. Evaluation of the nanoparticle stability. The stability of various
GON preparations was evaluated by mixing 10 mg of the nanoparticle
dispersion and 1 ml of phosphate-buffered saline (PBS) with fetal
bovine serum (FBS) and monitoring the particle size daily for 7 days
by DLS using a zeta potential and particle size analyzer (ELSZ-2000,
Otsuka Electronics, Japan).
2.2.4.7. Determination of the drug loading (DL) and encapsulation
efficiency (EE). The supernatant obtained during the preparation of
drug-loaded nanoparticles (described in Section 2.2.3) was collected
and analyzed by using a UV/visible spectrophotometer (Beckman
Coulter, DU® 730, Germany) to determine the amount of DOX that
was not encapsulated inside the GON. The DL and EE were determined
from this data. The amount of DOX in the nanoparticles was calculated
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N.M. Meghani et al.
InternationalJournalofPharmaceutics545(2018)101–112
Fig. 1. Mechanistic synthesis of gelatin-oleic-DBCO conjugates (GOC-DBCO) for click reaction.
of the control cells cultured in nanoparticle-free medium. Six replicates
of each experiment were performed.
conducted in triplicate. The results were expressed as the mean
fluorescence intensity and percentage uptake was calculated
compared with the fluorescence of the control (100%) by using
FlowJo™ software (Tree Star Inc. USA).
2.2.5.3. Confocal imaging of DOX-loaded GON (GON-DOX) and GON-
DBCO-DOX. A549 and MCF-7 cells (3 × 104 cells in 2 ml medium) were
seeded onto 35 mm glass-bottom dishes containing Ac4ManNAz
(50 µM) and incubated for 72 h. The inherent fluorescence of DOX
was visualized by using a confocal microscope. GON-DOX and GON-
DBCO-DOX and Caelyx® (equivalent DOX concentration of 2 µg/ml)
were added to the cells, which were then incubated for 4 h at 37 °C.
After incubation, the steps described in Section 2.2.5.1 were followed
for fixation and imaging using a confocal microscope (Nikon Eclipse Ti,
Nikon, Japan).
2.2.6. Statistical analysis
All results were expressed as the mean
standard deviation (SD).
The statistical significance (P value) of the observed differences was
analyzed by one-way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test computed by Prism software
(GraphPad Software Inc., San Diego, CA, USA). P values of < 0.05 were
considered to be statistically significant (ns = not significant,
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
2.2.5.4. Flow cytometry analysis. The cellular uptake and targeting of
the nanoparticles was quantitatively analyzed by using flow cytometry.
A549 and MCF-7 cells (3 × 104 cells in 2 ml medium) were seeded in
24-well plates with Ac4ManNAz (50 µM) and incubated for 48 h.
Nanoparticles containing 2 µg/ml DOX in each group, i.e., GON-DOX,
GON-DBCO-DOX, and Caelyx®, were added to the plates, which were
then incubated for 1 and 4 h. After incubation, the cells were washed
with DPBS (pH 7.4) and 100 µl of 0.25% (w/v) trypsin-
ethylenediaminetetraacetic acid (EDTA) was added to detach the
cells. The cells were then collected by centrifugation at 1000 rpm for
3 min and suspended in DPBS (pH 7.4). The DOX fluorescence in cells
was measured by using a flow cytometer (BD FACSCanto™ II, Becton
Dickinson, USA). The laser excitation wavelength was set to 488 nm
and the DOX fluorescence was recorded at 575 nm. The fluorescence of
10,000 cells was recorded for each sample and the experiments were
3. Results and discussion
3.1. Characterization of the GOC-DBCO conjugates
Although DOX is the first-choice treatment for various types of
cancers, the drawbacks include severe undesirable side effects, the re-
quirement for a higher dose to achieve the optimal therapeutic re-
sponse, and cancer cell resistance. Therefore, the design of novel drug
delivery systems to reduce these side effects and enhance the ther-
apeutic index has been desired since long time. In the present study, a
simple, effective, and targeted covalent chemistry-based drug delivery
system was designed by using novel, biocompatible, clickable nano-
particles, and characterized rigorously. Biocompatibility is one of the
most important parameters for a practical drug delivery system in order
to prevent unwanted immune response and tissue reactions to the drug
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N.M. Meghani et al.
InternationalJournalofPharmaceutics545(2018)101–112
Fig. 2. (a) ATR FT-IR analysis of gelatin, oleic and DBCO conjugates; (b) 1H NMR spectra of oleic acid, gelatin, GOC and GOC-DBCO.
especially for the preparation of biomaterials (Dvorakova et al., 2017).
In this study, we exploited the eCOOH group of OA and the eNH2
group of gelatin and conjugated them via the EDC/NHS reaction. The
FTIR data confirmed the formation of amide bonds between gelatin and
OA to form the GOC, and subsequently, between GOC and DBCO, to
form the GOC-DBCO (Fig. 2a). The ATR-FTIR spectrum of GOC showed
characteristic bands that corresponded to NeH stretching at
3288 cm−1, C]O stretching at 1636 cm−1, and NeH deformation at
1534 cm−1. The bands at 1628 cm−1 and 1522 cm−1 in the gelatin
spectrum had shifted to 1636 and 1534 cm−1 in the GOC spectrum,
respectively, which confirmed the presence of new amide bonds formed
by the reaction between the amino groups of gelatin and OA. These
peak changes confirmed the conjugation between gelatin and OA
(Gaihre et al., 2009; Park et al., 2015; Tran et al., 2013b). In the case of
GOC-DBCO, characteristic peaks were observed at 2173 cm−1, which
represented the C^C bond of the DBCO moiety, and 1437 cm−1, which
represented the C]C stretching in the benzene rings of DBCO. All other
characteristic peaks of the amide bonds in the GOC spectrum were also
visible in the GOC-DBCO spectrum, which confirmed the formation of
conjugates between GOC and DBCO. The conjugations between OA and
gelatin and between GOC and DBCO were also confirmed by using NMR
Table 1
Degree of substitution of conjugates as determined by TNBS method.
Samples
Amino concentration
(µM)*
Number of free
amino groups per
molecule
Degree of
substitution (%)
Gelatin
GOC
GOC-DBCO
103.14
54.16
37.02
4.11
2.69
2.67
∼29
∼15
∼10
–
48.27
33.33
* Data are presented as mean
standard deviation (SD) (n = 3).
carrier. Although several methods and materials have been used by
researchers to establish drug delivery systems, the problem of bio-
compatibility and lack of active targeting ability have hindered their
wider clinical application (Choi and Han, 2018; Naahidi et al., 2013).
The present study has combined the biocompatibility of gelatin and
oleic acid and employed novel bioorthogonal click chemistry for active
targeting.
The mechanistic scheme for the formation of gelatin-oleic-DBCO
conjugates (GOC-DBCO) is shown in Fig. 1. The activation of the che-
mical groups present in the molecules and their conjugation using EDC/
NHS chemistry are some of the most commonly used reactions,
Table 2
Physicochemical characterization of clickable GON systems.
System
Particle size (nm)
Polydispersity index (PDI)
Zeta potential (mV)
Drug loading (%)
Encapsulation efficiency (%)
GON
210.53
218.01
240.00
265.77
3.38
4.87
5.05
5.21
0.45
0.26
0.33
0.29
0.22
0.10
0.18
0.17
10.45
9.21
6.56
8.97
2.74
1.33
1.77
0.51
–
–
GON-DOX
GON-DBCO
GON-DBCO-DOX
12.29
–
10.35
1.04
0.62
73.77
–
62.10
6.28
3.77
Data are presented as mean
standard deviation (SD) (n = 3).
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Fig. 3. The physicochemical properties of clickable GON: (a) Particle size distribution of GON, GON-DBCO and GON-DBCO-Dox using DLS; (b) SEM micrographs of
GON, GON-DBCO and GON-DBCO-Dox; (c) Biostability of NPs in fetus bovine serum (FBS) (n = 3).
spectroscopy (Fig. 2b). The NMR spectrum of OA showed two char-
acteristic peaks at 5.31(eCH] CHe) and 11.95 (eCOOH) ppm (in-
dicated by red arrows in the spectrum), whereas the GOC spectrum
showed an alkene peak at 5.32 ppm (red arrow), which confirmed the
presence of OA. In addition, the proton peak of the carboxyl group of
OA at 11.95 was not found in the GOC spectrum, which was suggestive
of the formation of an amide bond between gelatin and OA (Tran et al.,
2013b). Further, the DBCO peak was found at 5.05 ppm (red arrow),
which indicated the presence of the DBCO moiety and, thus, the con-
jugation of DBCO with GOC (Koo et al., 2012). The number of OA
molecules conjugated to gelatin was identified by the TNBS assay
(Habeeb, 1966), as TNBS predominantly reacts with primary amines
present in the molecule. There are approximately 29 free amino groups
in a type A gelatin molecule of 1000 amino acid residues (Kale and
Bajaj, 2010). The degree of substitution of the amino groups and the
resulting amide bond formation were quantified as the percentage of
gelatin amino groups reacting with OA, which was determined from the
free primary amino groups available in gelatin and GOC as shown by
the standard calibration curve of L-lysine (1–8 µg/ml). The data in-
dicated that approximately 14 amino groups were estimated to parti-
cipate in the conjugation reaction of OA to form GOC and that five
amino groups were involved in the formation of GOC-DBCO (Table 1).
The number of free amino groups varied depending on the source and
molecular weight of gelatin. The molar concentration of free amino
groups in the gelatin, GOC, and GOC-DBCO was found to be 103.14,
54.16, and 37.02 µM, respectively (Table 1). Thus, the degree of sub-
stitution for GOC and GOC-DBCO was estimated to be 48.27 and
33.33%, respectively.
3.2. Characterization of the nanoparticles
The GON were prepared by a modified desolvation method by using
ethanol as an anti-solvent. The data from the physicochemical char-
acterization of the nanoparticles are shown in Table 2. GON-DOX and
GON-DBCO-DOX were prepared by using an incubation method. The
particle size of GON, GON-DBCO, GON-DOX, and GON-DBCO-DOX was
successfully optimized. The size of GON, GON-DOX, GON-DBCO, and
GON-DBCO-DOX was 210.53
3.38 (0.45
0.22), 218.01
4.81
5.21
(0.26
(0.29
0.10), 240.00
5.05 (0.33 0.18), and 265.77
0.17) nm, respectively. The DOX-loaded nanoparticles showed
an increase in particle size possibly due to increased hydrophilic affinity
(hydrophilic interaction) of DOX for the gelatin part of GON and thus
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Fig. 5. Cell viability (%) of (a) A549 cells, and (b) MCF-7 cells at 24 h,
Fig. 4. (a) Drug release profile of GON-DOX, GON-DBCO-DOX and Caelyx® at
(mean
S.D, n = 6).
pH 5.2, and (b) pH 7.4 (mean
S.D, n = 3).
size from day 4 may have been attributed to the degradation of glu-
taraldehyde cross-linking. The shift in the band at 1443.87 cm−1 in the
gelatin spectrum to 1444.52 cm−1 in the GOC spectrum suggested the
formation of a eC]N (aldimine linkage, not shown by arrow in Fig. 2a)
bond between the free amino groups of gelatin and the aldehyde
(eCHO) group of glutaraldehyde. As high amounts of glutaraldehyde
are known to cause toxicity, we attempted to optimize the amount of
the cross-linking agent without affecting the particle size and stability
(Azimi et al., 2014; Coester et al., 2000).
increased drug loading and encapsulation efficiency (Table 2). The data
further attested the successful loading of the drug into the nanoparticles
upon incubation. Fattigation-platform-based delivery system was
shown to increase the particle size after DOX loading into the nano-
particles (Amin et al., 2017). The optimal size range of 220–240 nm
nanoparticles was therapeutically suitable for diverse parenteral ad-
ministrations.
The GON surface was found to be mildly positively charged possibly
owing to the availability of few primary amine groups. The conjugation
of DBCO had a negligible impact on the charge of the nanoparticles,
which may have resulted from the few primary amine groups reacting
with the GOC. The nanoparticle systems with particle sizes below
300 nm and slightly positive or neutral surfaces have been found to be
ideal for the drug delivery for a variety of diseases (Blanco et al., 2015;
Champion et al., 2007). The zeta potential of GON, GON-DOX, GON-
3.3. DL, EE, and in vitro drug release
The DL and EE of GON and GON-DBCO were determined by using
UV spectroscopy. DOX was loaded into the nanoparticles by using the
incubation method. Both GON and GON-DBCO had a high DL content of
DBCO, and GON-DBCO-DOX was 10.45
2.74, 9.21
1.33,
12.29
1.04 and 10.35
0.62, respectively. The EE of GON and
6.28 and 62.10 3.77, re-
6.56 1.77 and 8.97 0.51 mV, respectively. The drug loading did
GON-DBCO was found to be 73.77
not affect the zeta potential of the GON. The DLS size distribution and
SEM images of GON, GON-DBCO, and GON-DBCO-DOX indicated that
the nanoparticles were dispersed over a narrow size range and were
spherical in shape (Fig. 3a and b). The stability of the nanoparticles is
the most important factor for effective targeted drug delivery. The
stability of the nanoparticles was monitored for 7 days in the presence
of FBS to mimic the physiological conditions. The particle size of GON-
DOX, GON-DBCO, and GON-DBCO-DOX was observed to be stable over
the study period, with no significant surge in the size (Fig. 3c). This
indicated that the drug-loaded nanoparticles were stable under phy-
siological conditions. All formulations were found to be generally
stable, with a negligible increase in particle size from day 4 onwards in
the cases of GON and GON-DBCO-DOX. This slight increase in particle
spectively (Table 2). Due to the amphiphilic nature of GON, DOX was
encapsulated via hydrophilic interactions with gelatin molecules. The
release mechanism of DOX from the nanoparticles was measured in two
pH conditions, i.e., pH 5.2 (cancer cell environment, Fig. 4a) and 7.4
(normal plasma, Fig. 4b) for 24 h. The drug-loaded nanoparticles and
Caelyx®, the reference product, exhibited a linear drug release rate at
both pH values. The percentage of DOX released at pH 5.2 was sig-
nificantly higher than that released at pH 7.4. Further, the DOX release
rate from GON-DBCO-DOX was slightly higher than that from GON-
DOX at pH 5.2. Over 50% of the DOX from GON-DBCO-DOX and GON-
DOX was released after 4 and 8 h, respectively. Approximately 80% of
the DOX from GON-DOX and GON-DBCO-DOX was released after 24 h,
which indicated a gradual release of the drug after the initial burst
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Fig. 6. Confocal imaging for generation of azide groups on cell surface. (a) A549 cells (b) MCF-7 cells at 5 µM, and 50 µM.
3.4. Cell viability analysis
release of up to 2 h. A burst release of Caelyx® occurred at the initial
time point, with approximately 60% of the DOX released within 1 h,
which was subsequently reduced to approximately 73% compared with
81% and 84% in the cases of GON-DOX and GON-DBCO-DOX, respec-
tively. At pH 7.4, all three systems gradually released DOX over 24 h. In
the case of GON-DBCO-DOX, up to 50% of the DOX was released within
2 h, which indicated an initial burst release compared with the gradual
release over 12 h of 50% of the DOX from GON-DOX and Caelyx®.
Despite the initial burst release, the release of DOX from GON-DBCO-
DOX was higher than that from GON-DOX and Caelyx® and gradually
reached 70% at 24 h. The gradual release of DOX from GON-DOX and
GON-DBCO-DOX indicated that a strong intermolecular force was in-
volved in the drug-carrier interaction. The higher drug release at pH 5.2
could be attributable to the hydrolytically unstable crosslinks that re-
sult in the generation of soluble polymer fragments (Heller, 1980;
Vandervoort and Ludwig, 2004). The pH dependency of DOX release
has been observed previously from polyelectrolyte polymers and dex-
tran-coated poly (lactic-co-glycolic acid) (PLGA) (Jeong et al., 2006;
Lee et al., 2014a). Significantly, the GON-DBCO-DOX exhibited ac-
celerated drug release (84.31% at 24 h) at pH 5.2, which indicated
superior control of drug release from the nanoparticles compared with
that of Caelyx®. It is observed that a drug is released by various pro-
cesses such as diffusion via a polymer matrix, polymer degradation, and
diffusion and solubilization through microchannels that exist in the
polymer matrix or are formed by erosion. Gelatin is known to exhibit
pH-dependent gelation properties. The hydrolysis of GON causes
swelling to increase with time and the gel becomes more pH-sensitive as
hydrolysis proceeds. A higher drug release in acidic pH is useful for the
development of drug delivery systems to target tissues with a low pH,
such as the tumor microenvironment. Therefore, the substantial release
of the drugs occurs around the tumor cells (pH 5.2); in the normal
physiological environment (pH 7.4), the majority of drugs remains in-
side the carriers, leading to lower exposure of normal tissues to cyto-
toxic drugs.
The viability of A549 and MCF-7 cells after treatment with the na-
noparticles was evaluated by using an MTT assay in order to establish
whether the GON-DBCO-DOX exhibited enhanced anticancer effects.
The viability of A549 cells and MCF-7 cells treated with DOX con-
centrations equivalent to 0.5, 1, 5, and 10 µg/ml in free DOX, GON
(without DOX), GON-DOX, GON-DBCO-DOX, and Caelyx® was de-
termined after 24 h of incubation (Fig. 5a and b). In both cell lines,
DOX-loaded nanoparticles caused a dose-dependent decrease of cell
viability. The IC50 values were computed by using GraphPad Prism®
software. The IC50 value for GON-DBCO-DOX (1.29 µg/ml) was found
to be approximately six times lower than that of Caelyx®
(IC50 = 10.54 µg/ml) in A549 cells. In MCF-7 cells, the IC50 value for
GON-DBCO-DOX (1.78 µg/ml) was lower than that of Caelyx® (2.84 µg/
ml). The MTT assay showed that the cytotoxic efficiency of GON-DBCO-
DOX was superior to that of free DOX and Caelyx® at all tested con-
centrations. Thus, a lower dose of DOX would be required to achieve
the same chemotherapeutic efficacy. In addition, the difference in the
cytotoxic efficiency after 24 h in A549 and MCF-7 cells could be attri-
butable to the difference in the expression of the azide group on the cell
surface via metabolic glycoengineering. Overall, the data indicated that
the antitumor activity of GON-DBCO-DOX was superior to that of free
DOX, GON-DOX, and Caelyx® owing to the selective and efficient in-
ternalization of GON-DBCO-DOX by the A549 and MCF-7 cells via
bioorthogonal click chemistry. Nanoparticles without DOX (GON-
DBCO) were not cytotoxic at any of the tested concentrations, which
further proved that the GON nanoparticle system was biocompatible
and had potential biomedical applications.
3.5. Confirmation of the presence of azide groups and nanoparticle targeting
by confocal microscopy
The generation of azide groups on the cell surface was evaluated by
confocal imaging of the A549 and MCF-7 tumor cells. Ac4ManNAz was
chosen as a precursor for metabolic glycoengineering because it
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InternationalJournalofPharmaceutics545(2018)101–112
Fig. 7. Confocal imaging for cellular uptake of (a) GON-DOX; (b) GON-DBCO-DOX, and (c) Caelyx® for A549 cell line.
selectively induces the expression of unnatural sialic acids on the tumor
cell surface (Prescher and Bertozzi, 2005). We selected Ac4ManNAz as
the precursor for the metabolic glycoengineering of sialic acids, as
Ac4ManNAz readily generates unnatural sialic acids on the tumor cell
surface via the natural glycoengineering process (intrinsic metabolism)
of each cell (Islam et al., 2012; Meghani et al., 2017). Thus, by taking
advantage of the natural cellular glycoengineering process, the dose-
dependent and temporal generation of chemical groups on the tumor
cell surface is generated in a similar manner to biological receptors and
therefore controls the tumor targetability of the nanoparticle delivery
system. This dose-dependent expression of unnatural targetable sialic
acids on the tumor cells is unique among other natural targeting moi-
eties, such as receptors for folate and RGD. The dose-dependent gen-
eration of azide groups on the cell surface was visualized in pink
fluorescence after the treatment of A549 and MCF-7 cells with TAMRA-
DIBO-alkyne dye. GON-DBCO, owing to its DBCO moiety, binds to this
unnatural glycolic acid via bioorthogonal click chemistry and thus acts
as a targeted drug delivery system. The cells treated with different
concentrations of Ac4ManNAz, i.e., 5 and 50 µM, showed a dose-de-
pendent increase in the generation of azide groups after 72 h in both
cell lines (Fig. 6a and b). These results indicated that Ac4ManNAz can
generate azide groups on the cell surface, thus confirming the success of
the metabolic engineering. Furthermore, the intracellular uptake of
DOX from GON-DBCO-DOX was observed in a time-dependent manner
by monitoring the intrinsic red fluorescence of DOX (DOX concentra-
tion equivalent to 2 µg/ml in each NP group) in A549 and MCF-7 tumor
cells.
In both cell lines, the intracellular uptake of DOX from GON-DBCO-
DOX was increased after 1 h, as indicated by the bright DOX fluores-
cence (Figs. 7 and 8). There is big difference between two cells in the
cellular uptake as indicated by the red color. Originally, we observed
confocal images in Fig. 8 that is in MCF-7 cell lines, there was less
internalization of DOX and the cellular uptake was reduced as visua-
lized by the difference in fluorescence of DOX in cell lines. However,
the cellular uptake in A549 cells was largely increased, showing higher
red color via cell-specific expression of the receptors. The observed
fluorescence intensity of GON-DBCO-DOX-treated cells was sig-
nificantly higher than that of GON-DOX- and Caelyx®-treated cells after
4 h of incubation (Figs. 7b and 8b) in A549 and MCF-7 cells. However,
the fluorescent intensity was higher in A549 cells than in MCF-7 cells.
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Fig. 8. Confocal imaging for cellular uptake of (a) GON-DOX; (b) GON-DBCO-DOX, and (c) Caelyx® for MCF-7 cell line.
The data also showed that the DOX fluorescence intensity was sig-
nificantly higher in the nucleus than in the cytoplasm. These results
indicated that GON-DBCO-DOX was efficiently internalized by the
cancer cells.
MCF-7 cells. The flow cytometry data showed that the DOX uptake was
approximately two times higher in GON-DBCO-DOX-treated cells than
in GON-DOX- and Caelyx®-treated cells, which indicated that the
bioorthogonal click reaction on the cell surface between the unnatural
azide-containing sialic acid residues and GON-DBCO-DOX. Further-
more, GON without DBCO also showed an increase in internalization, as
indicated by the increased DOX fluorescence intensity compared with
that of the Caelyx®-treated cells. This suggested that the EPR effect
mediated by the interaction between the positively charged nano-
particles and the negatively charged cell membrane may also contribute
to enhanced DOX uptake. However, in both cell lines, the fluorescence
intensity of GON-DOX-treated cells was not statistically significant
(P > 0.05) compared with that of Caelyx®-treated cells, which further
indicated that the DOX uptake was triggered by the EPR effect mediated
by the positively-charged nanoparticles, despite the modification of the
cell membrane surface via metabolic glycoengineering. These data
supported our hypothesis that the bioorthogonal click reaction- and
fattigation-based drug delivery system demonstrated potential selective
enhancement of the internalization of active cargo to cancer cells in
physiological conditions.
3.6. Flow cytometry analysis of the cellular internalization of nanoparticles
The intracellular uptake of DOX was measured quantitatively by
using flow cytometry. The mean DOX fluorescence was measured after
the incubation of A549 cells and MCF-7 cells with GON-DOX, GON-
DBCO-DOX, and Caelyx® (DOX concentration equivalent to 2 µg/ml) for
1 and 4 h. The mean fluorescence intensity of all three delivery systems
was comparable after 1 h (Fig. 9a) in the A549 cell line except GON-
DBCO-DOX, which had significantly (∗p < 0.05) higher uptake
(50.2%) than Caelyx®. However, the DOX fluorescence intensity was
significantly enhanced when the incubation time of the A549 cells with
the nanoparticles was increased. After incubation for 4 h, the fluores-
cence intensity of GON-DBCO-DOX-treated cells was significantly
higher (∗∗∗p < 0.001) than that of the Caelyx®- and GON-DOX-treated
cells (Fig. 9a). In MCF-7 cells, GON-DBCO-DOX treatment resulted in
significantly higher uptake of 46.5% (∗p < 0.05) and 83.1%
(
∗∗∗p < 0.001) at 1 and 4 h, respectively, compared with GON-DOX
4. Conclusions
(37.9% and 51%) and Caelyx® (39.4% and 61.1%, Fig. 9b). These data
show that DOX-loaded GON-DBCO-DOX was internalized in A549 and
We have developed a promising clickable drug delivery system for
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a period of 7 days. Both GON-DOX and GON-DBCO-DOX exhibited
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stability in the blood and the sustained action upon reaching the tumor.
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Acknowledgment
This work was primarily supported by a grant from Ministry of Food
and Drug Safety – Republic of Korea (16173MFDS542) in 2016.
Ngo, J.T., Champion, J.A., Mahdavi, A., Tanrikulu, I.C., Beatty, K.E., Connor, R.E., Yoo,
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labeling of proteins. Nat. Chem. Biol. 5, 715–717.
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Declaration of interest
The authors declare no conflicts of interest.
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