Novel naproxen-peptide-conjugated amphiphilic dendrimer self-assembly micelles for targeting drug delivery to osteosarcoma cells

Article abstract of DOI:10.1039/c6ra15022e

The aim of the current study was to synthesize and prepare novel self-assembly micelles loaded with curcumin (Cur) based on naproxen (Nap)-conjugated amphiphilic dendrimers. The apoptosis-inducing capacity of Nap-conjugated dendrimers and curcumin, the efficiency of uptake and the potential molecular mechanism on human osteosarcoma cells were investigated. The Nap-conjugated amphiphilic dendrimers were successfully synthesized, and the corresponding Cur-loaded micelles were conventionally prepared via self-assembly. These micelles showed good drug-encapsulating capacity, physiochemical properties, and drug-release profiles. The cellular proliferation and uptake assay suggested that the Cur-M-Nap induced more apoptosis of MG-63 human osteosarcoma cells. The western blot results suggested that these Nap-modified micelles could enhance the Cur-inducing apoptosis, mainly via intrinsic pathways and inhibition of the inflammation pathway. In summary, the Cur-loaded amphiphilic dendrimer-based micelles were successfully developed and they enhanced drug delivery to MG-63 cells. Mechanistic experiments suggested that Cur loaded into amphiphilic dendrimer-based micelles had a higher proliferation-inhibiting ability than free Cur, and could induce more apoptosis.

Full text of DOI:10.1039/c6ra15022e

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: Y. Zhao, Q. Zeng,
F. Wu, J. Li, Z. Pan, P. Shen, L. Yang, T. Xu, L. Cai and L. Guo, RSC Adv., 2016, DOI: 10.1039/C6RA15022E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Please do not adjust margins
RSC Advances
Page 1 of 9
View Article Online
DOI: 10.1039/C6RA15022E
RSC Advances
ARTICLE
Novel naproxen-peptide conjugated amphiphilic dendrimers self-
assembly micelles for targeting drug delivery to osteosarcoma
cells
Received 00th January 20xx,
Accepted 00th January 20xx
†
†
†
Yinbo Zhao,a, b, Qi Zeng,c, Fengbo Wu,a,c, Jing Li,c,* Zhaoping Pan,a Pengfei Shen,c Lu Yang,c Ting
DOI: 10.1039/x0xx00000x
Xu,c Lulu Cai,b,c,* and Li Guo,a,*
www.rsc.org/
The aim of current study was to synthesize and prepare a novel self-assembly micelles loading curcumin (Cur) based on the
naproxen (Nap) conjugated amphiphilic dendrimers. The apoptosis inducing capacity of Nap conjugated dendrimers and
curcumin, the efficiency of uptake as well as potential molecular mechanism on human osteosarcoma cells were
investigated. The Nap conjugated amphiphilic dendrimers were successfully synthesized, and the corresponding Cur
loaded micelles were conventional prepared via self-assembly. These micelles showed good drug encapsulating capacity,
physiochemical properties, as well as drug release profiles. The cellular proliferation and uptakage assay suggested that
the Cur-M-Nap induced more apoptosis of MG-63 human osteosarcoma cells. The western blot results suggested that
these Nap modified micelles could enhanced the Cur inducing apoptosis mainly via intrinsic pathway and inhibition of
inflammation pathway. In summary, the Cur loaded amphiphilic dendrimers based micelles were successfully developed
and enhanced drug delivery to MG-63 cells. Mechanistic experiments suggested that Cur loaded in amphiphilic
dendrimers based micelles had higher proliferation inhibiting ability than free Cur, and could induce more apoptosis.
layer groups. Moreover, dendrimers could be condensed into
nanoscale dendritic boxes, which are characterized by the
good encapsulation efficiency of guest molecules, regular
Introduction
As repetitively branched synthetic macromolecules,
dendrimers possess homogeneous structures from its interior
layers to the surface layers, especial in the branching units. It is
well established that dendrimers bear a series of unique
intrinsic properties, such as monodispersity molecular weight,
good biocompatibility, and spherical three-dimensional
structures with well-defined surface functionalities.1-6
Dendrimers have exhibited great application potentials in
many fields, including drug/gene delivery, catalysts as well as
bioimaging. Among them, applications of co-delivery of drugs
and/or genes by using dendrimers stand out and have been a
key focus of research in nanotechnology and nanobiology.7, 8
Because bioactive compounds could be trapped into the
interior cavities of dendrimers via hydrophobic or electrostatic
interactions, dendrimers could act as efficient nanocarriers of
bioactive compounds.9-12 On the other hand, the targeted
capacity of dendrimers predominantly depends on its exterior
shape, nanosize, and the formation of functional surfaces.6, 7,
13-17
Naproxen (Nap, see in figure 1) is a member of non-
steroidal anti-inflammatory drugs (NSAIDs). As one of the most
common used cyclooxygenase (COX) inhibitor, it has been used
for the treatment of many inflammation associated conditions,
e.g. arthritis, gout, tendinitis, bursitis, etc.18-20 Though some of
COX-2 inhibitors were demonstrated to be related to increases
the risk of cardiovascular events and gastrointestinal adverse
effects.21, an emerging body of data suggested that Nap rarely
increases the risk of adverse cardiovascular events compared
to other NSAIDs. Moreover, the development of urinary
bladder cancer has been demonstrated to be inhibited by Nap
treatment, but the detailed molecular mechanism as well as
binding targets remained unclearly.22-29 Recently, we designed
and synthesized several types of Nap modified peptide
dendrimers with good drug delivery properties.30-35 In addition,
the peptide dendrimers constituted by glutamic acid or
aspartic acid exhibited potent selectivity to bone tissues.31, 36-40
Herein we further designed and developed Nap and oligo-
aspartic acid modified dendrimers as an probable targeted
carrier of osteosarcoma.
a.Key Laboratory of Drug Targeting and Drug Delivery Systems of Ministry of
Education, Department of Medicinal Chemistry, West China School of Pharmacy,
Sichuan University, Chengdu 610041, P.R. China.
b.Department of Pharmacy, Hospital of the University of Electronic Science and
Technology of China and Sichuan Provincial People's Hospital, Chengdu 610041,
P.R. China.
Curcumin (alias as diferuloylmethane, see figure 1) is a
natural product extracted from of the herb Curcuma longa
(also named turmeric). It has been reported that the
therapeutic effects of Cur, such as anti-inflammation,
c. Department of Pharmacy and State Key Laboratory of Biotherapy, West China
Hospital, West china Medical School, Sichuan University, Chengdu 610041, P.R.
China.
† These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 2 of 9
View Article Online
DOI: 10.1039/C6RA15022E
ARTICLE
Journal Name
antioxidant, anticancer activity, and so on.41-45 It should be mixtue of water and methanol and the elute rate of 1.0
notable that Cur can efficiently suppress numbers types of mL/min was used. Naproxen and were obtained from energy
cancers, including breast cancer, colon cancer and chemical Co., Ltd.
47
osteosarcoma.46,
Moreover, Cur has regulated the
The L-aspartic acid, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-
bromide (MTT), 1-(3-
We also dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride
transcription factors, adhesion molecules, growth factors, diphenltetrazolium
apoptotic and autophagy-related proteins.42,
48-55
demonstrated synergistic antitumor effects of curcumin and a (EDCI) and O-Benzotriazole-N,N,N,N-tetramethyl-uronium-
H2S release prodrug of NSAIDs.56 In addition, the phase 2 hexafluorophosphate (HBTU) were purchased from GL
clinical trial of the prevent effect of Cur for colorectal cancer biochem. Co., Ltd. (Shanghai, China). The N-protected amino
treatment has achieved exciting results. However, its water acids were purchased from Chengdu Kaijie Co. Ltd. (Chengdu,
insolubility, instability for illumination, heat and alkaline China). The ultrapure water was prepared from Milli-Q water
obviously limits the further clinical application of Cur in cancer system without specification. The MG-63 human
therapy. The encapsulation of curcumin into nano- osteosarcoma cell lines were obtained from the ATCC
formulations has been report to rendered Cur dispersible in (American Type Culture Collection, Rockville, MD, USA). MG-63
aqueousand these nano-formulations displayed prior drug cells were incubated in the DMEM (Dulbecco's modified eagle
release behaviour and enhanced stability.45, 47, 49, 53, 56, 57
medium, Gibco, USA) and supplemented with 10% fetal bovine
serum (FBS, Gibco, USA). All cells were maintained at 37 °C in
humidified incubator containing 5% CO2.
Synthesis and characterization of Nap conjugated dendrimers
The synthesis process of Nap and oligo-aspartic acid
modified dendrimers were followed our previously reported
method and made some modifications, the synthetic route
was showed in Scheme 1. As shown in scheme 1A, the benzyl
acrylate was coupling with di-ethanol amine via a michael
Figure 1. Molecular structures of Naproxen and Curcumin.
Using mPEG-PLGA nanoparticels, the potential synergistic
effect of curcumin and H2S release prodrug of aspirin was
observed in the ovarian cancer cells. , However, the effective
therapeutic concentration of curcumin is far lower than that of
NSAIDs, which mades it difficult to assure the homogeneity
and stability of the co-delivery system. In this study, we
designed, synthesized and characterized a series of naproxen
conjugated amphiphilic dendrimers bearing a oligo-aspartic
acids tailor. The dendrimers are capable of rapidly transfering
into micelles via self-assembly and efficiently encapsulate
curcumin with satisfied loading amount. These Cur loaded
micelles displayed apoptosis inducing capacity inhibition on
the MG-63 osteosarcoma cells. The mechanism studies
suggested inflammation and apoptosis related proteins were
involved in the action mechanism of Cur loaded micelles.
addition reaction to form intermediate
hydroxyl groups were esterification by the carboxyl group of
naproxen to form compound , under the catalysis of EDCI and
4-N,N-dimethylaminopyrimidine. After the hydrogenation
catalyzed by 10% Pd-C, the benzyl of was removed to give
the intermediate Similar coupling and deprotection
reactions were performed on compounds 1 and 3 to form
intermediate . The structures of intermediates 1 to 4 were
determined by 1H NMR. The oligo-aspartic acid peptide and its
Nap-dendrimer conjugates and were synthesized by
1. Then the two
2
2
3.
4
5,
6
7
standard solid phase peptide synthesis (SPPS) protocols on the
CSBio 136XT peptide synthesis instrument. The peptide was
purified by high-performance liquid chromatography (HPLC) to
95% purity.
Determination of the critical micelle concentration (CMC)
The CMC of Compound 6 was determined according to the
58, 59
method from our previuos reports and literatures.56,
Materials and methods
Briefly, the so-called pyrene probe method had been used for
the determination of this important parameter in micelle
systems. Fluorescence spectra were recorded on a PerkinElmer
LS55 fluorescence spectrophotometer, and the pyrene was
used as a fluorescence probe. Samples for fluorescence
measurement were prepared, and the concentrations of
dendrimer 6 were 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01,
0.025, 0.05, 0.10, 0.20, 0.50 and 1.0 mg/mL. The pyrene
concentration in the aqueous solutions was 6.0×10−7 mol/L.
For the measurement of pyrene excitation and emission
spectra, the slit widths for excitation spectra was maintained
at 8 nm and the slit widths for emission sides was 2.5 nm, and
the emission spectra were recorded between 373 nm and 384
nm with the excitation wavelength at 334 nm. The intensity
ratios of the first (374 nm) to the third (384 nm) vibronic peaks
Materials and regents
All reagents were obtained at analysis grade from
commercial sources, and directly used unless special reminder.
The intermediates and compounds were purified by flash
chromatography using 200-300 mesh silica gel. 1H NMR
spectra were recorded on
a
Bruker Avance III-400
spectrometer at 400 MHz. The chemical shifts (δ) of NMR
relative to an internal control tetramethylsilane (TMS) were
recorded in parts per million (ppm) units. The mass spectrum
were performed on a Waters Bio-QTOF instrument. The
purities of all compounds were above 95.0% determined by
HPLC normalization method on the Shimadzu LC-20A
instrument with a Diamonsil C18 column (5.0 μm, 4.6 × 250
mm, 5µm, Dikma Technologies). The mobile phase was a
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 3 of 9
View Article Online
DOI: 10.1039/C6RA15022E
Journal Name ARTICLE
(I3/I1) were plotted as
concentration.
a
function of the dendrimer
6
The three Nap and oligo-aspartic acid heptapeptide
conjugated dendrimers were synthesized and tested as self-
assembling micelles. However, compound 7 was insoluble in
both PBS buffer and most of organic solutions, only slightly
soluble in DMSO. Compound 5 exhibited good water-solubility,
but poor solubility in organic solvents . Therefore, only
compound 6 was chosen for further subsequent investigations.
The curcumin loaded micelles of Nap conjugated dendrimers
(Cur-M-Nap) and Cur free micelles of Nap conjugated
dendrimers (M-Nap) were prepared as following: curcumin
and dendrimer (1:19, w/w) were fully dissolved in acetone,
then the solution was removed by vacuum evaporation. The
Nap conjugated amphiphilic dendrimers were re-dissolved into
normal saline to form M-Nap. For the preparation of Cur-M-
Nap, the curcumin was solved in acetone with Nap conjugated
dendrimers, and similar procedures were performed to
prepare M-Nap. The obtained micelles were filtered and
lyophilized into powder before use. The particle size and zeta
potential distribution of Cur-M-Nap were determined by
Malvern ZS-90 laser particle size analyzer (Worcestershire,
UK.) . The microscopic morphological characterization of Cur-
M-Nap was performed on the transmission electron
microscope (TEM, H-6009IV, Hitachi, Tokyo, Japan) after
negatively staining by phosphotungstic acid. The drug loading
(DL) and encapsulation efficiency (EE) were determined by
high performance liquid chromatography (HPLC) method on a
Shimadzu HPLC instrument (Shimadzu LC-20A, Milford, MA,
Japan) equipped a PDA (Photo-Diode Array) detector and a
reversed phase C18 column (4.6
× 150 mm, 5 μm,
Inertsil/WondaSil, Japan). The DL and EE of Cur-M-Nap were
calculated according to the following equations:
DL = Drug/(Drug + Polymer) × 100% (1)
EE = Drug in micelles/drug in feed × 100% (2)
The release profiles of curcumin and naproxen from Cur-M-
Nap or free curcumin were investigated by the dialysis method.
In brief, 1 mL of free curcumin solution or Cur-M-Nap were
placed into dialysis bags (molecular weight cut off = 3500),
then incubated at 37 °C with gentle shaking (100 rpm) in 50 mL
of neutral phosphate buffered solution (PBS) containing
Tween80 (0.5 wt %). After given time intervals, dialysis
medium was withdrawn and replaced with the same volume of
fresh buffer. The cumulative amount of released curcumin or
naproxen were analyzed and quantified by HPLC. All the
results were the mean value of three test runs and all data
were shown as the mean ± SD.
Cellular proliferation inhibition and uptake of the micelles
The cellular proliferation inhibition capacity of Cur-M-Nap,
M-Nap and free Cur were preformed in MG-63 cells by MTT
assay. In brief, the MG-63 cells were cultured in 96-well plates,
treated with Cur-M-Nap, M-Nap and free Cur under different
concentration gradient for 48 h, respectively. The mean
inhibition percentage of cell proliferation relative to that of
control groups were collected from data of three individual
experiments, and all the data were expressed as mean ± SD. In
the cellular uptake experiment, MG-63 cells were suspended
and seeded in the six-well plate (5 × 104 cells per well), and
incubated under 37 °C in humidified incubator containing 5%
Scheme 1. Synthetic route of Nap conjugated dendrimers.
Preparation and characterization of Cur loaded micelles
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 4 of 9
View Article Online
DOI: 10.1039/C6RA15022E
ARTICLE
Journal Name
CO2 for further 24 hours. Then the Cur-M-Nap or free Cur were aspartic acid α-hydrogen signals around δ 4.5 of compound 6
added into each well, respectively. The cultures MG-63 cells was found. As shown in Figure 1,compound 6 and 7 have
were further incubated for one hour and digested by trypsin, similar structure ,so there are something in common in their
re-suspended by washed with cold PBS twice. Finally, each 1H-NMR spectra which were shown in details as followed.The
sample was examined by fluorescent microscopy and flow multiplet signals at ꢀ 7.07–7.67 were attributed to the
cytometry to qualify the fluoresce strength of intracellular aromatic ring from naproxen. And the multiple signals at
curcumin of each group.
ꢀ3.98–4.06 came from the methylene(-CH2-O-) connected to
oxygen atom. The strong single peak at 3.89 was assigned to
Apoptosis assay by flow cytometry
ꢀ
The activities of Cur-M-Nap, M-Nap and free Cur to induce the methyl(CH3-O-) linked to the the oxygen atom. The weak
apoptosis in MG-63 cells were performed on a flow cytometry. peaks at 3.75–3.80 belonged to the methyne in naproxen.The
In brief, MG-63 cells were seeded in 6-well plates and strong peaks at 1.52–1.54 came from proton of methyl of
incubated for 24-48 hours. Cells in different groups were naproxen. The weak peaks at 2.79–2.94 in compound 6
ꢀ
ꢀ
ꢀ
exposed to DMEM culture containing Cur-M-Nap, M-Nap or belonged to the methylene(-CH2-N-) connected to the
free Cur for additional 48 hours, respectively. Then cells were nitrogen atom, while the same protons in compound 7 were
re-suspended by trypsin digestion, washed with PBS and migrated to
ꢀ
2.5. That is a normal phenomenon in 1H-NMR
stained with Annexin V/Propidium Iodide (BD PharMingen, San spectra. Because of compound 6 was insoluble in CDCl3, it was
Diego, CA, USA) kit. The cell counts of early apoptotic (Annexin dissolved in D2O, so the solvent peak was shifted from 7.25 to
V+/PI−) and late apoptoꢀc (Annexin V+/PI+) were quantified by 4.7. These data confirmed the chemical structure of compound
flow cytometry apparatus.
6. For the NMR spectra of compound 7, DMSO-d6 was used as
Western Blot analysis
solvent, because it was insoluble both CDCl3 and D2O. Its peaks
cells were treated by Cur-M-Nap, M-Nap and free Cur and were similar to those of compund 6, and the relative integral
their total proteins were extracted by protein extract kit buffer, areas of naproxen to oligo-aspartic acid heptapeptides were
respectively. The supernatant of cell lysates were collected about two fold than compound 6. The high resolution mass
after frozen centrifuged at 12,000 rpm for 10 min. (Pierce, spectrum results of compound 6 and 7 were 1405.5 (M-H, calc.
Waltham, MA, USA). Before SDS-PAGE gel electrophoresis, the 1405.4) and 2147.7 (M-H, calc. 2147.8), respectively.
O
OH
BCA protein assay kit was used for quantifing protein
O
N
OH
concentration, then the total protein were separate and
transferred into PVDF membranes. The primary antibodies
were added and incubated with PVDF membranes for
overnight at 4 °C or two hours at room temperature. To avoid
the non-specfic interactions, the PVDF membrane was
incubated with skimmed milk or bovine serum at room
temperature, washed with PBS several times and then
incubated with HRP-conjugated IgG (Abmart, Shanghai, China)
for several hours. The target proteins were visualized using
enhanced chemiluminescence (Millipore, Billerica, MA, USA)
according to the manufacturer’s protocols.
1
O
O
HO
N
O
O
O
O
O
3
(f)
(a)
O
O
(e)
(d)
(b)
N
N DDDDDDD
H
O
(c)
O
O
O
(c)
6
O
(f)
(a)
(e)
(b)
(d)
O
Results
O
O
O
O
O
N DDDDDDD
H
N
N
O
O
O
O
Characterization of Nap conjugated dendrimer
O
(e)
(c)
N
O
O
7
The synthesis of Nap and oligo-aspartic acid modified
dendrimers were partially referenced to our previous reports
(
e)
O
O
(f)
1
and made some modifications.22,28 In Figure 1 the H nuclear
(a)
O
magnetic resonance (1H NMR) spectrum of intermediates 1, 3
and dendrimer 6, 7 were represented, respectively. As shown
in Figure 2, the signal of phenyl hydrogen atoms, benzyl group
O
(b) (d)
hydrogen atoms and the other methylene group hydrogen Figure 2. The 1H NMR spectrum of intermediates and Nap
atoms in intermediate 1 were found as expected; The aromatic conjugated dendrimers.
hydrogen signals of the intermediate 3, including the single
peak of methyloxyl hydrogen atoms and the double peaks of Preparation and Characterization of Cur loaded micelles
methyl hydrogen atoms, suggested that the naproxens were
successfully conjugated to the dendrimer scaffold. The following a self-assembly method, the mean size of curcumin
intermediate was coupled with oligo-aspartic acid loaded micelles were about 35±6 nm (Figure 3A), with the
In general, the preparation of Cur-M-Nap was utilized by
3
heptapeptides to afford compound 6. Compared to the spectra optimum drug/polymer (w/w) ratio, the optimal drug loading
of intermediate 3, the multiple signals of aspartic acid β- (DL) and entrapped efficiency (EE) as 1:19, 4.6% ± 0.3% and
methylene group hydrogen signals at δ 2.8-3.0, and the 91.9% ± 3.1%, respectively. Furthermore, the polydisperse
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 5 of 9
View Article Online
DOI: 10.1039/C6RA15022E
Journal Name ARTICLE
index (PDI), and zeta potential of obtained curcumin loaded probe into the hydrophobic region of the micelles. According
Cur-m-nAP micelles were 0.24 ± 0.03, and -34.6 ± 7.2 mV, to figure 3E, the CMC of dendrimer 6 was around 10.6 g/mL,
µ
respectively (Figure 3B). The atomic force microscopy (AFM) which suggested that dendrimer 6 could self-assembled into
image of Cur-M-Nap was shown in Figure 3C, and it suggested micelles at the therapeutic concentration.
that the Cur-M-Nap was nearly spherical in shape and with a
In vitro release profile of curcumin and naproxen from Cur-
uniform diameter of 30-40 nm. The analysis of particle size and M-Nap were studied at 37 °C and pH 7.4 and pH 6.5. These
microscopic structure of micelles performed by AFM proved results suggested that curcumin can be well encapsulated in
that the homogenous and stable Cur-M-Nap aqueous disperse Cur-M-Nap, and released in an extended period. As shown in
could be achieved by loading curcumin into Nap conjuageted Figure 4, approximately 52% of total curcumin released after
dendrimer based micelles.
24 h, followed by release of 81% in 96 h, and there were not
significantly difference between pH 6.5 and 7.4. The free
curcumin could rapidly be released over 90% of total drugs
within two hours. And the naproxen was released from the
micelles with a slow manner. This cumulative drug release
suggests potential applicability of these micelles as drug
delivery system may result in a content pharmacokinetic
profile in vivo and reduce exposure of healthy tissues.
Figure 4. Time course of curcumin and naproxen releasing
from Cur-M-Nap at 37 °C and pH 7.4(A) and pH 6.5(B).
Released curcumin and naproxen were separated by dialysis
and quantified using HPLC.
Enhanced uptake of Cur after encapsulated in the micelles
The MG-63 osteosarcoma cells were treated with Cur-M-
Nap with or without oligo-aspartic acid heptapeptides, to
determine whether the increased drug uptake of Cur-M-Nap
to MG-63 cells. After incubation of free curcumin, Cur-M-Nap
and Cur-M-Nap with oligo-aspartic acid heptapeptides were
washed by PBS twice, then cells were collected and related
the curcumin-derived fluorescence was analysized by
fluorescent microscopy. The fluorescent microscopic images
(Figure 5 A-D) showed the difference of fluorescent intensity in
MG-63 cells treated with free curcumin, Cur-M-Nap and Cur-
M-Nap with oligo-aspartic acid heptapeptides, respectively.
The mean fluorescence intensity of Cur-M-Nap group was 7.2-
fold stronger (mean fluorescence intensity = 4.5 vs 32.5, p <
0.01) than free curcumin group on MG-63 cells. This result
suggested that the encapsulation into micelles increased the
uptake of the curcumin in MG-63 osteosarcoma cells.
Moreover, the uptake increase of Cur-M-Nap group was
reduced (mean fluorescence intensity = 32.5 vs 17.8, p < 0.05),
when the oligo-aspartic acid heptapeptides was added in the
culture. It was suggested that the binding of Cur-M-Nap to
MG-63 is a competitive manner.
Figure 3. Characterization of Cur-M-Nap. (A) particle size
distribution of Cur-M-Nap; (B) zeta potential of Cur-M-Nap; (C)
AFM image of Cur-M-Nap; (D) the TEM image of Cur-M-Nap; (E)
The determination of the CMC of dendrimer 6.
As shown in figure 3E, with the increase of concentration
of dendrimer 6, the intensity ratio exhibited a substantial
increase at a certain concentration, suggested that pyrene
probes were incorporated into the hydrophobic core upon
micelle formation. The critical micelle concentrations (CMC)
were therefore determined from the crossover point at the
low concentration range. It was well known that the plots of
the pyrene I3/I1 ratio as a function of the total surfactant
concentration show, around the CMC, a typical sigmoidal
decrease. Below the CMC, the pyrene I3/I1 ratio value
corresponding to a polar environment, on the other hand,
when the surfactant concentration increased, the pyrene I3/I1
ratio declined rapidly, indicated that the pyrene was
encapsulated into a more hydrophobic environment. When
concentration over the CMC, the pyrene I3/I1 ratio reached a
roughly constant value because of the incorporation of the
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 6 of 9
View Article Online
DOI: 10.1039/C6RA15022E
ARTICLE
Journal Name
1.2
1.0
0.8
0.6
0.4
0.2
M-Nap
Cur-M-Nap
Free curcumin
Free Cur + Free Nap
0.0
1
10
100
1000
Concentration (µg/mL)
Figure 6. The in vitro cytotoxicity of the free curcumin, free
curcumin combination with naproxen, M-Nap, and Cur-M-Nap
on MG-63 cells. The percentage of viable cells was quantified
using the methylthiazoletetrazolium (MTT) method.
In vitro apoptosis assay was preformed using flow
cytometry by Annexin V/PI staining to investigate the
apoptosis inducing potency of Cur-M-Nap, M-Nap and free
curcumin. Both early apoptosis (Annexin V+/PI−) and late
Figure 5. Different cellular uptake of free curcumin and Cur-M- apoptosis (Annexin V+/PI+) cells were included. According to
Nap on MG-63 cells. (A) Phase microscopic and fluorescent Figure 7, the percentage of apoptotic cells in Cur-M-Nap group
microscopic images of PBS group (Control); (B) Phase was 78.0% ± 7.2%, which was significantly higher than that of
microscopic and fluorescent microscopic images of MG-63 apoptotic cells in free curcumin (47.6% ± 4.1%), M-Nap (16.5%
cells treated by free curcumin (Free curcumin); (C) Phase ± 1.2%) and control (0.39 % ± 0.12%) groups. It was notably
microscopic and fluorescent microscopic images of MG-63 that M-Nap only slightly induced early apoptosis on MG-63
cells treated by Cur-M-Nap (Cur-M-Nap); (D) Phase cells. There were significant difference between the Cur-M-
microscopic and fluorescent microscopic images of MG-63 Nap and free curcumin groups. The late apoptosis cells in the
cells treated by Cur-M-Nap and oligo-aspartic acid Cur-M-Nap (58.9 % ± 6.7%) were more than that in free
heptapeptide (Cur-M-Nap + Asp heptapeptide); and (E) the curcumin (4.49%
± 1.07%,) groups. However, the early
quantification of the mean fluorescence intensities of each apoptotic cells in the free curcumin group (43.1% ± 3.8%) were
groups.
more than that of Cur-M-Nap group (19.1% ± 2.0%). The
results in FCM assay demonstrated that Cur-M-Nap could
induced strongly cellular apoptosis, probably due to the
In vitro cytotoxicity and apoptosis assay of the micelles
In vitro cellular proliferation assay were performed by MTT synergistic effects of curcumin and M-Nap.
method in MG-63 cells. After incubation 48 hours with the free
curcumin, M-Nap or Cur-M-Nap, the cell survive ratios were
measured following the absorbance of the degraded MTT at
570 nm. The cell survive ratios were decreased according to
the increase of curcumin concentration, and there were
significant difference between the Cur-M-Nap and free
curcumin groups as well as the combination of free curcumin
and naproxen (see Figure 6). Moreover, there was none
significant cell proliferation inhibition observed even in M-Nap
concentration up to 1.0mg/mL. As shown in Figure 6, the
median inhibitory concentrations (IC50) of curcumin in Cur-M-
Nap were about 21.2 μg/mL, which was less than that of free
curcumin (57.6 μg/mL).
Figure 7. Flow cytometric analysis of MG-63 cells stained with
Annexin V-FITC/PI after treatment with control group (A), 10
μM of M-Nap (B), free curcumin (C) or Cur-M-Nap (D).
Potential molecular mechanism of Cur-M-Nap
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 7 of 9
View Article Online
DOI: 10.1039/C6RA15022E
Journal Name ARTICLE
The expression level of apoptosis related proteins in MG-63
Curcumin is
a
promising proliferation inhibitor and
cells treated by Cur-M-Nap and free curcumin were evaluated apoptosis inducer in MG-63 osteosarcoma cells, but its poor
by western blot assays. Among tested proteins, the water solubility limited the further development and
mitochondrial pathway related apoptotic proteins were application in cancer therapy. In recent years, numbers of
concerned. Generally, the apoptotic cell death was usually nano-formulations provide effective methods to improve the
triggered by an intrinsic pathway, i.e. mitochondria pathway or aqueous solubility of hydrophobic drugs. Besides, as a classical
the extrinsic pathway involving death receptors. The death COX-2 inhibitor and NSAID, naproxen could mediated breast
receptor pathway could be activated by the ligands of the cancer cell death via inducing early apoptosis followed by
tumor necrosis factor super-family, such as Fas, TRAIL, IFN-γ or activation of intrinsic caspase-cascade. Furthermore, naproxen
TNF-α, etc. The mitochondrial pathway was involved in many could inhibite synthesis of PGE-2 and suppress cancer cell
apoptotic proteins such as cytochrome c, apoptosis inducing migration in vitro. Therefore, the combination of the curcumin
factor (AIF), caspase-3, caspase-9, the Bcl-2 family members. and naproxen might improve their therapeutic activities
According to literatures' reports, curcumin mainly induced synergistically. In our previous study, H2S release prodrug of
apoptosis by a mitochondria pathway. The initial mechanistic NSAID and Cur were encapsulated into mPEG-PLGA
studies showed that Cur-M-Nap and free curcumin increased nanoparticles and it showed synergistic anticancer effects.
the levels of Bax and decreased the levels of Bcl-2 (Figure 8).t However, the dosage of NSAID was higher than that of
Interestingly, the alterations in Cur-M-Nap groups were more curcumin, it was hardly encapsulated the NSAID and curcumin
apparent than that in free curcumin group, which indicated with the appropriate ratios into
a nano-formulation
free curcumin and Cur-M-Nap induced apoptosis mainly owing simultaneously and homogenously.60 In the current study, we
to mitochondrial pathway. Because mitochondria apoptosis reported the synthesis, characterization and in vitro evaluation
could activate the proapoptotic caspase cascade, we next of a curcumin loaded Nap conjugated peptide dendrimer
investigated whether caspases
3 and 9 were activated. based micelles through a modified self-assembling method.
Treatment of MG-63 cells with curcumin or Cur-M-Nap could The prepared micelles had a mean particle size of 35±6 nm and
efficently increase the expression levels of the cleaved forms were monodispersed (PDI =0.24±0.03) in PBS. The obtained
of caspases 3 and 9, and eliminated the levels of original Cur-M-Nap was electro negative because of the oligo-aspartic
caspase9 proteins (Figure 8). These results provided additional acid heptapeptides on the shell of micelles. The stability of the
evidence that curcumin induced apoptosis in the nanoparticles was increased by the negative zeta potential.
mitochondrial pathway, and the Nap conjugated dendrimer Hence, the Cur-M-Nap could form stable nano-formulation
could enhance the apoptosis-inducing capacity of curcumin to without mPEG segment or other steric stabilizer. In the Cur-M-
MG-63 human osteosarcoma cells.
Nap, the encapsulation efficiency of curcumin was 91.9% ±
3.1%, with a DL rate of 4.6%±0.3%. After encapsulation,
curcumin were molecularly dispersed in the naproxen core of
dendrimer based micelles, and they could be steady released
from the micelles in vitro. In the cytotoxicity and apoptosis
assay, the Cur-M-Nap retained the cytotoxicity of Cur on the
MG-63 human osteosarcoma cells in vitro, which shows its
better apoptotic inducing ability than that of either M-Nap or
free curcumin alone. Our results further suggested that the
Cur-M-Nap induced more late apoptosis in MG-63 cells by
triggering the mitochondria signaling pathway.
Nanotechnology provided alternative strategies to solve
the issue of poor water solubility of hydrophobic drugs.60-64
Moreover, there were great interests in multidrug co-delivery
that could encapsulate and deliver two or more types of
therapeutics simultaneously. Although the conventional
polymeric nanoparticles have many advantages, such as
reduced nonspecific cellular uptake and long circulation
lifetime, but there were still difficult on the precise dosage
control of multidrug encapsulation for combinatorial
treatment. In the present study, we applied a combination
therapy on human osteosarcoma cells with Cur-M-Nap.
Naproxen acted as hydrophobic core of the micelles, and the
curcumin was encapsulated in the micelles with high
entrapped efficiency. Compared to free curcumin, the micelles
not only retained the cytotoxicity and apoptosis inducing of
curcumin in the MG-63 cells, but also showed better apoptotic
inducing capacity. Furthermore, the relative fluorescence of
Figure 8. Free curcumin and Cur-M-Nap induces apoptosis via
mitochondrial pathways in MG-63 human osteosarcoma cells,
as detected by Western blot analysis, GAPDH (glyceraldehyde
phosphate dehydrogenase) served as a loading control.
Discussion
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 8 of 9
View Article Online
DOI: 10.1039/C6RA15022E
ARTICLE
Journal Name
curcumin absorbed into MG-63 was decreased significantly 8. S. Mignani, S. El Kazzouli, M. Bousmina and J. P. Majoral,
with addition of the free heptapeptide into the culture, which Advanced Drug Delivery Reviews, 2013, 65, 1316-1330.
suggested the oligo-aspartic acid heptapeptide might played 9. S. Mignani, S. El Kazzouli, M. Bousmina and J. P. Majoral,
uptake enhancing role for the Cur-M-Nap. Thus, the results Progress in Polymer Science, 2013, 38, 993-1008.
indicated that the co-delivery of naproxen and curcumin based 10. W. D. Tian and Y. Q. Ma, Chemical Society Reviews, 2013,
nanocarrier may provide a new vision in cancer therapy. The 42, 705-727.
combination of curcumin and naproxen resulted in increased 11. D. J. Bharali, M. Khalil, M. Gurbuz, T. M. Simone and S. A.
late apoptosis in MG-63 cells. The results of western blotting Mousa, International Journal of Nanomedicine, 2009, 4, 1-7.
suggested that the Cur-M-Nap mainly triggered the 12. W. D. Jang, K. M. K. Selim, C. H. Lee and I. K. Kang, Progress
mitochondrial-based intrinsic apoptosis pathway by activating in Polymer Science, 2009, 34, 1-23.
the cascade signaling pathway of caspase-3 and caspase-9. In 13. L. M. Kaminskas, V. M. McLeod, C. J. H. Porter and B. J.
summary, our developed Cur-M-Nap is an potential candidate Boyd, Molecular Pharmaceutics, 2012, 9, 355-373.
nanomedicine for the treatment of bone neoplasm, and 14. P. Kesharwani, K. Jain and N. K. Jain, Progress in Polymer
provide a novel platform for future design and discovery of Science, 2014, 39, 268-307.
bone-targeted drug delivery systems.
15. J. Lim and E. E. Simanek, Advanced Drug Delivery Reviews,
2012, 64, 826-835.
16. J. Liu, W. D. Gray, M. E. Davis and Y. Luo, Interface Focus,
Conclusion
2012, 2, 307-324.
17. R. E. Wang, F. Costanza, Y. H. Niu, H. F. Wu, Y. G. Hu, W.
Hang, Y. Q. Sun and J. F. Cai, Journal of Controlled Release,
2012, 159, 154-163.
In conclusion, a Nap-dendrimer micelles with loading
curcumin had been prepared for human osteosarcoma
chemotherapy. Its surfaces were modified with oligo-aspartic
acid heptapeptides. The prepared Cur-M-Nap possessed both
nanoscale particle size and potent negative zeta potential. In
vitro experiments, the drug-free M-Nap did not inhibited the
MG-63 cell proliferation and only slightly induced apoptosis.
Cur-M-Nap had higher cytotoxicity than that of free curcumin
in the cell proliferation assays, and induced more
mitochondrial apoptosis. Cellular uptake analysis showed that
the encapsulating of the micelles promoted the selective
uptake of curcumin by MG-63 cells.
18. D. Shcharbin, A. Shakhbazau and M. Bryszewska, Expert
Opinion on Drug Delivery, 2013, 10, 1687-1698.
19. R. M. Kannan, E. Nance, S. Kannan and D. A. Tomalia,
Journal of Internal Medicine, 2014, 276, 579-617.
20. G. Gasparini, R. Longo, R. Sarmiento and A. Morabito,
Lancet Oncology, 2003, 4, 605-615.
21. D. Y. Wei, L. W. Wang, Y. J. He, H. Q. Xiong, J. L. Abbruzzese
and K. P. Xie, Cancer Res, 2004, 64, 2030-2038.
22. E. F. J. de Vries, Curr Pharm Design, 2006, 12, 3847-3856.
23. J. L. Young, A. A. Jazaeri, C. J. Darus and S. C. Modesitt,
Gynecol Oncol, 2008, 109, 140-145.
24. L. Moreira and A. Castells, Current Drug Targets, 2011, 12
,
Acknowledgments
1888-1894.
This work was financially supported by the National Natural
Science Foundation of China (81402500 and 21472130), China
25. V. Jendrossek, Cancer letters, 2013, 332, 313-324.
26. A. Bhardwaj, J. Kaur, F. Wuest and E. E. Knaus,
Chemmedchem, 2014, , 109-116.
Postdoctoral
Science
Foundation
Funded
Project
9
(2014M560720), and Support Foundation of Science and
Technology Department of Sichuan Province (2014FZ0039).
27. M. Conda-Sheridan, S. S. Lee, A. T. Preslar and S. I. Stupp,
Chem Commun, 2014, 50, 13757-13760.
28. J. Deb, J. Majumder, S. Bhattacharyya and S. S. Jana, BMC
cancer, 2014, 14, 567.
References
29. J. A. Miranda, P. Stanley, K. Gore, J. Turner, R. Dias and H.
1. J. P. Tam, Journal of Immunological Methods, 1996, 196
,
Rees, Plos One, 2014, 9.
17-32.
30. T. M. Motawi, Y. Bustanji, S. El-Maraghy, M. O. Taha and M.
A. Al-Ghussein, Journal of enzyme inhibition and medicinal
chemistry, 2014, 29, 153-161.
2. R. Esfand and D. A. Tomalia, Drug Discovery Today, 2001, 6,
427-436.
3. A. R. Menjoge, R. M. Kannan and D. A. Tomalia, Drug
Discovery Today, 2010, 15, 171-185.
31. R. Kodela, N. Nath, M. Chattopadhyay, D. E. Nesbitt, C. A.
Velazquez-Martinez and K. Kashfi, Drug design, development
4. J. M. Oliveira, A. J. Salgado, N. Sousa, J. F. Mano and R. L.
Reis, Progress in Polymer Science, 2010, 35, 1163-1194.
5. M. A. Mintzer and M. W. Grinstaff, Chemical Society
Reviews, 2011, 40, 173-190.
and therapy, 2015, 9, 4873-4882.
32. S. La Maestra, F. D'Agostini, A. Izzotti, R. T. Micale, L.
Mastracci, A. Camoirano, R. Balansky, J. E. Trosko, V. E. Steele
and S. De Flora, Carcinogenesis, 2015, 36, 1531-1538.
6. S. Sadekar and H. Ghandehari, Advanced Drug Delivery
Reviews, 2012, 64, 571-588.
33. T. I. Oprea, L. A. Sklar, J. O. Agola, Y. Guo, M. Silberberg, J.
Roxby, A. Vestling, E. Romero, Z. Surviladze, C. Murray-Krezan,
A. Waller, O. Ursu, L. G. Hudson and A. Wandinger-Ness, Plos
7. K. Hatano, K. Matsuoka and D. Terunuma, Chemical Society
Reviews, 2013, 42, 4574-4598.
One, 2015, 10
.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 9 of 9
View Article Online
DOI: 10.1039/C6RA15022E
Journal Name ARTICLE
34. G. He and L. Guo, Chinese J Org Chem, 2008, 28, 1326- 58. S.-F. Peng, C.-Y. Lee, M.-J. Hour, S.-C. Tsai, D.-H. Kuo, F.-A.
1335. Chen, P.-C. Shieh and J.-S. Yang, International Journal of
35. L. Ouyang, W. C. Huang, G. He and L. Guo, Lett Org Chem, Oncology, 2014, 44, 238-246.
2009, , 272-277. 59. S.-J. Yang, S. A. Lee, M.-G. Park, J.-S. Kim, S.-K. Yu, C. S. Kim,
36. J. Z. Pan, L. Guo, L. Ouyang, D. Q. Yin and Y. Zhao, Synlett, J.-S. Kim, S.-G. Kim, J.-S. Oh, H.-J. Kim, H. S. Chun, Y. H. Kim and
2012, DOI: 10.1055/s-0031-1290403, 1937-1940. D. K. Kim, Oncology Reports, 2014, 31, 2286-2292.
37. J. Z. Pan, L. F. Ma, B. Li, Y. H. Li and L. Guo, Synthetic 60. L. Zhou, X. Duan, S. Zeng, K. Men, X. Zhang, L. Yang and X.
Commun, 2012, 42, 3441-3454. Li, Int J Nanomedicine, 2015, 10, 5205-5218.
38. J. Z. Pan, M. Wen, D. Q. Yin, B. Jiang, D. S. He and L. Guo, 61. R. Chang, L. Sun and T. J. Webster, International Journal of
Tetrahedron, 2012, 68, 2943-2949. Nanomedicine, 2014, , 461-465.
39. B. Jiang, J. Zhao, Y. H. Li, D. S. He, J. Z. Pan, J. H. Cao and L. 62. C. Gao, D. Wei, H. Yang, T. Chen and L. Yang, International
6
9
Guo, Lett Org Chem, 2013, 10, 594-601.
Journal of Nanomedicine, 2015, 10, 5139-5157.
40. G. He and L. Guo, Chinese Chem Lett, 2007, 18, 1505-1508.
63. J.-Y. Huang, Y.-K. Lai, F. Pan, L. Yang, H. Wang, K.-Q. Zhang,
41. G. He, L. Guo, L. F. Ma, Q. Q. Wang and Z. C. He, Acta Chim H. Fuchs and L.-F. Chi, Small, 2014, 10, 4865-4873.
Sinica, 2007, 65, 21-26. 64. T. Nhiem and T. J. Webster, Nanotechnology, 2013, 24
42. L. Ouyang, J. Z. Pan, Y. Zhang and L. Guo, Synthetic 65. S. Jain, T. J. Webster, A. Sharma and B. Basu, Biomaterials,
Commun, 2009, 39, 4039-4052. 2013, 34, 4891-4901.
43. L. Ouyang, L. F. Ma, B. Jiang, Y. H. Li, D. S. He and L. Guo, 66. N. G. Durmus, E. N. Taylor, K. M. Kummer and T. J.
.
Eur J Med Chem, 2010, 45, 2705-2711.
44. D. S. He, G. He and L. Guo, Lett Org Chem, 2011,
460.
Webster, Advanced Materials, 2013, 25, 5706-+.
8, 454-
45. Y. Li, J. Zhang, D. Ma, L. Zhang, M. Si, H. Yin and J. Li, Febs
Journal, 2012, 279, 2247-2259.
46. H. M. Collins, M. K. Abdelghany, M. Messmer, B. Yue, S. E.
Deeves, K. B. Kindle, K. Mantelingu, A. Aslam, G. S. Winkler, T.
K. Kundu and D. M. Heery, BMC cancer, 2013, 13
47. Q. Huang, L. Zhang, X. Sun, K. Zeng, J. Li and Y.-N. Liu, Rsc
Advances, 2014, , 59211-59217.
.
4
48. J. M. Moran, F. J. Rodriguez-Velasco, R. Roncero-Martin, V.
Vera and J. D. Pedrera-Zamorano, International Journal of
Nanomedicine, 2014,
49. P. Liu, L. Sun, D.-s. Zhou, P. Zhang, Y.-h. Wang, D. Li, Q.-h. Li
and R.-j. Feng, Scientific Reports, 2015,
50. C. Yao, M. Hedrick, G. Pareek, J. Renzulli, G. Haleblian and
9, 5273-5275.
5.
T. J. Webster, International Journal of Nanomedicine, 2013, 8,
3285-3296.
51. R. Chang, L. Sun and T. J. Webster, International Journal of
Nanomedicine, 2015, 10, 3351-3365.
52. D. K. Walters, R. Muff, B. Langsam, W. Born and B. Fuchs,
Investigational New Drugs, 2008, 26, 289-297.
53. S. S. Dhule, P. Penfornis, T. Frazier, R. Walker, J. Feldman,
G. Tan, J. He, A. Alb, V. John and R. Pochampally,
Nanomedicine-Nanotechnology Biology and Medicine, 2012, 8,
440-451.
54. A. Veprik, M. Khanin, K. Linnewiel-Hermoni, M. Danilenko,
J. Levy and Y. Sharoni, American Journal of Physiology-
Endocrinology and Metabolism, 2012, 303, E815-E824.
55. S. Chakraborty, K. Das, S. Saha, M. Mazumdar, A. Manna, S.
Chakraborty, S. Mukherjee, P. Khan, A. Adhikary, S. Mohanty,
S. Chattopadhyay, S. C. Biswas, G. Sa and T. Das, Journal of
Biological Chemistry, 2014, 289, 29074-29085.
56. Z. Chang, J. Xing and X. Yu, Tumor Biology, 2014, 35, 753-
758.
57. S. S. Dhule, P. Penfornis, J. He, M. R. Harris, T. Terry, V.
John and R. Pochampally, Molecular Pharmaceutics, 2014, 11
,
417-427.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins