Cathepsin B-sensitive cholesteryl hemisuccinate-gemcitabine prodrug nanoparticles: Enhanced cellular uptake and intracellular drug controlled release

Article abstract of DOI:10.1039/c4ra13870h

Gemcitabine [2′,2′-difluoro-2′-deoxycytidine (dFdC)], firstline treatment for pancreatic cancer in the clinic, is a cytotoxic nucleoside analogue. Nucleoside transporters are required in the transport of gemcitabine into cells since it is a hydrophilic compound. Actually, there are significant drawbacks for the application of gemcitabine in clinic, including a short half-life and serious side effects. In order to overcome the mentioned drawbacks, a novel prodrug, cholesteryl hemisuccinate-gemcitabine (CHSdFdC), was synthesized through covalently coupling the amino group of gemcitabine with the carboxylic group of cholesteryl hemisuccinate. The amphiphilic prodrug self-assembled spontaneously as nanoparticles in aqueous media confirmed by transmission electron microscopy (TEM). Dynamic light scattering (DLS) measurement revealed that the mean particle size is approximately 200 nm in aqueous media. The CHSdFdC nanoparticles displayed accumulative controlled drug release in simulated lysosome conditions (pH 5.0 NaAc buffer solution containing cathepsin B); the amount of drug release reached up to 80% within 10 h. However, there was almost no drug release in pH 7.4 PBS and pH 5.0 NaAc buffer solutions without cathepsin B. All these results indicated the intracellularly controlled drug release manner of the CHSdFdC nanoparticles. The controlled release of dFdC from the CHSdFdC nanoparticles was related closely to cleavage of amide bonds by cathepsin B. The CHSdFdC nanoparticles exhibited increased ability to inhibit cell growth compared with gemcitabine in vitro. Meanwhile, the CHSdFdC nanoparticles exhibited enhanced cellular uptake ability against Bxpc-3 cells, and the amount of CHSdFdC was about 15 fold of gemcitabine during the 2.5 h incubation. All these results showed that the CHSdFdC nanoparticle prodrug has great potential in the treatment of pancreatic cancer.

Full text of DOI:10.1039/c4ra13870h

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PAPER
Cathepsin B-sensitive cholesteryl hemisuccinate–
gemcitabine prodrug nanoparticles: enhanced
cellular uptake and intracellular drug controlled
release
Cite this: RSC Adv., 2015, 5, 6985
a
b
a
a
ab
a
c
Yanyun Xu, Jianqi Geng, Ping An, Yan Xu, Jin Huang, Wei Lu, Shiyuan Liu
a
and Jiahui Yu*
0
0
0
Gemcitabine [2 ,2 -difluoro-2 -deoxycytidine (dFdC)], firstline treatment for pancreatic cancer in the clinic,
is a cytotoxic nucleoside analogue. Nucleoside transporters are required in the transport of gemcitabine
into cells since it is a hydrophilic compound. Actually, there are significant drawbacks for the application
of gemcitabine in clinic, including a short half-life and serious side effects. In order to overcome the
mentioned drawbacks,
a novel prodrug, cholesteryl hemisuccinate–gemcitabine (CHSdFdC), was
synthesized through covalently coupling the amino group of gemcitabine with the carboxylic group of
cholesteryl hemisuccinate. The amphiphilic prodrug self-assembled spontaneously as nanoparticles in
aqueous media confirmed by transmission electron microscopy (TEM). Dynamic light scattering (DLS)
measurement revealed that the mean particle size is approximately 200 nm in aqueous media. The
CHSdFdC nanoparticles displayed accumulative controlled drug release in simulated lysosome
conditions (pH 5.0 NaAc buffer solution containing cathepsin B); the amount of drug release reached up
to 80% within 10 h. However, there was almost no drug release in pH 7.4 PBS and pH 5.0 NaAc buffer
solutions without cathepsin B. All these results indicated the intracellularly controlled drug release
manner of the CHSdFdC nanoparticles. The controlled release of dFdC from the CHSdFdC nanoparticles
was related closely to cleavage of amide bonds by cathepsin B. The CHSdFdC nanoparticles exhibited
increased ability to inhibit cell growth compared with gemcitabine in vitro. Meanwhile, the CHSdFdC
nanoparticles exhibited enhanced cellular uptake ability against Bxpc-3 cells, and the amount of
CHSdFdC was about 15 fold of gemcitabine during the 2.5 h incubation. All these results showed that the
CHSdFdC nanoparticle prodrug has great potential in the treatment of pancreatic cancer.
Received 12th November 2014
Accepted 15th December 2014
DOI: 10.1039/c4ra13870h
www.rsc.org/advances
Nucleoside transporter deciency would make it difficult for
gemcitabine to be transported into cells. In addition, as a
1
Introduction
0
0
0
Gemcitabine [2 ,2 -diuoro-2 -deoxycytidine (dFdC)] has been traditional chemotherapeutic drug, gemcitabine is difficult to
proved to be a potent cytotoxic nucleoside analogue which accumulate at tumor tissue selectively, which lead to undesired
demonstrated efficacy in the treatment of various solid tumors, side effects and inadequate drug concentrations reaching
5–8
including colon, lung, pancreatic, breast, bladder and ovarian tumor, despite it is a primary drug for cancer treatment. In
1
–3
cancers. In order to achieve therapeutic effects, gemcitabine this paper, in order to make it easier for gemcitabine to enter
must be transported into cells. Membrane proteins called cells, especially nucleoside transporter deciency cells, the
nucleoside transporters are required in the transport of gem- cholesteryl-hemisuccinate–gemcitabine conjugate was fabri-
citabine because it is a hydrophilic compound. The main cated into nanoparticles to change the approach that gemcita-
4
transporter types include hENT , hENT , hCNT and hCNT .
bine enter cells. As a result, gemcitabine would enter cells via
endocytosis rather than transport by nucleoside transporters. In
addition, nanodrug delivery system could use enhanced
permeability and retention (EPR) (Fig. 1) effect to promote the
drugs targeting tumor tissue selectively, leading to potentially
1
2
1
3
a
Institute of Drug Discovery and Development, Shanghai Engineering Research Center
of Molecular Therapeutics and New Drug Development, East China Normal University,
Shanghai 200062, PR China. E-mail: jhyu@sist.ecnu.edu.cn; Fax: +86 21 6223 7026;
5,9–14
enhanced antitumor effect and decreased side effects.
Tel: +86 21 6223 7026
b
College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, Numerous nanodrug or nanaodrug candidates have been
PR China
approved for clinical applications or under clinical trials at
c
13,15,16
Department of Diagnostic Imaging, ChangZheng Hospital, Shanghai, 200003, PR
different stages.
Aer being transported into cells, a part of
China
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Fig. 1 Illustration of EPR effect and intracellularly controlled release of dFdC from CHSdFdC.
gemcitabine is deaminized by cytidine deaminase into inactive
In this study, cholesteryl hemisuccinate was used to modify
uracil derivative (dFdU) intracellularly, hence resulting in a gemcitabine to give CHSdFdC prodrug. The prodrug could
17
4
short half-life. Acylation of N -amino group of the pyrimidine self-assemble into nanoparticles in aqueous media. The
ring of dFdC would protect the amino group from being rapidly properties of the nanoparticles were investigated, such as
degraded by cytidine deaminase. Taking the strategy of nano- critical micelle concentration (CMC), mean particle size and
drug delivery systerm, Couvreur's group covalently coupled size distribution, zeta potential, morphology and colloidal
gemcitabine with squalenic acid, and the resultant 4-(N)-tris- stability. The process of drug release from the nanoparticles
nor-squalenoyl-gemcitabine (SQdFdC) prodrug self-assembled and cellular uptake by Bxpc-3 cells in vitro were investigated in
into nanoparticles, which were shown to overcome gemcita- detail. Meanwhile, we assayed the ability of inhibiting the
1
8
bine resistance in murine leukemia cells (i.e., L1210 10K),
growth of Bxpc-3 cells of the prodrug in vitro. The nano-
18
human leukemia cells (i.e., CEM/ARAC8C), and human particles have the capability to realize intracellularly
19
pancreatic cancer cells (i.e., Panc-1). It was concluded that controlled release of gemcitabine and reduce side effects
SQdFdC nanoparticles enabled the partial circumvention of because amide bond of the prodrug nanoparticles can be
three well-known resistance mechanisms to gemcitabine, degraded by cathepsin B, an enzyme exists in lysosome.
including the deciency of nucleoside transporters, insufficient
activity of deoxycitidine kinase (dCK), and inactivation by
deaminases. Zhengrong Cui et al. have reported that 4-(N)-
stearoyl gemcitabine (GemC18), a stearic acid amide derivative
2
Experimental
20
2.1 Materials and cells
of gemcitabine, could effectively inhibit the growth of gemci- Cholesterol was purchased from J & K Chemical Ltd. Gemcita-
tabine resistance TC-1-GR tumors in mice, and in contrast, the bine hydrochloride was purchased from Shang Hai PuYi
molar equivalent dose of gemcitabine hydrochloride did not Chemical Co., Ltd. Gemcitabine base was purchased from
21
show any activity against the growth of the TC-1-GR tumors.
Shanghai Demo Medical Tech Co., Ltd. EDCI and DMAP was
Cholesterol is an indispensable substance in the formation purchased from Energy Chemical. Other chemicals were
of cell membranes. As the basic component of membrane, purchased from Sinopharm Chemical Reagent Co., Ltd. DCM,
cholesterol accounts for over 20% of plasma membrane lipids. DMF and THF were dried and redistilled before use.
Meanwhile, cholesterol is one of the important components of
The Bxpc-3 cell line is a kind of human pancreatic cancer cell
the liposome membranes. It has been found that cholesterol line, which was purchased from the Institute of Biochemistry &
22
can maintain the uidity of membrane. As a component of the cell Biology, Chinese Academy of Science.
23
biolm, cholesterol exist excellent biocompatibility. Because
of all these properties, cholesterol is important to membrane
fusion, which is an essential procedure for endocytosis.
2
1
.2 Characterization of compounds
24,25
H NMR spectra were recorded with a Bruker Avarice TM 400
NMR spectrometer. The Fourier transform infrared spectra (FT-
IR) were obtained with a Nicolet Nexus 670 spectrometer. The
samples were pressed into pellets with KBr.
Then, it is expected that the anti-tumor drug modied with
cholesterol will display enhanced ability of cellular uptake to
increase their therapeutic effect and decrease the side effects.
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2
.3 Synthesis of cholesteryl hemisuccinate–gemcitabine
emission spectra of pyrene was analyzed for the calculation of
the CMC. The experiment was performed in triplet. The mean
and corresponding standard deviations (mean ꢃ SD) are shown
in the results.
(
CHSdFdC)
Succinic anhydride (1.2 mmol) was added to stirred solution of
cholesterol (1.0 mmol) in toluene. The mixture was reuxed for
3
h, cooled to room temperature and ltered. The precipitate
2
.5 Formulation and characterization of CHSdFdC
was collected and recrystallized with ethanol for twice, the
cholesteryl hemisuccinate was obtained (yield: 82.8%). IR (neat,
cm ) 3442 (–OH), 2943–2860 (–CH –), 1712 (–C]O), 1176 CHSdFdC nanoparticles were prepared by nanoprecipitation.
nanoparticles
ꢀ
1
2
1
(C–O–C). H NMR (400 MHz, CDCl ) d: 0.68 (3H, s, 20 H), 0.8–2.4 Briey, CHSdFdC (5 mg) was dissolved in THF (5 mL) and the
3
(
28H, 1-H , 3-H , 6-H , 8-H , 9-H , 10-H , 11-H , 13-H , 14-H , solution was added dropwise into 10 mL ultrapure MilliQ®
2
2
2
2
1
1
1
2
2
15-H
2
, 16-H
2
, 17-H
1
, 21-H
1
, 22-H
2
, 24-H
2
, 25-H
2
, 26-H
1
), 0.87 water under constant stirring at 500 rpm. The form of CHSdFdC
(
1
4
6H, d, J ¼ 6.4 Hz, 27-H
3
, 28-H
), 2.61 (2H, m, COCH
), 5.37 (1H, m, 7-H ).
3
), 0.92 (3H, d, J ¼ 6 Hz, 23-H
3
), nanoparticles occurred spontaneously. THF was completely
CO), evaporated using a rotary evaporator at 37 C to obtain an
ꢁ
.02 (3H, s, 19-H
.64 (1H, m, 2-H
3
2
), 2.68 (2H, m, CH
2
1
1
aqueous suspension of pure CHSdFdC nanoparticles. The
Triethylamine (1.4 mmol) was added to stirred solution of nanoparticles suspension has been analyzed using RP-HPLC to
cholesteryl hemisuccinate (1.2 mmol) in anhydrous THF (3 mL), verify the actual concentration of CHSdFdC in the nal product.
ꢁ
under nitrogen. The mixture was cooled to ꢀ15 C, and the
The mean particle size, size distribution, polydispersity
solution of isobutyl chloroformate (1.2 mmol) in anhydrous index (PDI) and zeta potential were determined using dynamic
THF (3 mL) was added dropwise. The mixture was stirred at ꢀ15 light scattering (DLS) (ZetasizerNano ZS, Malvern Instruments,
ꢁ
C for 15 min and the solution of gemcitabine hydrochloride UK). The measurements were made aer dilution of the nano-
(
(
1.2 mmol) and triethylamine (1.4 mmol) in anhydrous DMF particles suspension with ultrapure MilliQ® water.
5 mL) was added dropwise to the mixture at the same
The morphology of the nanoparticles was observed by
temperature, then the mixture was stirred for another 0.5 h at transmission electron microscopy (TEM) (JM-2100, Japanese). A
ꢁ
ꢀ
15 C. Aer being stirred for 72 h at room temperature, the drop of aqueous nanoparticles suspension was deposited onto a
reaction mixture was concentrated. Aqueous sodium hydrogen 300 mesh copper grid coated with a thin carbon lm. The grids
carbonate was added and the mixture was extracted with DCM were dried at room temperature and observed by TEM.
(
3 ꢂ 50 mL). The combined extracts were washed with water,
dried over MgSO , and concentrated. The crude product was
4
2
.6 Colloidal stability of nanoparticles
puried by chromatography on silica gel eluting with 1–5%
methanol in dichloromethane to give cholesteryl hemi-
succinate–gemcitabine as amorphous white solid (yield: 32%).
The colloidal stability of nanoparticles was investigated by
measuring variation of mean particle size of the nanoparticles.
The nanoparticles were diluted with PBS to maintain the nal
concentration of CHSdFdC was 2 mg mL and stored at 4 C
for 28 days. Meanwhile, the nanoparticles were diluted with cell
culture medium (RPMI 1640 (Gibco BRI., Paris, France)) to
ꢀ1
IR (neat, cm ) 3500 (–CO–NH–), 3000 (–C]C–), 1665 (–CO–
ꢀ
1
ꢁ
1
NH–), 1400 (–CO–NH–), 1065 (C–O). H NMR (400 M, DMSO-d )
6
d: 0.65 (3H, s, 45-H ), 0.85 (6H, dd, 52-H , 53-H ), 0.90 (3H, d, 48-
3
3
3
H
H
H
3
2
1
), 0.96 (3H, s, 44-H
3
), 0.8–2.4 (28H, 28-H
2
, 31-H
2
, 32-H
2
, 34-
, 46-
ꢀ
1
maintain the nal concentration of CHSdFdC was 2 mg mL
, 35-H
, 47-H
CO), 3.66 (1H, m, 6-H
), 4.19 (1H, m, 11-H ), 4.45 (1H, m, 27-H
H ), 5.34 (1H, d, 8-H ), 6.18 (1H, t, 12-H ), 6.31 (1H, d, 9-H ),
1
, 36-H
1
, 37-H
1
, 39-H
2
, 40-H
), 2.56 (2H, m, COCH
), 3.80 (1H, d, 10-H ), 3.89 (1H, d,
), 5.28 (1H, t, 33-
2 2 2
, 41-H , 42-H , 43-H
1
ꢁ
and incubated at 37 C for 10 days. The experiment was per-
formed in triplet. The mean and corresponding standard devi-
ations (mean ꢃ SD) are shown in the results.
2
, 49-H
2
, 50-H
2
, 51-H
1
2
), 2.69 (2H,
m, CH
0-H
2
1
1
1
1
1
1
1
1
1
1
2.7 Cell line and culture
7
.24 (1H, d, 15-H ), 8.24 (1H, d, 14-H ), 11.08 (1H, s, 20-H ).
1 1 1
Human pancreatic cancer cell line Bxpc-3 was purchased from
the Institute of Biochemistry & cell Biology, Chinese Academy of
Science. Bxpc-3 cells were cultured in RPMI 1640 (Gibco BRL.,
Paris, France) supplemented with 10% fetal bovine serum (FBS,
2
.4 Nanoparticle fabrication and the critical micelle
concentration
The CHSdFdC (5 mg) was dissolved in 10 mL THF, and the
solution was added dropwise into 10 mL ultrapure MilliQ®
water with constant stirring at 500 rpm. Aer stirring, the
solution was loaded into a dialysis tube (MWCO 1000) and
dialyzed against 12 L (4 L ꢂ 3) deionized water for 24 h.
The critical micelle concentration (CMC) was determined
HvClone, Logan UT) and 1% penicillin and streptomycin. The
ꢁ
cell line was incubated at 37 C in humidied 5% CO
2
atmo-
sphere. When a cell conuence of 90% was reached, they were
routinely trypsinized and subcultured.
26
2.8 Drug release from the CHSdFdC nanoparticles
using pyrene as a uorescence probe. The concentration of
ꢀ
4
ꢀ1
CHSdFdC varied from 2 ꢂ 10 to 0.1 mg mL with a xed Release of gemcitabine from the CHSdFdC nanoparticles in an
ꢀ7
ꢀ1
pyrene concentration of 6 ꢂ 10 mol L . The uorescence aqueous buffer solution at different pH values (pH 5.0, pH 7.4)
spectra were measured on an F-4500 uorescence spectropho- was measured by RP-HPLC with an Agilent 1200 (Agilent
tometer (Hitchi F-4500) with an excitation wavelength of 335 Technologies INc., Shanghai Branch) using a Zorbax Eclipse
nm. The I373/I384 ratio of the uorescence intensity in the XDB-C18 column (5 mm, 4.6 mm ꢂ 250 mm) at 30 ꢁC. The
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1
CHSdFdC (1 mg mL in DMSO), 60 mL, was dispersed in 2 mL 2.10 Cellular uptake
of three different media including PBS (pH 7.4), sodium acetate
The cellular uptake of CHSdFdC nanoparticles was quantita-
tively measured by RP-HPLC. The Bxpc-3 cells were seeded in 6-
buffer solution (pH 5.0), sodium acetate buffer solution (pH 5.0)
ꢀ
1
contained 60 mL cathepsin B (U-activity unit ¼ 10 U mL ). The
5
wells plate at a density of 2.5 ꢂ 10 cells per well in 2 mL of
ꢁ
samples were kept in a THZ-C isothermal shaker at 37 C and
culture medium and incubated for 24 h. The original cell
culture medium was replaced with 2 mL fresh culture medium
150 rpm. At the predetermined time point, 100 mL of the sample
solution was withdrawn, and same amount of acetonitrile was
added immediately. The accumulative drug release was
measured by RP-HPLC using methanol as the mobile phase.
ꢀ1
contained dFdC and CHSdFdC (dFdC: 0.1 mg mL ). The cells
were cultured for another 0.5 h, 1.5 h and 2.5 h, respectively. At
the predetermined time point, the cell culture medium was
discarded, and 2 mL of 1% SDS was added to make cell
membrane lytic aer the wells were washed with cold PBS for
three times carefully. The cell lysate was dissolved in 200 mL
mixture of methanol and acetonitrile (v/v ¼ 1 : 1) aer freeze
drying. The concentrations of dFdC and CHSdFdC were
measured by RP-HPLC. Experiments were carried in triplicates.
Means and corresponding standard deviations (mean ꢃ SD)
were shown as results.
ꢀ
1
The ow rate of the mobile phase was 1 mL min . The Agilent
200 Uv/vis detector was set at 254 nm. The release percentage
1
of dFdC was calculated from the ratio of peaks area assigned to
free dFdC and CHSdFdC. The experiment was performed in
triplet. The mean and corresponding standard deviations
(
mean ꢃ SD) are shown in the results. The cathepsin B is a kind
27,28
of lysosomal enzyme which does not exist in human blood.
It
29
is reported that the pH value of lysosome is about 5.0. What we
expect is that the prodrug is stable when it circulates in human
blood whereas it can be degraded with presence of cathepsin B
to release controlled release of dFdC from CHSdFdC prodrug. In
2.11 Statistical data analysis
the paper, the PBS with pH 7.4 was used to simulate the pH Statistical data analysis was performed using Student's t-test.
value of human blood, pH 5.0 buffer solution with cathepsin B
was used to simulate the condition of lysosome, pH 5.0 buffer
solution without cathepsin B was used to clarify that cathepsin
B is an indispersable trigger in the controlled drug release.
3
Results and discussion
3.1 Synthesis of cholesteryl hemisuccinate–gemcitabine
(CHSdFdC)
Cholesteryl hemisuccinate was synthesized through esterica-
tion reaction between cholesterol and succinic anhydride.
During the reaction, DMAP was used as catalysis. The synthesis
scheme of cholesteryl hemisuccinate is shown in Scheme 1. The
structure of the product was conrmed by NMR, and the
detailed data of the chemical shis are displayed in part 2.3.
The structure was conrmed from the appearance of the peaks
at 2.61 and 2.68 ppm which belong to succinic anhydride, 0.68,
2
.9 In vitro ability of inhibiting the growth of Bxpc-3 cells
assay
The ability of inhibiting the growth of tumor cells of CHSdFdC
nanoparticles was investigated and compared with dFdC as
positive control by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide] assay against Bxpc-3 cell line.
Briey, 4000 cells per well were incubated in 100 mL of complete
culture medium (RPMI 1640 (Gibco BRL., Paris, France) sup-
plemented with 10% fetal bovine serum (FBS, HvClone, Logan
UT) and 1% penicillin and streptomycin) in 96-well plates for 24
h. The cells were then exposed to a series of concentrations of
CHSdFdC nanoparticles, free dFdC or free CHS in 100 mL fresh
complete culture medium for 72 h. The drug concentration in
the case of CHSdFdC nanoparticles is equivalent to dFdC
concentration. At the end of the incubation period, 20 mL of
MTT solution (5 mg mL ) in PBS was added to each well. The
culture medium was gently replaced by 100 mL of dime-
thylsulfoxide in order to dissolve the formazan crystals aer 4 h
incubation. The optical density (OD) was measured at 570 nm
with an automatic BIO-TEK microplate reader (Powerwave XS,
USA), and the cell viability was calculated through the following
equation:
0.87, 0.92, 1.02, 4.64, 5.37 ppm which belong to cholesterol. The
ratio of succinic acid to cholesterol was equal to 1 : 1, which was
conrmed by calculation of the integral ratios of the protons at
5
2
.37 ppm (multiplet) assigned to 7-H signal of cholesterol and
.61 ppm (multiplet) assigned to the methylene protons signal
of succinic acid. In order to further conrm the structure of the
product, Fourier transform infrared spectra were recorded. The
FT-IR spectra data showed a new absorption appearance located
ꢀ1
ꢀ1
ꢀ
1
at 1712 cm
assigned to carboxyl group and 3442 cm
assigned to hydroxyl group.
The CHSdFdC was synthesized through covalently coupling
the amino group of gemcitabine on pyridine ring and the
carboxyl group of cholesteryl hemisuccinate. The synthesis
scheme is shown in Scheme 1. The structure was conrmed by
NMR, and the detailed data of the shis are displayed in part
2.3. The structure was conrmed from the appearance of the
peaks at 11.08 ppm, which belong to amide bond and the
disappearance of the peaks at 7.41 ppm which belong to the
Cell viability (%) ¼ (ODsample/ODcontrol) ꢂ 100%.
(1)
The ODsample represents an OD value from a well that treated amino group of gemcitabine. The integral ratios of the protons
with samples, and ODcontrol comes from a well that treated with at 11.08 ppm (single) assigned to amide proton signal and 0.65
cell culture medium only. Each experiment was performed in ppm (single) assigned to the methyl protons signal of choles-
sextuplet. The mean and corresponding standard deviations teryl hemisuccinate were 1 : 3, indicating that the ratio of
(mean ꢃ SD) are shown in the results.
gemcitabine and cholesteryl hemisuccinate in the CHSdFdC
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Scheme 1 Synthesis scheme of cholesteryl hemisuccinate–gemcitabine (CHSdFdC).
media at room temperature. The mean particle size was 200 nm,
as shown in Fig. 3A. The main hydrodynamic diameter of the
CHSdFdC nanoparticles was between 40 and 500 nm. This
demonstrated that the nanoparticles were able to pass through
the large pores of the tumor blood vessel to target the tumor
tissue. The nanoparticles showed negative potential at around
ꢀ
0.06 mV in the measurement of zeta potential, suggesting
potential capacity for prolonging the circulation time in blood
because nanoparticles with positive surface charges are inclined
to agglomerate due to interaction with serum protein in human
33
blood.
The morphology of the nanoparticles was observed by
transmission electron microscopy (TEM), as shown in Fig. 3B
and C. The mean particle size was approximately 200 nm in a
dehydrated state. The size measured by TEM was very close to
that measured by DLS in aqueous media.
Fig. 2 Intensity ratios I373/I384 of the pyrene emission fluorescence
spectra as a function of the logarithm concentration of the CHSdFdC
(
means ꢃ SD, n ¼ 3).
was equal to 1 : 1. In order to further conrm the structure of
CHSdFdC, Fourier transform infrared spectra were recorded.
The FT-IR spectra data showed a new absorption appearance
3
.4 Colloidal stability of CHSdFdC nanoparticles
ꢀ1
The colloidal stability of the nanoparticles was investigated by
measuring the variation of mean particle size. The nano-
particles were diluted with PBS and stored at 4 C for 28 days.
located at 3500 cm assigned to the amide bond.
ꢁ
3.2 CMC measurement of CHSdFdC
This is the storage condition for CHSdFdC nanoparticles
The CHSdFdC formed nanoparticles in aqueous media due to
its amphiphilic structure. CMC was measured by uorospec-
trophotometer with pyrene as a probe. The plot of the intensity
ratio I373/I384 of the pyrene emission spectra against the loga-
rithm of the CHSdFdC concentration is shown in Fig. 2. The
CMC value can be determined at the CHSdFdC concentration of
onset of the I373/I384 ratio decrease. When the concentration of
CHSdFdC reaches the CMC, there is a sudden change of I373/I384
in the uorescence spectra due to the transfer of pyrene from a
polar environment to a non-polar environment caused by the
formation of nanoparticles. The CMC of CHSdFdC is 0.001 mg
ꢀ1
mL , as shown in Fig. 2. The CMC of CHSdFdC was very low,
indicating it is rather stable against dilution.
3.3 Characterization of the nanoparticles
Particle size is an important factor that affects their in vivo
performance and pharmacokinetics for nanoparticles. Tumors,
unlike most healthy tissues, possess a leaky vasculature that
allows the passage of colloidal particles with size in the range of
30–32
50–200 nm.
In this research, the mean particle size and size
Fig. 3 (A) Size and size distribution of CHSdFdC nanoparticles (B and C)
distribution, zeta potential were measured by DLS in aqueous transmission election microscopy (TEM) of CHSdFdC nanoparticles.
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suspension. Meanwhile, the nanoparticles were diluted with
cell culture medium (RPMI 1640 (Gibco BRI., Paris, France)) and
ꢁ
incubated at 37 C for 10 days. This condition was used to
simulate the human plasma, in order to assay the stability of
CHSdFdC nanoparticles when it circulates in human plasma. It
demonstrated that the CHSdFdC nanoparticles are stable at 4
ꢁ
ꢁ
C in PBS as well as at 37 C in cell culture medium (RPMI 1640
Gibco BRI., Paris, France)), with no signicant variation of the
mean particle size was observed, as shown in Fig. 4A and B.
(
3.5 Drug release from the CHSdFdC nanoparticles
The amide bond could be degraded by cathepsin B which exists
in lysosome. Thus, there is possibility for dFdC to release from
the CHSdFdC nanoparticles at the presence of cathepsin B. As
shown in Fig. 5, the release of dFdC was very slow under weakly
acid condition (pH 5.0 HOAc–NaOAc buffer solution) or neutral
pH condition (pH 7.4 PBS), the amounts were 0.64% and 1.84%
within 10 h, respectively. On the other hand, the release of dFdC
was much faster under the weakly acid condition (pH 5.0 HOAc–
NaOAc buffer solution) in the presence of cathepsin B, the
accumulative amount of drug release reached up to 80% within
Fig. 5 Accumulative drug release of dFdC from CHSdFdC nano-
particles at 37 C.
ꢁ
cells, especially at high concentration as shown in Fig. 6. It
has been reported that CHS could incorporate into cell
34
membrane to inhibit cell proliferation. Only when the
concentration of CHS reaches a certain high level, the cytotox-
icity could be observed. And the result in this paper is consistent
10 h. These results demonstrated the CHSdFdC nanoparticles
34
with the reported result.
had great potential to realize intracellular release of dFdC.
3.6 In vitro ability of inhibiting growth of Bxpc-3 cells assay
3.7 Cellular uptake
The in vitro ability of inhibiting growth of tumor cells of the
CHSdFdC nanoparticles was evaluated with a human pancreatic In order to conrm the hypothesis that the ability of cellular
cancer cell line Bxpc-3 via MTT assay. Representative concen- uptake of CHSdFdC nanoparticles is enhanced than dFdC, the
tration–growth inhibition curves showed the effects of treat- amount of cellular uptake of CHSdFdC nanoparticles was
ment with free dFdC and CHSdFdC nanoparticles on the growth measured by RP-HPLC, in the measurement, dFdC was used as
of Bxpc-3 cells aer 72 h. As shown in Fig. 6, both free dFdC and control. As shown in Fig. 7, the cellular uptake amount of
CHSdFdC nanoparticles inhibited cell growth in a dose- CHSdFdC nanoparticles varied apparently while that of dFdC
dependent manner, whereas the latter was more toxic than varied little, which is 15 folds of dFdC at the same culture
the former at the same concentration. It was thought that the period of 2.5 h. The conclusion was drawn that CHSdFdC
increased cytotoxicity of the CHSdFdC resulted from two nanoparticles could penetrate the cells much more easily than
reasons. On one hand, CHSdFdC nanoparticles possess free dFdC, which improved the efficacy of inhibiting growth of
enhanced ability of cellular uptake, which make it easier to tumor cells. The result in this paper showed that the CHSdFdC
enter tumor cells. This was conrmed by the cellular uptake nanoparticles could be internalized much more easily than free
experiment. On the other hand, the released CHS, due to the dFdC, this result is related to the different uptake mechanism of
degradation of CHSdFdC nonaparticles prodrug by cathepsin B, dFdC and CHSdFdC nanoparticles. But the cytotoxic activity of
also demonstrated ability of inhibiting the growth of Bxpc-3 the drug and the prodrug are similar, this result is due to the
ꢁ
Fig. 4 (A) Variation of the mean particle size of CHSdFdC nanoparticles at 4 C in PBS (B) variation of the mean particle size of CHSdFdC
ꢁ
nanoparticles at 37 C in cell culture medium (means ꢃ SD, n ¼ 3).
6990 | RSC Adv., 2015, 5, 6985–6992
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and Technology (11nm0506000), the National Natural Science
Foundation of China (81171333) and the EU-FP 7 project
(MARINA 263215).
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