Self-assembled targeted folate-conjugated eight-arm-polyethylene glycol-betulinic acid nanoparticles for co-delivery of anticancer drugs

Article abstract of DOI:10.1039/c5tb00042d

In this study, a targeted nanoparticle platform for co-delivery of anticancer drugs based on folate-conjugated eight-arm-polyethylene glycol-betulinic acid (F-8arm-PEG-BA) was first presented. F-8arm-PEG-BA was synthesized by introducing target molecules

Full text of DOI:10.1039/c5tb00042d

Journal of
Materials Chemistry B
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PAPER
Self-assembled targeted folate-conjugated
eight-arm-polyethylene glycol–betulinic acid
nanoparticles for co-delivery of anticancer drugs
Cite this: J. Mater. Chem. B, 2015,
, 3754
3
a
a
a
a
b
a
Lin Dai, Xin Cao, Ke-Feng Liu, Chun-Xiao Li, Gui-Feng Zhang, Li-Hong Deng,
c
a
a
Chuan-Ling Si, Jing He* and Jian-Du Lei*
In this study, a targeted nanoparticle platform for co-delivery of anticancer drugs based on folate-conjugated
eight-arm-polyethylene glycol–betulinic acid (F-8arm-PEG–BA) was first presented. F-8arm-PEG–BA
was synthesized by introducing target molecules (folate) and drug molecules (betulinic acid, BA) to
hydrophilic molecules (8arm-PEG). Then another anticancer drug, hydroxycamptothecin (HCPT), was
encapsulated into the self-assembled nanoparticles from the conjugate by a simple nanoprecipitation
method. These F-8arm-PEG–BA/HCPT nanoparticles (NPs) had a small size (B120 nm), acceptable critical
ꢀ
1
aggregation concentration (B64.8 mg mL ), and high drug loading (B18 wt% BA and B16 wt% HCPT).
Compared to the free drugs, the nanoparticles significantly improved the cellular cytotoxicity and exhibited
an obvious synergistic effect by the co-delivery of two different anticancer drugs, BA and HCPT.
Pharmacokinetics study revealed the nanoparticles could prolong the circulation of BA and HCPT in the
blood. In vivo studies indicated that the nanoparticles enhanced tumor targeting and antitumor activity
with lower systemic toxicity. In conclusion, F-8arm-PEG–BA/HCPT NPs have great potential for targeted
chemotherapy for cancer.
Received 8th January 2015,
Accepted 22nd March 2015
DOI: 10.1039/c5tb00042d
www.rsc.org/MaterialsB
However, there are still some challenges for nanoparticle
drug delivery, such as low drug loading capacity and low drug
Poor water solubility, rapid blood clearance, low tumor selectivity, targetability. Most of the polymeric systems use ‘‘inert’’ excipients
and severe side effects for healthy tissues are major problems in that lack therapeutic activity. Due to the large amount of the ‘no
clinical applications of many potent anticancer drugs such as medical effect’ carrier, the inherent drawback of these nanomedi-
1
. Introduction
7,8
1
betulinic acid (BA) and hydroxycamptothecin (HCPT). Recently, cines is their low drug loading capacity. More than that, the presence
2
3
4
various vehicles, such as hydrogels, liposomes, dendrimers,
of large amounts of carrier material not only adds to the cost, but
5
6
prodrugs, and polymeric nanoparticles, have been found to also imposes additional safety issues. Polymeric nanoparticles are
greatly enhance the water solubility and stability of anticancer formed from amphiphilic polymers having distinct hydrophilic and
drugs. In particular, self-assembled nanoparticles based on hydrophobic parts. Within each amphiphilic system, the formula-
poly(ethylene glycol) (PEG) are among the most studied systems tion of nanoscopic macromolecular core–shell structures is caused
due to its approved use in medical devices by the US FDA and by an aqueous phase separation. In fact, most anticancer drugs have
advantages in passive tumor targeting through leaky vasculature poor water solubility, but the strategy of using a highly hydrophobic
via the enhanced permeability and retention (EPR) effect, due to anticancer drug itself as the hydrophobic segment of polymeric
their small size ranging from 10 to 200 nm, which is small nanoparticles is rarely reported. For instance, Shen et al. prepared
enough to avoid filtration by the lungs and spleen.
amphiphilic prodrugs with camptothecin as the hydrophobic tails
and a very short oligomer chain of ethylene glycol with only eight
repeating units as the hydrophilic head. Notably, treatment
9
a
with low molecular weight PEG had been reported to result in
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,
1
0
Beijing 100083, P. R. China. E-mail: hejing2008@sina.com, ljd2012@bjfu.edu.cn;
toxic effects to the kidneys, so the safety and drug loading
capacity of self-assembled nanoparticles based on PEG were in
direct conflict. Therefore, it is necessary to develop higher drug
loading capacity of PEG–drug conjugates.
Fax: +86-10-62338152; Tel: +86-10-62338152
b
Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100090,
P. R. China
c
Tianjin Key Laboratory of Pulp & Paper, College of Material Science and Chemical
Recently, multiarm-PEGs have generated a great deal of
interest for drug delivery applications because of their
Engineering, Tianjin University of Science and Technology, Tianjin 300457,
P. R. China
3
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5
,11,12
unique structures.
Compared with linear PEG, multiarm- It has been proven that the increase of Top II activity occurs in
1
6
PEGs are well-defined highly branched macromolecules and cancer cells resistant to HCPT. BA has been reported to be a
have relatively high density of peripheral functional groups. In catalytic inhibitor of TOP I and II activity. Top II inhibitor
previous researches, some hydrophobic drugs were conjugated shows a collateral cytotoxicity on the HCPT adapted cancer
to multiarm-PEGs through either ester or amide linkages to cells. Therefore, the concomitant use of both Top I and Top II
form prodrug molecules. Multiarm-PEGs can not only improve inhibitors might elicit synergistic effects and prevent the emer-
the molecular weight but also improve the drug loading capacity, gence of drug resistance. Moreover, this targeted nanoparticle
thus leading to solubilization of very insoluble molecule com- drug delivery system could also improve the drug safety profile,
pounds, prolongation of the circulation time, and alteration of prolong the circulation of the drugs in blood compartments,
the biodistribution in tumors through the EPR effect of the and target cancerous tissues by active targeting and passive
1
1,12
parent drugs.
accumulation via tumors’ EPR effect (Scheme 1b). The structure
Although significant strides have been made in the design of of F-8arm-PEG–BA was characterized. We further determined
multiarm-PEG–drug conjugates, it should be noted that there the biophysical properties of drug-loaded nanoparticles includ-
are few reports on the development of nanoparticles based on ing particle size, loading capacity, and in vitro drug release
multiarm-PEGs for drug delivery. In addition, a more effective kinetics. Finally, the pharmacokinetics, side effects, the in vitro
and active targeting system is further needed to enhance the and in vivo antitumor activities of the nanoparticles were
intracellular uptake of drugs within cancerous cells at the tumor also investigated.
1
3,14
site.
achieving (i) a self-assembled nanoparticle for drug co-delivery,
ii) targeted chemotherapy for cancer cells, and (iii) combination
The objective of the present study was three-fold,
(
2
. Experimental details
therapy.
In this study, a folate-eight-arm-polyethylene glycol–betulinic
acid (F-8arm-PEG–BA) conjugate was synthesized, in which poorly Eight-arm-polyethylene glycol carboxylic acid (8arm-PEG–
water-soluble anticancer drug BA, 8arm-PEG, and folate were used COOH, M = 10 kDa) was received from JenKem Technology
2.1. Reagents and materials
w
as the hydrophobic segment, hydrophilic segment and target Co., Ltd (Beijing, China), and is an FDA and EU food grade
molecule, respectively. At the same time, they self-assembled into material. Betulinic acid (BA) and hydroxycamptothecin (HCPT)
nanoparticles (Scheme 1a), as an ideal carrier of another anti- were obtained from Chengdu Preferred Biotechnology Co., Ltd
cancer drug, HCPT, to achieve combination therapy. Both HCPT (Sichuan, China). 4-Dimethylaminopyridine (DMAP), 1-ethyl-3-
and BA are potent topoisomerase (Top) inhibitors, but HCPT just (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),
interferes with the action of Top I. Certain clinical limitations N-hydroxysuccinimide (NHS), and folate were purchased from
15
such as resistance of cancer cells impair its clinical application.
Sigma Aldrich. Vivaspin ultracentrifugation filters with 3500 Da
Scheme 1 Illustration of folate-8arm-PEG–betulinic acid nanoparticles for targeted delivery of BA and HCPT. The nanoparticles are prepared via a self
assembly process (a). In contrast to free drug, the nanoparticles are stable in circulation resulting in the EPR effect of solid tumors. The folate-conjugated
nanoparticles are efficiently internalized by the cancer cells via receptor-mediated endocytosis (b).
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5
MWCO were purchased from Fisher (Ottawa, ON, Canada). All previously reported. Briefly, 8arm-PEG–COOH (1.25 g, 0.25 mmol)
other reagents were purchased from Sigma Aldrich. and BA (0.91 g, 2.0 mmol) were dissolved in 25 mL of THF. The
Fetal bovine serum (FBS) was from HyClone. Penicillin and solution was cooled to 0 1C and EDC (0.23 g, 1.2 mmol) and
streptomycin, Gibco Dulbecco’s Modified Eagle’s Medium DMAP (0.24 g, 2.0 mmol) were added. The mixture was stirred at
(
(
(
DMEM), and Gibco Dulbecco’s Phosphate-Buffered Saline 0 1C for 1 h and at room temperature overnight. The solvent was
DPBS) were all bought from Invitrogen. Cell-Counting Kit-8 evaporated under vacuum. The residue was dissolved in 100 mL
CCK-8) kit was supplied by the Dojindo Laboratories. Murine of THF, and the crude product was precipitated with ethyl ether
Lewis lung carcinoma (LLC) cells and human lung cancer cells (100 mL). After filtration, the resulting solids were recrystallized
A549) were obtained from the Peking University Health Science with a mixture of DMF/IPA (24 mL/96 mL). Then, the solids
(
Center (Beijing, China) and were cultured in the listed medium: were filtered, washed with ethyl ether (2 ꢁ 100 mL), and dried
LLC by DMEM with 10% FBS, 1% streptomycin–penicillin and under vacuum at 40 1C to give 8arm-PEG–BA. The molar ratio
A549 by RPMI 1640 with 10% FBS, 1% streptomycin–penicillin. results indicated that approximately 1 or 2 functional groups of
All cell lines were maintained in an incubator supplied with 5% 8arm-PEG–COOH remained unconjugated.
CO
2
/95% air humidified atmosphere at 37 1C.
F-8arm-PEG–BA. The conjugation of folate to 8arm-PEG–BA was
adapted from Wang et al. Folate (44 mg, 0.10 mmol) was reacted
with EDC (38 mg, 0.20 mmol) and NHS (23 mg, 0.20 mmol) in
17
2.2. Animals and ethics
Female C57BL/6 mice, 6–7 weeks old, were purchased from 5 mL DMSO at 50 1C for 6 h. Although having two carboxylic
Beijing HFK BIOSCIENCE CO., LTD. All the animal experiments acid groups (a- and g-), the g-carboxylic acid of folate is more
18
were consistent with the guidelines set by the National Institutes selectively activated because of its higher reactivity. The resulting
of Health (NIH Publication No. 85-23, revised 1985) and were folate-NHS was reacted with pyridine (50 mg, 0.63 mmol) and
approved by the Experimental Animal Ethics Committee, Beijing. ethylenediamine (78 mg, 1.30 mmol) at room temperature for
2
2
4 h. The folate-NH was precipitated by the addition of an excess
2
.3. Synthesis of 8arm-PEG–BA and F-8arm-PEG–BA
of acetonitrile, and the precipitate was filtered off and washed
with diethyl ether, then dried under vacuum to get a yellow
conjugates
Scheme 2 shows the synthesis sequence of 8arm-PEG–BA and powder. The obtained folate-NH
F-8arm-PEG–BA conjugates. Synthesis of the intermediates and (1.03 g, 0.08 mmol), dissolved in 20 mL of MeCN, and activated by
structural characterizations are detailed below. EDC (38 mg, 0.20 mmol), NHS (23 mg, 0.20 mmol), and DMAP
arm-PEG–BA. The 8arm-PEG–BA conjugate was synthesized (3 mg, 0.02 mmol) for 8 h. The unreacted folate-NH was removed
2
was added to the 8arm-PEG–BA
8
2
via esterification reaction by using EDC as a coupling reagent by dialysis (MWCO 3500). The prepared F-8arm-PEG–BA was
and DMAP as an organic catalyst in tetrahydrofuran (THF), as lyophilized as a light yellow powder.
2
Scheme 2 Synthesis of folate-NH (a), 8arm-PEG–BA conjugates (b), and F-8arm-PEG–BA conjugates (c).
3
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.4. Preparation of F-8arm-PEG–BA/HCPT nanoparticles (NPs) with a UV detector, using a gradient of 15–100% of acetonitrile
ꢀ1 23
in 0.05% TFA at a flow rate of 1 mL min
.
Each stability
Nanoparticles were prepared by a precipitation method accord-
1
9
profile represents the average of three independent runs with
the same sampling schedules. The standard deviation of each
point is typically 2% or less. Drug loading efficiency (DLE) was
calculated according to the following equation: DLE (%) =
ing to previous reports. F-8arm-PEG–BA conjugate (0.20 g)
and free HCPT (0.10 g) in 0.2 mL of DMSO were mixed and
added dropwise to 3.8 mL normal saline solution, which was
then vortexed for 1 min. The resulting F-8arm-PEG–BA/HCPT NPs
solutions were transferred to a MWCO 3500 cartridge, and dialyzed
against normal saline (100 mL) for 3 h with two exchanges of
dialysate. The size of the particles was determined by dynamic light
scattering with a particle analyzer (Zetasizer Nano-ZS, Malvern
Instruments Ltd, Malvern, UK). 8arm-PEG–BA/HCPT NPs were
prepared in a similar fashion as F-8arm-PEG–BA/HCPT NPs.
(
(
weight of loaded drug/weight of nanoparticles) ꢁ 100%. Esterase
30 units) was added into the dialysis bag when the BA release in
the presence of esterase was studied.
2.7. Characterization of F-8arm-PEG–BA/HCPT NPs
8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs were
diluted 100ꢁ in deionized water, and a 2 mL aliquot of solution
was pipetted onto the surface of Formvar coated copper TEM
grids (Ted Pella, Redding, CA) and allowed to air dry. Analysis
was performed on a JEM-100CXa TEM at an acceleration voltage
of 100 kV. The hydrodynamic size and zeta potential of nano-
particles were measured by a Nano-ZS Zeta Sizer (Malvern
Instruments Ltd, Malvern, UK).
2
(
.5. Determination of critical aggregation concentration
CAC)
The CAC of 8arm-PEG–BA was determined by employing pyrene
2
0
as a fluorescence probe. A drug-free F-8arm-PEG–BA solution
in DPBS (2.5 mg mL ) was prepared via a solvent evaporation
method. A series of 2-fold dilutions were then made with 8arm-
ꢀ
1
ꢀ1
PEG–BA concentrations ranging from 0.8 to 400 mg L . All the 2.8. Hemolysis assay
aqueous sample solutions were mixed with pyrene at the same
The hemolytic activity of polymer solutions was described as
ꢀ
7
concentration of 6 ꢁ 10 M. The solutions were kept on a
shaker at 37 1C for 24 h to reach equilibrium before fluores-
cence measurement. The fluorescence intensity of samples was
measured at excitation wavelengths ranging from 240 to 360 nm
and an emission wavelength of 390 nm using a Synergy H1 Hybrid
Multi-Mode Microplate Reader (Winooski, VT). Both excitation
and emission bandwidths were 10 nm. From the pyrene emission
spectra, the intensities at 337 nm were analyzed as a function of
the polymer concentrations. The CAC was determined from the
threshold concentration, where a sharp increase in pyrene fluores-
cence intensity was observed.
24,25
follows.
Ten milliliters of fresh blood samples were col-
immediately to
lected from mice, and then added to EDTA–Na
2
prevent coagulation. The red blood cells (RBCs) were collected
by centrifugation at 1500 rpm for 10 min at 4 1C. After washing
in ice-cold DPBS until the supernatant was clear, erythrocytes
8
were diluted to 5 ꢁ 10 cells per mL in ice-cold DPBS. 1 mL
ꢀ1
ꢀ1
(1 mg mL
and 0.1 mg mL ) 8arm-PEG–BA/HCPT NPs,
F-8arm-PEG–BA/HCPT NPs or PEI25 K solution was mixed with
mL erythrocyte suspension. 1% Triton X-100 in DPBS and
1
DPBS were used as positive control (100% lysis) and negative
control (0% lysis), respectively. Samples were incubated under
constant shaking for 1 h at 37 1C. After centrifugation at 1500 rpm
for 10 min at 4 1C, the supernatant was analyzed for hemoglobin
2.6. Determination of drug loading and in vitro drug release
The mass and molar ratio of BA and folate were detected by release at 541 nm using an infinite M200 microplate spectro-
2
1,22
UV-vis spectrophotometry as reported previously.
BA and folate were dissolved in methanol. Then the UV calculated as (ODsample ꢀ ODnegative control)/(ODpositive control
absorbances of BA and folate were determined at 210 nm and ODnegative control) ꢁ 100%. Hemolysis was determined from
80 nm respectively, for five different concentrations. A known three independent experiments.
mass or molar ratio of 8arm-PEG–BA or F-8arm-PEG–BA
Conjug or nConjug) was diluted to a known concentration, and
absorbance and the concentration of BA or folate in the sample CCK-8 assay was used to evaluation the cell viability of different
Briefly, photometer (Tecan, Switzerland). Hemoglobin release was
ꢀ
2
2.9. In vitro cell cytotoxicity
(m
2
6
was used to obtain the mass and molar ratio of BA (mBA, nBA) or samples. Briefly, two types of lung cancer cells, LLC and A549
3
folate (m , n ). The mass and molar ratio of BA or folate-to- cells, were respectively seeded at a density of 3 ꢁ 10 and 4 ꢁ
F
F
3
carrier were thus calculated as mBA/mConjug and nBA/nConjug
,
10 cells per well in 180 mL culture medium and incubated for
m
F
/mConjug and n
F
/nConjug
.
24 h. Then, the cells were treated with various samples (BA,
The release of BA and HCPT from the F-8arm-PEG–BA/HCPT HCPT, 8arm-PEG–BA/HCPT NPs, and F-8arm-PEG–BA/HCPT
NPs was analyzed by a dialysis method. F-8arm-PEG–BA/HCPT NPs) at 37 1C in a humidified incubator with 5% CO for 72 h,
2
ꢀ
1
NPs PBS solution (1 mg mL , 5 mL) at pH 7.4 or 4.5 was loaded where the samples of the BA were dissolved in dimethylsulfoxide
into a dialysis bag (MWCO 3500). Then the dialysis bag was (Merck, Darmstadt, Germany) and diluted into tissue culture
immersed in 200 mL of PBS buffer (pH 7.4 or 4.5) at 37 1C with medium before assay. 8arm-PEG–BA/HCPT NPs and F-8arm-
gentle agitation. PBS medium (2 mL) was withdrawn at timed PEG–BA/HCPT NPs doses were equal to free HCPT. 20 mL of
intervals and the BA concentration in the medium was deter- CCK-8 solution was added to each well and incubated for 1 h at
mined by an Agilent 1200 (Agilent, USA) HPLC instrument. It 37 1C. The percentage of cell viability can be calculated by
employs a VYDAC 214TP54 (C18, 300A, 5 mm, 4.6 ꢁ 250 mm) measuring the absence of absorption at 450 nm. The IC50 was
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obtained as sample concentrations which inhibited the growth 5000 rpm for 5 min. The clear supernatant was dried under
of 50% of cells relative to non-treated cells according to Unger nitrogen, then reconstituted with 100 mL methanol before
2
7
29
et al. IC50 was calculated using the Boltzmann sigmoidal HPLC analysis. The HPLC employs a VYDAC 214TP54 (C18,
s
function from Origin 8.6 (OriginLab, Northampton, USA). 5 mm, 4.6 ꢁ 250 mm) with a UV detector, using a gradient of
ꢀ1
Data are representative of three independent experiments.
60% of acetonitrile in 0.05% TFA at a flow rate of 1 mL min .
We evaluated the synergistic effects between BA and Blood circulation data were plotted as the blood BA or HCPT
HCPT in the nanoparticles by applying the combination index levels with the units of percentage of injected dose per gram
(CI) = BACombined/BASingle + HCPTCombined/HCPTSingle, whereby (%ID per g) against time after injection.
BACombined and HCPTCombined represent the IC50 of drugs used
in the combination treatment, and BASingle and HCPTSingle
2.12. In vivo efficacy studies
represent single drug IC50 values. An index lesser than 1 Subcutaneous tumor xenograft models were induced in the
denotes drug synergism, while a value larger than 1 is an right axillary flank region of female C57BL/6 mice (6–7 weeks)
2
8
6
antagonistic effect.
after injection of 3 ꢁ 10 LLC cells. Treatments were initiated
3
when tumors reached an average volume of 100 to 150 mm ,
and this day was designated as day 0. On day 0, these mice were
2
.10. Cellular uptake study
In order to evaluate the selective cellular uptake of F-8arm- randomly divided into 5 groups (n = 6) and administered
ꢀ
1
PEG–BA/HCPT NPs via folate-receptor-mediated endocytosis, intravenous injection with PBS (control), free BA (10 mg kg ),
ꢀ1
LLC cells and A549 cells were placed in a 6-well plate at a free HCPT (10 mg kg ), 8arm-PEG–BA/HCPT NPs and F-8arm-
5
ꢀ1
density of 2.5 ꢁ 10 cells per well in medium and incubated for PEG–BA/HCPT NPs (10 mg HCPT-equivalent kg ), respectively,
4 h at 37 1C. The culture medium was then replaced with 1 mL on days 0, 2, 4, 6, and 8. It is important to note that the
of medium with 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/ concentrations of F-8arm-PEG–BA/HCPT NPs stated in this article
2
ꢀ
1
HCPT NPs for some time at 37 1C. The incubated cells were refer to HCPT equivalents. For example, a dose of 10 mg kg
washed three times with cold PBS to eliminate trace product of F-8arm-PEG–BA/HCPT NPs means that the dose contains
and detached with 0.02% EDTA–PBS and then suspended in 10 mg kg of HCPT and 63.25 mg kg of whole nanoparticles,
PBS containing 0.1% BSA. The suspended cells were directly assuming that the loading of HCPT in the whole nanoparticles
introduced to a FACSort flow cytometer (Becton Dickinson, USA). is 15.81%. In the observation phase, mice were monitored for
Cellular uptake and distribution of HCPT from nano- tumor sizes and body weights every other day. Tumor volume
ꢀ
1
ꢀ1
2
particles were observed by confocal laser scanning microscopy was calculated using the formula: (L ꢁ W )/2, where L is the
3
0,31
(
CLSM). After LLC cells achieved 70–80% confluency, the cells longest and W is the shortest tumor diameter (millimeter).
were trypsinized and seeded onto culture slides (BD Falcon, Relative tumor volume (RTV) was calculated at each measure-
5
Bedford, MA) at a density of 6.0 ꢁ 10 cells per well (surface ment time point (where RTV was equal to the tumor volume at a
2
area of 8 cm per well) and incubated for 24 h at 37 1C. HCPT, given time point divided by the tumor volume prior to initial
8
arm-PEG–BA/HCPT NPs, and F-8arm-PEG–BA/HCPT NPs (IC50
)
treatment). For efficacy studies, the percentage of tumor growth
were added and incubated for 4 h at 37 1C. After incubation, all inhibition (%TGI) was calculated using the following formula:
reagents were removed. Cells were washed with PBS (pH 7.4) [(C ꢀ T)/C] ꢁ 100, where C is the mean tumor volume of the
at least 3 times and fixed with 4% formaldehyde solution for control group at a specified time and T is the mean tumor
10 min. The liquid content was then dried completely. volume of the treatment group at the same time. To monitor
The nuclei were then counterstained by DMEM medium with potential toxicity, we measured the weight of each mouse. At
0
4
,6-diamidino-2-phenylindole (DAPI) (H-1200; Vector Labora- the end of the experiment (46 weeks) or when the implanted
3
tories, Inc., Burlingame, CA) for 15 min. The fixed cell mono- tumor volume reached 5000 mm , the mice were sacrificed for
layer was finally washed three times with PBS and observed by humane reasons. To further evaluate the hematological toxicity
CLSM (TCS SP5, Leica).
of the nanoparticles, 200 mL of blood was sampled from each
mouse after final administration to test the white blood cell
number (WBC) using a hematology analyzer (MEK-7222K,
2.11. Pharmacokinetic experiments in mice
Tumor-free healthy C57BL/6 female mice were injected intra- Nihon Kohden Celltac E).
venously via the tail vein (six mice per group) with free BA,
HCPT, 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT
2.13. Detection of allergic reaction
NPs. After intravenous administration, 200 mL blood samples Toxic side-effects of current chemotherapeutical drugs often
were collected at 0.083, 0.25, 0.5, 1, 2, 5, 10, 24, 48, 72 h and cause a severe reduction in the quality of life, so the detection
centrifuged immediately at 3000 rpm for 10 min at 4 1C. To of allergic reactions is very necessary and important. During the
determine the concentration of total BA and HCPT in each early development of drugs, type I hypersensitivity is the most
plasma sample, 100 mL of plasma was treated with 50 mL of common type of hypersensitivity reaction. Some of the natural
0.1 N NaOH in a water bath at 37 1C for 15 min, allowing the anti-cancer drugs, such as paclitaxel, docetaxel, and teniposide
hydrolysis of the nanoparticles. After that, 0.1 N HCl (50 mL) was cyclosporine, were usually associated with a high incidence of
added, followed by 100 mL methanol. After being vortexed for the type I hypersensitivity reaction. It has been demonstrated
2
min, the mixture was sonicated for 5 min and centrifuged at that IgE antibodies play an important part in mediating type I
3
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hypersensitivity responses. We thus selected IgE levels as peak protons of BA, those at 3.50–3.85 (4 nH, –(CH
2 2 n
CH O) –) and
the parameter for rapid evaluation of type I hypersensitivity 4.20 (2H, –CH OC(O)O–) to the methylene protons of PEG, and
2
reactions. Five groups of tumor bearing mice (26–28 g, n = 6) those at 6.8 and 7.7 ppm are attributed to the proton peaks of
were used in allergy testing studies (control, BA, HCPT, 8arm- folate. Due to the formation of ester bonds, the 4-methine proton
PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs). The peak d 3.00 (1H, t) of BA moved to d 3.84 (1H, t) and the terminal
samples were administrated via tail intravenous injection every methylene proton peak of PEG d 4.13 (1H, t) moved to d 4.17
ꢀ1
two days at the BA or HCPT dose of 10 mg kg body weight. (1H, t). There is a small terminal methylene proton peak of PEG
After administration with different samples for 10 days, orbit at d 4.13 (1H, t) which was due to the incompletely reacted
blood of mice in different groups was collected and centrifuged. functional group of PEG. By contrast, the signals of BA were
Serum samples were analyzed according to the procedure of significantly weakened and only that of PEG and folate were
mouse IgE ELISA.
detected in D O (Fig. 1d), further indicating a core–shell system
2
of the nanoparticles in water.
3
2,33
2
.14. Statistical analysis
The 8arm-PEG–BA and F-8arm-PEG–BA we generated are white
and light yellow powdery solids, respectively, readily soluble in
H O, THF, and MeCN, with no gelling properties. The conjugate is
2
All experiments in this study were performed at least three
times, and the data were expressed as the means ꢂ standard
deviation (SD). Statistical analyses were performed by analysis
of variance (ANOVA). In all analyses, p o 0.05 was taken to
indicate statistical significance.
a more soluble BA variant, owing to the hydrophilic ether bonds of
PEG, and enabled facile conjugation of BA to carrier compounds
34
with an ester bond susceptible to hydrolysis. The absorbance of
BA and 8arm-PEG–BA were measured by HPLC. The retention
times for BA and 8arm-PEG–BA were 19.4 and 15.5 min, respec-
tively. From the standard plot of concentration versus peak area,
3
3
. Results and discussion
.1. Synthesis of F-8arm-PEG–BA conjugate
ꢀ
7
ꢀ1
the coefficient (D) for BA was calculated to be 1.56 ꢁ 10 mg mL
.
8
arm-PEG–BA was dissolved in deionized water at an approximate
ꢀ1
Linear PEG is the most widely used because of the simple concentration of 200 mg mL (based on M
W
of 10 kDa), and the
synthetic steps and good water solubility, but the linear struc- peak area at 15.5 min was determined. Using this value, and
ture with only two functional groups limits the drug loading employing the coefficient D obtained above, the concentration of
capability of PEG. However, multiarm-PEG with a special mole- BA in 8arm-PEG–BA was determined. Thus, dividing this value by
cular structure can be a better choice for highly efficient incor- the 8arm-PEG–BA concentration provided the percentage of BA
poration of drugs on the polymer scaffold. In a previous study, a in the conjugate. The mean mass percentage of drug-to-carrier
conjugate made of hydrophilic PEG and hydrophobic drug BA for 8arm-PEG–BA was 22.23 ꢂ 1.86 and the molar ratio was
5
1
was successfully synthesized. Fig. 1a–c show the H-NMR 6.26 ꢂ 0.41. The mass/molar ratio indicates that approximately 1
(
CDCl ) spectra of BA, 8arm-PEG–BA and F-8arm-PEG–BA, where or 2 functional groups of 8arm-PEG–BA remained unconjugated.
3
the signals at d 0.70–2.18 are attributed to the most characteristic Fig. 2 presents the UV spectra of BA, folate, 8arm-PEG–BA,
1
Fig. 1 H-NMR spectra of BA in CDCl
3
(a), 8arm-PEG–BA in CDCl
3
(b), F-8arm-PEG–BA in D
2
O (c), and F-8arm-PEG–BA NPs in D
2
O (d).
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Fig. 2 Quantitative analysis of BA and folate content as determined by UV
method.
Fig. 3 Formation of 8arm-PEG–BA NPs and F-8arm-PEG–BA NPs. CAC
measurement of 8arm-PEG–BA (a) and F-8arm-PEG–BA (b) using pyrene
as a hydrophobic fluorescence probe. (c) Size of F-8arm-PEG–BA/HCPT
NPs formed with different concentrations of F-8arm-PEG–BA and HCPT.
and F-8arm-PEG–BA. Obviously, the characteristic peaks of folate
for F-8arm-PEG–BA appeared at 210 and 360 nm, and BA appeared
at 210 nm. For the similar mol% BA of F-8arm-PEG–BA
and 8arm-PEG–BA, the folate content in F-8arm-PEG–BA was
F-8arm-PEG–BA into aqueous medium (10ꢁ dilution). We have
encapsulated HCPT as another model chemotherapy drug into
the F-8arm-PEG–BA NPs for combination therapy. Hydrophobic
drug HCPT can be encapsulated into the hydrophobic core of
calculated using ABSFolate = ABSF-8arm-PEG–BA ꢀ ABS8arm-PEG–BA
,
assuming that the F-8arm-PEG–BA and 8arm-PEG–BA in methanol
have the same molar extinction coefficients and that both followed
Lambert–Beer’s law (Table 1).
8
arm-PEG–BA NPs, and the PEG outer shell could improve the
3
6
water solubility and particle stability. As determined by trans-
mission electron microscopy (TEM) and dynamic light scattering
(DLS), the F-8arm-PEG–BA/HCPT NPs exhibited slightly larger
3.2. CAC measurements
spherical shape than the 8arm-PEG–BA/HCPT NPs (Table 1).
Desired particles of F-8arm-PEG–BA/HCPT NPs were produced
provided the concentration of F-8arm-PEG–BA and HCPT in the
Fig. 3a and b show the results of CAC measurements using
pyrene as a fluorescence probe. Upon incorporation into the
nanoparticles, the fluorescence intensity of pyrene increases
substantially at concentrations of nanoparticles above the
CAC. On the basis of the partitioning of pyrene, the CAC of
F-8arm-PEG–BA could be obtained by plotting the fluorescence
intensity versus logarithm concentrations of the polymer. The
CAC of F-8arm-PEG–BA was determined from the crossover
point in the low concentration range. The CAC of the F-8arm-
ꢀ1
solvent solution ranged from 0.13 to 1.50 mg mL and 0.01 to
ꢀ1
1
.25 mg mL , respectively. It was observed that particles
ꢀ1
formed from 0.5 mg mL
F-8arm-PEG–BA conjugate and
HCPT were approximately 120 nm in size
ꢀ1
0
.10 mg mL
(
Fig. 3c, Table 1), and particles formed from other solutions
were smaller (90–110 nm) or the same size. The approximately
20 nm nanoparticle system suggested its potential for effective
1
ꢀ
1
PEG–BA conjugate was 64.8 mg mL , which is similar to most
reported nanoparticle delivery systems. As the F-8arm-PEG–BA
NPs were stable at the CAC, it is probable that these particles will
remain stable at high dilution in biological systems. For example,
37
tumor targeting in vivo.
The zeta potential is a key factor to consider when evaluating
the stability of a colloidal dispersion. Most charged functional
groups are responsible for active nanoparticle interaction with
cells. Cationic particles are well known to bind to negatively
charged groups on the cell surface. As positively charged
particles have the greatest efficiency in cell-membrane penetra-
ꢀ
1
in a 27 g mouse model treated at 10 mg kg BA, 200 mL of
F-8arm-PEG–BA/HCPT NPs administered by intravenous injec-
ꢀ1
tion (5.1 mg mL F-8arm-PEG–BA/HCPT NPs) would be diluted
ꢀ1
in B2 mL blood volume to 510 mg mL . Considering the
scenario when 90% of particles are out of circulation, the
concentration of nanoparticles would still be well above the CAC.
tion and cellular internalization, they form the primary plat-
38,39
form as carriers for drug delivery.
8arm-PEG–BA/HCPT NPs
and F-8arm-PEG–BA/HCPT NPs all have a positive surface
charge (x = 5.32 mV and 8.61 mV). The interparticle interactions
may be partly responsible for the charge of the nanoparticles to
3.3. Nanoparticle formulation
Nanoparticles were prepared using the well-established preci- easily disperse, which enables F-8arm-PEG–BA/HCPT NPs to be
3
5
40
pitation method by slow addition of a DMSO solution of easily resuspended after a period of sedimentation.
Table 1 Particle size and drug loading efficiency of nanoparticles
Compound
arm-PEG–BA
Folate (wt%)
—
4— .32 ꢂ 0.76
DLEBA (wt%)
DLEHCPT (wt%)
Size (nm)
Zeta potential (mV)
8
22.23 ꢂ 1.86
21.27 ꢂ 1.61
18.85 ꢂ 1.37
17.91 ꢂ 1.29
—
—
—
—
—
—
F-8arm-PEG–BA
arm-PEG–BA/HCPT NPs
F-8arm-PEG–BA/HCPT NPs
8
3.63 ꢂ 0.52
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.4. In vitro drug release
Journal of Materials Chemistry B
3
release of BA. The resulting release data appear in Fig. 4e and f.
The amounts of BA or HCPT loading are listed in Table 1.
Nanoparticles with suitable size (Fig. 4a and b) were first placed in
aqueous solutions that simulated biological fluids, and in vitro
BA release kinetic were quantified by HPLC analysis using a UV
detector at 210 nm (Fig. 4c and d). 8arm-PEG–BA/HCPT NPs and
F-8arm-PEG–BA/HCPT NPs very slowly hydrolyzed and released
the BA at a weakly acidic or neutral pH without the common
burst release phenomenon (Fig. 4c). In addition, the hydrolyzed
BA displayed a preferential release under weakly acidic condi-
tions (pH 4.5). This accelerated release under acidic conditions
was attributed to the re-protonation of the hydroxyl group of BA.
Providing the route that the nanocarriers were trapped in endo-
somes (pH 5–6) and lysosomes (pH 4–5) after cellular uptake, the
acid-facilitated release can be a benefit to the enhanced cyto-
toxicity. The resulting release data appear in Fig. 4d, suggesting
that HCPT encapsulated with nanoparticles revealed a slight
initial burst release. The slight initial burst release of HCPT from
the nanoparticles was credited to BA molecules located within
the shell or at the core and shell interface of the nanoparticles.
Furthermore, an initial burst of release may be attributed to
balancing the equilibrium between the inside and outside
release settings. The slow release of the drug from the nano-
particles could be attributed to the hydrophobic–hydrophobic
3
.5. Hemolysis study
Detrimental interaction of nanoparticles with blood constituents
such as RBCs must be avoided when these nanoparticles are
injected into the blood circulation as a carrier for drug delivery.
Erythrocytes were incubated with two concentrations of samples
as 1 mg mL and 0.1 mg mL , for 1 h at 37 1C. Hemolysis
was evaluated by measuring the amount of hemoglobin
released in the supernatant at 541 nm (Fig. 5). Triton X-100
was used as a positive control, which induced full hemoglobin
release. 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs
at concentrations of 1 mg mL and 0.1 mg mL showed a
comparable hemoglobin release to blank values (o5%), which
was significantly lower than similar concentrations of PEI25 K, a
cationic polymer known to have a significant hemolytic effect.
2
4
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
4
2
Although BA was cytotoxic to RBCs in a previous study, 8arm-
PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs released
little BA and HCPT during the short incubation period, suggest-
ing the excellent safety of the nanoparticles.
3.6. In vitro cytotoxicity
interactions between the HCPT molecules and the hydrophobic To ensure the effective of the drug delivery before their entry
4
1
BA inner shell of the nanoparticles. However, in the presence into human application, in vitro cytotoxicity should be consid-
of esterase, which is abundant in cytoplasm, all the nano- ered. The cytotoxicity of free BA, HCPT, 8arm-PEG–BA/HCPT
particles quickly hydrolyzed and released the BA and HCPT. NPs, and F-8arm-PEG–BA/HCPT NPs to LLC or A549 cancer
Therefore, these nanoparticles were prodrugs for intracellular cells was evaluated using the CCK-8 assay. Analysis of in vitro
Fig. 4 8arm-PEG–BA/HCPT NPs (a) and F-8arm-PEG–BA/HCPT NPs (b) observed by TEM (scale bar = 200 nm) and DLS. Drug release kinetics in PBS at
37 1C from the 8arm-PEG–BA/HCPT NPs (c: BA and e: HCPT) and F-8arm-PEG–BA/HCPT NPs (d: BA and f: HCPT).
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ꢀ1
Table 2 In vitro cytotoxicity analysis (IC50, mg mL
)
Compound
LLC
A549
BA
HCPT
6.56 (0.42735)
0.38 (0.02854)
0.23 (0.02278)
0.09 (0.00187)
8.31 (0.46351)
0.47 (0.03317)
0.16 (0.01225)
0.08 (0.00152)
8
arm-PEG–BA/HCPT NPs
F-8arm-PEG–BA/HCPT NPs
obtained from replicate experiments. The results showed that
the IC50 values of the samples are in the order BA 4 HCPT 4
8arm-PEG–BA/HCPT NPs
4
F-8arm-PEG–BA/HCPT NPs
Fig. 5 In vitro hemolysis assay of 8arm-PEG–BA/HCPT NPs and F-8arm-
PEG–BA/HCPT NPs compared to PEI25 K and Triton X-100 measured at (Table 2). Compared to free HCPT, the F-8arm-PEG–BA/HCPT
5
41 nm. Values are reported as the mean ꢂ SD for triplicate samples.
NPs treatment was 4.4ꢁ more toxic against LLC, and 5.8ꢁ more
toxic against A549. Compared to free BA, the F-8arm-PEG–BA/
HCPT NPs treatment was 76.3ꢁ more toxic against LLC, and
ꢀ1
cytotoxicity measurements showed that BA (10 mg mL ) and 102.6ꢁ more toxic against A549. The increased F-8arm-PEG–
ꢀ
1
HCPT (10 mg mL ) induced cell death which was dependent BA/HCPT NPs toxicity was approximately related to the chemo-
upon length of incubation. As shown in Fig. 6a and b, the time- sensitivity of each cell line and slow release of the drugs. In
dependent cytotoxic effect of the F-8arm-PEG–BA/HCPT NPs addition to the benefits of slow release, internalization of the
was evident, which indicated 23.3% LLC and 25.8% A549 graft copolymers and enhanced release in lysosomes may
survival after 24 h, 16.6% LLC and 16.5% A549 survival after 48 h, further enhance the action of drugs. Moreover, the combination
ꢀ
1
and 10.2% LLC and 8.0% A549 survival after 72 h at 10 mg mL
equivalent to native HCPT). The drug release from the F-8arm- efficacy versus free drugs. The F-8arm-PEG–BA/HCPT NPs
PEG–BA/HCPT NPs when incubated with cells is possibly from (17.91 wt% BA, 15.81 wt% HCPT) IC50 values of LLC and A549
therapy with BA and HCPT can induce superior in vitro therapeutic
(
ꢀ1
ꢀ1
the diffusion of drug molecules in the nanoparticles and the were 0.09 mg mL and 0.08 mg mL , the calculated combi-
degradation of nanoparticles, which is the main mechanism of nation index (CI) values of BA and HCPT in the F-8arm-PEG–BA/
4
3
drug release observed for polymer nanoparticles.
HCPT NPs were 0.25 (LLC) and 0.18 (A549), which are much
To compare the potency of the nanoparticles, the concentra- smaller than 1. This suggested that F-8arm-PEG–BA/HCPT NPs
tions of drug which killed 50% of the cells (IC50) were estimated achieves a significant synergistic effect by co-delivery of two
2
8
from survival curves as shown in Fig. 6c and d and Table 2, different anticancer drugs BA and HCPT.
Fig. 6 Cellular cytotoxicity of BA, HCPT, 8arm-PEG–BA/HCPT NPs, and F-8arm-PEG–BA/HCPT NPs in LLC and A549 cells. Cell viability of LLC (a) and
ꢀ1
A549 (b) cells treated with 10 mg mL of BA, HCPT, and nanoparticles (equivalent to native HCPT) was measured by CCK-8 assay (n = 3, error bars
represent standard deviation). CCK-8 assay of BA, HCPT, and nanoparticles with different concentrations in LLC (c) and A549 (d) cell lines (n = 3, error
bars represent standard deviation).
3762 | J. Mater. Chem. B, 2015, 3, 3754--3766
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.7. Folate competition
Journal of Materials Chemistry B
3
Compared with 8arm-PEG–BA/HCPT NPs, F-8arm-PEG–BA/
HCPT NPs could be delivered into cells more easily (the black
lines in Fig. 8a and b), which might be due to the more positive
In order to further evaluate the role of folate in the cellular
uptake of F-8arm-PEG–BA/HCPT NPs, LLC cells and A549 cells
were employed as folate receptor (FR) overexpressing cancer
cells and FR deficiency cancer cells, respectively.
were incubated with the nanoparticles (0.1 mg mL ) in culture
38,50
charge of F-8arm-PEG–BA/HCPT NPs (Table 1).
The positively
4
4,45
charged nanoparticles adsorbed much more on the negatively
charged cell-membrane surface and consequently show higher
levels of internalization, which may also contribute to the low level
of IC50 (A549) of F-8arm-PEG–BA/HCPT NPs.
The cells
ꢀ
1
medium containing increasing concentrations of free folate
4
6
as described previously with minor modification. The cyto-
toxicity of F-8arm-PEG–BA/HCPT NPs against LLC cells was
inhibited by excess free folate, and the cell viability increased
with increasing folate concentration (Fig. 7a). However, their
cytotoxicity against A549 cells did not change as a function of
folate concentration (Fig. 7b). For instance, the cell viability of
F-8arm-PEG–BA/HCPT NPs against LLC cells was approximately
The cellular uptake efficiencies of the free HCPT, 8arm-PEG–BA/
HCPT NPs, and F-8arm-PEG–BA/HCPT NPs were examined to
demonstrate the penetration and the targeting effects of the nano-
particles into the cells by CLSM. The internalization of different
samples incubated for 4 h was examined. In Fig. 9, the fluorescence
of HCPT (green) and DAPI (blue) can be observed. As seen, nearly no
uptake was observed in the HCPT group, as evidenced by the lack of
any green fluorescence associated with the cells. Free HCPT mole-
cules transported into the cytoplasm of the cell, in a passive
diffusion manner, were effluxed out by P-glycoprotein pumps. In
general, HCPT-loaded nanoparticulates, such as liposomes, micelles,
or polymer nanoparticles, have been known to be taken up by cells
through an endocytic pathway, thereby making them escape from
4
0.8% for F-8arm-PEG–BA/HCPT NPs and free folate concen-
ꢀ1
trations of 0.01 mg mL , but it was about 70% in the presence
ꢀ1
of 30 mg mL free folate. These findings suggest that free folate
molecules prevent the cellular uptake of the nanoparticles by
competitive binding to FR on the cell surface.
3.8. Cellular uptake
51
the effect of P-glycoprotein pumps. The endocytic delivery of HCPT
within cells by using nanoparticles maintains the intracellular HCPT
concentration to be high in the cytoplasm region. This can be seen
in Fig. 9. The cellular uptake extent of F-8arm-PEG–BA/HCPT NPs
was significantly higher than those of free HCPT and 8arm-PEG–BA/
HCPT NPs under the same conditions. The successful F-8arm-PEG–
BA/HCPT NPs delivery may be attributed to the FR which is
overexpressed in lung cancer cells and the positive charge of the
supported nanoparticles, which is attracted by the negatively
charged cell surface.
According to literature reports, cellular uptake could be affected
4
7
by many factors, such as particle size, different cell lines and
4
8
cell densities, surface properties (surface functional groups,
4
8,49
surface charge, and zeta potential),
and so on.
Flow cytometry results show that there was no difference in the
extent of cellular uptake of 8arm-PEG–BA/HCPT NPs between LLC
cells and A549 cells, when folate was absent (Fig. 8a), and a greater
fraction of F-8arm-PEG–BA/HCPT NPs was taken up within LLC
cells than A549 cells, when folate was present (Fig. 8b). The above
results suggest that F-8arm-PEG–BA/HCPT NPs were transported
within cells by a FR-mediated endocytosis process.
3.9. The pharmacokinetics in mice
Long blood circulation half-time of a drug carrier is desired to
improve the bioavailability of the drug. For most nanomedicines,
Fig. 7 Effect of free folate on viability of LLC (a) and A549 cells (b)
incubated with F-8arm-PEG–BA/HCPT NPs.
Fig. 9 Confocal microscopic pictures of LLC cells incubated with free HCPT
Fig. 8 Flow cytometric analysis of A549 cells and LLC cells treated with
(a), 8arm-PEG–BA/HCPT NPs (b), and F-8arm-PEG–BA/HCPT NPs (c) at an
ꢀ1
8
arm-PEG–BA/HCPT NPs (a) and F-8arm-PEG–BA/HCPT NPs (b).
equivalent HCPT concentration of 0.3 mg mL (IC50) for 4 h at 37 1C.
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this has been a worthwhile goal. Here, the pharmacokinetics of
nanoparticles was studied after intravenous (i.v.) administration
to C57BL/6 mice. The determined drug concentration after hydro-
lysis under basic conditions was actually the total BA in plasma,
the combination of both parent form and nanoparticles form. The
blood clearance profiles of free BA and nanoparticles in mice are
shown in Fig. 10a and b. Disappearance of free BA and free HCPT
from the blood circulation after intravenous administration of
injection was very rapid with the plasma concentration below 10%
of injected dose per gram (%ID per g) at 3 h and 2 h, respectively.
In contrast, 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT
NPs exhibited a remarkable prolonged clearance with the BA
levels of 6.9% and 11.2% ID per g, respectively, at 24 h after
administration. 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/
HCPT NPs could extend the blood circulation half-time of BA
from 0.8 h to 3.2 h and 3.8 h, respectively, which were far longer
(
4.50- and 4.7-fold compared with free BA) than values of BA. For
Fig. 10 Blood circulation curves and half-time of 8arm-PEG–BA/HCPT
NPs and F-8arm-PEG–BA/HCPT NPs compared with free BA (a, b) and free
HCPT (c, d). Error bars were based on six mice per group at each time
point.
HCPT levels, 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT
NPs exhibited 8.5% and 10.5% ID per g, respectively, at 24 h after
administration. The blood circulation half-time of free HCPT was
0.4 h. 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs
Fig. 11 Antitumor efficacy of BA, HCPT, 8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs in LLC-bearing mice model. (a) Tumor volumes of
mice with different treatment groups; dashed line: stop administration. (b) Therapeutic efficacy of different treatments. (c) Tumor photographs from each
treatment group excised on day 20 (i: control, ii: BA, iii: HCPT, iv: 8arm-PEG–BA/HCPT NPs, and v: F-8arm-PEG–BA/HCPT NPs). (d) The body weight
variation of mice with different treatments. (e) IgE levels of mice treated with different formulations for 30 min. (f) WBC change during different
administrations in normal mice. Blood samples were collected from mice on day 2 after the last dosage treatment. Data are shown as means ꢂ SD; n = 6.
3764 | J. Mater. Chem. B, 2015, 3, 3754--3766
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Journal of Materials Chemistry B
could extend the blood circulation half-time of HCPT from 3.11. Evaluation of the side effects
.4 h to 2.3 h (5.7-fold) and 3.0 h (7.3-fold), respectively. The
0
During the early development of a new drug, many drug formula-
tions, such as paclitaxel, docetaxel, and teniposide cyclosporine
are always associated with serious side effects including hemato-
logical toxicity and hypersensitivity reactions (usually type I
hypersensitivity). Therefore, we tested the potential side effects
of nanoparticles. To examine the type I hypersensitivity responses,
the IgE antibody level, the parameter for rapid evaluation, was
detected in each group. The blood IgE levels of mice in different
groups are shown in Fig. 11e. Mice treated with BA and HCPT
displayed higher IgE levels than the control group, which might
ꢀ1 be ascribed to the poor water solubility. As expected, no signifi-
cant change of IgE level was observed in the 8arm-PEG–BA/HCPT
NPs and F-8arm-PEG–BA/HCPT NPs groups, which explored the
idea that the use of these nanoparticles could reduce the risk of
hypersensitivity reactions substantially. The blood of mice after
treatment with different formulations was also collected to test
the WBC count, which is often used as an indicator of hemato-
logical toxicity. The total WBC count of mice treated with BA
showed a slight decrease over the normal group (Fig. 11f). No
discernible decreases in WBC number of the mice treated with
molecular weight and particle size of the nanoparticles, which
were much bigger than free BA, are important for obtaining long
circulation times.
3.10. In vivo studies
The results obtained above gave us a lot of motivation to
explore the antitumor efficacy of the F-8arm-PEG–BA/HCPT
NPs in a mouse tumor model. In vivo antitumor efficiency of
drug delivery nanoparticles was compared with free BA and
HCPT at equivalent doses of 10 mg kg BA and 10 mg kg
HCPT, respectively, on multiple-dose schedule in xenograft
models of lung tumor. For humane reasons, animals were
killed and regarded as dead if the implanted tumor volume
ꢀ
1
3
reached 5000 mm or at the end of the experiment (46 weeks).
Tumor-bearing mice treated with the nanoparticles showed a
clear survival advantage compared with the control treated
mice (Fig. 11b). As shown in Fig. 11a, the groups treated with
BA, HCPT, and different nanoparticles showed varied levels of
antitumor effects and they were ranked as F-8arm-PEG–BA/
HCPT NPs 4 8arm-PEG–BA/HCPT NPs 4 BA and HCPT.
The treatment with F-8arm-PEG–BA/HCPT NPs resulted in
8arm-PEG–BA/HCPT NPs and F-8arm-PEG–BA/HCPT NPs were
observed, indicating that the nanoparticles designed in this study
could avoid severe hematotoxicity.
9
0.5% TGI (day 20) and 83.3% survival of animals (day 30). In
contrast, multiple-dose free BA treatment resulted in 51.3% TGI
day 20), 0% survival of animals (day 30), and free HCPT
(
4. Conclusion
treatment resulted in 45.3% TGI (day 20) and 16.7% survival
of animals (day 30) (Fig. 11a and b, Table 3). Importantly, in In conclusion, we have developed F-8arm-PEG–BA nanoparticles
line with the literature, no signs of systemic toxicity were with a hydrophobic anticancer core of BA and a hydrophilic shell
observed by monitoring general behavior, appetite and mice of PEG (PEG-folate), which is capable of entrapping another anti-
body weight (Fig. 11d).
cancer drug, HCPT, to achieve a significant synergistic effect. These
As expected, when using the HCPT-encapsulated F-8arm- nanoparticles for co-delivery of anticancer drugs demonstrated a
PEG–BA/HCPT NPs for in vivo anti-tumor therapy, three effects series of attractive properties as an anticancer drug delivery carrier,
are anticipated to increase the uptake of the drug-loaded including ease of preparation, appropriate size, high loading capa-
nanoparticles by cancerous cells: (1) the appropriate size city of drugs, good stability, rapid cellular uptake by tumor cells,
(
B120 nm), the EPR effect of solid tumors would allow more high synergistic effects, and no side effects.
drug-loaded nanoparticles to be accumulated in the tumor
5
2
tissue; (2) high drug loading (B18 wt% BA and B16 wt%
Acknowledgements
HCPT); (3) drug-loaded nanoparticles can increase the solubi-
5
3
lity of the drug. In addition, the in vitro and in vivo experi- This study was funded by the Fundamental Research Funds
ments demonstrate that the F-8arm-PEG–BA/HCPT NPs have for the Central Universities (200-1244951), the State Forestry
better therapeutic efficacy than F-8arm-PEG–BA/HCPT NPs, Administration 948 Project of China (No. 2014-4-35), Beijing
which may be due to the active targeting effect of folic acid.
Natural Science Foundation of China (Grant No. 2142024), the
National High Technology Research and Development Program
of China (863 Program, No. 2014AA022109), and the National
Natural Science Foundation of China (No. 20976179).
Table 3 LLC xenograft model (q2d ꢁ 5): efficacy comparison
a
a
b
Mean TV ꢂ SD
TGI Cures
3
a
Compound
(mm )
RTV
(%) (%)
Notes and references
Control
BA
HCPT
4562 ꢂ 1872
2221 ꢂ 1296
2497 ꢂ 1297
936 ꢂ 456
34.6 ꢂ 14.2
0
0
0
17.6 ꢂ 10.3 51.3
1 E. Pisha, H. Chai, I.-S. Lee, T. E. Chagwedera, N. R. Farnsworth,
G. A. Cordell, C. W. W. Beecher, H. H. S. Fong, A. D. Kinghorn,
D. M. Brown, M. C. Wani, M. E. Wall, T. J. Hieken, T. K. Das
Gupta and J. M. Pezzuto, Nat. Med., 1995, 1, 1046–1051.
18.0 ꢂ 9.5 45.3 16.7
7.3 ꢂ 3.6 79.5 33.3
3.4 ꢂ 1.9 90.5 83.3
8
arm-PEG–BA/HCPT NPs
F-8arm-PEG–BA/HCPT NPs 432 ꢂ 240
a
Mean tumor volume (TV), RTV, and %TGI data were taken at day 20
2
H. Zou, W. Guo and W. Yuan, J. Mater. Chem. B, 2013, 1,
(
by day 20, a significant percentage of control animals were euthanized
b
due to excess tumor burden). %Cures were taken at day 30.
6235–6244.
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3
J.-H. Park, H.-J. Cho, H. Y. Yoon, I.-S. Yoon, S.-H. Ko, 29 B. Shi, C. Fang and Y. Pei, J. Pharm. Sci., 2006, 95,
J.-S. Shim, J.-H. Cho, J. H. Park, K. Kim, I. C. Kwon and
D.-D. Kim, J. Controlled Release, 2014, 174, 98–108.
S. Nigam, S. Chandra, D. F. Newgreen, D. Bahadur and
Q. Chen, Langmuir, 2014, 30, 1004–1011.
L. Dai, D. Li, J. Cheng, J. Liu, L.-H. Deng, L.-Y. Wang,
J.-D. Lei and J. He, Polym. Chem., 2014, 5, 5775–5783.
L. Dai, T. Yang, J. He, L. Deng, J. Liu, L. Wang, J. Lei and
L. Wang, J. Mater. Chem. B, 2014, 2, 6749–6757.
1873–1887.
30 K. Kim, J. H. Kim, H. Park, Y. S. Kim, K. Park, H. Nam,
S. Lee, J. H. Park, R. W. Park, I. S. Kim, K. Choi, S. Y. Kim,
K. Park and I. C. Kwon, J. Controlled Release, 2010, 146,
219–227.
31 M. J. Ernsting, M. Murakami, E. Undzys, A. Aman, B. Press
and S. D. Li, J. Controlled Release, 2012, 162, 575–581.
32 M. Ding, N. Song, X. He, J. Li, L. Zhou, H. Tan, Q. Fu and
Q. Gu, ACS Nano, 2013, 7, 1918–1928.
33 W. Tai, R. Mo, Y. Lu, T. Jiang and Z. Gu, Biomaterials, 2014,
35, 7194–7203.
34 W. S. Saari, J. E. Schwering, P. A. Lyle, S. J. Smith and
E. L. Engelhardt, J. Med. Chem., 1990, 33, 97–101.
4
5
6
7
8
9
M. S. Islam, H. N. Choi, W. S. Choi and H.-J. Lee, J. Mater.
Chem. B, 2015, 3, 1001–1009, DOI: 10.1039/C4TB01911C.
A. C. Anselmo and S. Mitragotri, J. Controlled Release, 2014,
190, 531–541.
Y. Shen, E. Jin, B. Zhang, C. J. Murphy, M. Sui, J. Zhao,
J. Wang, J. Tang, M. Fan, E. Van Kirk and W. J. Murdoch, 35 U. Bilati, E. All ´e mann and E. Doelker, Eur. J. Pharm. Sci.,
J. Am. Chem. Soc., 2010, 132, 4259–4265. 2005, 24, 67–75.
0 K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert, 36 Y. Sanada, I. Akiba, K. Sakurai, K. Shiraishi, M. Yokoyama,
1
1
Angew. Chem., Int. Ed., 2010, 49, 6288–6308.
E. Mylonas, N. Ohta, N. Yagi, Y. Shinohara and Y. Amemiya,
1 H. Zhao, B. Rubio, P. Sapra, D. C. Wu, P. Reddy, P. Sai,
J. Am. Chem. Soc., 2013, 135, 2574–2582.
A. Martinez, Y. Gao, Y. Lozanguiez, C. Longley, L. M. 37 Y. Huang, J. Lu, X. Gao, J. Li, W. Zhao, M. Sun, D. B. Stolz,
Greenberger and I. D. Horak, Bioconjugate Chem., 2008,
9, 849–859.
R. Venkataramanan, L. C. Rohan and S. Li, Bioconjugate
Chem., 2012, 23, 1443–1451.
1
12 L. Dai, L. Wang, L. Deng, J. Liu, J. Lei, D. Li and J. He, Sci. 38 A. Verma and F. Stellacci, Small, 2010, 6, 12–21.
Rep., 2014, 4, 5871, DOI: 10.1038/srep05871.
39 E. C. Cho, J. Xie, P. A. Wurm and Y. Xia, Nano Lett., 2009, 9,
13 S. D. Weitman, A. G. Weinberg, L. R. Coney, V. R. Zurawski,
1080–1084.
D. S. Jennings and B. A. Kamen, Cancer Res., 1992, 52, 40 H.-l. Ma, X.-r. Qi, Y. Maitani and T. Nagai, Int. J. Pharm.,
708–6711. 2007, 333, 177–186.
4 S. Chen, X.-Z. Zhang, S.-X. Cheng, R.-X. Zhuo and Z.-W. Gu, 41 H. K. Patra, N. U. Khaliq, T. Romu, E. Wiechec, M. Borga,
6
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
Biomacromolecules, 2008, 9, 2578–2585.
5 A. Saleem, T. K. Edwards, Z. Rasheed and E. H. Rubin, Ann.
N. Y. Acad. Sci., 2000, 922, 46–55.
6 Y. Sugimoto, S. Tsukahara, T. Oh-hara, L. F. Liu and
T. Tsuruo, Cancer Res., 1990, 50, 7962–7965.
7 Y. Wang, Y. Wang, J. Xiang and K. Yao, Biomacromolecules,
A. P. F. Turner and A. Tiwari, Adv. Healthcare Mater., 2014, 3,
526–535.
42 M. Gao, P. M. Lau and S. K. Kong, Arch. Toxicol., 2014, 88,
755–768.
43 S. Fredenberg, M. Wahlgren, M. Reslow and A. Axelsson, Int.
J. Pharm., 2011, 415, 34–52.
44 S. B. Gates, L. G. Mendelsohn, K. A. Shackelford,
L. L. Habeck, J. D. Kursar, L. S. Gossett, J. F. Worzalla,
C. Shih and G. B. Grindey, Clin. Cancer Res., 1996, 2,
1135–1141.
45 D. J. Bharali, D. W. Lucey, H. Jayakumar, H. E. Pudavar and
P. N. Prasad, J. Am. Chem. Soc., 2005, 127, 11364–11371.
46 K. S. Soppimath, L. H. Liu, W. Y. Seow, S. Q. Liu, R. Powell,
P. Chan and Y. Y. Yang, Adv. Funct. Mater., 2007, 17,
355–362.
2010, 11, 3531–3538.
8 S. Wang, R. J. Lee, C. J. Mathias, M. A. Green and P. S. Low,
Bioconjugate Chem., 1996, 7, 56–62.
9 M. J. Ernsting, W. L. Tang, N. MacCallum and S. D. Li,
Bioconjugate Chem., 2011, 22, 2474–2486.
0 S. B. La, T. Okano and K. Kataoka, J. Pharm. Sci., 1996, 85,
85–90.
1 J.-H. Choy, J.-S. Jung, J.-M. Oh, M. Park, J. Jeong, Y.-K. Kang
and O.-J. Han, Biomaterials, 2004, 25, 3059–3064.
2 G. Zhao, W. Yan and D. Cao, J. Pharm. Biomed. Anal., 2007, 47 W. Zauner, N. A. Farrow and A. M. R. Haines, J. Controlled
3, 959–962. Release, 2001, 71, 39–51.
3 H.-Y. Cheung and Q.-F. Zhang, J. Chromatogr. A, 2008, 1213, 48 T. Jung, W. Kamm, A. Breitenbach, E. Kaiserling, J. X. Xiao
31–238. and T. Kissel, Eur. J. Pharm. Biopharm., 2000, 50, 147–160.
4 R. Reul, J. Nguyen and T. Kissel, Biomaterials, 2009, 30, 49 K. A. Foster, M. Yazdanian and K. L. Audus, J. Pharm.
815–5824. Pharmacol., 2001, 53, 57–66.
5 J. Nguyen, T. W. J. Steele, O. Merkel, R. Reul and T. Kissel, 50 E. K. Park, S. B. Lee and Y. M. Lee, Biomaterials, 2005, 26,
J. Controlled Release, 2008, 132, 243–251. 1053–1061.
6 W. Wei, P. P. Lv, X. M. Chen, Z. G. Yue, Q. Fu, S. Y. Liu, 51 A. A. Gabizon, Cancer Res., 1992, 52, 891–896.
H. Yue and G. H. Ma, Biomaterials, 2013, 34, 3912–3923. 52 H. Maeda, J. Controlled Release, 2012, 164, 138–144.
7 F. Unger, M. Wittmar and T. Kissel, Biomaterials, 2007, 28, 53 O. P. Medina, N. Pillarsetty, A. Glekas, B. Punzalan,
4
2
5
1610–1619.
V. Longo, M. G o¨ nen, P. Zanzonico, P. Smith-Jones and
S. M. Larson, J. Controlled Release, 2011, 149, 292–298.
8 T.-C. Chou, Cancer Res., 2010, 70, 440–446.
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