Dendrimers with the protocatechuic acid building block for anticancer drug delivery

Article abstract of DOI:10.1039/c6tb01597b

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) is a well-known antioxidant compound and a potential antitumor drug that is commonly found in fruits and vegetables. This article describes the development of novel biodegradable dendrimers that contain

Full text of DOI:10.1039/c6tb01597b

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Dendrimers with protocatechuic acid building blocks for
anticancer drug delivery
Received 00th January 20xx,
Accepted 00th January 20xx
Xiujuan Xi†, Shiqi Hu†, Zhuxian Zhou*, Xiangrui Liu, Jianbin Tang, Youqing Shen*
DOI: 10.1039/x0xx00000x
Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) is a well-known antioxidant compound and a potential antitumor
drug that is commonly found in fruits and vegetables. This article describes the development of novel biodegradable
dendrimers that contain PCA as building blocks. The structures of the dendrimers were characterized by nuclear magnetic
resonance, gel permeation chromatography, and matrix-assisted laser-desorption ionization time-of-flight mass
spectrometry. PCA dendrimers could serve as potential anticancer drugs and also as nanocarriers for anticancer drug
delivery. PCA dendrimers can easily be loaded with hydrophobic drugs such as doxorubicin that benefit from the binding
interaction between PCA and the drug. Doxorubicin-loaded PCA dendrimers exhibited pH and redox-dual responsive drug
release in vitro. The antitumor effect of PCA dendrimers to which polyethylene glycol polymer chains have been attached
and doxorubicin-loaded dendrimers was preliminary evaluated both in vitro and in vivo.
www.rsc.org/
However, these dendrimers are typically inert, and their
degradation products have no effect on the treatment of tumors or
other diseases, so the carrier must be excreted in the form of
Introduction
additional waste, thus increasing the physical exertion required of
cancer patients.
Dendrimers are nanosized, highly branched macromolecules
characterized by well-defined monodisperse structures, internal
cavities for guest molecule encapsulations, ample surface groups
In this paper, we propose that the use of a bioactive molecule
as the building blocks of a polyester dendrimer would minimize the
use of other inert materials and increase the bioactivity of the
nanocarrier. Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA), a
phenolic compound isolated from plants, fruits, nuts, and black rice;
has demonstrated antioxidant, antibacterial, and antitumor
promotion effects. It has been reported that PCA may prevent
tumorigenesis15 and inhibit the proliferation and metastasis of a
series of cancer cells.16 PCA includes one carboxyl group and two
phenolic hydroxyl groups, and thus can be used as an AB2-type
building block for the preparation of polyester dendrimers with a
double-stage convergent approach. PCA dendrimers are designed
to hydrolyze under physiological conditions, and the unique
biological properties of the PCA compound may endow PCA
dendrimers with antitumor activity. Furthermore, PCA can also
serve as a drug-binding molecule because of its high π-π stacking
binding interaction with benzene-containing therapeutic drugs such
as doxorubicin (DOX).17, 18 The strong interaction of the dendrimers
with DOX may facilitate drug loading and reduce the initial drug
release in systemic administration. The antitumor activity of
PEGylated PCA dendrimers (PCA4-PEG) and DOX-loaded PCA
dendrimers (PCA4-PEG/DOX) was preliminary evaluated in vitro and
in vivo.
for functionalization, and an unusually low intrinsic viscosity that
2
allows easy transport in the blood.1,
These distinctive
characteristics make dendrimers ideal nanocarriers for drug and
gene delivery because they allow for control over the molecular
weight, hydrophilicity, multiplicity of therapeutic drugs, anchoring
with specific active targeting agents, and pharmacokinetic
behavior.3-5
Of the many available dendrimers, poly(amidoamine) (PAMAM)
dendrimers are the most widely studied for drug and gene
7
delivery.6, However, amine-terminated PAMAM dendrimers are
not biodegradable in vivo and are positively charged on the surface,
and they interact nonspecifically with negatively charged biological
membranes and cause a variety of problems in in vivo applications,
including cytotoxicity, hemolytic toxicity, rapid clearance from the
blood circulation, and
a
high level of uptake in the
reticuloendothelial system.8 These problems have hindered their
clinical application. As a result, efforts have focused on reducing the
toxicity and improving the pharmacokinetic behavior by developing
biodegradable and biocompatible dendrimers such as polyester
10
dendrimers,9,
polylysine dendrimers,11 and polyether
dendrimers.12 Polyester dendrimers are most attractive because of
their good biodegradability, which facilitates dendrimer clearance
by the hydrolysis of macromolecules into small molecules.10, 13, 14
Experimental
Materials
Reagent-grade dichloromethane (DCM) and tetrahydrofuran (THF)
were predried over calcium hydride (CaH2) and distilled under argon
immediately before use. DMF was distilled from CaH2 just before
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Journal Name
use. All other solvents and chemicals were used as received. DOX dissolved in toluene (15 ml) was added dropwise into the previous
hydrochloride salt (DOX·HCl) was supplied by Zhejiang Hisun solution over 10 min. The mixture was stirred at RT for 20 min and
Pharmaceutical Co. (Taizhou, China). PCA, tert-butyldimethylsilyl then heated to 50°C and stirred for another hour until gas evolution
had ceased. The yellow solution was evaporated, and the residue
was taken up in 20 ml of toluene at 70 °C. The product was
dissolved, and the yellow, viscous side product remained
undissolved. The product solution was decanted and precipitated
by adding 55 ml of cyclohexane to the hot toluene solution and
chloride,
(4-chlorocarbonylphenyl)
boronic
anhydride
，
tetrabutylammonium fluoride (TBAF), glutathione (GSH), and
PEG2000 were obtained from Aladdin Chemistry Co. (Shanghai,
China).
Instruments
separated to give a yellowish oil at a 86% yield (18 g)20
.
Nuclear magnetic resonance (1H NMR) spectra were recorded with
Synthesis of methyl protocatechuate (4)
a
Bruker Avance DRX-400 spectrometer (Bruker BioSpin
The synthesis of methyl protocatechuate was adapted from the
work of Alam et al.21 a PCA (9.0 g, 58 mmol) was dissolved in dry
methanol (150 ml), to which a catalytic amount (0.3 ml) of
concentrated sulfuric acid was carefully added. The solution was
heated under reflux for 24 h at 65 °C and then cooled to RT. To this
solution, sodium hydroxide (2.0 M) was added dropwise until the
excess acid had been neutralized. The solvent was removed using a
rotary evaporator to yield crude methyl protocatechuate as a white
crystalline solid. To separate the methyl protocatechuate from any
unreacted PCA impurities, the product was dissolved in a mixture of
ethyl acetate (60 ml) and pure water (20 ml). The organic layer
containing the methyl protocatechuate was separated, further
washed with pure water (2 × 20 ml) and brine (40 ml), and then
dried over anhydrous MgSO4. The solvent was removed under
vacuum, and the product was recrystallized from hot water, filtered
and dried on a high vacuum line to give methyl protocatechuate
Corporation, Billerica, MA) with deuterated chloroform (CDCl3) or
dimethyl sulfoxide (DMSO-d6) as a solvent and tetramethylsilane as
the internal standard. All chemical shifts are reported in parts per
million (δ, ppm). Gel permeation chromatography (GPC) was
performed on a Wyatt GPC/SEC-MALS (Wyatt Technology Corp.,
Santa Barbara, CA) system equipped with a DAWN® HELEOS® II 18-
angle static light scattering detector and an Optilab® T-rEXTM
refractive index detector, and a pair of Shodex KF-402.5 HQ and KF-
404 HQ columns at 30 °C using THF as the eluent at a flow rate of
0.80 ml/min. Data were recorded and processed with ASTRA v6.0
(Wyatt Technology Corp.) software. Matrix-assisted laser-
desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS) were recorded on a Bruker ultrafleXtreme™ MALDI-TOF MS
(Bruker Daltonics GmbH, Bremen, Germany) using α-cyano-4-
hydroxycinnamic acid diethylammonium (CCA) as the matrix. One
microliter of dendrimer solution (1 × 10−2 M PCA, methanol) was
mixed with 50 μL of matrix solution (10 gL−1 CCA, methanol). One
microliter of the mixture was deposited onto the sample stage and
allowed to dry in air. The laser intensity was set to the lowest value
possible to acquire high-resolution spectra. The spectra were
analyzed with FlexAnalysis v3.4 (Bruker Daltonics GmbH, Bremen,
Germany).
1
(8.9 g; 91%) as a white crystalline solid, H NMR (400 MHz; DMSO-
d6, 25 °C): δ (ppm) 9.53 (br s, 2H, 3-OH), 7.32 (d, 1H, 5-H of PCA),
7.29-7.27 (dd, 1H, 6-H of PCA), 6.78 (d, 1H, 2-H of PCA), 3.73 (3H, s,
-CO2CH3) (Figure S2).
Synthesis of protocatechuic acid tert-butyldimethylsilyl ether
dendrimers (PCAn-TBDMS) and protocatechuic acid dendrimers
(PCAn-OH)
Synthesis of 3,4-bis(tert-butyldimethylsiloxy) benzoic acid (2)
Methyl protocatechuate (4) (1.0 g, 6.0 mmol) and triethylamine (2.6
ml; 18.68 mmol) were dissolved in dry THF (20 ml) and stirred at RT.
To this solution, 3,4-bis(tert-butyldimethylsiloxy)benzoyl chloride (3)
(7.2 g, 18 mmol) in 10 ml of THF was slowly added under N2 at 0°C.
The solution was allowed to warm to RT and was stirred overnight.
The reaction mixture was filtered to remove insoluble fraction, and
the filtrate was concentrated by rotary evaporation. The product
was purified by column chromatography (SiO2, hexane/ethyl
acetate 10:1, Rf = 0.5) to give PCA1-TBDMS as a white powdery
PCA (15.4 g, 100 mmol) and imidazole (56.0 g, 800 mmol) was
dissolved in DMF (150 ml) and cooled to 0 °C. To this solution, tert-
butylchlorodimethylsilane (60.3 g, 400 mmol) in DMF (300 ml) was
slowly added under N2. The temperature of the solution was
allowed to increase to room temperature (RT) and the mixture was
stirred overnight. The reaction mixture was then poured into
saturated aqueous NaHCO3 (500 ml), extracted with ether, and
washed with brine. The combined organic extract was dried over
anhydrous Na2SO4 and concentrated on a rotary evaporator.
1
substance (4.2 g) at a 78% yield. H NMR (400 MHz, CDCl3, 25 °C): δ
The concentrate was then dissolved in a mixed solvent of THF
(80 ml) and MeOH (240 ml). To this solution, an aqueous solution of
K2CO3 (0.72 M, 80 ml) was added, and the mixture was stirred at RT
for 2 h. The reaction mixture was then concentrated to a 1/4
volume and diluted with brine (200 ml). The pH of the solution was
adjusted to 4-5 with KHSO4 (1.0 M), and the resulting mixture was
extracted with ether. The organic extract was combined, washed
with brine, dried over anhydrous Na2SO4, filtered and rotary
evaporated to dryness to give 2 as a white powdery substance (30 g)
at a 78% yield.19 1H NMR (400 MHz, CDCl3, 25°C): δ (ppm) 12.11 (br
s, 1H, COOH), 7.66-7.64 (dd, 1H, 6-H of PCA), 7.61 (d, 1H, 5-H of
PCA), 6.89 (d, 1H, 2-H of PCA), 1.01 (d, 18H, ArOSi(Me)2C(CH3)3),
0.26 (d, 12H, ArOSi(CH3)2 tBu) (Supporting information, Figure S2).
(ppm) 8.0 (d, 2H, Ar-H), 7.59-7.48 (m, 5H, Ar-H), 3.88 (s, 3H, -
CO2CH3), 0.94 (d, 36H -OSi(Me)2C(CH3)3), 0.22 (s, 12H, -OSi(CH3)2 t-
Bu), 0.13 (d, 12H, -OSi(CH3)2 t-Bu).
TBAF (7.07 g, 26.92 mmol) was added to a dry THF solution (20
ml) of PCA1-TBDMS (4 g, 4.4 mmol) under N2 at 0 °C, and the
mixture was stirred at 0 °C overnight. The reaction mixture was
then poured into saturated aqueous NH4Cl (50 ml) and extracted
with ethyl acetate. The combined organic extract was washed with
brine, filtered, and dried over anhydrous Na2SO4. The solution was
concentrated by rotary evaporation, and the residue was poured
into DCM to precipitate the crude product. The product was
purified by dissolving in methanol (2 ml) and precipitating in DCM.
The purified product was dried in a vacuum oven to give PCA1-OH
as a white powdery substance (1.96 g) at a 100% yield. MALDI-TOF
(CCA as matrix, m/z, [M+Na]+) 463.47 (obsd), 463.06 (calcd for
C22H16O10Na). 1H NMR (400 MHz, DMSO-d6, 25 °C): δ (ppm) 9.78 (br
s, 4H, -OH), 7.96 (d, 2H, Ar-H), 7.60 (d, 1H, Ar-H), 7.38 (s, 2H, Ar-H),
Synthesis of 3,4-bis(tert-butyldimethylsiloxy)benzoyl chloride (3)
A suspension of 3,4-bis(tert-butyldimethylsiloxy) benzoic acid (2)
(20 g, 52 mmol) in a mixture solvent of toluene (120 ml) and DMF
(0.2 ml) was stirred at RT. Thionyl chloride (12 ml, 165 mmol)
2 | J. Name., 2012, 00, 1-3
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7.35 (d, 2H, Ar-H), 6.77 (dd, 2H, Ar-H), 3.88 (s, 3H, -CO2CH3). PCA2-
The sizes (hydrodynamic diameters) and zeta potential of the
OH, PCA3-OH, PCA4-OH and PCA5-OH were synthesized by dendrimers were determined with a Nano-ZS Zetasizer (Malvern
repeating the reaction with 3 and followed by deprotection in TBAF Instruments Ltd., UK). Each measurement was performed in
solution.
triplicate, and the results were processed with DTS software version
6.20.
PEGylation of PCA4-OH dendrimer
In vitro hydrolysis and drug release
The synthesis of PCA4-OH PEG derivatives is summarized in Scheme
S1. A solution of MPEG (2KDa, 5 g, 2.5 mmol) and triethylamine (0.7 The degradation of PCA1-OH was studied by culturing PCA1-OH
ml) in 30 ml of DCM was added to the solution of (4- with dithiothreitol (DTT, 10 mM) and triethylamine (TEA, 1 mM). At
chlorocarbonylphenyl)boronic anhydride (0.9 g, 5 mmol) in 15 ml of different time interval, the solution was sampled for 1H NMR
DCM, and the mixture was stirred at RT for 6 h. The insoluble analysis. The release of DOX from the DOX-loaded dendrimers was
fraction was filtered, and the filtrate was concentrated with a rotary studied with a dialysis method. DOX-loaded dendrimers (DOX, 400
evaporator. The residual solution was dropped in cold diethyl ether μg/ml) were loaded into a dialysis bag (molecular weight cutoff,
to precipitate the crude product. The crude product was purified by 3500 Da) and immersed in 200 ml of HEPES buffer (0.01 M, pH 7.4)
dissolving in DCM and precipitating from cold diethyl ether. The with or without 10 mM of GSH or HEPES buffer (0.01 M, pH 5.5) at
purified product was dried in a vacuum to give PEG-B-OH as a white 37 °C with constant shaking. At timed intervals, the UV-Vis
powdery substance (4.67g) at a 86% yield. 1H NMR analysis absorbance at 480 nm of the liquid in dialysis bags was measured to
indicates that 75% of the end groups were modified with determine the DOX concentration.
phenylboronic acid (Figure S2 and S3). PEG-B-OH (2 eq. mol) and
PCA4-OH (1eq. mol) was dissolved in dry THF with anhydrous
Cell culture
A549 human lung cancer cells, BCap37 human breast cancer cells,
Na2SO4. The mixture was stirred at RT for 3 h and filtered, and the
and HepG2 liver cancer cells were obtained from the American Type
filtrate was precipitated in cold ether, filtered, and dried in vacuum
to obtain PCA4-PEG (Figure S3), 1H NMR analysis shows that 85% of
Culture Collection (Manassas, VA). All cell lines were maintained in
RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (Gibco), penicillin (100 units/ml), and streptomycin
(100 μg/ml) in a humidified atmosphere of 5% CO2 at 37 °C.
the dendrimer end groups were modified with PEG.
Determination of molecular weights by 1H NMR spectra
Two peaks of protons (a and b) with no overlap with any other
peaks were chosen for calculating and evaluating the molecular
Cellular uptake and intracellular distribution
A549 cancer cells were seeded onto glass-bottom petri dishes
weights of dendrimers. From the integral ratio of protons a and b in
1H NMR (χNMR) and the ratio of the number of the protons in the
(MatTek, Ashland, MA, USA, no. P35G-1.0-14-C) at a density of
100,000 cells per dish in 1 ml of medium and incubated for 24 h
before treatment. Cells were incubated with free DOX or DOX-
theoretical dendrimer structure (χT), the molecular weight of
dendrimers
can
be
determined
according
to:
ꢇ
ꢈ
loaded dendrimer at a DOX-equivalent dose of 2 μg/ml. LysoTracker
(Molecular Probes, Carlsbad, CA) was added to each dish at a
concentration of 200 nM 2 h before observation. DAPI (2-(4-
amidinophenyl)-6-indolecarbamidine; Beyotime, China) was added
to each dish at a concentration of 0.5 μg/ml 15 minutes before
observation. The cells were then thoroughly washed three times
with PBS; the images were taken at different times using a confocal
laser scanning microscope (CLSM, Nikon-A1 system, Japan). Nuclear
ꢅꢂꢃꢄ
ꢅꢆ
ꢇ
ꢊꢋ
ꢊꢋ
ꢉ
ꢈ
ꢉ
ꢀꢁ(ꢂꢃꢄ) =
· ꢀꢁ =
· ꢀꢁ (Equation 1)
1
where χNMR is the integral ratio of protons (a) and (b) in H NMR, χT
is the ratio of the number of protons in the theoretical dendrimer
structures, Sa and Sb are the peak areas of protons at a and b
positions, ΣHa and ΣHb are the sums of protons at the a and b
positions in the molecular formula of the PAn-TBDMS dendrimer,
and Wt is the theoretical molecular weight of the dendrimers.
staining was observed with
a 405-nm laser; the emission
wavelength was 425 to 475 nm and appeared blue. LysoTracker was
observed using a 488-nm laser; the emission wavelength was 510 to
540 nm and appeared green. The DOX-loaded dendrimer was
observed using a 543-nm laser; the emission wavelength was 560 to
610 nm and appeared red. Images were taken by using the lasers
sequentially with a 60× objective lens.
Preparation of DOX-loaded dendrimer
The DOX-loaded dendrimer was prepared as follows. Briefly,
DOX·HCl (5 mg) was dissolved in 1 ml of DMSO. Triethylamine (20 μl)
was added to the solution, and the mixture was stirred at RT
overnight. PCA4-PEG (30 mg) was added to the previous solution,
and the mixture was stirred for 30 min. Distilled water (9 ml) was
then dropped into the solution. The mixture was stirred at RT for
another 30 min and dialyzed (molecular weight cutoff, 3500 Da)
against pure water for 10 h (2 L × 3). The DOX content was
determined by measuring its UV-Vis absorbance at 480 nm and
calculated according to a calibrated linear curve of standard DOX
solutions.
In vitro cytotoxicity
The cytotoxicity of the DOX-loaded dendrimer was determined by
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT
assay. Briefly, cells (4000 cells per well) were evenly seeded onto
96-well plates and incubated overnight. The cells were incubated
with serial dilutions of free DOX, PCA4-PEG/DOX, and blank carriers
PCA4-PEG for 48 h. After treatment, the cells were incubated with
fresh medium containing 0.75 mg/ml of MTT for 3 h, which allowed
viable cells to reduce the yellow tetrazolium salt (MTT) into dark
blue formazan crystals. Finally, DMSO was added to dissolve the
formazan crystals. The absorbance at 562 nm was recorded with a
reference filter of 620 nm, using a microplate spectrophotometer
(SpectraMax M2e, Molecular Devices, Sunnyvale, CA). Each drug
concentration was tested in triplicate and in three independent
experiments.
The drug encapsulation efficiency (EE) and the drug loading
content (DLC) were calculated with the following formulas:
ꢍꢈꢎꢎ ꢏꢐ ꢑꢒꢓꢔ ꢕꢖꢁꢒꢈꢗꢗꢕꢑ
(
)
ꢌꢌ % =
× ꢘꢙꢙ
ꢍꢈꢎꢎ ꢏꢐ ꢐꢕꢕꢑ ꢑꢒꢓꢔ
ꢍꢈꢎꢎ ꢏꢐ ꢑꢒꢓꢔ ꢕꢖꢁꢒꢈꢗꢗꢕꢑ
ꢍꢈꢎꢎ ꢏꢐ ꢖꢈꢖꢏꢗꢈꢒꢁꢝꢞꢟꢕ
(
)
ꢚꢛꢜ % =
× ꢘꢙꢙ
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In vivo antitumor effect
weight distribution, as indicated by GPC traces (Figure 1). The GPC
traces of PCAn-TBDMS gradually shifted to the high–molecular-
weight region with the increase of dendrimer generations, and no
trace of low–molecular-weight fragments were found, which
indicates complete coupling and successful purification of each step.
Female BALB/c homozygous athymic nude mice (6 to 8 weeks of
age) were purchased from the Animal Center of Zhejiang University
and maintained under standard conditions. The experimental
procedures were approved by the Animal Ethics Committee of
Zhejiang University and were carried out in accordance with
institutional guidelines.
The in vivo antitumor efficacy of the drug-loaded dendrimers
was examined in nude mice with subcutaneous human lung
carcinoma (A549). Athymic nude mice were inoculated by the
subcutaneous injection of A549 cells (5 × 106) in the left auxiliary
flank. The tumor volume of each mouse was determined by
measuring two dimensions with calipers and calculated according to
the formula V = 1/2 ab2, wherein V is the volume of the tumor, a is
the longest diameter of the tumor, and b is the shortest diameter of
the tumor. When the tumors reached an average volume of
approximately 80 to 150 mm3, the mice were randomly divided into
their experimental groups (eight per group): (1) PBS (negative
control group), (2) PCA4-PEG, (3) PCA4-PEG/DOX, and (4) free DOX.
The DOX formulations were given by intravenous injection into the
tail vein in five doses of 4 mg/kg once every 3 days. Throughout the
study, the mice were weighed and their tumors were measured
with calipers. At the end of the experiments, all animals were
sacrificed according to institutional guidelines. The tumors were
resected, weighed, and fixed in formalin for paraffin embedding.
The tumor inhibition rate (TIR) was calculated with the following
equation: TIR = 100% × (mean tumor weight of control group −
mean tumor weight of experimental group)/mean tumor weight of
control group.
Histological examination of tumor tissues
Tumor and other tissue samples were fixed with 4% neutral
buffered paraformaldehyde, embedded in paraffin, and cross-
sectioned at a thickness of 5 μm. The sections were prepared and
stained with hematoxylin and eosin (Fisher Scientific, Waltham, MA)
for histologic examination and examined with light microscopy.
Scheme 1. Synthesis of PCA dendrimers.
Statistical analysis
The data are presented as means ± standard errors. Tumor volumes
over time were analyzed by one-way analysis of variance and then
by Student's t-test. All other statistical analyses were performed
using Student's t-test. Differences were considered to be
statistically significant at a level of p < 0.05.
Results and discussion
Synthesis and characterization of DOX-loaded protocatechuate
(PCA) dendrimers
PCA dendrimers were synthesized following the procedure as
shown in Scheme 1. To synthesize dendrimers with PCA as the
building blocks, we designed a pair of PCA-derived monomers, 3,4-
bis(tert-butyldimethylsiloxy)benzoyl chloride (3) as the repeating
units and methyl protocatechuate (4) as the dendrimer core. In the
propagation step, one molecule of
4 was reacted with two
molecules of 3 by the reaction of hydroxyl groups and acyl chloride
to form biodegradable ester bonds. The TBDMS-protected hydroxyl
groups were deprotected with TBAF. Repeating these two steps
produced PCA dendrimers with different generations.
Figure 1. GPC traces (THF, 30 °C, 0.8 ml/min) of PCAn-TBDMS (n =
1-5).
Moreover, the polydispersity of the dendrimers are mostly close to
1.00 in the range from 1.02 to 1.14, which indicates that the
obtained dendrimers have few defects and are of high purity. The
relative molecular weights of PCAn-TBDMS measured by GPC were
The molecular weight and structure of the PCA dendrimers was
characterized by GPC, 1H NMR, 13C NMR, and MALDI-TOF mass
spectra. All of the dendrimers showed monomodal molecular
4 | J. Name., 2012, 00, 1-3
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Table 1. Theoretical molecular weights (Mw(T)) and experimental
measurements of PCAn-TBDMS (n = 1-5) determined by GPC, 1H
NMR, and MALDI-TOF MS.
Mn
Mw/Mn
(GPC)
m/z[M+Na]+
( MALDI-TOF) a
PCA
dendrimers
Mw
(NMR)
Mw(T)
(GPC)
PCA1-TBDMS
PCA2-TBDMS
PCA3-TBDMS
PCA4-TBDMS
879.4
1898.8
3901.8
7907.6
734
1.02
1.03
1.06
1.14
1.10
879
1925
3821
7798
15722
919.6 [M+K]+
1920.8
1860
3460
5870
3925.5
7937.8
PCA5-TBDMS 15915.6 8740
16006.6
a
PCAn-TBDMS dendrimers with high generations are not stable at
high laser intensity; number represents found peak of
undecomposed fraction.
calculated according to polystyrene standards, which are lower
than the theoretical molecular weights, especially for dendrimers of
high generation (Table 1), because dendrimers exhibit a lower
hydrodynamic volume than their linear homologues due to
23
theircompact architecture.22,
The chemical structures of
synthesized PCAn-TBDMS dendrimers were further confirmed by 1H
NMR as shown in Figure 2A. Peaks on 1H NMR spectra of PCAn-
TBDMS dendrimers become broader as the dendrimer generation
increased, which is attributed to the densely packed structure of
the dendrimer arising from the AB2 substitution pattern and the
high tert-butyldimethylsilyl ether density generated at the
periphery. The 1H NMR spectra of the dendrimers can accurately
indicate protons at specific positions in the structure and can also
be used to determine molecular weight.24 The molecular weight of
Figure 2. 1H NMR spectrum of PCA5-TBDMS in (CD3)2CO (A) and
integration ratio of peak b (-Si-C(CH)3, TBDMS) to peak a (-COOCH3,
dendrimer core) in PCAn-TBDMS (n = 1-5) 1H NMR spectra (B).
1
PCAn-TBDMS can be calculated from the H NMR spectra from the
integration ratio of the peaks around 3.8 ppm (peak a, -COOCH3,
dendrimer core) and 0.9 ppm (peak b, -Si-C(CH)3, TBDMS) according
to Equation 1 in experimental section (Figure 2B). As the dendrimer
generation increased from G1 (PCA1-TBDMS) to G5 (PCA5-TBDMS),
the ratio of b:a increased from 36:3 to 569:3 and the calculated
Mw(NMR) increased from 879 Da to 15722 Da (Table 1). All of the
molecular weights calculated from the 1H NMR spectra are very
close to their theoretical values, with less than 2% error. This
further confirmed the successful synthesis of PCAn-TBDMS
dendrimers. The structures of the PCAn-TBDMS dendrimers were
further studied with MALDI-TOF MS (Figure S4). A product peak
with a molecular weight close to the theoretical value was found on
each spectrum. However, careful MS analysis revealed that the Figure 3. 1H NMR spectra of PCAn-OH (n = 1, 2, 3, 4, 5) in DMSO-d6.
degradation of phenol ester occurred upon ionization during MALDI
The interior of PCA4-PEG consisted of benzene rings that could
analysis and resulted in multiple peaks with molecular weight
form strong
π-π stacking interaction with the benzene ring of
differences (Δm/z) of 250 Da in the MS.25
DOX.17 Thus, DOX could be efficiently loaded into the dendrimer’s
cavity. The drug encapsulation efficiency and the drug loading
content were about 99% and 15%. The size and size distribution of
the PEGylated dendrimers were quantitated by dynamic light
scattering (Figure 4). The average diameter of PCA4-PEG was 30 nm,
with a polydispersity of 0.084. Loading DOX into PCA4-PEG did not
change the nanocarriers’ size. A nanocarrier between 10 and 100
nm is favorable for drug delivery that will not be cleared by renal
filtration and also has reduced hepatic filtration.29 Furthermore,
these small PEGylated dendrimers may have a long blood residence
time and a high rate of accumulation in the tumor tissue by passive
targeting via the enhanced retention and permeability effect of
tumor.30 The zeta potential of PCA4-OH and PCA4-PEG at pH 7.4 is -
11 mv and -6 mv, respectively. Loading DOX to PCA4-PEG did not
Hydroxyl terminated PCA dendrimers were obtained after the
deprotection of TBDMS by TBAF. The disappearance of TBAF peaks
1
between 0 and 1 ppm on the H NMR spectra indicated successful
deprotection (Figure 3), and the structure was further confirmed by
13C NMR (Figure S5). Product peaks of PCAn dendrimers with
molecular weights close to the theoretical values were also
identified in the mass spectra, accompanied by multiple
decomposed fragments (Figure S6). The fourth generation of PCA
dendrimer PCA4-OH was chosen for PEGylation and used as a
carrier for delivery of the anticancer drug DOX. PEGylation endows
nanocarriers with many favorable properties that include reduced
reticuloendothelial system clearance and prolonged blood
circulation time by avoiding protein opsonization.26-28
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Figure 6. Intracellular localization of free DOX (A) and DOX-loaded
dendrimers (B) in A549 cells was observed by CLSM after 2 h of
incubation, DOX: red; lysosomal dye LysoTracker: green; nuclear
dye DAPI: blue (scale bar, 20 ꢀm).
Figure 4. Size of PEGylated dendrimers measured by dynamic light
scattering in HEPES buffer (0.01 M; pH 7.4).
change the surface charge of the nanocarrier. The slightly negative
charged surface of nanocarrier may facilitate its accumulation and
deep penetration in tumor by reducing phagocytic uptake and
electrostatic adhesive interactions.31, 32
reduce the nonspecific drug release during blood circulation (pH
7.4) and accelerate drug release in the tumor tissue (pH < 7.0) or
intracellular compartments such as the endosome (pH 6.0 to 6.5)
and lysosome (pH 4.5 to 5.5).36 DOX release of PCA4-PEG/DOX was
also studied in a reductive environment with 10 mM of GSH (Figure
pH and redox-dependent drug release of PCA4-PEG/DOX
The degradation of PCA1-OH was studied and tracked by 1H NMR 5). Interestingly, DOX release was much faster in the presence of
spectra (Figure S7). When treated with dithiothreitol (DTT), a GSH than in its absence. About 78% of DOX was released in 48 h
reducing agent, along with triethylamine, PA1-OH was fully with the presence of 10 mM of GSH, which might be attributable to
degraded in three hours. The chemical shift of proton in –OCH3 the GSH-triggered thiolysis of phenol ester in the dendrimer.37 The
group gradually moved from 3.95 to 3.73 ppm in 3 hours. The GSH-dependent drug release profile of PCA4-PEG/DOX would
chemical shift of aromatic protons also changed with culturing time. enhance drug release in the strongly reducing intracellular
Drug release of PCA4-PEG/DOX was studied under different environment (approximately 0.5 to 10 mM of GSH), but the release
M GSH).38,
conditions to simulate different biological environments. As shown
would remain slow in the blood (approximately 20 to 40 ꢀ
39
in Figure 5, DOX was gradually released from the dendrimer at pH
The pH and redox dual-responsive PCA4-PEG/DOX is a promising
7.4 with reduced burst initial release, which may be attributed to
drug delivery system that can decrease side effects by reducing
the strong
π-π stacking interaction between the dendrimer and nonspecific drug release and meanwhile increase drug efficacy by
DOX.17 This is a great advantage over traditional nanocarriers, enhancing the delivery of active drug to the target sites.40-42
which are generally prone to severe burst release.33 It is feasible
Cellular uptake and intracellular localization of PCA4-PEG/DOX
and desirable to optimize the drug release profile by introducing an
optimal drug-binding molecule into the nanocarrier.34 After 48 h,
The cellular uptake and intracellular localization of PCA4-PEG/DOX
in A549 cancer cells were observed with confocal fluorescence
about 95% of the DOX was released at pH 5.0, whereas only 38% of
microscopy, and free DOX was used as a control (Figure 6). PCA4-
the DOX was released at pH 7.4. The loaded DOX was partially
PEG/DOX or DOX was incubated with cells for 2 h, and the
ionized at a lower pH, which may have reduced the binding
fluorescence of DOX appeared red. Late endosomes/lysosomes
interaction between the dendrimer and DOX, and thus triggered the
rapid release of DOX at a lower pH.17, 35 The hydrolysis of phenol
were labeled with LysoTracker, and the fluorescence appeared
green. Cell nuclei were labeled with nuclear dye, and the
ester and the destruction of PCA dendrimers at a lower pH may
fluorescence appeared blue. As shown in Figure 6, the overlay of
partly contribute to accelerated drug release. This acid-promoted
drug release is favorable for anticancer drug delivery, as it may
PCA4-PEG/DOX and Lysotracker images shows yellow spots that
indicate that PCA4-PEG/DOX was partly located in lysosomes within
the 2-h incubation period. The localization of PCA4-PEG/DOX in
lysosomes implies that PCA4-PEG/DOX might be taken up by
endocytosis, a possible pathway for nanocarriers. When cells were
incubated with free DOX, DOX was mainly found in the cell nuclei.
The fluorescent intensity in cells cultured with PCA4-PEG/DOX is
relatively higher than free DOX. These results demonstrate that
PCA4-PEG is an efficient carrier for delivery of DOX into the cell
cytoplasm, but also indicate that the internalization mechanism of
the DOX-loaded dendrimer differs from that of free DOX. This
finding is consistent with the reported DOX-loaded nanocarrier.43, 44
The results also suggest that free DOX can efficiently enter cells by
via passive diffusion rather than by receptor-mediated
endocytosis.45 Notably, some red fluorescence did not overlay with
Figure 5. DOX release kinetics were determined in HEPES buffer at
pH 7.4 or 5.0, or at pH 7.4 with 10 mM of GSH. Data are shown as
mean ± SD (n = 3).
green fluorescence and was observed in the cytoplasm but less so
in the nuclei. PCA4-PEG/DOX was internalized via an endocytosis
pathway and was located first in endosome/lysosomes. The acidic
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drug that acts by stabilizing the enzyme-DNA cleavable complex
leading to DNA breaks.48 Free DOX has a much higher nuclear
localization rate than PCA4-PEG/DOX as indicated by cellular
distribution study (Figure 6). However, the cytotoxicity of PCA4-
PEG/DOX is close to free DOX. The IC50 of DOX in PCA4-PEG/DOX to
A549, BCap37, and HepG2, is about 1.3, 5.2 and 3.2 times of free
DOX, respectively. PCA4-PEG/DOX could protect DOX from
deactivation and overcome the multidrug resistance of cancer cells,
but its time-consuming DOX-release and delayed delivery to nuclei
lower its cytotoxicity.
In vivo antitumor efficacy
The in vivo antitumor activity of the DOX-loaded dendrimers was
evaluated with a A549 xenograft model on nude mice (eight per
group) upon tail-vein injection. The antitumor efficacy of various
formulations is illustrated in Figure 8A. Blank PCA4-PEG showed
very low cytotoxicity in vitro, but can partially inhibit tumor growth
in vivo. It has been reported that PCA can prevent tumorigenesis by
reducing biomarkers of inflammation and angiogenesis49 and can
inhibit cell migration and invasion at non-cytotoxic
concentrations.47 Our finding indicates that PCA may have an
antitumor effect on A549 lung tumor by acting on the tumor
microenvironment. Both PCA4-PEG/DOX and free DOX (4 mg/kg)
Figure 7. Cytotoxicity of PCA4-PEG, PCA4-PEG/DOX, and free DOX
against A549 lung cancer cells, Bcap37 breast cancer cells, and
HepG2 liver cancer cells cultured for 48 h was estimated by MTT
assay. Data are presented as mean ± SD (n = 5).
environment of the endosome/lysosomes promoted the release of
DOX, and some DOX was transported into cytoplasm. When cells
were cultured with PCA4-PEG/DOX for 12 h (Figure S8), some DOX
had already entered or was close to the nuclei, which indicates that
DOX from PCA4-PEG/DOX can gradually enter nuclei to act as a DNA
toxin.
In vitro cytotoxicity
The in vitro cytotoxicity of PCA4-PEG, PCA4-PEG/DOX, and free DOX
was evaluated by MTT assays against a panel of human tumor cell
lines, including A549 lung cancer cells, BCap37 breast cancer cells,
and HepG2 liver cancer cells. Cells were cultured with each
treatment for 48 h, and the results are presented in Figure 7. Blank
PCA4-PEG had no effect on cell viability within the tested
concentrations as high as 100
ꢀg/ml, which indicates that the
carriers had low cytotoxicity.46 The antitumor activity of PCA
dendrimers may work by affecting the tumor microenvironment at
low concentrations.47 The IC50 (concentration that inhibits cell
growth to 50% of control) values of DOX in PCA4-PEG/DOX to A549,
Figure 8. Antitumor activities of DOX-loaded PCA dendrimers
against A549 human lung xenograft tumors. Nude mice with A549
tumors were treated with PBS, free DOX (4 mg/kg), PCA4-PEG, or
PCA4-PEG/DOX (equivalent to 4 mg/kg DOX) as indicated by the
arrows (q3d × 5; n = 6; data presented as average ± SE). (A) Tumor
volumes of mice as function of time (* p < 0.05, ** p < 0.005). (B)
Body weight of mice as function of time (** p < 0.005). (C)
Representative histologic images of tumor and heart tissue sections
(scale bar, 100 μm).
BCap37, and HepG2 were 0.88, 0.94, and 0.52
The IC50 value of DOX to these cancer cells was 0.66, 0.18, and 0.16
g/ml, respectively. Compared with DOX, PCA4-PEG/DOX showed
comparable cytotoxicity to A549 cells and about three to five times
less cytotoxicity to BCap37 and HepG2 cells. DOX is a DNA toxin
ꢀg/ml, respectively.
ꢀ
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treatments effectively suppressed tumor growth compared to that
seen in the control group. The TIR was calculated after the mice
were sacrificed at the end of treatment. The TIR of PCA4/PEG-DOX
was 48.2%, which was lower than that of free DOX (75.6%). The side
effects of the treatments were preliminarily evaluated by
monitoring the mice’s body weight. The body weight of the mice
injected with free DOX gradually decreased during treatment by
about 10% of the initial weight in 12 days. The final body weight of
the group treated with DOX was significantly lower than that of
mice treated with PBS (p = 0.005). In contrast, the group treated
with PCA4-PEG/DOX did not lose any obvious body weight (Figure
8B). No significant difference was found in the body weight
between the groups treated with PCA4-PEG/DOX and PBS, which
suggests that the encapsulation of DOX in PCA dendrimers
significantly reduced the general toxicity of DOX.
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The excised tumor and heart tissue was examined histologically
with hematoxylin-eosin staining. As shown in Figure 8C, the tumors
treated with PBS typically consisted of tightly packed tumor cells
and some necrotic regions due to their rapid growth. In contrast,
the tumors treated with PCA4-PEG/DOX or free DOX showed
extensive nuclear shrinkage and cellularity changes in many cancer
cells, which indicates that both PCA4-PEG/DOX and free DOX could
induce apoptosis. Histologic analysis of heart tissue was performed
to examine cardiomyocyte damage, which is the most serious side
effect of DOX. Severe myocardial alterations were observed in the
mice that were treated with free DOX. In contrast, the cardiac
muscle fibers were clear and vivid in the mice that were treated
with PCA4-PEG/DOX or PBS.
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Dendrimers with PCA as building blocks were synthesized,
characterized, and used as macromolecular drug carriers for cancer
therapy. PCA in the dendrimer also served as a drug-binding
molecule to facilitate drug loading and reduce burst release. The
DOX-loaded dendrimer PCA-PEG/DOX exhibited pH and GSH-dual
responsive drug release in vitro. PCA-PEG/DOX could be used to
gradually deliver DOX to cell nuclei and showed comparable
cytotoxicity to free DOX in vitro. The antitumor activity of PCA-PEG
and PCA-PEG/DOX was preliminary studied. PCA-PEG showed
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Acknowledgements
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This work was financially supported by the National Basic Research
Program, Grant Number: 2014CB931900, the Fundamental
Research Funds for the Central Universities (No.2016QNA4024),
and the China postdoctoral science foundation (No.2013T60595).
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