Self-assembled thermosensitive nanoparticles based on oligoethylene glycol dendron conjugated doxorubicin: Preparation, and efficient delivery of free doxorubicin

Article abstract of DOI:10.1039/c5ra22224a

An amphiphilic dendron-drug conjugate was synthesized via oligoethylene glycol (OEG) dendrons coupled with anticancer drug doxorubicin (DOX). The amphiphilic conjugate OEG-DOX (GD) self-assembled into spherical nanoparticles in aqueous solution, with mean

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PAPER
Self-assembled thermosensitive nanoparticle based on
oligoethylene glycol dendron conjugated doxorubicin:
Preparation, and efficient delivery of free doxorubicin
Received 00th January 20xx,
Accepted 00th January 20xx
Yanna Zhao,a Jing Zhao,b Chunying Hao,a Meihua Han,a Mincan Wang,b Yifei Guo,*,a and Xiangtao
DOI: 10.1039/x0xx00000x
10 Wang*,a
www.rsc.org/
An amphiphilic dendron-drug conjugate was synthesized via oligoethylene glycol (OEG) dendron coupled with
anticancer drug doxorubicin (DOX). The amphiphilic conjugate OEG-DOX (GD) self-assembled into spherical
nanoparticles in aqueous solution, with mean diameters of approximately 212.5 nm. As the effective drug carrier, DOX
was entrapped into GD to form drug-loaded nanoparticles (GD/D) with high drug loading content (24%, wt-%), due to
15 the enhanced hydrophobic/aromatic interactions between DOX moiety in GD and free DOX. Besides, GD nanoparticles
exhibited thermo-induced collapse and aggregation, therefore, the temperature-dependent drug release profiles of
DOX from GD/D nanoparticles were measured and the possible mechanism was proposed. Moreover, hemolytic activity
of GD/D revealed the good blood compatibility, and the cytotoxicity of DOX in GD/D was enhanced significantly in vitro
against HepG2 cells. Overall, amphiphilic conjugate GD was considered to be potentially feasible to overcome
20 formulation challenges for drug delivery and to be used in clinic.
5
temperature (LCST) to a compact hydrophobic structure above
LCST, which may result in destabilization of the nanoparticles,
45 accumulated in desired position and/or trigger a burst release of
the encapsulated drugs.22-24 As the thermosensitive compounds,
OEG dendrons with varying LCST are designed and synthesized by
Zhang and co-workers.25, 26 Based on the excellent water solubility,
biocompatibility, nontoxicity, and thermosensitive property, OEG
50 dendrons are utilized to prepare amphiphilic codendrimers which
are applied as drug carrier in our previous research.27, 28
1. Introduction
Benefiting from their unique merits, amphiphilic polymers
decorated with polyethylene glycol (PEG) have been researched
broadly to construct the drug delivery systems for hydrophobic
25 anticancer drugs.1-5 These drug delivery systems improve the
solubility of hydrophobic drugs, stabilize and protect drugs from
degradation, facilitate targeted drug delivery, enhance
accumulation in the tumor tissue via the enhanced permeability
and retention (EPR) effect, prolong circulation time by avoiding the
30 rapid renal clearance and reticuloendothelial systems (RES), and
decrease disruption in cellular uptake.6-11 However, the PEGylated
strategies present limitations, such as structural heterogeneity due
to the polydispersity of PEG chains, low drug loading content
(DLC), poor physical stability, drug leakage, and adverse side
35 effects.12-14 To overcome these limitations, oligoethylene glycol
(OEG) with well-defined structure and molar mass are synthesized
and applied as the drug carriers.15-18
For physical entrapment, the DLC and release profiles are
affected by the interactions between the drug and the
hydrophobic segments of amphiphilic polymers, therefore, the
55 composition of the polymers must be considered in the design and
evaluation of copolymer-based drug formulations.29-31 According
to the excellent compatibility, it is expected that the same or
similar structure between the hydrophobic segments of
amphiphilic polymers and the entrapped drug could enhance the
60 interactions, result in promoted DLC and controlled release. Park
and co-workers demonstrated that doxorubicin (DOX) conjugated
with PEG could assist to form and stabilize DOX nanoaggregates
due to the interaction between DOX moiety in the conjugate and
free DOX, this nanoaggregates showed high DLC (~25%), enhanced
65 cellular uptake, and increased cytotoxicity against KB cells.32
Besides, DOX was conjugated with PEGylated peptide to
encapsulate free DOX successfully by Byun and co-workers.33
Other researches performed by Allen and co-workers indicated
utilizing docetaxel (DTX) as the hydrophobic moiety to form PEG-
70 DTX conjugates could entrap free DTX with higher DLC, and the
morphologies of these nanoaggregates are influenced by the
Obviously, it should be more beneficial if the drug could be
delivered by a carrier that could respond to physiopathological
40 signals from an underlying disease.19-21 The nanoparticles with
thermosensitive feature could undergo a phase transition from an
expanded hydrophilic structure below the lower critical solution
a.Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing 100193, P. R. China.E-mail:
ffguo@163.com, xtaowang@163.com; Fax: +86-10-57833266; Tel: +86-10-
57833266
b.The College of Chemistry and Molecular Engineering, Zhengzhou University,
Zhengzhou, Henan Province 450052, P. R. China
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34
presence of conjugated DTX in the micelle core.30,
These
washed with brine and dried over MgSO4, the crude product was
researches demonstrated the enhanced DLC and controlled 55 purified by column chromatography with DCM/MeOH (20/1, v/v)
release profiles would be obtained using these amphiphilic carriers
with anticancer drug as hydrophobic segment, on account of the
strong hydrophobic interactions and excellent compatibility.
to yield compound 2 as a slight yellow oil (1.90 g, 95%). 1H NMR
(CDCl3): δ = 1.17-1.23 (t, 27H, CH3), 3.48-3.50 (m, 18H, CH2), 3.55-
3.67 (m, 72H, CH2), 3.70-3.74 (m, 18H, CH2), 3.82-3.86 (m, 30H,
CH2), 4.12-4.16 (m, 18H, CH2), 4.18-4.26 (m, 6H, CH2), 4.46-4.48 (s,
60 6H, CH2), 6.56-6.58 (s, 6H, CH), 7.45 (s, 2H, CH). 13C NMR (CDCl3): δ
= 15.10, 66.55, 68.82, 69.05, 69.30, 69.57, 69.71, 69.77, 70.35,
70.47, 70.54, 70.63, 70.65, 70.77, 72.25, 72.48, 73.19, 106.23,
109.38, 125.26, 133.69, 137.86, 144.38, 152.59, 167.07 ppm.
5
Considering the stimuli responsibility and composition, in the
work presented here, the amphiphilic OEG-DOX conjugate (GD) is
designed as the drug carrier and further loaded DOX by physical
entrapment (GD/D). As the drug carrier, the advantages of GD are
10 expected as follows: (1) based on the thermosensitive property,
this GD/D delivery system would be stable during the drug
Compound 3. The acid compound 2 (1.90 g, 0.78 mmol) and
transportation in the blood at normal temperature, and could 65 pentafluorophenol (0.22 g, 1.20 mmol) were dissolved in DCM (30
release a large amount of DOX to inhibit the proliferation of cancer
cells after arrived at tumor tissue with higher temperature. (2)
15 Utilizing OEG dendron as the hydrophilic segment results
amphiphilic molecule GD with well-defined structure and molar
mL) and stirred for 10 min. DCC (0.37 g, 1.80 mmol) was then
added. The mixture was stirred overnight at room temperature
before being washed with saturated NaHCO3 and brine
successively. After drying over MgSO4, the organic phase was
mass, which avoids structural heterogeneity in the amphiphilic 70 purified by column chromatography with DCM/MeOH (40/1, v/v)
molecules. (3) GD shows a good capacity to encapsulate the free
DOX due to the strong hydrophobic/aromatic interactions
20 between DOX moiety in GD and the free DOX. (4) The anticancer
efficacy of GD/D could be enhanced significantly in vitro. For these
to yield a slightly yellow oil (1.50 g, 74%). 1H NMR (CDCl3): δ = 1.19-
1.24 (t, 27H, CH3), 3.38-3.46 (m, 18H, CH2), 3.50-3.63 (m, 72H, CH2),
3.68-3.74 (m, 18H, CH2), 3.80-3.86 (m, 30H, CH2), 4.10-4.15 (m,
18H, CH2), 4.23-4.29 (m, 6H, CH2), 4.58-4.62 (s, 6H, CH2), 6.40-6.42
purposes, the physicochemical characteristics of conjugate GD and 75 (s, 6H, CH), 7.40 (s, 2H, CH). 13C NMR (CDCl3): δ = 15.10, 61.70,
GD/D such as solubility, morphology, thermosensibility,
cytotoxicity and release profiles are studied to evaluate the great
25 potential of GD as a thermosensitive drug carrier in long-acting
interventinal chemotherapy.
66.55, 68.82, 69.05, 69.30, 69.57, 69.71, 69.77, 70.35, 70.47, 70.54,
70.63, 70.65, 70.77, 72.25, 72.48, 73.19, 107.23, 110.38, 121.26,
133.69, 137.86, 144.38, 152.59, 162.07 ppm.
OEG-DOX conjugate (GD). The solution of compound 3 (0.40 g，
80 0.20 mmol) in DMF (10 mL) was added into a solution of DOX.HCl
(0.12 g, 0.03 mmol), TEA (67 mg, 0.09 mmol), and DMAP (20 mg) in
DMF (10 mL) at -5 °C with stirring, and the reaction temperature
was allowed to raise to room temperature. After stirring for 24 h,
the solvents were evaporated in vacuo at room temperature, the
85 residue was dissolved with DCM (30 mL). After the organic phase
had been washed with 0.2 M HCl solution (30 mL) and dried over
MgSO4, the crude product was purified by LH-20 gel column with
2. Experiment part
2.1 Materials
Compound 1 was synthesized according to previous reports.25, 26
30 Tetrahydrofuran (THF) was dried by refluxing with lithium
aluminum hydride (LAH) under N2 and distilled just before use.
Dichloromethane (DCM) was refluxed with calcium hydride and
distilled just before use. Doxorubicin hydrochloride (DOX.HCl,
purity > 98%), rhein (RHE, purity > 98%) were purchased from
35 Dalian Meilun Biotech Co., Ltd. (Liaoning, China).
Hydroxycamptothecine (HCPT, purity > 98%) was purchased from
Aladdin Co. Ltd. (China). Vitamin E (VE, purity > 98%) was
purchased from Jiakangyuan Technology Development Co., Ltd.
1
methanol to afford GD as a red oil (0.31 g, 70%). H NMR (CDCl3):
δ= 1.19 (t, 27H, CH3), 1.30 (d, 3H, CH3), 1.78-2.01 (m, 2H, CH2), 2.00
90 (br, 7H, OH, CH2, NH), 2.20 (m, 1H, CH2), 2.35 (d, 1H, CH2), 3.00-
3.09 (m, 2H, CH2), 3.25-3.29 (m, 2H, CH), 3.41-3.90 (m, 132H, CH2,
CH3), 4.01-4.26 (m, 26H, CH2, CH), 4.30-4.48 (m, 6H, CH2), 4.59-
4.68 (m, 3H, CH2, CH), 5.30 (br, 1H, CH), 5.52 (br, 1H, NH), 6.54 (s,
6H, CH), 7.15 (s, 2H, CH), 7.40 (d, 1H, CH), 7.75 (t, 1H, CH), 8.02 (d,
95 1H, CH); 13C NMR (CDCl3): δ = 14.70, 17.64, 26.81, 35.30, 43.77,
45.50, 56.32, 63.01, 66.15, 80.75, 70.09, 71.03, 72.15, 72.33, 72.51,
72.79, 72.87, 73.29, 73.43, 73.51, 73.62, 73.69, 73.72, 73.52, 73.84,
73.98, 95.00, 105.53, 108.09, 122.19, 123.46, 125.06, 129.39,
130.65, 131.29, 135.48, 145.08, 162.07, 150.81, 187.69 ppm.
100 HRMS: m/z: calcd for C145H231NO60 [M+Na]+ 2970.1697; found
2970.4153.
(Beijing, China). Podophyllotoxin (PPT, purity
> 98%) was
40 purchased from Nanjing Zelang Medical Technology Co., Ltd.
(Jiangsu, China). Other reagents and solvents were purchased as
reagent grade and used without further purification. Silica gel 300-
400 mesh and Sephadex LH-20 gel were used as the stationary
phase for column chromatography.
45 2.2 Syntheses
Compound 2. LiOH.H2O (0.10 g, 1.64 mmol) was added into a
solution of compound 1 (2.00 g, 0.82 mmol) in methanol (20 mL)
and water (10 mL) at -5 °C with stirring, and the reaction
temperature was allowed to raise to room temperature. After
50 stirring for 3 h, the solvents were evaporated in vacuo at room
temperature. The residue was dissolved with DCM, and the
solution pH was adjusted carefully with 10% KHSO4 aqueous
solution to approximately 5~6. After the organic phase had been
2.3 Hydrodynamic size measurements
The hydrodynamic sizes of drug-loaded nanoparticles were
determined by dynamic light scattering (DLS) using a Zetasizer
105 Nano-ZS analyzer (Malvern Instruments) with an integrated 4 mV
He-Ne laser, λ = 633 nm, which used backscattering detection
(scattering angle θ = 173°) at different temperature. Samples were
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prepared by dissolving in deionized water at a concentration of 2
mg mL-1.
temperature at which the transmittance at λ = 500 nm reached
50% of its initial value.
2.4 Transmission electron microscopy
30 2.7 Preparation of drug-loaded nanoparticles
Transmission electron microscopy (TEM) measurements were
performed with a JEM-1400 instrument at an accelerating voltage
of 100 KV. Samples were prepared by drop-casting GD and GD/D
solutions (0.2 mg mL-1) onto carbon-coated copper grids, air-drying
at room temperature and then dyeing with uranyl acetate.
Drug-loaded nanoparticles were prepared by the dialysis method.
5
Briefly, the amphiphilic compound GD (8 mg) and doxorubicin
(DOX),
hydroxycamptothecine
(HCPT),
rhein
(RHE),
podophyllotoxin (PPT), vitamin E (VE) (4 mg) (DOX.HCl neutralized
35 with two molar equivalent of TEA) was dissolved in methanol (2
mL) in a glass vial at room temperature separately. After that,
deionized water (5 mL) was added dropwise into these vials under
vigorous stirring. The mixture solution was transferred into a
dialysis membrane (MWCO 14000) and dialyzed against deionized
40 water (1 L) at room temperature, which was renewed every 4 h for
24 h to remove the organic solvent and the free drug. To quantify
2.5 Critical aggregation concentration
10 The CAC of the amphiphilic conjugate GD estimated by
a
fluorescence spectroscopic method using an F-4500 FL
Spectrophotometer and pyrene (Py) as the fluorescence probe.
Typically, Py solutions in acetone were added to each eppendorf
(EP) tube. The acetone was then evaporated, leaving 6.00 × 10−5
15 mol of Py in each tube. Aqueous solutions with various
concentrations (from 1.0 × 10−3 to 1.0 mg mL-1) were prepared and
added to the tubes. The mixtures were sonicated for 2 min and
stirred at room temperature for 12 h. The spectroscopy
measurements were conducted at an excitation wavelength of 334
20 nm.
the encapsulated drug,
1 mL solutions of the drug-loaded
nanoparticles were freeze-dried and then 1 mL MeOH were added,
the samples were analyzed by high-performance liquid
45 chromatography (HPLC) using a Dionex Ultimate 3000 with a UV
detector at the corresponding wavelength. The quantitative
analysis was carried out on a Thermo C18 (4.6 mm × 250 mm, 5
μm), and the sample injection volume was 20 μL. The details for all
hydrophobic drugs are shown in Table 1. The entrapment
50 efficiency (EE) and drug loading content (DL) were calculated as
follows:
2.6 Thermosensitive property
Turbidity measurements were conducted on a Cary 100 UV-Vis
spectrophotometer (Agilent Technologies) equipped with
a
thermostatically regulated bath. Aqueous compound solutions
25 were placed in the spectrophotometer (path length 1 cm) and
heated at a rate of 0.2 °C min-1. The absorption of the solution at λ
= 500 nm was recorded. The LCST was determined to be the
55 Table 1 Conditions for hydrophobic drugs measured by HPLC
EE = (weight of loaded drug/weight in feed) ×100%
DL = (weight of loaded drug/weight of drug-loaded nanoparticle)
×100%
Sample
DOX
PPT
λ (nm)
478
292
Eluent
Flow rate (mL)
Regression equation
y = 0.26x + 0.01
y = 0.18x – 0.02
y = 0.05x – 0.30
R2
KH2PO4 (2.5%)/ CH3OH 3/7
CH3CN/H3PO4 (0.1%) 4/6
CH3OH
0.8
1.0
1.0
0.9999
0.9999
0.9999
VE
285
2.8 Drug release in vitro
RBC suspension at 37 °C for 3 h with different concentration. The
unlysed RBCs were removed by centrifugation, 100 μL of the
supernatant was pipetted into a 96-microwell plate, and the
absorbance was measured at 540 nm using a microplate reader
80 (Versamax Tunable Microplate Reader). The results were
expressed as the percentages of hemolysis with the assumption
that deionized water caused 100% hemolysis and 150 mM NaCl
solution caused 0% hemolysis. Each experiment was done in triple.
The data were shown as the mean values plus standard deviation
85 (± SD). All experimental procedures were performed in accordance
with the Guidelines for Ethical and Regulatory for Animal
Experiments as defined by the Institutional Animal Care and Use
Committee of Peking University Health Science Center.
The DOX-loaded nanoparticles (GD/D) aqueous solution (2 mL)
was transferred into dialysis membrane (MWCO 14000),
a
immersed in 50 mL of PBS solutions. The release studies were
60 performed at 37 and 40 °C respectively with continuous magnetic
stirring at 100 rpm. At predetermined time intervals, 2 mL of the
external solution was withdrawn and replenished with an equal
volume of fresh media for analysis. The drug release study was
performed for 84 h. The amount of released DOX was analyzed
65 with HPLC using a Dionex Ultimate 3000 with a UV detector
operated at 478 nm. The release experiments were conducted in
triplicate, and the results were presented as the mean values plus
the standard deviation (±SD).
2.10 MTT assay
2.9 Hemolytic activity
90 The cytotoxicity against the human liver cancer cell line (HepG2
cells) was assessed by methyl thiazolyl tetrazolium (MTT) assay.
Briefly, cells were cultured in DMEM medium supplemented with
10% fetal calf serum, 1% L-glutamine, and 0.1 mg mL-1
streptomycin at 37 °C with 5% CO2 and then seeded in 96-well
95 plates at a density of 5000 cells per well. The GD, GD/D, and free
DOX solution were added into the wells. After incubation for 48 h
70 Approximately 5 mL of blood was taken from the ear artery of a
male New Zealand white rabbit and centrifuged at 3000 rpm for 5
min. The plasma supernatant was removed, and the erythrocytes
were re-suspended in normal saline solution. The red blood cells
(RBC) were again centrifuged at 3000 rpm for 5 min to collect
75 packed RBC. The GD/D solutions were incubated with the 2% (v/v)
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at 37 °C with 5% CO2, the growth medium was replaced with fresh
DMEM. Then, 10 μL MTT solution was added to each well and
incubation was continued for another 4 h. The medium was
removed and 200 μL DMSO was added into each well to dissolve
The results obtained from hydrodynamic diameter, drug-loading
content and release experiments, cytotoxicity were expressed as
the mean ± standard deviation (SD). Statistical analysis was
performed with SPSS 19.0 software. Student’s t-test was used to
5
the formazan, assisted by pipetting in and out several times. The 20 evaluate the differences between groups, and P < 0.05 was
absorbance of the solution in each well was measured using an
ELISA plate reader at a test wavelength of 570 nm to determine
the OD value. The cell inhibition rate was calculated as follows. Cell
inhibition = (1-ODtreated/ODcontrol) × 100%, where ODtreated was
considered statistically significant.
2.12 Instrumentation and measurements
1H NMR and 13C NMR spectra were recorded on a Bruker AV 600
spectrometers with CDCl3 as solvent, and chemical shifts were
10 obtained from the cells treated by the nanoparticles or free DOX,
ODcontrol was obtained for the cells treated by the culture medium, 25 reported as
δ values (ppm) relative to internal Me4Si. Molecular
and the other culture conditions were the same. Each experiment
was performed in quintuplicate. The data were shown as the mean
values plus the standard deviation (±SD).
weight was determined by Autoflex III MALDI-TOF mass
spectrophotometer with ESI detector. FT-IR spectra were recorded
on a FTIR-8400S (Shimadzu Japan) spectrometer. Samples were
pressed into potassium bromide (KBr) pellets.
15 2.11 Statistical analysis
30
Scheme 1 Synthetic route of the amphiphilic conjugate GD. Reagents and conditions: (a) LiOH, MeOH, -5 to 25 °C, 3 h (95%); (b) Pfp, DCC,
DCM, 25 °C, 12 h (74%); (c) DOX, TEA, DMAP, DMF, -5 to 25 °C, 24 h (70%). Pfp = pentafluorophenol, DCC = dicyclohexylcarbodiimide, DCM
= dichloromethane, TEA = triethylamine, DMAP = N, N-dimethylaminopyridine, DMF = dimethylformamide.
and 1.2 ppm corresponded to the proton resonance of methyl
terminals of OEG dendron and DOX respectively, the integral ratio
3. Results and discussion
55 of these two signals was 9.0. These results suggested the
successful synthesis of the amphiphilic compound GD
proposed structure of GD is further verified by the FT-IR
technique, meanwhile the DOX and compound are measured for
. The
35 3.1 Synthesis and characteristics of OEG-DOX conjugate
To construct the amphiphilic conjugate, the oligoethylene glycol
(OEG) dendron active ester was prepared, and the synthetic route
of OEG dendron conjugated DOX (GD) is shown in Scheme 1. The
1
comparing (Fig. 2). FT-IR results also confirmed the generation of
60 amphiphilic compound GD based on the characteristic stretching
vibration of carbonyl absorption at 1648 cm-1 from amide group,
the typical stretching C-O-C absorption at 1110 cm-1 from OEG
dendron, and the strong stretching N-H absorption at 3440 cm-1
from DOX. Then, the DOX content within GD (17.3%, wt-%) was
65 determined by UV-HPLC at 478 nm, which was consistent with the
theoretical value (17.1%). All these results proved that amphiphilic
conjugate GD had been successfully synthesized via amide
coupling reaction. Benefiting from its amphiphilic structure with
hydrophilic OEG and hydrophobic DOX, the self-assembly of GD
70 into nanoparticles can be expected in aqueous solution to
encapsulate the hydrophobic anticancer drugs into the core by
means of hydrophobic interaction, which is beneficial for further
biomedical applications.
compounds
1, 2 and 3 were synthesized following the procedures
40 in previous reports. DOX was conjugated with active ester
3
through the amide bond, after purification by Sephadex LH-20
column chromatography with methanol as eluent, amphiphilic
conjugate GD was obtained with a yield of 70%. The resultant
compound GD displayed a good solubility in common solvent, such
45 as MeOH, THF, DCM, DMF, and water, due to the excellent
solubility of the OEG dendron.
The structure of GD was confirmed by 1H NMR spectroscopy,
and the signals are ascribed separately and shown in Fig. 1.
Comparing with the compound 3
, the 1H NMR spectrum of GD
50 presented the characteristic peaks of DOX; the signals at 8.0-7.3
and 5.3 ppm were attributed to the benzene ring protons and
tetrahydropyran ring protons separately. The signals around 1.1
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Fig. 1 1H NMR spectra of compound
3 and GD in CDCl3.
15 3.1 nm (Fig. 3b). The diameter of GD nanoparticles observed by
TEM is smaller than that obtained from the DLS measurement. The
size of the nanoparticles measured by DLS reflects the
hydrodynamic diameter and the TEM image shows that of dried
particles, which is most likely due to shrinkage of the OEG
20 portion.31, 35
Fig. 3 DLS curves of amphiphilic compound GD in aqueous solution
(a); TEM image of self-assembled aggregates (b). Scale bar: 500
nm.
Fig. 2 FT-IR spectra of DOX (a), compound
1 (b), and GD (c).
5
Samples were pressed into potassium bromide pellets.
25 The aggregation behavior of amphiphilic compound GD was
further characterized by fluorescence spectroscopy using pyrene
as a probe, which was sensitive to the surrounding environment,
and the ratio of the intensity of the third (384 nm) and first (374
nm) peaks was used to confirm the CAC. It was apparent that the
30 ratio of I384/I374 remained constant below the CAC and then
changed substantially, and the CAC was dectected as 17.4 μg mL-1
(Fig. 4).
3.2 Self-assemble behavior
Due to the excellent solubility, amphiphilic conjugate GD was
dissolved in deionized water directly with concentration of 2 mg
mL-1 and self-assembled into the nanoparticles in the aqueous
10 solution, the particle size was detected by DLS (Fig. 3a). It was
found that GD formed nanoparticles with the mean hydrodynamic
diameter of approximately 212.5 ± 11.1 nm (Table 2). TEM
micrograph revealed that GD could aggregate into spherical
nanoparticles with the mean diameter of approximately 180.2 ±
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carriers, which is stable under physiological condition and
aggregates at the tumor tissue induced by slight higher
temperature to increase the cellular uptake due to the
30 hydrophobic interaction.36, 37
Fig. 4 Dependence of intensity ratio I384/I374 as a function of
amphiphilic compound GD concentration.
Fig. 6 Structures of hydrophobic drugs and images of drug-loaded
nanoparticle solutions.
Table 2 Results of the drug-loaded nanoparticlesa
b
Samples
Dh
ζc
Diameterd
(nm)
DLCe
(%)
(nm)
(mV)
-28.1
-26.7
-21.2
GD
GD/D
GD/P
212.5 ± 11.1
240.5 ± 16.7
252.1 ± 4.4
238.8 ± 0.8
180.2 ± 3.1
210.4 ± 2.7
n.d.f
0
24.7 ± 4.1
12.3 ± 1.2
6.5 ± 0.4
GD/V
-27.2
b
n.d.
a
35 Prepared by dialysis method. Dh: hydrodynamic diameter, 2 mg
mL-1, DLS detected, n = 3. ζ: zeta potential, 2 mg mL-1, DLS
c
d
detected, n = 3. The average diameters of 20 particles in TEM
images. e Detected by UV-HPLC. f Not detected.
3.3 Drug-loading capacity
40 To assess the influence of amphiphilic compound structure on
drug incorporation, doxorubicin (DOX), hydroxycamptothecine
(HCPT), rhein (RHE), podophyllotoxin (PPT), and vitamin E (VE)
were utilized as the model drugs to prepare drug-loaded
nanoparticles (Fig. 6). DOX.HCl was first neutralized by excess
45 triethylamine to remove the hydrochloride, and then these drugs
were incubated with GD in DMF, dialyzed against deionized water
to remove DMF and the free drugs.
5
Fig. 5 Plots of the transmittance (a) and Dh (b) vs temperature for
0.2 wt-% GD aqueous solution.
3.3 Thermosensitive behavior
Among the selected model drugs, DOX could be encapsulated in
amphiphilic GD successfully, and the drug-loading content (DLC)
50 was 24.7% (physical entrapment, Table 2), the DOX-loaded
nanoparticles (GD/D) solution was fully transparent red solution
(Fig. 6). RHE and HCPT formed precipitation after dialysis, although
PPT and VE could be entrapped into GD, the DLC of PPT-loaded
nanoparticles (GD/P) and VE-loaded nanoparticles (GD/V) were
55 12.3% and 6.5% separately (Table 2), significant lower than that of
GD/D. It seems that GD presented the excellent DOX-loading
capacity, which could be explained by the stronger hydrophobic
interactions induced by the similar structure between the
hydrophobic segments of GD molecules and the free DOX.38, 39
60 Besides, it was reported that DOX having an anthraycline ring
structure forms a dimmer in an aqueous solution due to π-π
interaction between the planar, aromatic anthracycline rings.32
Thus, DOX could be entrapped into the GD nanoparticles by the
hydrophobic and aromatic interactions between the DOX moiety
65 in GD and the free DOX; meanwhile, excellent drug-loading
capacity could be achieved.
Owing to the presence of OEG dendron, the GD nanoparticles
were assessed to have thermosensitive properties in aqueous
10 solution and undergo a phase transition from homogeneous
solution to heterogeneous aggregates around a lower critical
solution temperature (LCST). UV-vis spectrophotometry and
dynamic light scattering (DLS) technology were thus applied to
investigate the thermosensitive behavior of GD and to determine
15 its apparent LCST. The turbidity curve measured from 33 to 45 °C is
plotted in Fig. 5a. Compound GD showed typical thermosensitive
behavior, and its LCST is 39.2 °C. The thermally induced
aggregation was further investigated by DLS to record the
temperature-dependent aggregate size, the diameter curve
20 measured from 33 to 42 °C is plotted in Fig. 5b. The hydrodynamic
diameters (Dh) of GD nanoparticles (2 mg mL-1) were measured to
increase from 200 nm at 33 °C to 2800 nm at 42 °C, and the
appearance of GD solution was changed from fully transparent to
turbid. The increase in Dh could be attributed to the
25 internanoparticle aggregation. It is supposed that the novel
amphiphilic conjugate is of great potential use as smart drug
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3.4 Characterization of DOX-loaded nanoparticles
were shown to be temperature dependent. At 37 °C (< LCST),
approximately 80% of the loaded DOX was sustained release from
50 the GD/D nanoparticles for 84 h, which was most likely due to the
slow diffusion of DOX in the nanoparticles. At 40 °C (> LCST), the
release of DOX was enhanced dramatically on account of the
temperature-induced structural changes of the nanoparticles,
approximately 50% of DOX was released within the initial 4 h
55 followed by another slow release procedure (about 40%, from 4 to
84 h). The OEG dendron shell of GD/D nanoparticles becomes
hydrophobic above the LCST, which could lead to the partial
deformation of nanoparticles resulting in the faster drug release.45-
47 These results prompted that the GD/D delivery system would be
60 stable during the drug transportation in the blood, after arrived at
tumor tissue with higher temperature and acidic environment, it
could release a large amount of DOX to inhibit the proliferation of
cancer cells.
The particle size of GD/D nanoparticles with a concentration of 2
mg mL-1 in aqueous solution were investigated by DLS, and the
mean diameter of approximately 240.5 ± 16.7 nm (Table 2). The
stability of nanoparticles solution was investigated by monitoring
for nanoparticles size changes using DLS (Fig. 7a). It was apparant
that the particle sizes of DOX-loaded nanoparticles were
approximately 240 nm for 10 days incubation at 37 °C. These
results suggested that GD/D nanoparticles had the favorable
5
10 stability in aqueous solution, which was likely due to the OEG
dendron surface.
Fig. 7 Mean GD/D diameter as a function of time in deionized
water (a), and TEM micrograph (b). Scale bar: 500 nm.
15
The TEM micrograph revealed that GD/D nanoparticles were all
spherical with the mean diameter of approximately 210.4 ± 2.7 nm
(Fig. 7b). Similar as GD nanoparticles, the diameter of GD/D
nanoparticles observed by TEM is smaller than that obtained from
the DLS measurement. In addition, it was found that the sizes of
20 DOX-loaded nanoparticles were larger than the blank
nanoparticles, indicating that the DOX was successfully loaded into
the core of GD nanoparticles due to the hydrophobic/aromatic
interactions between the conjugated and the free DOX.40
65 Fig. 8 Cumulative DOX release in different conditions: free DOX in
pH 5.5 PBS at 37 °C (a), GD/D in pH 5.5 PBS at 40 °C (b), GD/D in
pH 5.5 PBS at 37 °C (c), GD/D in pH 6.8 PBS at 37 °C (d), and GD/D
in pH 7.4 PBS at 37 °C (e) (n = 3).
3.5 Release behavior of thermosensitive DOX-loaded
3.6 Cytotoxicity
25 nanoparticles
70 The hemolytic effect of GD/D on rabbit RBC was investigated to
verify the suitability via intravenous administration. The hemolysis
rates of GD/D were recorded as a function of concentration,
ranging from 0.1 to 20 mg mL-1. After incubation with the 2% (w/v)
RBC suspension at 37 °C for 3 h, the hemolysis rates of GD/D
75 solutions was below 5%, suggesting no RBC membrane damage
and the good blood compatibility.48, 49
After drug-loaded GD/D nanoparticles were prepared, the DOX
release profiles from the thermosensitive nanoparticles were
evaluated under a simulated physiological condition (PBS, pH 7.4)
at 37 °C and in an acidic environment (PBS, pH 5.5 and 6.8) at 37 °C
30 and 40 °C in separate trials to assess the feasibility of using GD/D
nanoparticles as an anticancer drug delivery system, as tumor
tissues have
a more acidic environment and slight higher
temperature than normal physiological conditions.41-44 A control
experiment using free DOX as also performed in pH 5.5 PBS at 37
35 °C, all the release results are shown in Fig. 8. For free DOX, the
burst release of up to 70% within the initial 2 h was observed, and
then followed by the complete release within 10 h. The release of
DOX from GD/D nanoparticles at physiological temperature (37 °C)
showed the pH-dependent property. Approximately 80% of DOX
40 was released from the nanoparticles at 84 h post-dialysis in PBS
5.5, while only 39% and 32% of DOX was released in PBS 6.8 and
7.4 respectively, since the solubility of DOX was enhanced
significantly in acidic aqueous solution. Although DOX could be
released at the extracellular environment of tumor tissues, a large
45 amount of DOX (>60%) was retained inside the nanoparticles.
Furthermore, under the tumor microenvironment (pH 5.5), the
DOX release rate was much faster, with approximately profiles
The cytotoxicities of GD, GD/D against a liver cancer line (HepG2
cells) were studied using an MTT assay, with the concentration
ranging from 0.1 to 100 μg mL-1 (DOX equivalent concentration)
80 (Fig. 9). The IC50 values for free DOX, GD, and GD/D were 27.1,
45.6, and 8.0 μg mL-1 (DOX equivalent concentration) respectively
after incubation for 48 h. Compared with free DOX, after
conjugation of DOX with OEG dendron showed significant
decrease in cytotoxicity due to the shielding effect (p < 0.001),
85 which could reduce severe side effects of DOX. GD/D exerted a
higher cytotoxicity effect against HepG2 cells at the same dose (p <
0.001), which suggested much more DOX was transferred into the
tumor cells and proved the activity of GD/D nanoparticles was
greater than that of the free drug. The enhanced cytotoxicity of
90 GD/D nanoparticles could be attributed to the facilitated
endocytotic transport and quick intracellular DOX release within
the endosomes, relative to passive diffusion of free doxorubicin
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through the cell membrane.15, 50 This was probably due to the
mechanism of endocytosis that might effectively circumvent the
multi-drug resistance (MDR) effect that occurs for free DOX.51, 52 In
the previous study of DOX-loaded nanoparticles, the similar
enhanced uptake of DOX against HepG2 cells was observed.53 It
was expected that GD/D nanoparticles could augment the
therapeutic effect and induce the severe side effects of DOX.33
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4. Conclusions
65
Anticancer drug DOX was conjugated with the hydrophilic OEG
dendron to form the amphiphilic thermosensitive conjugate GD
(LCST 39.2 °C), which could self-assemble into spherical
15 nanoparticles in aqueous solution with the mean diameter of
approximately 212 nm. The amphiphilic GD was utilized to entrap
DOX (GD/D) and the drug-loading content was approximately 24%,
due to the strong hydrophobic/aromatic interactions between
DOX moiety in GD and the free DOX. The release of DOX from
20 GD/D nanoparticles was enhanced dramatically on account of the
temperature-induced structural changes of the amphiphilic
dendron GD, which could maximize the anticancer efficacy of drug
delivery system. The drug-loaded GD/D nanoparticles presented
good blood compatibility (hemolysis rate
< 5%), and the
80
25 cytotoxicity was enhanced significantly in vitro via MTT assay
against HepG2 cells. Based on these advanced properties,
including high drug-loading content, temperature-dependent drug
release, and higher anticancer efficacy, it seems that amphiphilic
conjugate with anticancer drug as hydrophobic segment and
30 thermosensitive dendron as hydrophilic segment hold great
promise for efficient delivery and release of relative anticancer
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Acknowledgements
This work is financially supported by the National Natural
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