Hydrotropic polymer-based paclitaxel-loaded self-assembled nanoparticles: Preparation and biological evaluation

Article abstract of DOI:10.1039/c7ra04563h

The poor compatibility of carrier materials with drugs is one of the main obstacles in the drug encapsulation of nano-drug delivery system (NDDS), hindering the clinical translation of NDDS. In this study, using paclitaxel (PTX) as the insoluble model drug, we conjugated N,N-diethylniacinamide (DENA), a hydrotropic agent of PTX, to the backbone of poly(l-γ-glutamyl-glutamine) (PGG), a water-soluble polymer, to prepare the "hydrotropic polymer" PGG-DENA to improve its compatibility with PTX. By virtue of the hydrotropic effect of the DENA group, PTX was encapsulated by PGG-DENA to obtain the hydrotropic polymeric nanoparticles (PGG-DENA/PTX NPs). PTX-conjugated poly(l-γ-glutamyl-glutamine) acid (PGG-PTX) NPs previously reported were used as the control in the study. The PGG-DENA/PTX NPs showed a z-average hydrodynamic diameter of about 70 nm, and good long-term stability in PBS solution at 4 °C. The cumulative release rate of PTX from PGG-DENA/PTX NPs reached 79.10% at 96 h, while that of PGG-PTX NPs was 22.96%. PGG-DENA/PTX NPs showed significantly increased in vitro cytotoxicity on NCI-H460 lung cancer cells compared with PGG-PTX NPs. The hemolysis study proved that the PGG-DENA/PTX NPs has good biocompatibility. These results indicated that by introducing the hydrotropic agent DENA, the hydrotropic polymer PGG-DENA becomes an effective carrier material of PTX. This study provides a solution to increase the compatibility of carrier materials with insoluble drugs, and also may provide an effective way to develop a series of personalized carrier materials suitable for different insoluble drugs.

Full text of DOI:10.1039/c7ra04563h

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PAPER
Hydrotropic polymer-based paclitaxel-loaded
self-assembled nanoparticles: preparation and
biological evaluation
Cite this: RSC Adv., 2017, 7, 33248
a
a
a
a
a
a
Lipeng Gao, Liefang Gao, Mingxue Fan, Qilong Li, Jiyu Jin, Jing Wang,
b
a
a
a
Weiyue Lu, Lei Yu, Zhiqiang Yan * and Yiting Wang*
The poor compatibility of carrier materials with drugs is one of the main obstacles in the drug encapsulation of
nano-drug delivery system (NDDS), hindering the clinical translation of NDDS. In this study, using paclitaxel
(
PTX) as the insoluble model drug, we conjugated N,N-diethylniacinamide (DENA), a hydrotropic agent of
PTX, to the backbone of poly(L-g-glutamyl-glutamine) (PGG), a water-soluble polymer, to prepare the
hydrotropic polymer” PGG–DENA to improve its compatibility with PTX. By virtue of the hydrotropic effect
“
of the DENA group, PTX was encapsulated by PGG–DENA to obtain the hydrotropic polymeric
nanoparticles (PGG–DENA/PTX NPs). PTX-conjugated poly(L-g-glutamyl-glutamine) acid (PGG–PTX) NPs
previously reported were used as the control in the study. The PGG–DENA/PTX NPs showed a z-average
ꢀ
hydrodynamic diameter of about 70 nm, and good long-term stability in PBS solution at 4 C. The
cumulative release rate of PTX from PGG–DENA/PTX NPs reached 79.10% at 96 h, while that of PGG–PTX
NPs was 22.96%. PGG–DENA/PTX NPs showed significantly increased in vitro cytotoxicity on NCI-H460
lung cancer cells compared with PGG–PTX NPs. The hemolysis study proved that the PGG–DENA/PTX
NPs has good biocompatibility. These results indicated that by introducing the hydrotropic agent DENA, the
hydrotropic polymer PGG–DENA becomes an effective carrier material of PTX. This study provides
a solution to increase the compatibility of carrier materials with insoluble drugs, and also may provide an
effective way to develop a series of personalized carrier materials suitable for different insoluble drugs.
Received 23rd April 2017
Accepted 26th June 2017
DOI: 10.1039/c7ra04563h
rsc.li/rsc-advances
Accordingly, researchers introduced hydrotropic agents into
the hydrophobic side of the polymeric materials to increase the
1
. Introduction
3
–5
Micelles are widely studied because of their ability to increase drug encapsulation in micelles. Hydrotropic agents are a kind
drug solubility and stability, and their tumor targeting ability. of small molecules that can increase the water solubility of
Paclitaxel (PTX) encapsulated polyethylene glycol-b-polylactic insoluble drugs by forming a complex, association or complex
1
6
acid (PEG-b-PLA) micelles have been approved by the FDA. The salt. The micelles developed by combining the hydrotropic
traditional micelles are formed by self-assembly of amphiphilic agents with the traditional micelles are so-called hydrotropic
polymer materials (e.g., PEG-b-PLA, polyethylene glycol–phos- polymer micelles. Up to now, there have been several hydro-
phatidylethanolamine (PEG–PE)) via hydrophobic interaction. tropic agents reported, such as N-methylpyridinium nicotin-
7
4
However, the drug encapsulation in the traditional micelles amide (PNA),
N,N-dimethylbenzamide (DMBA),
N,N-
3,4
mainly depends on the non-specic hydrophobic interaction diethylniacinamide (DENA) and uorenylmethoxycarbonyl (a-
between the hydrophobic end of the polymer and the drug. Fmoc). For example, Kim group developed a PEG-b-(poly-N,N-
8
Thus the traditional micelles usually only have a good encap- diethylnicotinamide) (PEG-b-PDENA), which can self-assemble
sulating effect for a small amount of extremely hydrophobic to form micelles that showed good encapsulating effect for
4
drugs. But in fact most of drugs are oen mildly hydrophobic, PTX. The solubility of PTX was increased by 6000 times, which
resulting in most of the reported micelles having low drug is higher than the equivalent concentration of free DENA or
2
loading capacity and poor stability.
PEG-b-PLA does. The PTX encapsulated PEG-b-PDENA micelles
can be stably stored for at least 4 weeks. Because DENA has
9
a special solubilization mechanism for PTX (aromatic ring
a
Institute of Biomedical Engineering and Technology, Shanghai Engineering Research buildup and hydrogen bonding), it showed higher solubiliza-
Center of Molecular Therapeutics and New Drug Development, School of Chemistry
and Molecular Engineering, East China Normal University, Shanghai 200062,
China. E-mail: zqyan@sat.ecnu.edu.cn; ytwang@nbic.ecnu.edu.cn
4
tion effect on PTX than other cosolvents (DMBA or a-Fmoc) did.
More importantly, different form the free hydrotropic agents,
the polymerized hydrotropic agent cannot be easily taken up in
vivo, thereby avoiding the potential toxicity to body. In addition,
b
Department of Pharmaceutics, School of Pharmacy, Fudan University, Key Laboratory
of Smart Drug Delivery, Ministry of Education, Shanghai 201203, China
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the stability and drug release behavior of micelles can be altered cellular uptake by human NCI-H460 cancer cells in vitro and
4
by adjusting the molecular weight of the hydrotropic end. In hemolysis study in vivo. PGG–DENA/PTX NPs exhibited a desir-
spite of this, the hydrotropic polymer micelles are also decient: able drug release prole, and good biocompatibility and long-
the hydrophobicity of the polymerized hydrotropic agents is term stability.
weaker than that of the hydrophobic end of traditional micellar
materials (such as PLA), especially for DENA. This resulted in
a high critical micelle concentration (CMC) of these hydrotropic
2
. Materials and methods
polymers, which is disadvantageous for the formation and 2.1. Materials
2
further use of micelles. This problem hinders the further
development of the hydrotropic polymer micelles.
PGG (poly-(L-g-glutamyl-glutamine)) was synthesized by our
12
laboratory. 4-(Chloromethyl)benzoyl chloride, tert-butyldime-
PTX-conjugated poly(L-glutamic acid) (PGA–PTX, also known
as Xyotax™), a polymer drug conjugate, has entered the clinical
0
thylsilyl chloride (TBSCL), 2-hydroxynicotinic acid, N,N -car-
bonyldiimidazole (CDI) and 4-dimethylaminopyridine (DMAP)
10,11
trials due to its high water solubility and antitumor effect.
were purchased from Sigma-Aldrich, Inc. N-(3-Dimethylamino-
Poly(L-g-glutamyl-glutamine)–paclitaxel (PGG–PTX) is another
0
propyl)-N -ethylcarbodiimide (EDC) was purchased from EMD
polymer drug conjugate we previously developed on the basis of
Chemicals Inc. (Darmstadt, Germany). All other chemicals and
reagents were commercially available and directly used.
Human NCI-H460 carcinoma cell line was obtained from
Shanghai Institute of Cell Biology. This cell was cultured in
12–14
PGA–PTX,
which further increased the water solubility,
increased the tolerable dose, reduced the side effects and
15–18
improved the antitumor effects.
The results showed that
PGG–PTX could be directly dissolved in water and had a solu-
ꢀ
RPMI 1640 supplemented with 10% FBS at 37 C in a humidi-
ꢁ
1
bility of 50 mg mL , which was much higher than that of PGA–
0

ed atmosphere of 5% CO
2
and 95% air. DiO (3,3 -dio-
ꢁ
1
PTX (7 mg mL ). It can self-assemble to form nano core–shell
structure about 30 nm in water, with PTX as hydrophobic
nucleus and PGG skeleton as hydrophilic shell. However, PGG–
PTX is also decient: since PTX is covalently coupled to the PGG
backbone, its drug release performance is poor. The cumulative
release rate of PTX from PGG–PTX nanoparticles (NPs) in vitro is
ctadecyloxacarbocyanine, perchlorate) was purchased from
Tianjin Biolite Biotech Co., LTD. Hoechst 33342 was purchased
from Beyotime Institute of Biotechnology.
All experiments involving animals were performed in
accordance with the guidelines of the Institutional Animal Care
and Use Committee (IACUC) of East China Normal University.
All experimental protocols were approved by the IACUC of East
China Normal University. Male SD rats (6–8 weeks old) were
obtained from SLAC Ltd (Shanghai, China) and maintained
under SPF conditions. All efforts were made to minimize the
number of animals used and their suffering. Animal experi-
ments were reported in accordance with the ARRIVE (Animal
Research: Reporting In Vivo Experiments) guidelines.
12
only about 23% at 96 h.
Based on the above considerations, we here conjugated
DENA to the backbone of PGG to prepare the “hydrotropic
polymer” PGG–DENA. Then by virtue of the hydrotropic effect of
DENA group, PTX was encapsulated by PGG–DENA to obtain the
hydrotropic polymeric nanoparticles (PGG–DENA/PTX NPs)
(Fig. 1). We characterized the NPs by DLS, TEM, HPLC and
evaluated the PTX release, cytotoxicity, long-term stability and
2.2. Synthesis of PGG–DENA
The synthesis of PGG–DENA was illustrated in Fig. 2. The
starting material 4-(chloromethyl)benzoyl chloride (1) was
reacted with boron hydride to prepare compound 2, which was
protected with TBSCl to afford compound 3. The starting
material 2-hydroxynicotinic acid (4) was reacted with diethyl-
amine to produce compound 5. The compound 3 was reacted
with compound 5 to obtain compound 6, which was depro-
tected to afford compound 7. Then the compound 7 was
conjugated to PGG in the presence of EDC and DMAP to obtain
the nal “hydrotropic polymer” PGG–DENA, which was dialyzed
with
a
tangential ow ltration system followed by
lyophilization.
2.2.1 Synthesis of (4-(chloromethyl)phenyl)methanol (2).
The 4-(chloromethyl)benzoyl chloride (1) (1.34 g, 6 mmol) was
dissolved in 100 mL THF and ethanol (1 : 1, v/v). Then NaBH
0.908 g, 24 mmol) was added and stirred at room temperature
4
(
under N for 2 h. The solvent was removed by rotary evaporation
2
ꢀ
at 37 C. Ethyl acetate was added and washed with 0.3 M
NaHCO (aq). The organic layer was dried over Na SO , ltrated
3
2
4
and concentrated to give (4-(chloromethyl)phenyl)methanol (2)
1
Fig. 1 Schematics of the preparation process of the PGG–DENA/PTX. (867 mg, 92%) as white solid. H-NMR (400 MHz, CDCl
) d 4.50
3
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ꢀ
Fig. 2 Synthesis of PGG–DENA. Reagents and conditions: (a) NaBH
4
, THF/EtOH, rt; (b) TBSCl, DMAP, DCM, rt; (c) DEA, CDI, THF, 60 C; (d)
ꢀ
2
K CO
3
, anhydrous acetone, 60 C; (e) 2 M HCl (aq), rt; (f) PGG–COOH, EDC, DMAP, DMF, rt.
(
¼
d, J ¼ 4.8 Hz, 2H), 4.75 (s, 2H), 5.21 (t, J ¼ 4.8 Hz, 1H), 7.32 (d, J 6.6 Hz, 1H), 3.37 (q, J ¼ 7.0 Hz, 2H), 3.13 (q, J ¼ 7.0 Hz, 2H), 1.09
7.6 Hz, 2H), 7.39 (d, J ¼ 7.6 Hz, 2H). (t, J ¼ 7.1 Hz, 3H), 1.02 (t, J ¼ 7.1 Hz, 3H).
.2.2 Synthesis of tert-butyl((4-(chloromethyl)benzyl)oxy) 2.2.4 Synthesis of 2-((4-(((tert-butyldimethylsilyl)oxy)
2
dimethylsilane (3). Imidazole (95 mg, 1.4 mmol), (4-(chlor- methyl)benzyl)oxy)-N,N-diethylnicotinamide (6). N,N-Diethyl-2-
omethyl)phenyl)methanol (2) (157 mg, 1 mmol) and DMAP hydroxynicotinamide (5) (252.2 mg, 1.3 mmol), tert-butyl((4-
(85 mg, 0.7 mmol) were dissolved in 15 mL of CH
2 2
Cl . TBSCl (chloromethyl)benzyl)oxy)dimethylsilane (3) (271 mg, 1 mmol)
(
256 mg, 1.7 mmol) was added and stirred at room temperature and K CO (414 mg, 3 mmol) were dissolved in 20 mL anhy-
2
3
ꢀ
under N for 1 h. Then, 50 mL KHSO solution (1 M) was added drous acetone. The reaction mixture was stirred at 60 C under
2
4
into the reaction solution, followed by extraction with CH Cl . N for 24 h, and then cooled to room temperature. Then 0.3 M
2
2
2
The CH
rotary evaporation and dried under vacuum to obtain tert- extracted with EA (3 ꢂ 30 mL). The organic layer was dried over
butyl((4-(chloromethyl)benzyl)oxy)dimethylsilane (3) (262 mg, Na SO , ltrated and concentrated to give 2-((4-(((tert-butyldi-
) d 7.34 (m, 4H), 4.75 methylsilyl)oxy)methyl)benzyl)oxy)-N,N-diethylnicotinamide (6)
2 2 3
Cl layer was collected and the solvent was removed by NaHCO (aq) was added to the reaction solution, which was
2
4
1
9
(
7%) as clear oil. H-NMR (400 MHz, CDCl
s, 2H), 4.59 (s, 2H), 0.95 (s, 9H), 0.11 (s, 6H).
.2.3 Synthesis of N,N-diethyl-2-hydroxynicotinamide (5). (dd, J ¼ 6.7, 1.4 Hz, 1H), 7.50 (dd, J ¼ 6.8, 1.4 Hz, 1H), 7.30 (s,
3
1
3
(304 mg, 71%) as white solid. H-NMR (400 MHz, CD OD) d 7.79
2
The 2-hydroxynicotinic acid (4) (1 g, 7.2 mmol) was dissolved in 4H), 6.42 (t, J ¼ 6.8 Hz, 1H), 5.21 (s, 2H), 4.72 (s, 2H), 3.51 (q, J ¼
3
6
0 mL THF. CDI (1.17 g, 7.22 mmol) was added and stirred at 7.1 Hz, 2H), 3.22 (q, J ¼ 7.1 Hz, 2H), 1.22 (t, J ¼ 7.1 Hz, 3H), 1.09
ꢀ
0 C under N
2
for 1 h. Then, diethylamine (0.79 g, 10.8 mmol) (t, J ¼ 7.2 Hz, 3H), 0.93 (s, 9H), 0.08 (s, 6H).
was added to the system at room temperature, and the mixture
2.2.5 Synthesis of N,N-diethyl-2-((4-(hydroxymethyl)benzyl)
ꢀ
was stirred at 60 C under N
2
for 2 h. Aer the reaction system oxy)nicotinamide (7, DENA). The 2-((4-(((tert-butyldimethylsilyl)
was cooled to room temperature, the solvent was removed by oxy)methyl)benzyl)oxy)-N,N-diethylnicotinamide (6) (1 g, 2.33
rotary evaporation. 10 mL ethyl acetate was added. The mmol) was dissolved in 20 mL THF. Then, the pH of the reac-
suspension was ltered through sintered glass funnel with EA tion mixture was adjusted to 2–3 by 2 M HCl (aq) and stirred at
to give N,N-diethyl-2-hydroxynicotinamide (5) (1.18 g, 85%) as room temperature for 1 h. The solvent was removed by rotary
1
white solid. H-NMR (400 MHz, DMSO-d ) d 11.84 (s, 1H), 7.42 evaporation. 30 mL DCM was added. The organic layer was
6
(
dd, J ¼ 6.5, 1.9 Hz, 1H), 7.39 (dd, J ¼ 6.7, 2.0 Hz, 1H), 6.21 (t, J ¼ washed with 0.3 M NaHCO (aq) (3 ꢂ 30 mL) and dried with
3
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Na
2
SO
4
. The organic layer was concentrated by rotary evapora-
The morphology of PGG–DENA/PTX NPs were observed with
tion to give N,N-diethyl-2-((4-(hydroxymethyl)benzyl)oxy) transmission electron microscopy (TEM). The TEM study was
1
nicotinamide (7) (652 mg, 89%) as white solid. H-NMR (400 carried out using a JEM-2100 (Hitachi, Tokyo, Japan) electron
MHz, D O) d 7.75 (d, J ¼ 6.8 Hz, 1H), 7.58 (d, J ¼ 5.5 Hz, 1H), 7.29 microscope operating at an accelerating voltage of 75 kV.
2
(
1
7
d, J ¼ 7.9 Hz, 2H), 7.19 (d, J ¼ 7.9 Hz, 2H), 6.52 (t, J ¼ 6.8 Hz,
The drug loading capacity of PGG–DENA/PTX was deter-
H), 5.15 (s, 2H), 4.52 (s, 2H), 3.40 (q, J ¼ 7.1 Hz, 2H), 3.12 (q, J ¼ mined by HPLC (Agilent 1260 series). PGG–DENA/PTX (4 mg)
.0 Hz, 2H), 1.11 (t, J ¼ 7.2 Hz, 3H), 0.94 (t, J ¼ 7.1 Hz, 3H). was placed in a 5 mL centrifuge tube. Then, 2 mL of acetonitrile
.2.6 Synthesis of PGG–DENA. PGG (2.62 g, 16.5 mmol/ was added and the solution was shaken horizontally at 120
2
ꢁ
1
monomer-unit of polymer) was stirred in 30 mL anhy DMF for min
3
with an incubator shaker (HZ-8812S, Scientic and
ꢀ
0 min. Then EDC (2.53 g, 13.2 mmol), DMAP (0.42 g, 3.4 mmol) Educational Equipment plant, China) at 37 C. The supernatant
and DENA (7) (2.51 g, 8 mmol) were added into the reaction and solution was taken at 0.5 h, 3 h, 6 h, 9 h and 20 h, respectively,
stirred at room temperature for 24 h. The reaction was and ltered through a 0.22 mm pore-sized ltration membrane
3
quenched by 0.3 M NaHCO (aq). The solution was stirred for for HPLC determination.
1
5 min and dialyzed (MWCO 10k) for 24 h against water,
1
lyophilized to obtain PGG–DENA as a white solid. Then, the H-
NMR spectra of PGG and PGG–DENA in deuterated water (D O)
were recorded with a Bruker spectrometer at 400 MHz. The M
2.6. Long-term stability of PGG–DENA/PTX NPs
2
ꢁ
1
PGG–DENA/PTX NPs solution (2.0 mg mL ) was prepared in
PBS. The particle size and polydispersity index (PDI) of the
sample was monitored by DLS for 28 days.
w
of PGG and PGG–DENA were characterized by gel permeation
chromatography with a GPC-MALS system (Wyatt, Santa Bar-
bara, California).
2.7. In vitro release of PTX from PGG–DENA/PTX NPs
To investigate the in vitro release prole of PTX from PGG–
2
.3. Preparation of PGG–DENA/PTX NPs
DENA/PTX NPs, we used sodium salicylate solution (0.8 M, pH
PGG–DENA/PTX NPs were prepared by the emulsication-
21
6
.5) as the release medium as reported previously. The PGG–
19,20
solvent evaporation method.
solved in 2 mL of mixture of methylene chloride and acetone
3 : 1, v/v) as the organic phase, and 40 mg of PGG–DENA were
Briey, 12 mg of PTX was dis-
DENA/PTX NPs solution with the nal concentration of 2 mg
ꢁ1
mL was packed in 50 mL centrifuge tube and shaken hori-
(
ꢁ1
ꢀ
zontally at 120 min at 37 C. The sample was withdrawn at
predetermined time points and mixed with 1 mL ethyl acetate to
extract PTX. The ethyl acetate solution was dried with nitrogen
blowing instrument. Then the samples were resuspended with
acetonitrile and ltered through a 0.22 mm pore-sized ltration
membrane. The medium was refreshed at various time inter-
vals. The concentration of PTX was determined by HPLC
analysis.
suspended in 4 mL of sodium cholate solution as the water
phase. The mixture was emulsied by ultrasonic method in ice
bath and gently stirred at room temperature. The solution was
then opened to air overnight to allow slow evaporation of
organic solvent and formation of the PGG–DENA/PTX NPs,
which were puried by G50 gel column connecting AKTA puri-

er (GE Healthcare) to remove the free PTX.
2.4. PTX solubility
2.8. In vitro cytotoxicity assays
The solubility of PTX was determined by high performance
liquid chromatography (HPLC, Agilent 1260 series). DENA was
dissolved in deionized water and packed in centrifuge tube (2
mL). Excess PTX was added to each tube. The mixture was
The in vitro cytotoxicity of NPs was investigated by the CCK-8
assay according to the published protocols with modica-
22
3
tions. NCI-H460 cells (5 ꢂ 10 ) were seeded in 96-well plates
and incubated for 24 h in a humidied atmosphere with 5%
ꢀ
stirred using a magnetic stirring bar for 24 h at 37 C. The
CO . Then serial dilutions of PTX, PGG–PTX NPs, PGG–DENA/
2
sample was mixed with 1 mL ethyl acetate to extract PTX. The
ethyl acetate solution was dried with nitrogen blowing instru-
ment. Then the samples were resuspended with acetonitrile and
PTX NPs and PGG–DENA were added to the plate (100 mL per
well), respectively. Aer further incubation for up to 48 h, the
cells were treated with 10 mL of CCK-8 solution and cultured for

ltered through a 0.22 mm pore-sized ltration membrane. The
4
h. The absorbance was measured with a microplate reader
SpectraMax M5, Molecular Devices, USA) at 450 nm. The
survival rate was calculated using the following formula:
amount of PTX in each tube was measured by HPLC with a UV
detector at 228 nm.
(
viability rate ¼ [(OD
ꢁ ODblank)/(ODcontrol group ꢁ
test group
OD
)] ꢂ 100%, where OD
is the optical density (OD)
is the OD of control group,
2
.5. Characterization of PGG–DENA/PTX NPs
blank
test group
of experiment group, OD
and ODblank is the OD of blank group.
control group
The particle sizes of PGG–DENA/PTX NPs were measured by
dynamic light scattering (DLS) using a Mastersizer2000 (Mal-
vern Instruments Inc) equipped with He–Ne laser (4 mW, 633
ꢀ
2.9. In vitro cellular uptake
nm) light source and 90 angle scattered-light collection
conguration.
2.9.1 Preparations of PGG–DENA/PTX/DiO NPs. PGG–
The particle charge was quantied as zeta potential using DENA/PTX/DiO NPs were prepared by the emulsication-
19
a Mastersizer2000 (Malvern Instruments Inc).
solvent evaporation method. Briey, DiO was suspended in
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methylene chloride and acetone (3 : 1, v/v) as the organic phase, to remove cell aggregates. The cell suspensions were then
and PGG–DENA/PTX NPs were suspended in sodium cholate analyzed by ow cytometry (Guava easyCyte, USA).
solution as the water phase. The mixture was emulsied by
ultrasonic method in ice bath and rotary evaporated to remove
organic solvent and obtain the PGG–DENA/PTX/DiO NPs. PGG–
DENA/PTX/DiO NPs were then puried by G50 gel column
connecting AKTA purier to remove free DiO.
2
.10. Hemolysis study
Hemolysis study was carried out according to the published
23
procedure. Briey, freshly collected rat blood was washed
three times with saline by centrifugation at 1500 rpm for 15
minutes. The red blood cell suspension was diluted with saline
to obtain a 2% suspension (v/v). Various concentrations of PGG–
DENA/PTX NPs, Cremophor EL, Tween 80, saline were added
into the suspension, respectively. These samples were incu-
bated at 37 C for 1 hour, and centrifuged at 3000 rpm for 10
minutes. The supernatants were collected and analyzed for
hemoglobin content by spectrophotometric detection at
2.9.2 Cellular uptake. Cellular uptake studies were per-
formed on NCI-H460 cell line. Cells were seeded in glass bottom
4
dish or 6-well plates at a density of 3 ꢂ 10 cells per mL and
cultured for 24 h. Cells were washed with PBS and treated with
different concentrations of PGG–PTX/DiO and PGG–DENA/PTX/
DiO NPs for 2 h.
ꢀ
In order to observe the cellular uptake of NPs qualitatively,
the treated cells were washed three times with PBS, then xed
with 4% paraformaldehyde, stained with Hoechst 33342 and
observed using a confocal laser scanning microscope (CLSM,
TCS SP5, Leica).
545 nm. Analysis of each sample was performed in triplicate.
2.11. Statistical analysis
To quantitatively analyze the cellular uptake of NPs, the Statistical differences were evaluated by two-tailed student's t-
treated cells were washed with PBS, trypsinized and harvested test for two groups of data and one-way ANOVA for over three
by centrifugation at 1200 rpm for 5 min. The cells were resus- groups of data. The differences were considered to be signi-
pended in 200 mL PBS and ltered through a 40 mm nylon mesh cant at P < 0.05 and very signicant at P < 0.01.
3
. Results and discussion
3.1. PTX solubility
The solubilization of PTX by DENA was determined by HPLC.
The results (Fig. 3) showed that the solubility of PTX increased
gradually as the concentration of DENA increased. When the
concentration of DENA reached 6 M, the solubility of PTX was
ꢁ
1
increased to about 523 mg mL in water (the intrinsic solu-
ꢁ1
24
bility of PTX is 0.0003 mg mL ). The results indicating that
6
DENA increased the solubility of PTX by 1.7 ꢂ 10 -fold.
3.2. Characterization of PGG–DENA
1
3
.2.1 Characterization of H-NMR spectra. The chemical
structure of the PGG–DENA is shown in Fig. 4A. The green part
is PGG, and the red is DENA. The H-NMR spectra of PGG (I) and
Fig. 3 The solubility of PTX as a function of the molar concentration of
DENA. The solubility of PTX reached about 523 mg mL at 6 M of
1
ꢁ1
DENA. Data are means ꢃ SD from three measurements.
PGG–DENA (II) are shown in Fig. 4B. The characteristic peaks
1
Fig. 4 Characterization of the PGG–DENA. (A) The chemical structure of the PGG–DENA. The green part is PGG and the red is DENA. (B) H-
NMR spectra of PGG (I) and PGG–DENA (II). The characteristic peaks aromatic hydrogens of DENA at 7.80 (s, 1H), 7.59 (s, 1H), 7.21–7.30 (m, 4H)
and 6.53 (s, 1H) ppm (the black arrow) showed that DENA and PGG were successfully linked together.
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aromatic hydrogens of DENA at 7.80 (s, 1H), 7.59 (s, 1H), 7.21– particle size determined by DLS represents their hydrodynamic
.30 (m, 4H) and 6.53 (s, 1H) ppm (the black arrow) can be diameter, whereas that obtained by TEM represents the
7
1
found in the H-NMR spectrum of PGG–DENA. There was no collapsed micelles aer water evaporation. This result is also
1
25
peak found at 7–8 ppm in the spectra of PGG. The H-NMR consistent with previous report.
results showed that DENA and PGG had been successfully
linked together.
3.3.2 The long-term stability of PGG–DENA/PTX NPs. The
long-term stability of PGG–DENA/PTX NPs was evaluated by the
3.2.2 Characterization of M
w
. The results are shown in change of particle sizes in 28 days. As shown in Fig. 6, there are
Table 1. The M of PGG and PGG–DENA were about 51.29 (kDa) no obvious changes, suggesting the good stability of the NPs.
w
and 65.52 (kDa), and the PDI were 1.31 and 1.43, respectively. This results indicated the high stability of PGG–DENA/PTX NPs,
The PGG/DENA molar ratio was calculated to about 4.66 : 1. And which may be due to the increased compatibility of hydrotropic
the results also suggested the successful conjugated of DENA.
polymer material with PTX.
.3.3 Loading capacity of PTX in PGG–DENA NPs. The
3
anticancer drug, PTX, is difficult to encapsulate into traditional
micelles because it has the highly hydrophobic property. Thus,
we designed “hydrotropic polymer”, PGG–DENA NPs, as
a carrier for PTX to increase its aqueous solubility. The hydro-
phobic PTX was easily encapsulated into PGG–DENA NPs using
a emulsication-solvent evaporation method due to DENA has
a special solubilization mechanism for PTX (aromatic ring
buildup and hydrogen bonding). The schematic illustration of
PTX loading was showed in Fig. 1. The PTX loading capacity of
PGG–DENA/PTX was determined to be 11.7% (wt%) by HPLC
determination.
3
.3. Characterization of PGG–DENA/PTX NPs
.3.1 Characterization of DLS and TEM. The particle size of
PGG–DENA/PTX NPs was measured by DLS method. The result
Fig. 5A) showed that the average particle size of PGG–DENA/
3
(
PTX NPs was about 70 nm, with a PDI of 0.219. In addition,
the zeta potential of the PGG–DENA/PTX NPs was ꢁ30.3 mV. In
order to observe the morphology of NPs, the particles were
negatively stained with phosphotungstic acid and then visual-
ized using TEM. PGG–DENA/PTX NPs (Fig. 5B) showed well-
dened spherical morphology. The particle size of PGG–
DENA/PTX NPs observed by TEM was about 50 nm, which was
smaller than that determined by DLS. We speculated that the
3.4. In vitro release of PTX from PGG–DENA/PTX NPs
The PTX release from PGG–DENA/PTX NPs was performed in
sodium salicylate (0.8 M) at 37 C for 96 h. As shown in Fig. 7,
a
ꢀ
Table 1 Characterization of polymer conjugates
the results showed that the release of PTX from PGG–PTX NPs
and PGG–DENA/PTX NPs gradually increased with time.
Compared to PGG–PTX NPs (22.62%), the cumulative release
rate of PTX from PGG–DENA/PTX NPs reached 79.10% at 96 h.
PTX release from PGG–DENA/PTX NPs was remarkably higher
than that from PGG–PTX NPs. This results indicated that the
encapsulated PTX in PGG–DENA/PTX NPs is easier to be
released than chemically bonded PTX in PGG–PTX NPs.
Sample
M
w
(kDa)
w n
M /M (PDI)
PGG
PGG–DENA
51.29
65.52
1.31
1.43
a
All data are expressed as the mean M
w
of the samples (n ¼ 3).
3.5. In vitro cytotoxicity assays
The cell viability of NCI-H460 cells were evaluated following
incubation with PTX, PGG–PTX NPs, PGG–DENA/PTX NPs and
Fig. 5 Particle size and morphology characterization of PGG–DENA/
PTX NPs. DLS analysis (A) and TEM images (B) of PGG–DENA/PTX NPs.
The NPs exhibited uniform spherical morphology as displayed in the Fig. 6 Stability of PGG–DENA/PTX NPs in PBS solution at 4 C. PGG–
ꢀ
TEM image.
DENA/PTX NPs exhibited long-term stability.
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ꢁ
1
ꢁ1
mg mL and 2.0253 mg mL , respectively. PTX had the stron-
gest cytotoxicity. PGG–DENA/PTX NPs showed signicantly
increased cytotoxicity than PGG–PTX NPs, which should be
resulted from the increased PTX release from PGG–DENA/PTX
NPs compared with PGG–PTX NPs (Fig. 7).
3.6. In vitro cellular uptake
In order to determine the interaction of PGG–DENA/PTX NPs
with the tumor cells, we observed the cellular uptake of NPs by
NCI-H460 cell line using CLSM and ow cytometer. As shown in
Fig. 9, the percentages of uorescent cells in PGG–PTX/DiO NPs
and PGG–DENA/PTX/DiO NPs were 97.36% and 96.74%, and
the mean uorescent intensities for them were 183.40 and
Fig. 7 The kinetics of PTX release from PGG–DENA/PTX NPs and
PGG–PTX NPs in sodium salicylate at 37 ꢀC (n ¼ 3, bars represent
means ꢃ SD).
177.03, respectively. These data showed that the PGG–PTX/DiO
NPs and PGG–DENA/PTX/DiO NPs have similar cellular uptake
by NCI-H460 cells, which may be attributed to their similar
endocytic pathways.
3.7. Hemolysis study
To determine the biocompatibility of PGG–DENA/PTX NPs,
a hemolysis study was carried out. Our previous study showed
Fig. 8 The cytotoxicity of PTX, PGG–PTX NPs, PGG–DENA/PTX NPs
and PGG–DENA on NCI-H460 cells as measured by CCK-8 Kit. PGG–
DENA/PTX NPs showed significantly increased cytotoxicity than PGG–
PTX NPs.
PGG–DENA. As shown in Fig. 8, the results showed that the cell
viability of PTX, PGG–DENA/PTX NPs and PGG–PTX NPs
decreased gradually as the drug concentration increased. PGG–
DENA had no obvious cytotoxicity. The IC50 value for PTX, PGG–
Fig. 10 Hemolysis of red blood cells after incubation with PGG–
DENA/PTX NPs, Cremophor EL and Tween 80. Compared with Cre-
mophor EL and Tween 80, PGG–DENA/PTX NPs showed almost no
ꢁ1
DENA/PTX NPs and PGG–PTX NPs was 0.2304 mg mL , 0.7356 hemolytic activity.
Fig. 9 The CLSM images of cellular uptake and flow cytometry for PGG–PTX/DiO NPs (A) and PGG–DENA/PTX/DiO NPs (B). The numbers in
flow cytometry pictures represent percentages of DiO-positive cells and mean of fluorescence intensity, respectively. These data showed that
the PGG–PTX/DiO NPs and PGG–DENA/PTX/DiO NPs have similar cellular uptake by NCI-H460 cells.
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13
that the PGG polymer have good biocompatibility. As shown in release than chemical conjugation does. What is more, PGG–
Fig. 10, the surfactants Tween 80 caused signicant damage of DENA/PTX NPs can avoid the side effects of free DENA aer
ꢁ1
red blood cells at 2 mg mL , and Cremophor EL did not induce administration. We also found that the PGG–DENA/PTX NPs
ꢁ1
substantial hemolysis until reaching 4 mg mL . By contrast, showed signicantly increased cytotoxicity than PGG–PTX NPs
PGG–DENA/PTX NPs had no hemolytic activity even at high (Fig. 8), which should be resulted from the increased PTX
ꢁ
1
concentrations of 8 mg mL . These data suggested that PGG– release from PGG–DENA/PTX NPs compared with PGG–PTX
DENA/PTX has good compatibility with erythrocytes and can be NPs.
administered intravenously.
In summary, we have successfully developed a PGG–DENA/
PTX NPs delivery system which exhibited a high drug release
rate, good biocompatibility and long-term stability. This study
provide a solution to increase the compatibility of carrier
4
. Conclusions
In this work, using PTX as the model drug, we conjugated materials with insoluble drugs, and also may provide an effec-
DENA, the hydrotropic agents of PTX, to the backbone of tive way to develop a series of personalized carrier materials
a water-soluble polymer PGG to prepare the “hydrotropic poly- suitable for different insoluble drugs.
mer” PGG–DENA. Then by virtue of the hydrotropic effect of
DENA group, PTX was successfully encapsulated by PGG–DENA
to obtain the hydrotropic polymeric nanoparticles PGG–DENA/
Conflict of interest
PTX NPs. The results showed that the cumulative release rate of
PTX from PGG–DENA/PTX NPs reached 79.10% at 96 h. In vitro
cytotoxicity assays, PGG–DENA/PTX NPs showed signicantly
The authors declare no potential conicts of interest with
respect to the authorship and/or publication of this article.
increased cytotoxicity than PGG–PTX NPs. The NCI-H460 lung
cancer cells have similar cell uptake of PGG–PTX/DiO NPs and Acknowledgements
PGG–DENA/PTX/DiO NPs. Furthermore, the hemolysis study
This work was supported by National Basic Research Program of
proved that the PGG–DENA/PTX NPs has good compatibility
China (2013CB932500), National Natural Science Foundation of
with erythrocytes. The results indicated that the hydrotropic
China (60976004), “985” grants of East China Normal University
polymer PGG–DENA was an effective carrier material for PTX.
(ECNU).
The molecular structure of the hydrotropic agent is generally
composed of an aromatic ring system and an anionic group, in
which the aromatic ring moiety interacts with the drug and the
anionic group brings a high water solubility. The mechanism of
solubilization is much complicated, and the consensus on this
can be summarized as the four points: hydrophobic interaction,
aromatic ring accumulation, hydrogen bond formation and
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