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TPGS2k/PLGA nanoparticles for overcoming multidrug resistance by interfering mitochondria of human alveolar adenocarcinoma cells Dong-Fang Wang, Wen-Ting Rong, Yu Lu, Jie Hou, ShanShan Qi, Qing Xiao, Jue Zhang, Jin You, Shuqin Yu, and Qian Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/am508340m • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 6, 2015
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TPGS2k/PLGA nanoparticles for overcoming multidrug resistance by interfering mitochondria of human alveolar adenocarcinoma cells
3 4 5
Dong-Fang Wang a, Wen-Ting Rong b, Yu Lu a, Jie Hou a, Shan-Shan Qi b, Qing Xiao b
, Jue Zhang b, Jin You a, Shu-Qin Yu a, b, * and Qian Xu c,d, **
6 7
a. Jiangsu Key Laboratory for Supramolecular Medicinal Materials and Applications, College of Life
8
Sciences, Nanjing Normal University, Nanjing 210046, The People’s Republic of China
9
b. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life
10
Sciences, Nanjing Normal University, Nanjing 210046, The People’s Republic of China
11 12 13
c. Ministry of Education Key Laboratory of Environmental Medicine and Engineering, School of Public Health, Southeast University, Nanjing 210009, The People’s Republic of China
d. Suzhou Key Laboratory of Environment and Biosafety, Suzhou215123, People’s Republic of China
14 15
* Corresponding author: Shu-Qin Yu: Jiangsu Key Laboratory for Supramolecular Medicinal Materials
16
and Applications, College of Life Sciences, Nanjing Normal University, Wenyuan Road 1, Nanjing
17
210046, China. Tel/Fax: 0086-25-8589 1265. Email:
[email protected] 18 19
** Corresponding Author: Qian Xu: Ministry of Education Key Laboratory of Environmental Medicine
20
and Engineering, School of Public Health, Southeast University, DingJiaQiao 9, Nanjing 210009,
21
China. Tel/Fax: +86 25 8327 2563; E-mail:
[email protected] 22 23 24 25 26 27 28 29 30 31
Dong-Fang Wang and Wen-Ting Rong contributed equally to this work.
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ABSTRACT
34
This study successfully synthesized D-α-tocopheryl polyethylene glycol 2000
35
succinate (TPGS2k) and prepared TPGS2k-modified poly (lactic-co-glycolic acid)
36
nanoparticles (TPGS2k/PLGA NPs) loaded 7-ethyl-10-hydroxycamptothecin (SN-38),
37
designated TPGS2k/PLGA/SN-38 NPs. Characterization measurements showed that
38
TPGS2k/PLGA/SN-38 NPs displayed flat and spheroidal particles with diameters of
39
80 -104 nm. SN-38 was encapsulated in TPGS2k emulsified PLGA NPs with the
40
entrapment efficiency and loading rate of SN-38 being 83.6% and 7.85%, respectively.
41
SN-38 could release constantly from TPGS2k/PLGA/SN-38 NPs in vitro.
42
TPGS2k/PLGA/SN-38 NPs induced significantly higher cytotoxicity on A549 cells
43
and the multidrug resistance (MDR) cell line (A549/DDP cells and A549/Taxol cells)
44
compared with free SN-38. Further studies on the mechanism of the NPs in increasing
45
the death of MDR cells showed that following the SN-38 releasing into cytoplasm the
46
remaining TPGS2k/PLGA NPs could reverse the P-gp mediated MDR via interfering
47
with the structure and function of mitochondria and rather than directly inhibiting the
48
enzymatic activity of P-gp ATPase. Therefore, TPGS2k/PLGA NPs can reduce the
49
generation of ATP and the release of energy for the requisite of P-gp efflux
50
transporters. The results indicated that TPGS2k/PLGA NPs could become the
51
nanopharmaceutical materials (NPMs) with the capability to reversal MDR and
52
improve anti-cancer effects of some chemotherapy drugs as P-gp substrates.
53 54
KEY WORDS
55
TPGS2k, Nanopharmaceutical materials, Nanoparticles, Multi-drug resistance, P-
56
glycoprotein, Mitochondria, ATP
57 58 59 60 61 62 63 64
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INTRODUCTION
66
Multidrug resistance (MDR) is the course that carcinoma cell exposed to one
67
chemotherapy agent progress the cross-resistance to some chemical constitution and
68
biological effect unrelated cytotoxic medicines 1. MDR is the main obstacle for the
69
anticancer chemotherapy. It is generally recognized that MDR is apparently related to
70
the overexpression of some ATP-binding cassette (ABC) transporters, which consist
71
of P-glycoprotein (P-gp), MDR-related protein (MRP1) and other resistance proteins.
72
As a result, these ABC-transporters can reduce the intracellular concentration of some
73
chemotherapy drugs as the MDR substrates. Among these transporters, the
74
significance of P-gp mediated MDR has been most widely researched. P-gp
75
overexpression is often found in the cancer cells of patients with ineffective
76
chemotherapy. Small molecule P-gp inhibitor, including verapamil, had been applied
77
to co-delivery with anticancer drugs to achieve the aim of reversing MDR 2.
78
Verapamil can make the co-delivery drug get higher anticancer effective or better oral
79
bioavailability 3. However, the attempt to reverse MDR has not yet obtained
80
promising results because the dose of verapamil as a P-gp inhibitor is significantly
81
greater than that as anti-hypertension drug
82
more considerations have been recently concentrated on some novel delivery
83
materials to modulate P-gp 6.
4-5
. For fear of these undesirable effects,
84
Nanopharmaceutical materials (NPMs) applied in nano-drug delivery system
85
(NDDS) should have favorable biocompatible and biodegradable properties. At the
86
same time, NPMs can increase the absorption and the pharmacodynamics of loaded-
87
drug by adjusting the drug release rate and promoting membrane permeability.
88
Recently, the NDDS have raised widespread concern on overcoming MDR 7-9.
89
These NPMs include D-a-tocopheryl polyethylene glycol 1000 succinate 10
, poly (lactic-co-glycolic acid) (PLGA)
11
90
(TPGS1k)
91
solubility vitamin E derivative, is synthetized via trans-esterification of polyethylene
92
glycol 1000 and vitamin E succinate. Mu et al first proposed TPGS1k as a novel
93
emulsifier in solvent evaporation and extraction preparation for NDDS and proved
94
that TPGS1k could be an effective and safe emulsifier with easy preparation and
95
excellent physicochemical characterization
96
water-soluble derivative of natural vitamin E, named TPGS2k
12
and so on. TPGS1k, a water-
. Liu et al synthesized a long-chain
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. TPGS2k-emulsified
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PLGA nanoparticles (TPGS2k/PLGA NPs) proved preferable merit of the higher
98
cellular uptake over TPGS1k-emulsified PLGA nanoparticles (TPGS1k/PLGA NPs) 14.
99
TPGS2k/PLGA NPs conjugated with folic acid displayed outstanding advantages to
100
cellular uptake and therapeutically effective 13. Although TPGS1k or TPGS2k and PLGA as NPMs have been widely used in NDDS
101 102
15
103
characteristics, in vitro cytotoxicity and in vivo biological effects on loaded-drug
104
nanoparticles. Currently, it had become clear that TPGS2k/PLGA NPs had certain
105
inhibition effect on P-gp. However, the cellular and molecular mechanisms still
106
remain unclear. In our previous research, we had demonstrated that TPGS1k/PLGA
107
NPs can increase the cellular accumulation of loaded-drug by escaping the
108
recognition of P-gp and affecting efflux microenvironment of MDR cells
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efflux microenvironment is considered to include some subcellular structure and
110
biomolecules that influence the function of P-gp transporter, such as lipid raft in cell
111
membrane
112
microenvironment could impact the accumulation of chemotherapeutics in organelles.
113
Among NDDS relative to the efflux microenvironment, mitochondrial targeting is a
114
novel strategy for overcoming MDR 20.
, the emphases of investigation still were placed on preparation, physicochemical
17
, mitochondria
18
and P-gp Mg2+-ATPase
19
16
. The
. The effect on the efflux
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As an active metabolite, 7-Ethyl-10-hydroxy-camptothecin (SN-38) is also a
116
topoisomerase 1 inhibitor with anticancer effective 100 to 1000-times higher than its
117
raw compound irinotecan
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administrative doses, SN-38 had better to be administered directly for clinical
119
application to improve anticancer action. Irinotecan or SN-38 is also effect for human
120
small-cell lung cancer in single or combination with other anticancer drug, such as
121
cisplatin 22. Owing to the fact that SN-38 is the substrate of P-gp 23, we chose it as a
122
model drug in present research.
21
. To avoid systemic toxicity of irinotecan in large
123
Nevertheless, there continued to be no evidence to prove the relationship between
124
TPGS2k/PLGA NPs and efflux microenvironment. In order to clarify the mechanisms
125
of TPGS2k/PLGA NPs in reversing MDR, we hypothesized that TPGS2k/PLGA NPs
126
would change the P-gp efflux microenvironment by means of affecting mitochondrial
127
structure and function (Figure 1C). The aims of this investigation were to assess the
128
effects of TPGS2k/PLGA NPs overcoming MDR and its novel cellular and molecular 4
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mechanisms. For these objectives, we synthesized TPGS2K, prepared TPGS2k/PLGA
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NPs, and delivered anticancer drug SN-38 with the nanoparticles (named
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TPGS2k/PLGA/SN-38 NPs). Then, the effects of TPGS2k/PLGA NPs on mitochondria
132
of human alveolar adenocarcinoma cells need to be further evaluated, including the
133
mitochondrial membrane potential, ultra-microstructure and the respiration rate.
134 135
EXPERIMENTAL SECTION
136
Materials
137
Dichloromethane
(DCM),
Dicyclohexylcarbodiimide
(DCC)
and
4-
138
dimethylamiopryidine (DMAP) were obtained from Medpep (Shanghai, China).
139
PLGA (lactic acid: glycolic acid = 50:50, viscosity 1.13 dl/g, molecular weight 30
140
kDa) was ordered from Daigang Biomaterial (Jinan, China). Methoxypolyethylene
141
glycol 2000 (mPEG2k), D-α-tocopheryl succinate (α-TOS) and Rhodamine-123
142
(R123) were acquired from Sigma-Aldrich. Cisplatin (cisdiaminedichloroplatinum,
143
DDP), paclitaxel (Taxol) and SN-38 were obtained from Shanghai Knowshine
144
Pharma Chemicals. Sodium azide (NaN3) was purchased from Beyotime (Nantong,
145
China). Verapamil and Coumarin-6 (C6) were purchased from TCI (Tokyo, Japan).
146
Synthesis of TPGS2k
147
TPGS2k was synthesized based on previously published literature
14
. Briefly, α-
148
TOS, mPEG2k, DCC and DMAP were dissolved in DCM at a stoichiometric ratio of
149
1: 1: 2: 0.1. The solution was stirred overnight at nitrogen condition. Then, in order to
150
eliminate the by-products of reaction, the solution was filtered and precipitated with
151
precooled diethyl ether. After obtained precipitant was washed twice with diethyl
152
ether, it was subsequently dissolved in aqua distillate. The final emulsion was
153
gathered from dialyzing against water and filtering to remove impurities. TPGS2k
154
powder was got from the filtrate via freeze-drying (Figure 1A).
155
Preparation of TPGS2k/PLGA NPs, loaded-drug NPs and intracellular tracing NPs
156
TPGS2k/PLGA NPs were prepared utilizing oil-in-water (O/W) emulsion solvent 16
157
evaporation based on our previously report
. Briefly, PLGA solution (100 mg in 6
158
mL acetone) was added dropwise into TPGS2k aqueous solution (0.03%, pH 3.0) and
159
mixed under the homogenizer (IKA, German). After stirred at 1500 - 1600 rpm by a
160
magnetic stirrer (IKA, German) for 4 h at 25 °C and eliminated organic solvent by 5
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rotary vacuum evaporation, the mixture was centrifuged at 13500 rpm for 1 h at 4 °C.
162
Collected TPGS2k/PLGA NPs were washed twice in deionized water. Then, acquired
163
precipitation was freeze-dried using a lyophilizer (FL-60 system, China). The
164
resulting nanoparticles were stored at 4 °C for in-depth researching.
165
Nanoparticles loaded-drug were prepared using O/W emulsion solvent evaporation
166
as our reported previously 16. Briefly, SN-38 solution (10 mg, dissolved with dimethyl
167
sulphoxide) was mixed with PLGA solution. The mixture solution was ultra-sonicated
168
for 120 s in ice-water bath to gained a regular organic phase, which was then
169
unhurried injected into a homogeneous liquid of 0.03% TPGS2k. The acquired O/W
170
emulsion was mixed at 14000 – 15000 rpm for 5 min with a tissue homogenate
171
machine. After mixed emulsion was lightly stirred using a magnetic stirring apparatus
172
for 4 h at RT, the organic solvent was eliminated with a rotary vacuum evaporation
173
(Shanghai DongXi equipment, China) at 45 °C in a thermostatic water bath. A
174
refrigerated micro-centrifuge (Eppendorf 5417R, Germany) was employed to collect
175
the resultant sample at 13500 rpm for 45 min. The precipitation was then washed
176
triple with aqua distillate to eliminate surplus SN-38 and emulsifier. Finally, the
177
suspension was lyophilized to obtain a powdery loaded-SN-38 nanoparticles
178
(TPGS2k/PLGA/SN-38 NPs).
179
To research uptake and efflux mechanisms, fluorescent-labeled nanoparticles were
180
also prepared for tracing their intracellular distribution. They were coumarin-6 labeled
181
nanoparticles (TPGS2k/PLGA/C6 NPs) for uptake mechanism research and R123
182
fluorescent-labeled nanoparticles (TPGS2k/PLGA/R123 NPs) for efflux mechanism
183
study. Coumarin-6 or R123 substituted for SN-38 was mixed with the organic phase at
184
0.05% concentration to form TPGS2k/PLGA/C6 NPs or TPGS2k/PLGA/R123 NPs,
185
respectively.
186
Physicochemical characterization Surface morphology
187 188
The
surface
morphological
characteristics
of
TPGS2k/PLGA
NPs
and
189
TPGS2k/PLGA/SN-38 NPs were examined under a scanning electron microscope
190
(SEM, JEOL JSM-5900, Japan).
191
Particle size and Zeta potential
192
The size, size distribution and zeta potential of these nanoparticles were determined
193
with dynamic light scattering (DLS) (Malvern Zeta Potential Analyzer, UK). Before 6
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measurement, the particles were re-suspended into deionized water. All tested
195
nanoparticles were measured in triplicate.
196
Differential scanning calorimetry (DSC)
197
Thermal behavior of TPGS2k, PLGA, SN-38, TPGS2k/PLGA NPs and
198
TPGS2k/PLGA/SN-38 NPs were examined by DSC analysis (Pyris Diamond, Perkin
199
Elmer, U.S.A.). The thermal analysis was performed with a heating rate of 10 °C/min
200
from 25 °C to 300 °C under nitrogen atmosphere.
201
X-ray diffractometry (XRD)
202
Crystalline state of TPGS2k, PLGA, SN-38 and TPGS2k/PLGA NPs were evaluated
203
by XRD (D/max-Rc, Ricoh, Japan) using Cu-K radiation with 200 mA and 40 kV at a
204
scanning rate of 2°/min over the range from 3° to 40°.
205
Loading efficiency (LE%) and encapsulation efficiency (EE%)
206
LE% and EE% of TPGS2k/PLGA/SN-38 NPs were carried out with a ultraviolet
207
spectrophotometer (UV-2450, Shimadzu, Japan) as our reported previously
208
and EE% of SN-38 were calculated as follows formulas.
209 210 211
Loading efficiency (LE) (%) =
16
. LE%
The weight of SN-38 calculated × 100% The weight of drug-loaded nanoparticles
Encapsulation efficiency (EE) (%) =
The weight of SN-38 calculated × 100% The weight of SN-38 added
Drug release in vitro
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SN-38 release profile in vitro from TPGS2k/PLGA/SN-38 NPs was researched
213
using classical dialysis bag method. The nanoparticles equivalent to SN-38 were
214
dissolved in PBS (containing 0.1% polysorbate 80, v/v) that imitated physiological
215
condition (pH 7.4) and the microenvironment of carcinoma tissue or cancer cells (pH
216
5.0). After 5 mL solution mentioned was placed into a dialysis bag (cutoff molecular
217
weight 12 kDa, Millipore), both ends of the bag were solidly fixed with clamps. It was
218
slowly shaken (100 rpm) at 37 °C in a thermostatic water bath. Then, 5 mL release
219
solution was taken out to detect the concentration of SN-38 using an ultraviolet
220
spectrophotometer at 265 nm. At the same time, an isometric fresh solution was added
221
into the release solution at a designated time interval. The tests were repeated in
222
thrice. The accumulated release rate was calculated as follows formula.
223
drug released rate (%) =
SN - 38 released amount × 100% total amount of SN - 38 in NPs
224 225
Biological effect research on human lung adenocarcinoma cells 7
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Cell culture
227
HFL-I cells (a normal human lung fibroblasts cell line), A549 human lung
228
adenocarcinoma cells and its two types MDR cell lines (A549/DDP cells and
229
A549/Taxol cells) (Shanghai Cell Bank, Chinese Academy of Sciences) were used for
230
biological effect research. These cells were cultured in DMEM (Life Technologies,
231
U.S.A.) supplemented with 10% HyClone FBS (Thermo Scientific, U.S.A.) under a
232
humidified atmosphere containing 5% CO2 in a CO2 incubator (8000 WJ, Thermo
233
Scientific, U.S.A.). To retain MDR, A549/DDP cells or A549/Taxol cells were
234
maintained respectively with DDP (2 µg/mL) and Taxol (200 ng/mL) for 48 h, and
235
were further cultured with DDP-free DMEM or Taxol-free DMEM for 48 h before
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starting following experiments.
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Cell viability in vitro
238
The cell viability of TPGS2k and TPGS2k/PLGA NPs on HFL-I cells, a normal
239
human embryonic lung fibroblast, and other cancer cell lines (A549 cells, A549/DDP
240
cells and A549/Taxol cells) were studied in order to detect the cytotoxicity of NPMs.
241
The tests of NPMs were designed at different concentrations (0.125, 0.25, 0.5 and 1.0
242
mg/mL) and times (24, 48 and 72 h), respectively. The cytotoxicity of
243
TPGS2k/PLGA/SN-38 NPs and free SN-38 was studied on A549 cells, A549/DDP
244
cells and A549/Taxol cells for comparing the biological effect of two drug
245
formulations at different concentrations (12.5, 25, 50 and 100 nM, final concentration
246
of SN-38) or time intervals (24, 48 and 72 h). These cells were digested by trypsin for
247
5 min and harvested, and resuspended in DMEM. The cells were seeded in 96-well
248
plates (Corning, U.S.A.) at a density of 5 × 104 cells/mL. After these cells grew up to
249
attaching well wall until a confluence of 70%, the medium was replaced by 100 µL of
250
FBS-free medium containing above-mentioned tested materials at ranging
251
concentrations and times. The blank medium was used as a control. After treated cells
252
were incubated for 3 h in a CO2 incubator, WST-1 reagent was used for the
253
quantification of cellular viability based operating manual. The cell percentage
254
viability was calculated as the following formula.
255 256
All tests were repeated in triplicate. The viability was expressed as means ± 8
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standard deviation from the triplicate experiments.
258
Cell apoptosis
259
The apoptotic effects of TPGS2k/PLGA NPs on A549 cells and A549/DDP cells
260
were determined used Annexin V-FITC Apoptosis Detection Kit (Abcam, U.S.A.).
261
Tested cells were seeded in 6-well plates (Corning, U.S.A.) and cultured for 24 h to
262
make these cells to grow at adhesion wall. After treated with TPGS2k/PLGA NPs at
263
the concentrations of 0.25, 0.50 and 1.0 mg/mL for 12 h, the cells were digested with
264
0.25% trypsin and washed with PBS. Following procedure was completed according
265
to operating manual.
266
Intracellular accumulation related to uptake
267
A549/DDP
cell
line
was
respectively
incubated
medium
containing
268
TPGS2k/PLGA/C6 NPs and free coumarin-6 for 0.25, 0.5, 1 and 2 h. After the
269
medium was replaced, the cells were cultured sequentially with DMEM for 1 h. The
270
quantity of intracellular coumarin-6 was observed under confocal laser scanning
271
microscope (CLSM, Olympus Flowview FV1000, Japan).
272
Intracellular accumulation related to efflux pump
273
A549/DDP cells (1 × 106 cells/mL) were incubated with DMEM (blank control),
274
medium containing verapamil (positive control, 50 µM) and TPGS2k/PLGA NPs for 1
275
h, respectively. Then, after treating with DMEM containing free R123 or
276
TPGS2k/PLGA/R123 NPs for 2 h, A549/DDP cells were washed two times with ice-
277
PBS to remove the superfluous R123 or TPGS2k/PLGA/R123 NPs. Finally, incubated
278
with fresh medium for 1 h to allow the efflux of intracellular R123, the fluorescence
279
of these cells were observed under CLSM.
280
The mean fluorescence intensity (MFI) standing for the intracellular accumulation
281
of coumarin-6 or R123 was calculated on the fluorescence photographs using ImageJ
282
software 24.
283
Cell mechanization study
284
Mitochondrial membrane potential (MMP, ∆Ѱm)
285
MMP was assessed using JC-1 mitochondrial membrane potential assay kit
286
(Cayman Chemical, U.S.A.) under CLSM. A549/DDP cells were seeded in 6-well
287
plates (5×105 cells/mL). After reaching a confluence of 80%, these cells were treated
288
in DMEM (blank control), TPGS2k/PLGA NPs (0.25 and 0.5 mg/mL) and NaN3 (10 9
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mM, positive control) for 1 h or 4 h, respectively. Following operational process was
290
accomplished based on the manufacturer's protocol. The red to green fluorescence
291
ratio (R/G value) was calculated with ImageJ software. The relation between ∆Ѱm
292
and R/G value was expressed as following formula 25. R / G valuetreatment ∆ψ m = 1 − × 100% R / G valuecontrol
293 294 295
Analysis of respiration rate The cellular respiration rate was measured with XF96 cell mito-stress test kit 26, 27
296
(Seahorse Bioscience, U.S.A.)
297
determine the optimal seeding density of tested cells (A549 cells, A549/DDP cells and
298
A549/Taxol cells) and the adequate concentrations of all tested compounds for the
299
corresponding cell line. The three tested cell lines (3.5 × 105 cells/mL) were
300
respectively seeded on 96-well plates (Seahorse Bioscience, U.S.A.). As a
301
concentration dependent study, the cells were treated in TPGS2k/PLGA NPs (0.125,
302
0.25, 0.5 and 1.0 mg/mL) for 4 h. In the time dependent study, the cell line was dealt
303
with TPGS2k/PLGA NPs (0.5 mg/mL) for 1 h and 4 h, respectively. Then, the cells
304
were cultured with Seahorse assay medium (Seahorse Bioscience, U.S.A.) in
305
incubator without CO2 for 1 h to initiate succedent experiment. After measuring the
306
basal oxygen consumption rate (OCR), the following mitochondrial inhibitors were
307
added
308
trifluoromethoxyphenylhydrazone (FCCP), the mixture of 0.5 µM antimycin A and
309
0.5 µM rotenone) to assess the changes of OCR using the XF96 Extracellular Flux
310
Analyzer (Seahorse Bioscience, U.S.A.).
311
Mitochondrial ultrastructure morphology of A549/DDP cells
sequentially
(0.5
µM
. Firstly, preliminary test was performed to
oligomycin,
0.3
µM
carbonyl
cyanide-p-
312
To study the intracellular impact of TPGS2k/PLGA NPs, A549/DDP cells (3 mL)
313
treated with serum-free medium (as a negative control) or TPGS2k/PLGA NPs (0.25,
314
0.5 mg/mL) were plated in culture dish. After cultured for 24 h, the cells were
315
digested using trypsin and harvested, and then washed twice with PBS. Following,
316
treated samples were fixed using glutaric dialdehyde and osmic acid. The samples
317
were routinely prepared through dehydrating, embedding, cutting the ultrathin
318
sections and collecting on copper grids. Images were viewed under a transmission
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electron microscope (Hitachi H-7650 TEM, Japan).
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ATPase Assay 10
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The affect of TPGS2k/PLGA NPs on ATPase activity was measured using P-gp28
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Glo™ Assay System (Promega, U.S.A.)
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detected the effects of different tested compounds (TC) on ATPase activity, including
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sodium orthovanadate (Na3VO4, a P-gp ATPase inhibitor), verapamil (a P-gp
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inhibitor), and TPGS2k/PLGA NPs (0.125, 0.25, 0.5 and 1.0 mg/mL). The untreated
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group (NT) was treated with Pgp-Glo™ Assay Buffer. The final relative light unit
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(RLU) of every group was measured with a microplate reader (SpectraMax M2,
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U.S.A.). If ∆RLUTC (RLUNa3VO4 minus RLUTC) > ∆RLUbasal (RLUNa3VO4 minus
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RLUNT), the tested compound was a P-gp ATPase stimulator; otherwise, it was a P-gp
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ATPase inhibitor.
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Statistical analysis
. Following the Kit's instruction, we
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Data are expressed as means ± SD. The results were analyzed using differences
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among the means of groups with an unpaired and two-sided student’s t-test.
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Difference was confirmed significant at P < 0.05 and very significant at P < 0.01.
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RESULTS AND DISCUSSION
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Synthetized and structural confirmation of TPGS2k
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In the study, we have successfully synthetized TPGS2k and its composition was
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confirmed by 1H NMR spectra. Figure 1B shows a comparison of typical 1H NMR
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spectra of mPEG2k, α-TOS and TPGS2k. The generated carbonyl from esterification
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between mPEG2k and α-TOS enhanced local electronegativity and reduced the
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shielding effect, which brought about the augment of δ. Firstly, 1H NMR shows the
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characteristic peaks of methoxy groups and methylenes of mPEG2k (δ 3.14 and δ
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3.40). Due to the decrease of shielding effect, the relevant peaks move left slightly (δ
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3.35 and δ 3.66) in 1H NMR spectra of TPGS2k (Figure 1Ba). On the contrary, the
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decrease of shielding effect lead to the diminution of δ of methyls (δ 2.12) connecting
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to the benzene and methylenes (δ 2.17) close to generated carbonyl, which are 2.14
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and 2.19 in α-TOS (Figure 1Bb). Finally, the methyls and methylenes far away from
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the generated carbonyl are scarcely influenced, which means the peaks were obtained
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with negligible shift (Figure 1Bc). These results demonstrated the similarity as well as
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the change of chemical microenvironment between the structures of reactants and
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products, which suggested that TPGS2k has been successfully synthesized. 11
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Physicochemical characterization of the nanoparticles
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The diameter and size distribution of nanoparticles play a major effect on drug
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release behavior and cellular phagocytosis. According to SEM and its 3D models
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processed by ImageJ
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TPGS2k/PLGA/SN-38 NPs (Figure 2Ba, Figure 2Bb) had the global particles with
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smooth surface and the average diameter about 80 nm or 90 nm. The results of DLS
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(Figure 2Ac and Figure 2Bc) also confirmed mean diameter of TPGS2k/PLGA NPs,
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which was 104.1 nm, smaller than 109.8 nm of TPGS2k/PLGA/SN-38 NPs. Their size
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distributions displayed a cramped feature (50 – 110 nm under SEM and 50 – 120 nm
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by DLS). The polydispersity index (PDI) of TPGS2k/PLGA NPs was 0.037, which
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was slightly less than that of TPGS2k/PLGA/SN-38 NPs (0.05). Both of zeta potentials
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obtained from TPGS2k/PLGA NPs and TPGS2k/PLGA/SN-38 NPs were negative
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charge (-33.78 mV and -31.23 mV) 13. The results suggested that TPGS2k/PLGA NPs
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or TPGS2k/PLGA/SN-38 NPs were disperse and steadfast.
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, TPGS2k/PLGA NPs (Figure 2Aa, Figure 2Ab) or
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XRD was employed to settle the existing condition of NPMs and model drug in the
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NPs. Figure 3A shows the XRD prototype of TPGS2k, PLGA, SN-38, TPGS2k/PLGA
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NPs and TPGS2k/PLGA/SN-38 NPs. The characteristic peak (2θ = 21. 5° and 25.12°)
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of TPGS2k in both of nanoparticles was disappearance. The result suggested that
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TPGS2k was amorphously distributed on the surface of nanoparticles. At the same
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time, decrease or even disappearance of characteristic peak of SN-38 (2θ = 10.94°,
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18.60° and 26.24°) meant that drug had been loaded into these nanoparticles.
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Figure 3B presents the DSC curves of NPMs and preparative nanoparticles. For
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raw materials, the melting endothermic peak (Tm) of SN-38 is at 274.24 °C 16; there
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was a small peak at 49.83 °C from the curve of PLGA; and Tm of TPGS2k was 53.81
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°C according our detection. Nevertheless, Tm of SN-38 disappeared in
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TPGS2k/PLGA/SN-38 NPs. This result noted that SN-38 in TPGS2k/PLGA/SN-38
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NPs had been as a non-crystalline form. At the same time, there was no obvious peak
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to be seen at 45 - 55 °C based on the curve of TPGS2k/PLGA NPs and
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TPGS2k/PLGA/SN-38 NPs. The result indicated that TPGS2k scarcely affected
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thermogram property of these NPs and TPGS2k/PLGA/SN-38 NPs were successfully
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prepared. The EE% and LE% of SN-38 in TPGS2k/PLGA/SN-38 NPs were 83.6% and
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7.85%, respectively. The results indicated that TPGS2k/PLGA/SN-38 NPs possessed
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accredited efficiency of drug loading and encapsulation.
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Drug release profiles from TPGS2k/PLGA/SN-38 NPs in different pH environments
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in vitro are presented in Figure 4. PBS at pH 5.0 and pH 7.4 as the released solution
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was respectively simulated the tumor microenvironment and physiological condition.
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An initial burst of SN-38 could be up to 17.85% in the first 12 h at pH 5.0. In the
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following 168 h, the cumulative release of SN-38 was sustainable increased to
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49.03%. It possessed the ability to sustain treatment for the cancer. Moreover, less
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than 17.32% of SN-38 released from TPGS2k/PLGA/SN-38 NPs at 24 h at pH 7.4.
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The data indicated that TPGS2k/PLGA/SN-38 NPs were stable at physiological
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condition, implying the nanoparticles maybe have fewer toxicity and side effects
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during anticancer treatment in vivo.
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Biological effects
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A549/DDP cells and A549/Taxol cells are human lung adenocarcinoma cell line
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being resistance to cisplatin and Taxol, respectively. Then, they also are considered to
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be MDR cell line owing to the biological characteristic of P-gp overexpression 16, 29-31.
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The viability of tested cell lines incubation with NPMs (TPGS2k, TPGS2k/PLGA NPs)
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and model drug (SN-38 or TPGS2k/PLGA/SN-38 NPs) with variant concentrations
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and different times are shown in Figure 5.
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Both TPGS2k and TPGS2k/PLGA NPs exhibit slight toxicity at higher concentration
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(Figure 5A) or for a longer incubation time (Figure 5B). For TPGS2k/PLGA NPs,
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when the incubated concentration of TPGS2k/PLGA NPs was 0.50 mg/mL, the
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viability of these cell lines maintained higher level (82.9 ± 3.27%, 83.2 ± 2.37%, 83.0
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± 3.27% and 83.9 ± 3.30% in A549, A549/DDP, A549/Taxol and HFL-I cells,
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respectively) for 24 h. These results showed that the cytotoxicity of TPGS2k and
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TPGS2k/PLGA NPs had not obviously difference on normal cells and cancer cells at
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tested concentrations. Therefore, 0.50 mg/mL was chosen to explore time-related
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toxicity and even in-depth study.
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After human lung adenocarcinoma cell lines were incubated using free SN-38 for
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24 h, obvious cytotoxicity appeared only at the high concentration groups (50 and 100
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nM). The cell viability was respectively 71.7 ± 10.9% and 66.0 ± 3.12% for A549
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cells, 74.9 ± 3.58%, 64.5 ± 4.02% for A549/DDP cells and 75.9 ± 4.25%, 66.0 ±
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3.12% for A549/Taxol cells. Instead, when incubated with TPGS2k/PLGA/SN-38 NPs
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the cell viability decreased to 48.1 ± 2.63% and 33.9 ± 6.06% for A549/DDP cells and
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55.4 ± 2.77%, 33.9 ± 6.06% for A549/Taxol cells, respectively (Figure 5C). After
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treatment for 48 h and 72 h, the cell viability treated with TPGS2k/PLGA/SN-38 NPs
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achieved more obvious decrease compared to free SN-38 (Figure 5D). In fact,
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TPGS2k/PLGA/SN-38 NPs achieved higher cellular mortality at each concentration
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and incubation times than free SN-38.
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To verify the influence of these nanoparticles on human lung adenocarcinoma cells
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apoptosis, the Annexin V-FITC/PI apoptosis assay of A549 cells and A549/DDP cells
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was performed to distinguish apoptosis cells from normal cells. The effects
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of TPGS2k/PLGA NPs on apoptosis and necrosis of A549 cells and A549/DDP
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cells were quantified by total percentage of early apoptosis (Q3) and late apoptosis
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(Q2). As shown in Figure 6, compared to the apoptosis percentages of the blank
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control (4.06% for A549 cells and 3.59% for A549/DDP cells), after incubation with
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TPGS2k/PLGA NPs (0.25, 0.50 and 1.0 mg/mL) for 12 h, the induced apoptosis
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percentages were increased to 7.47%, 18.2% (p
