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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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ACS Applied Materials & Interfaces

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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, **

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a. Jiangsu Key Laboratory for Supramolecular Medicinal Materials and Applications, College of Life

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Sciences, Nanjing Normal University, Nanjing 210046, The People’s Republic of China

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b. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life

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Sciences, Nanjing Normal University, Nanjing 210046, The People’s Republic of China

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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

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* 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]

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** 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,

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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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ACS Paragon Plus Environment


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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

2


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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

3


13

. 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

109

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

115

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

118

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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129

mechanisms. For these objectives, we synthesized TPGS2K, prepared TPGS2k/PLGA

130

NPs, and delivered anticancer drug SN-38 with the nanoparticles (named

131

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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161

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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194

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

212

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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226

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

236

starting following experiments.

237

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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257

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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Page 10 of 38

289

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

319

electron microscope (Hitachi H-7650 TEM, Japan).

320

ATPase Assay 10


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321

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

324

sodium orthovanadate (Na3VO4, a P-gp ATPase inhibitor), verapamil (a P-gp

325

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,

328

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

330

ATPase inhibitor.

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Statistical analysis

. Following the Kit's instruction, we

332

Data are expressed as means ± SD. The results were analyzed using differences

333

among the means of groups with an unpaired and two-sided student’s t-test.

334

Difference was confirmed significant at P < 0.05 and very significant at P < 0.01.

335 336

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

339

confirmed by 1H NMR spectra. Figure 1B shows a comparison of typical 1H NMR

340

spectra of mPEG2k, α-TOS and TPGS2k. The generated carbonyl from esterification

341

between mPEG2k and α-TOS enhanced local electronegativity and reduced the

342

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

344

3.40). Due to the decrease of shielding effect, the relevant peaks move left slightly (δ

345

3.35 and δ 3.66) in 1H NMR spectra of TPGS2k (Figure 1Ba). On the contrary, the

346

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

350

with negligible shift (Figure 1Bc). These results demonstrated the similarity as well as

351

the change of chemical microenvironment between the structures of reactants and

352

products, which suggested that TPGS2k has been successfully synthesized. 11



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353

Physicochemical characterization of the nanoparticles

354

The diameter and size distribution of nanoparticles play a major effect on drug

355

release behavior and cellular phagocytosis. According to SEM and its 3D models

356

processed by ImageJ

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TPGS2k/PLGA/SN-38 NPs (Figure 2Ba, Figure 2Bb) had the global particles with

358

smooth surface and the average diameter about 80 nm or 90 nm. The results of DLS

359

(Figure 2Ac and Figure 2Bc) also confirmed mean diameter of TPGS2k/PLGA NPs,

360

which was 104.1 nm, smaller than 109.8 nm of TPGS2k/PLGA/SN-38 NPs. Their size

361

distributions displayed a cramped feature (50 – 110 nm under SEM and 50 – 120 nm

362

by DLS). The polydispersity index (PDI) of TPGS2k/PLGA NPs was 0.037, which

363

was slightly less than that of TPGS2k/PLGA/SN-38 NPs (0.05). Both of zeta potentials

364

obtained from TPGS2k/PLGA NPs and TPGS2k/PLGA/SN-38 NPs were negative

365

charge (-33.78 mV and -31.23 mV) 13. The results suggested that TPGS2k/PLGA NPs

366

or TPGS2k/PLGA/SN-38 NPs were disperse and steadfast.

24

, TPGS2k/PLGA NPs (Figure 2Aa, Figure 2Ab) or

367

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

369

NPs and TPGS2k/PLGA/SN-38 NPs. The characteristic peak (2θ = 21. 5° and 25.12°)

370

of TPGS2k in both of nanoparticles was disappearance. The result suggested that

371

TPGS2k was amorphously distributed on the surface of nanoparticles. At the same

372

time, decrease or even disappearance of characteristic peak of SN-38 (2θ = 10.94°,

373

18.60° and 26.24°) meant that drug had been loaded into these nanoparticles.

374

Figure 3B presents the DSC curves of NPMs and preparative nanoparticles. For

375

raw materials, the melting endothermic peak (Tm) of SN-38 is at 274.24 °C 16; there

376

was a small peak at 49.83 °C from the curve of PLGA; and Tm of TPGS2k was 53.81

377

°C according our detection. Nevertheless, Tm of SN-38 disappeared in

378

TPGS2k/PLGA/SN-38 NPs. This result noted that SN-38 in TPGS2k/PLGA/SN-38

379

NPs had been as a non-crystalline form. At the same time, there was no obvious peak

380

to be seen at 45 - 55 °C based on the curve of TPGS2k/PLGA NPs and

381

TPGS2k/PLGA/SN-38 NPs. The result indicated that TPGS2k scarcely affected

382

thermogram property of these NPs and TPGS2k/PLGA/SN-38 NPs were successfully

383

prepared. The EE% and LE% of SN-38 in TPGS2k/PLGA/SN-38 NPs were 83.6% and

384

12


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Page 13 of 38

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7.85%, respectively. The results indicated that TPGS2k/PLGA/SN-38 NPs possessed

386

accredited efficiency of drug loading and encapsulation.

387

Drug release profiles from TPGS2k/PLGA/SN-38 NPs in different pH environments

388

in vitro are presented in Figure 4. PBS at pH 5.0 and pH 7.4 as the released solution

389

was respectively simulated the tumor microenvironment and physiological condition.

390

An initial burst of SN-38 could be up to 17.85% in the first 12 h at pH 5.0. In the

391

following 168 h, the cumulative release of SN-38 was sustainable increased to

392

49.03%. It possessed the ability to sustain treatment for the cancer. Moreover, less

393

than 17.32% of SN-38 released from TPGS2k/PLGA/SN-38 NPs at 24 h at pH 7.4.

394

The data indicated that TPGS2k/PLGA/SN-38 NPs were stable at physiological

395

condition, implying the nanoparticles maybe have fewer toxicity and side effects

396

during anticancer treatment in vivo.

397

Biological effects

398

A549/DDP cells and A549/Taxol cells are human lung adenocarcinoma cell line

399

being resistance to cisplatin and Taxol, respectively. Then, they also are considered to

400

be MDR cell line owing to the biological characteristic of P-gp overexpression 16, 29-31.

401

The viability of tested cell lines incubation with NPMs (TPGS2k, TPGS2k/PLGA NPs)

402

and model drug (SN-38 or TPGS2k/PLGA/SN-38 NPs) with variant concentrations

403

and different times are shown in Figure 5.

404

Both TPGS2k and TPGS2k/PLGA NPs exhibit slight toxicity at higher concentration

405

(Figure 5A) or for a longer incubation time (Figure 5B). For TPGS2k/PLGA NPs,

406

when the incubated concentration of TPGS2k/PLGA NPs was 0.50 mg/mL, the

407

viability of these cell lines maintained higher level (82.9 ± 3.27%, 83.2 ± 2.37%, 83.0

408

± 3.27% and 83.9 ± 3.30% in A549, A549/DDP, A549/Taxol and HFL-I cells,

409

respectively) for 24 h. These results showed that the cytotoxicity of TPGS2k and

410

TPGS2k/PLGA NPs had not obviously difference on normal cells and cancer cells at

411

tested concentrations. Therefore, 0.50 mg/mL was chosen to explore time-related

412

toxicity and even in-depth study.

413

After human lung adenocarcinoma cell lines were incubated using free SN-38 for

414

24 h, obvious cytotoxicity appeared only at the high concentration groups (50 and 100

415

nM). The cell viability was respectively 71.7 ± 10.9% and 66.0 ± 3.12% for A549

416

cells, 74.9 ± 3.58%, 64.5 ± 4.02% for A549/DDP cells and 75.9 ± 4.25%, 66.0 ±

13



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Page 14 of 38

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3.12% for A549/Taxol cells. Instead, when incubated with TPGS2k/PLGA/SN-38 NPs

418

the cell viability decreased to 48.1 ± 2.63% and 33.9 ± 6.06% for A549/DDP cells and

419

55.4 ± 2.77%, 33.9 ± 6.06% for A549/Taxol cells, respectively (Figure 5C). After

420

treatment for 48 h and 72 h, the cell viability treated with TPGS2k/PLGA/SN-38 NPs

421

achieved more obvious decrease compared to free SN-38 (Figure 5D). In fact,

422

TPGS2k/PLGA/SN-38 NPs achieved higher cellular mortality at each concentration

423

and incubation times than free SN-38.

424

To verify the influence of these nanoparticles on human lung adenocarcinoma cells

425

apoptosis, the Annexin V-FITC/PI apoptosis assay of A549 cells and A549/DDP cells

426

was performed to distinguish apoptosis cells from normal cells. The effects

427

of TPGS2k/PLGA NPs on apoptosis and necrosis of A549 cells and A549/DDP

428

cells were quantified by total percentage of early apoptosis (Q3) and late apoptosis

429

(Q2). As shown in Figure 6, compared to the apoptosis percentages of the blank

430

control (4.06% for A549 cells and 3.59% for A549/DDP cells), after incubation with

431

TPGS2k/PLGA NPs (0.25, 0.50 and 1.0 mg/mL) for 12 h, the induced apoptosis

432

percentages were increased to 7.47%, 18.2% (p

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