Paclitaxel-Loaded Mixed Micelles Enhance ... - ACS Publications

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Paclitaxel-loaded mixed micelles enhance ovarian cancer therapy through extracellular pH-triggered PEG detachment and endosomal escape Haijun Zhao, Qian Li, and Zehui Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00164 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1

Paclitaxel-loaded mixed micelles enhance ovarian cancer

2

therapy through extracellular pH-triggered PEG detachment

3

and endosomal escape

4

Haijun Zhao,§ Qian Li,& Zehui Hong§,#,*1

5

§

6

Southeast University, Nanjing 210009, P. R. China,

7

Physics, Tsinghua University, Beijing 100084, P. R. China, #Department of Genetics and

8

Developmental Biology, Medical School of Southeast University, The key Laboratory of

9

Developmental Genes and Human Disease in Ministry of Education, Nanjing, P. R.

10

Department of Obstetrics and Gynecology, Zhongda Hospital, School of medicine, &

Department of Engineering

China.

11 12

ABSTRACT: Although PEGylation allows a drug delivery vehicle to have prolonged

13

blood circulation time, it faces the problem of reduced cellular uptake. Removal of the

14

polyethylene glycol (PEG)-shell at the appropriate time through tumor-microenvironment

15

triggers could be a feasible solution to this problem. Here, paclitaxel (PTX)-loaded mixed

16

micelles (PTX-mM) self-assembled from stearate-modified hyaluronic acid (SHA),

17

mPEG-b-poly(β-amino ester) (mPEG-b-PAE), and ethylene acetyl-b-poly(β-amino

18

ester)(EA-b-PAE) were developed. In the preparation of PTX-mM, SHA micelles were

19

coated with EA-b-PAE followed by co-loading of PTX and mPEG-b-PAE. PTX-mM

20

were capable of extracellular pH-triggered PEG-detachment, and poly(β-amino ester)

21

(PAE)-mediated endosomal escape. When the pH was changed from pH 7.4 to pH 6.8,

22

the particle size of PTX-mM significantly decreased from 97.5 ± 4.4 nm to 71.5 ± 2.3 nm.

23

It also resulted in rapid and complete release of mPEG-b-PAE from PTX-mM as

24

monitored using quartz crystal microbalance (QCM) technology. PTX-mM capable of

* Corresponding author: Z Hong, medical school of southeast university, Nanjing, Jiangsu, China. Tel/Fax: +86-025-52612185, Email: [email protected] -1-

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

25

PEG detachment provided significant enhancement of PTX accumulation in SKOV-3cells

26

compared to PEG non-detachable PTX-mM. Interestingly, intracellular transport studies

27

using confocal laser scanning microscopy (CLSM) showed that EA-b-PAE could

28

promote the escape of micelles from endo-lysosomes. The half-maximal inhibitory

29

concentration (IC50) of PTX-mM against SKOV-3 cells was 5.7 µg/mL, and PTX-mM

30

containing 20 µg/mL of PTX induced apoptosis in 53.0% of the cell population.

31

PTX-mM exhibited a highly prolonged elimination half-life (t1/2, 2.83 ± 0.37 h) and

32

improved area under the curve (AUC, 7724.82 ± 1190.75 ng/mL/h) than the PTX-loaded

33

SHA micelles (PTX-M). Furthermore, PTX-mM showed the highest tumor inhibition rate

34

(64.9%) and the longest survival time (53 days) against the SKOV-3 ovarian cancer

35

xenograft models among all formulations. Taken together, the results suggested that

36

PTX-mM have potential as an efficient anticancer formulation in treatment of ovarian

37

cancer.

38

KEYWORDS: pH-triggered mixed micelles; PEG detachment; endosomal escape;

39

paclitaxel; ovarian cancer therapy

40 41 42

INTRODUCTION Rational design of anticancer nano-sized formulations has greatly improved the

43

biodistribution

and

pharmacokinetics

of drugs

that

44

physiochemical properties, such as insolubility and instability, and reduced the side

45

effects caused by non-targeted chemotherapy.1-3 Among the drug delivery systems,

46

polymeric micelles are one of the most up-and-coming carriers that have realized the

47

tumor-targeted therapy, enhanced cellular uptake, molecular imaging, among others.4-6

48

Tumor-targeting can be achieved by either active or passive targeting approaches. The

49

most common active tumor targeting strategy is the conjugation of micelles with small

50

molecule ligands that facilitates specific uptake by tumor cells and thereby enhances the

51

intracellular drug delivery.7-9 However, only few tumor-targeted ligand-modified micelles -2-


suffered

from

undesired

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


52

have been successfully used for tumor targeting until now, because it has to overcome a

53

serious problem of the exogenous interference to tumor-specific recognition.10 On the

54

other hand, PEGylated nanomedicines (passive targeting) improve drug accumulation at

55

tumor sites due to prolonged circulation, and enhanced permeability and retention (EPR)

56

effect.11,12 However, PEGylation may impede cellular uptake because of the steric

57

hindrance of PEG segments, which reduces the internalization by tumor cells.13 Therefore,

58

it is necessary to address this PEG dilemma by seeking an optimal balance between

59

prolonged circulation time and promotion on cellular uptake.

60

In recent years, some delicate designs have been reported that guarantee the

61

detachment of PEG shell at the appropriate time, thus overcoming the PEG mediated

62

steric hindrance. For example, McNeeley et al. fabricated a smart nano-sized drug

63

delivery system capable of maintaining PEG-coating until the particles reached the tumor

64

tissues, where PEG-detachment occurred in a reduction sensitive manner that exposed the

65

masked targeting ligands to promote internalization.14 Furthermore, various other PEG

66

detachment

67

reduction-triggered,17,18

68

esterase-catalyzed20 approaches to remove the PEG layer on reaching the targeted sites.

69

Generally, incorporation of multiple functional moieties into a single drug delivery

70

system to obtain tumor microenvironment-sensitive characteristic is difficult. For

71

example, conjugation of hydrophobic moieties with hyaluronic acid offers a simple

72

approach to facilitate and enhance its practical applications in drug delivery.21,22 Due to

73

the finite sites for chemical modification, however, it is technically difficult to

74

incorporate different functional groups into one drug delivery system. Mixed micelles

75

constituted by two or more types of polymers are an alternative option to integrate

76

respective advantages into one system, potentially addressing the aforementioned

77

technical conundrum.23-26 For example, Pluronic block co-polymers, the most widely

78

investigated materials used to prepare amphiphilic micellar drug delivery systems, have

strategies

have matrix

been

reported

metalloproteinase

that

use

(MMP)

-3-


pH

sensitive,15,16

sensitive,19

and


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 34

79

shown benefits in the mixed micelle systems.27-29 Therefore, it is desirable to seek a

80

copolymer to fabricate a pH-responsive mixed micelle system that shows pH sensitivity

81

in tumor extracellular acidity (pHe, 6.8-7.2), avoids immunocapture by RES system, and

82

could be effectively internalized.

83

Among the promising pH-sensitive Pluronic polymers, methyl ether poly(ethylene

84

glycol)-b-poly(beta-amino ester) (mPEG-b-PAE) has received increasing attention due to

85

the

86

poly(beta-amino ester) (PAE) segments containing substantial tertiary-amine groups have

87

shown potential for endosomal escape through proton-sponge effect.32

capability

of

pHe-triggered

micellization-demicellization.30,31

Moreover,

88

Herein, we developed a paclitaxel (PTX)-loaded mixed micelles (PTX-mM) constituted

89

by PTX, stearate-modified hyaluronic acid (SHA), mPEG-b-poly(β-amino ester)

90

(mPEG-b-PAE) and ethylene acetyl-b-poly(β-amino ester) (EA-b-PAE) (Figure 1A),

91

which was capable of PEG detachment triggered by extracellular pH, and endosomal

92

escape offered by proton sponge effect of EA-b-PAE. It was hypothesized that this

93

self-assembling mixed micelle system developed from neutral SHA micelles

94

(EA-b-PAE-coated SHA micelles) and mPEG-b-PAE that shows pH-triggered PEG

95

detachment will not only reduce RES mediated clearance from blood circulation, but will

96

also enhance the intracellular uptake (Figure 1B). It can be suggested that PEG segments

97

detach from micelles through inherent protonation of mPEG-b-PAE in response to pHe.

98

The pH-sensitive PEG detachment was characterized using QCM with Dissipation

99

(QCM-D) and fluorescent probe techniques. The advantages of this design were

100

investigated through evaluation of cellular transport, bioavailability, and in vivo

101

therapeutic efficacy.

102

-4-


Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


103 104

Figure 1. (A) The illustration of preparation and potential detachment of PEG-detachable PTX-loaded

105

mixed micelles (PTX-mM). For preparation of PTX-mM, SHA micelles were coated with EA-b-PAE

106

followed by co-loading of PTX and mPEG-b-PAE. In this process, the most important step is

107

EA-b-PAE coating on the SHA micelles, which is vital to realize the further mPEG-b-PAE detachment.

108

EA-b-PAE coating is like an isolation layer to avoid potential charge attraction between highly

109

positive mPEG-b-PAE and highly negative SHA micelles in mildly acidity. (B) Schematic illustration

110

of potential mPEG-b-PAE detachment and endosomal escape at the cellular level. Due to the

111

hydrophilicity of mPEG-b-PAE segments triggered by mildly acidity, PEG segments could separate

112

when PTX-mM reached the vicinity of tumor cells. The remaining part (EA-b-PAE-coated PTX-M) -5-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 34

113

with mildly positive charge enhanced the delivery of PTX into the cells. Likewise, EA-b-PAE could

114

help micelles escape from endosomes through proton sponge effect by virtue of substantial amino

115

groups, and consequent release more PTX in the cytoplasma.

116 117

MATERIALS AND METHODS

118

Materials and Animals. SHA (15 kDa) was prepared by our laboratory, the

119

substitution degree of stearate groups was 12% (w%). PTX was bought from Zelang Co.,

120

Ltd. (Nanjing, China). Methoxy poly (ethylene glycol) (mPEG, 5kDa) and ethyl acrylate

121

(EA) were provided by Fluka Chemical Co. (USA). 4,4’-Trimethylene dipiperidine

122

(TDP),

123

3-(4,5-dimethylthiazol-2-yl)-2,5-iphenyltetrazolium bromide (MTT), ethyl acrylate,

124

coumarin 6 (C6) and rhodamine B isothiocyanate (RhB) were obtained from Aladdin

125

Chemical Co., Ltd. (Shanghai, China). RPMI-1640 incomplete medium and fetal bovine

126

serum (FBS) were offered by Thermo Fisher Scientific Inc. (Beijing, China). The water

127

was produced by using Millipore Elix® Essential 5 system (USA).

1,6-hexanediol

diacrylate

(HDD),

ethyl

acrylate,

acryloyl

chloride,

128

Female Sprague-Dawley (SD) rats (weight, 200 ± 20 g) and nude mice (weight, 23 ±2

129

g) purchased from Silaike Company (Shanghai, China) were used for the study. The

130

animals were determined to be free of disease prior to experimentation and had ad libitum

131

access to food and water. Animal experiments were conducted in accordance with the

132

Guidelines for Animal Experimentation of Southeast University (Nanjing, China).

133

Synthesis

134

(mPEG-b-PAE),

135

rhodamine B-labelled ethyl acrylate-b-poly(β-amino ester) (EA-b-PAE-RhB).

136

and

characterization

ethyl

of

mPEG-b-poly(β-amino

acrylate-b-poly(β-amino

ester)

(EA-b-PAE)

ester) and

The mPEG-b-PAE was synthesized exerting the method described in previous

137

papers.30-32 First, mPEG (1000 mg, 0.2 mmol) and triethylamine (TEA, 58 µL, 0.42 mmol)

138

were dissolved in 5 mL of anhydrous dicholoromethane (DCM) by using CaCl2 to protect -6-


Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


139

from moisture in an ice environment. Next, acryloyl chloride (29 mg, 0.32 mmol) was

140

slowly added in a dropwise manner. The mixture was stirred for further 2 h and then

141

allowed to return to room temperature with vigorous magnetic stirring for 24 h.

142

Monoacrylated mPEG was obtained through extraction with diluted HCl and

143

precipitation

144

monoacrylated mPEG (900 mg, 0.178 mmol) as a monoacrylate, HDD (402 mg, 1.78

145

mmol) as a diacrylate ester, and TDP (411 mg, 1.96 mmol) as a diamine. The chloroform

146

solution of HDD and TDP was mixed with monoacrylated mPEG and allowed to react for

147

48 h at 50 °C. Finally, a white power (1.53 g) with a yield of 89.3% was gained through

148

precipitation in the environment of diethyl ether.

in

hexane,

successively.

mPEG-b-PAE

was

synthesized

using

149

EA-b-PAE was synthesized by ethyl acrylate (17.8 mg, 0.178 mmol), HDD (402 mg,

150

1.78 mmol) and TDP (411 mg, 1.96 mmol) according to the similar method described

151

above. EA-b-PAE-RhB was synthesized as the following method: 50 mg of EA-b-PAE

152

and 5 µL of TEA were mixed in 5 mL of dichloromethane (DCM), and then 5 mg of RhB

153

was added into the mixture with magnetic stirring vigorously at 40 °C over night. At the

154

end of the time, the crude product was obtained after removing solvent under reduced

155

pressure and purified by precipitation in diethyl ether to gain EA-b-PAE-RhB.

156

The chemical structures of various polymers were characterized by 1H NMR using a

157

Bruker AVANCE-300 spectrometer. The molecular weight (Mw) and Mw distribution of

158

mPEG-b-PAE and EA-b-PAE was also determined by gel permeation chromatography

159

(GPC).

160

Preparation of PTX-M and PTX-mM. PTX-M preparation10,33: To prepare PTX-M,

161

a solution containing 20 mg of PTX in 400 µL of ethanol was dropped into 20 mL of

162

SHA solution (5 mg/mL) with vigorous magnetic (or mechanical) stirring at a

163

temperature of 25 °C. Once stirring was complete, the mixture was dialyzed against

164

deionized water for 12 h by using a dialysis membrane (10,000 molecular weight cut-off

165

range, 10 kDa cut-off) to remove the non-entrapped PTX. -7-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

166

EA-b-PAE-coated SHA micells: 10 mg of EA-b-PAE in 45 µL of ethanol was first

167

added in the SHA aqueous solution (100 mg, 5 mg/mL) at pH 7.4, and then successively

168

dialyzed against PBS with pH 6.8 and 7.4 to obtain EA-b-PAE-coated SHA micelles.

169

Likewise, EA-b-PAE-coated PTX-M was prepared using the similar method but adding

170

20 mg of PTX with EA-b-PAE.

171

PTX-mM preparation: a solution containing 20 mg PTX and 100 mg mPEG-b-PAE in

172

400 µL of ethanol was dropped into EA-b-PAE-coated SHA micells solution with

173

vigorous stirring. Likewise, the non-entrapped PTX was removed using the

174

above-mentioned method.

175

Non-detachable PTX-mM (control): 20 mg of PTX and 100 mg of mPEG-b-PAE in

176

400 µL of ethanol were dropped into 20 mL of SHA solution (5 mg/mL) with intense

177

stirring at 25 °C. And then, the non-entrapped PTX was removed through the

178

above-mentioned dialysis method.

179

Characterization of PTX-M and PTX-mM. Drug entrapment efficiency (DEE) of

180

each micellar solution was measured as follows. 4 mL of freshly prepared micelles were

181

firstly filtered using microfiltration membrane, and then adjusted the volume to 10 mL.

182

After 50-fold dilution with methanol, the content of PTX in micelles was finally tested

183

through HPLC. Likewise, the samples were freeze-dried to obtain drug loading efficiency

184

(DLE). The equations listed below were used for calculating DEE and DLE:

Ca × Va × 100 , Wa Cb × Vb DEE (%) = × 100 Wb

DLE (%)= 185

,

186

where Ca and Cb represent the concentration of PTX in freeze-dried micelles solution and

187

freshly prepared micellar solution, respectively; Va and Vb represent the volume of

188

freeze-dried micelles solution and freshly prepared micellar solution, respectively; Wa

189

and Wb represent the weight of freeze-dried power and the fed drug, respectively.

190

The average size and surface charge were tested by a zeta potential and particle size -8-


Page 8 of 34

Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


191

analyzer (Zeta Plus, Brookhaven, USA). Different pH environments were adjusted by

192

dilution with PB of various pH values. The morphology of PTX-mM was analyzed using

193

a transmission electron microscope (TEM, JEM-200CX, Japan). A total of 200 µL of

194

PTX-mM solution was dropped on the surface of the copper grid, and then stained with

195

50 µL of phosphotungstic acid (1%, w/v). The treated micelles were immediately

196

observed using the TEM after drying under the infrared lamp.

197

In vitro release. In this experiment, the release profile of PTX-M and PTX-mM was

198

studied using a modified dialysis method. For each of the two micelles, 1 mL of the

199

micelle with the same amount of PTX (1 mg) was immersed in 100 mL of PB (pH 7.4,

200

6.8) using a dialysis bag (10 kDa cut-off). In order to in accordance with the sink

201

condition, each of PB contained 0.1% Tween 80. At the observed time points (from 0.5 to

202

48 h), a volume of 1 mL of each sample was withdrawn, followed by filtration using a

203

polycarbonate membrane and replacement with an equivalent volume of the

204

corresponding blank buffer solution, successively. The concentration of PTX released

205

from each sample was quantified using HPLC.

206

Mechanism of reaction between PAE-related polymers and SHA micelles.

207

Characterization of mPEG-b-PAE detachment studied by QCM, and the pretreatment for

208

gold coated was performed in accordance with the previous papers.34,35 In this experiment,

209

the QCM (E1) instrument from Q-Sense was conducted at 24 °C and the third overtone

210

was used to record frequency change (∆F). PTX-M, EA-b-PAE-coated PTX-M and PEG

211

non-detachable PTX-mM were also employed as control groups for investigation of

212

mPEG-b-PAE detachment in response to weak acidity. Firstly, various micelles dissolved

213

in phosphate buffer (PB) of pH 8.0 were injected into QCM-D cells for adsorption to the

214

films until no further change of frequency and dissipation. Thereafter, PB with pH 7.4

215

and pH 6.8 were injected successively for 30 min to observe the changes in frequency.

216

Characterization of the mechanism of EA-b-PAE coating: In this study,

217

EA-b-PAE-RhB was used as a fluorescence probe to explore the mechanism of reaction -9-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

218

between EA-b-PAE and SHA micelles. EA-b-PAE-coated SHA micelles with

219

fluorescence were synthesized according to the similar process of EA-b-PAE-coated

220

PTX-M but with some modifications. Briefly, an ethanol solution containing 5 mg of

221

EA-b-PAE and equivalent EA-b-PAE-RhB was mixed with 20 mL of SHA solution (5

222

mg/mL) under vigorous stirring for 10 min. Next, ethanol was removed after 6 h-dialysis

223

against PBS with various pH values. The fluorescence intensity of various RhB-labeled

224

micellar solutions was detected using a fluorospectrophotometer (RF-5301PC,

225

SHIMADZU, Japan).

226

Cellular uptake studies. Cell culture: SKOV-3 cells (a type of human ovarian tumor

227

cells line) and L-02 cells (a type of normal human liver cell line) were obtained from the

228

cell bank of Chinese Academy of Sciences and used for culture. Both cell types were

229

cultured in RPMI-1640 complete medium. The cells were then sub-cultivated after

230

reaching 80% confluence.

231

Cellular uptake: A total of 1×105 SKOV-3 cells were seeded into each well of 24-well

232

plates. After reaching 80% confluence, the cells were rinsed thrice with 500 µL of PBS. A

233

total of 400 µL of various micelles containing 100 µg/mL of PTX were co-incubated with

234

cells at 37 °C for 2 h. Once the incubation was complete, the cells were washed with 4 °C

235

PBS thrice after removing test solutions, and co-incubated with 200 µL of SDS cell lysis

236

buffer (0.1%, w/v, KeyGen BioTECH) at 37 °C. The absorbed PTX was measured using

237

HPLC and the amount of cells was quantified using a BCA protein assay kit (KeyGen

238

BioTECH). The cellular uptake was calculated by the equation listed below: 5

239

Uptake ( µ g / mg ) =

240

where QPTX represents the PTX concentration in SKOV-3 cells, and Q protein represents the

241

amount of cells protein.

Q PTX , Q protein

242

Endocytosis pathways of various micelles: SKOV-3 cells were pre-treated with various

243

specific internalization inhibitors for 30 min as follows: (1) 0.45 µM sucrose (100 µL, - 10 -


Page 10 of 34

Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


244

clathrin-mediated endocytosis inhibitor; (2) 0.2 nM genistein (100 µL, caveolae-mediated

245

endocytosis inhibitor); (3) 0.43 nM amiloride (100 µL, macropinocytosis inhibitor) and

246

(4) 10 nM ammonium chloride (100 µL, endo-lysosome formation inhibitor).10 Next,

247

the cells were treated with freshly prepared micelles for an additional 2 h. The test

248

solutions were then removed and the cells in each well were rinsed with PBS (0.5 mL ×

249

3). The internalized PTX and cellular protein were quantified according to the process of

250

cellular uptake as mentioned above.

251

Intracellular distribution of PTX micelles: in order to validate the effect of PTX-mM

252

on endo-lysosomal escape, the organelle selective dye was carried out for observing

253

micelles distributed in cytoplasma using confocal laser scanning microscopy (CLSM).

254

The localization of micelles was visualized by C6 and the acidic endosome was labeled

255

with LysoTracker (Red, KeyGen BioTECH). 1×105 SKOV-3 cells were seeded in

256

confocal microscopy dish for 48 h. When cells reached 50% confluence, the culture fluid

257

was removed, followed by addition of C6/PTX-coloaded SHA micelles (C6/PTX-M),

258

C6/PTX-coloaded mixed micelles (C6/PTX-mM) and C6/PTX-coloaded non-detachable

259

mixed micelles (non-detachable C6/PTX-mM) respectively. The concentrations of all

260

C6-loaded micelles were adjusted to 150 ng/mL in this experiment. The culture medium

261

was removed at 4 h post-incubation, and then the cells were rinsed through ice-cold PBS

262

(0.5 mL × 3), followed by staining with 100 nM LysoTracker Red (KeyGen, China) for

263

0.5 h. After further washing thrice using PBS, the cells were observed by CLSM

264

(Olympus, Japan).

265

Cytotoxicity studies. A total of 5×103 SKOV-3cells were seeded in each well of a

266

96-well plate. When the cells grew up to 60% confluence, the culture medium was

267

replaced with different formulations or blank carriers. At 48 h after incubation, 20 µL of

268

PBS containing 5 mg/mL MTT was added into each well and co-incubated with the cells

269

for 4h. The mixture was then removed, followed by dissolution of the resulting formazan

270

crystal using DMSO. The absorbance was tested at 570 nm by anenzyme-linked - 11 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

271

immunosorbent assay (ELISA). The viability ratio was obtained as the following

272

equation:

273

Viability ratio (%) =

274 275

Absorbance test . Absorbance control

Likewise, the cytotoxicity of various blank carriers toward L-02 cells was evaluated using the above-mentioned method.

276

Cell apoptosis. Apoptosis of SKOV-3 cells was evaluated using an Annexin

277

V-FITC/PI apoptosis detection kit (Sigma-Aldrich, USA). First, 1×105 cells were briefly

278

seeded in each well of a 24-well plate and treated with various formulations (50 µg/mL)

279

for 5 h after reaching 80% confluence. Next, the cells in each well were collected through

280

trypsinization, rinsed using PBS thrice, and re-suspended in PBS, followed by mixing

281

with 5 µL of Annexin V-FITC and equivalent propidium iodide (PI). Finally, the cells

282

were analyzed using flow cytometry (Guava 6HT, Merck-Millipore) after staining in the

283

dark for 15 min.

284

Pharmacokinetic studies. Eighteen female SD rats (weight, 200 ± 20 g) were

285

randomly divided into four groups, with each group containing 6 rats, as follows: (1)

286

PTX commercial formulation; (2) PTX-M; (3) PTX-mM, and (4) non-detachable

287

PTX-mM. After intravenous administration of the test formulation (10 mg/kg), a 500 µL

288

blood sample was collected from the plexus venous in the eye ground from each rat at

289

prearranged time intervals. Plasma was prepared through centrifugation (8000 g × 10

290

min). The representative pharmacokinetic parameters were calculated by Kinetica 4.4

291

software (Thermo, USA), including area under the plasma concentration-time curve

292

(AUC0-∞), elimination half-life (t1/2), and mean residence time (MRT).

293

Antitumor efficacy in vivo. Nude mice bearing SKOV-3 xenograft was generated

294

by subcutaneous injection of SKOV-3 cells (1 × 107/mouse) in the armpit of left anterior

295

limb. When the tumor volume was around 60 mm3 on the 6th day after implantation, the

296

mice were randomly divided into six groups, with each group containing 8 nude mice, as - 12 -


Page 12 of 34

Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


297

follows: (1) saline, (2) Taxol®, (3) PTX-M, (4) PTX-mM, (5) EA-b-PAE-coated PTX-M

298

and (6) non-detachable PTX-mM. The administration route and dose were intravenous

299

injection and 10 mg PTX/kg, respectively. During the treatment, the volume of tumor was

300

measured by calipers, which was calculated by the formula:

301

Volume (cm 3 ) = 0.5 × length × w id th 2 .

302

The formula of tumor inhibition was listed below:

303

TW s TW t − B W s BWt Inhibition rate (%) = × 100%. TW s BW s

304

Where TWs and BWs are the tumoral weight and body weight in saline-treated group; 305

TWt and BWt represent the tumoral weight and body weight in the test groups. 306

Safety evaluation.

307

Mice were randomly selected from each group and euthanized 72 h after the last

308

treatment. The main normal organs were immediately collected and then rinsed thrice

309

with saline. The formulas listed below were used for calculating the liver and spleen

310

index: W liver × 100%; W body W spleen Spleen index = × 100%, W body Liver index =

311

312

where Wliver, Wspleen and Wbody represent the weight of liver, the weight of spleen and the

313

weight of body, respectively. The serum concentrations of IL-6 and TNF-α, as well as

314

aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea

315

nitrogen (BUN) were analyzed by using exclusive ELISA kits (KeyGen, China) and a

316

blood biochemical analyzer, respectively, at predetermined time points. At 24 h after

317

treatment, whole blood samples were collected to measure the hematologic parameters by

318

using a blood biochemical analyzer. In addition, the H&E staining of organs sections - 13 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

319

Page 14 of 34

were also observed using the method as described previously.36

320

Statistical analysis. All data in this study are shown as average value ± standard

321

variance. *P< 0.05 and **P< 0.01 represent statistical significance and extreme statistical

322

significance, respectively. A two-tailed Student’s t-test was used to determine

323

significance.

324 325

RESULTS AND DISCUSSION

326

Preparation

327

Amphiphilic mPEG-PAE block copolymer (mPEG-b-PAE) was polymerized through

328

classic Michael-type reaction of mPEG and PAE segment (Figure S1A). As shown in the

329

1

330

3.6 and 3.3), (δ (ppm) 1.23-2.01), and (δ (ppm) 2.70, 2.84-2.93 and 4.15), which probably

331

represented the segments of mPEG, HDD and TDP, respectively (see Figure S2A).30,31

332

EA-b-PAE, which could be considered as a hydrophobic segment of mPEG-b-PAE from

333

the view of chemical structure, was also synthesized using a similar method (Figure S1B).

334

The disappearance of mPEG signal and appearance of a new peak at δ (ppm) 4.01 in the

335

1

336

S2B). EA-b-PAE-RhB was synthesized by covalent linkage of isothiocyanate group of

337

RhB and secondary amine group of EA-b-PAE (Figure S1C). The 1H NMR spectrum

338

showed characteristic signal of aromatic ring attributed to rhodamine B (as shown in

339

Figure S2C). The average molecular weight of two PAE-containing derivatives

340

determined by GPC were 10 kDa and 5kDa, respectively (Figure S3).

and

characterization

of

mPEG-b-PAE

and

derivatives.

H NMR spectrum of mPEG-b-PAE, we observed the characteristic signals at (δ (ppm)

H NMR spectrum of EA-b-PAE confirmed the conjugation of EA with PAE (Figure

341

Characterization of PTX-loaded micelles. PTX-mM were prepared by

342

co-loading PTX and mPEG-b-PAE into EA-b-PAE-coated SHA micelles using the

343

dialysis method. The purpose of EA-b-PAE coating was to shield the highly negative

344

charge of SHA micelles, which could otherwise impede the detachment of mPEG-b-PAE

345

from mixed micelles. The results of physicochemical characterization of PTX micelle - 14 -


Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


346

formulations are presented in Table 1. PTX-mM had a size of 97.5 ± 4.4 nm, which was

347

clearly larger than PTX-M, and a surface charge of 4.2 ± 0.4 mV, which was also

348

significantly higher than PTX-M but comparable to EA-b-PAE-coated PTX-M. The DEE

349

of PTX-mM was 81.9 ± 2.8%, which was similar to that of non-detachable PTX-mM and

350

slightly higher than that of PTX-M and EA-b-PAE-coated PTX-M. According to the

351

previous papers, the optimal loading efficiency of PTX in single micelles, such as SHA

352

micelles and mPEG-b-PAE micelles, were only around 70% ~75%.21,30,31 In this

353

perspective, the mixed micelles we designed had an acceptable performance in the study

354

of loading efficiency. As presented in Figure 2A, the average size of PTX-mM sharply

355

decreased to approximately 65 nm (comparable to EA-b-PAE-coated PTX-M) when pH

356

was reduced to 6.8, suggesting that mPEG-b-PAE has separated from the micelles due to

357

pH-triggered amphiphilic-hydrophilic conversion. As described earlier, the preparation of

358

PTX-mM had two steps: EA-b-PAE coating on the surface of SHA micelles and

359

mPEG-b-PAE modification to the delivery system (see Figure 1A). The aim of the

360

EA-b-PAE coating was like an isolation layer to avoid potential charge attraction

361

between protonated mPEG-b-PAE (highly positive) and SHA micelles (highly negative)

362

in mildly acidity. In other words, only the neutralization of SHA micelles in advance by

363

protonated EA-b-PAE could mPEG-b-PAE detach from the system in the further pH

364

stimulation. Once the coating process was done, EA-b-PAE could be attracted on the

365

surface of the SHA micelle stably, resulting in that the zeta potential was around nearly

366

neutral and would not change even the pH turned back to 7.4. Therefore, the surface

367

charge of PTX-mM did not change regardless of the detachment of mPEG-b-PAE (Figure

368

2B), because the zeta potentials of PTX-mM and EA-b-PAE-coated PTX-M were almost

369

similar. However, non-detachable PTX-mM with highly negative charge (self-assembled

370

from mPEG-b-PAE and PTX-M simply) changed to mildly positive charge when exposed

371

to pH 6.8. This was perhaps due to lack of a shield against the highly negative charge of

372

SHA micelles in advance, and consequent adsorption of the protonated mPEG-b-PAE on - 15 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 34

373

the surface of PTX-M (failure to mPEG-b-PAE detachment). It suggested that the

374

EA-b-PAE coating was vital to realize mPEG-b-PAE detachment. Furthermore, the

375

morphology of PTX-mM visualized by transmission electron microscopy (TEM) is

376

shown in Figure 2C. PTX-mM displayed an approximately spherical shape and uniformly

377

dispersed size (approximately 90 nm), which was similar to the results obtained from

378

dynamic light scattering. The 48-hour cumulative PTX release from PTX-mM was 27.8 ±

379

1.2% and 46.9 ± 4.0% at pH 7.4 and pH 6.8, respectively. However, such pH-responsive

380

release was not observed in non-detachable PTX-mM (Figure 2D). It suggested that the

381

detachment of mPEG-b-PAE from mixed micelles could have helped to increase the drug

382

release rate. The stability of various formulations was also evaluated through incubation

383

with serum and PB of pH 7.4 for different time intervals (Figure S4). The average particle

384

size of all formulations had no obvious change during the observation time, expect for

385

EA-b-PAE-coated PTX-M, probably because of lack of PEG shell protection.

Table 1 Characteristics of various PTX-loaded micelles (n = 4)

Formulation

Size (nm)

PTX-M

60.3 ± 2.8

PTX-mM

PI

Zeta (mV)

DEE (%)

DLE (%)

0.181 ± 0.002

-46.5 ± 3.8

73.5 ± 3.2

13.6 ± 1.7

97.5 ± 4.4

0.173 ± 0.006

4.2 ± 0.4

81.9 ± 2.8

7.9 ± 0.5

64.5 ± 2.4

0.156 ± 0.002

3.5 ± 0.7

74.8 ± 1.8

11.4 ± 2.1

94.8 ± 3.2

0.131 ± 0.002

-28.9 ± 2.3

83.2 ± 4.2

7.6 ± 0.2

EA-b-PAE-coated PTX-M Non-detachable PTX-mM 386

- 16 -


Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


387 388

Figure 2. Characterization of various micelles. A) particle size and B) zeta potential of various

389

PTX-loaded micelles under different pH environments (n = 4). C) Hydrodynamic size of PTX-mM

390

measured by DLS, inset: TEM image of PTX-mM. D) Accumulative release profile of PTX from

391

PTX-mM and non-detachable PTX-mM under pH 7.4 and 6.8 at different time intervals (n = 4, **P

Paclitaxel-Loaded Mixed Micelles Enhance ... - ACS Publications

Recommend Documents