Theranostic, pH-Responsive, Doxorubicin-Loaded Nanoparticles

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Theranostic pH responsive doxorubicin loaded nanoparticles inducing active targeting and apoptosis for advanced gastric cancer Malcolm M.Q. Xing, Huanrong Ma, yuqing Liu, Min Shi, Xuebing shao, Wen Zhong, and Wangjun Liao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01039 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015

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.

Biomacromolecules 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 36

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

Biomacromolecules

Theranostic pH responsive doxorubicin loaded nanoparticles inducing active

1

Title:

2

targeting and apoptosis for advanced gastric cancer

3

Authors: Huanrong Maa,b,1, Yuqing Liub,d, 1, Min Shia, Xuebing Shao b, Wen Zhonge, Wangjun

4

Liao , Malcolm M.Q. Xing

5

Affiliations:

6

Guangzhou 510515, China

7

b

8

Canada

9

c

a,*

a

b, c, d,*

Department of Oncology, Nanfang Hospital, Southern Medical University,

Department of Mechanical Engineering, University of Manitoba, Winnipeg MB R3T 2N2,

Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg MB

10

R3T 2N2, Canada

11

d

Children’s Hospital Research Institute of Manitoba, Canada

12

e

Department of Biosystem Engineering, University of Manitoba, Canada

13

1

14

*: corresponding authors

15

Corresponding author: 1. Malcolm M.Q. Xing, a. Department of Mechanical Engineering,

16

University of Manitoba, Winnipeg MB R3T 2N2, Canada; b. Department of Biochemistry and

17

Medical Genetics, University of Manitoba, Winnipeg MB R3T 2N2, Canada; c. Children’s

18

Hospital Research Institute of Manitoba, Canada. Email: [email protected]

19

2. Wangjun Liao, Department of Oncology, Nanfang Hospital, Southern Medical University,

20

Guangzhou 510515, China. Email: [email protected].

: same contribution

21 22

1

ACS Paragon Plus Environment

Biomacromolecules

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

23

Abstract

24

This study developed a kind of magnetic-polymer nanocarriers with folate receptor-targeting

25

and pH-sensitive multi-functionalities to carry doxorubicin (DOX) for treatment of advanced

26

gastric cancer (AGC). Folate conjugated, pH-sensitive amphiphilic poly (β-aminoester)

27

self-assembled with hydrophobic oleic acid modified Iron oxide nanoparticles and the resulted

28

hydrophobic interaction area is a reservoir for lipophilic DOX (F-P-DOX). Confocal microscopy

29

illustrated that F-P-DOX treatment could keep higher DOX accumulation in cells than P-DOX

30

(without folate conjugated), and therefore get a higher efficiency of DOX internalization of at

31

pH 6.5 than at pH 7.4. Electron microscope characterization and real-time polymerase chain

32

reaction revealed cell apoptosis promoted by F-P-DOX. The better efficacy of F-P-DOX on GC

33

than free DOX and P-DOX was determined by MTT assay and xenograft model. Moreover the

34

accumulation of F-P-DOX in the tumor site was detected by MRI. All those observations

35

suggest F-P-DOX could be a promising theranostic candidate for AGC treatment.

36

Keywords:

37

Advanced gastric cancer, doxorubicin, theranostic nanoparticles, folate receptor–targeting, pH

38

sensitive polymer, apoptosis

39

2


Page 2 of 36

Page 3 of 36

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

Biomacromolecules

40

1. Introduction

41

Gastric cancer (GC) accounts for second cause of cancer death worldwide . More severely, in

42

developing countries, over 70% of GC patients are diagnosed with advanced gastric cancer

43

(AGC) and 40% of them occur in China

44

chemotherapy brings limited benefits for AGC patients, with an overall survival (OS) of 7~9

45

months 2. Among the classically active drugs used as chemotherapy agents for AGC treatment,

46

doxorubicin (DOX) is relatively effective but low in price. Nevertheless, the inherent

47

characteristics of poor water solubility, weak tissue penetration and adverse effect like

48

cardiotoxicity reduce its therapeutic efficacy and limit its systematic administration

49

Therefore, an efficient system for DOX delivery is desired to overcome its drawbacks and

50

improve its clinical therapy efficacy on GC.

51

Iron oxide nanoparticles (IONPs), as a primary kind of IONPs, have been rapidly developed as

52

promising candidates for cancer theranostic study 6. IONPs can be employed as drug delivery

53

carriers and simultaneously as magnetic resonance imaging (MRI) contrast agents

54

studies using IONPs to carry DOX showed positive anti-tumor effect and expected MRI quality

55

10,11

56

value

57

Usual methodologies include IONPs in the cores which are then coated with polymers such as

58

dextran, chitosan, and polyethylene glycol (PEG) and sometime with inorganic silica and gold

59

nanoparticles

60

phenotypes of tumor, folate receptor-ɑ (FR), a membrane-bound protein overexpressed in

61

various malignant tumors, has an exceedingly high affinity for folate, making FR a promising

1

2, 3

. As a primary means for treating AGC,

7

4, 5

.

8, 9

. Some

. Moreover, modified polymeric IONPs according to tumor characteristics such as low pH 12-14

or specific phenotype

15-18

further exhibited favorable efficacy in cancer therapy.

19, 20

. The drugs are embedded or conjugated with them. Among the specific

3


Biomacromolecules

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 36

21, 22

62

therapeutic target for cancers

63

nanoparticles were capable of improving chemotherapeutic efficacy and avoiding side effects

64

on normal tissues due to their specific attacks to FR-positive tumors and efficient cellular

65

internalization through FR-mediated endocytosis

66

prepared as both MRI contrast agent and FR-targeted stimuli responsive drug delivery system

67

in vitro and in vivo, and fewer as a strategy in GC treatment.

68

To this end, we synthesized multifunctional theranostic nanocarriers with magnetic imaging

69

function, pH-sensitivity and FR-targeting to deliver DOX (Schematic illustration). A novel

70

amphiphilic poly (β-aminoester) was synthesized via Michael addition from pentaerythritol

71

diacrylate monostearate, 4, 4'-trimethylenedipiperidine, poly (ethylene glycol) methyl ether

72

acrylate and folate (scheme 1 and 2). Poly (β-aminoester)s have been reported to present

73

weakly basic character due to their tertiary amines with a pKb value of about 6.5 , which leads

74

to water-soluble below pH 6.5 and non-soluble in water at a neutral pH

75

knowledge, it may be first time to use theranostic magnetic-polymer system to treat AGC. This

76

synthesized polymer hydrophobic side alkyl chain could self-assemble with modified IONPs for

77

DOX loading. After detecting the characteristics of the synthesized polymeric IONPs,

78

DOX-loaded polymeric IONPs were further synthesized for both cellular uptake, cytotoxicity

79

study in vitro and tumor growth suppression, tumor accumulation investigation in vivo, which

80

validated that F-P-DOX were effective on GC treatment, without significant side effects.

81

2. Materials and methods

82

2.1. Materials

83

All chemicals were bought from Sigma Aldrich. (St. Louis, MO, US) and used directly unless

. Previous studies demonstrated that folate-conjugated

23-26

. However, so far, only a few IONPs are

4


27

.To best of our

Page 5 of 36

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

Biomacromolecules

84

further purification was noted. RPMI-1640 media, fetal bovine serum (FBS), Trypsin/EDTA,

85

and phosphate buffered saline (PBS) were purchased from Hyclone (Logan, Utah, US).

86

Folate-free RPMI-1640 media was purchased from Giboco (California, US). The antibody of

87

FR and ɑ-Actin were purchased from Abcam (Cambridge Science Park, UK). Protein

88

Extraction Kit was purchased from KeyGEN BioTECH (Nanjing, China). All reagents for

89

western blotting were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

90

RNAiso and Reverse Transcription Kit were purchased from Takara Biotechnology (Dalian,

91

China). Real time polymerase chain reaction (rt-PCR) was performed using LightCycler480

92

SYBR Green I Master (Roche, Germany).

93

2.2. Preparation of polymers

94

2.2.1. Synthesis of poly (ethylene glycol) methyl ether acrylate (Mw: 5000 Da)

95

Acrylate mono-functionalized poly (ethylene glycol) (PEG-acrylate, Mw: 5000 Da) was

96

synthesized by acrylation of poly (ethylene glycol) methyl ether (PEG-OH, Mw: 5000 Da) with

97

acryloyl chloride, according to the reported procedure. In a typical reaction, 2.5 g of PEG-OH

98

(0.5 mmol) was heated in vacuum oven at 65 °C overnight, and then dissolved in 25 mL

99

anhydrous DCM in a single neck round flask capped with Suba Seal rubber septa. 0.28 mL

100

trimethylamine (TEA, 2 mmol, 0.203 g) was injected into the flask, and the solution was

101

degassed by nitrogen sparging for 20 min in ice bath. After that, 0.16 mL acryloyl chloride (2

102

mmol, 0.181 g) was slowly injected into the flask during stirring in the ice bath. The reaction

103

was shielded from light, and the temperature was naturally increased to room temperature.

104

After 24 h the reaction was terminated, and solvent was removed by rotational evaporation.

105

The product was dialyzed against DD water with a dialysis tube of 1000 Da molecular weight 5


Biomacromolecules

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 36

106

cut off (MWCO) to remove impurities. Water was removed at reduced pressure and the

107

product was recovered in vacuum oven to get white powder. Yield: 2.0 g (80%). H NMR (ppm)

108

in

109

-COO-CH2-CH2-O-), 5.75-6.50 (m, CH2=CH-COO).

110

2.2.2. Synthesis of PADM-TMDP copolymer

111

0.5107 g of pentaerythritol diacrylate monostearate (PADM, 1 mmol) was dissolved in 5 mL

112

DCM, and 0.2524 g of 4,4’-trimethylene dipiperidine (TMDP, 1.2 mmol) was dissolved in MeOH

113

respectively. Two solutions were mixed together in a 25 mL round bottom flask capped with

114

rubber septa, and the mixture was stirred at ambient temperature for 24 h. After reaction, the

115

solvent was removed by rotational evaporation, and the product was dried in vacuum oven at

116

room temperature. White wax-like solid was recovered. Yield: 0.7 g (92%). 1H NMR (ppm) in

117

chloroform-d:

118

-CH2-CH2-(CH2)14CH3), δ1.64 (m, -COO-CH2-CH2-(CH2)14CH3, -N(-CH2-CH2)2-CH-CH2-), δ1.95

119

(t,

120

-CH2-N(-CH2-CH2)2-CH-CH2-,

121

HN(-CH2-CH2)2-CH-CH2-, -CH2-N(-CH2-CH2)2-CH-CH2-), δ3.5

122

-OOC-CH2-CH2-N- ) , δ4.20 (m, -COO-CH2-) (see Supporting Figure 1B).

123

2.2.3. Synthesis of PEG-b-PADM-TMDP-b-PEG block copolymer

124

The PADM-TMDP copolymer was used directly for Michael addition reaction with

125

PEG-acrylate without further purification. 0.19 g of PADM-TMDP (containing 0.1 mmol

126

piperdine groups) and 0.75 g of PEG-acrylate (0.15 mmol) were dissolved in 10 mL of

127

DCM/MeOH (1/1 volume ratio), and stirred at room temperature for 3 days. After reaction, the

1

chloroform-d:

δ3.35

δ0.86

(t,

(s,

-O-CH3),

-(CH2)16-CH3),

-N(-CH2-CH2)2-CH-CH2-),

,

δ2.31

δ3.4-3.9

δ1.20

(m,

(s,

(m,

[-CH2-CH2-O-]n),

-NC5H5-(CH2)3-NC5H5),

-COO-CH2-CH2-(CH2)14CH3),

-OOC-CH2-CH2-,

δ4.30

-CH2-OH),

6


δ2.85

&

δ1.30

(m,

(s,

δ2.5-2.7

(m,

δ3.11

(m,

(s, -CH2-OH ), δ3.69 (s,

Page 7 of 36

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

Biomacromolecules

128

solvents were removed and the product was dissolved in DD water and purified by dialysis

129

with a dialysis tube of 6-8 kDa MWCO for 2 days. Water was removed at reduced pressure

130

and the product was recovered in vacuum oven to obtain white powder. Yield: 0.58 g (84.1%).

131

1

132

all other peaks referred to PADM-TMDP (see Supporting Figure 1C).

133

2.2.4 Conjugation of PEG-b-PADM-TMDP-b-PEG copolymer with folic acid

134

200mg of PEG-b-PADM-TMDP-b-PEG copolymer (0.0143mmol), 10mg of folic acid

135

(0.0226mmol), 10mg EDC.HCl (0.052mmol) and 5mg DMAP (0.041mmol) were dissolved in

136

10 mL of anhydrous DMF, and the solution was stirred at room temperature. After 24 h, 10 mL

137

of water were added into the solution to terminate the reaction. The solution was dialyzed

138

against DD water with a dialysis tubing of 6-8 kDa MWCO for 2 days at room temperature, and

139

DD water was changed 4 times per day. After dialysis, extra water was removed under

140

reduced pressure, and light yellow solid powder was recovered in vacuum oven at room

141

temperature. Yield: 0.17 g (81.0%). H NMR (ppm) in DMSO-d6: δ4.30, δ4.50, δ6.62, δ6.88,

142

δ7.41, δ7.62, δ8.65 are contributed to Folic acid (see Supporting Figure 1D), δ3.35 (s, -OCH3,

143

PEG),

144

PEG-b-PADM-TMDP-b-PEG.

145

2.2.5 NMR characterization

146

1

147

samples were dissolved in chloroform-d or DMSO-d6 at the concentration of 20 mg/mL, and

148

the relaxation delay (d1) was set as 2 s.

149

2.2.6 GPC characterization

H NMR (ppm) in chloroform-d: δ3.35 (s, -OCH3, PEG), δ3.40-3.9 (m, [-CH2-CH2-O-]n, PEG ),

1

δ3.4-3.6

(m,

[-CH2-CH2-O-]n,

PEG

),

all

other

peaks

referred

to

H NMR experiments were conducted on a Bruker Avance 300 Mhz NMR Spectrometer. The

7


Biomacromolecules

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

150

Molecular weight and molecular weight distribution of the polymers were determined by GPC

151

(Viscotek GPC system, equipped with a refractive index detector) using THF as eluent at a

152

flow rate of 1.0 mL/min at 22 °C.

153

2.3. Synthesis of modified IONPs

154

Fe(NO3)3•9H2O (2.3 g, 5.75 mmol) and FeSO4•7H2O (0.8 g, 2.88 mmol) were dissolved in

155

deionized water (20 mL). The resulting yellow solution was bubbled with nitrogen gas about

156

half hour, and then heated to 45 °C. Nitrogen degassed ammonium hydroxide (2.5 mL, 25%)

157

was added quickly into the solution by syringe. After 30 min oleic acid (1 mL) was added into

158

the mixture, which was stirred another one hour at 80 °C. Oleic acid modified magnetite

159

nanoparticles were collected from the solution by magnetic separation and washed several

160

times with deionized water, acetone and ethanol, then dried under vacuum conditions at 60 °C

161

overnight.

162

2.4. Preparation of DOX-loaded micelles

163

2mg DOX and 1.1 equivalent of trimethylamine were dissolved in 3 mL of chloroform in a 20mL

164

glass vial, and the red solution was stirred at room temperature for about 10 min. Then 2 mg of

165

oleic acid modified magnetite nanoparticles and 20 mg of PEG-b-PDAM-TMDP-b-PEG (PAE)

166

or Folate- PEG-b-PDAM-TMDP-b-PEG (F-PAE) were added into the solution, and stirred for

167

another hour. Then 5 mL of distilled water was added and the resulting mixture was bubbled

168

with nitrogen gas overnight to remove chloroform to obtain a clear brown solution. The solution

169

was dialyzed against DD water with a dialysis tubing of 6-8 kDa MWCO for 1day at 4°C. Then

170

DOX-loaded micelles were lyophilized to give DOX-loaded micelles as red powders with a

171

yield of 20.5mg (85%) and 19.7mg (82%) for EG-b-PDAM-TMDP-b-PEG (PAE) or Folate8


Page 8 of 36

Page 9 of 36

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

Biomacromolecules

172

PEG-b-PDAM-TMDP-b-PEG (F-PAE) respectively. For every sample, 1mg product was

173

dissolved in 1mL DMF to obtain a clear red solution, and the absorption intensity of the

174

solution at 480nm was measured by a UV-vis spectrometer, which was compared with

175

standard calibration curve to calculate the actual concentration of DOX. The calculated DOX

176

drug loading efficiency is 49%, and the actual entrapment efficiency of the IONPs is 4.1% for

177

PAE; while the drug loading efficiency is 54 %, and the actual entrapment efficiency of the

178

IONPs is 4.5% for F-PAE.

179

2.5. The properties of the micelles

180

The magnetic properties of the synthesized polymeric IONPs were recorded with a vibrating

181

sample magnetometer (VSM). The diameter and size distribution of the polymeric IONPs was

182

determined by transmission electron microscope (TEM) scanning and dynamic light scattering

183

(DLS) detection.

184

2.6. In vitro DOX release experiments

185

DOX-loaded micelles was dissolved into 3 mL of buffer solutions (pH = 7.4 or 6.5). The

186

solutions were quickly transferred into small dialysis bags. The bags were sealed and then

187

immersed in 15 mL of buffer solutions with different pH at 37 °C in dark. Periodically, 3 mL

188

incubation solutions were taken out and the same volumes of fresh buffer solutions were then

189

added to remain the incubation volume at 15 mL. The solutions taken out were tested by

190

UV-vis spectrometry around 480 nm, which is the characteristic maximum absorbance of DOX

191

in solutions. The release experiments were repeated three times and date were reported.

192

2.7. Cell culture

193

Two human gastric adenocarcinoma cell lines, BGC823 and SGC7901, were cultured in 9


Biomacromolecules

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 10 of 36

194

RPMI-1640 media supplemented with 10% FBS at 37 °C in 5% CO2. Cells were grown in

195

culture dish and were collected for all experiments at logarithmic growth phase.

196

2.8. Western blotting (WB) analysis

197

To explore the target effect of folate-conjugated micelles on GC cells, we identified the protein

198

expression of FR in GC cells by WB before the in vitro and in vivo experiments. BGC823 and

199

SGC7901 cells were lysed and the protein was collected with the Protein Extraction Kit. The

200

harvested protein was loaded to each lane with an amount of 20 µg for electrophoresis in 10%

201

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to

202

0.22 µm polyvinylidine difluoride (PVDF) membrane. After blocking with 5% skim milk for 1h at

203

room temperature, the membrane was incubated with primary antibodies of FR and α-Actin

204

overnight at 4 °C. On the second day, the membrane was incubated with fluorescent

205

secondary antibody (Odyssey) for 1 h and exposed with near infrared imaging system

206

(Odyssey).

207

2.9. Cellular uptake studies

208

SGC7901 cells were seeded in confocal dishes with folate-free culture media at a density of 4

209

× 104 cells per dish. After cultured overnight, cells were incubated with 2.5 µg/ml of free DOX,

210

P-DOX or F-P-DOX at pH 7.4 or pH 6.5 for 24 h. In this study, the concentrations of different

211

formulations were based on the amount of DOX contained. At the end of reaction time, the

212

cells

213

(4',6-diamidino-2-phenylindole). Finally, the fluorescent images were captured with confocal

214

microscope (OLYMPUS FV10C-W3).

215

2.10. MTT cytotoxicity assay

were

fixed

with

4%

paraformaldehyde

(PFA)

and

10


incubated

with

DAPI

Page 11 of 36

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

Biomacromolecules

216

SGC7901 cells were seeded in 96-well plates with folate-free culture media at a density of 5 ×

217

10 cells per well. On the second day, cells were treated with DOX, P-DOX or F-P-DOX at pH

218

7.4 or pH 6.5 at the concentrations of 1 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml, 15 µg/ml,

219

respectively. Twenty-four hours later, the media were removed and 200 µl of MTT solution (0.5

220

mg/ml) was added to each well. After incubating for 4 h, the MTT solution was removed and

221

150 µl of DMSO was added to each well to dissolve the formazan. Finally, the optical density of

222

each well was measured at 570 nm with microplate spectrophotometer (SpectraMax M5).

223

2.11. Cell apoptosis observation

224

After being treated with PBS or 5 µg/ml of F-P-DOX in folate-free media for 24 h, SGC7901

225

cells were fixed for transmission electron microscope (TEM) scanning (HITACH, H-7000FA, 75

226

kV).

227

2.12. Quantitative rt-PCR

228

The total RNA of SGC7901 cells treated with PBS or free DOX, P-DOX or F-P-DOX at the

229

concentration of 5 µg/ml in folate-free media for 24 h was extracted and prepared for reverse

230

transcription. The reaction product, cDNA, was used to perform rt-PCR using the

231

LightCycler480 instrument (G

232

human

233

5’-TACCAGGAAATGAGCTTGACAAAG-3’),

234

GCAGCAAACCTCAGGGAAAC-3’,

235

experimental procedures were performed according to the recommended protocols of

236

manufacturer.

237

2.13. Xenograft model and treatment

3

ermany). Specific primers sequences were as follows:

GAPDH

(5’-ACTTCAACAGCGACACCCACTC-3’,

5’-

Caspase

3

AACTGCTCCTTTTGCTGTGATCT

11


(5’-3’).

All

Biomacromolecules

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 12 of 36

238

BALB/c male nude mice aged 4 weeks were purchased from Laboratory Animal Center of

239

Southern Medical University. We choose BGC823 cells with higher tumorigenic ability to

240

construct xenograft model. Every mouse was subcutaneously injected with 5 × 106 BGC823

241

cells into the left flanks. When the tumors reached a volume of ~50 mm , the mice were

242

randomly divided into 4 groups (n = 3) and subjected to PBS, free DOX, P-DOX or F-P-DOX at

243

a dose of 5 mg/kg bodyweight through caudal intravenous injection. Every group was

244

administrated on day 1 and day 8. The bodyweight and tumor volume were measured every

245

another day. At the 17th day, the mice were euthanized with 1% pentobarbital sodium and

246

sacrificed for harvesting of tumors and organs including hearts, livers and kidneys, which were

247

fixed with 4% PA for the following experiments. The mice experiment was carried out

248

according to the National Guidelines for Animal Experimentation and approved by the Animal

249

Care and Use Committee of the Nanfang Hospital, Southern Medical University.

250

2.14. Hematoxylin & eosin (H&E) and Prussian blue staining

251

The above fixed tumors and organs were subjected to dehydration, paraffin embedding before

252

cutting into slices. The paraffin sections were routinely stained with H&E and Prussian blue.

253

The results were observed and photographed by light microscopy (OLYMPUS, BX51).

254

2.15. In vivo magnetic resonance imaging (MRI)

255

Two BALB/c male nude mice were subcutaneously injected with 5 × 106 BGC823 cells into the

256

left flanks. When the tumors reached a volume of ~100 mm , the mice were caudal

257

intravenous injected with P-DOX or F-P-DOX at a dose of 5 mg/kg bodyweight. Then MRI

258

scanning (GE Healthcare, 3.0 T superconducting unit, T2 sequence, ST 1.0 mm) was

259

employed for MNPs detection before, 12h and 36h after treatment, respectively.

3

3

12


Page 13 of 36

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

Biomacromolecules

260

2.16. Statistical analysis

261

All data were presented as means ± standard deviation (SD). The MTT findings, rt-PCR results,

262

and tumor volumes were analyzed using one-way analysis of variance (ANOVA), followed by

263

post hoc LSD test for intergroup comparisons. SPSS 16.0 software (SPSS Institute, Inc.,

264

Chicago, IL, USA) was used for statistical analyses, and p < 0.05 was regarded as statistically

265

significant.

266

3. Results and Discussion

267

3.1. Polymer synthesis and characterization of DOX-loaded micelles

268

Taking the advantages of Michael-type polymerization including forming degradable polymers

269

backbones, mild and undemanding reacting condition and conveniently introducing diverse

270

linkages in backbones and functional side groups

271

chains were prepared. Followed by conjugation with polyethylene glycol acrylate and then folic

272

acid, the expected tumor-targeting and pH sensitive amphiphilic poly(β-aminoester)

273

copolymers were obtained, which could self-assemble with IONPs to form nanocarriers for

274

drug delivery. The overall synthetic routes to prepare the desired polymers and nanoparticles

275

was described in Scheme 1, including synthesis of pH sensitive poly(β-aminoester),

276

preparation of amphiphilic copolymers by chain extension with hydrophilic poly (ethylene

277

glycol), conjugation of folic acids with amphiphilic copolymers for tumor targeting, as well as

278

preparation of oleic acids modified hydrophobic magnetic iron oxide nanoparticles.

28, 29

, the pH sensitive poly(β-aminoester)

13


Biomacromolecules

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 14 of 36

279 280

Scheme

281

(PEG-b-PDAM-TMDP-b-PEG) and iron oxide nanoparticles

282

In the first step, the pH sensitive poly(β-aminoester) chains were polymerized by

283

pentaerythritol diacrylate monostearate (PADM) and 4,4’-trimethylene dipiperidine (TMDP) via

284

AA (acrylate) + BB (piperidine) type step polymerization with a molar ratio of PADM:TMDP =

285

5:6. The polymer chains should be end-capped by TMDP units with piperidine groups on the

286

chain ends. In this reaction, hydrophobic monomer PDAM and hydrophilic monomer TMDP

1.

Synthesis

of

folate

conjugated

amphiphilic

14


poly(β-aminoester)

Page 15 of 36

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

Biomacromolecules

287

were dissolved in DCM and MeOH respectively to be miscible each other. The product is

288

wax-like solid due to the semi-crystallinity of long carbon side chains of PDAM units.

289

Supporting Figure 1A and 1B presented the 1H NMR spectra of unreacted PDAM and the

290

PDAM-TMDP copolymer after Michael addition reaction respectively. Compared with

291

Supporting Figure 1A, the peaks between 5.7 ppm and 6.5 ppm disappeared completely in

292

Supporting Figure 1B, which are assigned to the protons on -CH=CH2 double bonds, indicating

293

the 100% conversion and complete consumption of acrylate groups on PDAM. Because the

294

feeding molar ratio of PDAM: TMDP is set as 5:6, the theoretical average molecular weight of

295

PDAM-TMDP should be ~3800 Da by calculation according to the NMR result, and two ends of

296

the copolymer chain were capped with piperidine groups, which could further react with

297

acrylate groups of poly(ethylene glycol) mono ether acrylate (PEG-acrylate) for polymer chain

298

extension.

299

Without further purification, the PADM-TMDP copolymer reacted directly with excessive

300

amount of PEG-acrylate (Mw= 5000 Da, 1.5 molar excess to piperidine groups) to obtain

301

PEG-b-PADM-TMDP-b-PEG

302

synthesized by acrylation of poly (ethylene glycol) mono ether (PEG-OH, Mw= 5000 Da) with

303

acryloyl chloride in the existence of triethylamine, and introduced to two sides of PDAM-TMDP

304

polymer chains to provide hydrophilicity for the poly (β-aminoester) by Michael addition with

305

piperidine moieties. To obtain high conversion, the molar ratio of PEG-acrylate is 1.5 excess to

306

the amount of piperidine functionalities of PDAM-TMDP. The reaction was kept at room

307

temperature for 3 days, and then dialyzed against DD water with a dialysis tubing having a

308

molecular weight cut off of 6~8 kDa to remove any unreacted PEG-acrylate or PDAM-TMDP

amphiphilic

block

copolymers.

15


The

PEG-acrylate

was

Biomacromolecules

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 36

309

polymers.

310

PEG-b-PDAM-TMDP amphiphilic copolymers. As shown in Supporting Figure 1C, the H NMR

311

spectrum of PEG-b-PDAM-TMDP-b-PEG shows a strong broad peak at 3.4~3.9 ppm mainly

312

assigned with the protons of -O–CH2-CH2-O- of PEG and the peak at 0.9 ppm contributed to

313

the –CH3 of PDAM units, indicating the successful conjugation of PEG-acrylate and

314

PDAM-TMDP. The conjugation ratio of PEG and PDAM-TMDP can be calculated by

315

comparing the peak intensities of -O–CH2-CH2-O- (3.4~3.9 ppm) and –CH3 of PDAM at 0.9

316

ppm, which is around ~950: 15. For a complete conjugation reaction, one PDAM5-TMDP6 will

317

be covalently bonded with two PEG-acrylate chains. By calculation, the molar ratio of the

318

protons from -CH2-CH2-O- to that from –CH3 of PDAM should be around 908 (~228 repeat

319

units of C2H4O): 15 (5 repeat units of PDAM), which is similar to measured value from NMR

320

data. To further confirm the formation of amphiphilic copolymers, the molecular weight and

321

molecular weight distribution of PDAM-TMDP and PEG-b-PDAM-TMDP-b-PEG were

322

characterized by GPC, using THF as eluent. The GPC traces of PDAM-TMDP and

323

PEG-b-PDAM-TMDP-b-PEG were provided in Supporting Figure 2. The GPC trace of

324

PDAM-TMDP is comprised of continuous multiple peaks, indicating this step polymerization

325

product contains a series of polymers and oligomers with different molecular weight, which has

326

an average number molecular weight of ~1900 Da with a molecular weight distribution of 2.35.

327

While after conjugation with PEG-acrylate (5000) and sequent dialysis purification to remove

328

unconjugated low molecular weight polymers (molecular weight is less than 6~8kDa), the GPC

329

trace of PEG-b-PDAM-TMDP-b-PEG shows a single narrow peak with a molecular weight of

330

~11800 Da, indicating the successful conjugation of PEG blocks on PDAM-TMDP polymers.

The

final

product

should

mainly

be

PEG-b-PDAM-TMDP-b-PEG

or

1

16


Page 17 of 36

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

Biomacromolecules

331

Based on the combination of NMR and GPC data, it can be concluded that the conjugation of

332

PDAM-TMDP was successful and the main product should be PEG-b-PDAM-TMDP-b-PEG

333

amphiphilic copolymers. Although there may be some PEG-b-PDAM-TMDP polymers in the

334

product, they are also amphiphilic copolymers and could self-assemble with IONPs together

335

as well. The tertiary amines of PDAM-TMDP backbones offered the pH sensitivity for this

336

copolymer, which can be protonated to get better solubility, destabilize micelles and release

337

drug in weakly acidic environment.

338

Finally, folate groups were introduced into the polymer for tumor targeting by esterification of

339

folic acid and the hydroxyl groups of PDAM units, as shown in Scheme 2. The reaction took

340

place in anhydrous DMF for 24 h at room temperature with the existence of DMAP/EDC. After

341

reaction, the catalysts, unreacted folic acids, urea were removed by dialysis with a MWCO of

342

6~8 kDa as well. The light yellow color of the product indicates folic acids were grafted onto

343

polymer side chains. To further confirm successful conjugation of folate functionalities onto

344

polymer chain, folate conjugated copolymers were characterized by H NMR spectrometry

345

using DMSO-d6 as the solvent, and the spectrum is present in Supporting Figure 1D. The tiny

346

peaks at 4.30 ppm, 4.50 ppm, 6.62 ppm, 6.88 ppm, 7.41 ppm, 7.62 ppm, 8.65 ppm should be

347

assigned to corresponding protons of folates, as shown in Supporting Figure 1D, which

348

verified the covalently bonding of folate groups onto the amphiphilic copolymers. The graft

349

ratio of folate can be estimated by comparing intensity of the peaks at 6.62 ppm, 6.88 ppm,

350

7.41 ppm, 7.62 ppm (4 protons on the phenyl ring of folate) with that of the peak at

351

3.4ppm~3.6ppm (908 protons from -O–CH2-CH2-O- in DMSO-d6), which is 5.1: 908, indicating

352

1.27 pieces of folate per polymer backbone with a reaction ratio of 80%. After conjugation with

1

17


Biomacromolecules

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 18 of 36

353

folate, the solubility of polymer in THF became poor, and can not be characterized by GPC

354

using THF as eluent.

355

The tertiary amines of PDAM-TMDP backbones offered the pH sensitivity for this kind of

356

copolymers, which can be protonated to get better solubility, making the hydrophobic micelles

357

destabilized in a weakly acidic environment and the delivered drug released rapidly 27.

358

The IONPs were synthesized with ammonium hydroxide modifying and oleic acid capping via

359

a modified technique, and resulting IONPs have good solubility in nonpolar solvents, such

360

chloroform 30. The alkyl side chains of poly (β-amino ester) (PAE) / Folate-poly (β-amino ester)

361

(F-PAE) would self-assembly intercalated into the oleic acid layer of magnetite nanoparticles

362

though hydrophobic-hydrophobic interaction where the hydrophobic zone can be a reservoir of

363

DOX. Meanwhile the PEG chains would disperse the nanoparticles in aqueous media due to

364

their hydrophilicity, giving the obtained nanoparticles a hierarchical surface structure

365

PAE assembled IONPs denoted as PAE@IONPs and the F-PAE assembled MNPs denoted as

366

F-PAE@IONPs. The DOX-loaded micelles composed with PAE@IONPs were denoted as

367

P-DOX and the ones of F-PAE@IONPs were denoted as F-P-DOX.

368

We used a VSM to examine the magnetic properties of magnetic-polymer micelles and the

369

magnetization was shown as a function of the variation of magnetic field (Figure 1). The

370

saturation magnetization (σs) of PAE@IONPs and F-PAE@IONPs was around 5.4 emu/g and

371

3.2 emu/g respectively. The M-H curves (Figure 1) proved the magnetic nature of these

372

synthesized magnetic-polymer micelles. The saturation magnetization difference may be

373

related with the magnetic particles’ aggregation and size distribution of IONPs in neutral

374

solution.

11

. The

The DLS characterization showed the size of both PAE@IONPs and 18


Page 19 of 36

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

Biomacromolecules

375

F-PAE@IONPs is around 50~200nm, while F-PAE@IONPs had a higher size

376

distribution or aggregation according to Figure 2B and 2C.

377 378

Figure 1. M-H curves of the synthesized polymeric IONPs. The magnetic properties of

379

synthesized PAE@IONPs and F-PAE@IONPs were determined by vibrating sample

380

magnetometer.

381

The morphology of PAE@IONPs and F-PAE@IONPs was determined by TEM observation

382

(Figure 2A). Then DLS was employed to detect the size distribution and Zeta potential of the

383

micelles (Figure 2B, C and D). The intensity analysis indicated that the size of both

384

formulations was in the range of 50~200 nm at neutral condition, and the aggregation and

385

dissolution of the PAE@IONPs/F-PAE@IONPs were observed at the peak up to ~700

386

nm/~600 nm in diameter and the peak with only ~10 nm/~8 nm in diameter when the pH value

387

of the solution was adjusted to 6.5 (Figure 2B). Analogously, the number distribution also

388

demonstrated the disassociation of PAE@IONPs/F-PAE@IONPs at acidic condition (Figure

389

2C). The zeta potential of both formulations was increased about a fold at pH6.5 (Figure 2D).

390

With such a hydrodynamic size change in response to pH value, the system is suitable for drug

391

loading to treat cancer cells, which are always under acidic microenvironment.

19


Biomacromolecules

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

392 393

Figure 2. Transmission electron microscope imaging (TEM) and dynamic light scattering (DLS) 20


Page 20 of 36

Page 21 of 36

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

Biomacromolecules

394

measurement of the synthesized PAE@IONPs and F-PAE@IONPs. Diameter and size

395

distribution of the micelles were analyzed at the concentration of 2.5 mg/mL at pH 7.4 and pH

396

6.5, respectively.

397

After loading DOX to the above system, we utilized dialysis method to investigate the

398

pH-dependent DOX release performance from P-DOX and F-P-DOX micelles at pH 7.4 and

399

6.5, respectively. The release rates of both formulations were similar. As shown in Figure 3,

400

~50% of the DOX was released from both formulations at pH 7.4 within 48 h, with ~25% of

401

DOX released in the first 6 h. When the pH value of the solutions was adjusted to 6.5, evidently,

402

the incorporated DOX was released more effectively, with a ~35%/~40% release from

403

P-DOX/F-P-DOX micelles in the first 6 h and both amount to ~70% release in 48 h. These

404

results indicate that these DOX-loaded polymeric micelles conduct a slow and sustained

405

diffusion of the DOX from the micelles at neutral condition. However, in acidic environment, the

406

polymer depolymerized rapidly and the DOX entrapped in the micelles will be released

407

effectively. With such pH-responsive change of drug release rate, the delivery system can

408

enhance the DOX concentration in the acidic cancer microenvironment and the acidic cytosol.

21


Biomacromolecules

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

409 410

Figure 3. Drug release of the DOX-loaded micelles. The DOX release from P-DOX and

411

F-P-DOX micelles was investigated at pH 7.4 and 6.5, respectively.

412

3.2. Cellular internalization of DOX

413

Before evaluating the target effect of the F-P-DOX micelles, we carried out WB to examine the

414

FR expression on the membrane of GC cells and two FR-positive GC cells were screened out

415

(Figure 4). As the intracellular fluorescence intensity reflects the DOX amount absorbed by

416

cells, confocal microscope was used to assess the cellular internalization of DOX. We

417

incubated SGC7901 cells with free DOX, P-DOX and F-P-DOX at the equivalent DOX

418

concentration of 2.5 µg/ml at pH 7.4 and pH 6.5 respectively for 12 h. The result indicated that

419

the cells of both P-DOX and F-P-DOX treatments showed evidently stronger fluorescence at

420

pH 6.5 than at pH 7.4 (Figure 5A), owning to the pH-responsivity of the DOX-loading system. 22


Page 22 of 36

Page 23 of 36

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

Biomacromolecules

421

Furthermore, the fluorescence intensity of cells treated with F-P-DOX was stronger than that

422

treated with P-DOX under the same condition (Figure 5A). The DOX-loaded nanoparticles can

423

be ingested by cells and subsequently release drug in cytoplasm or directly release DOX

424

before entering cells. Since the DOX release rates of both formulations were quite similar

425

(Figure 3), it may be the way of nanoparticle internalization that made this difference. There

426

are three different processes of nanoparticle uptake into cells, including phagocytosis,

427

fluid-phase endocytosis, and receptor-mediated endocytosis

428

absorbed by GC cells through phagocytosis or fluid-phase endocytosis while targeted

429

F-P-DOX would be ingested by GC cells through FR-mediated endocytosis. Thereby, unlike

430

the way of P-DOX uptake, the high efficiency of receptor-mediated endocytosis would

431

guarantee the larger amount of F-P-DOX internalization, leading to larger amount of released

432

DOX in GC cells. Besides, as shown in Figure 5B, the red fluorescence intensity of DOX

433

enhanced and cell density decreased as the incubated time increased after F-P-DOX

434

treatment at pH 6.5. These results are in consistent with previous researches demonstrating

435

FR-targeted nanoparticles could be effectively taken up to inhibit growth of FR-positive tumor

436

cells

31

. Non-targeted P-DOX may be

18, 23, 24

.

437 438

Figure 4. Expression of FR in BGC823 and SGC7901 cells. Two GC cell lines were screened

439

out overexpressing FR by WB. ɑ-Actin was used as an internal reference. The left column of

440

black bands indicated marker; the middle and right bands indicated the FR protein of BGC823 23


Biomacromolecules

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

441

and SGC7901 cells, respectively.

442 443

Figure 5. Effective cellular internalization of DOX. Confocal microscope was applied to

444

observe the cellular influence intensity of DOX after treatment of free DOX, P-DOX or

445

F-P-DOX at the equivalent DOX concentration of 2.5 µg/ml. A: incubation with DOX, P-DOX

446

and F-P-DOX at pH 7.4 and pH 6.5 for 12 h, respectively. B: incubation with F-P-DOX at pH

447

6.5 for 2 h, 12 h and 24 h, respectively. The red fluorescence indicated DOX; the blue

448

fluorescence indicated DAPI. The images were photograghed under 400 × magnifications. 24


Page 24 of 36

Page 25 of 36

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

Biomacromolecules

449

3.3. Cytotoxicity of DOX, P-DOX and F-P-DOX

450

MTT assay was employed to investigate the cytotoxicity of different formulations on GC cell

451

lines. SGC7901 cells were exposed to different concentrations of blank IONPs (PAE@IONPs

452

and F-PAE@IONPs), free DOX, P-DOX and F-P-DOX, respectively. It turned out that the blank

453

IONPs were practically low cytotoxic (Figure 6A and B). An increase in cell inhibition rate of

454

free DOX (Figure 6C), P-DOX (Figure 6D) and F-P-DOX (Figure 6E) was observed as the

455

concentration of contained DOX increased at both pH 7.4 and pH 6.5. Both P-DOX (Figure 6D)

456

and F-P-DOX (Figure 6E) exhibited a trend of higher cytotoxicity at acidic condition compared

457

with neutral condition while free DOX presented no obvious distinction (Figure 6C), owing to

458

the pH-sensitivity of the DOX-loaded micelles. The half inhibitory concentration (IC50) for DOX,

459

P-DOX and F-P-DOX to SGC7901 cells after 24 h treatment was ~5 µg/ml, ~5 µg/ml and ~2.5

460

µg/ml at pH 7.4 and ~5 µg/ml, ~2.5 µg/ml and ~1.25 µg/ml at pH 6.5 (Figure 7C, D and E),

461

respectively. Poorer efficacy of free DOX compared with nanoparticle formulations may be due

462

to the multidrug and toxic compound extrusion (MATE) family transporters

463

pump

464

endocytosis and slow drug release may contribute to the better drug retention of DOX-loaded

465

micelles

466

to be the result of better internalization of F-P-DOX through FR-mediated endocytosis, which

467

was consistent with the above cellular uptake result. Herein, F-P-DOX was superior to both

468

P-DOX and free DOX in inhibiting GC cell growth.

23

32

or p-gly-coprotein

that facilitate the drug efflux from cytoplasm. On the contrary, the lysosome

33

. Additionally, the advantage of F-P-DOX over P-DOX on cytotoxicity was supposed

25


Biomacromolecules

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

469 470

Figure 6. Enhanced effect of F-P-DOX on GC cells. MTT assay was carried out to test the

471

cytotoxicity of PAE@IONPs (A), F-PAE@IONPs (B), free DOX (C), P-DOX (D) and F-P-DOX

472

(E), respectively at pH 7.4 and pH 6.5 after 24 h treatment. Each experiment was conducted in

473

triplicate.

474

3.4. Cell apoptosis induced by F-P-DOX 26


Page 26 of 36

Page 27 of 36

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

Biomacromolecules

475

TEM and rt-PCR were carried out to determine the degree of apoptosis induced by F-P-DOX

476

treatment. TEM observation revealed a sign of apoptosis that the compacted nuclear

477

chromatin formed chromatin clumps of varying sizes and shapes, with some clumps

478

aggregating to the nuclear membrane (Figure 7A a, short white arrows). Lipid droplet, another

479

indication for apoptosis, was captured as well (Figure 7A b, black arrow). Furthermore, some

480

nanoparticles were observed within lysosomes (Figure 7A c and d, long white arrows), which

481

was supposed to be a physiological response of cells to intrusion of the micelles. The acidic

482

lysosome (pH 4~6) would make the pH-sensitive F-P-DOX unstable, resulting in a release of

483

DOX

484

interpretation that lysosome endocytosis prevented DOX-loaded micelles from drug efflux.

485

Additionally, our rt-PCR analysis found that F-P-DOX treatment up-regulated the mRNA

486

expression level of Caspase 3 to ~8 times as much as that of PBS treatment, ~3 times as

487

much as that of DOX treatment and ~2 times as much as that of PBS treatment (Figure 7B).

488

This elevated expression of Caspase 3, an apoptosis associated gene, also hinted at the

489

occurrence of apoptosis. These results indicate that F-P-DOX could function through inducing

490

apoptosis of GC cells. It is well known that DOX causes cell apoptosis through a cytotoxic

491

mechanism of embedding DNA to inhibit the synthesis of nucleic acids

492

declare the apoptosis effect of DOX on cancer cells, which is consistent with the mechanism of

493

F-P-DOX micelles on GC cells

33

and a biodegradation of magnetite nanoparticles

34

, which also supported the above

36, 37

.

27


35

. Many studies

Biomacromolecules

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

494 495

Figure 7. Cell apoptosis induced by F-P-DOX. Transmission electron microscope was carried

496

out to observe the cells after 24 h incubation of F-P-DOX at the equivalent DOX concentration

497

of 5 µg/ml (A). The short white arrows pointed at the compacted nuclear chromatin (a); the

498

black arrow pointed at the lipid droplet (b); the long white arrows pointed at the nanoparticles in

499

lysosomes (c and d). The mRNA expression of Caspase 3 was examined by rt-PCR (B). Each

500

experiment was conducted in triplicate. *p < 0.05 vs PBS group, **p < 0.01 vs PBS group, ***p

501

< 0.001 vs PBS group;

502

3.5. Efficacy of DOX, P-DOX and F-P-DOX on xenograft model

503

To explore the anti-tumor efficacy in vivo, DOX, P-DOX and F-P-DOX was administrated into

504

gastric tumor-bearing nude mice separately. Compared with PBS treated control group, the

505

other three treated groups exhibited different degrees of tumor suppression efficacy (Figure 8A

§§

###

p < 0.001 vs DOX group; p < 0.01 vs P-DOX group.

28


Page 28 of 36

Page 29 of 36

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

Biomacromolecules

506

and B). The final tumor volume in F-P-DOX group was reduced to 252.76 ± 108.61 mm3 on

507

average, which was about a quarter of that in PBS group, one-third of that in free DOX group

508

and half of that in P-DOX group (Figure 8B). The tumor-targeting effect and FR-mediated

509

cellular internalization are probably responsible for the high therapeutic efficacy of F-P-DOX

510

on tumor regression. This finding is in good agreement with previous works which

511

demonstrated that FR-targeting nanoparticles had significant inhibition effect on tumor growth

512

23, 26

513

but less effective than F-P-DOX on tumor growth suppression in vivo (Figure 8B). Therefore, it

514

is important to enhance the tumor-targeting of nanocarriers in addition to improving their

515

sensitivity to local tumor microenvironment.

516

To assess side effects of the nanoparticles, we measured the bodyweight every two days and

517

no visible bodyweight dropping was noted (Figure 8C). H&E staining was performed and no

518

significant pathological change was detected in heart, liver and kidney, even in the free DOX

519

group (Figure 8D). This may due to the short duration of treatment course in the present study

520

or the poor sensitivity of H&E staining

521

least, didn’t increase the adverse effects of DOX. As shown from Prussian blue staining, there

522

was no iron deposition in tumors and organs at 17th day of both P-DOX and F-P-DOX

523

treatments (Figure 8E). As a result, P-DOX and F-P-DOX were low-toxic and well

524

biodegradable in systematic level, suggesting that DOX-loaded micelles may be safe for use

525

on AGC patients.

526

Taken together, with the advantages of tumor-targeting and FR-mediated cellular

527

internalization, the F-P-DOX had a better effect on GC treatment than both P-DOX and free

. Consistent with the anti-tumor efficacy in vitro, P-DOX was more effective than free DOX

38, 39

. Nevertheless, the nanoparticle modification, at

29


Biomacromolecules

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

528

DOX and caused no apparent adverse physiological consequences.

529 530

Figure 8. Improved anti-tumor efficacy of F-P-DOX on GC xenograft model without obvious

531

adverse effects. At the end of the course, tumors of different groups (A), the tumor growth

532

curves (B) and bodyweight curves (C) were all shown. H&E staining and Prussian blue results

533

were exhibited in D and E respectively. n = 3 for each group. **p < 0.01, ***p < 0.001 vs PBS

534

group; #p < 0.05, ##p < 0.01 vs DOX group; §p < 0.05 vs P- DOX group at the final day of

535

observation. The images in D and E were photograghed under 200× magnifications.

536

3.6. In vivo MRI detection of the DOX-loaded micelles

537

MRI scanning was performed to evaluate the imaging function of DOX-loaded micelles in vivo. 30


Page 30 of 36

Page 31 of 36

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

Biomacromolecules

538

Both the P-DOX and F-P-DOX contained magnetic iron oxide, which could be detected by low

539

signal in T2-weighted image. As shown in Figure 9, 12 h after injection of both micelles, slightly

540

decreased signal was observed in local area of the tumors (red arrows), suggesting a mild

541

accumulation of nanoparticles in tumors

542

region of both treatments presented enhanced signal in T2-weighted image, with some

543

masses remaining low signal. It was plausible that the enhanced signal was caused by edema

544

occurrence in tumor, which may be a response to anti-tumor therapy

545

sustained dark masses may be the presentation of accumulated MNPs in tumors, suggesting

546

a well retention of the nanoparticles in the tumor area. Hence, with noninvasive MRI detection,

547

P-DOX and F-P-DOX can be traced and the effect can be feasibly monitored. Combined with

548

the above anti-tumor effect, these findings indicated F-P-DOX was not only effective on

549

suppressing GC growth, but its therapy response could be monitored by safe imaging, which is

550

beneficial for clinical cancer treatment.

40, 41

. Unexpectedly, after another 24 h, the tumor

40

. In addition, the

551 552

Figure 9. MRI scanning of P-DOX and F-P-DOX treated xenograft tumors. When the tumors

553

grew to a volume of ~100 mm3, MRI was performed to monitor the accumulation of P-DOX and 31


Biomacromolecules

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

554

F-P-DOX in the tumor site before (pre), 12 h and 36 h after caudal intravenous injection. The

555

red arrows indicated the tumor sites.

556

4. Conclusion

557

In the present study, we developed versatile nanotherapeutic DOX-loaded micelles with

558

FR-targeting specificity and pH-sensitivity. It demonstrated that with a mechanism of apoptosis,

559

F-P-DOX was superior to free DOX and P-DOX in GC treatment both in vitro and in vivo

560

without causing obvious side effects. Besides, F-P-DOX could be feasibly detected by

561

noninvasive MRI scanning. Taken together, these findings suggest that F-P-DOX is a

562

promising theranostic candidate for GC, although more studies may be required to further

563

investigate its clinical application.

564

Acknowledgement

565

This work was supported by the NSERC Discovery Grant, CIHR-RPP and China 863 Project

566

(Grant N. 2012AA020504). We thank the help from Prof. Hongbo Zeng at University of Alberta

567

for GPC characterization.

568

Supporting Information

569

Fig. S-1 and Fig. S-2

570

This material is available free of charge via the Internet at http://pubs.acs.org.

571 572

References

573

(1) Soerjomataram, I.; Lortet-Tieulent, J.; Parkin, D. M.; Ferlay, J.; Mathers, C.; Forman, D.;

574 575 576

Bray, F. Lancet 2012, 380 (9856), 1840-1850. (2) Garrido, M.; Fonseca, P. J.; Vieitez, J. M.; Frunza, M.; Lacave, A. J. Expert Rev Anticancer Ther 2014, 14 (8), 887-900. 32


Page 32 of 36

Page 33 of 36

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

Biomacromolecules

577 578

(3) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J Clin 2011, 61 (2), 69-90.

579

(4) Henneberry, H. P.; Aherne, G. W. Br J Cancer 1992, 65 (1), 82-86.

580

(5) Konorev, E. A.; Vanamala, S.; Kalyanaraman, B. Free Radic Biol Med 2008, 45 (12),

581 582 583

1723-1728. (6) Boyer, C.; Whittaker, M. R.; Bulmus, V.; Liu, J.; Davis, T. P. NPG Asia Materials 2010, 2 (1), 23-30.

584

(7) Thuy T. T. N'Guyen; Hien T. T. Duong; Johan Basuki; Véronique Montembault; Sagrario

585

Pascual; Clément Guibert; Jérôme Fresnais; Cyrille Boyer; Michael R. Whittaker;

586

Thomas P. Davis; Laurent Fontaine. Angewandte Chemie 2013, 125 (52), 14402-14406.

587

(8) Basuki, J.; Jacquemin, A.; Esser, L.; Li, Y.; Boyer, C.; Davis, T.P. Polymer Chemistry

588 589 590

2014, 5 (7), 2611-2620. (9) Basuki, J. S.; Esser, L.; Duong, H. T. T.; Zhang, Q.; Wilson, P.; Whittaker, M. R.; Haddleton, D. M.; Boyer, C.; Davis, T. P. Chem. Sci 2014, 5 (2), 715-726.

591

(10) Basuki, J. S.; Duong, H. T.; Macmillan, A.; Erlich, R. B.; Esser, L.; Akerfeldt, M. C.; Whan,

592

R. M.; Kavallaris, M.; Boyer, C.; Davis, T. P. ACS Nano 2013, 7 (11), 10175-10189.

593

(11) Chen, J.; Shi, M.; Liu, P.; Ko, A.; Zhong, W.; Liao, W.; Xing, M. M. Biomaterials 2014, 35

594 595 596

(4), 1240-1248. (12) Chang, Y.; Meng, X.; Zhao, Y.; Li, K.; Zhao, B.; Zhu, M.; Li, Y.; Chen, X.; Wang, J. J Colloid Interface Sci 2011, 363 (1), 403-409.

597

(13) Fan, X.; Jiao, G.; Zhao, W.; Jin, P.; Li, X. Nanoscale 2013, 5 (3), 1143-1152.

598

(14) Unsoy, G.; Yalcin, S.; Khodadust, R.; Mutlu, P.; Onguru, O.; Gunduz, U. Biomed 33


Biomacromolecules

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

599

Pharmacother 2014, 68 (5), 641-648.

600

(15) Yang, L.; Mao, H.; Wang, Y. A.; Cao, Z.; Peng, X.; Wang, X.; Duan, H.; Ni, C.; Yuan, Q.;

601

Adams, G.; Smith, M. Q.; Wood, W. C.; Gao, X.; Nie, S. Small 2009, 5 (2), 235-243.

602

(16) Chen, H.; Wang, L.; Yu, Q.; Qian, W.; Tiwari, D.; Yi, H.; Wang, A. Y.; Huang, J.; Yang, L.;

603 604 605 606 607 608 609 610 611

Mao, H. Int J Nanomedicine 2013, 8, 3781-3794. (17) Wang, D.; Fei, B.; Halig, L. V.; Qin, X.; Hu, Z.; Xu, H.; Wang, Y. A.; Chen, Z.; Kim, S.; Shin, D. M.; Chen, Z. G. ACS Nano 2014, 8 (7):6620-6632. (18) Lv, Y.; Yang, B.; Jiang, T.; Li, Y. M.; He, F.; Zhuo, R. X. Colloids Surf B Biointerfaces 2014, 115, 253-259. (19) Chen, F. H.; Zhang, L. M.; Chen, Q. T.; Zhang, Y.; Zhang, Z. J. Chem Commun (Camb) 2010, 46 (45), 8633-8635. (20) Stephen, Z. R.; Kievit, F. M.; Veiseh, O.; Chiarelli, P. A.; Fang, C.; Wang, K.; Hatzinger, S. J.; Ellenbogen, R. G.; Silber, J. R.; Zhang, M. ACS Nano 2014, 8 (10), 10383-10395.

612

(21) Kelemen, L. E. Int J Cancer 2006, 119 (2), 243-250.

613

(22) Wibowo, A. S.; Singh, M.; Reeder, K. M.; Carter, J. J.; Kovach, A. R.; Meng, W.; Ratnam,

614

M.; Zhang, F.; Dann, C. R. Proc Natl Acad Sci U S A 2013, 110 (38), 15180-15188.

615

(23) Yoo, H. S.; Park, T. G. J Control Release 2004, 96 (2), 273-283.

616

(24) Esmaeili, F.; Ghahremani, M. H.; Ostad, S. N.; Atyabi, F.; Seyedabadi, M.; Malekshahi, M.

617

R.; Amini, M.; Dinarvand, R. J Drug Target 2008, 16 (5), 415-423.

618

(25) Kim, M.S.; Hwang, S.J.; Han, J.K.; Choi, E.K;

619

Macromol Rapid Comm 2006,27(6),447-451。

Park, H.J.; Kim, J.S.; and Lee, D.S.

620 621

(26) Ye, W. L.; Du JB; Zhang, B. L.; Na, R.; Song, Y. F.; Mei, Q. B.; Zhao, M. G.; Zhou, S. Y. 34


Page 34 of 36

Page 35 of 36

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

Biomacromolecules

622 623 624 625 626

PLoS One 2014, 9 (5), e97358. (27) Chen, J.; Chen, H.; Li, P.; Diao, H.; Zhu, S.; Dong, L.; Wang, R.; Guo, T.; Zhao, J.; Zhang, J. Biomaterials 2011, 32 (21), 4793-4805. (28) Sunshine, J.; Green, J. J.; Mahon, K. P.; Yang, F.; Eltoukhy, A. A.; Nguyen, D. N.; Langer, R.; Anderson, D. G. Adv Mater 2009, 21 (48), 4947-4951.

627

(29) Lu, C.; Xing, M. M.; Zhong, W. Nanomedicine 2011, 7 (1), 80-87.

628

(30) Namiki, Y.; Namiki, T.; Yoshida, H.; Ishii, Y.; Tsubota, A.; Koido, S.; Nariai, K.; Mitsunaga,

629

M.; Yanagisawa, S.; Kashiwagi, H.; Mabashi, Y.; Yumoto, Y.; Hoshina, S.; Fujise, K.;

630

Tada, N. Nat Nanotechnol 2009, 4 (9), 598-606.

631 632

(31) Yang, P. H.; Sun, X.; Chiu, J. F.; Sun, H.; He, Q. Y. Bioconjug Chem 2005, 16 (3), 494-496.

633

(32) Tanaka, Y.; Hipolito, C. J.; Maturana, A. D.; Ito, K.; Kuroda, T.; Higuchi, T.; Katoh, T.; Kato,

634

H. E.; Hattori, M.; Kumazaki, K.; Tsukazaki, T.; Ishitani, R.; Suga, H.; Nureki, O. Nature

635

2013, 496 (7444), 247-251.

636 637 638 639

(33) Wang, X.; Teng, Z.; Wang, H.; Wang, C.; Liu, Y.; Tang, Y.; Wu, J.; Sun, J.; Wang, H.; Wang, J.; Lu, G. Int J Clin Exp Pathol 2014, 7 (4), 1337-1347. (34) Pouliquen, D.; Le Jeune, J. J.; Perdrisot, R.; Ermias, A.; Jallet, P. Magn Reson Imaging 1991, 9 (3), 275-283.

640

(35) Blum, R. H.; Carter, S. K. Ann Intern Med 1974, 80 (2), 249-259.

641

(36) Wang, D.; Xu, Z.; Yu, H.; Chen, X.; Feng, B.; Cui, Z.; Lin, B.; Yin, Q.; Zhang, Z.; Chen, C.;

642 643

Wang, J.; Zhang, W.; Li, Y. Biomaterials 2014, 35 (29), 8374-8384. (37) Szwed, M.; Laroche-Clary, A.; Robert, J.; Jozwiak, Z. Chem Biol Interact 2014, 220, 35


Biomacromolecules

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

644

140-148 .

645

(38) Swarnakar, N. K.; Thanki, K.; Jain, S. Nanomedicine 2014, 10 (6), 1231-1241.

646

(39) Halupka-Bryl, M.; Asai, K.; Thangavel, S.; Bednarowicz, M.; Krzyminiewski, R.; Nagasaki,

647 648 649 650 651

Y. Colloids Surf B Biointerfaces 2014, 118, 140-147. (40) Lee, G. Y.; Qian, W. P.; Wang, L.; Wang, Y. A.; Staley, C. A.; Satpathy, M.; Nie, S.; Mao, H.; Yang, L. ACS Nano 2013, 7 (3), 2078-2089. (41) Song, X.; Gong, H.; Liu, T.; Cheng, L.; Wang, C.; Sun, X.; Liang, C.; Liu, Z. Small 2014, 10 (21), 4362-4370.

652 653

Table of Contents Graphic and Synopsis. pH-sensitive folate-amphiphilic poly(β-aminoester)

654

hydrophobic side chain self-assembled with lipophilic molecule modified IONPs to load

655

chemotherapeutic drug doxorubicin. This theranostic delivery system was used for gastric

656

cancer treatment and MRI detection.

657 658 659

36


Page 36 of 36

Theranostic, pH-Responsive, Doxorubicin-Loaded Nanoparticles

Recommend Documents