Novel Class of Ultrasound-Triggerable Drug Delivery Systems for the

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A Novel Class of Ultrasound-Triggerable Drug Delivery System for the Improved Treatment of Tumors Chaopei Zhou, Xiangyang Xie, Hong Yang, Shasha Zhang, Yinke Li, Changchun Kuang, Shiyao Fu, Lin Cui, Meng Liang, Chunhong Gao, Yang Yang, Chunsheng Gao, and Chunrong Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00194 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Molecular Pharmaceutics

The working scheme of DOX-loaded NGR/UT-L. Under the navigation effects of NGR, the DOX-loaded NGR/UT-L is accumulated in tumor sites. When the target sites are exposed to ultrasound, DOX should be released from the NGR/UT-L, which is destroyed by the sonodynamic effect. 119x87mm (300 x 300 DPI)

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1

A Novel Class of Ultrasound-Triggerable Drug Delivery

2

System for the Improved Treatment of Tumors

3

Chaopei Zhou

4

Changchun Kuang 2, Shiyao Fu 1, 3, Lin Cui 1, 3, Meng Liang 3, Chunhong Gao3, Yang

5

Yang 3, *, Chunsheng Gao 3, *, Chunrong Yang 1, *

6

1

College Pharmacy, Jiamusi University, Jiamusi 154007, China

7

2

Department of Pharmacy, General Hospital of Central Theater of the PLA, Wuhan 430070,

8

China

9

3State

1, 3, #,

Xiangyang Xie

2, #,

Hong Yang

4, #,

Shasha Zhang 5, Yinke Li 2,

Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of

10

Pharmacology and Toxicology, Beijing 100850, China

11

4

The 4th Affiliated Hospital of Harbin Medical University, Harbin 150001, China

12

5

The 1st Affiliated Hospital of Jiamusi University, Jiamusi 154003, China

13

# These

14

*Corresponding Author. Yang Yang [email protected]; Chunsheng Gao

15

[email protected];

authors contributed equally to this work.

Chunrong

Yang

1


[email protected].

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16

ABSTRACT:

17

The

18

release

19

anticancer drugs

20

at the tumor site

21

is

22

challenge

23

treating

24

To achieve this

25

goal, our strategy

26

was based on tumor-specific targeting and ultrasound-triggered release of an

27

anticancer agent from liposomal nanocarriers. To enhance the ultrasound-triggered

28

drug release, we incorporated a lipophilic sonosensitizer, chlorin e6 (Ce6) ester, into

29

the lipid bilayer of liposomes. Additionally, asparagine-glycine-arginine (NGR) that

30

binds to CD13, which is overexpressed in tumor cells, was introduced into these

31

liposomes. Under the navigation effects of the NGR, the novel ultrasound-triggerable

32

NGR-modified liposomal nanocarrier (NGR/UT-L) accumulates in tumor sites. Once

33

irradiated by ultrasound in tumor tissues, the sonodynamic effect produced by Ce6

34

could create more efficient disruptions of the lipid bilayer of the liposomal

35

nanocarriers. After encapsulating doxorubicin (DOX) as the model drug, the

36

ultrasound triggered lipid bilayer breakdown can spring the immediate release of

37

DOX, making it possible for ultrasound-responsive chemotherapy with great

38

selectivity. By combining tumor-specific targeting and stimuli-responsive controlled

39

release into one system, NGR/UT-L demonstrated perfect antitumor effect. Moreover,

40

this report provides an example of controlled-release by means of a novel class of

41

ultrasound triggering system.

42

KEYWORDS: sonodynamic effect; lipophilic sonosensitizer; ultrasound-responsive

43

release;

controlled

a

of

central in cancer.

dual-targeting;

drug

2


delivery

system;


44

Page 4 of 41

1. INTRODUCTION

45

In the past decades, several nanocarriers (e.g., polymeric nanoparticles) have

46

attracted significant attention for the delivery of drugs to tumor sites by an enhanced

47

permeability and retention (EPR) effect. 1 Nevertheless, the release behaviors in many

48

of these nanocarriers depend on the carriers' self-generated degradations in vivo and

49

cannot adjust the drug release patterns.

50

unexpected drug release during circulation 5 and, therefore, may produce undesirable

51

system side effects. To obtain the maximal therapeutic effects and minimal side

52

effects, an ideal nanocarrier should not release drugs in the noncancerous sites; rather,

53

once they arrive at the targeted sites, the drug should be released in a burst. 6, 7

2-4

These nanocarriers always suffer from

54

To date, various strategies have been successfully used in nanoparticles to

55

control the drug release at targeted sites. Among these strategies, a stimuli-responsive

56

nanocarrier that responds to changes in environmental conditions of targeted sites

57

such as enzymatic activity 8, pH 9, light

58

widely studied for their special superiorities in the accurate control of drug release.

59

However, precise control of drug release in a complicated physiological and

60

pathological environment at the suitable time with an endogenous trigger (e.g.,

61

enzymatic activity or pH) remains a huge challenge. Therefore, it would be reasonable

62

to explore an external triggering strategy which is free from the triggering conditions

63

of the internal tumor microenvironment. Light is a preferable choice for its

64

noninvasive property, obtainable tunability and high spatial resolution. In recent years,

65

several different groups have attempted to fabricate nanocarriers responsive to

10,

or electromagnetic fields

3


11

have been

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66

light-triggered photothermal or photodynamic effects in order to realize controlled

67

release. 12 In one example, Gao et al. developed a photosensitizer-decorated red blood

68

cells (RBCs)-based drug delivery system, which responded to light-induced

69

photodynamic effects to detach drugs encapsulated in cells. 13 Upon light illumination,

70

such photosensitizer-decorated RBCs could be immediately wrecked due to the

71

photodynamic disruption of the RBC membrane and thus release the drugs. However,

72

the above nanocarriers still need improvement. First, light-irradiation is commonly

73

restrained to superficial tissues, though internal tissues could be approached with the

74

help of laparoscopy. Hence, the application of light to trigger drug release in vivo may

75

be restricted. Second, although RBCs are recognized as autologous by the body, the

76

preparation and purification procedures for RBCs-based drug delivery systems are

77

inefficient, and their manufacture scale-up have difficulties in robust and reproducible

78

processes.

79

Unlike visible light, ultrasound is a kind of mechanical wave that can reach to the

80

cancer site buried deep in human tissues. In recent years, ultrasound-triggered drug

81

delivery systems have received extensively attentions for their usage in drug delivery

82

and release due to this feature. Nanobubbles, which consist of a gas-filled core

83

encapsulated by a shell made from biologically compatible materials, are widely

84

employed in biomedicine fields such as ultrasound imaging and drug delivery

85

system.14 Although conventional ultrasound-responsive bubbles could enhance the

86

drug release efficacy to a certain degree, its applications would be limited as the core

87

gas would diffuse out of the bubbles during the storing time. 15 Therefore, there is a

4



Page 6 of 41

88

requirement for a novel class of ultrasound-triggerable drug delivery system to have

89

reinforced controlled-release and stability of storage. Recently, sonodynamic therapy

90

(SDT) has become an option to the mature technology of photodynamic therapy (PDT)

91

in the field of anti-tumor therapy.

92

ultrasound and generate many singlet oxygen (1O2) and other reactive oxygen species

93

(ROS, such as superoxides, hydroxyl-radicals, and hydrogen peroxides).18

94

Consequently, Ce6 has been used for PDT or SDT in the clinic.

95

liposomes are one of the few nanomedicine products that have obtained approval for

96

clinical treatment of tumors. The aqueous lumen and lipid bilayer of liposomes

97

enables encapsulation of hydrophilic and hydrophobic molecules, respectively. The

98

above reports provide a novel theoretical foundation for ultrasound-triggered drug

99

release.

16, 17

Chlorin e6 (Ce6) can strongly absorb

19, 20

To date,

100

Herein, we developed a unique type of liposomal drug delivery system, which

101

can response to ultrasound-stimulated sonodynamic effects and thus efficiently

102

releasing the drugs. In this system, lipophilic Ce6 ester was mixed with lipids 21, 22

103

(according to the principle of similar miscibility) to prepare ultrasound-triggered

104

liposomes (UT-L). Upon exposure to ultrasound, the UT-L could disrupt immediately

105

due to the Ce6 ester induced destruction of the lipid bilayer of the liposome. After the

106

encapsulation of doxorubicin (DOX), a classical anti-tumor drug used in oncology, in

107

the inner hydrophilic capsule of the UT-L, the prepared DOX-loaded UT-L showed an

108

instant release feature of DOX when exposed to ultrasound. Although the UT-L could

109

enhance the drug delivery selectivity to some degree, surface modification for the

5


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110

UT-L with special ligands were required to achieve the active targeting. Many kinds

111

of tumors over-express the tumor vascular antigen aminopeptidase N (CD13).

112

Therefore,

113

Asparagine-Glycine-Arginine (NGR) peptide has been reported has specific affinity to

114

CD13.

115

DSPE-PEG2000-MAL, and then such conjugate was inserted into UT-L to prepare

116

NGR-modified ultrasound-triggered liposomes (NGR/UT-L) for loading DOX. The

117

working scheme of DOX-loaded NGR/UT-L is shown in Figure 1. Under the CD13

118

affinity of NGR, the DOX-loaded NGR/UT-L will accumulate within tumors. When

119

the target sites are exposed to ultrasound, DOX should be released from the

120

NGR/UT-L, which is destroyed by the products of Ce6 ester. In this work, the

121

physicochemical properties of NGR/UT-L were characterized, and its biological

122

characters and anti-tumor efficiencies were assessed in vitro and in vivo.

123

2. EXPERIMENTAL SECTION

24

CD13

In

is

this

a

suitable

paper,

the

target

NGR

for

peptide

tumor

was

drug

firstly

delivery.

coupled

23

The

with

124

2.1. Materials. NGR peptide (CYGGRGNG) with a cysteine on the N-terminal

125

(Cys-NGR) was provided by Cybertron medical technology Co. (Beijing, China).

126

Hydrogenated soy phosphatidylcholine (HSPC) and cholesterol (Chol) were procured

127

from

128

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy

129

(ammonium

130

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide (polyethylene glycol)

Lipoid

GmbH

(Mannheim,

salt)

Germany). (polyethyleneglycol)

(DSPE-mPEG2000)

6


and


131

(DSPE-PEG2000-Mal) were provided by Xi`an ruixi Biological Technology Co., Ltd

132

(Xi`an, China). Chlorin e6 (Ce6) ester was procured from J&K Scientific Inc (Beijing,

133

China). Doxorubicin hydrochloride (DOX) was purchased from Haizheng Co.

134

(Zhejiang, China). Other reagents were all of analytical grade and provided by

135

Millipore Sigma.

136

Human fibrosarcoma cells (HT-1080 cells) supplied by Cell Resource Centre of

137

IBMS (Beijing, China) were cultured in modified eagle’s medium (MEM)

138

supplemented with

139

mg/mL). These cell lines were cultured in a incubator at 37 °C in a 5% CO2

140

humidified atmosphere.

FBS (10%, V/V), penicillin (100 IU/mL), and streptomycin (100

141

Sprague-Dawley (SD) rats (male, 200 ± 20 g) and BALB/c nude mice (male, 20

142

± 2 g) were provided by the laboratory animal center of Jiamusi University (Jiamusi,

143

China). The animal experiments were approved by the Animal Care and Use Ethical

144

Committee of Medicine School in Jiamusi University. All animals used in this study

145

were handled in accordance with the guidelines of stated by this organization.

146

2.2. Synthesis and Characterization of Conjugates. NGR was mixed with

147

DSPE-PEG2000-Mal (1.5: 1 molar ratio) in chloroform included triethylamine (5 eq.),

148

kept stirring for 24 h under room temperature (20-25°C).10 The reactants were

149

dialyzed against deionized water in dialysis bag (molecular weight cutoff was 3.5 kDa)

150

for 48 h to remove the chloroform and excess NGR. Then the solutions were

151

lyophilized and kept at -20 °C for further usage. The product was confirmed by

152

assaying the molecular weight of the obtained DSPE-PEG2000-NGR through a 7


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153

matrix-assisted laser desorption ionization time of flight mass spectrometry

154

(MALDI-TOF MS).

155

2.3. Preparation of Liposomal Nanocarriers. To prepare the normal

156

liposome (N-L), a lipid mixture of HSPC (50%), cholesterol (43%) and

157

DSPE-PEG2000 (7%) was used (at molar ratio). All lipids were dissolved in

158

chloroform-methanol (3:1, v/v) in a flask, and then the mixture was evaporated under

159

reduced pressure to obtain a thin film on the flask. After this, citric acid buffer

160

solutions (300 mM) were added in the flask and kept rotating for 30 min under 50 °C.

161

The consequent dispersion was then extruded 11 times through polycarbonate

162

extrusion membranes (100 nm diameter pores) via an Avanti® Mini-Extruder (Avanti,

163

Canada). The ultrasound-triggered liposome (UT-L) was prepared following the same

164

procedures, except the Ce6 ester was added to these lipid mixtures at the required

165

molar ratio (1 %, 2 %, 4 %, 6 % or 8 % Ce6 ester of total lipid). Post-insertion method

166

was applied to prepare the modified liposomes (NGR/UT-L). Briefly, the UT-L

167

suspensions (4 % Ce6 ester of total lipids) were heated to 50 °C and kept for 30 min

168

in a water bath, then cooled to 20~25°C, added into the flask with

169

DSPE-PEG2000-NGR at the required molar ratio (1 %, 2 %, 4 %, 6 % or 8 %

170

DSPE-PEG2000-NGR of total lipid) in methanol. This flask with contents was kept 37

171

°C for 2 h in a water bath. Finally, DOX were encapsulated into the above prepared

172

liposomes through a pH gradient method with a drug/lipid mass ratio of 1:10 as

173

reported previously. 25 Briefly, blank liposomal suspensions were added into the citric

174

acid buffer solutions (300 mM), and DOX was added into this mixture system (2.0 8



175

mg/mL); then 100mM Na2CO3 solutions were used to adjusted the pH to 7.5, kept at

176

55 °C for 30 min. In the end, the drug-contented liposomal suspensions were filtrated

177

via a 100 nm filter and dispensed into a vials.

178

2.4. Evaluation of Ultrasound Sensitivity. 2.4.1 Choice of Acoustic

179

Parameters. DOX-loaded UT-L (with 4 % Ce6 ester) was diluted 20-fold with PBS

180

(0.1 M, pH 7.4) and kept at 37 °C for 5 min, then transferred quickly to the HUT-105

181

ultrasonic system (Huazhong Institute of Biomedical Engineering, China). After

182

treating with ultrasound irradiation for 200 s (1 MHz, 2 W.cm-2 power density,

183

sonicated 10 s and paused 10 s), the liposomal suspensions were allowed to stand for

184

5 min and then centrifuged, and the DOX contents were measured by an HPLC

185

method as described in a previous report.

186

methanol-water-acetic acid (70: 30: 0.25) was used to separate the drugs in a C18

187

column. The flow rate of the mobile phase was set to 1.0 mL·min-1, and the detection

188

wavelength was set to 233 nm.

26

The mobile phase consisted of

189

2.4.2. Ce6 Ester Proportion Screening. The released DOX from the

190

DOX-loaded UT-L, which contained the Ce6 ester at different proportions (1 %, 2 %,

191

4 %, 6 % or 8 % Ce6 ester of the total lipid) was determined followed the same

192

procedures as described above, except the acoustic parameters were partially

193

substituted by a total of 120 s.

194

2.5. Optimization of Targeting Peptide Density. To explore the influence of

195

NGR peptide amounts on cell uptake, NGR/UT-L labeled by Cy5.5 was prepared with

9


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196

various NGR peptide ratios (1 %, 2 %, 4 %, 6 % or 8 % DSPE-PEG2000-NGR of total

197

lipids). HT-1080 cells were seeded into a 6-well plate at a density of 5×105 cells per

198

well and incubated for 24 h at 37 °C. Then, the cells in wells were cultured with

199

various liposomes and cultured for 2 h. After that, cells were washed with cold PBS,

200

trypsinized and cold PBS (3 times). After centrifugation, the cells were separated and

201

resuspended with PBS. Finally, around 105 cells were sent into the BD-FACSCalibur

202

flow cytometry (FCM) with the Cy5.5 of 150 ng.mL-1.

203

2.6. Post insertion Efficiency of Targeting Peptides. The function material

204

with tumor targeting ability, DSPE-PEG2000-peptide-fluorescence probe, was

205

synthesized via the reaction between the carboxyl group (fluorescence probe) and the

206

primary amine group (DSPE-PEG2000-peptide). Accordingly, in the present study,

207

DSPE-PEG2000-NGR was labeled with the fluorescin group, 5-(6)-carboxyfluorescein

208

diacetate (CFDA), as our previously described.27 After dialysis and lyophilization,

209

powders of the final conjugates (DSPE-PEG2000-NGR-CFDA) with purification were

210

obtained. The fluorescence probe-labeled NGR-modified UT-L (NGR-CFDA/UT-L,

211

with 6% of DSPE-PEG2000-NGR-CFDA at molar ratio) was synthesized through

212

similar preparation process used in NGR/UT-L, apart from the fact that the

213

DSPE-PEG2000-NGR was replaced with DSPE-PEG2000-NGR-CFDA. After the

214

determination

215

ultraviolet-visible spectrophotometer was employed to measure the total absorbance

216

(ATotal) of NGR-CFDA/UT-L. After this, the samples were immediately centrifuged,

217

and the absorbance (AFree) of free DSPE-PEG2000-NGR-CFDA was detected. Ligand

the

characterizing

absorption

wavelength

10


of

CFDA,

the


218

connecting efficiency was calculated as follows:

219

Connection efficiency = (ATotal - AFree)/ATotal ×100 %

220

2.7. Characterization of Nanocarriers. Transmission electron microscopy

221

(TEM) was used to determine the morphologic properties of NGR/UT-L. The zeta

222

potential and mean diameter of prepared liposomes were characterized by dynamic

223

light scattering via a Nano ZS90 Malvern Zetasizer (Malvern Instruments Ltd., U.K.).

224

The DOX encapsulation efficiency (EE) of drug-loaded liposomes was analyzed by an

225

HPLC method reported previously. 26 The liposomes were ultra-centrifuged at 30,000

226

rpm (4°C) for 1 hour, then remove the supernatant and DOX quantity was measured

227

by the HPLC.

228

The long term storage stability of DOX-loaded NGR/UT-L was evaluated. The

229

liposomal nanocarriers were preserved in ampoule bottles under 4°C without any

230

other treatments and sample bottles were regularly taken out for assay. The analysis

231

parameters, such as diameter, PDI, EE and ultrasound stimulated release tests were

232

carried out every three months.

233

2.8. In Vitro Release Characterization. The in vitro DOX release patterns of

234

different DOX-loaded liposomal nanocarriers were determined in the dialysis bag.

235

Briefly, the liposomal DOX sample (1 mL) was added into a dialysis bag (the

236

molecular weight cut off was 12-14 kDa) and dialyzed in 20 mL of PBS (0.1 M, pH

237

7.4) with continuous gentle stirring at 37 °C, then imposed ultrasound treatment or not,

238

as described above. At predetermined time, 400 μL of samples were obtained from the

239

glass bottles for drug assessment, and then 400 μL of the fresh medium was 11


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240


replenished. The released DOX was measured by the HPLC as described above.

241

2.9. Cellular Uptake. To study the cell binding affinity of NGR/UT-L, various

242

liposomal formulations containing Cy5.5 (5 μM) (with or without pretreatment with

243

ultrasound, 1 MHz, 2 W.cm-2, sonicated 10 s and paused 10 s for a total of 120 s) were

244

applied (at 37 °C) to HT-1080 cells in petri dishes for 2 h. For free NGR pre-treated

245

group, 2 μM free NGR was added and for 30 min. Then, the petri dishes were washed

246

with PBS (~4 °C) for 3 times. After that, the cell suspensions were centrifugated and

247

resuspend in PBS, then the samples were determined by a confocal laser scanning

248

microscopy (CLMS) (UltraVIEW Vox, PerkinElmer) and assessed quantitatively

249

through the flow cytometer.

250

2.10. In Vitro Cytotoxicity Analysis. MTT assays were used to analyze the

251

cell viability of HT-1080 cells against different DOX-loaded liposomal formulations

252

and free DOX. Briefly, HT-1080 cells were cultured in 96-well plates at a density of

253

around 4000 cells per well, and incubated for 24 h at 37 °C. Then, different

254

DOX-loaded liposomal formulations or free DOX at various concentrations were

255

added into the cell wells. Two hours later, those liposomal formulations added groups

256

(DOX-loaded NGR/UT-L, DOX-loaded UT-L and DOX-loaded N-L) were treated

257

with or without ultrasound, as mentioned above. Then, 20 μL of MTT reagent (5 mg.

258

mL-1) were injected into the plates after 72 h. After a incubation of 4 h, the UV

259

absorbance of plate wells was measured by a Model 680 plate reader (BIO-RAD) and

260

the cell viability (%) was consequently calculated.

12



261

2.11. Pharmacokinetic Studies. The pharmacokinetics of DOX-loaded N-L,

262

DOX-loaded UT-L and DOX-loaded NGR/UT-L were performed and the same

263

concentration of free DOX was used as a control, using male SD rats. These samples

264

were administered via the vein with the DOX dose of 5 mg. kg-1. The blood samples

265

were withdrawn from rat retro-orbital sinus at predetermined time points. After

266

obtaining the blood samples, the plasmas were separated from the blood, and 0.1 mL

267

of daunorubicin hydrochloride solutions (as internal standard (I.S.) solution, 40 ng.

268

mL-1) were dropped into a 1.5 ml tube with 100 µL of plasma. Then, 0.8 mL of

269

methanol was added into to remove the proteins in plasma and vortexed for

270

approximately 1 min. Next, the mixture was centrifuged (12,000 rpm for 10 min) and

271

800 µL of supernatants were obtained. Then, the supernatants were treated with a

272

concentrator under 37 °C. The resulting residue in tube was added with mobile phase

273

(100 µL), vortexed, and centrifuged (12,000 rpm for 10 min). The supernatants (20

274

µL) from congregation were loaded into a LC/MS/MS for determination as described

275

previously.28 Briefly, the samples were analyzed on a C18 column under

276

reversed-phase gradient conditions (mobile phase A: water with 0.1% formic acid;

277

mobile phase B: methanol with 0.1% formic acid). Detection was performed on a

278

Thermo Scientific™ TSQ Vantage™ triple quadrupole mass spectrometer using

279

positive polarity, heated electrospray ionization (HESI) conditions operating in the

280

selected reaction monitoring (SRM) mode.

281

2.12. Concentration of DOX in the Solid Tumor. Tumor targeting properties

282

of DOX-loaded NGR/UT-L were performed on nude mice bearing tumor xenografts 13


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283

and, compared with that of DOX-loaded UT-L, DOX-loaded N-L and free DOX. The

284

xenograft tumor model was established by subcutaneously injecting HT-1080 cells as

285

reported previously.

286

treated as follows via intravenous administration: free DOX; DOX-loaded N-L;

287

DOX-loaded UT-L (with ultrasound); DOX-loaded NGR/UT-L (with ultrasound);

288

DOX-loaded UT-L (without ultrasound); and DOX-loaded NGR/UT-L (without

289

ultrasound) at the DOX dosage of 5 mg.kg-1, respectively. After all the injections (30

290

min later), the tumor bearing mice injected with DOX-loaded NGR/UT-L or

291

DOX-loaded UT-L were anesthetized, and their tumor locations of skin were daubed

292

with EcoGel 100 Imaging Ultrasound Gel (Eco-Med Pharmaceutical Inc. Mississauga,

293

Ontario, Canada) to 1.0 cm thickness. Next, the gel sites (0.8 cm2) were deal with the

294

ultrasound probe (1 MHz, 2 W.cm-2, sonicated 10 s and paused 10 s for a total of 120

295

s) of HUT-105 sonication system (Huazhong University of Technology, Wuhan,

296

China). And the other groups that were not subjected to ultrasound were used as

297

controls. An hour later, 3 mice were withdrawn from very group and sacrificed to

298

collect the tumor tissues, which were kept at −20 °C for further assay. To detect the

299

DOX concentrations of samples, the tumor tissues were mixed with 3-fold volumes of

300

deionized water and homogenized under 4°C. Then, added daunorubicin

301

hydrochloride solutions and methanol into the homogenate and vortexed for 1 min.

302

Finally, the mixtures were centrifuged and their supernatant were injected into the

303

LC-MS/MS for assessment as reported previously.28

304

10

The mice were randomly divided into 6 groups and were

2.13. In Vivo Antitumor Evaluation. Once the tumor volumes came to about 14



Page 16 of 41

305

200 mm3, the model animals were administrated with (via tail vein)

306

(control), free DOX, DOX-loaded N-L, DOX-loaded UT-L and DOX-loaded

307

NGR/UT-L (at the drug dosage of 10 mg/kg) on the day 6, 9, 12 and 15. When the

308

injection was completed 30 min later, the groups cured with DOX-loaded UT-L and

309

DOX-loaded NGR/UT-L were treated with the ultrasound described in "Section 2.12".

310

Tumor volumes and body weights were recorded. The estimated tumor volume was

311

reckoned as: tumor volume (mm3) = (tumor length×tumor width2)/2.

5% glucose

312

2.14. Statistical Data Treatment. All data are described as the averaged data

313

± standard deviations. Statistical significance (P 0.05). Thus, further experiments were

337

performed using 120 s of ultrasound stimulus.

29, 30

Different from the light,

338

To confirm the specific requirement of sonoactivated Ce6 for the observed

339

sonotriggering and DOX release from UT-L, we tested DOX-loaded N-L (with 0 %

340

Ce6 ester) and UT-L (with 4 % Ce6 ester) under identical conditions. As shown in

341

Figure S1 B, without ultrasound stimulus, the DOX encapsulated N-L (with 0 % Ce6

342

ester) or UT-L (with 4 % Ce6 ester) displayed a minor drug release (less than 2%) in

343

the testing medium during the initiate 5 min of incubation under 37 °C. In contrast,

344

UT-L (with 4 % Ce6 ester) showed a substantial amount (over 90 % of DOX) release

345

after treatment with ultrasound irradiation, whereas only a little of the DOX was

346

released from N-L (with 0 % Ce6 ester) confirming that Ce6 was essential for

347

sonotriggering. Since the content of Ce6 ester in liposomes was clearly a key factor

16



348

that influenced the release efficiency of UT-L, the release of DOX from different

349

liposomal formulations with various Ce6 ester proportions (1 %, 2 %, 4 %, 6 % and 8

350

% Ce6 ester of total lipid) were evaluated to optimize the liposomal formulations. For

351

the results exhibited in Figure 3, when the Ce6 ester proportion reached 2 % or below

352

in the UT-L there was an unsubstantial amount released after treating with ultrasound

353

irradiation. The results indicated that when the proportion of Ce6 ester in the UT-L

354

was lower, Ce6 could not generate a sufficient sonodynamic effect under

355

ultrasound-irradiation to effectively disrupt the lipid bilayer structures of the

356

nanocarriers, and thus, the release efficiency of UT-L was not ideal. When the Ce6

357

ester proportion reached 4 %, upon ultrasound irradiation, over 90 % of the DOX was

358

immediately released from the UT-L. However, the amount of release decreased

359

slowly as the molar ratio of Ce6 ester increased from 6 % to 8 %, indicating that the

360

acoustic parameters (1 MHz, 2 W.cm-2, 10 s of ultrasound and 10 s of stop repeated

361

for a total of 120 s) were insufficient to entirely trigger all of these Ce6. This implied

362

that Ce6 ester may possess a stabilizing effect on the liposomal wall. Excess Ce6

363

esters may absorb much of the ultrasonic energy, but cannot produce sufficient singlet

364

oxygen or other reactive oxygen species to disrupt the liposomal walls. In other words,

365

when a certain amount of sonosensitizer was added into the formulation of UT-L

366

(with 4 % Ce6 ester), it would bring about the maximal drug release after ultrasound

367

stimulation. The results demonstrated that the Ce6 ester proportion in the UT-L for 4

368

% appeared to be sufficient to generate a sonodynamic effect for those liposomes

369

under the acoustic conditions to allow for a burst release of the DOX loaded inside the

17


Page 18 of 41

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370

liposomes (Figure S2). Therefore, a molar proportion of 4 % for Ce6 ester was chosen

371

for subsequent studies.

372

3.3. Influence of Peptide Density. As the density of NGR in nanocarriers is a

373

crucial element influencing the tumor delivery efficiency of NGR/UT-L, the

374

formulation with different NGR ratios for the preparation of Cy5.5-labeled

375

NGR/UT-L were assayed in HT1080 cells, and the index of cellular uptake for

376

NGR/UT-L was utilize to gain the optimum liposomes. As shown in Figure 4, the

377

uptake index of fluorescein-labeled liposomes would be improved remarkably when

378

the

379

DSPE-PEG2000-NGR ratio kept increasing to 8%, no more notable improvement of

380

uptake was found as compared to that of 6% (P > 0.05). The saturable absorption of

381

CD13 to NGRs may explain this. Restrained by the receptor amount and recycling

382

times, receptor-mediated endocytosis is a saturable pathway31 that limits the cell

383

uptake

384

DSPE-PEG2000-NGR was a good choice for subsequent study.

DSPE-PEG2000-NGR

numbers

of

molar

NGR/UT-L.

ratios

In

increased

summary,

(0%-6%).

the

6%

When

molar

ratio

the

of

385

3.4. Connecting Efficiency of Modification Peptides onto Liposomes. In

386

order to improve their tumor targeting ability, UT-L were functionalized with NGR

387

peptides in this study. However, at present it is very difficult to directly count the

388

numbers of modified peptides that are connected onto the surfaces of nanoparticles,

389

due to the large molecular weights of carrier matrix. To overcome this problem, an

390

indirect but simple method was proposed here to quantitatively measure the insertion

391

efficiency of DSPE-PEG2000-NGR that stuck to the UT-L. After the comparing the 18



392

UV spectrums between DSPE-PEG2000-NGR-CFDA and UT-L, the maximum UV

393

absorption of DSPE-PEG2000-NGR-CFDA at 493 nm was found not interfere with

394

UT-L. Therefore, 493 nm was selected as the detective wavelength to perform the

395

assay. The equation of working curve was A = 0.1952C + 0.0063 (R2 = 0.9991, n = 5)

396

under the concentration rang of 1.1-4.3 μg·mL−1. Based on this standard curve, the

397

calculated connecting efficiency of NGR onto the NGR/UT-L was 70.87 %.

398

3.5. Characterization and In vitro Release of Liposomes. Figure 5 A

399

displayed the conventional parameters of the three prepared liposomal nanocarriers

400

after related analysis. The DOX encapsulation efficiency of the three liposomal

401

nanocarriers were larger than 90 %. The results indicated that the surface modification

402

with NGR and lipids mixed with Ce6 ester did not influence the final EE. For

403

nanocarriers, their particle size is an important factor that would greatly influenced the

404

behaviors of nanocarriers in vitro and in vivo. After the encapsulation efficiency assay,

405

the particle size of these liposomal nanocarriers was measured via a laser particle

406

analyzer. Also exhibited in Figure 5 A, the particle sizes of N-L, UT-L and

407

NGR/UT-L were between approximately 91.35 ± 1.08 nm and 93.23 ± 1.85 nm. This

408

implied that the particle sizes of N-L, UT-L and NGR/UT-L were not remarkably

409

influenced by the attached NGR peptide. The TEM image of NGR/UT-L in Figure 5

410

B showed that the particle size obtained from TEM was consistent with that from the

411

laser particle analyzer (Figure 5 C).

412

The in vitro release of DOX from different nanocarriers via sonication is

413

displayed in Figure 5 D. These data demonstrated that the DOX releases of 19


Page 20 of 41

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414

NGR/UT-L or UT-L exhibited an ultrasound-dependent characteristic. When

415

NGR/UT-L or UT-L were exposed to ultrasound, liposomes were effectively

416

disrupted by the products of Ce6 ester, leading to burst release of DOX from those

417

samples. In addition, the DOX release behaviors between the above two groups were

418

similar (P > 0.05), which showed that the attachments of NGR to UT-L did not

419

influence the final release results. In contrast, Figure S2 demonstrates that there was

420

minimal release (less than 6 %) from DOX-loaded NGR/UT-L or UT-L throughout

421

the course of a 24-h incubation in medium without ultrasound stimulus. However,

422

when these samples were subjected to ultrasound irradiation at the 25th h, complete

423

release occurred, implying the release controlling ability of the sonosensitizer. It was

424

illustrated that NGR/UT-L or UT-L could be stably presented until exposing to the

425

ultrasound irradiation. Therefore, it was anticipated that no drug would be released

426

into circulation from these ultrasound-responsive nanocarriers before the application

427

of ultrasound.

428

The stability tests showed that DOX-loaded NGR/UT-L was physically and

429

chemically stable at 4 °C up to 3 months. As illustrated in Figure S3 A, no remarkable

430

change in EE of DOX-loaded NGR/UT-L was observed during the course of the

431

stability study. The size of DOX-loaded NGR/UT-Ls was still near to 96 nm, and its

432

polydispersity index was also close to 0.07. These suggested that the tested carriers

433

remained its monodisperse properties. The ultrasound-triggered DOX release rate

434

from DOX-loaded NGR/UT-L remained relatively stable during storage. As shown in

435

Figure S3 B, upon ultrasound irradiation, over 90 % of the DOX was released from

20



436

DOX-loaded NGR/UT-L. The results indicated that the DOX-loaded NGR/UT-L kept

437

its ultrasound responsive profiles confirmed in the previous tests after the storage. The

438

stable DOX-loaded NGR/UT-L would thus be favorable for further applications in the

439

clinic.

440

To explore the drug release mechanism of UT-L, singlet oxygen and ROS were

441

detected in Ce6 ester (Figure S5). These results proved that Ce6 ester could absorb

442

ultrasound and produce singlet oxygen and ROS, which may reacted with the

443

liposome wall and undermine it, thus leading to the drug release from the liposome.

444

This mechanism was different from the conventional ultrasound responding liposomes,

445

which was determined by the effects of ultrasonic cavitation.

446

3.6. Analysis of Cellular Uptake. After confirming that the NGR/UT-L could

447

release drugs appropriately by ultrasonic irradiation, its capacity to transport payloads

448

into the tumor cells was verified in this section. According to the design strategy, the

449

NGR/UT-L could be efficiently accumulated in tumor sites through the NGR motif.

450

To test this hypothesis, the CD 13-positive cells of HT-1080 cells were chosen to

451

estimate the targeting efficiency of NGR. As exhibited in Figure 6 A, Cy5.5-labeled

452

NGR/UT-L (without ultrasound) displayed more fluorescence than that of N-L and

453

UT-L (without ultrasound) in HT-1080 cells, which illustrated the uptake

454

enhancement effects of NGR to liposomes. To evaluate the competitive affinity of

455

NGR/UT-L (without ultrasound) to HT-1080 cells, superfluous free NGRs (1 mg.mL-1)

456

were introduced into the culture media before the addition of the nanocarriers. Results

457

revealed that the cell uptake of liposomes was remarkably inhibited by the excess 21


Page 22 of 41

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458

NGR peptides, for the fluorescence of NGR/UT-L (without ultrasound) reduced to a

459

low levels as N-L and UT-L (without ultrasound) (Figure 6 B). This suggested that if

460

the surface CD 13 expression level of HT-1080 cells reduced, NGR/UT-L (without

461

ultrasound) would not be able to effectively attach with the HT-1080 cells by the

462

NGR ligands, hence the cell uptake efficiency of NGR/UT-L (without ultrasound)

463

would decreased. This experiment confirmed the NGR effects on the cellular uptake.

464

3.7. Cytotoxicity. The cytotoxicities of free DOX and various DOX-loaded

465

liposomal formulations with or without ultrasound stimulus were assessed in the

466

above mentioned cells by an incubation of 72 h. The IC50 of free DOX, DOX-loaded

467

N-L (without ultrasound), DOX-loaded UT-L (without ultrasound), DOX-loaded

468

NGR/UT-L (without ultrasound), DOX-loaded N-L (with ultrasound), DOX-loaded

469

UT-L (with ultrasound) and DOX-loaded NGR/UT-L (with ultrasound) were 18.72

470

ng/ml, 15817 ng/ml, 14564 ng/ml, 3089 ng/ml, 12592 ng/ml, 28.32 ng/ml, 11.17

471

ng/ml, respectively.

472

As displayed in Figure S4, without ultrasound irradiation, free DOX could bring

473

about the greatest anti-proliferative effect on HT-1080 cell. This phenomenon may be

474

explained as follows: compared with the other DOX formulations, the drug molecules

475

in free DOX formulation have less restriction in vitro, thus the DOX molecules could

476

rapidly spread into the cells via passive diffusion. In contrast, drug-loaded liposomal

477

formulations without ultrasound needed to undergo the drug release process (Figure 5

478

D and Figure S2) after their intracellular entry. Thus, free DOX showed the strongest

479

anti-proliferative effect among these liposomal formulations without ultrasound 22



480

irradiation, due to the higher cellular uptake efficiency of free DOX. Compared with

481

DOX-contained N-L and DOX-contained UT-L (without ultrasound), the delivery of

482

DOX by the NGR/UT-L (without ultrasound) significantly increased with its

483

cytotoxicity. This result revealed that the targeting contributions of NGR peptides to

484

the modified liposomes, it could promote the anti-proliferative effects of NGR/UT-L

485

on HT-1080 cell. These findings further support the findings about cell uptake of

486

liposomal nanocarriers shown in Figure 6. Following treatment with ultrasound

487

irradiation, compared with N-L regardless of ultrasound exposure, DOX-loaded

488

NGR/UT-L (with ultrasound) and DOX-loaded UT-L (with ultrasound) displayed a

489

significant improvement in cytotoxicity, which was near to the level as the free DOX

490

had. This suggested that DOX was burst released by ultrasound stimulus. The above

491

results demonstrated that the anti-proliferative activities of DOX-loaded NGR/UT-L

492

and DOX-loaded UT-L were greatly depended on the existences of ultrasound

493

irradiation. However, DOX-loaded N-L was not affected by treatment with ultrasound.

494

The above discoveries consisted with the facts found in the in vitro drug release as

495

exhibited in Figure S1 B.

496

So far, the results found in the in vitro experiments have approximately verified

497

several points of our strategy. To validate the practical targeting effects of the

498

designed drug delivery system, in vivo investigations are warranted.

499

3.8. Pharmacokinetic and Tumor Targeting Property. The blood

500

concentration-time curve and related pharmacokinetic parameters of DOX after

501

intravenous injection of free DOX and DOX-contained liposomal nanoparticles 23


Page 24 of 41

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502

without ultrasound are shown in Figure 7 A and Table S1. It was found that

503

DOX-loaded N-L, DOX-loaded UT-L (without ultrasound) and DOX-loaded

504

NGR/UT-L (without ultrasound) displayed resembling pharmacokinetic curves. Both

505

the

506

nonmodified liposomal nanocarriers (DOX-loaded UT-L and DOX-loaded N-L)

507

demonstrated relatively long lasting blood drug levels, while the blood drug

508

concentration of free DOX decreased rapidly indicated a quick clearance in vivo.

509

Furthermore, DOX-loaded liposomes demonstrated a notably smaller clearance rates

510

(CL) and larger AUC values than that of free DOX. As expected, no remarkable

511

difference between CL and AUC were found among these liposomal nanocarriers.

512

This suggests that the modification of NGR peptides of liposomes did not alter the

513

long-circulation characteristics of PEG.

NGR-modified

liposomal

nanocarriers

(DOX-loaded

NGR/UT-L)

and

514

To validate whether the prepared DOX-loaded NGR/UT-L possessed the tumor

515

targeting ability in vivo, concentrations of DOX in solid tumors were evaluated in

516

HT-1080 cell bearing nude mice following intravenous injection of free DOX,

517

DOX-contained N-L, DOX-contained UT-L and DOX-contained NGR/UT-L with or

518

without ultrasound, respectively. As displayed in Figure 7 B, the DOX concentration

519

in tumors of DOX-contained NGR/UT-L (with ultrasound) were 3.9 fold of free DOX,

520

2.5 fold of DOX-contained N-L (without ultrasound), 2.5 fold of DOX-contained

521

UT-L (without ultrasound), 1.8 fold of DOX-contained NGR/UT-L (without

522

ultrasound) and 1.3 fold of DOX-contained UT-L (with ultrasound). The above results

523

suggested that DOX-contained NGR/UT-L (with ultrasound) achieved a higher DOX

24



524

content in tumors than that of DOX-contained N-L (without ultrasound),

525

DOX-contained NGR/UT-L (without ultrasound) and DOX-contained UT-L (without

526

ultrasound), probably owing to the ultrasound triggered release at tumor sites. In other

527

words, DOX-contained NGR/UT-L combined with ultrasound could cause drug

528

targeted release at tumor sites. In addition, different from DOX-contained UT-L (with

529

ultrasound) was that DOX-contained NGR/UT-L (with ultrasound) modified with the

530

NGR peptides had a remarkably improved tumor site targeting ability, which led to a

531

higher DOX concentration in tumors. In addition, as showed in Figure S6, the

532

maximum DOX accumulation time for the prepared carriers to the tumor tissues was

533

around 30 min, thus this time was chosen to start the ultrasound treatment after i.v

534

injection.

535

Taken together, DOX-loaded NGR/UT-L (with ultrasound) possesses desirable

536

pharmacokinetic and tumor-distribution profiles, which makes it appropriative for the

537

targeted tumor delivery in vivo.

538

3.9. In Vivo Antitumor Activity. To determine whether DOX-loaded

539

NGR/UT-L (with ultrasound) possess the tumor inhibition effects in vivo, the

540

anti-tumor efficiency of free DOX, DOX-loaded N-L, DOX-loaded UT-L (without

541

ultrasound), DOX-loaded UT-L (with ultrasound), DOX-loaded NGR/UT-L (without

542

ultrasound) and DOX-loaded NGR/UT-L (with ultrasound) were investigated in

543

animal models. As exhibited in Figure 8 A, the tumor volumes of mice in the control

544

group (injected with 5% glucose) grew swiftly after 18 days, while the tumor volume

545

growth of free DOX or the liposomes demonstrated tumor suppression effects. 25


Page 26 of 41

Page 27 of 41 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


546

Compared with DOX-loaded N-L (without ultrasound) and DOX-loaded UT-L

547

(without ultrasound), DOX-loaded NGR/UT-L (without ultrasound) exhibited a mild

548

enhancement in tumor suppression, whereas DOX-loaded UT-L (with ultrasound)

549

exhibited a more potent tumor suppression efficiency. As expected, the maximal

550

tumor suppression effect was found in the group injected with DOX-contained

551

NGR/UT-L (with ultrasound). This was in accordance with the abovementioned

552

results illustrating the merit of NGR/UT-L (with ultrasound) over the other liposomal

553

nanocarriers estimated in cytotoxicity in vitro (Figure S4) and tumor-targeting

554

distribution in vivo (Figure 7 B), suggesting the mechanism efficacy of combining the

555

ultrasonic stimulation and NGR-mediated selectivity. Similar results were found in

556

the digital photos and H&E stain of the tumor (Figure S7 and S8).

557

The body weight alterations of the model mice were collected as an index of

558

safety evaluation. As displayed in Figure 8 B, there was no remarkable changes in the

559

body weights of different groups of mice treated with DOX-loaded liposomal

560

nanocarriers in this study (P > 0.05). This result implied that the acute or severe

561

toxicity of DOX-loaded liposomes at the present dose were minor. However, in the

562

end of this experiment, more than 16 % weight loss was observed in the mice of free

563

DOX group. This weight loss of the free DOX group may be owing to non-selective

564

bio-distribution of the DOXs and cachexia of tumors.

565

4. CONCLUSIONS

566

A novel, ultrasound-triggerable NGR-modified liposomal nanocarrier (NGR/UT-L)

26



567

was prepared and assessed in this paper. The NGR/UT-L accumulates in the tumor

568

tissues for the targeting effects of NGR and is then disrupted under ultrasound

569

irradiation due to a sonodynamic effect. As a result, the drug loaded inside the

570

liposomal nanocarriers could be burst released when triggered by ultrasound. The

571

study results implied that NGR/UT-L is a promising drug delivery system for

572

anti-tumor therapy. Meanwhile, NGR/UT-L may be a good alternative to the

573

sonodynamic therapy, which uses ultrasound to produce ROS specially in the tumor

574

site and induces the death of cancer cells. In the future, we will keep on performing

575

the in vivo investigations, including survival study, safety evaluation and detailed

576

validation for oncotherapy.

577



578

This work was support by the Beijing Science and Technology New Star (Grant No.

579

Z161100004916162), Beijing NSF (Grant No. 7172162), NSF (Grant No. 81874305),

580

Young & Middle-aged Medical Key Talents Training Project of Wuhan (Grant No.

581

2018-6) and Health & Family Planning Commission of Hubei Province (Grant No.

582

WJ2017Q031).

583



584

In vitro release of various liposomal nanocarriers with or without ultrasound; In vitro

585

release of various liposomal nanocarriers with ultrasound after a 24-h incubation;

586

Storage stability at 4 °C; The cytotoxicity of various liposomal formulations;

ACKNOWLEDGMENTS

SUPPORTING INFORMATION

27


Page 28 of 41

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587

Ultrasound-triggered generation of singlet oxygen; Concentration of DOX in the

588

major organs; Photographs of tumors; Histological staining. Annexin V-FI flow

589

cytometry investigation; Pharmacokinetic parameters.

590



591

(1) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: unique features of tumor

592

blood vessels for drug delivery, factors involved, and limitations and

593

augmentation of the effect. Adv. Drug Deliv. Rev. 2011, 63, 136-151.

REFERENCES

594

(2) Yuan, Q.; Zhang, Y.; Chen, T.; Lu, D.; Zhao, Z.; Zhang, X.; Li, Z.; Yan, C. H.;

595

Tan, W. Photon-Manipulated Drug Release From A Mesoporous Nanocontainer

596

Controlled By Azobenzene-Modified Nucleic Acid. ACS Nano. 2012, 6 (7),

597

6337-6344.

598

(3) van Rijt, S. H.; Bolukbas, D. A.; Argyo, C.; Datz, S.; Lindner, M.; Eickelberg, O.;

599

Konigshoff, M.; Bein, T.; Meiners, S. Protease-Mediated Release Of

600

Chemotherapeutics From Mesoporous Silica Nanoparticles To Ex Vivo Human

601

And Mouse Lung Tumors. ACS Nano. 2015, 9 (3), 2377-2389.

602

(4) Chen, Z.J.; Zhai, M.F.; Xie, X.Y.; Zhang, Y.; Ma, S.Y.; Li, Z.P.; Yu, F.L.; Zhao,

603

B.Q.; Zhang, M.; Yang, Y.; Mei, X.G. Apoferritin Nanocage for Brain Targeted

604

Doxorubicin Delivery. Mol. Pharm. 2017, 14, 3087-3097.

605

(5) Zhao C, Shao L, Lu J, Zhao C, Wei Y, Liu J, Li M, Wu Y. Triple Redox

606

Responsive Poly(Ethylene Glycol)-Polycaprolactone Polymeric Nanocarriers for

607

Fine-Controlled Drug Release. Macromol Biosci. 2017, 17, 1600295.

28



608 609

Page 30 of 41

(6) Khawar, I. A.; Kim, J. H.; Kuh, H. J. Improving Drug Delivery To Solid Tumors: Priming The Tumor Microenvironment. J. Controlled Release 2015, 201, 78-89.

610

(7) Blanquer, S. B.; Grijpma, D. W.; Poot, A. A. Delivery Systems For The Treatment

611

Of Degenerated Intervertebral Discs. Adv. Drug Delivery Rev. 2015, 84,

612

172-187.

613 614

(8) Andresen, T.L; Thompson, D.H.; Kaasgaard T. Enzyme-triggered nanomedicine: drug release strategies in cancer therapy. Mol. Membr. Biol. 2010, 27, 353-363.

615

(9) Ferreira Ddos, S.; Lopes, S.C.; Franco, M.S.; Oliveira, M.C. pH-sensitive

616

liposomes for drug delivery in cancer treatment. Ther. Deliv. 2013, 4, 1099-1123.

617

(10) Yang, Y.; Yang Y.F.; Xie, X.Y.; Wang, Z.Y.; Gong, W.; Zhang, H.; Li, Y.; Yu,

618

F.L.; Li, Z.P.; Mei, X.G. Dual-modified liposomes with a two-photon-sensitive

619

cell penetrating peptide and NGR ligand for siRNA targeting delivery.

620

Biomaterials 2015, 48, 84-96.

621

(11) Qiu, D.; An, X. Controllable release from magnetoliposomes by magnetic

622

stimulation and thermal stimulation. Colloids Surf. B: Biointerfaces 2013, 104,

623

326-329.

624

(12) Karimi, M.; Zangabad, P.S.; Baghaee-Ravari, S.; Ghazadeh, M.; Mirshekari, H.;

625

Hamblin, M.R. Smart Nanostructures for Cargo Delivery: Uncaging and

626

Activating by Light. J. Am. Soc. 2017, 5, 139(13), 4584-4610.

627

(13) Gao, M.; Hu, A.; Sun, X.Q.; Wang, C.; Dong, Z.L.; Feng, L.Z.; Liu, Z.

628

Photosensitizer

Decorated

Red

Blood

Cells

629

Light-Responsive Drug Delivery System. ACS Appl. Mater. Interfaces 2017, 9,

29


as

an

Ultrasensitive

Page 31 of 41 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

630 631 632


5855-5863. (14) Husseini, G.A.; Pitt W.G.; Martins A.M. Ultrasonically triggered drug delivery: Breaking the barrier. Colloids Surf. B: Biointerfaces 2014, 123, 364-386.

633

(15) Cachard, C.; Bouakkaz, A.; Gimenez, G. In vitro evaluation of acoustic

634

properties of ultrasound contrast agents: experimental set-up and signal

635

processing. Ultrasonics 1996, 34, 595-598.

636

(16) Rosenthal, I.; Sostaric, J.Z.; Riesz, P. Sonodynamic therapy e a review of the

637

synergistic effects of drugs and ultrasound. Ultrason. Sonochem. 2004, 11(6),

638

349-363.

639

(17) Costley, D.; Mc Ewan, C.; Fowley, C.; McHale, A.P.; Atchison, J.; Nomikou, N.;

640

Callan, J.F. Treating cancer with sonodynamic therapy: a review. Int. J. Hyperth.

641

2015, 31, 107-117.

642

(18) Jinadasa, R.; Hu, X.; Vicente, M.; Smith, K.M. Syntheses and Cellular

643

Investigations of 17 3 -, 15 2 -, and 13 1-Amino Acid Derivatives of Chlorin e 6. J.

644

Med. Chem. 2011, 54 (21), 7464-7476.

645 646 647 648

(19) Kessel, D.; Lo, J.; Jeffers, R.; Fowlkes, J.B.; Cain, C. Modes of photodynamic vs. sonodynamic cytotoxicity. J. Photochem. Photobiol. B 1995, 28, 219-221. (20) Misìk, V. Riesz, P. Free radical intermediates in sonodynamic therapy. Ann. N. Y. Acad. Sci. 2000, 899, 335-348.

649

(21) Namiki Y1, Namiki T, Date M, Yanagihara K, Yashiro M, Takahashi H.

650

Enhanced photodynamic antitumor effect on gastric cancer by a novel

651

photosensitive stealth liposome. Pharmacol Res. 2004, 50, 65-76.

30



Page 32 of 41

652

(22) Tan X, Pang X, Lei M, Ma M, Guo F, Wang J, Yu M, Tan F, Li N. An efficient

653

dual-loaded multifunctional nanocarrier for combined photothermal and

654

photodynamic therapy based on copper sulfide and chlorin e6. Int J Pharm.

655

2016 , 503, 220-228.

656

(23) Arap, W.; Pasqualini, R.; Ruoslahti, E. Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model. Science 1998, 279, 377-380.

657 658

(24)

Dunne,

M.;

Zheng,

J.Z.;

Rosenblat,

J.;

Jaffray,

D.A.;

Allen,

C.

659

APN/CD13-targeting as a Strategy to Alter the Tumor Accumulation of

660

Liposomes. J. Control. Release 2011, 154, 298-305.

661

(25) Zhang, Y.; Zhai, M.F.; Chen, Z,J.; Han, X.Y.; Yu, F.L.; Li, Z.P.; Xie, X.Y.; Han,

662

C.Y.; Yu, L.; Yang, Y.; Mei, X.G. Dual-modified Liposome Codelivery of

663

Doxorubicin and Vincristine Improve Targeting and Therapeutic Efficacy of

664

Glioma. Drug Deliv. 2017, 24(1), 1045-1055.

665

(26) Dong, X.Y.; Wang, W.; Qu, H.; Han, D.; Zheng, J.M.; Sun, G.R. Targeted

666

delivery of doxorubicin and vincristine to lymph cancer: evaluation of novel

667

nanostructured lipid carriers in vitro and in vivo. Drug Deliv. 2016, 23(4),

668

1374-1378.

669

(27) Cui, L.; Wang, Y.L.; Liang, M.; Chu, X.Y.; Fu, S.Y..; Gao, C.H.; Liu, Q.Q.;

670

Gong, W.; Yang, M.Y.; Li, Z.P.; Yu, L.; et al. Dual-Modified Natural High

671

Density Lipoprotein Particles for Systemic Glioma-Targeting Drug Delivery.

672

Drug Deliv. 2018, 25(1): 1865-1876.

673

(28) Gu, G.Z.; Xia, H.M.; Hu, Q.Y.; Liu, Z.Y.; Jiang, M.Y.; Kang, T.; Miao, D.; Tu,

31


Page 33 of 41 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


674

Y.F.; Pang, Z.Q.; Song, Q.X.; Yao, L.; Chen, H.Z. et al. PEG-co-PCL

675

nanoparticles modified with MMP-2/9 activatable low molecular weight

676

protamine for enhanced targeted glioblastoma therapy. Biomaterials 2013, 34,

677

196-208.

678

(29) Wan, G.Y.; Liu, Y.; Chen, B.W.; Liu, Y.Y.; Wang, Y.S.; Zhang, N. Recent

679

advances of sonodynamic therapy in cancer treatment. Cancer Biol. Med. 2016,

680

13(3), 325-338.

681

(30) Li, Q.; Wang, X.B.; Wang, P.; Zhang, K.; Wang, H.P.; Feng, X.L.; Liu, Q.H.

682

Efficacy of Chlorin e6-Mediated Sono-Photodynamic Therapy on 4T1 Cells.

683

Cancer Biother Radiopharm. 2014, 29(1), 42-52.

684

(31) Takara K, Hatakeyama H, Kibria G, Ohga N, Hida K, Harashima H.

685

Size-controlled, dual-ligand modified liposomes that target the tumor vasculature

686

show promise for use in drug-resistant cancer therapy. J Control Release. 2012,

687

162,

225-232.

32



Page 34 of 41

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Figure

1.

Schematic

illustration

of

DOX-loaded

33


NGR/UT-L.

Page 35 of 41 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


690 691

Figure 2. Principle of the preparation of DSPE-PEG2000-NGR (A). MALDI-TOF

692

mass spectra of DSPE-PEG2000-NGR (B). Red arrows represent the mass-charge

693

DSPE-PEG2000-NGR.

34



695 696

Figure 3. Effect of various Ce6 ester proportions in formulations on the efficiency of

697

ultrasound-triggered DOX release in PBS (0.1 M, pH 7.4) at 37 °C. The data are

698

presented as the means ± SD (n = 3).

35


Page 36 of 41

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700 701

Figure 4. Formulation optimization of NGR/UT-L modified with various

702

concentrations of DSPE-PEG2000-NGR. The cellular uptake of different

703

formulations of Cy5.5-labeled NGR/UT-L by HT-1080. The data are presented

704

as the means ± SD (n = 3).

36



706 707

Figure 5. Physicochemical characterization of NGR/UT-L. Characteristics of the

708

liposomal nanocarriers (A). Morphological appearance of DOX-loaded

709

NGR/UT-L based on TEM (B). Particle size distribution of DOX-loaded

710

NGR/UT-L (C). In vitro release of DOX from various liposomal formulations in

711

PBS (0.1 M, pH 7.4) at 37 °C (D). The data are presented as the means ± SD (n =

712

3).

37


Page 38 of 41

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714 715

Figure 6. Cellular uptake of different Cy5.5-labeled various liposome formulations by

716

HT-1080 cells. Intracellular fluorescence was captured by a CLSM (A), and

717

Cy5.5-positive cells were calculated by an FCM (B). Scale bars represent 20 μm.

38



718 719

Figure 7. Plasma DOX concentration-time profiles (A) after i.v. injection of different

720

formulations in rats (n=3). The distribution of DOX in tumors 0.5 h after i.v.

721

injection (B). The data are presented as the means ± SD (n = 3). * indicates P

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