Biomimetic Nanovesicles for Enhanced Antitumor Activity of

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Biomimetic Nanovesicles for Enhanced Antitumor Activity of Combinational Photothermal and Chemotherapy Tingting Wu, Dan Zhang, Qi Qiao, Xianya Qin, Conglian Yang, Miao Kong, Huan Deng, and Zhiping Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01142 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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

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Molecular Pharmaceutics

1

Biomimetic Nanovesicles for Enhanced Antitumor

2

Activity of Combinational Photothermal and

3

Chemotherapy

4 1

1

1

1

1

1

5

Tingting Wu , Dan Zhang , Qi Qiao , Xianya Qin , Conglian Yang , Miao Kong ,

6

Huan Deng , Zhiping Zhang

1

1,2,3*

7 8 9 10 11

1

Tongji School of Pharmacy

2

National Engineering Research Center for Nanomedicine

3

Hubei Engineering Research Center for Novel Drug Delivery System

HuaZhong University of Science and Technology, Wuhan, China 430030

12 13 14 15 16 17 18 19 20 21 22

ABSTRACT Combination of multiple modalities has shown great potential in cancer treatment 1

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

with improved therapeutic effects and minimized side effects. Here we fabricated a

24

kind of doxorubicin encapsulated biomimetic nanovesicles (NVs) by a facile method

25

with near-infrared dye insertion in the membrane for combinatorial photothermal and

26

chemotherapy. With innate biomimetic properties, NVs enhanced the uptake by tumor

27

cells while reduced the phagocytosis of macrophages. Upon laser irradiation, NVs can

28

convert the absorbed fluorescent energy into heat for effective tumor killing. The

29

hyperthermia can further induce membrane ablation of NVs to accelerate the release

30

of chemotherapeutic drug for potent cytotoxicity to tumor cells. The NVs improved

31

the drug accumulation and showed more efficient in vivo photothermal effect with

32

rapid temperature increase in tumor. Moreover, the NVs based combinational

33

photothermal and chemotherapy exhibited significant tumor growth suppression with

34

a high inhibitory rate of 91.6% and negligible systemic toxicity. The results indicate

35

that NVs could be an appealing vehicle for combinational cancer treatment.

36 37

KEY WORDS: Nanovesicles, photothermal therapy, chemotherapy, near-infrared,

38

cancer

39 40 41 42 43 44 2


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1. INTRODUCTION

46

With increasing incidence and mortality, cancer keeps as the major cause of death

47

all over the world.1 Chemotherapy, which can cause direct cytotoxicity to tumor cells,

48

is the commonly used treatment regimen in clinic cancer therapy. However, the low

49

selectivity of cytotoxic drugs always causes severe toxicity to normal tissues, which

50

would greatly limit the successful application of chemotherapy. Combinational

51

therapy with multiple modalities can improve the therapeutic efficiency as well as

52

minimize the side effects, which has shown great potential in cancer treatment.2-3 In

53

the past few decades, the combination of chemotherapy with other therapeutic

54

modalities has attracted much attention and achieved satisfied outcomes for tumor

55

killing.4-6 Photothermal therapy (PTT) is a well-developed technique which can utilize

56

the comparable heat generated by near-infrared (NIR) laser to directly destroy the

57

malignant tissues.7-8 Owing to the highly specific spatial-temporal selectivity and the

58

minimal toxicity to normal tissues, PTT has emerged as an appealing cancer

59

therapeutic modality.9 However, as heat distribution cannot be so uniform in whole

60

tumor, especially in the areas near large blood vessels where circulating blood can

61

rapidly dissipate the heat, PTT alone is unable to clear tumor cells.10 The combination

62

of PTT with chemotherapy can utilize multiple antitumor mechanisms and may work

63

cooperatively to suppress cancer development. On one hand, the hyperthermia

64

induced by PTT can disrupt extracellular matrix, relieve the tumor interstitial fluid

65

pressure and trigger the rapid release of cytotoxic drugs in tumor site.11-12 On the other

66

hand, the cytotoxic drugs can kill tumor cells which are unlikely to be cleared by PTT 3



67

owing to the nonuniform heat distribution. Such a multimodal approach may possess

68

the potential to eradicate tumor cells.

69

With rapid development of nanotechnology, nanosized drug delivery systems

70

have been extensively applied in cancer treatment. Encapsulating free drugs into

71

nanoformulations can protect the drug from degradation during blood circulation,

72

improve pharmacokinetics and pharmacodynamics as well as reduce side effects.13

73

The nanoformulations are usually prepared with synthesized materials. However,

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there are still several factors which impede the therapeutic potential of these

75

synthesized material based delivery systems. Firstly, as intruders to the host, foreign

76

materials can be easily detected by the immune system, which may cause some

77

immune disorder.14 Besides, the opsonization and nonspecific clearance by

78

reticuloendothelial system (RES) remain as a major challenge.15 Even the

79

modification with poly(ethylene glycol) (PEG) cannot completely prevent this

80

nonspecific clearance,16 which would then activate the complement system to induce

81

undesired immune response.17 Furthermore, the potential toxicity of foreign

82

nanomaterials with artificial nature has kept as a considerable concern for biomedical

83

applications.18 Given the limitations of synthetic materials, it is gaining popularity to

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develop biomimetic strategies for drug delivery. Cell based nanovesicles (NVs) are

85

spherical membrane vesicles consisting of various proteins, lipids and nucleic acids,19

86

which can mimic the natural properties of parent cells.20 As a top-down approach, the

87

biomimetic carriers can get rid of laborious and costly groups-modified engineering.21

88

With good biocompatibility, reduced uptake by macrophages, decreased blood 4


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clearance and specific targeting ability for enhanced tumor accumulation, NVs

90

showed great potential for drug delivery.22-23 Generally, NVs are spontaneously

91

secreted by cells and used as drug carriers. However, NVs generated in this way are

92

always suffered from low production yield, heterogeneity in size and composition,

93

and inefficient drug package.24 An alternative and facile method for generating

94

homogenous drug loaded-NVs is to subject cells and therapeutic agents to serial

95

extrusions using filters with diminishing pore sizes. Moreover, this approach can

96

promote the production of biomimetic vesicles, which has been reported to be more

97

than a 100-fold higher production yield as comparison to spontaneously generated

98

extracellular vesicles.25-26

99

In this work, we prepared a kind of biomimetic nanovesicles co-loading

100

near-infrared dye DIR and chemotherapeutic drug doxorubicin (DOX) for

101

combinational photothermal-chemotherapy. DIR, a lipophilic fluorescent probe, can

102

be embedded in the lipid bilayer of cell membrane with negligible influence on its

103

physiological properties and commonly used for in vivo fluorescence imaging and

104

tracking.12, 27 Notably, DIR has an absorption in NIR region and can convert absorbed

105

fluorescent energy into heat under NIR irradiation.28-29 Moreover, it can also work as

106

a photosensitizer to generate reactive oxygen species (ROS) upon light exposure.

107

Here we inserted DIR in the membrane of DOX loaded NVs (NV-DOX) for

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combinatorial photothermal and chemotherapy. This NV based combinational

109

platform (NV-DOX-DIR) can accumulate in tumor owing to the enhanced permeation

110

and retention (EPR) effect of nanoformulations and the specific targeting ability of 5



111

cell derived membrane vesicles. Upon NIR irradiation, DIR inserted in the membrane

112

of NVs can convert near-infrared fluorescence into thermal energy to kill tumor cells.

113

The induced hyperthermia can further destroy the membrane of NVs and release the

114

encapsulated DOX for direct cytotoxicity to tumor cells (Scheme 1). The biomimetic

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NVs can realize synergistic antitumor efficiency through the combination of

116

photothermal and chemotherapy.

117 118

Scheme 1. Schematic illustration for biomimetic NVs preparation and in vivo

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synergistic antitumor effect by combination of photothermal and chemotherapy. To

120

prepare NV-DOX-DIR, DC2.4 cells were firstly mixed with DOX and subjected to

121

serial extrusions by polycarbonate membranes with diminishing core size. After

122

removing the cells, debris and free DOX, NVs were then incubated with DIR to

123

obtain NV-DOX-DIR. When intravenously injected to the mice, NVs can accumulate

124

in tumor site owing to EPR effect and specific targeting ability of NVs. Upon

125

irradiation by NIR laser, NV-DOX-DIR can effectively convert the absorbed 6


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fluorescent energy into heat for tumor killing. The induced hyperthermia can further

127

destroy the NVs to release the encapsulated DOX for enhanced cytotoxicity.

128 129

2. EXPERIMENTAL SECTION

130

2.1. Materials and reagents

131

Doxorubicin hydrochloride was purchased from Beijing Huafeng United

132

Technology Co., China. 1, 1′-Dioctadecyl-3, 3, 3′, 3′- tetramethylindotricarbocyanine

133

iodide

134

5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was acquired from

135

Biosharp, South Korea. 4', 6-Diamidino-2-phenylindole (DAPI) was obtained from

136

Nanjing KeyGen Biotech. Inc., China. Radioimmunoprecipitation assay (RIPA) buffer，

137

dichlorodihydrofluorescein diacetate (DCFH-DA) and bicinchoninic acid (BCA)

138

protein quantitation kit were acquired from Beyotime Institute of Biotechnology,

139

China. 1, 1'-Dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DIL)

140

was purchased from Invitrogen Corporation, USA. Phosphatidylcholine was obtained

141

from Avanti Polar Lipids, Inc., USA. Cholesterol was purchased from Lipoid GmbH,

142

Germany. DSPE-PEG 2000 was purchased from Corden Pharma Switzerland LLC,

143

Switzerland. Pentobarbital sodium was obtained from Sigma-Aldrich, USA.

144

2.2. Cell lines and animals

(DIR)

was

acquired

from

AAT

Bioquest

Inc.,

USA.

3-(4,

145

Murine DC2.4 cells were maintained in RPMI-1640 containing 10% fetal bovine

146

serum (FBS), 100 µg/mL of streptomycin and 100 IU/mL of penicillin in a humidified

147

atmosphere incubator with 5% CO2 at 37 °C. Murine B16F10 melanoma cells were 7



148

cultured in Dulbecco's modified eagle medium (DMEM) with the same supplements

149

and culture condition as DC2.4 cells. Female C57BL/6 mice (6-8 week) were

150

obtained from the Experimental Animal Center of Hunan SJA, China. All mice were

151

maintained under specific pathogen-free (SPF) condition which keeps at 25 ± 1°C and

152

60% ± 10% humidity under a 12 h light/dark cycle in the Animal Center of Huazhong

153

University of Science and Technology (HUST), China. All animals were treated

154

according to the regulations of Chinese law, and the study was approved by the

155

Institutional Animal Care and Use Committee at Tongji Medical College, HUST,

156

China.

157

2.3. Preparation and characterization of NVs

158

NV-DOX were prepared as reported previously with minor modification.25

159

Briefly, DC2.4 cells were collected and resuspended in phosphate buffered saline

160

(PBS) at a concentration of 2.5×106 cells/mL. Then DOX was added to cell

161

suspensions and the resulted mixture were sequentially extruded through 10, 5, and 1

162

µm polycarbonate membrane (Whatman International Ltd, Maidstone, England) using

163

a mini-extruder (Avanti Polar Lipids, USA). To isolate the NVs, the suspension was

164

firstly centrifuged at 1500g for 15 min to get rid of the cells and debris. Then the

165

supernatant was centrifuged at 17, 000g for 45 min and NVs were obtained after

166

washing twice with PBS. To prepare DIR inserted NV-DOX, DIR was dissolved in

167

ethanol and added dropwise to the aqueous phase of NV-DOX. The mixture was then

168

incubated at 37 oC for 1 h and centrifuged at 17, 000 g for 45 min to remove the

169

unincorporated DIR. The resulting pellets were resuspended in PBS for further use. 8


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The hydrodynamic diameter of NVs was monitored by dynamic light scattering (DLS)

171

(Zeta Plus, Brookhaven Instruments, USA). To observe the morphology, the NVs

172

were fixed with 2% paraformaldehyde for 30 min, dropped on EM grids and stained

173

with 0.5% phosphotungstic acid for 2 min. The morphology of NVs was then

174

observed by TEM (JEM-1230, Japan). To monitor the successful package of DOX,

175

UV-Vis spectra of NVs were detected using a microplate reader (Multiskan MK3,

176

Thermo, USA).

177

2.4. Determination of drug loading efficiency in NVs

178

To detect the loading efficiency of DOX by NVs, DC2.4 cells were collected and

179

mixed with DOX in PBS. The drug loaded NVs were obtained as described above.

180

The NVs were lysed with RIPA buffer containing phenylmethanesulfonyl fluoride

181

(PMSF). The concentration of DOX was measured using a fluorescent microplate

182

reader and the total content of protein was detected by BCA protein quantitation kit.

183

To further determine the loading efficiency of DIR by NVs, different amounts of DIR

184

(w/w, DIR/total protein) were incubated with the NVs. After removing the free drug,

185

NVs were resuspended in DMSO to entirely dissolve the NVs and DIR. Then the

186

UV-Vis spectra were detected by the microplate reader. The amount of DIR insertion

187

in the membrane was calculated according to the linear standard curve of DIR in

188

DMSO.

189

2.5. In vitro cumulative release of DOX

190

The cumulative release behavior of DOX from NVs was performed in pH 7.4

191

PBS using an orbital shaker (37 °C, 100 rpm). To monitor the impact of NIR laser on 9



192

drug release, NV-DOX-DIR was pre-treated with or without 808 nm laser at a power

193

density of 2.5 W/cm2 for 2 min. The released drug was collected by centrifuge at

194

various time points and fresh medium were supplemented for continuous release. The

195

DOX content was then determined using fluorescent microplate reader.

196

2.6. In vitro photothermal profiles

197

The photothermal profiles of free DIR, DIR inserted blank NVs (NV-DIR) and

198

NV-DOX-DIR with equivalent DIR concentration of 100 µg/mL were treated with

199

808 nm laser at a power density of 2.5 W/cm2. At different time points, the images of

200

each group upon irradiation were photographed and corresponding temperature was

201

recorded using the infrared thermal imaging camera (FLIR System E40, Boston, MA,

202

USA). The thermal profiles of NV-DOX-DIR with different concentrations of DIR

203

were also evaluated.

204

2.7. The cell uptake of NVs by tumor cells

205

Confocal microscope and flow cytometer were used to determine the cell uptake

206

of NVs by tumor cells. Briefly, B16F10 cells were seeded into 6-well plate and

207

incubated overnight in the incubator. Afterward, all the medium were removed and

208

replaced with fresh medium containing DOX, NV-DOX and NV-DOX-DIR,

209

respectively. The cells were further incubated at 37 oC for different time intervals and

210

detected by flow cytometer (Accuri C6, BD, USA). To visualize the cell uptake, cells

211

were further monitored by confocal microscope at 4 h. After removing the supernatant,

212

cells were washed by cold PBS, fixed with 4% paraformaldehyde at room temperature

213

for 15 min and stained with DAPI. The cells were then rinsed with PBS and 10


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monitored by confocal laser scanning microscopy (710META, Zeiss, Germany).

215

2.8. Evasion of NVs from the uptake by macrophages

216

To investigate the evasion of NVs from phagocytosis by macrophages, DIL

217

labeled NVs were incubated with Raw 264.7 macrophages for 2 h. Instead of DIR,

218

DIL was used here owing to the visualized fluorescence of DIL for detection. A lipid

219

structure based liposome was prepared by filming-hydration method and used as

220

control. Briefly, phosphatidylcholine, cholesterol and DSPE-PEG 2000 (w/w, 4:4:1)

221

were dissolved in chloroform and evaporated by rotary evaporation to form a uniform

222

film. The liposome was obtained after hydration, labeled with DIL and incubated with

223

Raw 264.7 macrophages. Then the cells were rinsed with cold PBS, stained with

224

DAPI (5 µg/mL) for 5 min and imaged by confocal microscope. To quantitatively

225

determine the cellular uptake, the cells were resuspended in PBS and detected by flow

226

cytometer.

227

2.9. Intracellular temperature measurement and ROS generation

228

To measure the intracellular temperature, the photothermal profiles of B16F10

229

cells were evaluated after treating with free DIR, NV-DIR and NV-DOX-DIR.

230

B16F10 cells (1×104 cells/well) were seeded in 24-well plates and cultured overnight.

231

Then the medium were replaced with fresh medium containing 100 µg/mL free DIR,

232

NV-DIR or NV-DOX-DIR and incubated for 4 h. Then the cells were irradiated with

233

808 nm. The temperature of each time point was recorded using infrared thermal

234

imaging camera. To further detect the intracellular ROS generation, the cells treated

235

with various formulations were incubated with DCFH-DA at 37 °C for 20 min. Then 11



236

the cells were rinsed with PBS for three times, irradiated with NIR laser and collected

237

for detection by flow cytometer.

238

2.10. In vitro combinatorial effects of photothermal and chemotherapy

239

To investigate the enhanced antitumor effect in vitro, B16F10 cells were seeded

240

into 96-well plate and allowed for attachment overnight. Then the cells were treated

241

with different formulations. The concentrations of DOX and DIR range from 0.01 to 5

242

µg/mL and 0.008 to 4 µg/mL, respectively. After incubation for 4 h, the supernatant

243

was removed and fresh medium were added. Then the cells were irradiated with 808

244

nm laser for 2 min and cultured for another 20 h. The cell viability was determined by

245

MTT assay. The synergistic cytotoxicity of photothermal-chemotherapy was also

246

evaluated by Calcein AM/PI live/dead staining (Molecular Probes-Invitrogen).

247

B16F10 cells were seeded into 24-well plates and incubated overnight. The original

248

medium were replaced with the medium containing different formulations. After 4 h

249

incubation, the cells were irradiated with NIR laser. Twenty hours later, the cells were

250

stained with Calcein-AM/PI according to the manufacture’s instruction and then

251

monitored using the fluorescent microscope (CKX53, Olympus, Japan).

252

2.11. In vivo imaging and biodistribution of NVs

253

To study the biodistribution of NVs after systematic administration, free DIR and

254

NV-DIR were given to melanoma bearing mice by tail vein. The biodistribution of

255

injected formulations at various time points was monitored in live mice under

256

isoflurane anesthesia using the Pearl Impulse Small Animal Imager (LI-COR

257

Biosciences, Lincoln, NE). At 48 h post injection, all the mice were sacrificed. The 12


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258

major organs were collected, rinsed with PBS and imaged by imaging system.

259

2.12. In vivo temperature measurement and photothermal profiles

260

A well-established murine melanoma model was used to determine the in vivo

261

photothermal effect after systematic injection of NVs. PBS, free DIR, NV-DIR and

262

NV-DOX-DIR were intravenously injected to mice, respectively. After 24 h, the mice

263

were anesthetized by intraperitoneally injecting 50 µl 1% pentobarbital sodium. And

264

tumors were exposed to 808 nm laser at a power intensity of 2.5 W/cm2 and the

265

temperature was simultaneously monitored by infrared thermal imaging camera.

266

2.13. In vivo synergistic antitumor efficacy of NVs

267

To

investigate

the

in

vivo

synergistic

antitumor

effect

of

268

photothermal-chemotherapy, murine melanoma model was used for tumor inhibition

269

experiment. B16F10 cells were implanted on the left hind flank of female C57BL/6

270

mice on day 0. From day 11, the mice were intravenously injected with PBS, free

271

DOX (2.5 mg/kg), free DOX+DIR (2 mg/kg DIR), NV-DOX, NV-DIR and

272

NV-DOX-DIR every three days for 3 times, respectively. Twenty four hours after each

273

injection, the mice were irradiated with NIR laser. Tumor volume of each group,

274

calculated by (width)2 × length × 1/2, and body weight were recorded every other day.

275

All mice were sacrificed at day 21 and tumors were excised for imaging and

276

hematoxylin and eosin (HE) staining. Terminal-deoxynucleoitidyl transferase

277

mediated nick-end labeling (TUNEL) assay was conducted to evaluate the apoptosis

278

and necrosis of tumor after various treatments.

279

2.14. Systemic toxicity of NVs 13



280

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To investigate the systemic toxicity of NVs, C57BL/6 mice were treated with

281

PBS,

PBS+Laser,

DOX,

DOX+DIR+Laser,

NV-DOX,

NV-DIR+Laser

and

282

NV-DOX-DIR+Laser, respectively. Then the serum was sampled to measure the

283

blood biochemistry parameters. According to the manufacture's protocol (Nanjing

284

Jiangcheng Bioengineering Institute, China), the levels of alanine aminotransferase

285

(ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr)

286

and lactate dehydrogenase (LDH) were measured using a microplate reader. The

287

major organs were excised, stained with HE and observed under a microscope to

288

monitor the histopathological changes after systemic administration of NVs.

289

2.15. Statistical analysis

290

Data were represented as mean ± standard deviation (SD). The difference of the

291

mean was calculated by one-way ANOVA using SPSS software (version 19.0). There

292

was significantly statistical difference when the p value was less than 0.05.

293 294

3. RESULTS AND DISCUSSION

295

3.1. Preparation and characterization of NVs

296

NVs are commonly generated by spontaneous secretion from the cells under both

297

pathological and physiological conditions. However, this process is usually faced with

298

many challenges including low production yield, heterogeneity in size and

299

compositions and inefficient drug packaging. Here we introduced an alternative

300

method to generate a population of homogenous drug loaded-NVs for combinational

301

photothermal and chemotherapy. DC2.4 cells were detached from the culture plate 14


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302

and resuspended in PBS. The cells were then mixed with DOX and conducted with

303

serial extrusion through polycarbonate membranes (10 µm, 5 µm, and 1 µm). After

304

removing the cells and debris, the supernatant were centrifuged to collect DOX

305

loaded NVs (NV-DOX). To further prepare DIR incorporated NVs, NV-DOX were

306

incubated with DIR at 37 ℃ for 1 h following with centrifugation to remove the

307

unincorporated DIR. The morphology of NVs were observed by transmission electron

308

microscope (TEM). As shown in Figure 1A, NV-DOX showed regular morphology

309

with a near-spheric shape. As DIR showed negligible interference on the

310

physiological properties of cell membrane, no obvious morphology change was

311

monitored after inserting DIR in the membrane of NVs. After NIR irradiation, the

312

membrane structure of NVs was destroyed, which was mainly attributed to

313

photothermal ablation on cell membrane induced by NIR laser.30-32 To verify the

314

successful package of DOX and incorporation of DIR in NVs, the UV-Vis absorption

315

spectra of NV-DOX, free DIR, NV-DIR and NV-DOX-DIR were measured using a

316

microplate reader (Figure 1B). Free DIR in PBS showed very weak absorption while

317

NV-DIR and NV-DOX-DIR exhibited a peak absorption at 760 nm and remarkably

318

increased absorption in NIR region. It ensured the potential of NV-DIR and

319

NV-DOX-DIR for photothermal therapy induced by NIR laser. NV-DOX showed a

320

peak absorption at 490 nm of DOX, verifying the successful encapsulation of DOX by

321

NVs. After NIR irradiation, the absorption of DIR decreased owing to the consumed

322

DIR during the process of heat converting. The absorption of DOX was not

323

influenced by the irradiation, indicating DOX was in good stability during laser 15



324

illumination (Figure 1C). Then the quantitative DOX loading by NVs was determined.

325

As Figure 1D showed, the amount of DOX loaded in NVs was dependent on the

326

initial DOX concentration in cell suspensions before extrusion. With the increasing

327

concentration of DOX, the encapsulation of DOX by NVs was increased. To further

328

determine the incorporated amount of DIR into NVs, UV-Vis spectra of NVs with

329

different amounts of DIR insertion were recorded (Figure 1E). The absorption of DIR

330

in NVs increased with the elevating amount of DIR and became saturated when the

331

weight ratio of DIR/total protein raised to 11%. It seems that no more DIR could be

332

incorporated into the membrane of NVs. In contrast, free DIR with similar amount as

333

NVs in PBS showed very weak absorption (Figure 1F). According to DOX dosage in

334

our previous combinational therapy33 and effective DIR amount for PTT,29 200 μg/mL

335

initial DOX concentration was used to prepare NV-DOX and 11% DIR was

336

incorporated in the membrane to obtain NV-DIR in the following experiments. The

337

stability of drug carriers in physiological condition is critical for further biomedical

338

application. The stability of NVs was then monitored in commonly used physiological

339

media. As shown in Figure 1G and Figure S1, both NV-DOX and NV-DOX-DIR

340

showed good stability in PBS and FBS with negligible change of hydrodynamic size

341

during storage for 5 days at 4 oC. To further detect the drug retention during storage,

342

the leakage of DOX in the supernatant was detected using the fluorescent microplate

343

reader. As shown in Figure S2, 84.1 ± 1.1% and 83.0 ± 1.0% of DOX retained in

344

NV-DOX and NV-DOX-DIR after 24 h respectively. This may be attributed to the

345

good hydrophilicity of doxorubicin hydrochloride and the slight permeability of cell 16


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346

membrane.34 The cumulative release of DOX from NVs was also performed in PBS

347

(Figure 1H). There was no obvious difference in the release profiles of NV-DOX and

348

NV-DOX-DIR, which further confirmed the negligible interference on membrane of

349

NVs after DIR insertion. After laser irradiation, the release of DOX from

350

NV-DOX-DIR was accelerated and can reach to 88.6 ± 0.2% after 24 h while that of

351

NV-DOX and NV-DOX-DIR was 61.2 ± 0.7% and 62.3 ± 1.2%, respectively. This

352

may be caused by the NIR laser irradiation which can generate comparable localized

353

heat to destroy the membrane of NVs and thus trigger the accelerated DOX release

354

from the NVs.29

355 356

Figure 1．．Preparation and characterization of NVs. (A). TEM images of (a) NV-DOX

357

(b) NV-DOX-DIR and (c) NV-DOX-DIR with NIR laser (Scale bar, 500 nm). (B-C).

358

The UV-Vis spectra of NV-DOX, free DIR, NV-DIR and NV-DOX-DIR (B) before

359

and (C) after NIR irradiation. (D). DOX loading efficiency in NVs with the increasing 17



360

initial concentration of DOX (n = 3). (E). The UV-Vis absorption spectra of NVs after

361

different amount of DIR inserting into membrane. (F), The UV-Vis spectra of free

362

DIR with similar amount as NVs in PBS. (G). the physiologic stability of NV-DOX

363

and NV-DOX-DIR in PBS (n = 3). (H) In vitro cumulative DOX release of NV-DOX

364

and NV-DOX-DIR with or without NIR (n = 3).

365 366

3.2. In vitro photothermal effects of NVs

367

As described above, we have demonstrated that DIR incorporated in NVs showed

368

a remarkably increased absorbance in NIR region. To investigate the photothermal

369

effect of NVs, the real-time temperature changes with 808 nm NIR laser irradiation

370

were recorded by an infrared thermal imaging camera. As Figure 2A-B displayed,

371

upon laser illumination for 5 min, no obvious temperature change was observed in

372

PBS control. The temperature of free DIR raised slowly and reached maximum as

373

44.9 oC. However, the temperature of NV-DIR and NV-DOX-DIR could increase over

374

50 °C within a short period of irradiation for 1 min. After 2 min irradiation, the

375

temperature of NV-DIR and NV-DOX-DIR was raised up to 61.4 °C and 62.8 °C,

376

respectively. When the irradiation time was up to 5 min, the temperature of NVs can

377

reach above 70.0 °C with a dramatic temperature increase (△T) over 45 °C. This

378

could be resulted from the more concentrated status of DIR in NVs, which would lead

379

to higher energy converting efficiency under laser illumination.29 Besides, free DIR

380

exhibited very quick photodegradation with a rapid fluorescence quenching under

381

NIR laser irradiation while NV-DIR showed a much higher photostability (data not 18


Page 18 of 40

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382

shown).

The

photothermal

profiles

of

NV-DOX-DIR

exhibited

a

383

concentration-dependent pattern upon NIR laser irradiation (Figure 2C). These results

384

elucidated that DIR inserted in NVs could increase the photostability and meanwhile

385

enhance the heat converting efficiency compared with free DIR. Therefore, DIR

386

incorporated NVs could serve as a potent candidate for photothermal therapy.

387 388

Figure 2. The in vitro temperature profiles after NIR laser irradiation. (A). The

389

real-time photothermal images of PBS, free DIR in PBS (100 µg/mL), NV-DIR and

390

NV-DOX-DIR under NIR irradiation. (B). The corresponding photothermal profiles

391

of PBS, free DIR, NV-DIR and NV-DOX-DIR upon NIR laser illumination. (C). The

392

temperature profiles of NV-DOX-DIR at different DIR concentrations (808 nm, 2.5

393

W/cm2). 19



394 395

3.3. The uptake by tumor cells and evasion from macrophages

396

The effective internalization of therapeutic agents by tumor cells plays an

397

essential role in the subsequent tumor killing. Here, the intracellular uptake of NVs by

398

tumor cells was determined after incubation with free DOX, NV-DOX or

399

NV-DOX-DIR, respectively. For quantification of the cellular uptake, cells were

400

harvested for analysis by flow cytometer. As Figure 3A showed, the uptake behavior

401

of all the formulations displayed a time-dependent manner. Both NV-DOX and

402

NV-DOX-DIR showed enhanced cellular uptake with improved florescence intensity

403

compared to free DOX. This may be due to that the internalization of NVs was

404

mediated by the fusion of membrane components of NVs with the similar cell

405

membrane of tumor cells, which would promote the drug accumulation in the

406

cells.35-36 NV-DOX and NV-DOX-DIR exhibited comparable cellular uptake as the

407

DIR insertion did not influence the physiological properties on the membrane of NVs.

408

Furthermore, after incubation with different formulations for 4 h, the cells were

409

observed under the confocal microscope to visualize the internalization of NVs. It

410

showed the similar tendency as the results displayed in flow cytometer (Figure 3B).

411

RES clearance keeps as one of the major obstacles in the application of

412

nanomedicines as nanocarriers can be easily recognized as intruders to the host and

413

readily cleared by RES, which would greatly reduce the in vivo circulation time and

414

drug delivery efficiency.37 Surface modification by PEG is a commonly used strategy

415

to reduce the opsonization by RES but cannot effectively prevent this kind of 20


Page 20 of 40

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416

nonspecific clearance.16 With innate biomimetic nature, membrane vesicles may

417

evade the opsonization and clearance by RES and achieve prolonged in vivo

418

circulation time. To determine the evasion function of NVs from RES, we examined

419

the cellular uptake in Raw 264.7 macrophages. A liposome consisting of

420

phosphatidylcholine, cholesterol and DSPE-PEG 2000 was used as the control. As

421

shown in Figure 3C, DIL labeled NVs showed significantly decreased internalization

422

by RAW264.7 macrophages compared with the liposome, indicating the effective

423

evasion of NVs from RES clearance. The quantitative uptake of NVs by macrophages

424

detected by flow cytometer also showed a reduced mean fluorescence intensity (MFI)

425

in comparison to the liposome (Figure 3D). It may be attributed to that the

426

nanovesicles with similar functionality as cell membrane were not “non-self”

427

components after injection, which could effectively evade non-specific clearance from

428

the body.38 Taken together, NVs can effectively promote the internalization by tumor

429

cells while decrease the clearance by RES, which may be an ideal drug carrier in

430

cancer therapy.

21



431 432

Figure 3. The intracellular uptake of NVs by tumor cells and evasion from

433

macrophages. (A). The quantitative uptake of NVs by B16F10 cells detected by flow

434

cytometer (n = 3, *p < 0.05). (B). Confocal images of B16F10 cells after incubation

435

with free DOX, NV-DOX and NV-DOX-DIR for 4 h, respectively. Cell nucleus was

436

stained with DAPI (scale bar, 50 µm). (C). The cell uptake of DC2.4 derived NVs and

437

liposome by Raw 264.7 macrophages by confocal microscope (scale bar, 50 µm). (D).

438

The determination of quantitative cellular uptake by Raw 264.7 macrophages using

439

flow cytometer (n = 3, *p < 0.05).

440 441

3.4. In vitro combinational photothermal-chemotherapy

442

To investigate the potential photothermal effect on tumor cells, the intracellular

443

temperature of B16F10 cells upon irradiation was recorded using infrared thermal

444

camera after treating with different formulations. As Figure 4A showed, the 22


Page 22 of 40

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445

temperature change of PBS treated cells was only 5.5 ± 0.6 oC after NIR irradiation

446

for 3 min. Free DIR pretreated cells showed a maximum temperature increase as 10.5

447

± 1.1 oC and reduced to 8.2 ± 1.4 oC after 3 min illumination. However, after

448

treatment with NV-DIR or NV-DOX-DIR, the temperature of tumor cells was

449

dramatically increased within 30 s and then remained a relatively steady state during

450

the irradiation. To further investigate the impact of DIR inserted NVs on ROS

451

generation, the cells were stained with DCFH-DA and irradiated with NIR laser. As

452

shown in Figure 4B and Figure S3, NV-DIR and NV-DOX-DIR can remarkably

453

increase the level of intracellular ROS, which would induce irreversible damage or

454

apoptosis of cells. Therefore, incorporating DIR into NVs showed higher efficiency to

455

induce NIR triggered hyperthermia in B16F10 cells than free DIR, facilitating the

456

photothermal ablation of tumor cells. Then the cytotoxicity against tumor cells was

457

determined by MTT assay to evaluate the in vitro combinatorial antitumor effect of

458

photothermal-chemotherapy. B16F10 cells were treated with free DOX, free DOX

459

and

460

NV-DOX-DIR+Laser, respectively. The cytotoxicity induced by all the formulations

461

showed a concentration-dependent manner. Compared with free drugs (DOX or

462

DOX+DIR+Laser), the drug loaded NVs exhibited much lower cell viability, which

463

could be resulted from the enhanced drug accumulation and increased heat converting

464

efficiency by NVs. With a combinational effect of photothermal and chemotherapy,

465

NV-DOX-DIR showed the highest cytotoxicity to tumor cells compared with that of

466

single treatment with NV-DOX or NV-DIR+Laser (Figure 4C). To further visualize

DIR

with

laser

(DOX+DIR+Laser),

NV-DOX,

23


NV-DIR+Laser

and


467

the cytotoxic effect, B16F10 cells were incubated with different formulations and

468

treated with or without NIR laser. Afterward, the cells were stained with Calcein

469

AM/PI for visualizing the live/dead cells. Figure 4D showed that the NIR laser

470

irradiation alone did not significantly affect the cell viability, suggesting the safety of

471

laser illumination at 2.5 W/cm2. DOX and DOX+DIR+Laser showed higher

472

cytotoxicity to B16F10 cells than control, which was demonstrated by the reduced

473

cell density and increased PI staining. Monotherapy with NV-DIR+Laser or NV-DOX

474

also caused cytotoxicity to B16F10 cells. Notably, after treatment with

475

NV-DOX-DIR+Laser, the combinational therapy demonstrated higher tumor killing

476

effect as the presented much stronger PI signal for dead cells and rarely found Calcein

477

AM signal for live cells. Collectively, these results indicated that NVs could enhance

478

the cytotoxicity against tumor cells compared with free drugs and the

479

photothermal-chemotherapy based on NVs may possess great potential to realize

480

synergistic antitumor effect.

481 24


Page 24 of 40

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482

Figure 4. In vitro combinatorial photothermal-chemotherapy against tumor cells. (A).

483

The photothermal profiles of B16F10 cells after treating with free DIR, NV-DIR and

484

NV-DOX-DIR (n = 3, *p < 0,05). (B).The intracellular ROS generation after treating

485

with different formulations. The orange, black, green, blue and red line represented

486

CTRL, PBS, free DIR, NV-DIR and NV-DOX-DIR, respectively. (C) The cell

487

viability of B16F10 cells after treating with DOX, DOX+DIR+Laser, NV-DOX,

488

NV-DIR+Laser and NV-DOX-DIR+Laser, respectively. (D). Fluorescent images of

489

B16F10 cells after various treatments. The live cells were stained with Calcein AM

490

(green) and the dead cells were stained with PI (red). The scale bar is 50 µm.

491 492

3.5. In vivo imaging and biodistribution

493

We then observed the in vivo biodistribution of NVs after intravenous

494

administration on melanoma bearing mice to determine the feasibility of NVs as

495

effective drug carriers. Taking advantage of the intrinsic absorption of DIR in NIR

496

region, the distribution of free DIR and NV-DIR after administration can be easily

497

visualized.39 As shown in Figure 5A, the in vivo NIR fluorescence images were

498

monitored at various time points after intravenous injection of free DIR and NV-DIR.

499

It was found that NV-DIR showed gradually increased fluorescence of DIR with time

500

prolonging and reached the maximum after 24 h. However, free DIR showed much

501

weak fluorescence in tumor site, indicating the poor accumulation of free DIR in

502

tumor. The corresponding average fluorescence intensity also showed the similar

503

tendency (Figure 5B). The better distribution and accumulation of NV-DIR in tumor 25



504

may be attributed to the EPR effect of nano-sized drug carriers40 and the specific

505

targeting ability of cell derived NVs.22 To further investigate the distribution of free

506

DIR and NV-DIR, the mice were sacrificed, tumors and major organs were excised

507

for ex vivo imaging and fluorescence quantification at 48 h post injection. As shown

508

in Figure 5C, free DIR mainly distributed in liver, lung and spleen while showed a

509

weak accumulation in tumor. NV-DIR also showed a high accumulation in liver, lung

510

and spleen. However, the accumulation of NV-DIR in tumor site was much higher

511

than that of free DIR. The corresponding quantitative analysis also exhibited the

512

higher fluorescence intensity in tumor of NV-DIR than that of free DIR (Figure S4).

513

These results indicated that NVs showed an improved accumulation in tumor and may

514

realize the satisfied antitumor efficiency.

515 516

Figure 5. The in vivo biodistribution of free DIR and NV-DIR after systematic

517

administration. (A). The NIR fluorescent images of mice. (B). The quantitation of

518

fluorescence intensity of tumors (n = 3, *p < 0.05). (C). The ex vivo images of excised

519

tumor and major organs of C57BL/6 mice at 48 h after systematic injection of free 26


Page 26 of 40

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520

DIR and NV-DIR, respectively.

521 522

3.6. In vivo combinational photothermal and chemotherapy

523

Accoording to the results above, it has been demonstrated that incorporating DIR

524

into NVs could induce efficient hyperthermia in vitro. To further monitor the

525

photothermal effect of NVs in vivo, tumor bearing mice were injected with PBS, free

526

DIR, NV-DIR and NV-DOX-DIR, respectively. On the basis of accumulation of NVs

527

in tumor presented above, the mice were anesthetized and exposed to NIR laser after

528

24 h. As Figure 6A-B displayed, within 2 min irradiation, free DIR showed slightly

529

higher but no significant temperature increase compared to PBS, which may be

530

resulted from the restricted tumor accumulation and weak photothermal converting

531

efficiency of free DIR. However, for NV-DIR and NV-DOX-DIR treated mice, the

532

temperature of tumor was rapidly elevated over 50 °C, which would induce an

533

irreversible photothermal ablation of tumor cells.41 The results demonstrated the great

534

potential of incorporating DIR into NVs to realize effective PTT.

535

To further investigate the synergistic antitumor effect of combinational

536

photothermal and chemotherapy, C57BL/6 mice were inoculated with B16F10 cells

537

(5×104) at the left flank of back. At day 11, the mice were treated with PBS,

538

PBS+Laser, DOX, DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser

539

at an interval of 2 days for 3 times, respectively. The NIR irradiation was conducted at

540

24 h after injection. As Figure 6C showed, there is no significant difference in tumor

541

growth after treatment with laser alone compared with PBS, which further 27



Page 28 of 40

542

demonstrated the in vivo safety of laser illumination at 2.5 W/cm2. Comapred with

543

free DOX and DOX+DIR+Laser, the drug loaded NVs showed much better tumor

544

growth inhibition, suggesting the promising efficiency for NVs as drug carrier. As a

545

combination for photothermal and chemotherapy, NV-DOX-DIR achieved the best

546

tumor growth inhibition with the minimum tumor volume as 189.2 ± 53.4 mm3 at day

547

21. Moreover, NV-DOX-DIR showed the highest tumor inhibitory rate as 91.6%

548

compared with DOX, DOX+Laser, NV-DOX, NV-DIR+Laser of 38.7%, 45.6%, 76.8%

549

and 72.7%, respectively (Figure S5). During the treatment period, the body weight of

550

mice treating with different formulations all showed an increasing tendency (Figure

551

S6). Then all the mice were sacrificed, the direct image (Figure 6D) and weight of

552

excised tumors (Figure S7) showed the similar tendency as tumor growth curves.

553

Besides, the tumors were stained with HE and observed using a microscope. In PBS

554

and PBS+Laser treated groups, the tumor tissue showed compact and disordered cell

555

arrangement, indicating negligible tumor necrosis. DOX and DOX+Laser treated

556

tumors showed partial nucleus cracking while the necrosis region in NV-DOX and

557

NV-DIR+Laser

558

NV-DOX-DIR+Laser treated tumor, it showed a much larger area of tumor necrosis

559

compared with other groups (Figure 6E). To further indicate cell apoptosis at tumor

560

site, the tumors were then conducted with TUNEL assay. As Figure 6F showed,

561

NV-DOX-DIR+Laser displayed the highest expression of apoptosis fluorescence

562

compared with other groups. All in all, the combination of photothermal and

563

chemotherapy based on NVs may be a promising strategy for tumor therapy.

treated

groups

was

further

28


enlarged.

Notably,

for

Page 29 of 40 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


564 565

Figure 6. In vivo synergistic antitumor efficacy of NVs against murine melanoma. (A).

566

Infrared photothermal images of tumor-bearing mice upon exposure to NIR laser. (B).

567

In vivo photothermal profiles of the tumor tissues after treating with PBS, free DIR,

568

NV-DIR and NV-DOX-DIR, respectively (n = 3). (C). The tumor growth curves after

569

various treatments during a therapeutic period for 21 days (n = 7, *p < 0.05). (D).

570

Images of excised tumor at day 21 after treating with PBS, PBS+Laser, DOX,

571

DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 7,

572

*p < 0.05). (E). Representative HE staining images of tumor after various treatments

573

(scale bar, 250 µm). (F). TUNEL fluorescence examination images of tumor sections

574

after treating with PBS, PBS+Laser, DOX, DOX+Laser, NV-DOX, NV-DIR+Laser

575

and NV-DOX-DIR+Laser, respectively (scale bar, 50 µm). 29



576 577

3.7. Evaluation of systemic toxicity of NVs

578

To evaluate the systemic toxicity, the mice were treated with PBS, PBS+Laser,

579

DOX, DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively.

580

Afterward, the mice were sacrificed and the serum was collected to measure a series

581

of blood biochemistry parameters. ALT and AST have been considered as indicators

582

for liver pathology. After different treatment, the levels of ALT and AST were all

583

within normal range (Figure 7A-B). BUN and Cr are the commonly used indices to

584

assess the function of renal. As Figure 7C-D showed, no apparent difference was

585

observed in both BUN and Cr among all the groups. LDH is a kind of cytoplasmic

586

enzyme which can be found in all tissues. The increase of serum LDH is closely

587

related with cell damage, which directly reflects the tissue injury. In this experiment

588

setting, all the groups showed comparable level of LDH, indicating negligible damage

589

to major tissues. To further investigate the systematic toxicity, the major organs were

590

excised and stained with HE. As Figure 7F showed, there was no obvious

591

histopathological changes in all tissues after systematic treatment of various

592

formulations, respectively. The results suggested that NVs can be used as a kind of

593

safe vehicle for systematic drug delivery.

30


Page 30 of 40

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594 595

Figure 7. Systemic evaluation after intravenous administration of NVs. The

596

determination of blood biochemistry parameters including (A). ALT, (B). AST, (C).

597

BUN, (D). Cr and (E). LDH after treating with PBS, PBS+Laser, DOX, DOX+Laser,

598

NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 3). (F). The

599

representative HE staining images of the major organs after various treatments (scale

600

bar, 250 µm).

601 31



602

4. CONCLUSION

603

In this work, we fabricated a kind of DOX loaded and NIR dye inserted

604

biomimetic nanovesicles by a facile method for combinational photothermal and

605

chemotherapy. As a kind of membrane vesicles, NVs could effectively evade the

606

clearance by RES. Owing to the specific targeting ability of membrane vesicles and

607

EPR effect of nanosized vehicles, NVs can accumulate in tumor site after intravenous

608

injection. Upon NIR laser irradiation, the NVs with DIR insertion can convert

609

absorbed fluorescent energy into comparable heat with high efficiency to eradicate

610

tumor cells. Meanwhile, the NIR triggered hyperthermia can destroy the membrane of

611

NVs and thus induce the accelerated release of DOX to further cause direct

612

cytotoxicity to tumor cells. The combination of photothermal and chemotherapy could

613

achieve efficient in vitro tumor killing and satisfied in vivo tumor growth inhibition

614

against murine melanoma. The NVs demonstrated the great potential in the

615

combinatorial antitumor therapy with good biomimetic properties and feasibility for

616

clinic translation.

617 618

ASSOCIATED CONTENT

619

Supporting Information

620

The Supporting Information is available free of charge on the ACS Publications

621

website.

622

Supplementary Figures S1−S7

623

Figure S1. The relative diameter change of NV-DOX and NV-DOX-DIR in FBS for 5 32


Page 32 of 40

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624

days (n = 3).

625

Figure S2. The relative retained DOX in NVs for 5 days at 4 oC (n = 3).

626

Figure S3. Quantitative detection of ROS generation after treating with NIR

627

irradiation (n = 3).

628

Figure S4. The quantitation of fluorescent intensity of tumors and the major organs at

629

48 h after intravenous injection of free DIR and NV-DIR (n = 3, *p < 0.05).

630

Figure S5. The tumor inhibitory rate after treating with PBS+Laser, DOX,

631

DOX+Laser, NV-DOX, NV-DIR+Laser and NV-DOX-DIR+Laser, respectively (n = 7,

632

*p < 0.05).

633

Figure S6. The relative body weight change after various treatments (n = 7)

634

Figure S7. The weight of the excised tumors after different treatments (n = 7, *p

Biomimetic Nanovesicles for Enhanced Antitumor Activity of

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