In Vivo Analysis of Biodegradable Liposome Gold Nanoparticles as

Subscriber access provided by University of Otago Library

Communication

IN VIVO ANALYSIS OF BIODEGRADABLE LIPOSOME GOLD NANOPARTICLES AS EFFICIENT AGENTS FOR PHOTOTHERMAL THERAPY OF CANCER Aravind Kumar Rengan, Amirali B. Bukhari, ARPAN PRADHAN, Renu Malhotra, Rinti Banerjee, Rohit Srivastava, and Abhijit De Nano Lett., Just Accepted Manuscript • Publication Date (Web): 02 Jan 2015 Downloaded from http://pubs.acs.org on January 3, 2015

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

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

Page 1 of 24

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

Nano Letters

1

IN

2

NANOPARTICLES AS EFFICIENT AGENTS FOR PHOTOTHERMAL THERAPY

3

OF CANCER

VIVO

ANALYSIS

OF

BIODEGRADABLE

LIPOSOME

GOLD

4 5

Aravind Kumar Rengan1ǂ, Amirali B. Bukhari2ǂ, Arpan Pradhan1, Renu Malhotra2, Rinti

6

Banerjee1, Rohit Srivastava1* and Abhijit De2*

7

1 Department of Bioscience and Bioengineering, Indian Institute of Technology – Bombay,

8

Mumbai, INDIA

9

2 Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Navi Mumbai,

10

INDIA

11 12

ǂ Equal contribution

13

* Corresponding authors ([email protected]; [email protected])

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

* Corresponding Author: Dr. Abhijit De Scientific Officer 'F' Molecular Functional Imaging Laboratory Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai - 410210 INDIA Phone: +91-22-2740 5038 Fax: +91-22-2740 5085 Email: [email protected] * Co-corresponding Author: Dr. Rohit Srivastava Associate Professor Department of Bioscience and Bioengineering IIT Bombay, Powai, Mumbai, 400076, INDIA Phone: +91-22-2576 7746 Fax : +91-22-2572 3480 E-mail: [email protected]

1 Environment ACS Paragon Plus

Nano Letters

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

Page 2 of 24

39

ABSTRACT

40

We report biodegradable plasmon resonant liposome gold nanoparticles (LiposAu NPs)

41

capable of killing cancer cells through photothermal therapy. The pharmacokinetic study of

42

LiposAu NPs performed in small animal model indicates in situ degradation in hepatocytes

43

and further getting cleared through the hepato-biliary and renal route. Further, the therapeutic

44

potential of LiposAu NPs tested in mouse tumor xenograft model using NIR laser (750nm)

45

illumination resulting complete ablation of tumor mass, thus prolonging disease-free survival.

46 47

KEYWORDS:

Gold

nanoparticles,

48

Nanotechnology, Theranostics.

Liposomes,

Photothermal


Therapy,

Cancer

Page 3 of 24

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

Nano Letters

49

Many organic and inorganic nanosystems are being actively researched for their efficiency in

50

cancer diagnosis and treatment.1–6 Among them, plasmonic nanostructures for photothermal

51

therapy (PTT) gain considerable importance owing to the advent of two ongoing PTT based

52

clinical trials making use of gold nanoshells for the treatment of brain and metastatic lung

53

tumors.7 These plasmonic nanostructures also serve as efficient candidates for imaging, and

54

thereby bringing out their multifunctional capabilities.8–17 According to the Food and Drug

55

Administration (FDA) guidelines, any imaging agent (administered into the body) should be

56

capable of getting cleared completely from the body within a reasonable period of time.18,19

57

Gold based materials deployed in PTT are generally larger than 20nm in size.20–23

58

Accumulation of such metallic nanoparticles in body could serve as a potential health risk. In

59

2013, Melnik et al. reported the transfer of silver nanoparticles via placenta to the rat

60

foetuses, bringing out the gravity of risk involved in nanoparticle accumulation.24 Although

61

many of such materials could serve as efficient imaging agents, their larger size and non-

62

degradable nature prevents renal clearance, thus limiting their application in vivo. To achieve

63

renal clearance the size of inorganic nanoparticles will have to be < 5.5nm.18,25 Inorganic,

64

metal containing nanoparticles lesser than 5.5nm in size are capable of getting filtered

65

through the glomerular basement membrane (GBM),18 thereby serving as ideal candidates for

66

imaging (with renal route of clearance) but are unsuitable for PTT. Hence, a multifunctional

67

nanosystem capable of achieving good body clearance through both hepato-biliary and renal

68

route in addition to serving as effective agents for PTT is warranted. Our group synthesized a

69

liposome-gold nanoparticle hybrid system (henceforth referred to as LiposAu NPs) that has

70

such multifunctional capabilities.26 Since the core of this nanohybrid system is made up of

71

biodegradable lipid, the gold coating on the surface is capable of splitting into smaller

72

particles (≤5-8nm) and achieving both hepato-biliary and renal clearance. Such novel

73

nanoparticle systems also have an added advantage of getting accumulated specifically at the

74

tumor site due to the leaky vasculature of developing blood vessels and poorly developed

75

lymphatics (Enhanced Permeation and Retention - EPR effect).27 In other words, they could

76

be passively targeted to the tumor region.28 Alternatively, spatio-temporal control by external

77

trigger is another form of achieving specificity, wherein drug delivery is controlled to a

78

specific region by optical, magnetic or ultrasound modalities.29,30 In contrast to the passive

79

mode, active targeting involves antibody or affibody conjugation that binds with specific

80

antigen at the tumor site. However, the added bulk of the antibody protein size and the cost of

81

antibody limits its deployment on a larger scale.31 To overcome this limitation of increased


Nano Letters

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

82

cost factor, the spatio-temporal control of external trigger is an efficient and economical

83

alternate to the antibody mediated targeting.

84

Biodegradable plasmon resonant nanoparticles employing 1,2-Dipalmitoyl-sn-glycero-3-

85

phosphocholine (DPPC) gold hybrid nanostructures were synthesized by Troutman et al. in

86

2008.32 The transition temperature of DPPC being 41°C restricts its use only to drug delivery

87

rather than hyperthermic killing of cancer cells (as biological cells begin to die of

88

hyperthermia only when the temperature reaches ≥43oC).33 We had earlier reported the

89

synthesis of a nanoformulation using 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)-

90

cholesterol and further coating with gold (resulting in the formation of LiposAu NPs)

91

enabling the achievement of both drug delivery and photothermal therapy.26 Although

92

conceptual data was available with respect to such a lipid gold hybrid material, till date their

93

in vivo fate validation remains uncharted. In the current study, we demonstrate the in vivo

94

pharmacokinetics and photothermal efficacy of LiposAu NPs at the target site in a mouse

95

tumor xenograft model. Herein, we report the in vivo degradation of these LiposAu NPs and

96

their pharmacokinetic profile. The photothermal efficiency of these LiposAu NPs was tested

97

against cancer cell lines under in vitro and in vivo conditions. To the best of our knowledge,

98

this is the first report demonstrating in vivo degradation of these photothermally active

99

LiposAu NPs in hepatocytes and subsequent clearance of gold through both hepato-biliary

100

and renal route.

101

LiposAu NPs were synthesized as per established protocol with slight modification to

102

achieve a size range of 100-120nm.26 A schematic representing the synthesis and

103

photothermal effect on LiposAu NPs including their ability to cause DNA damage and self-

104

destruction achieving size reduction is shown in Figure 1. Representative transmission

105

electron microscope (TEM) and scanning electron microscope (SEM) images of these

106

LiposAu NPs have been shown in Figure 2A and 2B. Dynamic Light Scattering (DLS)

107

measurement indicates a size range of about 100nm (Figure 2C) and the polydispersity

108

index as 0.18. The lattice arrangement of Au is clearly visible in High Resolution-TEM (HR-

109

TEM) images of the surface region of these LiposAu NPs (Figure S1A). Such type of

110

plasmon resonant nanoparticles have shown to be responsive for specific wavelength of laser

111

light mediated excitation.34 Hence, the LiposAu NPs were tuned to an absorbance range of

112

750nm to achieve photothermal effect when subjected to a beam of 750nm laser light with

113

650mW power (Figure 2D). These LiposAu NPs when treated with lipase enzyme lost their


Page 4 of 24

Page 5 of 24

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

Nano Letters

114

NIR absorbance peak, confirming their degradable nature (Figure 2D). Also, when treated

115

with specific temperature increments (water bath mediated), these LiposAu NPs showed

116

corresponding reduction in NIR absorbance denoting their thermo-sensitivity (Figure S1B).

117

The in vivo biodistribution and pharmacokinetic study was performed using Swiss albino

118

mice. The analysis of various tissues, plasma and urine was performed at varying time

119

periods (Day 1, 7, and 14) after intravenous injection of LiposAu NPs (~110µg/400µl)

120

through the tail vein. It was found that majority of the injected particles were accumulated in

121

liver, followed by spleen and kidney of the mouse. As these NPs were not targeted, they were

122

directly taken up by the reticulo-endothelial system (i.e. liver and spleen) of the mouse. As

123

liver is the major metabolizing organ of the body playing an important role in lipid

124

metabolism,35 the probability of LiposAu NPs to undergo enzymatic degradation gets

125

maximized owing to their greater accumulation in liver region. The accumulation and

126

metabolic degradation of these NPs in the liver was qualitatively confirmed by TEM analysis

127

of the liver tissue. As revealed from Figure 3A, 1 day after intravenous delivery, these NPs

128

were found to be in an aggregated state, but their original spherical morphology was

129

completely lost. This indicates that under in vivo condition the LiposAu NPs present in

130

systemic circulation would undergo metabolic degradation due to enzymatic activity in

131

hepatocytes. Further, to confirm the aggregation observed in TEM, Energy Dispersive X-ray

132

Spectroscopy (EDAX) analysis was performed (Figure S2A). Inductively Coupled Plasma –

133

Atomic Emission Spectroscopy (ICP-AES) analysis of mouse liver revealed considerable

134

accumulation of gold, right from day 1 end point analysis. But there was a considerable

135

decrease in the %ID/g on subsequent long term study. The observed reduction in the %ID

136

from about 52% (day 1) to 9.8% (day 7) and further declined to about 3% (day14) (P =

137

0.0037) (Figure 3E). The percentage reduction of Au in the liver at 14 days was further

138

confirmed by TEM analysis (Figure S2B). Also, HR – TEM images of liver and kidney

139

samples confirms the presence of degraded Au NPs showing lattice arrangement which

140

further indicates their metallic nature (Figure S2B). The negligible Au values detected in the

141

liver of the normal saline treated controls were subtracted from those of the treated samples

142

as background corrections. Similar observations were noted for spleen, kidneys, and intestine.

143

We also performed TEM analysis of blood plasma at 2 hours end point that showed Au NPs

144

of size 2-8nm [Figure 3G (i) and (ii)]. Presence of such small Au NPs in blood suggests that

145

the disintegrated NPs (from the larger LiposAu NPs) reach the circulation after their

146

enzymatic degradation in liver.


Nano Letters

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

147

It was observed that majority of the injected particles get accumulated in liver and spleen and

148

the smaller percentage of 2-8nm particles circulating in blood could have resulted in

149

accumulation in kidney and further clearance through urine. TEM analysis of kidney

150

identified presence of such smaller particles (Figure 3C). The Inductively Coupled Plasma –

151

Mass Spectroscopy (ICP-MS) analysis of kidneys indicated an accumulation of about 2.7% at

152

day 1 which reduced to an approximate 0.25% at day 7 and further to about 0.22% on day 14

153

(Figure 3E). Though the current percentage of accumulation in kidney is small in

154

comparison to liver we expect an improvement in renal clearance when the LiposAu NPs are

155

subjected to both photothermal and enzymatic degradation. The current study has limited

156

scope of understanding the real-time in vivo biodistribution and enzymatic degradation of

157

LiposAu NPs. Though some amount of particles are getting cleared through the hepato-biliay

158

route (Table S1), we find that small amount of Au in urine even on day 7 and 14 indicating

159

the possibility of renal excretion (Table S2). We however speculate that the excretion of Au

160

through the renal route is a constant process overtime and determination of this complete

161

excretion in real time remains a limitation. Generally, individual small molecules like

162

albumin, get repelled by the negatively charged glomerular basement membrane (GBM) of

163

the nephrons.36 This charge based repulsion prevents any accumulation of negatively charged

164

particle/molecule in kidney that in turn restricts their excretion through urine.37 The higher

165

accumulation of gold in kidney at day 1 time period indicates that gold NPs owing to their

166

positively charged surface were able to overcome the charge based repulsion in the GBM.

167

The ICP-MS analysis of mice plasma showed significant reduction of gold between day 1

168

and 14 end point analysis (P < 0.0001) (Figure 3F). The value of gold was reduced from

169

390±13.7ng/ml to 105.4±5.11ng/ml. As already pointed out, the size range of the gold NPs

170

observed in mice blood plasma was also falling under the range of renal excretion. Also, the

171

ICP-MS analysis of urine samples (collected at day 1, 7 and 14) revealed the presence of gold

172

in varying concentrations confirming its excretion through the renal route (Table S2).

173

Furthermore, we studied biodistribution in tumor bearing mice using Indocyanine Green

174

(ICG; NIR dye) coated LiposAu NPs to facilitate their short term tracking in vivo. In order to

175

understand if they possess any tumor homing properties after intravenous injection, we made

176

use of the HT1080 xenograft model. Data suggests that these particles are not capable of any

177

specific homing at the tumor site owing to their non-targeted nature. Also, in order to achieve

178

successful homing, several parameters play a key role, one such being the leaky vasculature

179

of the tumor bed. However, unlike only ICG injected control mice, we were able to obtain

180

considerable signal from the bladder region for up to a period of 24 hours (Figure S3A) 6 Environment ACS Paragon Plus

Page 6 of 24

Page 7 of 24

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

Nano Letters

181

suggesting possible renal clearance. ICP-MS validations also confirm no LiposAu NPs

182

uptake in the tumor (Figure S3B).

183

To understand the toxic effect of these gold NPs accumulation in liver and kidney, the blood

184

plasma serum glutamic pyruvic transaminase (SGPT or ALT) and creatinine levels were also

185

analyzed. No significant difference was observed between the control and the LiposAu NPs

186

treated groups (Day 1) (Figure 3B and 3D) confirming that no specific acute toxicity to the

187

liver or kidney was exhibited. Qualitative urine dipstick analysis was also performed in mice

188

urine (LiposAu NPs treated and controls). There was no detectable blood or protein observed

189

in the urine samples (Figure S4). In the absence of human data availability, such

190

biodistribution studies involving experimental animals stands as the most reliable approach to

191

determine the toxicity properties of our chemically synthesized NPs. The close resemblance

192

of anatomy and physiology of mice to that of humans enables us to predict the likely

193

pharmacokinetics of these NPs in future human clinical transition. Additionally, in vitro

194

biocompatibility on NIH-3T3 cells revealed no indications of toxicity associated with

195

LiposAu NPs (Figure S5).

196

In vitro photothermal efficacy studies were performed using MCF-7 (breast) and HT1080

197

(fibrosarcoma) cancer cell lines. Both cell types were engineered for overexpressing a fusion

198

reporter i.e. firefly luciferase 2 (fluc2) and turboFP fluorescent protein. Optimization of the

199

laser irradiation time suggests a lethal effect on the breast cancer cells beyond 4 minute of

200

continuous exposure (Figure S6). Hence, 4 minute was chosen as the ideal irradiation time

201

for a concentration of 15µg/ml LiposAu NPs photothermal treatment. Qualitative assessment

202

of the photothermal efficacy was studied in vitro using MCF-7-fluc2-turboFP cells by

203

fluorescence microscopy. Combination treatment of LiposAu NPs and laser showed loss of

204

fluorescence (suggesting cell death) at the area of contact as indicated by the arrow in Figure

205

4A. Such ablation of cancer cells is due to heat generation in the region of laser contact

206

where LiposAu NPs are also present. Additionally, no change was observed in the

207

fluorescence signal of either the untreated control or the internal control groups (LiposAu

208

only and laser only). This result was further supported by the significant reduction of fluc2

209

luminescence in MCF-7-fluc2-turboFP (P = 0.0034) and HT1080-fluc2-turboFP (P =

210

0.0024) cells when compared to laser treated control (Figure 4B). Herein, the luciferase light

211

output is a direct measure of cell viability as the fluc2 enzyme catalyzes its substrate D-

212

luciferin only in the presence of cellular ATP.


Nano Letters

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

213

As PTT is known to cause DNA damage mediated cell death,38,39 we also monitored the

214

formation of γH2A.X foci, a marker for DNA double strand breaks. Minimal or no γH2A.X

215

foci were observed in the untreated control and only LiposAu treated cells. The only laser

216

treated control cells revealed a slightly higher number of the γH2A.X foci in comparison.

217

However, it was seen that the cells that received the combination treatment with both

218

LiposAu and laser, demonstrated the presence of highly significant number of γH2A.X foci

219

formations in both the MCF-7 (P < 0.0001) and HT1080 (P = 0.0007) cells (Figure 4C and

220

4D). This validates that the photothermal therapy mediated DNA double strand break was

221

responsible for cancer cell ablation.

222

In vivo temperature increment was critically determined by creating subcutaneous blebs of

223

normal saline or LiposAu NPs in varying volumes (25µl, 50µl, and 100µl) in hairless

224

(BALB/c Nude) mice. Each of the blebs was treated with the NIR laser for 4 minutes. The

225

temperature of the blebs was continuously monitored by an IR thermometer pre- and post-

226

treatment. The blebs injected with normal saline did not show any specific temperature

227

increment after 4 minutes of continuous laser irradiation. However, the blebs injected with

228

LiposAu NPs showed a temperature increment up to 7°C with 4 minutes of laser irradiation.

229

There was also eschar formation on the treated area (noticed after 24 hours of treatment),

230

indirectly indicating temperature increment (Figure S7). Such eschar formation has been

231

previously reported for gold nanoshells based PTT as well.40

232

Further, in vivo photothermal efficacy was determined using HT1080-fluc2-turboFP tumor

233

xenograft model in BALB/c NUDE mice. On the 20th day with growing tumors (average size

234

of about 70mm3), mice were randomly segregated into 3 groups (n=5 per group). Group I

235

received normal saline (30µl) as vehicle control; group II animals were treated with laser

236

only while group III animals were given the combination treatment of LiposAu NPs

237

(0.5µg/µl in 30µl) and laser. Group II and III animals were subjected to laser irradiation for a

238

period of 4 minutes. The treatment cycle was divided into two rounds between day 20 and 30.

239

Two days interval was kept between the treatment cycles to avoid any therapy burden on the

240

animals. All animals were imaged by injecting D-luciferin substrate every 10th day starting

241

from day 0 till the end of the experiment. Our study reveals a significant reduction in the

242

bioluminescence signal (3.36 x 106 ± 1.52 x 106 p/sec/cm2/sr) on day 30 of the group III

243

animals when compared to group I (5.47 x 1010 ± 2.08 x 1010 p/sec/cm2/sr) (P = 0.0302) or

244

the group II (4.95 x 1010 ± 1.75 x 1010 p/sec/cm2/sr) (P = 0.0068) animals (Figure 5A and


Page 8 of 24

Page 9 of 24

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

Nano Letters

245

5B). Additionally, the group III animals demonstrated complete regression of tumor and a 4

246

out of 5 animal survived until 6 months (P = 0.003) in contrast to the group I and group II

247

animals, which all died within 35-40 days due to tumor burden (Figure 5C). At the end point

248

of monitoring, non-invasive in vivo bioluminescence imaging of group III showed no signal

249

output at the therapy site (Figure 5D). It was noted that the combination treatment of

250

LiposAu NPs with laser results in a 4.63 fold reduction of the bioluminescence signal with

251

respect to the respective controls (Figure 5E). Histopathological analysis revealed that the

252

combination of LiposAu NPs and laser resulted in the most extensive necrotic response in

253

that region. Due to this combination treatment, the tumor cells in the underlying region were

254

completely ablated, whereas about 95% of the tumor mass remained in laser treated animals

255

(Figure 5F).

256

The current study involves understanding the degradation dynamics of plasmon resonant

257

LiposAu NPs under physiological condition. It was observed that these particles were able to

258

degrade by the enzymatic reaction into smaller particles whose size range was ideal for renal

259

excretion in addition to hepato-biliary route. The long term in vivo analysis also confirmed

260

the bimodal clearance of these NPs. LiposAu NPs proved to be efficient candidate for in vivo

261

photothermal mediated ablation of cancer. Their ability to generate massive amount of

262

γH2A.X foci is a strong indicator of the mode of tumor ablation by DNA double strand

263

breaks. Applicability of such novel biodegradable hybrid nanoparticle system holds great

264

promise in cancer nanotherapeutics.

265 266

SUPPORTING INFORMATION

267

Supporting Information Available: Details of all experimental procedures, and materials used

268

during the study can be found in the supplementary information. This material is available

269

free of charge via the Internet at http://pubs.acs.org.

270

ACKNOWLEDGEMENTS

271

The authors would like to acknowledge TMC Seed-in-Air Intramural funding to AD and

272

Molecular optical imaging equipment (IVIS Lumina II) support from DBT Bioengineering

273

research grant to AD; IIT-B Healthcare initiative for funding the project and SAIF-IITB,


Nano Letters

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

274

IRCC for characterization studies. ACTREC TEM facility is also acknowledged for sample

275

processing. This work is part of the doctoral thesis of AKR at IIT-Bombay.

276

CONFLICT OF INTEREST

277

The authors declare no competing financial interest.

278 279

REFERENCES

280 281

(1)

Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Adv. Mater. 2011, 23, H18–H40.

282 283

(2)

Timko, B. P.; Dvir, T.; Kohane, D. S. Adv. Mater. Deerf. Beach Fla 2010, 22, 4925– 4943.

284

(3)

Kim, J.; Piao, Y.; Hyeon, T. Chem. Soc. Rev. 2009, 38, 372–390.

285

(4)

Sailor, M. J.; Park, J.-H. Adv. Mater. 2012, 24, 3779–3802.

286 287

(5)

Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat Nano 2007, 2, 751–760.

288 289

(6)

Yezhelyev, M. V; Gao, X.; Xing, Y.; Al-Hajj, A.; Nie, S.; O’Regan, R. M. Lancet Oncol. 2006, 7, 657–667.

290 291

(7)

Thakor, a S.; Jokerst, J.; Zavaleta, C.; Massoud, T. F.; Gambhir, S. S. Nano Lett. 2011, 11, 4029–4036.

292

(8)

Alivisatos, P. Nat. Biotechnol. 2004, 22, 47–52.

293

(9)

Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Nano Lett. 2005, 5, 709–711.

294 295

(10)

Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Chem. Rev. 2008, 108, 494–521.

296 297

(11)

Chen, J.; Wang, D.; Xi, J.; Au, L.; Siekkinen, A.; Warsen, A.; Li, Z.-Y.; Zhang, H.; Xia, Y.; Li, X. Nano Lett. 2007, 7, 1318–1322.

298 299

(12)

Khlebtsov, N. G.; Dykman, L. A. J. Quant. Spectrosc. Radiat. Transf. 2010, 111, 1– 35.

300

(13)

Rozanova, N.; Zhang, J. Sci. China Ser. B Chem. 2009, 52, 1559–1575.

301 302

(14)

Rengan, A. K.; Kundu, G.; Banerjee, R.; Srivastava, R. Part. Part. Syst. Charact. 2014, 31, 398–405.


Page 10 of 24

Page 11 of 24

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

Nano Letters

303 304

(15)

Oldenburg, S. J.; Jackson, J. B.; Westcott, S. L.; Halas, N. J. Appl. Phys. Lett. 1999, 75, 2897.

305 306

(16)

Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115–2120.

307

(17)

Cheng, F.-Y.; Chen, C.-T.; Yeh, C.-S. Nanotechnology 2009, 20, 425104.

308 309

(18)

Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2007, 25, 1165–1170.

310

(19)

Agdeppa, E. D.; Spilker, M. E. AAPS J. 2009, 11, 286–299.

311

(20)

Huang, X.; El-Sayed, M. A. J. Adv. Res. 2010, 1, 13–28.

312 313

(21)

Ryu, J. H.; Koo, H.; Sun, I.-C.; Yuk, S. H.; Choi, K.; Kim, K.; Kwon, I. C. Adv. Drug Deliv. Rev. 2012, 64, 1447–1458.

314 315

(22)

Hwang, S.; Nam, J.; Jung, S.; Song, J.; Doh, H.; Kim, S. Nanomedicine (Lond). 2014, 9, 2003–2022.

316

(23)

Zhao, J.; Wallace, M.; Melancon, M. P. Nanomedicine (Lond). 2014, 9, 2041–2057.

317 318

(24)

Melnik, E. A.; Buzulukov, Y. P.; Demin, V. F.; Demin, V. A.; Gmoshinski, I. V; Tyshko, N. V; Tutelyan, V. A. Acta Naturae 2013, 5, 107–115.

319 320

(25)

Zhang, X.-D.; Wu, D.; Shen, X.; Liu, P.-X.; Fan, F.-Y.; Fan, S.-J. Biomaterials 2012, 33, 4628–4638.

321 322

(26)

Rengan, A. K.; Jagtap, M.; De, A.; Banerjee, R.; Srivastava, R. Nanoscale 2014, 6, 916–923.

323 324

(27)

Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H. Drug Discovery Today, 2006, 11, 812– 818.

325 326

(28)

Bertrand, N.; Wu, J.; Xu, X.; Kamaly, N.; Farokhzad, O. C. Adv Drug Delivery Rev, 2014, 66, 2–25.

327

(29)

Urban, C.; Urban, A. S.; Charron, H.; Joshi, A. Transl. Cancer Res. 2013, 2, 292–308.

328

(30)

Ahmed, N.; Fessi, H.; Elaissari, A. Drug Discovery Today, 2012, 17, 928–934.

329

(31)

Firer, M. A.; Gellerman, G. J. Hematol. Oncol. 2012, 5, 70-85.

330

(32)

Troutman, T. S.; Barton, J. K.; Romanowski, M. Adv. Mater. 2008, 20, 2604–2608.

331

(33)

Issels, R. D. Eur. J. Cancer 2008, 44, 2546–2554.

332

(34)

Troutman, T. S.; Leung, S. J.; Romanowski, M. Adv. Mater. 2009, 21, 2334–2338.


Nano Letters

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

333 334

(35)

Nguyen, P.; Leray, V.; Diez, M.; Serisier, S.; Bloc’h, J. Le; Siliart, B.; Dumon, H. J. Anim. Physiol. Anim. Nutr. (Berl). 2008, 92, 272–283.

335

(36)

Haraldsson, B.; Nyström, J.; Deen, W. M. Physiol. Rev. 2008, 88, 451–487.

336

(37)

Brenner, B. M.; Hostetter, T. H.; Humes, H. D. N. Engl. J. Med. 1978, 298, 826–833.

337 338

(38)

Choi, Y. J.; Kim, Y. J.; Lee, J. W.; Lee, Y.; Lee, S.; Lim, Y.-B.; Chung, H. W. J. Nanosci. Nanotechnol. 2013, 13, 4437–4445.

339

(39)

Roti Roti, J. L. Int. J. Hyperth. 2008, 24, 3–15.

340 341

(40)

Stern, J. M.; Stanfield, J.; Kabbani, W.; Hsieh, J.-T.; Cadeddu, J. A. J. Urol. 2008, 179, 748–753.

342


Page 12 of 24

Page 13 of 24

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

Nano Letters

343

Figures and Figure Legends:

344

Graphical Abstract

345 346 347


Nano Letters

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

348 349 350

Figure 1: Schematic diagram representing the principle of synthesis of LiposAu NPs

351

and their mode of action to perform photothermal treatment causing intracellular DNA

352

damage.

353


Page 14 of 24

Page 15 of 24

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

Nano Letters

354 355 356

Figure 2: Characterization of LiposAu NPs. A) TEM image of LiposAu NPs. (Scale =

357

50nm) B) SEM image of LiposAu NPs. (Scale = 100nm) C) DLS size distribution of

358

LiposAu NPs. D) UV-Vis absorbance spectra of lipase, liposome, and LiposAu NPs treated

359

with lipase enzyme in comparison with (untreated) LiposAu NPs.

360


Nano Letters

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

361 362 363

Figure 3: In vivo biodistribution and clearance of LiposAu NPs. A) TEM image of liver

364

tissue showing (i) control hepatocytes, (ii) & (iii) hepatocyte containing LiposAu NPs in

365

aggregated state. B) Mice plasma levels of ALT (U/L) at the end of 24 hours. C) TEM

366

images of kidney tissue showing (i) control tissue, (ii) & (iii) kidney tissue containing

367

LiposAu NP in its dissociated state with it less than 5nm sized gold seeds. D) Mice plasma

368

values of creatinine (mg/dl) at the end of 24 hours. E) Graph represents tissue biodistribution

369

of Au in vivo as determined by ICP-MS and ICP-AES analysis at various end points. F) ICP-

370

MS based mice blood plasma levels of Au (ng/ml). G) TEM image of blood plasma showing

371

small gold particles of varying size range as represented in (i) and (ii). Significance is

372

designated as * indicates P < 0.05, ** indicates P < 0.005 and **** indicates P < 0.0001.

373

Scale bar: A(i & ii) 2µm; A(iii) 1 µm; C(i) 1µm; C(ii & iii) 20nm; G(i) 5nm; and G(ii) 2nm.

374


Page 16 of 24

Page 17 of 24

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

Nano Letters

375 376 377

Figure 4: In vitro photothermal ablation of cancer cells by LiposAu NPs. A)

378

Fluorescence micrograph images of photothermal therapy mediated cell death in MCF-7-

379

fluc2-turboFP cancer cell line. Red color represents the fluorescence of the turboFP (635 nm

380

emission) protein. B) Quantitative analysis of bioluminescence based photothermal cell death

381

in MCF-7-fluc2-turboFP (P = 0.0034) and HT1080-fluc2-turboFP (P = 0.0024) cancer cells.

382

Representative images for qualitative assessment are given below the graph representing the

383

various groups. Pseudocolor bar indicates the photons captured by the CCD camera. C)

384

Representative images showing the formation of γH2A.X foci after treatment in MCF-7 and

385

HT1080 cancer cells. D) Quantitative assessment of γH2A.X foci in MCF-7 (P < 0.0001) and

386

HT1080 (P = 0.0007) cancer cells.

387


Nano Letters

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

388 389 390

Figure 5: In vivo photothermal ablation by LiposAu NPs in tumor xenograft. A)

391

Representative pre- and post-treatment in vivo bioluminescence images of mice bearing

392

HT1080-fluc2-turboFP tumor xenografts. B) Quantitative assessment of bioluminescence to

393

demonstrate the increase in tumor volume. The highlighted region indicates the treatment

394

period (* indicates P < 0.05 and ** indicates P < 0.01). C) Kaplan-Meier survival curve of

395

the tumor bearing mice (P = 0.003). D) Representative photographic and bioluminescence

396

image of LiposAu NPs and laser treated mouse post 6 months of treatment reveals no signs of

397

regression. E) Bar diagram represents the fold change in bioluminescence between the laser,

398

and LiposAu NPs + laser treated tumors. F) Hematoxylin and Eosin (H&E) stained

399

histological evaluation of tumor tissue after PTT.

400


Page 18 of 24

Page 19 of 24

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

Nano Letters

Graphical Abstract 39x19mm (300 x 300 DPI)

ACS Paragon Plus Environment

Nano Letters

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

Schematic diagram representing the principle of synthesis of LiposAu NPs and their mode of action to perform photothermal treatment causing intracellular DNA damage. 109x68mm (300 x 300 DPI)


Page 20 of 24

Page 21 of 24

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

Nano Letters

Characterization of LiposAu NPs. A) TEM image of LiposAu NPs. (Scale = 50nm) B) SEM image of LiposAu NPs. (Scale = 100nm) C) DLS size distribution of LiposAu NPs. D) UV-Vis absorbance spectra of lipase, liposome, and LiposAu NPs treated with lipase enzyme in comparison with (untreated) LiposAu NPs. 79x75mm (300 x 300 DPI)


Nano Letters

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

In vivo biodistribution and clearance of LiposAu NPs. A) TEM image of liver tissue showing (i) control hepatocytes, (ii) & (iii) hepatocyte containing LiposAu NPs in aggregated state. B) Mice plasma levels of ALT (U/L) at the end of 24 hours. C) TEM images of kidney tissue showing (i) control tissue, (ii) & (iii) kidney tissue containing LiposAu NP in its dissociated state with it less than 5nm sized gold seeds. D) Mice plasma values of creatinine (mg/dl) at the end of 24 hours. E) Graph represents tissue biodistribution of Au in vivo as determined by ICP-MS and ICP-AES analysis at various end points. F) ICP-MS based mice blood plasma levels of Au (ng/ml). G) TEM image of blood plasma showing small gold particles of varying size range as represented in (i) and (ii). Significance is designated as * indicates P < 0.05, ** indicates P < 0.005 and **** indicates P < 0.0001. Scale bar: A(i & ii) 2µm; A(iii) 1 µm; C(i) 1µm; C(ii & iii) 20nm; G(i) 5nm; and G(ii) 2nm. 153x132mm (300 x 300 DPI)


Page 22 of 24

Page 23 of 24

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

Nano Letters

In vitro photothermal ablation of cancer cells by LiposAu NPs. A) Fluorescence micrograph images of photothermal therapy mediated cell death in MCF-7-fluc2-turboFP cancer cell line. Red color represents the fluorescence of the turboFP (635 nm emission) protein. B) Quantitative analysis of bioluminescence based photothermal cell death in MCF-7-fluc2-turboFP (P = 0.0034) and HT1080-fluc2-turboFP (P = 0.0024) cancer cells. Representative images for qualitative assessment are given below the graph representing the various groups. Pseudocolor bar indicates the photons captured by the CCD camera. C) Representative images showing the formation of γH2A.X foci after treatment in MCF-7 and HT1080 cancer cells. D) Quantitative assessment of γH2A.X foci in MCF-7 (P < 0.0001) and HT1080 (P = 0.0007) cancer cells. 134x101mm (300 x 300 DPI)


Nano Letters

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

In vivo photothermal ablation by LiposAu NPs in tumor xenograft. A) Representative pre- and posttreatment in vivo bioluminescence images of mice bearing HT1080-fluc2-turboFP tumor xenografts. B) Quantitative assessment of bioluminescence to demonstrate the increase in tumor volume. The highlighted region indicates the treatment period (* indicates P < 0.05 and ** indicates P < 0.01). C) Kaplan-Meier survival curve of the tumor bearing mice (P = 0.003). D) Representative photographic and bioluminescence image of LiposAu NPs and laser treated mouse post 6 months of treatment reveals no signs of regression. E) Bar diagram represents the fold change in bioluminescence between the laser, and LiposAu NPs + laser treated tumors. F) Hematoxylin and Eosin (H&E) stained histological evaluation of tumor tissue after PTT. 124x87mm (300 x 300 DPI)


Page 24 of 24

In Vivo Analysis of Biodegradable Liposome Gold Nanoparticles as

Recommend Documents