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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
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1
IN
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NANOPARTICLES AS EFFICIENT AGENTS FOR PHOTOTHERMAL THERAPY
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OF CANCER
VIVO
ANALYSIS
OF
BIODEGRADABLE
LIPOSOME
GOLD
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Aravind Kumar Rengan1ǂ, Amirali B. Bukhari2ǂ, Arpan Pradhan1, Renu Malhotra2, Rinti
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Banerjee1, Rohit Srivastava1* and Abhijit De2*
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1 Department of Bioscience and Bioengineering, Indian Institute of Technology – Bombay,
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Mumbai, INDIA
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2 Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Navi Mumbai,
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INDIA
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ǂ Equal contribution
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* Corresponding authors (
[email protected];
[email protected])
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* 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:
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ABSTRACT
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We report biodegradable plasmon resonant liposome gold nanoparticles (LiposAu NPs)
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capable of killing cancer cells through photothermal therapy. The pharmacokinetic study of
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LiposAu NPs performed in small animal model indicates in situ degradation in hepatocytes
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and further getting cleared through the hepato-biliary and renal route. Further, the therapeutic
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potential of LiposAu NPs tested in mouse tumor xenograft model using NIR laser (750nm)
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illumination resulting complete ablation of tumor mass, thus prolonging disease-free survival.
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KEYWORDS:
Gold
nanoparticles,
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Nanotechnology, Theranostics.
Liposomes,
Photothermal
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Therapy,
Cancer
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Many organic and inorganic nanosystems are being actively researched for their efficiency in
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cancer diagnosis and treatment.1–6 Among them, plasmonic nanostructures for photothermal
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therapy (PTT) gain considerable importance owing to the advent of two ongoing PTT based
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clinical trials making use of gold nanoshells for the treatment of brain and metastatic lung
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tumors.7 These plasmonic nanostructures also serve as efficient candidates for imaging, and
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thereby bringing out their multifunctional capabilities.8–17 According to the Food and Drug
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Administration (FDA) guidelines, any imaging agent (administered into the body) should be
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capable of getting cleared completely from the body within a reasonable period of time.18,19
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Gold based materials deployed in PTT are generally larger than 20nm in size.20–23
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Accumulation of such metallic nanoparticles in body could serve as a potential health risk. In
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2013, Melnik et al. reported the transfer of silver nanoparticles via placenta to the rat
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foetuses, bringing out the gravity of risk involved in nanoparticle accumulation.24 Although
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many of such materials could serve as efficient imaging agents, their larger size and non-
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degradable nature prevents renal clearance, thus limiting their application in vivo. To achieve
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renal clearance the size of inorganic nanoparticles will have to be < 5.5nm.18,25 Inorganic,
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metal containing nanoparticles lesser than 5.5nm in size are capable of getting filtered
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through the glomerular basement membrane (GBM),18 thereby serving as ideal candidates for
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imaging (with renal route of clearance) but are unsuitable for PTT. Hence, a multifunctional
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nanosystem capable of achieving good body clearance through both hepato-biliary and renal
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route in addition to serving as effective agents for PTT is warranted. Our group synthesized a
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liposome-gold nanoparticle hybrid system (henceforth referred to as LiposAu NPs) that has
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such multifunctional capabilities.26 Since the core of this nanohybrid system is made up of
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biodegradable lipid, the gold coating on the surface is capable of splitting into smaller
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particles (≤5-8nm) and achieving both hepato-biliary and renal clearance. Such novel
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nanoparticle systems also have an added advantage of getting accumulated specifically at the
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tumor site due to the leaky vasculature of developing blood vessels and poorly developed
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lymphatics (Enhanced Permeation and Retention - EPR effect).27 In other words, they could
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be passively targeted to the tumor region.28 Alternatively, spatio-temporal control by external
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trigger is another form of achieving specificity, wherein drug delivery is controlled to a
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specific region by optical, magnetic or ultrasound modalities.29,30 In contrast to the passive
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mode, active targeting involves antibody or affibody conjugation that binds with specific
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antigen at the tumor site. However, the added bulk of the antibody protein size and the cost of
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antibody limits its deployment on a larger scale.31 To overcome this limitation of increased
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cost factor, the spatio-temporal control of external trigger is an efficient and economical
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alternate to the antibody mediated targeting.
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Biodegradable plasmon resonant nanoparticles employing 1,2-Dipalmitoyl-sn-glycero-3-
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phosphocholine (DPPC) gold hybrid nanostructures were synthesized by Troutman et al. in
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2008.32 The transition temperature of DPPC being 41°C restricts its use only to drug delivery
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rather than hyperthermic killing of cancer cells (as biological cells begin to die of
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hyperthermia only when the temperature reaches ≥43oC).33 We had earlier reported the
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synthesis of a nanoformulation using 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)-
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cholesterol and further coating with gold (resulting in the formation of LiposAu NPs)
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enabling the achievement of both drug delivery and photothermal therapy.26 Although
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conceptual data was available with respect to such a lipid gold hybrid material, till date their
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in vivo fate validation remains uncharted. In the current study, we demonstrate the in vivo
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pharmacokinetics and photothermal efficacy of LiposAu NPs at the target site in a mouse
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tumor xenograft model. Herein, we report the in vivo degradation of these LiposAu NPs and
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their pharmacokinetic profile. The photothermal efficiency of these LiposAu NPs was tested
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against cancer cell lines under in vitro and in vivo conditions. To the best of our knowledge,
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this is the first report demonstrating in vivo degradation of these photothermally active
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LiposAu NPs in hepatocytes and subsequent clearance of gold through both hepato-biliary
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and renal route.
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LiposAu NPs were synthesized as per established protocol with slight modification to
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achieve a size range of 100-120nm.26 A schematic representing the synthesis and
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photothermal effect on LiposAu NPs including their ability to cause DNA damage and self-
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destruction achieving size reduction is shown in Figure 1. Representative transmission
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electron microscope (TEM) and scanning electron microscope (SEM) images of these
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LiposAu NPs have been shown in Figure 2A and 2B. Dynamic Light Scattering (DLS)
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measurement indicates a size range of about 100nm (Figure 2C) and the polydispersity
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index as 0.18. The lattice arrangement of Au is clearly visible in High Resolution-TEM (HR-
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TEM) images of the surface region of these LiposAu NPs (Figure S1A). Such type of
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plasmon resonant nanoparticles have shown to be responsive for specific wavelength of laser
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light mediated excitation.34 Hence, the LiposAu NPs were tuned to an absorbance range of
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750nm to achieve photothermal effect when subjected to a beam of 750nm laser light with
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650mW power (Figure 2D). These LiposAu NPs when treated with lipase enzyme lost their
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NIR absorbance peak, confirming their degradable nature (Figure 2D). Also, when treated
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with specific temperature increments (water bath mediated), these LiposAu NPs showed
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corresponding reduction in NIR absorbance denoting their thermo-sensitivity (Figure S1B).
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The in vivo biodistribution and pharmacokinetic study was performed using Swiss albino
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mice. The analysis of various tissues, plasma and urine was performed at varying time
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periods (Day 1, 7, and 14) after intravenous injection of LiposAu NPs (~110µg/400µl)
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through the tail vein. It was found that majority of the injected particles were accumulated in
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liver, followed by spleen and kidney of the mouse. As these NPs were not targeted, they were
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directly taken up by the reticulo-endothelial system (i.e. liver and spleen) of the mouse. As
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liver is the major metabolizing organ of the body playing an important role in lipid
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metabolism,35 the probability of LiposAu NPs to undergo enzymatic degradation gets
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maximized owing to their greater accumulation in liver region. The accumulation and
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metabolic degradation of these NPs in the liver was qualitatively confirmed by TEM analysis
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of the liver tissue. As revealed from Figure 3A, 1 day after intravenous delivery, these NPs
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were found to be in an aggregated state, but their original spherical morphology was
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completely lost. This indicates that under in vivo condition the LiposAu NPs present in
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systemic circulation would undergo metabolic degradation due to enzymatic activity in
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hepatocytes. Further, to confirm the aggregation observed in TEM, Energy Dispersive X-ray
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Spectroscopy (EDAX) analysis was performed (Figure S2A). Inductively Coupled Plasma –
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Atomic Emission Spectroscopy (ICP-AES) analysis of mouse liver revealed considerable
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accumulation of gold, right from day 1 end point analysis. But there was a considerable
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decrease in the %ID/g on subsequent long term study. The observed reduction in the %ID
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from about 52% (day 1) to 9.8% (day 7) and further declined to about 3% (day14) (P =
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0.0037) (Figure 3E). The percentage reduction of Au in the liver at 14 days was further
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confirmed by TEM analysis (Figure S2B). Also, HR – TEM images of liver and kidney
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samples confirms the presence of degraded Au NPs showing lattice arrangement which
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further indicates their metallic nature (Figure S2B). The negligible Au values detected in the
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liver of the normal saline treated controls were subtracted from those of the treated samples
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as background corrections. Similar observations were noted for spleen, kidneys, and intestine.
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We also performed TEM analysis of blood plasma at 2 hours end point that showed Au NPs
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of size 2-8nm [Figure 3G (i) and (ii)]. Presence of such small Au NPs in blood suggests that
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the disintegrated NPs (from the larger LiposAu NPs) reach the circulation after their
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enzymatic degradation in liver.
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It was observed that majority of the injected particles get accumulated in liver and spleen and
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the smaller percentage of 2-8nm particles circulating in blood could have resulted in
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accumulation in kidney and further clearance through urine. TEM analysis of kidney
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identified presence of such smaller particles (Figure 3C). The Inductively Coupled Plasma –
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Mass Spectroscopy (ICP-MS) analysis of kidneys indicated an accumulation of about 2.7% at
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day 1 which reduced to an approximate 0.25% at day 7 and further to about 0.22% on day 14
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(Figure 3E). Though the current percentage of accumulation in kidney is small in
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comparison to liver we expect an improvement in renal clearance when the LiposAu NPs are
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subjected to both photothermal and enzymatic degradation. The current study has limited
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scope of understanding the real-time in vivo biodistribution and enzymatic degradation of
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LiposAu NPs. Though some amount of particles are getting cleared through the hepato-biliay
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route (Table S1), we find that small amount of Au in urine even on day 7 and 14 indicating
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the possibility of renal excretion (Table S2). We however speculate that the excretion of Au
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through the renal route is a constant process overtime and determination of this complete
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excretion in real time remains a limitation. Generally, individual small molecules like
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albumin, get repelled by the negatively charged glomerular basement membrane (GBM) of
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the nephrons.36 This charge based repulsion prevents any accumulation of negatively charged
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particle/molecule in kidney that in turn restricts their excretion through urine.37 The higher
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accumulation of gold in kidney at day 1 time period indicates that gold NPs owing to their
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positively charged surface were able to overcome the charge based repulsion in the GBM.
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The ICP-MS analysis of mice plasma showed significant reduction of gold between day 1
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and 14 end point analysis (P < 0.0001) (Figure 3F). The value of gold was reduced from
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390±13.7ng/ml to 105.4±5.11ng/ml. As already pointed out, the size range of the gold NPs
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observed in mice blood plasma was also falling under the range of renal excretion. Also, the
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ICP-MS analysis of urine samples (collected at day 1, 7 and 14) revealed the presence of gold
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in varying concentrations confirming its excretion through the renal route (Table S2).
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Furthermore, we studied biodistribution in tumor bearing mice using Indocyanine Green
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(ICG; NIR dye) coated LiposAu NPs to facilitate their short term tracking in vivo. In order to
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understand if they possess any tumor homing properties after intravenous injection, we made
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use of the HT1080 xenograft model. Data suggests that these particles are not capable of any
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specific homing at the tumor site owing to their non-targeted nature. Also, in order to achieve
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successful homing, several parameters play a key role, one such being the leaky vasculature
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of the tumor bed. However, unlike only ICG injected control mice, we were able to obtain
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suggesting possible renal clearance. ICP-MS validations also confirm no LiposAu NPs
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uptake in the tumor (Figure S3B).
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To understand the toxic effect of these gold NPs accumulation in liver and kidney, the blood
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plasma serum glutamic pyruvic transaminase (SGPT or ALT) and creatinine levels were also
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analyzed. No significant difference was observed between the control and the LiposAu NPs
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treated groups (Day 1) (Figure 3B and 3D) confirming that no specific acute toxicity to the
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liver or kidney was exhibited. Qualitative urine dipstick analysis was also performed in mice
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urine (LiposAu NPs treated and controls). There was no detectable blood or protein observed
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in the urine samples (Figure S4). In the absence of human data availability, such
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biodistribution studies involving experimental animals stands as the most reliable approach to
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determine the toxicity properties of our chemically synthesized NPs. The close resemblance
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of anatomy and physiology of mice to that of humans enables us to predict the likely
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pharmacokinetics of these NPs in future human clinical transition. Additionally, in vitro
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biocompatibility on NIH-3T3 cells revealed no indications of toxicity associated with
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LiposAu NPs (Figure S5).
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In vitro photothermal efficacy studies were performed using MCF-7 (breast) and HT1080
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(fibrosarcoma) cancer cell lines. Both cell types were engineered for overexpressing a fusion
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reporter i.e. firefly luciferase 2 (fluc2) and turboFP fluorescent protein. Optimization of the
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laser irradiation time suggests a lethal effect on the breast cancer cells beyond 4 minute of
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continuous exposure (Figure S6). Hence, 4 minute was chosen as the ideal irradiation time
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for a concentration of 15µg/ml LiposAu NPs photothermal treatment. Qualitative assessment
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of the photothermal efficacy was studied in vitro using MCF-7-fluc2-turboFP cells by
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fluorescence microscopy. Combination treatment of LiposAu NPs and laser showed loss of
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fluorescence (suggesting cell death) at the area of contact as indicated by the arrow in Figure
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4A. Such ablation of cancer cells is due to heat generation in the region of laser contact
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where LiposAu NPs are also present. Additionally, no change was observed in the
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fluorescence signal of either the untreated control or the internal control groups (LiposAu
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only and laser only). This result was further supported by the significant reduction of fluc2
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luminescence in MCF-7-fluc2-turboFP (P = 0.0034) and HT1080-fluc2-turboFP (P =
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0.0024) cells when compared to laser treated control (Figure 4B). Herein, the luciferase light
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output is a direct measure of cell viability as the fluc2 enzyme catalyzes its substrate D-
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luciferin only in the presence of cellular ATP.
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As PTT is known to cause DNA damage mediated cell death,38,39 we also monitored the
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formation of γH2A.X foci, a marker for DNA double strand breaks. Minimal or no γH2A.X
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foci were observed in the untreated control and only LiposAu treated cells. The only laser
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treated control cells revealed a slightly higher number of the γH2A.X foci in comparison.
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However, it was seen that the cells that received the combination treatment with both
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LiposAu and laser, demonstrated the presence of highly significant number of γH2A.X foci
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formations in both the MCF-7 (P < 0.0001) and HT1080 (P = 0.0007) cells (Figure 4C and
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4D). This validates that the photothermal therapy mediated DNA double strand break was
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responsible for cancer cell ablation.
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In vivo temperature increment was critically determined by creating subcutaneous blebs of
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normal saline or LiposAu NPs in varying volumes (25µl, 50µl, and 100µl) in hairless
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(BALB/c Nude) mice. Each of the blebs was treated with the NIR laser for 4 minutes. The
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temperature of the blebs was continuously monitored by an IR thermometer pre- and post-
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treatment. The blebs injected with normal saline did not show any specific temperature
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increment after 4 minutes of continuous laser irradiation. However, the blebs injected with
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LiposAu NPs showed a temperature increment up to 7°C with 4 minutes of laser irradiation.
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There was also eschar formation on the treated area (noticed after 24 hours of treatment),
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indirectly indicating temperature increment (Figure S7). Such eschar formation has been
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previously reported for gold nanoshells based PTT as well.40
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Further, in vivo photothermal efficacy was determined using HT1080-fluc2-turboFP tumor
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xenograft model in BALB/c NUDE mice. On the 20th day with growing tumors (average size
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of about 70mm3), mice were randomly segregated into 3 groups (n=5 per group). Group I
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received normal saline (30µl) as vehicle control; group II animals were treated with laser
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only while group III animals were given the combination treatment of LiposAu NPs
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(0.5µg/µl in 30µl) and laser. Group II and III animals were subjected to laser irradiation for a
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period of 4 minutes. The treatment cycle was divided into two rounds between day 20 and 30.
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Two days interval was kept between the treatment cycles to avoid any therapy burden on the
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animals. All animals were imaged by injecting D-luciferin substrate every 10th day starting
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from day 0 till the end of the experiment. Our study reveals a significant reduction in the
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bioluminescence signal (3.36 x 106 ± 1.52 x 106 p/sec/cm2/sr) on day 30 of the group III
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animals when compared to group I (5.47 x 1010 ± 2.08 x 1010 p/sec/cm2/sr) (P = 0.0302) or
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the group II (4.95 x 1010 ± 1.75 x 1010 p/sec/cm2/sr) (P = 0.0068) animals (Figure 5A and
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5B). Additionally, the group III animals demonstrated complete regression of tumor and a 4
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out of 5 animal survived until 6 months (P = 0.003) in contrast to the group I and group II
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animals, which all died within 35-40 days due to tumor burden (Figure 5C). At the end point
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of monitoring, non-invasive in vivo bioluminescence imaging of group III showed no signal
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output at the therapy site (Figure 5D). It was noted that the combination treatment of
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LiposAu NPs with laser results in a 4.63 fold reduction of the bioluminescence signal with
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respect to the respective controls (Figure 5E). Histopathological analysis revealed that the
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combination of LiposAu NPs and laser resulted in the most extensive necrotic response in
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that region. Due to this combination treatment, the tumor cells in the underlying region were
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completely ablated, whereas about 95% of the tumor mass remained in laser treated animals
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(Figure 5F).
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The current study involves understanding the degradation dynamics of plasmon resonant
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LiposAu NPs under physiological condition. It was observed that these particles were able to
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degrade by the enzymatic reaction into smaller particles whose size range was ideal for renal
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excretion in addition to hepato-biliary route. The long term in vivo analysis also confirmed
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the bimodal clearance of these NPs. LiposAu NPs proved to be efficient candidate for in vivo
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photothermal mediated ablation of cancer. Their ability to generate massive amount of
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γH2A.X foci is a strong indicator of the mode of tumor ablation by DNA double strand
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breaks. Applicability of such novel biodegradable hybrid nanoparticle system holds great
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promise in cancer nanotherapeutics.
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SUPPORTING INFORMATION
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Supporting Information Available: Details of all experimental procedures, and materials used
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during the study can be found in the supplementary information. This material is available
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free of charge via the Internet at http://pubs.acs.org.
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ACKNOWLEDGEMENTS
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The authors would like to acknowledge TMC Seed-in-Air Intramural funding to AD and
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Molecular optical imaging equipment (IVIS Lumina II) support from DBT Bioengineering
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research grant to AD; IIT-B Healthcare initiative for funding the project and SAIF-IITB,
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IRCC for characterization studies. ACTREC TEM facility is also acknowledged for sample
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processing. This work is part of the doctoral thesis of AKR at IIT-Bombay.
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CONFLICT OF INTEREST
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The authors declare no competing financial interest.
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Figures and Figure Legends:
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Graphical Abstract
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Figure 1: Schematic diagram representing the principle of synthesis of LiposAu NPs
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and their mode of action to perform photothermal treatment causing intracellular DNA
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damage.
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Figure 2: Characterization of LiposAu NPs. A) TEM image of LiposAu NPs. (Scale =
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50nm) B) SEM image of LiposAu NPs. (Scale = 100nm) C) DLS size distribution of
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LiposAu NPs. D) UV-Vis absorbance spectra of lipase, liposome, and LiposAu NPs treated
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with lipase enzyme in comparison with (untreated) LiposAu NPs.
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Figure 3: In vivo biodistribution and clearance of LiposAu NPs. A) TEM image of liver
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tissue showing (i) control hepatocytes, (ii) & (iii) hepatocyte containing LiposAu NPs in
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aggregated state. B) Mice plasma levels of ALT (U/L) at the end of 24 hours. C) TEM
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images of kidney tissue showing (i) control tissue, (ii) & (iii) kidney tissue containing
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LiposAu NP in its dissociated state with it less than 5nm sized gold seeds. D) Mice plasma
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values of creatinine (mg/dl) at the end of 24 hours. E) Graph represents tissue biodistribution
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of Au in vivo as determined by ICP-MS and ICP-AES analysis at various end points. F) ICP-
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MS based mice blood plasma levels of Au (ng/ml). G) TEM image of blood plasma showing
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small gold particles of varying size range as represented in (i) and (ii). Significance is
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designated as * indicates P < 0.05, ** indicates P < 0.005 and **** indicates P < 0.0001.
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Scale bar: A(i & ii) 2µm; A(iii) 1 µm; C(i) 1µm; C(ii & iii) 20nm; G(i) 5nm; and G(ii) 2nm.
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Figure 4: In vitro photothermal ablation of cancer cells by LiposAu NPs. A)
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Fluorescence micrograph images of photothermal therapy mediated cell death in MCF-7-
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fluc2-turboFP cancer cell line. Red color represents the fluorescence of the turboFP (635 nm
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emission) protein. B) Quantitative analysis of bioluminescence based photothermal cell death
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in MCF-7-fluc2-turboFP (P = 0.0034) and HT1080-fluc2-turboFP (P = 0.0024) cancer cells.
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Representative images for qualitative assessment are given below the graph representing the
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various groups. Pseudocolor bar indicates the photons captured by the CCD camera. C)
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Representative images showing the formation of γH2A.X foci after treatment in MCF-7 and
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HT1080 cancer cells. D) Quantitative assessment of γH2A.X foci in MCF-7 (P < 0.0001) and
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HT1080 (P = 0.0007) cancer cells.
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Figure 5: In vivo photothermal ablation by LiposAu NPs in tumor xenograft. A)
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Representative pre- and post-treatment in vivo bioluminescence images of mice bearing
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HT1080-fluc2-turboFP tumor xenografts. B) Quantitative assessment of bioluminescence to
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demonstrate the increase in tumor volume. The highlighted region indicates the treatment
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period (* indicates P < 0.05 and ** indicates P < 0.01). C) Kaplan-Meier survival curve of
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the tumor bearing mice (P = 0.003). D) Representative photographic and bioluminescence
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image of LiposAu NPs and laser treated mouse post 6 months of treatment reveals no signs of
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regression. E) Bar diagram represents the fold change in bioluminescence between the laser,
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and LiposAu NPs + laser treated tumors. F) Hematoxylin and Eosin (H&E) stained
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histological evaluation of tumor tissue after PTT.
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Graphical Abstract 39x19mm (300 x 300 DPI)
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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)
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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)
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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)
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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)
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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)
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