Biodegradable Radiofrequency Responsive Nanoparticles for

Mar 25, 2016 - Here, we report a biodegradable nanoparticle (NP) system showing excellent RF hyperthermic response together with the ability to locall...
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Biodegradable Radio-frequency responsive nanoparticles for augmented thermal ablation combined with triggered drug release in liver tumor Vijay Harish Somasundaram, Rashmi Pillai, Giridharan Malarvizhi, Anusha Ashokan, Siddaramana Gowd, Reshmi Peethambaran, Shanmugasundaram Palaniswamy, AKK Unni, Shantikumar V Nair, and Manzoor Koyakutty ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00511 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 29, 2016

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Article type: Full paper Title: Biodegradable radiofrequency responsive nanoparticles for augmented thermal ablation combined with triggered drug release in liver tumor Authors: Vijay Harish Somasundaram, Rashmi Pillai, Giridharan Malarvizhi, Anusha Ashokan, Siddaramana Gowd, Reshmi Peethambaran, Shanmugasundaram Palaniswamy, Unni AKK, Shantikumar Nair*, Manzoor Koyakutty* Address: Amrita Vishwavidyapeetam, Amrita Institute of Medical Sciences & Research Center, Ponekkara P.O. Kochi, Kerala – 682041. India. E-mail: [email protected], [email protected] Keywords: radiofrequency responsive nanoparticle; biodegradable nanoparticles; RF triggered drug release; doxorubicin; HCC Abstract Radio-frequency ablation (RFA) and doxorubicin (Dox) chemotherapy are separately approved for liver cancer therapy; however, both have limited success in clinics due to suboptimal/non-uniform heating and systemic side-effects respectively. Here, we report a biodegradable nanoparticle (NP) system showing excellent RF hyperthermic response together with the ability to locally deliver Dox in liver under RF trigger and control. The nanosystem was prepared by doping a clinically permissible dose (~ 4.3wt%, 0.03 ppm) of stannous ions in alginate nanoparticles (~ 100nm) co-loaded with Dox at ~ 13.4wt% concentration and surface conjugated with galactose for targeting asialo-glycoprotein receptors in liver tumors. Targeted NP-uptake and increased cytotoxicity when combined with RF exposure was demonstrated in HEPG2 liver cancer cells. Following in vitro (chicken liver phantom) demonstration of locally augmented RF thermal response, in vivo

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scintigraphic imaging of 99Tc labeled NPs was performed to optimize liver localization in Sprague-Dawley (SD) rats. RF ablation was performed in vivo using cooled-tip probe and uniformly enhanced (~ 44%) thermal ablation demonstrated with magnetic resonance imaging; along with RF controlled Dox release. In orthotopic rat liver tumor models, real-time infrared imaging revealed significantly higher (~ 200C) RF thermal response at tumor site, resulting in uniform augmented ablation (~ 80%) even at a low RF power exposure of 15W for just 1minute duration. Being a clinically acceptable, biodegradable material, alginate nanoparticles holds strong translational potential for augmented RF hyperthermia combined with triggered drug-release. Introduction Hepatocellular carcinoma (HCC) is considered the leading cause of death among patients with cirrhotic liver disease and its incidence in developing countries is on the rise.1 Surgical resection and orthotopic liver transplantation are considered the most effective treatment options for HCC,2 but nearly 70% of patients at initial presentation are not eligible for surgical resection.2,3 Among alternative treatment methods for unresectable liver tumors; radiofrequency ablation (RFA) has emerged as the most widely practiced procedure.4 Conventionally, RFA uses an alternating current of radiofrequency waves passed from an uninsulated electrode inserted into the tumor. The rapidly alternating electric field produces ionic agitation and frictional heating which trigger a series of events in the tissue which finally culminates in coagulative necrosis and thus tumor ablation.5 Achieving tissue heating > 60OC is considered ideal to cause thermal ablation and usually a ~ 0.5cm margin of normal tissue beyond the tumor margin is also included in ablation

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field for better therapeutic outcome.2 A major challenge faced with conventional RFA is to achieve uniform ablation in large sized tumors, and for this the RF power needs to be increased beyond 100 watts (W) (473 KHz generator) for an extended duration of > 30 minutes. This invariably causes charring of tissue immediately adjacent to the electrode, which impedes transfer of energy to rest of the tumor.5 Due to this inefficient heating effect, only tumors < 3cm in diameter can be confidently treated with conventional RFA.4,5,6 Various techniques such as modifications in electrode type/geometry have been attempted to overcome this shortcoming, with no significant success. Injection of saline into the tumor before or during RF application had been reported to improve tissue coagulation and thus increase size of ablated area.7,8 But the amount and concentration of saline that could be injected was restricted; as it was not contained at the tumor site, often entering the general circulation and causing systemic side-effects. Combining RFA with other established treatment modalities like chemotherapy and chemoembolization has showed synergistic effect in disease control.5,6,9,10 Combining low RF hyperthermia and chemotherapy was recently tested using Low Temperature Sensitive Liposomes (LTSL) which burst-released doxorubicin (chemotherapeutic agent) at temperatures greater than 39.50C.11,12,13 However this formulation fails to address the basic underlying deficiency of conventional RFA: sub-optimal and non-uniform tissue heating. Here RF is used only for drug release and not for hyperthermic tumor damage. With the evolution of novel nanoparticles (NPs) for biological applications, RF responsive metallic and carbon based NPs were extensively studied by Steven Curley et.al., using the Kanzius RF system.13-16 The RF thermal response of single wall carbon nanotubes (SWNTs) due to their resistive conductivity and high aspect ratios was initially

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exploited. Despite the absence of acute toxicity of SWNTs being demonstrated in preclinical studies,14 their complete safety can only be assured after evaluation of chronic toxicity in preclinical models. Gold (Au) nanoparticles, where the RF to thermal energyconversion is assumed to be via Joule heating was also extensively studied and is now under clinical trial. Recent works however, suggest the primary role of Au electrolyte contaminants in Joule heating process, thus leading to some uncertainty regarding role of Au NPs in RFA.17,18 A newer addition to the list of RF responsive nanomaterials is functionised graphene.19 The primary problem with all these NPs reported so far is their relatively inferior biodegradability, which may potentially produce long-term toxicity issues and thus hinder clinical translation. We identified stannous (Sn2+), which is being used in nuclear medicine clinics, as a potential RF hyperthermia agent. Here we describe, for the first time, development of a biodegradable RF nano probe based on stannous (Sn2+) crosslinked alginate nanoparticles for augmented RF hyperthermia. Use of these polymeric NPs gave us an excellent opportunity to co-load a clinically used chemodrug; doxorubicin (Dox), for RF triggered drug delivery in liver tumor. In addition, since these NPs have been developed for systemic

administration,

they

were

galactosylated

for

targeted

delivery

to

asialoglycoprotein receptor overexpressing liver cancer cells. Promising effect of this single NP agent producing a dual function of RF hyperthermia along with RF triggered Dox release was demonstrated in vitro, in liver tissue phantoms and in vivo using normal and orthotopic liver tumor models in Sprague Dawley rats.

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Experimental Section Doxorubicin hydrochloride (98%; Molecular Weight: 579.98 g/mol) was procured from A. K. Scientific, USA. Sodium Alginate (Low viscosity) and Galactose (C6H12O6; Molecular Weight: 180.16 g/mol) were purchased from Sigma Aldrich, India. Stannous chloride dihydrate (225.6 g/mol) and Polyethyleneimine (60kDa) were purchased from Merck, India. Molisch’s Reagent was bought from Nice Chemicals, India. Other solvents and chemicals were procured from various manufacturers and were of analytical grade. For the animal experiments, Sprague-Dawley rats were obtained from the Amrita Central Animal Research Facility, and all procedures conducted were approved by the university scientific and ethical committees. Preparation of GAD nanoparticles Stannous doped alginate NPs co-loaded with doxorubicin (Dox) were prepared using ionic crosslinking technique. Sodium alginate dissolved in distilled water with constant stirring [5MHL DX (REMI, India)] to a final concentration of 1% w/v. Cross linker solution of stannous chloride and doxorubicin in 1:1 proportion prepared in a low-light ambience by dissolving in distilled water which was acidified with 0.1N HCl to bring the pH to ~ 4. Crosslinker solution was loaded in 10ml syringe (BD Biosciences), fitted with a 26 Gauge needle. Syringe was then fitted on infusion pump [KD-100 (KD Scientific, USA)] with flow rate set at 0.4 ml/hr, and the cross-linker added drop-wise into alginate solution (4ml) kept under continuous stirring (1000 rpm), over 1 hour. This was followed by 1ml polyethyleneimine (PEI) (0.003%) being added to the above reaction mixture which now contained Alginate-Dox (AD) NPs and stirred for 1 hour (200 rpm), to achieve polyethyleneimine (PEI) coating of the AD NPs, which were retrieved by

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centrifuging [Sorvall Legend 1XR (Thermo Fischer Scientific, Germany)] at 6000 rpm (210C) for 5 minutes and repeatedly washed with distilled water to remove unbound regents. Galactosylation of AD NPs carried out using a modification of the technique described by Aswathy Jayasree et.al.20 It was performed in two steps: Initially, D-galactose (0.5µM) was dissolved in 0.1M sodium acetate buffer at pH 4 and 600C for 2 hours, resulting in ring opening (activation) of the galactose molecules. Next, the activated galactose solution was added drop-wise into PEI coated AD NPs suspension and incubated for 15 minutes at room temperature. The galactosylated AD NPs (GAD NPs) were retrieved by centrifuging (as described earlier) and washed repeatedly to remove any unbound galactose. Characterization of GAD NPs The AD and GAD NPs was characterized using dynamic light scattering [Zetasizer NanoZS (Malvern Instruments, USA)] and Atomic Force Microscopy [AFM; JEOL-JSPM5200, Japan]. Dox encapsulation efficiency (EE) in GAD NPs was estimated by first measuring the absorbance (at 485nm) using a multiplate spectrophotometer [Biotek, Power Wave XS, USA], of a known volume of NPs as well as that of supernatant obtained after washing the NPs; and then absolute value derived from a predetermined Dox standard graph. Loading Efficiency of GAD NPs determined by; estimating the absorbance of a known concentration of NPs in suspension (using a spectrophotometer), and LE calculated using the formula: [weight of drug loaded in NPs (mg) / total weight of dried NPs (mg)] x 100 %. Concentration of Stannous in the NPs was estimated using

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quantitative inductively coupled plasma – optical emission spectroscopy (ICP) analysis. All the above tests were conducted in triplicate and average values taken. Galactosylation of NPs was confirmed with Molisch’s test. 2ml of GAD NPs taken in a test tube, Molisch’s reagent (2-4 drops) added to it and mixed thoroughly. Finally 2ml of concentrated sulphuric acid (H2SO4) was added slowly along the side of the tube. Formation of a purple ring at interface of H2SO4 and NPs suspension was considered positive for galactosylation. Further, Fourier transform infrared (FT-IR) spectra of alginate NPs before & after PEI coating also after galactosylation were recorded (PerkinElmer Spectrum RX-1, USA). Each set of NPs separately mixed with dried KBr and characterized for wave numbers ranging between 4000 - 400cm-1; using the transmission mode of operation. Thermo gravimetric analysis (TGA) of the NPs was also performed using to Exstar SII 6200 TG/DTA system to demonstrate the drug loading into the beads (0oC to 550oC). RF thermal response evaluation: Comdel (USA) model CDX1000, RF generator producing a uniform 13.56 MHz RF field was used as the non-invasive RF source. Gold Chloride (HAuCl4), calcium chloride (CaCl2 anhydrous), sodium chloride (NaCl) and iron chloride (FeCl3) were procured from Sigma Aldrich, India. Separate sets of solutions of HAuCl4, CaCl2, NaCl, FeCl3 & SnCl2 (concentrations ranging from 0.0312 – 10 mg/ml) were prepared. Similar volumes of each solution, (4ml volume in glass petri plate) was exposed to a uniform RF field of 100W power for 1 minute. Temperature of the solutions was measured before and immediately after RF exposure using a thermocouple temperature probe (Eutech Instruments, USA) and the difference (∆T) was plotted against concentration of the solution. Similarly, RF thermal response for different

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concentrations of GAD NPs was measured and plotted. Further, RF thermal response of individual reagents in the GAD NPs (at concentrations similar to that contained in them) was also similarly estimated. The effect of RF exposure on the morphology of the NPs was studied using the DLS system, procedure similar to as described earlier. Drug release studies: Drug release from the GAD NPs was tested under two specific conditions; with and without RF exposure. Known concentration of NPs was dispersed in fixed volume of PBS (triplicate) and incubated at 37oC in a shaking incubator (KS4000 E&K Scientific, USA). Amount of drug released was estimated by recording the absorbance of the supernatant periodically at 485 nm using a multiplate spectrophotometer. This procedure was then replicated on a different sample of GAD NPs (similar concentration), which had been exposed to a uniform RF (13.56 MHz, 100 watts power) field for 1 minute. Effect of the RF exposure on drug release was evaluated. This was followed by evaluating the effect of increasing durations of RF exposure on drug release from GAD NPs (fixed concentration and 100W RF power); using the same technique as mentioned above. Cell culture experiments Human hepatocellular carcinoma cells (HEPG2) was procured from National Centre for Cell Sciences, Pune, India and maintained in Minimum Essential Medium (GIBCO) supplemented with 10 % FBS, penicillin (50 IU/ml) and streptomycin (50µg/ml) under humidified 5% CO2 atmosphere at 370C. Cellular

uptake

studies:

HEPG2

cells

were

cultured

in

a

24-well

plate

(10000cells/1ml/well). Following 24 hours attachment period, the wells were classified into separate groups and treated with any one of the following: 1µM free Dox, AD NPs

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or GAD NPs (with the NPs containing an effective concentration of 1µM of Dox). Dox uptake was estimated at 2 time points; 30 minutes and 2 hours after incubation. For this, the cells were trypsinized, washed thrice with PBS (1500 rpm/ 3 min), and subjected to flowcytometry analysis using excitation energy of 488nm wavelength and recording emission at 585±21 nm. Further, confocal microscopy of unfixed cells was performed to confirm the intracellular visualization Dox. Cytotoxicity studies: Cytotoxicity produced by GAD NPs, with and without RF exposure was studied using Alamar Blue assay (in triplicate). Similarly cytotoxicity produced by free Dox, non galactosylated NPs and RF exposure alone was also studied and compared. HEPG2 cells were seeded at a density of 10,000 cells/well in a 24-well plate. After 24 hours, the cells were treated with either 1µM free Dox or AD NPs or GAD NPs (containing 1µM Dox) or RF exposure (100watts, 1 min). A separate set of cells was treated with both GAD NPs and RF exposure. Untreated HepG2 cells served as negative control and HEPG2 cells treated with 0.1% Triton X-100 served as positive control. The cells were incubated for 48 hours following which their viability assessed using Alamar blue assay (Invitrogen, USA); optical absorbance (570-600nm) quantified using a microplate spectrophotometer (Biotek, PowerWave XS, USA). In Vitro (liver phantom) experiments Augmentation of RF thermal response was evaluated in chicken liver phantoms. A fixed dose of GAD NPs (100µg in 0.1ml distilled water) was instilled at a depth of 1cm over a marked area on the liver phantom. Then, the entire liver phantom was exposed to a uniform RF field of 100 watts power for 1 minute. Following this the liver phantom was cut across the site of NP injection to observe for ablation. Cut sections were stained with

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haematoxylin & eosin stains to observe histological changes. Similar sections also made at points away from site of NP injection on the phantom, which were compared (visually & microscopically) with the NP injected sites to observe difference in changes produced. Instillation of NPs alone in liver phantoms with no exposure to RF field was also performed (as control) and after 15 minute of incubation in room temperature, phantom samples cut across site of NPs injection to assess changes. In Vivo experiments Radiolabeling of GAD & AD NPs and their in vivo imaging: 1ml volume of normal saline containing ~ 2mg of GAD NPs was mixed with 500µCi (microcurie) of 99m-Technetium (as sodium pertechnetate solution) which was freshly eluted from a 99m-Technetium (99mTc) generator (Mon.tek Mo99/Tc99m generator from Monrol, Turkey). Reaction mixture was incubated at room temperature for 20 minutes followed by separation of the NPs by centrifugation; (6000 rpm for 5 minutes) at 210C and repeated washing with 0.9N saline to remove the unbound 99mTc. Paper chromatography was performed (with normal saline as liquid phase) to test the efficiency of radiolabeling. Using similar technique, the non-galactosylated (AD) NPs were also radiolabeled. All radiation safety precautions were taken during these tests. Adult Sprague-Dawley (SD) rats of either sex, weighing 250-350g were used. ~ 2mg of the 99mTc labeled GAD NPs was dispersed in ~ 150 µl of normal saline (0.9% NaCl) and loaded into a 1ml syringe fitted with 26G needle. After intravenous (via tail-vein) injection of the NPs, the animals were anaesthetized with an intramuscular injection of Ketamine 50mg/ml + Xylazine 20mg/ml in 3:2 ratio; 2.5microlitres/g of animal body weight. Animals were positioned for ventral projection images on a Kodak in vivo

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imaging station (Kodak In-vivo Multispectral imaging system FX, Carestream, USA) and imaged (2 minute acquisition) at every 10-minute interval for up to 60 minutes. Distribution of the radiolabeled GAD NPs was assessed visually; & quantitatively by estimating intensity of signal from the region of liver and plotting it over different time points to get the time-activity curve. This was followed by evaluation of distribution of the radiolabeled AD NPs in another set of healthy SD rats, using the same technique as mentioned above. The time-activity curves for GAD and AD NPs were compared. In vivo evaluation of RF thermal response and drug release: Was performed after confirming that GAD NPs show peak accumulation in liver 40 minutes after injection. GAD NPs (~ 2mg) were injected intravenously in adult SD rats and they were prepared by anaesthetizing them and shaving the fur over their dorsal aspect (to allow better contact with grounding pads of RF system). 40 minutes after intravenous injection of NPs, animals (under anaesthesia) were placed supine over the grounding pad of a Cooltip RF ablation system (Cool-tip E series, Covidien, USA). Under sterile conditions, a central incision was made over abdomen and flaps lifted to expose the liver. 17 gauge sterile RF electrode was introduced into the liver and RF energy was administered (figure 6B). The different combinations of RF power and duration evaluated were: 5W, 10W, 15W and for durations of 30 seconds and 1 minute. This was followed by intracardiac blood draw and euthanasia of the animal (with overdose of anaesthesia) to harvest its liver. The procedure was also performed in control animals without the NPs injection. Dox content in the drawn blood was quantified from the plasma of each sample by measuring specific absorbance at 485 nm. Magnetic resonance imaging of the harvested livers were performed on a Biospec 7 Tesla MRI system (Bruker, Germany). Besides

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gross evaluation of ablated area in the fresh and formalin fixed liver samples, tetrazolium chloride (TTC) staining was also performed to assess accurate size of ablated tissue. TTC was procured from Sigma Aldrich India. Liver samples, cut (sectioned into 5mm slices) and uncut were incubated in freshly prepared 1% (w/v) solution of TTC in normal saline, for 20 minutes. Following this the tissue samples were fixed in 10% formalin for 20 minutes. Size of ablated areas (recognized as regions remaining colorless) were measured and compared between study and control animals. Testing of GAD NPs in N1S1 liver tumor models: Syngenic orthotopic N1S1 rat liver tumor cell line was obtained from National Centre for Cell Sciences, India. The cells were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% FBS. Adult, male SD rats weighing 200 – 350g were used. For tumor induction, the rats were anesthetized with a gas mix of 2 - 5% isoflurane and 95% O2 (for induction) which was maintained with 0.5 – 4% isoflurane via nose cone. Rats placed supine and abdominal area shaved and disinfected with betadine. Subcostal incision made and 200µl of inoculation mix instilled into thickest part of left lobe of liver. Inoculation mix contained ~ 2 x 106 cells (N1S1) in DMEM. Abdominal wall was closed in layers and animals were started on prophylactic antibiotic and analgesic for the following 5 days. 1 week after cell inoculation, the animals were imaged (serially every alternate day) using the MRI system to identify and estimate tumor growth. By 2 weeks the tumors achieved ~ 2cm diameter; at this point ~ 2mg of GAD NPs were injected intravenously and 15 minutes later open RFA of liver tumor and adjacent normal liver lobe was performed (as described earlier). During the ablation procedure, thermal changes occurring at the site of RF probe insertion (tumor and normal liver tissue) was

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measured in real-time using a non-contact infrared thermometer (VTO4 Visual IR Thermometer, Fluke technologies, USA). The difference in heating profile between the tumor and the normal liver in each animal was recorded. The size of ablated area measured using TTC staining (as described earlier). Results Preparation of GAD NPs Schematic (Figure 1), depicts the technique of preparing Sn2+ doped alginate NPs coloaded with Dox (AD NPs): by ionic precipitation resulting from controlled dropping of a mixture of stannous chloride (SnCl2) and Dox solutions, into sodium alginate (Alg) solution under constant stirring. The NPs were then coated with polyethyleneimine (PEI), to provide necessary amine groups for galactosylation. Galactose activation (ring opening) was performed by incubating D-galactose solution in sodium acetate buffer (pH 4.0) at 60OC for 2 hours. Ring opened galactose was added drop-wise into the PEI coated Alg-Dox NPs suspension and kept under constant stirring for 15 minutes at room temperature. The galactosylated NPs (referred hereafter as GAD NPs) were finally retrieved by centrifugation. Characterization of GAD NPs DLS analysis of PEI coated AD NPs (Figure 2A) showed particle size ranging between 70 - 100nm (zeta potential -37mV) which increased to 100 - 200nm (Zeta potential 26mV) upon galactosylation (GAD NPs) (Figure 2B). AFM evaluation confirmed these findings (Figure 2C and 2D). Besides providing amine groups for galactosylation; PEI coating of NPs served a dual purpose in that they produced a 54% greater retention of Dox within the NPs (Figure 2E, spectroscopic analysis). Figure 2E inset image clearly

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indicates that; the supernatant in PEI coated NPs (tube i) contained lesser free Dox than the supernatant in PEI uncoated particles (tube ii). Loading and encapsulation efficiency of doxorubicin in the GAD NPs was estimated to be ~ 13.4% and ~ 63 ± 2% respectively. Successful galactosylation of the NPs (GAD) was confirmed using the Molisch’s test which showed formation of a purple ring at interface of sulphuric acid and the reagent treated NPs (Figure 2F). The test was negative for non-galactosylated NPs (AD). ICP analysis of GAD NPs revealed the concentration of stannous ions to be ~ 43µg per mg of NPs. FT-IR spectra of the NPs (plain alginate, PEI coated & galactosylated) is represented in Figure 2G. Carbohydrate peak near 1032 cm-1 originates from the coupling of C-O, C-C & C-O-H vibrations. Decreased intensity of this peak upon PEI coating and its regain after galactosylation indicates interaction with NH groups and successful galactosylation, respectively. Peaks in the 3500 – 2800 cm-1 region seen upon PEI coating which increase in intensity and width in the galactosylated NPs. An increase in C=O stretching (1630 cm-1) is seen upon PEI coating and galactosylation. Further, an increase in 1420 cm-1 peaks for CH2, CH3 bending is confirmatory of galactosylation. The TGA analysis of the NPs further confirmed the drug loading (Supplementary Figure S1). In vitro RF thermal response and drug release evaluation of GAD NPs The RF thermal response (∆T) of equal volumes of different clinically used ionic solutions (over a range of concentrations); when they were exposed to a uniform RF field, produced a very interesting result (Figure 3A). Regardless of the type of ions in solution; peak RF heating happened around 0.2mg/ml (0.02% w/v) concentration, beyond which the thermal response actually started reducing with increasing ion concentration.

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The RF thermal response of distilled water, which was the solvent for all the above solutions, was 3oC (± 1.3oC). Unlike the SnCl2 solution, RF thermal response of the GAD NPs, showed peak heating at around 1mg/ml concentration, beyond which no further increase in temperature (∆T) was noted (Figure 3B). Thermal response of a fixed concentration of the NPs was compared to the RF thermal response of its individual components (Alg, Galactose and SnCl2) at concentrations identical to that contained in the NPs (Figure 3C). Temperature increase (∆T) of approximately 6.10C, 4.80C and 5.70C (above baseline temperature of 37oC) were seen with the individual solutions respectively. While ∆T seen for the NPs was significantly higher at ~ 120C (p < 0.001), thus reaching a temperature of ~ 500C within 1 minute. Distilled water (DW) showed a ∆T of ~ 4.00C. Dox release from the GAD NPs was studied under two different conditions: with and without exposure (for 1 minute), to a uniform RF field of 15W power. Without RF exposure, there was only a small initial release (~ 20%) of Dox into phosphate buffered saline at 370C, followed by ~ 50% of it being released over a period of 12hrs, and beyond that ~ 15% of contained Dox was released every 10hrs (Figure 3D). The RF exposure however, triggered an instantaneous release of

~ 40% of drug, followed by an

exponential release wherein ~ 75% of drug is released by 1hr and ~ 98% of Dox released within 4hrs. An instant release of ~ 100% of drug was observed when the NPs were exposed continuously to a 100W RF field for 3 minutes. Following RF exposure it was observed that the GAD NPs tended to clump to each other and form bigger aggregates in vitro (Supplementary Figure S2).

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In vitro cellular uptake and cytotoxicity evaluation of GAD NPs Flowcytometric analyses of HEPG2 cells treated with doxorubicin solution (free Dox), PEI coated AD (non galactosylated) NPs and GAD NPs (all containing identical concentrations of Dox), is presented in Figure 4A. Intracellular uptake of free Dox was seen in ~ 74% of cells at half hour and in ~ 85% cells at 2 hours. While nongalactosylated NPs were taken up in ~ 81% cells at half hour, the targeted (galactosylated) GAD NPs were taken up by ~ 91% of the cells within same time and this further increased to ~ 94% cells by 2 hours. Fluorescence confocal microscopy of the cells incubated with GAD NPs (Figure 4B) shows the characteristic nuclear uptake of Dox from the NPs. Next, influence of this preferential uptake of GAD NPs on the toxicity to HEPG2 cells was evaluated. The cytotoxicity produced by free Dox (FD), plain alginate NPs (A), Dox loaded NPs - non-galactosylated (AD) & galactosylated (GAD), RF exposure alone (RF), and finally cytotoxicity upon combining NPs with RF exposure (GAD+RF) was evaluated. Figure 4C depicts the results of Alamar blue viability assays performed on the HEPG2 cells, 48 hours after the treatments. Highest cell death (~ 81%) was always seen in cells treated with both, GAD NPs and RF exposure (GAD+RF). Relatively lower and almost identical levels of cell death was seen upon treatment with either GAD NPs or RF exposure alone; ~ 51% and ~ 52.5% respectively. Again, no statistically significant differences were seen between the cytotoxicity produced by non-galactosylated AD NPs and free Dox (~ 35%). No significant cytotoxicity was produced by plain alginate NPs (A).

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In vitro (chicken liver phantom) RF thermal response evaluation of GAD NPs RF thermal response of GAD NPs was evaluated using chicken liver phantoms. The NPs were injected at a specific point in the liver phantoms; marked with black arrow in Figure 5A. Following uniform RF exposure of the entire phantom, they were cut along a plane crossing the point where NPs were injected. As expected, areas of whitish discoloration, indicating the ablated tissue were seen restricted to focal regions around the point of NPs injection (Figure 5B); while rest of the tissue (which can be considered as the control) appeared grossly unaffected. Histopathological evaluation (haematoxylin & eosin staining) of the liver phantoms confirmed that sites away from point of NPs injection retained normal histological properties and structure (Figure 5C) despite the RF exposure, whereas sites around point of NPs injection (Figure 5D) showed destruction of hepatocyte morphology and arrangement (clumping of cells). Injection of NPs into liver phantoms, without RF exposure did not produce any appreciable changes. Having demonstrated this, the next step was to test our system in vivo. In vivo biodistribution, RF triggered drug release & thermal response evaluation of GAD NPs Upon incubation of GAD and AD NPs in 99m-Technetium (99m-Tc) containing sodium pertechnetate solution, we could radiolabel them with ~ 80% efficiency (confirmed by paper chromatography). Following intravenous (i.v.) injection of these radiolabeled NPs into Sprague-Dawley (SD) rats, their scintigraphic images were acquired at different time points and superimposed on their corresponding X-ray images to aid in anatomic localization. As seen in Figure 6A, a gradual and progressive accumulation of GAD NPs in the liver was observed with peak accumulation consistently occurring by ~ 40th minute.

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The accumulation in liver was however slower for the AD NPs. The time-activity curves for GAD and AD NPs accumulation in liver over 60 minutes (Figure 6B), confirms this with a significantly larger area under the curve (AUC) for the GAD NPs. As the maximum number of GAD NPs accumulated in the liver by 40th minute, this time point was marked for RF application. Both groups of animals; study (with NPs injection) and control (without NPs injection) survived the open (laprotomic) RF application to liver (Figure 6C). After application of RF energy (fixed power and duration) to the study animals, intra-cardiac blood was drawn and liver harvested for analyses. Spectroscopic evaluation of the plasma (Figure 6D) revealed; no quantifiable Dox released into systemic circulation for both 30 second and 1 minute RF exposure, when power was 5W. At 10W, systemic Dox release after 30 second exposure was negligible (< 0.3 µg/ml), while after 1 minute the Dox release was ~ 4 µg/ml. Upon increasing the RF power to 15W; Dox concentration in circulation was ~ 4 µg/ml and ~ 16 µg/ml after 30 second and 1 minute exposure respectively. Thus a RF controlled drug release in vivo was observed. Harvested livers from both the control and study animals were subjected to magnetic resonance imaging (MRI). Livers of control animals (Figure 6E-i) showed the size of ablated areas (seen as area of hyperintense T2 signal) to be ~ 9mm in diameter. Upon gross examination (Figure 6E-ii) these ablated zones had irregular margins with normal appearing tissue interspersed within the ablated (discolored) regions. MRI images of study animals (Figure 6E-iv) showed the ablated areas to measure ~ 13mm, with gross examination showing more uniform and intense discoloration of the ablated areas with a well-defined margin (Figure 6E-v). Triphenyl Tetrazolium Chloride (TTC) staining of liver samples from control (Figure 6E-iii) and study animals (Figure 6E-vi) correlated

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with the MRI images in that the livers containing GAD NPs had larger and more uniform areas of necrosis. The final step was to evaluate whether these positive findings are reproduced in orthotopic liver tumor models. Serial MR images following intrahepatic inoculation (in Sprague-Dawley rats) of N1S1 rat liver tumor cells showed that the developed tumors reached sizes of ~ 2cm by 15th day (Figure 7A). After this confirmation, ~ 2mg of GAD NPs was injected intravenously and 15 minutes later open RF ablation of the tumor was performed. The adjacent normal liver lobe was also ablated using exactly the same amount of RF energy and duration, so as to have a control for comparison. Infrared (IR) in vivo thermal images (Figure 7B & 7C) acquired during the ablation process clearly revealed a higher thermal signal (~ 620C) from the tumor area compared to the normal liver (~ 420C). Post ablation, gross (Figure 7D) and TTC stained (Figure 7E) evaluation of the liver samples showed larger areas of discoloration (necrosis) at tumor sites when compared to normal liver. Further, cut sections of the TTC stained liver tumors showed ablation of ~ 80% of ~ 2cm diameter tumor with just 1 minute of 15W RF exposure (Figure 7F). Discussion Minimally invasive treatment techniques for HCC like RFA, have been gaining popularity in recent times. However, the important challenge that still remains is to achieve uniform tissue ablation over larger areas. Nanoparticles investigated so far for enhanced for RFA have been non-biodegradable and potentially toxic.13-16,19 Though intratumoral injection of biocompatible salt solutions during RF procedure seemed promising initially7,8; later workers identified that apart from producing systemic side-

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effects, uneven spread of the ionic solutions resulted in non-uniform impedance, causing irregular and unpredictable areas of ablation.20 This highlighted the fact that RF enhancing agents need to be uniformly contained within the tumors. Considering the prior demonstration of RF thermal response of ionic solutions7 and the synergistic effect of combining RFA with chemotherapy,6 we proposed a biodegradable, targeted nanoformulation doped with a clinically acceptable level of metallic ion and co-loaded with doxorubicin. To produce this we used a simple, inert, linear polysaccharide – alginate, crosslinked it with bivalent cation, stannous which is clinically used in nuclear medicine. An interesting observation made while evaluating the RF thermal response of ionic solutions was that regardless of the type of ions in solution (Ca2+, Na+, Au2+, Sn2+), the ∆T versus salt concentration plot followed a common pattern with peak heating around 0.02% (w/v). This correlated with the work of Kruse et al, who identified peak heating of NaCl solution at 0.019% w/v under an RF field of 13.56 MHz.23 From our observations, we propose that probably, all ionic solutions have a relatively similar maximization of loss tangent at the tested frequency range. However, it has to be understood that these ∆T versus ion concentration plots may not follow the same patterns in vivo due to different tissue density and ionic characteristics. Among the various ions studied, we selected stannous chloride (SnCl2.2H2O), for it is regularly used in the clinics as a reducing agent in cold-kits of Technetium-99m labeled radiopharmaceuticals.24 Additionally, Sn2+ being a bivalent cation, readily crosslinks with alginate thereby providing a means for its encapsulation. Single largest dose of Sn2+ injected intravenously for a nuclear imaging procedure in humans (70kg average weight) is 2mg, which is approximately 1/500th the

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LD50 reported in rats.24 ICP results showed the Sn2+ content in GAD NPs to be ~ 43µg / mg; means that nearly 50mg of GAD NPs will contain the same amount of Sn2+ routinely administered in clinical practice. Therefore up to 50mg of GAD NPs can be given as a single dose in humans. Chemically, alginate is a (1-4)-linked block co-polymer composed of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G). Sn2+ ions will link adjacent alginate chains by either of two mechanisms; each Sn2+ ion binding 4 G units to produce the popularly known ‘egg-box’ model (2/1 helical conformation) of alginate gelation,25 or by the 3/1 helical conformation (slow gelation) which also has a ratio of 4:1 between amount of G units and bivalent cation.26 However, in our NPs, amount of stannous ions contained was significantly lower than the alginate content (instead of the expected 1:4 ratio). This indicated that some other agent in the GAD NPs was behaving as a crosslinker and actively participating in the gelation reaction. We have co-loaded Dox as chemotherapeutic agent in alginate as it is clinically used for HCC. Dox is known to exist almost entirely in cationic form at pH 5 to 9.27 This therefore indicates that Dox molecules may be behaving as cationic crosslinkers in alginate gelation (as depicted in Figure 1). This theory is further supported by the fact that concentration of Dox while preparing the NPs had to be titrated to achieve desirable size; because higher concentration of Dox caused precipitation of larger particles. Thus the possibly covalent interaction of Dox with alginate apart from just weak hydrogen linkages ensured better drug retention within the NPs and a more steady release profile. Size of the GAD NPs ranged between 100-200nm with no appreciable aggregation seen upon their storage. In agreement with previously reported works,28 PEI coating which was primarily done to aid galactosylation, served another purpose too; it improved Dox

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retention within alginate NPs (as indicated in Figure 2E). Molisch’s test, which is a sensitive technique to detect presence of carbohydrates, indicated the successful galactosylation of the NPs and this was further corroborated by the FT-IR absorbance spectra. In clinical practice, RFA on HCC patients who were already on Dox therapy has shown ‘more complete tumor necrosis’.11,29 Cytotoxic effect of Dox is attributed to: intercalation of drug into DNA, induction of DNA breaks/chromosomal aberrations, free radical formation and finally induction of alterations in cell membrane.26 Functional and structural integrity of doxorubicin is known to remain unchanged even upon exposure to temperatures of 60oC for 3 months.[28] In vitro Dox release from GAD NPs, in absence of RF exposure, followed zero order kinetics with only a short initial burst. Influence of RF exposure on the Dox release was demonstrated in vitro when ~ 70% of the contained Dox was released almost instantly upon exposure to a 13.56 MHz RF field. This RF triggered Dox release here could be attributed to the augmented thermal response producing expansion of the porous alginate NPs and also breaking of ionic bonds between Dox and polymer chains. The RF triggered drug release is also reflected in vivo; where drug release observations clearly indicate that the amount of Dox entering into systemic circulation could be controlled by RF power and duration (Figure 6C). Thus, RF exposure could be optimized to locally deliver a therapeutic dose of drug around the point of RF application (within the tumor). Conventional chemoembolization with Dox eluting microbeads produces a tissue Dox concentration of 0.5 - 0.65 µM with maximum tissue penetration of only 1.2 mm around the embolised arteries.31 Our targeted GAD NPs will achieve better tissue penetration as they can travel even up to microvasculature level and

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specifically be taken up by tumor cells. Further, the local Dox concentration can be actively controlled using RF and tailored to achieve higher or lower levels. RFA procedures in clinical practice often extend over several minutes (~ 30 minutes) depending on size of the tumor. In vitro RF exposure of the GAD NPs (1mg/ml) showed the temperature to increase from ~ 370C to ~ 490C within 1 minute. The RF thermal response for GAD NPs is similar to the thermal response reported in literature for gold NPs, carbon nanotubes as well as graphene; for similar concentrations and duration.32 However, in comparison to the other systems, the GAD NPS are more biodegradable. This augmented RF thermal response of the NPs suggested that when contained in tissues, these NPs would produce areas of ablation even with short durations of relatively lower energy RF exposure. This hypothesis was tested and proved right in chicken liver phantoms. A one-minute RF exposure, which was not long enough to cause ablation in rest of the liver phantom, produced uniform areas of necrosis exactly around the site of NPs injection (Figure 5B). We tested the targeting as well as the combined effect of augmented RFA with Dox, produced by GAD NPs, in HEPG2 cells which are known to overexpress asialoglycoprotein receptors (ASGPR) (~ 76,000/cell).33,34 In our earlier works, we have demonstrated the importance of targeted delivery of therapeutic NPs against leukemic34,35 as well as liver cancer cells.36 Though previously we had exploited the overexpression of transferrin receptors on malignant cells to target HCC, in this study we used a more specific approach by targeting ASGPR. As predicted, a relatively more rapid and preferential uptake (p value < 0.005) of galactosylated NPs (~ 91% at ½ hr) by the cells was noted when compared to the non-galactosylated ones (~ 81% at ½ hr). This indicates

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that galactosylation of the NPs will ensure their efficient localization at tumor sites in vivo; due to their preferential uptake by the ASGPR overexpressing liver cancer cells.33 Cytotoxicity of the GAD NPs was similar to that produced by 1 minute RF exposure (~ 50%). Combining RF exposure along with NPs treatment killed ~ 81% of the HEPG2 cells in culture. From a translational point-of-view this finding indicates a two-fold advantage: one being the obvious improved efficiency in tumor destruction using a single agent. Another advantage will be, reduction in treatment-associated side effects, since higher therapeutic effects could be achieved with lower systemic drug concentrations, lower RF power and shorter RF exposure. In vivo biodistribution of GAD and AD NPs in normal Sprague-Dawley rats was studied by radiolabelling them with reduced 99m-Technetium (99m-Tc). Linkages between 99mTc and NPs are more likely to be electrostatic interactions than covalent ones. However, since negligible amounts of unbound 99m-Tc was seen in the excretory system (kidneys & urinary bladder) of the animals imaged (Figure 6A), it can safely be assumed that the radiolabelling was strong enough to confidently evaluate biodistribution of the NPs for at least up to 1 hour. We observed a gradual and progressive uptake of GAD NPs into the liver which points towards their selective uptake into hepatocytes rather than the more rapid uptake of Kuffer cells (reticuloendothelial system). In comparison, accumulation of the non-galactosylated (AD) NPs in liver was significantly slower (Figure 6B), thus demonstrating the effect of targeting the NPs to ASGPR overexpressing cells. In clinical translation, this efficient uptake into the liver will effect in shorter duration of the NPs in circulation, lesser non-specific uptake and ultimately lesser systemic side effects of doxorubicin released from these NPs. The peak accumulation of the radiolabelled GAD

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NPs was seen at 40th minute; hence the laprotomic RF procedure was performed only after a minimum period of 40 minutes following intravenous NPs injection. Although the highest RF power applied was only 15 watts and for a maximum duration of 1 minute (considering the small size of rat livers), MR imaging of the livers following RFA could clearly delineate areas of ablation and also identify augmented hyperthermic effects in animals injected with the NPs. ~ 44% increase in size of ablated area was seen with NP injection, compared to ablated areas in animals without NP injection. Apart from confirming this finding, gross examination of the ablated livers also revealed more uniform areas of ablation in the study animals. To objectively validate these findings we used TTC, an indicator of cellular respiration (viability). Absence of red staining in regions of the tissue samples indicates absence of live cells. (Figures 6D iii & vi). Uniform ablation throughout the tumor will ensure a more efficient treatment and lower chances of recurrence in clinical practice. This augmented RF thermal response was finally tested in the N1S1 orthotopic rat liver tumor models. We used MR imaging to confirm the successful development of tumors (Figure 7A). Open RFA was performed 15 minutes after intravenous injection of GAD NPs. We chose 15 minutes post intravenous injection because of our earlier observation that peak NPs accumulation in healthy liver happened by 40th minute. Thus we hypothesized that, because of enhanced permeation and retention (EPR) effect, there will be early accumulation of NPs in tumor; whereas the healthy liver tissue will show minimal uptake. It was for this same reason that the healthy liver lobe, beside the one containing the tumor, was used as control to compare RF thermal response. An accurate real-time evaluation of the tissue heating was not easy when using a temperature probe;

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since (being a conducting surface) it always interfered with the RF field resulting it altered heating patterns. Therefore we resorted to using an IR thermal imaging camera to assess the RF thermal response in the tissue. Our observations (Figure 7B & 7C) convincingly showed a ~ 200C higher heating at the tumor when compared to the normal liver. Gross evaluation of the excised tumors with TCC staining proved beyond any doubt that major parts (~ 80%) of the tumor (~ 2cm diameter) tissue were rendered non-viable within 1 minute of the relatively lower energy RF exposure. This is a significant data because in human clinical practice up to 100W RF needs to be applied for ~ 20 - 30min for ablating 3-5 cm tumors. Therefore use of GAD NPs will considerably reduce the duration and power of RF application while remaining efficient in disease ablation; thus ensuring more patient comfort and lesser side effects. In addition, compared to our recent studies with graphene where augmented RF response was observed at 100W exposure for 5 minutes,32 the present data is more promising because alginate nanoparticles are better biocompatible and biodegradable than carbon nanostructures. Conclusion In conclusion, we have demonstrated for the first time, a biodegradable RF nano probe based on stannous-doped alginate produced using a simple wet-chemistry technique, for targeted augmentation of RF thermal ablation; which could be co-loaded with Dox for combined RF triggered doxorubicin release in liver tumors. Encapsulated form of doxorubicin in NPs targeted specifically to the liver, where their release can be locally triggered, will drastically reduce incidence of the side effects commonly encountered with systemic administration of the drug alone. Being a material that is already used in humans as part of many pharmaceutical formulations, alginate holds a strong potential for

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clinical translation of nanoparticle mediated augmented RFA, in comparison to the nonbiodegradable systems. These results may stimulate more investigations on simple biodegradable nanosystems and RFA, which would be strong contenders for clinical translation. Acknowledgements The authors are grateful for the financial assistance from the Department of Science & Technology (DST), Government of India; for supporting this work under the project Thematic Unit of Excellence. The authors acknowledge Mr. Sajin P Ravi, Mr. Sarath S and Mr. Shivashanmugam for their technical assistance. Supporting Information Thermo gravimetric analysis of GAD NPs to confirm Dox loading has been described. The effect of RF exposure on the morphology NPs included along with DLS results in support of the same.

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References 1. Thorgeirsson S.S, Grisham J.W. Molecular Pathogenesis of human hepatocellular carcinoma. Nat. Genet. 2002;31(4):339-346. 2. Minami Y, Kudo M. Radiofrequency ablation of hepatocellular carcinoma: a literature review. Int. J Hepatol. 2011; 104685. 3. Schwartz M, Roayaie S, Konstadoulakis M. Stratergies for the management of hepatocellular carcinoma. Nat Clin Pract Oncol. 2007;4(7):424-432. 4. Delis S.G, Dervenis C. Selection criteria for liver resection in patients with hepatocellular carcinoma and chronic liver disease. World J Gastroentrol. 2008; 14(22):3452-3460. 5. Goldberg S.N. Radiofrequency tumor ablation: principles and techniques. Eur J Ultrasound. 200;13(2):129-147. 6. Yamakado K, Nakatsuka A, Takaki H, Yokoi H, Usui M, Sakurai H, Isaji S, Shiraki K, Fuke H, Uemoto S, Takeda K. Early-stage hepatocellular carcinoma: rediofrequency ablation combined with chemoembolization versus hepatectomy. Radiology, 2008;247(1):260-266. 7. Curley M.G, Hamilton P.S. Creation of large thermal lesions in liver using salineenhanced RF ablation. Proc 19th Internat. Conference IEEE/EMBS, 1997, 25162519. 8. Livraghi T, Goldberg S.N, Monti F, Bizzini A, Lazzaroni S, Meloni F, Pellicanò S, Solbiati L, Gazelle G.S. Saline-enhanced radio-frequency tissue ablation in the treatment of liver metastases. Radiology, 1997; 202(1):205-210.

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9. Seegenschmiedt M.H, Brady L.W, Sauer R. Interstitial thermoradiotherapy: review on technical and clinical aspects. Am J Clin Oncol, 1990;13:352-363. 10. Trembley B.S, Ryan T.P, Strohbehn J.W, in Hyperthermia and Oncology, vol 3. (Eds: Urano M, Douple E), Utrecht: VSP, 1992, 11-98. 11. Hong C.W, Libutti S.K, Wood B.J. Liposomal doxorubicin plus radiofrequency ablation for complete necrosis of a hepatocellular carcinoma. Curr Oncol. 2013, 20(3), e274-277. 12. Landon C.D, Park J.Y, Needham D, Dewhirst M.W. Nanoscale drug delivery and Hyperthermia: The materials design and preclinical and clinical testing of Low Temperature-Sensitive Liposomes used in combination with mild hyperthermia in the treatment of local cancer. Open Nanomed J. 2011;3:38-64. 13. Celsion corporation, Celsion announces results of Phase III HEAT study of ThermoDox

in

primary

liver

cancer,

January

13,

2013,

radiofrequency-induced

targeted

http://investor.celsion.com/releasedetail.cfm?ReleaseID=737033 14. Raoof

M,

Curley

S.A.

Non-invasive

hyperthermia for the treatment of hepatocellular carcinoma. Int J Hepatol. 2011; 676957. 15. Gannon C.J, Cherukuri P, Yakobson B.I, Cognet L, Kanzius J.S, Kittrell C, Weisman R.B, Pasuali M, Schmidt H.K, Smalley R.E, Curley S.A. Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field. Cancer, 2007;110(12):2654-2664. 16. Raoof M, Corr S.J, Kaluarachchi W.D, Massey K.L, Briggs K, Zhu C, Cheney M.A, Wilson L.J, Curley S.A. Stability of antibody-conjugated gold nanoparticles

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in

the

endolysosomal

nanoenvironment:

implications

Page 30 of 41

of

noninvasive

radiofrequency-based cancer therapy. Nanomedicine, 2012;8(7):1096-1105. 17. Glazer E.S, Massey K.L, Zhu C, Curley S.A. Pancreatic carcinoma cells are susceptible to noninvasive radio frequency fields after treatment with targeted gold nanoparticles. Surgery, 2010;148(2):319-324. 18. Sassaroli E, Li K.C.P, O’Neill B.E. Radiofrequency absorption in gold nanoparticle suspensions: a phenomenological study. J Phys D: Appl Phys. 2012, 45:075303. 19. Li D, Jung Y.S, Tan S, Kim H.K, Chory E, Geller D.A. Negligible absorption of radiofrequency radiation by colloidal gold nanoparticles. J Colloid Interface Sci. 2011;358(1):47-53. 20. Jayasree A, Sasidharan S, Koyakutty M, Nair S, Menon D. Mannosylated chitosan-zinc sulphide nanocrystals as flouroscent bioprobes for targeted cancer imaging. Carbohydrate Polymers. 2011;85:37-43. 21. Sasidharan A, Sivaram A.J, Retnakumari A.P, Chandran P, Malarvizhi G.L, Nair S, Koyakutty M. Radiofrequency ablation of drug-resistant cells using molecularly targeted carboxyl-functionalized biodegradable graphene. Adv Healthc Mater. 2015;4(5):679-684. 22. Merkle E.M, Goldberg S.N, Boll D.T, Shankaranarayanan A, Boaz T, Jacobs G.H, Wendt M, Lewin J.S. Effects of superparamagnetic iron oxide on radiofrequency-induced

temperature

distribution:

in

vitro

measurements

in

polyacrylamide phantoms and in vivo results in rabbit liver model. Radiology, 1999;212(2):459-466.

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23. Kruse D.E, Stephens D.N, Lindfors H.A, Ingham E.S, Paoli E.E, Ferrara K.W. A radio-frequency coupling network for heating of citrate-coated gold nanoparticles for cancer therapy: design and analysis. IEEE Trans Biomed Eng. 2011;58(7): 2002-2012. 24. Kowalsky R.J, Falen S.W, in Radiopharmaceuticals in nuclear pharmacy and nuclear medicine, American Pharmacista Association, 2004, Ch 9, 235-236. 25. Morris E.R, Rees D.A, Thom D, Boyd J. Chiroptical and stoichiometric evidence of a specific, primary dimerisation process in alginate gelation.

Carbohydr

Res. 1978;66:145–154. 26. Li L, Fang Y, Vreeker R, Appelqvist I, Mendes E. Reexamining the egg-box model in calcium-alginate gels with X-ray Diffraction. Biomacromolecules, 2007; 8(2):464-468. 27. Brulé S, Levy M, Wilhelm C, Letourneur D, Gazeau F, Ménager C, Visage C.L. Doxorubicin release triggered by alginate embedded magnetic nanoheaters: a combined therapy. Adv Mater. 2011;23(6):787-790. 28. DHHS/National Toxicology Program; Eleventh report on carcinogens: Adriamycin

(Doxorubicin

Hydrochloride)

(23214-92-8),

http://toxnet.nlm.nih.gov/cgibin/sis/search/a?dbs+hsdb:@term+@DOCNO+3070, Jan 2005. 29. Matricardi P, Di Meo C, Coviello T, Alhaique F. Recent advances and perspectives on coted alginate microspheres for modified drug delivery. Expert Opin Drug Deliv. 2008;5(4):417-425.

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Page 32 of 41

30. Aristide V, Martin J.W, in "Analytical profiles of drug substances" (Ed. Klaus F), "Doxorubicin", Academic Press, New York, 2005, 245-74. 31. Namur J, Citron S.J, Sellers M.T, Dupuis M.H, Wassef M, Manfait M, Laurent A. Embolization of hepatocellular carcinoma with drug-eluting beads: doxorubicin tissue concentration and distribution in patient liver explants. J. Hepatol. 2011;55(6):1332-1338. 32. Bijukumar D.R, Girish C.M, Sasidharan A, Nair S.V, Koyakutty M. Transferrin conjugated biodegradable graphene for targeted radiofrequency ablation of hepatocellular carcinoma. ACS Biomater. Sci. Eng, 2015;1(12):1211-1219. 33. Li Y, Huang G, Diakur J, Wiebe L.I. Targeted delivery of macromolecular drugs: asialoglycoprotein receptor (ASGPR) expression by selected hepatoma cell lines used in antiviral drug development. Curr Drug Deliv. 2008:5(4):299-302. 34. Retnakumari A, Jayasimhan J, Chandran P, Menon D, Nair S, Mony U., Koyakutty M. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for

targeted

flow-cytometric

detection

of

acute

myeloid

leulaemia.

Nanotechnology, 2011;22(28): 285102. 35. Chandran P, Kavalakatt A, Malarvizhi G.L, Vasanthakumari D.R, Retnakumari A.P, Sidharthan N, Pavithran K, Nair S, Koyakutty M. Epigenetics targeted protein-vorinostat nanomedicine inducing apoptosis in heterogenous population of primary acute myeloid leukaemia cellc including refractory and relapsed cases. Nanomedicine, 2014;10(4):721-732.

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36. Malarvizhi G.L, Retnakumari A.P, Nair S, Koyakutty M. Transferrin targeted core-shell nanomedicine for combinatorial delivery of doxorubicin and sofafenib against hepatocellular carcinoma. Nanomedicine. 2014;10(8):1649-1659.

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Figure 1: Schematic showing preparation of stannous doped alginate (Alg) nanoparticles co-loaded with doxorubicin (Dox), and further surface modified with activated galactose to produce the GAD NPs.

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Figure 2: (A) and (C) represent DLS analysis and AFM images (respectively), showing characteristics of AD NPs before galactosylation; (B) and (D) show increase in average size of the NPs post galactosylation. (E) absorbance spectrum of free drug (Dox) in supernatant of NPs with (i) and without (ii) PEI coating; inset image showing the higher drug retention in PEI coated NPs (ii). (F) Molisch’s test results; positive in GAD NPs, and negative in non galactosylated (AD) NPs. (G) FTIR spectrum of stannous doped Alg NPs with and without PEI coating and NPs after galactosylation showing successful binding of galactose to the PEI residues.

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Figure 3: (A) RF thermal response of different ionic solutions at varying concentrations, showing a common peak heating at ~ 0.2 mg/ml concentration. (B) concentration dependent RF thermal response of GAD NPs differs from that of SnCl2 solution and plateaus at 1 mg/ml. (C) comparison of RF thermal response of GAD NPs versus the thermal response of each individual component contained within it reveals the significantly higher heating of the nanoparticle formulation. Effect of RF exposure on drug released from the GAD NPs into PBS is depicted in (D). The linear release pattern seen without RF exposure (blue curve), changes to a rapid release pattern (red curve) when the NPs are exposed to a uniform RF field; thus confirming the influence of RF exposure on drug release.

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Figure 4: (A) flowcytometric analysis of: free Dox (FD), non-galactosylated NPs (AD) & Galactosylated NPs (GAD) uptake by HEPG2 cells at 1/2 and 2 hours. Confocal fluorescence microscopy image (B) demonstrating the characteristic intranuclear uptake of Dox released from the GAD NPs. (C) Alamar assay data showing the additive cytotoxic effect seen in HEPG2 cells upon combining GAD NPs treatment with RF exposure (GAD+RF). The plain alginate NPs (A) show negligible toxicity. Treatment with only NPs or RF exposure alone is significantly less toxic than the combined approach.

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Figure 5: (A) chicken liver phantom with arrow indicating site of instillation of GAD NPs, before exposing the whole phantom to a uniform RF field. (B) cut section of the phantom post RF exposure showing ablation only around site of NPs injection. (C) & (D) represent H&E stained liver phantom sections following RF exposure; from sites without and with (respectively) NPs instillation. Loss of histological integrity and morphology is seen only at the site of NPs injection (D) despite the fact that both regions (whole phantom) were exposed to the RF field.

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Figure 6: (A) nuclear scan images showing biodistribution of radiolabeled (99m Technetium) GAD NPs in Sprague Dawley rats; its accumulation in liver peaks at around 40th minute. (B) is a time-activity plot showing the concentration of GAD and AD NPs in liver over time (60 min). Area under curve for GAD significantly larger than for that of AD NPs (C) depicts the open RF ablation procedure technique. (D) the spectroscopic evaluation of Dox concentration in circulation following open RF ablation (with varying RF energies and durations) of the liver, where we see an RF dependent release of the drug. (E) are images representing the RF ablated livers in control (i, ii, iii), and study (iv, v, vi) animals. Images (i) & (iv) depict MR studies showing larger areas of ablation in livers containing the NPs, which is also seen on gross examination; (ii) & (iii). TTC stained liver samples undoubtedly confirmed the larger and more uniform necrotic areas in the study animals (vi) as compared to controls (iii).

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Figure 7: (A) MRI imaging confirming the successful development of the N1S1 tumor (red arrow) in liver (green arrow) of Sprague Dawley rats. Infrared imaging performed during the RF ablation procedure showed higher heating (> 20 OC difference) at the tumor region (B) than in the adjacent normal liver (C), which was used as the control. This augmented heating was confirmed by gross examination and TTC staining which showed larger areas of ablation at the tumor sites in liver (red arrow) when compared to normal control (green arrow). (F) depicts cut section of TTC stained liver tumor post RF ablation, which shows ≥ 80% tumor necrosis (black arrow) even with the short & low energy RF exposure. Blue arrow indicates the viable tumor tissue.

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Content Abstract (TOC Graphic)

Title: Biodegradable radiofrequency responsive nanoparticles for augmented thermal ablation combined with triggered drug release in liver tumor Authors: Vijay Harish Somasundaram, Rashmi Pillai, Giridharan Malarvizhi, Anusha Ashokan, Siddaramana Gowd, Reshmi Peethambaran, Shanmugasundaram Palaniswamy, Unni AKK, Shantikumar Nair*, Manzoor Koyakutty*

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