Plasmonically Enhanced Galactoxyloglucan Endowed Gold

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Plasmonically Enhanced Galactoxyloglucan Endowed Gold Nanoparticles Exposed Tumor Targeting Biodistribution Envisaged in a Surface-Enhanced Raman Scattering Platform Manu M Joseph, Jyothi B Nair, Kaustabh Kumar Maiti, and Sreelekha Therakathinal T Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01109 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 20, 2017

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Biomacromolecules

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Plasmonically Enhanced Galactoxyloglucan Endowed Gold Nanoparticles

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Exposed Tumor Targeting Biodistribution Envisaged in a Surface-

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Enhanced Raman Scattering Platform

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[1, 2]

, Jyothi B. Nair

[1, 3]

, Kaustabh Kumar Maiti*

[1, 3]

and Sreelekha

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Manu M. Joseph

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Therakathinal T* [1]

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[1] Laboratory of Biopharmaceutics & Nanomedicine, Division of Cancer Research,

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Regional Cancer Centre (RCC), Thiruvananthapuram-695011, Kerala, India

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[2] Chemical Sciences & Technology Division (CSTD), CSIR-National Institute for

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Interdisciplinary Science & Technology (CSIR-NIIST), Thiruvananthapuram-695019,

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Kerala, India.

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[3] Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

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*Address for correspondence

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E-mail: [email protected]

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E-mail: [email protected]

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ABSTRACT

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Biopolymer capped gold nanoparticles (AuNPs) were perceived for tracing biodistribution in

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a solid tumor mice through surface-enhanced Raman scattering (SERS) fingerprinting. In this

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strategy, a robust and ecofriendly green chemistry approach was adopted to construct

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galactoxyloglucan (PST001) endowed AuNPs (PST-GNPs) with cancer-cell-selective toxic

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nature and excellent biocompatibility. Plasmonically enhanced light scattering properties

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facilitated PST-GNPs to be a superior SERS substrate with high Raman signal enhancement.

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In this context, PST-GNPs were scrutinized for the non-invasive label-free SERS live cell

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spectral imaging to evaluate the fingerprint molecular details of cellular processes.

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Consequently, the inherent SERS feature of PST-GNPs enabled us to investigate the dynamic

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and complex nature with NP biodistrubution in tumor-bearing mice on a SERS platform

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which illustrated the tumor targeting nature. Henceforth, the present findings emphasized a

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futuristic clinically relevant scenario for tracing the in vivo NP dissemination in a label-free

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fashion for providing vital biochemical details in molecular level.

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KEYWORDS

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Surface-enhanced Raman scattering, galactoxyloglucan, cancer, biodistribution, gold nanoparticles

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Biomacromolecules

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INTRODUCTION

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The rapid progress in nanomedicine and nanotechnology has ignited the assembly of

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countless metallic, non-metalic and hybrid nanosystems for various biomedical applications.

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Owing to the medial applications of nanoparticles (NPs), metal nanoparticles gained

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progressive attention accounting to their trusted physiochemical features including high

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surface to volume ratio, facile synthetic strategies, tunable size, shape and the capacity to

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meritoriously carry biological targets1. Since ancient times, gold has been considered as the

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“elixir of life” and hence it is not amazing to envision the ubiquity of gold nanoparticles

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(AuNPs) in diverse applications in several bio-diagnostic systems and targeted drug delivery

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systems for managing complex, often intractable disease like cancer2. The exceptional

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features of AuNPs are enormously reliant to their size, shape, crystal facets and surface

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chemistry3. Tumor microenvironment is endowed with the feature of enhanced permeation

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and retention (EPR) effect, which ultimately provides easier access by NPs to tumor tissues4.

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Although AuNPs are embedded with salient features, the currently employed fabrication

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strategies impedes inherent toxicity not only because of the presence of toxic capping agents

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but also due to the interaction of NPs with blood counter parts and normal cells which

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hampers the biocompatibility and biodegradability5 thereby limiting the extensive clinical

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translation. Hence the need for the improvement of more effective approaches in this

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direction is imperative.

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In the wave of bio-analyte detection and imaging, surface-enhanced Raman scattering

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(SERS) has been rewarded as a fascinating phenomenon wherein weak Raman signals was

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amplified for tapping precise information of biomolecules based on their Raman fingerprint.

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By virtue of the ability to provide single molecular detection, SERS-based applications are

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not limited to biosensors, environmental monitoring, medical diagnostics, chemical imaging

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and theranostics6. SERS signals strongly depend on the presence of ‘hot spots’ generated by 3 ACS Paragon Plus Environment


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the high localized surface plasmon resonance (LSPR) occurring within interstitial crevices in

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metal NPs7. In SERS, generation of hot spots in the near vicinity of NPs can provide

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electromagnetic field Raman enhancement thereby providing a platform to detect even

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minute changes8. Notwithstanding, Raman spectroscopy has been extensively explored for

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illuminating bio molecular fingerprint of the sub-cellular components in a non-invasive

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manner. This approach enabled a real time monitoring of the biochemical changes in sub-

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cellular compartments towards different treatments such as chemotherapy, radiation and

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further employed for early detection of cancer using -specific marker assisted Raman

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nanoprobes9–11.

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On account of their tunable size, shape and high LSPR, AuNPs evolved as a flexible tool in

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nano-biophotonics and acts as emerging SERS substrates12 when used in label-free

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immunoassays, bio-sensing, imaging of living cells and microbes in ultralow level of limit of

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detection (LOD). Engineering AuNPs with green chemistry approach using biodegradable

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natural polymers as either reducing or capping agent or as both often generates

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biocompactable NPs which will subside conventional chemical approaches13. The structural

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diversity, aqueous solubility, biodegradability and chemical stability render polysaccharides a

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crowning position among natural polymers for the design of functional materials and nano-

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carrier delivery systems14. Also polysaccharides are naturally abundant and bio eliminable,

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and most of them are approved as excipients for oral, transcutaneous, and parenteral drug

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administration. Tamarindus indica (Ti) seed kernel polysaccharide (PST001) exposed wide

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range of physiochemical features including high viscosity, wide range of pH tolerance,

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biocompatibility, biodegradability, thermal stability and adhesiveness15. PST001 was

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characterized to be a galactoxyloglucan demonstrating cancer cell specific cytotxicity through

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the TRAIL-DR4/DR5 apoptotic pathways with impacable immunomodulatory potential in

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animal models16 without any systemic toxicity. The presence of galactose moiety with the 4 ACS Paragon Plus Environment

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Biomacromolecules

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PST001 could be selectively recognized by asialoglycoprotein receptor, over expressed in

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malignancies17 and hence PST001 capped NPs could be easily engineered to target tumor

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cells for receptor mediated endocytosis18. The presence of hydroxyl groups and hemiacetal

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reducing end played important roles in the facile fabrication of stable AuNPs using PST001

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(PST-GNPs) which not only demonstrated cancer cell selective toxicity both in vitro and in

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vivo but also executes superior immune-stimulatory effects19,20.

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At this juncture, with the perspective of unravelling the molecular fingerprints of various

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tissues upon intra peritoneal (i.p.) administration of PST-GNPs, we explored the enhanced

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plasmonic scattering property of the NPs to monitor the real-time biodistribution in a solid

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tumor-bearing syngraft mice model. A single nano construct utilized for simultaneous read

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out with chemotherapy and performed as a perfect SERS substrate for in vivo distribution

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studies (Scheme). In this perspective, we have aimed to exhilarate PST-GNPs to generate

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sufficient high intensity Raman substrate for tracing the NP distribution in a non-invasive

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manner. Hence, the current nanoconstruct PST-GNPs will definitely be a futuristic nanoprobe

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with real-time in vivo SERS monitoring topology for compacting the complexities associated

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with management of cancer.

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2. MATERIALS AND METHODS

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2.1 Preparation and Characterization of PST-GNPs

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The detailed procedure adopted for the isolation, purification and characterization of the

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galactoxyloglucan (PST001) was followed as reported earlier16,19 and is described in SI- 1.

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Preparation of AuNPs utilizing the galactoxyloglucan as both a reducing and capping agent

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was described previously19,20. In brief, AuNPs were prepared using a one-step approach by

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the reduction of HAuCl4 (1 mM) by PST001. Reaction conditions were standardized (Table

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S1) and the excess unreacted reactants were separated from the NPs by repeated dialysis 5 ACS Paragon Plus Environment


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using a cellulose membrane (Spectra/Por, MWCO 12–14,000 Daltons, Spectrum

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Laboratories, Rancho Dominguez, CA, USA). PST-GNPs were characterized by means of

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UV–vis spectroscopy (Bio Spec-1601, Shimadzu, Kyoto, Japan), transmission electron

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microscopy (Hitachi TEM system) at an accelerated voltage of 80 kV (Hitachi TEM system)

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and the hydrodynamic diameter and surface charge was determined with the help of SZ-100

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nanopartica (HORIBA Instruments, Kyoto, Japan). In order to access the stability of the thus

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fabricated NPs, the size and zeta potential was tested in different physiological solutions such

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as DMEM (Dulbecco’s modified Eagle’s medium), FBS, and PBS with a pH of 7.4.

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2.2 Cell lines and Cell Culture

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Human cancer cell line HeLa (cervical carcinoma) and murine myoblast cell line H9c2 was

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obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SKOV3

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(ovarian carcinoma) cells were generously provided by the Rajiv Gandhi Centre for

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Biotechnology (RGCB, Thiruvananthapuram, India). The fibroblast-like murine pre-

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adipocyte cell line 3T3-L1 was gifted from the Inter-University Centre for Genomics and

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Gene Technology, University of Kerala (Thiruvananthapuram, India). Human lung fibroblast

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cell line WI-38 was kindly gifted form Indian Institute of Chemical Biology (CSIR-IICB),

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Kolkata, India. Cells were maintained in DMEM with 10% FBS and 5% CO2 at 37ºC. The

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murine transplantable lymphoma cell lines, Ehrlich ascites carcinoma (EAC) was maintained

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in the peritoneal cavity of mice by intraperitoneal (ip) transplantation of 1x106cells per

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mouse. All animals used for the study were handled carefully without making pain or distress

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and with due care for their welfare. Animal experiments were performed according to the

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CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on

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Animals) guidelines and protocols were reviewed and approved by the Institutional Animal

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Ethics Committee (IAEC) of the Regional Cancer Centre, Trivandrum, India (No.

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657/Go/Re/02/CPCSEA). 6 ACS Paragon Plus Environment

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Scheme: Preparation of SERS active gold nanoparticles (PST-GNPs) in a green chemistry approach using PST001 for cancer cell targeted cytotoxicity and Raman imaging.

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Evaluating the efficacy of PST-GNPs as a Raman substrate was done with the aid of a

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confocal Raman microscope (WI-Tec, Inc., alpha 300R,Germany) using 4-Aminothiophenol

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(ATP) and Rhodamine 6G (R6G) as Raman reporter molecules and data was analysed using

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WI-Tec Project plus (v 2.1) software package. Specimens under investigation were excited

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using a 633 nm excitation wavelength laser with 7mW powers and the instrumental setups

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were maintained as described before11,21. Calibration with a silicon standard (Raman peak

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centred at 520 cm-1) was performed prior to each measurement. The “magnitude’’ of the

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enhancement in SERS was estimated at by taking the Raman signal of the reporter

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(ATP/R6G) alone (1 mM) and various dilutions up to limit of detection (LOD) with a

2.3 SERS Measurements for Analytical Enhancement Factor



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constant concentration (1µg/ mL) of PST-GNPs. Accordingly, to a clean glass slide, 10 µL of

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Raman reporter is mixed with 10 µL of NPs and the spectra were collected after background

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correction to exclude fluorescence interference. Dilutions were made with the Raman reporter

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up to no visible spectra are observed, the analytical enhancement factor (AEF) and LOD was

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estimated21,22.

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2.4 Cytotoxicity Assays

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PST-GNPs was initially evaluated for cytotoxicity on cancer and normal cell lines by 3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay as reported19 previously.

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The cytotoxicity of the NPs was compared with that of PST001 and standard citrate stabilized

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AuNPs over a wide range of concentration (0.01 ng to 100 µg/ mL) for 24, 48 and 72 h and

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later the absorbance was measured at 570 nm by means of a microplate spectrophotometer

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(BioTek, Power Wave XS). The cytotoxicity was further confirmed with BrdU assay kit

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(colorimetric -11647229001, Roche Diagnostics, IN, USA) and the experiments were carried

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out with the instructions given in the kit, measurements were made at 450/690 nm. Also the

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effect of PST-GNPs on isolated peripheral lymphocytes was also evaluated by BrdU assay.

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The lymphocytes were isolated from peripheral blood as described in SI-2.

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2.5 Haemolysis Assay

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Haemolysis assay was employed to analyse the outcome of PST-GNPs and PST001 on

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peripheral red blood cells (RBCs) as reported earlier8. Accordingly, RBCs were isolated from

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EDTA-stabilized human blood samples. Test compounds at various concentrations in PBS

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(0.8 mL) were then added to RBC suspension (0.2 mL). Positive and negative control

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samples were also prepared by adding 0.8 mL 2% Triton X-100 and PBS, respectively.

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Samples were then subjected for incubation at room temperature for 2 h, centrifuged at 700 g,


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and supernatants (100 µL) were transferred to a 96-well plate and absorbance was measured

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at 570 nm with the aid of a microplate reader.

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2.6 Splenocyte Proliferation Assay

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In order to further substantiate the immunostimulatory potential of PST-GNPs, splenocyte

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proliferation assay was performed on male BALB/c mice with four groups (6 animals each)

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as described23. Animals were given repeated i. p. administration of NPs at days 1, 3 and 5

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(100 mg/kg), a negative control group with PBS administration was also maintained.

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Experimental mice were subjected to sacrifice (7, 14 and 21 days post exposure) and the

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spleen was dissected, washed and collected in an ice cold Roswell Park Memorial Institute

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(RPMI) media. Splenocytes (single cell suspension) was obtained using Ficoll-PaqueTM Plus

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solution (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden), re-suspended in complete

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(10% FBS) RPMI media. Cells were counted and 20,000 cells were seeded onto each well of

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96 micro well plates and were allowed to grow for 48 h in presence of 5% CO2 at 37 ºC, cell

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proliferation was evaluated by BrdU assay upon comparison and normalization with vehicle

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control.

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2.7 Raman Imaging of Live Cells

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Raman imaging of cells was done with the aid of a confocal Raman microscope (WI-Tec,

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Inc., Germany). For cellular imaging, 20 µL (10 ng/mL) of PST-GNPs was added to HeLa

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cells and was incubated at 37 ºC for 30 minutes; a negative control well without NPs was also

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maintained. SERS mapping was recorded by focusing the laser beam on the cell surface

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selected at a position z = 0 µm using 0.5 as integration time, 150 x 150 as points per line and

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50 × 50 µm mapping area along X and Y directions over a motorised scan stage. The Raman

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images were subjected to data interpretation and cluster mapping8,21.



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2.8 Apoptotic Assays

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Estimation of programmed cell death by morphological assays was conducted in HeLa and

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SKOV3 cells treated with PST-GNPs (10 µg/ mL) for 24 h. Initially, cells were observed for

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any visible gross morphological changes under phase contrast objective (Olympus 1X51,

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Singapore) to view the apoptotic or non-apoptotic cells. Live-dead assay using acridine

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orange-ethidium bromide was performed as described earlier19,24 and the cells were observed

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under a FITC filter (Olympus 1X51, Singapore). Observation for any apoptosis related

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changes with Hoechst 33342 staining (Olympus 1X51, Singapore) was performed as

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described before8. TUNEL assay (DeadEnd™ fluorometric TUNEL system-G3250, Promega,

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USA) was used to detect the incorporation of the fluorescein-12-dUTP in the fragmented

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DNA of apoptotic cells, using the terminal-deoxynucleotidyl- transferase recombinant (rTdT)

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enzyme as per the manufacturer’s instructions using propidium iodide as counter-stain.

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Furthermore, evaluation of apoptosis by FITC-Annexin V staining (BD Pharmingen

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#556547, BD Biosciences, San Jose, CA) was also performed by flow cytometry, using kit

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specified instructions on a FACS Calibur flow cytometer (BD Bio-sciences, San Jose, CA)

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and the data was analysed with the Cell QuestPro software25. Early onset of programmed cell

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death was further observed using APO Percentage™ dye (Biocolor, Belfast, Northern

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Ireland) as per manufacturer’s instructions as described before26. Light microscopic images of

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APO Percentage dye-labelled cells, which stained pink under a light microscope, were used

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to quantify the extent of apoptosis. The dye uptake was further quantified using colorimetric

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method according to the manufacturer’s instruction. The cells were lysed and the absorbance

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was measured at 550 nm using a microplate reader (Biotek, USA).

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2.9 Caspsae

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The influence of both executioner caspases (caspase 3) and initiator caspases (caspases 8,9,

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and 2) on the cell death imposed by cytotoxic gents was determined using Apo AlertTM 10 ACS Paragon Plus Environment

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Biomacromolecules

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Caspase Profiling kit (Clontech, CA, USA) as per the manufacturer’s protocol8,26. Cells were

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treated with PST-GNPs (10 µg/mL) for 24 h, and samples were transferred to 96-well plates

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for fluorimetric reading (λex 380 nm, λem 460 nm), and signals were recorded using a

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spectrofluorimeter (FLx800, BioTek).

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2.10 Biodistribution Studies on Solid tumour Mice Syngraft

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Female BALB/c mice were used for the study and were maintained in well-ventilated cages

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with free access to normal mouse food and water. Temperature (25 ± 2°C) and humidity (50±

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5%) was regulated and the illumination cycle was adjusted to 12 h light/dark. EAC cells

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(1×106 cells/mouse) were injected subcutaneously with a fine needle (31G) in the hind limb

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of mice (n = 6/group). The experiments were performed after the tumour has attained

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palpable volume20. The distribution of PST-GNPs in EAC solid tumour bearing mice was

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evaluated at various time intervals (2, 4, and 6 h) after i.p. administration of NPs in a SERS

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platform. Consequently, mice were sacrificed by cervical dislocation; blood, tumour, liver,

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kidney, spleen, lungs and heart were collected and the tissue level NP distribution was

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evaluated using Raman fingerprinting as described before21. A minimum of three independent

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measurements were made out from each sample and the spectra were subjected to baseline

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subtraction, normalization and later the Raman intensities were converted to counts per

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second (cps) and the biodistribution in each tissue was tabulated. In order to further re-

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establish the biodistribution of PST-GNPs in a fluorescence platform, the NPs were labelled

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with FITC (SI-3) as described previously 21,27. EAC solid tumor mice were administered with

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PST-GNP-FL and the tissue supernatants were collected as described above and was

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examined with the aid of a spectrofluorometer (FLx800, BioTek, USA) with an excitation

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and emission λ485 nm and 528 nm, respectively.

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2.11 Statistical Analysis



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The data were expressed as the mean ± standard deviation (SD) of three replicates and

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analysed using Graph Pad PRISM software version 5.0 (San Diego, USA). One-way analysis

3

of variance was used for the repeated measurements and the differences were considered to

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be statistically significant if p < 0.05. The IC50 values were calculated by using the Easy Plot

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software (Spiral Software, MA, USA). MATLAB 8.3 (MATLAB R2014a, MA, USA) was

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employed for spectral normalisation and plotting.

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RESULTS AND DISCUSSION

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3.1 Preparation of Colloidal PST-GNPs

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Galactoxyloglucan

(PST001),

isolated

and purified

by high

performance

liquid

10

chromatography have a neutral pH, with around 93% total sugar content. The current

11

fabrication strategy of PST-GNPs didn’t require any chemicals as PST001 itself acts as both a

12

capping and reducing agent. PST-GNPs of different morphology and surface charge were

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constructed by varying the stoichiometry of the reaction mixture (Table S1) and were

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screened in terms of Raman signal enhancement efficiency (discussed later). The most

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promising SERS substrate (serial number #1) was selected for detailed characterization and

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further studies. TEM evaluation clearly indicated that the particles are anisotropic and

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heterogeneous in nature with an average size around 40 nm (Figure S1a, b). The presence of

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PST001 as a capping layer over the gold core could be clearly observed under the magnified

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TEM image (Figure S1a inserts). The NPs possessed hydrodynamic size of 45 nm (Figure

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S1c) with a negative surface charge of 32 (Figure S1d). The fruitful assembly of PST-GNPs

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was later confirmed with UV–vis spectroscopy (Figure S1e) wherein an intense peak around

22

570 nm was observed. Evaluation of the size, stability and surface charge of PST-GNPs in

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water, FBS, DMEM and PBS at physiological pH emphasized the stability of the biogenic

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colloidal NPs. The size was increased and surface charge was marginally decreased in FBS


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upon prolonged incubation of 72 h, suggesting the chances of slight aggregation (Table S2).

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However, the parameters were largely unaltered in water, media and PBS highlighting the

3

efficacy of galactoxyloglucan as a good capping agent. Evaluation of the stability using UV–

4

vis spectroscopy proved that PST-GNPs existed as a stable colloidal solution in water for

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more than 3 months (Figure S2a), FBS (Figure S2b), media (Figure S2c) and PBS (Figure

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S2d). The strategic fabrication of the colloidal AuNPs using PST001 without any hazardous

7

chemicals turned into a “green” synthesis without the aid of external capping agent. PST-

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GNPs selected for the current study was not previously examined for any activity and hence

9

the detailed investigations holds specific scientific merits.

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3.2 Evaluation of Raman Enhancement of PST-GNPs

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To concurrently explore the efficiency of PST-GNPs as SERS substrate for enhancing the

13

molecular Raman vibrations of the analytes, two commonly used Raman reporters were

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employed. Spectral analysis of 4-Aminothiophenol (Figure 1a, insert) with PST-GNPs

15

indicated a most distinct peak around 1020 cm-1 along with other prominent peaks (Figure

16

1a, Table S3) corresponding to the molecular structure of the reporter. Similarly, Rhodamine

17

6-G (Figure 1b, insert) was characterised with most intense peaks around 1365 and 1514 cm-1

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(Figure 1b, Table S4). The signal intensity of Rhodamine 6-G was more intense than 4-

19

Aminothiophenol with a fixed concentration (1µg/mL) of PST-GNPs. It is to be noted that

20

only at a higher concentration (1M) both the reporters alone could produce detectable

21

fingerprints in a Raman platform (Figure S3a, c) but in the presence of PST-GNPs even lower

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concentrations (1mM) generated high intense Raman spectral signatures (Figure S3b, d)

23

corresponding to the chemical structures. The noticeable increment in the Raman signal

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intensity upon addition of PST-GNPs postulates the potential of this biogenic nanofabrication

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as a Raman substrate. In the case of 4-Aminothiophenol, an achievable limit of detection 13 ACS Paragon Plus Environment


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(LOD) of 100 pM was obtained whereas for Rhodamine 6-G, a much higher LOD of 50 pM

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was obtained using PST-GNPs as a SERS substrate. The enhanced Raman signature peak at

3

1020 cm-1 of 4-Aminothiophenol (Figure 1c) and 1365cm-1 of Rhodamine 6-G (Figure 1d)

4

which corresponds to the strong C=S bonding and C-C stretching vibrations respectively was

5

evaluated by lowering dilutions starting form 1M up to LOD. An enhancement factor of 3.7

6

x10

7

Rhodamine 6-G indicated the potential of PST-GNPs as SERS substrate. The LSPR spectral

8

shifts induced by tight inter-particle junctions of plasmonic nanostructureswill create

9

dramatically improved signal amplification for Raman molecules28. The good spectral

10

reproducibility and locally enhanced electromagnetic field could be due to the presence of

11

PST001 as the capping agent. Plasmonic NPs surface caged with biomaterials execute

12

defined optical and structural properties thereby generating stable and reproducible Raman

13

signals29. Enhancement factors in the order of 107-108 are in fact adequate for the detection of

14

single molecule-SERS signals30, the relatively high enhancement factor of PST-GNPs

15

enabled its future exploration in SERS platform for bio-imaging purposes.

5

and 2 x 10

8

at an excitation wavelength of 633 nm for 4-Aminothiophenol and


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Biomacromolecules

1 2 3 4 5 6

Figure 1: PST-GNPS as a potential SERS nanotag. (a) Raman spectra of the analytes (1mM) 4-Aminothiophenol and (b) Rhodamine 6G with PST-GNPs (1µg/mL). The insert figure represents the respective chemical structures. SERS enhancement by PST-GNPs (1µg/mL) with decreasing concentrations from 1mM of (c) 4-Aminothiophenol and (d) Rhodamine 6G up to the limit of detection.

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Page 16 of 35

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3.3 Evaluation of Cytotoxicity and Biocompatibility Profile

2

Assessment of the cytotoxicity of PST-GNPs on cancer and normal cells by MTT and BrdU

3

assay over a time span of 24-72 h displayed a cancer cell selective toxicity pattern. Cervical

4

carcinoma cell line HeLa was growth arrested with an IC50 of 2.34 and 0.74 µg/mL at 48 and

5

72 h respectively by PST-GNPs but the parent polymer, PST001 produced IC50s at a

6

relatively higher concentration (76.5 and 22 µg/mL) at 48 and 72 h. Both polysaccharide and

7

the NPs displayed a dosage and time-dependent increase in the cytotoxicity but standard

8

citrate stabilized AuNPs didn’t exhibit any toxicity (Figure 2a, b, Figure S4a, b). Although

9

PST001 failed to produce an IC50 on SKOV3, PST-GNPs produced the same at 72 h (2.5

10

µg/mL). Concentration and time dependent increase in cytotoxicity was demonstrated by

11

both PST001 and NPs but standard AuNPs was nontoxic (Figure S4c-f). Human lung

12

fibroblast cell line WI-38 was used as a source of normal cells of human origin. It was

13

observed that the growth of normal human lung fibroblasts was not influenced by any of the

14

administered agents even at a higher dosage and incubation time (Figure 2c, d. Figure S5a,

15

b). Similarly, murine fibroblast-like pre-adeposite cell line 3T3-L1 was also not affected by

16

any of the administered agents even at higher concentrations and incubation period (Figure

17

S5c-f). Similar trend of cytotoxic behaviour was delivered by PST001, PST-GNPs and

18

standard AuNPs on murine myoblast cell line H9c2 with no signs of toxicity (Figure S5g-j).

19

In order to evaluate the biocompatibility of the NPs, its cytotoxicity on isolated peripheral

20

lymphocytes was evaluated using BrdU assay. Both the polysaccharide and NPs were not

21

only observed to be safer towards lymphocytes but also exhibits lymphocyte proliferation

22

suggesting the immunostimulatory potential of the agents (Figure 2e). Next, hemolysis assay

23

was performed to determine the toxicity towards red blood cells (RBCs) under three different

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pH conditions to substantiate the biocompatibility wherein both PST001 and PST-GNPs was

25

found to be totally non-hemolytic in nature event at higher concentrations (Figure 2f) upon 16 ACS Paragon Plus Environment

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Biomacromolecules

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comparison

with

positive

control.

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Figure 2: Cytotoxicity evaluation on HeLa cells at 48 h by (a) MTT and (b) BrdU assay, WI38 cells at 48 h by (c) MTT and (d) BrdU assay. Biocompatibility testing on isolated peripheral lymphocytes (e) and on red blood cells (f). (g) Splenocytes proliferation in PSTGNP exposed mice on comparison with vehicle control. Data represent mean ± SD, statistically significant differences at *P

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