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Exploration of Biogenic Nano-Chemobiotics Fabricated by Sliver Nanoparticle and Galactoxyloglucan with an Efficient Bio distribution in Solid Tumor Investigated by SERS Fingerprinting Manu M Joseph, Jyothi B Nair, Ramya N Adukkadan, Neethu Hari, Raveendran Pillai, Ananthakrishnan J Nair, Kaustabh Kumar Maiti, and Sreelekha Therakathinal T ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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ACS Applied Materials & Interfaces

Exploration of Biogenic Nano-Chemobiotics Fabricated by Sliver Nanoparticle and Galactoxyloglucan with an Efficient Bio distribution in Solid Tumor Investigated by SERS Fingerprinting Manu M. Joseph[a,b], Jyothi B. Nair[a,c], Ramya N. Adukkadan[a,c], Neethu Hari[d], Raveendran K. Pillai[e], Ananthakrishnan J. Nair[d], Kaustabh Kumar Maiti*[a,c], Sreelekha Therakathinal T *[b] a) Chemical Sciences & Technology Division (CSTD), Organic Chemistry Section, CSIR-National Institute for Interdisciplinary Science & Technology (CSIR-NIIST), Thiruvananthapuram-695019, Kerala, India. b) Laboratory of Biopharmaceutics &Nanomedicine, Division of Cancer Research, Regional Cancer Centre (RCC), Thiruvananthapuram-695011, Kerala, India. c) Academy of Scientific and Innovative Research (AcSIR), New Delhi, India d) Department of Biotechnology, University of Kerala, Thiruvananthapuram-695581, Kerala, India e) Clinical Laboratory Services, Regional Cancer Centre (RCC), Thiruvananthapuram695011, Kerala, India Corresponding Authors * T.T. Sreelekha. Email: [email protected] * K.K Maiti. Email: [email protected] 1 ACS Paragon Plus Environment


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ABSTRACT: An incredible exploration of a dual modality nano composite wherein, chemotherapy in fusion with antibacterial efficacy obtained in a biogenic fabrication, which transformed as a novel nanochemobiotics (NCB) prevailing fundamental molecular level investigation by surface enhanced Raman scattering (SERS) platform. The nano composite is a facile, robust and eco-friendly constitution between silver nanoparticles (SNPs) and a naturally occurring galactoxyloglucan (PST001) denoted as SNP@PST which displayed biocompatibility with an upgraded selective cytotoxicity towards cancer cells. The relatively non-toxic nature of the SNP@PST on normal cells and RBCs was further proved by detailed toxicological profiling on BALB/c mice. As a unique outcome, we observed an excellent anti-bacterial activity which is in complementary to the greater cytotoxicity by the NCB. In diagnostic aspect, SNP@PST was revealed to be a superior SERS substrate with multiscale Raman signal enhancement contributed by homogenous hot-spot distribution. Finally, the inherent SERS feature enabled us to investigate the biodistribution of the NCB in tumor-challenged mice using Raman fingerprinting and mapping analysis. Hence, the unrevealed SNP@PST orchestrated with the surfactant-free green method resembled as a potential theransonstic NCB-construct with synergistic anticancer and antibacterial potential in a single platform.

KEYWORDS: cancer, galactoxyloglucan, surface enhanced Raman scattering, nanochemobiotics, antimicrobial

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INTRODUCTION The advent of nanotechnology has ignited human mind with its distinctive features at the nanoscale dimension. Metal nanoparticles found extensive applications owing to their unique size, shape and surface to volume ratio that transformed their physio-chemical and optical properties. Among various nanoparticles (NPs) being explored, silver nanoparticles (SNPs) are embedded with unique features1 but currently employed fabrication approaches impedes inherent toxicity which limits the extensive biomedical applications2,3. Metallic silver as well as SNPs are widely demonstrated as an excellent anti-microbial agent since ancient times4. The bio-active molecules involved in the ‘green’ synthesis of SNPs have rendered the NPs with sustained stability and bio-compatibility making them suitable for biomedical applications. SNPs evolved as an adaptable tool in nano-biophotonics due to their high localized surface plasmon resonance (LSPR) which causes electromagnetic field enhancement at the NP surface5 known as “hot spots”.In the present scenario, surface-enhanced Raman scattering (SERS) provides the most promising advantages like multi-parameter molecular analysis and multiplexing potential due to the narrow “fingerprint” Raman spectrum unique to the chemical species making it possible to detect even minute biochemical changes based on vibrational Raman fingerprint6,7. In this contest, SNPs with varying size and shapes revealed as SERS substrates8,9 by the LSPR of silver10. As of now, amazing applications involving SERS have been developed which includes label-free immunoassays, bio-sensing, live cell imaging and tracing molecular changes occurred during apoptotic events6,11.The in vivo SERS12 phenomenon is realized by SERS-active NPs known as SERS nanotags, which are constructed by attaching Raman-active reporter molecules onto strong Raman substrates13.



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Scheme 1 (A) Preparation of silver nanoparticles (SNP@PST) in a green chemistry approach using PST001. (B) SNP@PST was evaluated for anti-bacterial and anticancer potential and was later utilized as SERS substrate for tracing cellular internalization and bio-distribution of the NPs. 4 ACS Paragon Plus Environment

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The enhanced permeation and retention (EPR) effect in the tumor microenvironment, provides easier access to a wide range of nano-sized delivery systems including liposomes, micelles and dendrimers14. In the recent time, functional modification of nano-drug delivery15 system with naturally occurring biocompatible polymeric materials such as polysaccharides, proteins and lipids gains scientific attention. Among the group of bio-degradable natural polymers, polysaccharides have been extensively used in the design of functional material for nano-carrier delivery systems owing to their excellent chemical stability, biodegradability and aqueous solubility16. Polysaccharide (PST001) isolated from the seed kernel of Tamarindus indica exposed wide range of physiochemical features17 and was found to be a galactoxyloglucan demonstrating cancer cell specific cytotxicity with impacable immunomodulatory potential in animal models18. The cytotoxic action of PST001 investigated through gene expression analysis revealed the induction of apoptosis selectively on cancer cells through the TRAIL-DR4/DR5 pathways19. The galactose moiety of PST001 could be recognized and internalized by asialoglycoprotein receptor, over expressed in malignancies. Therefore, PST001 capped NPs could be selectively and easily targeted to tumor cells and then internalized by receptor mediated endocytosis20. Cancer cell-selective uptake21 and superior tumor-specific cytotoxicity coupled with immunostimulatory effects22,23 of the NP formulations of PST001 was well established. So far, there is no report on a single nanoconstruct that could be used for simultaneous read outs with chemotherapy in fusion with anti-bacterial efficacy and utilized as a perfect SERS substrate for in vivo bio-distribution studies. In this perspective, we have aimed to exhilarate a smart and simple nano-chemobiotics (NCB) based on SNPs and PST001 by adopting the principles of green chemistry. The facile synthetic strategy for NCB has been taken for in depth investigation with (i) photo-physical characterization, anti-microbial, and cytotoxicity profile in order to select 5 ACS Paragon Plus Environment


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the best biogenic fabrication of SNP@PST; (ii) mechanistic mode of cytotoxicity has been evaluated in various cancer, normal cells and animal models to establish the cancer-specific toxicity and bio-compatibility of the NCB; (iii) finally the significant Raman fingerprinting of SNP@PST has been utilized in SERS platform for tracing NP cellular internalization and precise molecular level bio-distribution in tumor challenged animals (Scheme 1). Hence, the current nanoconstruct (SNP@PST) will definitely be a futuristic multi-therapeutic nanoprobe with realtime in vivo SERS monitoring topology for efficient management of bacterial infections and cancer.

RESULTS AND DISCUSSION Biogenic Colloidal SNP@PST Initially, PST001 was isolated, purified by high performance liquid chromatography and later used as both reducing and capping agent for the fabrication of our biogenic NCB (SNP@PST) by varying the stoichiometry of the reaction mixture (Table S1). Various NPs thus fabricated were screened in terms of stability and selective cytotoxicity and the most promising SNP@PST was selected (serial number #7) for further studies. The successful production of SNP@PST was evaluated using UV–vis spectroscopy (Figure 1a, Figure S2a) in which PST001 acted as both reducing and capping agent, imparting stability to the SNP@PST (Figure S2b). PST001 successfully reduced AgNO3 to form SNPs with observable variation in colours (FigureS2c). TEM evaluation clearly indicated that the majority of the particles were circular in shape with an average size around 100 nm (Figure 1b, Figure S2d, e). The presence of PST001 as a capping layer over the Ag core could be clearly observed under the magnified TEM image (Figure 1b inserts). The NPs bears hydrodynamic size of 90 nm (Figure 1c) with a negative surface charge of 17 (Fig. 1d). The chemical interactions that occurred during the formation of the SNP@PST 6 ACS Paragon Plus Environment

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were characterized by FTIR spectroscopy (Figure 1e) .Evaluation of the size, stability and surface charge of SNP@PST in water, FBS, media and PBS with physiological pH highlighted the stability of the biogenic colloidal NPs. The size and surface charge was changed only in FBS, where a slight increase in size of about 5 nm is observed after 72h along with a change in zeta value from -17 to -14. Evaluation of the stability using UV–vis spectroscopy proved that SNP@PST is stable in all the physiological solutions (FigureS2.1a-d; Table S1.1). The preparation of proposed NCB using PST001 does not include any environmental hazardous chemicals and turned into a “green” synthesis without the aid of external capping agent. Negatively charged NPs in the range of 50–100 nm are reported to exhibit higher rates of cellular endocytosis via caveolae pathways24. The ability of PST001 to act as both reducing and capping agent has already been demonstrated from our group with the preparation of highly stable AuNPs22,23. From the FTIR spectrum of the SNP@PST, the peak around 1364 cm-1 indicates the presence of symmetric stretching vibration of NO2 group confirming the formation of polysaccharide capped NPs. The other prominent bands coming from the IR spectrum are characteristics to the pure polysaccharide, such as 2123, 1640 and 3388 cm-1 corresponding to the presence of C≡C symmetric stretching, –HC=O stretching and intra-molecular hydrogen bonding respectively. From the merged spectra the weaker bands near 1340 and 1390 cm-1 as well as the weak absorption around 1448 cm-1 represents the symmetric frequencies of AgNO325from which the NP is formed. Evaluation of Cytotoxicity Profile The cytotoxicity of SNP@PST was initially evaluated on cancer and normal cell lines by MTT assay which displayd selective cytotoxicity towards cancer cells (Table S2). It was observed that the growth of melanoma cells A375 and lung adenocarcinoma cells A549 were arrested by the



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SNP@PST with a lower IC50 but AgNO3 and PST001 required higher dosage (Figure S3a-f). SNP@PST exhibited marked cytotoxicity against cervical carcinoma cell line HeLa in a dosedependent manner where AgNO3 demonstrated severe toxicity at higher concentration but PST001 displayed a regulated cytotoxic profile (Figure 2a-c). Even though PST001 fails to generate an IC50 on ovarian carcinoma cell line SKOV3, the SNP@PST produced a favourable response even at lower dosages (Figure S4a-c). Murine fibroblast-like pre-adipocyte cell line 3T3L1 proliferation was arrested in a dosage dependent fashion by AgNO3 with IC50s even at lower concentrations but PST001 and SNP@PST displayed a non-toxic behaviour (Figure 2d-f). Similarly in murine myoblast cell line H9c2 only AgNO3 displayed toxicity (Figure S4d-f). SNP@PST demonstrated significant cytotoxic profile against murine ascetic carcinoma cell lines DLA and EAC cells in a dosage and time dependent manner (Figure S5a-f). The cancer cell selective toxic behaviour displayed by SNP@PST over PST001 and AgNO3 was further confirmed by BrdU assay wherein, all the three agents produced cytotoxicity in harmony with MTT assay (Figures S6a-d, S7a-d). Haemolysis assay was performed to determine the toxicity on red blood cells (RBCs) under three different pH conditions (Figure 2g) in which AgNO3 demonstrated concentration-dependent lysis but SNP@PST showed reasonably lower haemolysis and PST001 was found to be totally non-haemolytic in nature. The cytotoxicity was further checked on isolated normal peripheral lymphocytes wherein SNP@PST was proved to be non-toxic (Figure S8) but AgNO3 illustrated marked cytotoxicity in a dosage dependent fashion. The increased surface to volume ratio of SNP@PST provided optimal activities by allowing better penetration of the cell membranes and greater access into various cellular compartments after endocytosis26. The highlighted cytotoxicity of SNP@PST might be attributed to the nanosize and the presence of PST001 as a capping agent which resulted in bridging the sensitivity and


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selectivity towards cancers cells. The preferential non-toxic nature of PST001 could be responsible for the observed bio-compatibility of the NPs. The immuno-stimulatory effects of PST00118 and its AuNPs22 was previously reported and hence the non-toxic behaviour of SNP@PST towards lymphocytes is credited to PST001. Despite the fact that conventionally prepared SNPs are accountable for haemolysis27 the presence of PST001 in the SNP@PST makes it blood-compactable for enabling safer in vivo administration.



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Figure 1. Preparation and characterization of SNP@PST. (a) UV-visible spectra of the selected SNP@PST, insert picture represent the photographic image of the colloidal suspension. (b) TEM image of the selected NPs, insert figures showed magnified view of a single nanoparticle. (c) DLS and (d) Zeta potential measurements of selected SNP@PST. (e) FTIR spectra of PST001 and SNP@PST over a wave number range of 4000–400 cm−1.


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Figure 2. Evaluation of cytotoxicity by MTT assay on HeLa (a) at 24 h, (b) at 48 h, (c) at 72 h and 3T3-L1 (d) at 24 h, (e) at 48 h and (f) at 72 h. (g) Haemolysis experiment performed. The insert figures represent (a) PST001, (b) SNP@PST, (c) AgNO3and (d) Triron X at 1 µg/ mL. Data represent mean ± SD, statistically significant differences at *P < 0.05, ** P < 0.01, *** P < 0.001 and ns, non-significant, for SNP@PST when compared with negative control.



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Antibacterial Properties of SNP@PST SNP@PST failed to produce noticeable antioxidant potential against experimentally generated free radicals (Figures S9a-c, 10). Hence, the antibacterial activity was evaluated against Escherichia coli, Salmonella typhi and Staphylococcus aureus by well-diffusion method in which PST001 displayed no antibacterial property (Figure S10a-c). Anti-bacterial potential of SNP@PST (FigureS10d-f) against both Gram positive and negative organisms is predominant than positive control streptomycin and AgNO3 (Table S3). The minimum inhibitory concentration and minimum bactericidal concentration values of SNP@PST against E. coli were 3.90 µg/mL and 15.625 µg/mL whereas streptomycin created the same at 7.8 and 15.6 µg/mL respectively. The primary mechanism of antibacterial action of SNPs is release of silver ion (Ag+) in a sustainable fashion28. Even though the exact mechanism of antibacterial effects of SNPs is still unknown, the plausible reason is due to the formation of free radicals produced by the NPs which interact with the membrane lipids and finally spoil the membrane functions29 of the microbe. The outstanding anti-bacterial potential of SNP@PST could be due to the optimal release of Ag+ which will interacts with negatively charged bacterial cell membranes. Apoptosis as the Mode of Cytotoxicity Morphological evaluation of HeLa cells using phase-contrast microscopy (Figure 3a), acridine orange-ethidium bromide staining (Figure 3b) and Hoechst 33342 nuclear staining (Figure 3c) displayed progressive apoptotic features by the SNP@PST. The mechanism of cell death induced by SNP@PST was further confirmed by TUNEL assay wherein, cells displayed a green color, indicating TUNEL positivity upon treatment and control cells were found to be largely TUNEL negative (Figure 3d, e). A similar pattern of apoptosis induction was also observed with A375 (Figure S11a-f), A549 (Figure S12a-f) and SKOV3 (FigureS13a-f) cells by the above mentioned


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assays. Since caspases are the effective mediators of apoptosis, expression of caspases 3, 8, 9, and 2 was estimated. There was a significant (p

Exploration of Biogenic Nano-chemobiotics ... - ACS Publications

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