Silver Nanocluster Embedded Composite ... - ACS Publications

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Silver nanoclusters embedded composite nanoparticles for targeted prodrug delivery in cancer theranostic Amaresh Kumar Sahoo, Upashi Goswami, Deepanjalee Dutta, Subhamoy Banerjee, Arun Chattopadhyay, and Siddhartha Sankar Ghosh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00334 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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ACS Biomaterials Science & Engineering

Silver

nanoclusters

embedded

composite

nanoparticles for targeted prodrug delivery in cancer theranostic Amaresh Kumar Sahoo1, Upashi Goswami1, Deepanjalee Dutta1, Subhamoy Banerjee2, Arun Chattopadhyay 1,3,* and Siddhartha Sankar Ghosh1,2* 1

Centre for Nanotechnology, 2Department of Biosciences and Bioengineering, 3Department of

Chemistry. Indian Institute of Technology Guwahati, Guwahati, Assam, India KEYWORDS: Ag NC, theranostics, prodrug and targeted delivery

ABSTRACT

Cancer therapy with theranostic nanoparticles having the dual properties of concurrent delivery of therapeutics and its tracking, offers huge prospect to overcome the limitations of conventional therapy. Delivery of the nontoxic prodrug, which converts into toxic drug due to the cellular stimuli, offers great deal of scope in the cancer therapy. Paracetamol dimer (PD) generally considered as nontoxic is encapsulated with the fluorescent silver nanoclusters (Ag NCs) embedded composite nanoparticles where it acts as a prodrug. This is possibly converted to toxic metabolite due to elevated reactive oxygen species (ROS), leading to apoptosis mediated cell death. Conjugation of folic acid with these composite NPs offers the credibility of distinguishing 1 ACS Paragon Plus Environment


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between two different cancer cell lines such as HeLa which over expresses folic acid receptors and A549 down regulates its expression, probed by fluorescence intensity of Ag NCs. Importantly, Ag NCs along with PD synergistically induces prodrug mediated targeted cell death at much reduced concentration of silver. Thus, theranostic nanocarriers have been developed offering the dual property of therapy and imaging based on the differential uptake.

INTRODUCTION The latest developments in medical science are facing the challenge of complete cure of cancers along with poor survival outcome of conventional and existing therapies.1-3 Stimuli responsive intracellular conversion of prodrug into therapeutic drug offers great deal of scope in the cancer therapy. Recently, our group has reported the synthesis of nanocomposites of paracetamol dimer (PD) by the reaction of silver4 as well as gold salt5 with paracetamol (para-hydroxy-acetanilide) which is commonly used as an anti-pyretic and analgesic drug. It has been reported that in case of E.coli, paracetamol dimer (PD) acts as a prodrug which gets converted into N-acetyl-pbenzoquinone imine (NAPQI), a potent DNA gyrase inhibitor, by elevated reactive oxygen species (ROS) caused by Ag NPs. The NAPQI causes linearization of the plasmid DNA.4 Akin to DNA gyrase, mammalian cells contain topoisomerase II which is also poisoned by NAPQI leading to apoptosis.6-7 Moreover, there are no reports available till date on adversary activity of PD towards mammalian cells, except the plausibility of the non-covalent interaction with macrophage migration inhibitory factor (MIF).6,8 Thus, to explore its anti-cancer activity the Ag NCs embedded composite NPs were employed to deliver PD into HeLa cells with the anticipation that PD may be converted into NAPQI, resulting in the synergistic effects along with Ag NCs. As recent studies have manifested that Ag NPs and its composite(s) destabilize the

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integrity of the plasma membrane and induces reactive oxygen species (ROS), causing mitochondria dependent apoptosis.9-10 However, higher dose of silver is cytotoxic towards normal cells. This inherent demerit limits its direct use as cancer therapeutic agents because of the unavoidable association of cancer cells with normal cells. Various reports from several laboratories, including ours, have demonstrated that nanocarrier mediated delivery of Ag NPs could reduce the effective concentration of silver.11-13 The nanocarrier offers likelihood of size explicit passive targeting and/or cell specific ligand receptor interaction mediated active targeting - which improves the bioavailability of the therapeutics at the programmed sites and as a consequence lower the effective concentration of drug.14-16Moreover, while active targeting offers the plausibility of improved efficacy of therapeutics owing to its considerable amount of selectivity and specificity as compared to the non-specific delivery which deals with likelihood of uninvited distribution of therapeutics all over the body, thus gains edge over the passive targeting.17Generally, cancer cells over express the receptors for growth factors such as glucose and vitamins (e.g. folic acid) to promote their growth and differentiation.13 Thus, by targeting this specific ligand-receptor interaction one could easily deliver the Ag NPs into predetermined sites. Another aspect is the size of the Ag NPs, smaller the size better is the activity of apoptosis mediated cell death. For instance, Ag NPs particularly size less than 10 nm were always found to have enhanced activities than its bigger counterparts.18-19 In this pursuit, the cytotoxic effect of silver nanoparticles size less than 2 nm, termed as silver nanoclusters, offer huge promises.20 Owing to its critical size, metal nanoclusters do not support the surface plasmon resonance (SPR) which is a general signature of the metal NPs. On the other hand, the continuous energy band of the metal discretize in this size realm, hence exhibits bright fluorescence emission.21 Apart from this, it possesses a large Stokes shifted emission and high photostability which is the inherent



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limitation of organic fluorophores and thus offers the advantages of using it as multi-coloured dye for in vivo imaging.22-23 Herein, we have synthesized fluorescent silver nanoclusters (Ag NCs) by using bovine serum albumin (BSA) which was further converted into targeted composite NPs by using folic acid (FA) conjugated chitosan. Folate plays a very crucial role in the synthesis of nucleotides and cell division.24 It has been found that folate receptors are available in almost all types of cell but cancer cells produce higher numbers of folate receptors as compared to normal cells mainly because of their rapid proliferation. Among different cancer cell lines also there lies a variation in their expression level which can be exploited for selective targeting. For example, HeLa cells (human cervical carcinoma) overexpresses folate receptors as compared with A549 cells (human lung adenocarcinoma).25Thus, folic acid was covalently linked with fluorescent composite NPs (Ag NC- FA-CS NPs) with the keenness that it will facilitate receptor-mediated endocytosis and thus provide a great deal of scope for both therapy as well as detection of the folic acid over expressed cancer cells by using a single module i.e., tumour ‘theranostics’.”Further, these fluorescent composite NPs were employed to deliver PD into the cancer cells to study the synergistic effect of Ag NCs and the PD. The overall idea is projected in the scheme1.


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Scheme 1. Overall representation of the synthesis of the fluorescent Ag NCs and folic acid conjugated composite NPs (Ag NC- FA-CS NPs) for targeted cancer theranostic which induces apoptosis mediated cells death.

MATERIALS AND METHODS Chemicals. Paracetamol, silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from Merck India, Ltd. The 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide 5 ACS Paragon Plus Environment


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hydrochloride (EDC), anhydrous dimethyl sulfoxide (DMSO), N-Hydroxysuccinimide (NHS), chitosan (viscosity averaged molecular weight, Mν,672kDa and degree of deacetylation >75%), folic acid, sodium tripolyphosphate (TPP) were procured from Sigma-Aldrich and used as received. Conjugation of the folic acid and chitosan. Chitosan (CS) was conjugated with folic acid (FA) via formation of the amide bond.26 For that, the folic acid was first activated by EDC and NHydroxysuccinimide, in 20 mL anhydrous DMSO (molar ratio of FA, EDC and NHS was 1:1:0.5) and stirred at room temperature for 2 h. Into this 0.5% (w/v) chitosan in acetic acid solution (0.1% v/v, pH 4.5) was added and stirred at room temperature in the dark for 24 h to complete reaction. The conjugated product of FA-CS was collected by adjusting the pH of the solution to 9.0 by addition of NaOH (10 M), which precipitates the products. Then, the precipitate was repeatedly washed with water to remove DMSO and dried in oven at 65 ⁰C for overnight. Finally, the required amount of FA-CS conjugate was redispersed in acidic water and is used for the synthesis of the composite NPs. Synthesis of the fluorescent silver nanoclusters (Ag NCs). Fluorescent Ag NCs were synthesized based on the previous method with slight modification.27-28 For 20 mL reaction, 2 mL of bovine serum albumin (BSA 96%; 50 mg/mL) was taken in a conical flask and 400 µL of 10 mM Ag NO3 was added into it. Then, 40 µL of a freshly prepared aqueous solution of NaBH4 (10 mM) was added along with the addition of 100 µL of 0.3 M NaOH and incubated in the dark with ice (4 ⁰C) under vigorous stirring for 12 h for complete reduction of the silver to silver nanoclusters. In the basic pH, excess amount of BSA served as an additional reducing agent for complete reduction of silver ions as well as template for NCs. The yield of the formation of the


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NCs completely relies on the reaction environment, minute variation leads to formation of Ag NPs. Synthesis of the fluorescent Ag NCs embedded folic acid-chitosan composite NPs (Ag NC – FA-CS NPs) and paracetamol dimer (PD) loaded composite NPs. 0.5 mL of (0.5% w/v) FACS conjugated product was mixed with aforementioned BSA-Ag NCs with a ratio of 2.5:1 (w/w). Then, 0.2 mg/mL of sodium tripolyphosphate (TPP) was added drop wise for the synthesis of composite NPs (Ag NC- FA CS NPs), by using the ionotropic gelation method, originally developed by Calvo et al.29 For the synthesis of the drug loaded composite NPs, 10 mg/mL pre-synthesized paracetamol dimer (PD) were added into the reaction mixture prior to addition of the TPP (0.2 mg/mL). After the addition of TPP the clear solution turned into milky white indicating the formation of the composite NPs which was collected by centrifugation at 10,000 rpm for 5 min. The PD loading was calculated by probing the absorbance of PD at 320 nm. The loading efficiency was calculated based on the following formula. % of drug loading efficiency of NPs = {(Amount drug added into the NPs – Amount of free drug)/ Amount of drug added into the NPs} ×100 Analytical measurements.UV–Vis spectra and fluorescence spectra were recorded by a Perkin– Elmer lambda 45 spectrophotometer (Perkin–Elmer, Fremont, CA) and Perkin–Elmer fluorescence spectrophotometer (model: LS 55) respectively. Atomic Absorption Spectroscopy. The amount of silver present in the composite NPs was determined by using Atomic absorption spectrophotometer AA 240-Varian Inc. Transmission electron microscopy (TEM).To observe, the nanocarriers and Ag NCs under a high-resolution transmission electron microscope (TEM; JEM 2100; Jeol, Peabody, MA, USA) 7 ACS Paragon Plus Environment


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10 µL of each samples were drop coated onto a carbon coated copper TEM grid followed by air drying. The voltage was fixed at 200 keV during TEM analysis. Selected area electron diffraction (SAED) and energy dispersive X-ray (EDX) spectroscopy were carried out using the same instrument and for same samples. Dynamic light scattering study.The zeta potential and hydrodynamic diameter of the composite NPs and drug loaded composite NPs were measured by dynamic light scattering-based analysis (Malvern Zetasizer Nano ZS). Fourier transform infrared (FTIR) spectroscopy. Lyophilized samples were mixed with KBr to make the pellets, and characterized by FTIR spectroscopy (Perkin-Elmer) in the range 400−4000 cm−1. Drug release study. For the release study, PD loaded composite NPs were redispersed into two different buffers such as PBS (pH 7.4) and acetate buffer (pH 4.5) and incubated at 37 ⁰C. After specific time intervals the sample were withdrawn followed by centrifugation at 10,000 rpm for 10 min. The absorbance of supernatant was probed at 320 to find out the amount of release PD. Epi fluorescence microscopy. To visualize the cells after treatment with Ag NCs and composite NPs epi fluorescence microscopy was done in HeLa cell line. 1 × 105 cells were seeded into 6 wells plate for overnight followed by addition of the Ag NCs-CS NPs. Same microscope was also used to observed acridine orange and ethidium bromide (AO/EtBr) double staining after treatment of the IC50 dose of drug loaded composite NPs for 24 h. Also for lysosomal staining assay the same microscope was used after 1h staining of the lysosomes of 3h composite NPs treated HeLa cells.


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Cell culture. HeLa cells (human cervical carcinoma) and A549 cells were procured from National Center for Cell Sciences (NCCS), Pune, India, and cultivated in Dulbecco’s Modified Eagle’s Medium supplemented with L-glutamine (4 mM), penicillin (50 units/mL), streptomycin (50 mg/mL), and 10% (v/ v) foetal bovine serum (obtained from PAA Laboratories, Austria) in 5% CO2 humidified incubator at 37 ⁰C. Cell viability assay. To perform MTT based cell viability assay, 1×104 HeLa and A549 cells/well were seeded in a 96- well microtiter plate and grown overnight by maintaining the same medium and condition as described previously. Then, different amount composite NPs (Ag NC- FA CS NPs) and PD loaded composite NPs were added into it and incubated for 24 h prior to addition of MTT (3-(4,5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) dye to find out the number of viable cells. Viable cells reduce MTT into colour formazan which has absorbance at 550 nm. The % of cell viability was calculated as below. While absorbance at 690 nm is due to background interference. %viable cells = [(A550- A690) of composite NP treated cells / (A550- A690) of untreated cells]×100 Cell cycle analysis. Propidium iodide (PI) based cell cycle analysis was carried out in flow cytometer (FACS) which measures the DNA content of the cells at different stages of cell division. For that, 1× 105 cells were seeded into 6 well plates and incubated for 12 h under the same conditions as mentioned above. Then, the cells were treated with drug loaded composite NPs for 24 h along with their respective controls. Cells were collected after trypsinization and were fixed by addition of 1 mL of cold 70% ethanol, and kept it at -20 ⁰C for 1 h. Finally, the fixed cells were collected by centrifugation and redispersed in PBS followed by RNase A (100 µg/mL) treatment at 37 ⁰C for 1 h. Prior to doing analysis, the cells were incubated in dark with 9 ACS Paragon Plus Environment


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PI (30 µg/mL) for 30 min and flow cytometry assay FACS Calibur (BD Biosciences, NJ) was carried out . The PI fluorescence data were collected in FL2 channel (band pass filter, 585/42 nm) which corresponds to red emission for 15,000 cells in each sample and data were analyzed by the CellQuest pro. ROS measurement. Reactive oxygen species (ROS) generation was determined by fluorescence activated cells sorter (FACS) using 2, 7-dichlorofluoresceindiacetate (DCFH-DA; SigmaAldrich, USA) staining method. For this, 1×105 cells were seeded into 6 -well plates and incubated for 12 h. The cells were then treated with IC50 dose of drug loaded composite NPs with their respective controls for 3 h and 10 µL of 1 mM DCFH-DA was added and kept at 37 ⁰C for 10 min. Then, the cells were collected after trypsinization and suspended in 1 mL of DMEM before analyzed in a flow cytometer (FacsCalibur, BD Biosciences, NJ). The emission of the DCF was collected in FL1-H channel (530/30 nm) which corresponds to green emission. The fluorescence data were recorded with the CellQuest program (BD Biosciences) for 15, 000 cells in each sample. Caspase 3-assay.To probe apoptotic mediated cell death, FACS based caspase-3 assay was performed. For that, 1×105 cells were seeded into 6 -well plates and kept for overnight incubation. Cells were treated with IC50 dose of drug loaded composite NPs along with their respective controls, and incubated for another 24 h. After incubation the cells were collected in PBS and were fixed with 0.1% (v/v) of formaldehyde for 15 min. The fixed cells were harvested by centrifugation at 650 rcf, followed by incubation in dark with 1 mL of membrane permealization solution (0.5% of Tween 20 in PBS) for 20 min in dark. Cells were washed twice with PBS to remove the surfactant prior to the addition of phycoerythrin (PE) conjugated Rabbit Anti –Active Caspase-3 (BD Biosciences) for 30 min in dark. The fluorescence data were 10 ACS Paragon Plus Environment

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recorded for 15, 000 cell in FL2 channel (band pass filter, 585/42 nm) of FACS and analyzed by CellQuest program (BD Biosciences). Statistical test. Statistical significance of the folic acid receptors (FR) expression level for two different cancer cell line such as HeLa and A549 were done by Student’s t-test. The values are represented as mean ± SD of results from three individual experiments. Results of MTT assays were statistically analyzed by two-way analysis of variance (ANOVA). The values are represented as mean ± SD of results from three individual experiments. The ANOVA test with pairwise comparison was carried out to find out the statistical significance for each concentration. The

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Silver Nanocluster Embedded Composite ... - ACS Publications

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