Glutathione conjugated superparamagnetic Fe3O4-Au core shell nanoparticles for pH controlled release of DOX

Materials Science & Engineering C 100 (2019) 453–465

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Glutathione conjugated superparamagnetic Fe3O4-Au core shell nanoparticles for pH controlled release of DOX Nimisha Singh1, Jyotsnamayee Nayak1, Suban K. Sahoo, Rajender Kumar

T

⁎

Department of Applied Chemistry, S.V. National Institute of Technology, Surat 395007, Gujarat, India

ARTICLE INFO

ABSTRACT

Keywords: Superparamagnetic Core shell Cytotoxicity Cellular uptake Cancer theranostics

Glutathione (GSH) coated gold‑iron oxide core shell nanoparticles (GS-Au-Fe3O4) were prepared by coating glutathione shell on nanoparticles to reduce the dose dependent behaviour of anticancer drug, doxorubicin (DOX). The resultant nanoparticles were characterized using XPS, FTIR, HR-TEM with STEM profile to analyze the GSH shell over the surface. The GS-Au-Fe3O4 nanoparticles were loaded with DOX and maximum drug entrapment capacity of 54% was observed in 48 h. In-vitro drug release were evaluated using UV–vis and Fluorescence spectroscopy. The results show that drug release is facilitated under acidic conditions as well as by extracellular glutathione spiking. Cytotoxicity and cellular uptake was studied on HeLa cells where GS-Au-Fe3O4 nanoparticles lead to significantly higher uptake of DOX as compared to free drug. The use of glutathione conjugation thus act as an efficient drug delivery vehicle which requires significantly low concentration of GSAu-Fe3O4 nanoparticles for DOX release besides triggering drug release by using redox active GSH.

1. Introduction One in every four person suffering from cancer dies in United States due to unavailability of effective cancer cure [1]. Cancer cells are very different in terms of morphology and cell growth, different intracellular interaction within the cells and with extracellular matrix in comparison with normal cells [2,3]. In recent time, significant attentions have been paid to develop the nanocarrier for increasing the potency and efficacy of the cancer drug delivery and theranostics. The designed nanoparticles to be used effectively in cancer theranostics require biodegradability and friendly interaction between the drug and nanoparticles besides lower toxicity towards healthy cells. In this class, gold (Au) and iron oxide (Fe3O4) based nanoparticles have been most extensively studied. The Au nanoparticles have high chemical stability and ease of surface functionalization with several functional groups. Au nanoparticles based drug delivery vehicles have been reported for the delivery of several drugs like paclitaxel [4], ciprofloxacin [5], Doxorubicin (DOX) [6–8], curcumin [9], and chloroquine [10]. Also there are reports that Au nanoparticles helps in overcoming the multidrug resistance thus making them effective nanocarriers [11,12]. Fe3O4 based nanocarrier commonly called as SPIONs (superparamagnetic iron oxide nanoparticles) are also studied extensively for

cancer treatment, Magnetic resonance imaging and drug delivery [13,14]. The main advantage of developing magnetic core for the cancer therapy are that it can be easily manipulated/fabricated and guided to the targeted sites using external magnetic field, visualised by magnetic resonance imaging and can be used for hyperthermia for cancer therapy [15]. Particularly, SPIONs are biocompatible and thus are successfully employed for in-vivo applications as the ions produced by SPIONs after degradation can be easily assimilated by body's physiological mechanism [16]. Thus, several magnetic fluids carrying anticancer drugs such as epidoxorubcin and mitotoxantrone are potentially used for the locoregional treatment of the cancer in phase I clinical trials [17]. However, SPIONS generally have poor stability because of its tendency to get oxidised Although, these limitations are reduced to certain extent by developing the functional coatings on the SPIONs with polymers like PEG, PEI etc. [18,19], but these coatings create complex architecture and chemistry leading to the unpredictable interactions of these carriers within intracellular environment and causing long term toxicity. In order to overcome these limitations, the core shell nanoparticles with Fe3O4 core and Au shell have emerged as excellent choice. The coating of Au nanoparticle not only provides the various possibility of surface functionality for increasing biodegradability but also overcomes

Corresponding author. E-mail address: [email protected] (R. Kumar). 1 Contribution of Nimisha Singh and Jyotsnamayee Nayak is major and equal. ⁎

https://doi.org/10.1016/j.msec.2019.03.031 Received 27 November 2018; Received in revised form 12 February 2019; Accepted 9 March 2019 Available online 10 March 2019 0928-4931/ © 2019 Published by Elsevier B.V.

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the limitation of surface oxidisability of Fe3O4 to Fe2O3 and minimise the particle aggregation [20]. The functionalization of the core shell nanoparticles with the thiol containing ligands is one of the best available choices due to the very strong interaction between sulphur and Au creating the redox sensitive carriers [21,22]. Among various thiols, glutathione (GSH, γ-glutamyl-cysteinyl-glycine), is one of the most important tripeptide which is directly related to the cancerous cell environment besides affecting almost every physiological function in body [23]. GSH have been reported to be involved in the many cellular functions, protection of healthy cells and plays a major role in regulating various physiological mechanisms of cells. GSH, being one of the most important redox species present in the human body is known to have highest potential to trigger the release of anticancer drugs from carriers [24,25]. The addition of free GSH have been reported to increase the efficacy of drug release and cellular mortality [25,26]. Despite of this fact, there is a scarcity of scientific research describing GSH coated nanoparticles for anticancer drug release. Only few reports describes the conjugation of GSH to the Au shell for the DOX release for anticancer treatment [27,28]. Nevertheless, none of the report describes the GSH coated super magnetic Fe3O4-Au core shell nanoparticles for the DOX release at tumour sight. Thus, in this report we mimic the GSH environment of the cancerous cells by conjugating the tripeptide GSH on to the magnetic core shell particles (Fe3O4-Au) using strong covalent bond like bonding between –SH group of the GSH and Au shell of Fe3O4-Au. The GSH conjugated carriers lead to effective and controlled release of the anticancer drug to the cancer cells. Besides, the designed nanoparticles facilitate the pH specific drug release to the target cells in addition to redox triggered drug release. The DOX is a first line of defence in cancer treatment and have several side effects on healthy cells. The presence of conjugated GSH on the shell may help to reduce the side effects of DOX in addition to controlled release and thus providing higher anti-tumour efficacy [29]. The designed nanoparticles thus incorporate several features in one carrier like, magnetic properties useful for magnetic hyperthermia, surface plasmon resonance, biocompatibility, pH sensitive drug release, Redox active antitumor efficacy and healthy cell friendly environment. The designed nanoparticles have the increasing ability to cause cancerous cell death and apoptosis by natural mimicry mechanism. Not only this, the designed nanoparticles are safe to the normal cells as GSH coated nanoparticles are nontoxic towards the normal cells. In summary, stealth magnetic core shell nanoparticles containing Fe3O4 core and first shell as Au and second shell of tripeptide GSH was prepared and characterized using XPS,HR-TEM, XRD, ATR-FTIR etc., and was loaded with DOX. The resulting drug loaded nanoparticles were then analyzed for its cytotoxicity using MTT assay and apoptosis studies using flow cytometry. The designed drug delivery nanoparticles proved to be of high efficacy and brought significant mortality in cancer cells when compared with the control DOX. According to best of our knowledge this is the first report where stealth magnetic nanoparticles with GSH shells have been designed for DOX delivery in to the cancer cells with excellent efficacy and efficiency.

and were used without further purification. All the experiments were performed using deionised water. 2.2. Preparation of Fe3O4 nanoparticles A facile chemical co-precipitation method [30,31]was adopted for the preparation of Iron Oxide (Fe3O4) nanoparticles (IONPs). Iron salts, FeCl3 (3.24 g) and FeSO4·7H2O (2.78 g) were initially mixed and dissolved in 1.2 mM HCl (200 mL) solution using ultrasonication for uniform dispersion. NaOH (1.25 M, 300 mL) was added dropwise under vigorous shaking at 60 °C to obtain black precipitates of Fe3O4. The precipitates were then washed with deionised water till the solution reach at pH ~ 7, to ensure the complete removal of unreacted chemicals. 2.3. Preparation of gold coated Iron oxidecore shell (Fe3O4-Au) nanoparticles Au shell were coated on IONPs using reported method [32], where Au (III) is reduced to Au by using sodium citrate as reducing and capping agent, much similar to the Turkewich method [33]. Briefly, HAuCl4 (20 mL, 0.1%) was added into 250 mL round bottom flask containing 40 mL DI water. The solution was refluxed and Fe3O4 (28 mg in 10 mL) were then added to the solution. The mixture was allowed to boil for 30–45 min. Sodium citrate (1 wt%, 4 mL) was then rapidly added to the boiling mixture under continuous stirring. Addition of the citrate leads to the colour change of the solution from brown to red. The solution was then allowed to boil for more 15 min and was stirred further until the solution comes to room temperature. 2.4. Functionalization of gold coated IONPs (Fe3O4-Au) with glutathione (GS) Functionalization of GSH over Fe3O4-Au core shell nanoparticles was done using the self-assembly method, where 300 μL of GSH (5 mM) was added in to 1.0 mL of Fe3O4-Au core shell nanoparticles solution and allowed to self-assemble for 4 h at room temperature in dark and air tight conditions [34]. GSH was rapidly coated on the surface of Fe3O4-Au core shell nanoparticles as sulphur forms strong dative (AueS) bonds with Au shell that enhance the reactivity of the nanoparticles towards GSH. 2.5. Drug loading and kinetics To load DOX in to the as prepared GS-Au-Fe3O4 core shell nanoparticles, the nanoparticles (10 mg/mL) were in the distilled water and to it were added DOX (0.6 mg/mL) with continuous stirring for 48 h at room temperature under nitrogen environment in dark. DOX loaded nanoparticles were then magnetically separated and washed twice with PBS buffer and deionised water to remove the superficially adsorbed DOX. The removal of superficial DOX was verified by UV–vis spectroscopy. DOX loaded nanoparticles were then dispersed in PBS buffer (10 mM, pH 7.4) and drug release studies were carried out using double diffusion method. The donor cell was filled with drug loaded nanoparticles (1 mg/mL) and the receiver chamber was filled with PBS buffer of the required pH. The cells were separated by a dialysis membrane having a pore size of 2.4 nm and a molecular weight cut off between 12,000 and 14,000 Da. The cell was stirred continuously at 37 °C to monitor the drug release. Loaded drug can diffuse through the cell easily but nanoparticles could not cross the membrane. This was confirmed using the fluorescence spectroscopy, where buffer from the receiver chamber were continuously analyzed at different time interval. The extracted samples were analyzed by measuring fluorescence intensity at λex = 470 nm and λem = 590 nm with Ex. slit (nm) = 5 and Em slit (nm) = 10 having a scan rate of 600 nm/min. A standard

2. Materials and methods 2.1. Chemicals and reagents Chloroauric acid (HAuCl4), Glutathione (GSH) and Doxorubicin. HCl were purchased from Sigma Aldrich (St. Louis, MO, USA). FITC Annexin V Kit, Propidium Iodide, 10× Annexin V Binding Buffer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2 used for biochemical assay and were purchased from Sigma AldrichTri‑sodium citrate, Iron (III) chloride (FeCl3), Iron (II) Sulphate (FeSO4·7H2O), HCl (0.1 N) and NaOH (0.1 M) to maintain the pH, were purchased from Finar (India) 454

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calibration curve was plotted to quantify the loading kinetics.

ATR-FTIR spectra were carried out in Shimadzu Infinity 1S-Wl spectrophotometer with resolution of 4 cm−1in the range 400–4000 cm−1 and a total scan of 45 per measurement. HR-TEM with EDAX measurements were performed on Tecnai G2 20 (FEI) S-Twin, 200 kV high resolution transmission electron microscope. DLS and Zeta potential measurements were analyzed using Malvern Zeta size NanoZS90 (Malvern, UK) to calculate the hydrodynamic diameter of the particles and charge developed on the nanoparticles. X-ray photoelectron spectroscopy was done using micron ESCA (Electron Spectroscope for Chemical Analysis) from Oxford Instrument Germany.

2.6. Study of effect of extracellular spiked glutathione on DOX release GSH has been reported to facilitate the DOX release from nanoparticles at enhanced pace resulting in the faster drug release kinetics [35,36]. In order to verify this on as prepared GS-Au-Fe3O4 nanoparticles, drug release studies were carried out in presence and absence of GSH. Briefly DOX loaded nanoparticles were dispersed in PBS buffer (10 mM, 7.4 pH) in the donor chamber and to it was added 2 mL solution of GSH of different concentrations(2 mM, 5 mM,10 mM & 20 mM). Receiving chamber was filled with PBS buffer (10 mM) and maintained at two different pH 7.4 and 5.0.The cell was then stirred, maintained at 37 °C and analyzed at different time interval using Fluorescence spectroscopy for drug release kinetics.

3. Results and discussion 3.1. Preparation of GSH conjugated magnetic (GS-Au-Fe3O4) nanoparticles

2.7. MTT and apoptosis assay

GS-Au-Fe3O4 nanoparticles were prepared as per Scheme 1. The optical properties of prepared nanoparticles were studied using UV–visible spectroscopy. The Fe3O4 nanoparticles can be prepared and coated easily with oxidation resistant shell with enhanced functionalization potential. Therefore, gold shell were coated on IONPS by following reduction method whereby the Au3+ was reduced to Au+ using citrate as reducing agent as depicted in the reaction (1).

HeLA cells were used for the biological assays and were first propagated till the density reached to 80% confluence. Medium was then aspirated and cells were washed at room temperature with 1× PBS. 2 mL of TrypLE was then added and the cells were monitored for detachments. To the culture, 3 mL of prepared complete medium was then added and aspirated by gentle pipetting. It was then centrifuged and supernatant was aspirated and discarded, subjecting cells to 1 mL of media and further cell count. After count cells were split (approx 2 × 105 cells in each well of 12 well plates (Annexin V assay)) inoculants were further cultured at 37 °C in 5% CO2 humidified incubator. Medium was changed each day until cells reached 70% confluency. Test compounds were exposed to the cells for 24 h duration in different concentration as per the requirements. The medium was aspirated and washed with 1XPBS. 0.5 mL of TrypLE was added to each well and cells were monitored for detachments. 1.5 mL media were added and cells were collected in 2 mL microfuge tubes. Centrifuged at low rpm and supernatant were discarded. After 24 h, the cell uptake was analyzed under phase contrast microscope and images were captured. Staining procedure was then performed where cells were washed twice with cold PBS in 2 mL microfuge tubes and then re-suspended in 1× binding buffer at a concentration of 1 × 106 cells/mL. After that 100 μL of the solution (1 × 105 cells) was transferred to a 5 mL culture tube where 5 μL of FITC Annexin V and 5 μL PI was added. The cells were then gently vortexed incubated for 15 min at RT (25 °C) in the dark. Binding buffer, 400 μL was then added to each tube. It was then analyzed using flow cytometry within 1 h. For MTT assay the HeLa cells were cultured in 96 well plates with confluence level of 80%. To analyze the nanoparticles at different concentration, the culture media was replaced with the fresh media and incubated at various time intervals. Afterwards, media having the nanoparticles was eliminated from the culture wells and washed twice with the buffer to ensure the complete removal of unbound nanoparticles in the culture. The culture containing 100 μL of media and 20 μL of MTT solution in PBS was incubated for 3 h at 37 °C. The culture was then washed and aspired to remove the unreacted MTT. The absorbance was then measured at 630 nm after removing all water insoluble compounds like formazon by adding 100 μL DMSO.

AuCl3 + 2e

AuCl + 2Cl

(1)

−

The 2e generated from the oxidation of citrate leads to the reduction of Auric salt to Aurus salt which further followed the disproportionation reaction of Aurous salt to neutral Au shell [37] as shown in reaction (2).

3AuCl 2Au0 + AuCl3

(2)

The solution of Fe3O4 exhibits brown colour, which on coating with Au (Fe3O4-Au) turns to red and on further functionalised with GSH turns purple (GS-Au-Fe3O4) as shown in Scheme 1. The UV–vis spectra of Fe3O4-Au core shell nanoparticles shows surface plasmon resonance peak at 530 nm (Fig. 1A). It is likely due to the surface plasmon band which is highly dependent on size of the particles, as the collective oscillation of the free electron is sensitive to the particles size. So as the diameter increase the energy required to collectively excite the motion of the surface plasmon electrons decreases [38]. Thus, the change in band gap results in colour change from red to purple as shown in Fig. 1B. In order to study the effect of pH on shell formation, GSH (5 mM) was made to react with Fe3O4-Au core shell nanoparticles at different pH (pH 2.0 to 5.0). On decreasing the pH, the interparticle distance reduces resulting in the broadening of the peak at 530 nm as shown in the Fig. 1B. At a very low pH (pH 2–3), an extra small peak appears at around 650 nm suggesting the aggregation in acidic medium which flattened at pH = 2.0 [39]. To confirm the stability, charge and bioavailability of the prepared nanoparticles, DLS, Zeta potential and polydispersity index were measured and is shown in Table 1. It was observed that on reacting GSH with Fe3O4-Au core shell nanoparticles, the hydrodynamic diameter decreases which is attributed to the formation of tight self-assembled monolayers of GSH on Au shell by replacing the loosely held water and citrate molecules. Thus, the GSH functionalization not only reduced the diameter making the particles stable but also reduces the PDI values in the appropriate range for biomedical application [40]. The DLS measurements were comparable with the HR-TEM results (Fig. 2) which shows that upon GSH functionalization, the morphology of the nanoparticles changed and appeared to be quite stable. In order to confirm the formation of GSH shell and its constitution, STEM-EDX (Scanning TEM-energy dispersive X-ray spectroscopy) line profiling was done as shown in Fig. 3 to get the elemental composition and features of the nanoparticles. During the scan, a large number of particles were studied and within each particle both Fe and

2.8. Characterization techniques UV–visible spectroscopic measurements were performed using Cary 50 Varian UV–vis spectrophotometer and absorption spectra were recorded within the range of 200–800 nm at room temperature with quartz cuvette of 1 cm path length. Fluorescence spectroscopy were recorded using Cary Eclipse Fluorescent spectrophotometer with fluorescence intensity at λex = 470 nm and λem = 590 nm with Ex. slit (nm) = 5 and Em. slit (nm) = 10, having a scan rate of 600 nm/min. 455

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Scheme 1. Schematic Representation of the synthesis step showing A. Fe3O4 nanoparticles B. coating of gold (Au) shell over the magnetic core forming Fe3O4-Au nanoparticles, C. Conjugation of GSH on the Au surface to stabilize the nanoparticle and allow the conjugation of D. Doxorubicin drug onto the nanoparticle to act as a nanocarriers where the release of the drug was studied in E. presence of GSH and F. in absence of GSH.

Au were found (Fig. 3C and D) in addition to presence of C, N,O and S (Fig. 3E–H) which indicates the presence of GSH shell. The evidence also suggests the functionalization of GSH on the Au surface which was further supported by the EDAX mapping (Fig. 4) and observing individual elements on position count spectra. Additionally, zeta potential (ζ) of the prepared nanoparticles decreases upon formation of GSH shell on the Fe3O4-Au nanoparticles (Table 1). According to Chow and Zukoski, the increase in the concentration of citrate increases the zeta potential of particles [41]. However, ζ is affected by several factors such as surface morphology, chemical composition and electrical conductivity of surface. Thus, Fe3O4-Au nanoparticles, when coated with GSH show decrease in the potential values to −19.2 mV as it introduces a new group –SH on the nanoparticle surface that exchanges the citrate present on the Au surfaces [42,43]. Thus, decrease in ζ further confirms the successful bonding of GSH to the Au surface. Although ζ is generally independent

Table 1 Showing the hydrodynamic diameter and zeta potential of the prepared nanoparticles. Sr.·no 1 2

Samples Fe3O4-Au GS-Au-Fe3O4

Hydrodynamic diameter

Zeta potential

PDI

123.4 ± 4.7 70.8 ± 3.1

−27.6 ± 2.6 −19.2 ± 1.4

0.723 ± 0.1 0.42 ± 0.1

of the van der wall forces nevertheless, it is influenced by the Hamaker constant [44], which states that even the low zeta potential values ( ± 15 mV) can exhibit good stability as exhibited by colloidal silica [45]. Since GSH offers weak van der Waal forces, it produces stable nanoparticles despite of having low ζ values as weakening of the van der wall forces arise from low Hamaker constant leading to reduced ζ. Thus, functionalization of GSH over the nanoparticles may enhance the stability of the colloidal dispersion and can be of potential application

Fig. 1. UV–vis spectra showing A. core shell Fe3O4-Au nanoparticles and B. Fe3O4-Au with Glutathione at different pH (GS-Au-Fe3O4). 456

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Fig. 2. HRTEM images of the prepared nanoparticles; A-B. Fe3O4-Au nanoparticles and C-D. GSH functionalised nanoparticles (GS-Au-Fe3O4).

Fig. 3. STEM-EDX Line profile of glutathione conjugated Fe3O4-Au nanoparticles (GS-Au-Fe3O4).

457

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Fig. 4. EDX spectra and mapping showing the elemental composition of GS-Au-Fe3O4 nanoparticles.

as anticancer drug carrier because of its efficient biocompatibility. To further confirm the conjugation of GSH onto the surface of Fe3O4-Au nanoparticles, ATR-FTIR measurements were performed as shown in the Fig. 5. Main features of GSH, that ensure the functionalization on nanoparticles surface observed are the peptidic bond at 3460 cm−1 (–NH stretching), a weak thiol band, SeH at 2688 cm−1 and CeS stretching vibrations in region of 650–700 cm−1 [46]. It was observed that, on adding DOX, the NeH band of GSH was shifted and a broad peak was observed at 3142 cm−1. This might be due to the introduction of DOX ring structure leading to (CeH, ring) vibration [47]. A weak carboxylic peak was also observed at 1368 cm−1 of CeOeH bending which was unaffected by DOX addition. An additional broad peak was observed at 1590 cm−1 of NH bending due to the conjugation of DOX on the surface [48].

The nature of bonding was then analyzed in detail using XPS where each element peak was deconvoluted to understand the bonding between the conjugated molecules. XPS measurements are highly sensitive and provides specific information about the chemical constitution of the species of interest. It was thus performed on the prepared nanoparticles to ensure the bonding of GSH onto the surface of the Fe3O4Au nanoparticles and also the loading of DOX molecule on the nanoparticles. The XPS elemental composition of prepared core shell nanoparticles (Table 2) demonstrates the successful coating of Au onto the surface of Fe3O4 nanoparticles. Presence of Sulphur and Nitrogen in the GS-Au-Fe3O4 nanoparticles and its absence in the Fe3O4 and Fe3O4-Au nanoparticles confirms the presence of GSH shell. The XPS survey scan and accompanying deconvoluted XPS peaks for as prepared nanoparticles are shown in Figs. 6 and 7 which reveals the complete chemical constitution. The binding energy of deconvoluted Fe peaks were observed at 710.2 eV and 723.9 eV because of Fe 2p3/2 and Fe 2p1/2 which are the characteristic peaks of core levels spectra of Fe3O4 nanoparticles (Fig. 6A). Also, a relatively small peak was observed at around 59 eV as shown in Fig. 6A of Fe 3p that confirms the oxidation state of iron oxide [49,50]. Additionally, for Fe3O4 nanoparticles, three types of O 1s peaks were observed at 530.8 eV, 531.5 eV [49], and 532.5 eV. The formation of Au shell on Fe3O4 shows a minor shift in Fe peak towards Table 2 Showing the XPS elemental composition and percentage of the prepared nanoparticles. Sr.·no

1. 2. 3. 4.

Fig. 5. Infrared spectra showing the conjugation of Glutathione and DOX drug onto the surface of Fe3O4-Au nanoparticles. 458

Samples

Fe3O4 Fe3O4-Au GS-Au-Fe3O4 Dox-GS-Fe3O4-Au

Elemental composition C

N

O

S

Fe

Au

38.87 30.75 35.75 52.19

– – 1.80 3.96

45.96 39.02 44.28 26.40

– – 1.05 4.12

15.17 28.28 15.04 11.67

– 1.94 1.82 1.6

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Fig. 6. XPS spectra showing the survey and deconvoluted spectra of A. Fe3O4 nanoparticles and B. Fe3O4-Au nanoparticles.

higher binding energies to 726.2 eV and 712.9 eV. The Au shell coating on Fe3O4 serves as a stable coating to prevent oxidation to maghemite or haematite [51,52].The deconvoluted XPS spectra of Au shows two major peaks at 89.8 eV and 86.0 eV with higher intensities as shown in Fig. 6B. These peaks are attributed to the nonoxidised Au nanoparticles arising from 4f7/2 and 4f5/2 spin-orbit components. Peak at 89.8 eV corresponds to the lower energy 4f7/2 spin orbit component and peak observed at around 95.4 eV and 88.5 eV is attributed to Au 4f5/2 showing the presence of metallic Au [53]. Additionally, a well-defined peak that appears at 86.0 eV may arises due to the ionic Au species [54] as shown in the inset of Fig. 6B. The GSH bonding with the Au shell shows the peak for sulphur and nitrogen in XPS survey scan (Fig. 7) while XPS survey scan of nanoparticles without GSH coating shows absence of S or N peak (Fig.6).

Further deconvolution analysis of the Nitrogen and Carbon peaks of GSAu-Fe3O4 nanoparticle shows the presence of CeN and NeH/NH2 peaks (Fig. 7) which were not observed in XPS spectra of Fe3O4 and Fe3O4-Au nanoparticles (Fig. 6) confirming the GSH functionalization of Fe3O4Au nanoparticles. The deconvoluted XPS spectra of Carbon in GSH coated nanoparticles (Fig. 7B) shows three peaks with binding energies at 286.5 eV (CeN/CeC), 287.6 eV (C]O) and 289.8 eV (OeC]O). The GSH binding to the shell of the nanoparticles via AueS strong dative bonding was further confirmed on observing the deconvoluted XPS spectra of S which shows the peak at binding energy of 162.1 eV (data not shown). The deconvolution of nitrogen (Fig. 7C) shows two major peaks at 400.6 eV and 401.6 eV that arises due to the presence of NH2 and NH groups contributed by GSH [55]. This all confirms the successful

Fig. 7. A. XPS survey Scan and (B-C) deconvoluted spectra of Glutathione functionalised nanoparticles (GS-Au-Fe3O4) showing types of bonding present in Carbon (B) and Nitrogen (C) containing groups. 459

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Fig. 8. Deconvoluted Spectra of DOX loaded Glutathione functionalised (Glu-Fe3O4-Au) nanoparticles showing types of bonding present in A. Carbon and B. Nitrogen containing groups.

functionalization of GSH onto the surface of Fe3O4-Au nanoparticles. XPS spectra were also recorded for DOX loaded nanoparticles to ensure the successful drug loading and decipher the interactions upon loading. DOX has the complex structure and consist of many rings and variety of functional groups that may interact/conjugate with the GSH. Thus, deconvoluted peak of carbon helps to analyze the bonding/interaction of DOX with GSH shell as shown in Fig. 8A. It is observed that the C 1s peak for C]O and OeC]O were significantly shifted towards the lower binding energy when compared with similar peak in nanoparticles without DOX (Fig. 7A) which implies a significant interaction between DOX and GSH coated nanoparticles. The DOX loaded nanoparticles shows additional peak at 283.3 eV which can be attributed to the CeNH2/CeH groups. Other C1s peaks for DOX loaded nanoparticles were observed at 284.0 eV, 285.1 eV and 287.O eV for C]C, CeO and C]O respectively. The atomic percentage of the C]O groups has been found to increase significantly from 33% to 51% upon DOX loading (Figs. 7B and 8A). The source for such high increase in the C]O percentage is only possible with the DOX as the drug is rich in the quinone groups. The deconvoluted N1s spectra of DOX loaded nanoparticles (Fig. 8B) shows three peaks at B.E of at 398.6 eV, 399.7 eV and 400.6 eV which were attributed to the NeCH3, NHeR and NH2eR functional groups respectively [56] compared to the two N1s peak for the GSH coated nanoparticles without DOX (Fig. 7C). The presence of three different N1s peaks with DOX loaded nanoparticles shows significant chemical interactions between the GSH shell and DOX. Thus, deconvoluted spectra of C and N of DOX loaded nanoparticles give further evidence for coherent interaction/conjugation of the drug to the GSH coated nanoparticles (GS-Au-Fe3O4). This is further supported with the EDX results obtained from HRTEM where the atomic percent for N have been found to increase from 9.42 to 9.53 after DOX loading onto the nanoparticles. In-vitro Drug loading was analyzed using UV–visible spectroscopy to evaluate the adsorption of drug onto the surface of nanoparticles. DOX is used as an anticancer model drug to check the loading and releasing behaviour as described in Section 2.5. DOX can easily incorporate in to the nanoparticles as shown by the adsorption study using UV–vis spectroscopy (Fig. 9A) due to the electrostatic interaction with the positively charged amino and carbonyl groups of DOX with the positively charged amino and negatively charge carboxylic group of GSH. Additionally, it has been reported that drug molecules possessing aromatic rings can be efficiently loaded on the nanoparticle surface utilising the delocalised π electrons via hydrophobic interaction and π-π stacking [57].

From the spectra (Fig. 9A), it is observed that the absorbance intensity of DOX after loading in to nanoparticles decreases compared to free DOX (peak position of λmax = 490 nm) as a reference parameter. The decrease in intensity is continuous with increase in loading time. The percent loading of DOX was calculated from the calibration curve obtained from the UV–vis spectroscopy with different concentration of DOX loading as shown in Fig. 9B. Drug loading efficiency was calculated at different interval of time ranging from 12 to 48 h. Maximum drug loading capacity or entrapment efficiency (EE) of 54% was calculated after 48 h of drug loading using eq. 3. On the other hand, the lower adsorption time of 12 and 24 h shows low loading capacity of 27% and 31% respectively. The drug loading efficiency was found to be linear with time of loading with good R2 values (R2 = 0.99). Thus, 48 h drug loading time was taken as standard time for all loading experiments.

%EE =

Concentration (Drug added–Free unentrapped Drug) × 100 Drug Added (3)

In-vitro Drug Release were measured using Fluorescence spectroscopy under physiological conditions at 37 °C by dialysing DOX loaded Glu-Fe3O4-Au nanoparticles. DOX loaded nanoparticles were dispersed in PBS buffer (10 mM, pH 7.4) and poured into the dialysis bag as a donor chamber. After that, 2 mL of the medium from the receiving chamber was withdrawn at each interval of time as described in the Section 2.5 to check the release kinetics of the drug (Fig. 10), and added back into the medium to maintain the concentration of the total volume before taking next measurement. The profile for drug release exhibited various changes when observed at different pH (2.5, 3.5, 5.0 and 7.4) and shows the maximum DOX release at pH 3.5 as shown in Fig. 10B. It is observed that, the release of DOX was much faster under acidic condition when compared with physiological pH 7.4. This may be due to the breaking of the AueS linkages at acidic pH which facilitate the drug release, as AueS bond are pH sensitive [58]. However, since the drug release difference is not much at pH of 2.5 and 3.5 it can be concluded that the breakage of AueS bond is not extensive but a slow process which leads to much less difference in amount of drug released at two increasingly acidic pH of 2.5 and 3.5. On the other hand decrease in acidity leads to significant decrease in amount of drug release as can be observed from Fig. 10C and D showing drug release profile at increasing pH. Beside, other factor contributing towards higher drug release at acidic pH, may be the reduction in the π-π stacking interaction of DOX 460

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Fig. 9. UV–vis spectra showing the A. successive loading of DOX in to GS- Au-Fe3O4 nanoparticles at different time interval and B. Calibration curve obtained at room temperature.

molecules with decrease in pH and thus facilitating the drug release under acidic pH [59,60]. Since the pH of the tumour cells is acidic, present designed model offers a suitable platform for DOX release to the tumour cells under controlled conditions of pH. It has been reported that extracellular addition of GSH significantly increases the release of DOX from nanoparticles [61]. The extracellular pH and the level of GSH within the normal tissues are 7.4 and 10 μM. Nonetheless, inside the endosomes and lysosomes in the extracellular tumour tissues, the observed average pH is ~5.0 and the level of GSH is upto 10 mM. Thus, in-vitro release studies of DOX were also performed in presence (Fig. 11) and absence (Fig. 10) of GSH by spiking GSH in to the donor chamber. Different concentrations of GSH (2 mM, 5 mM,

10 mM and 20 mM) were spiked along with the DOX loaded nanoparticles and DOX release was measured at pH 5.0 and 7.4 at 37 °C as shown in the Fig. 11(A–H) thus attempting to mimic the acidic GSH stimulated environment of endosomal compartments of cancer cells [62,63]. It was observed that the DOX release in absence of GSH was low compared to release in presence of spiked GSH as shown in Fig. 11. Also, with increase in the concentration of spiked GSH the release of DOX increases. This may be attributed to the exchange of spiked GSH with the DOX containing GSH shell of nanoparticles thus resulting in exchange of the shell GSH with spiked GSH facilitating the DOX release during exchange process. Hence, it can be interpreted that the developed DOX carriers with

Fig. 10. Fluorescence Spectra showing the release of DOX at 37 °C from Glu-Fe3O4-Au nanoparticles at A. pH 2.5, B. pH 3.5, C. pH 5.0 and D. pH 7.4. 461

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Fig. 11. Fluorescence spectra showing the release of DOX on adding different concentration of GSH A. 2 mM at 5.0 pH, B. 5 mM at 5.0 pH, C. 10 mM at 5.0 pH, D. 20 mM at 5.0 pH, E. 2 mM at 7.4 pH, F. 5 mM at 7.4 pH, G. 10 mM at 7.4 pH and H. 20 mM at 7.4 pH.

controlled drug release capability are worthy platforms to overcome the drawback of the toxicity and severe side effect of the anticancer drugs to the healthy cells.

directly dependent on the number of cells treated. The effect of nanoparticles with and without loaded drug on HeLa cell lines is shown in the Fig. 13. It was observed that there is significant decrease in the cell viability when cells were treated with DOX loaded nanoparticles compared to the nanoparticles without loaded DOX. At the nanoparticle concentrations of 80 and 100 μg/mL, the statistical difference in cell viability observed was huge which suggests that the inhibitory effect of the prepared nanoparticles is directly proportional to the dosage of the injected nanoparticles. Thus, this data provides clear evidence that DOX loaded nanoparticles provide an efficient platform towards the anticancer drug carrier and has the potential for use in targeted drug delivery.

3.2. In-vitro cytotoxic assay and cellular uptake The intracellular drug uptake and morphology changes were studied with HeLa cells to check the response of HeLa cells towards the prepared nanoparticles. Behaviour of both the GSH functionalised nanoparticles (GS-Au-Fe3O4) without DOX and DOX loaded GS-Au-Fe3O4 nanoparticles was checked to understand the convolution of the developed nanoparticles as a nanocarrier. Two different concentrations of nanoparticles were used to check the behaviour towards HeLa cells as shown in Fig. 12. It was observed that cell shows aggregation on using the higher concentration of the nanoparticles in both the cases (Fig. 12C and E). However, the DOX loaded nanoparticles shows abrupt changes in the cell size and the cell wall seems to be damaged as can be seen in Fig. 12E. However, the same HeLa cells when analyzed under green and red fluorescence panel didn't show major changes as observed in blue fluorescence panel, but the cell size increases with increase in the DOX concentration. Also, with DOX loaded nanoparticles, the cell shows small cluster formations which were not visible with nanoparticles without loaded DOX (Fig. 12E).This gives an inference that cells treated with DOX loaded nanoparticles induces more intense fluorescence at any concentration when compared to nanoparticles without loaded DOX suggesting the greater uptake of the DOX loaded nanoparticles on HeLa cells.

3.4. Cell apoptosis using Flow cytometry Apoptosis analysis using Flow cytometry further confirms the enhanced cellular uptake of the DOX loaded nanoparticles as can be seen in the Fig. 14. The killing ability of the developed nanoparticles on HeLa cells with free DOX as a positive control and at different concentration of DOX loaded nanoparticles was studied using flow cytometry. The flow cytometry spectra as shown in Fig. 14 is generally divided into four zones according to the population of the cells, i.e., M1, M2, M3 and M4 which defines the cell population as live cells, early apoptotic cells, late apoptotic cells and necrotic cells in their different zones respectively. As observed in Fig. 14(C–F), the cell population moves towards the necrotic (M4) and apoptotic region (M3) of the spectra when the concentration of DOX loaded nanoparticles has been increased subsequently from 20 μg/mL to 80 μg/mL. For the blank samples, the cell population in M1 zone was 92.27% which is subsequently reduced to 84.37%, 67.42%, 42.39% and 17. 09% when treated with DOX loaded nanoparticles at different concentration of 200, 400, 600 and 800 μg/mL. Additionally, the percent for late apoptotic cells (M3) and necrotic cells (M4) gradually increases when exposed to DOX loaded nanoparticles to 10.86%, 14.98%, 20.02%, 19.95% and 4.77%, 17.60%, 37.59%, 62.95% for 200, 400,

3.3. MTT assay To check the cytotoxicity of the prepared nanoparticles, MTT assay were performed with DOX loaded nanoparticles (DOX-GS-Au-Fe3O4) and compared with the nanoparticles without loaded DOX (GS-AuFe3O4) as shown in the Fig. 13. MTT assay is an important method to evaluate the in-vitro cytotoxicity, where the observed absorbance is 462

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Fig. 12. Cellular uptake on HeLa cells showing the A. Positive control images, B. Glutathione functionalised Fe3O4-Au nanoparticles (GS-Au-Fe3O4) at 2 and 80 μg/ mL concentration and DOX loaded Glutathione functionalised Fe3O4-Au nanoparticles at 2 and 25 μg/mL.

apoptosis are alternative and may occur simultaneously. However, necrosis is considered more toxic process and results in significantly larger change in the cell morphology compared to apoptosis [64]. Necrosis may lead to cell swelling and karyolysis while the apoptosis may result in the pyknosis and Cell shrinkage. The observed necrosisis due to the presence of DOX which has the potential to induce oxidative stress with significant production of malondialdehyde and thus resulting in oncosis parallel to the apoptosis [65]. Thus, the in-vitro studies confirm that the DOX loaded nanoparticles induces the cell apoptosis in a dose dependent manner when compared with the blank treatment (Fig. 14A). Moreover, the cellular uptake of nanoparticles is efficiently enhanced by DOX thereby improving the cytotoxicity of the prepared nanoparticles against the HeLa cells on one hand and preventing damage to the healthy cells on account of biocompatible GSH shell on another hand. Additionally, significantly lower amount of DOX carried through nanoparticles which induces higher cell killing compared to free DOX may lead to minimal side effects and better bioavailability of drug for efficient cancer treatment.

Fig. 13. MTT assay showing the percent inhibition of HeLa cells on treatment with A. GS-Au-Fe3O4 nanoparticles and B. DOX-GS-Au-Fe3O4-nanoparticles.

600 and 800 μg/mL concentration respectively. However, slight increase is observed in percent of necrotic cells (M4) when compared with late apoptotic cells (M3). It has been suggested that necrosis and 463

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Fig. 14. Apoptosis study using Flow cytometry analysis of Hela cells A. Negative control, B. Positive control, C. 200 μg/mL, D. 400 μg/mL, E. 600 μg/mL and F. 800 μg/mL.

4. Conclusion

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GS-Au-Fe3O4 nanoparticles, as prepared in this study, were used for successful delivery of DOX to the cancer cells (HeLa cell lines). The nanoparticles were found to show significant DOX uptake compared to free drug even in low concentrations. The drug loaded nanoparticles triggered the significant drug release in-vitro under acidic and redox environment stimulated by spiking external GSH. Thus the nanoparticles proved effective in releasing DOX by mimicking the prevalent conditions in cancer cells which may lead to the increased efficacy of prepared nanoparticles for drug delivery. The significant release of DOX by GSH exchange not only triggers the controlled drug release but may also reduce the Side effects of DOX towards healthy cells on account of presence of GSH shell which protects healthy cells from cytotoxic effects. Induction of apoptosis in the HeLa cells by drug loaded nanoparticles was demonstrated using flow cytometry which clearly indicates the efficiency of prepared nanoparticles in low concentrations. The results are significant and may prove to be of therapeutic applications as the drug release and cell death could be achieved in controlled manner by optimising concentration of nanoparticles, pH and redox environment using spiked GSH. Thus, the present findings may lead to possible application of GSH based superparamagnetic nanoparticles for DOX delivery to cancer cells where synergistic behaviour of GSH may lead to efficient desired apoptosis and therapeutic potential. Acknowledgements This work was made possible by a Fellowship to N.S. from the SVNIT, Surat and SERB, New Delhi (YSS/2015/00184) financial support for project. We would like to thank MNIT, Jaipur for providing us HRTEM and XPS and also Genexplore Research and diagnostics pvt. Ltd. for carrying out the biological study. References [1] K.T. Yong, I. Roy, M.T. Swihart, P.N. Prasad, J. Mater. Chem. 19 (2009) 4655–4672. [2] J. Gao, S.-S. Feng, Y. Guo, Nanomedicine (7) (2012) 465–468.

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