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Erlotinib conjugated gold nanocluster enveloped magnetic iron oxide nanoparticles–A targeted probe for imaging pancreatic cancer cells
Sensors and Actuators B 257 (2018) 1035–1043
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Research Paper
Erlotinib conjugated gold nanocluster enveloped magnetic iron oxide nanoparticles–A targeted probe for imaging pancreatic cancer cells John Nebu, J.S. Anjali Devi, R.S. Aparna, K. Abha, George Sony ∗ Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom Campus, Thiruvananthapuram, 695581, Kerala, India
a r t i c l e
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Article history: Received 29 September 2017 Received in revised form 4 November 2017 Accepted 4 November 2017 Available online 6 November 2017 Keywords: Gold nanocluster Iron oxide nanoparticle Erlotinib Bioimaging Bovine serum albumin
a b s t r a c t A specific targeting, biocompatible, magnetic nanoprobe for bioimaging of pancreatic cancer cell lines was developed by simple three-step route in aqueous solution. Herein, the nanocomposite consists of superparamagnetic iron oxide core having gold nanocluster adsorbed around it through electrostatic attraction. The Au NC corona layer is further conjugated with positively charged erlotinib. The Fe3 O4 nanoparticles ensures the magnetic enrichment, Au NCs exhibit intense red fluorescence property and erlotinib act as a specific targeting agent for pancreatic cancer cells. The resultant targeted nanoparticles had a diameter of 24 ± 2.5 nm with relatively high fluorescence life (av = 1.17 s @ em = 640 nm) and can kill EGFR over expressed pancreatic cancer cells. Confocal laser scanning microscope images of the normal cells (L-929) and pancreatic cancer cells (PANC-1) treated with probe is conducted. The results shows that the morphology of the normal cells remains intact attesting its non toxicity while PANC-1 which is over expressed with EGFR exhibit cytotoxicity towards the probe proving the potential theranostic ability of the nanocomposite. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Advances in the earlier diagnosis and improved methods for the treatment of cancer has enhanced the survival rate of all cancers [1]. Despite this, the diagnosis of pancreatic cancer is extremely difficult and accounts about fourth leading cause of all cancer deaths. The ‘Pancreatic Cancer Action Network’ estimates that pancreatic cancer will become the second leading cause of death due to cancer by 2020 [2,3]. The effective treatment at the time of prognosis is surgical resection, but only 15–20% of patients are undergoing it and the other options left with later stages disease are chemotherapy and radiation therapy [4]. The clinical response of pancreatic cancer patients to chemotherapy is poor [5]. In surgical resection, possibilities of retention of residual tumor tissues are a major concern and are reported to be 40% in pancreaticoduodenectomies. This necessitates the requirement of intraoperative identification of cancer margins in real time which will reduce the possibilities of overzealous resection comprising adjacent vital tissues and organs leading to poor functional outcomes [6]. Targeting receptor protein which is over expressed in pancreatic cancer cell can improve the diagnosis of pancreatic cancer.
∗ Corresponding author. E-mail address: [email protected] (G. Sony). https://doi.org/10.1016/j.snb.2017.11.017 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Pancreatic cancer cells have been found to over express the epidermal growth factor receptor (EGFR), which may cause resistance to chemotherapy [7,8]. EGFR is a glycoprotein which act as tyrosine kinase ATP-binding site that play a major role in cell proliferation, carcinogenis, angiogenisis and invasion [9–11]. The dysfunction of EGFR signalling occurs in 50% of the pancreatic cancer cells leading to poor prognosis and tumor progression, which signifies the importance of a suitable targeting agent for EGFR [12]. Erlotinib has been clinically approved as an inhibitor of EGFR and is effective than other tyrosine kinase receptor. It can suppress the trans-phosphorylation of EGFR, thereby potentiates the significant inhibition of tumor growth [13,14]. The combination of targeting ability with imaging technique in a single entity is an important tool for the early detection and treatment of pancreatic cancer. Currently a number of non-invasive optical and magnetic imaging technique such as fluorescent imaging, ultra sound imaging, X-ray computed tomography, confocal microscopy imaging, positron emission tomography (PET) and magnetic resonance imaging (MRI) are used in clinical practice for monitoring, grading and evaluating prognosis of cancer [15,16]. Among various techniques, fluorescence imaging has been widely adapted for invitro analysis. In addition to the advantages, each technique has its own limitations, and so researchers are focussed to develop probes which potentiate a combination of different imaging techniques via multimodal imaging [17–19]. The optical imaging technique employs the use of contrast probes with fluorescent proper-
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Fig. 1. (A) UV/Visible spectra of (a) HAuCl4 , (b) BSA, (c) Au NCs and (d) Fe3 O4 @Au NCs. (B) Excitation spectra of (a) Au NCs and (b) Fe3 O4 @Au NCs. Emission spectra of (c) Au NCs and (d) Fe3 O4 @Au NCs. (C) Photographs of Au NCs under (a) 330 nm UV light, (b) 365 nm UV light and (c) Visible light. (D) TEM image of the Au NCs.
Scheme 1. Schematic representation highlighting the possible potential application of Fe3 O4 @Au NC@erlotinib.
ties such as metal nanoclusters, carbon dots, quantum dots and organic dyes [20,21]. In contrast with the liability of photobleaching of organic dyes and cytotoxicity of quantum dots, noble metal nanoclusters are emerging as promising material with thermal stability, low cytotoxicity, high fluorescence intensity and good biocompatibility [22]. To overcome the limitations of poor contrast images, MRI imaging is also carried out with appropriate contrast agents [23–25]. Apart from Gadolinium complexes the MRI contrast agents, consisting of metal oxide nanoparticle having paramagnetic nature such as iron oxide, gadolinium oxide and manganese oxide have been established. The Fe3 O4 nanoparticles with superparamagnetic behaviour and better biocompatibility have proven to be more versatile by virtue of its simultaneous T1 and T2 relaxation behaviour [24,26–28]. The luminescent gold nanocluster possess excellent photostability, ultrafine size, large stoke shift, size-dependent optical
properties and good biocompatibility. It can find potential application in drug delivery, optical imaging, sensor for biomolecules, ions and had become favoured candidate for theranostic applications [28,29]. Herein, we synthesized Iron oxide-gold nanoclusters (Fe3 O4 @AuNC) core shell nanocomposite probe, which shows hybrid features including superparamagnetic properties and excellent red fluorescence. Iron oxide nanoparticles are often synthesized in the organic phase with complex procedure or in aqueous phase with aggressive reducing agents which may be cytotoxic in nature. In the present work, we synthesized magnetic iron oxide (Fe3 O4 MNPs) using Arginine, an essential amino acid as reducing agent and by making use of the electrostatic attraction with Au NCs, Fe3 O4 @Au NC nanocomposite were prepared. Here, we present a targeted theranostic probe Fe3 O4 @Au NC conjugated to erlotinib, which has the potential for molecular imaging of pancreatic cancer cells and
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Fig. 2. (A) Photographs showing HAuCl4 , Au NCs and Fe3 O4 @Au NC (a, b & c) under Visible light and (d, e & f) under UV radiation. (B) Photographs showing the response of (a) Fe3 O4 MNPs solution towards a strong hand held magnet, (b) dried Fe3 O4 MNPs and (c) response of dried Fe3 O4 MNPs towards a strong hand held magnet. (C) XRD spectrum of Fe3 O4 . (D) TEM image of Fe3 O4 MNPs. (E) HRTEM image of Fe3 O4 MNPs. (F) SAED pattern of the selected single Fe3 O4 nanoparticle.
Scheme 2. Schematic representation of the formation of Fe3 O4 @AuNC@erlotinib.
drug delivery (Scheme 1). The proven T1 /T2 contrast ability of the Fe3 O4 @Au NC [22] can be employed with erlotinib conjugation to precisely determine the epicentre of pancreatic cancer prior to its surgical resection enabling visual determination of pancreatic cancer cells in real time surgery by virtue of fluorescent property of Au NCs (Scheme 2).
2. Experimental 2.1. Materials The following chemicals were purchased from Sigma-Aldrich: Bovine serum albumin (BSA), Erlotinib hydrochloride and Ferrous ammonium sulphate (FeSO4 (NH4 )2 SO4 .6H2 O). Hydrogentetra-
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Fig. 3. (A) Photographs showing the fluorescence of Au NCs (left) and Fe3 O4 @Au NC (right) of the dried sample. (B) TEM image showing the core shell structure of Fe3 O4 @Au NC (Inset: enlarged image of it). (C) Magnetic hysteresis loops of (a) Fe3 O4 , (b) Fe3 O4 @Au NC deposited on a cover slip by heating at 100 ◦ C for 1 min.
chloroaurate (HAuCl4 ·3H2 O) and Arginine were bought from Alfa Aesar. PANC-1 cells and L-929 cells were received from National Centre for Cell Science, Pune. The chemicals, otherwise not mentioned were obtained from Merck. All the reagents were of analytical grade and used without further purification. Millipore water was used in all the experiments.
2.2. Synthesis of Au NCs The Au NCs were synthesized according to a previously reported method with slight modification [30]. Briefly, 250 mg of BSA dissolved in 5 ml of water was transferred to 5 ml of HAuCl4 (10 mM) with continuous stirring (600 rpm) and add 0.5 ml of 1 M NaOH after 2 min. After stirring (600 rpm) for 5 min, the solution was irradi-
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Fig. 4. (A) High resolution XPS spectra of (A) Au 4f5/2 , Au 4f7/2 , (B) C 1s, (C) O 1s; and (D) high resolution and its deconvolution XPS spectra for the Fe 2p3/2 and Fe 2p1/2 signal of the sample containing Fe3 O4 @Au NCs.
ated with microwave radiation (800 W) for about 50 s in a domestic microwave oven. The formation of BSA-Au NCs was indicated by the change in color to deep brown with the emission of deep red light under a UV lamp at 365 nm and the solution is used for the synthesis of Fe3 O4 @Au NCs nanocomposite. 2.3. Synthesis of Fe3 O4 The Fe3 O4 magnetic nanoparticles (MNPs) were synthesized using previously reported method with minor modification [31]. In a typical procedure, 50 ml of 1 mM aqueous solution of Arginine was added to 20 ml of 80 mM FeSO4 (NH4 )2 SO4 ·6H2 O under stirring (1800 rpm). After 1 min, 10 ml of 25% ammonia was added to the mixture with constant stirring (1800 rpm) for 1 h at 25 ◦ C. The synthesized solution was used for the preparation of Fe3 O4 @Au NCs nanocomposite and Fe3 O4 MNPs were separated magnetically, and after decantation dried in vacuum oven at a temperature of 50 ◦ C for further analysis. 2.4. Synthesis of Fe3 O4 @Au NCs In brief, 200 l of Fe3 O4 MNPs were added to 5 ml phosphate buffer (PBS) at pH = 7.4 with vigorous stirring (2500 rpm), and mixed with 5 ml of Au NCs. The mixture was sonicated for 1 h, dried in hot air oven at 100 ◦ C (1 min) for AFM analysis.
2.6. Release of erlotinib from Fe3 O4 @Au NCs@erlotinib The release of erlotinib was done in PBS buffer of different pH = 7.4 and pH = 5.0. Briefly, 250 l of Fe3 O4 @Au NCs@erlotinib were suspended in 0.5 ml PBS buffer in cellulose dialysis membrane tied at both ends. The cellulose dialysis membrane was then immersed in a 10 ml different pH buffered solutions at 37 ◦ C with continuous stirring (300 rpm). The amount of erlotinib released was estimated at =247 nm in a regular interval of time with erlotinib as control.
2.7. Cell viability assay Cell viability study was done using MTT assay and apoptosis assay [22] towards L-929 (mouse lung fibroblast cell line) and PANC-1 (pancreatic cell line). Briefly, either 4 × 103 L-929 and 3 × 103 PANC-1 cells were seeded into 96-well plates and incubated for 24 h. The Fe3 O4 @AuNCs@erlotinib was added to the cells in a 24 well plates to give effective erlotinib concentration of 5, 10, 25, 50 and 80 M. Then the cells were incubated for 24 h and then washed it with PBS. After treating the cells with 200 l DMEM, 20 ml MTT stock solution were added to each well and incubated for 5 h at 37 ◦ C. The culture medium was removed and 150 l of DMSO was added. After shaking the mixture for 5 min, the cell viability was studied and expressed by comparing with the cells incubated in culture medium alone.
2.5. Synthesis of Fe3 O4 @Au NCs@erlotinib Briefly, 250 l of 20 M erlotinib was added to 5 ml of Fe3 O4 @Au NC with constant stirring (1800 rpm) for 1 day at a temperature of 25 ◦ C. The non-conjugated erlotinib was removed from the solution and the solution is sterilized for further analysis. The drug loading content and loading efficiency was determined spectrophotometrically.
2.8. Confocal microscopy imaging The Fe3 O4 @Au NCs@erlotinib solution (50 g mL−1 ) was dispersed in a serum-free DMEM cell solution and incubated for 4 h at 37 ◦ C in the presence of 5% CO2 . The cells in the culture medium after washed three times with PBS, fixed with 4% paraformaldehyde in PBS medium for 1 h.
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Fig. 5. (A) Time-resolved fluorescence life time analysis of (a) Au NC and (b) Fe3 O4 @AuNC. (B) Release profiles of erlotinib from the Fe3 O4 @AuNC@erlotinib in the PBS buffer mimicking extracellular environment of pH = 7.4 and pH = 5.0. (C) Cell viability of L-929 mouse lung fibroblast cells and PANC-1 pancreatic cells in cell medium for 24 h after incubation of different concentrations of Fe3 O4 @AuNC@erlotinib as analyzed by MTT assay.
3. Results and discussion The synthesis process for Fe3 O4 @Au NCs@erlotinib is illustrated in Scheme 1. The overall synthesis process involved four steps consisting of (i) synthesis of Au NCs, (ii) synthesis of Fe3 O4 , (iii) their combination and (iv) conjugation of erlotinib. We first synthesized Au NCs by microwave assisted heating following the previously reported method with slight modification using bovine serum albumin (BSA) as the template. The as-prepared Au NCs exhibit emission peak at ∼ 640 nm via excitation at ∼ 480 nm. The intensity of the emission and excitation peak first increases and then decreases as the power of the microwave increases (Fig.S1 and Fig.S2). The decrease in intensity at high microwave power is due to the evaporation of the BSA template during microwave irradiation. Here a microwave power of 800 W for a time 50 s is optimized for further experiments. The positively charged amino acids like Arginine and Lysine present in the BSA makes the co-ordination of AuCl4 − ions with BSA, the trypsin/tryptophan residues reduce the Au ions and the stability is due to the presence of thiol groups containing 35 cysteine residues present in it [32].
Fig. 1A show the UV–vis absorbance spectra of BSA, HAuCl4 , Au NCs and Fe3 O4 @Au NCs, in which BSA has a sharp peak at 280 nm, Au NCs and Fe3 O4 @Au NCs have a broad absorbance at 510 nm. Fig. 1B shows excitation and emission spectra of Au NCs (a, c) and Fe3 O4 @Au NCs (b, d) respectively. Even though, Fe3+ ion is well known as a quencher of fluorescence, incorporation of Fe3 O4 cause only slight quenching of the fluorescence of Au NCs. The deep brown solution of Au NCs emitted an orange red colored fluorescence (Fig. 1C (a)) and an intense red colored fluorescence (Fig. 1C (b)) under UV radiation at 330 nm and 365 nm respectively. The TEM images as shown in Fig. 1(D) indicates that the Au nanoclusters are monodispersed and exhibit an average size of 4.0 ± 0.3 nm without aggregation of the cluster. The size distribution and the hydrodynamic diameter (Fig.S2(A)) from the DLS measurement reveals a mean size of 18.5 ± 2.5 nm further indicates the formation of well dispersed nanoclusters [33]. The stability of Au NCs in solution phase is monitored for 30 days and found that there is not much decrease in intensity, ensuring the stability of Au NCs for prolonged storage. We synthesized Fe3 O4 nanoparticles by previously reported method using the amino acid, Arginine as the reducing agent. The amino acid, Arginine has isoelectric point pI = 10.76, as it has a positive charge at pH = 7.4, which is made use for the synthesis of Fe3 O4 @Au NCs, where the isoelectric point of BSA is pI = 4.7 [34] and hence it has a negative charge at pH = 7.4. It ensures the required electrostatic attraction between Fe3 O4 enveloped by positively charged arginine and negatively charged BSA capped Au NC. Fig. 2(A) shows the photograph of the HAuCl4 , Au NCs and Fe3 O4 @Au NCs in the Visible light (a, b & c) and UV radiation (d, e & f), in which it is clear that the presence of Fe3 O4 does not diminishes the fluorescence property of Au NCs. Fig. 2(B) shows the photograph illustrating the magnetic property of Fe3 O4 in the synthesized wet state (a), its dried state (b) and magnetic property of it in the dried state in the vicinity of an external magnetic field (c). As depicted in Fig. 2(C), the XRD pattern shows peak at 30.29◦ , 35.59◦ , 43.25◦ , 47.26◦ , 53.84◦ , 57.13◦ and 62.79◦ 2 that can be assigned to the 220, 311, 400, 331, 422, 511 and 440 reflections (JCPDS 65–3107) respectively. All the diffraction peaks of the samples corresponds to a face-centred cubic phase with the lattice constant a = b = c = 8.425 Å and ␣=  = ␥ = 90◦ . The average particle size of Fe3 O4 nanoparticles based on the Fe (311) peak of the XRD pattern was calculated with the Scherrer’s formula to be 12.3 nm, which is in close agreement with the size as shown in the TEM image. TEM image of Fig. 2(D) demonstrate that these nanoparticles have diameter of 11.2 nm to 13.5 nm. The lattice spacing in the HRTEM (Fig. 2(E)) image of the nanoparticle measures at 0.23 nm corresponding to the (220) plane of the Fe3 O4 nanoparticle. SAED pattern of the sample as shown in Fig. 2(F) displays several diffraction rings indexed to 111, 220, 311, 400, 331, 422, 511, 440 and 533, indicative of the polycrystalline nature of Fe3 O4 [35]. The size distribution and the hydrodynamic diameter of Fe3 O4 as shown in Fig.S2 (B) indicate a mean diameter of 14.6 ± 2 nm. The Zeta potential measurement reveals that the charge of Fe3 O4 is about +7.6 which are suitable for the formation of the core shell structure of Fe3 O4 @Au NCs (Fig.S3 (B)). As shown in Fig. 3(A), the dried Fe3 O4 @Au NCs show intense red fluorescence under 365 nm UV light. On prolonged storage, Fe3 O4 @Au NCs leads to sedimentation and has to be sonicated and dispersed prior to use. The TEM image as shown in Fig. 3(B) shows a core shell structure with Fe3 O4 as the core and Au NCs as the corona. The size distribution from the DLS measurement is narrow with a mean diameter of 32.3 ± 3.5 nm. The TEM image and the DLS data indicates a monodispersed Fe3 O4 @Au NC nanoparticle. Fig. 3(C) shows the magnetic M-H hysteresis loop of Fe3 O4 and Fe3 O4 @Au NCs, which suggests that Fe3 O4 has a saturation magnetization of 48.0 emug−1 and has almost zero coercivity and remanence
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Fig. 6. Confocal laser images of L-929 and PANC-1 cell lines. First row (a) fluorescence image, (b) bright field image of control L-929 cells (mouse lung fibroblast cell). Second row (c) fluorescence image, (d) bright field image, (e) overlapped image of L-929 cells treated with Fe3 O4 @AuNC@erlotinib. Third row (f) fluorescence image, (g) bright field image of control PANC-1 cells (pancreatic cancer cell). Fourth row (h) fluorescence image, (i) bright field image, (j) overlapped image of PANC-1 cells treated with Fe3 O4 @AuNC@erlotinib.
indicating the superparamagnetic nature of Fe3 O4 . Moreover the saturation magnetization of Fe3 O4 @Au NCs decreases slightly to 34.168 emug−1 and the coercivity and remanence remains zero. The slight decrease in the saturation magnetization occurs due to the changes in surface spin effect on Fe3 O4 caused by the electrostatic attraction of Au NCs around it. Even after enveloping with Au NCs, the Fe3 O4 retains its superparamagnetic behaviour evidenced by the fact that the Fe3 O4 @Au NCs can be magnetically collected using hand held external magnet. The AFM 3D images as shown in Fig. 3(D) indicate the random distribution of spike-like features owing to Au NCs. The spike density reveals the stability of Au NCs in the BSA template and the height of the AFM images indicates the agglomeration of the BSA stabilized Au NCs which occurs due to
the denaturation of the protein while heating and drying. The morphology of Fe3 O4 @Au NCs is further analyzed using AFM analysis (Fig. 3(E) which reveals the appearance of more spikes attributed to the increase in the density of the particles by the formation of coreshell structure [36]. Moreover the height of the spikes is around 19.7 nm indicating the BSA stabilized Au NCs retains its properties after heating and drying at 80 ◦ C which is in agreement with the size distribution of the Fe3 O4 @Au NCs from the TEM image and DLS measurements. To analyze the detailed electronic structure of Fe3 O4 @Au NCs, XPS was done to explore the oxidation state of Au, C, O and Fe atoms and the results are shown in Fig. 4. The peak centred at 83.69 eV as a result of the binding energy of 4f7/2 is close to the characteristic
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peak of Au(0) in metallic gold, attributed to the presence of Au(0) core of the gold nanocluster. The second peak appears at 87.35 eV which arises as a result of the 4f5/2 orbital. The peak at 87.35 eV suggests the presence of Au(I) on the surface, which favours the stability offered by 32 cysteine residues of the BSA as capping agent. The results indicate the existence of Au(0)-Au(I) core-shelled structure in the Fe3 O4 @AuNC nanocomposite. The C 1 s peaks at 284.67 eV (sharp), 287.86 eV (satellite peak) and 289.11 eV (sharp) corresponds to C C (sp2 ), C O and O C O or C N bonds of the Arginine as well as the amino acid residues of the BSA[37]. Here O 1 s peaks at 523.21 eV (broad), 531.02 eV (sharp) and 535.56 eV(broad, satellite peak) occurs due to hydroxyl group, C O bonding and metal-oxygen bond respectively. The Fe 2p deconvoluted peaks at 707.53 eV and 709.88 eV are due to Fe(II)-O and Fe(III)-O bonds in magnetite. The sharp peak at 711.15 eV is due to 2p3/2 and the broad peaks at 713.11 eV and 714.38 eV indicates the presence of Fe-OH or Fe-OOC interactions at the surface of magnetite nanoparicle. Here a couple of broad peaks in the energy interval of 715–720 eV are the satellite peaks of Fe(II) and Fe(III) ions, which may be due to the interaction of Fe3 O4 with Au NCs. The characteristics binding energy observed at 723.5 eV are attributed to 2p1/2 orbital[38]. As per the XPS analysis results, the atomic concentration and mass concentrations of Fe based on the intensity of the corresponding peaks are 0.11% and 1.6% and that of gold are 0.13% and 1.81% respectively. In contrast to this, the atomic and mass concentrations of Fe determined based on the area of the peaks are 0.18% and 2.53% and that of Au are 0.20% and 2.76% respectively. To investigate the effect of Fe3 O4 on the fluorescence properties of Au NCs, the fluorescence life time measurements were conducted. Fig. 4(A) shows the fluorescent emission life time decay curve depicting a bi-exponential pattern. The fluorescence life time of Au NCs was 1.2 s and Fe3 O4 @Au NCs is 1.17 s. The relatively high life time of Au NCs [39] has attributed to the existence of slow component of triplet formation and fast component of Au (I) surface with Au (0) core of the BSA capped Au NCs [32]. The incorporation of Fe3 O4 only marginally shifts the fluorescence life time of Au NCs from 1.2 s to 1.17 s. The core-shell Fe3 O4 @Au NCs with negatively charged shell are conjugated to the positively charged erlotinib by electrostatic attraction. The amount of erlotinib loaded in the Fe3 O4 @Au NCs nanocomposites determined by spectrophotometrically is 8.093 g/mL and the loading efficiency is about 94.12%. The high loading efficiency attained due to the strong electrostatic interaction between positively charged erlotinib and negatively charged Fe3 O4 @Au NCs nanocomposite. The size of the Fe3 O4 @Au NCs@erlotinib as indicated by the DLS measurement is about 41.8 ± 2.4 nm. The in-vitro drug release study of erlotinib was done in physiological environments mimicking PBS buffers with pH = 7.4 and 5.0 at 37 ◦ C by measuring the absorbance at = 247 nm following dialysis. Fig. 4(B) exhibits the in-vitro release profiles of Fe3 O4 @Au NCs@erlotinib for erlotinib. At pH of 5.0, about 78.3% of drugs (erlotinib) get released in comparison with 52.1% release at pH of 7.4. The two release profile are significant, owing to the reduce release of drug while circulating in the blood till it reaches the targeted site where the pH of the cancer cells is less. Moreover, the result suggests that the Fe3 O4 @Au NCs@erlotinib exhibit a pH triggered drug release, indicating the ability of the material for drug delivery to the cancer cells. To investigate the targeting ability of Fe3 O4 @Au NCs@erlotinib, we tested the viability of a normal cell, L-929 and a pancreatic cancer cell, PANC-1 using MTT assay and apoptosis assay. PANC1 cells over expresses the EGFR where L-929 cells do not over expresses the EGFR. As shown in Fig. 4 (C), erlotinib exhibit a cytotoxic effect on PANC-1 in a dose dependent manner but it does not cause much cytotoxic effect to L-929 cell even at high doses of Fe3 O4 @Au NCs@erlotinib. Here the specific cytotoxic nature of
Fe3 O4 @Au NCs@erlotinib nanocomposites on PANC-1 and nontoxicity of it on L-929 normal cell indicate that the cytotoxicity arises due to erlotinib, where it is a targeting agent of PANC-1 cells. This result suggests that Fe3 O4 @Au NCs@erlotinib is effective for the selective targeting of EGFR over expressed pancreatic cancer cells and the free Fe3 O4 @Au NCs is biocompatible in nature. To validate the effectiveness of Fe3 O4 @Au NCs@erlotinib, after 12 h of incubation, we examined the intracellular distribution of it in cells using confocal fluorescence microscopy and the images are shown in Fig. 5. From the bright field image, it is clear that L-929 cell maintained their normal morphology with some bright red spot of fluorescence arising from Fe3 O4 @AuNC@erlotinib appear outside the cells in the fluorescence image. The overlapped image consists of red fluorescent spots outside the cells, indicating the biocompatibility of the nanoparticles towards the normal cells. On the other hand, the bright field image of PANC-1 cells shows that the morphology of the cells are affected and in the fluorescence image, the intense red fluorescence is observed in the cytoplasm and nucleus of the cells. It further confirmed by observing the overlapped image, indicating the cellular uptake of EGFR expressed PANC-1 cells. All these results show that Fe3 O4 @Au NCs@erlotinib can be adaptable for imaging of cancer cell prior to its surgical removal (Fig. 6). 4. Conclusion This study investigates three main properties (i) superparamagnetic behaviour (ii) fluorescence property (iii) targeting ability of Fe3 O4 @Au NCs@erlotinib. Herein, we used the amino acid, Arginine as the reducing agent for Fe3 O4 formation and BSA for Au NC formation which maintains the biocompatible nature of the material for bioimaging. Moreover, the presence of erlotinib ensures the targeting ability of the composite (Fe3 O4 @Au NCs@erlotinib) towards pancreatic cancer cells. The relatively high life time of the probe shows the promising nature of the nanocomposite for optical imaging and the currently employed superparamagnetic nature of Fe3 O4 ensures its potential for simultaneous T1 and T2 contrast agents. Acknowledgement The authors thank the Professor and Head, Department of Chemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram for pursuing the platform to conduct the research. The authors also thank the Director, SICC, University of Kerala, Kariavattom campus, Thiruvananthapuram; Director, SAIF-STIC-CUSAT, Kochi; RGCB, Thiruvananthapuram, DST-SAIF, M.G. University, Kottayam. The author N.J. acknowledge support for this work by University Grants Commission, Bangalore, India through the teacher fellowship (F.No.FIP/12th plan/KLMG035, TF: 03) under faculty development programme during XIIth plan period. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2017.11.017. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA: A Cancer Journal for Clinicians 67 (2017) (2017) 7–30. [2] C.G. England, R. Hernandez, S.B.Z. Eddine, W. Cai, Molecular imaging of pancreatic cancer with antibodies, Mol. Pharm. 13 (2016) 8–24. [3] L. Rahib, B.D. Smith, R. Aizenberg, A.B. Rosenzweig, J.M. Fleshman, L.M. Matrisian, Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States, Cancer Res. 74 (2014) 2913–2921. [4] J. Long, Y. Zhang, X. Yu, J. Yang, D.G. LeBrun, C. Chen, et al., Overcoming drug resistance in pancreatic cancer, Expert Opin. Ther. Targets 15 (2011) 817–828.
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Biographies Nebu John is an Assistant professor of chemistry at Mar Thoma college, Tiruvalla, Kerala, India. He is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. His research interest includes magnetoplasmonic nanoparticle and bioanalytical applications. Anjali Devi J.S. (DST-INSPIRE fellow) is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Aparna R.S. is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Abha K. is an Assistant professor of chemistry at Bishop Moore College, Mavelikkara, Kerala, India. She is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Sony George had his Ph.D. at Department of Chemistry, University of Kerala, Kerala, India and he is currently working as an Assistant Professor in the same institution. His area of interest is magnetic and fluorescent nanomaterials for sensoronic and bioimaging. He had been a visiting professor at A. A. Athinoula centre for Biomedical imaging, MGH affiliated to Harward Medical school. He had sabbatical stint at Department of Chemical physics, LUND University, Sweden.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Research Paper
Erlotinib conjugated gold nanocluster enveloped magnetic iron oxide nanoparticles–A targeted probe for imaging pancreatic cancer cells John Nebu, J.S. Anjali Devi, R.S. Aparna, K. Abha, George Sony ∗ Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom Campus, Thiruvananthapuram, 695581, Kerala, India
a r t i c l e
i n f o
Article history: Received 29 September 2017 Received in revised form 4 November 2017 Accepted 4 November 2017 Available online 6 November 2017 Keywords: Gold nanocluster Iron oxide nanoparticle Erlotinib Bioimaging Bovine serum albumin
a b s t r a c t A specific targeting, biocompatible, magnetic nanoprobe for bioimaging of pancreatic cancer cell lines was developed by simple three-step route in aqueous solution. Herein, the nanocomposite consists of superparamagnetic iron oxide core having gold nanocluster adsorbed around it through electrostatic attraction. The Au NC corona layer is further conjugated with positively charged erlotinib. The Fe3 O4 nanoparticles ensures the magnetic enrichment, Au NCs exhibit intense red fluorescence property and erlotinib act as a specific targeting agent for pancreatic cancer cells. The resultant targeted nanoparticles had a diameter of 24 ± 2.5 nm with relatively high fluorescence life (av = 1.17 s @ em = 640 nm) and can kill EGFR over expressed pancreatic cancer cells. Confocal laser scanning microscope images of the normal cells (L-929) and pancreatic cancer cells (PANC-1) treated with probe is conducted. The results shows that the morphology of the normal cells remains intact attesting its non toxicity while PANC-1 which is over expressed with EGFR exhibit cytotoxicity towards the probe proving the potential theranostic ability of the nanocomposite. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Advances in the earlier diagnosis and improved methods for the treatment of cancer has enhanced the survival rate of all cancers [1]. Despite this, the diagnosis of pancreatic cancer is extremely difficult and accounts about fourth leading cause of all cancer deaths. The ‘Pancreatic Cancer Action Network’ estimates that pancreatic cancer will become the second leading cause of death due to cancer by 2020 [2,3]. The effective treatment at the time of prognosis is surgical resection, but only 15–20% of patients are undergoing it and the other options left with later stages disease are chemotherapy and radiation therapy [4]. The clinical response of pancreatic cancer patients to chemotherapy is poor [5]. In surgical resection, possibilities of retention of residual tumor tissues are a major concern and are reported to be 40% in pancreaticoduodenectomies. This necessitates the requirement of intraoperative identification of cancer margins in real time which will reduce the possibilities of overzealous resection comprising adjacent vital tissues and organs leading to poor functional outcomes [6]. Targeting receptor protein which is over expressed in pancreatic cancer cell can improve the diagnosis of pancreatic cancer.
∗ Corresponding author. E-mail address: [email protected] (G. Sony). https://doi.org/10.1016/j.snb.2017.11.017 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Pancreatic cancer cells have been found to over express the epidermal growth factor receptor (EGFR), which may cause resistance to chemotherapy [7,8]. EGFR is a glycoprotein which act as tyrosine kinase ATP-binding site that play a major role in cell proliferation, carcinogenis, angiogenisis and invasion [9–11]. The dysfunction of EGFR signalling occurs in 50% of the pancreatic cancer cells leading to poor prognosis and tumor progression, which signifies the importance of a suitable targeting agent for EGFR [12]. Erlotinib has been clinically approved as an inhibitor of EGFR and is effective than other tyrosine kinase receptor. It can suppress the trans-phosphorylation of EGFR, thereby potentiates the significant inhibition of tumor growth [13,14]. The combination of targeting ability with imaging technique in a single entity is an important tool for the early detection and treatment of pancreatic cancer. Currently a number of non-invasive optical and magnetic imaging technique such as fluorescent imaging, ultra sound imaging, X-ray computed tomography, confocal microscopy imaging, positron emission tomography (PET) and magnetic resonance imaging (MRI) are used in clinical practice for monitoring, grading and evaluating prognosis of cancer [15,16]. Among various techniques, fluorescence imaging has been widely adapted for invitro analysis. In addition to the advantages, each technique has its own limitations, and so researchers are focussed to develop probes which potentiate a combination of different imaging techniques via multimodal imaging [17–19]. The optical imaging technique employs the use of contrast probes with fluorescent proper-
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Fig. 1. (A) UV/Visible spectra of (a) HAuCl4 , (b) BSA, (c) Au NCs and (d) Fe3 O4 @Au NCs. (B) Excitation spectra of (a) Au NCs and (b) Fe3 O4 @Au NCs. Emission spectra of (c) Au NCs and (d) Fe3 O4 @Au NCs. (C) Photographs of Au NCs under (a) 330 nm UV light, (b) 365 nm UV light and (c) Visible light. (D) TEM image of the Au NCs.
Scheme 1. Schematic representation highlighting the possible potential application of Fe3 O4 @Au NC@erlotinib.
ties such as metal nanoclusters, carbon dots, quantum dots and organic dyes [20,21]. In contrast with the liability of photobleaching of organic dyes and cytotoxicity of quantum dots, noble metal nanoclusters are emerging as promising material with thermal stability, low cytotoxicity, high fluorescence intensity and good biocompatibility [22]. To overcome the limitations of poor contrast images, MRI imaging is also carried out with appropriate contrast agents [23–25]. Apart from Gadolinium complexes the MRI contrast agents, consisting of metal oxide nanoparticle having paramagnetic nature such as iron oxide, gadolinium oxide and manganese oxide have been established. The Fe3 O4 nanoparticles with superparamagnetic behaviour and better biocompatibility have proven to be more versatile by virtue of its simultaneous T1 and T2 relaxation behaviour [24,26–28]. The luminescent gold nanocluster possess excellent photostability, ultrafine size, large stoke shift, size-dependent optical
properties and good biocompatibility. It can find potential application in drug delivery, optical imaging, sensor for biomolecules, ions and had become favoured candidate for theranostic applications [28,29]. Herein, we synthesized Iron oxide-gold nanoclusters (Fe3 O4 @AuNC) core shell nanocomposite probe, which shows hybrid features including superparamagnetic properties and excellent red fluorescence. Iron oxide nanoparticles are often synthesized in the organic phase with complex procedure or in aqueous phase with aggressive reducing agents which may be cytotoxic in nature. In the present work, we synthesized magnetic iron oxide (Fe3 O4 MNPs) using Arginine, an essential amino acid as reducing agent and by making use of the electrostatic attraction with Au NCs, Fe3 O4 @Au NC nanocomposite were prepared. Here, we present a targeted theranostic probe Fe3 O4 @Au NC conjugated to erlotinib, which has the potential for molecular imaging of pancreatic cancer cells and
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Fig. 2. (A) Photographs showing HAuCl4 , Au NCs and Fe3 O4 @Au NC (a, b & c) under Visible light and (d, e & f) under UV radiation. (B) Photographs showing the response of (a) Fe3 O4 MNPs solution towards a strong hand held magnet, (b) dried Fe3 O4 MNPs and (c) response of dried Fe3 O4 MNPs towards a strong hand held magnet. (C) XRD spectrum of Fe3 O4 . (D) TEM image of Fe3 O4 MNPs. (E) HRTEM image of Fe3 O4 MNPs. (F) SAED pattern of the selected single Fe3 O4 nanoparticle.
Scheme 2. Schematic representation of the formation of Fe3 O4 @AuNC@erlotinib.
drug delivery (Scheme 1). The proven T1 /T2 contrast ability of the Fe3 O4 @Au NC [22] can be employed with erlotinib conjugation to precisely determine the epicentre of pancreatic cancer prior to its surgical resection enabling visual determination of pancreatic cancer cells in real time surgery by virtue of fluorescent property of Au NCs (Scheme 2).
2. Experimental 2.1. Materials The following chemicals were purchased from Sigma-Aldrich: Bovine serum albumin (BSA), Erlotinib hydrochloride and Ferrous ammonium sulphate (FeSO4 (NH4 )2 SO4 .6H2 O). Hydrogentetra-
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Fig. 3. (A) Photographs showing the fluorescence of Au NCs (left) and Fe3 O4 @Au NC (right) of the dried sample. (B) TEM image showing the core shell structure of Fe3 O4 @Au NC (Inset: enlarged image of it). (C) Magnetic hysteresis loops of (a) Fe3 O4 , (b) Fe3 O4 @Au NC deposited on a cover slip by heating at 100 ◦ C for 1 min.
chloroaurate (HAuCl4 ·3H2 O) and Arginine were bought from Alfa Aesar. PANC-1 cells and L-929 cells were received from National Centre for Cell Science, Pune. The chemicals, otherwise not mentioned were obtained from Merck. All the reagents were of analytical grade and used without further purification. Millipore water was used in all the experiments.
2.2. Synthesis of Au NCs The Au NCs were synthesized according to a previously reported method with slight modification [30]. Briefly, 250 mg of BSA dissolved in 5 ml of water was transferred to 5 ml of HAuCl4 (10 mM) with continuous stirring (600 rpm) and add 0.5 ml of 1 M NaOH after 2 min. After stirring (600 rpm) for 5 min, the solution was irradi-
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Fig. 4. (A) High resolution XPS spectra of (A) Au 4f5/2 , Au 4f7/2 , (B) C 1s, (C) O 1s; and (D) high resolution and its deconvolution XPS spectra for the Fe 2p3/2 and Fe 2p1/2 signal of the sample containing Fe3 O4 @Au NCs.
ated with microwave radiation (800 W) for about 50 s in a domestic microwave oven. The formation of BSA-Au NCs was indicated by the change in color to deep brown with the emission of deep red light under a UV lamp at 365 nm and the solution is used for the synthesis of Fe3 O4 @Au NCs nanocomposite. 2.3. Synthesis of Fe3 O4 The Fe3 O4 magnetic nanoparticles (MNPs) were synthesized using previously reported method with minor modification [31]. In a typical procedure, 50 ml of 1 mM aqueous solution of Arginine was added to 20 ml of 80 mM FeSO4 (NH4 )2 SO4 ·6H2 O under stirring (1800 rpm). After 1 min, 10 ml of 25% ammonia was added to the mixture with constant stirring (1800 rpm) for 1 h at 25 ◦ C. The synthesized solution was used for the preparation of Fe3 O4 @Au NCs nanocomposite and Fe3 O4 MNPs were separated magnetically, and after decantation dried in vacuum oven at a temperature of 50 ◦ C for further analysis. 2.4. Synthesis of Fe3 O4 @Au NCs In brief, 200 l of Fe3 O4 MNPs were added to 5 ml phosphate buffer (PBS) at pH = 7.4 with vigorous stirring (2500 rpm), and mixed with 5 ml of Au NCs. The mixture was sonicated for 1 h, dried in hot air oven at 100 ◦ C (1 min) for AFM analysis.
2.6. Release of erlotinib from Fe3 O4 @Au NCs@erlotinib The release of erlotinib was done in PBS buffer of different pH = 7.4 and pH = 5.0. Briefly, 250 l of Fe3 O4 @Au NCs@erlotinib were suspended in 0.5 ml PBS buffer in cellulose dialysis membrane tied at both ends. The cellulose dialysis membrane was then immersed in a 10 ml different pH buffered solutions at 37 ◦ C with continuous stirring (300 rpm). The amount of erlotinib released was estimated at =247 nm in a regular interval of time with erlotinib as control.
2.7. Cell viability assay Cell viability study was done using MTT assay and apoptosis assay [22] towards L-929 (mouse lung fibroblast cell line) and PANC-1 (pancreatic cell line). Briefly, either 4 × 103 L-929 and 3 × 103 PANC-1 cells were seeded into 96-well plates and incubated for 24 h. The Fe3 O4 @AuNCs@erlotinib was added to the cells in a 24 well plates to give effective erlotinib concentration of 5, 10, 25, 50 and 80 M. Then the cells were incubated for 24 h and then washed it with PBS. After treating the cells with 200 l DMEM, 20 ml MTT stock solution were added to each well and incubated for 5 h at 37 ◦ C. The culture medium was removed and 150 l of DMSO was added. After shaking the mixture for 5 min, the cell viability was studied and expressed by comparing with the cells incubated in culture medium alone.
2.5. Synthesis of Fe3 O4 @Au NCs@erlotinib Briefly, 250 l of 20 M erlotinib was added to 5 ml of Fe3 O4 @Au NC with constant stirring (1800 rpm) for 1 day at a temperature of 25 ◦ C. The non-conjugated erlotinib was removed from the solution and the solution is sterilized for further analysis. The drug loading content and loading efficiency was determined spectrophotometrically.
2.8. Confocal microscopy imaging The Fe3 O4 @Au NCs@erlotinib solution (50 g mL−1 ) was dispersed in a serum-free DMEM cell solution and incubated for 4 h at 37 ◦ C in the presence of 5% CO2 . The cells in the culture medium after washed three times with PBS, fixed with 4% paraformaldehyde in PBS medium for 1 h.
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Fig. 5. (A) Time-resolved fluorescence life time analysis of (a) Au NC and (b) Fe3 O4 @AuNC. (B) Release profiles of erlotinib from the Fe3 O4 @AuNC@erlotinib in the PBS buffer mimicking extracellular environment of pH = 7.4 and pH = 5.0. (C) Cell viability of L-929 mouse lung fibroblast cells and PANC-1 pancreatic cells in cell medium for 24 h after incubation of different concentrations of Fe3 O4 @AuNC@erlotinib as analyzed by MTT assay.
3. Results and discussion The synthesis process for Fe3 O4 @Au NCs@erlotinib is illustrated in Scheme 1. The overall synthesis process involved four steps consisting of (i) synthesis of Au NCs, (ii) synthesis of Fe3 O4 , (iii) their combination and (iv) conjugation of erlotinib. We first synthesized Au NCs by microwave assisted heating following the previously reported method with slight modification using bovine serum albumin (BSA) as the template. The as-prepared Au NCs exhibit emission peak at ∼ 640 nm via excitation at ∼ 480 nm. The intensity of the emission and excitation peak first increases and then decreases as the power of the microwave increases (Fig.S1 and Fig.S2). The decrease in intensity at high microwave power is due to the evaporation of the BSA template during microwave irradiation. Here a microwave power of 800 W for a time 50 s is optimized for further experiments. The positively charged amino acids like Arginine and Lysine present in the BSA makes the co-ordination of AuCl4 − ions with BSA, the trypsin/tryptophan residues reduce the Au ions and the stability is due to the presence of thiol groups containing 35 cysteine residues present in it [32].
Fig. 1A show the UV–vis absorbance spectra of BSA, HAuCl4 , Au NCs and Fe3 O4 @Au NCs, in which BSA has a sharp peak at 280 nm, Au NCs and Fe3 O4 @Au NCs have a broad absorbance at 510 nm. Fig. 1B shows excitation and emission spectra of Au NCs (a, c) and Fe3 O4 @Au NCs (b, d) respectively. Even though, Fe3+ ion is well known as a quencher of fluorescence, incorporation of Fe3 O4 cause only slight quenching of the fluorescence of Au NCs. The deep brown solution of Au NCs emitted an orange red colored fluorescence (Fig. 1C (a)) and an intense red colored fluorescence (Fig. 1C (b)) under UV radiation at 330 nm and 365 nm respectively. The TEM images as shown in Fig. 1(D) indicates that the Au nanoclusters are monodispersed and exhibit an average size of 4.0 ± 0.3 nm without aggregation of the cluster. The size distribution and the hydrodynamic diameter (Fig.S2(A)) from the DLS measurement reveals a mean size of 18.5 ± 2.5 nm further indicates the formation of well dispersed nanoclusters [33]. The stability of Au NCs in solution phase is monitored for 30 days and found that there is not much decrease in intensity, ensuring the stability of Au NCs for prolonged storage. We synthesized Fe3 O4 nanoparticles by previously reported method using the amino acid, Arginine as the reducing agent. The amino acid, Arginine has isoelectric point pI = 10.76, as it has a positive charge at pH = 7.4, which is made use for the synthesis of Fe3 O4 @Au NCs, where the isoelectric point of BSA is pI = 4.7 [34] and hence it has a negative charge at pH = 7.4. It ensures the required electrostatic attraction between Fe3 O4 enveloped by positively charged arginine and negatively charged BSA capped Au NC. Fig. 2(A) shows the photograph of the HAuCl4 , Au NCs and Fe3 O4 @Au NCs in the Visible light (a, b & c) and UV radiation (d, e & f), in which it is clear that the presence of Fe3 O4 does not diminishes the fluorescence property of Au NCs. Fig. 2(B) shows the photograph illustrating the magnetic property of Fe3 O4 in the synthesized wet state (a), its dried state (b) and magnetic property of it in the dried state in the vicinity of an external magnetic field (c). As depicted in Fig. 2(C), the XRD pattern shows peak at 30.29◦ , 35.59◦ , 43.25◦ , 47.26◦ , 53.84◦ , 57.13◦ and 62.79◦ 2 that can be assigned to the 220, 311, 400, 331, 422, 511 and 440 reflections (JCPDS 65–3107) respectively. All the diffraction peaks of the samples corresponds to a face-centred cubic phase with the lattice constant a = b = c = 8.425 Å and ␣=  = ␥ = 90◦ . The average particle size of Fe3 O4 nanoparticles based on the Fe (311) peak of the XRD pattern was calculated with the Scherrer’s formula to be 12.3 nm, which is in close agreement with the size as shown in the TEM image. TEM image of Fig. 2(D) demonstrate that these nanoparticles have diameter of 11.2 nm to 13.5 nm. The lattice spacing in the HRTEM (Fig. 2(E)) image of the nanoparticle measures at 0.23 nm corresponding to the (220) plane of the Fe3 O4 nanoparticle. SAED pattern of the sample as shown in Fig. 2(F) displays several diffraction rings indexed to 111, 220, 311, 400, 331, 422, 511, 440 and 533, indicative of the polycrystalline nature of Fe3 O4 [35]. The size distribution and the hydrodynamic diameter of Fe3 O4 as shown in Fig.S2 (B) indicate a mean diameter of 14.6 ± 2 nm. The Zeta potential measurement reveals that the charge of Fe3 O4 is about +7.6 which are suitable for the formation of the core shell structure of Fe3 O4 @Au NCs (Fig.S3 (B)). As shown in Fig. 3(A), the dried Fe3 O4 @Au NCs show intense red fluorescence under 365 nm UV light. On prolonged storage, Fe3 O4 @Au NCs leads to sedimentation and has to be sonicated and dispersed prior to use. The TEM image as shown in Fig. 3(B) shows a core shell structure with Fe3 O4 as the core and Au NCs as the corona. The size distribution from the DLS measurement is narrow with a mean diameter of 32.3 ± 3.5 nm. The TEM image and the DLS data indicates a monodispersed Fe3 O4 @Au NC nanoparticle. Fig. 3(C) shows the magnetic M-H hysteresis loop of Fe3 O4 and Fe3 O4 @Au NCs, which suggests that Fe3 O4 has a saturation magnetization of 48.0 emug−1 and has almost zero coercivity and remanence
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Fig. 6. Confocal laser images of L-929 and PANC-1 cell lines. First row (a) fluorescence image, (b) bright field image of control L-929 cells (mouse lung fibroblast cell). Second row (c) fluorescence image, (d) bright field image, (e) overlapped image of L-929 cells treated with Fe3 O4 @AuNC@erlotinib. Third row (f) fluorescence image, (g) bright field image of control PANC-1 cells (pancreatic cancer cell). Fourth row (h) fluorescence image, (i) bright field image, (j) overlapped image of PANC-1 cells treated with Fe3 O4 @AuNC@erlotinib.
indicating the superparamagnetic nature of Fe3 O4 . Moreover the saturation magnetization of Fe3 O4 @Au NCs decreases slightly to 34.168 emug−1 and the coercivity and remanence remains zero. The slight decrease in the saturation magnetization occurs due to the changes in surface spin effect on Fe3 O4 caused by the electrostatic attraction of Au NCs around it. Even after enveloping with Au NCs, the Fe3 O4 retains its superparamagnetic behaviour evidenced by the fact that the Fe3 O4 @Au NCs can be magnetically collected using hand held external magnet. The AFM 3D images as shown in Fig. 3(D) indicate the random distribution of spike-like features owing to Au NCs. The spike density reveals the stability of Au NCs in the BSA template and the height of the AFM images indicates the agglomeration of the BSA stabilized Au NCs which occurs due to
the denaturation of the protein while heating and drying. The morphology of Fe3 O4 @Au NCs is further analyzed using AFM analysis (Fig. 3(E) which reveals the appearance of more spikes attributed to the increase in the density of the particles by the formation of coreshell structure [36]. Moreover the height of the spikes is around 19.7 nm indicating the BSA stabilized Au NCs retains its properties after heating and drying at 80 ◦ C which is in agreement with the size distribution of the Fe3 O4 @Au NCs from the TEM image and DLS measurements. To analyze the detailed electronic structure of Fe3 O4 @Au NCs, XPS was done to explore the oxidation state of Au, C, O and Fe atoms and the results are shown in Fig. 4. The peak centred at 83.69 eV as a result of the binding energy of 4f7/2 is close to the characteristic
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peak of Au(0) in metallic gold, attributed to the presence of Au(0) core of the gold nanocluster. The second peak appears at 87.35 eV which arises as a result of the 4f5/2 orbital. The peak at 87.35 eV suggests the presence of Au(I) on the surface, which favours the stability offered by 32 cysteine residues of the BSA as capping agent. The results indicate the existence of Au(0)-Au(I) core-shelled structure in the Fe3 O4 @AuNC nanocomposite. The C 1 s peaks at 284.67 eV (sharp), 287.86 eV (satellite peak) and 289.11 eV (sharp) corresponds to C C (sp2 ), C O and O C O or C N bonds of the Arginine as well as the amino acid residues of the BSA[37]. Here O 1 s peaks at 523.21 eV (broad), 531.02 eV (sharp) and 535.56 eV(broad, satellite peak) occurs due to hydroxyl group, C O bonding and metal-oxygen bond respectively. The Fe 2p deconvoluted peaks at 707.53 eV and 709.88 eV are due to Fe(II)-O and Fe(III)-O bonds in magnetite. The sharp peak at 711.15 eV is due to 2p3/2 and the broad peaks at 713.11 eV and 714.38 eV indicates the presence of Fe-OH or Fe-OOC interactions at the surface of magnetite nanoparicle. Here a couple of broad peaks in the energy interval of 715–720 eV are the satellite peaks of Fe(II) and Fe(III) ions, which may be due to the interaction of Fe3 O4 with Au NCs. The characteristics binding energy observed at 723.5 eV are attributed to 2p1/2 orbital[38]. As per the XPS analysis results, the atomic concentration and mass concentrations of Fe based on the intensity of the corresponding peaks are 0.11% and 1.6% and that of gold are 0.13% and 1.81% respectively. In contrast to this, the atomic and mass concentrations of Fe determined based on the area of the peaks are 0.18% and 2.53% and that of Au are 0.20% and 2.76% respectively. To investigate the effect of Fe3 O4 on the fluorescence properties of Au NCs, the fluorescence life time measurements were conducted. Fig. 4(A) shows the fluorescent emission life time decay curve depicting a bi-exponential pattern. The fluorescence life time of Au NCs was 1.2 s and Fe3 O4 @Au NCs is 1.17 s. The relatively high life time of Au NCs [39] has attributed to the existence of slow component of triplet formation and fast component of Au (I) surface with Au (0) core of the BSA capped Au NCs [32]. The incorporation of Fe3 O4 only marginally shifts the fluorescence life time of Au NCs from 1.2 s to 1.17 s. The core-shell Fe3 O4 @Au NCs with negatively charged shell are conjugated to the positively charged erlotinib by electrostatic attraction. The amount of erlotinib loaded in the Fe3 O4 @Au NCs nanocomposites determined by spectrophotometrically is 8.093 g/mL and the loading efficiency is about 94.12%. The high loading efficiency attained due to the strong electrostatic interaction between positively charged erlotinib and negatively charged Fe3 O4 @Au NCs nanocomposite. The size of the Fe3 O4 @Au NCs@erlotinib as indicated by the DLS measurement is about 41.8 ± 2.4 nm. The in-vitro drug release study of erlotinib was done in physiological environments mimicking PBS buffers with pH = 7.4 and 5.0 at 37 ◦ C by measuring the absorbance at = 247 nm following dialysis. Fig. 4(B) exhibits the in-vitro release profiles of Fe3 O4 @Au NCs@erlotinib for erlotinib. At pH of 5.0, about 78.3% of drugs (erlotinib) get released in comparison with 52.1% release at pH of 7.4. The two release profile are significant, owing to the reduce release of drug while circulating in the blood till it reaches the targeted site where the pH of the cancer cells is less. Moreover, the result suggests that the Fe3 O4 @Au NCs@erlotinib exhibit a pH triggered drug release, indicating the ability of the material for drug delivery to the cancer cells. To investigate the targeting ability of Fe3 O4 @Au NCs@erlotinib, we tested the viability of a normal cell, L-929 and a pancreatic cancer cell, PANC-1 using MTT assay and apoptosis assay. PANC1 cells over expresses the EGFR where L-929 cells do not over expresses the EGFR. As shown in Fig. 4 (C), erlotinib exhibit a cytotoxic effect on PANC-1 in a dose dependent manner but it does not cause much cytotoxic effect to L-929 cell even at high doses of Fe3 O4 @Au NCs@erlotinib. Here the specific cytotoxic nature of
Fe3 O4 @Au NCs@erlotinib nanocomposites on PANC-1 and nontoxicity of it on L-929 normal cell indicate that the cytotoxicity arises due to erlotinib, where it is a targeting agent of PANC-1 cells. This result suggests that Fe3 O4 @Au NCs@erlotinib is effective for the selective targeting of EGFR over expressed pancreatic cancer cells and the free Fe3 O4 @Au NCs is biocompatible in nature. To validate the effectiveness of Fe3 O4 @Au NCs@erlotinib, after 12 h of incubation, we examined the intracellular distribution of it in cells using confocal fluorescence microscopy and the images are shown in Fig. 5. From the bright field image, it is clear that L-929 cell maintained their normal morphology with some bright red spot of fluorescence arising from Fe3 O4 @AuNC@erlotinib appear outside the cells in the fluorescence image. The overlapped image consists of red fluorescent spots outside the cells, indicating the biocompatibility of the nanoparticles towards the normal cells. On the other hand, the bright field image of PANC-1 cells shows that the morphology of the cells are affected and in the fluorescence image, the intense red fluorescence is observed in the cytoplasm and nucleus of the cells. It further confirmed by observing the overlapped image, indicating the cellular uptake of EGFR expressed PANC-1 cells. All these results show that Fe3 O4 @Au NCs@erlotinib can be adaptable for imaging of cancer cell prior to its surgical removal (Fig. 6). 4. Conclusion This study investigates three main properties (i) superparamagnetic behaviour (ii) fluorescence property (iii) targeting ability of Fe3 O4 @Au NCs@erlotinib. Herein, we used the amino acid, Arginine as the reducing agent for Fe3 O4 formation and BSA for Au NC formation which maintains the biocompatible nature of the material for bioimaging. Moreover, the presence of erlotinib ensures the targeting ability of the composite (Fe3 O4 @Au NCs@erlotinib) towards pancreatic cancer cells. The relatively high life time of the probe shows the promising nature of the nanocomposite for optical imaging and the currently employed superparamagnetic nature of Fe3 O4 ensures its potential for simultaneous T1 and T2 contrast agents. Acknowledgement The authors thank the Professor and Head, Department of Chemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram for pursuing the platform to conduct the research. The authors also thank the Director, SICC, University of Kerala, Kariavattom campus, Thiruvananthapuram; Director, SAIF-STIC-CUSAT, Kochi; RGCB, Thiruvananthapuram, DST-SAIF, M.G. University, Kottayam. The author N.J. acknowledge support for this work by University Grants Commission, Bangalore, India through the teacher fellowship (F.No.FIP/12th plan/KLMG035, TF: 03) under faculty development programme during XIIth plan period. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2017.11.017. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA: A Cancer Journal for Clinicians 67 (2017) (2017) 7–30. [2] C.G. England, R. Hernandez, S.B.Z. Eddine, W. Cai, Molecular imaging of pancreatic cancer with antibodies, Mol. Pharm. 13 (2016) 8–24. [3] L. Rahib, B.D. Smith, R. Aizenberg, A.B. Rosenzweig, J.M. Fleshman, L.M. Matrisian, Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States, Cancer Res. 74 (2014) 2913–2921. [4] J. Long, Y. Zhang, X. Yu, J. Yang, D.G. LeBrun, C. Chen, et al., Overcoming drug resistance in pancreatic cancer, Expert Opin. Ther. Targets 15 (2011) 817–828.
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Biographies Nebu John is an Assistant professor of chemistry at Mar Thoma college, Tiruvalla, Kerala, India. He is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. His research interest includes magnetoplasmonic nanoparticle and bioanalytical applications. Anjali Devi J.S. (DST-INSPIRE fellow) is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Aparna R.S. is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Abha K. is an Assistant professor of chemistry at Bishop Moore College, Mavelikkara, Kerala, India. She is currently working under the guidance of Dr. Sony George as a Ph.D. student at University of Kerala. Sony George had his Ph.D. at Department of Chemistry, University of Kerala, Kerala, India and he is currently working as an Assistant Professor in the same institution. His area of interest is magnetic and fluorescent nanomaterials for sensoronic and bioimaging. He had been a visiting professor at A. A. Athinoula centre for Biomedical imaging, MGH affiliated to Harward Medical school. He had sabbatical stint at Department of Chemical physics, LUND University, Sweden.