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Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery
Journal Pre-proof Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery
Srinivasan Ayyanaar, Mookkandi Palsamy Kesavan, Chandrasekar Balachandran, Swetha Rasala, Perumal Rameshkumar, Shin Aoki, Jegathalaprathaban Rajesh, Thomas J. Webster, Gurusamy Rajagopal PII:
S1549-9634(19)30218-7
DOI:
https://doi.org/10.1016/j.nano.2019.102134
Reference:
NANO 102134
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Revised date:
19 November 2019
Please cite this article as: S. Ayyanaar, M.P. Kesavan, C. Balachandran, et al., Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/ 10.1016/j.nano.2019.102134
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© 2019 Published by Elsevier.
Journal Pre-proof
Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery Srinivasan Ayyanaar, MSca, Mookkandi Palsamy Kesavan, PhDb, Chandrasekar Balachandran, PhDc, Swetha Rasala, BPharmd, Perumal Rameshkumare, Shin Aoki, PhDd,e, Jegathalaprathaban Rajesh, PhDf, *, Thomas J. Webster, PhDg,*, and Gurusamy Rajagopal, PhDa, * PG and Research Department of Chemistry, Chikkanna Government Arts College, Tiruppur 641 602, Tamilnadu, India.
b
Department of Chemistry, Hajee Karutha Rowther Howdia College, Uthamapalayam-625 533, Tamil Nadu, India.
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a
c
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
e
Department of Chemistry, Kalasalingam Academy of Research and Education, Krishnankoil-626 126, Tamil Nadu, India.
Research Institute of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
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f
Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway H91 W2TY, Ireland.
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d
Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai-623 806, Tamil Nadu, India.
h
Department of Chemical Engineering, Northeastern University, Boston, MA 02115 USA.
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* Corresponding authors. E-mail addresses: [email protected] (J. Rajesh), [email protected] (G.
Word count for abstract: 140
Number of figures: 7
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Number of tables: 1
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Number of references: 60
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Word count for manuscript: 3944
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Rajagopal), [email protected] (T.J. Webster)
Number of Supplementary online-only files, if any: 1
Conflicts of Interest: There are no conflicts to declare.
This work was supported by DST-SERB (Ref. No: SB/FT/CS-130/2012) and the authors sincerely thank the Chikkanna Government Arts College, Tiruppur and Mohamed Sathak Engineering College, Kilakarai for their lab and instrumental facilities.
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Journal Pre-proof Abstract This study describes a sensitive reactive oxygen species (ROS)-responsive lecithin (LEC) incorporated iron oxide nanoparticle (Fe3O4 NP) system with potent anti-inflammatory properties and even more so when the antioxidant drug curcumin (CUR) is encapsulated in the PLGA hybrid magnetic microsphere system (Fe3O4@LEC-CUR-PLGA-MMS). The delivery system
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is responsive to ROS including an H2O2 environment to release the payload (CUR) drug. Greater cytotoxicity properties were observed in the presence of Fe3O4@LEC-CUR-PLGA-MMS against
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A549 and HeLa S3 cells with IC50 values after 24 h of 10 and 12 µg/mL and 10 and 20 µg/mL,
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respectively. The present Fe3O4@LEC-CUR-PLGA-MMS system demonstrated much better in
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vitro cytotoxicity, cellular morphological changes and moreover an ability to limit colony
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formation for A549 and HeLa S3 cancer cell lines than non-cancerous cells, and thus, should be
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further studied for a wide range of medical applications.
Keywords: poly(D, L-lactide-co-glycolic acid); lecithin; magnetic core microsphere; cytotoxicity; targeted drug delivery.
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Background
The survival of reactive oxygen species (ROS) homeostatically in the human body plays a key role in various human physiological processes [1-3]. Oxidative stress related diseases can be diagnosed and cured with the aid of ROS since ROS can serve both as pathological markers [4-6] and as therapeutic agents [7-12]. This motivates the development of various ROS responsive
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biological and chemical sensors, prodrugs, imaging agents, and ROS responsive polymeric
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systems [13-20]. Moreover, ROS causes harm to macromolecular substances such as nucleic
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acids, lipids and can cause cell apoptosis by delaying cell differentiation [21]. The main
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consequence of ROS includes the presence of oxidative stress and reduction of pH [22, 23]. ROS
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including H2O2 derived oxidants are highly reactive with genetic molecules of DNA, plasma proteins, and lipids, which cause significant damage to cells [24]. The toxic effect of excessive
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ROS is consequently associated with an array of pathological conditions, including cancer,
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aging, diabetes, cardiovascular diseases, infection, as well as neurodegenerative diseases [25].
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Furthermore, biogenic methods utilizing bio-waste materials have recently been reported for the production of Fe3O4 NP [26,27]. One of them is a watermelon rind which is a rich source of polyphenols, citrulline (amino acid), cellulose (carbohydrate), as well as carotenoids [28,29] and they can function as both reducing and capping agents in the formation of Fe3O4 NP [30,31]. It is known that the magnetic Fe 3O4 NP can carry a drug to a specific target site rapidly based on an external magnetic field and has the potential to release drugs in a controlled manner. Magnetic targeted therapy has associated advantages because of its effectiveness, low toxicity, biocompatibility, targeting and high drug carrying capacity [32-35].
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Journal Pre-proof Among others, Fe3O4 NP are reactive towards ROS (including H2O2) and play an important role in biomedical applications including targeted drug delivery. Similarly, curcumin (CUR), isolated from the rhizome of turmeric, possesses potent antiinflammatory and antioxidant activities and it is hydrophobic and fluorescent in nature. Even though CUR molecules were found to be unstable under different physical and chemical
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environments like pH, alkali medium, heat or light [36-38], they have interesting clinical applications for various therapeutic interventions. Moreover, CUR encapsulated polymeric
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particles which are lipophilic, hydrophilic and composed of phospholipids have demonstrated
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potential applications for controlled drug release systems [39, 40].
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In recent years, polyethylene glycol (PEG) [41], polylactic acid (PLA), polyvinyl alcohol
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(PVA) [42] and chitosan (CS) [43] have been widely used in biomedical applications owing to their excellent biodegradation properties inside biological systems. Specifically, PLGA has been
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widely used as a bioactive and biocompatible material for different biomedical applications,
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such as controlled release drug delivery applications [44]. Compared to other polymers, PLGA
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microspheres are highly preferred for drug delivery applications because of their lower toxicity, high stability and higher encapsulation efficiency [45]. Herein, we report for the first time the biogenic synthesis of Fe3O4 NP using watermelon rind extract as a reducing and stabilizing agent, and the incorporation of the NP into ROS sensitive LEC for drug delivery applications. As a potent anti-inflammatory drug, CUR was encapsulated in the PLGA-MMS system of Fe3O4@LEC-CUR-PLGA-MMS and were fabricated using a double emulsion (water-in-oil-inwater) method. The Fe3O4@LEC-CUR-PLGA-MMS showed magnetic targeting ROS (including H2O2) activated drug release in cancer cells at different pH values. Moreover, an AO/EB staining
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Journal Pre-proof assay showed the cytotoxicity of A549 and HeLaS3 cancer cells treated with CUR and the Fe3O4@LEC-CUR-PLGA-MMS system. Methods Chemicals Iron (III) chloride hexahydrate (FeCl3·6H2O), iron (II) chloride tetrahydrate (FeCl2·4H2O),
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sodium acetate, lecithin (LEC), and sodium oleate were obtained from Alfa Aesar. Poly (D, L-
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lactide-co-glycolic acid) (PLGA, MW = 24,000 Da), poly ethylene glycol (PEG, MW = 50,000 Da)
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and curcumin (CUR) were received from Sigma Aldrich. Watermelon was collected from a local market. Deionized Millipore water was used throughout the experiments to prepare solutions.
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Organic solvents of analytical grade were used in the experiments. Preparation of watermelon rind extract
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A fresh ripen watermelon fruit was washed with milli-Q water to remove dust particles
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on its surface. Subsequently, the pulp from watermelon pieces was separated to obtain the
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watermelon rind. The rinds were sliced into small portions and dried at room temperature for 20 days under a dust free zone. The dried watermelon rinds were smashed and carefully weighed (15 g) using an analytical balance, and were transferred into 100 mL of water contained in a neat 250 mL round-bottom flask. The above mixture was allowed to reflux at 70 o
C until it became yellow [46]. The obtained yellow colored mixture was cooled down to normal
temperature, and then filtered using cheesecloth. The mixture was carefully stored at ice-cold conditions until needed for further analysis. Biogenic synthesis of Fe3O4 NP
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Journal Pre-proof The synthesis of Fe3O4 NP involved the solidification of FeCl3∙6H2O (4 mmol, 1.08 g), FeCl2·4H2O (2 mmol, 0.4 g) and sodium acetate (3.28 g, 40 mmol) in 40 mL of instantly prepared rind extract with continuous stirring for 2 h at 80 °C until the mixture turned black. Sodium acetate can serve as an oxygen source for the formation of Fe 3O4 NP and the solution may act as a reducing and capping agent. The obtained product was washed with ethanol followed by
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water and then was dried under vacuum at 95oC overnight. The product was primarily confirmed by using an external magnetic field.
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Synthesis of Fe3O4@LEC NP
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The surface of Fe3O4 NP was functionalized with oleate by vigorously stirring an aqueous
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solution of sodium oleate (3.0 g) and Fe3O4 NP at room temperature for 2 h as the surface
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became hydrophobic. Furthermore, the lecithin polymer (1.5 g) was slowly dropped onto the surface of the modified Fe3O4 NP suspension. The suspension was washed with copious
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amounts of water and ethanol to remove unreacted particles and then the pH of the
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under vacuum.
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suspension was adjusted to neutral. Finally, the Fe3O4@LEC NP (oil–base surface) were dried
Synthesis of Fe3O4@LEC-CUR-PLGA-MMS Fe3O4@LEC-CUR-PLGA-MMS was prepared using a double emulsion formation method (water-in-oil, oil-in-water) (Scheme 1). Firstly, 5.5 mL of dichloromethane (DCM) (organic phase, O) containing dissolved PLGA (0.6 g) and CUR (10 mg) was mixed with 0.2 mL doubly distilled water containing 12.5 mg Fe3O4@LEC NP (oil–base surface) (inner aqueous phase, W1). Subsequently, the W1/O emulsion was transferred into 100 mL of a 1% PEG solution (outer aqueous phase, W2) under magnetic stirring at 2200 rpm for 30 min. The resulting W1/O/W2
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Journal Pre-proof emulsion was continuously stirred for 5 h to evaporate DCM. Finally, the MMS were repeatedly washed with distilled water with the aid of centrifugation at 5000 rpm for 10 min each. The excess unabsorbed drug molecules present on the surface of the microspheres were removed while washing with deionized water. The Fe3O4@LEC-CUR-PLGA-MMS was collected in powder form for further analyses [47].
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Characterization of Fe3O4@LEC-CUR-PLGA-MMS An X-ray crystallographic study of Fe3O4NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-
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MMS was performed using an X-ray diffractometer, D/Max-IIIC, Japan (Cu-kα radiation, λ =
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1.5406 Å). The position of the crystal peaks was matched to the standard JCDPS files to identify
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their crystalline structure. For functional group identification, Fourier transform Infra-Red (FTIR)
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spectra were recorded for Fe3O4 NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-CURPLGA-MMS on an Avator360, America, spectrophotometer. The surface structure of
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Fe3O4@LEC-CUR-PLGA-MMS was studied by using scanning electron microscopy (SEM, FEI
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QuanTa200, Holland). The magnetic microsphere-coated aluminum foil underwent gold–
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palladium mixture sputtering for SEM analysis. The morphology of Fe 3O4 NP and Fe3O4@LEC NP was analyzed by using a Philips CM12 TEM instrument at an accelerating voltage of 120 kV. The content of Fe3O4@LEC-CUR-PLGA-MMS was measured by Thermo gravimetric analysis (TGA) (Pyris 1, America). The measurement was performed under a N2 atmosphere with a heating rate of 20 °C/min between 50 and 700 °C. The magnetic properties of the Fe3O4@LEC-CURPLGA-MMS was studied using a vibrating sample magnetometer (VSM) (XL5 SQUID, America) and the magnetism values were calculated based on the results. Determination of drug encapsulation efficiency
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Journal Pre-proof The weight of CUR encapsulated in Fe3O4@LEC-CUR-PLGA-MMS was determined by dissolving it in DCM and was measured by using UV-Vis absorption characteristics. About 10 mg of Fe3O4@LEC-CUR-PLGA-MMS was placed in a sealed glass tube and was dissolved in DCM (1 mL); the solution was mixed with 10 mL of phosphate buffer (pH 7.4) under magnetic stirring at room temperature for 2 h. The solution was centrifuged at 5000 rpm for 10 min in order to
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separate the aqueous phase after the complete evaporation of DCM. The concentration of CUR in the buffer solution was determined using the UV-Vis absorption peak at 421 nm from a
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standard calibration curve. The drug content and encapsulation efficiency were calculated from
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In vitro drug release studies
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the following equations:
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A dialysis protocol was adopted for the determination of in vitro CUR release from the
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nanoparticles. For this, about 100 mg of Fe3O4@ LEC-CUR-PLGA-MMS was transferred into dialysis tubing (MWCO: 10 KDa) quickly and the tubing was immersed in 10 mL of phosphate buffer (pH 7.4/ 5.4 and 7.4/ 5.4 with 1.25 μM H2O2) under stirring at 37 oC in the dark. At different time intervals, 1.0 mL of the dialysis solution was removed and the concentration of CUR released from Fe3O4@LEC- CUR-PLGA-MMS was determined by using UV-Vis measurements (at 421 nm). The total volume was maintained by refilling with the same volume of fresh buffer. Moreover, the drug releasing efficiency of the Fe 3O4@LEC-CUR-PLGA-MMS was determined as follows [48]:
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Journal Pre-proof ( ) where,
is the concentration of CUR released from Fe3O4@LEC-CUR-PLGA-MMS at time .
is
the initial concentration of CUR encapsulated in the Fe3O4@LEC-CUR-PLGA-MMS. In vitro cytotoxicity studies MTT assay
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MTT assays were carried out using the following method [49-52]. Briefly, A549, HeLa S3,
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IMR-90 and MCF-7 cells were preserved in Dulbecco’s Modified Eagle’s Medium (DMEM)
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containing 5% CO2 at 37 ºC. In order to adhere the cells, a seeding density of 20,000 cells per
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well were plated in 96 well plates and incubated overnight. Then, the cells underwent a treatment with different concentrations of nanoparticles (0.78 to 50 µg/mL) for 24 h. After the
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24 h treatment, to determine cell viability, 10 µL of the MTT (5 mg mL -1) (3-(4,5-dimethylthiazol-
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2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well and incubated for 3-4
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h. 100 μL of a formazanlysis solution (10% SDS in 0.1N HCl) was introduced and the data was measured (at 570 nm) (BIO-RAD).
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Fluorescence images using AO/EB stain Blebbing and nuclear condensation was carried out using A549 and HeLa S3 cells in order to investigate morphological changes due to the nanoparticles. A549 and HeLa S3 cells with a density of 1×105 were plated in 35 cm plates and incubated overnight to allow for cell adhesion. Then, cells were treated with Fe3O4@LEC-CUR-PLGA-MMS (15 µg/mL) for 24 h and were incubated with 10 µL of a AO/EB stain for 15 min in dark. A typical fluorescence image of A549 and HeLa S3 cells after treatment with Fe3O4@LEC-CUR-PLGA-MMS were captured using a Biorevo, BZ-9000, Keyence (20x) fluorescence microscope system [27]. 9
Journal Pre-proof Colony formation assay A549 and HeLa S3 cells/well at a density of 5×103 were plated into 24 well tissue culture plates and were incubated with 5% CO2 at 37 ºC overnight. The cells underwent treatment with Fe3O4@LEC-CUR-PLGA-MMS at different concentrations. Then, each well was washed twice with PBS after a 24 h treatment and was incubated with freshly prepared DMEM for 10 days.
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Staining of the cells was completed using a crystal violet solution for 15 min and washed with H2O. The data was interpreted by ImageJ software using colony area.
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Statistical analysis
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Results are summarized as the mean ± SD and Origin Pro 8.1 using P <0.05 as statistically
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significant.
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Results
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FTIR and crystalline phase characterization studies The successful grafting of Fe3O4NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-
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CUR-PLGA-MMS as confirmed by FTIR analysis is shown in Fig. 2 (A). In order to prove the
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successful fabrication, the corresponding functional groups on each sample were determined. A band obtained at 584 cm−1 for the Fe3O4 NP is characteristic of Fe-O stretching. The bands observed at 2945, 2834 and 1350 cm−1 for Fe3O4@LEC NP are attributed to the stretching vibration of –CH3 (or –CH2–) and bending vibration of–N–H bonds, respectively. The FTIR spectrum of the PLGA revealed the -OH stretching band at 3451 cm−1, the ester group (C-O-C) stretching band at 2828 cm −1and the methyl C-H band at 1613 and 954 cm −1 [53]. The spectrum from pure CUR exhibited the characteristic C=O and C-O-C stretching vibrations at 1345 and 1245 cm-1, respectively. The FT-IR analysis of Fe3O4@LEC-CUR-PLGA-MMS confirmed the
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Journal Pre-proof presence of Fe3O4 NP, Fe3O4@LEC NP, pure CUR and PLGA by showing the respective bands corresponding to the functional groups in the IR spectra. XRD analysis was performed to elucidate the crystallanity of Fe 3O4@LEC-CUR-PLGAMMS. The diffraction patterns of Fe3O4 NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS are shown in Fig. 2(B). The characteristic peaks of cubic spinel Fe3O4 NP were observed at 2θ values
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of 30.15°, 35.54°, 43.59°, 57.04°, and 63.09° which were assigned to the (220), (311), (400), (511), and (440) lattice planes of Fe3O4, showing a lattice parameter of 8.3 Å. The crystalline
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property of Fe3O4 NP in Fe3O4@LEC NP was retained even after loading of the lipophilic LEC on
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the surface of Fe3O4 NP. However, the polycrystalline nature of Fe3O4@LEC-CUR-PLGA-MMS is
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evidenced from the peaks that appeared at 20.63°and 25.35° [54]. On decorating the
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Fe3O4@LEC NP surface with CUR, PLGA and PEG increased the intensity of the polycrystalline peaks and further broaden the diffraction peaks of Fe 3O4 NP. This also provides evidence for
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the successful formation of Fe3O4@LEC-CUR-PLGA-MMS.
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Morphological characterization of Fe3O4@LEC-CUR-PLGA-MMS
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The surface topography of the Fe3O4@LEC-CUR-PLGA-MMS prepared by W1/O/W2 was analyzed using SEM (Fig. 3 (A)). The SEM micrographs showed spherical shaped Fe 3O4@LECCUR-PLGA-MMS exhibiting a smooth surface with an average diameter of 0.5 µm. As illustrated in Fig. 3 (B), EDS analysis showed the peaks for C, N, Fe, and O elements present in Fe 3O4@LECCUR-PLGA-MMS. The morphology of Fe3O4@LEC-CUR-PLGA-MMS was studied using TEM. The Fe3O4 NP were roughly spherical in shape with an average crystal diameter of 0.2 µm (Fig. 4 (A)). The TEM image in Fig. 4 (B) confirms the spherical shape of Fe3O4 NP and Fe3O4@LEC NP, and the successful incorporation of Fe3O4 NP into the polymer. Nevertheless, the synthesized
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Journal Pre-proof system showed that the Fe3O4 NP were adsorbed onto the surface of Fe3O4@LEC NP. Fig. 4 (C) shows the SAED pattern of Fe3O4@LEC-CUR-PLGA-MMS. The dotted ring patterns observed in SAED all matched well to the d-spacing values corresponding to the (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0) diffraction planes of spinel Fe3O4 NP [56]. This supports the formation of Fe3O4 NPs with high crystallanity.
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Thermal and magnetic property analysis To further characterize the novel NPs, TGA was performed on the Fe 3O4 NP, Fe3O4@LEC
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NP and Fe3O4@LEC-CUR-PLGA-MMS from an ambient temperature to 700 ºC with a 20 ºC min -1
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heating rate under a nitrogen atmosphere (Fig. 4 (D)). The removal of physically adsorbed
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water from Fe3O4 NP was observed up to 150 ºC and significant weight loss occurred at ~350 ºC
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[57]. In the case of Fe3O4@LEC NP, a slow weight loss (about 12 wt%) at <150 ºC was observed due to the removal of the solvent and a slight increase in weight loss which occurred between
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150 and 350 ºC. However, a greater weight loss was observed for Fe 3O4@LEC-CUR-PLGA-MMS
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between 150 and 500 ºC because of the decomposition of LEC, PLGA, PEG and CUR. The
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magnetic properties of Fe3O4 NP and Fe3O4@LEC-CUR-PLGA-MMS were studied using VSM at room temperature and the data is represented in Fig. 3 (C). The saturated values of magnetization of the Fe3O4 NP and the Fe3O4@LEC-CUR-PLGA-MMS were 86.05 emu/g and 47.09 emu/g, respectively. Such results indicate that the Fe3O4 NP retained their magnetism even after forming the composite material Fe3O4@LEC-CUR-PLGA-MMS. Fe3O4 NP have shown advantages in preventing the aggregation of polymer molecules and in assembling molecules rapidly in the absence of a magnetic field [55]. The appreciable higher magnization value of the Fe3O4@LEC-CUR-PLGA-MMS enables such materials to be applied for magnetically targeted
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Journal Pre-proof drug-delivery. As indicated in Fig. 3 (D), even though the Fe3O4@LEC-CUR-PLGA-MMS microspheres had relatively low saturation magnetization, they still possessed good magnetic mobility. This behavior ensures that drug loading would not significantly affect the magnetic properties of the Fe3O4@LEC-CUR-PLGA-MMS. In vitro drug releasing study
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The release profile of CUR from Fe3O4@LEC-CUR-PLGA-MMS was determined in PBS at both physiological pH (pH 7.4) and acidic pH (pH 5.4) values (Fig. S1 (A)). The release rate of
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CUR from Fe3O4@LEC-CUR-PLGA-MMS at pH 5.4 is faster than at pH 7.4 which confirmed the
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improved solubility of CUR in acidic medium [58,59]. Moreover, the cumulative CUR release
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was 63.18% at pH 5.4 and 39.40% at pH 7.4. The increased CUR release at pH 5.4 can be
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attributed to protonation and consequent degradation of hydrogen bonded PLGA and PEG polymer chains addicted to the monomers [38]. These processes facilitated the easy release of
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CUR from the PLGA layer. Therefore, Fe3O4@LEC-CUR-PLGA-MMS had a greater ability at pH 5.4
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than at pH 7.4 for drug release to cancer cells over a shorter period of time. Furthermore,
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under acidic conditions, the released CUR was biologically active and exhibited enhanced hydrophobic action under polar conditions because of its prolonged chain.
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concludes that the Fe3O4@LEC-CUR-PLGA-MMS system can be a suitable candidate for delivering hydrophobic drugs to pH targeted cancer cells in the presence of a magnetic field without any assistance from other parts of the body [60]. The ROS-responsiveness of Fe3O4@LEC-CUR-PLGA-MMS was evaluated in the presence of H2O2 in PBS, with an acidic pH of 5.4 and neutral pH of 7.4 at 37 °C. Fig. S1 (B) shows a CUR release in the presence of H2O2 at 89.28% at pH 5.4 compared to 69.70% at pH 7.4 of CUR. It is
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Journal Pre-proof assumed that only the PLGA layer degraded at pH 5.4 which led to the release of CUR, but in the presence of H2O2, the LEC layer also underwent degradation which enabled the abundant release of CUR from Fe3O4@LEC-CUR-PLGA-MMS. These results clearly showed that Fe3O4@LEC-CUR-PLGA-MMS exhibited a positive CUR release response capacity with the aid of ROS. Furthermore, the CUR release from Fe3O4@LEC-CUR-PLGA-MMS increased with respect to
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the increase in the blended concentration of PEG in the presence of PLGA. It was observed that the CUR released from Fe3O4@LEC-CUR-PLGA-MMS at pH 5.4 (H2O2
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at PBS) was high and in a controlled manner (Fig. S2). The CUR release in cancerous cells was
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found to be biologically active with preferential conformation under acidic conditions and in a
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prolonged chain form. Moreover, they served as powerful scavengers of free-radical oxidants
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via H+ donation as well as electron transfer, exerting antioxidant activity. However, the Fe3O4@LEC-CUR-PLGA-MMS exhibited CUR release at pH 5.4, indicating the nature of the
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loaded CUR, suggesting that Fe3O4@LEC-CUR-PLGA-MMS are potential drug carriers to deliver
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CUR on target to cancerous cells. Conversely, the CUR release mechanism from the Fe 3O4@LEC-
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CUR-PLGA-MMS at the targeted site needs further in vivo experiments for validation of these exicting in vitro results. Anticancer studies Cytotoxic properties of the present nanoparticles (Fe 3O4@LEC NP, CUR and Fe3O4@LECCUR-PLGA-MMS) against A549, HeLa S3 and MCF-7 cells were completed via MTT assays at various concentrations (1.5 to 50 µg/mL) for 24 h at 37º C. As shown in Fig.5 and table 1, the viability of A549, HeLa S3 and MCF-7 cells in the presence of Fe3O4@LEC-CUR-PLGA-MMS showed a superior cytotoxic property against A549 and HeLa S3 cells with IC 50 values at 24 h of
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Journal Pre-proof 10 and 12 µg/mL, respectively. However, CUR alone revealed only a modest cytotoxic activity against A549 and HeLa S3 cells with IC 50 values of 47.1 and 47.2 µg/mL, respectively. In addition, the viability of A549, HeLa S3 and MCF-7 cells did not change by using Fe3O4@LEC NP. A further toxicity study of CUR and Fe3O4@LEC-CUR-PLGA-MMS was conducted against human normal lung IMR-90 cells by an MTT assay (Fig. S3). Interestingly, both CUR and Fe3O4@LEC-
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CUR-PLGA-MMS did not show toxicity up to >50µg/mL in IMR-90 cells, thus, providing evidence
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of their selective killing of cancer not healthy cells.
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Furthermore, based on the cytotoxic study, nuclear fragmentation was studied by costaining with acridine orange and ethidium bromide (AO/EB) in A549 and HeLa S3 cells in order
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to detect the morphological changes of the nanoparticles. AO and EB were used as the
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indicators of live cells and dead cells, respectively. A549 and HeLa S3 cells were treated in the
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presence of Fe3O4@LEC-CUR-PLGA-MMS at 15 µg/mL for 24 h and images were taken with the help of AO/EB using a fluorescence microscope. As shown in Fig. 6, in the presence of
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Fe3O4@LEC-CUR-PLGA-MMS, a significantly augmented death of A549 and HeLa S3 cells was
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observed as indicated in red which is co-staining of EB. Moreover, the ability of Fe3O4@LECCUR-PLGA-MMS in controlling A549 and HeLa S3 colony formation was determined here. As shown in Fig.7, complete inhibition of colony formation was observed in the presence of Fe3O4@LEC-CUR-PLGA-MMS against A549 and HeLa S3 cells at 10 and 20 µg/mL, respectively. However, Fe3O4@LEC-CUR-PLGA-MMS did not affect colony formation at the lowest concentration (such as 5 and 10 µg/mL) in both cell lines, respectively (Fig. S4). Finally, herein, we concluded that Fe3O4@LEC-CUR-PLGA-MMS not only show cytotoxicity to cancer cells
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Journal Pre-proof (including morphological changes and an ability to control the colony formation in A549 and HeLa S3 cells) but were not toxic to healthy cells. Discussion Here, Fe3O4 NP were successfully prepared by a biogenic synthetic process using a watermelon rind extract without using a toxic chemical surfactant and reducing agent. The
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Fe3O4@LEC-CUR-PLGA-MMS were successfully synthesized by a double emulsion (water-in-oil,
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oil-in-water) method. The resultant product of Fe3O4@LEC-CUR-PLGA-MMS was analyzed by
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various spectral as well as microscopic techniques. Along with the CUR from Fe3O4@LEC-CURPLGA-MMS, is the ROS (including H2O2) triggered pH responsiveness for drug release at an
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acidic pH of 5.4 and basic pH of 7.4. Meanwhile, experiments from the MTT assay, fluorescence
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staining and colony formation assay revealed that the Fe3O4@LEC-CUR-PLGA-MMS can be a
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potential anticancer agent showing toxicity to cancer cells but cytocompatibilty to healthy cells. In summary, despite the existence of great challenges in the design of an ROS-responsive drug
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release mechanism, here one is demonstrated to efficiently deliver therapeutic agents to a
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disease site, with more cellular interactions to enhance therapeutic efficacy for suitable biomedical applications.
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Tables Table 1 IC50 values for CUR, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS against IMR-90, A549, MCF-7 and HeLa S3 cells. A549 (µg/mL)
HeLa S3 (µg/mL)
47.1
47.2
Fe3O4@LEC NP
>50
>50
Fe3O4@LEC-CUR-PLGA-MMS
10.9
12
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na
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Note- nt: not tested
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CUR
25
MCF-7 (µg/mL)
IMR-90 (µg/mL)
51.9
>50
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Compound
>50
Nt
30.1
>50
Journal Pre-proof Figure captions Fig. 1 Schematic illustrations showing the synthesis of Fe3O4@LEC-CUR-PLGA-MMS. Fig.2 (A) FT-IR analysis of Fe3O4 NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-CUR-PLGAMMS. (B) XRD patterns of Fe3O4 NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS. Fig.3 (A) SEM image and (B) EDS spectrum of Fe3O4@LEC-CUR-PLGA-MMS. (C) Room temperature hysteresis curves of magnetite with Fe 3O4NP and Fe3O4@LEC-CUR-PLGA-MMS. (D)
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Magnetic mobility of the microspheres with and without the drug.
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Fig.4 (A) TEM image of Fe3O4 NP and (B) Fe3O4@LEC NP (C) single sphere SAED pattern of
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Fe3O4@LEC-CUR-PLGA-MMS. (D) TGA curves of Fe3O4 NP, Fe3O4@LEC NP, and Fe3O4@LEC-CUR-
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PLGA-MMS.
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Fig. 5. Cytotoxic studies of Fe3O4@LEC NP, CUR and Fe3O4@LEC-CUR-PLGA-MMS against A549, HeLa S3 and MCF-7 cells. The experiment was conducted in triplicate and date was expressed
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in mean±SD with help of GrapPad Prism 6 software.
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Fig. 6 Typical luminescence microscopy images (Biorevo, BZ-9000, Keyence) of A549 and HeLa
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S3 cells treated with Fe3O4@LEC-CUR-PLGA-MMS (15 µg/mL) for 24 h at 37 oC. Scale bar 50µm. Fig. 7 Effect of Fe3O4@LEC-CUR-PLGA-MMS on colony formation studies using A549 and HeLa S3 cells at different concentrations. The data was calculated by ImageJ software using colony area. The figure is a representative of three replications. Data is expressed as mean ± SD from three replications, **** p < 0.0001 and ** p<0.01 when compared with the untreated control group.
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Journal Pre-proof Graphical abstract text
The present Fe3O4@LEC-CUR-PLGA-MMS system demonstrated much better in vitro cytotoxicity, cellular morphological changes and moreover ability to control colony formation for A549 and HeLa S3 cancer cell lines than non-cancerous cells, and thus, should be further
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studied for a wide range of medical applications.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Srinivasan Ayyanaar, Mookkandi Palsamy Kesavan, Chandrasekar Balachandran, Swetha Rasala, Perumal Rameshkumar, Shin Aoki, Jegathalaprathaban Rajesh, Thomas J. Webster, Gurusamy Rajagopal PII:
S1549-9634(19)30218-7
DOI:
https://doi.org/10.1016/j.nano.2019.102134
Reference:
NANO 102134
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Revised date:
19 November 2019
Please cite this article as: S. Ayyanaar, M.P. Kesavan, C. Balachandran, et al., Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/ 10.1016/j.nano.2019.102134
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© 2019 Published by Elsevier.
Journal Pre-proof
Iron oxide nanoparticle core-shell magnetic microspheres: Applications toward targeted drug delivery Srinivasan Ayyanaar, MSca, Mookkandi Palsamy Kesavan, PhDb, Chandrasekar Balachandran, PhDc, Swetha Rasala, BPharmd, Perumal Rameshkumare, Shin Aoki, PhDd,e, Jegathalaprathaban Rajesh, PhDf, *, Thomas J. Webster, PhDg,*, and Gurusamy Rajagopal, PhDa, * PG and Research Department of Chemistry, Chikkanna Government Arts College, Tiruppur 641 602, Tamilnadu, India.
b
Department of Chemistry, Hajee Karutha Rowther Howdia College, Uthamapalayam-625 533, Tamil Nadu, India.
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a
c
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
e
Department of Chemistry, Kalasalingam Academy of Research and Education, Krishnankoil-626 126, Tamil Nadu, India.
Research Institute of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
-p
f
Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway H91 W2TY, Ireland.
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d
Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai-623 806, Tamil Nadu, India.
h
Department of Chemical Engineering, Northeastern University, Boston, MA 02115 USA.
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g
* Corresponding authors. E-mail addresses: [email protected] (J. Rajesh), [email protected] (G.
Word count for abstract: 140
Number of figures: 7
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Number of tables: 1
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Number of references: 60
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Word count for manuscript: 3944
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Rajagopal), [email protected] (T.J. Webster)
Number of Supplementary online-only files, if any: 1
Conflicts of Interest: There are no conflicts to declare.
This work was supported by DST-SERB (Ref. No: SB/FT/CS-130/2012) and the authors sincerely thank the Chikkanna Government Arts College, Tiruppur and Mohamed Sathak Engineering College, Kilakarai for their lab and instrumental facilities.
1
Journal Pre-proof Abstract This study describes a sensitive reactive oxygen species (ROS)-responsive lecithin (LEC) incorporated iron oxide nanoparticle (Fe3O4 NP) system with potent anti-inflammatory properties and even more so when the antioxidant drug curcumin (CUR) is encapsulated in the PLGA hybrid magnetic microsphere system (Fe3O4@LEC-CUR-PLGA-MMS). The delivery system
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is responsive to ROS including an H2O2 environment to release the payload (CUR) drug. Greater cytotoxicity properties were observed in the presence of Fe3O4@LEC-CUR-PLGA-MMS against
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A549 and HeLa S3 cells with IC50 values after 24 h of 10 and 12 µg/mL and 10 and 20 µg/mL,
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respectively. The present Fe3O4@LEC-CUR-PLGA-MMS system demonstrated much better in
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vitro cytotoxicity, cellular morphological changes and moreover an ability to limit colony
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formation for A549 and HeLa S3 cancer cell lines than non-cancerous cells, and thus, should be
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further studied for a wide range of medical applications.
Keywords: poly(D, L-lactide-co-glycolic acid); lecithin; magnetic core microsphere; cytotoxicity; targeted drug delivery.
2
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Background
The survival of reactive oxygen species (ROS) homeostatically in the human body plays a key role in various human physiological processes [1-3]. Oxidative stress related diseases can be diagnosed and cured with the aid of ROS since ROS can serve both as pathological markers [4-6] and as therapeutic agents [7-12]. This motivates the development of various ROS responsive
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biological and chemical sensors, prodrugs, imaging agents, and ROS responsive polymeric
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systems [13-20]. Moreover, ROS causes harm to macromolecular substances such as nucleic
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acids, lipids and can cause cell apoptosis by delaying cell differentiation [21]. The main
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consequence of ROS includes the presence of oxidative stress and reduction of pH [22, 23]. ROS
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including H2O2 derived oxidants are highly reactive with genetic molecules of DNA, plasma proteins, and lipids, which cause significant damage to cells [24]. The toxic effect of excessive
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ROS is consequently associated with an array of pathological conditions, including cancer,
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aging, diabetes, cardiovascular diseases, infection, as well as neurodegenerative diseases [25].
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Furthermore, biogenic methods utilizing bio-waste materials have recently been reported for the production of Fe3O4 NP [26,27]. One of them is a watermelon rind which is a rich source of polyphenols, citrulline (amino acid), cellulose (carbohydrate), as well as carotenoids [28,29] and they can function as both reducing and capping agents in the formation of Fe3O4 NP [30,31]. It is known that the magnetic Fe 3O4 NP can carry a drug to a specific target site rapidly based on an external magnetic field and has the potential to release drugs in a controlled manner. Magnetic targeted therapy has associated advantages because of its effectiveness, low toxicity, biocompatibility, targeting and high drug carrying capacity [32-35].
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Journal Pre-proof Among others, Fe3O4 NP are reactive towards ROS (including H2O2) and play an important role in biomedical applications including targeted drug delivery. Similarly, curcumin (CUR), isolated from the rhizome of turmeric, possesses potent antiinflammatory and antioxidant activities and it is hydrophobic and fluorescent in nature. Even though CUR molecules were found to be unstable under different physical and chemical
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environments like pH, alkali medium, heat or light [36-38], they have interesting clinical applications for various therapeutic interventions. Moreover, CUR encapsulated polymeric
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particles which are lipophilic, hydrophilic and composed of phospholipids have demonstrated
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potential applications for controlled drug release systems [39, 40].
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In recent years, polyethylene glycol (PEG) [41], polylactic acid (PLA), polyvinyl alcohol
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(PVA) [42] and chitosan (CS) [43] have been widely used in biomedical applications owing to their excellent biodegradation properties inside biological systems. Specifically, PLGA has been
na
widely used as a bioactive and biocompatible material for different biomedical applications,
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such as controlled release drug delivery applications [44]. Compared to other polymers, PLGA
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microspheres are highly preferred for drug delivery applications because of their lower toxicity, high stability and higher encapsulation efficiency [45]. Herein, we report for the first time the biogenic synthesis of Fe3O4 NP using watermelon rind extract as a reducing and stabilizing agent, and the incorporation of the NP into ROS sensitive LEC for drug delivery applications. As a potent anti-inflammatory drug, CUR was encapsulated in the PLGA-MMS system of Fe3O4@LEC-CUR-PLGA-MMS and were fabricated using a double emulsion (water-in-oil-inwater) method. The Fe3O4@LEC-CUR-PLGA-MMS showed magnetic targeting ROS (including H2O2) activated drug release in cancer cells at different pH values. Moreover, an AO/EB staining
4
Journal Pre-proof assay showed the cytotoxicity of A549 and HeLaS3 cancer cells treated with CUR and the Fe3O4@LEC-CUR-PLGA-MMS system. Methods Chemicals Iron (III) chloride hexahydrate (FeCl3·6H2O), iron (II) chloride tetrahydrate (FeCl2·4H2O),
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sodium acetate, lecithin (LEC), and sodium oleate were obtained from Alfa Aesar. Poly (D, L-
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lactide-co-glycolic acid) (PLGA, MW = 24,000 Da), poly ethylene glycol (PEG, MW = 50,000 Da)
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and curcumin (CUR) were received from Sigma Aldrich. Watermelon was collected from a local market. Deionized Millipore water was used throughout the experiments to prepare solutions.
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Organic solvents of analytical grade were used in the experiments. Preparation of watermelon rind extract
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A fresh ripen watermelon fruit was washed with milli-Q water to remove dust particles
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on its surface. Subsequently, the pulp from watermelon pieces was separated to obtain the
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watermelon rind. The rinds were sliced into small portions and dried at room temperature for 20 days under a dust free zone. The dried watermelon rinds were smashed and carefully weighed (15 g) using an analytical balance, and were transferred into 100 mL of water contained in a neat 250 mL round-bottom flask. The above mixture was allowed to reflux at 70 o
C until it became yellow [46]. The obtained yellow colored mixture was cooled down to normal
temperature, and then filtered using cheesecloth. The mixture was carefully stored at ice-cold conditions until needed for further analysis. Biogenic synthesis of Fe3O4 NP
5
Journal Pre-proof The synthesis of Fe3O4 NP involved the solidification of FeCl3∙6H2O (4 mmol, 1.08 g), FeCl2·4H2O (2 mmol, 0.4 g) and sodium acetate (3.28 g, 40 mmol) in 40 mL of instantly prepared rind extract with continuous stirring for 2 h at 80 °C until the mixture turned black. Sodium acetate can serve as an oxygen source for the formation of Fe 3O4 NP and the solution may act as a reducing and capping agent. The obtained product was washed with ethanol followed by
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water and then was dried under vacuum at 95oC overnight. The product was primarily confirmed by using an external magnetic field.
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Synthesis of Fe3O4@LEC NP
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The surface of Fe3O4 NP was functionalized with oleate by vigorously stirring an aqueous
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solution of sodium oleate (3.0 g) and Fe3O4 NP at room temperature for 2 h as the surface
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became hydrophobic. Furthermore, the lecithin polymer (1.5 g) was slowly dropped onto the surface of the modified Fe3O4 NP suspension. The suspension was washed with copious
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amounts of water and ethanol to remove unreacted particles and then the pH of the
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under vacuum.
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suspension was adjusted to neutral. Finally, the Fe3O4@LEC NP (oil–base surface) were dried
Synthesis of Fe3O4@LEC-CUR-PLGA-MMS Fe3O4@LEC-CUR-PLGA-MMS was prepared using a double emulsion formation method (water-in-oil, oil-in-water) (Scheme 1). Firstly, 5.5 mL of dichloromethane (DCM) (organic phase, O) containing dissolved PLGA (0.6 g) and CUR (10 mg) was mixed with 0.2 mL doubly distilled water containing 12.5 mg Fe3O4@LEC NP (oil–base surface) (inner aqueous phase, W1). Subsequently, the W1/O emulsion was transferred into 100 mL of a 1% PEG solution (outer aqueous phase, W2) under magnetic stirring at 2200 rpm for 30 min. The resulting W1/O/W2
6
Journal Pre-proof emulsion was continuously stirred for 5 h to evaporate DCM. Finally, the MMS were repeatedly washed with distilled water with the aid of centrifugation at 5000 rpm for 10 min each. The excess unabsorbed drug molecules present on the surface of the microspheres were removed while washing with deionized water. The Fe3O4@LEC-CUR-PLGA-MMS was collected in powder form for further analyses [47].
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Characterization of Fe3O4@LEC-CUR-PLGA-MMS An X-ray crystallographic study of Fe3O4NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-
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MMS was performed using an X-ray diffractometer, D/Max-IIIC, Japan (Cu-kα radiation, λ =
-p
1.5406 Å). The position of the crystal peaks was matched to the standard JCDPS files to identify
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their crystalline structure. For functional group identification, Fourier transform Infra-Red (FTIR)
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spectra were recorded for Fe3O4 NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-CURPLGA-MMS on an Avator360, America, spectrophotometer. The surface structure of
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Fe3O4@LEC-CUR-PLGA-MMS was studied by using scanning electron microscopy (SEM, FEI
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QuanTa200, Holland). The magnetic microsphere-coated aluminum foil underwent gold–
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palladium mixture sputtering for SEM analysis. The morphology of Fe 3O4 NP and Fe3O4@LEC NP was analyzed by using a Philips CM12 TEM instrument at an accelerating voltage of 120 kV. The content of Fe3O4@LEC-CUR-PLGA-MMS was measured by Thermo gravimetric analysis (TGA) (Pyris 1, America). The measurement was performed under a N2 atmosphere with a heating rate of 20 °C/min between 50 and 700 °C. The magnetic properties of the Fe3O4@LEC-CURPLGA-MMS was studied using a vibrating sample magnetometer (VSM) (XL5 SQUID, America) and the magnetism values were calculated based on the results. Determination of drug encapsulation efficiency
7
Journal Pre-proof The weight of CUR encapsulated in Fe3O4@LEC-CUR-PLGA-MMS was determined by dissolving it in DCM and was measured by using UV-Vis absorption characteristics. About 10 mg of Fe3O4@LEC-CUR-PLGA-MMS was placed in a sealed glass tube and was dissolved in DCM (1 mL); the solution was mixed with 10 mL of phosphate buffer (pH 7.4) under magnetic stirring at room temperature for 2 h. The solution was centrifuged at 5000 rpm for 10 min in order to
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separate the aqueous phase after the complete evaporation of DCM. The concentration of CUR in the buffer solution was determined using the UV-Vis absorption peak at 421 nm from a
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standard calibration curve. The drug content and encapsulation efficiency were calculated from
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In vitro drug release studies
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the following equations:
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A dialysis protocol was adopted for the determination of in vitro CUR release from the
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nanoparticles. For this, about 100 mg of Fe3O4@ LEC-CUR-PLGA-MMS was transferred into dialysis tubing (MWCO: 10 KDa) quickly and the tubing was immersed in 10 mL of phosphate buffer (pH 7.4/ 5.4 and 7.4/ 5.4 with 1.25 μM H2O2) under stirring at 37 oC in the dark. At different time intervals, 1.0 mL of the dialysis solution was removed and the concentration of CUR released from Fe3O4@LEC- CUR-PLGA-MMS was determined by using UV-Vis measurements (at 421 nm). The total volume was maintained by refilling with the same volume of fresh buffer. Moreover, the drug releasing efficiency of the Fe 3O4@LEC-CUR-PLGA-MMS was determined as follows [48]:
8
Journal Pre-proof ( ) where,
is the concentration of CUR released from Fe3O4@LEC-CUR-PLGA-MMS at time .
is
the initial concentration of CUR encapsulated in the Fe3O4@LEC-CUR-PLGA-MMS. In vitro cytotoxicity studies MTT assay
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MTT assays were carried out using the following method [49-52]. Briefly, A549, HeLa S3,
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IMR-90 and MCF-7 cells were preserved in Dulbecco’s Modified Eagle’s Medium (DMEM)
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containing 5% CO2 at 37 ºC. In order to adhere the cells, a seeding density of 20,000 cells per
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well were plated in 96 well plates and incubated overnight. Then, the cells underwent a treatment with different concentrations of nanoparticles (0.78 to 50 µg/mL) for 24 h. After the
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24 h treatment, to determine cell viability, 10 µL of the MTT (5 mg mL -1) (3-(4,5-dimethylthiazol-
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2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well and incubated for 3-4
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h. 100 μL of a formazanlysis solution (10% SDS in 0.1N HCl) was introduced and the data was measured (at 570 nm) (BIO-RAD).
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Fluorescence images using AO/EB stain Blebbing and nuclear condensation was carried out using A549 and HeLa S3 cells in order to investigate morphological changes due to the nanoparticles. A549 and HeLa S3 cells with a density of 1×105 were plated in 35 cm plates and incubated overnight to allow for cell adhesion. Then, cells were treated with Fe3O4@LEC-CUR-PLGA-MMS (15 µg/mL) for 24 h and were incubated with 10 µL of a AO/EB stain for 15 min in dark. A typical fluorescence image of A549 and HeLa S3 cells after treatment with Fe3O4@LEC-CUR-PLGA-MMS were captured using a Biorevo, BZ-9000, Keyence (20x) fluorescence microscope system [27]. 9
Journal Pre-proof Colony formation assay A549 and HeLa S3 cells/well at a density of 5×103 were plated into 24 well tissue culture plates and were incubated with 5% CO2 at 37 ºC overnight. The cells underwent treatment with Fe3O4@LEC-CUR-PLGA-MMS at different concentrations. Then, each well was washed twice with PBS after a 24 h treatment and was incubated with freshly prepared DMEM for 10 days.
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Staining of the cells was completed using a crystal violet solution for 15 min and washed with H2O. The data was interpreted by ImageJ software using colony area.
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Statistical analysis
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Results are summarized as the mean ± SD and Origin Pro 8.1 using P <0.05 as statistically
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significant.
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Results
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FTIR and crystalline phase characterization studies The successful grafting of Fe3O4NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-
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CUR-PLGA-MMS as confirmed by FTIR analysis is shown in Fig. 2 (A). In order to prove the
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successful fabrication, the corresponding functional groups on each sample were determined. A band obtained at 584 cm−1 for the Fe3O4 NP is characteristic of Fe-O stretching. The bands observed at 2945, 2834 and 1350 cm−1 for Fe3O4@LEC NP are attributed to the stretching vibration of –CH3 (or –CH2–) and bending vibration of–N–H bonds, respectively. The FTIR spectrum of the PLGA revealed the -OH stretching band at 3451 cm−1, the ester group (C-O-C) stretching band at 2828 cm −1and the methyl C-H band at 1613 and 954 cm −1 [53]. The spectrum from pure CUR exhibited the characteristic C=O and C-O-C stretching vibrations at 1345 and 1245 cm-1, respectively. The FT-IR analysis of Fe3O4@LEC-CUR-PLGA-MMS confirmed the
10
Journal Pre-proof presence of Fe3O4 NP, Fe3O4@LEC NP, pure CUR and PLGA by showing the respective bands corresponding to the functional groups in the IR spectra. XRD analysis was performed to elucidate the crystallanity of Fe 3O4@LEC-CUR-PLGAMMS. The diffraction patterns of Fe3O4 NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS are shown in Fig. 2(B). The characteristic peaks of cubic spinel Fe3O4 NP were observed at 2θ values
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of 30.15°, 35.54°, 43.59°, 57.04°, and 63.09° which were assigned to the (220), (311), (400), (511), and (440) lattice planes of Fe3O4, showing a lattice parameter of 8.3 Å. The crystalline
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property of Fe3O4 NP in Fe3O4@LEC NP was retained even after loading of the lipophilic LEC on
-p
the surface of Fe3O4 NP. However, the polycrystalline nature of Fe3O4@LEC-CUR-PLGA-MMS is
re
evidenced from the peaks that appeared at 20.63°and 25.35° [54]. On decorating the
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Fe3O4@LEC NP surface with CUR, PLGA and PEG increased the intensity of the polycrystalline peaks and further broaden the diffraction peaks of Fe 3O4 NP. This also provides evidence for
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the successful formation of Fe3O4@LEC-CUR-PLGA-MMS.
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Morphological characterization of Fe3O4@LEC-CUR-PLGA-MMS
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The surface topography of the Fe3O4@LEC-CUR-PLGA-MMS prepared by W1/O/W2 was analyzed using SEM (Fig. 3 (A)). The SEM micrographs showed spherical shaped Fe 3O4@LECCUR-PLGA-MMS exhibiting a smooth surface with an average diameter of 0.5 µm. As illustrated in Fig. 3 (B), EDS analysis showed the peaks for C, N, Fe, and O elements present in Fe 3O4@LECCUR-PLGA-MMS. The morphology of Fe3O4@LEC-CUR-PLGA-MMS was studied using TEM. The Fe3O4 NP were roughly spherical in shape with an average crystal diameter of 0.2 µm (Fig. 4 (A)). The TEM image in Fig. 4 (B) confirms the spherical shape of Fe3O4 NP and Fe3O4@LEC NP, and the successful incorporation of Fe3O4 NP into the polymer. Nevertheless, the synthesized
11
Journal Pre-proof system showed that the Fe3O4 NP were adsorbed onto the surface of Fe3O4@LEC NP. Fig. 4 (C) shows the SAED pattern of Fe3O4@LEC-CUR-PLGA-MMS. The dotted ring patterns observed in SAED all matched well to the d-spacing values corresponding to the (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0) diffraction planes of spinel Fe3O4 NP [56]. This supports the formation of Fe3O4 NPs with high crystallanity.
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Thermal and magnetic property analysis To further characterize the novel NPs, TGA was performed on the Fe 3O4 NP, Fe3O4@LEC
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NP and Fe3O4@LEC-CUR-PLGA-MMS from an ambient temperature to 700 ºC with a 20 ºC min -1
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heating rate under a nitrogen atmosphere (Fig. 4 (D)). The removal of physically adsorbed
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water from Fe3O4 NP was observed up to 150 ºC and significant weight loss occurred at ~350 ºC
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[57]. In the case of Fe3O4@LEC NP, a slow weight loss (about 12 wt%) at <150 ºC was observed due to the removal of the solvent and a slight increase in weight loss which occurred between
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150 and 350 ºC. However, a greater weight loss was observed for Fe 3O4@LEC-CUR-PLGA-MMS
ur
between 150 and 500 ºC because of the decomposition of LEC, PLGA, PEG and CUR. The
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magnetic properties of Fe3O4 NP and Fe3O4@LEC-CUR-PLGA-MMS were studied using VSM at room temperature and the data is represented in Fig. 3 (C). The saturated values of magnetization of the Fe3O4 NP and the Fe3O4@LEC-CUR-PLGA-MMS were 86.05 emu/g and 47.09 emu/g, respectively. Such results indicate that the Fe3O4 NP retained their magnetism even after forming the composite material Fe3O4@LEC-CUR-PLGA-MMS. Fe3O4 NP have shown advantages in preventing the aggregation of polymer molecules and in assembling molecules rapidly in the absence of a magnetic field [55]. The appreciable higher magnization value of the Fe3O4@LEC-CUR-PLGA-MMS enables such materials to be applied for magnetically targeted
12
Journal Pre-proof drug-delivery. As indicated in Fig. 3 (D), even though the Fe3O4@LEC-CUR-PLGA-MMS microspheres had relatively low saturation magnetization, they still possessed good magnetic mobility. This behavior ensures that drug loading would not significantly affect the magnetic properties of the Fe3O4@LEC-CUR-PLGA-MMS. In vitro drug releasing study
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The release profile of CUR from Fe3O4@LEC-CUR-PLGA-MMS was determined in PBS at both physiological pH (pH 7.4) and acidic pH (pH 5.4) values (Fig. S1 (A)). The release rate of
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CUR from Fe3O4@LEC-CUR-PLGA-MMS at pH 5.4 is faster than at pH 7.4 which confirmed the
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improved solubility of CUR in acidic medium [58,59]. Moreover, the cumulative CUR release
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was 63.18% at pH 5.4 and 39.40% at pH 7.4. The increased CUR release at pH 5.4 can be
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attributed to protonation and consequent degradation of hydrogen bonded PLGA and PEG polymer chains addicted to the monomers [38]. These processes facilitated the easy release of
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CUR from the PLGA layer. Therefore, Fe3O4@LEC-CUR-PLGA-MMS had a greater ability at pH 5.4
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than at pH 7.4 for drug release to cancer cells over a shorter period of time. Furthermore,
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under acidic conditions, the released CUR was biologically active and exhibited enhanced hydrophobic action under polar conditions because of its prolonged chain.
This result
concludes that the Fe3O4@LEC-CUR-PLGA-MMS system can be a suitable candidate for delivering hydrophobic drugs to pH targeted cancer cells in the presence of a magnetic field without any assistance from other parts of the body [60]. The ROS-responsiveness of Fe3O4@LEC-CUR-PLGA-MMS was evaluated in the presence of H2O2 in PBS, with an acidic pH of 5.4 and neutral pH of 7.4 at 37 °C. Fig. S1 (B) shows a CUR release in the presence of H2O2 at 89.28% at pH 5.4 compared to 69.70% at pH 7.4 of CUR. It is
13
Journal Pre-proof assumed that only the PLGA layer degraded at pH 5.4 which led to the release of CUR, but in the presence of H2O2, the LEC layer also underwent degradation which enabled the abundant release of CUR from Fe3O4@LEC-CUR-PLGA-MMS. These results clearly showed that Fe3O4@LEC-CUR-PLGA-MMS exhibited a positive CUR release response capacity with the aid of ROS. Furthermore, the CUR release from Fe3O4@LEC-CUR-PLGA-MMS increased with respect to
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the increase in the blended concentration of PEG in the presence of PLGA. It was observed that the CUR released from Fe3O4@LEC-CUR-PLGA-MMS at pH 5.4 (H2O2
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at PBS) was high and in a controlled manner (Fig. S2). The CUR release in cancerous cells was
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found to be biologically active with preferential conformation under acidic conditions and in a
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prolonged chain form. Moreover, they served as powerful scavengers of free-radical oxidants
lP
via H+ donation as well as electron transfer, exerting antioxidant activity. However, the Fe3O4@LEC-CUR-PLGA-MMS exhibited CUR release at pH 5.4, indicating the nature of the
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loaded CUR, suggesting that Fe3O4@LEC-CUR-PLGA-MMS are potential drug carriers to deliver
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CUR on target to cancerous cells. Conversely, the CUR release mechanism from the Fe 3O4@LEC-
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CUR-PLGA-MMS at the targeted site needs further in vivo experiments for validation of these exicting in vitro results. Anticancer studies Cytotoxic properties of the present nanoparticles (Fe 3O4@LEC NP, CUR and Fe3O4@LECCUR-PLGA-MMS) against A549, HeLa S3 and MCF-7 cells were completed via MTT assays at various concentrations (1.5 to 50 µg/mL) for 24 h at 37º C. As shown in Fig.5 and table 1, the viability of A549, HeLa S3 and MCF-7 cells in the presence of Fe3O4@LEC-CUR-PLGA-MMS showed a superior cytotoxic property against A549 and HeLa S3 cells with IC 50 values at 24 h of
14
Journal Pre-proof 10 and 12 µg/mL, respectively. However, CUR alone revealed only a modest cytotoxic activity against A549 and HeLa S3 cells with IC 50 values of 47.1 and 47.2 µg/mL, respectively. In addition, the viability of A549, HeLa S3 and MCF-7 cells did not change by using Fe3O4@LEC NP. A further toxicity study of CUR and Fe3O4@LEC-CUR-PLGA-MMS was conducted against human normal lung IMR-90 cells by an MTT assay (Fig. S3). Interestingly, both CUR and Fe3O4@LEC-
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CUR-PLGA-MMS did not show toxicity up to >50µg/mL in IMR-90 cells, thus, providing evidence
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of their selective killing of cancer not healthy cells.
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Furthermore, based on the cytotoxic study, nuclear fragmentation was studied by costaining with acridine orange and ethidium bromide (AO/EB) in A549 and HeLa S3 cells in order
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to detect the morphological changes of the nanoparticles. AO and EB were used as the
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indicators of live cells and dead cells, respectively. A549 and HeLa S3 cells were treated in the
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presence of Fe3O4@LEC-CUR-PLGA-MMS at 15 µg/mL for 24 h and images were taken with the help of AO/EB using a fluorescence microscope. As shown in Fig. 6, in the presence of
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Fe3O4@LEC-CUR-PLGA-MMS, a significantly augmented death of A549 and HeLa S3 cells was
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observed as indicated in red which is co-staining of EB. Moreover, the ability of Fe3O4@LECCUR-PLGA-MMS in controlling A549 and HeLa S3 colony formation was determined here. As shown in Fig.7, complete inhibition of colony formation was observed in the presence of Fe3O4@LEC-CUR-PLGA-MMS against A549 and HeLa S3 cells at 10 and 20 µg/mL, respectively. However, Fe3O4@LEC-CUR-PLGA-MMS did not affect colony formation at the lowest concentration (such as 5 and 10 µg/mL) in both cell lines, respectively (Fig. S4). Finally, herein, we concluded that Fe3O4@LEC-CUR-PLGA-MMS not only show cytotoxicity to cancer cells
15
Journal Pre-proof (including morphological changes and an ability to control the colony formation in A549 and HeLa S3 cells) but were not toxic to healthy cells. Discussion Here, Fe3O4 NP were successfully prepared by a biogenic synthetic process using a watermelon rind extract without using a toxic chemical surfactant and reducing agent. The
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Fe3O4@LEC-CUR-PLGA-MMS were successfully synthesized by a double emulsion (water-in-oil,
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oil-in-water) method. The resultant product of Fe3O4@LEC-CUR-PLGA-MMS was analyzed by
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various spectral as well as microscopic techniques. Along with the CUR from Fe3O4@LEC-CURPLGA-MMS, is the ROS (including H2O2) triggered pH responsiveness for drug release at an
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acidic pH of 5.4 and basic pH of 7.4. Meanwhile, experiments from the MTT assay, fluorescence
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staining and colony formation assay revealed that the Fe3O4@LEC-CUR-PLGA-MMS can be a
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potential anticancer agent showing toxicity to cancer cells but cytocompatibilty to healthy cells. In summary, despite the existence of great challenges in the design of an ROS-responsive drug
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release mechanism, here one is demonstrated to efficiently deliver therapeutic agents to a
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disease site, with more cellular interactions to enhance therapeutic efficacy for suitable biomedical applications.
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Journal Pre-proof
Tables Table 1 IC50 values for CUR, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS against IMR-90, A549, MCF-7 and HeLa S3 cells. A549 (µg/mL)
HeLa S3 (µg/mL)
47.1
47.2
Fe3O4@LEC NP
>50
>50
Fe3O4@LEC-CUR-PLGA-MMS
10.9
12
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ur
na
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re
-p
Note- nt: not tested
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CUR
25
MCF-7 (µg/mL)
IMR-90 (µg/mL)
51.9
>50
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Compound
>50
Nt
30.1
>50
Journal Pre-proof Figure captions Fig. 1 Schematic illustrations showing the synthesis of Fe3O4@LEC-CUR-PLGA-MMS. Fig.2 (A) FT-IR analysis of Fe3O4 NP, Fe3O4@LEC NP, pure CUR, PLGA and Fe3O4@LEC-CUR-PLGAMMS. (B) XRD patterns of Fe3O4 NP, Fe3O4@LEC NP and Fe3O4@LEC-CUR-PLGA-MMS. Fig.3 (A) SEM image and (B) EDS spectrum of Fe3O4@LEC-CUR-PLGA-MMS. (C) Room temperature hysteresis curves of magnetite with Fe 3O4NP and Fe3O4@LEC-CUR-PLGA-MMS. (D)
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Magnetic mobility of the microspheres with and without the drug.
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Fig.4 (A) TEM image of Fe3O4 NP and (B) Fe3O4@LEC NP (C) single sphere SAED pattern of
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Fe3O4@LEC-CUR-PLGA-MMS. (D) TGA curves of Fe3O4 NP, Fe3O4@LEC NP, and Fe3O4@LEC-CUR-
re
PLGA-MMS.
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Fig. 5. Cytotoxic studies of Fe3O4@LEC NP, CUR and Fe3O4@LEC-CUR-PLGA-MMS against A549, HeLa S3 and MCF-7 cells. The experiment was conducted in triplicate and date was expressed
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in mean±SD with help of GrapPad Prism 6 software.
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Fig. 6 Typical luminescence microscopy images (Biorevo, BZ-9000, Keyence) of A549 and HeLa
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S3 cells treated with Fe3O4@LEC-CUR-PLGA-MMS (15 µg/mL) for 24 h at 37 oC. Scale bar 50µm. Fig. 7 Effect of Fe3O4@LEC-CUR-PLGA-MMS on colony formation studies using A549 and HeLa S3 cells at different concentrations. The data was calculated by ImageJ software using colony area. The figure is a representative of three replications. Data is expressed as mean ± SD from three replications, **** p < 0.0001 and ** p<0.01 when compared with the untreated control group.
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Journal Pre-proof Graphical abstract text
The present Fe3O4@LEC-CUR-PLGA-MMS system demonstrated much better in vitro cytotoxicity, cellular morphological changes and moreover ability to control colony formation for A549 and HeLa S3 cancer cell lines than non-cancerous cells, and thus, should be further
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studied for a wide range of medical applications.
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Figure 1
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