Ascorbic Acid-Assisted Microwave Synthesis of Mesoporous Ag

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Ascorbic Acid-Assisted Microwave Synthesis of Mesoporous Ag-doped Hydroxyapatite Nanorods from Bio-Waste Seashells for Implant Applications Gopalu Karunakaran, Eun-Bum Cho, Govindan Suresh Kumar, Evgeny Kolesnikov, Gopinathan Janarthanan, Mamatha Muraleedharan Pillai, Selvakumar Rajendran, Selvakumar Boobalan, Mikhail Vladimirovich Gorshenkov, and Denis V. Kuznetsov ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00239 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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ACS Applied Bio Materials

Ascorbic Acid-Assisted Microwave Synthesis of Mesoporous Ag-doped Hydroxyapatite Nanorods from Bio-Waste Seashells for Implant Applications

Gopalu Karunakarana,*, Eun-Bum Choa,**, Govindan Suresh Kumarb, Evgeny Kolesnikovc, Janarthanand,e,

Gopinathan

Mamatha

Muraleedharan

Pillaid,

Selvakumar

Rajendrand,

Selvakumar Boobalanf, Mikhail Vladimirovich Gorshenkovg, and Denis Kuznetsovc aBiosensor

Research Institute, Department of Fine Chemistry, Seoul National University of

Science and Technology, Gongneung-ro 232, Nowon-Gu, Seoul, 01811, Republic of Korea bDepartment

of Physics, K.S. Rangasamy College of Arts and Science (Autonomous),

Tiruchengode 637 215, Tamil Nadu, India cDepartment

of Functional Nanosystems and High-Temperature Materials, National University

of Science and Technology “MISiS,” Leninskiy Pr. 4, Moscow 119049, Russia dTissue

Engineering Laboratory, PSG Institute of Advanced Studies, Peelamedu, Coimbatore-

641004, India eDepartment

of Chemical & Biomolecular Engineering, Seoul National University of Science

and Technology (Seoul Tech), Gongneung-ro 232, Nowon-Gu, Seoul, 01811, Republic of Korea fDepartment

of Biotechnology, K.S. Rangasamy College of Arts and Science (Autonomous),

Tiruchengode 637 215, Tamil Nadu, India gDepartment

of Physical Materials Science, National University of Science and Technology

“MISiS,” Leninskiy Pr. 4, Moscow, 119049, Russia

Corresponding Authours: E-mail address: *[email protected] , **[email protected] 1 ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tel: +82-2-970-9976, +82-2-970-6729

Abstract Implant-related infection is one of the most challenging issues in orthopedics, which is mainly developed by infective micro-organisms. A potential approach to overcome this issue is developing biomaterials with efficient antibacterial activity. The main intention of this present research is devoted to ascorbic acid-assisted microwave synthesis of mesoporous silver Agdoped hydroxyapatite (HAp) nanorods using bio-waste seashells with antibacterial properties. XRD, FTIR, and Raman spectroscopy results revealed that the synthesized nanoparticles are hexagonal crystalline HAp. Further, the silver doped HAp was also successfully produced without affecting the HAp crystalline phase by forming electrostatic interaction with PO43- ions during the synthesis. The morphological features confirm that the pure HAp is elongated mesoporous nanorods with 20 nm width and 300-500 nm length. However, silver doped HAp nanoparticles such as AgHA-1, AgHA-2, and AgHA-3 are found to be similar mesoporous rods but with different aspect ratios in sizes of 15, 10–15, 5–10 nm width and 80-100, 10–15, 20-30 nm length. The BET specific surface areas were obtained as 29±3, 84±2, 87±2, and 128±3 m2 g1, and pore diameters were 4.68, 4.18, 9.30 and 3.77 nm, respectively, for pure HA, AgHA-1, AgHA-2, and AgHA-3. Therefore, HAp nanoparticles with different dimensions and mesoporous structures could be rapidly prepared using microwave assisted method and ascorbic acid as a supporting material. In addition, the synthesized HAp nanoparticles are analyzed for its antibacterial and cytotoxicity studies. The antibacterial and cytotoxicity study clearly reveals that the Ag doped HAp nanorods are efficient antibacterial and non-toxic in nature. Hence, it is clear that ascorbic acid enabled microwave-assisted method will be one of the best methods for the

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rapid production of HAp nanoparticles with different dimensions and mesoporous structures for its application as an implant material.

Keywords: Bio-waste, Seashells, Ascorbic acid, Microwave-assisted synthesis, Mesoporous hydroxyapatite, Implant applications.

1. Introduction The recent research in the area of regenerative medicine, calcium phosphate based inorganic biomaterials finds the foremost superiority for its wide applications for bone tissue rejuvenation.1 The most commonly used calcium phosphates in diverse biomedical applications are amorphous calcium phosphates (ACP), monocalcium phosphate (Ca(H2PO4)2), dicalcium phosphate (CaHPO4), octacalcium phosphate (Ca8H2(PO4)6), α- and β-tricalcium phosphate (TCP) and hydroxyapatite (HAp) (Ca10(PO4)6(OH)2).2 Among the several calcium phosphates used, the most broadly applied biomaterial is HAp due to its inherent bioactive properties which easily form interfaces with the bone and its surrounding tissues.3 HAp structure also plays a major role in the reactivity with the living cells.4 Among the different structures available mesoporous HAp possess excellent bioactivity, better osteoblast attachments and with eminent cell proliferation activity.5 Hence, it is one of the principal thrust areas of research to improve HAp nanoparticles with mesoporous structures. The major applications of HAp are in dentistry, maxillofacial surgeries, and orthopedic defects.6 As the demand of HAp is higher an ecofriendly and easy large scale production method is the major task in the present day research. Many different researches are focused on the synthesis of HAp using chemicals.7 However, it is proven that when the HAp is synthesized



using bio based source, the produced HAp are found to be non-toxic and shows excellent inflammatory repair.8 Some recent researchers focused on the use of bio based sources such as coral, 9 egg shells, 10 fish bone 11 and seashells.12 Among the above sources seashells are the most easily available alternative materials which are rich in calcium content. Seashells are one of the most common and widely consumed as sea foods in different Asian countries. Thus, the seashells are originated as a bio-waste and are being dumped in the landfills. Hence, seashells can be easily used as a bio-resource for the synthesis of HAp nanoparticles, which in turns can minimize the pollutants.13 In spite of rich in calcium source till now no description on the usage of Cerastoderma edule (Common Cockle) seashells for the synthesis of HAp.14 Hence, in this study we used Cerastoderma edule (Common Cockle) sea shell as a calcium source for synthesis of HAp nanoparticles. The widest applications of HAp are in dental and orthopedic surgery as bone fillers and coating materials during metallic implantation.15,

16

However, post-surgical infections are the

major problem arises after the biomedical implantation.17 Thus, it leads to the implant failure. The implant failure starts via microorganism’s adhesion on implant exterior portion and forms a biofilm which leads to develop resistance towards the antimicrobial agents. In order to solve the implant failure different types of antimicrobial agents are proposed which includes antibiotics, sulphate, metal ions and fluorine.18-20 However, the major drawback of using antibiotics is that it is easily washing from the body through kidney and also it leads to the development of antibiotic resistant strains, thus, it cannot inhibit the infections after surgery. The metal ions which are basically used as medicines now days are magnesium (Mg2+),21 zinc (Zn2+),22 copper (Cu2+),23 and silver (Ag+).24 Among the metal ions used silver ions has a tremendous antimicrobial


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property towards pathogens as well as antibacterial resistant strains with minimal effects towards human cells. Recent findings have proved that silver ions for coating implant materials prevent the bacterial adhesion. Some recent progress on the preparation of silver doping on the HAp by using the precursors of ammonium and nitrates origin. Kim et al. produced silver doped HAp nanoparticles through the wet chemical technique via substituting silver ions over the nitrate apatite.25 Similarly, sol-gel mediated synthesis also leads to the formation of nitrate apatite phase besides the formation of HAp due to the incorporation of silver ions.26 Oh et al. doped silver over the HAp using the ammonium phosphates as a precursor.27 However, the above recent studies fail to obtain exact doping of silver over the HAp due to attachment of NO3− and NH4+ ions on the surface of HAp which greatly influences the structure of HAp. The major complex impurities formation during the synthesis of silver doped HAp is the formation of stable [Ag(NH3)2]+ ions which greatly influence the assimilation of silver on HAp.28 Thus, there is a necessity of alternative research to find a solution for the incorporation of silver over the HAp as silver doped without any impurities. The present work is designed to produce highly crystalline silver doped HAp nanoparticles for implant failures. The doping is performed in the presence of ascorbic acid as an organic modifier. As ascorbic acid is one of the well-known capping agents in tuning the structural properties under controlled conditions. The addition of silver ions in the ascorbic acid containing calcium ions leads to the formation of stable silver doped HAp with the addition of phosphate ions without the formation of [Ag(NH3)2]+ ions. Hence, it will be very useful for excellent doping of silver on HAp. Further, the silver doped HAp is analyzed for its bioactivity using human osteoblast cell line and also its antibacterial properties using standard methods.



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2. Experimental procedures 2.1. Materials Dipotassium hydrogen orthophosphate (K2HPO4), ascorbic acid (C6H8O6), silver nitrate (AgNO3), and potassium hydroxide (KOH) were bought by the chemical company Reachem (Russia). Fetal bovine serum (FBS), 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM), Nutrient broth, and MullerHinton

Agar

were

purchased

from

Himedia,

India.

Hoechst

stain

33258,

1,9-

dimethylmethyleneblue (DMMB) was procured from Sigma-Aldrich, USA. Dimethyl sulfoxide (DMSO), methanol, ethyl acetate, and acetic acid were acquired from Merck, India.

2.2. Synthesis of Ag-doped HAp using bio-waste seashells Silver doped HAp nanoparticles were synthesized using ascorbic acid enabled microwave mediated method. At the very first step, the seashells was washed using tap water followed by using deionized water and kept for drying at 100 oC, in order to remove the surface dust and other surface impurities. Further, the seashells were converted into fine powder via mortar and pestle. Then, about 15 gram of fine seashell powder was added in 80 ml (1000 ml beaker) of 0.1 M concentrated HCl solution under the aid of magnetic stirring, the experiments are performed under the laminar air hood. As when seashell is added to the HCl solution evolution of carbon dioxide takes place in the form of air bubbles which shows foam like appearance. After the seashell powder was dissolved in HCl, the solution was filtered 3 times using whatman filter paper to completely remove the dust particles present in the solution. Further, the solution is making up to 100 ml with the addition of deionized water. To this solution 3.5 g of ascorbic acid is added at the rate of 0.1M. After the addition of ascorbic acid the solution turns to


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light yellow colour. Further, to this 100 ml of 0.6 M K2HPO4 solution was added dropwise under the aid of magnetic stirring. The solution was adjusted to basic pH 13 with the use of KOH solution. After pH 13 is attained the mixture solutions were transferred equally in two 250 ml beakers and kept inside the microwave oven for processing. The microwave oven was set with the microwave power of 700 W for a period of 20 mins. Once the reaction was completed, the precipitate was obtained by washing and centrifugation process. The precipitate was washed until pH 7 is attained using deionized water. The centrifugation was performed at 4000 rpm for about 1 h to collect the final precipitate. Hot air oven was used to dry the precipitate by maintaining the temperature to about 120 oC for 6 h. After the drying process the dried HAp was further crushed into fine powders. Hear after as prepared sample was named as pure HA. In the same way, three silver doped HAp samples were synthesized by altering molar concentrations of seashell Ca: Ag as 0.99 M: 0.01M, 0.97 M: 0.03M and 0.90 M: 0.1M. Briefly, appropriate amount of silver nitrate was mixed with calcium solution under the magnetic stirring. After mixing 0.1M of ascorbic acid and 0.6 M of K2HPO4 were added drop wise and the other procedure was performed similar to the samples which was prepared previously. The newly formed samples are named as AgHA-1, AgHA-2, and AgHA-3, respectively.

2.3. Characterization The morphological features and elemental composition of seashell was analyzed using Tescan Vega 3, (TESCAN, Czech Republic) attached with SDD - XMAS (Japan). Three different types of silver doped HAp samples along with un-doped Pure HAp were synthesized and the nanoparticles are characterized by different techniques. The formation of crystalline phase and the occurrence of silver on the doped HAp (AgHA-1, AgHA-2, and AgHA-3) is



analyzed by X-ray diffraction (XRD) technique via Difray-401 X-ray diffractometer (JSC Scientific Instruments, Russia), with X-ray source chromium (Cr) (λ = 2.2909 Å). The occurrences of the phases in all the samples are compared using the standard ICCD - XRD pattern available. The unit cell constraints (a, c, and V) of synthesized HAp samples was deliberate via the relation specified below: 1 4(h 2  hk  k 2 ) l 2   2 d2 3a 2 c

where d denotes the presence of the inter planner spacing (which was calculated through Bragg’s equation i.e., 2dsinθ = nλ) and volume (V) was acquired via the relation of V = 0.866 a2c.29 Further, the functional groups existence in pure HAp and silver doped HAp samples were analyzed by Fourier transform infrared (FT-IR) spectrometer (Nicolet 380 instrument, Thermo Scientific, USA). The synthesized HAp particles are also further analyzed by Raman spectrometer (Thermo DXR, Raman Microscope) under the laser excitation at 532 nm with the aid of laser power of 2 mW. The spectrum of the samples was recorded with an exposure time of 2 s up to 16 exposures at 100x objective. The elemental composition of the pure HAp and AgHAp nanoparticles was analyzed by Energy-dispersive X-ray (EDX) analyzer (Tescan Vega 3, (TESCAN, Czech Republic) attached with SDD - XMAS, Japan). Nova 1200e (Quantachrome Instruments, USA) analyzer is applied to characterize the nitrogen adsorption-desorption isotherms of the produced HAp nanoparticles. The specific surface area (SBET) of the samples were derived by Brunauer–Emmett–Teller (BET) method using adsorption data under relative pressure (P/P0) between 0.04 to 0.2.30 The Kruk– Jaroniec–Sayari (KJS) method was used for calculating the pore size distribution (PSD) based on the adsorption isotherms.31 The pore volume (Vt) of samples was identified based on the adsorbed relative pressure of 0.90 along with the amalgamation of the PSD curve. 8 ACS Paragon Plus Environment

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Morphological characteristics like shape, size and selected area electron diffraction (SAED) pattern were explored using Transmission electron microscope (TEM, JEOL, Japan) of the produced samples. The zeta potential of all the samples was also analyzed using the Zetasizer (Malvern, UK).

2.4. Cytotoxicity assay using MTT and DNA estimation The assay namely MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) was used to characterize the influence of HAp samples on MG63 (human osteoblasts cell lines) cellular viability. This assay relies on the conversion of MTT by the mitochondrial dehydrogenases enzyme to a purple formazan crystal which is measured spectrophotometrically. For this study, 96 well plate were seeded with 10,000 cells/well, supplemented with Dulbecco's modification of Eagle medium with 10% Fetal bovine serum and 1% antibiotics and maintained under standard conditions (37 ºC, 5% CO2) in a CO2 incubator (Galaxy 170S, Eppendorf, Germany). After cell attachment, cells were treated with different concentrations (25- 250 µg/ml) of HAp samples. Control cells were maintained without any treatment. After 48 h, bright field images were taken (Nikon Ti-S Eclipse, Japan) to analyze the cellular morphological changes, followed by MTT assay. Briefly, culture mediums were pipette out and further, washed with phosphate buffer saline (PBS) and treated with 20 µl of MTT reagent (5 mg/ml in PBS) and then it was incubated for 3 h in a CO2 incubator, followed by dissolving formazan crystal via addition of 150 µl DMSO into the wells. After 15 min of continuous stirring and incubation, spectrophotometric reading was taken at 570 nm via 96 well Multiskan™ GO Microplate reader (Thermo Scientific, USA). Viability of cells was calculated against control (untreated cells).



The amount of DNA in the cell was estimated using DNA binding dye (Hoechst dye 33258). MG63 cells were trypsinised from culture plates and counted using hemocytometer. Approximately, 10,000 cells/well were seeded in the 96 well plates, and maintained under standard conditions. After MG63 cell attachment, HAp samples treatment were given at different concentrations (25- 250 µg/ml). Control cells were maintained separately without any treatment. After 48 h, medium was removed followed by PBS wash. Further, cells were fixed using Carnoy’s fixative (methanol: glacial acetic acid 1:3) for 10 min. Followed by 100 µl of Hoechst dye was added and incubated for 15 min. Fluorescence was measured at emission and excitation wavelengths at 461 and 358 nm, using Fluoroskan Ascent Microplate Fluorimeter (Thermo Scientific). Further, the same wells with cells were used for imaging under UV filter of phase contrast microscope.

2.5. Antibacterial susceptibility test The antibacterial activity was tested against bacterial human pathogens which are mostly involved in implant failures. Bacterial pathogens include gram positive bacteria namely Streptococcus pneumoniae (MTCC 1935), Bacillus subtilis (MTCC 1133), and gram negative bacteria such as Escherichia coli (MTCC 1692), and Klebsiella pneumoniae (MTCC 7407) were considered for the present study. Nutrient broth was prepared and sterilized. The bacterial human pathogens were inoculated separately and kept for incubation at 37 °C for 8 h to obtain freshly bacterial inoculum suspension. The disk diffusion assay as per Kirby-Bauer 32 was adopted in the present assay. Each bacterial suspension was spreaded over the surface of Muller-Hinton Agar Plates. The plates containing disk of 5mm diameter were filled with respective nanoparticles samples in disk B (pure HA), disk C (AgHA-1), disk D (AgHA-2) and disk E (AgHA-3). In


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addition, the disk A was loaded with commercial HAp powder as negative control to confirm the activity of HAp. The plates were then incubated at 37 °C for 24 h. The results were expressed in terms of the diameter of the inhibition zone.

2.6. Statistical analysis The experiments were carried out in triplicates and the mean was calculated by using Microsoft Office Excel 2003. For the statistical analysis Statistical Package for Social Sciences software (Version 16.0; SPSS Inc., USA) was used. The observed variation between the control and tested groups were verified by one way analysis of variance (ANOVA), followed by multiple comparisons among groups using Tukey's post hoc test at p

Ascorbic Acid-Assisted Microwave Synthesis of Mesoporous Ag

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