Mechanistic approach to study conjugation of nanoparticles for biomedical applications

Accepted Manuscript Mechanistic approach to study conjugation of nanoparticles for biomedical applications

Imran Uddin PII: DOI: Reference:

S1386-1425(18)30432-3 doi:10.1016/j.saa.2018.05.042 SAA 16076

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

24 March 2018 8 May 2018 9 May 2018

Please cite this article as: Imran Uddin , Mechanistic approach to study conjugation of nanoparticles for biomedical applications. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi:10.1016/ j.saa.2018.05.042

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ACCEPTED MANUSCRIPT Mechanistic approach to study conjugation of nanoparticles for biomedical applications Imran Uddin*

Interdisciplinary Nanotechnology Centre, Aligarh Muslim University, Aligarh 202002, India. *

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Corresponding author, Telephone; (+91) 8445503438; E-mail: [email protected]

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Abstract

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Interaction of nanoparticles with biological systems turns out to be vibrant for their efficient

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application in biomedical field. Here, we have shown antibiotic amakicin loaded nanoparticles are responsible for the dual role as reducing and stabilizing the silver

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nanoparticles without the use of any undesired chemicals. Synthesized nanoparticles are welldispersed having quasi spherical morphology with an average particle size around 10-11 nm.

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Crystallinity of nanoparticles was measured using selected area electron diffraction (SAED)

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and powder XRD analysis which show that particles are perfectly crystalline with cubic phase of geometry. UV-Vis, FTIR and circular dichroism (CD) analysis explained the

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presence and interaction of antibiotic on the nanoparticle’s surface. Amakicin functionalized Ag nanoparticles used in this study have shown enhanced antibacterial activity against E.

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Coli. These studies will help in designing an in-depth understanding that how nanostructures can possibly interact with biological systems.

Keywords: Amikacin; Silver; Interaction; Nanoparticles; Circular dichroism; Antibacterial

ACCEPTED MANUSCRIPT Introduction Interaction of inorganic nanoparticles with drug or biomolecules is fascinating for several pharmaceutical and biomedical applications [1]. Conjugation of inorganic nanoparticles to biomolecules generates hybrid materials that can be used to let the nanoparticles interact specifically with biological systems [2]. Nanostructured surfaces offer a new route to study

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protein interactions [3]. Nanomaterial’s provide large surface areas for biomolecule

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adsorption and can be engineered to present multiple surface functional groups for interaction

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with biomolecules, such as enzymes and antibiotics [4-8]. However, interaction of drug molecules like antibiotics on inorganic nanoparticles surfaces has not yet been explored and it

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required numerous steps.

Many types of antibiotics are commercially available to combat different bacteria; however,

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the broad use of these antibiotics has caused bacteria to develop resistance against them. This situation can be overcome by conjugating antibiotics with nanoparticles. Nanomaterial-

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protein interactions have been optimized for a variety of biomedical applications [9-11].

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Nanomaterials have been conjugated with small molecules such as DNA, drugs and proteins for delivery applications [12-15]. Nanoparticles have a very large surface-to-volume ratio, so

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that even minor quantities of particles present extremely large surface areas available for the interaction [16]. As it is very well known that changes in morphology of the particles such as

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the particles become smaller, the composition and organization of the associated biomolecules will change dramatically [17]. Indeed, direct determination of curvature effects on biomolecular interaction can be made, as with some material types, the size of the particle can be increased until the curvature effects vanish [18-19]. This offers several possibilities in terms of high-throughput or mass screening of nanoparticle-biomolecules interactions. Nanotechnology offers approaches to control a wide variety of biological and medical processes that occur at nanometer length and are believed to have a successful impact on

ACCEPTED MANUSCRIPT biology and medicine [20-21]. Therefore, considering the interaction of nanomaterials with biological systems becomes vital for their safe and efficient application. It is well-known that nanosilver can interact with proteins and amino acids. Nanosilver can cause the formation of protein corona, protein unfolding, and altered protein functions [22]. The interaction of nanosilver with proteins is believed to be an important mechanism of toxicity for silver

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nanoparticles [23]. All these proteins interact with nanosilver and as a result, cause protein

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conformational changes or even protein damages. Biosynthesis of Ag nanoparticles using

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antibiotics; amikacin as reducing and capping agent was done in ambient condition. These antibiotic capped silver nanoparticles have potential applications in the fabrication of

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bioactive and biocompatible nanoparticles. However, it is believed that even the antibiotic molecules may have enhanced antibacterial activity, have a longer shelf life and increase

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bioavailability once they are tagged onto Ag nanoparticle surfaces which are due to conformational changes as well as change in the surrounding electronic environment [24].

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These conformational changes of biomolecules with amikacin as model system in this case

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are transparent to their biological activity and potency. Amikacin is a class of aminoglycoside antibiotic; induces the cell death by its inhibitory

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effect on bacterial proteins. Its mechanism involved binding with 30S subunits of 16 rRNA which mislead the central dogma [25-27].

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In this study we report the reduction of silver ions using antibiotics amikacin for the synthesis of silver nanoparticles. We assume, this study add up the probability of conjugation or functionalization of required drug molecule onto the nanoparticle surfaces, which further heighten the biomedical applications. We have performed detailed characterizations of as synthesized nanoparticles using UV-Vis spectroscopy, XRD and TEM techniques. The interaction studies to analyses the functional groups responsible for binding and reduction was done using FTIR studies. Conformational changes of amikacin on silver (Ag)

ACCEPTED MANUSCRIPT nanoparticle surface were investigated using circular dichroism (CD) spectroscopy. This amakicin reduced silver nanoparticles were used for evaluation of their antibacterial property. This study can come out with significant results in synthesis of antibiotic capped metal nanoparticles which can inhibit microbial growth with lower antibiotic doses. We assume that this study will explore the fundamental research wherein we can badge any aspire

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biomolecules onto the nanoparticle surfaces.

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Experimental

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Materials/ Chemicals details

Silver nitrate (AgNO3) was purchased from Sisco Research Lab (SRL). Antibiotic was

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procured from Himedia, India. All chemicals were of analytical grade. All other reagents and

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solvents were obtained from commercial suppliers and used as received. All aqueous solutions were prepared using ultra-pure water obtained from a Mili-Q Water system.

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Aligarh Muslim University.

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Escherichia Coli. bacterial strain was obtained from Interdisciplinary Biotechnology Unit of

Material synthesis / Reactions

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In a typical experiment for the synthesis of silver nanoparticles, 1mM concentration aqueous AgNO3 solution mixes with antibiotics amikacin at room temperature. The reduction of Ag+

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ions to Ag0 can be easily achieved by carrying out their reaction with antibiotics and maintaining steady conditions during the reaction. The particle solution was quite stable without aggregation or precipitation. Bioreduction of Ag+ was monitored by recording the UV-Vis absorption spectra of reaction mixture, synthesis of nanoparticles was checked by Transmission electron microscopy (TEM), Selected Area Electron Diffraction (SAED) analysis, X-ray diffraction (XRD) analysis and Fourier transform infrared (FTIR) spectroscopy.

ACCEPTED MANUSCRIPT Characterizations UV-Vis spectroscopy measurements of Ag nanoparticles were carried out on an Agilent dual beam spectrophotometer operated at a resolution of 1 nm in the wavelength range between 200-800 nm. The size and shape analysis of the Ag nanoparticles were done by TEM and HR-TEM analysis using JEOL, JEM-2010 operated at an acceleration voltage of 200 kV with

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a lattice resolution of 0.14 nm and a point image resolution of 0.20 nm. For this purpose, we

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prepared the samples by drop coating the particles suspended in aqueous medium on carbon

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coated copper grids. Selected area electron diffraction (SAED) analysis was carried-out on the same grids. To confirm the crystallinity of synthesized Ag nanoparticles, powder XRD

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patterns were recorded using Rigaku Miniflex-II X-ray diffractometer equipped with high intense Cu-Kα radiations (λ= 1.5406 Å) operated at a voltage of 30kV and current 15mA at

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scan rate of 20/min in the 2θ range of 20-800. FTIR spectroscopy measurement on Ag nanoparticles powder taken in KBr pellet was carried out using Perkin-Elmer Spectrum

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instrument. The spectrometer was operated in the diffuse reflectance mode at a resolution of

of 450-4000 cm−1.

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2 cm−1. To obtain good signal to noise ratio, 128 scans of the powder were taken in the range

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Escherichia Coli. were used for antibacterial tests of Ag nanoparticles using disc diffusion method. In these Pre-inoculums of all the bacterial strains were inoculated separately in 10

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mL of Luria Bertini medium and incubated at 30 °C for 24 hours. Approximately 106 CFU/mL of above stated bacterial strains was inoculated on Luria-Bertani (LB) agar plates. Filter papers with a diameter of 1.5 cm sucked with antibiotic functionalized Ag nanoparticles were placed on the surface of seeded agar plate respectively. After 24 hours of incubation at 37 °C, diameters of the inhibition zones were measured and optical images of the plates were captured.

ACCEPTED MANUSCRIPT Circular dichroism spectra of antibiotic and Ag antibiotic functionalized Ag nanoparticles were obtained using a JASCO CD (J-815) spectrometer; spectra were recorded from measuring range 320 - 200 nm, using a quartz cell. Results and discussion

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In the Fig.1, we have shown UV-Vis spectra of the antibiotic functionalized silver nanoparticles. It was found that interaction of antibiotic with the 1 mM AgNO3 leads to the

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reduction of Ag+, resulting in the formation of silver nanostructures. The absorption spectrum

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of silver nitrate in distilled water shows a characteristic band peaking at 378 nm [28]. The addition of amikacin antibiotic to this solution causes a change in the absorption spectrum;

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the surface plasmon band appearing at 413 nm reveals the synthesis of Ag nanoparticles [29-

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30]. This peak is arising due to formation of Ag nanoaprticles. The UV-Vis absorption spectrum clearly indicates that absorbance appeared in pure antibiotics in the region of 320 and 270 nm [31]. The amino group present in amikacin attributed to the formation of a charge

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transfer complex between amikacin and Ag nanoparticles, which may be associated with a π → π * and n→ π * transitions [32]. The inset in Fig.1 represents the solutions of Ag nanoparticle, which reveals the dark brownish yellow, confirming the synthesis of silver (Ag)

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nanoparticles. We further observed that the amikacin-reduced silver nanoparticles are very

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stable and do not aggregate even after several days. Additionally, the dried nanoparticles are re-dispersible in water indicating the presence of capping molecules at their surface [33-34]. To investigate the formation, morphology and size range of Ag nanoparticles, TEM imaging was carried out for the reaction aliquots, collected after 72 hours of reaction of antibiotic and silver nitrate. In Fig.2a, we have presented representative TEM micrographs of amikacin reduced silver nanoparticles. The particles in these images are irregular in shape, predominantly quasi-spherical. We observe that all the particles are well separated and stabilized. The particle size histogram of the as-synthesized Ag nanoparticles shows that the

ACCEPTED MANUSCRIPT particles are in the range of 7-13 nm with an average size of 10 nm (Fig.2b). Synthesis of nanoparticles using biomolecules approach always confers relatively smaller particle size in comparison with to other wet-chemical techniques. Synthesis of smaller size nanoparticles in this approach occur due to the antibiotic which bind to the surface of the nanoparticles during their growth and hence thereby restricting any further growth of nanoparticles.

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To investigate the crystallinity of nanoparticles, SAED pattern was obtained from Ag

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nanoparticles Fig. (3a). SAED pattern shows a spot pattern confirming that the structures seen in TEM are nanocrystalline in nature. The diffraction spots could be indexed on the

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basis of Ag crystal structure where the value of ‘d’ spacing obtained is well matched with

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standard literature values. We indexed the SAED spots <311>, <220>, <200> and <111> for cubic phase [35].

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For precise investigation of crystallinity at the single particle level, we used the high resolution transmission electron microscopy (HR-TEM). In Fig.3 (b), we have respectively

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shown the HR-TEM image of as-synthesized Ag nanoparticles after approximately 72 hours

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of reaction. The extent of single-crystallinity observed here in nanoparticles synthesized by biological approach, which occurs in ambient conditions is quite remarkable. The lattice

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planes exhibit a spacing of ~1.23Å and ~2.04 Å for the as-synthesized Ag nanoparticles sample having the lattice planes {311} and {200} respectively.

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To verify the crystallinity of biosynthesized Ag nanoparticles In Fig.4, we have plotted the XRD data as obtained from drop cast films of as-synthesized Ag nanoparticles on glass substrate showing intense peaks corresponding to plane {111}, {200}, {220}, {311} and {222}. The peak position and 2θ values concur with those reported for Ag nanoparticles (Fig.3). Almost all peaks in the pattern could be indexed to cubic Ag nanoparticles with cell parameters of a=b=c=4.086 and α=β=γ=900 which are close to the reported literature [35-37]. The broadening of these diffraction peaks indicates that the nanoparticles are having a small

ACCEPTED MANUSCRIPT size. Moreover, XRD pattern exhibit a broad diffraction hump towards lower angle region which is due to capping or bimolecular functionalization arise due to the amorphous nature of antibiotic. Broader XRD pattern may signify some kind of interaction between the nanoparticles and antibiotics. Furthermore, to investigate the presence of stretching/vibration bands and antibiotic molecule

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interact with silver (Ag) nanoparticles, diffuse reflectance mode of FTIR were used. In Fig.5,

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we have shown Fourier transform infrared (FTIR) spectra for the pure antibiotic and antibiotic functionalized Ag nanoparticles, which prove the functionalization of carboxylic

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group as well as presence of amide linkage at the surface of the silver (Ag) nanoparticles. It’s

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showing that broad band in the region 3450 cm-1, which comes is due to the presence of O-H stretching vibration of carboxylic acid functional group as well as N-H stretching vibration as

1

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shown in the curve 1 and 2 in Fig. 5 [38]. Besides this two absorption bands around 1646 cmand 1538 cm-1 are present in both pure antibiotic and antibiotic functionalized Ag

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nanoparticles which are due to the amide I and II bands which arise due to the carbonyl

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stretch and –N-H stretch vibrations respectively in the amide linkages of the antibiotic and attributed to the formation of amide bond on the surface of the Ag nanoparticles which arises

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from the aminoglycoside moiety [39-40]. In continuation of this presence of peak around 1646 cm-1 showing the contribution of both anionic carboxylate group and aromatic C=C

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stretching vibrational modes, there is presence of broad peak at 1381 cm-1which corresponds to stretching vibration of C-N bond [41]. There is presence of out of plane bending occurring in the region of 500-800 cm-1, which happens due to the N-H bond vibration [42]. There is shifting of out of plane bending of N-H group in amakicin functionalized Ag nanoparticles which may be attributed to the interaction of nanoparticles with antibiotic molecules. FTIR spectrum of amikacin functionalized Ag nanoparticles clearly confirm the attachment of the amikacin to the surface of nanoparticles with various functional groups present in

ACCEPTED MANUSCRIPT antibiotic, which carry the amine as a functional group with aromatic moieties. In conclusion we can conclude that amine group and aromatic moiety may induce reduction of Ag+ ions to Ag0 state, which further give the stability to Ag nanoparticles by functionalization and antibiotic interaction. FTIR analysis reveals considerable changes in the vibrational frequencies of different functional groups reacting with silver nitrate, could be because of the

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interaction of antibiotics molecule. However, due to the complex structure of amikacin, it is

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very tough to precisely monitor the exact functional group that donates the electrons or

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interacts directly to initiate the stable synthesis of silver (Ag) nanoparticles. To determine the antimicrobial effect of Ag nanoparticles [43-46], for this zone of inhibition

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assay were carried out on LB plates, against Escherichia Coli. It’s showing in Fig.6, Ag nanoparticles functionalize with antibiotic showing very nice clearance zones around the

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paper disc. In the control reaction in the disc which contains distilled water doesn’t show any zone of clearance and there was faint zone of inhibition seen in case of pure antibiotic. A

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well-defined zone of inhibition was observed in antibiotic functionalized Ag nanoparticles at

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lower concentration (1 wt %) and zone of inhibition further increased at the concentration 10 wt % (Fig.3d). This is evident that Ag nanoparticle carrying antibiotic moiety enhances the

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antimicrobial effect and required fewer doses for a variety of antimicrobial applications. Antibiotic amikacin conjugated with as-synthesized Ag nanoparticles were studied using

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circular dichroism (CD) spectroscopy analysis. It is a useful technique which gauges structural changes of biomolecules (as amikacin in this case) which are often necessary to their biological activity. It is very well documented that CD signals appear where absorption of specific wavelength of radiation happen and due to this given CD spectrum can allocate the structural characteristic of a specific molecules. We found that that amikacin biomolecule interact with Ag nanoparticles as shown by signals in the UV region (below 220 nm) as shown in Fig. 7 (curve b), while there is absence of significant signals in case of pure

ACCEPTED MANUSCRIPT amikacin in CD spectra Fig. 7 (curve a). These results show that Ag nanoparticles in some way interact with amikacin, and produce conformation changes as a consequently induce CD signals. There are few reports that explain that phenomenon like chemisorption help in the interaction of Ag nanoparticles with biomolecules [47-49]. In case of antibiotic, absorption occur below 220 nm due to peptide linkage, we deduce that amikacin which carry the amine

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as a functional group may interact with Ag nanoparticles and create the CD signal, which is

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also shown in FTIR analysis. Antibiotic molecule interacts with the Ag nanoparticle due to the electronic interaction between metal electron and amine group moiety as well as aromatic

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side chains, which occur due to the n→ π * and π → π * transitions.

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Conclusion

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In conclusion, broad use of this antibiotic has developed resistance against microorganisms. The interaction of antibiotics capped nanoparticles has possible high effect on microorganism than normal antibiotics. Further, nanoparticles with lower antibiotic concentration can be

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used in medicated bandages/fabrics [50-52]. Interaction of Ag nanoparticles possibly induces conformational changes and folding characteristics of amikacin molecules. Mechanistic approach of interaction of Ag nanoparticles well explained using FTIR analysis and CD

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spectroscopy. Ag nanoparticles interact with amikacin by electronic interaction of metal

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surface and functional group like amine present in the antibiotic molecules. It is clearly showing induce CD signals in CD spectrum of amikacin functionalize Ag nanoparticles. Further this may help to understand the antimicrobial mechanism of antibiotic capped Ag nanoparticles. Acknowledgements Imran Uddin would like to extend thanks to the Council of Scientific and Industrial Research (CSIR), India for Senior Research Associateship (Scientists’ Pool Scheme).

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Fig. 1. UV-Visible spectra (1) Antibiotic (2) silver nitrate (3) as-synthesized Ag nanoparticles after 72 hours reaction with herbal extract. Inset showing solutions of Ag nanoparticles synthesize using antibiotic

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Fig. 2.(a) TEM images of Ag nanoparticles obtained after 96 hours of reaction between antibiotic and AgNO3 (b) Particle size histogram.

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Fig. 3. SAED pattern for as-synthesized nanoparticles recorded from Ag nanoparticles shown in TEM micrograph (a); HR-TEM images of Ag nanoparticles synthesized using antibiotic (b) showing interplanar distance.

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Fig. 5. FTIR spectra recorded from pure antibiotic molecules (curve 1) and antibiotic functionalized Ag nanoparticles (curve 2).

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Fig. 6. Luria-Bertini-Agar plates showing zone of inhibition of bacterial colonies (Escherichia Coli); (a) control (b) pure antibiotic (c) antibiotic functionalized Ag nanoparticles against different concentrations (c) 1% and (d) 10%.

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Fig. 7. Circular dichroism (CD) of (a) pure antibiotic (b) antibiotic functionalized Ag nanoparticles.

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Graphical abstract

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Antibiotic amakicin loaded nanoparticles are responsible for the dual role as reducing and stabilizing the silver nanoparticles.

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Interaction of antibiotics capped nanoparticles has possible high effect on microorganism than normal antibiotics. Interaction of Ag nanoparticles possibly induces conformational changes and folding

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characteristics of amikacin molecules.

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This study will explore the fundamental research wherein we can badge any aspire

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biomolecules onto the nanoparticle surfaces.

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Graphics Abstract

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