Synthesis and characterization of Fe₃O₄@SiO₂@PDA@Ag core–shell nanoparticles and biological application on human lung cancer cell line and antibacterial strains

Abstract

Novel magnetic and metallic nanoparticles garner much attention of researchers due to their biological, chemical and catalytic properties in many chemical reactions. In this study, we have successfully prepared a core–shell Fe₃O₄@SiO₂@PDA nanocomposite wrapped with Ag using a simple synthesis method, characterised and tested on small cell lung cancer and antibacterial strains. Incorporating Ag in Fe₃O₄@SiO₂@PDA provides promising advantages in biomedical applications. The magnetic Fe₃O₄ nanoparticles were coated with SiO₂ to obtain negatively charged surface which is then coated with polydopamine (PDA). Then silver nanoparticles were assembled on Fe₃O₄@SiO₂@PDA surface, which results in the formation core–shell nanocomposite. The synthesised nanocomposite were characterized using SEM-EDAX, dynamic light scattering, XRD, FT-IR and TEM. In this work, we report the anticancer activity of silver nanoparticles against H1299 lung cancer cell line using MTT assay. The cytotoxicity data revealed that the IC₅₀ of Fe₃O₄@SiO₂@PDA@Ag against H1299 lung cancer nanocomposites cells was 21.52 µg/mL. Furthermore, the biological data of nanocomposites against Gram-negative ‘Pseudomonas aeruginosa’ and Gram-positive ‘Staphylococcus aureus’ were carried out. The range of minimum inhibitory concentration was found to be 115 µg/mL where gentamicin was used as a standard drug. The synthesized AgNPs proves its supremacy as an efficient biomedical agent and AgNPs may act as potential beneficial molecule in lung cancer chemoprevention and antibacterial strains.

Key Messages

1. In the present study, we have successfully prepared a core-shell Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

2. We have investigated the dose-dependent cellular toxicity of silver nanocomposite in the nonsmall cell lung cancer cell line H1299 using MTT assay.

3. Also, we have evaluated the mode of cell death using apoptosis.

4. We have also evaluated the bioactivity of AgNPs on both Gram-positive and Gram-negative bacterial cells with highly efficient antibacterial potency.

GRAPHICAL ABSTRACT

Keywords:

• lung cancer
• silver nanocomposite
• polydopamine
• biomedical application
• antibacterial strains
• silver nanoparticles (AgNPs)

1. Introduction

Lung cancer is dominant and second most commonly diagnosed cancer among men and women which is largely widespread around the globe. It has a high morbidity and mortality rates worldwide, causing 1.8 million deaths [1]. However, the nanoparticles have proven as an effective tools from its diagnosis to cancer therapy [2]. The size of synthetic nanoparticle derived from polymers, metals etc. having a diameter of <100 nm are particularly useful as size is similar to most of the biological structures and molecules. Hence, they confer functional properties for both in vitro and in vivo cancer research [3]. Silver NPs (AgNPs) show significant features like high catalytic activity, chemical stability, easy fabrication, good conductivity and potential therapeutical application such as antibacterial [4], anticancer [5,6], anti-inflammatory activities [7,8], nanoelectronic devices, water purification, and environmental pollution control [9,10]. Despite from other metal nanoparticles, silver nanoparticles are nontoxic at lower dosage for a human body [11,12]. The high surface to volume ratios of silver nanoparticles have high reactivity and hence provide its usage in biomedical applications. Recently, core shell AgNPs has been studied for effective gene delivery and it showed that it can completely condensate DNA and protect it from degradation [13]. Also hybrid Fe₃O₄@ AgNPs were synthesised via green route and demonstrated selective cytotoxicity against tumour cell line [14]. Silver nanoparticles (AgNPs) have now been recognised as significant class of nanomaterials for various biomedical applications and more attention are focused on AgNPs because they have risen up as promising therapeutic molecule in cancer treatment [15]. AgNPs have exhibited potential cytotoxicity against various cancer cell lines like lung cancer A549 [16–18], Hela cells [19], breast cancer MCF-7 cells [20–22] and so on. Furthermore, as we all are aware that bacterial infections are major threat to public health globally. Several nanocomposites based on Au [23], Ag [24,25], Cu [26,27] and ZnO [28] have been used as antibacterial agents [29]. As we know, various processes have now been established to synthesise highly dispersed AgNPs using solution based methods, therefore in order to address this challenge, increasing efforts has been aimed towards AgNPs introduction on solid supports (polymers, metal oxides) using different nanostructures like spheres, mesoporous silica to generate composite catalysts, which could be the best approach to avoid aggregation of small-sized AgNPs without affecting properties [30,31].

A new trend in field of material sciences is coating of materials and modifying their surface which can provide novel surface functionalities and properties [32]. Recently, three layered core-shell nanocomposites have gained much attention in cancer application. Among the other preferred carrier materials, silica coated with Fe₃O₄ magnetic carrier have emerged as a powerful catalytic support as they have excellent thermal stability, low toxicity, ease of synthesis, effortless separation from the reaction through external magnetic sources [33]. The SiO₂ core have some advantages like ease in controlling particle size, high colloidal stability and has potential application in drug delivery and biosensors [34,35]. However, the surface properties of silica nanoparticles have been found tuneable for various purposes like multi-functionalization, drug loading, drug release and delivery [36]. Usually, a nanomaterial using Fe₃O₄ core and SiO₂ shell has a good magnetic response and chemically modifiable surface. Polydopamine (PDA) has been extensively used as a versatile functionalization tool since 2007, having been inspired by adhesive nature of amines and catechols in mussel adhesive proteins [37]. These PDA could self-polymerize and form PDA layers which can be subsequently applied to variety of substrates to improve biocompatibility and to tolerate other bio-functionalization [38,39]. PDA can readily be deposited under slightly basic conditions by dopamine polymerization, to form a biocompatible layer that will stick to range of material surfaces [40,41]. The metallic nanoparticles like Ag, Au Pd in combination with other metal oxides like (SiO₂, TiO₂), polymer matrices like polyaniline, PDA, pyrrolidone, polypyrrole have been exponentially explored due to high conductivity, thermal stability and redox potential nanocomposites [42].

Recently, Zhang et al. [43] reported the green synthesis of AgNPs decorated on Fe₃O₄@PDA as effective antimicrobial agents. Wang et al. [44] have synthesised PDA coated magnetic nanocellulose AgNP for inactivation of E. coli bacteria in waste water. Nikmah et al. [45] have reported the synthesis of Fe₃O₄@SiO₂@Ag nanocomposites and promising antibacterial activity using agar diffusion method. Baoliang et al. [46] have reported a composite of Fe₃O₄@SiO₂@Ag and their application in Surface enhanced Raman Scattering. Similarly, novel cube like Fe₃O₄@SiO₂@Au@Ag magnetic nanocomposites have been reported as efficient SERS substrate for pesticide detection [47]. Here, Fe₃O₄@SiO₂@PDA core has been used as the support for Ag nanoparticles. In order to improve the biocompatibility of the nanoplatform, for the first time we report the fabrication and characterization of the synthesised ‘Fe₃O₄@SiO₂@PDA@Ag’ nanocomposites where SiO₂ nanoparticles were synthesised by hydrolysis of TEOS followed by thermal decomposition and its used as core for synthesis for core–shell nanoparticles. Various studies reveal that Ag nanocomposites decorated with ferrite-silica core have the potential to destroy cancer cells without affecting the normal cells. Therefore, in the present study, we have investigated the dose-dependent cellular toxicity of silver nanocomposite in the non-small cell lung cancer cell line H1299 by the MTT. Also we have evaluated the mode of cell death using apoptosis. We have also evaluated the bioactivity of AgNPs on both Gram-positive and Gram-negative bacterial cells with highly efficient antibacterial potency and can be exploited as drugs next door in potential healthcare settings.

2. Experimental

2.1. Materials and reagents

FeCl₃.6H₂O was purchased from Central Drug House Pvt. Ltd, FeSO_4.7H₂O, tetraethyl orthosilicate (TEOS) from Sigma Aldrich. The ethyl alcohol (C₂H₅OH, 95 %), and aqueous ammonia (25 wt %) were supplied by SRL Co., Ltd. All chemical reagents were directly used without further purification. MTT dye was purchased from Sigma Aldrich. Double deionised water was used throughout the study. All reagents were used without purification.

2.2. Synthesis of ferrite (Fe₃O₄) nanoparticles

Initially, FeCl₃.6H₂O, 6.0 g and FeSO_4.7H₂O, 4.2 g were dissolved in 250 ml of deionised water and were allowed to stir at 50 °C until appearance of yellow-orange solution. After, we added 25% ammonium hydroxide dropwise to slowly adjust the pH to 10 and then the mixture was allowed to stir for another 1h at 50 °C [48]. The ferrite nanoparticles were precipitated as a black material which was separated by an external magnet and washed several times with water and ethanol. This was dried overnight in oven at 50 °C to obtain the nanoparticles.

2.3. Synthesis of Fe₃O₄@SiO₂

The SiO₂ coating over ferrite nanoparticles was done using sol–gel approach. A suspension of 0.5 g of ferrite nanoparticles and 0.1 M HCl (2.2 ml) was prepared in water (50 ml) and ethanol (200 ml) mixture under sonication for 1 h at RT. After, 25 % NH₄OH (5 ml) was added to the mixture followed by dropwise addition of TEOS (1 ml) and resulting mixture was stirred at 50 °C for 6 h. The resulted silica coated magnetic nano particles Fe₃O₄@SiO₂ were separated magnetically and washed with ethanol and water several times and then dried in oven at 40 °C.

2.4. Synthesis of Fe₃O₄@SiO₂@PDA

The Fe₃O₄@SiO₂ nanoparticles (0.18 g) were dispersed via sonication into ethanol (100 ml) for half hour. Then dopamine hydrochloride (100 mg) and triethylamine (74 μL) were added to the reaction mixture. The mixture was then stirred at 50 °C for 24 h and then nanoparticles were purified by three wash cycles of centrifugation/redispersion in ethanol [32,49].

2.5. Synthesis of Fe₃O₄@SiO₂@PDA@Ag

Fe₃O₄@SiO₂@PDA nanospheres (100 mg) were dispersed in 100 ml water. In another beaker, dissolve 50 mg silver nitrate in 100 ml water. Mix the above two solutions and allowed to stir for 24 h. The mixture was washed with water and ethanol several times and then dried at 50 ^oC

3. Characterization of synthesised Fe₃O₄@SiO₂@PDA@Ag

3.1. Electron microscopy

To determine the size and morphology of synthesised nanocomposite, Fe₃O₄@SiO₂@PDA@Ag was subjected to transmission electron microscopy using TEM (TALOSTEM from Thermo Scientific). For TEM analysis, carbon-coated Cu grids were used, and the sample was deposited on them using the drop-casting method. Image J software was used to calculate the average size of nanoparticles.

0.5 mg of nanocomposite was diluted with 70% ethanol (1 ml), sonicated for 30 min before placing onto carbon coated copper grid.

The Fe₃O₄@SiO₂@PDA@Ag was subjected to FESEM-EDAX using Zeiss GeminiSEM 500 thermal field emission type microscope.

3.2. Particle size and zeta potential

Nanoparticle samples were dispersed, sonicated for 5 min in milliQ water and 1 ml of this was taken in quartz cuvette before analysis. The particle size was measured using dynamic light scattering (DLS), Nanoplus particle size analyser from Particulate Systems and surface charge measurement of nanocomposite were measured by Zetasizer Nano-ZS (Malvern Intsruments, UK). All measurements were carried out in triplicate (n = 3).

3.3. Fourier-transforms infra-red (FT-IR) spectroscopy

FTIR was performed using Shimadzu II FT-IR Spectrometer. In brief, spectrum for physical mixture of Fe₃O_4, Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂@PDA as well as Fe₃O₄@SiO₂@PDA@Ag was recorded.

3.4. Powder X-ray diffraction pattern (PXRD)

The polymorphic state of the drug in lipid matrix was confirmed using Rigaku Ultima IV X-ray Diffractometer. The Ni-filtered, Cu Kα-radiation, voltage of 40 Kv and current of 40 mA was used. The scanning rate was 1º/min over 10 º–50º diffraction angle (2θ) range. The crystal lattice of Fe₃O₄@SiO₂@PDA@Ag was accounted.

3.5. Stability data

The stability analysis of Fe₃O₄@SiO₂@PDA@Ag nanocomposites was performed using DLS measurements at different time intervals. The suspension of AgNPs were kept at 4 ^oC and size measurements at different time intervals was observed using DLS at 1, 7, 14, and 21 days. Also, we have prepared the suspension of AgNPs in different PBS buffers (neutral, acidic and basic) and size was recorded [50,51].

4. Cellular experiments

Human lung adenocarcinoma H1299 cancer cells were purchased from the Cell Bank of Type Culture Collection of ACBR and cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin in a cell culture incubator maintained under a 5% CO₂ atmosphere at 37 °C.

Gram positive ‘Staphylococcus aureus’ (S. aureus, MTCC 741) and Gram negative ‘Pseudomonas aeruginosa’ (P. aeruginosa) were the microbes used in this study. Both S. aureus and P. aeruginosa were grown and cultured in Brain Heart Infusion (BHI) media procured from HiMedia at 37$\mathrm{°C}$ and 180 rpm for 12 h. Furthermore, pure colony of these bacteria were collected with a sterile inoculating loop from agar plate and sub cultured in BHI broth and stored at 4$\mathrm{°C}$ for future experimental purpose.

4.1. MTT assay

H1299 lung adenocarcinoma cell line was cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics penicillin and streptomycin (100 µg/mL). Cells were maintained in humidified incubator at 5% CO₂ and 37 °C. Cells were passaged and harvested at ∼80% confluency; they were seeded in 96 well plates at a cellular density of 7 × 10³ cells per well. The seeded cells were grown overnight and treated with various concentrations of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag nanocomposites in triplicates. A 2 mg/mL stock solution of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag was prepared in MilliQ water and sonicated to achieve a homogenised solution. Carefully calculated amount of the nanocomposite was added to the 96 well plates for the following final concentrations- 5, 10, 20, 40, 60, 80, 100 µg/mL. The plates were then incubated at 37 ^oC, 5% CO₂ humidified incubator for 48 h. Post-incubation, the effect of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag nanocomposite was evaluated using a freshly prepared Methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution at a final concentration of 0.5 mg/mL each well. Next, 100 µl of DMSO was added to each well to dissolve the formazan crystals and reading was taken using a multiplate reader at 570 nm and reference at 630 nm. $$\%\text{ Viability }=\text{ Average reading at}\hspace{0.17em}570\text{nm}/\text{Average reading of control at}\hspace{0.17em}\text{57}0\text{ nm }*\text{1}00$$

4.2. Apoptosis

The mode of cell death upon treatment with Fe₃O₄@SiO₂@PDA@Ag on H1299 cancer cell line was determined using Flow cytometer. The Annexin-FITC Apoptosis Detection Kit was used and the protocol was followed as per the manufacturer’s protocol. In brief, H1299 cells were seeded separately in six-well cell culture with a total concentration of about 1 × 10⁵ cells per well. Cells were grown for 24 h in DMEM medium containing 10% FBS prior to all experiments. The dosing solution was prepared in MilliQ the concentration close to IC₅₀ of Fe₃O₄@SiO₂@PDA@Ag was added to the well. Sample were incubated with cultured H1299 cells for 48 h. After predetermined incubation period, the treated cells were extensively rinsed with ice-cold PBS and harvested with Trypsin-EDTA, followed by centrifugation and washing with ice-cold PBS twice and incubated with Annexin/PI dye for 30 min. Then the analyses of the samples were performed on a flow cytometer at FL1 (FITC) and FL2 (PI) channels using BD FACS CALIBUR^TM. And data was further analysed using Cell Quest pro software.

4.3. Minimum inhibitory concentration (MIC)

MIC test is widely used in clinical microbiology as it provides the lowest concentration of the test antimicrobial agent required to inhibit the visible growth of the microorganism. In a 96-well sterile microtitration plate (Corning), we performed this microbroth dilution procedure with escalating dosages of the as-synthesised Fe₃O₄@SiO₂@PDA@Ag nanocomposites. The final volume of 200 μL was achieved with 10 μL of mid-log phase bacterial inoculum and BHI broth with the desired nanoparticle concentration. Wells with only media and S.aureus or P.aeruginosa served as growth controls. The 96-well microtitration plates after inoculation was subjected to well-mixing and incubated in incubator shaker at 180 rpm and 37$\mathrm{°C}$ for 16–18 h. The growth of the treated cells was monitored by measuring the absorbance at 600 nm in the TECAN Infinite M200 Pro, micro-plate reader.

4.3.1. SEM analysis of bacterial cell rupture on P. Aeruginosa cells

The structural changes in the bacterial morphology induced by as-synthesised Fe₃O₄@SiO₂@PDA@Ag nanocomposites were examined by Scanning Electron Microscopy. Pseudomonas aeruginosa was grown overnight at 37 °C under aerobic conditions. Then,10⁶ CFU/mL of the fresh cultures was used to carry out subsequent experiments. The bacterial suspensions were centrifuged at 4000 rpm for 10 min to separate bacterial pellets. The aqueous dispersion of nanoparticles (at MIC value) was incubated with freshly prepared culture of P. aeruginosa. The pellet appeared was thoroughly washed with 1X PBS solution (3 × 1 ml). After centrifugation, each bacterial pellet was fixed with 4% paraformaldehyde solution, and the pellet was kept at 4 °C overnight. After centrifugation, again the washing of each pellet was done with 1× PBS (3 × 1 ml). Ultimately, serial dehydration of bacterial cells was carried out by washing with increasing concentrations of ethanol (10%, 20%, 40%, 60%, 80% and 100%). The pellet was dried and deposited on SEM stub for analysis.

4.4. In vitro Haemolysis assay

Haemolysis assay was done to ascertain the lytic interactions of as synthesised nanocomposites with the mammalian membrane. The whole blood sample was washed with 1× PBS solution (pH = 7.4) and resuspended in PBS solution. 100 μL of diluted RBC suspension in 1× PBS solution was exposed to various 100 μL of the nanoparticle suspension at concentration of 100, 250, 350, 450, 550 and 650 μg/mL (test group), PBS (positive group), and 1% TritonX-100 (negative group). After incubation at 37 °C for 1 h and centrifugation for 5 min at 800 g, the supernatant of all samples was transferred to a 96-well plate and the absorbance was measured by a microplate reader (BioTek 800 TS absorbance reader) at 405 nm. The percent haemolysis was expressed by the following formula and plotted as a function of percent haemolysis with concentration (μg/mL): $$\text{\% Haemolysis }=\mathrm{ }\frac{\mathrm{OD} \left(\mathrm{test}\right)\text{-}\mathrm{OD} \left(\mathrm{negative}\right)}{\mathrm{OD} \left(\mathrm{positive}\right)\text{-}\mathrm{OD} \left(\mathrm{negative}\right)}\mathrm{ }\times \mathrm{ }100$$

5. Result and discussion

5.1. Characterization of synthesised Fe₃O₄@SiO₂@PDA@Ag

5.1.1. Electron microscopy

The TEM images suggested that particles were roughly spherical in shape as show in the figure below at 50 nm scale with the size ranging from 10 to 30 nm (Figure 1). However, TEM image at 100 nm suggested that the nanocomposites are present in chain-like confirmation.

Figure 1. (a) TEM image of synthesised Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

(a) 50 nm (b) 20 nm (c) 100 nm (d) 200 nm. (b) Fe-SEM image of synthesised Fe₃O₄@SiO₂@PDA@Ag nanocomposite (a)100 nm (b) 200 nm (c) Elemental mapping image of synthesised Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

5.1.2. DLS and zeta potential

The formation of Fe₃O₄@SiO₂@PDA@Ag was established by DLS study and the results revealed the hydrodynamic size of the nanostructures was found to be ∼ 139.1 nm at 25 ^oC in water as solvent with polydispersity index of 0.237 (Figure 2). This could be due to the aggregation of the nanostructures in water. However, the average size difference in TEM and DLS could be due to the hydrodynamic diameter with swollen nanostructures in DLS [52,53]. The low zeta potential value of 5.02 mV indicates that nanostructures could agglomerate. (Figure 3).

Figure 2. DLS of synthesised Fe₃O₄@SiO₂@PDA@Ag (medium: Water; size, d: 139.1 nm).

Figure 3. Zeta potential distribution image of synthesised Fe₃O₄@SiO₂@PDA@Ag.

5.1.3. FTIR spectra

FT-IR was used to study the successful immobilization of various functionalities at each step of nanocomposite preparation. The stretching frequency of Fe₃O₄, Fe₃O₄@SiO₂, Fe₃O₄@SiO₂@PDA as well as Fe₃O₄@SiO₂@PDA@Ag have been recorded. The peak at 565 cm⁻¹ is attributed to Fe–O vibration of the bulk Fe₃O_4. The immobilization of the silane on the magnetic nanoparticle surface was confirmed by the appearance of peaks at 455 cm⁻¹ corresponding to Si–O–Si bending modes and peak at 1083 cm⁻¹ corresponding to Si-O bending. However, the peak at 3385 cm⁻¹ is due to O–H stretching vibrations of PDA and the peak displayed at 2372 cm⁻¹ corresponds to C–H stretching vibrations. The appearance of peak at 1662 cm⁻¹ is attributed to N-H group and the peaks at 1088 is associated with C-N stretching [53]. Hence, FT-IR spectra postulated that Fe₃O₄, SiO₂ and PDA persisted its characteristics in the lipid matrix (Figure 4).

Figure 4. FT-IR of (a) Fe₃O₄, (b) Fe₃O₄@SiO₂, (c) Fe₃O₄@SiO₂@PDA, and (d) Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

5.1.4. Powder X-ray diffraction (PXRD) pattern

In order to determine the chemical composition of the so-formed nanocomposites, XRD analysis of Fe₃O₄, @SiO₂@PDA@Ag was carried out. The peaks at 30.32^o, 35.58^o, 46.19^o, 57.33^o, 62.86^o indicates the presence of magnetite nanoparticles.The position and relative intensities of all the peaks in the XRD pattern of Fe₃O₄@SiO₂@PDA@Ag nanocomposite displayed the peaks at 38.11^o, 43.25^o, 62.86^o, 77.46^o that correspond to 111, 200, 220 and 311 planes of Silver nanoparticles [54] (Figure 5).

Figure 5. XRD of Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

5.1.5. Elemental analysis

The EDX analysis of Fe₃O₄@SiO₂@PDA@Ag nanocomposite displayed the presence of carbon, nitrogen, oxygen, silicon, silver and Iron in 6.71 wt %, 1.92 wt %, 32.36 wt %,13.49 wt %,11.15 wt % and 34.38 wt %, respectively (Figure 6).

Figure 6. EDAX of Fe₃O₄@SiO₂@PDA@Ag nanocomposite.

5.2. Stability data of the Fe₃O₄@SiO₂@PDA@Ag nanocomposite

The stability analysis of AgNPs was performed using DLS measurements at different time intervals. The suspension of AgNPs in water were made and kept at 4 ^oC and size was measured using DLS at 1, 7, 14, 21 days’ time intervals. The size of AgNPs in water was found in the range of 120–150 nm. Also, size at different pH was measured and found to be in range of 130–300 nm in pbs and basic buffer, but in acidic buffer size increased to 500 nm after 14 days.

It can be concluded that particles have high stability despite pH and long storage (Figure 7).

Figure 7. Stability data of Fe₃O₄@SiO₂@PDA@Ag nanocomposite in water, PBS, acidic and basic buffers.

5.3. Biological analysis

5.3.1. Evaluation of cytotoxic effect of nanoparticles using MTT assay

MTT assay is among the few preliminary assays to assess the effectiveness or toxicity of small molecules, biomaterials etc. on cultured cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) is a yellow-coloured water-soluble dye that gives purple crystals on its reduction concept on which MTT assay is based upon. In a live cell, cellular respiration produces NADPH which reduces the yellow-coloured dye to purple formazan crystals after incubation; the amount of crystals formed or the amount of colour is directly proportional to levels of NADPH which in turn is proportional to cell viability. The formazan crystals are solubilised in DMSO and measured spectrophotometrically.

Herein, to assess the anticancer activity of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag nanocomposite in non-small cell lung cancer cell line H1299, each cell line was treated with Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag nanocomposite with different concentration—5, 10, 20, 40, 60, 80, 100 µg/mL for 48 h. The % viability was calculated and plotted using the spectrophotometric data for each concentration along with standard deviation (Figure 8). The graphs depict a concentration-dependent effect of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag nanocomposite on H1299 cells lines. The IC₅₀ for H1299 were calculated as 62.84 µg/mL for Fe₃O₄@SiO₂@PDA. However, the IC₅₀ for Fe₃O₄@SiO₂@PDA@Ag nanocomposites were calculated as 21.52 µg/mL. Figure 9 displayed the microscopic images of the effect of the Fe₃O₄@SiO₂@PDA@Ag nanoparticles on H1299 cancer cell line. A large population of nanoparticles (black dots) are internalised by target H1299 cancer cells.

Figure 8. MTT assay at various concentrations on H1299 cancer cell line; (A) % cell viability of Fe₃O₄@SiO₂@PDA (B) % cell viability of Fe₃O₄@SiO₂@PDA@Ag C) comparison of % cell cytotoxicity of Fe₃O₄@SiO₂@PDA and Fe₃O₄@SiO₂@PDA@Ag.

Figure 9. Microscopic images of the effect of the Fe₃O₄@SiO₂@PDA@Ag nanoparticles on H1299 cancer cell line. (A) Untreated/control (B) at 50 µg/mL.

5.3.2. Apoptosis assay of Fe₃O₄@SiO₂@PDA@Ag nanoparticles using Flow cytometry

The mechanism of cell death that occurs in H1299 cells upon treatment with Fe₃O₄@SiO₂@PDA@Ag nanoparticles using Annexin-V FITC/PI dual staining assay was performed, and cells were analysed using FACS. After treatment with increasing concentration for 48 h the apoptosis index in H1299 cells was increased to 21% in early apoptosis & 46 % in late apoptosis at 30 µg/mL (Figure 10).

Figure 10. Cell apoptosis assay at different concentrations of Fe₃O₄@SiO₂@PDA@Ag on H1299 cancer cell line (A) control; 0 µg/mL, (B) 20 µg/mL, (C) 30 µg/mL.

5.3.2. Evaluation of antibacterial activity of nanoparticles

To comprehend the potency of the Fe₃O₄@SiO₂@PDA@Ag nanocomposites as antibacterial agents, we performed the standard microbroth dilution assay with both Gram-positive and Gram-negative bacteria. The results showed that the nanoparticles were effective in inhibiting the growth of these bacteria at a concentration of 115 μg/mL, for both S. aureus and P. aeruginosa, indicating another beneficial property of these nanocomposites. This activity could be attributed to the interaction of the nano-sized particles with the larger surface area of the microorganisms. The relative survival percentage of these bacteria is endorsed in Figure 11 as compared to growth controls. It is evident from the findings that the antibacterial propensity of the nanocomposite (due to 11.15 wt% of silver atom) against both Gram-positive or Gram-negative is concentration-dependent. The presence of thick peptidoglycan layer around Gram-positive bacterial cells leads to less suppression in bacterial growth at the same concentration for S. aureus as compared to P. aeruginosa.

Figure 11. Antibacterial activity of Fe₃O₄@SiO₂@PDA@Ag nanocomposites with increasing concentrations against (A) S. aureus and (B) P. aeruginosa.

Additionally, the SEM images displayed in Figure 12 demonstrated morphologically induced changes like rupture of the cell membrane, cell shrinkage, aggregation of bacterial cells in Fe₃O₄@SiO₂@PDA@Ag nanocomposites treated samples, whereas in control cells displayed normal, smooth and well-preserved cell membrane. The nanocomposite treated cell had deformed and distorted cell structure indicating leakage of intracellular components. SEM images clearly indicated morphological alterations along with complete lysis of the outer membrane of bacteria. EDX analysis also confirmed the presence of strong Ag metallic signals in treated cells. Other signals like Si and Fe were observed due to the Fe₃O₄@SiO₂ shell present in the nanocomposite, in addition to the C, N, O present attributed to the bacterial biomolecules.

Figure 12. Scanning electron micrographs of (A) and (B) untreated P. aeruginosa cells and its EDAX composition (C) and (D) cells treated with Fe₃O₄@SiO₂@PDA@Ag nanocomposites at MIC and its EDAX composition.

5.4. In vitro Haemolysis assay

Haemolysis study was conducted with 100, 250, 350, 450, 550 and 650 μg/ml concentrations of Fe₃O₄@SiO₂@PDA@Ag nanoparticles. The haemolysis was found to be minimal at a concentration of 100 μg/mL. This concentration is relatively higher than the observed IC₅₀ value (21.52 µg/mL) for this nanocomposite. At maximum concentration (650 μg/mL), which was 30 times the observed IC₅₀, the haemolysis was negligible. These observations demonstrated that these nanoparticles can be used directly or can be used as a drug delivery agent, which can reduce their toxicity on human cells (Figure 13).

Figure 13. Haemolysis assay of Fe₃O₄@SiO₂@PDA@Ag nanoparticle at various concentrations.

6. Conclusion

Lung cancer is the most aggressive human cancer and a leading cause of death worldwide. Here, we have reported a simple protocol to synthesise Ag-wrapped Fe₃O₄@SiO₂@PDA nanocomposites. The anticancer and antibacterial activity displayed by the Ag nanocomposites reported here represent a better alternative to available options. We have also shown the mode of cell death using apoptosis assay and biocompatibility of synthesised nanocomposites using haemolysis assay. The nanocomposites have effusively demonstrated their antibacterial activity against Gram-positive and Gram-negative bacteria but with different susceptibilities at the same concentration range. Thus, the ordinance of these nanocomposites serves as a lucrative option in this ever-evolving era of bacterial infections.

Author’s contribution

R.C, S.S., and T.G. contributed to the idea or design of the work. S.S, A.S., T.G. and H.C. performed the nanoparticle synthesis, characterization, analysis and interpretation of data for the work. H.C. and T.G. performed the anticancer activity and did the interpretation of the results. T.G. and M.T. did the apoptosis experiment and analysed the data. N.C. and I.R. did the antibacterial experiments and analysed the data. R.C., S.S. and M.T. supervised the project. S.S., A.S., T.G, H.C., N.C., I.R., M.T. and R.C. contributed to the drafting of the work, reviewing the intellectual content of the manuscript. All authors have agreed to the final version, helped in analysis of the data and provided critical feedbacks during the writing and revision of the manuscript.

Acknowledgements

Prof. Ramesh Chandra would like to acknowledge University of Delhi for providing support and necessary facilities to carry out research work. Snigdha Singh is grateful to DST (Indo-Russia: INT/RUS/RFBR/389) for providing the financial assistance. Tanya Goel is thankful to DST-Inspire Fellowship for their assistance. Heerak Chugh and Aarushi Singh are thankful to ICMR for providing Senior Research Fellowship.

Disclosure statement of interest

The authors declare no potential competing interest.

Data availability statement

Raw data available on request.

Additional information

Funding

R.C. and S.S. are grateful to DST (Indo-Russia: INT/RUS/RFBR/389) for providing the financial assistance. R.C. and A.S. are grateful to ICMR-SRF (45/10/2020-Nan/BMS) for providing the fellowship.