Synthesis and characterisation of novel biopolymer stabilised organic Pt‐nanocomposite: assessment of its antioxidant and antitumour properties

Abstract

Green synthesis of organic Pt‐nanocomposite was accomplished using carboplatin as a precursor and novel biopolymer – gum kondagogu (GK) as a reducing agent. The synthesised GK stabilised organic Pt‐nanocomposite (GKCPt NC) was characterised by different analytical techniques such as ultraviolet–visible spectroscopy, nanoparticle analyser, scanning electron microscopy and energy dispersive X‐ray analysis, X‐ray diffraction (XRD), Fourier‐transform infrared spectroscopy, transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS) and inductively coupled plasma optical emission spectrophotometer. The XRD pattern established the amorphous nature of GKCPt NC. TEM analysis revealed the homogeneous, monodisperse and spherical nature, with Pt metal size of 3.08 ± 0.62 nm. The binding energy at 71.2 and 74.6 eV show the presence of metallic platinum, Pt(0) confirmed by XPS studies. Further, in vitro radical scavenging and antitumour activity of GKCPt NC have been investigated. In comparison to GK and carboplatin, GKCPt NC showed superior 1, 1‐diphenyl‐2‐picrylhydrazyle activity of 87.82%, whereas 2, 2‐azinobis‐(3‐ethylbenzthinzoline‐6‐sulphonic acid) activity was 38.50%, respectively. In vitro studies of the antitumour property of GK, GKCPt NC and carboplatin were evaluated by potato disc tumour bioassay model. The efficacy of synthesised GKCPt NC concentration (IC₅₀) on tumour inhibition was found to be 2.04‐fold lower as compared to carboplatin. Overall, the synthesised GKCPt NC shows both antitumour and antioxidant properties when compared to the original drug – carboplatin and might have promising applications in cancer therapy.

1 Introduction

The application of green chemistry in the synthesis of metal nanoparticles (NPs) has opened new avenues in the area of research and the past decade has seen an upsurge of literature in the field of nano‐biotechnology. Biopolymer stabilised metal NPs have found their applications in diverse fields such as pharma, food, biosensors, medical, bioremediation, removal of radioactive effluents and antibacterial agents [1, 2, 3, 4, 5]. The best example for biopolymers includes tree exudate gums since they are non‐toxic, biodegradable, inexpensive, biocompatible and could be manipulated effortlessly for nanomaterial research [6, 7, 8, 9]. Tree gums are heterogenous polysaccharides which include gum arabic, tragacanth, karaya, ghatti and kondagogu, composed of salts of hexuronic acids, rich in hydroxyl groups, presence of various neutral monosaccharides often esterified in highly branched structures and extremely high molecular weight [10, 11, 12, 13, 14]. Metal NP synthesis and encapsulation of drugs for controlled drug delivery are the versatile applications of tree gums since they are rich in various functional groups, thus serving as superior reducing and stabilising agents [15, 16, 17, 18, 19, 20]. Apart from this property, tree gums could sustain high temperatures, pH changes and chemical additive addition making easy pathways for the synthesis of NP which is a prerequisite for green chemistry.

The novel biopolymer, gum kondagogu (GK) is a heteropolysaccharide derived as an exudate from the bark of Cochlospermum gossypium and other species of Cochlospermum belonging to the Bixaceae family and has been well characterised. Recent research articles showed that either total GK [21, 22, 23] or soluble GK [24] could be used for the synthesis of metal NP which is the unique characteristic feature of this gum. This novel biopolymer is non‐toxic [25] with high mass and intrinsic viscosity compared to other tree gums [26], and non‐allergic or zero immunologic response in nature [27], approving it to be safe for numerous applications in food and pharma industries. Other properties such as morphological, structural, physicochemical, compositional, conformational, rheological, emulsifying and metal‐biosorption properties of GK have been elucidated for its use in commercial applications [11, 28, 29, 30]. GK is a good emulsifying agent at very low concentrations (0.4–0.6% w/v GK) [31] and carrier matrix for controlled delivery of drugs like diclofenac by reducing dosing frequency and gastric toxicity [32], showing its application in the pharmaceutical industry. Extensive studies have been carried out where GK has been used as a matrix for the synthesis of any metal NP [17, 21, 24, 33]. GK shows biosorbent properties that can reduce environmental metal pollutants from effluent discharges by the industries [34, 35].

Drugs synthesised in the form of nanomaterial either in the form of metal NP or polymeric lipid NPs are superiorly effective than original parental drugs [36]. This concept has been adapted in cancer drug research to decrease the side effects of the original drug and increase bioavailability, solubility and stability [37, 38, 39]. Further, nano‐formulation of conventional anti‐cancer drugs, enhance their delivery to tumour sites through increased permeability and retention effect and decrease the toxicity of the drugs to non‐targeting organs thereby providing safer treatment of cancer [40, 41]. According to the World Health Organization (WHO) and World Cancer Report, there will be 15 million new cases of cancer worldwide in 2020 (WHO, 2019, WHO media centre, www.who.int/cancer). More than 90% of cancer‐related deaths occur by the spread of malignant cells to vital organs, a process called metastasis [42, 43]. Noble metal NP such as Au, Ag or a combination of both has been widely studied for cancer therapeutics and diagnosis [44]. Carboplatin is one of the drugs used in chemotherapy and used for the treatment of a wide number of cancers such as ovarian, lung, head and neck, brain, neuroblastoma, endometrial, oesophageal, bladder, breast and cervical; central nervous system or germ cell tumours; osteogenic sarcoma and as preparation for a stem cell or bone marrow transplant [45, 46]. For patients undergoing chemotherapy, it is given intravenously with 5% dextrose. Carboplatin binds to DNA efficiently, thereby inhibiting both replication and transcription followed by inducing cell death [47]. This, in turn, leads to changes that generate errors in replication, with the accumulation of cells in the G2/M phase and the induction of apoptosis [48]. The presence of higher levels of reactive oxygen species (ROSs) in cancer cells may lead to tumour development and progression [49] thereby inducing DNA damage and genetic instability [50]. Oxidative stress‐mediated signalling events in cancer could induce cell cycle progression and proliferation, cell survival and apoptosis, energy metabolism, cell morphology, cell–cell adhesion, cell motility, angiogenesis and maintenance of tumour stemness [51, 52, 53, 54]. Resistance to carboplatin is a major problem in cancer therapy and has attracted the attention of researchers. Carboplatin encapsulated in sodium alginate showed improved therapeutic efficacy and bioavailability than free drug carboplatin thereby minimising the dose required for therapy plus dose‐related side effects [55]. Porcel et al. [56] reported that a combination of fast ion irradiation therapy with platinum NPs (PtNPs) coated with polyacrylic acid (PAA) showed effective lethal damage in the DNA of tumour cells when compared to dispersed platinum atoms. PtNPs are extensively studied for their antimicrobial, antioxidant and anticancer properties apart from their established catalytic applications [57].

There are relatively a number of published works on the synthesis of metal NPs using metal inorganic salts as precursors and biopolymers as a reducing agent [21, 22, 24, 33]. The present manuscript describes the synthesis of GK stabilised organic Pt‐nanocomposite (GKCPt NC) using carboplatin as a precursor and GK as stabilising or reducing agent. Characterisation studies of the synthesised GKCPt NC have been performed to evaluate the physical and chemical properties. Further, in vitro radical scavenging and antitumour activity of GKCPt NC have been investigated. Radical scavenging activity was measured by 1, 1‐diphenyl‐2‐picrylhydrazyle (DPPH) and 2, 2‐azinobis‐(3‐ethylbenzthinzoline‐6‐sulphonic acid) (ABTS) assay in comparison with ascorbic acid. Antitumour activity has been evaluated by in vitro potato disc tumour induction assay using Agrobacterium tumifaciens since this gram‐negative bacterium can induce crown gall disease in plants. This assay detects the antitumour properties of a drug [58, 59] and the validity of this assay was based on the observations that certain tumourigenic mechanisms were similar in plants and animals [60, 61, 62]. Henceforth, the current research article explores the green synthesis of tree gum stabilised organic Ptnanocomposite including the antioxidant and antitumour properties, in comparison with carboplatin.

2 Materials and methods

2.1 Materials

GK samples procured from Girijan Co‐operative Corporation, Hyderabad, Government of Andhra Pradesh undertaking and M/s. D.K. Enterprises, Hyderabad, India. GK is available in three grades viz – Grade I, Grade II and Grade III. Grade I was selected for the metal NP preparation since it is free of extraneous material [27]. Carboplatin (TCI chemicals, Japan), ABTS (Sigma‐Aldrich, Bengaluru, India), ethanol absolute, methanol (Merck, Mumbai, India), DPPH (Sigma‐Aldrich), potassium persulphate (SD Fine Chemical Ltd., Mumbai, India), were used for the current research work. Milli‐Q water was used for the preparation of the solutions. All other chemicals and reagents used were of analytical grade.

2.2 Preparation of soluble GK

GK sample was powdered using a high‐speed mechanical blender (Philips, Mumbai, India). The powdered sample was sieved (mesh size – 45 μm) to obtain a uniform sample size. The preparation of soluble GK was done according to Kalaignana Selvi et al. [24].

2.3 Synthesis of GKCPt NC

For the preparation GKCPt NC using soluble GK and carboplatin, the method described in Vinod et al. [21] and Godugu and Sashidhar [22] was followed with minor modifications. The synthesis was carried out by the following procedure.

2.3.1 Optimisation of GKCPt NC synthesis

The GK and carboplatin solutions were mixed and the pH was adjusted (ELICO, Hyderabad, India) to 8 using 0.1N NaOH. This solution was subjected to probe sonication (ENUP‐500; Lifecare Equipment, Mumbai, India) for 5 min with 50% power, pulse 10 s on and off. Later, this solution was subjected to autoclave (Ketan, semi‐automatic autoclave, Hyderabad, India) process at 121°C and 15 psi for 30 min. The solution turned into a dark colour indicating the formation of Pt NC. The concentration effect of gum (0.1–0.5%) and carboplatin (0.125–1.0 mM) on NC synthesis was investigated. Later the colloidal solution was subjected to sonication for 10 min with 50% power and 10 s pulse on and off. The black colloidal solution turned to a homogeneous black solution. To solubilise the nanocomposite, the colloidal solution was subjected to probe sonication.

2.3.2 Purification of GKCPt NC

The black coloured solution was further subjected to a spin column (Sartorius, Gottingen, Germany) of 5000 Da cut off and centrifuged at 4000g for 15 min. A clear colourless solution was collected down in the spin column while the black colour solution remained on top of the membrane of the spin column. At each of the mentioned steps, samples were analysed in the ultraviolet (UV)–visible spectrophotometer (Cary 60 UV–Vis, Agilent, California, USA) and NP Analyser (DelsaMax PRO; Beckman Coulter, Indianapolis, USA). The black colour solution was collected from the spin column which is GKCPt NC and further subjected to characterisation studies to evaluate chemical and biological properties. Stability studies of purified GKCPt NC were also evaluated, upon storage at 4°C over a period of 270 days by dynamic light scattering (DLS) method (NP analyser).

2.4 Characterisation studies of GKCPt NC

2.4.1 UV–vis spectrophotometry

To assess the formation of GKCPt NC, UV–vis spectrophotometer was used. This was the first step to judge the formation of metal NP since each metal has unique surface plasmon resonance (SPR). Both concentration and temperature effects were studied on GKCPt NC synthesis.

2.4.2 NP analyser – Zeta potential and particle scattering diffusometry (PSD)

PSD and zeta potential were analysed using NP analyser. This instrument performs with high accuracy, less time consuming, reproducible data and low sample volume are required. The instrument utilises PSD to evaluate molecular size simultaneously with phase analysis light scatter, to determine zeta potential and has an inbuilt 32 detector array which allows sensitive detection of particle motion in response to the applied electric field. The principle is based on the measurement of the Brownian motion of the molecules present in the solution [63, 64, 65]. PSD defines the uniformity, spherical nature of the sample and hydrodynamic diameter of the particles which includes organic core plus molecules adsorbed on the surface, while zeta potential defines the stability by measuring the net surface charge [66, 67]. The measurements were made by directly adding the solution in the cuvette provided by the instrument and the data was analysed using DelsaMax software supplied by the manufacturer (DelsaMax PRO; Beckman Coulter).

2.4.3 Scanning electron microscopy and energy dispersive X‐ray analysis (SEM–EDXA)

EDXA is a chemical microanalysis technique used in conjunction with SEM (Zeiss EVO SEM, New York, USA). The samples were analysed in their natural state and were directly coated on the carbon tape. The EDXA technique detects X‐rays emitted from the sample during bombardment by an electron beam to characterise the elemental composition of the sample.

2.4.4 Fourier‐transform infrared spectroscopy (FTIR)

The functional groups involved in the formation of GKCPt NC were analysed by the FTIR spectrophotometer (JASCO Model 420 FTIR, Tokyo, Japan). The pre‐set parameters were of 16 scans with 4 cm⁻¹ resolution and a measurement range of 250–4000 cm⁻¹. Since the samples were in liquid form, they were directly placed between infrared plates for the analysis.

2.4.5 Transmission electron microscopy (TEM)

TEM (Tecnai F 12 TEM, Michigan, USA) was performed to study the metal radius. TEM images were recorded at an acceleration voltage of 15 kV. The emission current (‘emission’ in the ‘filament’ control) was stabilised to ∼0.5–1 µA with spherical aberration (Cs) at 2.0 mm. The samples were viewed in CCD camera pixel size of 7.4 µm, (2.1 Å/pixel @ 50,000×) and the nominal magnification was set at 50,000×: 35,238× provided in the instrument. For TEM measurements, samples were first sonicated and a drop (∼100 μl) of the sample was added on a carbon‐coated copper grid and allowed to air dry.

2.4.6 X‐ray diffraction (XRD) analysis

XRD (Axis D8 advance, Bruker, Wisconsin, USA) was performed using Cu Kα radiation at wavelength 1.5406 A⁰. The measurements were made using a step‐scanning program with 0.02° per step and acquisition time of 5 s per step and the data were analysed using X'Pert High Score software. XRD is a rapid analytical technique primarily used for phase identification of a crystalline material and could provide information on unit cell dimensions. The sample was first lyophilised and then finely ground, homogenised for XRD analysis.

2.4.7 X‐ray photoelectron spectroscopy (XPS)

XPS (Kratos Analytical Axis SUPRA, Manchester, UK) measurements were carried to study the binding energy (BE) and oxidation states of the elements in the NC. Monochromatic X‐ray sources were in the range of AlKa (1486.6 eV) with a hemispherical energy analyser of diameter 165 mm. Samples were first lyophilised and then subjected to XPS analysis.

2.4.8 Inductively coupled plasma optical emission spectrophotometer (ICP‐OES)

ICP‐OES analyses the concentration of metals in the NPs. The concentration of Pt ions in the GKCPt NC and Pt ions in carboplatin was determined by using ICP‐OES (Ultima 2, Horiba Scientific, Tokyo, Japan). Platinum was analysed at a wavelength of 224.552 nm. The samples were diluted appropriately with MilliQ water for the ICP‐OES analysis.

2.5 Bioassays for the validation of in vitro antioxidant and antitumour activity of the synthesised GKCPt NC

2.5.1 In vitro antioxidant activity

Both DPPH and ABTS were performed to assess the antioxidant activity of GKCPt NC.

DPPH assay: The scavenging activity of GKCPt NC was assayed by measuring the decrease in absorbance at 517 nm of the stable free radical DPPH^. (1, 1‐diphenyl‐2‐picrylhydrazyl). The purple‐coloured free radical reacts with scavenger to yield the yellow colour product 1,1‐diphenyl‐2‐picrylhydrazine which is read at 517 nm. The method was adapted to a 96 well microplate scale [68]. The working standard of DPPH was 100 μM prepared in absolute methanol. GKCPt NC, soluble GK, carboplatin and ascorbic acid concentrations were in the range of 5–25 μg/ml. DPPH reagent and samples were mixed in a 1:1 ratio and incubated in dark at room temperature for 30 min. Samples were run in triplicates and absorbance was recorded at 517 nm. Ascorbic acid served as positive control while GK was used as the negative control. The DPPH scavenging activity was calculated as follows:

$$\mathrm{DPPH}\mathrm{scavenging}\left(\text{\%}\right)=\frac{\mathrm{DPPH}\mathrm{absorbance}-\mathrm{Sample}\mathrm{absorbance}}{\mathrm{DPPH}\mathrm{absorbance}}\times 100$$

ABTS assay: The ABTS⁺ [2,2‐ azino bis (3‐ethyl‐benzothiazoline‐6‐sulfonic acid)] scavenging activity of GKCPt NC was assayed by measuring the decrease in absorbance at 734 nm. This assay measures the relative ability of an antioxidant to scavenge ABTS generated in the aqueous phase. 7.4 mM ABTS solution is added in 2.5 mM potassium persulphate and incubated under dark at room temperature for 12–18 h for obtaining a stable oxidative state. Then, the solution was diluted with sodium phosphate buffer (10 mM, pH 7.4) to get an absorbance of 0.8 at 734 nm. This is the working solution of ABTS⁺ radical cation and is greenish‐blue in colour. This, in turn, becomes a colourless solution upon the activity of antioxidants which is read at 734 nm [69]. The method was adapted to a 96 well microplate scale. GKCPt NC, soluble GK, carboplatin and Ascorbic acid concentrations were in the range of 5–25 μg/ml. ABTS reagent and samples were mixed in a 1:1 ratio and incubated in dark at room temperature for 60 min. Samples were run in triplicates and absorbance was recorded at 734 nm. Ascorbic acid served as positive control while GK was used as the negative control. The ABTS scavenging activity was calculated as follows:

$$\mathrm{ABTS}\mathrm{scavenging}\left(\text{\%}\right)=\frac{\mathrm{ABTS}\mathrm{absorbance}-\mathrm{Sample}\mathrm{absorbance}}{\mathrm{ABTS}\mathrm{absorbance}}\times 100$$

2.5.2 In vitro antitumour assay: potato disc tumour induction assay

Assay performed in two steps and was executed as per the method reported by Suresh et al. [70]. The first step comprises, evaluation of the antibacterial activity of GK, GKCPt NC, carboplatin while Pen‐Strep was used as a positive control. Milli Q water was used as a negative control since all the samples were soluble in Milli Q water. The second step involves the analysis of antitumour activity by potato disc tumour bioassay procedure. Milli Q water and GK were negative controls while GKCPt NC and carboplatin, as samples to be assessed.

2.6 Statistical analysis

The experimental data were analysed using GraphPad Prism software 8 version for the analysis of antioxidant and antitumour activity. The study was done in triplicates and all the values were expressed as mean ± standard error. Further, antioxidant activity was analysed by two‐way analysis of variance (ANOVA) and p ‐value ≤ 0.0001 was accepted as significant. For evaluation of antitumour activity, linear regression followed by IC₅₀ analysis was carried out using GraphPad Prism software 8 version.

3 Results and discussion

3.1 Characterisation studies of GKCPt NC

3.1.1 UV–vis spectroscopy analysis

The aqueous solution of GK–carboplatin was transformed from colourless to black (Pt) colour solution due to the excitation of surface plasmon vibrations in the formed NPs (Fig. 1 a inset). The first observation of GKCPt NC formation is the visually distinct black coloured solution of NC within the GK bio‐template. These observations were confirmed by UV–vis spectrophotometer analysis and no SPR was observed in GKCPt NC in agreement with earlier reports [21, 71] that Pt has strong d‐electron excitations which increase the complex dielectric constants ɛ 1 and ɛ 2. The UV spectrum of carboplatin showed a strong one absorbance peak corresponding to the Pt²⁺ ions at 230 nm (Fig. 1 b inset graph). After the addition of GK, the absorption peak at 230 nm disappears corresponding to a steady reduction of Pt²⁺ resulting in NP formation (Figs. 1 a and b). The clear colourless solution from the spin column was the unreacted portion of carboplatin residue as confirmed from UV–vis analysis since the absorption spectra were similar to native carboplatin [Fig. 1 b (inset graph)].

Fig. 1.

UV–vis absorption spectra of GKCPt NC

(a) Different concentrations of GK (0.1–0.5%) at constant 1 mM carboplatin concentration. Inset: GK stabilised Pt NC formation (A) before and (B) after autoclave process, (b) Different concentrations of carboplatin (0.125–1.0 mM) at constant 0.1% GK concentration. Inset: absorption spectrum of GKCPt NC, unreacted portion and native carboplatin, wherein the peak at 230 nm disappears in GKCPt NC and remains prominent in native carboplatin and unreacted portion solution, (c) Study of different temperature effects (25–95°C) on GKCPt NC. Inset: absorption scan of GKCPt NC before and after heat treatment, showing the stability of the GKCPt NC

The synthesis was studied by changing the gum concentrations (0.1%–0.5%) while maintaining the constant concentration of carboplatin at 1 mM (Fig. 1 a). It was observed from the UV–vis studies that with an increase in the concentration of gum, there was not much enhancement in the Pt²⁺ ion reduction, as shown by gradual intensity increase from visible to UV region in broad continuous absorption spectra. Visually, the intensity of the black colour solution decreased from 0.1 GK to 0.5% GK. Since visually 0.1% GK solution showed deep black colour, 0.1% GK concentration was selected for PtNP synthesis. Based on these observations, optimisation studies were further carried out by changing the carboplatin concentrations (0.125–1 mM) while maintaining the constant concentration of GK at 0.1%. It was observed from the UV–vis data that the intensity of the absorption band increased with an increase in metal precursor concentration (Fig. 1 b). This might be as a result of a greater number of NP formation due to an increase in the nuclei formation. These observations suggest the abundance of reducing functional groups in GK. UV–vis spectra results were in accordance with previous reports of Pt NP synthesis studies [72, 73]. Henceforth, observations based on the UV–vis spectra, the optimal condition for GKCPt NC synthesis was found to be 0.1% soluble GK, 1 mM carboplatin with pH 8 at 30 min of autoclave time. Further, the effect of temperature (25–95°C) on GKCPt NC was carried out and portrayed in Fig. 1 c. The data indicated that temperature differences did not influence absorption intensity, deducing that the synthesised nanocomposite is thermally stable.

3.1.2 Dynamic light scattering and zeta potential analysis

NP analyser observations of GKCPt NC synthesis at each step are given in Table 1. At each synthesis step of GKCPt NC, the readings were taken to evaluate the formation of the metal nanocomposite. The freshly prepared solution showed a mean particle size of 27.90 nm plus zeta potential of −39.35 mV. This solution was subjected to probe sonicator treatment to increase the solubility of GKCPt NC. After treatment, the mean particle size decreased to 24.60 nm while zeta potential increased to −41.68 mV. By subjecting to probe sonication, the particle size in the solution becomes more uniform and homogeneous due to the action of pulse vibrations. Finally, after purification steps, the obtained GKCPt NC solution showed a mean particle size of 20.40 nm (Fig. 2 a) and zeta potential of −43.67 mV (Fig. 2 b), while the unreacted clear colourless solution showed 13.60 nm and zeta potential of −1.65 mV. The zeta potential of GKCPt NC gradually increased from −39.35 to −43.67 mV due to probe sonication treatment. Hence, probe sonication is a prerequisite step for maintaining the uniformity and stability of the metal nanocomposite. The higher negative charge means more stability of NP in the colloidal solution due to the intrinsic capping by polysaccharides and proteins of the gum [3, 23]. The present results are in agreement with the findings of Kora [23] and Godugu and Sashidhar [74]. The results could be attributed to the findings of Roy et al. [75] where the DLS pattern of the suspension of Ag NP synthesised using parsley (Petroselinum crispum) leaf extract was found to be in the range of 25–30 nm. The GKCPt NC solution was stored at 4°C and was analysed every fortnight for nine months to study the stability (Table 2). The observations showed a slight increase in mean particle size (20.40–26.40 nm) due to the aggregation properties of Pt NP than other noble metals [76]. The zeta potential marginally decreased from −43.67 to −21.81 mV (Figs. 2 b and d) and this could be attributed to the aggregation properties of Pt NPs. Figs. 2 c and d shows mean particle size and zeta potential of GKCPt NC solution stored after 240 days, at 4°C. The DLS pattern of the particles shows uniformity with no significant variation, suggesting its stability at 4°C over a period of 240 days (Figs. 2 a and c). NP analyser is a useful analytical tool to study the stability of the synthesised NP as it measures the uniformity, spherical shape and hydrodynamic diameter of the particles. From the observations, it could be inferred that GKCPt NC was stable at 4°C for an extensive time period.

Table 1.

Particle size and zeta potential values of GKCPt NC determined by NP analyser

S No.	Sample name	Particle size, nm	Zeta potential, mV
1	total GKCPt NC	27.90	−39.35
2	probe sonicated total GKCPt NC	24.60	−41.68
3	purified GKCPt NC	20.40	−43.67
4	unreacted colourless solution collected in spin column	13.60	−1.65

Fig. 2.

Particle size and Zeta potential analysis

(a) DLS (particle size) of GKCPt NC – fresh solution, (b) Zeta potential of GKCPt NC – fresh solution, (c) DLS (particle size) of GKCPt NC after 240 days storage period, (d) Zeta potential of GKCPt NC after 240 days storage period

Table 2.

Stability studies of GKCPt NC as determined by NP analyser at different time points

S No.	GKCPt NC sample stability (d)	Particle size, nm	Zeta potential, mV
1	‘0’	20.40	−43.67
2	07	19.90	−43.18
3	30	23.20	−41.42
4	60	24.10	−42.69
5	90	18.60	−45.13
6	120	27.80	−53.31
7	135	27.90	−55.62
8	150	28.50	−48.18
9	180	33.90	−25.70
10	210	32.70	−23.78
11	240	27.70	−19.58
12	270	26.40	−21.81

3.1.3 SEM and EDXA analysis

Fig. 3 shows SEM photo‐micrographs with corresponding EDXA spectra of soluble GK and GKCPt NC. The highly filamentary nature of soluble GK was observed from the SEM micrograph (Fig. 3 a). EDXA shows the presence of different minerals such as Na, K, Mg, Cl, Ca, P (Fig. 3 b). Similar observations were reported by Vinod et al., [21] for total GK and Kalaignana Selvi et al., [24] for soluble GK. SEM micrograph of GKCPt NC showed the uniform distribution of nanocomposite (Fig. 3 c). EDXA of GKCPt NC (Fig. 3 d) showed that the Pt atoms are present within the gum matrix, which could be attributed to the strong interaction of organo‐metal with the gum matrix to form metal‐nanocomposite. This evidence suggests that the reduced metal ions are bound to the surface of the gum matrix. Strong signals from Pt atoms in the nanocomposite were observed, while weaker signals from C, O, Na, P, Mg, K, Ca and Na atoms were also recorded. Comparable observations were reported by Mavukkandy et al. [71] in Pt NPs.

Fig. 3.

SEM photo‐micrographs and EDXA spectra

(a) SEM photo‐micrograph of soluble GK, (b) EDXA spectra of soluble GK, (c) SEM photo‐micrograph of GKCPt NC, (d) EDXA spectra of GKCPt NC (arrow) showing Pt atoms embedded within the gum matrix

3.1.4 XRD analysis

The observations of XRD showed the amorphous nature of GKCPt NC (Fig. 4 a) while the crystalline nature of carboplatin (Fig. 4 b). The XRD data of carboplatin shows crystalline nature, with a face‐centred cubic structure. The broad peaks were observed at 39.889° (111), 46.95° (200) and 67.61° (220) assigned to JCPDS Card No. 04‐0802 demonstrating the presence of Pt (Fig. 4 b). The other diffraction peaks are due to organic ligands present in carboplatin. Much of the literature reported the crystalline nature of Pt NPs [21, 22, 71, 73] where the precursor of platinum is hydrochloroplatinic acid. The current research article reports the amorphous nature of GKCPt NC where the precursor of Pt is carboplatin.

Fig. 4.

XRD spectra

(a) XRD of native soluble GK and GKCPt NC showing amorphous nature, (b) XRD of carboplatin, showing crystalline nature of with face centred cubic structure

3.1.5 TEM analysis

The TEM investigation of native soluble GK shows a branched leaf‐like structural morphology as depicted in Fig. 5 a and a similar observation was reported by Vinod et al. [21] in total GK. Selected area electron diffraction (SAED) of soluble GK shows a diffused ring pattern suggesting the amorphous nature of the soluble GK. The TEM image of GKCPt NC depicts the nature of the Pt nanocomposite to be spherical and highly mono‐dispersed (Fig. 5 b). The SAED showed a diffused ring pattern which suggests the amorphous nature of the Pt nanocomposite (Fig. 5 b inset). The particle size distribution showed the GKCPt NC ranging from 1.672 to 4.515 nm, with an average size of 3.08 ± 0.62 nm (Fig. 5 c).

Fig. 5.

TEM image micrograph of soluble GK

(a) Showing branched network structure. Inset: corresponding SAED pattern showing amorphous nature of GK, (b) TEM image micrograph of GKCPt NC showing uniform distribution of small spherical particles. Inset: corresponding SAED pattern showing diffused ring pattern which suggests the amorphous nature of the GKCPt NC, (c) Histogram shows particle size distribution and the average size of Pt was in the range of 3.08 ± 0.62 nm (mean ± SD)

The present results are in agreement with Vinod et al., [21] and Godugu and Sashidhar [22] who reported GKPt NPs to be 2.4 ± 0.7 nm and 2–4 ± 0.50 nm, respectively. Sreedhar et al. [72] reported 2–3 nm with gum acacia stabilised Pt NPs while Kora and Rastogi [73] reported 4.4 ± 0.5 nm with gum olibanum stabilised Pt NPs. It is imperative to note that with tree gums as reducing agent the Pt NPs size has been maintained to <5 nm.

3.1.6 FTIR analysis

FTIR spectroscopic measurements of soluble GK and GKCPt NC showed the involvement of various functional groups in the formation of metal NPs (Fig. 6). The soluble GK showed prominent absorbance bands at, 2934, 2116, 1723, 1621,1417, 1255, 1046 and 631 cm⁻¹, respectively. The major functional groups present in FTIR spectrum of soluble GK are 3442 (‐OH), 2934 [–C(=0)H, aldehyde], 1723 (C=O), 1621 asymmetric stretching frequency (C–O), 1417 (C–N) stretching frequency, 1255 (−CH3CO), 1046 (C–0) stretching frequency methylene acetal and 664 C–H out‐of‐plane bend cis RHC=CHRʹ. These results were in accordance with the observations of Kalaignana Selvi et al. [24].

Fig. 6.

FTIR data of native soluble GK and GKCPt NC indicating the involvement of various functional groups in the formation of metal nanocomposite

In the case of GKCPt NC, a large shift in the absorbance peak was observed from 3442 to 3287;1621 to 1632 cm⁻¹, 1255 to 1260 cm⁻¹ and 664 to 631 cm⁻¹, suggesting the binding of Pt atoms with hydroxyl and carboxylate groups of the gum, respectively. In total, 2116 cm⁻¹ corresponds to NH₂ ⁺–NH⁺ stretching frequency suggesting the stabilisation of GKCPt NC by the gum proteins. It is pertinent to note that the nanocomposite shows prominent peak shifts from 1621 to 1632 cm⁻¹, 1255 to 1260 cm⁻¹ and 664 to 631 cm⁻¹ corresponding to amide I and amide II linkages.

3.1.7 XPS analysis

XPS survey scan of GKCPt NC showed the presence of oxygen, O 1 s (at 535 eV); carbon, C 1 s (at 285 eV) and platinum, Pt 4f (at 72 eV) (Fig. 7 a). Curve fitting analysis showed that the Pt 4f spectra for GKCPt NC resulted from two pairs of spin‐orbit components. BE was observed at 71.2 and 74.6 eV, respectively, and have been attributed to metallic platinum Pt(0). The second set of peaks for Pt 4f_7/2 and Pt 4f_5/2 were observed at BE = 73.2 and 76.4 eV, respectively, occurring at higher BE values could be attributed due to Pt atoms with lower charge density. Although the presence of Pt in two oxidation states as observed from the graph, the higher intensity in Pt nanocomposite indicates the metallic form of the sample. The BE value at ∼73.2 eV could be attributed to the presence of Pt atoms on the surface capping on PtNPs. The BE at 71.2 and 74.6 eV, shows the presence of metallic platinum Pt(0) confirmed by XPS studies [77]. XPS high‐resolution narrow scans of C 1 s are depicted in Fig. 7 b. The observed C 1 s peaks could be deconvoluted into five peaks, and the binding energies are attributed due to the energies distinct for substituted carbon moieties. The observed C 1 s peaks at 283.9, 284.6, 285.4, 286.6 and 287.5 eV are characteristic of Pt–C, –C–C–/–CH–, a carbon singly bonded to one oxygen atom (–C–O–), –O–C–O– and carboxylic acid group (–O–C=O–). The current XPS observations are in agreement with Sreedhar et al. [72].

Fig. 7.

XPS high‐resolution spectra

(a) XPS high‐resolution narrow scans of Pt 4f for GKCPt NC. BE = 71.2 and 74.6 eV, represent metallic platinum Pt (0). The second set of peaks for Pt 4f 7/2 and Pt 4f 5/2 observed at BE = 73.2 and 76.4 eV, (b) XPS high resolution narrow scans of C 1 s. The observed C 1 s peaks at 283.9, 284.6, 285.4, 286.6 and 287.5 eV are characteristic of Pt–C, –C–C–/–CH–, (–C–O–), –O–C–O– and (–O–C=O)

3.1.8 ICP‐OES studies

The concentration of Pt in carboplatin and GKCPt NC was determined by ICP‐OES. The concentration of Pt in GKCPt NC was determined to be 200 μg ml⁻¹, while the concentration of Pt in carboplatin was determined to be 235 μg ml⁻¹. The percent conversion of Pt ions to NPs was 85.11% suggesting the excellent reductive property of GK in the formation of GKCPt NC. Earlier, Godugu and Sashidhar [22] reported percent conversion of Pt ions to NPs at 56.06% in GKPt NP.

3.2 Bioassays investigation studies

3.2.1 Analysis of antioxidant assay of the synthesised GKCPt NC

Both DPPH and ABTS assays have been standardised in 96‐well enzyme linked immunosorbent assay (ELISA) plates. DPPH activity checked at 517 nm while ABTS activity at 734 nm. In comparison to GK and carboplatin, GKCPt NC showed superior DPPH activity of 87.82% while ABTS activity was only 38.50%, respectively. Since the DPPH activity was high, the IC₅₀ value was evaluated and was found to be 9.215 µg/ml with Hillslope of 3.893 and R ²  = 0.9877. Ascorbic acid was used as a reference standard (Fig. 8 a and b).

Fig. 8.

Assessment of antioxidant potential of GKCPt NC

(a) DPPH radical scavenging activity of GKCPt NC, soluble GK, carboplatin with corresponding positive control Ascorbic acid. Values are mean ± SD for n  = 3 and p  < 0.0001 compared with the positive control and ascorbic acid, (b) ABTS radical scavenging activity of GKCPt NC, soluble GK, carboplatin with corresponding positive control ascorbic acid. Values are mean ± SD for n  = 3 and p  < 0.0001 compared with the positive control and ascorbic acid

The scavenging activity may perhaps be explained by the negative zeta potential (−43.27 mV) of GKCPt NC, where an electron of PtNPs could transfer to DPPH radical. Since the NC is electron‐rich, it could transfer its electrons to hydrogen peroxide and DPPH radical thereby neutralising the free radicals as shown below:

$$\mathrm{DPP}{\mathrm{H}}^{\cdot }+2{\mathrm{e}}^{-}+2{\mathrm{H}}^{-}\to \mathrm{DPP}{\mathrm{H}}_2$$

Hence, GKCPt NC could act as a reducing agent and this property could be applied in treating diseases due to high ROSs. The present results could be attributed to the observations of Aiuchi et al. [78] who reported that colloidal Pt NP's could reduce H₂ O₂, DPPH and DCIP radicals. On the other hand, GKCPt NC showed 38.50% ABTS activity when compared to ascorbic acid, which showed 99.12% ABTS activity (Fig. 8 b). Even though GKCPt NC is reactive towards ABTS⁺ radical cation, it displayed slow kinetics when compared to ascorbic acid and requires strong antioxidants such as phenolics, thiols and vitamin C to neutralise ABTS⁺ radical cation [79]. Carboplatin, a chemotherapy drug does not show antioxidant activity but the GKCPt NC displayed the property (Figs. 8 a and b). Watanabe et al. [80] demonstrated superoxide dismutase (SOD)/catalase mimetic property and activity like mitochondrial electron transfer complex I of PAA‐protected Pt NP species (PAA‐Pt) and further stressed its applications in the medical treatment of diseases related to oxidative stress and ageing. Pt NPs could scavenge superoxide anion radical $\left({\mathrm{O}}_2^{-\bullet }\right)$ effectively while bimetallic Au and Pt NP could scavenge both ${\mathrm{O}}_2^{-\bullet }$ and H₂ O₂ [81]. The peroxidase activity and bioremediation properties of gum olibanum stabilised Pt NPs were reported by Kora and Rastogi [73]. GK stabilised PtNPs (GKPt NP) showed antifungal activity by destabilisation of the fungal outer membrane through the mechanism of oxidative stress [22].

3.2.2 Potato disc tumour induction assay

Potato disc tumour bioassay model has been successfully employed in screening the antitumour property of GKCPt NC and carboplatin. It is a screening test to assess the antineoplastic activity of the drugs [59]. The results are explained in two steps as mentioned below, antibacterial and potato tumour bioassay.

Antibacterial assay analysis: An important prerequisite for potato disc tumour induction assay is that the GKCPt NC should not inhibit the growth of A. tumifaciens, as its growth is essential for the infection and tumour formation in potato discs. The purpose of this experiment was to examine the sensitivity of A. tumifaciens to GKCPt NC (antibiotic effect). The antibacterial activity of GK, carboplatin and pen‐strep (positive control) was also evaluated along with GKCPt NC. Milli Q water was used as a negative control since all the samples were soluble in Milli Q water. The results reveal that the A. tumifaciens was not sensitive to GKCPt NC even at the highest concentration of 100 µg/ml and the viability of the bacterium was unaffected (zero zones of inhibition), indicating that the bacteria can induce tumour in potato discs. However, only positive control, pen‐strep could inhibit A. tumifaciens as observed from the formation of a clear zone of inhibition (Fig. 9). MilliQ water, GK and carboplatin had no inhibitory effect on A. tumifaciens growth (Fig. 9). Based on the outcome of these results, the potato tumour bioassay experiment was carried out to evaluate the antitumour property of GKCPt NC and carboplatin.

Fig. 9.

Antibacterial assay: Anti‐ A. tumifaciens activity of positive control – pen‐strep; soluble GK; GKCPt NC and carboplatin. Assay clearly indicates that GK, GKCPt NC and carboplatin have no inhibitory effect on bacterial growth

Potato Tumour Bioassay analysis: Tumour induction by A. tumifaciens was significantly evident in negative controls, MilliQ water and GK. Tumours were visible after 15 days of incubation and counted using a magnifying glass. The objective was to verify the antineoplastic activity in the potato disc tumour induction assay, regardless of the mode of antineoplastic drug action. The concentration range of all the samples was from 20 to 100 μg/ml. The experiment was performed in triplicates. Tumour induction by A. tumefaciens in potato disc was significantly apparent in the negative control and GK at a concentration of 20–100 μg/ml. In the case of GKCPt NC, the tumours were observed at 20–40 μg/ml and the complete tumour inhibition was observed at the concentration of 60 μg/ml. In discs treated with carboplatin, the formation of tumours was observed from 20 to 60 μg/ml, but the tumour induction was completely inhibited at a concentration of 80 μg/ml. It is inferred that, as the concentration of GKCPt NC and carboplatin increases, the tumour count decreased, i.e. the concentration of drugs is inversely proportional to the number of tumours. Fig. 10 illustrates the IC₅₀ graph of GKCPt NC and carboplatin. IC₅₀ of GKCPt NC was found to be 7.84 µg/ml (Hillslope of 1.479 and R ²  = 0.9905), while 15.95 µg/ml (Hillslope of 1.903 and R ²  = 0.9766) for carboplatin, respectively. The efficacy of synthesised GKCPt NC concentration (IC₅₀) on tumour inhibition was found to be 2.04‐fold lower as compared to the native drug molecule, suggesting its efficacy.

Fig. 10.

IC₅₀ analysis of Potato tumour bioassay

(a) IC₅₀ graph of GKCPt NC, (b) IC₅₀ graph of carboplatin

The present experimental results validate the antioxidant and anticancer properties of the synthesised GKCPt NC. The mechanism of antitumour activity could be inhibition of topoisomerase, interference with tubulin function or prevention of microtubule reorganisation and the results are comparable with mammalian cell lines [59, 70]. Godugu and Sashidhar [74] reported antitumour activity of GKPt NP through potato tumour assay and the IC₅₀ was found to be 10.1 μg ml⁻¹. It could be imperative to note that drugs with both antitumour and antioxidant activity have higher therapeutic efficiency in treating cancer and GKCPt NC shows both properties. Li et al. [82] reported a positive linear relationship between antioxidant activity and anticancer effect of the five Chinese medicinal herbal water extracts. Plant extract of Tecoma stans showed both free radical scavenging and anticancer property against lung cancer cell lines and henceforth, both might be positively correlated [83]. Likewise, AL‐Faifi [84] reported both antioxidant and anticancer activity of methanolic extract of Euphorbia cactus Ehrenb plant. Overall, GKCPt NC has enhanced antitumour activity than the original drug carboplatin and in addition, shows excellent antioxidant activity when compared to carboplatin.

4 Conclusion

The novel natural biopolymer and biocompatible matrix GK was used for the synthesis of organic Pt nanocomposite. The synthesis of GKCPt NC was confirmed by characterisation studies such as UV–vis spectrophotometer, NP analyser, SEM–EDXA, XRD, TEM, FTIR, XPS and ICP‐OES studies. The UV–vis studies showed the optimal concentration for the synthesis of Pt nanocomposite to be 0.1% soluble GK and 1 mM carboplatin at pH 8 with 30 min of autoclave time. The XRD pattern established the amorphous nature besides, TEM analysis revealed the homogeneous, monodisperse and spherical nature of synthesised GKCPt NC with Pt metal size at 3.08 ± 0.62 nm. NPs or Nanocomposites as vehicles for drug delivery or as a drug is a significant research field combining the life sciences with nanotechnology. The current research article explored the GKCPt NC synthesis through the precursor carboplatin and biopolymer GK as both reducing and stabilising agent. GKCPt NC exhibited excellent scavenging and antitumour property when compared to its counter original drug carboplatin. This could be an indication that GKCPt NC could be effective in treating cancer due to both antioxidant and anti‐cancer properties in comparison to the original drug carboplatin. In the future perspective, in vitro assays would be carried out using human cancer cell lines to establish the correlation between antioxidant and anticancer properties of the synthesised GKCPt NC. Pharmacokinetic and pharmacodynamic studies of the GKCPt NC are yet to be explored.