γ-radiation induced synthesis of antibacterial silver nanocomposite scaffolds derived from natural gum Boswellia serrata

Journal of Drug Delivery Science and Technology 56 (2020) 101550

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γ-radiation induced synthesis of antibacterial silver nanocomposite scaffolds derived from natural gum Boswellia serrata

T

Amit Kumar Sharmaa,∗, Balbir Singh Kaitha, Uma Shankera, Bhuvanesh Guptab a b

Department of Chemistry, Dr B R Ambedkar National Institute of Technology, Jalandhar, 144 011, Punjab, India Department of Textile Technology, Indian Institute of Technology, New Delhi, 110016, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanocomposite Semi-IPN Biodegradable Antibacterial Crosslinked

This paper introduces a novel approach for the fabrication of antibacterial silver nanocomposite scaffolds using the plant gum exudate of Boswellia serrate. The fabrication of the nanocomposites was done through the formation of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) matrices under the impact of high energy γ-radiations. The γ-radiations induced in-situ reduction of Ag+ ions into Ag0 nanoparticles also limits the use of chemical reducing agents and adds novelty to the present study. The semi-IPN and IPN were found to uptake 8574% and 4493% water in comparison to their initial dry weight in the basic medium at 80 °C. The biodegradability analysis of the semi-IPN and IPN suggested 73% and 61% degradation in garden soil and 75% and 64% degradation in bio-compost, respectively after 70 days of time interval. The average particle size was found to be 11.3 nm and 8.6 nm in case of Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm, respectively. The antibacterial assay suggested that both the nanocomposite scaffolds possessed sufficient antibacterial potential against different bacterial strains including P. aeruginosa, S. aureus, B. cereus, and E. coli. The promising fluid absorptivity, biodegradability and antibacterial activity of the hydrogel nanocomposite scaffolds suggested their applicability towards biomedical applications.

1. Introduction Superabsorbent polymers have a unique property to show volume phase transition from swollen to un-swollen state under the impact of external stimuli like temperature, pH and pressure. Such type of behavior of the hydrogel makes them useful for designing smart materials to be used in agriculture, pharmaceutical and biomedical sector [1]. Hydrogels can be derived from synthetic or natural sources depending upon their application. Hydrogels derived from synthetic sources possess high strength, but they are usually non-biodegradable in nature [2]. On the other hand, natural backbone based hydrogels are more environment-friendly due to their highly biodegradable nature and thus such materials are more beneficial compared to other materials derived from synthetic monomers. The backbones derived from natural sources such as starch, cellulose, gelatin, chitosan, psyllium, gum ghatti, gum xanthan and gum dammar can be crosslinked with synthetic monomers to design 3-D crosslinked network structures possessing hydrophilic properties [3–5]. To further enhance the applicability of hydrogels in agriculture, pharmaceutical and electronic equipment industries, they can be modified

by the imbibement of organic/inorganic nanoparticles resulting in polymer nanocomposites [6,7]. Silver nanoparticles are the most widely used inorganic nanomaterials having a wide range of applications in the biomedical industry. Qiao et al. 2019 have synthesized silver nanoparticles decorated with carboxyl betaine groups ((AgNPsLA-OB) and used it as antibacterial nano-medicine against bacterial biofilms [8]. In another study, κ-carrageenan capped silver nanocomposites were synthesized using microwave radiations. CRG-Ag hydrogels exhibited excellent stability and antibacterial properties [9]. The prime importance of hydrogel nanocomposites is that the morphology and particle size of the nanomaterial can be easily controlled merely by controlling the concentration of monomer, crosslinker and polymer functionality [10]. In the last few decades, there has been an increasing interest in synthesizing hydrogels by γ-irradiation technique. γ- Radiations have sufficient energy to crosslink the polymeric chains onto the backbone without using any toxic reaction initiator [11,12]. Irradiation of samples using γ-radiations generates hydroxyl radicals and macro-radicals which further forms a crosslinked structure [13]. Synthesis of hydrogel using γ-radiations has several advantages, such as environmental

∗

Corresponding author. Department of Chemistry, Dr B R Ambedkar National Institute of Technology, Jalandhar, Punjab, India. E-mail addresses: [email protected] (A.K. Sharma), [email protected] (B.S. Kaith), [email protected] (U. Shanker), [email protected] (B. Gupta). https://doi.org/10.1016/j.jddst.2020.101550 Received 12 December 2019; Received in revised form 23 January 2020; Accepted 25 January 2020 Available online 29 January 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.

Journal of Drug Delivery Science and Technology 56 (2020) 101550

A.K. Sharma, et al.

was dried in a hot air oven at 60 ± 2 °C. The obtained residue (15 g) was finally grinded in pestle mortar to give a fine powder. 0.5 g of Boswellia serrate (Bs) aqueous fraction was dissolved in 21 mL double distilled water and this was further followed by the gentle addition of 0.8039 mol L−1 of acrylamide monomer. The reaction mixture was stirred over mechanical stirrer under slight heating conditions (50 °C). After homogeneous mixing 1.8532 × 10−2 mol L−1 crosslinker (N,N′Methylenebisacrylamide) was added in small installments to the above reaction mixture with continuous stirring. The reaction mixture was put into 80 mL beaker and placed in 60Co gamma radiation chamber to attain 3 KGy radiation dose at a dose rate of 3.7 kGy/h. After attaining optimum dose, the beaker was taken outside from the gamma radiation chamber and obtained semi-IPN (Bs-cl-polyAAm-Gm) was washed with distilled water and dried in a hot air oven at 60 °C.

friendliness, high penetration ability, ability to run at room temperature and simple process control [14]. In addition, this technique does not require the use of toxic initiators such as ammonium persulphate, potassium persulphate, hydrogen peroxide etc. Therefore, the hydrogels synthesized through this technique are highly applicable in developing scaffolds for wound healing treatments. Metal nanoparticles can be synthesized from metal salts using chemical reducing agents, but, chemical toxicity and agglomeration are some common limitations of such methods. Therefore, metal nanoparticles can be in situ synthesized inside the hydrogel matrix using the swelling-shrinking process. The reduction of metal ions using gamma radiations further limits the use of toxic reducing agents and adds novelty to the present work [15,16]. Silver nanoparticles have greater toxicity against different bacterial strains, but, their poor binding surface interactions limits their applicability as an antibacterial agent against the infected bacterial surface site. Therefore, silver-nanocomposite scaffolds based on hydrogel matrices are of great importance to inhibit bacterial infections [17]. In this paper, we have developed an eco-friendly method for the synthesis of silver nanocomposite scaffolds based on gum Boswellia serrate (Bs). The gum is obtained from the tree of Boswellia serrate which usually grows in dry rocky hills. The water-soluble fraction of gum is mainly composed of different types of polysaccharide fractions which were crosslinked with biocompatible and biodegradable polyacrylamide chains. Low cost of the starting materials and initiator free green synthesis of hydrogels, which limits the use of toxic reaction initiators adds novelty to the present work. The present method also limits the use of toxic reducing agents for the reduction of silver ions into silver nanoparticles. The synthesized scaffolds possess sufficient antibacterial activity and can be of great importance in the biomedical sector.

2.2.2. Conversion of semi-IPN (Bs-cl-polyAAm-Gm) into IPN (Bs-clpolyAAm-IPN-AA-Gm) The obtained semi-IPN (Bs-cl-polyAAm-Gm) was grinded to give a fine powder. 21 mL distilled water was added to 0.5 g semi-IPN (Bs-clpolyAAm-Gm) in a beaker followed by the addition of 2.4307 mol L−1 acrylic acid. The beaker was left as such overnight for the interpenetration of acrylic acid molecules inside the semi-IPN matrix (Bs-clpolyAAm-Gm). The pre-optimized amount of N,N′Methylenebisacrylamide (1.8532 × 10−2 mol L−1) was added slowly to the reaction mixture and finally, the reaction was carried out in gamma radiation chamber to achieve 3 KGy radiation dose. The synthesized IPN (Bs-cl-polyAAm-IPN-AA-Gm) was washed with distilled water to separate unreacted chemicals and homopolymer. Finally, the sample was dried in a hot air oven until constant the weight was attained. 2.2.3. γ-Radiations induced synthesis of silver nanocomposite scaffolds Green synthesis of silver nanocomposite scaffolds was carried out by immersing 1.0 g sample of each semi-IPN (Bs-cl-polyAAm-IPN-AA-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) in 200 mL distilled water to open up the crosslinked pores of hydrogels. After complete swelling, the samples were taken out, wiped off gently with tissue paper and then placed in freshly prepared silver nitrate aqueous solution of 1 × 10−4 mol L−1 concentration for 24 h. The access silver ions loaded over the surface of semi-IPN (Bs-cl-polyAAm-IPN-AA-Gm) and IPN (Bscl-polyAAm-IPN-AA-Gm) were removed and the samples were irradiated in 60Co γ-radiation chamber to achieve 50 KGy radiation dose at 3.7 kGy/h dose rate. High energy γ-radiations have sufficient potential to reduce silver ions without the use of any toxic chemical reducing agent. Finally, the nanocomposite scaffolds (Ag0/Bs-cl-polyAAm-IPNAA-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm) were dried in hot air oven at 60 °C.

2. Experimental 2.1. Materials and methods Gum Boswellia serrate (Bs) was purchased from MP Herbal Products, Shivpuri (India). Acrylamide (AAm), N, N′-methylene bisacrylamide (MBA) and acrylic acid (AA) were purchased from S D Fine Chemical Limited, India. Silver nitrate was procured from Merk. All the procedures were precisely followed using high purity double distilled water. Different samples were characterized using different techniques including FTIR (Agilent Technologies Carry 630 spectrophotometer), XRD (PAN ANALYTICAL X-ray diffractometer), FE-SEM (FE-SEM QUANTA 200 model), and HR-TEM {Tecnai G220 (FEI) S-Twin}. 60Co γ-radiation chamber {(GC-1200, BRIT, India) at IUAC (Inter University Accelerator Centre), New Delhi, India} was used as a source of gamma radiations. The weighing of the samples was done in Sartorius analytical balance (CPA225D). Optimization of reaction parameters was done on the basis of maximum % swelling (Ps) which was calculated for a 0.5 g sample immersed in 100 ml of double distilled water. The sample was repeatedly taken out, wiped gently and weighed after a definite time interval. Eq. (1) was used to calculate % swelling.

Ps =

Ws − Wd × 100 Wd

2.3. Biodegradation studies Eco-friendliness of the synthesized samples was investigated from the biodegradation behavior. The degradation of semi-IPN (Bs-clpolyAAm-IPN-AA-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) after their disposal was studied with respect to composting and soil burial methods. The soil for testing the biodegradability was collected from the campus of NIT, Jalandhar (India) and an equal amount of soil was placed in different well-leveled pots of equal size. 10 dry samples each of weight 1.0 g were placed in their respective pots in a circular fashion along the perimeter of the pot. Each sample was placed 6 cm deeper inside the soil surface and it was placed 3 cm distance apart from the other sample. Everyday, 250 mL of normal tap water and microbial concentration collected from sewage treatment plant (NIT Jalandhar) were added separately in the respective pots to maintain the moisture content of soil through soil burial and composting method, respectively. All the studies were carried out for 10 weeks and every week one sample from the respective pot was taken out, washed with water and

(1)

where Ps = swelling percentage, Ws = weight of swollen sample and Wd = weight of dry sample [18]. 2.2. Green synthesis of silver nanocomposite scaffolds 2.2.1. Synthesis of semi-IPN (Bs-cl-polyAAm-Gm) 100 g gum Boswellia serrate (Bs) was dissolved in double distilled water and the aqueous fraction of the gum was extracted with ether in order to remove ether soluble resin fraction. The water-soluble fraction 2

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semi-IPN showed 1450% maximum swelling percentage, whereas, the maximum swelling percentage was found to be 980% in case of IPN. The optimum value of γ-radiation dose was observed by varying the radiation dose from 2 KGy to 4 KGy with an increment of 0.5 KGy and keeping rest of the reaction parameters constant (solvent amount: 15 mL, pH: 7.0, N,N′-methylenebisacrylamide: 1.8532 × 10−2 molL−1 and acrylamide: 0.8039 molL-1). Initially, with the increase in γ-radiation dose, the swelling percentage was found to increase. But, after attaining the maxima (PS = 779.25), a decrease in swelling percentage was observed (Fig. 2a). The initial increase in swelling percentage with the increase in γ-radiation dose was due to the increase in free radical species which generates a large number of radical centers onto the backbone Boswellia serrate and monomeric moieties leading to graft copolymerization reaction. But, beyond the optimum γ-radiation dose, the swelling percentage decreases due to the excessive generation of free radicals leading to chain termination through intermolecular collision. Similarly, the water absorptivity increases initially with the increase in solvent amount but beyond an optimum value, a gradual decrease in water absorptivity was observed due to the increased number of *H free radicals and *OH free radicals (Fig. 2b) [24]. The optimum solvent amount was found to be 21 mL. The optimization was done by varying the solvent amount from 12 mL to 24 mL with an increment of 3 mL and keeping other reaction parameters constant (γradiation dose: 3 KGy, pH: 7.0, N,N′-methylenebisacrylamide: 1.8532 × 10−2 molL−1 and acrylamide: 0.8039 molL-1). The pH of reaction medium was varied from 3.0 to 11.0 to analyze its impact on swelling percentage under constant reaction conditions i.e. γ-radiation dose = 3 KGy, water volume = 21 mL, N,N′-methylenebisacrylamide = 1.8532 × 10−2 molL−1 and acrylamide = 0.8039 molL-1. The maximum swelling percentage = 1181% was observed at pH 7.0. Beyond this value, the swelling percentage decreases with the decrease or increase in pH of reaction medium (Fig. 2c). Osmotic pressure theory depicts the osmotic pressure of a weekly charged superabsorbent as per Eq. (7).

dried in a hot air oven at 60 °C until it attains constant weight. Biodegradation percentage (% BD) was evaluated at each stage from the weight loss of hydrogel sample Using Eq. (2) [19].

BD =

Wi − Wf Wi

× 100

(2)

where Wi = initial weight and Wf = final weight of the sample after every 7 days' time interval. Biodegraded samples were further analyzed qualitatively using FTIR and FESEM techniques. 2.4. Antibacterial properties The antibacterial properties of synthesized silver nanocomposite scaffolds were studied in nutrient agar type-III media which was prepared by mixing 5.0 g peptone, 5.0 g sodium chloride, 2.0 g yeast extract in 1000 mL double distilled water at pH 7.0 [20]. The mixture was sterilized in an autoclave at 15 psi pressure and 121 ± 2 °C temperature. The sterilized agar media in an appropriate amount was transferred to sterilized petri plates and allowed to solidify under laminar air-flow. Under sterilized conditions, different bacterial strains were placed over the well-leveled petriplates. For the examination of antibacterial activity, 3 different wells were created with the help of cork borer on the surface of solidified agar media of each petriplate. The drug amoxicillin was used as a reference sample against hydrogels and hydrogel nanocomposites. 40 μL of each sample (sample composition = 50 gL-1) was added to the respective well and left as such for 15 min for the diffusion of samples. 100 μL bacterial inoculation (OD = 1.0) was spread over each petriplate and the pertiplates were covered with parafilm in order to avoid any external contamination. Finally, the petriplates were incubated at 37 °C for 48 h in order to observe the growth pattern. 3. Results and discussion

ᴨion = RT∑(Cig - Cis)

Semi-IPN (Bs-cl-polyAAm-IPN-AA-Gm) and IPN (Bs-cl-polyAAmIPN-AA-Gm) was synthesized through γ-radiation induced graft copolymerization reaction of polyacrylamide and polyacrylic acid chains onto the backbone Boswellia serrate. Under the impact of γ-radiations, water molecules produces *OH free radicals and *H free radicals which further propagate the chain reaction and crosslinks the polyacrylamide and N,N′-methylenebisacrylamide to give a crosslinked semi-IPN [21,22]. Under similar reaction conditions, semi-IPN was converted into IPN through the interpenetration of polyacrylic acid chains. The synthesized semi-IPN and IPN were further loaded with Ag+ ions and irradiated with gamma radiations. On interacting with water molecules, γ-radiations also produces a large number of hydrated electrons {Eq. (3)}. The redox potential value of *H free radical {E0 (H+/H*) = −2.3 VNHE} and hydrated electrons {E0 (H2O/eaq‒) = −2.87 VNHE} is sufficiently high and they can effectively reduce silver ions into silver nanoparticles {Eq. (4), (5)} (Fig. 1) [23]. − ⋅ + H2 O+ ϒ − radiation eaq , H,H 3 O ,H2 O2

(3)

− Ag+ + eaq Ag 0

(4)

Ag+ + H* Ag0 + H+

(5)

(7)

Where ᴨion = osmotic swelling pressure of a weakly charged superabsorbent, R = gas constant, T = absolute temperature, Cig = Molar concentration of mobile ion in the swollen state and Cis = Molar concentration of mobile ion in the external solution. The carboxylate functional groups (-COO-) which are responsible for increased swelling percentage gets protonated in acidic medium resulting in a decreased swelling percentage. In a neutral medium, the sufficient number of carboxylate functional groups (-COO-) present onto the backbone repels one another resulting in an increased value of swelling percentage. In basic medium, the decreased swelling percentage was due to the screening effect of Na+ cations onto the -COOanions which suppresses inter-anionic repulsions [25]. N,N′-methylenebisacrylamide concentration was by varied from 1.2355 × 10−2 molL−1 to 3.7064 × 10−2 molL−1 under constant reaction conditions (γ-radiation dose = 3 KGy, solvent amount = 15 mL, pH = 7.0, and acrylamide = 0.8039 molL-1). The optimized value of N,N′-methylenebisacrylamide (Ps = 1147.20%) was found to be 1.8532 × 10−2 molL−1. Initially, the swelling percentage increases due to the formation of a large number of crosslinks. But, after optimium concentration of N,N′-methylenebisacrylamide, the swelling percentage decreases due to the increased crosslink density among the different polymeric chains (Fig. 2d) [26]. The optimum value of acrylamide (primary monomer) was determined by varying its concentration from 0.4020 molL-1 to 1.2059 molL-1. The constant value of other reaction parameters was γ-radiation dose: 3 KGy, solvent amount: 15 mL, pH: 7.0, and N,N′-methylenebisacrylamide: 1.8532 × 10−2 molL−1. The maximum swelling percentage (1450%) was observed with 0.8039 molL-1 acrylamide concentration (Fig. 2e). IPN (Bs-cl-polyAAm-IPN-AA-Gm) was synthesized

3.1. Optimization of reaction parameters The optimized reaction parameters for the synthesis of semi-IPN and IPN were calculated with respect to the maximum swelling percentage. Under optimized reaction conditions (γ-radiation dose: 3 KGy, solvent amount: 21 mL, pH: 7.0, N,N′-methylenebisacrylamide: 1.2355 × 10−2 molL−1, acrylamide: 0.8039 molL-1 and acrylic acid: 2.4307 molL−1) 3

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Fig. 1. Mechanism for the synthesis semi-IPN, IPN and silver nanocomposites.

swelling percentage of semi-IPN and IPN under pre-optimized time and neutral pH. Maximum swelling percentage Ps = 2649% and 1371% was observed at 80 °C for semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-clpolyAAm-IPN-AA-Gm), respectively. The increase in swelling percentage with the increase in swelling medium temperature was due to the expansion of pores of hydrophilic polymer matrix which allowed the encapsulation of a large number of water molecules. Beyond 80 °C, indefinite expansion of the hydrogel matrices resulted into dissociation of crosslinked structure [29]. pH-dependent swelling studies for semi-IPN and IPN were performed under pre-optimized swelling medium temperature and preoptimized time. Both the samples showed maximum swelling response in basic medium. Maximum swelling percentage equal to 8574% and 4493% was observed for semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-clpolyAAm-IPN-AA-Gm), respectively in pH = 13.0 (Fig. 2i). The enhancement of swelling percentage with the increase in pH of the swelling medium was due to the de-protonation of –COOH and –OH functional moieties producing –COO- and –O- anions. The inter-anionic repulsions among these ions increase the pore size of hydrogel matrices resulting in increased swelling percentage [30].

by varying the concentration of acrylic acid (secondary monomer) from 1.3890 molL-1 to 3.1252 molL-1 and the maximum swelling percentage = 980% was observed at 2.4307 molL-1 acrylic acid concentration (Fig. 2f). The swelling percentage (Ps) initially increases with the increase in concentration of primary and secondary monomers due to the increase in accessibility of monomeric moieties in the locality of Backbone (Bs) which resulted in graft copolymerization reactions. Beyond the optimum concentration of acrylamide and acrylic acid, the swelling percentage decreases due to the increase in reaction medium viscosity hindering the movement of activated free radicals and polymeric chains [6,7,27]. 3.1.1. Swelling studies in deionized water Fig. 2g–i represents the effect of time, temperature and pH on the swelling behavior of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-clpolyAAm-IPN-AA-Gm). In both the cases, swelling percentage increases initially with the increase in contact time due to the hydrophilic interactions of –OH, –COOH and –NH2 functional groups with water molecules, but after optimum time, no appreciable increase in swelling percentage was observed. This was due to the saturation of hydrophilic polymer matrix. In case of semi-IPN (Bs-cl-polyAAm-Gm), the saturation in swelling percentage was observed after 192 h with Ps = 1707%. On the other hand, IPN (Bs-cl-polyAAm-IPN-AA-Gm) showed maximum swelling percentage Ps = 982.4% after 288 h (Fig. 2g). The lower swelling percentage of IPN (Bs-cl-polyAAm-IPN-AA-Gm) compared to semi-IPN (Bs-cl-polyAAm-Gm) was due more crosslinked and compact structure of the IPN matrix [28]. Fig. 2h depicts the effect of swelling medium temperature on the

3.1.2. Salt resistant swelling studies The effect of ionic strength and cationic charge on the swelling behavior of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAmIPN-AA-Gm) was studied in different salt solutions of NaCl, KCl, CaCl2, BaCl2 and FeCl3 (Fig. 2j and k). The swelling percentage of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) was found to increase with the decrease in cationic charge (i.e. Fe3+ ˂ Ba2+ ˂ 4

Journal of Drug Delivery Science and Technology 56 (2020) 101550

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Fig. 2. Optimization of different reaction parameters (a) effect of γ-radiation dose (b) solvent amount (c) pH (d) N,N′-methylenebisacrylamide concentration (e) acrylamide concentration and (f) acrylic acid concentration on the swelling percentage of semi-IPN and IPN; effect of (g) time (h) temperature (i) pH and (j,k) ionic strength/cationic charge on the swelling percentage of semi-IPN and IPN.

Ca2+ ˂ K+ ˂ Na+) and salt concentration (9% ˂ 7% ˂ 5% ˂ 3% ˂ 1%). The lower value of swelling percentage with the increase in ionic strength was due to the shielding effect of cations onto the anionic moieties located over the polymeric chains of semi-IPN and IPN. The shielding effect resulted into a reduction of anion-anion repulsions among different –COO- and –O- ions and lone pair-lone pair repulsions between various –CONH2 functional groups located onto the semi-IPN and IPN matrix. The maximum swelling response was observed in the

absence of such cations due to the increased repulsive forces operating among different anions and lone pairs resulting in increased pore size of the hydrogel matrices. This can further be supported with osmotic pressure theory according to which an increase in the ionic concentration of the external solution reduces the osmotic pressure and decreases the swelling percentage [31]. The decrease in swelling percentage with the increase in cationic charge was due to the enhancement of the magnitude of shielding effect.

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Fig. 3. SEM micrographs of (a) Bs (b) Bs-cl-polyAAm-Gm (c) Bs-cl-polyAAm-IPN-AA-Gm (d) Ag0/Bs-cl-polyAAm and (e) Ag0/Bs-cl-polyAAm-IPN-AA-Gm; EDS spectra of (f) Bs (g) Bs-cl-polyAAm-Gm (h) Bs-cl-polyAAm-IPN-AA-Gm (i) Ag0/Bs-cl-polyAAm and (j) Ag0/Bs-cl-polyAAm-IPN-AA-Gm.

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Table 1 EDS results of Bs, Bs-cl-polyAAm-Gm, Bs-cl-polyAAm-IPN-AA-Gm, Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm. S. No.

Sample Code

C atomic% (weight %)

N atomic% (weight %)

O atomic % (weight %)

Ag atomic % (weigh %)

1. 2. 3. 4. 5.

Bs Bs-cl-polyAAm-Gm Bs-cl-polyAAm-IPN-AA-Gm Ag0/Bs-cl-polyAAm-Gm Ag0/Bs-cl-polyAAm-IPN-AA-Gm

68.79 56.57 67.43 58.32 61.30

2.35 (2.49) 13.23 (13.75) 8.43 (9.99) 18.42 (17.77) 13.52 (13.86)

28.86 30.20 24.14 21.94 24.77

0 (0) 0 (0) 0 (0) 1.32 (9.18) 0.41 (3.26)

(62.55) (50.41) (61.63) (48.24) (53.88)

(34.96) (35.84) (29.39) (24.17) (29.00)

Fig. 4. (a) FTIR and (b) XRD spectra of (i) Bs (ii) Bs-cl-polyAAm-Gm (iii) Bs-cl-polyAAm-IPN-AA-Gm (iv) Ag0/Bs-cl-polyAAm-Gm and (v) Ag0/Bs-cl-polyAAm-IPNAA-Gm.

(35.84%), respectively. Similarly, in case of IPN (Bs-cl-polyAAm-IPNAA-Gm), the elemental composition of carbon, nitrogen and oxygen becomes 67.43% (61.63%), 8.43% (9.99%) and 24.14% (29.39%), respectively. The nanocomposites Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-clpolyAAm-IPN-AA-Gm showed the presence of addition peak corresponding to silver nanoparticles with 1.32% (9.18%) and 0.41% (3.26%), atomic % (weight %) of silver.

3.2. Material characterizations 3.2.1. FE-SEM analaysis SEM-EDS analysis was done in order to see the structural morphology and elemental composition of the samples. Fig. 3a–e represents the SEM images of Bs, Bs-cl-polyAAm-Gm, Bs-cl-polyAAm-IPN-AA-Gm, Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm. It is clear that the smooth and plane surface of the backbone (Bs) becomes nonhomogeneous and rough after crosslinking with the acrylamide molecules (Fig. 3a and b). The SEM image of IPN (Bs-cl-polyAAm-IPN-AAGm) indicated that the convexity and irregularity of surface roughness increases after the incursion of polyacrylic acid chains into the semi-IPN matrix (Fig. 3c). The presence of Ag0 nanoparticles inside the 3Dcrosslinked matrices of nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm) was observed with their SEM images at higher resolution (Fig. 3d and e). Quantitative elemental analysis of the samples was done through EDS studies and the results are presented in Fig. 3f–j and Table 1. In case of backbone Bs, the atomic % (weight %) of carbon, nitrogen and oxygen was found to be 68.79% (62.55%), 2.35% (2.49%) and 28.86% (34.96%), respectively. After the formation of semi-IPN (Bs-clpolyAAm-Gm), the elemental composition of carbon, nitrogen and oxygen becomes 56.57% (50.41%), 13.23% (13.75%) and 30.20%

3.2.2. FTIR The FTIR spectral data of Bs, Bs-cl-polyAAm-Gm, Bs-cl-polyAAmIPN-AA-Gm, Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AAGm indicated the presence of different functional groups (Fig. 4a). A broad peak at 3281 cm−1 was found to appear in the FTIR spectrum of the backbone (Bs). The peak represents the –O-H stretching frequency of carbohydrates. A spectral peak observed at 2920 cm−1 corresponds to –CH2 asymmetric stretching vibrations. The FTIR spectral peak at 1597 cm−1 and 1418 cm−1 indicates in plane bending and wagging vibrations of –CH and –CH2 moieties. The spectral peaks at 1248 cm−1 and 1023 cm−1 corresponds to –COH deformation modes and solvolysis of –COH groups. All the spectral peaks which appeared in the FTIR spectrum of backbone Bs were also found to be observed in the FTIR spectra of semiIPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) with 7

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Fig. 5. (i) SAED patterns (ii) TEM image and (iii) histogram depicting particle size distribution of (a) Ag0/Bs-cl-polyAAm-Gm and (b) Ag0/Bs-cl-polyAAm-IPN-AAGm.

mode and –C]O amide stretching, respectively. The FTIR data of the nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPNAA-Gm) represented all the characteristic peaks of semi-IPN (Bs-clpolyAAm-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) with a slight shift in the FTIR spectral peaks confirming the formation of nanocomposites. This was further supported by XRD and TEM analysis [32].

some additional spectral peaks. In case of semi-IPN (Bs-cl-polyAAmGm), the spectral peaks at 3334 cm−1 and 3190 cm−1 represent –N-H stretching frequencies in primary amide. A sharp peak at 1650 cm−1 and peak at 1411 cm−1 corresponds to –C]O stretching in amide and –N-H in plane bending modes. In case of IPN (Bs-cl-polyAAm-IPN-AAGm), the additional spectral peaks were found to appear at 2935 cm−1, 1697 cm−1, 1406 cm−1 and 1160 cm−1 corresponding to –OH stretching of acid, –C]O stretching of carboxylic acid, -C-O-H bending 8

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Fig. 6. Biodegradation studies of (a) Bs-cl-polyAAm-Gm (b) Bs-cl-polyAAm-IPN-AA-Gm; FTIR analysis of biodegraded (c) Bs-cl-polyAAm-Gm and (d) Bs-cl-polyAAmIPN-AA-Gm matrices through soil burial method; (e) Bs-cl-polyAAm-Gm and (f) Bs-cl-polyAAm-IPN-AA-Gm matrices through composting method.

nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPNAA-Gm) have shown the presence of [111], [200], [220] and [311] characteristic crystallographic planes of FCC Ag crystal [33]. The XRD data was further supported by TEM analysis.

3.2.3. XRD analysis Fig. 4b represents the X-ray diffraction patterns of backbone Bs, semi-IPN (Bs-cl-polyAAm-Gm), IPN (Bs-cl-polyAAm-IPN-AA-Gm) and nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPNAA-Gm). The percentage crystallinity of the samples was calculated as per the following standard equation (8) [26].

Ac Xc = × 100 Ac + Aa

3.2.4. TEM analysis The particle size analysis was done with Transmission Electron Microscope (TEM) and the results are presented in Fig. 5. Face centered cubic crystal structure of Ag0 nanoparticles was confirmed from the SAED patterns representing [111], [200], [220] and [311] crystallographic planes (Fig. 5a and b). In both the samples i.e. Ag0/Bs-clpolyAAm-Gm and Ag0/Bs-cl-polyAAm-IPN-AA-Gm, the shape of nanoparticles was found to be spherical. The average particle size was found to be 11.3 nm and 8.6 nm in case of Ag0/Bs-cl-polyAAm-Gm and Ag0/ Bs-cl-polyAAm-IPN-AA-Gm, respectively. Compared to semi-IPN, the smaller particle size in case of IPN matrix was due to its compact

(8)

here Xc, Ac and Aa represent percentage crystallinity, area of crystalline region and area of amorphous region, respectively. The percentage crystallinity of the backbone (Bs) was found to be 38.23%. After the formation of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAmIPN-AA-Gm), the crystallinity of the sample increases due to the formation of a more ordered structure and it becomes 56.23% and 60.12%, respectively. The X-ray diffraction patterns of the 9

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Fig. 7. SEM images of the biodegraded semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-cl-polyAAm-IPN-AA-Gm) samples though soil burial and composting method.

compost, respectively after seven weeks (Fig. 6a and b). The degradation of the samples in garden soil and bio-compost takes place due to the microbial attack in the presence of water breaking chemical and physical bonds of the 3-D crosslinked semi-IPN and IPN matrices. The lower degradation percentage observed in case of IPN matrix as compared to semi-IPN matrix was due to its highly compact crosslinked structure which allows a lower fraction of water molecules to enter inside the IPN matrix and the growth of micro-organisms takes place with a lower rate which ultimately resulted into lower degradation percentage. Similarly, the higher degradation percentage in case of composting method as compared to soil burial method was due to the rich supply of micro-organisms which can directly trigger the degradation of crosslinked hydrogel matrices [19,36]. Different biodegradation stages were further analyzed by FTIR and SEM analysis.

structure and availability of smaller pore size for the stabilization of silver nanoparticles [34,35].

3.3. Biodegradation studies Biodegradation studies of semi-IPN and IPN were carried out in garden soil (using soil burial method) and in bio-compost (using composting method). The degradation was done upto 70 days of time interval with three distinct degradation stages. Stage-I, stage-II and stageIII degradation was assumed to 14 days, 42 days and 70 days. The semi-IPN (Bs-cl-polyAAm) was found to degrade upto 73% and 75% in garden soil and biocompost, respectively after 70 days’ time interval. In case of IPN (Bs-cl-polyAAm-IPN-AA-Gm), the percentage degradation was found to be 61% and 64% in garden soil and bio10

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Table 2 Zone of bacterial growth inhibition of synthesized samples against different bacterial strains. Bacterial strain

Zone of Bacterial Growth Inhibition (mm) (S1)

P. aeruginosa (gram -ve)

B. cereus(gram + ve)

S. aureus(gram + ve)

E. coli(gram -ve)

M ± SD ± SE M ± SD ± SE M ± SD ± SE M ± SD ± SE

(S2)

1

2

3

i

ii

iii

32 1 0.7 30 0.57 0.4 26 1 0.7 30 1.5 1.1

– – – 12 1.5 1.1 – – – – – –

17 0.57 0.4 24 1.5 1.1 14 1 0.7 14 0.57 0.4

29 0.57 0.4 31 0.57 0.4 19 0.57 0.4 26 0.57 0.4

– – – 11 0.57 0.4 12 0.57 0.4 – – –

25 1 0.7 22 1.5 1.1 17 1 0.7 13 1 0.7

Fig. 8. Antibacterial activity of semi-IPN (Bs-cl-polyAAm-Gm), IPN (Bs-cl-polyAAm-IPN-AA-Gm) and silver nanocomposites (Ag0/Bs-cl-polyAAm and Ag0/Bs-clpolyAAm-IPN-AA-Gm) against (a) P. aeruginosa, (b) B. cereus, (c) S. aureus and (d) E. coli

3.4. Antibacterial activity

A three-stage degradation of semi-IPN and IPN in garden soil and bio-compost involves the breakdown of covalent bonds which are responsible for the stabilization of 3-D crosslinked networks. Fig. 6c–f represents the FTIR spectra of biodegraded samples. Biodegradation of the samples resulted in the decrease in intensity of the initially present functional groups. The decrease in the intensity was found to be more remarkable in the stage-II and stage-III compared to stage-I which was due to the enzymatic degradation of covalent bonds resulting in depolymerization of the matrix and formation of the byproducts including H2O and CO2 [36]. The scanning electron micrographs also gave clear evidence for the degradation of the semi-IPN and IPN matrices (Fig. 7). In the degradation stage-I, samples were found to show the destruction of the crosslinked morphology. Pits and cracks were clearly found to be observed in the degradation stage-II. Finally, the samples showed complete destruction of the surface morphology in the degradation stage-III with enhanced heterogeneity of the surface. Fissures and holes are clearly visible at this stage of biodegradation [36].

Semi-IPN (Bs-cl-polyAAm-Gm), IPN (Bs-cl-polyAAm-IPN-AA-Gm) and the nanocomposites of silver (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bscl-polyAAm-IPN-AA-Gm) were tested for antibacterial activity against different bacterial strains including Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus and Escherichia coli. The samples were analyzed for the calculation of inhibited bacterial zone and a comparative study of the results was done against the standard drug amoxicillin (Table 2, Fig. 8).S1 = semi-IPN and semi-IPN based nanocomposite; S2 = IPN and IPN based nanocomposite; (1) = Amoxicillin; (2) = Sg-cl-polyAAm-Gm; (3) = Sg-cl-polyAAm-Gm-Ag0; (i) = Amoxicillin; (ii) = Sg-cl-polyAAm-IPN-AA-Gm; E = Sg-cl-polyAAm-IPN-AAGm-Ag0; M = mean value; ± SD = standard deviation and ± SE = standard error. Silver nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-clpolyAAm-IPN-AA-Gm) were found to possess higher antibacterial activity as compared to semi-IPN and IPN samples. The zone of bacterial growth inhibition for the standard drug amoxicillin was found to be 32 ± 0.7 mm, 30 ± 0.4 mm, 26 ± 0.7 mm and 30 ± 1.1 mm against P. aeruginosa, B. cereus, S. aureus and E. coli, respectively. In case 11

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Fig. 9. Mechanism of antibacterial activity (a) cell membrane of gram + ve and gram −ve bacteria, (b) lysis of cell membrane through its interaction with Ag0 nanoparticles.

of semi-IPN based nanocomposite (Ag0/Bs-cl-polyAAm-Gm) the zone of bacterial growth inhibition was found to be 17 ± 0.4 mm, 24 ± 1.1 mm, 14 ± 0.7 mm and 14 ± 0.4 mm against P. aeruginosa, B. cereus, S. aureus and E. coli, respectively. Similarly, in case of IPN based nanocomposite (Ag0/Bs-cl-polyAAm-IPN-AA-Gm), the zone of bacterial growth inhibition was found to be 25 ± 0.7 mm, 22 ± 1.1 mm, 17 ± 0.7 mm and 13 ± 0.7 mm against P. aeruginosa, B. cereus, S. aureus and E. coli, respectively. The semi-IPN (Bs-clpolyAAm-Gm) was found to possess antibacterial activity only in the case of B. cereus with 12 ± 1.1 mm of an inhibited bacterial growth zone. Similarly, IPN (Bs-cl-polyAAm-IPN-AA-Gm) was found to possess antibacterial activity against B. cereus and S. aureus with 11 ± 0.4 mm and 12 ± 0.4 mm zone of bacterial growth inhibition (Table 2). The inhibition mechanism for bacterial cell growth is presented in Fig. 9. The overall negative charge onto the cell wall of gram-negative bacteria is due to the presence of lipopolysaccharides. Whereas, the outer cell surface of gram-positive bacteria is composed up of peptidoglycan cell wall and teichoic acid producing an overall positive charge onto the cell membrane [37]. The antibacterial activity of silver nanocomposites (Ag0/Bs-cl-polyAAm-Gm and Ag0/Bs-cl-polyAAm-IPNAA-Gm) was due to the diffusion of silver nanoparticles through the bacterial cell membranes by hindering the functions of respiratory

chain proteins and transport proteins. After diffusion, the smaller size silver nanoparticles interfere with cell membrane functions including respiration and permeability [38,39]. The bacterium can be destroyed by the silver nanoparticles through the breakage of Cys-Cys sulfur bridges present inside protein chains. They can also interfere with the sulfur-containing compounds such as DNA [40–42]. The oxidation of silver nanoparticles inside the bacterial cell membrane produces Ag+ ions which can interfere with the functioning of cytoplasm, nucleic acid and respiratory enzymes. A very low concentration of Ag+ ions can produce massive proton leakage through the cell membrane of bacteria resulting in loss of proton motive force [43]. The diffusion of silver nanoparticles inside the cell membrane can show hindrance towards cell division. After their interaction with the dissolved oxygen, they can produce reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals and superoxide radicals. The excessive assembly of ROS can produce oxidative stress and can bring free radical attacks onto the membrane lipids. This could results in the death of bacteria through the breakdown of cell membrane [44–46]. The antibacterial effect of semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bscl-polyAAm-IPN-AA-Gm) hydrogels against the gram-positive bacterial strains was due to the presence of –NH2 functional moieties that can easily be protonated leading to the hydrolysis of bacterial cell 12

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membrane constituents and finally the death of bacteria [47]. [9]

4. Conclusion An efficient procedure was developed successfully for a morphologically controlled green synthesis of antibacterial nanocomposite scaffolds under gamma radiations. Natural backbone Boswellia serrate was chemically modified through the crosslinking of acrylamide and acrylic acid to generate semi-IPN (Bs-cl-polyAAm-Gm) and IPN (Bs-clpolyAAm-IPN-AA-Gm) matrices which possessed high water uptake potential (8574% in case of semi-IPN and 4493% in case of IPN). The degradation results suggested excellent degradability of the materials in garden soil as well as biocompost. The size of silver nanoparticles can be regulated through the choice stabilizing matrix. It was found to be 11.3 nm in case of semi-IPN matrix and 8.6 nm in case of IPN matrix. The nanocomposite hydrogels showed excellent antibacterial potential against P. aeruginosa, S. aureus, B. cereus, and E. coli bacterial strains and can find applications for the treatment of infected effluents.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

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One of the authors is highly thankful to Inter University Accelerator Centre, New-Delhi, India, for providing fellowship and gamma radiation facility. The authors are grateful to DST-FIST, New Delhi for receiving financial support to acquire instrumentation facility at NIT Jalandhar (grant no. SR/FST/CSI-228/2011 (C) dated March 02, 2012). The authors are also thankful to MHRD, New Delhi for developing institute instrumentation center to carry out the research work, IIT Roorkee and MNIT Jaipur for the characterization of samples.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2020.101550.

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