Kokkarachedu
Varaprasad
*ab,
G.
Siva Mohan Reddy
b,
J.
Jayaramudu
b,
Rotimi
Sadiku
b,
Koduri
Ramam
a and
S. Sinha
Ray
c
aDepartamento de Ingeniería de Materiales-DIMAT, Facultad de Ingeniería Universidad de Concepción, Concepción, Chile. E-mail: varmaindian@gmail.com; kvaraprasad@udec.cl
bDepartment of Polymer Technology, Tshwane University of Technology, CSIR Campus, Building 14D, Private Bag X025, Lynwood Ridge 0040, Pretoria, South Africa
cDST/CSIR Nanotechnology Innovation Centre, National Centre for Nano-Structured, Materials, Council for Scientific and Industrial Research, Pretoria 0001, South Africa
First published on 25th October 2013
The present scientific research resulted in the development of novel microbial resistant inorganic nanocomposite hydrogels, which can be used as antibacterial agents. They are promising candidates for advanced antimicrobial applications in the field of biomedical science. Novel inorganic nanocomposite hydrogels were developed from Carbopol® 980 NF and acrylamide. Dual-metallic (Ag0–Au0) nanoparticles were prepared (via a green process) by the nucleation of silver and gold salts with mint leaf extract to form a hydrogel network. The Carbopol nanocomposite hydrogels contain (Ag0–Au0) nanoparticles ∼5 ± 3 nm in size, which was confirmed by transmission electron microscopy. The developed hydrogels were characterized using Fourier transform infrared (FTIR) spectroscopy, thermo-gravimetric analysis (TGA), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and transmission electron microscopy (TEM). The pure and inorganic nanocomposite hydrogels developed were tested against Bacillus and E. coli, for their antibacterial properties. The results indicate that the inorganic nanocomposites show significantly greater antimicrobial activity than the pure hydrogels. Therefore, novel microbial resistant Carbopol nanocomposite hydrogels can be used as potential candidates for antibacterial applications.
So far, many researchers have synthesized inorganic nanocomposite hydrogels using a physical and chemical cross-linking method,14 whereby toxic chemicals, which are harmful to living systems, were used for the reduction of the inorganic particles.13 To solve this problem, researchers have introduced ‘green processes’.13 In a few of the green processes researchers have used plant leaf extracts as reducing agents for inorganic nanoparticles, since they (leaf extracts) are cost-effective and also require ambient conditions for the reduction reaction.15 The green reduction process of inorganic ions to inorganic nanoparticles can be facilitated with a high degree of efficiency in a hydrogel network and by controlling the inorganic salts, the formation of the nanoparticles and the way in which the nanoparticles embed in the hydrogel networks was controlled.16 This special character of hydrogels mainly depends on the hydrogels’ composition4 and hence by varying their composition; hydrogels were fabricated to be employed in biomedical applications.17 Varaprasad et al.4 have reported the fabrication of various types of hydrogels by varying their compositions.
The present work explores the development of microbial resistant Carbopol nanocomposite hydrogels from Carbopol® 980 NF (C980) and acrylamide (AM) via a green process. C980 is a water soluble vinyl polymer, which is a non-toxic and non-irritant material with no evidence of its hypersensitivity in humans when used topically.18 C980 and its derivative products have a variety of uses in biomedical fields, such as for: vaginal drug delivery, anti-cancer and anti-HIV-1 applications.19–21 Due to its characteristics that are suitable for biomedical applications, C980 was selected for the preparation of the inorganic core–shell nanocomposite hydrogels. In this regard, we have utilized (as a natural reducing agent) mint leaf extract, which is a highly biologically active compound.22 Mint leaf is naturally-occurring, non-toxic and is an excellent bioactive agent for humans (wound healing properties).23 Structural, thermal and morphological studies of the hydrogels and their corresponding inorganic core–shell nanocomposite hydrogels were carried out by using Fourier transform infrared (FTIR) spectroscopy. The content and distribution of the dual nanoparticles in Poly(Carbopol® 980 NF-Acrylamide) P(C980-AM) hydrogels were determined by thermogravimetric analysis (TGA), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and transmission electron microscopy (TEM). The effect of dual nanoparticles on the antibacterial activity of the P(C980-AM) hydrogels was studied. Herein, a study on designing P(C980-AM) core–shell inorganic nanocomposite hydrogels for their significant antibacterial applications is presented.
Hydrogel code | AM (mM) | C980 (g) | MBA (mM) | APS (mM) |
---|---|---|---|---|
P(C980-AM) 0 | 8.837 | 0.0 | 0.648 | 2.191 |
P(C980-AM) 1 | 8.837 | 0.05 | 0.648 | 2.191 |
P(C980-AM) 2 | 8.837 | 0.10 | 0.648 | 2.191 |
P(C980-AM) 3 | 8.837 | 0.15 | 0.648 | 2.191 |
The swelling ratios of the hydrogel samples were measured at ambient temperature, using a gravimetric method.24,25 The dried hydrogels were immersed in a 50 ml beaker containing double distilled water until their weights became constant. The hydrogels were then removed from water and their surfaces were blotted with filter paper before being weighed. Furthermore, swollen hydrogels were treated with a dual-metallic aqueous solution and subsequently with mint solution; a green process, as explained in the experimental section. The swelling ratio or swelling capacity (Sg/g) of the hydrogel developed or the respective nanocomposite was calculated using eqn (1):
Swelling ratio (Sg/g) = [Ws − Wd]/Wd | (1) |
Fig. 1 Swelling behavior of hydrogels with different concentrations of C980 and dual-metallic nanocomposite hydrogels. |
Fig. 2 FTIR spectra of pure P(C980-AM)3 hydrogel, P(C980-AM)3 + Ag0, P(C980-AM)3 + Au0 and P(C980-AM)3 + Ag0 + Au0 nanocomposite hydrogels. |
Fig. 3 SEM images of: (a) P(C980-AM)1 + Ag0 + Au0, (b, c) P(C980-AM)3 + Ag0 + Au0 nanocomposite hydrogels, EDS images of: (d) P(C980-AM)3 + Ag0 + Au0 nanocomposite hydrogel. |
Fig. 5 TGA curves of: pure P(C980-AM)3 and P(C980-AM)3 + Ag0, P(C980-AM)3 + Au0 and P(C980-AM)3 + Ag0 + Au0 nanocomposite hydrogels. |
The core–shell nanoparticle formation depends principally on the swelling behaviour of hydrogels. The results in Fig. 1 show that the values of the swelling characteristics were influenced by the hydrogel concentration; with an increase in the C980 concentration resulting in increases in the swelling ratio values of the conventional and dual-metallic nanocomposite hydrogels. This is due to the hydrophilic nature of C980. However, the formed dual-metallic nanocomposite hydrogels have higher swelling ratios, when compared to the conventional P(C980-AM) hydrogels. The reason being that when Ag+–Au3+ ion-loaded hydrogels were treated with mint extract, the addition of many Ag+–Au3+ ions led to the formation of the nanoparticles within the hydrogel, which expanded the hydrogel networks and promoted a higher water uptake capacity. This interesting phenomenon could play a significant role in biomedical applications, particularly in wound care applications.
Fig. 2 shows the FTIR spectra of pure [P(C980-AM)], single metallic [P(C980-AM) + Ag0, P(C980-AM) + Au0] and dual metallic [P(C980-AM) + Ag0 + Au0] nanocomposite hydrogels. The spectrum of the P(C980-AM) hydrogel shows a broad absorption band at 3338 cm−1 that is related to the –NH asymmetric and –OH symmetric stretching vibrations, the bands at 2928 cm−1 are attributed to stretching vibrations of the –CH3 unit and the absorption band at 1646 cm−1 is from the carbonyl groups of the P(C980-AM) hydrogel. The other main characteristic peaks of C980 at 1184 and 1121 cm−1 were assigned to the carbonyl group.28 However, these peaks shifted in the case of the single metallic [P(C980-AM) + Ag0, P(C980-AM) + Au0] and dual metallic nanocomposite hydrogels [P(C980-AM) + Ag0 + Au0]. Table 2 illustrates the important peaks observed for the single metallic [P(C980-AM) + Ag0, P(C980-AM) + Au0] and dual metallic nanocomposite hydrogels [P(C980-AM) + Ag0 + Au0]. Overall, the shifting of the peaks confirms the formation of core–shells nanoparticles with Carbopol, which provides significant stabilization for the core–shells nanoparticles formed.
Hydrogel code | FTIR bands (cm−1) |
---|---|
P(C980-AM) | 3338.3, 2928.0, 1646.5, 1415.1, 1184.2, 1121.3 |
P(C980-AM) + Ag0 | 3331.6, 2925.5, 1643.3, 1415.3, 1184.6, 1121.8 |
P(C980-AM) + Au0 | 3340.6, 2932.6, 1650.2, 1416.2, 1184.2, 1122.5 |
P(C980-AM) + Ag0 + Au0 | 3333.9, 2923.6, 1646.4, 1418.1, 1171.6, 1117.0 |
The structure and elemental composition of the core–shell nanoparticles were investigated by means of SEM-EDS. The hydrogel morphology (stabilization of core–shells) is obviously dependent on the C980 content in the hydrogels (Fig. 3). Fig. 3a shows that the dual-metallic loaded nanocomposite hydrogel [P(C980-AM)1] has fewer nanoparticles in its structure, whereas from Fig. 3b and c it is obvious that a huge quantity of dual-metallic nanoparticles was dispersed on the [P(C980-AM)3] hydrogel surface. This is due to the C980 concentration in the hydrogel network, which considerably stabilizes the core–shell nanoparticles. Furthermore, EDS was employed to confirm the presence of core–shell nanoparticles in the P(C980-AM) hydrogel. The EDS spectra of the dual-metallic loaded nanocomposite hydrogels were then investigated. The spectrum of the dual-inorganic nanocomposite hydrogels (Fig. 3d) shows clearly the peaks of Au0 and Ag0. The intensity of the Au0 and Ag0 peaks are proportional to the metal concentration in the hydrogel composites. Therefore, the existence of core–shell nanoparticles on the hydrogel is confirmed by the EDS spectra. The C980 in the networks is used to stabilize the core–shell nanoparticles formed in the hydrogel networks.
In order to analyze the structure and size of the core–shell nanoparticles, transmission electron microscopy (TEM) measurements are also performed. As shown in Fig. 4, the different images of the particles indicate the formation of core–shell (Ag0–Au0) nanoparticles, which have different shapes and their average size is ∼5 ± 3 nm. It is evident that Ag0 nanoparticles are highly stabilized by using C980 in the hydrogel network. These results are mainly due to the strong interaction between the Ag0–Au0 nanoparticles and P(C980-AM) hydrogels.
Furthermore, thermogravimetric analysis was used to study the formation of core–shell nanoparticles and the thermal stability of the different hydrogels. As shown in Fig. 5, the thermal decomposition of pure [P(C980-AM)] and single metallic nanocomposite hydrogels [P(C980-AM) + Ag0 and P(C980-AM) + Au0], occurred at 625 °C with a significant mass loss of 99.81%, 98.12%, 96.59%, respectively. For the dual metallic nanocomposite hydrogel [P(C980-AM) + Ag0 + Au0], a comparatively very low mass loss (94%) was observed at 625 °C, which was due to the partial decomposition of the Ag0 and Au0 nanoparticles. Moreover, according to the TGA results, the dual metallic nanocomposite hydrogels [P(C980-AM) + Ag0 + Au0] showed a higher thermal stability when compared with the other hydrogels.
Inorganic nanoparticles inherently possess bacteria-killing properties, but by modifying the inorganic nanoparticles these properties can be improved. Recently in the biomedical field, a synthesis of much smaller core–shell nanoparticles by a green process was developed to enhance the inactivation of bacteria in wounds.
The mechanism of the inhibitory effect of AgNPs towards the growth of microorganisms is not clear.29 However, among the various possible mechanisms proposed,29 the ‘Inhibition by formation of pits’ mechanism was considered at this point. The initial contact of the core–shell inorganic hydrogels with the microbes allows the core–shells to interact with the cell wall of the microbe membrane to form pits. These pits cause polymer molecules and membrane proteins of the bacteria to leak, leading to microbial death.30Fig. 6A,B support this conclusion. The dual nanoparticles enter into the bacteria cell more effectively, causing damage to the nuclei and resulting in faster bacterial death. The antimicrobial efficacy of the bimetallic hydrogels developed from core–shell nanoparticles was examined against Bacillus and E. coli model bacteria. The effects of the P(C980-AM)], P(C980-AM) + Ag0, P(C980-AM) + Au0 and P(C980-AM) + Ag0 + Au0 hydrogels on bacteria are shown in Fig. 6. The diameters of the inhibition zones for the P(C980-AM) + Ag0 + Au0 hydrogel (Fig. 6Ad 1.85 cm, Fig. 6Bd 1.81 cm) are larger than those for the P(C980-AM) + Ag0, P(C980-AM) + Au0 hydrogels (Fig. 6Ac 1.1 cm and Fig 6Ab 1 cm, Fig 6Bb 0.9 cm), whereas the pure P(C980-AM) hydrogels (Fig. 6A,Ba 0.0 cm) showed no inhibition ability. The dual inorganic nanoparticles that were formed, contain hydrogels that have two kinds of metallic surfaces and charges, and these dual inorganic nanoparticle nanohydrogels interact more effectively with bacteria in comparison to other nanohydrogels. Therefore, C980 in combination with core–shell nanocomposite hydrogels exhibits excellent antibacterial activity. Above all, the mechanism was further supported by the studies of Singh et al., which postulated that nanoparticles formed in the range 1–10 nm attach to the surface of the cell membrane and drastically disturb its proper function by forming pits.31 The core–shell nanoparticles formed using the present approach, were in the range of 2–8 nm, which strongly supports the proposed mechanism.
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