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Hydrogel beads bio-nanocomposite based on Kappa-Carrageenan and green synthesized silver nanoparticles for biomedical applications
International Journal of Biological Macromolecules 104 (2017) 423–431
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Hydrogel beads bio-nanocomposite based on Kappa-Carrageenan and green synthesized silver nanoparticles for biomedical applications Susan Azizi a,∗ , Rosfarizan Mohamad a,b,∗∗ , Raha Abdul Rahim c , Reza Mohammadinejad d , Arbakariya Bin Ariff a,e a Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Laboratory of Biopolymer and Derivatives, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia c Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran e Bioprocessing and Biomanufacturing Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e
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Article history: Received 29 October 2016 Received in revised form 22 May 2017 Accepted 2 June 2017 Available online 4 June 2017 Keyword: Hydrogel Silver nanoparticles Green method Citrullus colocynthis
a b s t r a c t This paper describes the fabrication and characterization of bio-nanocomposite hydrogel beads based on Kappa-Carrageenan (-Carrageenan) and bio-synthesized silver nanoparticles (Ag-NPs). The silver nanoparticles were prepared in aqueous Citrullus colocynthis seed extract as both reducing and capping agent. Cross-linked -Carrageenan/Ag-NPs hydrogel beads were prepared using potassium chloride as the cross-linker. The hydrogel beads were characterized using XRD and FESEM. Moreover, swelling property of the hydrogel beads was investigated. The Ag release profile of the hydrogels was obtained by fitting the experimental data to power law equation. The direct visualization of the green synthesized Ag-NPs using TEM shows particle size in the range of 23 ± 2 nm. The bio-nanocomposite hydrogels showed lesser swelling behavior in comparison with pure -Carrageenan hydrogel. Regardless the slow Ag release, -Carrageenan/Ag-NPs presented good antibacterial activities against Staphylococcus aureus, Methicilin Resistant Staphylococcus aurous, Peseudomonas aeruginosa and Escherichia coli with maximum zones of inhibition 11 ± 2 mm. Cytotoxicity study showed that the bio-nanocomposite hydrogels with non-toxic effect of concentration below 1000 g/mL have great pharmacological potential and a suitable level of safety for use in the biological systems. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels derivative from natural polysaccharides are highwater content polymeric materials which have a number of characteristics such as biodegradability, biocompatibility, stimuliresponsive characteristics and biological functions promising for biomedical applications such as tissue engineering, drug delivery and biosensor [1,2].
∗ Corresponding author. ∗∗ Corresponding author at: Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. E-mail addresses: [email protected] (S. Azizi), [email protected] (R. Mohamad). http://dx.doi.org/10.1016/j.ijbiomac.2017.06.010 0141-8130/© 2017 Elsevier B.V. All rights reserved.
The -Carrageenan is a sulphated linear polysaccharide of D-galactose and 3,6-anhydro-d-galactose obtained by alkaline extraction from red algae. Because of their biocompatibility and high capacity to form hydrogels, -Carrageenan has been widely used in food and pharmaceutical industries [3]. The use of complexation between oppositely charged macromolecules to generate -Carrageenan beads can attract great attention as a drug-controlled release formulation because of the simplicity and gentleness of this manner and production of small size and uniform shapes in compared with the conventional hydrogels [4–7]. Owing to the greater biomedical relevance, there is an increasing attention to develop the antibacterial hydrogels [8]. Among the antimicrobial hydrogels, the antibacterial inorganic-based nanocomposite hydrogels are especially favorable to inhibit the bacterial growth, consequently making them attractive in the fields of biomedical and biotechnology [9]. Several types of inorganic
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2. Materials and methods 2.1. Materials -Carrageenan (300 000 g/mol Fluka Chemie), AgNO3 (䊐99.98%, Merck), potassium chloride (KCl) (>99%, Sigma-Aldrich). Ripe fruits of C. colocynthis were collected from Koohzar district, Khuzestan, Iran in May–June 2015. The plant specimens were identified and authenticated by Department of Botany, Shahid Chamran University, Iran. 2.2. Preparation of C. colocynths seed extract
Fig. 1. Photograph of dried Citrullus colocynthis.
nanoparticles with antimicrobial activities have come up such as titanium [10], zinc oxide [11], magnesium, gold [12] and silver (Ag) [13]. Among the above nanoparticles, silver has established to be favorable candidates for antimicrobial activity against a broad spectrum of pathogenic bacteria [14], as well antibiotic-resistant bacteria [15,16]. Sliver nanoparticles also well known for its antiseptic, anti-inflammatory and multilevel antimicrobial activities (multidrug resistance) [17,18] accompanied by low systemic toxicity [18]. Hence, many studies had been done on the development of silver nanocomposite hydrogels for biomedical applications during the last few years [19–22]. However, in most of these studies chemical methods have been applied to synthesize silver nanoparticles, which restrict the medical applications of hydrogel nanocomposites, due to toxicity of starting materials. Considering to the above concern, this study was undertaken to develop silver nanocomposite hydrogels with green chemistry to enhance their level of safety for use in biological systems. Recently, plant-mediated biological synthesis of nanoparticles is gaining significance owing to its simplicity, eco-friendliness and extensive pharmaceutical effects [23]. Bio-constituents of different plant extracts were known as potential synthesizers and stabilizers of metal nanoparticles. In addition, the plant metabolites with medicinal effects have the potential to be attached on the surface of nanoparticles during the synthesis process which finally leads to the occurrence of subsequent varied surface effects during their medicinal applications [24]. Citrullus colocynthis (C. colocynthis) (Fig. 1) a member of the Cucurbitaceae family, is an important and extensively used plant in traditional medicine distributed throughout Asia. It has been reported to have numerous important biological properties such as antioxidant [25] anticancer [26] anti-inflammatory [27] and antimicrobial [28–30] activities. The seed contains phytochemicals such as glycosides, flavonoids, alkaloids, carbohydrates, fatty acids and essential oil [31] which are capable of reduction and stabilization with high biological activities that motivate the our interest to utilize it in the synthesis of Ag-NPs. In this study, we have first demonstrated a simple one-step green process to synthesizing Ag-NPs using C. colocynthis seed extract as the both reducing and stabilizing agent. Secondly, we designed a high safety hydrogel based antimicrobial bionanocomposite using biosynthesized Ag-NPs as an antimicrobial agent and -Carrageenan as hydrogel matrix. The effect of the concentration of the as synthesized Ag-NPs on the morphology, swelling behavior, cytotoxicity and antibacterial activity was examined.
The seeds were removed from the fruits and dried at room temperature and then milled to fine powder using grinder. The material that passed through 80-mesh sieve was used for extraction purpose. Briefly, the fine material was extracted with ethanol for 8 h in a Soxhlet apparatus. The solvent was removed by rotary evaporation. The dried, crude concentrated extract then stored in a refrigerator (−4◦ C). 2.3. Biosynthesis of Ag-NPs Silver nitrate (0.003 M) was dissolved in 100 mL of distilled water under magnetic stirring. After complete dissolution, 0.2 g of the dried C. colocynths seed extract was added to the above solution under continuous stirring at 45 ◦ C and allowed to react over 1 h. The reducing of silver ions to silver nanoparticles was observed by the changing color of solution mixture from light yellow to dark brown. For the purification of Ag-NPs, the fully reduced solution was centrifuged at 8000 rpm for 15 min. The supernatant liquid was discarded and the residue was dispersed in Millipore water. The samples were centrifuged five times to remove any constituents that had been absorbed onto the surface of the Ag-NPs. The final product was dried at 60 ◦ C overnight. The resulting dried sample preserved in air-tight bottles for further studies. 2.4. Determination of percentage yield (%Y) of Ag-NPs Concentration of Ag+ ions before and after addition of extract was measured using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) model Perkin Elmer 1000. Equation (1) was used to calculate percentage yield (%Y) using initial concentration (IC ) and final concentration (FC ) of Ag+ ions: %Y =
Ic − Fc × 100% Ic
(1)
2.5. Preparation of -Carrageenan/Ag-NPs hydrogel beads A series of nanocomposites were prepared by blending the biosynthesized Ag-NPs with the -Carrageenan matrix as follows. 2 gr of -Carrageenan was added to 80 mL of distillated water under magnetic stirring, at 80 ◦ C. After complete dissolution, 0.0, 0.1, 0.3, 0.5, 0.7 and 1.0 mL were taken from stock solution of silver nanoparticles (1 mg/mL) and added to 9 mL of -Carrageenan solutions under magnetic stirring until homogeneous viscose solutions obtained. The bio-nanocomposite beads were formed by dropping the hydrogel bio-nanocomposite solutions from a needle with 2 mL internal diameter into 1 M of KCL solution at room temperature. After 30 min, the beads were separated and washed with distilled water, then dried under vacuum at room temperature. The prepared beads based on the amount of Ag-NPs were called -Ca/Ag0, -Ca/Ag1, -Ca/Ag2, -Ca/Ag3, -Ca/Ag4 and -Ca/Ag5 mean -Carrageenan/Ag-NPs bio-nanocomposite hydrogel beads,
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which have 0.0, 0.01, 0.03, 0.05, 0.07 and 1.0 mg/mL of Ag-NPs content, respectively. 2.6. Characterization and analysis The X-ray diffraction analysis was carried out by Philips X’pert PXRD, with Cu-Ka radiation at 40 kV in the scan range of 2 from 2◦ to 80◦ . Infrared spectra were obtained on an FTIR spectrometer (PerkinElmer 1725X) in the 4000–400 cm−1 range. Transmission electron microscopy (TEM) of Ag nanoparticles was performed using a Hitachi H-700 transmission microscope. UV–vis spectroscopy was carried out on a Lambda 25-PerkinElmer UV–vis spectrophotometer. The morphology of the hydrogel samples was observed using a field emission scanning electron microscope (FESEM) (JSM-6360LA Philips). 2.7. Swelling behavior The swelling ratio were carried out by immersion of 100 mg of hydrogel beads in PBS 0.01 M, pH 7.4 at 37 ◦ C. At various time points, the samples were lifted from the solution and excess water was removed from the surface by blotting on wet filter paper before being weighted. The swelling ratio of bio-nanocomposite beads was calculated according to Eq. (2): Swelling Ratio (%) =
(w2 − w1 ) × 100 w1
at 37 ◦ C. The 100 L of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbent compared to control. The 50% inhibitory concentration (IC50 ) value, defined as the amount of sample that inhibits 50% of cell growth, was calculated from the concentration-response curves. 2.10. Antimicrobial assessment Antibacterial activity of the bio-nanocomposite hydrogels beads was evaluated against Gram positive (Staphylococcus aureus (S.aureus) S276 and Methicilin Resistant Staphylococcus aurous (MRSA)) and Gram negative (Peseudomonas aeruginosa (P. aeruginosa) ATCC 15442 and Escherichia coli (E.coli) E266) pathogens by using agar diffusion method. The agar plates were inoculated with 100 l spore suspensions of bacteria. For each sample, one swelled bead (∼10 mg) was placed on the agar plate away from the antibiotic (Streptomycin) and incubated at 37 ◦ C for 24 h. After incubation, the zone of whole inhibition was measured. All tests were replicated three times. 3. Results 3.1. Characterization of Ag-NPs
(2)
where w1 is the initial weight of the sample and w2 is the weight of swollen sample. The equilibrium swelling ratio (ESR) was determined at the point the hydrated hydrogel reached to a constant weight value. The swelling capacities were measured in triplicate. 2.8. Release study of silver content from hydrogel nanocomposites Ag-NPs release profile from -Carrageenan hydrogel nanocomposites was studied in phosphate buffer saline (PBS 0.01 M, pH 7.4) at 37 ◦ C. Hydrogels beads (∼30 mg) immersed in 10 mL PBS in an incubator shaker at 120 rpm. 0.5 mL solution were taken out after regular time intervals of (6, 12, 24, 48, 72 and 96 h) and analyzed for the amount of Ag released using Inductively Coupled Plasma Atomic Emission Spectrometry(ICP-AES) (Perkin Elmer 1000, USA), and then the same volume of fresh phosphate buffered saline solution was added. The release profile of Ag-NPs from -Carrageenan hydrogel nanocomposites was studied by release kinetics RitgerPeppas model [32], a semi-empirical power law described by Eq. (3) Ct = Kt n Ceq
425
(3)
where Ct and Ceq are the cumulative concentrations of Ag released from the hydrogel at a specified time and at equilibrium, respectively, k is a characteristic constant of the hydrogel and n is the diffusional coefficient used to interpret the release mechanism. 2.9. Cytotoxicity assay The in vitro cytotoxicity of hydrogel beads was evaluated by the method using 3-(4,5-dimethylthiazol-2-yl)-2,5- dephenyltetrazolium bromide (MTT) assay. Briefly, VERO cells were seeded at a density of 2 × 105 cells/mL in 96-well microplates and incubated for 24 h. Subsequently, the cells were treated with 100 L of the suspension of hydrogels in FBS (1 mg/mL) for 24 h. The samples were suspended separately in a stock solution at 5 g/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 L of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were incubated for another 4 h
The use of plant extract for synthesis and stabilization nanoparticles is well proven [33–36]. The use of seed extract of C. colocynths was significantly effective on the synthesis of Ag nanoparticles. The seed extract contains some biomolecules such as Isosaponarin and Isovitexin with a huge number of hydroxyl (O H) and carbonyl (C O) groups in their structures which facilitate the complexation of silver ions to these molecules. By transforming of free electron from the electrons of carbonyl (C O) or hydroxyl groups to the free orbital of metal ions in a Red/Ox system silver ions convert to silver nanoparticles [23]. The color change of Ag+ /C. colocynths solution from yellow to dark brown after 20 min indicates formation of silver nanoparticles (inset in Fig. 2A). In fact, C. colocynths extract acts as both reducing and stabilizing agent. The stabilization of nanoparticles by bio-compounds prevent from their aggregations. The UV–vis spectroscopy of sample shows a typical absorption peak at 440 nm (Fig. 2.A) which corresponds to the typical of surface plasmon resonance (SPR) of Ag-NPs. Based on the SPR, the spherical Ag-NPs displays the characteristic SPR at the wavelength between the ranges of 400–450 nm [37]. From TEM image of AgNPs (Fig. 2B) observed that the nanoparticles are spherical in shape with the mean particle diameter of 23 ± 2 nm. The XRD spectrum of the Ag-NPs (Fig. 2C) shows four distinct crystalline reflections at 2 = 38.24◦ , 44.51◦ , 64.32◦ and 77.45◦ and its corresponding lattice plane value was indexed at (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic nature. The FTIR spectrum of pristine Ag-NPs (Fig. 2Da) shows the corresponding peaks of Ag-O at 1625 and 668 cm−1 [38]. Fig. 2Db demonstrates the structure of C. colocynths extract with bands at 3384, 1722, 1426, 1230 and 1108, cm−1 . Fig. 2Dc represents the FTIR spectrum of C. colocynths extract reduced Ag-NPs, where signals’ intensities at 3384 (O H stretching), 1722 (C O), 1230 and 1108 (C O stretching) cm−1 decreased after synthesis of Ag-NPs, indicating the involvement of carbonyl and hydroxyl groups in the reduction process. The FTIR spectrum clearly shows that the C. colocynths acted as both reducing and capping agent. The zeta potential value of nanoparticles was −28.5 mV which shows that the NPs were stable and warped with anionic organic phases and responsible for electrostatic stabilization. ICPAES analysis of Ag+ content after formation of Ag-NPs by extract was found to be 189 ppm at pH 7. The percent yield (%Y) was calculated as ∼96%.
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Fig. 2. (A) UV spectrum and (inset) color change of seed extract after formation of Ag-NPs, (B) TEM image and (C) XRD diffraction pattern of bio-formed Ag-NPs, (D) FTIR spectrum of (a) pristine Ag-NPs, seed extract (b) before and (c) after formation of NPs.
3.2. Characterization of -Carrageenan/Ag-NPs bio-nanocomposite hydrogel beads 3.2.1. XRD analysis Nanocomposites hydrogel beads were prepared by homogeneous dispersion of different ratio of bio-synthesized Ag-NPs within -Carrageenan solutions, followed by gelled beads formation of the mixture in the presence of K+ ions, through electrostatic interactions between negatively charged -Carrageenan chains and positively charged K+ ions. The XRD patterns of the original Carrageenan hydrogel and -C/Ag3 bio-nanocomposite hydrogel in the 2 range of 10–80◦ are shown in Fig. 3. The -C/Ag3 shows two sets of diffraction peaks corresponding to -Carrageenan and AgNPs. The peaks corresponds to Ag were almost the same as that of the bio-synthesized of Ag-NPs (Fig. 2C). The halo typical peak of -Carrageenan at a 2 of 21.35◦ was broader than that of the pure -Carrageenan, this event may be due to the presence of biomolecules of C. colocynths which were introduced on the surface of silver nanoparticles.
Fig. 3. (a) XRD pattern of original -Carrageenan hydrogel and (b) -C/Ag3 bionanocomposite hydrogel.
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Fig. 4. (A) Digital photo of -C/Ag bio-nanocomposite hydrogel beads with different ratio of Ag-NPs, (B) FESEM micrographs of (a) original -Carrageenan and (b-f) -C/Ag (1-5) bio-nanocomposite hydrogel beads.
3.2.2. Morphology of -C/Ag bio-nanocomposite hydrogel beads The dried bio-nanocomposite hydrogel beads were spherical with a diameter of about 1 mm which changed their color from yellow to dark brown and surfaces from smooth to rough with the increase in Ag-NPs content (Fig. 4A ).
The surface morphology of the original -Carrageenan and C/Ag bio-nanocomposite hydrogel beads are shown in Fig. 4B(a–f). FESEM images clearly show that the spherical Ag-NPs with particle size less than 25 nm were well dispersed within hydrogel -Carrageenan up to 0.05 mg/mL (Fig. 4B(a–c)) and generated a smooth and tight surface as compared with the pure -Carrageenan
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Fig. 5. Swelling behavior of -C/Ag bio-nanocomposite hydrogel beads.
which possesses uneven surface with many cavities. Beyond this level, with the increase in the Ag-NPs content, the nanoparticles mostly agglomerated and the surface morphology of hydrogel beads became rough and undulant (Fig. 4e–f), as was observed by visual inspection of samples. The Ag-NPs at low concentrations loading could possibly act as intermolecular cross-linker, which restrict the mobility of the -Carrageenan chains and then enhanced the surface morphology of -Carrageenan to a smooth and tight surface. By increasing in nanoparticles loading, Ag-NPs due to the small dimensions, high surface activity, and large specific surface area were agglomerated which results in the decrease in interfacial interactions between -Carrageenan chains and Ag-NPs, this event makes an undulant surface and pitted be created. The porous structure could make the capillary forces which allowed the diffusion of fluids into the beads and thereby the drug easily released into water [39]. Therefore from FESEM results can conclude that those nanocomposites hydrogels which contain 0.01–0.05 mg/mL silver nanoparticles have appropriate morphology to use in prolonged and more controlled drug releases in a biological system. 3.2.3. Swelling behavior The equilibrium swelling ratio of the hydrogel beads was shown in Fig. 5. Equilibrium swelling ratio decreased in case of Ag-NPs loaded hydrogel than pure -Carrageenan hydrogel. The swelling capacity is directly proportional to the porosity of the hydrogel networks. The composition of the network -Carrageenan polymer chains and the crosslinking density are the important factors which effect on the pore volume fraction, pore sizes and their interconnection [40]. As was observed by the FESEM results, the Ag-NP acts as an effective cross-linker which improves the surface tight with the decrease in porosity of the hydrogel networks compared to pure -Carrageenan. This might be the reason for decreased swelling properties in Ag-NPs loaded hydrogels compared to pure -Carrageenan. However in -Ca/Ag4 and -Ca/Ag5 hydrogels, the swelling property due to forming some cavities in hydrogel structure somewhat increased compared to those nanocomposites which have smooth and tight surfaces. Another possible explanation for this swelling enhancement may be related to the negative surface charge of Ag-NPs used in the hydrogel. The immobilization of surface charged Ag-NPs within -Carrageenan results in the afflux of water to balance the osmotic pressure buildup caused by the immobilization of the surface charge NPs. The degree of osmotic swelling depends on the amount of charges immobilized within the gel and is proportional to the surface charge and concentration of the nanoparticles. Thus, hydrogel nanocom-
Fig. 6. In vitro Ag releasing properties of -Ca/Ag nanocomposites in PBS buffer solution: (A) release quantity changes of silver ion in PBS and (B) log(release quantity of Ag) vs log(times) curve.
posites with high load of Ag-NPs swell more and these results are in agreement with previous observation in hydrogel containing Fe3 O4 NPs [41]. From the graphs was also observed that swelling increased with time and reached to a maximum whitin12 h and 16 h in the cases of pure -Carrageenan and Ag-NPs loaded hydrogels respectively and then it reached equilibrium almost after 16 h. 3.2.4. In vitro Ag releasing properties of -Ca/Ag bio-nanocomposite beads Ag releasing profiles of -Ca/Ag bio-nanocomposite in PBS solution were presented in Fig. 6(A) and (B). The power law exponent, n, from slopes of the logarithmical curves of Ct/Ceq as a function of time and fitting coefficient (R2 ) calculated and listed in Table 1. The results show that n approximately is varied for samples which is indicative of different transport mechanism. When n = 0.45 means that the drug release mechanism is controlled by Fickian diffusion. The power law of Ag releasing rate of -Ca/Ag1 and -Ca/Ag 2 hydrogels is near to 0.45 with R2 ≥ 0.903, which seems the Ag release mechanism is controlled by Fickian diffusion. Anomalous transport mechanism, which is the contribution of both Fickian diffusion and macromolecular relaxation is observed when 0.45 ≤ n ≤ 0.89. In the -Ca/Ag3 hydrogel nanocomposite, n increased to 0.507 ± 0.010, proposing a trend towards the Fickian diffusion. The release mechanism for samples -Ca/Ag4 (n = 0.624 ± 0.03) and -Ca/Ag5
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Table 1 Release quantity and release ratio of Ag from -Ca/Ag bio-nanocomposite (∼30 mg) after 4 day; slope of fitting and fitting coefficient of log (release quantity of Ag) vs log (times) curve. Release quantity (g) Sample
1day
4 day
Slope of fitting (n)
Fitting coefficient (R2 )
-Ca/Ag 1 -Ca/Ag 2 -Ca/Ag 3 -Ca/Ag 4 -Ca/Ag 5
28.1 ± 2 33.4 ± 3 39.3 ± 2 42.4 ± 3 59.2 ± 4
35.2 ± 3 43.1 ± 4 57.4 ± 2 79.1 ± 4 90.3 ± 3
0.441 ± 0.02 0.466 ± 0.05 0.507 ± 0.01 0.624 ± 0.03 0.628 ± 0.02
0.903 0.926 0.953 0.964 0.939
(n = 0.628 ± 0.02) hydrogels became anomalous which indicates the contribution of both Fickian diffusion and macromolecular relaxation. In such cases, the agglomerated Ag-NPs in the hydrogel nanocomposites are very nearby to each other. Consequently, anion-anion electrostatic repulsion forces due to negative charges on their surface are generated, throughout the polymer network and the macromolecular relaxation begins to act [42]. 3.2.5. Cytotoxicity evaluation The 50% inhibitory concentration (IC50 ) value of hydrogel nanocomposites for a cut-off of 1000 g/mL was calculated as the dose required to inhibit the cell growth by 50% and shown in Fig. 7. None of hydrogels considerably inhibited the cell growth indicating that IC50 is higher than 1000 g/mL. The values of growth inhibition at 1000 g/mL were much lower than 50%, signifying that the hydrogels do not have any toxic, damaging elements for living cells. 3.2.6. Antimicrobial assessment Agar diffusion assay was employed to probe the antibacterial activity of bio-nanocomposite hydrogels against Gram positive (S.aureus and MRSA) and Gram negative (P. aeruginosa and E.coli) bacteria. The results obtained are shown in Fig. 8. The results
Fig. 7. Cytotoxic effect of hydrogel -Ca/Ag (1-5) nanocomposites on the growth inhibition of VERO cells.
demonstrated that the all bio-nanocomposite hydrogels had toxic effect on bacteria with the highest effect for sample of -Ca/Ag 3. The -Ca/Ag 3 bio-nanocomposite hydrogel showed inhibition zones of 11 ± 2 mm against all Gram positive and negative bac-
Fig. 8. Inhibition zone of -Ca/Ag (1-5) hydrogels (a-e) and chemically synthesized Ag hydrogel beads (f) against S. aureus (A), MRSA (B), P. aeruginosa (C) and E. coli (D) pathogens.
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teria compared to inhibition zones of 6 ± 2 mm for other -Ca/Ag bio-nanocomposite hydrogels. The reason for decreasing of antimicrobial activity with the increase in Ag loading in hydrogels is probably due to the form of large agglomerates of nanoparticles which are less probable to diffuse into the cell membrane to damage the bacteria from the inside. In the case of -Ca/Ag 3 nanocomposite, the diameter of the zone of inhibition was favorable with respect to positive control i.e. Streptomycin (25 mm). The reason of the lesser ring of inhibition in comparison with Streptomycin is owing to that slow release rate of silver into the agar plate due to their loading into the -Carrageenan matrices. The silver release result also supports the above observation. Furthermore, a chemically synthesized silver nanoparticle by a precipitation method with a particle size of 25 nm containing hydrogel bead was tested and compared with the equal Ag-NPs loading hydrogel bead (Ca/Ag 3). The growth inhibition zones of the chemically synthesized containing hydrogel were 4 ± 2 mm (Fig. 7(f)), which were ∼50% lower than the inhibition zones of -Ca/Ag 3. A significant aspect of the antimicrobial activity of Ag-NPs is the synergistic effect which occurs when these particles are functionalized with others natural and synthetic compounds. The capping of Ag-NPs with C. colocynthis probably enhanced the antimicrobial activity of the bio-formed Ag-NPs containing hydrogel beads in comparison with chemically formed Ag-NPs loading hydrogel beads. 4. Conclusion To conclude, -Ca/Ag-NPs hydrogel beads were successfully prepared through ex-situ synthesis of silver nanoparticles by a green method using of C.colocynthis seed extract, dispersion into -Carrageenan hydrogel and then followed by the formation of beads. Bio-nanocomposite hydrogel beads showed a lesser swelling capacity in comparison to that of the pure -Carrageenan hydrogel which was dependent on the porosity of the hydrogel networks. -Ca/Ag-NPs bio-nanocomposite hydrogels offered excellent and sustainable controllability of Ag release, following an exponential power law, with exponent n dependent of Ag content. Silver release from -Ca/Ag-NPs (∼10 mg) bio-nanocomposite hydrogels was slow in PBS solution with (∼9.3–19.5 g) of Ag released in 24 h. Regardless the slow Ag release, -Ca/Ag-NPs 3 with only ∼13 g still showed strong antibacterial activity with inhibition zone of 11 ± 2 against S. aureus, MRSA, P.aeruginosa as well as E. coli, and the antimicrobial effects of, -Ca/Ag-NPs 3 were ∼50% higher to chemically synthesized silver-containing hydrogel beads. This opens new windows to research challenges for designing the nanocomposites hydrogels with strong antimicrobial activity by using green synthesized nanoparticles in hydrogels. Cytotoxicity results showed that -Ca/Ag-NPs bio-nanocomposite hydrogels have an acceptable level of toxicity, representing great pharmacological potential. Acknowledgment The authors are grateful to the Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences for the laboratory facilities. References [1] A. Jain, Y. Gupta, S.K. Jain, Perspectives of biodegradable natural polysaccharides for site specific drug delivery to the colon, J. Pharm. Pharm. Sci. 10 (2007) 86–128. [2] D. Buenger, F. Topuz, J. Groll, Hydrogels in sensing applications, Prog. Polym. Sci. 37 (2012) 1678–1719. [3] A.M. Stephen, G.O. Philips, P.A. Williams, Food Polysaccharides and Their Applications, Marcel Dekker, New York, 1995, pp. 205–217. [4] A. Polk, B. Amsden, K.D. Yao, T. Peng, M.F.A. Goosen, Controlled release of albumin from chitosan-alginate microcapsules, J. Pharm. Sci 83 (1994) 178–185.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Hydrogel beads bio-nanocomposite based on Kappa-Carrageenan and green synthesized silver nanoparticles for biomedical applications Susan Azizi a,∗ , Rosfarizan Mohamad a,b,∗∗ , Raha Abdul Rahim c , Reza Mohammadinejad d , Arbakariya Bin Ariff a,e a Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Laboratory of Biopolymer and Derivatives, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia c Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran e Bioprocessing and Biomanufacturing Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 29 October 2016 Received in revised form 22 May 2017 Accepted 2 June 2017 Available online 4 June 2017 Keyword: Hydrogel Silver nanoparticles Green method Citrullus colocynthis
a b s t r a c t This paper describes the fabrication and characterization of bio-nanocomposite hydrogel beads based on Kappa-Carrageenan (-Carrageenan) and bio-synthesized silver nanoparticles (Ag-NPs). The silver nanoparticles were prepared in aqueous Citrullus colocynthis seed extract as both reducing and capping agent. Cross-linked -Carrageenan/Ag-NPs hydrogel beads were prepared using potassium chloride as the cross-linker. The hydrogel beads were characterized using XRD and FESEM. Moreover, swelling property of the hydrogel beads was investigated. The Ag release profile of the hydrogels was obtained by fitting the experimental data to power law equation. The direct visualization of the green synthesized Ag-NPs using TEM shows particle size in the range of 23 ± 2 nm. The bio-nanocomposite hydrogels showed lesser swelling behavior in comparison with pure -Carrageenan hydrogel. Regardless the slow Ag release, -Carrageenan/Ag-NPs presented good antibacterial activities against Staphylococcus aureus, Methicilin Resistant Staphylococcus aurous, Peseudomonas aeruginosa and Escherichia coli with maximum zones of inhibition 11 ± 2 mm. Cytotoxicity study showed that the bio-nanocomposite hydrogels with non-toxic effect of concentration below 1000 g/mL have great pharmacological potential and a suitable level of safety for use in the biological systems. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels derivative from natural polysaccharides are highwater content polymeric materials which have a number of characteristics such as biodegradability, biocompatibility, stimuliresponsive characteristics and biological functions promising for biomedical applications such as tissue engineering, drug delivery and biosensor [1,2].
∗ Corresponding author. ∗∗ Corresponding author at: Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. E-mail addresses: [email protected] (S. Azizi), [email protected] (R. Mohamad). http://dx.doi.org/10.1016/j.ijbiomac.2017.06.010 0141-8130/© 2017 Elsevier B.V. All rights reserved.
The -Carrageenan is a sulphated linear polysaccharide of D-galactose and 3,6-anhydro-d-galactose obtained by alkaline extraction from red algae. Because of their biocompatibility and high capacity to form hydrogels, -Carrageenan has been widely used in food and pharmaceutical industries [3]. The use of complexation between oppositely charged macromolecules to generate -Carrageenan beads can attract great attention as a drug-controlled release formulation because of the simplicity and gentleness of this manner and production of small size and uniform shapes in compared with the conventional hydrogels [4–7]. Owing to the greater biomedical relevance, there is an increasing attention to develop the antibacterial hydrogels [8]. Among the antimicrobial hydrogels, the antibacterial inorganic-based nanocomposite hydrogels are especially favorable to inhibit the bacterial growth, consequently making them attractive in the fields of biomedical and biotechnology [9]. Several types of inorganic
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2. Materials and methods 2.1. Materials -Carrageenan (300 000 g/mol Fluka Chemie), AgNO3 (䊐99.98%, Merck), potassium chloride (KCl) (>99%, Sigma-Aldrich). Ripe fruits of C. colocynthis were collected from Koohzar district, Khuzestan, Iran in May–June 2015. The plant specimens were identified and authenticated by Department of Botany, Shahid Chamran University, Iran. 2.2. Preparation of C. colocynths seed extract
Fig. 1. Photograph of dried Citrullus colocynthis.
nanoparticles with antimicrobial activities have come up such as titanium [10], zinc oxide [11], magnesium, gold [12] and silver (Ag) [13]. Among the above nanoparticles, silver has established to be favorable candidates for antimicrobial activity against a broad spectrum of pathogenic bacteria [14], as well antibiotic-resistant bacteria [15,16]. Sliver nanoparticles also well known for its antiseptic, anti-inflammatory and multilevel antimicrobial activities (multidrug resistance) [17,18] accompanied by low systemic toxicity [18]. Hence, many studies had been done on the development of silver nanocomposite hydrogels for biomedical applications during the last few years [19–22]. However, in most of these studies chemical methods have been applied to synthesize silver nanoparticles, which restrict the medical applications of hydrogel nanocomposites, due to toxicity of starting materials. Considering to the above concern, this study was undertaken to develop silver nanocomposite hydrogels with green chemistry to enhance their level of safety for use in biological systems. Recently, plant-mediated biological synthesis of nanoparticles is gaining significance owing to its simplicity, eco-friendliness and extensive pharmaceutical effects [23]. Bio-constituents of different plant extracts were known as potential synthesizers and stabilizers of metal nanoparticles. In addition, the plant metabolites with medicinal effects have the potential to be attached on the surface of nanoparticles during the synthesis process which finally leads to the occurrence of subsequent varied surface effects during their medicinal applications [24]. Citrullus colocynthis (C. colocynthis) (Fig. 1) a member of the Cucurbitaceae family, is an important and extensively used plant in traditional medicine distributed throughout Asia. It has been reported to have numerous important biological properties such as antioxidant [25] anticancer [26] anti-inflammatory [27] and antimicrobial [28–30] activities. The seed contains phytochemicals such as glycosides, flavonoids, alkaloids, carbohydrates, fatty acids and essential oil [31] which are capable of reduction and stabilization with high biological activities that motivate the our interest to utilize it in the synthesis of Ag-NPs. In this study, we have first demonstrated a simple one-step green process to synthesizing Ag-NPs using C. colocynthis seed extract as the both reducing and stabilizing agent. Secondly, we designed a high safety hydrogel based antimicrobial bionanocomposite using biosynthesized Ag-NPs as an antimicrobial agent and -Carrageenan as hydrogel matrix. The effect of the concentration of the as synthesized Ag-NPs on the morphology, swelling behavior, cytotoxicity and antibacterial activity was examined.
The seeds were removed from the fruits and dried at room temperature and then milled to fine powder using grinder. The material that passed through 80-mesh sieve was used for extraction purpose. Briefly, the fine material was extracted with ethanol for 8 h in a Soxhlet apparatus. The solvent was removed by rotary evaporation. The dried, crude concentrated extract then stored in a refrigerator (−4◦ C). 2.3. Biosynthesis of Ag-NPs Silver nitrate (0.003 M) was dissolved in 100 mL of distilled water under magnetic stirring. After complete dissolution, 0.2 g of the dried C. colocynths seed extract was added to the above solution under continuous stirring at 45 ◦ C and allowed to react over 1 h. The reducing of silver ions to silver nanoparticles was observed by the changing color of solution mixture from light yellow to dark brown. For the purification of Ag-NPs, the fully reduced solution was centrifuged at 8000 rpm for 15 min. The supernatant liquid was discarded and the residue was dispersed in Millipore water. The samples were centrifuged five times to remove any constituents that had been absorbed onto the surface of the Ag-NPs. The final product was dried at 60 ◦ C overnight. The resulting dried sample preserved in air-tight bottles for further studies. 2.4. Determination of percentage yield (%Y) of Ag-NPs Concentration of Ag+ ions before and after addition of extract was measured using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) model Perkin Elmer 1000. Equation (1) was used to calculate percentage yield (%Y) using initial concentration (IC ) and final concentration (FC ) of Ag+ ions: %Y =
Ic − Fc × 100% Ic
(1)
2.5. Preparation of -Carrageenan/Ag-NPs hydrogel beads A series of nanocomposites were prepared by blending the biosynthesized Ag-NPs with the -Carrageenan matrix as follows. 2 gr of -Carrageenan was added to 80 mL of distillated water under magnetic stirring, at 80 ◦ C. After complete dissolution, 0.0, 0.1, 0.3, 0.5, 0.7 and 1.0 mL were taken from stock solution of silver nanoparticles (1 mg/mL) and added to 9 mL of -Carrageenan solutions under magnetic stirring until homogeneous viscose solutions obtained. The bio-nanocomposite beads were formed by dropping the hydrogel bio-nanocomposite solutions from a needle with 2 mL internal diameter into 1 M of KCL solution at room temperature. After 30 min, the beads were separated and washed with distilled water, then dried under vacuum at room temperature. The prepared beads based on the amount of Ag-NPs were called -Ca/Ag0, -Ca/Ag1, -Ca/Ag2, -Ca/Ag3, -Ca/Ag4 and -Ca/Ag5 mean -Carrageenan/Ag-NPs bio-nanocomposite hydrogel beads,
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which have 0.0, 0.01, 0.03, 0.05, 0.07 and 1.0 mg/mL of Ag-NPs content, respectively. 2.6. Characterization and analysis The X-ray diffraction analysis was carried out by Philips X’pert PXRD, with Cu-Ka radiation at 40 kV in the scan range of 2 from 2◦ to 80◦ . Infrared spectra were obtained on an FTIR spectrometer (PerkinElmer 1725X) in the 4000–400 cm−1 range. Transmission electron microscopy (TEM) of Ag nanoparticles was performed using a Hitachi H-700 transmission microscope. UV–vis spectroscopy was carried out on a Lambda 25-PerkinElmer UV–vis spectrophotometer. The morphology of the hydrogel samples was observed using a field emission scanning electron microscope (FESEM) (JSM-6360LA Philips). 2.7. Swelling behavior The swelling ratio were carried out by immersion of 100 mg of hydrogel beads in PBS 0.01 M, pH 7.4 at 37 ◦ C. At various time points, the samples were lifted from the solution and excess water was removed from the surface by blotting on wet filter paper before being weighted. The swelling ratio of bio-nanocomposite beads was calculated according to Eq. (2): Swelling Ratio (%) =
(w2 − w1 ) × 100 w1
at 37 ◦ C. The 100 L of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbent compared to control. The 50% inhibitory concentration (IC50 ) value, defined as the amount of sample that inhibits 50% of cell growth, was calculated from the concentration-response curves. 2.10. Antimicrobial assessment Antibacterial activity of the bio-nanocomposite hydrogels beads was evaluated against Gram positive (Staphylococcus aureus (S.aureus) S276 and Methicilin Resistant Staphylococcus aurous (MRSA)) and Gram negative (Peseudomonas aeruginosa (P. aeruginosa) ATCC 15442 and Escherichia coli (E.coli) E266) pathogens by using agar diffusion method. The agar plates were inoculated with 100 l spore suspensions of bacteria. For each sample, one swelled bead (∼10 mg) was placed on the agar plate away from the antibiotic (Streptomycin) and incubated at 37 ◦ C for 24 h. After incubation, the zone of whole inhibition was measured. All tests were replicated three times. 3. Results 3.1. Characterization of Ag-NPs
(2)
where w1 is the initial weight of the sample and w2 is the weight of swollen sample. The equilibrium swelling ratio (ESR) was determined at the point the hydrated hydrogel reached to a constant weight value. The swelling capacities were measured in triplicate. 2.8. Release study of silver content from hydrogel nanocomposites Ag-NPs release profile from -Carrageenan hydrogel nanocomposites was studied in phosphate buffer saline (PBS 0.01 M, pH 7.4) at 37 ◦ C. Hydrogels beads (∼30 mg) immersed in 10 mL PBS in an incubator shaker at 120 rpm. 0.5 mL solution were taken out after regular time intervals of (6, 12, 24, 48, 72 and 96 h) and analyzed for the amount of Ag released using Inductively Coupled Plasma Atomic Emission Spectrometry(ICP-AES) (Perkin Elmer 1000, USA), and then the same volume of fresh phosphate buffered saline solution was added. The release profile of Ag-NPs from -Carrageenan hydrogel nanocomposites was studied by release kinetics RitgerPeppas model [32], a semi-empirical power law described by Eq. (3) Ct = Kt n Ceq
425
(3)
where Ct and Ceq are the cumulative concentrations of Ag released from the hydrogel at a specified time and at equilibrium, respectively, k is a characteristic constant of the hydrogel and n is the diffusional coefficient used to interpret the release mechanism. 2.9. Cytotoxicity assay The in vitro cytotoxicity of hydrogel beads was evaluated by the method using 3-(4,5-dimethylthiazol-2-yl)-2,5- dephenyltetrazolium bromide (MTT) assay. Briefly, VERO cells were seeded at a density of 2 × 105 cells/mL in 96-well microplates and incubated for 24 h. Subsequently, the cells were treated with 100 L of the suspension of hydrogels in FBS (1 mg/mL) for 24 h. The samples were suspended separately in a stock solution at 5 g/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 L of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were incubated for another 4 h
The use of plant extract for synthesis and stabilization nanoparticles is well proven [33–36]. The use of seed extract of C. colocynths was significantly effective on the synthesis of Ag nanoparticles. The seed extract contains some biomolecules such as Isosaponarin and Isovitexin with a huge number of hydroxyl (O H) and carbonyl (C O) groups in their structures which facilitate the complexation of silver ions to these molecules. By transforming of free electron from the electrons of carbonyl (C O) or hydroxyl groups to the free orbital of metal ions in a Red/Ox system silver ions convert to silver nanoparticles [23]. The color change of Ag+ /C. colocynths solution from yellow to dark brown after 20 min indicates formation of silver nanoparticles (inset in Fig. 2A). In fact, C. colocynths extract acts as both reducing and stabilizing agent. The stabilization of nanoparticles by bio-compounds prevent from their aggregations. The UV–vis spectroscopy of sample shows a typical absorption peak at 440 nm (Fig. 2.A) which corresponds to the typical of surface plasmon resonance (SPR) of Ag-NPs. Based on the SPR, the spherical Ag-NPs displays the characteristic SPR at the wavelength between the ranges of 400–450 nm [37]. From TEM image of AgNPs (Fig. 2B) observed that the nanoparticles are spherical in shape with the mean particle diameter of 23 ± 2 nm. The XRD spectrum of the Ag-NPs (Fig. 2C) shows four distinct crystalline reflections at 2 = 38.24◦ , 44.51◦ , 64.32◦ and 77.45◦ and its corresponding lattice plane value was indexed at (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic nature. The FTIR spectrum of pristine Ag-NPs (Fig. 2Da) shows the corresponding peaks of Ag-O at 1625 and 668 cm−1 [38]. Fig. 2Db demonstrates the structure of C. colocynths extract with bands at 3384, 1722, 1426, 1230 and 1108, cm−1 . Fig. 2Dc represents the FTIR spectrum of C. colocynths extract reduced Ag-NPs, where signals’ intensities at 3384 (O H stretching), 1722 (C O), 1230 and 1108 (C O stretching) cm−1 decreased after synthesis of Ag-NPs, indicating the involvement of carbonyl and hydroxyl groups in the reduction process. The FTIR spectrum clearly shows that the C. colocynths acted as both reducing and capping agent. The zeta potential value of nanoparticles was −28.5 mV which shows that the NPs were stable and warped with anionic organic phases and responsible for electrostatic stabilization. ICPAES analysis of Ag+ content after formation of Ag-NPs by extract was found to be 189 ppm at pH 7. The percent yield (%Y) was calculated as ∼96%.
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Fig. 2. (A) UV spectrum and (inset) color change of seed extract after formation of Ag-NPs, (B) TEM image and (C) XRD diffraction pattern of bio-formed Ag-NPs, (D) FTIR spectrum of (a) pristine Ag-NPs, seed extract (b) before and (c) after formation of NPs.
3.2. Characterization of -Carrageenan/Ag-NPs bio-nanocomposite hydrogel beads 3.2.1. XRD analysis Nanocomposites hydrogel beads were prepared by homogeneous dispersion of different ratio of bio-synthesized Ag-NPs within -Carrageenan solutions, followed by gelled beads formation of the mixture in the presence of K+ ions, through electrostatic interactions between negatively charged -Carrageenan chains and positively charged K+ ions. The XRD patterns of the original Carrageenan hydrogel and -C/Ag3 bio-nanocomposite hydrogel in the 2 range of 10–80◦ are shown in Fig. 3. The -C/Ag3 shows two sets of diffraction peaks corresponding to -Carrageenan and AgNPs. The peaks corresponds to Ag were almost the same as that of the bio-synthesized of Ag-NPs (Fig. 2C). The halo typical peak of -Carrageenan at a 2 of 21.35◦ was broader than that of the pure -Carrageenan, this event may be due to the presence of biomolecules of C. colocynths which were introduced on the surface of silver nanoparticles.
Fig. 3. (a) XRD pattern of original -Carrageenan hydrogel and (b) -C/Ag3 bionanocomposite hydrogel.
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Fig. 4. (A) Digital photo of -C/Ag bio-nanocomposite hydrogel beads with different ratio of Ag-NPs, (B) FESEM micrographs of (a) original -Carrageenan and (b-f) -C/Ag (1-5) bio-nanocomposite hydrogel beads.
3.2.2. Morphology of -C/Ag bio-nanocomposite hydrogel beads The dried bio-nanocomposite hydrogel beads were spherical with a diameter of about 1 mm which changed their color from yellow to dark brown and surfaces from smooth to rough with the increase in Ag-NPs content (Fig. 4A ).
The surface morphology of the original -Carrageenan and C/Ag bio-nanocomposite hydrogel beads are shown in Fig. 4B(a–f). FESEM images clearly show that the spherical Ag-NPs with particle size less than 25 nm were well dispersed within hydrogel -Carrageenan up to 0.05 mg/mL (Fig. 4B(a–c)) and generated a smooth and tight surface as compared with the pure -Carrageenan
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Fig. 5. Swelling behavior of -C/Ag bio-nanocomposite hydrogel beads.
which possesses uneven surface with many cavities. Beyond this level, with the increase in the Ag-NPs content, the nanoparticles mostly agglomerated and the surface morphology of hydrogel beads became rough and undulant (Fig. 4e–f), as was observed by visual inspection of samples. The Ag-NPs at low concentrations loading could possibly act as intermolecular cross-linker, which restrict the mobility of the -Carrageenan chains and then enhanced the surface morphology of -Carrageenan to a smooth and tight surface. By increasing in nanoparticles loading, Ag-NPs due to the small dimensions, high surface activity, and large specific surface area were agglomerated which results in the decrease in interfacial interactions between -Carrageenan chains and Ag-NPs, this event makes an undulant surface and pitted be created. The porous structure could make the capillary forces which allowed the diffusion of fluids into the beads and thereby the drug easily released into water [39]. Therefore from FESEM results can conclude that those nanocomposites hydrogels which contain 0.01–0.05 mg/mL silver nanoparticles have appropriate morphology to use in prolonged and more controlled drug releases in a biological system. 3.2.3. Swelling behavior The equilibrium swelling ratio of the hydrogel beads was shown in Fig. 5. Equilibrium swelling ratio decreased in case of Ag-NPs loaded hydrogel than pure -Carrageenan hydrogel. The swelling capacity is directly proportional to the porosity of the hydrogel networks. The composition of the network -Carrageenan polymer chains and the crosslinking density are the important factors which effect on the pore volume fraction, pore sizes and their interconnection [40]. As was observed by the FESEM results, the Ag-NP acts as an effective cross-linker which improves the surface tight with the decrease in porosity of the hydrogel networks compared to pure -Carrageenan. This might be the reason for decreased swelling properties in Ag-NPs loaded hydrogels compared to pure -Carrageenan. However in -Ca/Ag4 and -Ca/Ag5 hydrogels, the swelling property due to forming some cavities in hydrogel structure somewhat increased compared to those nanocomposites which have smooth and tight surfaces. Another possible explanation for this swelling enhancement may be related to the negative surface charge of Ag-NPs used in the hydrogel. The immobilization of surface charged Ag-NPs within -Carrageenan results in the afflux of water to balance the osmotic pressure buildup caused by the immobilization of the surface charge NPs. The degree of osmotic swelling depends on the amount of charges immobilized within the gel and is proportional to the surface charge and concentration of the nanoparticles. Thus, hydrogel nanocom-
Fig. 6. In vitro Ag releasing properties of -Ca/Ag nanocomposites in PBS buffer solution: (A) release quantity changes of silver ion in PBS and (B) log(release quantity of Ag) vs log(times) curve.
posites with high load of Ag-NPs swell more and these results are in agreement with previous observation in hydrogel containing Fe3 O4 NPs [41]. From the graphs was also observed that swelling increased with time and reached to a maximum whitin12 h and 16 h in the cases of pure -Carrageenan and Ag-NPs loaded hydrogels respectively and then it reached equilibrium almost after 16 h. 3.2.4. In vitro Ag releasing properties of -Ca/Ag bio-nanocomposite beads Ag releasing profiles of -Ca/Ag bio-nanocomposite in PBS solution were presented in Fig. 6(A) and (B). The power law exponent, n, from slopes of the logarithmical curves of Ct/Ceq as a function of time and fitting coefficient (R2 ) calculated and listed in Table 1. The results show that n approximately is varied for samples which is indicative of different transport mechanism. When n = 0.45 means that the drug release mechanism is controlled by Fickian diffusion. The power law of Ag releasing rate of -Ca/Ag1 and -Ca/Ag 2 hydrogels is near to 0.45 with R2 ≥ 0.903, which seems the Ag release mechanism is controlled by Fickian diffusion. Anomalous transport mechanism, which is the contribution of both Fickian diffusion and macromolecular relaxation is observed when 0.45 ≤ n ≤ 0.89. In the -Ca/Ag3 hydrogel nanocomposite, n increased to 0.507 ± 0.010, proposing a trend towards the Fickian diffusion. The release mechanism for samples -Ca/Ag4 (n = 0.624 ± 0.03) and -Ca/Ag5
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Table 1 Release quantity and release ratio of Ag from -Ca/Ag bio-nanocomposite (∼30 mg) after 4 day; slope of fitting and fitting coefficient of log (release quantity of Ag) vs log (times) curve. Release quantity (g) Sample
1day
4 day
Slope of fitting (n)
Fitting coefficient (R2 )
-Ca/Ag 1 -Ca/Ag 2 -Ca/Ag 3 -Ca/Ag 4 -Ca/Ag 5
28.1 ± 2 33.4 ± 3 39.3 ± 2 42.4 ± 3 59.2 ± 4
35.2 ± 3 43.1 ± 4 57.4 ± 2 79.1 ± 4 90.3 ± 3
0.441 ± 0.02 0.466 ± 0.05 0.507 ± 0.01 0.624 ± 0.03 0.628 ± 0.02
0.903 0.926 0.953 0.964 0.939
(n = 0.628 ± 0.02) hydrogels became anomalous which indicates the contribution of both Fickian diffusion and macromolecular relaxation. In such cases, the agglomerated Ag-NPs in the hydrogel nanocomposites are very nearby to each other. Consequently, anion-anion electrostatic repulsion forces due to negative charges on their surface are generated, throughout the polymer network and the macromolecular relaxation begins to act [42]. 3.2.5. Cytotoxicity evaluation The 50% inhibitory concentration (IC50 ) value of hydrogel nanocomposites for a cut-off of 1000 g/mL was calculated as the dose required to inhibit the cell growth by 50% and shown in Fig. 7. None of hydrogels considerably inhibited the cell growth indicating that IC50 is higher than 1000 g/mL. The values of growth inhibition at 1000 g/mL were much lower than 50%, signifying that the hydrogels do not have any toxic, damaging elements for living cells. 3.2.6. Antimicrobial assessment Agar diffusion assay was employed to probe the antibacterial activity of bio-nanocomposite hydrogels against Gram positive (S.aureus and MRSA) and Gram negative (P. aeruginosa and E.coli) bacteria. The results obtained are shown in Fig. 8. The results
Fig. 7. Cytotoxic effect of hydrogel -Ca/Ag (1-5) nanocomposites on the growth inhibition of VERO cells.
demonstrated that the all bio-nanocomposite hydrogels had toxic effect on bacteria with the highest effect for sample of -Ca/Ag 3. The -Ca/Ag 3 bio-nanocomposite hydrogel showed inhibition zones of 11 ± 2 mm against all Gram positive and negative bac-
Fig. 8. Inhibition zone of -Ca/Ag (1-5) hydrogels (a-e) and chemically synthesized Ag hydrogel beads (f) against S. aureus (A), MRSA (B), P. aeruginosa (C) and E. coli (D) pathogens.
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teria compared to inhibition zones of 6 ± 2 mm for other -Ca/Ag bio-nanocomposite hydrogels. The reason for decreasing of antimicrobial activity with the increase in Ag loading in hydrogels is probably due to the form of large agglomerates of nanoparticles which are less probable to diffuse into the cell membrane to damage the bacteria from the inside. In the case of -Ca/Ag 3 nanocomposite, the diameter of the zone of inhibition was favorable with respect to positive control i.e. Streptomycin (25 mm). The reason of the lesser ring of inhibition in comparison with Streptomycin is owing to that slow release rate of silver into the agar plate due to their loading into the -Carrageenan matrices. The silver release result also supports the above observation. Furthermore, a chemically synthesized silver nanoparticle by a precipitation method with a particle size of 25 nm containing hydrogel bead was tested and compared with the equal Ag-NPs loading hydrogel bead (Ca/Ag 3). The growth inhibition zones of the chemically synthesized containing hydrogel were 4 ± 2 mm (Fig. 7(f)), which were ∼50% lower than the inhibition zones of -Ca/Ag 3. A significant aspect of the antimicrobial activity of Ag-NPs is the synergistic effect which occurs when these particles are functionalized with others natural and synthetic compounds. The capping of Ag-NPs with C. colocynthis probably enhanced the antimicrobial activity of the bio-formed Ag-NPs containing hydrogel beads in comparison with chemically formed Ag-NPs loading hydrogel beads. 4. Conclusion To conclude, -Ca/Ag-NPs hydrogel beads were successfully prepared through ex-situ synthesis of silver nanoparticles by a green method using of C.colocynthis seed extract, dispersion into -Carrageenan hydrogel and then followed by the formation of beads. Bio-nanocomposite hydrogel beads showed a lesser swelling capacity in comparison to that of the pure -Carrageenan hydrogel which was dependent on the porosity of the hydrogel networks. -Ca/Ag-NPs bio-nanocomposite hydrogels offered excellent and sustainable controllability of Ag release, following an exponential power law, with exponent n dependent of Ag content. Silver release from -Ca/Ag-NPs (∼10 mg) bio-nanocomposite hydrogels was slow in PBS solution with (∼9.3–19.5 g) of Ag released in 24 h. Regardless the slow Ag release, -Ca/Ag-NPs 3 with only ∼13 g still showed strong antibacterial activity with inhibition zone of 11 ± 2 against S. aureus, MRSA, P.aeruginosa as well as E. coli, and the antimicrobial effects of, -Ca/Ag-NPs 3 were ∼50% higher to chemically synthesized silver-containing hydrogel beads. This opens new windows to research challenges for designing the nanocomposites hydrogels with strong antimicrobial activity by using green synthesized nanoparticles in hydrogels. Cytotoxicity results showed that -Ca/Ag-NPs bio-nanocomposite hydrogels have an acceptable level of toxicity, representing great pharmacological potential. Acknowledgment The authors are grateful to the Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences for the laboratory facilities. References [1] A. Jain, Y. Gupta, S.K. Jain, Perspectives of biodegradable natural polysaccharides for site specific drug delivery to the colon, J. Pharm. Pharm. Sci. 10 (2007) 86–128. [2] D. Buenger, F. Topuz, J. Groll, Hydrogels in sensing applications, Prog. Polym. Sci. 37 (2012) 1678–1719. [3] A.M. Stephen, G.O. Philips, P.A. Williams, Food Polysaccharides and Their Applications, Marcel Dekker, New York, 1995, pp. 205–217. [4] A. Polk, B. Amsden, K.D. Yao, T. Peng, M.F.A. Goosen, Controlled release of albumin from chitosan-alginate microcapsules, J. Pharm. Sci 83 (1994) 178–185.
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