A novel and green biomaterial based silver nanocomposite hydrogel: Synthesis, characterization and antibacterial effect

Journal of Inorganic Biochemistry 117 (2012) 367–373

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A novel and green biomaterial based silver nanocomposite hydrogel: Synthesis, characterization and antibacterial effect Ghasem R. Bardajee ⁎, Zari Hooshyar, Habib Rezanezhad Department of Chemistry, Payame Noor University, PO BOX 19395–3697, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 24 February 2012 Received in revised form 23 June 2012 Accepted 25 June 2012 Available online 1 July 2012 Keywords: Silver nanoparticles Drug delivery Tetracycline hydrochloride Hydrogel Antibacterial effect

a b s t r a c t In the present study, we report a facile and eco-friendly method for the preparation of a novel silver nanocomposite hydrogel (SNH) based on poly(acrylic acid) (PAA) grafted onto salep as a water soluble polysaccharide backbone. The presence of inorganic silver nanoparticles (nano-Ag) in the hydrogel was confirmed by thermo-gravimetric (TG) analysis. The TEM images illustrated the presence of embedded nano-Ag throughout the hydrogel matrix. In addition, the transmission electron microscopy (TEM) images showed that the formed nano-Ag had an average particle size of 5–10 nm. The potential of obtained SNH was examined for Tetracycline hydrochloride (TH) release in simulated colon conditions. Lastly, the in vitro antibacterial properties of the obtained optimum sample were successfully evaluated against gram-negative and gram-positive bacteria. © 2012 Elsevier Inc. All rights reserved.

1. Introduction In recent years, the study and preparation of metal particles on the nanometer scales have attracted considerable interest from both fundamental and applied researches [1–3]. It is due to different physical and mechanical properties of metal nano-sized particles from those of macroscopic materials. They are mainly utilized in solving the problems of water purification, catalysis, electronics, sensors, hydrogen storage [4–9], luminescence devices [10], photonics [11], pharmaceuticals [12], biotechnology, and medicine [13]. The silver nanoparticles (nano-Ag) have proved to be most effective as they exhibit potent antimicrobial efficacy against bacteria, viruses and eukaryotic micro-organisms [14,15]. It has been demonstrated that nano-Ag can inhibit viral replication of viruses, such as human immunodeficiency virus type 1, hepatitis B virus, respiratory syncytial virus, herpes simplex virus type 1, and monkey pox virus [16]. Nano-Ag have been studied as a medium for antibiotic delivery [17], and to synthesize composites for use as disinfecting filters [18] and coating materials [19]. To receive a better stabilization or dispersion of nano-Ag in aqueous media, various protective agents have been used for the synthesis and controlling the size of nano-Ag [14,17,18]. For this purpose, different polymeric stabilizing agents, dendrimers, latex particles and microgels have exclusively been studied. In addition, many researches are heading for exploiting the in situ synthesis of nano-Ag within the polymeric network architectures which leading to new hybrids or composite systems in chemistry and engineering sciences [19–21]. In this way the carrier

⁎ Corresponding author. Tel.: +98 281 3336366; fax: +98 281 3344081. E-mail address: [email protected] (G.R. Bardajee). 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2012.06.012

systems, for example dendrimers or microgels, act as ‘nanoreactors’ that immobilize the particles and provide an easy handling. Hydrogels offer large free spaces between the crosslinked networks in the swollen stage that can act as a nanoreactor for the nucleation and growth of the nano-Ag [22–28]. This approach due to the long reaction times, the use of chemical and usually toxic reagents, low efficiency in converting the silver cations (Ag+) to nano-Ag, and lack of control over the size of nano-Ag has not been welcomed. The biopolymers applied for the synthesis of silver nanocomposites due to their almost limitless availability, low price, and biocompatibility. As the purpose of silver nanocomposites is mostly toward biomedical treatment, different hydrogels have been applied in this field. For example, Vimala et al. [29] report the preparation of semi-interpenetrating hydrogel networks based on cross-linked poly(acrylamide). The polymer was prepared through a redox polymerization of N,Nmethylenebisacrylamide in the presence of different carbohydrate polymers such as gum acacia and carboxymethylcellulose. They reduced silver cations inside the network using sodium borohydride. The amide and hydroxyl functional groups of the network were projected to enhance the stability of the silver nanoparticles inside the matrix; however their size distribution was broad. Their antimicrobial effectiveness was tested against E. coli. In addition, silver nanoparticles have been obtained with hydrogel networks as nanoreactors via in situ reduction of silver nitrate using sodium borohydride as a reducing agent. However, the antimicrobial activity was not high, obviously due to the limited ion release from the capped and protected silver nanoparticles. In addition, other hydrogel–silver nanocomposites have been synthesized by a synthetic route involving the formation of silver nanoparticles within swollen poly(acrylamide-co-acrylic acid) hydrogels by using citrate ions. The formation of silver nanoparticles was

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confirmed by TEM and they demonstrated good antimicrobial effects against E. coli strains [29–31]. The present investigation involves the preparation of a novel and eco-friendly silver nanocomposite hydrogel (SNH) based on poly(acrylic acid) (PAA) grafted onto salep. The nano-Ag was synthesized in situ in the presence of salep backbones before hydrogel formation in the absence of any chemical reducing agent. Salep is a kind of water-soluble and totally eco-friendly polysaccharide which can be obtained from dried tubers of certain natural terrestrial orchids and referred as a good source of glucomannan (Fig. 1) [32–34]. In addition to glucomannan as a main constituent of salep, it also contains starch (2.7%), nitrogenous substance (5%), moisture (12%) and ash (2.4%). Glucomannans are natural, neutral and water-soluble fibers, which can assist to normalize blood sugar, relieve stress on the pancreas and discourage blood sugar abnormalities such as hypoglycemia. They also act as a preventative of chronic diseases and as a weight control agent. PH-sensitivity of the SNH was discussed and the controlled drug delivery behavior of hydrogel was investigated by using tetracycline hydrochloride (TH) as a model drug. TH is primarily bacteriostatic and exerts their antimicrobial effect by the inhibition of protein synthesis. The TH is active against a wide range of gram-negative and gram-positive organisms. It is soluble in water, and easily destroyed by strong acidic or alkaline hydroxide solutions (Fig. 2) [35,36]. Therefore, it causes vomiting, upset stomach (dyspepsia), lack of appetite and difficulty swallowing. The problems can be eliminated by protecting dug from absorption in the environment of the upper gastrointestinal tract and then its release into the colon. Based on this, we incorporated TH into SNH to investigate its application for TH release in colon site and improve significantly the therapeutic antibacterial efficacy of the drug. 2. Experimental section 2.1. Materials Salep was purchased from a supplier in Kordestan, Iran (Mn = 1.17 × 10 6 g/mol, Mw = 1.64 × 10 6 g/mol (high Mw), PDI = 1.39, eluent = water, flow rate = 1 mL/min, acquisition interval = 0.43 s from GPC results). Silver nitrate (AgNO3, from Fluka, St. Louis, MO ), methylenebisacrylamide (MBA, from Merck, Darmstadt, Germany) as a crosslinker, ammonium persulfate (APS, from Fluka, St. Louis, MO) as a water soluble initiator, and acrylic acid (AA, from Merck) as a monomer, were analytical grades and used without further purification. TH was received from Alborz Darou Co., Tehran, Iran as a gift. All other chemicals were also analytical grade. Gram-negative Escherichia coli (E. coli, ATCC 51813) and gram-positive Staphylococcus aureus (S. aureus, ATCC 27661) bacteria were prepared from NIGEB Bacterial Bank (Tehran, Iran). Luria Bertani (LB) broth (peptone 10 g, yeast extract 5 g, NaNO3 10 g, distilled water 1000 mL) and agar powder were purchased from Sigma (SIGMA Chemical Co., St. Louis, MO). In through experiments, double distilled water (DDW) was used for preparing solutions.

OH O

OH O OH

O NH2

OH

, HCl

OH N

Fig. 2. The chemical structure of TH.

A Shimadzu UV–visible 1650 PC spectrophotometer was used for recording absorption spectra in solution. All samples were placed in a 1.00 cm quartz cuvette for UV measurements. The dynamic weight loss tests were conducted on a TA instrument 2050 thermo-gravimetric (TG) analyzer. All tests were conducted under N2 atmosphere (25 mL/min) using sample weights of 5–10 mg over a temperature range of 25–700 °C at a scan rate of 20 °C/min. The mass of the sample pan was continuously recorded as a function of temperature. The morphology of the dried samples was examined using a scanning electron microscope (SEM) (Philips, XL30) operated at 10 kV after coating the dried samples with gold films. Transmission electron microscopy (TEM) was taken on a Zeiss TEM at an acceleration voltage of 80 kV. Samples for TEM were prepared by putting a drop of solution on a carbon-coated copper grid. 2.3. Preparation of SNH In general, salep (1.0 g) was added to DDW (80 mL) at a three-neck reactor equipped with a mechanical stirrer while stirring (200 rpm). After homogenization, 5 mL of the different concentrations of AgNO3 (0.002, 0.005, 0.01 and 0.05 M) was added to the reaction mixture and it was stirred for further 30 min. The reactor was placed in a thermostated water bath preset at desired temperature (80 °C) and 5 mL of the AA monomer (0.12 M), 5 mL of the MBA crosslinker (6.5×10−3 M), and 5 mL of APS initiator (1.65×10−2 M) were added and the reaction mixture was stirred until a gel-like product was observed after around 20 min. Finally, the reaction mixture was cooled to room temperature. The product was poured into 100 mL of ethanol, remained for 2 h and then chopped to small pieces for further drying. To remove the sol fraction of mixture (uncrosslinked and not grafted PAA, uncrosslinked salep and possibly also some unreacted monomer), the dewatered hydrogel was allowed to completely swell for overnight in plenty of DDW (400 mL) and then dewatered in ethanol (200 mL, 2 h). The non-solvent ethanol was decanted and then 100 mL fresh ethanol was added. The chopped particles were further remained for 24 h in ethanol to completely dewater. The dewatered gel particles were filtered and dried in oven (at 50 °C) for 24 h. After grinding, the powdered superabsorbent hydrogels were stored in the absence of moisture, heat, and light. In remaining of the manuscript, SNH0, SNH1, SNH2, SNH3, and SNH5 means SNH which have 0.0, 0.002, 0.005, 0.01 and 0.05 M of AgNO3 content, respectively. 2.4. Swelling measurements using tea bag method [37]

2.2. Instrumental analysis Fourier transform infrared (FT-IR) spectra of samples in the form of KBr pellets were recorded using a Jasco 4200 FT-IR spectrophotometer.

The swelling of SNH was evaluated by considering certain amounts of the SNH samples (0.5 ±0.001 g) in 100 mL of DDW and different pH buffered solutions. The various buffered solutions were prepared

Fig. 1. The general structure of glucomannan repeating units.

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according to Merck Co. procedure. Buffered solutions of pH 2, 4, and 6 were prepared by taking certain amount of 0.2 M KCl and 0.2 N HCl in volumetric flask to make volume 200 ml with DDW. Buffered solutions of pH 8 and 10 were prepared by taking certain amount of 0.2 M KH2PO4 and 0.2 N NaOH in volumetric flask to make volume 200 ml with DDW. The tea bag was hung up for 5 min in order to remove the excess solution. The SNH swelling was calculated according to following equation (Eq. 1) and reported as grams of water per grams of resin (g/g). Swellingðg=gÞ ¼ ðWeight of swollen SNH=0:5Þ–1

ð1Þ

2.5. Drug loading TH loading in the SNH was performed by diffusion method. 0.1 g of powdered SNH was added in tea bag (i.e., a 100 mesh nylon screen) and was immersed entirely in 60 ml of different concentrations of drug (TH) solution (0.01, 0.05, 0.1 and 0.2 M) for 2 h to complete drug loading. At specific time intervals, 1 μl of solution was withdrawn and assayed for the amount of loaded drug as a function of time. The amount of loaded drug was determined by UV spectroscopy at characteristic λmax using a calibration curve. 2.6. Drug releasing The release rate experiments were performed in simulated gastric and colon conditions (2 and 8 buffered solutions at 37 °C under unstirred conditions). The various buffered solutions were prepared according to Merck Co. procedure. The drug (TH) loaded hydrogel was put in a 100 ml beaker containing buffered solutions. At a given time intervals, 1 mL of filtered samples were withdrawn and assayed for the amount of released drug to the solution. The amount of released drug was determined by UV spectroscopy at characteristic λmax using a calibration curve constructed from a series of drug solutions with known concentrations. 2.7. Antibacterial assays The antibacterial assays were done against gram-negative E. coli and gram-positive S. aureus bacteria by disk diffusion and dilution techniques. The LB broth/agar medium was used to cultivate bacteria. 3. Results and discussion 3.1. Mechanism of SNH formation In this study, a SNH has been prepared via sunlight UV-irradiation mediated synthesis of salep capped nano-Ag, followed by graft copolymerization of AA onto salep- (nano-Ag) composite, and crosslinking in the presence of MBA as a crosslinking agent. It is well known that

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UV-irradiation can promote the reduction of the Ag+ into nano-Ag in the presence of a capping agent. Sunlight-UV can promote reductionoxidation reaction in the presence of salep as a reducing agent and cause facile and cheap preparation of the nano-Ag particles. Furthermore, salep as a biocompatible, biodegradable, non-toxic effective capping agent, and multifunctional material was utilized to inhibit the agglomeration of the freshly prepared nano-Ag in solution. In this approach, the coordinated Ag + was reduced into the nano-Ag by photochemical reactions. The schematic representation of this conversion was indicated in Scheme 1. In detail, in-situ reduction of Ag+ and stabilization of particles can be explained as (a) first metal ions are anchored by functional groups of salep backbones (hydroxyl groups of glucomannan repeating units), and (b) metal reduction process takes place in the presence of sunlight UV-irradiation, to give salep capped nano-Ag. In the second step, APS, as an initiator, is decomposed under heating and produced sulfate anion-radicals that remove hydrogen from anomeric carbon or OH groups of salep backbones. This persulfate– saccharide redox system results in active centers capable to radically initiate polymerization of AA monomers and leading to a graft copolymer. Since the crosslinking agent, MBA, is accessible in the system, the copolymer comprises a crosslinked nanocomposite structure (Scheme 1). The hydrogel networks around nano-Ag effectively inhibit their aggregation for longer periods and can be extracted into water whenever they are required for usage. This method excludes restrictions such as using chemical reducing agents which cause toxicity or biological hazards. 3.2. Swelling study Water swelling occurs when a nanocomposite hydrogel is placed in an aqueous solution and water molecules will penetrate into the polymer networks. In this regard, some spaces are occupied by entering water molecules, and as a result some meshes of the network will start expanding, allowing other water molecules to enter within the network. This property is one of the most important parameters for utilizing of nanocomposite hydrogels as drug delivery system. While drug delivery is accomplished by the swelling of the nanocomposite hydrogel, drug release is also related to this swelling behavior. Many structural factors including electrostatic interactions and hydrogen bonds affect the swelling characteristics of nanocomposite hydrogels. For ionic nanocomposite hydrogels, the swelling capacity depends not only on the chemical composition but also on the pH of the surrounding medium [22–25]. In this experiment, capacity of swelling was highly dependent to the amount of AgNO3 and pH media. So, the swelling behavior of the SNH1, SNH2, SNH3 and SNH4 were studied at various pH buffer solutions after 60 min at 25 °C. As one can see in Fig. 3, the swelling increases with rising pH up to 8 for all samples and then it decreases at pH =10. It has been observed that maximum degree of swelling was observed at pH 8 for the SNH3 sample. It seems that at lower pHs, most of the carboxylate anions are protonated, so the main anion-anion repulsive forces are eliminated and consequently swelling values are decreased. However

Scheme 1. Proposed mechanism pathway for the synthesis of SNH.

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350

Swelling ( g/g)

300

SNH3 SNH2 SNH4

250

SNH1

200 150 100 50 1

2

3

4

5

6

7

8

9

10

11

12

13

pH Fig. 3. Swelling dependency of the SNH1, SNH2, SNH3 and SNH4 samples on pH value after 60 min at 25 °C. Fig. 5. FT-IR spectra of (a) SNH0, (b) SNH3, (c) salep, and (d) PAA.

at pH 8, some of the carboxylate groups are ionized and the electrostatic repulsions between –COO- groups cause an enhancement in the swelling capacity. Furthermore, in higher acidic or basic solutions (pH=2, 4 and 10), ionic strength of the medium is increased and the repulsions between aforementioned groups shielded by the counter ions from solution and results in lower absorbency. In the remainder of this manuscript, the SNH3 sample was chosen as the optimum sample and used for further investigations. Moreover, the results indicate that the presence of nano-Ag in the hydrogel networks caused less swelling compared to SNH0 [32]. It is probably due to the chelation of some hydroxyl and carboxylate groups of the hydrogel networks with nano-Ag which neutralize the repulsions in the networks. In continue, the reversible swelling–deswelling behavior of SNH3 in solutions with pH 2 and 8 was examined (Fig. 4). At pH 8, the SNH3 swells due to anion-anion repulsive electrostatic forces, while at pH 2, it shrinks within a few minutes due to protonation of the carboxylate anions. Because of the pH change occurs at many specific or pathological body sites, this swelling– deswelling behavior of the SNH3 makes it as a suitable candidate for drug delivery systems.

OH and COOH functional groups in the polymer networks. The SNH3 (Fig. 5b) has shown all the above characteristic peaks with a slight shift to higher wavelengths (1645 cm-1 and 1594 cm-1 relating to C=O stretching vibrations of PAA and glucomannan repeating units, respectively). This shifting can be attributed to the formation of coordination bond between the nano-Ag and the electron rich groups (such as C=O and OH) present in the hydrogel network. 3.4. TG analysis The TG analysis was applied to confirm the presence of nano-Ag in SNH3 structure. Fig. 6 shows the percentage decomposition of SNH0 and SNH3. The hydrogel has followed two main decomposition steps

110 100

a

90 80

10 0

pH 2 200

100

200

300

400

500

600

700

Temperature (0C) 160

250

SNH0

20

pH 8

300

salep

30

b

400 300

140

TG % / DTA (uV)

Swelling (g/g)

50 40

400 350

SNH3

60

200

DTG

120

100 100

0

TG

80

SNH0

-100

60

SNH3

-200 -300

40

150 20

DTG

To provide some information about the existence of Ag in SNH3 structure and the presence of desired functional groups from salep and PAA in final hydrogels, the FT-IR spectra of SNH0, SNH3, salep, and PAA are shown in Fig. 5. The SNH0 (Fig. 5a) has shown absorption peaks at 1630 cm-1 and 1575 cm-1 relating to C=O stretching vibrations of PAA (Fig. 5d) and glucomannan repeating units (Fig. 5c) from salep. The peak observed at 3300 cm-1 is due to the stretching vibrations of

TG %

70

3.3. FT-IR spectra

-400

DTA

-500

100

0 0

30 60 90 120 150 180 210 240 270 300 330 360 390 420 450

Time (min) Fig. 4. pH-reversibility of SNH3 with 60 min time intervals between the pH changes at 25 °C.

100

200

300

400

500

600

-600 700

0

Temperature ( C) Fig. 6. (a) TG analysis of salep, SNH0 and SNH3, and (b) TGA/DTG/DTA curves of SNH0 and SNH3.

G.R. Bardajee et al. / Journal of Inorganic Biochemistry 117 (2012) 367–373

a

371

b

Fig. 7. SEM images of (a) SNH0, and (b) SNH3.

confirmed that the nano-Ag formed in the cross-linked networks are spherical and highly dispersed within 5–10 nm in size.

and near 80% of the SNH0 has decomposed at 500 °C. However, two degradation steps with 95% weight loss at 560 °C are occurred in the case of SNH3.The difference in decomposition between the SNH0 and SNH3 is found about 11%.The presence of nano-Ag in the hydrogel networks can catalyze CO2 elimination from polymer chains and accelerate the degradation process. For the better study of the thermal behaviors of SNH0 and SNH3 samples, derivative thermogravimetric (DTG) and differential thermal analysis (DTA) curves of the hydrogel are provided in Fig. 6b. The DTG curve shows that the maximum decomposition rate of the SNH0 occurs in the broad peak at 301.5 °C, while the maximum decomposition rate of the SNH3 occurs via a sharp peak at 504.8 °C. According to DTA, at aforementioned temperatures for these two hydrogels, endothermic reactions cause their decompositions. Other minor decomposition points of the hydrogels are also endothermic decompositions.

3.6. TH loading To determine the potential application of SNH3 in drug delivery, at first we have investigated the TH loading behavior of SNH3 at 25 °C. The effect of the initial concentration of TH solution on the adsorption capacities of SNH3 is shown in Fig. 9. As can be seen from the Fig. 9, the loading efficiency increased with growing drug concentration up to 0.1 M and after that it decreased. These results might be due to the higher diffusion of drug from solution to biopolymer nanocomposite in higher concentrations of drug (for concentrations below 0.1 M). The lower loading in 0.15 and 0.2 M concentrations can be attributed to the salt effect of TH. This salt effect leads to the lower swelling of the SNH3 and consequently makes lower loading of TH drug. The amount of the loaded drug in SNH3 was significantly affected by the impregnation times (Fig. 10). It is obvious that with increasing the loading time, the amount of drug loading is initially increased and then begins to level-off. The initial increment in the amounts of the drug loading with time increasing can be ascribed to the increasing drug diffusion into the swollen matrix. The most efficient time of TH loading is around 160 min, where a major amount of TH was encapsulated.

3.5. SEM and TEM images SEM images of SNH0 and SNH3 are shown in Fig. 7. The images of SNH0 and SNH3 show a porous structure which is the characteristic of hydrogel networks. The more clear images from SEM to prove the presence of nano-Ag in the polymer matrix was failed. TEM image (Fig. 8) was utilized for more precise study of the presence of nano-Ag in the SNH3 structures. The TEM image has shown a highly uniform distribution of nano-Ag in the biopolymer matrix. It is

b 10

8

Number of particles

a

6

4

2

0

4

5

6

7

8

9

Particle diameter(nm)

Fig. 8. (a) TEM image and (b) the particle size histogram of SNH3.

10

11

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Amount of TH released ( %)

Amount of TH loadeed ( %)

100

80

60

SNH3

40

SNH0

20 0.00

0.05

0.10

0.15

0.20

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SNH3 SNH0

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60

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20

0

0.25

a

0

50

100

150

Concentration of TH ( mol/L)

200

250

300

350

400

450

500

Time (min) 50

Fig. 9. Concentration dependency of TH loading into SNH3 and SNH0 after 100 min at 25 °C.

The release of water-soluble drugs from hydrogels occurs only after water penetrates into the polymer networks to dissolve the drug, followed by drug diffusion out to the surface of the device through the aqueous pathways. Fig. 11 shows the time dependency of the cumulative amount of TH release profile of the drug loaded into SNH3 at various pHs in simulated conditions (buffered solutions and 37 °C). From the percent release profile of the hydrogel, it can be suggested that the amount of TH release in pH 8 is higher than trivial release of pH 2 for both hydrogels. The higher amount of TH release in pH 8 can be correlated with the higher swelling behavior of the SNH3 (Fig. 3) at this pH. The results show that the 99% of TH is gradually released from SNH3 after 375 min in simulated colon conditions. Furthermore, the negligible TH release in strong acidic media (Fig. 6b) can protect the drug from destruction as well as absorption in the upper gastrointestinal tract media and support its successful release in the colon.

Amount of TH released ( %)

3.7. TH releasing

b 40

30

20

SNH3

10

SNH0 0

0

50

100

150

200

250

300

350

400

450

500

Time (min) Fig. 11. Simulated comparative release of TH from SNH3 and SNH0 as a function of time in (a) pH 8 and (b) pH 2 at 37 °C.

3.8. Antimicrobial Activity The in vitro antibacterial properties of SNH3 and SNH0 were evaluated comparatively against gram-negative E. coli and gram-positive S. aureus bacteria by disk diffusion technique and the antibacterial effectiveness of our hydrogel is compared with ampiciline as a well- established drug and AgNO3, as a starting reagent (Fig. 12). Fig. 12 exhibits the typical antibacterial test results of SNH3 and SNH0 by the disk diffusion method which antibacterial activity is measured by the diameter of the inhibition zone under and around the tested samples. In this test, firstly, the tested bacteria were cultivated on the surface of LB nutrient agar plates, then ampicilin, AgNO3, nanocomposite SNH3 and SNH0 samples were placed

Amount of TH loadeed ( %)

100

SNH3 SNH0

80

60

40

20 0

20

40

60

80

100

120

140

160

180

200

Time ( min) Fig. 10. Time dependency of the drug loading amount into SNH3 and SNH0 at 25 °C (source drug concentration = 0.1 M).

Fig. 12. Comparative antibacterial activity of SNH0 and SNH3 against (a) gram-negative E. coli and (b) gram-positive S. aureus by disk diffusion method (In both plates, number 1 indicates ampicilin as a positive control, 2 is AgNO3, 3 is SNH0 and 4 is SNH3; and the concentrations were kept 60 μg/ml for tested solutions).

G.R. Bardajee et al. / Journal of Inorganic Biochemistry 117 (2012) 367–373 Table 1 MIC and MBC of SNH3 against two different bacteria. Sample

E. coli bacteria

S. aureus bacteria

MIC (μg/ml) MBC (μg/ml)

6 7.5

8 10

on the surface. After 24 h in 37 °C incubation, no S. aureus and E. coli bacteria colonies could grow around SNH3, ampiciline and AgNO3 samples on the LB agar plates, whereas SNH0, as a control, showed no inhibition ability. Our results show that the antibacterial activity of SNH3 is less when compared with ampicilin. On the other hand the antibacterial activity of SNH3 and AgNO3 are similar. It is important to note that antibacterial function of elemental silver has been believed to be either as a contact-active material or release system of silver cations. In the present study, the SNH3 seems to inhibit the bacteria only through contact-active way. The diffusing ability of the silver ions on agar plate might have been limited by the formation of secondary silver compounds and entrapment the Ag+ ions into the hydrogel. Therefore, LB medium method was used to determine the antimicrobial activity of SNH3 quantitatively. In this test, bacterial growth was evaluated by visually inspecting turbidity of the LB broth. The LB broth without bacterial growth is clear and after growing the bacteria, the color becomes turbid. The SNH3 and SNH0 samples were diluted in different concentrations (2.0, 5.0, 8.0, 10.0, 12.0, 15.0 and 20.0 μg/mL) and added to 6 mL of LB medium with 105– 106 cfu/ml of tested bacterial concentrations and were incubated at 37 °C for 24 h (AgNo3 and ampiciline were used as control). After this time the turbidity of cultures were monitored. The transparency of LB broth is related to inhibition of bacterial growth (bacteriostatic) or killing bacterial completely (bactericidal). To determine whether the composites were bacteriostatic or bactericidal, 100μL aliquots from the incubated LB broth were taken and spread on nutrient agar plates. After 24 h incubating the plates at 37 °C, colonies were counted. No bacterial colonies would be observed if the tested material concentration is bactericidal. The results are shown in Table 1. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) are the lowest concentrations (μg/mL) at which a compound can inhibit bacterium growth or kill more than 99% of the added bacteria respectively. It is observed that the effective antibacterial concentration of SNH3 is less (10 μg/ml) in comparison with ampicilin (50 μg/ml). However the bactericidal concentration of AgNO3 and SNH3 are nearly similar. 4. Conclusions In summary, we have developed a highly facile and inexpensive approach to prepare novel SNH based on PAA grafted onto salep as a backbone. It is predicted that this green synthesis procedure can be easily extended to other similar systems, which is valuable for the utilization of other bioresource materials. The TEM image showed that the inorganic nano-Ag with average particle size of 5–10 nm was almost dispersed uniformly in the biopolymer based hydrogel. The SNHs showed excellent bacterial inhibition growth capabilities and they were successfully examined for the release of TH in simulated colon environment. List of abbreviations AA Acrylic acid Ag + Silver cations AgNO3 Silver nitrate APS Ammonium persulfate DDW Double distilled water DTA Differential thermal analysis DTG Derivative thermogravimetric E. coli Escherichia coli FT-IR Fourier transform infrared

LB MBA MBC MIC nano-Ag PAA SEM TEM TH TG SNH S. aureus cfu

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Luria Bertani Methylenebisacrylamide Minimum bactericidal concentration Minimum inhibitory concentration Silver nanoparticles Poly(acrylic acid) Scanning electron microscopy Transmission electron microscopy Tetracycline hydrochloride Thermo-gravimetric Silver nanocomposite hydrogel Staphylococcus aureus colony forming units

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