The present contribution deals with size controlled synthesis of silver nanoparticles through green route by using sulfated polysaccharide isolated from marine red algae by controlling the pH. These nanoparticles were well characterized by UV/Visible spectroscopy, HRTEM, X-ray diffraction and zeta potential measurement. The obtained silver nanoparticles showed surface plasmon resonance (SPR) centered at 404 nm with average particle size measured to be 15±3 nm. The dose dependent effect of silver nanoparticles was more pronounced against gram-negative bacteria than gram-positive bacteria. Rheological behavior of silver nanoparticles containing gel formulation was studied for topical application of these nanoparticles. Further, we have carried out in-vivo wound healing activity of silver nanoparticle gel formulation and compared with control group. In conclusion, the synthesized silver nanoparticles were revealed not only strong antibacterial activity but also in-vivo wound healing activity.
Key words - sulfated polysaccharide, marine red algae, silver nanoparticles, gel, rheology, antibacterial activity and wound healing activity.
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Now a day, human are contaminated by several microorganisms such as bacteria and virus . Thus, ammonium salts, metal salts and antibiotics like numerous antibacterial agents have been used to control these infections, but these are toxic and weakly effectual in nature . Therefore, research has been concentrated to find out antibacterial material containing different inorganic substances from various natural sources. Among them, silver or silver ions are used as a strong antibacterial and antifungal agent when used in reasonable amounts showed positive effects on the human body against many pathogens like bacteria, viruses and fungi etc . But, it is expected that the high surface area and large portion of surface atoms of silver nanoparticles (AgNps) will escort to show high antibacterial activity compared to bulk silver compound . Consequently there is a continuous need to build up eco-friendly processes for the synthesis of such silver nanoparticles.
From past few years, several researches have been made sincere attempts to develop various techniques for synthesis of AgNps including chemical reduction [5, 6], electrochemical reduction , photochemical reduction  and heat evaporation . In most of these techniques, aggregation of nanoparticles was prevented by using surface stabilizers such as thiophenol , thiourea , marcapto acetate  are toxic in nature to pollute the environment during large scale production of nanoparticles. Thus, the purpose for such synthesis has shifted from physical and chemical approaches to 'green' chemistry and bioprocess approaches . Natural sources like green tea (Camellia sinensis) , neem (Azadirachta indica) leaf broth , natural rubber , starch , aloe vera plant extract , lemongrass leaves extract [18, 19] leguminous shrub (Sesbania drummondii)  were used as a reducing and stabilizing agent for green synthesis of AgNps.
Recently, Wei et al. reported that biodegradable chitosan polysaccharide reduced AgNps showed strong antibacterial activity as compared to silver salt . Kathiresan et al. reported the marine fungus synthesized AgNps . Dadosh et al. reported size controlled synthesis of AgNps using tannic acid . Kora et al. reported the superior bactericidal activity of SDS capped AuNps due to easy insertion into lipid bi-layer of gram negative bacterial cell wall .
For our interest, we have used sulfated polysaccharide (SP) for size controlled synthesis of AgNps, SP is an important food source in many parts of the world. This polysaccharide comprises the hot-water soluble portion of cell wall, is the main component of SP. SP is mainly related to agarose, which contains disaccharide units consisting of 3-linked-d-galactosyl residues alternating with 4-linked 3, 6- anhydro-l-galactose, but differs in that is the residue of the 6-sulfate. From last few years, many studies have been made on the pharmaceutical effects of SP such as antioxidant  and anticancer activity .
In the present study, we have carrier out novel size controlled synthesis of AgNps using isolated SP from marine red algal collected from western costal area of Maharashtra state (Harihareshwar), India by adjusting pH and temperature conditions. The synthesized AgNps were well characterized by UV/ Visible spectroscopy, fourier transform infrared spectroscopy, high resolution transmission electron microscopy, X-ray diffractometer and zeta potential measurement. The effect of pH and electrolyte on AgNps was also studied. Six month stability study of synthesized AgNps was carried out at ambient temperature. The minimum bacterial count of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria were carried out at various concentrations of AgNps. Further, minimum inhibitory concentration of AgNps was found against E. coli and S. aureus bacteria. In-vivo wound healing activity of 1 % carbopol gel embedded 0.1 mg g-1 AgNps was carried out on Wister rat and compared with control group.
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Silver nitrate (AgNO3) was purchased from Sigma Aldrich Ltd. India. Two bacterial strains and one fungal strain, namely Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were subjected to this analysis respectively. The components of the Luria-Bertani (LB) medium and different antibiotics used in the study were supplied by Hi-Media Laboratories, India. Analytical grade reagents used from Merck India Ltd., India. Deionized water was used for the synthesis of AgNps.
Isolation of sulfated polysaccharide from marine red algae
SP was isolated as per the previously reported method [28-29]. Briefly, the dried marine red algae were soaked in 7.5 % w/v formalin for 12 h at ambient temperature. An equal volume of water was added and solution refluxed on boiling water for 8 h. The mixture was centrifuged at 10,000 rpm for 20 minutes and supernatant was filtered through diatomite. The resulting filtrate was adjusted to pH 7 with 1M sodium hydroxide solution and evaporated 75% volume at 65 0C on water bath. Four fold volume of methanol was added to residual solution to precipitated polysaccharide content. The mixture was centrifuged at 10,000 rpm for 20 min and supernatant was discarded. The precipitated polysaccharide was washed with 80% aqueous methanol. The residue was freeze dried to yield white powdered polysaccharide.
Synthesis of silver nanoparticles
Size controlled synthesis of silver nanoparticles was carried out by using isolated SP. In brief, an aqueous solution of AgNO3 (1X10-3 M, 100 µL) was reduced to AgNps by heating in 100 mL aqueous solution of polysaccharide (0.01% w/v). After addition of AgNO3 the pH of the solution was adjusted with sodium hydroxide to 11, which yielded yellow colored AgNps on boiling. The AgNps dispersion was thoroughly dialyzed (dialysis tubing 12 kDa cut off) for 24 h to remove ionic impurities used during the reduction. After dialysis, the pH of the AuNps solution was measured to be 7.
Preparation of silver nanoparticles containing gel (AgNps-Gel)
In order to use AgNps in the form of a topical hydrophilic formulation, the 1% w/v carbopol based gel formulations. Where, final concentration of silver was 0.1 mg g-1. This AgNps gel formulation was packaged under sterile conditions, labeled with appropriate details and stored at room temperature for further use.
Characterization of silver nanoparticles
2.4.1. UV/Visible spectroscopy
The UV/Visible spectrum of isolated sulfated polysaccharide fraction (0.1% W/V) was monitored by using Jasco dual beam UV/Visible spectrophotometer (Model V-570, Japan). The change in surface plasmon resonance (SPR) of size controlled AgNps was monitored by UV/Visible spectroscopy measurements.
2.4.2. Fourier transform infrared spectroscopy (FTIR) measurement
FTIR spectra of native polysaccharide was recorded in KBr pellets using FTIR spectrophotometer (Jasco, Japan). The scan was performed in the range 400 to 4000 cm-1.
2.4.3. High resolution transmission electron spectroscopy (HRTEM)
Samples for HRTEM analysis were prepared by drop casting of AgNps dispersion on carbon coated copper grids and allowed to dry at room temperature. Measurements were done on a TECHNAI G2 F30 S-TWIN instrument operated at an accelerated voltage of 300 kV with a lattice resolution of 0.14 nm and point image resolution of 0.20 nm. The particle size analysis was carried out using Gattan software (Pleasanton, CA, USA).
2.4.4. X-ray diffractometer (XRD) measurement
Films were prepared on glass substrates by simple solvent evaporation of AgNps at room temperature. The diffraction measurements were carried out on X'pert Pro X-ray diffractometer (Netherlands) instrument operating at 40 kV and a current of 30 mA at a scan rate of 0.388/min.
2.4.5. Zeta potential (ZP) measurement
The surface charge of AgNps was determined by using a zeta potential analyzer (Brookhaven Instruments Corporation, NY). The average zeta potential of nanoparticulate dispersion was determined as such without any dilution.
2.4.6. pH and electrolytic stability study
The pH of dialyzed AgNps was measured using calibrated pH meter (Delux pH meter 101, India). In the pH stability study, the pH of dialyzed AgNps dispersion was adjusted using 0.1N hydrochloric acid (pH 2, 3, 4, 5 and 6) and 0.1M sodium hydroxide (pH 7, 8, 9 and 10). Change in SPR of AgNps dispersion was recorded after 24 h using a UV/Visible spectrophotometer. Also, the effect of electrolyte was studied by adding varying molar concentration of sodium chloride (101 to 10-6 M) to AuNps dispersion. The change in SPR was recorded after 24 h using UV/Visible spectrophotometer.
2.4.7. Stability study
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The stability study of dialysized AgNps was carried out at ambient temperature. The SRP was recorded at the end of six month using UV/Visible spectroscopy.
2.4.8. Antibacterial activity
The effect of AgNps on gram-negative and gram-positive bacteria was investigated by minimum bactericidal concentration (MBC) method. Where the organisms were cultured in LB agar plates (106 colony forming units (CFU) of each strain per plate) supplemented with nanoparticles at concentrations of 5, 10 and 15 μg ml−1. Plates without AgNps were used as controls. Plates were incubated for 24 h at 370C and the number of colonies was counted. The counts on three plates corresponding to a particular sample were averaged.
In another study, minimum inhibitory concentration (MIC) was carried on two standard bacterial strains (E. coli, S. aureus). The AgNps (5, 10 and 15 μg mL−1) were added to 10 mL of Luria broth (LB) medium with tested bacterial concentrations of 106 CFU mL-1 (CFU = colony forming units) in separate test tubes. Positive control tubes contained 10 mL of Luria broth medium with tested bacterial concentrations of 106 CFUmL-1. Negative control tubes contained only inoculated broth. The tubes were incubated at 37 0C up to 24 h in a constant temperature incubator. The MIC was read by the visual turbidity of bacterial growth and determined by measuring optical density at 600 nm at regular time interval (0, 6, 12, 18 and 24 h).
2.4.9. Rheological analysis of silver nanoparticle containing gel
Rheological analysis of AgNps gel and blank gel were performed using a stress control rheometer, Viscotech Rheometer (Rheologica Instruments AB, Lund, Sweden), equipped with Stress Rheologic Basic Software, version 5, using cone-plate geometry with the diameter of the cone being 25 mm and a cone angle of 1°, operating in the oscillation and static mode. The gap was maintained at 0.5 mm. Rheological analysis of blank gel was performed at 25°C. All experiments were performed in triplicate. In this study, dynamic oscillation stress sweep test was performed to determine the linear viscoelastic region and the viscoelastic properties of the blank and AgNps gel. The linear viscoelastic region gives information about the critical stress beyond which the sample may show significant structural changes, and therefore the consequent choices of the stress value to be used in other oscillation tests (frequency sweep and creep recovery tests). Dynamic oscillation frequency sweep test was used to determine the capability of AgNps gel to resist structural changes under the increased frequency. The creep recovery test was used to determine the viscoelastic properties of the AgNps gel samples. The samples were exposed to the selected averaged stress of the stress sweep mode for 100 s. It was followed by relaxation period for 200 s for recovery. The creep compliance Jc (defined as the ratio of measured strain to the applied stress) is monitored against time.
In-vivo wound healing activity of silver nanoparticle containing gel formulation
In-vivo wound healing activity was performed at Poona College of Pharmacy, Pune, India. Animal handling was performed according to Good Laboratory Practice. The study protocol was approved by IAEC (Institutional Animal Care and Ethics Committee) constituted as per guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), India. Twelve to thirteen weeks old Wistar male rats weighing 180-200 g each were provided by Lupin Research Park, Pune, India. The animals were housed under standard conditions of temperature (250C), in 12/12 h light and dark cycles and fed with standard pellet diet and water ad libitum. The animals were divided in groups containing six animals each. The rats were anesthetized by administering ketamine (0.5 ml/kg b. w. i.p.). A full thickness of the excision wound of circular area (approx. 500 mm2) and 2 mm depth was made on the shaved back of the rats 30 min later the administration of ketamine injection. The wounding day was considered as day 0. The wounds were treated with topical application of the AgNps gel formulation (0.1 mg mL-1) till the wounds were completely healed . The wounds were monitored and the area of wound was measured on 0, 4, 8 and12 post-wounding days and the mean % wound closure was measured by following formula as
Where n =number of days 4th, 8th and 12th day.
Result and discussion
In our study, sulfated polysaccharide was isolated successfully from marine red algae. Further, absence of protein and nucleic acid in the isolated polysaccharide was confirmed by UV/Visible spectra of SP showed no peak was found at 260 to 280 nm (data not shown). The FTIR spectra revealed the presence of sulfate group and 3, 6-anhydrogalactose unites in the polysaccharide (data not shown) and such finding was compared with previous report .
This isolated SP was used for synthesis of AgNps (Scheme 1). Figure 1 depicted that the UV/Visible spectra of 0.01% w/v SP reduced AgNps and band corresponds to SPR centered at 404 nm and the reduction of AgNO3 occurred on boiling as indicated by the formation of yellow color (inset Figure 1). It can be attributed to a narrow size distribution of the AgNps formed in the solution at 1 X 10-3 M concentration of AgNO3. Several reports justified that the absorption peaks in the range of 380-500 nm (UV/Vis spectra) can be assigned to AgNps because of the SPR of AgNps .
Scheme 1. Schematic diagram represented the sulfated polysaccharide reduced silver nanoparticles.
Figure 1. UV/Visible spectra of sulfated polysaccharide reduced AgNps and inset photograph represented the AgNps dispersion.
HRTEM image represented the morphology of synthesized AgNps revealed that AgNps was found to be spherical in shape (Figure 2 (A)) with narrow size distribution nature, having an average particle size measured to be 15±3 nm (Figure 2 (B)). Figure 3 depicted the XRD pattern of AgNps shows a number of Bragg reflections with 2θ values of 380, 460, 630 and 770 correspond to the (111), (200), (220) and (311) sets of lattice planes was observed which may be indexed as the band for face centered cubic (fcc) structures of silver in agreement with the literature value of fcc crystal structure of silver [33, 34]. The XRD pattern thus clearly illustrates that the synthesized AgNps was crystalline in nature.
Figure 2. (A) HRTEM image of AgNps and (B) graph of particle size distribution.
Figure 3. XRD pattern of AgNps.
Zeta potential measurement is an important parameter to find out the surface change on nanoparticles justified the electrostatic stability of dispersed nanoparticles. Previous report suggested that the particle aggregation is less likely to occur for charged particles with optimum zeta potential (~ ±30 mV) due to electrostatic repulsions . Thus, surface change of AgNps was found to be - 35.05 mV. This clearly revealed that the obtained AgNps were properly coat with the SP, helps nanoparticles to retain stability by electrostatic means.
The therapeutic applicability of these AgNps required much more stability under different pH condition and electrolytic concentrations. Therefore we have carried out pH and electrolytic stability study for synthesized AgNps. In case of pH study, Figure 4 (A) depicted the insignificant change in peak intensity and SPR shift was observed in pH range of 2 to 10 after 24 h incubation. Also, UV/visible spectra revealed that the aggregation of AgNps was not observed after addition of electrolyte (NaCl) up to 1 X 10-1 M (Figure 4 (B)). The slight red shift in SRP is due to the surface neutralization and adsorption of chloride ion on the surface of nanoparticles such findings was also observed for chitosan reduced AgNps . These results are meeting the stipulations laid out for the utility of these particles for several applications.
In stability study, AgNps were found to be stable for over six month was confirmed by monitoring the SRP on UV/Visible spectroscopy (Figure 4 (C)) indicating the synthesized AgNps is highly stable at ambient temperature condition. Inset photograph of Figure 4 (C) revealed that no change in color of AgNps solution after six month stability. Thus the stability could be attributed to the SP being wrapped around AgNps which helped in maintaining the shape and size of the nanoparticles during stability period without any aggregation.
Figure 4. UV visible spectra of AgNps (A) at different pH, (B) at different concentration of electrolyte (NaCl) and (C) stability sample up to six month stability.
The antibacterial effect of silver ions on micro-organisms is very well known, silver ions have been demonstrated to be useful and effective in antibacterial applications, but due to the unique properties of nanoparticles, nanotechnology presents a reasonable alternative for development of new antibacterial with wide applications. Thus, we have determined the kinetics of antibacterial activity of SP capped AgNps toward E. coli and S. aureus were investigated by minimum bacterial count (MBC) method. Where, colonies formed after incubation was counted and these corresponded to the number of live bacteria in each suspension at the time of aliquot withdrawal. The negative control (without AgNps) plate of each bacteria strain showed more than 300 colonies up to 8 hour incubation. Different concentrations of AgNps showed more than 300 bacteria colony at 0 hour incubation against E. coli and S. aureus. No bacterial colony of E. coli was observed after 8 h incubation (Figure 5(A)) revealed that lover concentration of AgNps successfully killed the gram negative bacterial strain (E. coli). In case of S. aureus, countable colonies were found after 1 hour incubation but as the concentration of AgNps increased the colonies were decreased up to 8 h incubation (Figure 5 (B)). Obtained results revealed that the gram negative bacteria were killed more effectively than the gram positive bacteria.
Figure 5. Representative photographs of bactericidal activity of porphyran capped AgNps against (A) E. Coli, and (B) S. aureus.
Further, the antibacterial activity was tested by using the MIC is defined as the lowest concentration at which there is no visible growth . Three different concentrations of AgNps (5, 10 and 15 µg/ml) were tested for MIC. In this experiment, the progressive growth inhibition of E. coli was observed at 5 µg mL-1 concentration of AgNps (Figure 6 (A)). The lag phase was found to be more prolonged up to 24 hours than that described in the earlier reports . In contrast, these nanoparticles were less sensitive against growth of S. aureus till the concentration of nanoparticles rose to15 µg mL-1 (Figure 6 (B)). It took about 6 h to instigate the noticeable growth of bacteria. Previously, Shrivastava et al. reported that D-glucose and hydrazine blend reduced AgNps showed strong inhibitory effect against E. coli at 25 µg mL-1 concentration and in contrast no reduction of bacterial growth was observed against gram positive bacteria at 25 µg mL-1. These results indicated that the synthesized AgNps are more effective against E.coli than S. aureus.
Figure 6. Minimum inhibitory concentration of AgNps against (A) E.coli and (B) S. aureus.
The mechanism of bacterial cell growth inhibition on application of silver ions is due to the inactivation of protein and loss of replication ability of DNA . Also, several report suggested that the AgNps improved the antibacterial activity may be due to the electrostatic attraction between positively charged AgNps and negatively charged bacterial cells [37,39,40]. But, as per the zeta potential results AgNps used in this study were negatively charged and it has showed effective antibacterial activity against negatively charged bacterial cell as compared to positively charged bacterial cell. Therefore, mechanism behind this inhibition was unclear. But, the improvement in antibacterial activity against gram-negative bacteria may be due to fast uptake of nanoparticles from cell wall of bacteria which leads to fast inactivation of protein structure, causing structural changes to cell death. Sondi et al. reported that negatively charged ascorbic acid reduced AgNps showed strong antibacterial activity against gram-negative E.coli bacteria . Previous report suggested that S. aureus is having strong defensive property as compared to E. coli because this bacteria is having a thicker peptidoglycan containing cell wall, which is observed in the center of cells where DNA molecules are present. Therefore, this thicker cell wall prevented the cells from uptake of silver ions in the cytoplasm . This may be the main resone that the synthesized AgNps showed less antibacterial effect at 15 µgmL-1 concentration on S. aurous as compared to E.coli.
Silver nitrate and silver sulfadiazine being used for topical applications . Among these, silver sulfadiazine is the most popular topical antimicrobial agent in use due to non availability of other safer alternatives for delivery of silver. An alternative silver delivery system trough an AgNps based formulation will be ideal for various topical applications. In this study, AgNps containing gels were prepared by using carbopol as gelling agent. Carbopol as gelling agent is effective at low concentrations and simply gels on cooling. Here, 1% w/v concentration of carbopol was used for preparation of AgNps containing gels without addition of any ions. Carbopol gels were found to be clear with good transparency. Further, we evaluated the rheology behavior to obtain information about viscous and elastic nature of AgNps containing gel system compared with blank carbopol gel (1% w/v).
Where, oscillatory measurements give the information about viscous and elastic properties of gel was performed. An oscillation frequency sweep test is a dynamic test measuring the response of a system as a function of frequency at constant stress amplitude. It reveals the storage modulus G' (elastic response) which is a measure of energy stored and the loss modulus G'' (viscous response) which reflects the energy lost. If performed within the linear viscoelastic region a frequency sweep provides a fingerprint of a viscoelastic system under non-destructive conditions (Figure 7 (A)). The AgNps containing gel system had shown weak dependency of both G' and G'' on the applied frequency which is a typical result for a viscoelastic solid. The storage modulus is higher than the loss modulus over the whole frequency range, indicating the presence of a gel-like structure. The higher values of the storage modulus show that the investigated system is more elastic than viscous in the investigated frequency range. Thus, AgNps based gel system was found to be more stable and considered for further studies. In the creep test a constant stress within LVR is applied for a fixed time (100S) and then removed (200S). In this, the strain of a sample is determined as a function of time. Thus we can predict that system had more viscous property when compared with blank gel system (Figure 7 (B)). Percentage creep recovery of bank gel and AgNps based gel were 72.35% and 54.31% revealed the embedded AgNps in gel was reduced the percent creep recovery as compared to blank gel system. These rheology results revealed that the prepared gel formulation of AgNps is suitable for topical application.
Figure 7. Represented graph of rheological study where, (A) effect of frequency on G'/G'' of 0.2 mgmL-1 of AgNps gel and (B) creep recovery of 0.2 mgmL-1 of AgNps gel.
Silver in the form of nanoparticles is very effective antimicrobial and is also used in wound dressings [44, 45]. Previously, Wong et al. reported the wound healing properties of AgNps in an animal model and it was rapid healing and better cosmetic appearance in a dose dependent manner . Tian et al. also reported that after application of AgNps on excised wounds healed fastly (16±0.41 days) after injury as compared to control group (18.5±0.65 days) . In this study, the AgNps containing gel (0.1 mg g-1) system was applied on the wound ones a day for 12 days. The results of wound healing activity by excision wound model are presented in Figure 8. Figure 8 (A) represented the percentage wound closer at 4, 8, 12 days of the test groups and compared with control group. It was observed that wound contracting ability of animals treated with AgNps containing gel formulation was found to be significantly higher on days 4 and 8 as compared to the control group. Figure 8 (B) showed images of wounds contraction after treatment of AgNps containing gel formulation and compared with control group. This improvement in wound healing process may be due to cytokines which play an important role in wound healing and it may help to maintained levels of IL-6 mRNA in the wound areas treated with AgNps throughout the healing process .
Figure 8. (A) Graph represented the time verses percent wound closer and (B) photographs of time dependent animal wounds after application of AgNps gel compared with control group.
From this study, it was interesting to note that novel synthesized AgNps have strong antibacterial activity against gram-negative bacteria as compared to gram-positive bacteria and which improved the wound healing process justified suitability for topical application of AgNps.
In summary, the isolated SP contained sulfur group and 3,6-anhydrogalactose were confirmed through FTIR results. The controlled synthesis of AgNps by using SP were demonstrated for pH stability, electrolytic stability and six month stability of AgNps revealed the AgNps were properly wrapped with SP and it gave stability to AgNps by electrostatic means justified by zeta potential. These AgNps showed both fast and long-lasting antibacterial effectiveness toward E. coli and S. aureous. Further, AgNps prepared in our present system used for a wide variety of therapeutic applications by preparing the gel formulation containing AgNps such as in vivo wound healing activity and it was observed that wound contracting ability of animals treated with AgNps containing gel formulation was found to be significantly higher than the control group.