Escolar Documentos
Profissional Documentos
Cultura Documentos
www.elsevier.com/locate/jcis
Abstract
In this study, hydrogel–silver nanocomposites have been synthesized by a unique methodology, which involves formation of silver nanoparti-
cles within swollen poly (acrylamide-co-acrylic acid) hydrogels. The formation of silver nanoparticles was confirmed by transmission electron
microscopy (TEM) and surface plasmon resonance (SPR) which was obtained at 406 nm. The TEM of hydrogel–silver nanocomposites showed al-
most uniform distribution of nanoparticles throughout the gel networks. Most of the particles, as revealed from the particle-size distribution curve,
were 24–30 nm in size. The X-ray diffraction pattern also confirmed the face centered cubic (fcc) structure of silver nanoparticles. The nanocom-
posites demonstrated excellent antibacterial effects on Escherichia coli (E. coli). The antibacterial activity depended on size of the nanocomposites,
amount of silver nanoparticles, and amount of monomer acid present within the hydrogel–silver nanocomposites. It was also found that immersion
of plain hydrogel in 20 mg/30 ml AgNO3 solution yielded nanocomparticle–hydrogel composites with optimum bactericidal activity.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Hydrogel; Silver nanoparticles; Surface plasmon resonance; Nanocomposites; E. coli; Transmission electron microscopy; Silver nitrate;
Antibacterial activity; Scanning electron microscopy; Colony-forming unit
have opened a new skylight for different applications in bio- The antibacterial property of silver has been studied by a
medical engineering and these approaches are most effective number of microbiologists. For example, Deitch et al. [28] eval-
and safe because they are compatible with most of biolog- uated the antibacterial activity of silver nylon fabrics on the
ical molecules, cells, tissues, etc. Current studies have ex- growth of S. aureus and C. albicans to develop these fabrics
ploited in situ synthesis of metal nanoparticles within the as wound dressings. In another study, Sondi and Salopek-Sondi
swollen hydrogel networks [17] and the resulting products are [7] evaluated the antimicrobial activity of silver nanoparticles
leading to new hybrid or composite systems that have ver- against E. coli. The results confirmed that the treated E. coli
satile applications in bioengineering and biomedical fields. cells were damaged, showing pit formation on bacterial cell
The most advanced feature of this novel approach is that walls. Recently, Jain and co-workers [10] tested the bacter-
we can alter the size and morphology of the nanoparticles ial action of silver nanoparticle-coated polyurethane foam and
by changing the monomer and crosslinker concentrations in suggested its use as an antibacterial water filter. In another
the gel formulations. Well-defined gold nanoparticles embed- work, Hu et al. [29] treated cotton fabrics with a suspension
ded inside thermosensitive hydrogel matrices were reported of silver oxide in chitosan and studied its antibacterial action
by Wang et al. [18,19]. In their work, the templates of gel against S. aureus. Attempts have also been made to coat urinary
networks were prepared using a combination of two crosslink- catheters with silver compounds to prevent infection [30]. Apart
ers, namely N,N -methylenebisacrylamide (MBA) and N ,N - from silver, polycationic groups like alkylammonium have also
cystaminebisacrylamide (CBA). CBA is highly responsible in been employed to provide antimicrobial surfaces [31].
controlling the morphology of the nanoparticles by control- The real significance of this work is that if the surface
ling the crosslinking density of the networks. In another study, of medical prostheses is grafted with polymeric chains, then
Zhang et al. [20] produced polymeric microgels which can act this proposed novel approach may be used to incorporate Ag
as versatile nanoreactors for semiconductor, metal, and mag- nanoparticles into the grafted polymer network, followed by
netic nanoparticles. Recently, microgel formulations based on citrate reduction, to yield devices with Ag-impregnated antimi-
N -isopropylacrylamide with nanoparticles have been effec- crobial surfaces.
tively employed for photonic, electronic, and electroanalytical
application purposes [21–23]. Hydrogels have also been em- 2. Experimental
ployed for magnetic nanoparticle entrapment and the result-
ing ferronanohydrogel composite properties have been stud- 2.1. Materials
ied [24]. Gels constructed with methyl methacrylate (MMA)
and ethylenedimethacrylate (EDMA) have been utilized as tem- The two monomers used in this study, namely acrylamide
plates to control the size and morphology of Pd nanoparticles. (AAm) and acrylic acid (AAc), and the crosslinking agent
The developed microgel–metal nanoclusters are appropriate N ,N -methylenebisacrylamide were obtained from HiMedia
for catalysis applications [25]. More recently, silver nanopar- Laboratories, Mumbai, India. The salts silver nitrate (AgNO3 ),
ticles of ∼35 nm are embedded in a novel hydrogel system trisodium citrate (SC), and potassium persulfate (KPS) were
based on poly(vinyl alcohol)/poly(styrene)-co-poly(ethylene obtained from E. Merck, Mumbai, India, and used as received.
glycol methacrylate) [PVA/PS-PEGMA] [26]. A recent study Standard cultures of the organisms were provided by the De-
demonstrates that hydrogels are capable of producing nanosil- partment of Biotechnology. Nutrient broth and Nutrient and
ver particles about ∼4 nm in size [27]. In these studies, dif- m-Endo agars were obtained from HiMedia Chemicals, In-
ferent strategies have been followed to prepare hydrogel–silver dia. Double-distilled water was used throughout the investiga-
nanocomposites. These include (1) polymerization of monomer tions.
in the presence of initiator-funtionalized metal nanoparticles;
(2) shrinking of a swollen gel in acetone followed by its 2.2. Synthesis of poly(acrylamide-co-acrylic acid) hydrogels
reswelling in solution of metal nanoparticles and then again its
shrinking in acetone, thus finally resulting in the formation of The hydrogel disks were prepared by carrying out free rad-
nanoparticle-loaded gels; (3) surfactant-free emulsion polymer- ical aqueous copolymerization of AAm and AAc using MBA
ization (SFEP) to encapsulate metal nanoparticles within larger as the crosslinker and KPS as the initiator. In brief, in order to
spherical hydrogel particles; and (4) mixing a colloidal solution prepare a sample (HG2), 14.08 mM AAm, 5.55 mM AAc and
of metal nanoparticles with aqueous polymer solution, followed 0.32 mM crosslinker MBA were dissolved in water and the final
by solvent evaporation. However, the method used in our study volume was made 5 ml. Then, 0.11 mM KPS was dissolved and
is very simple and economic and does not involve the use of the whole reaction mixture was transferred in the test tube (in-
organic solvents. The use of organic solvents and other toxic ternal diameter 1.5 cm) and kept in an electric oven (Tempstar,
reagents, harsh synthesis conditions, limit their use in preparing India) at 60 ◦ C for a period of 2 h. After the polymerization was
hydrogel/silver nanocomposites for biomedical applications. over, the test tube was broken and the resulting almost trans-
Contrary to this, we hereby report a universal approach for parent hydrogels were cut into slices of same thickness. The
synthesizing nanocomposites. In the present work, we have hydrogels were equilibrated in the distilled water for a period
reported a novel approach for synthesizing Ag nanoparticles of 2 days to remove the unreacted monomers, crosslinker, and
containing poly(acrylamide-co-acrylic acid) hydrogels and in- initiator and finally dried in a dust-free vacuum chamber till the
vestigated their antimicrobial activity against E. coli. gels attained constant weight. A blank poly (AAm) hydrogel,
V. Thomas et al. / Journal of Colloid and Interface Science 315 (2007) 389–395 391
2.4. Characterization
Fig. 2. Photographs of (a) hydrogel, (b) hydrogel–silver nitrate salt composite, (c) and hydrogel–silver nanoparticle composite, respectively.
Fig. 4. X-ray diffraction pattern of (A) hydrogel and (B) hydrogel–silver nano-
composite.
Fig. 6. (A) TEM image of HG2 (inset SAED pattern) and (B) silver nanoparti-
cles distribution in the HG2 hydrogel.
particle size from the SEM analysis because the study was done
on dry nanocomposite sample.
Fig. 6A shows the TEM image of the silver nanoparticles
prepared within the hydrogel with 4.16 mM monomer acid in
the feed mixture. The image indicates nearly uniform distri-
bution of silver nanoparticles. In addition, a typical selected
area electron diffraction (SAED) pattern of a collection of sil-
ver nanoparticles is also shown (see inset). The pattern appears
to be a little diffused due to smaller particle size, but three dif-
fraction rings are clearly visible and they can be indexed to the
face-centered cubic structure of silver as follows. The strongest
Fig. 5. (A) SEM image of HG1 hydrogel, (B) silver nanoparticles on the surface ring and the one closest to the center is probably a combina-
of HG1, and (C) silver nanoparticles in the hydrogel networks of HG1 (cross tion of the {111} and {200} reflections. The second ring is
section). Bar indicates 1 µm in all the SEM images. likely the {222} reflection whereas the outermost and the weak-
est third ring are either the {420} and/or the {422} reflections.
The reason for the presence of silver nanoparticles inside the Almost similar results have also been reported elsewhere [33].
gel networks may be attributed to the fact that when silver ions, The size distributions were obtained by measuring the diame-
present on the surface of the swollen gel, are reduced by sodium ter of 35 particles in an arbitrarily chosen area of TEM image
citrate, the silver nanoparticles, so produced, increase the gel (see Fig. 6B). As can be seen, nearly 40% of particles have an
porosity, thus providing a pathway for reducing agents to enter average diameter of 26 nm and moreover, the distribution curve
into the bulk to produce nanoparticles. We could not judge the appears to be more or less symmetrical with all the nanopar-
394 V. Thomas et al. / Journal of Colloid and Interface Science 315 (2007) 389–395
Fig. 7. Photographs showing bacterial colonies in petri plates containing nano- Fig. 8. Photographs of growth of bacterial colonies is the petri dishes containing
composites with (A) 0.00 and (B) 5.55 mM acrylic acid. (A) 200 and (B) 100 µm sized particles.
ticles falling within the narrow range of 12–42 nm. One more a key factor governing the bactericidal property of the resulting
interesting feature is that the nanoparticles do not seem to form nanocomposites.
aggregates. This may be due to excellent stabilization of silver
nanoparticles by carboxylate anions present in the gel macro- 3.5. Effect of particle size on biocidal action
molecular chains.
It is expected that size of the Ag hydrogel particles should in-
3.4. Antibacterial property of Ag–hydrogel nanocomposites fluence their antibacterial action because change in particle size
results in change in surface area which is in contact with the
As noted in earlier sections, the formation of silver nanopar- bacterial species. To investigate this, hydrogel–silver nanocom-
ticles within the swollen hydrogel is mainly due to an ion- posites of 100 and 200 µm sized particles were tested for their
exchange process. So, it is expected that the sample, prepared antibacterial action against E. coli. The results, as depicted in
with a higher content of monomer acid in the feed mixture, Fig. 8, clearly indicate that 100 µm sized particles show greater
should contain more silver nanoparticles, and hence should tendency to inhibit growth of bacterial colonies as compared
demonstrate greater antibacterial properties. In order to inves- to the particles with size 200 µm. There are nearly 200 CFUs
tigate this, we examined the antibactericidal property of two in the control set while this number reduces to 53 and 93 for
samples, prepared with 0.00 and 5.55 mM acrylic acid in the the petri dishes containing Ag–hydrogel particles with diameter
feed mixture, (i.e., samples HG1 and HG2, respectively) under 100 and 200 µm, respectively. Thus, it can be seen that 200 µm
identical conditions. Fig. 7 clearly demonstrates that formation sized hydrogel–Ag composite particles are able to reduce the
of bacterial colonies in the presence of nanocomposite HG2 is bacterial growth up to 50% while 100 µm sized particles re-
almost negligible whereas more population of bacterial colonies duce the colonies growth upto 75% as compared to the control
appears in the presence of sample HG1. This may simply be set. The results may simply be attributed to the fact that 100 µm
attributed to the fact that Ag nanoparticles are present in less sized particles possess greater surface area and hence they are in
number in the sample HG1 and hence the sample HG1 is inca- contact with a greater number of bacteria, thus inhibiting their
pable in retarding the growth of bacterial colonies to a great ex- growth more effectively. Therefore it may be inferred that small
tent while the sample HG2, which contains a sufficient number sized particles are preferable for obtaining greater inhibition of
of silver nanoparticles, shows great potential for reducing the bacterial colony growth.
bacterial growth under similar conditions. The results also sup-
port the proposed mechanism that ion exchange between Ag+ 3.6. Effect of silver content on bacterial action
ions present in the external AgNO3 solutions and H+ ions pro-
duced due to ionization of –COOH groups within the swollen The amount of silver nanoparticles within the hydrogel can
phase is a key factor for the entrapment of Ag+ ions within the be varied by immersing plain hydrogel disks in silver nitrate
polymer network. Here it is worth noting that the presence of solutions of different concentrations followed by citrate reduc-
silver nanoparticles within the sample HG1 (i.e., without con- tion. This will of course affect the bactericidal action of Ag
taining monomer acid) shows that silver ions, to some extent, hydrogel nanocomposites. To investigate this, plain hydrogel
must have entered into the swollen network and later on were samples HG2 were dipped in silver nitrate solutions, varying
reduced by sodium citrate. However, the presence of –COO− in concentration from 10 to 30 mg/30 ml. A definite amount
groups (due to acrylic acid as in sample HG2) becomes a ma- of nanocomposites so obtained was tested for their antibacte-
jor driving force for Ag+ ions to enter into the swollen network rial action against E. coli. The results as depicted in Fig. 9
through Ag+ –H+ ion-exchange process. Hence, it may be con- clearly indicate that as the concentration of AgNO3 solutions
cluded that composition of monomer acid in the feed mixture is increases the antibacterial activity of resulting nanocomposites
V. Thomas et al. / Journal of Colloid and Interface Science 315 (2007) 389–395 395
Acknowledgment
References
[1] J. Dutta, H. Hofmann, in: H.S. Nalwa (Ed.), in: Encyclopedia of Nano-
science and Nanotechnology, vol. 9, American Scientific Publishers, 2004,
p. 617.
[2] Y. Yeo, H.J. Lee, S.H. Jeong, J. Mater. Sci. 38 (2003) 2143.
[3] S.Y. Yeo, S.H. Jeong, Polym. Int. 52 (2003) 1053.
[4] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, J. Biomed.
Mater. Res. 52 (2000) 662.
[5] C.N. Kraft, M. Hansis, S. Arens, J. Biomed. Mater. Res. 49 (2000) 192.
[6] A. Gupta, S. Silver, Nat. Biotechnol. 16 (1998) 888.
[7] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177.
[8] D.G. Maki, P.A. Tambyah, Emerg. Infect. Dis. 7 (2001) 342.
[9] A.B. Lansdown, I. Silver, J. Wound Care 11 (2005) 125.
Fig. 9. Photograph showing growth of bacterial colonies in (A) silver-free con- [10] P. Jain, T. Pradeep, Biotechnol. Bioeng. 90 (2005) 59.
trol plate; and in plates containing Ag–hydrogel particles, prepared by immer- [11] M. Bosetti, A. Masse, E. Tobin, M. Cannas, Biomaterials 23 (2002) 887.
sion in AgNO3 solution of concentration (B) 10, (C) 20, and (D) 30 mg per [12] J.F. Hillyer, R.M. Alrecht, J. Pharm. Soc. 90 (2001) 1927.
30 ml water. [13] J. Zuhuang, patent number CN 1387700, 2003.
[14] M. Chen, S. Chen, patent number CN 1355335, 2002.
also increases. It is clear that for the nanocomposites, prepared [15] J.L. Elechiguerra, J.L. Burt, J.R. Morones, A. Camacho-Brabado, X. Gao,
by immersion in 20 and 30 mg/30 ml solutions of AgNO3 , H.H. Lara, M.J. Yacaman, J. Nanotechnol. 3 (2005) 1.
[16] Y.M. Mohan, K.J. Lee, T. Premkumar, K.E. Geckeler, Polymer 48 (2006)
the growth of bacterial colonies is almost nil, thus suggest- 158.
ing a fair bactericidal activity of their nanocomposites. This [17] Y.M. Mohan, T. Premkumar, K.J. Lee, K.E. Geckeler, Macromol. Rapid
indicates that optimal concentration of AgNO3 solutions to ob- Commun. 27 (2006) 1346.
tain fair antibacterial activity of Ag hydrogel nanocomposites [18] C. Wang, N.T. Flynn, R. Langer, Adv. Mater. 16 (2004) 1074.
is 20 mg/30 ml. It is also evident that the hydrogel–silver [19] C. Wang, N.T. Flynn, R. Langer, Mater. Res. Soc. Symp. Proc. 820 (2004)
R 2.2.1.
nanocomposites, prepared with 10 mg/30 ml AgNO3 solution,
[20] J. Zhang, S. Xu, E. Kumacheva, J. Am. Chem. Soc. 126 (2004) 7908.
are not so effective in inhibiting bacterial growth. [21] S. Xu, J. Zhang, C. Paquet, Y. Lin, E. Kumacheva, Adv. Mater. 13 (2003)
468.
4. Summary [22] X. Zhao, X. Ding, Z. Deng, Z. Zheng, Y. Peng, X. Long, Macromol. Rapid.
Commun. 26 (2005) 1784.
From the results of the above study it may be concluded [23] E.A. Kazimierska, M. Ciszokowska, Electroanalysis 17 (2005) 1384.
[24] C.R. Mayer, V. Cabuil, T. Lalot, R. Thouvenot, Adv. Mater. 12 (2000) 417.
that silver nanoparticles can be produced within the swollen [25] A. Biffis, N. Orlandi, B. Corain, Adv. Mater. 15 (2003) 1551.
polymer network. The developed silver–hydrogel nanocompos- [26] Y. Lu, P. Spyra, Y. Mei, M. Ballauff, A. Pich, Macromol. Chem. Phys. 208
ite demonstrates fair antibacterial activity against E. coli. Their (2007) 254.
bacterial action depends on size of the particles, amount of sil- [27] P. Saravanan, M. Padmanabha Raju, Sarfaraz Alam, Mater. Chem. Phys.
ver nanoparticles within the hydrogel, and amount of monomer (2007), available at: doi:10.1016/j.matchemphys.2007.02.025.
[28] E.A. Deitch, A.A. Marino, V. Malakanok, J.A. Albright, J. Trauma 27
acid in the feed mixture. The formation of Ag nanoparticles
(1987) 301.
with in the gel takes place due to entrapment of Ag+ ions [29] Z. Hu, J. Zhang, W.L. Chan, Y.S. Szeto, Mater. Res. Soc. Symp. Proc. 920
into the swollen hydrogel network via Ag+ –H+ ion-exchange (2006), 0920-S02-03.
mechanism, followed by citrate reduction. The X-ray diffrac- [30] S. Saint, J.G. Elmore, S.D. Sullivan, S.S. Emerson, T.D. Koepsell, Am. J.
tion analysis confirms the fcc structure of Ag nanoparticles. Med. 105 (1998) 236.
[31] N.M. Milovic, J. Wang, K. Lewis, A.M. Klibanov, Biotechnol. Bioeng. 90
In the next part of the study, we propose to graft monomer
(2005) 715.
onto the surface of urinary catheters and then incorporate sil- [32] S.K. Bajpai, S. Johnson, React. Funct. Polym. 62 (2005) 271.
ver nanoparticles to finally study the antibacterial action of the [33] M. Andersson, V. Alfredsson, P. Kjellin, A.E.C. Palmqvist, Nano Lett. 12
catheter surface. (2002) 1403.