RESEARCH PAPER

Stabilizer specific interaction of gold nanoparticleswith a thermosensitive polymer hydrogel

A. Murugadoss Æ Aslam Khan ÆArun Chattopadhyay

Received: 2 February 2009 / Accepted: 29 May 2009 / Published online: 27 June 2009

� Springer Science+Business Media B.V. 2009

Abstract We report the experimental results on

temperature-dependent studies of interactions between

a novel biocompatible thermosensitive polymer hydro-

gel and different stabilizing agent capped gold nano-

particles (Au NPs) with particle size ranging from 5 to

20 nm. Stabilizing agents such as thioglycolic acid,

tryptophan, and phenylalanine have been used as

capping agents for Au NPs. The poly-N-isopropyl

acrylamide-co-acrylic acid (pNIPAm-AAc) with

3.0 ± 0.7 lm in size was synthesized by radical

polymerization of a selected mixture of N-isopropyl

acrylamide (NIPAm), methylene-bis-acrylamide and

acrylic acid (AAc). The capped Au NPs were mixed

with a solution of pNIPAm-AAc hydrogel. The

temperature-dependent properties of the mixture were

studied by UV–vis spectroscopy, dynamic light

scattering based particle size analysis, and transmis-

sion electron microscopy (TEM). The observations

indicated change in the lower critical solution

temperature (LCST) depending on the nature of the

stabilizer, with hydrophobic ones lowering the value

while hydrophilic stabilizers increasing the same.

Also, the optical absorption due to Au NPs, when

stabilized with hydrophobic groups, reduced signifi-

cantly at above LCST along with significant blue shift

of wavelength maximum.

Keywords Gold � Nanoparticles �Hydrogel � Thermosensitive � Spectroscopy �Drug delivery

Introduction

Stimuli responsive materials such as hydrogels are

important as delivery vehicles for futuristic nanoscale

drugs. The changes in size, shape, and solubility in a

solvent in response to changes in stimuli such as

temperature, pH, ionic, or other chemical and biolog-

ical species make hydrogels ideal for drug delivery and

controlled release of bioactive molecules (Juodkazis

et al. 2000; Chen et al. 2007; Langer and Tirrell 2004).

The advent of nanoscale materials in therapeutics and

medical diagnostics has brought about newer chal-

lenges in conjugating hydrogels with nanomaterials for

targeted delivery (Retama et al. 2007; Robinson et al.

2000; Francois et al. 2007). In this regard, nanoparti-

cles (NPs) could be chemically bonded to the three-

dimensional structure of the gel and thus the hybrid gel

Electronic supplementary material The online version ofthis article (doi:10.1007/s11051-009-9668-0) containssupplementary material, which is available to authorized users.

A. Murugadoss � A. Chattopadhyay (&)

Department of Chemistry, Indian Institute of Technology

Guwahati, Guwahati 781039, India

e-mail: arun@iitg.ernet.in

A. Khan � A. Chattopadhyay

Centre for Nanotechnology, Indian Institute

of Technology Guwahati, Guwahati 781039, India

123

J Nanopart Res (2010) 12:1331–1348

DOI 10.1007/s11051-009-9668-0

would act as both the delivery vehicle as well as the

drug itself. On the other hand, chemically or biolog-

ically functionalized NPs could also be encapsulated to

hydrogels that are held by electrostatic or van der

Waals forces and the functionalized NPs could thus be

released in response to a stimulus. Understanding the

functioning of both kinds of hybrids is important for the

development of better drugs and delivery systems.

An important thermosensitive polymer that has

been extensively studied is poly (N-isopropyl acryl-

amide) (pNIPAm), the lower critical solution temper-

ature (LCST) of which can be changed from 32 �C up

to 60 �C by various degrees of copolymerization with

acrylic acid (AAc) (Das et al. 2007; Jones and Lyon

2003; Berndt et al. 2005; Nayak and Lyon 2005). In

addition to tunable LCST and large volume change

accompanying transition, AAc copolymerized in the

backbone of pNIPAm offers a robust architecture

which responds very well to environmental changes in

temperature, pH, ionic strengths, and solvents (Berndt

et al. 2005). These advantages have consequently

contributed to the significant scientific and techno-

logical literature related to pNIPAm-AAc copolymers

as environmentally sensitive delivery system (Heras

Alarcon et al. 2005; Gan and Lyon 2001).

On the other hand, recent developments in the

controlled syntheses of inorganic NPs such as Au

NPs and quantum dots (Qdots) bring additional

potential for their use as drugs such as in hyperther-

mia treatment or in imaging at the cellular level (Jain

and Stroh 2004; Storh et al. 2005). However, these

materials also need appropriate delivery vehicle in

order for their controlled release and targeted deliv-

ery. In this respect, pNIPAm has been proposed as the

stimuli responsive carrier of the NPs (Lu et al. 2006).

There have been some recent reports on the temper-

ature-dependent study of Au functionalized pNIPAm

using the surface plasmon resonance (SPR) of Au

NPs as the probe (Shan and Tenhu 2007; Yusa et al.

2007; Karg et al. 2007). However, these studies were

limited to chemical attachment of the NPs on the

polymer by appropriately modifying the backbone of

the polymer. On the other hand, there are no reports

of temperature-dependent interactions between func-

tionalized Au NPs and pNIPAm polymer or its

derivative. This is important as understanding the

interaction between the functionalized NPs and the

carrier polymer, and its temperature sensitivity may

play pivotal roles in futuristic drug delivery and

chemical sensors (Kim et al. 2006). Also, if a simple

technique such as UV–vis spectroscopy could indi-

cate the interaction between stabilized NPs and the

hydrogel that would broaden the scope of such

studies in future. This is all more important against

the backdrop of substantial literature reports on

interactions of organic molecules and ions with

hydrogels and accompanying volume phase transi-

tions (Zhang et al. 2002; Matsukaa and Ando 1996).

Herein, we report the study of temperature-

dependent interactions of pNIPAm-AAc with Au

NPs, which were functionalized by different stabiliz-

ing agents, to study their effect on the volume phase

transitions of the hydrogel. The principal aim of the

studies has been to understand the stabilizer specific

interactions between the polymer and stabilized Au

NPs and their temperature dependence. The temper-

ature-dependent properties of the polymer alone,

polymer functionalized Au NPs, and tryptophan,

thioglycolic acid, or phenylalanine-capped Au NPs

dispersed in the polymer medium were investigated.

The results indicate that the SPR of the stabilized Au

NPs could act as a sensitive indicator for the change

in the environment of such systems. In addition, the

absorption characteristics of the NPs could them-

selves act as clear indicators of LCST. Essentially,

the results demonstrate that functionalized Au NPs

could be used for probing LCST transition of the

polymer. In addition, knowledge about the details of

immediate environment of the polymer, before and

after the LCST transition could possibly be obtained.

Experimental section

Materials

N-isopropyl acrylamide (NIPAm, 97% purchased

from Sigma-Aldrich Chemical Co.), acrylic acid

(AAc, 99% from Sigma-Aldrich), N, N0-methylene-

bis-acrylamide (MBA, 99% from Sigma-Aldrich),

potassium perdisulphate (KPS, 98% from Merck),

hydrogen tetrachloroaurate (HAuCl4, 17% Au in HCl

from Sigma-Aldrich), L-tryptophan (99% from

Sigma-Aldrich), thioglycolic acid (98% from

Sigma-Aldrich), L-phenylalanine (95% from Merck)

and sodium borohydride (95%, from Merck) were

used as received without further purification. Milli-Q

grade water was used in all the experiments.

1332 J Nanopart Res (2010) 12:1331–1348

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Methods

Synthesis of PNIPAm-AAc hydrogel

Typically, 50 mL of aqueous NIPAm (987 mg) was

taken in a 100 mL three-necked round-bottom flask.

To this solution 63.0 lL of acrylic acid (0.126% V/V)

and 300.0 lL (4.9% W/V) of methylene-bis-acryl-

amide (molar ratio of NIPAm: AAc = 93:7) were

added under magnetic stirring. These mixtures were

kept in an oil bath at 70 �C and were carefully

degassed by bubbling nitrogen for 30 min. When the

reaction mixtures were turned into homogeneous

solution, 50.0 lL (5% W/V) of potassium perdisul-

phate was introduced to this solution under the same

condition and the reaction was allowed to proceed for

4 h. In the end, the product was centrifuged at

20,000 rpm to remove the unreacted monomers and

excess potassium per disulphate followed by washing

with Milli-Q water at above LCST. Interestingly, we

found that at above LCST the pNIPAm-AAc had

settled as a gel. This gel again was kept at room

temperature which got converted into milky white

solution of hydrogel, and this solution was put into a

dialysis tube (Hi-media dialysis bag 12,000 cut off)

and was subjected to dialysis against distilled water

for 2/3 days. The medium was replaced every 1 h for

the first 6 h. The resultant solution was freeze-dried

for storage.

Synthesis of thioglycolic acid-capped Au NPs

In a typical experiment, 50 mL aqueous solution

containing 15.0 mg of trisodium citrate was kept in

ice-water bath under constant magnetic stirring. To

this solution, 600.0 lL of 17.3 mM HAuCl4 solution

was added. This was followed by addition of

1.2 mL of 20.0 mM freshly prepared sodium boro-

hydride (NaBH4) solution. The resultant solution

was dark red in color, indicating formation of Au

NPs. This solution was kept under the same

condition for 2 h.

In a 5 mL beaker containing 14.0 mM of sodium

hydroxide solution, 120.0 lL of thioglycolic acid was

added. The solution was then slowly poured into

citric acid-capped Au NP solution prepared as above.

The mixture was then stirred for 1 h after which

purple colored precipitation appeared. The precipitate

along with the solution was subjected to centrifuga-

tion (1000 rpm at 4 �C). The pellet was washed with

copious amount of ethanol to remove the excess of

uncapped thioglycolic acid, redispersed in 5 mL

water, and was used as the stock solution for further

characterization studies.

Tryptophan- and phenylalanine-capped Au NPs

One hundred micro liters aqueous solution containing

0.1 mM of HAuCl4 was mixed with 0.01 g of solid

NaBH4. This mixture was kept under constant

magnetic stirring for 2 h at room temperature. The

color of the solution was ruby red, indicating the

formation of Au NPs. This was followed by addition

of 10 mL aqueous solution containing 1.0 mM of

L-tryptophan to 90 mL of the above Au NP solution.

The mixture was stirred for 2 h, after which the

solution color turned red indicating possible forma-

tion of tryptophan-capped Au NPs (Selvakannan

et al. 2004). Finally, the solution was subjected to

ultra-speed centrifugation (70,000 rpm at 15 �C), and

the resulting pellet was washed with copious amount

of water to remove the excess, uncoordinated tryp-

tophan. The pellet was redispersed in 5 mL water; the

resulting solution was stable for weeks. In the

preparation of phenylalanine-capped Au NPs, the

same procedure was followed, taking 10 mL of

9.7 mM of L-phenylalanine solution instead of tryp-

tophan solution, which was added into 90 mL ruby

red Au NP solution. All other conditions were the

same as above.

Synthesis of pNIPAm-AAc stabilized Au NPs

composite

Three hundred micro liters of 17.3 mM HAuCl4solution was added to 50 mL of 0.04% (W/V)

polymer hydrogel solution. To this solution, 600 lL

of 20 mM NaBH4 solution was added. This mixture

was then allowed for 3 h stirring at room tempera-

ture. The solution was then centrifuged at a high

speed (20,000 rpm at 37 �C) and lyophilized to

obtain powdered of composite Au NP-hydrogel

products. Finally, 10 mg of composite Au NP-

hydrogel powder was dissolved in 50 mL water,

and subsequent solution was used for further charac-

terization studies.

J Nanopart Res (2010) 12:1331–1348 1333

123

Preparation of different stabilizing agent capped Au

NP-hydrogel composite

Typically, in a 100 mL beaker containing 50 mL of

0.02% polymer hydrogel solution, 2 mL of a partic-

ular stabilizing agent capped Au NP solution as

prepared above was added. The resulting solution

was stirred for 2 h which was used for further

experiments. Finally, the pH of all the solutions was

measured. The pH of the polymer hydrogel solution

was measured to be 5.60; bare Au NP-hydrogel

composite solution to be 5.40, tryptophan-capped Au

NP composite solution to be 5.22, thioglycolic acid-

capped Au NP composite solution to be 4.69, and that

for phenylalanine-capped Au NP composite hydrogel

solution was measured to be 5.27.

Analytical measurements

UV–vis spectra of the polymer hydrogel, composites

of polymer-Au NPs hydrogel, and different stabiliz-

ing agent capped Au NPs with polymer hydrogel

samples were recorded in the range of 250–800 nm,

using a Perkin-Elmer Lambda-45 spectrophotometer

with temperature controlled sample compartment.

The phase transition for polymer hydrogel, compos-

ites of polymer-Au NP-hydrogel, and different sta-

bilizing agent capped Au NPs with polymer hydrogel

were examined in the temperature range of 20–45 �C

with a heating rate of 0.1 �C/min. FTIR spectra of

the samples were recorded using a Perkin-Elmer

Spectrum one spectrometer. The different stabilizing

agent capped Au NPs were dried as a powder and

mixed with KBr, the resulting mixtures were made as

a pellet for FTIR measurements. The 1H-NMR

spectra of the polymer hydrogel in DMSO-d6

(20 mg/mL) at 20 �C were recorded using a Varian

400 MHz spectrometer, with TMS as the internal

reference. The molar mass and polydispersity of the

polymer hydrogel were determined by gel perme-

ation chromatography (GPC) using Waters GPC

2410 equipment fitted with a refractive index detec-

tor. The HPLC grade of tetrahydrofuran was used as

an eluent, and calibrations were carried out with

polystyrene standard. The dynamic light scattering

(DLS) measurements of the polymer hydrogel,

composites of polymer-Au NPs hydrogel, and differ-

ent stabilizing agent capped Au NPs with polymer

hydrogel samples were performed with an LB-550

(HORIBA) DLS measurement system. The volume

phase transition of the above liquid samples was

examined with increasing temperature at heating rate

of 0.1 �C/min. The average values of the particle

sizes obtained from DLS measurements were calcu-

lated in the following way. When the distribution was

uniform and there was only one particle size

distribution, then the values reported by the instru-

ment were reported as it was. On the other hand,

when two type particle size distributions were

obtained, then higher and lower particle size distri-

butions were individually calculated from the data

obtained from the instrument and then reported.

Transmission electron microscopy (TEM) of the

particles was performed using a JEOL-2100 equip-

ment operating at a maximum acceleration voltage of

200 kV. The TEM samples were prepared by placing

a drop of liquid sample on carbon-coated copper grid

(300 mesh) followed by evaporation of the solvent at

room temperature. The grid was then observed under

the TEM.

Results and discussion

The polymer pNIPAm undergoes a coli-to-globule

transition in aqueous solution at an LCST of ca.

32 �C. It is known that LCST can be altered by co-

polymerization with different monomers containing

hydrophilic or hydrophobic groups. The choice of

AAc monomer for co-polymerization here was due

to its easy solubility in water, bioadhesivenesss, and

biocompatibility (Kim et al. 2007). The synthesis of

pNIPAm-AAc was achieved from the free radical

precipitation polymerization reaction (Khan 2008).

The copolymerization of pNIPAm with AAc has

been confirmed by 1H-NMR using DMSO as the

solvent (refer to supplementary information, SI).

The molecular weight of the polymer and their

polydispersity index (PI) were measured by gel

permeation chromatography. The ratio of weight

averaged and number averaged molecular weight

was close to unity (refer to SI), meaning that

molecular size distribution in the hydrogel was

narrow.

Analytical and other studies were pursued by

dissolving pNIPAm-AAc as well as pNIPAm-AAc

stabilized Au NPs in water at fixed compositions. The

polymer only solution was colorless and transparent

1334 J Nanopart Res (2010) 12:1331–1348

123

at room temperature (below 30 �C), while the

solution consisting of NP-polymer composite

assumed characteristic color of Au NPs due to their

SPR. The solution became progressively opaque with

increase in temperature. Temperature-dependent opti-

cal transmission of the polymer and the composite

measured at the wavelength of 450 nm (Zhang et al.

2007) (Fig. 1a, b) did not vary till about 30 �C.

However, a sharp decrease in transmittance could be

observed between 32 and 35 �C, and the transmission

was constant beyond 35 �C in both the samples. The

sharp decrease in transmission is associated with

phase transition of the hydrogels i.e. transparent coil

to opaque globule transition. Interestingly, the phase

transition temperature of pNIPAm-AAc stabilized Au

NPs (32.5 �C) was slightly lower than that of

pNIPAm-AAc (34.3 �C). Also, the phase transition

of the composite was not as sharp as that of the

polymer alone. The temperature-dependent particle

size measurements of the two species indicated that

the collapse of the diameters of the composite started

at a temperature lower than that of the polymer. Also,

the reduction in diameter of the composite occurred

at a slower rate than the polymer alone. The results of

the particle size analysis by DLS measurements, of

the pNIPAm-AAc as well as pNIPAm-AAc stabilized

Au NPs, at different temperatures are shown in

Fig. 1c and d, respectively. Interestingly, at room

temperature the hydrodynamic diameter of pNIPAm-

AAc polymer was measured to be 3.0 ± 0.7 lm

20 25 30 35 40 45 5070

75

80

85

90

95

100

)T

%( ecnati

msn ar T

Temperature (°C)25 30 35 40 45

45

50

55

60

65

70

75)

T%( ec

nattims

narT

Temperature (oC)

26 28 30 32 34 36 38 40 42 440

400

800

1200

1600

2000)mn( rete

maiD c i

m anydordy

H

Temperature (oC)24 26 28 30 32 34 36 38 40 42

0

500

1000

1500

2000

2500

3000

3500

)m

n( retemai

D cima

nyd

ordy

H

Temperature (oC)

(b) (a)

(d)(c)

Fig. 1 a Optical transmittance of the pNIPAm-AAc and bpNIPAm-AAc stabilized Au NPs at different temperatures.

Plots in c and d are dynamic light scattering based particle size

analysis results of pNIPAm-AAc and pNIPAm-AAc stabilized

Au NPs hydrogel at various temperatures

J Nanopart Res (2010) 12:1331–1348 1335

123

(at 28 �C), while that of pNIPAm-AAc stabilized Au

NPs was found to be 1.8 ± 0.1 lm (at 28 �C). The

decrease of the hydrodynamic diameter of the

polymer stabilized Au NPs compared to that of

polymer could be ascribed to the reduction of the

electrostatic repulsion among the free –COO- moiety

(Lin and Chiu 2006) in the polymer hydrogel in the

presence of Au NPs. The slow and early change in

volume phase transition of the composite in compar-

ison to the polymer is consistent with the results of

optical transmission studies. The results of the DLS

studies also indicated that Au NPs connected to the

polymer lead to change in collapse behavior of the

polymer and also hasten the volume phase transition

in the composite. It is plausible that the electrostatic

interaction between the carboxylate moieties of the

polymer and Au NPs leads to a structure that favors

coil to globule transition at a temperature lower than

that of the polymer. UV–vis spectra of the pNIPAm-

AAc stabilized Au NPs at different temperatures,

shown in Fig. 2a and b (after normalization of

Fig. 2a), consist of a single peak with absorption

maximum at ca. 530 nm. As is clear from the figure,

kmax did not vary with temperature. At above the

transition temperature, the intensity of the absorption

(after due consideration of the change in background)

decreased, which is possibly due to change in the

immediate environment (and hence refractive index

of the medium) (Morones and Frey 2007) of the Au

NPs upon collapse of the original structure. However,

the lack of shift in the absorption maximum probably

indicates that the interaction between the carboxylate

moieties and Au NPs did not change significantly

upon transition to globule form. It is also interesting

to note that upon increase in temperature and

resulting volume phase transition of the composite

there was no agglomeration of the NPs as is clear

from the UV–vis spectra. This implies that the

interaction between Au NPs and the carboxylate

moieties of the polymer is sufficiently strong so as to

prevent agglomeration due to increase in temperature.

It is important to mention here that the temperature-

dependent optical as well as hydrodynamic behavior

of the composite was completely reversible, indicat-

ing that the composite could be a good model for the

study of the behavior of Au NPs in thermosensitive

hydrogels.

Further, in order to understand the effect of

swelling and de-swelling of the polymer hydrogel

on the structural aspects of Au NPs, TEM measure-

ments of the polymer stabilized Au NPs were carried

out. In this regard, two samples were prepared; one

from a solution that was evaporated at below the phase

transition temperature (at 24.3 �C), while the other

sample was evaporated at above the phase transition

temperature (at 40 �C). Figure 3a–c shows different

views of the TEM images of the polymer stabilized

Au NPs with the sample prepared at below the LCST.

The composite consisted of blobs of particles of about

200 nm in diameters interconnected with each other.

The Au NPs (darker spots) were of 10–20 nm in sizes

and were separated well from each other in the

composite. The particles present in between the blobs

evidenced the presence of polymer in the intervening

region, too. Overall, small and individual NPs

connected to the polymer were present in the

composite. On the other hand, the sample prepared

at above the LCST also consisted of individual NPs

without the presence of any significant agglomeration

(Fig. 3d–f). However, the density of the particles (Au

NPs) appears to be higher in comparison to the sample

prepared at below LCST. This is because at above the

phase transition temperature, the diameters of the

polymer particles and thereby volumes are signifi-

cantly reduced. This increases the apparent density of

the particles. The overall structure of the polymer

350 400 450 500 550 600 650 700 750 350 400 450 500 550 600 650 700 750

0.1

0.2

0.3

0.4 45oC

).u.a( sb

A

Wavelength (nm)

25oC

0.2

0.4

0.6

0.8

1.0

1.2

1.4

).u.a( s

bA

Wavelength (nm)

(a) (b)Fig. 2 a The UV–visible

absorption spectra of

pNIPAm-AAc stabilized Au

NPs at different

temperatures and bnormalized absorption

spectra of (a)

1336 J Nanopart Res (2010) 12:1331–1348

123

seems to be similar, with a slight decrease in the

diameter of the blobs at higher temperature. The

increases in density of the NPs on the blobs of the

composite at higher temperature supports that the

structure indeed collapsed into more compact globule

form at the higher temperature. It may also be

mentioned that at higher temperatures the dispersions

of NPs attached to the polymer might become

inhomogeneous (accompanying volume phase transi-

tion), and hence the particles appear to be close to

each other at certain locations (Fig. 3).

Further probe of the effect of additional agents on

the volume phase transition was pursued using

thioglycolic acid; tryptophan or phenylalanine

Fig. 3 The transmission

electron microscopy (TEM)

images of pNIPAm-AAc

stabilized Au NPs at 25 �C

(a), while b and c are the

expanded views of (a). The

plot in d is TEM picture of

the same at 40 �C, while eand f are the expanded

views of (d)

J Nanopart Res (2010) 12:1331–1348 1337

123

stabilized Au NPs. Thioglycolic acid-capped Au NPs

were generated from citrate stabilized Au NPs using

the legend exchange method. The typical sizes of the

Au NPs generated were 5.0 ± 1.5 nm. The interac-

tion of the –SH group of the thioglycolic acid with

Au NPs resulted in the loss of a peak due to –SH

stretching frequency at 2541 cm-1, which was con-

firmed by FTIR spectroscopy (Lin et al. 2005) (refer

to SI, Figure S2). The acid functionalized Au NPs

were then mixed with pNIPAm-AAc in aqueous

solution. We shall henceforth refer to the composite

of thioglycolic acid-capped Au NPs and pNIPAm-

AAc as pPA-thio-Au for simplicity. The temperature-

dependent UV–vis spectra of pPA-thio-Au are shown

in Fig. 4a. The characteristic SPR band of Au NPs is

present in all the spectra (at all temperatures

recorded). As is clear from the spectra, the spectra

changed little from 25 �C to ca. 34 �C. Also, the

absorption due the NPs did not change in this

temperature range. However, the background scatter-

ing increased significantly at above this temperature

(LCST) indicating phase transition to have occurred.

Interestingly, the absorption due to Au NP only

(obtained after appropriate background correction)

also changed significantly at above the LCST. The

normalized absorption values were obtained as fol-

lows. The absorption values at 350 nm for all

temperatures were first measured. That value was

made to be unity for all temperatures and all other

values were adjusted by dividing them with the value

of absorption at 350 nm for a particular temperature.

The absorption at the maximum wavelength was then

taken as the absorption. Sample normalized absorp-

tion versus wavelength plots is shown in Figure S3

(a) (refer to SI). This method of normalization is

based on the works of Tenhu and coworkers. A plot

of normalized Au NP absorbance at 529 nm (Fig. 4b)

indicated significant decrease in absorbance due to

Au NPs at above the LCST. The value of absorbance

(at kmax) leveled off slowly at above LCST. Also

interesting to note is the blue shift in absorption

maximum (kmax) with as much as 6 nm change with

temperatures (Fig. 4c). For example, the absorption

maximum was 529 nm at 25 �C, while at 36 �C the

absorbance maximum shifted to 525 nm. The blue

shift of absorption maximum could be ascribed to the

changing dielectric constant of the medium surround-

ing the capped Au NPs (Ung et al. 1997). In other

words, when thioglycolic acid-capped Au NPs were

mixed with pNIPAm-AAc in aqueous solution there

can be several types of interaction between the

polymer and thioglycolic acid-capped Au NPs. This

interaction might be either van der Waals or

350 400 450 500 550 600 650 700 750

0.1

0.2

0.3

0.4

0.5

0.646oC

).u.a( s

bA

Wavelength (nm)

25oC

24 26 28 30 32 34 36 38 40 42 44 46

522

523

524

525

526

527

528

529

λxa

m)

mn(

Temperature (oC)

24 26 28 30 32 34 36 38 40 42 44 46

0.60

0.65

0.70

0.75

0.80

0.85

).u.a( s

bA

Temperature (oC)

(b)

(c)

(a)

Fig. 4 a UV–visible spectra of pPA-thio-Au NPs with

increasing temperature. b The plot of normalized maximum

absorbance of the SPR band at different temperatures, while cis the temperature dependence absorption maximum (wave-

length) of SPR band of Au NPs in pPA-thio-Au NPs

1338 J Nanopart Res (2010) 12:1331–1348

123

hydrogen bonding interaction (inter or intra molec-

ular H-bonding). The polymer itself could form H-

bonding among its own –COOH groups and with

water molecules. Similarly, the –COOH groups of the

thioglycolic acid could form H-bonds with water as

well as with the polymer at below LCST. At above

the LCST, the polymer shrinks to a form where water

molecules are expelled from the network, and when

thioglycolic acid-stabilized Au NPs are part of the

polymer structures then the hydrogen bonding would

primarily be between the –COOH groups of the

stabilizer and those (along with amide groups or

–COOH group of AAc moieties) of the polymer.

Thus, a change in the environment might well be

reflected in the observed change in the absorption

characteristic of the Au NPs at above LCST. The shift

in the UV–vis absorption of Au NPs indicated that the

phase transition temperature here was 36 �C which is

different from pure polymer. Interestingly, this

observation implies that the SPR absorption of Au

NPs could act as a good probe for measuring the

phase transition temperature of thermosensitive

polymers.

Further, to understand better the volume phase

transition of the pPA-thio-Au NPs, DLS measure-

ments of the composite hydrogel were carried out at

different temperature. The results are shown in Fig. 5.

At temperatures below LCST, there were two types of

particles sizes: one corresponding to the polymer with

a value of 1.4 ± 0.5 lm, while the other one possibly

was due to thioglycolic acid-stabilized Au NPs with

particles sizes of 116 ± 26 nm when measured at

27 �C. Thus, the average particle sizes mentioned in

the figures are the averages of the values, automati-

cally reported by the software used in measuring the

particle sizes. On other hand at 35.6 �C, the sizes were

reduced down to 116 ± 26 nm. The gradual change in

the particle sizes at above the LCST indicates gradual

de-swelling of the composite in comparison to the

polymer alone. The above results suggest that at

temperatures below LCST, thioglycolic acid-stabi-

lized Au NPs and pNIPAm-AAc hydrogel remain as

individual species in aqueous medium, where inter-

molecular H-bonding is predominant. When the

temperature of the medium is increased, the intramo-

lecular H-bonding in polymer and intermolecular

H-bonding between the polymer and thioglycolic acid-

stabilized Au NPs are favored. At above the LCST,

these H-bondings provide the main force for the

formation of a composite structure consisting of Au

NPs (with the stabilizer) and the polymer. The results

are the formation of particles of uniform sizes and

change in the SPR spectral characteristics of the Au

NPs. Further, the attractive forces leading to composite

formation, at above LCST, between the functionalized

Au NPs and the polymer might be responsible for the

0 1000 2000 3000 4000 5000 60000

5

10

15

)%( yc

neu

qerF

Hydrodynamic diameter (nm)

60 80 100 120 140 160 180 2000

5

10

15

20

)%( yc

neu

qerF

Hydrodynamic diameter (nm)40 60 80 100 120 140 160

0

5

10

15

20

)%( yc

neu

qerF

Hydrodynamic diameter (nm)

28 30 32 34 36 38 40 42

200

400

600

800

1000

1200

1400

)m

n( retemai

D cima

nyd

ordy

H

Temperature (oC)

(b)(a)

(c) (d)

Fig. 5 a Temperature-

dependent hydrodynamic

diameters of the PPA-thio-

Au NPs as measured by

dynamic light scattering

technique. b The histogram

of hydrodynamic diameters

of pPA-thio-Au NPs at

27 �C, c 35.6 �C, and d39 �C obtained by dynamic

light scattering

measurements

J Nanopart Res (2010) 12:1331–1348 1339

123

changes in the SPR peak of the Au NPs. However, the

above investigation of the DLS measurements shows

that there will be a marked attraction between the

thioglycolic acid-capped Au NPs and pNIPAm-AAc

polymer network and this leads to the decreasing of

the hydrodynamic radius of the polymer network gel

toward to the thioglycolic acid-capped Au NPs sizes

with increasing the temperature of the system, which

also leads to the changing the kmax of the SPR toward

to the lower wavelength. In other words, at higher

temperature the water molecules are gradually

removed from polymer hydrogel, and the removal

induces a decrease in total volume of the polymer gel

and brings thioglycolic acid-capped Au NPs very

close to the polymer network leading to the blue shift

of the SPR absorption of the Au NPs in the system. It

must be mentioned here that all of the above behaviors

have been found to be reversible with respect to

change in temperature. Further, TEM analysis of the

pPA-thio-Au NP samples prepared at below the phase

transition temperature (25 �C) and at above the phase

transition temperature (40 �C) was pursued to have a

better understanding of the structural transitions due

to temperature increase. The results are shown in

Fig. 6. At a temperature below the LCST, the Au NPs

could be observed to have been dispersed rather

uniformly all over the polymer (Fig. 6a). Individual

Au NPs could be seen spread over the network of

polymers (Fig. 6c) indicating uniform distribution of

particles in the medium, which upon evaporation

formed such structures. On the other hand, at above

the LCST (at 40 �C), the density of the particles is

much higher and clusters of particles could be seen

deposited over the polymer (Fig. 6d–f). The average

diameter of the Au NPs was found to be 8.0 ± 0.3 nm

in all cases, indicating that there was no agglomera-

tion of particles. The increase in density of the

particles clearly indicates the shrinking of polymer

along with the functionalized Au NPs. This means that

the Au NPs were bound to the polymer at above

LCST, possibly through electrostatic interaction

between the polymer and the stabilizers of Au NPs.

The apparent inconsistency between the particle sizes

as measured from DLS and TEM could originate due

to two factors. First of all, the microviscosity for the

polymer particles and those due to stabilized NPs

would not be identical. They have been assumed to be

the same in the measurement. This would significantly

affect the exact particle size calculation (of the NPs)

from the DLS measurements. In addition, as is clear

from the TEM images there are significant presence of

associated NPs even at lower temperatures. This could

make the apparent particle sizes of the NPs appear

larger than they are.

In addition to thioglycolic acid, we were interested

in studying the effect of amino acids on the volume

phase transition by incorporating of amino acid-

capped Au NPs into pNIPAm-AAc hydrogel solution.

Sastry and coworkers have reported that amino group

can bind more strongly to the surface of Au NPs than

carboxylic acid group (Joshi et al. 2006; Selvakannan

et al. 2003). In addition to their studies, there has been

several report of synthesis of different amino acid-

capped Au NPs, which are useful for biological

applications. The motivation has been that since the

amino acids could have different side chain group—

such as hydrophilic or hydrophobic, polar or non-

polar—interactions of the stabilizer with biological

molecules would also be different. In the present

study, we have taken two different amino acids such

as tryptophan and phenylalanine. Amino acid trypto-

phan contains hydrophilic indole moiety, while

phenylalanine consists of hydrophobic phenyl ring.

Thus, the phase behaviors of the two different amino

acid-stabilized Au NPs with the polymer, at different

temperature, would be potentially interesting. The

capping of Au NPs by amino acids has been confirmed

by FTIR spectroscopy (refer to SI, Figure S4 and S5).

Tryptophan- and phenylalanine-capped Au NPs have

absorption maxima at 543 and 519 nm, respectively.

For subsequent studies, the amino acid-capped Au

NPs were mixed in aqueous solutions containing

pNIPAm-AAc hydrogel. We shall henceforth refer the

resultant composites hybrid hydrogels as pPA-try-Au

NPs (for pPA-AA-tryptophan-capped Au NPs) and

pPA-phe-Au NPs (for pPA-AA-phenylalanine capped

Au NPs). Our observations as detailed below suggest

that the phase transition as well as optical behavior of

the two differently stabilized Au NPs are different.

The temperature-dependent UV–vis spectral changes

and hydrodynamic diameters of pPA-try-Au NPs and

pPA-phe-Au NPs are shown in Figs. 7 and 8, respec-

tively. The as-observed UV–vis spectra are shown in

Figs. 7a and 8a, respectively. The normalized absor-

bances at the maxima are shown in Figs. 7b and 8b,

respectively. It may be mentioned here that to obtain

normalized absorbance from Fig. 7a, the method

similar to the calculations in Fig. 4 was followed

1340 J Nanopart Res (2010) 12:1331–1348

123

(Refer to SI, Figure S3 (b)). On the other hand, the

normalized absorbance values at the maxima from

Fig. 8a were obtained by drawing a baseline between

475 and 555 nm and then measuring the value of

absorbance from the baseline to the maximum

absorption at the kmax (refer to SI, Figure S3 (c)).

For the pPA-try-Au NPs composite, the SPR band of

Au NPs hardly changed with temperature in terms of

intensity (at kmax, Fig. 7c), while there was discern-

ible change in the values of kmax (Fig. 8c). For

example, at below the transition temperature the kmax

of the pPA-try-Au NPs was 543 nm, while at above

the phase transition temperature the kmax was

observed to be 539 nm. The results, however, indicate

Fig. 6 a TEM image of

pPA-thio-Au NPs at 25 �C,

while b and c are the

expanded views of the

picture from (a). The image

in d is the TEM picture of

the same at 40 �C, while eand f are the expanded

views of the picture in (d)

J Nanopart Res (2010) 12:1331–1348 1341

123

behavior similar to the ones of thioglycolic acid as the

stabilizer, although they are less remarkable. Further,

the phase transition temperature as measured from the

UV–vis studies (Fig. 7b and c) occurred at 39 �C,

which is significantly higher than that of the pure

polymer. In addition, the results of measurements of

hydrodynamic diameters (Figs. 7d and 8d, respec-

tively) also support the above results. Essentially,

although the general behavior of the hydrophilic

tryptophan moiety (in the composite) is similar to that

of thioglycolic acid, there are significant differences

as described above, which indicate that the interaction

between the polymer and the stabilized Au NPs could

be substantially different at different temperatures.

The H-bond that could be formed between the indole

group of tryptophan and the carboxylate or amide

moieties of the polymer could possibly be the reason

for the behavior described above. Further evidence in

support of the above conclusion came from the

temperature-dependent UV absorption due to G–G*

transition of the indole moieties of the tryptophan

occurring at 265 nm. It is worth pointing out that the

tryptophan has absorption maxima at 278 nm, which

shifted to 265 nm after the formation of stabilized Au

NPs. This indicates interaction between the Au NPs

and the p-electron cloud of tryptophan (Selvakannan

et al. 2004). Figure 9a shows the temperature-depen-

dent absorption spectrum of a mixture of pPA-try-Au

NPs composites solutions, recorded in the range of

190–420 nm. As is evident from the figure, the band at

265 nm vanished at phase transition temperature

(39 �C) of the pPA-try-Au NPs. It should be men-

tioned here that the band could again be observed

when the temperature reverted back to a temperature

30 32 34 36 38 40 42 44

0

200

400

600

800

1000

1200

1400)m

n( retemai

D cima

n yd

ord y

H

Temperature (oC)30 32 34 36 38 40 42 44 46

539

540

541

542

543

544

λxa

m)

mn(

Temperature (oC)

350 400 450 500 550 600 650 700 750

0.2

0.3

0.4

30oC).u.a( s

bA

Wavelength (nm)

45oC

32 34 36 38 40 42 44

0.90

0.91

0.92

0.93

0.94

0.95

0.96

0.97

0.98

).u.a( s

bA

Temperature (oC)

(b)(a)

(c) (d)

Fig. 7 a Temperature-dependent UV–visible spectra of pPA-

try-Au NPs, while b is the plot of normalized absorbance at

different temperatures obtained from [Figure S3 (b)], and c is

the change of kmax of the surface plasmon band of the pPA-try-

Au NPs from normalized graph of Figure S3 (b). The plot in dis the dynamic light scattering measurement results (hydrody-

namic diameters) of the pPA-try-Au NPs at different

temperatures

1342 J Nanopart Res (2010) 12:1331–1348

123

below LCST. This observation clearly indicates that

there was a significant interaction between the poly-

mer hydrogel and tryptophan-capped Au NPs, which

is reversible with respect to change in temperature. It

might be that encapsulation of the tryptophan moieties

inside the globule of the polymer leads to vanishing of

the absorption.

The results involving pPA-phe-Au NPs were

remarkably different from those mentioned above.

The UV–vis absorption due to SPR of Au NPs

virtually disappeared with increase in temperature

(Fig. 8a, b). Moreover, the changes were more

gradual than in the cases with the other stabilizers.

The changes in the wavelength maxima were also

significant. For example, at room temperature

(25 �C), the kmax was 517 nm, while that at 36 �C

was shifted to 508 nm. Further, the kmax shifted

gradually to still lower wavelengths with increasing

temperatures (Fig. 8c). The phase transition temper-

ature was found to be 36 �C (Fig. 8b, c). It is

important to mention here that the volume phase

transition temperatures of the systems pPA-try-Au

NPs and pPA-phe-Au NPs were found to be 39 and

36.3 �C, respectively, as obtained from temperature-

dependent DLS measurements (Figs. 7d and 8d).

These results are consistent with phase transition

temperatures obtained from temperature-dependent

absorption maxima of the stabilized Au NPs. The

above results suggest that SPR absorption of Au NPs

could act as a probe for understanding the nature of

26 28 30 32 34 36 38 40 42

0.004

0.005

0.006

0.007

0.008

0.009

).u.a( s

bA

Temperature (oC)

26 28 30 32 34 36 38 40 42

506

508

510

512

514

516

λxa

m)

mn(

Temperature (oC)26 28 30 32 34 36 38 40 42

0

200

400

600

800

1000

1200)m

n( retemai

D cima

nyd

ordy

H

Temperature (oC)

300 350 400 450 500 550 600 650 700

0.20

0.24

0.28

0.32

43oC

).u.a( s

bA

Wavelength (nm)

25oC

(a) (b)

(d)(c)

Fig. 8 a Temperature-dependent UV–visible spectra of pPA-

phe-Au NPs. The plot b of normalized absorbance at different

temperatures is based on figure shown in Figure S3 (c) (refer to

SI), while c is the plot of the kmax of the surface plasmon band

of the pPA-phy-Au NPs also calculated from Figure S3 (c).

Here d is the average hydrodynamic diameters of the pPA-phy-

Au NPs at different temperature, measured by dynamic light

scattering method

J Nanopart Res (2010) 12:1331–1348 1343

123

interactions between the stabilizer molecules and

polymer hydrogel. This is especially true in estab-

lishing the LCST of the polymer hydrogel in the

presence of NPs with different stabilizers. Further, a

question may arise about the volume phase transition

temperature of pPA-Try-Au NP composite hydrogel

being higher than that of pPA-phe-Au NP composite

hydrogel. This could be explained in terms of side

chains of the individual amino acids. For example,

tryptophan has more hydrophilic moiety of the indole

ring, which can make the stronger intermolecular H-

bond with polymer hydrogel, as well as with the

surrounding water molecules. When the temperature

is increased gradually, the intermolecular H-bond

between the water and polymer and between the

water and tryptophan may gradually be lost due to the

thermal motion of the water molecules and conse-

quently the volume of the pPA-try-Au NP composite

hydrogel slowly shrinks (Fig. 7d). Stoller et al.

(2004) reported that H-bonding interaction between

the indole-water is more energetically favorable than

that of aromatic ring. They considered that H-

bonding interaction between the heterocyclic ring

and water is much stronger than simple aromatic

benzene ring (Fredericks et al. 1996; Carney et al.

1998). This could be responsible for higher volume

phase transition temperature of the pPA-Try-Au NPs

than that of pPA-phe-Au NPs composite hydrogel.

This was further supported by the results of temper-

ature-dependent UV–vis spectra of NPs in both the

systems. At the phase transition temperature, the SPR

peak of the pPA-phe-Au NPs was more affected than

that of pPA-try-Au NP composite hydrogel. This

could be ascribed to the more hydrophobic nature of

the phenyl ring than indole. Further, the hydrophobic

interaction with the polymer is more dominant in the

phenylalanine capped Au NPs, which leads to

significant shift of the SPR peak in comparison to

tryptophan-capped NPs (Mulvaney 1996; Ung et al.

2001). In addition, phenylalanine contains a highly

hydrophobic side residue that would have different

interaction with the polymer than the highly hydro-

philic thioglycolic acid or tryptophan. Also, the coil

to globule transition of the polymer at above the

transition temperature involves reinforced intramo-

lecular hydrogen bonding and strong van der Waals

interactions (attraction) among its hydrophobic moi-

eties (Zhu et al. 2004). This would lead to localiza-

tion of the stabilized Au NPs into the hydrophobic

pockets of the globule. This is probably the reason for

reduced optical absorption as the NPs that are buried

within the ‘‘particles’’ of the globule. The same may

be the reason for changes in the absorption maxi-

mum. Raula et al. (2003) have shown that both kmax

and absorption of the SPR peak of thermosensitive

hydrogel-coated Au NPs, at above the LCST changes

similar to the above. The reason here has also been

proposed to be due to formation of hydrophobic

pocket after the collapse of the structure into globule

form.

Temperature-dependent particles size histogram

plot of DLS measurements of pPA-try-Au NPs and

pPA-phe-Au NPs indicated specific interactions

between the stabilized Au NPs and the polymer.

The results are shown in Fig. 9 (pPA-try-Au NPs)

and Figure S6 (refer to SI) (pPA-phe-Au NPs). In

these cases, the particles consisted of two distribu-

tions of sizes. The higher particle diameters with

average values of 1.3 ± 0.8 and 1.15 ± 0.9 lm for

pPA-try-Au NPs and pPA-phe-Au NPs, respectively,

in addition to lower particle diameters of values

62 ± 18 and 50 ± 15 nm, respectively for the above.

This means that polymer particles were probably

dispersed separately in the medium from the stabi-

lized Au NPs. However, since the average value of

the polymer particles herein was smaller than those of

polymer alone or polymer stabilized Au NPs, it is

plausible that there were at least some interactions

between the two. Further, at higher temperatures the

particle collapsed into sizes, which are considerably

less than those of all three cases discussed before. For

example, at 39 �C the hydrodynamic diameter of the

pPA-try-Au NPs was 62 ± 18 nm while pPA-phe-Au

NPs showed a value of 52 ± 15 nm at 36 �C. This

means the interactions between phenylalanine-capped

NPs and tryptophan-capped NPs and the polymer led

to globular structures with diameters smaller than that

of the polymer (in its globular form) at above the

LCST. Seida and Nakano (1996) reported the effect

of various organic molecules on the nature of

interaction with the polymer at above and below the

phase transition temperatures. They observed that

organic molecules (phenol and benzoic acid and

aniline) are absorbed much into the polymer at above

the phase transition due to the domination of

hydrophobic interaction. This could be the reason

that when the phenylalanine-capped Au NPs inter-

acted with polymer hydrogel that led to the

1344 J Nanopart Res (2010) 12:1331–1348

123

decreasing of the hydrodynamic diameter of the pPA-

phe-Au NPs than that of pPA-try-Au NPs at above

the phase transition temperature. On the other hand,

strong hydrophobic interactions between phenyl

groups and the hydrophobic backbone of the polymer

may lead to a collapsed structure that is smaller in

diameter than the polymer itself. Temperature-depen-

dent TEM measurements pPA-try-Au NPs are shown

in Fig. 10. At below the LCST, the particles were

uniformly distributed all over the solution and hence

the evaporated materials had more or less uniform

distributions of particles (Fig. 8a–c). The NPs had

typical diameters of 8.0 ± 0.6 nm. On the other

hand, when the temperature was increased above

LCST, the particles seem to agglomerate into the

collapsed structure of the polymer. This behavior is

different from the other samples where either poly-

mer or thioglycolic acid-stabilized Au NPs were

used. The reversibility of the structure in solution

(with respect to changes in temperature) indicates

that these particles might not have agglomerated into

lumps of particles only. On the contrary, the inter-

actions between the globular polymer and the stabi-

lizers lead to some sort of collapsed structure with

particles coming very close to each other in compar-

ison to the structures obtained from polymer or

thioglycolic stabilized NPs. Similar results were also

observed in the case of the pPA-phe-Au NPs system

(refer to SI). The TEM pictures are shown in Figure

S7.

Qualitatively, the results mentioned above could

be explained primarily by invoking two factors:

hydrophobic and H-bonding interactions. In the

system involving polymer-capped Au NPs, there is

no extraneous stabilizing agent present and hence no

additional interaction between the species. Thus, the

20 40 60 80 100 120 140 1600

5

10

15

)%( yc

neu

qerF

Hydrodynamic diameter (nm)

0 1000 2000 3000 40000

4

8

12

16

)%( yc

neu

qerF

Hydrodynamic Diameter (nm)

0 500 1000 15000

3

6

9

12

)mn( ycne

uqer

F

Hydrodynamic Diameter (nm)

250 300 350 400

0.4

0.6

0.8

).u.a( s

bA

Wavelength (nm)

(d)(c)

(b)(a)

Fig. 9 a The UV–visible absorption spectra of pPA-try-Au

NPs at different temperatures, measured in the region of 225–

420 nm. b The histogram of hydrodynamic diameters of pPA-

try-Au NPs at 30 �C, c 36 �C, and d 39 �C obtained by

dynamic light scattering measurements

J Nanopart Res (2010) 12:1331–1348 1345

123

results would be similar to those of polymer alone

with minor variations due to the presence of Au NPs.

When thioglycolic acid is used as the stabilizer at

below the LCST, there would be hydrogen bonding

between the stabilizer and water as well as the

polymer. However, at above LCST, the hydrogen

bonding between the carboxylate moieties of the

stabilizer and the hydrophilic groups of the polymer

would be favored over that with the solvent as the

stable globular structure would have hydrophobic

pocket and hydrophilic surfaces. Hence the final

structure at above LCST may form H-bonds between

the stabilizer and the polymer with stabilized Au NPs

being in the pocket of the globule and the results

Fig. 10 a TEM pictures of

pPA-try-Au NPs at 25.4 �C,

while b and c are the

expanded views of (a). dThe TEM image of pPA-

try-Au NPs at 40 �C, while

e and f are the expanded

views of (d)

1346 J Nanopart Res (2010) 12:1331–1348

123

would be different. In the case of phenylalanine

stabilized Au NPs, the final structure in the globule

would possibly involve the stabilized NPs deeply

embedded in the pocket of the globule thereby

reducing even the optical absorption of Au NPs,

which is different from the other cases. Finally, the

general behavior involving the interactions between

stabilized Au NPs and the polymer at below and

above LCST is described schematically in Fig. 11.

Essentially, when Au NPs are stabilized by hydro-

philic moieties then particles are either exposed to the

aqueous medium even after the collapse (at above

LCST) or embedded in the collapsed structure being

bonded to the hydrophilic groups of the polymer. On

the other hand, in the presence of hydrophobic

stabilizers, the particles are embedded deeply inside

the polymer collapsed structure at above LCST.

Conclusion

In the present article, we have been able to report the

use of various functional group stabilized Au NPs on

the volume phase transitions of a thermosensitive

polymer hydrogel. The optical properties of Au NPs

stabilized with different functional groups have been

used as one of the probes to understand the behavior

of volume phase transitions in mixed systems.

Particle size analysis and TEM measurements were

also made to substantiate the conclusions of the

observations. Essentially, when hydrophilic groups

were used in the stabilizer the collapsed structures

consisted of H-bonding between the stabilized Au

NPs and the polymer, with little effect on the

hydrophobic pocket of the globule. On the other

hand, when hydrophobic group was used in the

stabilizer then the observations indicated that the Au

NPs along with the stabilizer were embedded deep

inside the hydrophobic pocket of the globule, dras-

tically changing the optical absorption due to SPR of

Au NPs. That amino acids were used as the stabiliz-

ing agents and their effect on the volume phase

transitions of a biofriendly hydrogel would support

future endeavors involving Au NPs in the drug

delivery systems.

Acknowledgment We thank Central Instrumentation Facility

(IITG) for help in recording TEM. We also thank the

Department of Science and Technology (DST) (No. SR/S5/

NM-01/2005, 2/2/2005-S�F., SR/FT/L-39/2004), Council of

Scientific and Industrial Research (CSIR, 01(2172)/07/EMR-

II), Government of India for financial support. Mr. A.

Murugadoss thanks CSIR for a fellowship.

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