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Biomaterials 26 (2005) 67436753 www.elsevier.com/locate/biomaterials

Hollow gold nanoparticles encapsulating horseradish peroxidase

Rajiv Kumara, A.N. Maitraa, P.K. Patanjalib, Parvesh Sharmac,
Department of Chemistry, University of Delhi, Delhi 110 007, India b Institute of Pesticide Formulation Technology, Gurgaon, India c Department of Chemistry, St. Stephens College, University of Delhi, India Received 12 February 2005; accepted 5 April 2005 Available online 13 June 2005
a

Abstract Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from AushellAgClcore nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows MichaelisMenten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles. r 2005 Elsevier Ltd. All rights reserved.
Keywords: Hollow gold nanoparticles; Aushell AgClcore nanoparticles; Enzyme entrapment; Horseradish peroxidase (HRP); Dextran-o-dianisidine

1. Introduction The potential applications of mesoporous gold lie in the eld of catalysis [1], biosensing electrodes [26], analytical chemistry [7], solid oxide fuel cells [8], detection of biomolecules using ellipsometry [6], and plasmonics [910]. Nanoshells of gold typically exhibit much higher optical sensitivity than solid core gold nanoparticles [11]. This feature has been extensively explored for use in fabricating optical lters [12], photon energy transport devices [13,14], probes for scanning near-eld optical microscopy [15,16], active surface for surface-enhanced Raman [1720] and uorescence scattering [21,22], rapid immunoassay [23], and photothermal ablation of tumor cells in vivo [24]. But gold
Corresponding author. Tel.: +91 9 8682 44469;

fax: +91 011 2766 6593. E-mail address: parveshsharma@ststephens.edu (P. Sharma). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.04.045

nanoparticles, being completely benign material in the living system [2527], have seldom been explored as carriers for enzyme therapeutic purposes. Hollow gold nanoparticles with porous shell structure can best be used as carriers for therapeutic compounds in vivo. This is more particularly important when therapeutic compounds are enzymes. Enzymes can be used as agents for specic degradation of unwanted metabolites, including toxic compounds in diseased individuals. Exogenous enzymes being antigenic in nature, it is desirable that the enzyme should be entrapped permanently within the biocompatible and non-antigenic carriers such as nanoparticles made of suitable ceramic and polymeric materials, and the undesirable metabolites should reach the enzyme through the porous shells of the carriers. The metabolism of excessive urea accumulated in the liver or kidney by the enzyme urease, or the reduction of blood L-asparagine concentration to a minimum level by

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using L-asparaginase are expected to be best examples of possible applications of enzyme therapy using nanoparticles as enzyme carriers. However, biodegradable nanoparticles cannot be used for such a purpose, as these carrier materials, after degradation, would enable the entrapped enzyme to come out in the body system causing immunogenic reaction. To overcome this problem, we have reported in this paper the synthesis of hollow gold nanoparticles entrapping a marker enzyme, HRP, by using the softchemical method at room temperature, and have demonstrated that the entrapped enzyme is chemically and biologically active. Hollow nanoshells of gold have been prepared up till now either by heating coreshell nanoparticles with organic polymer as core at high temperatures, or by dissolving the suitable core materials with strong acid or alkali or an organic solvent. All the above methods are unsuitable when the encapsulated material has thermal and/or chemical sensitivity such as an enzyme.

micelles), which in this case was 15. The solution B was then added to A dropwise with constant stirring. After complete addition, the solution was left to stir for nearly 4 h, and at the end of which a faint translucent solution (without any precipitation) was obtained due to the formation of HRPdoped AgCl nanoparticles in the aqueous core of the reverse micellar droplets. 2.2.2. Growth of Au shell on the surface of HRP-doped colloidal AgCl particles To 50 ml of AgCl nanoparticles dispersed in reverse micelles, as obtained by the above process, 200 ml of 5% HAuCl4 solution was added drop by drop with constant stirring. The W o of the resultant reverse micellar solution was 17. The solution was distinctly homogeneous and clear, and acquired a distinct yellow coloration. It was stirred further for 1 h at room temperature. To this reverse micellar solution, 200 ml of 0.05 M aqueous hydrazine hydrate was added. Hydrazine hydrate reduced the Au3+ to metallic Au. With the progressive addition of hydrazine hydrate to reverse micelles, the solution acquired a purple to red colour due to the formation of colloidal gold. The solution was then stirred for another 2 h. 2.2.3. Extraction of particles from reverse micelles and removal of AgCl core To the above 50 ml reverse micelles containing HRP-doped AgCl nanoparticles coated with a gold layer 5 ml of absolute ethanol was added. The solution was then stirred for a few minutes. This led to the complete breakdown of reverse micelles with the formation of two immiscible layers of aqueous ethanol and iso-octane, and the nanoparticles along with the surfactant molecules were accumulated at the interface of iso-octane and ethanol. The ethanol was carefully removed using a separating funnel. The particles thus obtained were washed four times with iso-octane and centrifuged to remove any residual AOT. The pelleted particles were then dispersed in 10 ml water by vigorous stirring for 1015 min and the dispersed system was dialysed against distilled water for 2 h using a 12 kDa cut-off dialysis bag. The resultant aqueous solution was lyophilised to obtain dry HRP-doped AgCl nanoparticles coated with gold. The total amount of lyophilised powder of HRP-doped gold-coated AgCl nanoparticles (HRP@AushellAgClcore) was then redispersed in 10 ml 0.1 M aqueous NH3 solution with constant stirring for 2 h and the solution was dialysed for 12 h at room temperature. After dialysis, the nanoparticles solution in aqueous dispersion was again lyophilised to obtain the dry powder. The lyophilised particles were characterised by DLS, TEM, XRD and optical spectroscopy. 2.2.4. Preparation of void hollow gold nanoparticles The placebo nanoparticles containing no HRP were prepared following the above protocol, using equal volumes of water in place of an aqueous solution of HRP so that the same W o was maintained. 2.2.5. Preparation of core gold (Aucore) nanoparticles Core gold nanoparticles containing HRP were obtained by a method similar to that described in Section 2.2.2. For

2. Experimental 2.1. Materials Chloroauric acid was purchased from S.D. Fine Chemicals, India. Aerosol-OT (AOT), i.e. sodium bis-(2-ethylhexyl) sulphosuccinate (99%), was purchased from Acros Organics (Belgium). HRP of Rz 2:9, o-dianisidine and dextran were procured from Sigma, USA. Iso-octane (AR grade) and sodium periodate were obtained from Spectrochem Pvt. Ltd. Sodium borohydride was obtained from E. Merck (India) Ltd. Hydrazine hydrate was obtained from SRL India and AgNO3 and NaCl were procured from Ranbaxy Fine Chemicals Ltd. All the reagents were used without further purication. Water used was deionised and double distilled. 2.2. Method of preparation of hollow gold nanoparticles encapsulating HRP The overall procedure for the preparation of HRP-doped hollow gold nanoparticles consisted of three major steps: (i) preparation of AgCl nanoparticles entrapping HRP; (ii) growth of gold layer on the surface of AgCl nanoparticles; and (iii) leaching out AgCl core to get hollow gold nanoparticles containing HRP entrapped into the cavity of the nanoparticles. 2.2.1. Preparation of HRP-doped AgCl nanoparticles To prepare HRP-loaded AgCl nanoparticles, 475 ml of 0.05 M AgNO3 solution and 200 ml of 1:5 104 M aqueous solution of HRP were added to 25 ml of 0.1 M AOT in isooctane to give a clear reverse micellar solution A. Similarly, reverse micelle B was prepared from 25 ml 0.1 M AOT in isooctane, containing 475 ml of 0.05 M NaCl solutions, and 200 ml of HRP of the above concentration. Excess water was added to maintain W o (the molar ratio of water to AOT in the reverse

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example, to 25 ml of 0.01 M AOT in iso-octane, 200 ml of 5% HAuCl4 and 400 ml of 1:5 104 M aqueous solution of HRP were added to give a clear reverse micellar solution. Finally, 165 ml of double distilled water was added to obtain a resultant reverse micellar solution of W o 17. The solution was distinctly homogeneous and clear, and acquired a distinct yellow coloration. It was stirred further for 1 h at room temperature. In all, 200 ml of 0.05 M aqueous hydrazine hydrate was added to this reverse micellar solution with constant stirring. Hydrazine hydrate reduced the Au3+ to metallic Au and the solution acquired purple-red colour due to the formation of colloidal gold. The solution was then stirred for another 2 h. 2.2.6. Preparation of dextran-o-dianisidine (DexoDA) conjugate DexoDA conjugate was prepared by a parallel scheme as reported earlier [2829]. Briey, 250 mg of dextran was rst treated with 50 ml of 0.03 M sodium periodate overnight in the dark. The polyaldehyde dextran thus formed was dialysed extensively. To the dialysed polyaldehydic dextran solution, 250 mg o-dianisidine was added. The sample was stirred for 2 h in the dark at room temperature to form Schiff base, and dialysed overnight. The Schiff base was then nally reduced by using sodium borohydride (29 mg in 30 ml double distilled water) with constant stirring for 2 h and then dialysis was performed overnight. The dialysed sample was lyophilised to obtain a light brown powder. 2.3. Physical characterisation of HRP-doped hollow gold nanoparticles 2.3.1. Dynamic laser light scattering (DLS) DLS measurement to determine the size of the nanoparticles was performed using a Brookhaven 8000 instrument with a BI200 SM goniometer. All measurements were made at a scattering angle of 901. An air-cooled argon ion laser operating at 488 nm was used as a light source. The time-dependent intensity autocorrelation function of the scattered light was derived using a 128-channel digital correlator. The size of the particles was determined from their diffusion using the StokeEinstein equation. 2.3.2. Transmission electron microscopy (TEM) TEM measurements were performed with a JEOL Model JEM 2000E 200 electron microscope. Samples were prepared by placing small drops of dispersed particles in water on formvar-coated copper grids and allowing the solvent to slowly evaporate at room temperature. 2.3.3. XRD measurements Powder X-ray diffraction measurements were performed on a Bruker AXS D8 diffractometer using pressed pellets as ( samples with Ka radiation (l 1:5418 A. 2.3.4. Optical studies All spectrophotometric studies were carried out using a UV1601 Shimadzu UV-VIS spectrophotometer. While measuring the absorbance in reverse micelles, AOT reverse micelles of the same W o value were used as reference.

2.4. Enzyme kinetic studies of HRP-loaded hollow gold nanoparticles with o-dianisidine and its dextran derivative The enzymatic activity of HRP for the catalytic oxidation of o-dianisidine by H2O2 was determined, as reported earlier [30], from the initial slope of the time-dependent absorbance of the oxidised product of o-dianisidine measured spectrophotometrically at 445 nm. Enzyme kinetic studies were performed as follows. A total of 29 mg of HRP-loaded, hollow gold nanoparticles was dispersed in 10 ml of phosphate buffer (0.1 M, pH 7.2). The enzyme kinetics of HRP-catalysed odianisidine oxidation reaction by H2O2 was studied by adding 5 ml of dispersed particles, 5 ml of o-dianisidine solution (1 w/v%) and 530 ml 1 M H2O2 to 2.5 ml 0.1 M phosphate buffer. The estimation of the concentration of the coloured oxidised product of o-dianisidine was done spectrophotometrically by measuring the absorbance at 445 nm, and the rates of the reactions were evaluated from the initial slopes of the time-dependent absorbance curves. All the enzymatic activities reported in the paper were measured in a 0.1 M phosphate buffer of pH 7.2, unless otherwise stated. Kinetic parameters such as Km and kcat were calculated from LineweaverBurk plots using the MichaelisMenten equation. The enzyme kinetic studies with DexoDA conjugate were done as follows: 29 mg of HRP-loaded hollow gold nanoparticles were dispersed in 10.0 ml of 0.1 M phosphate buffer, as described above. For measuring the activity of the HRP encapsulated in the cavity of the hollow gold nanoparticles with DexoDA conjugate, 5 ml of dispersed particles, 5 ml of DexoDA conjugate (1 w/v%) and 5 ml of 1 M H2O2 were added to 2.5 ml 0.1 M phosphate buffer and the activity was measured spectrophotometrically at 420 nm, in the same manner as above. For comparison, the enzyme kinetic studies using free HRP in aqueous buffer in place of entrapped enzyme were also determined for the oxidation of o-dianisidine and its dextran conjugate by H2O2.

3. Results and discussion 3.1. Synthesis of nanoparticles and gold nanoshells In the rst stage of preparation, AgCl nanoparticles entrapping HRP were precipitated in the aqueous core, which were partially protected by the surfactant monolayer (Reaction I below). After Ostwald ripening (2 h stirring), HAuCl4 solution was added to the above reverse micellar system, where HAuCl4 reached the aqueous core of the micellar droplets and accumulated around the AgCl nanoparticles (Reaction II). The solution acquired a distinct yellowish coloration, similar to that of HAuCl4 solution. Subsequent addition of an aqueous solution of hydrazine hydrate reduces the Au3+ ions to metallic Au (Reaction III). At this stage, the reverse micellar system acquired a typical red-purple coloration of colloidal gold shell particles. HRP@AushellAgClcore nanoparticles

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were extracted from the reverse micelles and the dried particles were dispersed in dilute NH4OH solution in which AgCl was dissolved and was leached out of the gold shell, leaving HRP inside the cavity of hollow gold nanoparticles (Reaction IV). 3.2. Optical properties The progress of the formation of a gold shell over AgCl core nanoparticles was followed by the spectrophotometric method. As shown in Fig. 1, while no characteristic absorption band was observed in the AgCl nanoparticles encapsulated in reverse micelles (curve A), a distinct absorption band at and around 530 nm was observed when gold shells were deposited on AgCl particles and the intensity of the band was increased with the progressive deposition of gold (curves CF). The absence of any band at and around 400 nm, which was the characteristic surface plasmon resonance (SPR) band for silver colloids [31], suggests that no AgCl was reduced to metallic silver during the hydrazine hydrate addition process. Noble metal nanoparticles exhibit a highly tunable plasmon resonance band, whereby light induces collective oscillations of the conductive metal electrons at the nanoshell surface. By varying the shell thickness, core diameter and total nanoparticles diameter, one can allow the optical properties of the nanoshell to be tuned. The SPR bands have been found to be characteristically different for Aucore, AushellAgClcore and Aushell nanoparticles and this has been reected in the optical spectra

of these particles in aqueous dispersions as shown in Fig. 2(a). While AushellAgClcore nanoparticles exhibited maximum SPR absorption band at 530 nm in reverse micelles, this band has been red shifted to 590 nm with an apparent broadening between 500 and 800 nm when dispersed in water. Leaching of AgCl core from AushellAgClcore nanoparticles by aqueous NH3 with the formation of hollow gold nanoparticles caused a blue shift in the SPR band from 590 nm to 534 nm in aqueous dispersion. Replacement of AgCl with water within the cavity of the gold nanoshells caused a considerable decrease of refractive index of the core of the nanoparticles. Liang et al. [32] have reported that in the nanoshells of gold, a substitution of polystyrene in the core by tetrahydrofuran caused a blue shift of the SPR band. Of course, the size and thickness of the gold nanoshell also modulate the SPR band of gold. Fig. 2(b) shows the absorption spectra for Aushell nanoparticles dispersed in water (curve B), supernatant obtained after treatment of AushellAgClcore nanoparticles with dilute NH3 (curve A) and the solution obtained by the addition of hydrazine hydrate to the supernatant solution (curve C). Clearly, curve B shows absorption maxima for the gold shell nanoparticles with no sign of the presence of any colloidal Ag. While the supernatant solution (dilute solution of Ag(NH3)+Cl) shows no 2 characteristic absorption maxima (curve A), the appearance of an absorption peak at lmax 400 nm on the addition of reducing agent to the supernatant solution conrmed the formation of colloidal Ag. The 400 nm

HRP + AgNO3

HRP + NaCl

HRP in AgCl

(Reaction I) HAuCl 4 (Reaction II)

HRP in AgCl

N2 H 4
HRP in AgCl

(Reaction III)

Extraction process

dil. NH 3 aq.
HRP in AgCl HRP

Ag(NH 3 )2 Cl

(Reaction IV)
Reaction I-IV. Reaction scheme.

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AgCl

Aucl3

50uL HH

170 uL HH

270uL HH

1% HH

Absorbance (a.u.)

(F) (E) (D) (C) (B) (A)

350 400 450 500 Wavelength (in nm) 550 600 650

Fig. 1. Absorption spectra at various stages of the synthesis. Curves AF show absorption for A: AgCl nanoparticles in reverse micelles; B: absorption after addition of HAuCl4 solution; CF: formation of metallic gold on the surface of AgCl nanoparticles on periodic addition of reducing agent.

peak is red-shifted on further addition of hydrazine hydrate. The absence of any peak at 400 nm for Aushell nanoparticles and subsequent appearance in supernatant solution (obtained by washing the AushellAgClcore nanoparticles with dilute NH3) upon addition of a reducing agent unambiguously shows that aqueous ammonia diffuses inside the core shell to form the corresponding metal complex and diffuses out of the gold shell. The porous nature of the nanoparticles is further conrmed by the enzyme kinetics. 3.3. Enzyme kinetics studies The ultimate goal of this work was to predict whether an enzyme can be used in vivo for therapeutic purposes without any degradation, and exhibiting no immunological reactions. This would be possible if we see that (i) the entrapped enzyme is not leached out of the hollow gold nanoparticles and (ii) the enzyme exhibits its activity while it is entrapped. HRP is a robust enzyme and its lyophilised powder does not show any decrease of activity. Moreover, HRP entrapped in nanoparticles showed enzymatic activity even after lyophilisation. An aqueous dispersion of lyophilised, HRP-doped, hollow gold nanoparticles was ltered through Millipore lter (100 kDa cutoff) so that free enzyme (molecular weight 40 kDa) was ltered but not the nanoparticles. The ltrate containing free enzyme was treated with o-dianisidine and H2O2 and the reaction was allowed to complete in 2 h. The enzyme concentration in the ltrate was measured spectro-

photometrically from the concentration of the oxidised product of o-dianisidine by measuring the OD at 445 nm. The experiment was continued for 45 days and it was observed that no detectable concentration of HRP was found in the ltrate from zero to 45 days (data not shown), indicating that the enzyme had not leached out through the pores of gold nanoshells during the experimental period. The analysis of the MichaelisMenten parameters of free HRP, HRP entrapped in hollow gold nanoparticles and Aucore nanoparticles was done from the LineweaverBurk (LB) plots (data not shown), which revealed that the kinetics is diffusion dependent. The relatively high afnity of free enzyme for the substrate is noted from the Michaelis constant, K m ; of 76 mmol/ml, while the enzyme entrapped in the cavity of hollow gold nanoparticles is 737 mmol/ml. However, the HRP did not show any activity when trapped inside Aucore nanoparticles. The kcat values calculated from the LB plots showed 1:23 107 s1 for free enzyme, and 1:90 105 s1 for entrapped enzyme in hollow gold nanoparticles. The reduced afnity for the substrate, as revealed from the comparison of K m values, may be accounted for by factors such as constrained diffusion of the substrate through the pores of the gold layer [33] (strained diffusion of the oxidised product outside the pores of nanoshell of gold), conformational distortion of the entrapped enzyme in the restricted volume of the cavity, etc. [34]. These results clearly highlight that the surface of the hollow gold nanoparticles is porous to the substrate and allows the reaction

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Intensity (a.u.)

(C) (B)

(A)

200

300

400

500

600

700

800

900

1000

1100

(a)

Wavelength (nm)

(C)

Intensity (a.u.)

(B)

(A)

300

400

500

600

700

800

900

1000

1100

(b)

Wavelength (nm)

Fig. 2. (a) Absorption spectra of solid gold nanoparticles (curve A), gold-coated AgCl nanoparticles (curve B), and hollow gold nanoparticles obtained after treatment of core-shell particles with dilute ammonia solution (curve C). All are dispersed in water; (b) Absorption spectra of supernatant obtained after treatment of AushellAgClcore nanoparticles with dilute NH3 (curve A), Aushell nanoparticles dispersed in water (curve B), and the solution obtained by addition of reducing agent (hydrazine hydrate) to the supernatant solution (curve C).

between HRP and substrate within the cavity of hollow gold nanoparticles. The enzyme entrapped into the cavity of gold nanoshell should be safe from proteases degradation inside the body. This is possible if any large molecule is prevented from entering the cavity of the nanoshell. To observe this, the activity of the entrapped enzyme was also studied against a large substrate molecule,

DexoDA conjugate (molecular weight 410 kDa), and compared with the activity of free HRP with the same conjugate (Fig. 3). It is clear from the gure that the entrapped enzyme showed absolutely no activity with the conjugate. The diffusional constraint of such a big molecule across the pores of the nanoshell and consequent inability to reach the proximity of the enzyme is thought to be responsible for no reaction. Moreover, it

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0.02

6749

0.015

(A)

Intensity (a.u.)

0.01

0.005

(B)
0 0 10 20 30 Time (sec.) 40 50 60

-0.005

Fig. 3. Time-dependent absorbance of the oxidised product of Dextran-o-Dianisidine conjugate in the presence of free horseradish peroxidase (HRP) (A) and HRP entrapped in hollow gold nanoparticles (B).

becomes reasonably clear that since DexoDA conjugate (molecular weight 410 kDa) cannot diffuse through the pores of the Au surface, HRP molecule with a larger size (molecular weight 40 kDa) cannot be leached out from the Au shell. 3.4. Size determination 3.4.1. Dynamic light scattering (DLS) A representative DLS spectrum of AushellAgClcore colloids in the reverse micelles (Fig. 4) shows that the average diameter of the particles formed was about 4550 nm. As it is known that the particle size is always dependent on the size of the aqueous core of the reverse micelles in which the particles have been prepared, we also prepared AushellAgClcore nanoparticles in the reverse micelles at W o 42 using the same protocol as above. The particles size was found to be around 90 nm diameter. 3.4.2. TEM image Fig. 5a shows the resulting cluster of hollow nanoparticles of gold. It was observed that gold nanoparticles each of about 5070 nm average diameters were present in an aggregated manner. A close look at the TEM picture of hollow gold particles (Fig. 5b) showed that the hollow gold particles were well dispersed with a mean diameter of 5070 nm, and consisted of void cores, i.e. the central portion is lighter than the edges. The TEM image of bigger particles prepared in reverse micelles of W o 42 showed that the average particle diameter was around 9095 nm. The particles in this case were also spherical in shape with a narrow size distribution (Fig. 6a). It is interesting to

note that the particles are hollow from inside, seen as a lighter region, with a surrounding shell of nearly 15 nm thickness. The hollow feature of these nanoparticles could be more evident from the comparison of this TEM image with that of AushellAgClcore nanoparticles as shown in Fig. 6b. The inset of Fig. 6a representing the TEM picture of hollow particles showed very distinctly that ca. 90 nm diameter particles were hollow from inside with a ca. 15 nm thick shell. 3.4.3. XRD To determine the crystallographic nature of the hollow gold nanoparticles, electron diffraction and XRD experiments were carried out. XRD can address the structural information for a large portion of the sample, while transmission electron diffraction can provide structural information for the selected areas of the sample. Fig. 7 shows the powder XRD pattern obtained from hollow Au nanoparticles. The peaks were found to be broad, as was normally observed for ultra low size nanoparticles. The particle size was calculated from the XRD peak at d 1:438 using the Scherrer equation [35] t 0:9l=B cos yB , where t is the particle diameter, l is the X-ray wavelength, B is the full width at half maximum (FWHM) of the diffraction peak and yB is the diffraction angle. The average particle diameter calculated using the Scherrer equation came out to be nearly 45 nm (2y 64:75), which corresponded well with those determined from TEM (50 nm) and DLS (45 nm). Similarly, the average particle diameter for sample II (W o 42), as determined from XRD, DLS, and TEM,

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turns out to be 105, 90 and 95 nm, respectively. The average particle diameter as determined from the various different methods is almost the same. The XRD pattern can be successfully indexed at facecentered cubic (FCC) packing with a 7:46 nm. However the similar XRD pattern can also be successfully indexed as hexagonal close packing of gold nanoparticles with the parameter of a 5:25 nm. Wang et al. made a very similar observation recently during the characterization of colloidal gold nanoparticles pre-

Intensity (a.u.)

pared in aqueous solution [36]. The above XRD experiments did not uniquely determine the colloidal crystal with either hexagonal close packing or cubic close packing. Therefore, a selected area transmission electron diffraction (TED) technique was adopted to collect further crystallographic data for structural determination. Selected area TED was performed on randomly sampled individual (hollow) gold particles. The TED pattern showed one strong ring and a few isolated spots (Fig. 8). This is consistent with the presence of nano-sized particles, which do not diffract strongly. The TED pattern originating from a single gold sphere showed rings indicating that the sphere was made out of polycrystalline gold. As the micrograph was ( taken at 100 kV (l 0:0386 A) using a camera constant of 100.0 cm, the radius of the rst ring is nearly 1.70 cm, which may be indexed to (1 1 1) reection of FCC gold (2.29 A). Further, considering two spots at almost identical distance of average radius r 1:95 cm, they may be indexed to (200) reection in cubic gold. The TED thus proved the hollow Au to be FCC.

4. Conclusion In the reverse micellar system, hollow gold nanoparticles of sizes less than 100 nm diameter entrapping enzyme HRP have been synthesised using the softchemical method. Optical spectra clearly showed the gradual growth of gold layer on silver chloride surface with the progressive addition of reducing agent, hydrazine hydrate. AgCl core was removed by treatment

50 Diameter (nm)

500

5000

Fig. 4. Representative dynamic laser light scattering spectrum of goldcoated AgCl colloids in reverse micelles for sample prepared at W o 17.

Fig. 5. Transmission electron microscopy (TEM) images of (a) a cluster of hollow gold nanoparticles prepared at W o 17 and (b) hollow gold nanoparticles (closer picture).

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Fig. 6. TEM images of (a) hollow gold nanoparticles prepared at W o 42 and (b) gold-coated AgCl nanoparticles. Inset: TEM picture of an isolated hollow particle.

Fig. 7. Powder X-ray diffraction of hollow gold nanoparticles.

with dilute ammonia solution to obtain Auhollow nanoparticles. Enzyme entrapped in the hollow cavity of Auhollow nanoparticles shows enzymatic activity and it follows MichaelisMenten kinetics, showing that the direct encapsulation of the enzyme is possible without causing its degradation. Further, the inactivity of the entrapped enzyme against DexoDA conjugate showed that large substrate molecules, however, are being

prevented from entering into the cavity of the nanoparticles probably due to the small pore size. The porous nature of the Auhollow nanoparticles was also established by reducing the silver-amine complex leached out of the nanoshells and observing the characteristic SPR of silver at 416 nm and a blue shift of Au nanoparticles. TEM pictures indicated the cavity inside the core of the gold particles, and therefore justify the formation of hollow

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Fig. 8. Transmission electron diffraction pattern of randomly sampled hollow gold nanoparticles.

nanoparticles. FCC close packing arrangement of gold atoms has been evidenced from the XRD and TED studies. Since the enzyme is not leachable out of the nanoparticles, and the substrate can enter into the cavity of the nanoparticles through the pores of the nanoshell, it may be presumed that these systems can be effectively used in enzyme therapy without causing any untoward immunological reaction in the living system.

Acknowledgement PS gratefully acknowledges the Principal, St. Stephens College, Delhi for extending all facilities. PKP thanks the Director, IPFT, Gurgaon for being allowed to do this work at Delhi University. References
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