INTRODUCTION:

Photodynamic therapy (PDT) is one of the emerging treatment modalities for cancer that takes advantage of the interaction between light and a photosensitizing agent to initiate cell death^1,2. The conventional photosensitizers viz., diheaematoporphrin ester, photoporphyrin – IX, ALA induced endogenous porphyrin and the second generation photosensitizers such as phthalocyanines and nanpahthalocyanines^3,4 have their own limitations^5,6 and hence their photodynamic activity is affected considerably. In this context, to overcome such drawbacks of conventional photosensitizers, many researchers in recent years have considered the possibility of using the non-toxic semi conducting nanoparticles in PDT^7,8 and it is often referred for example as nano-PDT. It is a promising route to overcome many difficulties associated with traditional PDT. This is because the nanoparticles are having the following advantages over the conventional photosensitizers. They are photostable and nontoxic. They have also high emission efficiency, large absorption cross section, good photoluminescence quantum yields and long emission lifetimes.

Due to their adjustable surface chemistry, they can be modified to become water soluble, biocompatible⁹and target specific. However the use of nanoparticles in nano-PDT is still under primitive stage.

One of the ways to make metal NPs stable at extreme conditions is to protect them with stable and chemically inert shells such as oxides. The catalytic properties of the oxide surfaces modified with the metal core especially photocatalysis are an important aspect^10,11. Nanoshell particles are highly functional materials with tailored properties which are quite different from either of the core or shell material. Their properties can be modified by changing either the constituting materials or core to shell ratio¹². Hence the present study is aimed to synthesize the core-shell type Ag@TiO₂and Au@TiO₂ nanoparticles, characterize them and compare their photodynamic activities on human erythrocytes. Further attempt was also made to understand the nature of mechanism involved in nano-PDT by using these core-shell NPs as photosensitizers.

MATERIALS AND METHODS:

Titanium (IV) isopropoxide was purchased from Sigma Aldrich. Tetrachloroauric acid trihydrate and AgNO₃were obtained from CDH chemicals. All the other chemicals used were of Analar grade. Milli Q water was used. The core-shell type Ag@TiO₂ and Au@TiO₂ were prepared by slight modification of the method described by Renjis. T. et al¹³ In brief 20mM each of Ti (IV) iso propoxide and acetylacetone in 30ml of isopropanol was prepared by sonicating the mixture for 15 minutes. Now 10 mM solutions of AgNO₃ or HAuCl_4.3H₂O in 5ml of milli -Q water was prepared and 20ml of DMF was added and stirred well. To this solution 30ml of the above said sonicated solution was added and strring continued for 10 more minutes. The final mixture was refluxed at 60-70°C for 1 hour. The solution became pink in the case of gold and greenish black in the case of Ag. The refluxing was continued for 1 more hour resulted in the formation of a precipitate which is sonicated for 2 hours to disperse. On adding Toluene the colloidal material was precipitated and washed several times with toluene and redissolved in isopropanol. The solvent was evaporated at room temperature to get a red powder in the case of Au@ TiO₂ and greenish black powder in the case of Ag@ TiO₂ nanoparticles.

UV–visible spectra were measured using a Perkin Elmer Lambda-35 spectrophotometer. The fluorescence spectra were recorded using spectrofluorimeter (FluoroMax-2). X-ray diffraction (XRD) patterns were recorded by X^’pert PRO PANalytical diffractometer operating with CuKa radiation (λ=1.5406A°) source. High resolution transmission electron microscopy (HR-TEM) photographs were taken using a JEOL JEM -3010 Electron microscope operating at 300 KeV. The magnifying power used was 600 and 800K times.

Erythrocyte separation:

Fresh human blood was obtained from healthy volunteers, Health Centre, Anna University and mixed with anticoagulant EDTA in the ratio 3:1. The erythrocytes were allowed to settle for an hour and the plasma leukocytes and thrombocytes were separated by aspirating the supernatant. The sediment was washed 4-5 times with PBS to remove any left out plasma. A stock solution of 0.5% erythrocyte suspension was prepared by diluting 1ml of solution with 39ml of PBS.

Phosphate buffered saline was prepared by mixing 280ml of 0.2M monobasic sodium phosphate and 720ml of 0.2M dibasic sodium phosphate along with 9g of sodium chloride and the pH was found to be 7.3.All the solutions used were prepared in PBS.

Sample Irradiation:

Light from Xenon source filtered at 515nm with 20nm band pass filter was used as the light source for irradiating the sample in the case of Au@TiO₂ and 445nm in the case of Ag@TiO₂. The microtitre plate having wells (2.5cm dia) containing 1ml of the sample was irradiated at different times using different concentrations of Au@TiO₂/Ag@TiO₂nanoparticles. The irradiated cell suspension was centrifuged at 1500 rpm for 10min and the supernatant was pipetted out and its O.D at 413nm was measured using spectrophotometer (Perkin Elmer Lamda 35) to quantify the percentage hemolysis. The same procedure was repeated to study the role of scavengers such as sodium azide¹⁴ and GSH¹⁵ by adding 1ml of each scavenger separately with 1ml of the core-shell nanoparticles in PBS during hemolysis.

RESULTS AND DISCUSSION:

UV – Visible Spectrum:

The absorption spectrum of Ag@TiO₂ (Fig.1) shows band with maximum close to 425 nm. The absorption spectrum of Au@TiO₂is shown in Fig.(2). Here again there is enhanced absorption due to the presence of gold between 400 and 600 nm. The absorbance maximum corresponding to TiO₂ shell is not well resolved. The red shift observed with respect to the core metals in both the cases may be due to increase in the particle dimension and /or change in the dielectric constant of the surrounding matrix upon encapsulation¹³.

Fig.1. UV-Visible absorption spectrum of Ag@TiO₂-NPs

Fig.2. UV-Visible absorption spectrum of Au@TiO₂-NPs

XRD Analysis:

Fig (3a and b) show the X-ray diffraction pattern of as prepared and annealed Ag@TiO₂ (at 650°C in air for 5hrs). The Fig. 3(a) shows that the as-prepared Ag@TiO₂ core-shell nanoparticle is fully amorphous. Fig. 3(b) shows 4 characteristic peaks of nanocrystalline pure Ag@TiO₂ of monoclinic structure (JCPDS # 52-1202). The major diffraction intensity peaks at 2θ, 25.42, 32.182, 37.995, 47.839° were identified to originate from (220), (420) (112) and (040) planes of Ag@TiO₂ respectively. The XRD patterns could be indexed to the C2/c (15) space group, end centered monoclinic structure with cell parameters a = 16.77, b = 7.594, c = 5.044 and β = 102.01. The patters are very well comparable to those reported in literature⁽¹³⁾. The average grain size using Scherrer equation was found to be 39 nm.

Fig.3(a and b).XRD patterns of Ag@TiO₂-NPs.

Fig.4(a) shows the XRD pattern of as prepared Au@TiO₂.There are 3 characteristic peaks at 2θ about 37.77, 43.89 and 63.63 which were found to be originated from (111), (200) and (220) planes of Au@TiO₂ respectively. The intensity of the peaks is very much less as both Au and TiO₂ particles are in nano form. The peaks are characteristic of TiO₂-NPs of face centered cubic structure (JCPDS # 89-3077). The XRD patterns could be indexed to the Fm 3m (225) space group, face centered cubic with cell parameters a = 4.166. The XRD pattern of the as prepared Au@TiO₂ has no peak corresponding to Au metal. The peak widths correspond to an average particle diameter of 37 nm as derived from Scherrer formula.

Fig.4(a and b).XRD patterns of Au@TiO₂-NPs.

Fig 4(b) shows the X-Ray diffraction pattern of Au@TiO₂ annealed at 650°C in air for 5hrs. It shows two characteristic peaks resembling a mixture of anatase form of TiO₂ and gold. The peak at 2θ about 25.45 corresponds to anatse form of TiO₂ (JCPDS# 89-4921) Tetragonal structure with body centered lattice. The peaks at 38.04, 44.05 and 64.06 were identified to originate from (111) ,(200) and (220) planes of Au respectively (JCPDS # 89 – 3697). These XRD patterns could be indexed to the space group Fm 3m (225) space group, face-centered cubic structure with cell parameter a = 4.079. The patters are very well comparable to those reported in literature ⁽¹³⁾. The average particle size calculated was 44 nm.

HR-TEM:

The HR-TEM analysis of single Ag@TiO₂ particle illustrated in Fig.5 shows the formation of nearly spherical core particles and appear to be associated with TiO₂.The boundary between core (Ag) and shell (TiO₂) is very much distinct. The average particle size is found to be35-40nm.The TiO₂ shell thickness ranges from 3-5 nm.

Fig.5.HR-TEM image of Ag@ TiO₂ –NPs.

The HR-TEM image of Au@TiO₂ single particle is shown in Fig.6. A nearly well defined spherical morphology is observed. This image illustrates absence of GNPs aggregation. The particle size is found to be close to 40 nm. The shell thickness is found to be 2-3nm.

Fig.6.HR-TEM image of Au@ TiO₂ –NPs.

Photohemolysis using core-shell Ag@TiO₂ and Au@TiO₂nanoparticles:

As human erythrocytes are considered as semi model cellular systems, photohemolysis studies were carried out in vitro under two different experimental conditions in order to i) understand the dependency of light dose (7.2, 14.3, 21.5J/cm²) at fixed concentrations of both Ag@TiO₂ and Au@TiO₂ core-shell type nanoparticles, and ii) to study the dependency of nano-sensitizer concentrations (50, 100 and 150µg/ml) at fixed light dose

Photohemolysis as the function of fluence:

The percent hemolysis increases with increase influence at fixed concentration of NPs (Fig.7 and 8). The LD₅₀(leathal for 50% hemolysis) is found to be 7J/cm² for Ag@TiO₂ NPs and 13 J/cm² for Au@TiO₂ NPS for 150 µg/ml. Similarly for 100µg/ml the LD₅₀are found to be for 17 J/cm² and 19 J/cm² forAg@TiO2NPs and Au@TiO2 NPs respectively. From this it is clearly observed that the light dose is reduced to half when the sensitizer concentration is increased from 100µg/ml to 150µg/ml in each case. Comparing the LD50 values, Ag@TiO₂ NPs require less amout of fluence for the same concentrations. 100% was achieved in both cases.

Fig.7. Effect of light dose on photohemolysis of Ag@TiO2-NPs

Fig.8. Effect of light dose on photohemolysis of Au@TiO₂-NPs

Photohemolysis as the function of concentration:

Fig.9 and10 show the variation of photohemolysis with concentration of Ag@TiO₂ and Au@TiO₂ NPs respectively at fixed light dose. The LC₅₀ (Leatheal concentration of NP for 50% hemolysis) are found to be 100µg/ml in the case of Ag@TiO₂ and 145µg/ml in the case of Au@TiO₂ for 14.3, J/cm² respectively. As the lysis depends both on light dose as well as concentration of nanosensitizer the nature of photohemolysis is purely a drug mediated one i.e. by Type I/II mechanism.

Fig.9. Effect of concentration on photohemolysis of Ag@TiO₂-NPs

Fig.10. Effect of concentration on photohemolysis of Au@TiO₂-NPs

Effect of Scavengers:

In order to know the mechanism of photohemolysis we carried out hemolysis in the presence of scavengers such as GSH and NaN₃ which are the best known quenchers for superoxide anion and ¹O₂ respectively. The inhibition of photohemolysis was calculated by taking the corresponding percent hemolysis without scavenger as 100. Fig.11 shows the role of scavengers such as GSH for different concentrations of Ag@TiO₂ at a fixed fluence of 14.3 J/cm². When the photohemolysis with 50µg/ml Ag@TiO₂ was carried out with 1ml of 20mM GSH, the percent hemolysis is reduced to 29% whereas with 40mM GSH with same concentration of NP percent hemolysis is reduced to 15 %.Similarly in the case of Au@TiO₂ the percent hemolysis is reduced to 57.4% with 20mM GSH and 24.5% with 40 mM GSH for 50µg/ml concentration. This indicates that the formation of considerable amount of super oxide anion during hemolysis in both cases and among the two sensitizers Ag@TiO₂produces more amount of singlet oxygen than the other. A control experiment was carried out with different concentrations of both NPs without irradiation and it is confirmed that the non-irradiated nano-sensitizers were not toxic to erythrocytes. Similarly non-irradiated core shell NPs with scavengers GSH and NaN₃ showed no cell killing effect.

Fig.11. Effect of scavenger GSH on photohemolysis of Ag@TiO₂-NPs

Fig.12. Effect of scavenger GSH on photohemolysis of Au@TiO₂-NPs

CONCLUSION:

Core shell type Ag@TiO₂ and Au@TiO₂ NPs were synthesized, characterized and confirmed as nanoparticles. In the present study human erythrocyte cells were effectively lysed by photoexcitation of core-shell NPs. The percent hemolysis increased considerably with the increase in the sensitizer concentration as well as the fluence. When the concentration was increased from 100 to 150µg/ml, 100% hemolysis was achieved at 21.5J/cm². Comparing the values of percentage hemolysis for the same concentration of nano-photosensitizersAg@TiO₂ has more photokilling power than Au@TiO₂.The effect of singlet oxygen is more than that of other ROS. Hence though photodynamic activity of both the NPs favours both Type I and Type II mechanisms, the latter one was predominated. The unexposed nano-photosensitizers were found to be non-toxic towards red blood cells. The study concludes that light irradiated Ag@TiO₂ NPs and Au@TiO₂ NPs may be convenient substitutes for the classical photosensitizers such as organic dyes. Further studies are to be carried out on human cell lines and animal tumour models to understand the biopharmacology of those NPs.

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Received on 05.05.2010 Modified on 19.05.2010

Asian J. Research Chem. 4(3): March 2011; Page 387-391