INTRODUCTION:

Photodynamic therapy (PDT) is a minimally invasive treatment modality for cancer and utilizes a photosensitizer which is preferably localized in solid tumours. The photosensitizer is then irradiated with light of suitable wavelength and the subsequent photochemical reaction results in oxygen mediated destruction or modification of the target tissue^1,2. The conventional photosensitizers viz., dihaemato porphyrin ester, photoporphyrin – IX, ALA induced endogenous porphyrin and the second generation photosensitizers such as phthalocyanines and nanpahthalocyanines^3,4have their own limitations⁵. In this context, to overcome such drawbacks of conventional photosensitizers, many researchers in recent years have considered the possibility of using non-toxic semi conducting nanoparticles in PDT^6,7 and it is often referred for example as nano-PDT. It is a promising route to overcome many difficulties associated with traditional PDT. The nanoparticles are photostable and nontoxic. They have 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, biocompatible⁸and target specific. Metal NPs display fascinating optical properties due to the resonance of surface plasmons with visible light at well-defined frequencies⁹. They are emerging as important colorimetric reporters due to their high extinction coefficients, which are several orders of magnitude larger than those of organic dyes¹⁰. They have unusual chemical and physical properties which make them attractive for applications in catalysis, electronics, optics and bio-technology^11,12.

Currently gold nanoparticles find potential applications in biodiagnostics¹³ and therapeutics¹⁴. Modifying colloidal gold nanoparticles with DNA is a new and interesting approach in the development of genetic biosensors¹⁵. Gold and silver nanoparticles have high light-scattering power, as first reported by Yguerabide et.al¹⁶. These particles, also called plasmon – resonant particles, are quench resistant and generate very high signal intensities. A 60nm Au particle is equivalent to 3.3x10⁵ fluoresecin molecules (PDT agent). Attaching bio molecules such as antibodies and DNA to these nanoparticles does not affect their optical properties. The NPs have thus been used successfully as labels in nucleic acid¹⁷ and protein detection¹⁸. GNPs can also function both as a scaffold and fluorescent quencher for the homogenous detection of nucleic acids¹⁹.

However the use of nanoparticles in nano-PDT is still under primitive stage. Hence in the present study gold nanoparticles were synthesized, characterized and used as nanosensitizer for photodynamic therapy.

MATERIALS AND METHODS:

Tetracholoauric acid trihydrate HAuCl₄.3H₂O was purchased from CDH chemicals. Sodium citrate, Sodium borohydride were obtained from Merck and all were Analar grade and Milli-Q water was used.

Gold nanoparticles (GNPs) of 5nm size were prepared by the method reported in the literature²⁰. 18ml of Milli-Q water, 0.5ml of 0.01mM aqueous tetrachloauric acid and 0.5ml of 0.01mM aqueous sodium citrate were taken in a clean Erlenmeyer flask and the resulting yellow solution was stirred for 10minutes 0.5ml of 0.1M aqueous sodiumborohydride was added to it. The colour of the solution changed to burgundy red. The contents of the flask were filtered through a 0.2 micron sortorius cellulose filter paper.

The absorption spectrum of GNPs was taken in a Perkin Elmer Lamda 35 spectro photometer. The fluorescence spectra were recorded using spectrofluorimeter (FluoroMax-2). High resolution transmission electron microscope (HRTEM) images were taken using a JEOL JEM-3010 Electron microscope operating at 300 KeV. The magnifying powers used were 600 and 800 k times. AFM images were taken by a VECO Nanoscope-III atomic force microscope.

Erythrocyte Seperation:

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. The stock solution of 0.5% erythrocyte suspension was prepared by diluting 2ml of solution with 38ml of PBS.

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

Sample irradiation:

Light from Xenon source filtered at 515nm with 20nm band pass filter was used for irradiating the sample. The microtitre plates having wells (2.5cm dia) containing 1ml of the sample were irradiated at different exposure times using different concentrations of Gold nanoparticles. 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 a spectrophotometer (Perkin Elmer Lamda 35) to quantify the percentage hemolysis. The same procedure was repeated to study the role of scavengers such as sodium azide²¹ and glutathione reduced (GSH)²²by adding 1ml of each scavenger separately with 1ml of gold nanoparticles during hemolysis.

RESULTS AND DISCUSSION:

UV-Visible Absorption Spectrum of GNPs:

The UV-Visible absorption spectrum of GNPs is shown in Fig.1. The maximum absorption occurred at about 520nm. As the absorption band was broadened, the GNPs were suggested to have varying sizes.

Fig.1.UV-Visible absorption spectrum of GNPs.

The absorption spectrum was similar to the one reported in literature²⁰. The final concentration of Au particles was calculated from the absorbance value at 519nm assuming 100% yield in the citrate reduction of HAuCl₄. This assumption led to an extinction coefficient of 2.39 x 10⁸ M^-1 cm^-1 at 519 nm for GNPs which is in good agreement with previously reported data²³. Au-nanoparticle aggregation results in visible colour changes. These colour changes were due to a combination of absorption and scattering of light by the GNP solution²⁴.

Steadystate Fluoresence Spectroscopic Characteristics of GNPs:

The fluorescence spectrum of GNPs excited at 470nm is shown in Fig 2. There were two emission maxima one at about 525nm and other at about 560nm. The 525 nm band was more intense and resolved than the 560 band which appeared as a shoulder to 525 nm band. The appearance of two absorption bands is ascribed to the presence of two groups of particles with different sizes, but there might not be much difference in size as the higher wavelength maxima was not resolved. It is also clearly evident from the HR-TEM images, shown below. Hence the nanoparticles corresponding to 525nm might be in more concentration than that of 560nm band.

Fig.2.Fluorescence spectrum of GNPs excited at 470nm.

HR-TEM:

The HR-TEM images of gold nanoparticles are shown in Fig (3a-3c). Fig. 3(a) clearly illustrates particles of nearly uniform size. The particle size was in the range 2 to 6 nm. Two or three particles appear to be associated, but there is not so much agglomeration. The association of particles is due to dipole-induced dipole interactions in addition to Vander Wall’s force.

Fig.3(a).High Resolution Transmission electron microscopy picture of GNPs.

Fig.3(b).High Resolution Transmission electron microscopy picture of GNPs

Fig.3(c).High Resolution Transmission electron microscopy picture of GNPs

Atomic Force Microscopy:

The AFM images of GNPs are shown in Fig. (4a-4c).The particles appear to have different sizes. The particles of size below 25nm are dominating.

Fig.4 (a).AFM picture of GNPs.

Fig.4(b).AFM picture of GNPs.

Fig.4(c).AFM picture of GNPs.

Photohemolysis using GNPs as nanophotosensitizer:

Photohemolysis studies were carried out under two different experimental conditions in order to understand i)the dependence of light dose (7.2,14.3,21.5J/cm²) at fixed concentrations of GNPs and ii) the effect of concentration (25, 50, 75 and 100µg/ml) at fixed light dose.

Photohemolysis as a Function of Light Dose:

Fig. (5) shows the variation of the percentage hemolysis with light dose at fixed concentration of GNPs. During photohemolysis using 25µg/ml the percentage hemolysis increased from 7.2 to 21.5J/cm² i.e. from 18 to 58.8%. The lethal dose LD₅₀ (50% hemolysis) was found to be 19.8 J/cm². On increasing the concentration to 50µg/ml the percentage hemolysis increased. But the increase with light dose from 7.2J/cm² to 21.5J/cm² was very small. At the concentration 75µg/ml, the PDA increased slightly. The LD₅₀ was found to be 7.5J/cm². LD₅₀ is reduced to one third when the concentration was increased from 25 to 75µg/ml. When the concentration of GNP was increased to 100 µg/ml the percentage hemolysis decreased with light dose. At this concentration the LD₅₀ was found to be 7.5 J/cm². The light dose has little effect on increasing the concentration from 75-100µg/ml at this concentration. At higher concentration GNPs (above 100µg/ml) the PDA decreased. This might be due to the shielding effect as reported in the case of conventional dyes²⁵. As the concentration increased the number of GNPs per unit volume increased. It enhanced agglomeration of GNPs and suppressed PDA.

Photohemolysis as a Function of Sensitizer Concentration:

The erythrocytes were also irradiated as a function of concentration of NPs and the hemolysis was watched at three different fluences viz. 7.2, 14.3 and 21.5 J/cm².

Fig.6. shows the variation of photohemolysis with the concentration of GNPs at fixed light dose. At 7.2J/cm²light dose with the increase of concentration of GNPs from 25 to 100µg/ml the photohemolysis increased gradually but from 75-100 µg/ml the increase was small. The LC₅₀ (lethal concentration for 50% hemolysis) at this fluence was found to be 47, 75 and 95 µg/ml which showed that increase of concentration had no effect on hemolysis. At 14.3 J/cm²the lysis percentage increased from 25 to 75µg/ml but at 100µg/ml the percent hemolysis decreased. The LC₅₀ at this fluence was found to be 45 and 95µg/ml. Similarly with 21.5 J/cm²light dose the percent hemolysis increased at lower concentrations and at 100µg/ml it decreased. The LC₅₀ value was found to be 95µg/ml. Comparing LC₅₀ values as the fluence increased from 7.2 to 21.5 the LC₅₀ values were almost same. The results are somewhat different when compared to other nanosensitizers⁽⁵⁾. The nature of cell killing is purely a chemical mediated one i.e. Type I / Type II mechanism.

Fig.5.The Effect of light dose on photohemolysis of GNPs

The Effect of Scavengers:

In order to understand the mechanism, the photohemolysis was carried out in the presence of scavengers such as GSH and NaN₃ which are the best known quenchers for reactive superoxide anion radical and ¹O₂ respectively. The inhibition of photohemolysis was calculated by taking the corresponding percent hemolysis without scavenger as 100%. Fig .7 shows the role of scavenger NaN₃ for various GNP concentrations. When the photohemolysis was carried out with GNPs of 25µg/ml and NaN₃of 45mM the percent hemolysis reduced to 61.8%, but with 90mM NaN₃ the percent hemolysis reduced to 46.5%. Even at higher GNP concentration (50,75,100µg/ml) also NaN₃ reduced percent hemolysis. This clearly indicates the formation of singlet oxygen during photohemolysis.

Fig.7. Effect of scavenger NaN₃ on photohemolysis of GNPs

With 25µg/ml GNPs and 1ml of 20mM GSH the percent hemolysis reduced to 34.8% but with 1ml of 40mM GSH the percent hemolysis reduced to 27.3% (Fig.8). With higher concentration of GNPs (50, 75, 100µg/ml) and in the presence of GSH (20mM and 40mM) the inhibition was same as with 25µg/ml GNPs. At concentrations of GNPs 75µg/ml and 100µg/ml and in the presence of 1ml of GSH the inhibition was less than that at lower concentrations of GNPs. Hence, increase in the concentration of scavengers decreased the percent hemolysis to a considerable amount.

The inhibition effect of GSH was higher than that with NaN₃. Therefore it is confirmed that even though both superoxide anion radical and ¹O₂ were formed during photohemolysis which are the major cause for cell death, the former might be formed more since the inhibition of photohemolysis was more with GSH than NaN₃. Therefore, the photohemolysis of human erythrocytes using GNPs followed both type I and type II mechanisms and of these the former one predominates more than the latter.

Fig.8. Effect of scavenger GSH on Photohemolysis of GNPs

A control experiment was carried out with different concentrations of GNPs without irradiation and it was confirmed that non-irradiated GNPs were not toxic to erythrocytes. Similarly the non-irradiated GNPs with scavengers also showed no cell killing effect.

CONCLUSION:

In the present study the human erythrocyte cells were effectively killed by photo-excited GNPs in vitro. The percent hemolysis unlike other nano photosensitizers showed a decrease in PDA above a particular GNPs concentration. The effect of scavengers such as GSH and NaN₃ indicated formation of considerable amount of ROS during photoexcitation of GNPs. The photohemolysis by GNPs as nano-sensitizer favours both Type I and Type II mechanisms and the former one predominates more. The unexposed GNPs were found to be non toxic towards red blood cells. Similarly the unexposed GNPs with scavengers also showed no cell killing effect. Hence it is concluded that light irradiated GNPs could be a convenient substitute for the classical photosensitizers such as dyes.

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Received on 24.06.2010 Modified on 08.07.2010

Asian J. Research Chem. 4(1): January 2011; Page 58-63