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«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»

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Characterization of Overall doxorubicin-PDPH-PEG Coating Complete adsorption of doxorubicin-PDPH onto gold nanoparticle After verifying maximum loading of doxorubicin-PDPH onto gold nanoparticle with UV-vis absorption spectra, fluorescence of resulting complex was measured. The emission wavelength ranges 500-800nm with excitation wavelength at 471nm.

Background noise was subtracted from the original spectra to result in doxorubicin signal. Then, for further verification of complete adsorption of doxorubicin-PDPH onto gold nanoparticle, supernatant was collected after centrifugation at 2000g for 20minutes and fluorescence of any unbound doxorubicin was measured. As a control, equal concentration of pure doxorubicin was mixed with equivalent amount of gold nanoparticles. Then, fluorescence of pure doxorubicin-gold nanoparticle and its supernatant was measured as above.

Full coverage of doxorubicin-PDPH-gold nanoparticle with PEG without replacing bound doxorubicin-PDPH To confirm any replacement of bound doxorubicin-PDPH by addition of CH3OPEG-SH, fluorescence of Au-DOX-PEG with various percentages of PEG coating was compared to fluorescence of free doxorubicin-PDPH and doxorubicin-PDPH-gold nanoparticle (Au-DOX) complex. Furthermore, optimum concentration for full coverage of Au-DOX by PEG was determined by zeta-potential and Dynamic Light Scattering (DLS) measurements. Gradually, increasing amount of PEG was added to Au-DOX then zeta-potential and size of the resulting complex were measured at each adding step.

Various concentrations of PEG were added to Au-DOX until saturation point was reached that there was no more changes in numerical values for both zeta-potential and size.

SERS Measurements SERS spectra were measured on HoloLab Raman microscopy. The excitation wavelength was at 785 nm from a diode laser. Laser power was 20 mW with the focus area of 15 μm in diameter. SERS spectra of pure gold nanoparticle, pure doxorubicin, pure doxorubicin-PDPH, AU-DOX, Au-DOX-PEG, and gold with free doxorubicin were measured. Each sample was placed on a glass slide and laser beam was focused 300 s for each sample. Resulting SERS spectra was corrected by subtracting the background.

pH-dependent drug release test Au-DOX and Au-DOX-PEG, synthesized from the same batch according to the method listed above, was divided equally in volume for each time point and placed in pH 4 citric acid or pH 7.4 PBS buffer. All release study was carried out at 37°C. At each time point of 24, 48, 72, 96 hr, Au-DOX and Au-DOX-PEG were centrifuged at 2000g for 20 minutes and supernatant was collected. Comparing to concentration (equivalent to 100% doxorubicin release) of pure doxorubicin-PDPH in each buffer, concentration of released doxorubicin (from collected supernatant) was quantified against pure doxorubicin-PDPH fluorescence spectra.

In vitro drug delivery study Tu686 cells were cultured on four different 96 well plates designated for 24, 48, 72, and 96 hour time points. For each time point, triplicates of Au-DOX-PEG (0.3 μg DOX/ mL and 0.2nM of gold nanoparticle), pure doxorubicin (0.3 μg DOX/ mL), and Au-PEG (0.2 nM), synthesized from the same batch, were added to cells and incubated at 37°C accordingly with time. At each designated time point, MTT assay kit (Sigma) was used to measure cell viability. MTT assay measures the cellular reduction of MTT by the mitochondrial dehydrogenase of viable cells to form blue formazan crystals as product.

These crystals can be measured spectrophotometrically by obtaining absorbance with a scanning multiwell spectrophotometer. Detailed procedure was followed from information sheet provided by Sigma. Briefly, MTT powder was reconstituted with 1X PBS and added to 10% of culture medium volume. 150uL of reconstituted MTT solution was added to each well and continued to culture for 2 hours in the incubator. After incubation, 150uL of MTT solubilization solution was added to the original culture to dissolve crystals. Dissolved blue formazan crystals were detected at a wavelength of 570nm, and background absorbance of 96 well plates at 690nm was subtracted from the original 570nm readings.

3.4 RESULTS AND DISCUSSION Characterization of doxorubicin-gold nanoparticle system Doxorubicin-PDPH conjugate (dox-PDPH) was synthesized in methanol (Figure 3.1(a)). PDPH acts as a linker and introduces thiol functional group to ensure adsorption of doxorubicin onto gold surface. Doxorubicin itself contains an amine group, but it has been reported that bonds between gold and sulfur group (~50 kcal/mol) [131-133] is much stronger than bonds between gold and amine group (3-6 kcal/mol) [133]. PDPH linker also contains acid-sensitive hydrazone bond that chemically bound doxorubicin is released under slightly acidic conditions of pH ~ 5 [130, 134], which resembles intracellular endosomal and lysosomal pH conditions. The doxorubicin-PDPH conjugate was water-soluble and was stable when stored at 4°C in dimethyl sulfoxide.

Figure 3.1.

Chemical Synthesis and Self Assembly of Gold Nanoparticle System (a) Chemical synthesis of doxorubicin-hydrazone linker conjugate (DOX-PDPH); (b) Schematic illustration for synthesis of multifunctional drug delivery system and its pHdependent doxorubicin release Concentrations of dox-PDPH and gold nanoparticle were quantified by UV-vis spectroscopy. For dox-PDPH, standard curve at 495nm was created. The 60nm gold nanoparticle had the maximum absorption peak at 531nm and Beer-Lambert law was used to calculate the concentration of gold nanoparticles (extinction coefficient of 3.531 x 1010 M-1 cm-1). The UV-vis spectra of dox-PDPH-gold nanoparticle resembled the spectra of pure gold nanoparticle that fluorescence spectra was used to further assist in analysis.





Adsorption of dox-PDPH onto gold nanoparticle was studied via UV-vis and fluorescence spectra. Similar to the method used by Cheng, Y. et al., considering planar geometry for dox-PDPH on gold surface and inherent chemical bond lengths of the system, it was found that single dox-PDPH molecule has a theoretical footprint of ~1.08nm2. Thus, a single 60nm gold nanoparticle can hold 10513 dox-PDPH molecules for complete surface coverage. However, experimental findings indicate that a single 60nm gold nanoparticle holds maximum of ~2147 dox-PDPH molecules and be colloidally stable. This is equivalent to coating 20% of available surface area (~0.1 wt-%) for 60nm gold nanoparticle. It was found that increasing the surface area coverage to 25% and furthermore to 33% resulted in aggregation of gold nanoparticles, as indicated by slight bump in red-wavelength region of UV-vis spectra (Figure 3.2(a)). Thus, there is a concentration dependence of doxorubicin coating that dox-PDPH can occupy maximum 20% of total surface area of 60nm gold nanoparticle. The resulting dox-PDPH-gold nanoparticle system (Au-DOX) was soluble in water and was stable without any aggregation.

Figure 3.2.

Characterization of Dox-PDPH Loading (a) UV-vis spectra indicate that maximum of 20% of gold surface can be coated with dox-PDPH for colloidal stability;

(b) Fluorescence spectra indicate quenching of dox-PDPH on gold surface compared to partial quenching of pure doxorubicin with gold nanoparticles Fluorescence spectra were used to further verify adsorption of dox-PDPH onto gold. When dox-PDPH was conjugated to gold nanoparticles in water, it was quenched on gold surface (Figure 3.2(b)). Previous studies also report quenching of fluorescent dyes on metallic particles when they are chemisorbed onto the surface [135-137].

Furthermore, fluorescence quenching on metallic surface is observed for distance of few nanometers [138, 139], which suggests proximity of doxorubicin onto gold surface linked via short PDPH linker.

When Au-dox was centrifuged, the supernatant did not contain any free doxorubicin (Figure 3.2(b)). In contrast, when equal concentrations of pure doxorubicin was added to the same amount of gold nanoparticles in water, fluorescence spectra (Figure 3.2(b)) indicate that doxorubicin was partially quenched and free doxorubicin was detected in the supernatant. Thus, this indicates that dox-PDPH is completely bound onto gold surface. A measurable change in fluorescence intensity resulted from dynamic displacement of adsorbed citrate on gold nanoparticle by dox-PDPH. We believe that dox-PDPH has formed a covalent bond, or chemisorbed, with gold surface via thiols from PDPH linker, whereas pure doxorubicin was loosely bound onto gold surface through weak electrostatic interactions, or physisorbed, between positively-charged amine group of doxorubicin and negatively-charged gold nanoparticle [140].

Characterization of PEGylated doxorubicin-PDPH-gold nanoparticle system PEG is commonly used in biomedical applications to increase solubility in water and enhance biocompatibility of nanoparticles. PEG provides colloidal stability for Audox system that PEG protects gold nanoparticles from physiological conditions and prevents aggregation [141, 142]. PEG also serves as a protective barrier for bound doxPDPH on gold surface. Furthermore, PEG reduces adsorption of cellular proteins and increases the circulation time of nanoparticles [143].

To find the optimum PEG density for colloidal gold stability, DLS and zeta potential measurements were used. After subtracting the gold surface area (20%) occupied by dox-PDPH, the free, available surface area was coated with various concentrations of PEG. As we increased the PEG concentration, saturation point was reached for both DLS and zeta potential measurements (Table 3.1).

Table 3.1.

Change in Surface Charge (ζ-potential) and Size of Au-dox as More PEG is Added to Result in Au-dox-PEG System

–  –  –

Indicates the amount of excess PEG added to free, available surface area on gold surface † after adsorbing dox-PDPH For DLS measurements, Au-dox-PEG kept increasing in size until it reached ~75nm. As more PEG is added to Au-dox, PEG initially binds in a mushroom conformation then changes its conformation to brush mode for full coverage [144, 145].

Mushroom mode is characterized by low surface grafting density and polymer tends to “lie” close to the surface that multiple points of a single polymer is covering the surface.

On the other hand, brush conformation is characterized by high surface grafting density and polymer tends to “stand up” that polymer is attached by a single point on the surface.

Thus, as the conformation changes from mushroom to brush modes, nanoparticle size will increase and the size will stop increasing until all the anchoring sites on the gold surface are saturated. For zeta potential measurements, Au-dox-PEG became more positive in charge as more PEG was added to the system. PEG has neutral charge and gold nanoparticle has negative charge. As more PEG is added to Au-dox, negative charge of gold nanoparticle is shielded and offset by more neutral charge of PEG. Both DLS and zeta potential measurements agreed in saturation PEG value to be between 100% and 150% coverage of the free, available surface area. This saturation PEG value resulted in PEG footprint of ~ 0.35 nm2, which is consistent with the literature footprint value [146]. Thus, this also supports our dox-PDPH footprint to be a good approximation on 60nm gold nanoparticle. Centrifuging Au-dox-PEG did not affect the values for DLS and zeta potential. Findings in DLS and zeta potential measurements were supported with salt stability test that fully PEGylated Au-dox-PEG was stable in 1X PBS and 0.5M NaCl solution (data not shown).

Besides Au-dox-PEG colloidal stability, we wanted to ensure that bound doxPDPH is not affected by addition of PEG. After conjugating dox-PDPH onto gold, various concentrations of PEG were added to Au-dox then fluorescence measurements were taken. Fluorescence spectra (Figure 3.3) indicate that addition of PEG, especially excess amount of PEG, did not affect bound dox-PDPH and there was no detectable replacement of bound dox-PDPH. Au-dox-PEG fluorescence spectra overlapped with Au-dox fluorescence spectrum.

Figure 3.3.

Fluorescence Spectra of Au-dox-PEG with Various Concentrations of PEG Indicate No Detectable Replacement of Bound Doxorubicin-PDPH on Gold Surface Compared to Au-dox and Pure Dox-PDPH If there was replacement of bound dox-PDPH, there should be an increase in fluorescence intensity due to presence of free doxorubicin in Au-dox-PEG solution. As seen in Figure 3.2(b), when free doxorubicin is present in the mixture of gold nanoparticle, fluorescence of free doxorubicin is not completely quenched by the gold.

Thus, increase in concentration of free doxorubicin in solution will result in increase in overall fluorescence intensity.

Surface Enhanced Raman Scattering (SERS) and Doxorubicin When 20% of gold surface was covered by dox-PDPH, SERS signal was present in both Au-dox and Au-dox-PEG (Figure 4.4). 60nm gold nanoparticle is an appropriate size for SERS at near-infrared excitation as previous research reports 60-80nm in diameter gold nanaoparticles exhibit most efficient SERS at red (630-650nm) and near infrared (785nm) excitations [147]. Doxorubicin SERS spectrum was characterized by major peaks at 1242, 1261, 1438, and 1603 cm-1 for Au-DOX-PEG system, where the Raman shift (cm-1) values were consistent to those in previous studies [148, 149].

However, when equivalent concentration of free doxorubicin was added to gold nanoparticles, no SERS signal was present. Thus, in contrast to physisorbed free doxorubicin, only covalently tethered dox-PDPH induces SERS for non-aggregated gold nanoparticles. SERS is also distance dependent that SERS is only present when SERS tag is placed within few nanometer of metallic surface [150, 151]. For Au-dox, doxorubicin is conjugated to PDPH linker ( 1nm) to gold surface, which is well within the specification to induce SERS.

Figure 3.4.

Surface Enhanced Raman Scattering (SERS) Spectra of Doxorubicin-PDPH on Non-Aggregated Gold Nanoparticle (Au-DOX and Au-DOX-PEG) Compared to SERS for Pure Doxorubicin and Gold Nanoparticle Mixture (spectra are shifted on purpose for better visualization).



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