«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
It is important to note that doxorubicin SERS spectrum (Figure 3.4) resulted from colloidally stable singlets of Au-dox and Au-dox-PEG. Furthermore, doxorubicin was covalently bound to gold surface via an acid-sensitive linker, thus resulting in fluorescence quench. Previous research demonstrated SERS spectrum for mixture of free, unmodified doxorubicin and aggregated metallic sols or films. [148, 152-154] Metallic sols were aggregated by salts or DNA complexation and metallic surface was modified to trap doxorubicin to induce SERS.
The ability to induce SERS for Au-dox-PEG provides several advantages. First, doxorubicin itself can serve as SERS tag for spectroscopic detection of tumor. SERS utilizes the intrinsic SERS of the bound molecule, mostly with delocalized pi electrons, onto metallic particle and it does not require any labeling . Au-dox-PEG SERS spectrum was measured with 785nm laser, allowing near-infrared (NIR) window detection of SERS tag for reduced in vivo background noise . Combining the enhanced permeability and retention (EPR) effect of tumor cells and NIR window detection, Au-dox-PEG provides the potential to spectroscopically locate tumor cells.
Second, in conjunction with UV-vis and fluorescence spectra, SERS spectra of Au-doxPEG provides additional piece of evidence to support chemisorption of dox-PDPH onto gold surface. Third, SERS spectra of Au-dox-PEG can be used for real-time monitoring of doxorubicin release. As shown in Figure 3.4, non-covalently bound free doxorubicin does not induce SERS. This indicates that when hydrazone bond from PDPH is cleaved to release doxorubicin to the surroundings, there will be decrease in SERS intensity over time.
pH dependent release study of doxorubicin Figure 3.5 shows pH-dependent release profile of doxorubicin linked to hydrazone bond of PDPH. For Au-dox, ~80% and ~20% of bound doxorubicin were released at pH 4 and pH 7.4 in 24 hours, respectively. More doxorubicin was released over time, especially for pH 4 condition, which led to four times more doxorubicin release at the end of 96 hours for pH 4 condition compared to that of neutral condition.
Previous studies also reported increased hydrolysis of hydrazone bond and rapid release of doxorubicin in acidic conditions compared to neutral conditions [130, 156].
pH-Dependent Doxorubicin Release Over Time Au-dox-PEG system also demonstrated pH-triggered release of doxorubicin, but there was a delayed release kinetics compared to Au-dox. Release profile for pH 4 and pH 7.4 conditions were similar up to 24 hrs; however, at 48 hr, there was a difference in release profile that ~17% and ~9% of bound doxorubicin were released at pH 4 and pH 7.4, respectively. At the end of 96 hr, approximately four times more doxorubicin was released at acidic pH compared to neutral pH for Au-dox-PEG. There are two possible reasons for the delayed release of doxorubicin: (1) is due to diffusion barrier created by PEG coating and (2) is due to interactions between the polymer and the drug. PEG chains interact with one another that complex is formed amongst PEG chains by hydrogen bonding . As polymer chain length increases and more inter-polymer complexes are formed, the release rate of the drug is decreased. Also, complexation affects PEG conformation that polymer coils provide additional diffusion barrier for more tortuous path for drug release [157, 158]. Because PEG is a hydrophilic polymer, as the hydrophobicity of the drug increases, the diffusion rate of the drug decreases.
There are several reasons why hydrolysis of hydrazone bond under acidic condition was responsible for doxorubicin release. First, dox-PDPH was completely bound onto gold surface (Figure 3.2(b) and Figure3. 3) unlike pure doxorubicin mixed with gold nanoparticles. In order to observe fluorescence in the supernatant, quenched doxorubicin on gold surface must be cleaved away from the gold surface. Bound doxorubicin could be released via cleavage of hydrazone bond or Au-S bond of PDPH linker. However, latter is highly unlikely due to strong Au-S bond energy [131-133].
Second, there was a high pH-dependent drug release that doxorubicin was released at a faster rate (four fold increase) in pH 4 buffer than in pH 7.4 buffer. Although increased aqueous solubility of doxorubicin in acidic pH may contribute to doxorubicin release, change in pH from neutral to acidic for a pH-insensitive system (micelle containing only pure doxorubicin) did not significantly increase the release rate of the drug [159-161].
Thus, hydrolysis of hydrazone bond is responsible for release of doxorubicin into surroundings.
The pH-dependent Au-dox-PEG is an optimal system for minimizing the drug release during circulation and maximizing the drug release under mildly acidic conditions of endosomal or lysosomal vesicles.
In vitro drug delivery study MTT assay with Tu686 cell line was used to study the anticancer efficacy of Audox-PEG system. Cell viability was inversely related to doxorubicin activity that absence or minimal efficacy of doxorubicin resulted in increased cell survival. Cell viability for each group (pure doxorubicin, Au-PEG, and Au-dox-PEG) was compared to the control group which was free of doxorubicin. Initially, pure doxorubicin had higher anticancer efficacy compared to Au-dox-PEG. Figure 6 shows that pure doxorubicin had an immediate effect during 24 hour period where ~74% of cells were alive compared to the control. There was a gradual cell viability decrease for pure doxorubicin treated cells that about 21 % of cells survived at the end of 96 hour period.
In vitro Drug Release Study on TU 686 Cells via MTT Assay [0.3 μg DOX/ mL] According to Figure 3.6, Au-dox-PEG had minimal efficacy until 48 hour that approximately 80% of cells survived. Beyond 48 hours, there was a significant decrease in cell viability for Au-dox-PEG treated cells. Interestingly, In vitro cell viability results were consistent with the data obtained from the release profile (Figure 3.5), indicating 48 hour is a critical time point for doxorubicin release from Au-dox-PEG. This time delay is advantageous to elongate in vivo circulation time of drug delivery system in the future.
Furthermore, compared to the control, cell rounding and decrease in cell density were apparent under the microscope (data not shown) at 48 hour time point. At the end of 96 hour period, ~34% cells survived with Au-dox-PEG treatment.
Compared to the pure doxorubicin 96 hour toxicity (~21% cell viability), the slight lower toxicity of Au-dox-PEG could have resulted from slow release of doxorubicin within PEG shell and different cellular localization of Au-dox-PEG system compared to pure drug. However, Au-dox-PEG provides potential to significantly increase the accumulation and dosage at target sites by EPR effect and eventually increase the anticancer efficacy compared to pure drug.
To eliminate any uncertainty stemming from gold nanoparticle toxicity itself, we used PEGylated gold nanoparticle (Au-PEG), free of doxorubicin, as another control.
When equivalent gold concentration for Au-PEG was used to treat cells, Au-PEG had minimal toxicity on Tu686 cells and had average of 93% cell viability throughout the 96 hour incubation period.
Overall, Au-dox-PEG is an effective drug delivery system. Even though Au-doxPEG has slower release rate of doxorubicin, gold nanoparticle successfully delivered doxorubicin to cells to release it under acidic conditions. Au-dox-PEG’s anticancer efficacy caught up with that of pure doxorubicin after 48 hours and we observed sustained release of doxorubicin over time.
3.5 CONCLUSION Here, we have shown the feasibility of developing and characterizing a pHsensitive multifunctional drug-gold delivery system for treatment and SERS spectroscopic detection of tumor. Multifunctional delivery system, comprising of poly(ethylene glycol), doxorubicin, pH-sensitive linker, and gold nanoparticle (Au-doxPEG), successfully delivered anticancer agent to tumor cells and displayed surface enhanced raman scattering (SERS) for spectroscopic detection. Doxorubicin, modified with pH-sensitive hydrazone linker and attached to gold nanoparticle, served as therapeutic agent and spectroscopic detection agent. There was a concentration dependence of doxorubicin binding to the gold surface that doxorubicin can occupy maximum 20% of total surface area of gold nanoparticle. SERS spectra were detected for non-aggregated Au-dox-PEG at near-infrared wavelength. Also, Au-dox-PEG displayed pH and time dependent release of doxorubicin. Decrease in pH to acidic condition resulted in increased release of doxorubicin compared to neutral condition. It took approximately 48 hours to see significant anticancer efficacy of Au-dox-PEG.
Consistency in release profile and in vitro cell viability results supports the therapeutic efficacy of Au-dox-PEG. Anticancer efficacy of Au-dox-PEG caught up with that of pure doxorubicin after 48 hours that we observed controlled release of doxorubicin over time.
Thus, the development of Au-dox-PEG multifunctional nanoparticle raises exciting opportunities for simultaneous spectroscopic detection and therapy of tumors in the future.
4.1 ABSTRACT Our results demonstrated that functionalized 5nm gold nanoparticle-based drug delivery system represents a highly attractive candidate as a potential drug delivery carrier for cancer nanotherapy. Our smart design of combining prodrug approach (drughydrazone linker) with PEGylation renders controlled release of anticancer agent and colloidal stability characteristics to the system, thus making gold nanoparticle-anticancer agent-PEG (Au-DOX-PEG) system a promising drug delivery platform for in vivo applications. Due to its size, Au-DOX-PEG accumulated at a high concentration at the tumor site via enhanced permeability and retention (EPR) effect and displayed therapeutic efficacy against tumor. In contrast to pure doxorubicin which resulted in heart, kidney, and lung toxicities, passively targeted Au-DOX-PEG system did not display any apparent toxicity in vital organs.
4.2 INTRODUCTION The ultimate goal of drug delivery is 1) to increase the bioavailability of the drug and 2) to reduce the toxicity to healthy cells. Nanoscopic systems, such as gold nanoparticles, can alter the pharmacological and therapeutic properties of the drugs being incorporated and overcome any intrinsic toxicity or poor bioavailability of the drug.
Normally, tumor cells are characterized by leaky vasculature and defective lymphatic drainage that results in enhanced permeability and retention (EPR) effect .
EPR effect prolongs nanoparticle residence time and also selectively “traps” nanoparticles for improved efficacy of therapeutic agents. By taking advantage of these size and EPR effect, one can increase the efficacy of drug at the tumor site.
The idea of using gold nanoparticle as a carrier for drug delivery is recent [50, 123, 124, 143]. Previously, gold nanoparticles have been mainly used for chemical sensing, photothermal therapy, and diagnostic purposes. Gold nanoparticle makes an ideal candidate as a drug delivery platform for cancer therapy due to its several attractive physical and chemical properties. Gold nanoparticles are inert and have low in vivo toxicity compared to the other metallic materials [93, 94]. Gold nanoparticles are easily synthesized with various shapes and sizes that it confers size and shape-tunability .
Furthermore, chemical properties are easily altered by attaching various ligands via covalent thiol-gold bond interactions for surface modification. Also, gold nanoparticle is generally insoluble and rarely present in the biological tissues that it makes easy to detect even at low concentrations using methods such as ICP-MS . Finally, its unique optical property allows spectroscopic detection (SERS) [102-104] and microscopic visualization (transmission electron and darkfield microscopy) [97, 162].
Currently, only a few gold nanoparticle-based anticancer drug delivery systems have been studied, compared to the polymer-based delivery systems [124, 143, 163-165].
Few cases are reported for delivery of small molecule therapeutic agents by gold nanoparticles for cancer therapy, which relies on passive (EPR effect) and active targeting [50, 78, 166]. For example, 13nm gold nanoparticle was coated with methotrexate (MTX) for treatment of lung cancer . Similarly, 26nm gold nanoparticle was coated with TNFα, thiolated poly (ethylene glycol), and paclitaxel and resulted in 10-fold more delivery of TNFα and paclitaxel to the tumor site for effective treatment . When anticancer agent gemcitabine was conjugated to gold nanoparticle with VEGF antiangiogenic molecule, it also showed therapeutic efficacy . Finally, 30nm gold nanoparticle coated with PEG, arginine-glycine-aspartic acid (RGD) peptide, and nuclear localization signal peptide resulted in nuclear-targeting gold nanoparticles that causes apoptosis of cancer cells .
To this date, most of the gold nanoparticle-based drug delivery system utilizes gold nanoparticles that are larger than 10nm, with an exception of recent study on 2nm gold nanoparticle loaded with paclitaxel . However, this 2nm gold nanoparticlepaclitaxel system study only focuses on characterization and loading efficiency, omitting any biological effects of the system. Moreover, it has been reported that gold nanoparticles of 1-2nm are highly toxic to both healthy and cancerous cells [45, 46].