«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
Numerous types of ligands with thiolated terminals can be used to for place exchange reaction on a single gold nanoparticle. Thus, the capability to coat gold surface with various ligands has brought gold nanoparticle as a promising “multifunctional” agent for biomedical applications.
Rationale for Using AuNP in this study Model System for Biomedical Applications in Nanotechnology There are several reasons why gold nanoparticle is suitable for study of size effect in nanotechnology. First, gold nanoparticles are widely used in biomedical research such as cancer diagnostics, cellular imaging, photothermal therapy, and targeted drug delivery applications [56, 65-70, 119], which are representative fields of nanotechnology. Second, gold nanoparticle can represent the other hard surface metallic nanoparticles such as quantum dots and iron oxide nanoparticles inside the biological system. Third, unlike the liposomes or polymeric particles, gold nanoparticles can be synthesized with greater size (1-100nm) and shape variabilities (rods, cages, spheres, shells) in a controlled manner.
Fourth, due to its unique optical properties, various analytical methods such as UV-vis spectrophotometry, TEM, SERS, darkfield microscopy, fluorescence, ICP-MS, etc. can be used. Polymeric nanoparticles are difficult to trace within the biological system that usually fluorescence tags are applied for detection, whereas gold nanoparticle itself can be used as a tag for detection due to its unique optical property. Fifth, unlike the other metallic nanoparticles such as quantum dots, gold is biocompatible that there is minimal toxicity. Sixth, gold nanoparticle is inert and does not degrade inside the biological system that it can be easily identified with various analytical techniques. Polymeric nanoparticles used for size studies can change its property (via interactions with biological molecules) when applied to the biological system and it can further degrade over time, making it difficult to trace them for size study. Seventh, chemical and physical properties of gold nanoparticle can be easily modified via surface coatings with various ligands that it does not require complicated chemical synthesis steps as seen for polymeric nanoparticles. Finally, the biggest strength of using gold nanoparticle for size effect study is that the amount of gold in the biological system can be easily “quantified”, rather than qualitatively referring to the presence and absence of the nanoparticle in various organs, by ICP-MS since it is not a readily available element in the biological systems. Other biocompatible metallic particle such as iron oxide nanoparticle exists, but this nanoparticle degrades over time and iron (Fe) is prevalent in biological system that can lead to false results for ICP-MS-based size effect studies. Similarly, it is difficult to quantify the different amount of polymer in various organs since polymer nanoparticle or polymer-fluorescence tag nanoparticle can degrade over time and change its property.
Gold Nanoparticle Design In This Study Figure 2.11 illustrates the general design of the gold nanoparticle system used in this thesis. There are four types of gold nanoparticle systems used in this thesis (in order of appearance): 1) 60nm gold core-drug-PEG system, 2) 5nm gold core-drug-PEG system, 3) 5nm gold core-PEG system, and 4) 60nm gold core-PEG system. For the size effect study for drug delivery applications, pro-drug approach was taken where chemotherapeutic agent (i.e. doxorubicin) was modified with acid-sensitive hydrazone linker. It was designed in a such way that drug will be intact during circulation and will be released in an appropriate condition (pH 5.5) for efficient delivery of the chemotherapeutic agent. PEG layer was added to give colloidal stability and non-fouling surface for longer circulation time and biocompatibility in the biological system.
Schematic Design of Gold Nanoparticle Used In This Thesis
2.4 CONCLUSION In conclusion, gold nanoparticle is an ideal candidate for studying the size effect in cancer nanotechnology. Gold nanoparticle represents many research fields in the cancer nanotechnology, and its unique optical properties render multiple analytical methods for characterization. By applying different sizes of gold nanoparticle (5nm versus 60nm) in various studies in this thesis, we can find the ideal size candidate that can be applied universally in many research fields in cancer nanotechnology. Here, we will particularly focus on the drug delivery application of gold nanoparticle and the size effect on therapeutic efficacy, toxicity, biodistribution, and clearance.
3.1 ABSTRACT We report development and characterization of multifunctional drug delivery system (Au-dox-PEG) for treatment and SERS spectroscopic detection of tumor.
Doxorubicin, a therapeutic agent and a SERS tag, was chemically conjugated to gold nanoparticle via pH-sensitive hydrazone linker then PEG was added to develop Au-doxPEG. Doxorubicin occupied maximum of 20% of total surface area of gold nanoparticle to result in colloidal stability. SERS spectra were detected for non-aggregated Au-doxPEG at near-infrared wavelength, and doxorubicin release was time and pH dependent.
Consistency in release profile and in vitro cell viability results supports the efficacy of Au-dox-PEG system. Thus, the development of Au-dox-PEG multifunctional system raises exciting opportunities for simultaneous spectroscopic detection and therapy for tumors in the future.
3.2 INTRODUCTION Cancer nanotechnology has gained great interest during recent years [4, 12, 13, 19, 20, 104, 120, 121]. Adopting a size scale equivalent to biological molecules, nanometer-scale particles (1~100 nm in diameter) contain large surface area for modification with targeting ligands, anticancer agents, imaging agents, and other small molecules. Due to its small size, there is an increased uptake of nanoparticles by cells , ultimately improving the availability of particular agent. Recently, semiconductor quantum dots [4-7] and iron oxides [9-11] have been used to detect and image tumor cells for diagnostics, whereas polymer based nanoparticles [12-14] have been used for treatment of cancer. 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 or imaging agents.
Here, we report development and characterization of a multifunctional nanoparticle consist of PEGylated colloidal gold and anticancer agent. The multifunctional delivery system demonstrated therapeutic effect on tumor cells along with in vitro spectroscopic detection based on surface enhanced Raman scattering (SERS). The idea of using gold nanoparticle as a carrier for drug delivery is recent [50, 123, 124]. Gold nanoparticles confer several advantages such as biocompatibility  and size-tunability (synthesizing various sizes) . Furthermore, chemical properties are easily altered by attaching various ligands for surface modification. Finally, due to its unique optical properties, Raman signal from adsorbed reporter molecules can be increased up to 1014~1015 orders in magnitude, allowing single molecular level spectroscopic detection [102-104]. This phenomenon, well known as SERS, is an ultrasensitive analytical method. SERS provides characterization and spectroscopic detection of reporter molecules and further allows dynamic monitoring of small chemical changes occurring at the interfaces of gold nanoparticles [103, 105].
Recently, Qian, X. et al. reported successful in vivo tumor detection through SERS obtained from targeted PEGylated gold nanoparticle with raman tag (malachite green isothiocyanate). For our system, raman tag was replaced by doxorubicin, serving a dual function of chemotherapeutic agent and SERS reporter molecule. Eliminating the possibility of adding any other SERS tag, sole presence of doxorubicin increases the loading efficiency of chemotherapeutic agent onto gold surface.
Doxorubicin, an anthracycline derivative, is commonly used chemotherapeutic agent for various malignancies such as solid tumors of breast, esophagus, liver, and softtissue sarcoma . Despite its high anti-tumor activity, doxorubicin presents side effects by not only inducing tumor cell death but also affecting normal, healthy cells, especially leading to irreversible cardiotoxicity [127, 128]. Furthermore, doxorubicin exhibits poor water solubility and narrow therapeutic index that it is difficult to significantly increase the dosage at target sites . To overcome these side effects along with addition of SERS spectroscopic detective function, doxorubicin conjugate systems has been developed: doxorubicin was modified with pH-sensitive hydrazone linker and attached to gold nanoparticle. Hydrazone linker, PDPH, was also chosen due to its pH sensitivity. Hydrazone bond is stable under neutral pH conditions, but it is cleaved under mild acidic conditions of less than pH 5 , resembling the endosomal and lysosomal environment. Furthermore, hydrazone linker provides thiol bond for adsorption of doxorubicin onto gold nanoparticle surface. To increase biocompatibility and stability of gold colloids, resulting doxorubicin-gold conjugates were coated with PEG. This self-assembled, biocompatible model system was characterized by various techniques and SERS signal was measured. PEGylated drug-gold system was stable in salt solutions (0.5M NaCl solution and 1X phosphate buffered saline) and released doxorubicin in pH and time dependent manner. Also, the resulting drug-gold model system not only demonstrated SERS signal but also had similar cytotoxicity effect on tumor cells compared to equivalent concentrations of free doxorubicin. Thus, this multifunctional system raises exciting opportunities for simultaneous spectroscopic detection and therapy for tumors in the future.
3.3 MATERIALS AND METHODS Materials Chemical Reagents Doxorubicin hydrochloride was purchased from Polymed Science (Houston, TX).
Citrate-stabilized gold colloid, 60nm in size, was obtained from Ted Pella, Inc. (Redding, CA). Hydrazone linker, 3-[2-Pyridyldithio]propionyl hydrazide (PDPH), was acquired from Pierce (Rockford, IL). Poly(ethylene glycol) (CH3O-PEG-SH) of molecular weight 5000 was purchased from Rapp Polymere (Germany). Methanol, acetonitrile, dimethyl sulfoxide, citric acid, and MTT based in vitro toxicology assay kit were all obtained from Sigma (St. Louis, MO). Mili-Q deionized water (Millipore, 18.2 MΩ cm-1) was used throughout the experiments. All of the products were used without modification or purification unless as noted.
Instrumentation Nanoparticle surface charge (zeta potential) and size were measured by ZetaSizer Nano-ZS90 (Malvern Instrument). Adsorption spectra were obtained through ultravioletvisible spectrophotometer (Beckman Coulter DU530). Fluorescence of nanoparticle was evaluated by Fluoromax-2 (Jobin Yvon-Spex, Horiba Group), equipped with xenon arc lamp. SERS spectra were obtained from HoloLab Series 5000 VPT sytem (Kaiser Optical Systems, Inc.) with the excitation wavelength at 785nm. NMR spectra were obtained from INOVA 600. Finally, scanning multiwell spectrometer, Synergy 2 (Biotek), was used to read absorption of blue formazan crystals for MTT assay.
Cell Line Tu686 (human head and neck carcinoma cell line) was a gift from Dr. XiangHong Peng (Emory University). Tu686 cells were cultured in DMEM/F-12 (Mediatech, Inc.; Manassas, VA) containing 10% fetal bovine serum (American Type Culture Collection; Manassas, VA) and penicillin-streptomycin solution (Mediatech, Inc.;
Manassas, VA). Cells were grown in a 37°C humidified incubator containing 5% CO2.
1X phosphate buffered saline (1X PBS) was purchased from Mediatech, Inc.
Synthesis of doxorubicin-PDPH Doxorubicin was conjugated to hydrazone linker, PDPH, in a similar method reported previously by Greenfield, R. et al. with slight modifications . Briefly, doxorubicin-HCl (11.340 mg, 0.017 mmol) and excess PDPH (10.340 mg, 0.045 mmol) were dissolved in methanol (7 mL) and stirred at room temperature in the dark for 6 days.
Methanol from reaction mixture was evaporated by rotary evaporator and acetonitrile was added to obtain a precipitate. Precipitate was collected through centrifugation and reprecipitated twice with the same procedure indicated above to remove excess PDPH.
Final product of 9.450 mg (71 %) was dissolved in dimethyl sulfoxide and stored at 4°C.
Obtained 1H NMR spectrum was consistent with the values reported in literature .
Maximum Loading of doxorubicin-PDPH onto gold nanoparticle Maximum loading of doxorubicin-PDPH was governed by colloidal stability and surface area coverage of gold nanoparticle. Initially, total surface area of 60nm gold nanoparticle was calculated (4*pi* radius2). Then, doxorubicin-PDPH footprint (~1.08 nm2) was estimated based on chemical structure and known chemical bond lengths.
Various concentrations of doxorubicin-PDPH solution (corresponding to 20, 25, and 33% surface coverage; diluted in deionized water) were added drop-by-drop manner into 300uL of stirring gold nanoparticle solution (2.6E10 particles/mL). UV-vis absorption spectra were used to verify the colloidal stability of resulting doxorubicin-PDPH-gold nanoparticle complex.
PEGylation of doxorubicin-PDPH-gold nanoparticle complex Level of PEGylation for doxorubicin-PDPH-gold nanoparticle complex was determined based on surface area coverage and salt stability of resulting complex. Based on the results from maximum doxorubicin-PDPH loading onto gold surface, available surface area for binding can be calculated. For example, when 20% of gold nanoparticle surface area was covered by doxorubicin-PDPH, then there is 80% surface area available for other molecules to bind onto gold surface. 25, 50, 75, 100, 150, and 175 percent of the remaining (80%) gold surface area was coated with 5K CH3O-PEG-SH. PEG solution (12.1 μM) was added very slowly drop-by-drop into stirring doxorubicin-PDPH-gold nanoparticle complex solution. PEG-doxorubicin-PDPH-gold nanoparticle (Au-DOXPEG) complex was centrifuged at 2000g for 20 minutes to remove any unbound CH3OPEG-SH. Finally, Au-DOX-PEG complex was incubated in 1X PBS and 0.5M sodium chloride solutions to test salt stability. Au-DOX-PEG colloidal stability in salt solutions was determined by absorption spectra from UV-vis spectrometer.