«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
This work is significant by providing an insight on a potential ideal candidate for gold nanoparticle-based drug delivery system that uses small (5nm) gold nanoparticle to study therapeutic efficacy on solid tumor. To our knowledge, we are the first team to investigate in detail for 5nm gold nanoparticle drug delivery system in vivo and its behavior for better understanding of the gold nanoparticle-based drug delivery. To date, most of the gold nanoparticle-based drug delivery systems have been focusing on a size scale of 20~35nm.
Here, the application of small-sized 5nm gold nanoparticle as a drug delivery system (Au-DOX-PEG) demonstrated 1) successful accumulation of Au-DOX-PEG at the tumor site via passive targeting by taking advantage of the EPR effect for therapeutic efficacy and 2) resulted in no apparent toxicity to vital organs.
4.3 MATERIALS AND METHODS Materials Chemical Reagents Doxorubicin hydrochloride was purchased from Polymed Science (Houston, TX).
Citrate-stabilized gold colloid, 5 nm 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. Scanning multiwell spectrometer, Synergy 2 (Biotek), was used to read absorption of blue formazan crystals for MTT assay. Gold content was analyzed by ICP-MS (HP 4500, Agilent Technologies). TEM were taken by using Hitachi H7500 highmagnification electron microscope. Finally, Olympus IX71 inverted microscope was used to take brightfield and darkfield images.
Cell Line and Mouse Model Murine breast cancer cell line 4T1 was a gift from Dr. Lily Yang (Emory University). 4T1 cells were cultured in RPMI-1640 (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. 6-7 week old female Balb/C mice were obtained from commercial vendor (Jackson Laboratories). The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University.
Statistical Analysis Statistical analysis was performed using one-way ANOVA followed by multiple comparison Bonferroni’s test. Data were collected from at least three different animals and P0.05 was considered statistically significant.
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 .
Drug Loading Efficiency Adsorption of DOX-PDPH onto gold nanoparticle was studied via UV-vis and fluorescence spectra. Concentrations of DOX-PDPH and gold nanoparticle were quantified by UV-vis spectroscopy. For DOX-PDPH, standard curve at 495nm was created. The 5nm gold nanoparticle had the maximum absorption peak at 514 nm and Beer-Lambert law was used to calculate the concentration of gold nanoparticles (extinction coefficient of 9.696 x 106 M-1 cm-1). 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. When surface coverage per 5nm gold nanoparticle was increased from 25, 100, 150, and 200 %, the corresponding loaded doxorubicin weight percents (wt-%) were 1.42, 5.58, 8.35, 11.12 wt-%, respectively. UV-vis absorption spectra and fluorescence were used to verify the colloidal stability of resulting doxorubicin-PDPH-gold nanoparticle complex.
PEGylation of Gold Nanoparticle-Anticancer Agent System To find the optimum PEG density for colloidal gold stability, dynamic light scattering measurement was used for size change. After subtracting the gold surface area occupied by modified doxirubicin, available surface area was coated with various concentrations of PEG. As we increased the PEG concentration, saturation point was reached for gold-drug-PEG size measurement (Table 1). PEG-doxorubicin-PDPH-gold nanoparticle (Au-DOX-PEG) complex was ultra-centrifuged at 100,000g for 1 hour to remove any unbound CH3O-PEG-SH. Then the resulting gold system was freezed-dried to a powder form. 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.
pH-dependent drug release test Colloidal gold system, 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 5 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-PEG were ultra-centrifuged at 100,000g for 1 hour 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 cytotoxicity study 4T1 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 (7 μg DOX/ mL equivalent), pure doxorubicin (7μg DOX/ mL), and Au-PEG, 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. 150μL 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.
Cellular Uptake of Colloidal Gold Study 4T1 cellular uptake of doxorubicin, gold-drug-PEG, and gold-PEG will be determined by fluorescence and darkfield microscopy. Control, doxorubicin, gold-drugPEG, and gold-PEG groups will be created. Equal concentration of doxorubicin (5 μg DOX/ mL) will be used for doxorubicin and gold-drug-PEG groups, whereas same amount of gold will be used for gold-drug-PEG and gold-PEG groups. Control group will be left untreated. Cells will be treated with according groups and incubated at 37°C for
2.5 hours. Then, cells will be fixed with 3.7% formaldehyde and stained with DAPI for nucleus detection. Cover-slipped cell slide will be then imaged with fluorescence microscope to confirm the uptake of drug and particles by cells.
In vivo study of gold system Murine breast cancer cell line 4T1 will be injected into Balb/c mouse at mammary fat pad, subcutaneously with 2 X 106 tumor cells. When tumor size was approximately 100mm3, mice were divided randomly into group of four (n=5) of control (untreated), pure doxorubicin, polymer-drug-gold conjugate, and polymer-gold conjugate groups.
Drug will be administered through tail-vein injection (2mg DOX/kg and 0.04g Au/kg) and all groups will be treated four times total (every 3 days) except for the control group, which is left untreated. Tumor size and body weight will be measured every 2 days. In addition to tumor size and body weight changes, effect of polymer-drug-gold conjugate on tumor treatment is further evaluated by weighing the harvested tumor and comparing the “actual” size of the tumor amongst various groups. After the sacrifice, various organs including heart, kidney, lung, liver, spleen, brain, and tumor will be collected for various analyses. To verify the uptake of polymer-drug-gold conjugate via EPR effect, concentration and location of gold nanoparticle within tumor tissue will be studied by ICP-MS, histology, TEM, and dark-field microscopy. Evaluations of any toxicity exerted by polymer-drug-gold conjugate on healthy, normal tissue will be mainly investigated by histological analysis. For serum biochemical analysis, whole blood was centrifuged twice at 3000 rpm for 10 minutes in order to separate serum. Using a biochemical analyzer (Type 7170, Hitachi), serum biochemical analysis was carried out to determine the serum level of various proteins. A certified pathologist will compare the stained tissue section from all four groups for any morphological changes occurring in various organs harvested from the mouse.
4.4 RESULTS AND DISCUSSION Chemical Synthesis and Characterization of Au-DOX-PEG 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. Furthermore, doxorubicin exhibits poor water solubility and narrow therapeutic index that it is difficult to significantly increase the dosage at the target sites. To overcome these side effects, doxorubicin conjugate system has been developed: doxorubicin was modified with pH-sensitive hydrazone linker (PDPH) and attached to gold nanoparticle. Hydrazone bond is stable under neutral pH conditions, but it is cleaved under mild acidic conditions of pH less than 5.5 , resembling the endosomal and lysosomal environment. In addition to providing pH sensitivity, hydrazone linker PDPH provides thiol bond for adsorption of modified doxorubicin onto gold nanoparticle surface.
Gold nanoparticle-anticancer agent-PEG (Au-DOX-PEG) drug delivery system was synthesized by first coating the gold nanoparticle with modified doxorubicin (DOXPDPH) then with thiolated methoxy-PEG (Figure 4.1(a)). PEG provides colloidal stability for gold-doxorubicin conjugate (Au-dox) system that PEG protects gold nanoparticles from physiological conditions and prevents aggregation. Furthermore, PEG reduces adsorption of cellular proteins and increases the circulation time of nanoparticles . The self-assembly of gold nanoparticle-anticancer agent-PEG system is a spontaneous process, which resulted in water-soluble, colloidally stable Au-DOX-PEG system with a size of ~17.8 ± 1.3 nm and -2.16 ± 0.217 mV for zeta potential.
Chemical Synthesis and Self Assembly of AU-DOX-PEG To find the maximum drug loading capacity of 5nm gold nanoparticle while maintaining the colloidal stability of gold nanoparticle, UV-vis spectroscopy and fluorescence spectra were used to test the adsorption of series of different concentrations of DOX-PDPH onto gold. When DOX-PDPH was conjugated to gold nanoparticles in water, the fluorescence of doxorubicin was quenched on gold surface (Figure 4.2 “Before Centrifuge”). 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 gold nanoparticle-anticancer agent-PEG system was centrifuged for purification, the supernatant did not contain any detectable amount of unbound doxorubicin up to 5.58 wt-%. However, when excess amount of DOX-PDPH was added to the gold nanoparticle solution, we observed fluorescence of unbound DOX-PDPDH in the supernatant (Figure 4.2 (c)). Thus, the maximum drug loading capacity of 5nm gold nanoparticle was ~5.5 wt-%.