Synthesis of Iron Oxide Nanocrystals
The iron oxide nanocrystals (NCs) were synthesized by the thermal decomposition of iron–oleate complexes in a solution of oleic acid surfactants and octadecene solvent.13
First, 2.2 g of iron(III) chloride hexahydrate (Sigma, 98%) and 7.4 g of sodium oleate (TCI, 95%) were dissolved in a mixture of 16.3 mL of absolute ethanol and 12.2 mL of water and mixed with 28.5 mL of hexane. The solution was refluxed for 4 h. The mixture was then washed with water several times in a separatory funnel, and the hexane was removed from the mixture by using rotary evaporation. The synthesized iron–oleate complex was then dried under vacuum overnight. One gram of the iron–oleate complex was dissolved in a solution of 177.3 μL of oleic acid (Aldrich, 90%) and 7.1 mL of octadecene (Aldrich, 90%). The mixture was placed under vacuum and heated at 80 °C for 30 min. It was then stirred vigorously under nitrogen flow, heated to 320 °C at a rate of 3 °C/min, and kept at that temperature for 1 h. After the mixture had cooled to room temperature, 5 mL of hexane was added, and the NCs were precipitated by adding an excess of ethanol. The NCs were separated from the solution by centrifugation. The NCs were then washed twice in a solution of 1:3 hexane–ethanol and dried under vacuum.
Mesoporous Silica Formation
The dried oleate-capped iron oxide NCs were dissolved in chloroform. Two milliliters (10−20 mg/mL) of the NCs solution was mixed with 0.4 g of cetyltrimethylammonium bromide (CTAB, Aldrich, 95%) and 20 mL of water. The mixture was then sonicated and stirred vigorously, and the chloroform solvent was boiled off from the solution. The aqueous CTAB–iron oxide NCs solution was filtered through a 0.44 μm syringe filter to remove any large aggregates or contaminants. One milligram of fluorescein isothiocyanate (FITC, Sigma, 90%) was dissolved in 545 μL of absolute ethanol and mixed with 2.2 μL of aminopropyltriethoxysilane (APTS, Aldrich, 99%) for 2 h. Five milliliters of the aqueous CTAB-stabilized NCs solution was added into a solution of 43 mL of distilled water and 350 μL of sodium hydroxide (2 M) and heated to 80 °C. For higher concentration of iron oxide materials, the solution may need to be heated at lower temperature (65−70 °C) in order to avoid the coalescence of the mesoporous silica in forming large clumps of materials. After the temperature had stabilized, 0.6 mL of the ethanolic FITC–APTS solution was mixed with 0.5 mL of tetraethylorthosilicate and added slowly into the aqueous solution containing the CTAB-stabilized NCs. After 15 min of stirring, 127 μL of 3-(trihydroxysilyl)propyl methylphosphonate (Aldrich, 42%) was added into the mixture, and the solution was stirred for another 2 h. The synthesized materials were centrifuged and washed with methanol. The CTAB surfactants were removed from the mesopores by dispersing the as-synthesized materials in a solution of 160 mg of ammonium nitrate (Fisher) and 60 mL of 95% ethanol and heating the mixture at 60 °C for 15 min. The materials were then centrifuged and washed with ethanol.
Gold– and Silver–Mesoporous Silica
Gold NCs were synthesized by following the Brust method.26
First, 180 mg of gold(III) chloride trihydrate (Aldrich, 99.9%) was dissolved in 15.3 mL of water and mixed with 40.6 mL of toluene solution containing 1.1 g of tetraoctylammonium bromide (Aldrich, 98%). The solution was stirred vigorously for 30 min before addition of 102.3 μL of dodecanethiol (Aldrich, 98%). Next, 12.7 mL of aqueous solution of 192.1 mg of sodium borohydride (Alfa Aesar, 97%) was added slowly to the mixture. After further stirring for 3 h, the aqueous layer was removed using a separatory funnel, and the toluene was removed using rotary evaporation. The solids were dissolved in a minimal amount of toluene, precipitated with absolute ethanol, and collected by centrifugation. After the process was repeated two more times, the solids were dried under vacuum.
Silver NCs were synthesized by following the method developed by Hiramatsu and Osterloh.27
First, 50 mg of silver acetate (Aldrich, 99%) was dissolved in 2.5 mL of oleylamine (Aldrich, 70%) and added quickly into a boiling toluene solution. The mixture was refluxed and stirred vigorously for 12 h. Most of the solvent was removed using rotary evaporation until ~5 mL remained in the container. The silver NCs were precipitated by adding methanol into the mixture and recovered by centrifugation. The process was repeated two more times using a minimal amount of hexane and an excess of methanol before drying the solids under vacuum.
To synthesize the gold–mesoporous silica NPs and silver–mesoporous silica NPs, a procedure similar to that used to make the iron oxide–mesoporous silica NPs was followed.
Folic Acid Modification
To attach folic acid to the iron oxide–mesoporous silica NPs, 20 mg of the materials (after removing the CTAB using the ion-exchange method) were washed with dimethyl sulfoxide (DMSO) and resuspended in DMSO. In a flask, 0.1 mg of folic acid (Sigma, 98%) and 0.05 μL of APTS were mixed in 1 mL of DMSO. Next, 0.03 mg of N-hydroxysuccinimide (Aldrich, 98%) and 0.05 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Alfa Aesar, 98%) were added into the mixture and stirred for 2 h. In a separate flask containing 4 mL of toluene and the NPs–DMSO suspension, the folate–APTS solution was added, and the mixture was stirred for 20 h at room temperature. The materials were recovered by centrifugation, washed twice with toluene, and dried under vacuum.
The modified materials were loaded with either camptothecin (CPT, Sigma, 95%) or paclitaxel (TXL, Sigma) by incubating 10 mg of the materials in a solution of 1 mg of drugs and 0.25 mL of DMSO for 4 h. After the drug-loaded NPs were removed from the suspension by centrifugation and the supernatant was removed completely, the materials were then dried under vacuum. The drug-loaded NPs were washed and sonicated with water before being resuspended in aqueous solution.
In order to determine the amount of drugs that were inside the NPs, the aqueous drug-loaded NPs suspension was incubated at 4 °C for 6 h before centrifugation to show that the drugs were not being slowly released from the mesopores. The resulting supernatant was mixed with the previous supernatant solution from the washing process and measured using UV/vis absorption spectroscopy. The drug-loaded NPs pellet was resuspended and sonicated in DMSO (or methanol for TXL-loaded NPs) and collected by centrifugation. The process was repeated two more times (~15 min total time) to ensure that the drugs were completely removed from the pores. The DMSO (or methanol) supernatants were then measured using UV/vis absorption.
Human cancer cell lines PANC-1 and BxPC3 were obtained from the American Type Culture Collection, and human foreskin fibroblasts (HFF) were a generous gift from Dr. Peter Bradley at UCLA. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal calf serum, 2% l-glutamine, 1% penicillin, and 1% streptomycin stock solutions. The media were changed every three days, and the cells were passaged by trypsinization before confluence.
The cellular uptake of the NPs was confirmed by fluorescence microscopy. The cells were incubated in an eight-well cell culture chamber with the NPs and then washed with DMEM and PBS to remove the NPs that did not enter the cells. The cells were then stained with DAPI solution and/or WGA-Alexa Fluor 594 before being monitored using the fluorescence microscope.
Cell Viability Assay
The cytotoxicity assay was performed by using a cell-counting kit from Dojindo Molecular Technologies, Inc. Cells were seeded in 96-well plates (5000 cells/well) and incubated in fresh culture medium at 37 °C in a 5% CO2/95% air atmosphere for 24 h. The cells were then washed with PBS, and the medium was replaced with a fresh medium containing the NPs or the drug-loaded NPs. After 24 h, the cells were washed with PBS and incubated in fresh medium for an additional 48 h. The cells were washed with PBS and incubated in DMEM with 10% WST-8 solution for another 2 h. The absorbance of each well was measured at 450 nm with a plate reader. Since the absorbance is proportional to the number of viable cells in the medium, the viable cell number was determined by using a previously prepared calibration curve (Dojindo Co.).
Western Blot Analysis
Cell lysate was separated by gel electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate and then transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline (TBS) containing 5% (w/v) skimmed milk. After being washed with TBS containing 0.1% Tween 20 (Sigma), the membranes were incubated overnight at room temperature with α-folate receptor (F-15) antibody (Santa Cruz Biotechnology) diluted with TBS. After being washed, the membranes were incubated for 2 h at room temperature with the second antibody (Santa Cruz Biotechnology). Bands were detected with an ECL system (Amersham Pharmacia Biotech.)
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed using a platinum taq DNA polymerase high-fidelity RT-PCR kit. The cells were harvested from culture dishes. RNA was extracted using TRIzol reagents (Invitrogen), and 1 μg of RNA was reverse-transcribed. The resulting cDNAs were amplified by PCR reaction using primers for human folate receptor (forward, AACACAGCTGCTGCTCCTTCTAGT; reverse, AACAGGGCAGGGATTTCCAGGTAT). The PCR reaction was conducted for 40 cycles. Each cycle consisted of 30 s at 94 °C, 30 s at 57 °C, and 1 min at 72 °C. The reaction products were electrophoresed in 1% TAB agarose gel. The gel was stained by ethidium bromide and then photographed.
Magnetic Resonance (MR) Imaging
The MR imaging experiments were performed on a Siemens Avanto 1.5-T MR system. An extremity coil was used for the data acquisition, and the pulse sequence used was a T2-weighted turbo spin–echo sequence with the following parameters: TR = 4620 ms, slice thickness = 3 mm, TE = 98 ms, field of view = 157 × 180 mm, number of acquisitions = 1. For the experiments to observe the MR contrast effect of the NPs within the cells, PANC-1 cells were incubated with either the iron oxide–mesoporous silica NPs or plain mesoporous silica NPs29
for 1 and 4 h periods, trypsinized, and then placed in a 0.2 mL PCR tube. Each tube contained approximately 105