Synthesis of Ac-EEC peptide
The Ac-EEC peptides were synthesized by standard solid-phase methodology using N9-fluorenyl-methyloxycarbonyl chemistry (Fmoc)-amino acids with acid-labile side chain protecting groups. Solid phase syntheses were carried out on a Prelude automatic peptide synthesizer (Protein Technologies, Inc., Tucson, AZ) using commercially available Rink resin. The resin (0.05–0.1 g) was swollen and washed 5 times with 1.5 mL of dimethylformamide/dichloromethane (DMF)/CH2Cl2. Fmoc groups were removed by 3 treatments with 1.5 mL of 20% piperidine/DMF for 5 min each. For coupling, 3-fold excesses of Fmoc-amino acids, diisopropylcarbodiimide, and 1-hydroxybenzotriazole were added to 3 mL of DMF/CH2Cl2. This procedure was repeated once. After coupling and deprotection steps, the resins were washed 3 times with 3 mL of DMF/CH2Cl2. On completion of the peptide chain elongation, the N-terminal was capped by acetic anhydride, and resins were again washed 3 times with 3 mL of CH2Cl2 and were treated with a mixture of trifluoroacetic acid:triisopropylsilane:H2O (95:2.5:2.5) for 15 min. The combined filtrates were left at room temperature for 1–2 h, and the volumes were reduced in vacuum. Peptides were precipitated in ice-cold ethyl ether, collected by centrifugation, washed 2 times with ethyl ether. The peptides were purified by reverse-phase high-performance liquid chromatography on an Agilent 1200 system (C-18, Vydac, 10 × 250 mm, 10 µm; Agilent Technologies, Santa Clara, CA). Liquid chromatography–high-resolution electrospray ionization mass spectrometry (LC–MS) acquired in the positive ion mode was used to identify the peptides.
Synthesis and characterization of PEG-Ac-EEC(Pt)-HGNPs and F-Ac-EEC(Pt)-HGNPs
HGNPs were synthesized according to our previously reported procedure (Melancon et al. 2008). Briefly, cobalt nanoparticles were first synthesized by a chemical reduction method in the presence of sodium borohydride. The resulting cobalt nanoparticles served as a sacrificial template for the reduction of chloroauric acid and subsequent deposition of metallic gold on their surfaces. The obtained HGNPs nanoparticles were stabilized with sulfhydryl methoxy polyethylene glycol (MeO-PEG-SH, 5 kDa). Ac-EEC peptide (4 mg/mL, 125 µL) was added to PEG-coated HGNPs (1 mg) in 1 mL of water overnight at room temperature. The mixture was centrifuged and washed with deionized water three times to remove free peptide; the resulting Ac-EEC-HGNPs were mixed with 1 mL of aqueous cisplatin (2.5 mg/mL) and the mixture subjected to shaking at 60 °C in a water-bath for 2 h. The solution was further shaken for 72 h at room temperature and then centrifuged at 7000 rpm for 15 min. The product, Ac-EEC(Pt)-HGNPs, was washed 3 times with deionized water and collected for further characterization.
Folic acid was introduced to the gold surface of Ac-EEC-HGNPs through a thioctic acid–terminated PEG linker (F-PEG-TA) according to previously reported procedures (Lu et al. 2010a). Briefly, Ac-EEC-HGNPs (1 mg) was mixed with both PEG thiol (PEG-SH; 200 µg) and F-PEG-TA (50 µg) in 1 mL water at room temperature overnight to afford the folic acid–conjugated product F-Ac-EEC-HGNPs. Similarly, the non-targeting counterpart PEG-Ac-EEC-HGNPs was synthesized by mixing 250 µg of PEG-SH with Ac-EEC-HGNPs without F-PEG-TA. The resulting conjugates, F-Ac-EEC-HGNPs and PEG-Ac-EEC-HGNPs, were finally complexed with cisplatin to yield F-Ac-EEC(Pt)-HGNPs and PEG-Ac-EEC(Pt)-HGNPs by following the same procedures used for the synthesis of Ac-EEC(Pt)-HGNPs.
Drug payload (Pt to Au ratio) was calculated by determining the amounts of Au and Pt following dissolution of the samples in aqua regia (Cheng et al. 2009). Au and Pt contents were quantified by inductively coupled plasma mass spectroscopy (ICP-MS; Galbraith Laboratories, Inc., Knoxville, TN) on a Varian 810 model (Varian, Inc., Walnut Creek, CA). HNO3 (2% v/v) was recorded as a blank solution prior to analysis of each individual sample to monitor memory effects.
High-resolution transmission electron microscopy (HRTEM) micrographs were obtained using JEOL 2100 field emission TEM gun operating at 200 kV (JEOL, Tokyo, Japan). TEM specimens were made by evaporating one drop of sample solution onto ultrathin carbon type-A 200 mesh copper grids (Ted Pella Inc., Redding, CA). Measurements of dynamic light scattering (DLS) were conducted with a Zetaplus (Brookhaven, Inc, Holtsville, NY) in low volume disposable cuvettes and the mean of at least three measurements was taken. The energy-dispersive X-ray spectroscopy (EDS) mapping of gold and platinum from HRTEM was obtained by using a Gatan Imaging Filter (Gatan, Inc., Pleasanton, CA). X-ray photoelectron spectroscopy (XPS) measurements were collected by using a PHI Quantera SXM XPS/ESCA system (ULVAC-PHI, Inc., Kanagawa, Japan). A monochromatic Al X-ray source at 100 W was used with an analytical spot size of 0.15 mm × 1.4 mm and a 45° takeoff angle, with pass energy of 26.00 eV. Unless noted, samples were referenced against an internal Au 4f7/2 line at 84.0 eV or Pt 4f7/2 line at 71.2 eV. Powder XPS photoelectron lines were referenced against the C 1 s signal at 284.50 eV.
Release of Pt from PEG-Ac-EEC(Pt)-HGNPs
PEG-Ac-EEC(Pt)-HGNPs (0.4 mg/mL) were dispersed in 2.0 mL deionized water or saline solution in a 5-mL test tube at room temperature. An NIR laser with a peak centered at 808 nm was generated from a master oscillator power amplifier Ti3+:sapphire tunable laser (LT-2211A, LOTIS TII, Minsk, Republic of Belarus) pumped by a pulsed Q-switched Nd:YAG laser system (LS-2137/2, LOTIS TII) with the following parameters: wavelength, 532 nm; pulse duration, 15 ns; pulse energy, 400 mJ; laser beam diameter, 7.0 mm. The laser beam was expanded through a concave lens to a spot size of about 1 cm in diameter. A PM50-10 analog optical power meter with an S212A sensor (Thorlabs, Newton, NJ) was used to measure the power of the laser beam at the sample position. At predetermined time intervals, the samples were irradiated with the NIR laser. The HGNPs solutions were subjected to centrifugation (14,000 rpm, 20 min) and the supernatants withdrawn for analysis of Pt before and after NIR laser irradiation by ICP-MS.
In a separate experiment, PEG-Ac-EEC(Pt)-HGNPs were dispersed in 2.0 mL deionized water or saline solution without laser irradiation at room temperature. The Pt concentration was analyzed at predetermined time intervals.
Identification of Pt-containing compounds released from PEG-Ac-EEC(Pt)-HGNPs
To identify the Pt derivatives released after laser irradiation, Ac-Glu-Glu-Cysteic acid-NH2 (Ac-EECya) was synthesized using Rink amide resin and Fmoc chemistry as described above. The complex of cisplatin and Ac-EECya peptide, Ac-EECya(Pt), was synthesized by mixing cisplatin and Ac-EECya in water at 37 °C for 72 h. LC–MS analysis was applied to aliquots collected from both PEG-Ac-EEC(Pt)-HGNPs and F-Ac-EEC(Pt)-HGNPs after laser treatment, and the resulting chromatograms and mass spectra were compared with that of the authentic Ac-EECya(Pt) to confirm the structure of the released Pt-containing species.
Cytotoxicity
Folate receptor-expressing KB cells were seeded in 96-well plates (2.0 × 104 cells/well) and incubated for 24 h to allow the cells to attach. The cells were exposed to free cisplatin, PEG-Ac-EEC(Pt)-HGNPs, or F-Ac-EEC(Pt)-HGNPs with various Pt concentrations. After 4 h, the cells were irradiated with 15-ns pulsed NIR laser at an output power of 50 mW/cm2 (1 min/treatment, 3 treatments in 2 h). The cells were then incubated at 37 °C for an additional 24 or 48 h without washing steps. Cell survival efficiency was measured by the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), or MTT, assay (Sigma, St Louis, MO) according to manufacturer-suggested procedures. The data are reported as the means of triplicate measurements.
In a separate experiment, KB cells were seeded onto a 96-well plate at a density of 20,000 cells/well 24 h before the experiment. Cells were washed 3 times with Dulbecco modified essential medium (DMEM) without phenol red. Cells were incubated with 100 µL of DMEM containing F-Ac-EEC-HGNPs (17 µM of Au) or F-Ac-EEC(Pt)-HGNPs (17 µM of Au and 5 µM of Pt) at 37 °C for 4 h. The cells were then washed 3 times with phosphate-buffered saline solution to remove unbound nanoparticles. After cells were resupplied with DMEM containing 10% fetal bovine serum, they were irradiated with 15-ns pulsed NIR laser light centered at 808 nm at an output power of 50 mW/cm2 (1 min/treatment, 3 treatments in 2 h) and then incubated at 37 °C. Twenty after laser treatment, cells were washed 3 times with Hanks balanced salt solution and stained with calcein AM (Life Technologies, Grand Island, NY) for visualization of live cells. Untreated cells were used as controls. Cells were examined on a Zeiss Axio Observer. Z1 fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) equipped with a filter set specific for excitation/emission wavelengths at 494/517 nm for calcein staining.