Synthesis and characteristics of CSO-SA
CSO-SA was prepared from a modified procedure developed in the laboratory (College of Pharmaceutical Science, Zhejiang University, China) by Fu-qiang Hu. CSO-SA was synthesized by esterification reaction between amine groups of CSO (molecular weight 18 kDa) and the carboxyl group of SA (C18
, Shanghai Chemical Reagent Co, Shanghai, China) in the presence of carbodiimide (EDC). Briefly, SA and EDC (SA:EDC = 3:20, w/w) were dissolved in ethanol. The solution was stirred and heated to 80°C in order to activate the carboxylic acid of SA. After CSO (CSO:SA = 2.4:1.0, w/w) solution was added, the reaction was carried out at 80°C under stirring for 4 hours. The reaction solution was dialyzed using a dialysis membrane (molecular weight cutoff [MWCO] 7.0 kDa) against pure water and then lyophilized. Finally, CSO-SA was obtained, and the degree of amino substitution was tested as described previously.27
The critical micelle concentration (CMC) of the synthesized CSO-SA was determined by fluorescence methods. In brief, using pyrene as a probe, fluorescence spectra of CSO-SA micelles were measured by fluorometer (F-2500, Hitachi, Tokyo, Japan), excitation wavelength of which was set at 337 nm. The intensities of the emission were recorded at a range of 360–450 nm. The value of CMC was determined by the emission intensity ratio of the first peak (I1, 374 nm) to the third peak (I3, 385 nm).
Preparation of OXA-loaded CSO-SA micelles
CSO-SA/OXA micelles were prepared through thin film dispersed as previously reported.31
A volume of 2.5 mL of 1 mg/mL OXA (Sigma-Aldrich, St Louis, MI) solution was added into CSO-SA micelle solution (OXA:CSO-SA = 1:10, w/w). The mixture was stirred for 30 minutes and added into 2.5 mL lecithin ethanol solution. The solvent was removed by rotary evaporation at 45°C. CSO-SA/OXA micelles were prepared by dissolving in 5% glucose.
Physicochemical properties of OXA-loaded CSO-SA micelles
The size and its distribution of CSO-SA/OXA micelles were measured by dynamic light scattering (DLS) using a Zetasizer (3000 HS, Malvern Instruments Ltd, Worcestershire, UK). The zeta potential of the micelles was detected by the Zetasizer.
The morphological examinations of CSO-SA/OXA micelles were determined by transmission electron microscopy (TEM) (JEM-1230, JEOL, Tokyo, Japan). The samples were stained with 2% (w/v) phosphotungstic acid and placed on copper grids for viewing by TEM.
The OXA content in the drug-loaded micelles was detected using inductively coupled plasma mass spectrometer (ICP-MS) (Hitachi). In brief, the platinum (Pt) content in CSO-SA/OXA micelle solution and ultrafiltrated solution were measured, respectively. The latter solution was obtained from the ultracentrifugation of CSO-SA/OXA solution, using an ultrafilter centrifuge tube (MWCO 10 kDa; Millipore, Bedford, MA) centrifuged at 10,000 rpm for 10 minutes. Pt content in solution was determined by ICP-MS, and then the OXA content was calculated. OXA encapsulation efficiency (EE) was calculated using the following equation: EE = (mass of OXA encapsulated in micelle/mass of OXA added) × 100%.
In-vitro drug release-assay
In-vitro release profiles of OXA from the CSO-SA/OXA micelles were examined using the dialysis method. CSO-SA/OXA micelles were mixed with fetal bovine serum (FBS) (CSO-SA/OXA:FBS = 1:9, v:v), resulting in a final OXA concentration of 50 μg/mL. The solution was then added into a dialysis bag (MWCO 7.0 kDa), and sealed to a plastic tube containing 10 mL of FBS. The test was conducted in an incubator shaker at 37°C, which was shaken horizontally at 60 rpm. At various time points, FBS in the tube was collected and replaced with the fresh media. The amount of released drug was determined by ICP-MS. All drug-release tests were performed thrice.
Cell culture and spheroid formation
CRC cell lines HT29 and SW620 were purchased from the cell bank of the Chinese Academy of Medical Science (China). The HT29 cells were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA) and SW620 cells in Leibovitz’s L-15 medium (Invitrogen). Media were supplemented with 10% FBS, 100 unit/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in a 5% CO2 humidified incubator.
For the formation of spheroids, cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Invitrogen) basal serum-free medium supplemented with B27, 20 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor (Invitrogen), 2 mg/mL bovine serum albumin, 100 unit/mL penicillin, and 100 μg/mL streptomycin. Single cells prepared from enzymatic dissociation were seeded in 6-well ultra low attachment plates at low densities (500–1000 cells/well) or in 96-well ultra low attachment plates at a density of 100–200 cells/well. After 10–14 days of suspension culture with or without drug, the number of spheroids was counted per 200 cells in primary seeded culture.
Intracellular uptake of OXA-loaded CSO-SA micelles
The cellular uptake studies were done as previously described.18
In brief, CSO-SA/OXA micelles were labeled with fluorescein isothiocyanate (FITC) via the reactive amino group of chitosan and the isothiocyanate group of FITC (CSO-SA/OXA:FITC = 1:8, mol/mol). Unreacted FITC was removed using dialysis. HT29 spheroids were seeded at a density of 100 cells/well in a 24-well low adhesion plate (Corning, New York, NY). When grown for 24 hours, cells were then incubated with FITC-labeled CSO-SA/OXA micelle solution in growth medium for an additional 30 minutes, 1 hour, and 2 hours, respectively. After incubation, the cells were washed with phosphate-buffered saline (PBS) thrice, and then the cellular uptake was visualized by confocal microscope (Zeiss LSM 710, Carl Zeiss, Dublin, CA).
Pt accumulation studies of OXA-loaded CSO-SA micelles
For drug-accumulation studies, dissociated HT29 and SW620 spheroid cells were seeded into 5 × 104 cells/well in a 24-well low adhesion plate. When grown for 24 hours, cells were exposed to OXA and CSO-SA/OXA micelles for various periods (2 hours, 4 hours, or 8 hours). After incubation, all cells were washed with ice-cold PBS thrice, harvested, and dissolved in pure water. Total cellular Pt content was determined by ICP-MS, and drug accumulation was then calculated.
The drug-accumulation studies of tumor tissues were investigated in xenograft tumors. When the tumors reached a mean diameter of 10 mm, mice were injected in the tail vein with OXA or CSO-SA/OXA micelle solution. At various time points (2 hours, 8 hours, or 24 hours), mice were humanely euthanized and tumors were harvested. The cancer tissues were thoroughly washed four times in PBS solution and crushed in liquid nitrogen. Pt content was detected as above.
Fluorescence-activated cell analyzing and sorting
Fluorescent-activated cell analyzing or sorting was performed using standard protocols. Single cells obtained from cell cultures or xenograft tumors were labeled with mouse antihuman CD133/2-PE (Miltenyi-Biotec GmbH, Bergisch Gladbach, Germany) and CD24-FITC (BD Biosciences, San Diego, CA). Mouse immunoglobulin G was used as isotype control. Cells were analyzed by FACSCalibur machine ( Becton Dickinson, Franklin Lakes, NJ) or sorted by FACSAriaII (Becton Dickinson).
The cytotoxicities of CSO-SA micelles, OXA, and CSO-SA/OXA micelles were assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTS) assay using the CellTiter 96 Aqueous MTS (Promega, Fitchburg, WI). HT29 and SW620 cells were seeded in 100 μL of medium at a density of 2 × 104 to 5 × 104 cells/well in 96-well plates. In order to preserve stemness of CSLCs, dissociated spheroids and CD133+/CD24+ cells from HT29 and SW620 were cultured in 96-well low adhesion plates (Corning) containing stem-cell medium. After each treatment for 48 hours, the MTS assay was performed according to the manufacturer’s instructions.
In-vivo tumorigenicity assay and mouse treatment
All experiments involving mice were conducted in accordance with the standard protocol approved by the local animal care committee. Dissociated HT29 and SW620 spheroid cells were injected subcutaneously into the flank of 4–5-week-old female nude mice. Two weeks after the injection, mice carrying xenograft tumors were randomly divided into four groups. Each group with six mice was treated intravenously once a week for 4 weeks with CSO-SA/OXA micelles (OXA content 2 mg/kg), free OXA (2 mg/kg), CSO-SA micelles, or 5% glucose, respectively. Each tumor was measured with a caliper, and the volume calculated using the formula: V = 1/2 (width2 × length).
At the end of each treatment, tumors were collected from mouse xenografts and washed four times in PBS solution. After being minced into approximately 1 mm3 in size, enzymatic digestion was performed using 1.5 mg/mL collagenase type IV and 1 mg/mL hyaluronidase in medium RPMI (Roswell Park Memorial Institute) 1640 (Invitogen) at 37°C for about 1 hour. Cells were then obtained through a 40 μm mesh and resuspended in PBS, which was used for flow cytometry.
TdT-mediated dUTP nick end labeling (TUNEL) assay
TUNEL-reaction was performed on 10-mm thick frozen tissue sections using the one-step TUNEL apoptosis assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In brief, samples were fixed with paraformaldehyde for 30 minutes and permeabilized with 0.1% Triton® X-100 (Dow Chemical Company, Midland, MI) for 10 minutes. The sections were then washed in PBS and incubated with 5-bromo-4-chloro-3-indolyl-phosphate substrate labeled with Cy-3 for 40 minutes at 37°C. Randomly chosen fields were examined at ×400 magnification using confocal microscope.
Statistical analysis described in the experimental sections was done with GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA) software. Statistical significance was determined by one-way analysis of variance. P-values < 0.05 were regarded to be statistically significant in all cases.