Paclitaxel conjugated with BODIPY® 564/570, a red–orange fluorescent dye with an excitation of 564 nm and emission of 570 nm used in this study was obtained from Invitrogen (Carlsbad, CA). Wheat germ agglutinin (WGA) Oregon Green 488 conjugate was obtained from Invitrogen, Molecular Probes (Eugene, OR). All regents used for the chitosan–niosomes were obtained from the sources described in our previous study (Williams et al. 2012).
Preparation of thermo-sensitive cross-linked chitosan solutions
Synthesis of hydrogel and process of encapsulation of niosomes was previously described by us (Williams et al. 2012). The following methodology was used. A 3 mL solution of 65% (w/v) b-glycerophosphate was added to 9 mL of 2.78% (w/v) chitosan solution (in 0.1 M HCl) drop-wise, stirring continuously over an icebath. The final solution was stirred for additional 10 min to ensure complete mixing. The final solution contained a molar ratio of 4:1 of b-glycerophosphate:chitosan. Niosomes were embedded into the chitosan network by adding niosomes into the prepared chitosan-b-glycerophosphate solution, mixing them thoroughly at 25C, and heating the resulting solution to 37C to facilitate cross-linking.
Preparation of niosomes
Niosomes preparation was done using thin-film hydration of Span-60 (sorbitan monostearate surfactant), cholesterol and dicetyl phosphate in 1:1:0.1 molar ratios in chloroform as described by Williams et al. (2012) and Williams (2012). Briefly, this solution was transferred to a round bottom flask, and the solvent was evaporated in a Buchi rotary evaporator overnight. Hydration of the film was then done with paclitaxel solutions with concentrations ranging from 2 nM to 20 mM, maintaining the evaporator’s temperature at 60 °C for 1 h. Then, the solution was extruded using a polycarbonate membrane with 100 nm porous size maintaining the warm temperature and passing it through for 12 times. The hydrophobic lipophilic balance (HLB) for the niosomes prepared here falls between 4 and 8. The critical packing parameter (CPP), which is a parameter that measures the aggregation ability of amphiphiles, was 0.5–1.0 (dimensionless). The niosomes were then separated using ultracentrifugation (60,000 rpm for 40 min). The niosome was added to the preparation of the thermos-sensitive cross-linked chitosan using 0.1 M HCL chitosan solutions under ice and adding 65% v/v β-glycerophosphate cross-linker stirring for 10 min to obtain cross-link molar ratios ranging from 3:1 to 5:1.
Human epithelial carcinoma OV2008 cells recently reclassified from ovarian to cervical carcinoma cell line (Korch et al. 2012) and two primary cultures of normal human surface epithelial cells used in this study were derived from ovaries of women with no disease and no family history of breast and/or ovarian cancer that were transfected with SV-40 large T-antigen: IOSE-121 and IMCC3. These cells were kindly provided by Dr. Patricia Kruk, Department of Pathology and Cell Biology, College of Medicine, University of South Florida, Tampa. They were grown in tissue culture flasks in a 1:1 mixture of MCDB105/M199 medium (Sigma-Aldrich, MO). In addition, human glioblastoma grade III astrocytoma U373-MG (American Type Culture Collection ATCC; Manassas, VA) grown in Minimum Essential Medium was used in this study. Both growth media were supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, UT), 2 mM l-glutamine, penicillin, and streptomycin. All cells were incubated at 37 °C in 5% CO2.
In vitro study using a novel experimental model
We designed a novel in vitro system using glass-bottom microwell culture dishes (P35G-1.5 20-C.S., MatTek Corp. Ashland, MA) referred to as MatTek’s plates that allow for a live cell observation using differential interference contrast (DIC) and confocal microscopy while either the chitosan or the DDS-paclitaxel BODIPY 564/570 diffusion was in progress from the outer chamber into the inner chamber with the adherent cells.
First, the cells were trypsinized and seeded in MatTek’s plates according to the manufacturer’s procedure at 10 × 103 cells/per MatTek’s dish insert and incubated in 2 mL growth medium at 37 °C in 5% CO2 for 24 h allowing cells to attach. The following day, the MatTek’s glass-bottom culture dishes containing adherent cell cultures were removed from the incubator and either used for short-term studies (up to 1 h incubation) or for long-term studies (up to 24 h incubation).
The media at the outer edge (outside chamber—as per Figs. 1b and 3a) of the dish was pipetted out and either replaced with fresh medium in the controls or with 300 µL of liquid chitosan polymer alone or chitosan–niosome polymer system with encapsulated fluorescently tagged BODIPY 564/570 paclitaxel. The polymer was added using a syringe directly to the outer rim (in the short-term studies up to 1 h) or the sterile aluminum insert was placed first in the outer rim of the MatTek’s plate and 300 µL of liquid chitosan–niosome BODIPY 564/570 paclitaxel polymer was added with the syringe (in the long-term studies). The dishes were then placed in the incubator for 3 min to facilitate cross-linking of chitosan resulting in its gelling.
For the short-term observations chitosan–niosome BODIPY 564/570 paclitaxel complex at various concentrations (0.01, 0.04 and 0.4 µM) was placed directly into the outer chamber, and followed by the addition of 400 µL of media into the inside chamber of the MatTek’s plate covering the cells, as well as the polymer. The cells used for the short-term (up to 1 h) observations were OV2008 cervical carcinoma, U373 glioma, and normal ovarian epithelial cells IOSE-121 and IMCC3.
For the long-term studies, the aluminum inserts containing chitosan–niosome BODIPY 564/570 paclitaxel at 0.01 µM concentration were removed after 15 min and 400 µL of growth medium was added to the inside chamber of the MatTek’s plate to cover the cells and the polymer. With time, the small fragments of the polymer complex were gradually breaking off from the polymer located in the outside chamber and diffused toward the center of the MatTek’s plate which contained seeded cells. The cell fluorescence intensity was evaluated at 5 min, and 24 h using confocal microscopy and images were captured of the cells growing both at the edge and at the center of the inner plate. Following a confocal evaluation, MatTek’s dishes were returned to 37 °C at 5% CO2 incubator.
The 15 min pulse treatment of cells with the DDS in these long-term studies (up to 24 h) limited the exposure of cells growing in this enclosed in vitro system to the DDS-containing paclitaxel–BODIPY 564/570 and therefore allowing for a gradual diffusion of chitosan polymer from an outside chamber towards the inside chamber with the cells. Direct fluorescence intensity was measured at the start of the incubation (5 min) and again at 24 h using Leica LAS AF Lite. The cells evaluated in the long-term studies were OV2008 epithelial carcinoma and normal epithelial IMCC3 cells.
Confocal microscopy and differential interference contrast (DIC) microscopy
Images of each sample (adherent cells in the MatTek’s glass-bottom culture dishes) were obtained with a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Germany) through a 63×/1.4NA or 100×/1.4 NA. 405 Diode, argon, and helium neon (HeNe) 594 laser lines were applied to excite the samples and tunable filters were used to minimize crosstalk between fluorochromes. Gain, offset, and pinhole setting were identical for all samples within each experiment. Differential interference contrast (DIC) microscopy images were also captured using the Argon laser line. All images were taken at room temperature. The magnification for these images is 630× or 1000×. To obtain enlarged views of the cells a 3× optical zoom was applied. The scale bar shown on all images is 10 µm.
Quantitation of the extracellular chitosan alone
Measurements of the in vitro chitosan accumulation at the immediate vicinity of cells compared to the media controls were performed using the differential interference contrast (DIC) microscopy. Images were quantified with the ImageJ software (National Institutes of Health, Maryland, USA). Multiple radial directions from the cell center were taken into consideration, and grey value light intensity in the space around the cells (normalized by the background values) was measured in the line segments of 4 µm each. The results were exported to Microsoft Excel for statistical analysis.
Measurement and quantitation of extracellular and intracellular paclitaxel fluorescent light intensity
Analysis of the extracellular diffusion of paclitaxel BODIPY 564/570 around OV2008 cells was performed by quantifying the fluorescence intensity with ImageJ software (National Institutes of Health, Maryland, USA) using the confocal images. The extracellular space immediately around the cells was divided into three zones of intensity, 24 µm width each, and labeled Z1–Z3 with Z1 being the region closest to the cell. The fluorescent intensity signal was evaluated using multiple radial line segments of 30 pixels in thickness to constitute a circular pattern around each cell. The results were exported to Microsoft Excel and IBM SPSS Statistics 24 for statistical analysis.
Analysis of each cell’s fluorescence intensity resulting from the uptake of paclitaxel BODIPY 564/570 was performed and quantified with Leica LAS AF Lite software (Leica Microsystems, Germany) using the confocal images. The control plates with medium alone were used to establish “Gain” for the paclitaxel’s BODIPY 564/570 fluorescence to exclude the auto fluorescence. Individual cells were segmented using the “Region of Interest” (ROI) selection tool and mean intensity of the paclitaxel signal was measured in pixels within the ROI. Results were exported to Microsoft Excel for statistical analysis.
Wheat germ agglutinin staining of the cell membrane
Wheat germ agglutinin (WGA) Oregon Green 488 conjugate from Molecular Probes with excitation at 496 nm and emission at 524 nm was used for the staining of cell membrane in live cells. Two MatTek’s plates for each cell line OV2008 and IMCC3 were seeded for the long-term experiments as described above. The cells in one plate were non-treated and used as a control and the cells in the second plate were treated with chitosan–niosome–paclitaxel BODIPY 564/570 at 0.01 µM concentration with a 15 min pulse treatment in the long-term studies as described above. Following over 24 h incubation at 37 °C in 5% CO2, the 300 μL volume of growth medium was removed from above the cells in the inner chamber of the MatTek’s plate and replaced with 300 μL of WGA Oregon Green 488 conjugate solutions made in Hanks Balanced Salt Solution (HBSS) at 5 μg/mL as recommended by the manufacturer or HBSS alone for the control plates. Next, cells were incubated for 10 min at 37 °C in 5% CO2, the labeling solution was removed, and cells were washed twice in HBSS. The cells were then observed by confocal microscopy and images were captured.
Microsoft Excel Analysis Tool Pack (Redmond, WA) and IBM SPSS Statistics (IBM Corporation, New York, USA) was used to evaluate the fluorescence intensity data.
For chitosan alone diffusion evaluation: Descriptive statistics with mean and standard deviation (SD) were obtained for intensity values for each cell at the 1 h time point of the experiment. The total number of evaluated cells in the experiment was as follows: for OV2008 n = 14 and IOSE-121 n = 5 (incubation with chitosan). Also OV2008 n = 7 and IOSE-121 n = 7 were evaluated (medium control). Determination of F value for two sample variances was followed by the appropriate (assuming unequal or equal variances) two tail Student’s t test. Differences between the groups were considered statistically significant at p < 0.05.
For intracellular fluorescence intensity evaluation: Descriptive statistics with mean and standard deviation (SD) were obtained for fluorescence intensity measured for each cell at time points 5 min and 24 h in three separate experiments. The total number of evaluated cells in all experiments was as follows: for OV2008 n = 55 (5 min time point); n = 49 (24 h time point), and IMCC3 n = 17 (5 min time point), n = 16 (24 h time point), Determination of F value for two sample variances was followed by the appropriate (assuming unequal or equal variances) two tail Student’s t-test. Differences between the groups were considered statistically significant at p < 0.05.
For the extracellular paclitaxel fluorescent signal evaluation: Descriptive statistics with mean and standard deviation (SD) were obtained for fluorescence intensity measured at time point 5 min, in two experiments. The total number of evaluated extracellular spaces defined by an area adjacent to one cell or a group of OV2008 cells was: n = 12. Determination of normality (normally distributed or not normally distributed sample) of each zone at each time point was completed using the Shapiro–Wilk test and followed by the appropriate analysis of multiple means (ANOVA or Kruskal–Wallis test). Differences between the zones were considered statistically significant at p < 0.05.