Cellular Uptake Mechanism of Paclitaxel Nanocrystals Determined by Confocal Imaging and Kinetic Measurement
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Nanocrystal formulation has become a viable solution for delivering poorly soluble drugs including chemotherapeutic agents. The purpose of this study was to examine cellular uptake of paclitaxel nanocrystals by confocal imaging and concentration measurement. It was found that drug nanocrystals could be internalized by KB cells at much higher concentrations than a conventional, solubilized formulation. The imaging and quantitative results suggest that nanocrystals could be directly taken up by cells as solid particles, likely via endocytosis. Moreover, it was found that polymer treatment to drug nanocrystals, such as surface coating and lattice entrapment, significantly influenced the cellular uptake. While drug molecules are in the most stable physical state, nanocrystals of a poorly soluble drug are capable of achieving concentrated intracellular presence enabling needed therapeutic effects.
KEY WORDScellular uptake confocal microscopy nanocrystals paclitaxel pharmacokinetics
Our in vivo experiments have demonstrated that nanocrystal formulations of chemotherapeutic agents are capable of eliciting similar and better anticancer efficacy—compared with solubilized or encapsulated delivery systems—but exhibiting much reduced side effects (1,2). This may be contributed by the absence of helper chemicals that are used in the conventional formulations for solubilizing and/or encapsulating poorly soluble drugs (3, 4, 5, 6, 7). Any side effects associated with carrier chemicals are completely eliminated in (pure) nanocrystal formulations. Formulated as nanosized particles, a chemotherapeutic drug may also take advantage of possible leaky vasculatures found in tumor and augment local drug retention, a hallmark of the EPR (enhanced permeability and retention) effect. Importantly, nanocrystals are physically stable—albeit possible particle aggregation, which can be remedied or minimized by surface coating with surfactants—and unlikely to undergo drastic phase transition and structural change. Our recent study of paclitaxel nanocrystals (PTX-NCs) showed that the delivery system had similar but extended tumor accumulation with Taxol®, possibly due to the localization of drug nanocrystals (1). In addition, it was observed that more than 40% of injected nanocrystals were taken up by liver, considerably surpassing that by Taxol. It remains, however, enigmatic how drug nanocrystals exhibit anticancer effects to cancer cells, whether through dissolution followed by membrane diffusion into the cells or being taken up directly through endocytosis. It is further interesting to explore the impact by surface-coating nanocrystals with hydrophilic surfactants on the cellular uptake, shedding light on ways to improving pharmacokinetics and biodistribution of drug nanocrystal formulations and to particularly reduce possible liver toxicities due to significant uptake of drug nanocrystals by the mononuclear phagocyte system.
As such, the purpose of this report is to unveil cellular uptake mechanisms of drug nanocrystals and understand their therapeutic effects. Specifically, we tested in cells hybrid PTX-NCs, in which a fluorescent dye was physically integrated (2,8), as well as surface-coated nanocrystals with polymers and surfactants. Through chemical analyses and confocal imaging, it was found that the drug nanocrystals could be internalized by cells directly and at much higher concentrations than those in the cells treated by the solubilized formulation.
Paclitaxel (PTX) (>99.5% purity) was purchased from LC Laboratories (Woburn, MA, USA). Sulforhodamine B (SRB), folic acid (FA), dopamine, Pluronic® F68, and F127 were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). FA-PEG3400-NH2, mPEG2000-NH2, fluorescein(FITC)-PEG2000-NH2, and mPEG-FITC were purchased from Nanocs Inc. (New York, NY, USA). Hoechst 33258 and Lysotracker® Deep Red were obtained from Life Technologies Inc. (Grand Island, NY, USA).
Preparation of PTX Nanocrystals
PTX-NCs were grown from solution by an anti-solvent crystallization method reported in our previous studies (8). In general, 1 mL of 3 mg/mL PTX dissolved in ethanol was introduced into 20 mL of deionized water in a flask. The solution was sonicated and under high-speed stirring. The suspension was then filtered with a 50-nm polycarbonate filter paper and the retentate was re-suspended in deionized water by homogenization.
Preparation of Fluorescein Hybrid Paclitaxel Nanocrystals
To produce SRB-integrated hybrid nanocrystals, the same procedure was used but with the dye dissolved in the deionized water prior to the crystallization process (8). After crystallization and filtration, the retentate was re-suspended in water and underwent additional centrifugation-re-suspension cycles to remove any loosely bounded dye molecules on the surface of nanocrystals. PEG and FA-PEG-integrated PTX hybrid nanocrystals were also prepared with the same approach where PEG or FA-PEG was present in the crystallization medium.
Preparation of PEG Hybrid Paclitaxel Crystals
To verify the possibility of PEG integration in the crystal lattice of PTX, larger hybrid crystals were prepared with a similar procedure but without sonication and high-speed stirring. Briefly, 1 mL of 3 mg/mL paclitaxel in ethanol was added into 20 mL of deionized water in vial and, subsequently, FITC-PEG was introduced to the solution. The FITC-PEG-PTX hybrid crystals were collected by centrifugation-re-suspension cycles for three times. The crystals were observed by a confocal microscope (Nikon A1) at 488 nm.
Preparation of Surface-Treated PTX Nanocrystals
PTX-NCs were treated with several different polymers in order to modify the surface chemistry of the nanocrystals. Polydopamine(Dp)-coated nanocrystals were prepared according to the reported method with some modification (9,10). Three milligrams PTX nanocrystals was added into 20 mL of Tris-HCl solution (10 mmol/L, pH = 8.5) under sonication. Dopamine was added into the mixture and self-polymerization was allowed to proceed for 3 h at the room temperature. The Dp-PTX-NCs were collected by centrifugation. In addition, Dp-PTX-NCs were dispersed in PBS solution (1 mmol/L, pH = 7.4) under sonication and then incubated with 2 mg/mL FA-PEG3400-NH2 for 2 h at the room temperature. The FA-PEG-Dp-PTX-NCs were collected by centrifugation. PEG-Dp-PTX-NCs were prepared with the same method but with 2 mg/mL mPEG2000-NH2 instead.
To verify the surface-coating technique, larger crystals of PTX were prepared with SRB integrated in the crystal in accordance with the similar crystallization method for preparing hybrid nanocrystals. Briefly, 1 mL of 3 mg/mL paclitaxel in ethanol was added into 20 mL of deionized water with SRB in the vial without stirring and sonication. The SRB-PTX crystals were then treated with polydopamine as described above and then conjugated with FITC-PEG2000-NH2. The FITC-PEG-SRB-PTX crystals were obtained by three centrifugation-re-suspension cycles to remove loosely bounded polymers and fluorescent molecules from the surface of the crystals. The crystals were observed by a confocal microscope (Nikon A1).
Physically coated PTX-NCs were prepared by treating nanocrystals with Pluronics F68 and F127. Three milligrams PTX nanocrystals was re-suspended in 3-mL deionized water under sonication. Two percent (m/m) Pluronics F68 or F127 was added to the solution under mechanical shaking for 3 h at 4°C. F68/F127-PTX-NCs were collected by centrifugation-washing cycles for three times to remove loosely bounded polymers.
Characterization of PTX Nanocrystals
Particle size and zeta potential of nanocrystals were determined by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern) at 25°C. Each measurement was done in triplicate. Particle morphology was detected by a scanning electron microscope (SEM, Nova NanoSEM, FEI) at an accelerating voltage of 5 kV. The samples were completely dried under vacuum and coated with conductive layers of gold palladium (Au/Pd) for 1 min at a current of 20 mA prior to the SEM measurement.
Chemical quantification of PTX was determined by a high-pressure liquid chromatography (HPLC, Agilent 1100) with a C18 column (5 μm, 4.6 × 150 mm) and a UV detector at 227 nm. The mobile phase of acetonitrile and water (50:50 v/v) were pumped at a total flow rate of 1 mL/min. The column was kept in 25°C and the injection volume of samples was 20 μL.
Human KB nasopharyngeal epidermal carcinoma cells were obtained from ATCC (Manassas, VA, USA). Cells were cultured in folic acid (FA)-deficient RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell culture was done at 37°C in a humidified atmosphere containing 5% CO2.
Cellular Uptake Imaging of Nanocrystals
KB cells were seeded in confocal petri dishes at a density of 2 × 105 cells/well and cultured for 24 h. After adhering to dish walls, the KB cells were incubated in FA-deficient RPMI 1640 medium with 100 μg/mL SRB-PTX-NCs at different durations (15, 30 min, 1, 2, and 3 h) or incubated for 3 h with the concentration of the nanocrystals varied (1, 5, 25, and 100 μg/mL). Additionally, the KB cells were incubated for 3 h in FA-deficient RPMI1640 medium with 100 μg/mL bare or surface-modified SRB-PTX-NCs (PEG-Dp-SRB-PTX-NCs, FA-PEG-Dp-SRB-PTX-NCs, PEG-SRB-PTX-NCs, FA-PEG-SRB-PTX-NCs, F127-SRB-PTX-NCs, and F68-SRB-PTX-NCs). At the end of incubation, cells were gently washed with cold PBS for three times. Lysotracker Red (red) was added at the concentration of 100 nM and the cells were further incubated at 37°C for 30 min. After being collected and washed, the cells were fixed with 4% paraformaldehyde in PBS at the room temperature for 10 min, followed by cell nuclei staining with 2 mL Hoechst 33258 (2 μg/mL) for 15 min (blue). The cells were imaged by a laser scanning confocal microscope (Nikon A1).
Cellular Uptake Kinetics
KB cells were treated with nanocrystals and drug concentrations in the medium and inside cells were analyzed by HPLC. KB cells were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured for 24 h. Upon adhesion, the KB cells were incubated in FA-deficient RPMI 1640 medium with 100 μg/mL pure PTX-NCs and Taxol, respectively, for different durations (0.5, 1, 2, and 3 h) or for 3 h with different drug concentrations (12.5, 25, 50, and 100 μg/mL). Temperature effect was studied by incubating with the drug formulations in 37 and 4°C, respectively. Influence of FBS (fetal bovine serum) on cellular uptake was also studied by treating KB cells with 100 μg/mL pure PTX-NCs and 0, 10, and 20% FBS, respectively, in FA-deficient RPMI 1640 medium. In addition, polymer-treated nanocrystals were used for treating KB cells at 25 μg/mL in FA-deficient RPMI1640 medium for 3 h. At the end of incubation, 1 mL of the culture medium was collected and centrifuged at 10,500 rpm for 15 min. The supernatant, 0.5 mL, was removed and analyzed for the extracellular solubilized drug concentration. The rest of the culture medium was collected and cells were washed three times with cold PBS. The total extracellular drug concentration, which is of both the extracellular solubilized drug and the extracellular drug nanocrystals, was then obtained from the analyses of the remaining liquid in the centrifugation tube, the rest of the culture medium, and the PBS liquid used for washing the cells, in addition to the concentration already determined from the supernatant. The cells were treated with 0.25% trypsin-EDTA and lysed by a Selecta Sonopuls (Nikon A1, Nikon Co. Ltd., Tokyo, Japan) for determining the drug concentration (i.e., total intracellular drug concentration). All samples were diluted properly with acetonitrile and PTX was quantified by HPLC.
The Taxol formulation used in treating cells was prepared by dissolving PTX into a 50:50 mixture of ethanol and Cremophor EL at room temperature.
Cellular uptake data were represented as the mean with standard deviation (SD). One-way analysis of variance (ANOVA) was performed to determine the null hypothesis among groups, followed by Bonferroni post hoc test. All statistical tests were two-sided, and P values less than 0.05 were considered to be statistically significant.
RESULTS AND DISCUSSION
Characterization of PTX Nanocrystals
Particle sizes of PTX-NCs were measured by DLS. Immediately after the crystallization process, PTX-NCs showed a wider particle size distribution ranging with a major peak at 300 nm and a small one at 5441 nm (Figure S1A in Supporting Information). The distribution narrowed to 100–500 nm with one major peak at 239 nm (Figure S1B), illustrating the effect of high-speed homogenization.
Particle Size and Zeta Potential of PTX Nanocrystals (Mean ± SD; n = 3)
Particle size (nm)
Zeta potential (mV)
240 ± 11
0.16 ± 0.06
−10.7 ± 1.1
231 ± 11
0.08 ± 0.05
−27.5 ± 1.3
235 ± 17
0.12 ± 0.04
−28.5 ± 0.8
233 ± 4
0.10 ± 0.02
−16.5 ± 0.9
276 ± 52
0.10 ± 0.08
−9.0 ± 0.3
825 ± 151
0.43 ± 0.35
6.1 ± 0.8
334 ± 23
0.19 ± 0.05
−8.3 ± 0.6
234 ± 21
0.06 ± 0.03
−5.9 ± 0.1
243 ± 22
0.16 ± 0.04
−12.9 ± 0.9
Confocal Imaging of Cellular Uptake of Drug Nanocrystals
Cellular Uptake Kinetics
In conclusion, our cellular uptake studies of paclitaxel nanocrystals in KB cells explored cellular uptake mechanism of drug nanocrystals. By utilizing confocal imaging and quantifying drug internalization kinetics, our in vitro investigation supports the notion that the drug nanocrystals could be taken up directly by the cells as solid particles. The intracellular drug concentrations were much higher than those of the solubilized drug formulation (Taxol); more than half of co-cultured drug nanocrystals were taken up by KB cells, as compared to about 2% of Taxol. The confocal imaging and temperature-dependent internalization results indicate that endocytosis may be responsible for the nanocrystal uptake. In addition, our studies examined polymer-treated nanocrystals and the impact on drug internalization by the polymer coating or physical integration to the nanocrystals. While no clear trend can be identified, the polymer treatment exhibited significant influences, both positive and negative. All polydopamine-treated nanocrystals including FA conjugation drastically reduced the intracellular drug concentrations. And among the physical integration and surfactant adsorption, the nanocrystals incorporated with PEG and coated with F68 enabled additional cellular uptake compared with pure PTX-NCs. The F68-treated PTX-NC formulation is currently tested in vivo.
The cellular uptake studies provide further support to our pursuit of using drug nanocrystals for delivering poorly soluble drugs. The nanocrystal formulation of a chemotherapeutic drug can exert sufficient and likely stronger therapeutic effects against cancer cells than conventional, solubilized delivery systems. As a drug nanocrystal slowly dissolves inside a cell, the drug concentration builds up intracellularly, achieving a much lethal microenvironment for the cell, which may be difficult to achieve by solubilized delivery systems via drug diffusion through cell membrane. Undissolved nanocrystals can be further recycled by other cells. Our preliminary cytotoxicity results suggest that drug nanocrystals are much more potent than solubilized drug formulations in the KB cells (data not shown and to be published elsewhere). Direct cellular uptake and intracellular drug release thus make nanocrystal formulations appealing for cancer therapy.
This work was financially supported by Lilly Endowment Seed Grant Program.
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