Bioconjugate Chem. 2008, 19, 1319–1325
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Hyaluronic Acid-Paclitaxel Conjugate Micelles: Synthesis, Characterization, and Antitumor Activity Hyukjin Lee, Kyuri Lee, and Tae Gwan Park* Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea. Received February 11, 2008; Revised Manuscript Received April 8, 2008
Chemical conjugates of paclitaxel and hyaluronic acid (HA) were synthesized by utilizing a novel HA solubilization method in a single organic phase. Hydrophilic HA was completely dissolved in anhydrous DMSO with addition of poly(ethylene glycol) (PEG) by forming nanocomplexes. Paclitaxel was then chemically conjugated to HA in the DMSO phase via an ester linkage without modifying extremely hydrophilic HA. A series of HA-paclitaxel conjugates with different conjugation percentages were synthesized and characterized. HA-paclitaxel conjugates self-assembled in aqueous solution to form nanosized micellar aggregates, as characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). An intact form of paclitaxel was regenerated from HA-paclitaxel conjugate micelles at acidic pH conditions. HA-paclitaxel conjugate micelles exhibited more pronounced cytotoxic effect for HA receptor overexpressing cancer cells than for HA receptor deficient cells, suggesting that they can be potentially utilized as tumor-specific nanoparticulate therapeutic agents.
INTRODUCTION Paclitaxel is a powerful anticancer drug originally isolated from the bark of pacific yew (Taxus breVifolia) and is wellknown for its antimitotic activity that promotes tubulin assembly into stable microtubules (1–3). Paclitaxel prevents the depolymerization of polymerized microtubules, thus inhibiting cell replication in the late G2/M phase of the cell cycle (4, 5). Although paclitaxel has shown remarkable potential as an anticancer drug, its poor solubility in water has limited widespread use in cancer therapy. Current clinical formulation such as Taxol contains a 50:50 mixture of Cremophore EL and ethanol to enhance the solubility of paclitaxel in water. However, a large number of studies have reported various side effects of Cremophore EL such as hypersensitivity, neurotoxicity, and neuropathy (6–8). Previous attempts to overcome the serious side effects include formulating paclitaxel in various carriers such as polymer conjugates, liposomes, polymeric micelles, emulsions, and nanospheres (9–12). Among them, drug-polymer conjugates have distinctive advantages over conventional polymeric nanosized carriers by demonstrating high drug content and good solubility in water, increasing drug half-life in the body, and enhancing antitumor effects (13). Although various water-soluble synthetic polymers have been widely exploited for conjugation of hydrophobic drugs, naturally occurring polymers with intrinsic cell specific binding capacity have tremendous potential as a target specific drug carrier. For example, hyaluronic acid (HA), a naturally occurring polysaccharide composed of N-acetyl-D-glucosamine and D-glucuronic acid, has a strong affinity with cell-specific surface markers such as CD44 and RHAMM (14). HA plays important roles in biological functions, such as stabilizing and organizing the ECM, regulating cell adhesion and motility, and mediating cell proliferation and differentiation (15–17). HA is also closely related to angiogenesis in many types of tumors, in which HA receptors (CD44 and RHAMM) are abundantly overexpressed on the surface. Thus, malignant cells with high metastatic activities often exhibit enhanced binding and uptake * Corresponding author. Tel: +82-42-869-2621; Fax: +82-42-8692610; E-mail address:
[email protected] (T.G. Park).
of HA (18–20). More recently, HA and its derivatives have been popularly used as target-specific drug delivery vehicles for various therapeutic agents (21–23). Chemical modification and conjugation of HA have been achieved in aqueous solution by utilizing reactive functional groups in HA such as carboxylic groups and hydroxyl groups and a reducing end (24–26). However, its extreme hydrophilicity and poor solubility in most organic solvents restrict the direct conjugation of HA with hydrophobic polymers, drugs, and lipids. To circumvent the above problems, various solubilization methods were utilized to obtain a homogeneous mixture of HA and hydrophobic reactants in a common solvent. For example, a mixture of polar organic solvent and water (i.e., DMSO/H2O or DMF/H2O) was used to codissolve N-hydroxysuccinimideactivated paclitaxel and adipic dihydrazide functionalized HA for conjugation (27). Polyanionic HA was physically complexed with aliphatic quaternary ammonium compounds or chemically blocked to enhance the solubility of HA in the mixed organic solvents for further modification (28). However, the above methods have shown some limitations such as multiple activation steps required for conjugation in mixed water/organic solvent phase and utilizing environmentally cytotoxic ammonium surfactants that are not preferred for biomedical and drug delivery applications. Previously, we reported the solubilization of DNA in organic solvents using biocompatible poly(ethylene glycol) (PEG) by forming nanoscale complexes (29). More recently, other hydrophilic biomacromolecules such as proteins and carbohydrates could also be solubilized in organic solvents by using PEG as a nanocomplexing agent (30). Since PEG can act as both hydrogen bonding donor and acceptor, it was hypothesized that the addition of PEG in organic solvents could also facilitate the formation of HA/PEG nanocomplexes by inter- and intrahydrogen bonds (31). In the present study, HA was solubilized in anhydrous DMSO by complexing with dimethoxy poly(ethylene glycol) (dmPEG). It was expected that HA soluble in DMSO could be readily conjugated by various hydrophobic drugs, thus generating HA-drug bioconjugates with high yield without using any salts and blocking agents. A typical water-insoluble anticancer drug, paclitaxel, was chosen for HA-drug conjuga-
10.1021/bc8000485 CCC: $40.75 2008 American Chemical Society Published on Web 05/16/2008
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Table 1. HA-Paclitaxel Conjugates Prepared by Different Conditions sample number
HA (mg)
DCC/DMAP molar ratio to -COOH
Paclitaxel in feed (mg)
Paclitaxel amount in the conjugates (mg)
Paclitaxel conjugation yield (%)
Paclitaxel loading amount (w/w %)
1 2 3 4
100 100 100 100
0.5 1.0 3.0 3.0
10 10 10 20
1.18 ( 0.03 3.26 ( 0.05 5.96 ( 0.08 12.16 ( 0.16
11.8 32.6 59.6 60.8
1.17 3.16 5.62 10.84
tion in organic solvents. HA-paclitaxel conjugate was synthesized and their self-assembly properties in aqueous solution were characterized using dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Critical aggregation concentration (CAC) was also determined by employing hydrophobic fluorescence probes. Target-specific antitumor activities were comparatively evaluated using HA-receptor positive and negative cells.
EXPERIMENTAL PROCEDURES Materials. Hyaluronic acid (HA) sodium salt (MW: 64 kDa) was purchased from Lifecore Biomedical (Chaska, MN). Poly(ethylene glycol) dimethyl ether (dmPEG) (MW: 2000 Da), 1,3-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), propidium iodide (PI), pyrene, and anhydrous dimethyl sulfoxide (DMSO) were all obtained from Sigma-Aldrich (St. Louis, MO). Paclitaxel was purchased from TCI (Tokyo, Japan). Cell counting kit-8 (CCK-8) was obtained from Dojindo laboratories (Kumamoto, Japan). All other chemicals and reagents were of analytical grade and used as received. Preparation of HA/dmPEG Complexes and Solubilization of HA/dmPEG in DMSO. For desalting process, 1 g of HA (64k Da) was dissolved in 100 mL of deionized water, dialyzed (MWCO: 1k Da) for 24 h, and lyophilized. A blend mixture of desalted HA and dmPEG at four different dmPEG/ HA weight ratios (0.5, 1, 5, and 10) was added in 25 mL of deionized water. The solution was well-stirred and lyophilized to obtain dry HA/dmPEG complex powder. A total of 600 mg of lyophilized HA/dmPEG complexes was added in 5 mL of anhydrous DMSO under dry N2. After the solution was vigorously stirred at 80 °C for 2 h, the transmittance value was measured at 400 nm using a spectrophotometer (UV-1601, Shimadzu, Japan) to verify the solubilization of HA/dmPEG complexes in DMSO as a function of added dmPEG amount. Synthesis of HA-Paclitaxel Conjugate. A total of 600 mg of HA/dmPEG powder prepared at 5 weight ratios of dmPEG/ HA was dissolved in 5 mL of anhydrous DMSO with addition of DCC and DMAP at different molar ratios. The solution was stirred for 1 h to activate carboxylic groups of HA. Paclitaxel with different amounts (10 and 20 mg) was dissolved in 1 mL of anhydrous DMSO and slowly added to the above solution using a syringe under dry N2. The mixture was then stirred for 2 days at 40 °C. The resultant solution was dialyzed against DMSO for 1 day and deionized water for 3 days using a dialysis membrane (MWCO: 3500 Da) to remove unreacted paclitaxel and dmPEG. HApaclitaxel conjugate was collected and lyophilized for 3 days and analyzed. Five different HA-paclitaxel conjugates were synthesized depending on the molar ratio of paclitaxel, DCC, and DMAP. 1 H NMR spectra of HA-paclitaxel conjugate was obtained by a Bruker 400 MHz NMR spectrometer (Bruker, Germany) using D2O and DMSO-d6. The amount of paclitaxel conjugated to HA was determined by an HPLC (1100 series, Agilent Technologies, Palo Alto, CA, USA) using a reversed-phase column (Waters Spherisorb ODS2: 4.6 mm ID × 250 mm). The mobile phase consisting of acetonitrile/water (50/50 (v/v)) was used to fully expose conjugated paclitaxel molecules in the cosolvent phase for UV detection. The solvent was delivered at a flow rate of 0.8 mL/min for the HPLC analysis and eluted peaks were monitored (λmax: 227 nm, ) 2.81
× 104 M-1cm-1). Free paclitaxel was used to generate a standard curve for the estimation of paclitaxel content in HA-paclitaxel conjugates. There was no difference in the molar extinction coefficient of paclitaxel molecule before and after the conjugation. Characterization of Self-Assembled HA-Paclitaxel Conjugate Micelles in Aqueous Solution. The formation of HA-paclitaxel conjugate micelles in aqueous solution was characterized by measuring their hydrodynamic diameter using a dynamic light scattering instrument (Zeta-Plus, Brookhaven, NY). The size measurement was carried out (n ) 6) at a concentration of 5.0 mg/mL of HA-paclitaxel conjugate (sample #4 in Table 1) in deionized water at 25 °C. Nanosized HA-paclitaxel micelles were also visualized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). For AFM image, 100 µL of HA-paclitaxel micelle solution (5.0 mg/mL) was placed on a clean mica surface and then air-dried overnight. The image was obtained with a PSIA XE-100 AFM system (Santa Clara, CA) in a noncontact mode with a scanned area of 5 × 5 µm. For TEM image, HA-paclitaxel micelles were analyzed by Zeiss Omega 912 transmission electron microscope (TEM) (Carl Zeiss, Germany). TEM sample was prepared by depositing 20 µL of HA-paclitaxel solution (5.0 mg/mL) on a 300 mesh copper TEM grid with a carbon film and air-dried at room temperature. Critical aggregation concentration (CAC) of HA-paclitaxel conjugate was estimated by employing a hydrophobic fluorescence probe, pyrene, as described previously (32). A pyrene stock solution prepared in deionized water was prepared at a concentration of 6.0 × 10-7 M. Different amounts of HA-paclitaxel conjugate ranging from 0.1 µg to 1 mg/mL were dissolved in the pyrene stock solution. At an emission wavelength at 390 nm, excitation spectra of HA-paclitaxel micelle solution with pyrene were monitored by a spectrofluorometer (Shimadzu, Japan). pH Dependent Release of Paclitaxel from HA-Paclitaxel Conjugate Micelles. HA-paclitaxel conjugate micelles were formed in deionized water at a concentration of 10 mg/mL. pH of the sample solution was adjusted from 1 to 7 using 0.1 M NaOH or HCl. After gentle mixing for 3 h at 37 °C, the solution was filtered using a 0.45 µm syringe filter. The amount of paclitaxel cleaved from the conjugate was monitored by HPLC as described above using a reverse-phase column (Waters Spherisob ODS2: 4.6 mm ID × 250 mm) with a refractive index (RI) detector (RI-71, Shodex, Japan). In Vitro Antitumor Activities of HA-Paclitaxel Conjugate Micelles. HCT-116 and MCF-7 cells were used as CD44 overexpressing cancer cell lines to verify in Vitro cell-specific targeted delivery of HA-paclitaxel conjugate micelles (21, 27). NIH-3T3 cells were used as a CD44 deficient cell line. Cells were plated in a 96-well plate at a density of 1 × 104 cells per well and grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum for 24 h at 37 °C. Cytotoxicity of HA-paclitaxel conjugate micelles was evaluated by treating cells with different concentrations of HA-paclitaxel conjugates for 2 days at 37 °C. For comparison, Taxol was prepared as described elsewhere and used as a positive control (32). Cell viability were determined by a CCK-8 cell viability assay kit (33). An apoptotic event such as DNA fragmentation was observed using a confocal laser microscope (LSM 510, Carl-Zeiss Inc., USA) and a flow cytometer (FACSCalibur, USA). HCT-116 cells were
Technical Notes
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Figure 1. Transmittance of HA/dmPEG solubilized DMSO after 2 h of mixing at 80 °C as a function of dmPEG amount. Note that the inset shows the photographic image of DMSO solution at the pointed samples.
plated in a chamber slide at a density of 1.0 × 105 cells/mL and treated with Taxol formulation or HA-paclitaxel conjugate micelle formulation at an equivalent paclitaxel concentration of 1 µg/mL for 24 h at 37 °C. The cells were fixed with 1% paraformaldehyde and then incubated with a propodium iodide (PI) staining solution (0.25 mg/mL PI and 0.1 mg/mL RNase A in PBS solution) at a concentration of 1 × 106 cells/mL for 30 min at 37 °C. Stained cells were visualized at an excitation wavelength of 543 nm using a confocal microscope. For flow cytometric analysis, the stained cells were collected and resuspended in 1.5 mL PBS solution (pH 7.4). The PI fluorescence of each nucleus was analyzed using CELLQUEST software (PharMingen, USA).
RESULTS AND DISCUSSION The formulation of paclitaxel with biocompatible materials has been examined in depth to increase its solubility in aqueous solution. Among the formulations using nanoscale particulates and polymer conjugates, polyglutamic acid and polypeptides were previously used to chemically conjugate paclitaxel to enhance the solubility (34–36). Paclitaxel-conjugated polyglutamic acid, as a prodrug, showed promising antitumor effect in ViVo and is now in clinical trial. HA, a naturally occurring biocompatible polysaccharide, was also used for the conjugation of paclitaxel. Adipic dihydrazide functionalized HA was reacted with active ester derivatized paclitaxel in DMF/H2O mixture for HA-paclitaxel conjugation (27). Although HA-paclitaxel conjugates prepared in the mixed-solvent system showed enhanced solubility of paclitaxel in aqueous medium with good cytotoxicity results, their synthetic method required multiple cumbersome steps for direct conjugation of paclitaxel on the backbone of HA. This is because HA is not soluble in most organic solvents mainly due to extremely hydrophilicnaturewithstronginter/intramolecularhydrogenbonding(14,15). To make HA soluble in polar organic solvents, HA was physically complexed with cationic salts and surfactants to shield highly anionic carboxylic acid groups and to disrupt inter/ intramolecular hydrogen bonds in HA. Alternatively, functional groups in HA were partially protected by hydrophobic blocking agents and deprotected after the conjugation with hydrophobic molecules (25, 28). We recently discovered a novel solubilization method of various biomacromolecules, such as plasmid DNA, proteins, and carbohydrates, in selected organic solvents by nanocomplexing with poly(ethylene glycol) (PEG) (29, 30). In this study, HA was solubilized using dmPEG as nanoscale complexes in anhydrous DMSO. Figure 1 shows transmittance value of the DMSO solution as a function of dmPEG/HA weight ratio. The inset displays photographs of the DMSO solution containing dmPEG/HA mixture taken at the indicated points. The dmPEG/
Figure 2. Synthetic scheme of HA-paclitaxel conjugation.
HA/DMSO solution at a dmPEG/HA weight ratio of 5 became transparent with a transmittance value greater than 90%, while the HA/DMSO solution without adding dmPEG was turbid with a much lower transmittance value as expected. It is likely that HA and dmPEG interacted together to form HA/dmPEG nanocomplexes in DMSO phase via hydrogen bonding as reported earlier (30). The size of HA/dmPEG complexes was 120 ( 6.3 nm in DMSO, as determined by dynamic light scattering technique, sufficiently small to not scatter visible light, resulting in a homogeneous and clear solution. Paclitaxel was then conjugated to HA/dmPEG nanocomplexes finely dispersed in the DMSO phase. A synthetic scheme for the HA-paclitaxel conjugate is shown in Figure 2. Hydroxyl groups of paclitaxel were directly conjugated to carboxylic groups of HA in a single organic phase using DCC/DMAP as coupling agent (37, 38). The resultant HA-paclitaxel conjugate had an acid-cleavable ester linkage. In this study, dimethoxy-PEG (dmPEG) was used for solubilization of HA in DMSO instead of dihydroxyl-PEG to avoid any unfavorable conjugation of hydroxyl group terminated PEG to HA during the HA-paclitaxel conjugation. It should be noted that HA in the HA/dmPEG nanocomplexes was also partially cross-linked by the DCC/DMAP coupling process because it has both hydroxyl and carboxylic acid groups. However, it was most likely that paclitaxel was conjugated to HA mainly at the surface of compactly formed nanocomplexes, limiting self-crosslinking between dmPEG-bound HA molecules. The synthesis of HA-paclitaxel conjugate was verified by 1H NMR analysis (Figure 3). Two different solvents were used to obtain characteristic proton peaks of paclitaxel and HA. In the case of D2O, a strong acetyl (-NHCOCH3) peak was identified at 1.86 ppm along with glucosidic H (10H) at 3.0-4.0 ppm and anormeric H (2H) at 4.30 and 4.40 ppm, while characteristic peaks of paclitaxel such as multiplets in aromatic rings (7.37-7.58 and 7.74-7.85 ppm) were minimal. When DMSO-d6 was used as a solvent, the characteristic peaks of paclitaxel strongly appeared in the 1H NMR spectra along with the disappearance of HA specific acetyl peaks at 1.86 ppm and reduced anormeric H (2H) peak at 4.33 ppm. However, the specific conjugation site in paclitaxel could not be identified. According to the previous reports, it appears that the 2′-hydroxyl group in paclitaxel was preferentially conjugated to HA rather than the 7′-hydroxyl group because it is less sterically hindered (34). From the 1H NMR results, it is conceivable that HA-paclitaxel conjugate micelles were formed in aqueous solution by hydrophobic interaction between paclitaxel molecules conjugated to the HA backbone. Although the paclitaxel specific peaks were observed
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Figure 3. 1H NMR spectra of HA-paclitaxel in (A) D2O and (B) DMSO-d6. HA and paclitaxel specific peaks are indicated with arrows.
in 1H NMR in the polar organic phase, the shielding and deshielding effects of paclitaxel and HA peaks made it difficult to accurately estimate the conjugation extent of paclitaxel to HA. Therefore, the conjugation percent of paclitaxel to HA and the yield of each batch sample were determined by an HPLC using a 50:50 mixture of acetonitrile and water as a cosolvent, and the results were summarized in Table 1. As the molar excess of DCC/DMAP increased, the yield of HA-paclitaxel conjugate increased and reached about 60%. The most paclitaxel conjugated sample exhibited ca. 10.8% of paclitaxel loading content (w/w %), which means approximately nine paclitaxel molecules
Lee et al.
were bound to each HA chain. These data demonstrate that the feed ratio of DCC/DMAP was a critical factor in conjugating paclitaxel to HA. The size of HA-paclitaxel conjugate micelles was measured by DLS (Figure 4A). The HA-paclitaxel conjugate micelles showed an average diameter of 196 ( 9.6 nm with a narrow size distribution. It is well-known that amphiphilic copolymers, such as Pluronic copolymers, poly(lactic-co-glycolic acid) (PLGA)-PEG block copolymers, and PLGA-poly(L-lysine) graft copolymers self-assemble to produce nanosized micelles in aqueous solution via hydrophobic interactions (39–41). To visualize HA-paclitaxel conjugate micelles, AFM and TEM images were obtained (Figure 4B,C). From AFM images, roundshaped HA-paclitaxel conjugate micelles were observed with an average diameter of 232.0 ( 28.2 nm (n ) 30), which corresponds well to the size as measured by DLS. TEM image also provided evidence for the spherical morphology (183 ( 17.8 nm, n ) 30). HA-paclitaxel conjugate micelles were likely to have a core/shell structure composed of a hydrophobic inner core containing aggregated paclitaxel molecules and a hydrophilic HA shell layer. The self-assembly behavior of HA-paclitaxel conjugate in aqueous solution was characterized by measuring the critical aggregation concentration (CAC) of HA-paclitaxel conjugate using pyrene as a hydrophobic fluorescence probe (32, 42). It is known that pyrene can partition into the hydrophobic core of polymer micelles and exhibits aggregation-induced enhanced emission (AIEE) in aqueous solution. Excitation spectra were measured and the intensity ratio of I339/I334 of pyrene was evaluated as a function of HA-paclitaxel conjugate concentration (Figure 4D). A rapid increase of fluorescence intensity and a red shift of pyrene were observed when increasing the concentration of HA-paclitaxel conjugate. From the plot of intensity ratios (I339/I334), the CAC value was estimated to be 7.8 µg/mL. This value is similar to those of polymeric micelles such as poly(L-lactic acid) grafted chondroitin sulfate (5.0 µg/ mL) and PLGA grafted poly(L-lysine) (1.0-9.6 µg/mL), but significantly lower than those of Pluronic copolymers (1-24
Figure 4. Measurement of HA-paclitaxel conjugate micelles by (A) DLS, and the images of HA-paclitaxel nanoparticle by (B) AFM and (C) TEM. (D) Critical aggregation concentration (CAC) measurement by pyrene: plot of intensity ratio between I339 and I334 as a function of concentration.
Technical Notes
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Figure 5. HPLC measurement of released paclitaxel from HA-paclitaxel conjugate micelles after 3 h incubation at different pH values. Paclitaxel is used as a positive control.
mg/mL) (41, 43). Since paclitaxel was conjugated to HA via an acid-cleavable ester linkage, an intact form of paclitaxel was cleaved from the conjugate at slightly acidic environments such as extracellular solid tumor tissues and intracellular endosomal and lysosomal vesicles. After 3 h incubation of the HA-paclitaxel conjugate at four different pH conditions, the release of paclitaxel was monitored using reversed-phase HPLC. As shown in Figure 5, an intact paclitaxel peak appears to a greater extent at more acidic pH. In addition, the gradual left-shift of the HA-paclitaxel conjugate peak was also observed at acidic pH, because the hydrophobicity of the HA-paclitaxel conjugate started to decrease with the gradual cleavage of paclitaxel. It is likely that the release of free paclitaxel from the conjugate micelles was also dependent on the incubation pH. Since acidic physiological condition is in the range of pH 5.0 to 6.0, slow release of paclitaxel from HA-paclitaxel conjugates was observed. Therefore, the pH-dependent release behavior would play a favorable role in enhancing the cytotoxicity effect of free paclitaxel to acidic tumor cells. In Vitro cytotoxicity of HA-paclitaxel conjugate micelles was monitored by a cell viability assay using CCK-8 (Figure 6) (21, 33). As compared to the commercially available Taxol formulation, HA-paclitaxel conjugate micelles exhibited more cytotoxicity for HCT-116 and MCF-7 cells, but reduced cytotoxicity for NIH-3T3 cells. HCT-116 and MCF-7 cells are known to overexpress HA recognizable CD44 receptors on the cell membrane, while NIH-3T3 cells do not overexpress CD44 receptors. Our data suggested that HA-paclitaxel conjugate micelles were internalized to a greater extent within HCT-116 and MCF-7 cells via HA receptor-mediated endocytosis than paclitaxel in the Taxol formulation, resulting in higher cytotoxic effect. Taxol is supposed to transport into cells by a passive diffusion mechanism, but HA-paclitaxel conjugate micelles are likely to be internalized within cells via a HA-receptor-mediated endocytosis process. The cytoxicity effect of HA-paclitaxel conjugate micelles was lower than that of the Taxol formulation for NIH-3T3 cells, probably due to their reduced extent of cellular uptake. It seems that negatively charged HA prohibits the anticancer effect of paclitaxel to HA receptor-deficient cells like NIH-3T3 by reducing nonselective uptake of HA-paclitaxel conjugate micelles. Previously, we showed that HA nanogels physically encapsulating small interfering RNA (siRNA) could be delivered to HCT-116 cells in a HA receptor-mediated, target-specific
Figure 6. In Vitro cytotoxicity of HA-paclitaxel conjugate micelles and Taxol against three different cell lines (A) HCT-116, (B) MCF-7, and (C) NIH-3T3.
manner (21). Similarly, it is conceivable that HA-paclitaxel conjugate micelles could be utilized for targeted delivery of paclitaxel to many tumor cells overexpressing HA receptors. To verify the apoptosis-inducing effect of HA-paclitaxel conjugate micelles, confocal microscopy and flow cytometric analyses were performed for HCT-116 cells. Figure 7 illustrates the confocal microscopic images of HCT-116 cells followed by incubation with various paclitaxel formulations at an equivalent paclitaxel concentration of 1 µg/mL. Morphological change of the cell nuclei such as DNA fragmentation was visualized by propodium iodide (PI) staining to confirm paclitaxel-induced apoptotic cell death. Since HCT-116 cells treated with the control formulation (20 µg/mL of HA) without
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Figure 7. Confocal images of PI stained HCT-116 cells: (A) control, (B) Taxol, and (C) HA-paclitaxel. FACS analysis of apoptotic effects on HCT-116 cells: (D) control, (E) Taxol, and (F) HA_-paclitaxel conjugate micelles. All paclitaxel formulations have 1 µg/mL of equivalent paclitaxel concentration.
paclitaxel were nonapoptotic, strong red fluorescence was detected homogeneously within the nucleus (Figure 7A). In contrast, after incubation with Taxol or HA-paclitaxel conjugate micelles, HCT-116 cells displayed apparent evidence of apoptosis such as segregation and fragmentation of cell nucleus into dense and tiny granules (Figure 7 B,C). It was thought that the activation of endogenous nucleases was associated during the apoptosis which cleaved chromosomal DNA into oligonucleosomal fragments (44, 45). The extent of apoptosis was quantitatively assessed by flow cytometric analysis. Since the cytotoxic activity of paclitaxel is mainly due to its stabilizing effect on polymerized microtubules necessary for spindle formation and cell division, paclitaxel has been shown to cause a cell cycle arrest in the G2/M phase and finally cell death through an apoptotic mechanism (4, 5). As shown in Figure 7D-F, HA-paclitaxel conjugate micelles induced a significant increase in G2/M cell population (64.9%), compared to that of the control (16.1%) and Taxol (31.2%). Consequently, these results demonstrate that HA-paclitaxel conjugate micelles were readily taken up by CD44 overexpressing cells and significantly enhanced the extent of apoptosis-inducing cell death. Nanosized and self-assembled HA-paclitaxel conjugate micelles could be utilized as efficient all-in-one carriers not only for the solubilization of paclitaxel, but also for its passive and active targeted delivery to cancer cells.
CONCLUSIONS In this study, paclitaxel, a very hydrophobic anticancer drug, was directly conjugated to HA in DMSO by forming HA/PEG nanocomplexes without using any ionic surfactants, salts, and blocking agents to functional groups in HA. HA-paclitaxel conjugates were self-assembled to form nanosized spherical
micelles in aqueous solution. HA-paclitaxel conjugate micelles exhibited greater cytotoxicity to HA recognizable CD44 overexpressing cells than the conventional paclitaxel formulation, suggesting that they could be efficiently delivered to cancer cells that overexpress HA receptors.
ACKNOWLEDGMENT This study was supported by the National Research Laboratory grant from the Ministry of Science and Technology, Republic of Korea.
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