Apoptosis induced by paclitaxel-loaded copolymer PLA–TPGS in Hep-G2 cells

Lactide (3,6-dimethyl-1,4-dioxane, C₆H₈O₄), stannous octotate (C₁₆H₃₀O₄Sn), sulforhodamine B, fetal bovine serum (FBS), fetal bovine serum-minimal essential medium (FBS-MEM) were purchased from Sigma-Aldrich. TPGS, toluene, dichloromethane, methanol and dimethyl sulfoxide were obtained from Merck. Paclitaxel was purchased from Dabur Pharma Limited, India. All chemicals were of analytical grade and used without further purification. Antitumor promotion and apoptosis assays in vitro on human hepatocellular carcinoma cell line (Hep-G2) (the cell line obtained from National Institute of Hygienic Epidemy—NIHE) have been performed at Experimental Biology Lab—Institute of Natural Products Chemistry of Vietnam Academy of Science and Technology.

PLA–TPGS copolymer was synthesized by ring-opening polymerization of PLA and TPGS in the presence of stannous octotate as catalyst. The experimental details and the mechanism of reaction have been described in our previous report [24]. Briefly, a given amount of stannous octotate, PLA and TPGS (50:50 w/w) was dissolved in distilled toluene in an ampoule. The reaction was carried out for 10 h at 130 °C under inert gas atmosphere in a silicone oil bath. After the reaction time, the resultant mixture was gently stirred overnight to evaporate the organic solvent. The product was dissolved in dichlomethane and then precipitated in cold methanol in excess to remove unreacted lactide monomers and TPGS. The final product was obtained by filtration.

A modified solvent extraction/evaporation technique was used to fabricate paclitaxel-loaded PLA–TPGS nanoparticles [25]. In brief, a given amount of copolymer PLA–TPGS was dissolved in 20 ml double distilled water containing a small amount of TPGS as an emulsifier. After that, paclitaxel dissolved in dichloromethane was added dropwise into this mixture under vigorously stirring to form an emulsion. After a period of time, the solvent was evaporated and the resulting mixture was centrifuged at 5000 rpm for 15 min to eliminate un-encapsulated paclitaxel and free surfactants. The supernatant-containing paclitaxel-loaded PLA–TPGS nanoparticles were collected by lyophilization.

Lyophilized paclitaxel-loaded PLA–TPGS nanoparticles were redispersed into distilled water to saturation level. The amount of entrapped paclitaxel in nanoparticles was determined by the high-performance liquid chromatography (HPLC) method.

Cell survival cytotoxicity experiments using sulforhodamine B method were performed in order to determine the maximal doses of the testing materials. Soft agar colony assay antitumor promoting activity was estimated based on the inhibition of soft agar colony induction in the Hep-G2 cell line. The cells were cultured in 10% FBS-MEM medium at 36.5 °C in an incubator with 5% CO₂ and 95% air. Cells growing logarithmically in a monolayer culture were trypsinized and suspended in 0.33% agar medium containing 10% FBS with or without samples at the concentrations of 25 μg ml⁻¹. For antitumor promoting assay, using duplicate 6-well plates, 500 μl of the suspension (1 × 10⁴ cells) was poured onto an agar layer containing the same concentration of sample (10 μg ml⁻¹) in 5% dimethyl sulfoxide solution. Soft agar colonies of cells were investigated after 2 weeks of incubation under an inverted microscope with camera to compare the visual cells in their tumor formation, the tumor size and morphology. The inhibitory activities were the average of triplicated experiments and expressed as a percentage of that of the control.

Molecular structure of synthesized copolymer was characterized by Fourier transform infrared spectroscopy (FTIR, SHIMADZU spectrophotometer) using KBr pellets in the wave number region of 400–4000 cm⁻¹. Surface morphology of paclitaxel-loaded PLA–TPGS nanoparticles was investigated by field-emission scanning electron microscopy (FE-SEM) on a Hitachi S-4800 system while size distribution and Zeta potential were determined by dynamic light scattering (DLS) method on a Malvern's Zetasizer–NanoSeries.

Figure 1 shows FTIR spectra of pure paclitaxel, copolymer PLA–TPGS and paclitaxel-loaded nanoparticles. It can be seen from the FTIR spectra that C–H and ${\rm C}=\!\!\!={\rm O}$ stretching bands of PLA–TPGS obtained at 2974 and 1756 cm⁻¹ were observed to shift to 2940 and 1716 cm⁻¹, respectively, in paclitaxel loaded PLA–TPGS. Compared with pure paclitaxel, in IR spectrum of paclitaxel-loaded copolymer nanoparticles there were band shifts from 1072 (C–O stretching of ester bond) to 1069 cm⁻¹, from 1247 (CO–O– stretching vibration) to 1244 cm⁻¹ and from 1650 (amide stretching band of ${\rm C}=\!\!\!={\rm O}$) to 1642 cm⁻¹. The peak at 1718 cm⁻¹, assigned to carbonyl stretching vibration of ester bond in TPGS, changed to 1716 cm⁻¹ in paclitaxel-loaded copolymer. Especially, there was a substantial change in the region of 400–950 cm⁻¹. This may be due to the direct interaction between the hydrophobic core (paclitaxel) and the hydrophobic segment of copolymer (PLA) which created changes in vibrational frequency of C–H out-of-plane deformation (or C–${\rm C}=\!\!\!={\rm O}$ deformation) (689 cm⁻¹) and C–H in-plane deformation (803–941 cm⁻¹). All of these confirmed the success in formation of polymeric micelles loaded with paclitaxel.

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Surface morphology of paclitaxel-loaded PLA–TPGS nanoparticles is shown in figure 2. It can be seen clearly from FE-SEM images that paclitaxel-load PLA–TPGS nanoparticles were round-shape with mean diameter about 10 nm. This result is quite compatible with the size distribution revealed in figure 3. The size distribution is rather narrow and the average size of nanoparticles is about 44 nm. This size is much smaller in comparison with 300 nm of nanoparticles which were fabricated by Zhiping Zhang and Si-Shen Feng [26]. The difference in size between the results of FE-SEM and DLS was because of the different states. Data from FE-SEM showed the dried state of nanoparticles, while DLS method measured the size of nanoparticles suspended in aqueous environment.

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Zeta potential is the electrostatic potential on the slip surface of a particle dispersed in liquid environment. It is a very important index to assess the stability of nanoparticle suspensions. The higher the absolute value of Zeta potential is, the more stable the system will be. The Zeta potential of our paclitaxel-loaded PLA–TPGS nanoparticles was found to be about −18 mV.

The poor solubility of paclitaxel in water is very low (0.4 μg ml⁻¹). After being entrapped in polymeric micelles, the solubility of paclitaxel was found to be 0.2 mg ml⁻¹ increasing 500-fold compared to pour paclitaxel.

To investigate chemotherapeutic potent of paclitaxel-loaded PLA–TPGS nanoparticles, their proliferation inhibition and apoptosis enhancement to Hep-G2 in vitro was studied.

The results in antitumor promoting assay show that there were remarkable changes in size and morphology of tumor in all the samples tested (paclitaxel-free and paclitaxel-loaded PLA–TPGS) as compared with the control. Especially, the tumor size decreased significantly in the case treated with paclitaxel-loaded PLA–TPGS (figure 4). Thus, it can be concluded that paclitaxel-loaded PLA–TPGS has positive effects on tumor promotion of Hep-G2 cell line in vitro.

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Because proliferation and apoptosis are the most important manners of cancer cells, when the proliferation is in a dysregulated condition, cancer cells may become resistant to antiproliferative factors and uncontrollable in their proliferation rate. The apoptosis pathway in many cases can regulate the cell proliferation [27, 28].

Under the phase-contrast microscope, the cells exposed to 0.1, 0.2, 0.4, 0.5 and 1 μg ml⁻¹ of paclitaxel-loaded PLA–TPGS exhibited obvious changes in morphologic characteristics. There were more shrinking cells and floating cells that had lost their adhibit ability in the paclitaxel-loaded PLA–TPGS than in the controls (figure 5). With lower concentration of paclitaxel-loaded PLA–TPGS (0.1 μg ml⁻¹), the cell morphologic had been changed and the cells began to separate from each other. It seems that there was a loss of surface receptors of the cell, resulting in their losing their adherence onto the bottom of wells or losing their cell–cell connective signals needed for their angiogenic forming. From figure 5 we can see that their apoptosis increased with increasing of tested complex concentration (0.2, 0.4, 0.5 and 1 μg ml⁻¹). Besides the apoptosis pathway with small size ranging from 40 to 50 nm, paclitaxel-loaded PLA–TPGS nanoparticles may penetrate through cell membrane and may interfere in the metabolite actions of cells, and at last cause cell death. This should also be one of the most typical apoptotic pathways while the larger size of paclitaxel-free PLA–TPGS makes it hard to penetrate through the cell membrane and it cannot interfere in metabolism of the cells. In this case, the cross-talking of the antitumor promotion and pro-apoptotic signaling pathways caused by paclitaxel-loaded PLA–TPGS may suggest an ultimate determination for further research at the molecular level. These pro-apoptotic or anti-apoptotic factors are spotlighted in both the clinical treatments of malignancy and cancer chemoprevention strategy as well.

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