Enhanced cellular uptake and cytotoxicity of folate decorated doxorubicin loaded PLA-TPGS nanoparticles

Copolymer PLA-TPGS was obtained from Laboratory of biomedical nanomaterials, Institute of Materials Science, Vietnam Academy of Science and Technology. Vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate) was obtained from Merck. Doxorubicin hydrochloride (DOX.HCl), 1-(ethyl-3-(3-dimethylamino) propylcarbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), glutaric acid, folic acid, ethylene diamine and triethylamine were obtained from Sigma-Aldrich. All solvents used are HPLC grade, which include toluene, dichloromethane (DCM), methanol and dimethyl sulfoxide (DMSO, anhydrous) from Aldrich. Distilled water was used throughout all experiments. Human cervical carcinoma (HeLa) and human colon adenocarcinoma (HT-29) cell lines were obtained from Lab of Department of Biology, Hanoi University of Science. Solvents and chemicals for bioassays were purchased from Invitrogen.

Folate was covalently attached to TPGS molecules through a modified process described by Pan and Feng [16]. Firstly, TPGS was activated by reaction with aspartic acid in the presence of DCC and NHS as catalysts at the molar ratio of TPGS/aspartic acid/DCC/NHS of 1/1/1.2/1.2. Aspartic acid was attached to TPGS through the esterification of its carboxyl group with hydroxyl group of TPGS. The reaction was carried out at room temperature for 24 h. The product was filtered to remove unreacted parts and by-product. Secondly, folic acid was animated by reaction with NHS using DCC as catalyst in DMSO solvent at the molar ratio of folic acid/DCC/NHS of 1/1.2/1.2 and stirred for 6 h at 50 °C. The product was then reacted with ethylene diamine and precipitated with acetonitrile. Finally, activated TPGS was reacted with animated folic acid at a molar ratio of 1/1.2 for 6 h at 37 °C. The product was filled and then precipitated with acetonitrile. The final product was lyophilized to obtain dry TPGS-Fol.

DOX loaded nanoparticles were prepared by an emulsion solvent evaporation method. DOX.HCl (15 mg) was dissolved in dichloromethane (15 ml) and then deprotonated by the addition of triethylamine (1.5 ml). The dichloromethane solution of DOX was stirred in a closed flask for 6 h. Copolymer PLA-TPGS or mixture of PLA-TPGS and TPGS-Fol (9:1, w/w) (40 mg) was dissolved in double distilled water (60 ml). The dichloromethane solution of DOX was added dropwise into the water solution of copolymer under vigorous stirring. The mixture was stirred for 24 h in a closed flask and then dichloromethane was evaporated under vacuum pressure. The obtained mixture was centrifuged at 5600 rpm for 10 min to remove free DOX. The red transparent solution was collected. A half of this solution was lyophilized and stored at 4 °C.

Molecular structure of DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs was characterized by Fourier transform infrared spectroscopy (FTIR, SHIMADZU spectrophotometer) using KBr pellets in the wave number region of 400–4000 cm⁻¹. Their morphology was investigated by field emission scanning electron microscopy (FE-SEM) on a Hitachi S-4800 system. Size distribution was measured by dynamic light scattering (DLS) method.

Drug loading content (LC) and drug encapsulation efficiency (EC) were determined with a UV–vis spectrophotometer at 480 nm. A calibration curve was obtained with DOX.HCl/water solution at different concentrations of DOX. The LC and EC were calculated based on the following equations

$$\begin{eqnarray*}&&{\rm LC}(\%)=\frac{{{W}_{{\rm drug}}}}{{{W}_{{\rm total}}}}\times 100,\end{eqnarray*}$$

$$\begin{eqnarray*}&&{\rm EC}(\%)=\frac{{{W}_{{\rm drug}}}}{{{W}_{0,{\rm drug}}}}\times 100\end{eqnarray*}$$

with W_drug is the weight of loaded drug, W_total is the total weight of polymers, W_0,drug is the initial weight of fed drug.

In vitro drug release of drug delivery systems were performed in phosphate buffer saline (PBS) solution at pH 7.4 at 37 °C. 5 mg lyophilized nanoparticle was dispersed in 20 ml PBS. After each period of time, a 3 ml sample was taken and 3 ml distilled water was added. The taken sample was centrifuged at 5600 rpm for 10 min to remove released DOX. DOX concentration in obtained solution was determined based on absorbance intensity at 480 nm. DOX release was calculated by following equation:

$$\begin{eqnarray*}&&{\rm DOX}\;{\rm release}\;(\%)=\frac{{{C}_{0,{\rm DOX}}}-{{C}_{t,{\rm DOX}}}}{{{C}_{0,{\rm DOX}}}}\times 100\end{eqnarray*}$$

with C_0,DOX is initial concentration of DOX, C_t,DOX is concentration of DOX at time t.

Two cancer cell lines (HT29 and HeLa) were chosen to investigate cytotoxicity and targeting effect of drug delivery systems. Cancer cells were activated and cultured under atmosphere of 5% CO₂ and 95% air at 37 °C. Each cell line was cultured by an appropriate medium. The media were refreshed every 2 days to ensure sufficient nutrients and remove dead cells. After being cultured, cells were placed on a 96-wells plate and kept stable for 48 h.

In vitro cytotoxicity of drug delivery systems was examined by MTS assay. A cell was exposed to free DOX, DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs at the concentration of DOX ranging from 0.017 to 5.172 μM. After 48 h incubation with drug formulations, the cell was incubated with the mixture of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) in the presence of PMS (phenazinemethosulfate). MTS will be converted to formazan by dyhedrogenase in a viable cell. The absorbance of produced formazan, which correlated with the number of viable cells in each well, was measured by a UV–vis spectrophotometer at 490 nm. The absorbance of treated groups was compared with the control group to obtain cell survival percentage. All experiments were run in triplicate and the results were recorded as mean ± standard deviation.

In vitro cellular uptake experiments were performed on HT29 cell lines. The cells were exposed to free DOX, DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs at a concentration of 10 μM for 2 h. Cells were washed three times with deionized water, stained with Hoechst 33 342 fluorescent stain for 15 min and washed three times with deionized water. Cell images were taken by laser scanning confocal microscope (LSCM) at 346 and 480 nm.

LC and EC were determined through the absorbance of DOX solutions at 480 nm. The calculated LC and EC of DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs were 30.68 ± 1.18, 81.80 ± 4.73% and 25.81 ± 0.80, 68.82 ± 2.13%, respectively. The slight decrease of LC and EC may be due to the reduction in emulsification efficiency of copolymer PLA-TPGS when mixed with TPGS-Fol.

Chemical structures of DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs were investigated by FTIR spectroscopy. Figure 1(a) shows the FTIR spectra of PLA-TPGS, DOX and DOX/PLA-TPGS NPs. After loading DOX, the characteristic bands of PLA-TPGS and DOX were observed to be shifted. Typically, the bands at 2974 and 1756 cm⁻¹ are assigned to the C–H and C=O stretching vibrations of PLA-TPGS [11] which, respectively, shifted to 2920 and 1760 cm⁻¹ in spectrum of DOX-PLA-TPGS. Besides, characteristic bands at 1630 cm⁻¹ corresponding to N–H bending vibrations, at 1427 cm⁻¹ corresponding to C–C stretching vibrations, at 1010 cm⁻¹ corresponding to C–O stretching vibration of DOX [17] were observed at 1620, 1400 and 1023 cm⁻¹ in DOX-PLA-TPGS NPs spectrum. The appearance of these characteristic bands proved the loading of drug into the micelle system of PLA-TPGS.

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Figure 1(b) shows the changes in FTIR spectra of DOX/PLA-TPGS NPs, folic acid and Fol/DOX/PLA-TPGS NPs. Characteristic bonds of DOX/PLA-TPGS NPs at 2920, 1760, 1620, 1400, 1216 and 1023 cm⁻¹ were shifted to 2927, 1703, 1615, 1393, 1234 and 1011 cm⁻¹, respectively, in the FTIR spectrum of Fol/DOX/PLA-TPGS NPs. The peaks at 1703 and 1615 cm⁻¹ were also attributed to the C=O stretching vibrations of folic acid (at 1695 and 1608 cm⁻¹). In addition, the presence of peak at 1512 cm⁻¹ related to the bending mode of N–H vibration of folic acid [18].

To evaluate the sizes of DOX loaded PLA-TPGS nanoparticles, field emission scanning electron microscope (FESEM) images were taken of DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs and are shown in figure 2. We can see that nanoparticles have spherical shape with a uniform diameter of about 50–60 nm.

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The hydrodynamic diameter of the polymeric system was determined by DLS method (figure 3). The obtained results from DLS were slightly larger, about 90 nm for DOX/PLA-TPGS and 110 nm for Fol/DOX/PLA-TPGS. The size distribution followed the standard curve with one peak distribution. The difference in size between the results of FESEM and DLS was because of the different states. Data from FESEM showed the dried state of nanoparticles, while DLS method measured the size of nanoparticles suspended in aqueous environment.

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Figure 4 shows the DOX release from DOX.HCl, DOX/PLA-TPGS and Fol/DOX/PLA-TPGS NPs. In the case of DOX.HCl, the drug was absolutely soluble in PBS solution right after dissolving into buffer solution. For both cases of DOX/PLA-TPGS and Fol/DOX/PLA-TPGS NPs, the DOX release from nanoparticles displayed a biphasic release profile. The initial burst associated with the fast release of drug molecules took place in the first 8 h. In the second phase, DOX was progressively released and reached about 60% release. The difference in DOX release rate from DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs was not noticeable.

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Figures 5(a) and (b) show the HT29 and HeLa dose response curve of free DOX, DOX/PLA-TPGS and Fol/DOX/PLA-TPGS NPs, which were determined using MTS assay. For both cancer cell lines we can see that as the DOX concentration increased, the cell survival decreased which indicates that the effect of free drug and loaded drug are dependent on the concentration. From these results, the half maximal inhibitory concentration (IC₅₀) was calculated. For HT29 cells, IC₅₀ values of free DOX, DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs were 0.64 ± 0.03, 1.39 ± 0.05 and 0.39 ± 0.04 μM, respectively, while for HeLa cells, IC₅₀ values of those were 0.46 ± 0.03, 1.22 ± 0.07 and 0.24 ± 0.02 μM, respectively. From these values, we suggest that the DOX loaded nanoparticles with folate decoration significantly decreased the IC₅₀ values for both HT29 and HeLa cells while the values of those without folate were much higher compared to free DOX. Similar results were also obtained by quantitatively comparing the cell density from the cytotoxicity images shown in figure 6.

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Figure 7 shows the cellular uptake of Free DOX, DOX/PLA-TPGS NPs and Fol/DOX/PLA-TPGS NPs. Cells after incubation with nanoparticles were stained with nuclei staining Hoechst 33 342. The blue fluorescent light of Hoechst (excited at 346 nm) shows the nuclei position of cell in the samples while the red fluorescent light of DOX (excited at 480 nm) expresses the position of drug inside the cells. From these images, we can see that DOX was taken into the nuclei of the cells in all cases. Quantitatively, Fol/DOX/PLA-TPGS NPs exhibited the best cellular uptake via the highest fluorescent intensity. In contrast, it is hard to observe the red fluorescent light in cells incubated with DOX/PLA-TPGS NPs. This shows that the cellular uptake of DOX/PLA-TPGS NPs is lowest.

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