Folate-modified, curcumin and paclitaxel co-loaded PLA-TPGS nanoparticles: preparation, optimization and in vitro cytotoxicity assays

Ring-opening polymerization method was used to synthesize 5 formulations of PLA-TPGS as described in the previous work [25]. Briefly, exact amount of the lactide monomer and TPGS was weighted and dissolved in toluene. Lactide monomer, TPGS (in toluene) and stannous octoate were added in an ampoule and allowed to react at 130 °C in nitrogen atmosphere. After reacting for 7 h, the solvent was removed to obtain raw product. Then this mixture was purified by dissolving in dichloromethan followed by precipitating in cold methanol for 3 times. Afterthat, copolymer PLA-TPGS was obtained by centrifugation and dried in oven at 45 °C for 48 h.

In order to optimize the structure of this delivery system, we carried out modifying ratios between PLA and TPGS as 1:3, 1:2, 1:1, 2:1, 3:1 (w/w) to find out which ratio has the best Cur and PTX loading and biological effect.

Initially, the hydroxyl groups of TPGS were reacted with carboxyl groups of aspartic acid. The esterification was performed at room temperature for 24 h with the catalystic activity of EDC and NHS (molar ratio of 1.2:1.2). Next, folic acid (Fol) was also activated with EDC and NHS in NH₄OH 2 M for 6 h at 50 °C to establish an identified mixture of Fol-NHS. The activated TPGS-COOH solution above was dropped into the Fol-NHS solution, and then the obtained mixture was adjusted pH to 8, before being stirred at 37 °C for 4 h.

First of all, each 10 mg PLA-TPGS copolymer with different ratios of monomer was dissolved in 10 ml dichloromethan and magnetically stirring for 2 h. Then 10 ml H₂O was added and the mixture was continuously stirred until all the copolymer transferred to the aqueous phase. Then this aqueous phase was collected. Simultaneously, a solution of Cur and PTX was also prepared by dissolving completely 4 mg Cur and 4 mg PTX in 10 ml ethanol. In the next step, the solution of Cur and PTX was dropped into water layer separated from the copolymer mixture above. The obtained solution was stirred for 24 h at room temperature. Finally, 1 ml activated folate solution was added and repeatedly stirred for 2 h to form (Cur  +  PTX)-PLA-TPGS-Fol NPs. After centrifugation, the nanosystem was obtained from the supernatant while the pellet was used to determine the nonencapsulated amount of PTX and CUR.

The encapsulation efficiency ($EE\left( \% \right)$) of PTX and Cur in micelle was calculated by the following equation:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle &E{{E}_{{\rm Cur}~}}\left( \% \right)\nonumber \\ &=\frac{{\rm Total}\,{\rm amount}\,{\rm of}\,{\rm Cur{\text -}nonencapsulated}\,{\rm amount}\,{\rm of}\,{\rm Cur}}{{\rm Total }~ \,{\rm amount}\,{\rm of}\,{\rm Cur}}~\times 100,\nonumber \end{align} \tag{ 1 }$$

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle &E{{E}_{{\rm PTX}}}\left( \% \right)\nonumber \\ &=\frac{{\rm Total}\,{\rm amount}\,{\rm of}\,{\rm PTX{\text -}nonencapsulated}\,{\rm amount}\,{\rm of}\,{\rm PTX}}{{\rm Total}\,{\rm amount}\,{\rm of}\,{\rm PTX}}~\times 100.\nonumber \end{align} \tag{ 2 }$$

FTIR spectra of PLA-TPGS copolymer, Cur, PTX, folic acid and nanoparticles were determined by using compressed KBr pellet method in Perkin Elmer FTIR spectrophotometer (Perkin Elmer Spectrum Two, Waltham, Massachusetts, USA). Size and morphology of nanoparticles was obtained by FESEM images. Dynamic size, polydispersity and zeta potential of (Cur  +  PTX)-PLA-TPGS-Fol were performed using a Zetasizer version 7.03, Malvern instruments Ltd, Malvern, UK [16].

In the first cytotoxicity assays, five (Cur  +  PTX)-PLA-TPGS-Fol nanosystems based on different PLA-TPGS copolymers were studied for their anticancer activity on 4 cell lines: Hep-G2, HeLa, LU-1 and Vero. This assay was carried out as described by Skehan et al and Likhitwitayawuid et al [26, 27]. In brief, cells were cultured in Dulbecco's modified eagle medium DMEM, a medium consisting of 1% penicillin-streptomycine and 10% bovine calf serum, at 37 °C under 95% air and 5% CO₂ atmosphere for 24 h. Next, the medium containing (Cur  +  PTX)-PLA-TPGS-Fol nanosystems was replaced the initial medium. After 48 h of incubation, trichloroacetic acid was added to stop the reaction and incubated for 1 h. After being washing with distilled water, the plates were dyed with SRB for 30 min. The results were read by ELISA. The cell survival value (CS) was calculated according to the following formula:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle CS\left( \% \right)=\frac{OD\left( {\rm sample} \right)-OD\left( {\rm day }~{\rm 0} \right)}{OD\left( {\rm DMSO} \right)-OD\left( {\rm day }~{\rm 0} \right)}~~\times ~100,\nonumber \end{align}$$

in which OD(sample) is the optical density of the plates incubated with (Cur  +  PTX)-PLA-TPGS-Fol; OD (day 0) is the optical density of the plates before incubation; OD(DMSO) is the optical density of the plates incubated with dimethyl sulfoxide (DMSO, control sample).

In the second cytotoxicity, the best nanosystem chosen from these above results was used to test its activity on A549, MCF7, Hep-G2 and HacaT cell lines in comparison with free drugs and nanosystem encapsulating single drug (Cur or PTX). This assay was carried out by MTS method. In brief, each cancer cell line was cultured in a 96 well cell plate and then was added with accurate concentration of drug solution or nanosystems, incubated for 48 h. After that, the cells were incubated with MTS in 3 h and the results were read by ELISA. Half maximal inhibitory concentration ($I{{C}_{50}}$) was calculated as the concentration of drug or drug delivery nanosystems that was required to inhibit 50% of cancer cells.

The efficacy of 5 drug delivery nanosystems based on 5 copolymers with different ratio of PLA and TPGS were tested on 3 cancer cell lines (HepG2, HeLa, LU-1) and Vero cell line. The results were presented in table 2.

From table 2 we can see that different formulations had different effects on cancer cell lines. All of them were cytotoxic to HepG2 cells with very high effect at low concentration of $0.0625\,\mu {\rm g}\times {\rm m}{{{\rm l}}^{-1}}$. Besides, all of the formulation had positively effect on HeLa cells but with different levels of action: the nanosystems based on copolymer with higher ratio of TPGS (1:3, 1:2, 1:1) performed a very strong effect on HeLa cells when that of formulation with high ratio of PLA (2:1, 3:1) still existed but weaker. LU-1 cancer cells seem hard to be influenced by the nanosystems except the formulation of PLA-TPGS (3:1) and (1:1) at high drug concentration of $0.5\,\mu {\rm g}\times {\rm m}{{{\rm l}}^{-1}}$. Surprisingly, the highest loading content of Cur and PTX was achieved on the 3:1 nanosystem, however, the nanocarrier-based formulation with the ratio 1:1 of PLA/TPGS was the most effective system on all cancer cell lines, especially on HepG2 and HeLa. This observation could be explained by 2 reasons. One reason is the smaller size helps 1:1 formulation easier cellular uptake. Another reason is the faster rate of Cur or PTX release resulted from high percent of hydrophilic TPGS in 1:1 formulation cause higher toxicity on cancer cells than 2:1 or 3:1 formulation [28]. Therefore we chose the copolymer PLA-TPGS 1:1 for further study.

From these above results, we chose copolymer PLA-TPGS (1:1 w/w) to study the cytotoxicity of the drug delivery nanosystem encapsulating both Cur and PTX compared to the nanosystems with only one drug (PLA-TPGS-Cur-Fol or PLA-TPGS-PTX-Fol). The outcomes were expressed in table 3, figures 4 and 5.

Table 3. $I{{C}_{50}}$ of the drug delivery nanosystems on A549, MCF7, HepG2 and HacaT cell line.

Cell	IC50  ±  SD
	A549	MCF7	HepG2	HacaT
PLA-TPGS-Cur-Fol	$6.5\pm 2.2$	$6.1\pm 0.5$	$5.0\pm 1.2$	$3.3\pm 0.8$
PLA-TPGS-PTX-Fol	$3.4\pm 0.5$	$4.6\pm 0.9$	$3.2\pm 0.3$	$3.3\pm 1.7$
(Cur  +  PTX)-PLA-TPGS-Fol	$0.3\pm 0.1$	$4.5\pm 1.3$	$3.4\pm 0.3$	$2.0\pm 0.8$

Zoom In Zoom Out Reset image size

Zoom In Zoom Out Reset image size

As shown in figure 4, the cells of all the 4 cell lines were shrunk and distorted when treated with (Cur  +  PTX)-PLA-TPGS-Fol nanosystem. In addition, significant cell surface disruption and fragments of organelles were observed. Especially, at the same concentration of the drug, the co-formulation of (Cur  +  PTX)-PLA-TPGS-Fol show better effect on the cancer cells than monodrug formulation.

The free Cur, free PTX and PLA-TPGS had $I{{C}_{50}}$ of more than $200\,\, \mu {\rm g}\times {\rm m}{{{\rm l}}^{-1}}$, so they were considered as non-cytotoxic agents (data not shown). Meanwhile, the drug delivery nanosystems had significantly enhanced cytotoxicity on 4 cancer cell lines with very low $I{{C}_{50}}$ values.

The combination of Cur and PTX in the same drug delivery nanosystem showed a meaningful improvement in inhibiting A549 cancer cells when compared with the drug delivery systems with only Cur or PTX ($I{{C}_{50}}$ of $0.3\,\, \mu {\rm g}\times {\rm m}{{{\rm l}}^{-1}}$ compared to 6.5 or $3.4\, \mu {\rm g}\times {\rm m}{{{\rm l}}^{-1}}$). To explain for this result, besides anticancer efficacy, Cur assists to prevent PTX from turning inactive nuclear factor kappa B ($NF-\kappa B$) into active form. To be more specific, this factor is inactive in healthy cells, ensuring apoptosis process happen in normal way. Almost anticancer agents including PTX activate $NF-\kappa B$, which might lead to uncontrolled growth of tumor [29]. Moreover, Cur could down regulate the expression of permeability glycoprotein (P-gp) and multidrug resistance protein 1 (MDR1) in cancer cells [30], had reverse effect on enzyme system mediated tumor multidrug-resistance [31], reverse effect on apoptosis proteins mediated tumor multidrug-resistance [32], and reversal effect on DNA repair mechanisms mediated tumor multidrug-resistance [33].

For other cancer cell lines, the combination did not improve the cytotoxicity of the nanosystems. HacaT cells also have folate receptors on their surface so it is still impacted by the folate-nanosystems.

The results point out that the loading of PTX and Cur by copolymer block enhances the cytotoxic effects of drugs. Besides, it can be implicated that sustained release of drug from nanocarriers along with the possibly higher cellular uptake could be major reason behind the enhanced inhibitory effect of nanoparticles.