Targeted drug delivery nanosystems based on copolymer poly(lactide)-tocopheryl polyethylene glycol succinate for cancer treatment

Nanotechnology has brought about many breakthrough advances in human society. Its applications cover many scientific areas such as chemistry, biology, medicine, material, physics, etc. In medicine, nanotechnology helps in making modern tools for diagnosing and treating disease. Among them, drug delivery nanosystems (DDNSs) exhibit huge potential in treating cancer. DDNSs enable precise delivery of drugs to disease sites through targeting mechanisms; namely passive and active targeting. In addition, DDNSs play a crucial role in protecting drugs from the physiological activities of the body.

Passive targeting uses the unique properties of the tumor microenvironment, most notably: (i) leaky tumor vasculature, which is highly permeable to macromolecules relative to normal tissue; and (ii) a dysfunctional lymphatic drainage system, which results in enhanced fluid retention in the tumor interstitial space [1]. The tumor blood vessels are irregular in shape, dilated, leaky or defective, and the endothelial cells are poorly aligned or disorganized with large fenestrations. The pore cutoff size of these fenestrations has been reported to be between 400–600 nm [1]. This allows macromolecules or particles with a size below 400 nm to easily penetrate into the tumor. Moreover, the clearance of macromolecules from tumors is lower than that from normal tissue, leading to the longer retention at the tumor. This phenomenon has been characterized and termed the tumor-selective enhanced permeability and retention (EPR) effect [2]. However, to escape capture by fixed macrophages that are lodged in the reticuloendothelial system, such as the liver and spleen, their size must be below 200 nm [3].

In comparison with passive targeting, active targeting is achieved by delivering drug-encapsulated nanoparticles to uniquely identified sites while having minimal undesired effect elsewhere. Targeting ligands are conjugated on the particle surface that can recognize and bind to specific receptors that are unique to cancer cells. Targeting ligands may be antibodies, aptamers, peptides or small molecules [4].

Materials used for DDNSs must be biocompatible, biodegradable and non-toxic. Among them, biocompatible and biodegradable polymers have been extensively studied for fabricating nanocarriers, for example, polymeric micelles. Polylactide acid (PLA) is a polymer synthesized from mono lactide which is approved for biomedical applications. Its hydrophobicity allows the carrying of hydrophobic anticancer drugs. However, its hydrophobicity also limits the dispersion of drugs into the blood circulation. Therefore, many hydrophilic polymers such as polyethylene glycol (PEG) and d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) were combined with PLA to produce amphiphilic copolymers [5, 6]. These copolymers form polymeric micelles having a core–shell structure in an aqueous environment. The core composed of PLA serves as a microenvironment for incorporating hydrophobic drugs, and the shell composed of hydrophilic polymers stabilizes the micelles and protects the drugs from the physiological environment of the body [7]. The polymeric micelles possess many advantages such as small size, high drug-loading capacity, highly structural stability and good bioavailability [8].

Previously we have synthesized the copolymer PLA-TPGS in order to fabricate a DDNS carrying curcumin, paclitaxel and doxorubicin [9]. To improve the selective targeting effect to cancer cells, folate as an active targeting ligand was decorated onto the DDNS. In this article we present the synthesis and characterization methods as well as testing results in in vitro and in vivo models. The results showed that micelles composed of copolymer PLA-TPGS were a good DDNS for loading drugs and targeting cancer cells and tumors.

The copolymer PLA-TPGS was synthesized by ring-opening polymerization in toluene at a high temperature. In the presence of stannous octoate (Sn(Oct)₂) as a catalyst, mono lactide acid was polymerized to form PLA chains which then combined with polymer TPGS through estefication between the carboxyl group of PLA chains and hydroxyl group of TPGS to produce the copolymer PLA-TPGS. Control of size and drug-loading capacity is based on controlling the length of the PLA chain.

Active targeting ligand (folate) was attached to the TPGS molecule through a modified process reported by Pan and Feng [10]. Firstly, TPGS was activated with aspartic acid instead of glutamic acid through the esterification between the hydroxyl group of TPGS and carboxyl group of aspartic acid. Next, folic acid was animated with ethylene diamine through the formation of an amide bridge. Finally, the activated TPGS was reacted with the animated folic acid to form folate-attached TPGS (TPGS-Fol) molecules. All reactions were performed in dimethyl sulfoxide, under an ambient temperature and in the presence of N-hydroxysuccinimide and N,N'-dicyclohexylcarboiimide as catalysts.

Anticancer drugs including curcumin, paclitaxel and doxorubicin were chosen to fabricate DDNSs in our studies. Drugs were loaded onto the delivery systems by the emulsification and solvent evaporation method. Briefly, drugs were dissolved in organic solvents such as ethanol, methanol and dichloromethane. The emulsification process took place by slowly adding drug solutions into water solutions containing copolymer PLA-TPGS or a mixture of copolymer and TPGS-Fol. After a determined period of time, the solvent was evaporated. The final transparent solution was obtained by centrifugation at 5600 rpm to remove unencapsulated drugs and then stored at 4 °C for further use.

Nuclear magnetic resonance (¹HNMR) and Fourier transform infrared (FTIR) spectroscopies were used to analyze the structure of the synthesized copolymer and also DDNSs. In the ¹HNMR spectra (figure 1), characteristic signals of proton vibrations corresponding to –CH (5.043 ppm), –CH₃ (1.630 ppm) of PLA and –CH₂CH₂– (3.641 ppm) of TPGS were shifted to 5.166, 1.562 and 3.639 ppm in the ¹HNMR spectrum of  PLA-TPGS copolymer [9].

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The copolymer PLA-TPGS is a polyester; thus, ester linkage is its characteristic linkage. Figure 2 shows vibrations at 1756 cm⁻¹ which are attributed to ester linkages in the synthesized copolymers. The C-H banding of PLA at 2930 cm⁻¹ was shifted to 2974 cm⁻¹ in PLA-TPGS [2].

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For hydrophobic drugs, their solubility after loading onto the delivery system is a concerning issue. First of all, a good drug delivery system must have a high drug-loading capacity. After encapsulation by copolymer PLA-TPGS, the solubility of curcumin and paclitaxel increased remarkably. The solubility in water of paclitaxel is 0.4 μg ml⁻¹; after encapsulation by copolymer PLA-TPGS, its solubility increased 500-fold to 0.2 mg ml⁻¹ [11]. In the case of curcumin, its water solubility is 6.79 μg ml⁻¹, which increased about 350-fold to 2.4 mg ml⁻¹ [9] (figure 3).

The physiochemical characteristics of drug delivery systems were investigated by FTIR. The interaction between drugs and copolymer PLA-TPGS was assessed by analyzing the changes in characteristic vibrations of components. For example, figures 4 and 5 show the FTIR spectra of paclitaxel-loaded copolymer PLA-TPGS (PTX/PLA-TPGS NPs) and folate-decorated paclitaxel-loaded copolymer PLA-TPGS nanoparticles (Fol/PTX/PLA-TPGS NPs) [12]. In the FTIR spectrum of PTX/PLA-TPGS, the peak at 1716 cm⁻¹ was attributed to the overlap of C=O stretching of PTX at 1712 cm⁻¹ and PLA-TPGS at 1756 cm⁻¹. Characteristic peaks of PTX at 1648 cm⁻¹ (C-C stretching), 1245 cm⁻¹ (C-N stretching) and 1074 cm⁻¹ (C-O stretching) [13] were shifted to 1640, 1240 and 1069 cm⁻¹, respectively. The presence of a peak at 1531 cm⁻¹ was assigned to the absorption band at 1546 cm⁻¹ of the aromatic ring of PLA-TPGS. In the FTIR spectrum of Fol/PTX/PLA-TPGS, compared to that of PTX/PLA-TPGS, characteristic peaks of PTX/PLA-TPGS at 1716, 1640, 1240 and 1069 cm⁻¹ were shifted to 1677, 1616, 1230 and 1064 cm⁻¹, respectively. In addition, the presence of peaks at 1590 cm⁻¹ and 1480 cm⁻¹ was attributed to N-H bending (1605 cm⁻¹) and the absorption of the phenyl ring (1485 cm⁻¹) of folic acid [14]. All these data provided evidence of the success in fabricating Fol/PTX/PLA-TPGS NPs.

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Field emission scanning electron microscopy (FESEM) was used to investigate the size of DDNSs, which is one of the critical factors determining the therapeutic efficacy of drugs.

All drug-loaded copolymer PLA-TPGS nanoparticles have a very small size of 50 to 100 nm (figure 6). This size range is very suitable for application in a drug delivery system in cancer treatment for two reasons. Firstly, nanoparticles with a size below 100 nm are able to escape from the elimination of macrophages in the reticulendothelical system. Secondly, nanoparticles in this size range can easily penetrate into the tumor through the EPR effect. These reasons contribute to increasing the therapeutic efficacy of DDNS.

3.1. In vitro enhanced cellular uptake and cytotoxicity

Cellular uptake of an object depends on many factors such as size, surface properties, state (crystalline or solute), and hydrophobic or hydrophilic nature. In our studies, the cellular uptake of drug-loaded copolymer PLA-TPGS nanoparticles was investigated by taking advantage of the natural fluorescence of curcumin and doxorubicin.

In the case of curcumin, the green signal in figure 7 exhibits the presence of curcumin inside the cancer cell. Compared to free curcumin (figure 7(a)), curcumin-loaded copolymer PLA-TPGS nanoparticles (Cur/PLA-TPGS NPs) (figure 7(b)) clearly showed higher cellular uptake. To explain this, we investigated the size of free curcumin and Cur/PLA-TPGS NPs. Curcumin is hydrophobic; thus, when dispersed in an aqueous environment, large curcumin crystals will be formed (figure 8(a)). In contrast, Cur/PLA-TPGS NPs have excellent water solubility with a very small size below 100 nm (figures 6(b) and 8(b)). It is clear that with the small size, drugs will easily penetrate into the cancer cell.

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In the case of doxorubicin, cellular uptake of three systems including free doxorubicin (free DOX), doxorubicin-loaded PLA-TPGS copolymer nanoparticles (DOX/PLA-TPGS NPs) and folate-decorated doxorubicin-loaded copolymer PLA-TPGS nanoparticles (Fol/DOX/PLA-TPGS NPs) was investigated (figure 9) [15]. The blue fluorescent light of Hoechst (excited at 346 nm) shows the nuclei position of the cells in the samples while the red fluorescent light of DOX (excited at 480 nm) shows the position of the 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. The results could be explained based on the cell internalization mechanism. It is reported that the cellular uptake of nanoparticles happens via the endocytosis process [16] while free DOX in the form of water-soluble molecules are internalized into the cell via passive diffusion [17]. Because they exist in the form of single molecules, the entry of DOX.HCl molecules into the cell via passive diffusion may be easier and faster than the endocytosis of DOX/PLA-TPGS NPs. For Fol/DOX/PLA-TPGS NPs, folic acid induced the specific binding to the folate receptor overexpressed on the HeLa and HT29 cell surface. This binding facilitated folate receptor-mediated endocytosis, resulting in a better cellular uptake of Fol/DOX/PLA-TPGS NPs.

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3.2. In vitro increased cytotoxicity in cancer cells

Growth inhibition of cancer cells was assessed in cancer cell lines including Hep-G2, HeLa and HT29. Cancer cells were exposed to drug delivery systems at different concentrations of the drug. Morphology changes and cell density were observed and calculated to assess the impact of drug delivery systems.

Cell growth inhibition of PTX/PLA-TPGS NPs was studied on the liver cancer cell line Hep-G2 (figure 10). The results showed that PTX/PLA-TPGS NPs had a strong effect on cell growth. After exposure to nanoparticles, cells clearly changed their morphology from their typical shape (in the control sample) to a rounded shape. The cell survival density decreased when the concentration of the drug increased. Meanwhile, at the same concentrations of drug, free PTX did not inhibit cell growth notably (data not shown). The morphological changes of cells demonstrated that nanoparticles may interact with cancer cells through the apoptosis pathway [11].

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The cell growth inhibition of nanoparticles is better in the presence of active targeting ligand. In our studies, folate (folic acid) was chosen as the active targeting ligand to attach to the drug delivery systems (Fol/PTX/PLA-TPGS NPs [12] and Fol/DOX/PLA-TPGS NPs [15]). The targeting effect of folate was assessed through the morphological change and the half-maximal inhibitory concentration (IC₅₀). From the cell images (figures 11 and 13), we can see that all folate-decorated drug delivery systems have the best effect on cancer cells compared to other systems. The results were confirmed quantitatively in dose response curves (figures 12 and 14(a), (b)) and the IC₅₀ table (table 1). In the case of doxorubicin, DOX/PLA-TPGS NPs induced lower cytotoxicity than that of free DOX. This can be explained by the different cellular uptake mechanisms of soluble DOX and DOX/PLA-TPGS NPs, as discussed in the previous section. Soluble DOX can penetrate into the cell more easily than DOX/PLA-TPGS through ion channels, leading to higher cytotoxicity. However, cellular uptake of Fol/DOX/PLA-TPGS NPs was facilitated by folate receptor-mediated endocytosis, resulting in the best cytotoxicity.

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Table 1.  IC₅₀ of different PTX and DOX formulations.

	IC50
Formulation	for HeLa	for HT29
Free PTX	1.35 ± 0.17 (μg ml−1)	ND
PTX/PLA-TPGS NPs	0.59 ± 0.12 (μg ml−1)	ND
Fol/PTX/PLA-TPGS NPs	0.26 ± 0.07 (μg ml−1)	ND
Free DOX	0.46 ± 0.03 (μM)	0.64 ± 0.03 (μM)
DOX/PLA-TPGS NPs	1.22 ± 0.07 (μM)	1.39 ± 0.05 (μM)
Fol/DOX/PLA-TPGS NPs	0.24 ± 0.02 (μM)	0.39 ± 0.04 (μM)

ND: not determined.

3.3. Improved tumor growth inhibition in tumor-bearing nude mice

The in vivo targeting effect of nanoparticles was evaluated by the tumor growth inhibition in colorectal tumor-bearing nude mice. PTX in different formulations (free PTX, PTX/PLA-TPGS NPs and Fol/PTX/PLA-TPGS NPs) was intravenously administrated into mice. The concentration of PTX was the same in all formulations. Drug administration started on day 0 and was repeated every 7 days, for a total of six times. The tumor volume was measured every 7 days until day 42. After 7 days of treatment, the tumor volume of mice in groups treated with PTX formulations started to differ from those in the saline-treated control group. Groups treated with PTX/PLA-TPGS NPs and Fol/PTX/PLA-TPGS NPs showed a better tumor inhibitory effect compared with the group treated with free PTX during the treatment time (figure 15). After 42 days of treatment, the Fol/PTX/PLA-TPGS NPs exhibited the best tumor growth inhibition (figures 16 and 17) [12].

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Significant tumor growth inhibition was probably due to the combination of two potential mechanisms: (i) the EPR effect; and (ii) selective uptake by cancer cells through receptor-mediated endocytosis. The EPR effect was mediated by the small size of nanoparticles promoting passive permeability at the tumor site and the PEG residue on the nanoparticles causing the nanoparticles to remain in the circulatory system for a longer duration. The receptor-mediated endocytosis was mainly due to the presence of folate moiety on Fol/PTX/PLA-TGPS NPs. After cell surface binding, the Fol/PTX/PLA-TPGS NPs might be internalized into the targeted cell via a folate receptor-mediated endocytosis instead of passive diffusion through the cell membrane. Consequently, a higher PTX concentration would be achieved inside the cell, causing better tumor growth inhibition.

Current anticancer drugs have many disadvantages such as low aqueous solubility, a short half-life and non-selective targeting to cancer cells/tumors, which lower the therapeutic efficacy and increase unwanted side effects. In order to overcome these limitations, DDNSs were fabricated and demonstrated as a potential tool.

In the case of hydrophobic anticancer drugs, their low water solubility seriously impacts on the biodistribution and bioavailability of drugs in the body. Polymeric micelles have been reported to have a high drug-loading capacity, which increases the water solubility of hydrophobic anticancer drugs. Our polymeric micelles composed of copolymer PLA-TPGS significantly improved the solubility of curcumin (350-fold) and paclitaxel (500-fold). Furthermore, the particle size of DDNS is an important factor affecting the action of drugs in the body.  A small particle size (below 200 nm) reduces the capture of microphases which prolong the circulation of nanoparticles. Long-circulating nanoparticles with a small size have the ability to locate at tumor sites at a higher concentration through the well-known EPR effect. In addition, DDNS enhances the cellular uptake, resulting in increasing cytotoxicity of the drug. After encapsulation by the copolymer PLA-TPGS, curcumin was much smaller than free curcumin. Cur/PLA-TPGS NPs exhibited a higher penetration into cancer cells and tumors. PTX/PLA-TPGS NPs induced better efficiency in preventing cell growth.

Folic acid (folate) is a small molecule required for essential cell functions, and has a high binding affinity to the folate receptor, which is overexpressed on the cell surface of many human tumors. The conjugation of folate to nanocarriers can specifically target the tumor site, which can avoid their non-specific attacks on normal tissues as well as increase their cellular uptake within target cells. The mechanism of cell internalization and trafficking inside the cell has been debated. However, regardless of the route of entry, folate conjugates clearly pass through the plasma membrane into cytoplasm via a folate receptor-mediated endocytosis. In our studies, the cellular uptake and cytotoxicity of Fol/DOX/PLA-TPGS NPs were notably higher than those of free DOX and DOX/PLA-TPGS NPs. Most notably, the therapeutic efficacy of free PTX, PTX/PLA-TPGS NPs and Fol/PTX/PLA-TPGS NPs was investigated in tumor-bearing nude mice. The results showed that both PTX/PLA-TPGS NPs and Fol/PTX/PLA-TPGS NPs exhibited better tumor growth inhibition than free PTX. The targeting role of folate was clearly observed, with the best tumor inhibition growth belonging to Fol/PTX/PLA-TPGS NPs.

In this article we presented synthetic, characteristic methods for targeted DDNSs based on copolymer PLA-TPGS. Their biological effects such as cellular uptake, cytotoxicity and tumor growth inhibition were assessed in cancer cells, tumors and tumor-bearing nude mice. The results showed that copolymer PLA-TPGS was an excellent carrier for loading and delivering hydrophobic anticancer drugs (curcumin and paclitaxel). The fabricated DDNS had a very small size of 50 to 100 nm. This size range is suitable for increasing the drug concentration at the tumor site through the EPR effect and enhanced the cellular uptake and cytotoxicity. In particular, active targeting ligand folate exhibited the important role of inducing better cellular uptake and higher cytotoxicity than those of the counterparts without folate. Most notably, Fol/PTX/PLA-TPGS NPs were tested in tumor-bearing nude mice. The results showed that Fol/PTX/PLA-TPGS NPs exhibited the best tumor growth inhibition compared to free PTX and PTX/PLA-TPGS NPs. All results demonstrated the huge potential of targeted DDNSs composed of copolymer PLA-TPGS in cancer treatment.

The authors respectfully thank Academician Nguyen Van Hieu for his concern and encouragement in the research field 'Drug Delivery Nanosystems'. The authors also express sincere thanks to the Laboratory of Biomedical Nanomaterials and the National Key Laboratory for Electronic Materials and Devices, Institute of Materials Science, Vietnam Academy of Science and Technology. This work was financially supported by the Vietnam Academy of Science and Technology under Grant No. VAST03.03/13-14 (HPT) and the National Foundation for Science and Technology Development of Vietnam-NAFOSTED under Grant No. 106.99-2012.43 (HPT).