Nanoparticles Consisting of Tocopheryl Succinate Are a Novel Drug-Delivery System with Multifaceted Antitumor Activity

Abstract

Tumor heterogeneity hampers the clinical efficacy of cancer chemotherapy. Therefore, it is necessary to develop a multifaceted, rational treatment strategy with the potential to modulate overall tumor heterogeneity. Since combination therapy using several drugs has been shown to have enhanced therapeutic effects compared with monotherapy, combining agents with different antitumor effects would be a multifaceted form of therapy to overcome tumor heterogeneity. Therefore, the development of effective drug-delivery system (DDS) carriers for combination therapy is required. The ideal DDS carrier for combination therapy should itself have antitumor activity in addition to the ability to deliver drugs to tumors. α-Tocopheryl succinate (TS), a succinic acid ester of α-tocopherol, has attracted attention as a unique antitumor agent, and TS itself can form nanoparticles. In this review, we introduce nanoparticles consisting of TS as a novel DDS carrier with multifaceted antitumor effects for combination therapy.

1. INTRODUCTION

The effectiveness of antitumor drugs is suggested to be dependent on tumor heterogeneity such as the presence of drug-resistant cells and a complex microenvironment containing aberrant angiogenesis and local immunosuppression.¹⁾ Various agents such as antiangiogenic drugs, immune modulators, and inhibitors of various growth factors and their signaling pathways have been developed to combat each limited process in tumor heterogeneity.²⁾ Because there is room to improve the effectiveness of monotherapy using these agents, it is necessary to develop a multifaceted, rational treatment strategy making it possible to modulate overall tumor heterogeneity.

In past clinical research, combination therapy using several drugs, each with a different mechanism of action, was shown to enhance the therapeutic effect compared with monotherapy.^3,4) Therefore, to expand the availability of combination therapy, drug carriers for multidrug therapy were developed.^5–7) Liposomes can encapsulate several drugs at a suitable ratio and be passively delivered to tumor tissue via the enhanced permeability and retention (EPR) effect, not only for greater synergy but also to decrease side effects. Although liposomes encapsulating hydrophobic antitumor agents in their membranes have been studied in attempts to enhance their capacity by co-encapsulation of hydrophilic materials, such as anticancer drugs or functional nucleic acids, in the aqueous compartment,⁸⁾ they showed insufficient biodistribution and bioavailability due to their high affinity for biogenic substances and low stability.^9,10) On the other hand, anionic substances, including liposomes, are known to diminish interactions with biogenic substances.¹¹⁾ Thus, liposomes consisting of anionic compounds would be expected to be ideal drug carriers for multidrug therapy.

Recently, it has been suggested that α-tocopheryl succinate (TS), a succinic acid ester of α-tocopherol (α-T), could serve as a material for nanoparticles with antitumor activity. TS is an anionic agent at physiologic pH and exerts multifaceted antitumor activity. In this review, we focus on the multifaceted antitumor activities and in vivo applications of TS.

2. MULTIFACETED ANTITUMOR ACTIVITIES OF TS

Although TS has no antioxidative activity, unlike α-T that is a well-known lipophilic antioxidant (Fig. 1), TS was reported to have various biological activities, such as the enhancement of nitric oxide production induced by lipopolysaccharide and interferon-γ in vascular smooth muscle cells,¹²⁾ inhibition of the function of transcriptional factor nuclear factor κB,¹³⁾ promotion of differentiation,¹⁴⁾ arrest of the cell cycle,¹⁵⁾ and induction of apoptosis in various tumor cell lines.^16,17)

Fig. 1. Structure of TS and Its Multifaceted Antitumor Activity

TS is a succinyl ester of α-T, has cancer cell-specific apoptosis-inducing activity, and inhibits tumor angiogenesis and MDR.

Since tumor cells are more susceptible to TS than normal cells in the induction of apoptosis,^16,17) TS is expected to be a novel anticancer agent (Fig. 1). It is considered that the high susceptibility of tumor cells to TS-induced apoptosis is due to their poor antioxidative defense system.¹⁷⁾ It is known that the activities of antioxidative enzymes such as superoxide dismutase and catalase in various cancer cells are lower than those in normal cells.¹⁸⁾ Alternatively, it was reported that TS-induced apoptosis was associated with an increase in extracellular O₂⁻ generation by the activation of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.¹⁹⁾ Moreover, Dong et al. reported that TS increased mitochondrial O₂⁻ generation via the direct binding of TS to the ubiquinone-binding sites in mitochondrial respiratory complex II.²⁰⁾ Therefore it is suggested that TS activates the cellular O₂⁻ generation system, which induces potent apoptosis of cancer cells with lower antioxidative enzyme activities compared with normal cells. Since TS can induce cancer cell-specific apoptosis, it is expected to become an anticancer agent without negative side effects.

In addition to the induction of apoptosis, TS has attracted much attention as an angiogenesis inhibitor for tumor therapy (Fig. 1). It is known that tumor angiogenesis makes rapid tumor growth possible by supplying oxygen and nutrients and promotes metastasis through intra/extravasation. Thus, the inhibition of angiogenesis is a promising strategy for tumor therapy.²¹⁾ Various angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) secreted from tumor cells induces the proliferation of angiogenic endothelial cells.²²⁾ Dong et al. reported that TS prevents angiogenesis by selective induction of apoptosis in angiogenic endothelial cells.²³⁾ On the other hand, it was found that the mechanism of the antiangiogenic activity of TS is associated with the inhibition of VEGF expression and disruption of paracrine FGF2 signaling.^24,25) Moreover, TS promotes tumor dormancy via the inhibition of angiogenesis. Therefore, TS prevents tumor metastasis as well as growth by inhibiting angiogenesis.²⁶⁾

Although various antitumor drugs such as doxorubicin (DOX) and cisplatin are in widespread clinical use, the presence of tumor cells that have acquired resistance to them hampers the efficacy of cancer chemotherapy.²⁷⁾ It is accepted that high levels of the multidrug resistance (MDR) protein in tumor cells is involved in the DOX resistance of tumor cells.²⁸⁾ Since MDR is responsible for the cellular efflux of DOX, the level of DOX decreases in tumor cells expressing MDR, leading to the prevention of DOX-induced cell death. Zhang et al. reported that TS enhanced DOX-induced cell death via the inhibition of MDR-mediated DOX efflux as well as the promotion of DOX influx.²⁹⁾ Therefore the combination of TS with DOX may be effective against DOX-resistant tumors.

There is no cumulative toxicity with TS as distinct from many anticancer agents because it is hydrolyzed to α-T, silencing apoptotic activity.³⁰⁾ Consequently, TS is expected to be an ideal antitumor agent with multifaceted antitumor activity (Fig. 1).

3. APPROACHES TO THE CLINICAL APPLICATION OF TS

As described above, TS has been expected to become a promising antitumor agent. However, for the clinical application of TS it will be necessary to improve its poor water solubility and develop an efficient delivery system to tumor tissue. To increase the water solubility of TS, its derivative conjugated with polyethylene glycol (PEG) (TPGS) was developed. Youk et al. reported that TPGS induces more potent apoptosis in human lung tumor A549 cells than TS by increasing the generation of reactive oxygen species (ROS),³¹⁾ suggesting that the modification of TS with PEG both enhances the antitumor activity of TS and improves its water solubility. Moreover, TPGS is a water-miscible preparation that forms micelles at low concentrations (0.04–0.06 mmol/L).³²⁾ Due to this property, TPGS increases the water solubility of the hydrophobic antitumor drug paclitaxel³³⁾ in addition to that of the TS molecule itself. Hence, TPGS is expected to act as a solubilizer with antitumor activity of hydrophobic antitumor agents. It is likely that TPGS could be developed as assembled nanoparticles like micellar structures containing antitumor drugs.

On the other hand, to deliver TS specifically to tumor tissue, Wang et al. reported that epidermal growth factor receptor type 2 (erbB2)-binding peptide-conjugated TS efficiently suppressed the growth of erbB2-positive breast tumors.³⁴⁾ In an attempt to improve both the water solubility of TS and its specific delivery to tumor tissue, Hrzenjak et al. found that high-density lipoprotein (HDL)-associated TS showed more potent antitumor activity than the TS molecule alone in C57BL6 mice inoculated with murine Lewis lung carcinoma cells overexpressing scavenger receptor class B, type I, the prime receptor mediating selective lipid uptake from HDL.³⁵⁾ However, it is difficult for these formulations to be applied in a co-delivery system with other drugs.

4. TS AS A NOVEL DRUG CARRIER MATERIAL

TS has unique physicochemical properties that can resolve some of the problems in its clinical application. TS disperses in water as vesicles due to its amphipathic structure (Fig. 2). Nanovesicles consisting solely of TS (TS-NVs) are formed by suspending or sonicating it under alkaline conditions.³⁶⁾ Since TS-NVs have a barrier function like phospholipid bilayers with lamellar structures,³⁶⁾ various agents such as other anticancer drugs and functional nucleic acids can be encapsulated into their inner-water phase. Because the terminal carboxylic moiety of TS is ionized at physiologic pH, the surface charge of TS-NVs is anionic, which is useful for effective biodistribution like PEG-modified nanoparticles. Moreover, the particle size of TS-NVs can be controlled at about 100 nm, leading to the efficient delivery of TS to tumor tissues via the EPR effect. It was previously reported that intravenous administration of TS-NVs significantly suppressed tumor growth in mouse melanoma B16-F1 cell-bearing mice,³⁷⁾ indicating that TS-NVs have the potential to act as a drug carrier with anticancer effects like PEGylated liposomes. In addition, Ramanathapuram et al. demonstrated that the combination of TS-NVs with dendritic cells (DCs) showed more potent tumor growth inhibition than TS-NVs alone via both cancer cell killing and activation of DCs.³⁸⁾ Thus it was suggested that TS-NVs have an adjuvant effect, which is useful for tumor immunotherapy. The vesicular formulation of TS is therefore suitable for specific delivery to tumors and multifaceted tumor treatment in addition to the increase in its water solubility.

Fig. 2. Improvement of NVs Consisting of TS

Charge neutralization of negatively charged TS by divalent cations causes a membrane structural change from lamellar to hexagonal II. TS-EPC-NVs constructed by mixing TS and EPC have higher stability in the presence of divalent cations and serum compared with TS-NVs.

However, TS-NVs have technical limitations due to their collapse or structural conversion from lamella to a hexagonal II structure in response to divalent cations, acidic pH, or serum (Fig. 2). Thus TS-NVs lack the stability required for a clinical drug carrier. Recently, a novel drug-delivery system (DDS) consisting of TS and egg phosphatidylcholine (EPC), which can form a stable lamellar structure,³⁹⁾ has been developed as TS-EPC-NVs⁴⁰⁾ (Fig. 2). TS-EPC-NVs maintain a more stable vesicular structure compared with TS-NVs, even in the presence of divalent cations and serum. The enhanced vesicular stability of TS-EPC-NVs under in vivo conditions contributes to the potent antitumor activity, i.e., TS-EPC-NVs showed more potent in vitro and in vivo antitumor activity than TS-NVs⁴⁰⁾ (Fig. 3). The reason for the potent effect of TS-EPC-NVs could be explained by differences in biodistribution, such as tumor accumulation and intratumoral distribution, and the cytotoxicity of the NVs. It is known that the tumor accumulation of NVs by the EPR effect, its intratumoral penetration, and cellular uptake depend heavily on particle size.⁴¹⁾ The diameter of TS-EPC-NVs was maintained below 200 nm even under in vivo conditions, leading to its efficient tumor accumulation and homogenous intratumoral distribution and cellular uptake. Moreover, TS-EPC-NVs taken up via the endocytotic pathway were effectively delivered to the cytosol.⁴⁰⁾ Previously, it was reported that TS-induced apoptosis is triggered by interaction with mitochondrial or cytosolic proteins.^20,42) Thus, the enhanced anticancer activity of TS-EPC-NVs was suggested to be due to its efficient cytosolic delivery. Alternatively, Gu et al. reported that small unilamellar vesicles consisting of TS and phosphatidylcholine (TS-SUVs) induced more potent apoptosis than TS molecules alone in hamster cheek pouch carcinoma cells, and the mechanism of the potent activity of TS-SUVs was involved in enhancing ceramide-mediated apoptosis,⁴³⁾ which participates in the apoptosis induced by TS molecules.⁴⁴⁾ It is therefore suggested that NVs prepared by mixing TS with phospholipid enhances the apoptotic pathway induced by TS molecules alone.

Fig. 3. Tumor Growth Inhibition by TS-NVs and TS-EPC-NVs in B16-F1 Cell-Bearing Mice⁴⁰⁾

TS-NVs and TS-EPC-NVs were injected into melanoma-bearing mice via the tail vein root 5 times every 3 d, as indicated by arrows in the graph. Data are shown as the ratio to tumor volume in each mouse 4 d after tumor inoculation. *p<0.05, **p<0.01.

To evaluate the suitability of TS-EPC-NVs as drug carriers, encapsulation of small interfering RNA (siRNA) into TS-EPC-NVs was also attempted.⁴⁰⁾ Following the method in a previous report,⁴⁵⁾ siRNA was condensed with a polycation, and then the polyplex was encapsulated into TS-EPC-NVs. As shown in Fig. 4, in B16-F1 cells stably expressing luciferase, TS-EPC-NVs encapsulating anti-luciferase siRNA showed significant knockdown activity similar to the well-known transfection reagent lipofectamine 2000. It therefore appears that TS-EPC-NVs with antitumor activity would be useful DDS carriers capable of delivering encapsulated drugs to the cytosol.

Fig. 4. Knockdown Efficiency of siRNA-Encapsulated TS-EPC-NVs⁴⁰⁾

Specific knockdown efficiency was evaluated using anti-luciferase siRNA-encapsulated TS-EPC-NVs (20 pmol/40000 cells) in stably luciferase-expressing B16-F1 cells. Positive controls for transfection applied Lipofectamine 2000 (LFN2000). Luciferase activities were measured 48 h after transfection. Data are shown as relative light units (RLU)/mg protein. Values and bars represent mean and S.D., respectively. **p<0.01.

5. CONCLUSION AND FUTURE PERSPECTIVES

This review article introduces the multifaceted antitumor activity of TS and its in vivo application in nanoparticle form. The multifaceted activity of TS includes the induction of cancer cell-specific apoptosis, inhibition of tumor angiogenesis, and enhancement of the therapeutic effect of other antitumor agents via the inhibition of MDR. Moreover, the vesicular formulation of TS not only shows potent antitumor activity but also can encapsulate agents such as siRNA. Thus nanoparticles consisting of TS are unique DDS carriers that have antitumor activity. In the future, TS nanoparticles encapsulating other agents with different antitumor activity are expected to be applied to multifaceted tumor therapy, possibly overcoming tumor heterogeneity.

Acknowledgment

The authors would like to express thanks to our colleagues for their helpful advice and support. This work was supported in part by the Japan Society for the Promotion of Science and by the Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research.

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