Elsevier

Journal of Controlled Release

Volume 246, 28 January 2017, Pages 79-87
Journal of Controlled Release

Tumor regression after intravenous administration of targeted vesicles entrapping the vitamin E α-tocotrienol

https://doi.org/10.1016/j.jconrel.2016.12.014Get rights and content

Abstract

The therapeutic potential of tocotrienol, a member of the vitamin E family of compounds with potent in vitro anti-cancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study is to determine whether a novel tumor-targeted vesicular formulation of tocotrienol would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo.

In this work, we demonstrated that novel transferrin-bearing multilamellar vesicles entrapping α-T3 resulted in a dramatically improved (by at least 52-fold) therapeutic efficacy in vitro on A431 cell line, compared to the free drug. In addition, the intravenous administration of tocotrienol entrapped in transferrin-bearing vesicles resulted in tumor suppression for 30% of A431 and 60% of B16-F10 tumors, without visible toxicity. Mouse survival was enhanced by > 13 days compared to controls administered with the drug solution only.

This tumor-targeted, tocotrienol-based nanomedicine therefore significantly improved the therapeutic response in cancer treatment.

Graphical abstract

Tumor growth studies in a mouse A431 xenograft model after intravenous administration of transferrin-bearing vesicles encapsulating α-T3 (10 μg/injection) (green) (controls: control vesicles entrapping α-T3 (orange), α-T3 solution (red), untreated tumors (black)) (n = 10).

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Introduction

Tocotrienol, a member of the vitamin E family of compounds, is currently receiving increased attention because of its promising anti-tumor activity [1], [2]. It has been reported to exert its anti-cancer effects through various mechanisms, such as activation of p53, modulation of Bax/Bcl-2 ratio, decrease of oxidative stress and induction of apoptosis [1], [2], [3], [4]. It can also inhibit angiogenesis by downregulating the expression of the vascular endothelial growth factor (VEGF) receptor and blocking intracellular VEGF signaling. In addition, it is able to potentiate apoptosis through inhibition of DNA polymerase and telomerase and of NF-κB activation pathway [1], [2], [3], [4]. This wide range of anti-cancer effects therefore makes tocotrienol a very promising therapeutic molecule.

However, its efficacy against cancer cells in vivo is hindered by its inability to specifically reach cancer cells at a therapeutic concentration, without affecting normal cells. Given its anti-proliferative properties, it is of the utmost importance to find a strategy to deliver this therapeutic drug specifically to its site of action.

On the basis that iron is essential for tumor cell growth and can be effectively carried to tumors by using transferrin receptors overexpressed on cancer cells [5], [6], we recently demonstrated that the conjugation of transferrin (Tf) to vesicles entrapping tocotrienol-rich fraction (TRF) extracted from palm oil could lead to tumor regression, and even tumor suppression, on both the tested cancer cell lines, following intravenous injection to a murine model [7], [8], [9]. It resulted in complete tumor eradication for 50% of B16-F10 tumors and 20% of A431 tumors [7]. The treatments were well tolerated by the animals, without weight loss or apparent signs of toxicity.

These previous studies, however, used TRF but did not evaluate the anti-cancer potency of each individual tocotrienols and tocopherols forming TRF, which is a mixture of α, γ-, δ-tocotrienol, α-tocopherol and other tocotrienol-related compounds. Structurally, tocotrienols and tocopherols consist of a chroman ring linked to a phytyl side chain, which is saturated only for tocopherols. Furthermore, tocotrienols and tocopherols compounds differ in the number and position of methyl substituents attached to their chroman ring [10]. Hence, the therapeutic efficacy resulting from tocotrienol formulations could be further enhanced by entrapping only the most effective tocotrienol compound(s) within optimized tumor-targeted vesicles.

The objectives of this study are therefore 1) to evaluate the efficacy of the individual tocotrienols and tocopherols entrapped in transferrin-bearing vesicles against the in vitro viability of various cancer cells and 2) to optimize the delivery system carrying tocotrienol by a) increasing the amount of drug entrapped within the vesicles, b) optimizing the amount of transferrin grafted to the vesicles and c) optimizing the overall structure of the vesicles.

Section snippets

Cell lines and reagents

α-tocotrienol (α-T3), γ-tocotrienol (γ-T3), δ-tocotrienol (δ-T3), α-tocopherol (α-Toc), human holo-transferrin, dimethylsuberimidate and all other chemicals that are not specifically mentioned below were purchased from Sigma Aldrich (Poole, UK). Solulan C24 came from Amerchol (Edison, NJ). d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was obtained from Eastman Chemical Company (Kingsport, TN). A431 human epidermoid carcinoma and Bioware® B16-F10-luc-G5 mouse melanoma that expresses

Preparation and characterization of transferrin-bearing vesicles entrapping tocotrienol and tocopherol compounds

Tf-bearing and control unilamellar vesicles entrapping tocotrienol compounds have been successfully prepared, as confirmed by TEM imaging (Fig. 1). These vesicles were able to entrap 69.9 ± 1.8% of α-T3, 83.3 ± 3.0% of γ-T3, 83.3 ± 2.0% of δ-T3 and 68.9 ± 3.1% of α-Toc. An amount of 10.1 ± 0.5 mg transferrin was conjugated to the vesicles, which corresponds to an increase compared to the conjugation of about 6 mg transferrin obtained in our previous studies [8]. As expected, the conjugation of Tf to the

Discussion

The use of the vitamin E tocotrienol for the treatment of cancer has so far been limited by the inability of this drug to specifically reach tumors following systemic injection. To overcome this issue, we hypothesized that entrapping only the most efficacious tocotrienol compound(s) within a tumor-targeted delivery system that has been optimized to enhance the anti-proliferative effect of the carried drug, would increase the therapeutic efficacy in vivo.

Transferrin receptor is an attractive

Conclusions

In this study, we have demonstrated for the first time that a novel formulation of α-T3 entrapped in tumor-targeted vesicles can lead to complete tumor eradication for up to 60% of the tested tumors, following intravenous administration.

Among the various formulations investigated, multilamellar vesicles conjugated to 12 mg Tf and entrapping 2 mg of α-T3 were found to give the highest efficacy against A431 cancer cell viability in vitro.

In vivo, α-T3 entrapped in Tf- bearing multilamellar vesicles

Acknowledgments

This work was financially supported by the University of Strathclyde. The IVIS was funded with an equipment grant (No. ME0442) from The Wellcome Trust.

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