Abstract B34: Resveratrol in transferrin-modified liposomes for eliminating both, bulk tumor cells and tumor-initiating cells in glioblastoma
Authors: Jhaveri Aditi , Torchilin Vladimir 
Published: March 2017

Abstract
Purpose: The purpose of this study was to develop transferrin (Tf)-modified liposomes loaded with resveratrol (RES) (Tf-RES-L) to achieve an effective therapy for glioblastomas (GBMs) by eliminating both, the bulk tumor cell (BTC) and tumor-initiating cell (TIC) populations within GBMs, and to provide an effective delivery system for RES to overcome its limitations as a free drug. We hypothesize that Tf-RES-L will show an improved efficacy versus the free drug or non-targeted liposomes and will act as a suitable platform for effective delivery of RES into GBM cells.
Background: GBMs harbor a sub-population of extremely chemo and radio-resistant cells known as TICs or cancer stem-like cells which are highly tumorigenic and can sustain the growth, invasiveness and recurrence of GBMs. Conventional chemotherapeutics act on the rapidly dividing BTCs but leave behind the quiescent TICs, which can lead to tumor relapse. They also exhibit severe toxicities to the surrounding normal tissues. RES, a natural polyphenol is well-tolerated, acts on both these populations of cells, and has proven chemo-preventive effects in all the major cancer stages including initiation, promotion and progression. However, its low aqueous solubility and bioavailability, chemical instability, and poor pharmacokinetics severely limit its use as a free drug. We have developed a liposomal formulation of RES (RES-L) to counter these drawbacks and to eradicate the BTC and TIC populations in GBMs. Tf-receptors (TfRs) are over-expressed on GBM BTCs and TICs compared to normal cells. We exploit this feature for tumor-specific delivery by modifying the liposome surface with a natural ligand Tf, to develop Tf-RES-L which can target the TfRs.
Methods: The neurosphere (NS) assay was used to develop TIC models from GBM cell lines. CD133 expression studies and in vitro limiting dilution assays (LDAs) were carried out to characterize the NS. Liposomes were prepared using the thin-film hydration method and RES was loaded passively into liposomes. Tf was attached to the liposome surface using a simple in-house conjugation protocol. Formulations were characterized for their size, charge, morphology and drug-loading efficiency. Rhodamine labeled, Tf-targeted liposomes (Rh-TfLs) were tested for their association with and internalization into cells using flow cytometry and confocal microscopy, respectively. Cytotoxicity assays, cell-cycle analyses, apoptosis studies and oxidative stress measurements were performed to assess the general mechanisms of action of RES on GBM cells.
Results: NS cultures showed the presence of TICs as determined from LDAs and CD-133 expression. RES inhibited the anchorage-independent growth of NS in two GBM cell-lines. The liposome formulations had a narrow size distribution and a good drug loading efficiency. All RES formulations induced a time and dose-dependent cytotoxicity in cells. At low concentrations, Tf-RES-L were significantly more cytotoxic compared to free RES or RES-L. Rh-TfLs showed a significantly higher association with and internalization into cells compared to the non-targeted liposomes. RES formulations arrested GBM cells in the S-phase of the cell-cycle at concentrations ≤ 75μM and exhibited a pro-oxidant effect at concentrations ≥100 μM inducing significant oxidative stress on GBM cells. Tf-RES-L also induced significantly higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells compared to the free drug and non-targeted RES-L.
Conclusions: Our studies showed that RES is effective in eliminating both, the BTCs and TICs in glioblastoma, and its encapsulation in Tf-modified liposomes still further improves its efficacy compared to the free drug or drug in non-targeted liposomes. Tf-RES-L thus seem like a very promising nanomedicine candidate for further development to treat GBM.
Citation Format: Aditi Jhaveri, Vladimir Torchilin. Resveratrol in transferrin-modified liposomes for eliminating both, bulk tumor cells and tumor-initiating cells in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B34.