The Formation of Morphologically Stable Lipid Nanocarriers for Glioma Therapy

Abstract

Cerasomes are a promising modification of liposomes with covalent siloxane networks on the surface that provide outstanding morphological stability while maintaining all the useful traits of liposomes. Herein, thin film hydration and ethanol sol injection methods were utilized to produce cerasomes of various composition, which were then evaluated for the purpose of drug delivery. The most promising nanoparticles obtained by the thin film method were studied closely using MTT assay, flow cytometry and fluorescence microscopy on T98G glioblastoma cell line and modified with surfactants to achieve stability and the ability to bypass the blood-brain barrier. An antitumor agent, paclitaxel, was loaded into cerasomes, which increased its potency and demonstrated increased ability to induce apoptosis in T98G glioblastoma cell culture. Cerasomes loaded with fluorescent dye rhodamine B demonstrated significantly increased fluorescence in brain slices of Wistar rats compared to free rhodamine B. Thin film hydration with Tween 80 addition was established as a more reliable and versatile method for cerasome preparation. Cerasomes increased the antitumor action of paclitaxel toward T98G cancer cells by a factor of 36 and were able to deliver rhodamine B over the blood-brain barrier in rats.

Keywords: T98G cells; Tween 80; blood–brain barrier; cerasome; cytotoxicity; liposome; paclitaxel; surfactant.

Figure 1

Structural formulas of the compounds used in this work.

Figure 2

TEM images of CFL16 cerasomes prepared via sol injection method.

Figure 3

TEM images of 50% CFL16 50% PC cerasomes prepared via thin film hydration method.

Figure 4

Apoptotic effects measured by flow cytometry using annexin V Alexa Fluor 647 staining protocol. Nanoparticle composition is as follows: (A) paclitaxel (PTX); (B) CFL 1 mM PC 1 mM PTX 0.08 mM; (C) CFL 1 mM PC 1 mM 14-6-14(Et) 0.057 mM and PTX 0.08 mM. Samples were diluted and tested at 0.1 mM of CFL16 (column 1) and 0.2 mM of CFL16 (column 2) or 4 µM and 8 µM for paclitaxel, respectively.

Figure 5

Cellular uptake of cerasomes with and without PTX evaluated using flow cytometry at 6 h and 24 h of incubation: (a) comparison of 10% Tween 80 effect on 50% CFL 50% PC cerasomes; (b) comparison of 14-6-14(Et) effect on 50% CFL 50% PC cerasomes with additional 10% Tween 80. *—p < 0.01 comparing samples at 24 h; #—p < 0.01 comparing samples at 6 h. Comparisons were made with first sample in each group.

Figure 6

Internalization of CFL PC 1:1 cerasomes into T98G cells at 2, 6 and 24 h after incubation visualized with fluorescence microscopy. Green channel is coumarin 6 and blue channel is DAPI. Separate channel microphotographs are provided in Section S3 of the Supplementary Information.

Figure 7

Rat brain slices after administration of (a) free rhodamine B; (b) plain rhodamine B-loaded cerasomes; (c) Tween 80 cerasomes labeled with rhodamine B.