Natural Compounds in Liposomal Nanoformulations of Potential Clinical Application in Glioblastoma

Abstract

Glioblastoma (GBM) is the most common malignant neoplasm in adults among all CNS gliomas, with the 5-year survival rate being as low as 5%. Among nanocarriers, liposomal nanoformulations are considered as a promising tool for precise drug delivery. The herein presented study demonstrates the possibility of encapsulating four selected natural compounds (curcumin, bisdemethoxycurcumin, acteoside, and orientin) and their mixtures in cationic liposomal nanoformulation composed of two lipid types (DOTAP:POPC). In order to determine the physicochemical properties of the new drug carriers, specific measurements, including particle size, Zeta Potential, and PDI index, were applied. In addition, NMR and EPR studies were carried out for a more in-depth characterization of nanoparticles. Within biological research, the prepared formulations were evaluated on T98G and U-138 MG glioblastoma cell lines in vitro, as well as on a non-cancerous human lung fibroblast cell line (MRC-5) using the MTT test to determine their potential as anticancer agents. The highest activity was exhibited by liposome-entrapped acteoside towards the T98G cell line with IC₅₀ equal 2.9 ± 0.9 µM after 24 hours of incubation. Noteworthy, curcumin and orientin mixture in liposomal formulation exhibited a synergistic effect against GBM. Moreover, the impact on the expression of apoptosis-associated proteins (p53 and Caspase-3) of acteoside as well as curcumin and orientin mixture, as the most potent agents, was assessed, showing nearly 40% increase as compared to control U-138 MG and T98G cells. It should be emphasized that a new and alternative method of extrusion of the studied liposomes was developed.

Keywords: cancer; drug delivery system; glioblastoma; liposomes; natural compounds.

Figure 1

Chemical structures of natural products used in this study: curcumin (CUR); bisdemethoxycurcumin (BDMC); Acteoside, and Orientin.

Figure 2

Types of liposomes: SUV—small unilamellar vesicles, LUV—large unilamellar vesicles, MLV—multilamellar large vesicles, MVV—multilamellar large vesicles.

Figure 3

Schematic representation of liposomes preparation protocol.

Figure 4

The size of the liposomes (with CUR, ORI, and mixture of CUR + ORI) was measured with dynamic light scattering (DLS).

Figure 5

An example recovery of the magnetization as a function of time t for empty liposomes DOTAP:POPC.

Figure 6

The pulse sequence for the measurement of the spin–spin relaxation time T_2.

Figure 7

The decay of the magnetization obtained in the experiment for the measurement of the spin–spin relaxation time T₂ for all liposome samples.

Figure 8

X band measurement results performed to investigate the presence of the stable free radicals in the liposome samples; CUR—curcumin, BDMC—bisdemethoxycurcumin.

Figure 9

(A) L band radical scavenging results. (B) The number of remaining radicals of the 3CPy spin probe after mixing with the liposomal formulations (in arbitrary units). (C) The amount of the swept-off radicals of the 3CPy spin probe after mixing with the liposomal formulations (in arbitrary units). (D) The scavenging reaction time in seconds; CUR—curcumin, BDMC—bisdemethoxycurcumin.

Figure 10

The cytotoxic activity of liposomal formulation containing CUR, BDMC, ACT, ORI and their combination against T98G, U-138 MG, and MRC-5 cells. Data are expressed as the mean ± SEM from three independent experiments; BDMC—bisdemethoxycurcumin. Statistical significance was assessed by Tukey’s test (*** p < 0.001).

Figure 11

The effect of ACT and CUR+ORI on the protein level of p53 in panels (A) and (B) and caspase-3 in panels (C) and (D) in GBM cells. Data are presented as mean ± SEM. * represents p < 0.05, ** represents p < 0.01, compared with the untreated group. The values in the x axes are concentration in µM of ACT or the 1:1 molar mixture of CUR + ORI. The representative immune blot respectively for Figure 11 show in Figures S1–S4.

Figure 12

Schematic representation of set for the alternative method of liposome extrusion (A). The size of the liposomes with curcumin was measured with dynamic light scattering (DLS)—hydrated and untreated (B) and extruded using an ultrasonic UV 2070 homogenizer with an MS 73 head (C).