Liposomal Carrier Conjugated to APP-Derived Peptide for Brain Cancer Treatment

Abstract

Brain tumors are hard to treat with the currently available therapy. The major obstacle in the treatment of brain tumors is the lack of therapeutic strategies capable to penetrate the blood-brain barrier (BBB). The BBB is an endothelial interface that separates the brain from the circulatory blood system and prevents the exposure of the central nervous system (CNS) to circulating toxins and potentially harmful compounds. Unfortunately, the BBB prevents also the penetration of therapeutic compounds into the brain. We present here a drug-delivery liposomal carrier, conjugated to a peptide inserted in the liposomal membrane, which is putatively recognized by BBB transporters. The peptide is a short sequence of 5 amino acids (RERMS) present in the amyloid precursor protein (APP). This APP-targeted liposomal system was designed specifically for transporting compounds with anti-cancer activity via the BBB into the brain in an effective manner. This drug-delivery liposomal carrier loaded with the anti-cancer compounds temozolomide (TMZ), curcumin, and doxorubicin crossed the BBB in an in vitro model as well as in vivo (mice model). In the in vitro model, the targeted liposomes crossed the BBB model fourfold higher than the non-targeted liposomes. Labeled targeted liposomes penetrated the brain in vivo 35% more than non-targeted liposomes. Treatment of mice that underwent intracranial injection of human U87 glioblastoma, with the targeted liposomes loaded with the three tested anti-cancer agents, delayed the tumor growth and prolonged the mice survival in a range of 45% -70%. It appears that the targeted liposomal drug-delivery system enables better therapeutic efficacy in a SCID mouse model of glioblastoma compared to the corresponding non-targeted liposomes and the free compounds.

Keywords: APP-targeted liposomes; Blood–brain barrier (BBB); Curcumin; Doxorubicin; Glioblastoma; Temozolomide (TMZ).

Fig. 1

Design of BBB in vitro model, a primary porcine brain endothelial cells, b primary newborn rat glial cells

Fig. 2

Fluorescence of Texas-Red-labeled liposomes with or without the targeter after 24 h of exposure. Medium in contact with endothelial cells (pre-BBB) and medium in contact with glial cells (post-BBB) were collected from the corresponding compartments. In addition, cells were washed with PBS and fixated with 4% paraformaldehyde (PFA). Fluorescence intensity (arbitrary units) of medium and cell compartments (endothelial and glial layers) was measured at 480 nm excitation and 590 nm emission by a plate reader. Two-way ANOVA was performed. ***p < 0.001, **p < 0.01, *p < 0.05 of liposomes with targeter vs. liposomes without targeter

Fig. 3

Ratio of post-BBB/pre-BBB fluorescence levels of Texas-Red (TR). TR was conjugated to the liposome membrane and they were exposed for 24 h to the in vitro BBB system. One-way ANOVA was performed followed by Dunnett's test ***p < 0.0001, *p < 0.05 vs. non-targeted liposomes

Fig. 4

Left panel: Representative whole brain fluorescence of PE-cy-7 of the 4 studied groups (Control group mice (a), liposomes without the targeter (b), with the targeter (c), and with the targeter and loaded with TMZ (d)). Fluorescence intensity of the slides was quantified by IVIS LUMINA 5 (excitation 460 nm, emission 570 nm) and is presented in the right panel. One-way ANOVA was performed. ***p < 0.0001 vs. control, ^###p < 0.0001 vs. liposomes without targeter

Fig. 5

Left panel—representative parafilm slides from mouse brain scanned with VM fluorescence microscope (excitation 460 nm, emission 570 nm). a control, b non-targeted liposomes conjugated with PE-cy-7, c targeted liposomes conjugated with PE-cy-7, d targeted liposomes loaded with TMZ conjugated with PE-cy-7. Right panel—quantified fluorescence of PE-cy-7 labeled liposomes in parafilm slides. As expected, fluorescence of control slides was 0. For each sample, fluorescence intensity and area were measured and the ratio of intensity/mm³ was calculated. One-way ANOVA was performed. ***p < 0.0001 vs. non-targeted liposomes

Fig. 6

MRI of SCID mice, 8 days (left) and 22 days (right) after intra brain U87cell injection

Fig. 7

Survival rate analysis of SCID mice in the various treatment groups. One-way ANOVA followed by Bonferroni post hoc test, p < 0.05 treatments (liposomes-TMZ, liposomes-curcumin and liposomes-doxorubicin) vs. control (saline treated). Also, p < 0.05 vs. the corresponding free compounds

Fig. 8

The luminescence emitted by U87 glioblastoma cells in the various treatment groups measured as radiance (p/s/str/cm²; mean ± SEM). a 3 groups: Control (saline), Targeted liposomes loaded with TMZ (4 mg/kg), Free TMZ (4 mg/kg), and Non-targeted liposomes loaded with TMZ (4 mg/kg). b 3 groups of treatment, Control (saline), Targeted liposomes loaded with curcumin (4 mg/kg), Free curcumin (4 mg/kg), and Non-targeted liposomes loaded with curcumin (4 mg/kg). c 3 groups of treatment, Control (saline), Targeted liposomes loaded with doxorubicin (1 mg/kg), Free doxorubicin (1 mg/kg), and Non-targeted liposomes loaded with doxorubicin (1 mg/kg). The number of mice in each group was 6