Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy

Abstract

Cell penetrating peptides (CPPs) were widely used for drug delivery to tumor. However, the nonselective in vivo penetration greatly limited the application of CPPs-mediated drug delivery systems. And the treatment of malignant tumors is usually followed by poor prognosis and relapse due to the existence of extravascular core regions of tumor. Thus it is important to endue selective targeting and stronger intratumoral diffusion abilities to CPPs. In this study, an RGD reverse sequence dGR was conjugated to a CPP octa-arginine to form a CendR (R/KXXR/K) motif contained tandem peptide R8-dGR (RRRRRRRRdGR) which could bind to both integrin αvβ3 and neuropilin-1 receptors. The dual receptor recognizing peptide R8-dGR displayed increased cellular uptake and efficient penetration ability into glioma spheroids in vitro. The following in vivo studies indicated the active targeting and intratumoral diffusion capabilities of R8-dGR modified liposomes. When paclitaxel was loaded in the liposomes, PTX-R8-dGR-Lip induced the strongest anti-proliferation effect on both tumor cells and cancer stem cells, and inhibited the formation of vasculogenic mimicry channels in vitro. Finally, the R8-dGR liposomal drug delivery system prolonged the medium survival time of intracranial C6 bearing mice by 2.1-fold compared to the untreated group, and achieved an exhaustive anti-glioma therapy including anti-tumor cells, anti-vasculogenic mimicry and anti-brain cancer stem cells. To sum up, all the results demonstrated that R8-dGR was an ideal dual receptor recognizing CPP with selective glioma targeting and efficient intratumoral diffusion, which could be further used to equip drug delivery system for effective glioma therapy.

Keywords: Anti-glioma; C-end Rule; Cell penetrating peptides; Glioma targeting; Tumor penetration.

Fig 1

Schematic illustration of PTX-R8-dGR-Lip. After transporting across the BBB and accumulating in glioma foci, liposomes could achieve an efficient synergetic glioma tissue penetration through three pathways and accomplish a synthesized anti-glioma therapy by combined treatment of tumor cells, VM and BCSCs.

Fig 2

Characterization of liposomes. Size distribution graph (A) and Zeta potential distribution graph (B) of PTX-R8-dGR-Lip were measured by Malvern Zetasizer Nano ZS90. (C) The variations of transmittance of liposomes in 50% FBS over 48 h (n = 3, mean ± SD). (D) PTX release behaviors of PTX-loaded liposomes and free PTX in release media for 48 h (n = 3, mean ± SD).

Fig 3

SPR response curve of different modified liposomes with integrin α_vβ₃.

Fig 4

(A) Expression level of Neuropilin-1 and integrin β₃ on C6, bEnd.3 and Hela cells. (B) Quantitative 4 h cellular uptake of CFPE-labeled liposomes on C6, bEnd.3 and Hela cells determined by a flow cytometer (n = 3, mean ± SD), ** and *** indicates p < 0.01 and p < 0.001 respectively, # represents p < 0.05 versus R8-Lip group. (C) Confocal images of 4 h cellular uptake of CFPE-labeled liposomes on C6, bEnd.3 and Hela cells.

Fig 5

Confocal images of the uptake of CFPE-labeled liposomes on C6 tumor spheroids with different depth. Scale bars represent 100 μm.

Fig 6

BBB model study in vitro. Uptake of CFPE-labeled liposomes on bEnd.3 cells on the transwell membrane (A) and on C6 cells in the presence of bEnd.3 monolayers (B) determined by a flow cytometer (n = 3, mean ± SD), ** and *** indicates p < 0.01 and p < 0.001 respectively, # represents p < 0.05 versus R8-Lip group. (C) Confocal images of the liposomal uptake on C6 cells in the presence of bEnd.3 monolayers.

Fig 7

(A) The cytotoxicity study of different PTX formulations on C6 cells (n= 3, mean ± SD). *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001 versus PTX-R8-dGR-Lip group, respectively. (B) The percentage of apoptosis and necrotic cells after different PTX formulations treatment (n= 3, mean ± SD), * represents p < 0.05 versus other groups.

Fig 8

(A) Destruction of C6 VM channels in vitro after 6 h and 24 h treatment of free PTX and PTX-loaded liposomes, untreated group was used as negative control. a = Saline, b = free PTX, c = PTX-PEG-Lip, d = R8-Lip, e = PTX-R8-RGD-Lip, f = PTX-R8-EGR-Lip, g = PTX-R8-dGR-Lip. Scale bars represent 200 μm. (B) Morphological identification of C6 stem cell tumor spheres. Scale bar represents 50 μm. (C) CD133 expression level on C6 cells and C6 stem cells. (D) The cytotoxicity study of different PTX formulations on C6 stem cells (n= 3, mean ± SD). *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001 versus PTX-R8-dGR-Lip group, respectively.

Fig 9

(A) In vivo images of intracranial C6 glioma bearing mice at 1 h, 4 h, 8 h and 24 h post-injection of DiD-loaded liposomes. (B) Ex vivo images of the glioma bearing brains. In (A) and (B), a = blank, b = PEG-Lip, c = R8-Lip, d = R8-RGD-Lip, e = R8-EGR-Lip, f = R8-dGR-Lip. (C) Confocal images of glioma sections of C6 bearing mice 24 h after systemic administration of DiD-loaded liposomes.

Fig 10

The anti-glioma assay of PTX-loaded liposomes on intracranial C6 bearing mice. (A) Kaplan-Meier survival curve of intracranial C6 glioma bearing mice treated with different PTX formulations (n= 9). Saline group was used as negative control. Red arrows indicate times of treatment. (B) HE staining of brain glioma tissues. Scale bars represent 400 μm. (C) CD34-PAS dual staining of brain glioma tissues. Scale bars represent 200 μm. Black arrows indicate pink vasculogenic mimicry in vivo. (D) CD133 staining of brain glioma tissues. Scale bars represent 50 μm. Brain cancer stem cells with CD133 positive expression were shown in brown. In (B)-(D), a = Saline, b = free PTX, c = PTX-PEG-Lip, d = R8-Lip, e = PTX-R8-RGD-Lip, f = PTX-R8-EGR-Lip, g = PTX-R8-dGR-Lip.