All-stage precisional glioma targeted therapy enabled by a well-designed D-peptide

Abstract

Uncontrollable cell proliferation and irreversible neurological damage make glioma one of the most deadly diseases in clinic. Besides the multiple biological barriers, glioma stem cells (GSCs) that are responsible for the maintenance and recurrence of tumor tissues also hinder the therapeutic efficacy of chemotherapy. Therefore, all-stage precisional glioma targeted therapy regimens that could efficiently deliver drugs to glioma cells and GSCs after overcoming multiple barriers have received increasing scrutiny. Methods: A polymeric micelle-based drug delivery system was developed by modifying a "Y-shaped" well-designed ligand of both GRP78 protein and quorum sensing receptor to achieve all-stage precisional glioma targeting, then we evaluated the targeting ability and barrier penetration ability both in vitro and in vivo. In order to achieve all-stage precisional therapy, we need kill both GSCs and glioma related cells. Parthenolide (PTL) has been investigated for its selective toxicity to glioma stem cells while Paclitaxel (PTX) and Temozolomide (TMZ) are widely used in experimental and clinical therapy of glioma respectively. So the in vivo anti-glioma effect of combination therapy was evaluated by Kaplan-Meier survival analysis and immunohistochemical (IHC) examination of tumor tissues. Results: The "Y-shaped" well-designed peptide, termed ^DWVAP, exhibited excellent glioma (and GSCs) homing and barrier penetration ability. When modified on micelle surface, ^DWVAP peptide significantly enhanced accumulation of micelles in brain and glioma. In addition, ^DWVAP micelles showed no immunogenicity and cytotoxicity, which could guarantee their safety when used in vivo. Treatment of glioma-bearing mice with PTL loaded ^DWVAP modified PEG-PLA micelles plus PTX loaded ^DWVAP modified PEG-PLA micelles or PTL loaded ^DWVAP modified PEG-PLA micelles plus TMZ showed improved anti-tumor efficacy in comparison to PTL and PTX loaded unmodified micelles or PTL loaded unmodified micelles plus TMZ. Conclusion: Combination of all-stage targeting strategy and concomitant use of chemotherapeutics and stem cell inhibitors could achieve precise targeted therapy for glioma.

Keywords: DWVAP; all-stage targeting; drug delivery; glioma; glioma stem cells; multiple biological barriers.

Figure 1

Schematic illustration of the main obstacles to anti-glioma drug delivery and the concept of the all-stage precisional glioma targeting process. Peptide ligand functionalized micelles are designed to transverse the blood-brain barrier (BBB), blood-brain tumor barrier (BBTB) and destruct vasculogenic mimicry (VM), then target glioma cells and glioma stem cells.

Figure 2

Designation and characterization of ^DWVAP peptide. (A) Schematic illustration of the design of ^DWVAP peptide. (B) The HPLC and MS spectrum of ^DWVAP-Cys. (C) Interaction of GRP78 protein with ^DVAP and ^DWVAP. Data were collected with Biacore control software version 2.0 and analyzed by Biacore T200 evaluation software. All peptides were prepared in HBS-N buffer in a 2-fold serial dilution and injected onto the GRP78 protein-immobilized CM5 sensor chip at a flow rate of 30 µL/min. (D) Targeting ability of various peptides in BCEC cells, U87 cells and HUVEC cells evaluated by confocal microscope, scale bar = 10 µm. (E) Targeting ability of various peptides in OPC cells observed by confocal microscopy, scale bar is 10 µm. (F) In vivo imaging of biodistribution of Cy7-labeled peptides in brains and ex vivo imaging of dissected tumor 2 h after injection. (G) Normalized fluorescence intensity of brains in each group at 15, 30, 45 and 120 min. Mean ± SD, n = 3. *** p < 0.001.

Figure 3

Evaluation of the BBB and BBTB penetrating capacity in vitro and the brain and glioma targeting ability in vivo in mice. (A) Schematic illustration of the BBB using BCEC monolayer and U87 tumor spheroid co-culture model. Uptake of Coumarin-6-loaded plain micelle, ^DVAP Micelle, ^DWSW Micelle and ^DWVAP Micelle was imaged by confocal microscopy. (B) Schematic illustration of the BBTB using HUVEC monolayer and U87 tumor spheroid co-culture model. Uptake of Coumarin-6-loaded plain micelle, ^DVAP Micelle, ^DWSW Micelle and ^DWVAP Micelle was imaged by confocal microscopy. (Bar = 250 µm). (C) In vivo distribution of DiD labeled plain micelles and peptides modified micelles in the excised brain of normal mice 2 h, and 24 h after injection. (D) In vivo distribution of DiD labeled plain micelles and peptides modified micelles in the excised brain of intracranial tumor bearing mice 2 and 24 h after injection (mean ± SD). Distribution of various micelle formulations in the brains of nude mice bearing U87 glioma determined by a confocal laser microscope. Red: anti-CD31 antibody (E), anti-GRP78 antibody (F), anti-CD133 antibody (G). Blue: DAPI. Green: Coumarin-6 labeled micelles. (H) Statistical analysis of different micelle formulations co-localized with two different markers CD31, GRP78 and CD133. The co-localization rate was calculated by Image-Pro Plus software. Scale bar is 100 µm. Mean ± SD, n = 3. ** p < 0.05, *** P < 0.001.

Figure 4

Safety evaluation of ^DWVAP peptide modified micelles. (A) Serum IgG, (B) IgM production in ICR mice at day 4, day 8 and day 14 after injection of various formulations at day 0, day 3 and day 6 (Mean ± SD, n=3), ** p < 0.01, *** p < 0.001. Effect of various functional micelle materials on the proliferation of U87 cells (C) and HUVEC cells (D) by MTT assay.

Figure 5

In vitro anti-glioma efficacy of drug loaded ^DWVAP peptide modified micelles. Effect of various formulations on the proliferation of U87 cells (A and C) and HUVEC cells (B and D) by MTT assay. Tube formation assay was performed to assess the effect of various formulations on U87 VM formation and HUVEC tube formation (E). The quantitative data of the U87 VM formation (F) and HUVEC tube formation (G) treated by various formulations. Cells treated with drug-free DMEM served as the control. Data were presented as the percentages of the control group, which was set at 100%. *p < 0.05. (H) Apoptosis analysis of U87 cells after different treatments.

Figure 6

Kaplan-Meier survival curves of intracranial glioblastoma-implanted nude mice treated with different regimens. (A) Mice (n = 8) were treated with TMZ (10 mg/kg) alone or together with ^DWVAP-M/PTL (5 mg/kg with respect to PTL). TMZ was administered through oral gavage while PTL was given by caudal vein on the 6th, 8th, 10th, 12th and 14th day after implantation. (B) Mice were treated with ^DWVAP-M/PTX (6 mg/kg with respect to PTX) alone or together with ^DWVAP-M/PTL (5 mg/kg with respect to PTL). All formulations were administered through caudal vein for all mice on the day on the 6th, 9th, 12th, 15th and 18th day after implantation. Mean ± SD, n = 8. ** p < 0.01, *** p < 0.001. Immunohistochemical analysis of the tumor tissues of the mice bearing intracranial U87 glioma cells after various treatment. Anti-CD31 antibody was used to stain neovasculature (C). Quantification of angiogenesis inhibition in tumors with different formulations in comparison to saline group (D). TUNEL kit was used to label apoptosis cells (E). Quantification of TUNEL positive cells in tumors with different formulations in comparison to saline group (F). Anti-CD133 antibody was used to label tumor stem cells (G). Quantification of tumor stem cell inhibition in tumors with different formulations in comparison to saline group (H). Three random fields were selected under a light microscope. Mean ± SD, n = 3. *p < 0.05, **p < 0.01.

Figure 7

Rationality analysis of combination therapy. Safety evaluation of various treatment regimen. (A) Blood routine parameters of mice with different teatment. Mice (n = 3) were treated with five injections (5 mg/kg for PTL, 10 mg/kg for TMZ and 6 mg/kg for PTX each time) of equal drug dose (at 0, 3, 6, 9 and 12 days). Blue area indicates normal range of each parameter. WBC (white blood cell), RBC (red blood cell), PLT (platelet). Biochemical analysis of blood samples from various formulation treated groups. ALT (Alanine aminotransferase) and AST (Aspartate transaminase) were used to evaluate the liver functions (B and C). Parthenolide used alone or in combination with paclitaxel inhibited the NF-κB activation by inhibiting p65 nuclear translocation. (D) NF-κB subcellular localization was investigated by immunofluorescence staining with anti-p65 antibody (red), the nuclei were stained with Hochest (blue). (E) The fluorescence intensity was used to measure the expression level of p65 by the Image J software. (F) The co-localization rate of blue and red was used to measure the p65 nuclear translocation by the Image Pro Plus software. Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** P < 0.001.