Peptides Mediated Drug Delivery Systems For Enhanced Targeted Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most common and malignant form of brain tumors, accounting for approximately40%of all primary brain tumors. The conventional clinical treatment for glioblastoma involves surgical debulking of the accessible tumor from the patient’s brain, but the proximity to critical regions for brain function makes the complete removal of tumor very difficult and tumor re-growth from residual tumor very possible. Therefore, chemotherapy is very critical among the therapeutic regimens for the treatment of glioblastoma, which directly influence the prognosis and life quality of patients. However, the therapeutic effects of present chemotherapy are very limited, but often causing systemic side effects, mainly due to the low concentration of most drugs in glioma cells resulting from their low permeability across the blood-brain barrier (BBB) and blood-brain tumor barrier (BTB), and poor cell internalization ability. Therefore, the development of a drug delivery strategy which can mediate efficient glioblastoma targeting together with high cellular internalization is essential and important for glioblastoma treatment.In the present study, we explored two novel methods that might have the potential use in enhancing the therapy for glioblastoma:(1) to endow cell penetrating peptides low molecular weight protamine (LMWP) with glioblastoma tarteting ability, we constructed an activatable low molecular weight protamine (ALMWP) and conjugated it to PEG-PCL nanoparticles to develop a ’smart’ drug delivery system for enhanced targeted glioblastoma therapy;(2) an active targeting DDS was constructed by conjugating MTl-AF7p to poly (ethyleneglycol)-poly (lactic acid) nanoparticles, and iRGD was co-administrated to enhance glioblastoma-specific drug accumulation and penetration.In the first part, a protease-sensitive drug delivery system was developed, using activatable low molecular weight protamine (ALMWP) as the targeting ligand. Low molecular weight protamine (LMWP) has attracted growing interest as it possess similar potency as TAT in mediating cellular translocation of its payloads, and can be produced in mass quantities direct from native protamine, while others must be chemically synthesized. However, its lack of selectivity in cellular association and tissue biodistribution could increase the risk of drug-induced toxic effect on normal tissues. By taking advantage of the dramatically upregulated expression of matrix metalloproteinases MMP-2and MMP-9in glioblastomas and the powerful transport ability of low molecular weight protamine (LMWP), we constructed the ALMWP in which the positive charges on the LMWP necessary for transduction were initially masked by a polyanionic peptide (E10) via a MMP-2/9-cleavable peptide linker sequence PLGLAG to endow LMWP with glioma homing ability. Using PTX as a model drug, the ALMWP was conjugated it to poly (ethylene glycol)-poly (ε-caprolactone) nanoparticles to develop a ’smart’ drug delivery system (ALMWP-NP) for enhanced targeted glioblastoma therapy. When ALMWP-NP has circulating through the tumor site, the linker sequence PLGLAG could be efficiently cleaved by glioma-specific expressed MMP-2/9, leading to the anion part dissociated out, and cationic part exposed to play strong penetrating ability and tumor tissue infiltration capacity, resulting in nanoparticle uptake by glioma cells.The MPEG-PCL and Maleimide-PEG-PCL block copolymers were synthesized by ring-opening polymerization of ε-CL and MPEG-OH (Maleimide-PEG-OH). Paclitaxel-loaded PEG-PCL nanoparticle (NP-PTX) was prepared using an emulsion/solvent evaporation technique, LMWP and ALMWP were functionalized to the surface of NP-PTX via a maleimide-mediated covalent binding procedure, respectively. The obtained ALMWP-NP-PTX showed a particle size of121nm, and Zeta potential of-21.8mV. Cellular experiments showed that ALMWP-NP exhibited significantly elevated MMP-dependent cellular accumulation in C6cells. The uptake amount of ALMWP-NP was significantly more than that of NP, almost at the same level with that of LMWP-NP, consistent with previous reports that claimed proteases are catalytic enzymes, and one protease molecule can specifically activate hundreds, or even thousands of its substrates. In vitro C6tumor spheroid uptake confirmed ALMWP-NP could also possess an excellent ability to penetrate through the multicellular layers of glioma cells to penetrate deeper into the tumor. Endocytosis inhibition experiments demonstrated that the mechanism of C6cellular uptake of ALMWP-NP should be shared between lipid raft-mediated endocytosis and energy-dependent macropinocytosis, involving Golgi apparatus and lysosome in the intracellular transport. Pharmacokinetic results indicated that ALMWP-NP-PTX demonstrated significantly slower clearance rate (CL)(p<0.001) and higher AUC (4.7folds, p<0.001) when compared with LMWP-NP-PTX. Meanwhile, ALMWP-NP-PTX and NP-PTX showed similar blood concentration-time curves, suggesting the conjugation of an adequate amount of ALMWP on the surface of NP did not impair the long-circulation characteristic of PEG. Tissue biodistribution experiments showed that the concentrations of PTX in the tumor of ALMWP-NP-PTX at0.5,1,3,6,12,24h were2.31,3.73,2.90,2.20,2.77,2.86-fold over that of Taxol,2.02,2.49,2.40,1.79,2.49,2.74-fold over that of NP-PTX, and1.65,1.90,2.18,2.28,3.07,3.21-fold over that of LMWP-NP-PTX, respectively. Furthermore, ALMWP-NP exhibited a similar biodistribution profile with the unmodified NP in the non-targeted tissues. The active protease-dependent glioma targeting effects of ALMWP-NP was further confirmed by in vivo imaging experiment which showed that accumulation of ALMWP-NP in intracranial tumor site was much higher than that of NP and LMWP-NP at all the time points. In vivo glioma distribution of the functinalized nanoparticles demonstrated that ALMWP-NP showed the highest distribution selectively in the glioma regions, indicating ALMWP could provide a notable glioma-targeted delivery of nanoparticles with high efficacy. Significantly enhanced cytotoxicity was achieved for ALMWP-NP-PTX (IC503.95times lower than that of Taxol,3.07times lower than that of NP-PTX). The improved anti-glioma efficacy of the ALMWP-NP was also confirmed in vivo in nude mice bearing intracranial C6glioma. ALMWP-NP-PTX significantly prolonged animal survival when compare with saline (p<0.01log-rank analysis), Taxol (p<0.01), NP-PTX (p<0.01) and LMWP-NP-PTX (p<0.05). However, no significant difference was observed between the LMWP-NP-PTX group and the NP-PTX one (p>0.05).The second part is the combination of MT1-AF7p-conjugated PEG-PLA nanoparticles with tumor homing and penetrating peptide iRGD for enhanced anti-glioblastoma therapy. Recent findings witnessed the tumor-specific vascular extravasation and tissue penetration activity of iRGD that contains a RGD motif with a protease site and a cryptic C-end Rule (CendR) motif (R/KXXR/K). The RGD motif of iRGD firstly binds to av integrins that specific expressed in tumor endothelium, and then is subjected to a proteolytic cleavage, exposing the CendR motif (RGDK/R) that binds to neuropilin-1(NRP-1) and triggers extravasation and tissue penetration. Co-administration of iRGD was more effective in delivering therapeutic agents into tumor parenchyma than conjugation. Membrane type-1matrix metalloproteinase (MT1-MMP), also referred to as MMP-14, was of central importance in glioma invasion and angiogenesis. It is predominantly expressed in angiogenic blood vessels and glioma cells, and increased with the elevation of glioma grade. MT1-AF7p peptide, identified via phage display, possesses high specificity to MT1-MMP. Therefore, in this part, encapsulating paclitaxel (PTX) as the model drug, an active targeting DDS was constructed by conjugating MT1-AF7p to poly (ethyleneglycol)-poly (lactic acid) nanoparticles, and iRGD was co-administrated to enhance glioblastoma-specific drug accumulation and penetration.PTX loaded PEG-PLA nanoparticle (NP-PTX) was prepared using an emulsion/solvent evaporation technique and MT1-AF7p was functionalized to the surface of nanoparticle via a maleimide-mediated covalent binding procedure. After modification with MT1-AF7p, PEG-PLA nanoparticles displayed a uniformly spherical shape with particle size of131nm and Zeta potential of-31.43mV. MT1-AF7p peptide density on the nanoparticle surface was317under our experiment conditions. Cellular association of PEG-PLA nanoparticles in C6cells was enhanced following the MTl-AF7p functionalization, and was time-, temperature-and concentration-dependant, suggesting that the cellular internalization of MT1-NP was an active endocytosis process. Endocytosis inhibition experiments demonstrated that the cellular internalization pathways utilized by MT1-NP included both energy-dependent macropinocytosis and lipid raft-mediated endocytosis, with Golgi apparatus involved in the intracellular transport. In vitro cytotoxicity of PTX to C6glioma cells was effectively enhanced following its encapsulation in MT1-NP. The IC50value of MT1-NP-PTX was2.81and2.47folds lower than that of Taxol and NP-PTX, respectively. Flow cytometry analysis of cell apoptosis also demonstrated that MT1-NP-PTX increased the induction of both early and late apoptosis in C6glioma cells. In vitro C6glioma spheroid assays showed that MT1-NP effectively penetrated into the glioma spheroids and significantly improved the inhibitory effects of PTX on the growth of glioma spheroids. In vivo imaging and glioma distribution together confirmed that MT1-AF7p functionalization and iRGD co-administration significantly improved the nanoparticles extravasation across BTB and accumulation in glioma parenchyma, benefiting from the MTl-AF7p-mediated transcytosis and iRGD-facilitated extravasation and tumor penetration. The anti-glioma effect was evaluated in nude mice bearing intracranial C6glioma. The median survival of mice treated with MT1-NP-PTX+iRGD (60days) was significantly longer than those of mice treated with physiological saline (21days, P<0.001), Taxol (24days, P<0.001), NP-PTX (32days, P<0.001), NP-PTX+iRGD (40days, P<0.01) and MT1-NP-PTX (48days, P<0.05).