Peptides Mediated Targeted Drug Delivery Systems For Gliomas

Glioblastoma multiforme (GBM) is the most frequent primary malignant brain tumors and accounts for approximately 40%. The conventional clinical treatment for GBM 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. Consequently chemotherapy seems essential in the treatment of GBM.RGD sequence has been identified as an essential binding motif to facilitate interaction between drug delivery systems including micelles and some integrins which connect cells to proteins of the extracellular matrix. Among these integrins,αv integrin receptors, which are found to be highly expressed on activated endothelial cells and tumor cells (such as U87 glioblastoma cells) but not in resting endothelial cells and most normal organ systems, constitute a potential target for tumor imaging and therapy. At the early stage of gliomas, BBB is normal integral to impair drug transport to the brain. However, nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction. It is widely expressed in the brain, including the brain capillaries endothelial cells (BCECs). Therefore, nAChRs mediate brain transport may be a promising strategy. Neurotoxins from snake venoms are well known to bind with high affinity and selectivity to nicotinic acetylcholine receptors. They belong to a family of proteins called "three-finger toxins", which adopt a flat, leaf-like shape formed by three adjacent loops that emerge from a small globular core. The tips of the loopⅡof neurotoxins are thought to be nAChRs binding domain. Thus, we synthesized some peptides derived from snake neurotoxin, which had been further conjugated with drug delivery systems to overcome BBB.The recent reports demonstrated that gene therapy had much potential for the treatment of gliomas. Paclitaxel has been proven effective in the treatment of GBM, acting by promoting the assembly and stabilization of microtubules inhibiting cellular division, preventing de-polymerization of the assembled microtubules and thereby halts mitosis or cell division and binding to Bcl-2 which normally blocks the process of apoptosis, allowing apoptosis to proceed. Combination of TRAIL and paclitaxel would effectively improve the anti-tumor efficacy in vitro and in vivo of the latter.The present work includes three parts:(1)Targeted drug delivery system for the therapy of GBM mediated byαvβ3 Integrin.In this part, we prepared c(RGDyK) modified PEI and PEG-PLA micelle for the targeted chemotherapy and gene therapy.RGD is widely chosen as a ligand to improve the specific gene targeting transfection efficiency of PEI in vivo. However, the optimal RGD conjugating mode, c(RGDyK)-poly(ethylene glycol)-polyethylenimine (c(RGDyK)-PEG-PEI) or c(RGDyK)-polyethylenimine-methoxyl poly(ethylene glycol) (c(RGDyK)-PEI-PEG) still remains controversial. Here, c(RGDyK)-PEG-PEI was proven to have better performance than c(RGDyK)-PEI-PEG in the glioblastoma targeting gene transfer in vivo. The anti-glioblastoma effect of c(RGDyK)-PEG-PEI/pORF-hTRAIL complex had also been investigated. After 4 doses treatment, the median survival time of intracranial GBM mice was significant prolonged compared with treatment by saline or PEG-PEI/pORF-hTRAIL complex.C(RGDyK)-PEG-PLA, which had high binding affinity with U87 cell, enhanced the cytotoxicity of encapsulated paclitaxel by 2.5 folds. It was significantly accumulated in the subcutaneous U87 tumor via RGD-Integrin binding. As to the intracranial GBM model, encapsulated DiR was notably distributed in the brain domain. PTX encapsulated c(RGDyK)-PEG-PLA micelle had higher anti-glioblastoma effect both in the subcutaneous and intracranial GBM model compared with the normal PTX formulations.(2) Brain targeted drug delivery system for the therapy of GBM mediated by nAChRs.The loopⅡof the three-finger snake neurotoxins is considered as the binding domain with nAChRs, so they maybe have the potential to enhance drug or drug delivery system intracranial transport. In the present work, binding of the synthetic peptides to the neuronal nAChRs was assessed by measuring their ability to inhibit the binding of 125I-α-bungarotoxin to the receptor. The synthetic peptides demonstrated high binding affinities and the IC50 values were 301.99nM (CDX) and 32.51nM (KC2S), respectively. The specific uptake by brain capillary endothelial cells (BCECs) demonstrated that KC2S could be cell endocytic after binding with nAChRs. In vivo, the qualitative and semi-quantitative biodistribution results indicated KC2S modified could overcome the BBB.The present study also constructed CDX and KC2S modified drug delivery system. DiR encapsulated KC2S-PEG-PLA micelle increased the fluorescent intensity in the brain during 24h. We prepared coumarin-6 encapsulated CDX-PEG-PLA, KC2S-PEG-PLA or PEG-PLA micelles to evaluate the brain uptake profiles. The AUC of coumarin-6 in the brain of CDX-PEG-PLA and KC2S-PEG-PLA micelle were about 2.11 and 2.60 folds compared with that of PEG-PLA micelle, respectively. And the Cmax were about 1.91 and 1.98 folds. The results indicated that CDX or KC2S modified micelle did increase the accumulation of encapsulated coumarin-6 in the brain, which might result from the fact that the KC2S induced active transport of drug delivery system through the BBB.We also investigated the pharmacodynamics of CDX-PEG-PLA-PTX micelle. The CDX-PEG-PLA could overcome BBB at the early stage of GBM, and with the progress of GBM, it could accumulate in tumor. After treatment with CDX-PEG-PLA-PTX, the median survival time was significantly prolonged.(3) anti-GBM effects of co-delivery of paclitaxel and pORF-hTRAIL.As we all known, paclitaxel had high anti-glioblastoma effect during long time. In this part, we tried to shorten the duration of action and enhance the anti-glioblastoma effect. TRAIL gene was chosen to co-deliver with paclitaxel for its conclusive mechanism. After co-dilivery with TRAIL gene, the IC50 of paclitaxel was reduced by 5.4 folds during 48h. On the other hand, the co-delivery enhanced cell apoptosis percent and the U87 cells were almost totally located in G2-M stage. Paclitaxel also enhanced the gene transfection efficiency by the FACS and ELIAS tests. Compared with solitary usage of paclitaxel or TRAIL gene, co-delivery also dramatically prolonged the median survival time of intracranial GBM model mice.In the present work, we had designed the GBM targeted drug delivery system and set the therapeutic scheme. RGD modified drug delivery systems had been also prepared to overcome blood tumor barrier (BTB).We had screened the brain targeted peptides based on nAChRs to overcome BBB.The brain targeted peptides CDX and KC2S were further conjugated with PEG-PLA micelle, which had much potential in the diagnosis and therapy of brain diseases. The tumor targeted delivery systems would be useful protocols for the chemotherapy, gene therapy and molecular imaging of GBM.At last, the anti-glioblastoma effect of paclitaxel was further enhanced by co-delivery of TRAIL gene, and the therapeutic scheme also obtained satisfactory anti-glioblastoma effects.