Stable Peptides Mediated Dual Targeting Drug Delivery System For Gliomas

Gliomas, the most frequent primary brain cancer, account for 29% of all primary brain and CNS tumors and 80% of malignant tumors. The five-year survival rates are still low after current multimodal treatment-aggressive surgical resection followed by concurrently or sequential radiation and temozolomide chemotherapies. Since gliomas possess many distinctive characteristics from peripheral tumors, the oncogenesis and developments are complicated with various barriers preventing drug from reaching the tumor sites. Therefore, gliomas at specific development stages require corresponding targeting treatment strategies. At the early stage of glioma, the blood-brain barrier (BBB) which is the key challenge for drug delivery system to the brain tissue remains intact. With the deterioration of brain tumors, angiogenesis and gradual impairment of BBB, tumor neovasculature has formed to support the growth of gliomas and blood-brain tumor barrier (BBTB) locates between brain tumor tissues and microvessels formed by highly specialized endothelial cells (ECs), limiting the drug delivery to tumor tissue. Even though BBB is compromised under this situation, the infiltrating glioma especially around the tumor edge still utilize the available brain vasculature and BBB still limits the transport of the chemotherapeutic agents. Hence, based on distinctive characteristics of glioma at different stages, brain tumor targeting drug delivery systems aiming at corresponding barriers are constructed to achieve a better therapeutic effect of gliomas.Obviously, brain targeting drug delivery systems which circumvent the BBB play an important part in the glioma targeting treatment. Peptides are common BBB targeting moleculars. Due to the challenges of various enzymes in the blood after systemic administration and in the process of BBB transporting, the stability of the brain targeting moiety has direct effects on the brain targeting efficiency. Firstly, a stable brain targeting peptide was designed and decorated on the surface of nanocarriers for the study of glioma targeting drug delivery and its mechanism. Secondly, nano drug delivery system modified with both stable BBB targeting molecular and BBTB and tumor cells dual targeting moiety was constructed for the research on drug delivery to circumvent various barriers and finally target tumor cells.In the first chapter, a stable D configuration brain targeting molecular named DCDX was designed using retro-inverso isomersation technique and the comparative reseach with LCDX was conducted. Compared to the parent L-peptide (LCDX), D- peptide ligand (DCDX) displayed fivefold higher binding affinity to nAChRs which are highly expressed on the BBB. The cellular uptake by brain capillary endothelial cells increased by 2.6 times and DCDX revealed 2.2-fold higher transport efficiency versus LCDX through in vitro BBB cell culture model. Moreover, it exhibited a quick distribution in the brain tissue after systematic administration. It was demonstrated that DCDX was colocalized with lysosome in brain capillary cells, indicating its lysosomal pathway when crossing BBB. As a result, DCDX was endowed with higher brain-targeting efficiency in vitro and in vivo due to its higher binding affinity and stability.In the second part, BBB targeting functional material-DCDX-PEG-DSPE was prepared, and DCDX mediated brain targeting liposome (DCDX-LS) was constructed. DCDX-LS demonstrated effective transport across the in vitro BBB model, further consistent with the results of cellular uptake studies, exhibiting 1.5 fold and 5.1 fold higher than LCDX modified liposome (LCDX-LS), respectively. Furthermore, DCDX-LS also generated much higher brain distribution using ex vivo fluorescence imaging technique when compared with LCDX-LS. After administration of DCDX decorated liposome encapsulating doxorubicin (DOX), the survival of intracranial glioblastoma-bearing model mice was significantly prolonged, registering a median survival of 33.5 days, increasing by 20% compared with LCDX-LS group. Consequently, with the modification of DCDX, the brain targeting ability of liposomes was significantly improved, accompanied by the enhancement of the glioma treatment effects.In chapter 3, based on distinctive characteristics of glioma at different stages, glioma targeting drug delivery system modified with both DCDX and c(RGDyK)(DCDX/c(RGDyK)-LS), which holds targeting ability to BBB, BBTB and tumor cells, was constructed. In vitro cellular uptake and three-dimensional tumor spheroid penetration studies demonstrated that DCDX/c(RGDyK)-LS could not only target endothelial cells and transport across in vitro BBB monolayer but also target tumor cells and penetrate tumor spheroid. Ex vivo imaging further demonstrated that this glioma targeting liposome provided the high glioma distribution at different stages. The distribution was also reconfirmed by fluorescent images of the brain slides. As a result, this DOX-loaded DCDX/c(RGDyK)-LS presents the best anti-glioma effect in vivo by significantly improving the survival of glioma bearing nude mice.In chapter 4, two D-peptides were adopted as the brain targeting moiety and the influences of their stability on targeting ability were assessed. The studies reveal that: DCDX binding with nAChRs and DAngiopep which binds with low density lipoprotein-related protein 1 (LRP-1), displayed high stability in rat serum and lysosome homogenate. But the former exhibits a much higher binding affinity with receptors compared to LCDX, consistent to the celluar uptake. On the contrary, the binding affinity of DAngiopep was lower than its L parent, as evidenced by lower cellular uptake. Since they all experience blood circulation and the process of lysosomal pathway, both of DCDX and DAngiopep elicit a better brain targeting efficiency of nanocarriers. Consequently, the stability of the targeting peptide exerts an important influence on its brain targeting ability.In conclusion, according to distinctive characteristics of glioma, a stable peptide mediated brain targeting drug delivery system was developed to circumvent BBB at the early stage of glioma. And then, glioma targeting drug delivery system was constructed to target both BBB and BBTB to improve the targeting ability and treatment effects. Finally, it is verified that, as brain targeting moieties, D-peptides exhibit some advantages at mediating BBB transporting of drug delivery system.