Angiopep2-Functionalized Lipid Cubosomes for Blood-Brain Barrier Crossing and Glioblastoma Treatment

<b>Background: </b>Glioblastoma multiforme is an exceptionally aggressive form of brain cancer known for its high malignancy and resistance to traditional treatments, leading to a bleak prognosis for patients. Various treatment methods, such as surgery, radiotherapy, and chemotherapy, have been developed to address brain diseases; however, they often come with significant side effects on healthy tissues. Additionally, delivering drugs from the bloodstream to the brain is hindered by the presence of the blood-brain barrier (BBB). Lipid nanoparticles have gained substantial interest because of their inherent benefits, including biocompatibility, facile surface functionalization for targeted drug delivery, and improved solubility for poorly soluble drugs. These distinctive features make them promising carriers for bioactive agents to cross the BBB.<br/><br/><b>Aims:</b> In this study, we have pioneered the development of Ang2-conjugated cubosomes, an approach aimed at enhancing BBB penetration and improving the uptake of glioblastoma cells. Our previous research successfully yielded a range of amphiphilic block copolymers, synthesized through RAFT polymerization, which included polyethylene glycol (PEG), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), and poly(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate) (PTBA). These polymers served as effective stabilizers for monoolein cubosomes with pH nad H₂O₂ responsiveness. In this study, we selected PEG114-b-PDMAEMA17-b-PTBA9-RAFT polymer to craft Ang2-conjugated monoolein-based cubosomes, loaded with the anti-cancer drugs cisplatin (CDDP) and temozolomide (TMZ), which are known for their poor solubility.<br/><br/><b>Methods: </b> A comprehensive analysis of these developed nanoparticles was conducted to assess their particle size, surface charge, and internal structures through dynamic light scattering and synchrotron Small-Angle X-ray Scattering. Furthermore, their potential to enhance BBB penetration was evaluated using three in vitro models: a Transwell BBB model based on the hCMEC/D3 human brain endothelial cell line, a 3D hCMEC/D3 spheroid model, and an innovative microfluidic BBB/GBM-on-a-chip model. This microfluidic model featured a unique setup with hCMEC/D3 cells in one channel (representing the blood channel) and U87 glioblastoma cells in an adjacent, interconnected channel (representing the brain channel). This design enabled the simultaneous investigation of the cubosomes' ability to cross the BBB and enter U87 cells. To further evaluate the anticancer effectiveness of CDDP/TMZ-loaded cubosomes, in vitro cytotoxicity studies on U87 glioblastoma cells was conducted.<br/><br/><b>Results: </b>These developed lipid cubosomes exhibited a particle size of approximately 300 nm, possessed an internally ordered inverse primitive cubic phase, achieved a high conjugation efficiency of Ang2 on the particle surface, and displayed an encapsulation efficiency exceeding 70% for both CDDP and TMZ. We employed various in vitro BBB models, including hCMEC/D3 cell Transwell assays, 3D spheroid cultures, and microfluidic BBB/GBM-on-a-chip models, to evaluate the effectiveness of these cubosomes in crossing the BBB. The results demonstrated that Ang2-functionalized cubosomes exhibited superior BBB penetration. Furthermore, these modified cubosomes exhibited significantly increased uptake by U87 glioma cells, with a notable three-fold enhancement observed in the BBB/GBM-on-a-chip model compared to unmodified cubosomes. Moreover, CDDP-loaded Ang2-functionalized cubosomes exhibited a heightened toxic effect on U87 glioma spheroids.<br/><br/><b>Coclusion: </b>These findings suggest that the Ang2-functionalized cubosomes hold great promise for improving BBB penetration and enhancing the delivery of therapeutics to glioblastoma.