Modifying layered double hydroxide nanoparticles with peptide to penetrate blood brain barrier for gene delivery

Abstract

Blood–brain barrier (BBB) is a formidable barrier interface that exists between blood and brain, therefore, preventing therapeutic drugs or particles to enter the brain tissue. Nano-medicine is emerging as a prospective way for enhancing pharmaceutical product delivery to the brain with the development of various nano-biotechnologies. In particular, non-viral inorganic layered double hydroxide (LDH) nanoparticles have unique advantages over others in terms of various instinctive properties, such as low cytotoxicity, good biocompatibility and high drug loading capacity. Importantly, previous studies have demonstrated that LDHs can efficiently deliver siRNAs into the neuronal cells, resulting in the knockdown of the target protein. Despite the previous results, two major hurdles still exist before LDHs can be employed as an efficacious central nervous system (CNS) drug delivery system: (i) LDH aggregation upon exposure to the serum environment and (ii) non-specificity in the BBB penetration.This thesis focuses on developing novel engineered LDH delivery system with enhanced suspension stability, redispersion capability and brain targeting ability based on the bovine serum albumin (BSA) coating strategies. Angiopep2 (Ang2) and rabies virus glycoprotein peptide (RVG) are selected as targeting ligands as they can target to the low-density lipoprotein receptor-related protein (LRP) over-expressed on U87 glioma cells and nicotinic acetylcholine receptor (nAchR) on Neura 2a (N2a) cells, respectively. These two ligands can also target the endothelial cells which form the BBB. Moreover, the manganese ions are incorporated into the LDHs system to form magnetic nanoparticles that can act as the potential theranostic agent for simultaneous brain imaging and delivery.First of all, BSA coated LDHs are further crosslinked by glutaraldyhyde (GTA) to improve the suspension stability and residpersion capability based on previous BSA coating strategies. Enhanced colloidal stability and excellent redispersity have been achieved compared with uncrosslinked BSA-LDHs and pristine LDH nanoparticles, with negligible effect on cell cytotoxicity and cell uptake. Thus, the newly developed GTA crosslinking strategy provides a solid LDH-based platform for the following in vitro and in vivo studies.Subsequently, LDHs-based targeting delivery system has been developed by conjugating ligand Ang2 or RVG with LDHs through BSA (Ang2-NPs and RVG-NPs) for brain tumour targeting. In vitro cell uptake shows that the Ang2-NPs and RVG-NPs have enhanced delivery efficiency and demonstrated specificity to relative cells. MTT assay and hemolysis study shows that the newly fabricated various LDH formulations have good blood compatibility and less cytotoxicity. Moreover, incorporating anticancer drug 5-FU with the LDH targeting system results in increased cell growth inhibition in comparison with the unmodified LDHs. Thus, this research provides a facile approach for ligand modification and synchronous maintenance of colloidal stability with narrow batch-to-batch variation.Then, in vivo and in vitro brain targeting ability of ligand modified LDHs has been evaluated. Endothelial cells (bEnd.3 cells) are the major cells that constitute BBB with both Ang2 and RVG receptors overexpressed. In the in vitro cellular uptake study and in the in vitro BBB model, the Ang2/RVG-NPs demonstrate increased cellular uptake and BBB penetration compared with NPs. In vivo confocal neuroimaging (ICON) results clearly show that the ligand modified LDH nanoparticles have longer retention time in the retina vessel. The in vivo optical imaging study confirms that the Ang2 ligand better target the mouse brain than RVG, which is consistent with the ICON observation that RVG-NPs disappear much more quickly than Ang2- NPs.Moreover, we have synthesised a new manganese-based bifunctional LDHs (Mn-LDHs) for brain cancer theranostics. The newly designed Mn-LDHs exhibits similar biocompatibility and excellent gene delivery efficacy to normal LDHs. Incorporating the contrast agent Mn into the LDHs system endows themselve as contrast agents for MRI imaging. Compared with commercial Gd (DTPA)-based contrast agent (r1 ≈ 3.4 mM −1 s −1), the Mn-LDHs demonstrate relatively high r1 value (4.47 mM −1 s −1). In consistence, the Mn-LDHs loaded with the cell death siRNA (CD-siRNA) show higher cytotoxicity to cancer cells than the free CD-siRNA, and cause significant brain tumour cell growth inhibition. Supportively, the amount of MnLDHs used for CD-siRNA delivery has been examined to be enough for MRI imaging of the cancer cells. Collectively, the obtained bifunctional Mn-LDHs show great potential applications as highly effective bimodal imaging and delivery for cancer diagnosis and chemotherapy simultaneously.