Poly(β-amino ester) Nanoparticles Modified with a Rabies-Virus-Derived Peptide for the Delivery of ASCL1 across a 3D In Vitro Model of the Blood–Brain Barrier

Abstract

Gene editing has emerged as a therapeutic approach to manipulate the genome for killing cancer cells, protecting healthy tissues, and improving immune response to a tumor. The gene editing tool achaete-scute family bHLH transcription factor 1 CRISPR guide RNA (ASCL1-gRNA) is known to restore neuronal lineage potential, promote terminal differentiation, and attenuate tumorigenicity in glioblastoma tumors. Here, we fabricated a polymeric nonviral carrier to encapsulate ASCL1-gRNA by electrostatic interactions and deliver it into glioblastoma cells across a 3D in vitro model of the blood−brain barrier (BBB). To mimic rabies virus (RV) neurotropism, gene-loaded poly(β-amino ester) nanoparticles are surface functionalized with a peptide derivative of rabies virus glycoprotein (RVG29). The capability of the obtained NPs, hereinafter referred to as RV-like NPs, to travel across the BBB, internalize into glioblastoma cells, and deliver ASCL1-gRNA is investigated in a 3D BBB in vitro model through flow cytometry and CLSM microscopy. The formation of nicotinic acetylcholine receptors in the 3D BBB in vitro model is confirmed by immunochemistry. These receptors are known to bind to RVG29. Unlike Lipofectamine which primarily internalizes and transfects endothelial cells, RV-like NPs are capable to travel across the 3D BBB in vitro model, preferentially internalizing glioblastoma cells, and delivering ASCL1-gRNA at an efficiency of 10%, causing noncytotoxic effects.

Hydrogel nanoparticles as new drug delivery system for CNS disorders

“…In addition, several drug delivery methods, such as liposomes, microspheres, nanoparticles, nanogels, and bionanocapsules, have been used to enhance the transportation of medicine to the brain. Recently, the use of biodegradable polymers and microchips has become more important in the treatment of brain tumors [47,48] . By the end of this section, numerous medication delivery strategies will be discussed, including:…”

Peptide-Hitchhiking for the Development of Nanosystems in Glioblastoma

“…These receptors are ligand-gated ion channels that, once bound, can reduce the transvascular delivery of drugs into the brain . Based on the understanding that nAChRs serve as a molecular target for rabies virus (RV), a rabies virus glycoprotein (RVG)-derived peptide was employed to mimic RVs transient pathway across the BBB for targeted delivery to GBM. , Additionally, α7 nAChR, a member of this receptor family, is being explored as a potential target for brain tumors . Therefore, the D CDX (GREIRTGRAERWSEKF) peptide was designed to bind to this receptor, and two different liposomal approaches were successfully functionalized, demonstrating that D CDX facilitated BBB penetration and increased tumor accumulation, thus improving therapeutic efficacy. , …”

Exploration of the Noncoding Genome for Human-Specific Therapeutic Targets—Recent Insights at Molecular and Cellular Level

“…These first experimental therapeutic studies hold promise for further developments with translational potential employing highly sophisticated and safe drug delivery systems. These should have well-established molecular and cellular mechanisms of action, including carrier ligand-cell surface receptor [184][185][186][187][188][189][190] interactions, as well as clearly characterized in vivo behavior [191][192][193]. the availability of a safe and efficient hepatocyte-specific ligand-receptor system.…”

“…Molecular and cellular basis of liver targeting: Similar to the situation about a decade ago, when efficient and hepatocyte-selective in vivo delivery systems (Figures 3 and 5) for experimentally already well established targets in the liver were still unavailable, brain or even brain cell-specific drug delivery is still in early infancy today. While hepatocytespecific genetic drug delivery is clinically applied, for none of a range of brain-targeting approaches based on synthetic nanoparticles [189,191,192,[263][264][265][266][267][268][269][270][271], or rabies virus proteins [272,273] or vectors [274][275][276][277][278][279][280], the key therapeutic efficacy requirements (Figure 5) have been established so far. Nonetheless, they are most useful for experimental pathogenetic research purposes already.…”

“…Challenges of brain targeting: Targeted and safe delivery of any nucleic acid-based (siRNA, ASO) drug to specific regions of the brain appears to be a far greater challenge than liver targeting or ex vivo blood stem cell modulation (Figure 7). A remarkable spectrum of brain-targeting approaches encompasses synthetic nanoparticles [189,191,192,[263][264][265][266][267][268][269][270][271], rabies virus proteins [272,273], or vectors [274][275][276][277][278][279], yet none of these are established with respect to key efficacy requirements (Figure 5). Nonetheless, they are already most useful for pathogenetic research.…”