Delivery of imaging agents and pharmaceutical payloads to the central nervous system (CNS) is essential for efficient diagnosis and treatment of brain diseases. However, therapeutic delivery is often restricted by the blood-brain barrier (BBB), which prevents transport of clinical compounds to their region of interest. For example, MRI contrast agents such as DOTA-Gd3+ are unable to enter the brain for imaging purposes unless the BBB has been compromised. Therefore, an innovative approach to facilitate transport of these molecules across the BBB and into the brain is a crucial area of research. A multifunctional nanoparticle (NP) system is needed that 1) can be loaded with functional molecules, 2) transport across the BBB, and 3) localize to specific regions in the brain.
Here, we investigate a delivery system based on the MS2 bacteriophage capsid. MS2 self-assembles into a protein cage structure with hundreds of reactive groups on its surface for conjugation of targeting moieties, making it an ideal NP platform. Angiopep-2 (AP2) is a 3kDa synthetic peptide capable of receptor-mediated transcytosis across the BBB. We report on the development of our multifunctional NP utilizing AP2 conjugated to MS2 bacteriophage capsids and evaluate transport over an in vitro BBB model, investigate the feasibility of dual modification of the MS2 surface with both AP2 and anti-NMDAR2D IgG, and evaluate nucleotide-mediated loading of a MRI contrast agent based on psoralen and DOTA-Gd3+ into MS2 NPs.
The targeting moieties were conjugated to the MS2 surface using the heterobifunctional reagent SMCC. Traut’s Reagent was used to activate IgG before conjugation to the MS2 capsid surface. The dually functionalized NP constructs were tested for the presence of the two targeting moieties using a dot blot. The particles were also tested for their ability to recognize NMDAR2D receptors in brain cell lysate using a Western blot. Confluent 2D monolayers of primary rat brain microvascular endothelial cells (RBMECs) on Transwell inserts were used to evaluate the ability of AP2-conjugated MS2 NPs to cross an in vitro BBB model. NP transport over the BBB model was monitored over 24 hours using real-time reverse transcriptase PCR. Samples were taken from the bottom compartment and amplified using probe sequences for MS2 RNA. Loading of the MRI contrast agent DOTA-Gd3+ via nucleotide driven interactions was evaluated using inductively coupled plasma mass spectrometry (ICP-MS).
Preliminary results show successful conjugation of the targeting moieties to the MS2 capsid surface and that the antibody is functional after reaction. Studies are currently ongoing to confirm in vitro BBB transport of MS2-AP2 NPs, the potential for using PCR in MS2 NP detection, and the ability to load MS2 with MRI contrast agent.