(E-163) Characterization of mechanically tuned liposomal nanocarriers for cell and phenotype-specific endocytosis using Atomic Force Microscopy

Introduction:: In the central nervous system, circulating blood and brain extracellular fluids (BECFs) are separated by semipermeable barriers called blood-brain barriers (BBBs) that protect the brain from potentially dangerous circulatory substances, keeps the brain's neuronal environment steady, and controls the entry of nutrients and other essential molecules. Nanocarriers can overcome the blood-brain barrier's (BBB) selective permeability and carry medications directly to the brain, making them effective as drug delivery systems in the BBB. For the treatment of a variety of brain illnesses, including as Alzheimer's disease, Parkinson's disease, and brain tumors, nanocarriers have demonstrated significant promise as drug delivery vehicles. As the efficiency of nanocarriers (NCs) to enable drug transport over the BBB has been intensively studied and appreciated, our goal is to investigate the mechanical (stiffness) and molecular (surface ligands) features by nanomanufacturing hybrid liposomal nanocarriers (LNCs) to regulate cell-specific targeting, cellular uptake, and therapeutic action.

Materials and Methods:: PLL slides coated with tetrakis (hydroxymethyl) phosphonium chloride (THPC) gold nanoparticles and citrate-capped gold nanoparticles were employed in the AFM experiment. Before the glass slide sample was placed on the AFM stage, the spring constant and deflection sensitivity of the SCAN-ASYST FLUID probe were calibrated, yielding results of 0.64020 N/m for the spring constant and 15.866 nm/mV for the deflection sensitivity, respectively, where the tip radius was 20 nm. The spring constant and deflection sensitivity values of the SCAN-ASYST AIR were 0.53960 N/m and 30.822 nm/mV, respectively, after the calibration and the tip radius was 5 nm. All force curves were fitted using a Hertzian (spherical) model.

Results, Conclusions, and Discussions:: Bruker BioScope Resolve Atomic Force Microscopy (Bio-AFM) was used in our experiment to probe gold nanoparticles coated on glass slides to first obtain topographical images, measure the particle sizes, and then investigate the nanomechanical properties (Young's modulus, deformation, dissipation, and adhesion) of these gold nanoparticles at room temperature. By tuning the Young's modulus of LNCs and surface ligands simultaneously, kinetics of uptake will be controlled more efficiently, and cell-specific targeting will be achieved more effectively than with individual tuning. Many AuNPs synthesis protocols have been tried, tested, and our recent most AFM data demonstrates that our most recent experiment's average Young's modulus (THPC AuNPs) is 45 MPa, significantly lower than the average Young's modulus of our prior experiment (citrate capped AuNPs), which was 65 MPa. These will be utilized to finish the study as liposomes can be effectively coated on AuNPs. As a result, individual lipidcoated gold nanoparticles with varying coating thicknesses will be investigated simultaneously in Peak force QNM mode. A total of nine different combinations of LNCs with changes in the gold shell thickness, ligand type, and density will finally be used to measure the modulus. We will also examine how the type and density of surface ligands correlate to the Young's modulus of LNCs to determine their stability. The preliminary results demonstrate that ligands can remain attached to gold nanoparticles even after the gold shell stiffness is changed from KPa to GPa. Overall, we will nanomanufacture hybrid liposomal nanocarriers (LNCs) with varying gold shell thicknesses and functionalize the LNCs with three different ligands that target the Transferrin receptors in brain microvascular endothelial cells (BMECs), excitatory amino acid transporter 2 (EAAT2) receptors in astrocytes, and cluster of differentiation 40 (CD40) that are specific to M1 polarized microglia to increase their stability, circulation time, and targeting capabilities. Besides providing sustained therapeutic effects, these modifications will also allow controlled delivery of the drug at the desired site.

Acknowledgements (Optional): : This research was supported by Exploratory Research Project (ERP) grant at Iowa State University.

References (Optional): : Rade, Jaydeep, Juntao Zhang, Soumik Sarkar, Adarsh Krishnamurthy, Juan Ren, and Anwesha Sarkar. "Deep learning for live cell shape detection and automated afm navigation." Bioengineering 9, no. 10 (2022): 522