A Model System for Studying Biologically Compatible Colloidal Probes for Cancer Resarch

Colloidal Probe;Hyaluronic Acid;TIRM;CD44;Chemical & Biomolecular Engineering

Brandon, Samantha M.

Johns Hopkins University

关键词: Colloidal Probe;    Hyaluronic Acid;    TIRM;    CD44;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39419/BRANDON-THESIS-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

A key challenge in the development of targeted drug delivery techniques has been to further understand the weak, specific interactions between proteins and carbohydrates. The intermolecular forces between these biomolecules influence critical biological functions such as cell-cell adhesion, motility, signaling, proliferation, and metastasis. In the scope of cancer research, the CD44-Hyaluronic Acid (HA) complex is tightly coupled with many essential cellular functions and has been pointed to as a marker in cancer metastasis. While this complex has been studied thoroughly, there still lacks substantial measurements of the multivalent binding properties. This work attempts to fill that knowledge gap by utilizing lipid bilayers as a model cell system to study CD44-Hyaluronic Acid interactions. In order to develop a controlled model of a cellular system, we utilized lipid bilayers integrated with a polymer cushion. With confocal microscopy, we were able to verify that the lipid bilayers formed planar monolayers on glass slides. Fluorescence Recovery After Photobleaching (FRAP) confirmed that the lipid bilayers maintained fluidity similar to that of in vivo cellular systems. We then determined the colloidal stability of the system by studying the interaction potentials of PEG (Polyethylene Glycol) coated and HA coated particles with the bilayer. These results demonstrate that the lipid bilayer system is suitable for studying CD44 interactions with HA modified particles. Next, to study the multivalent interactions between CD44 and HA we integrated CD44 proteins into our lipid bilayer model. After isolating CD44 proteins from MDA-MB-231 breast cancer cells, we were able to integrate the transmembrane protein into our lipid bilayer. We confirmed this with confocal imagining and reassessed the stability of our system with PEG particles. The specific binding of HA modified particles to CD44 was observed through TIRM. 100 kDa HA coated particles appear to formed clusters of stuck particles over regions of CD44 proteins, as was expected. This could indicate that not only is HA binding to CD44, but the CD44 proteins are also clustered together in lipid rafts. The robustness of this system was further demonstrated by inducing conditions that would inhibit HA-CD44 binding. The addition of 2mg/ml and 5mg/ml free 100 kDa HA to the bulk solution created competition of the HA binding domains. The TIRM data under these conditions resulted in potential energy profiles that show HA modified particles were stable above the lipid bilayer. Based on the findings in this work, a supported lipid bilayer based model cell system can be used to study the kT scale interactions of CD44 and HA.