Tricia Lin
Biotinylated-Streptavidin Conjugation for Tumor Targeting


To this day, research has progressed in developing various versatile carrier systems for delivering active molecules down to the nano-scale. In particular, several carrier systems are focused on using liposomes and nanoparticles as colloidal particles to enhance the efficacy compared to traditional drug delivery. Currently, research on this aim has mainly focused on the design of the liposomes and nanoparticles that allow the attachment of targeting ligands on the transport system as well as enabling the delivery of active compounds itself. However, there are several obstacles with this delivery system such as the stability and coupling efficiency between the carrier system and targeting ligands and the in vitro capabilities for active molecules to bind and be delivered.

With this rationale, the subsequent objective is to develop such carrier system utilizing past ideas and present materials. Specifically, a carrier of interest is using a liposome attached to the protein, streptavidin, as the targeting ligand. Vesicles are optimal chemical transports due to their large surface area, high flexibility and curvature. Past research investigated the crystallization of streptavidin on the surfaces of large, unilamellar lipid bilayer vesicles that are greater than 10 micrometers in diameter through hydration and rapid evaporation. These streptavidin-coated vesicles displayed spherical and ellipsoidal shapes, demonstrating a resistance to curvature. (Ratanabanangkoon P. et al.) Streptavidin is a tetrameric protein produced by the bacteria, Streptomyces avidinii. Streptavidin is a widely used protein because of its high binding affinity to biotin, a component necessary for cell growth. Due to this high binding affinity, this streptavidin-biotin complex system is an ideal cross-linker for other biomolecules to be delivered into the cell.

:labmembers:avidin-biotin.gif
Figure 1: An illustration of streptavidin as a tetrameric protein with 4 biotin molecules embedded within each binding site. Notice that the binding sites are opposite to one another. (http://www.iob.uio.no/forskning/celler/teknikk/IH-bakgrunn.html)

In another study performed by Ratananbanangkoon et al., two-dimensional crystallization of streptavidin molecules on lipid bilayer vesicles was explored. Using a confocal microscope, images of the streptavidin-bound vesicles exhibited rigid and distorted spherical surface when compared to simple avidin-coated vesicles (see Figure below)From this study, researchers also identified that the optimal conditions for proper growth of streptavidin crystals such as high concentration of sucrose as a buffer for pH control. Although this study acknowledges the possibility of existence streptavidin-coated vesicles, no information or hypotheses were indicated regarding the functionality of these streptavidin-bound vesicles and its ability to endocytose into cells and delivering proteins [2].

:labmembers:streptavidinvesicle.jpg
Figure 2: Confocal microscopic images of two shapes observed among streptavidin-coated vesicles. (A) Rough surfaces have many ridges and edges compare to avidin-coated vesicles. (B) Ellipsoids are smoother with ridges running parallel to the major axis

Following similar procedures from past research findings, the first aim is to produce appropriate amounts of streptavidin, enough to cover ample surface area on the lipsome. Currently, the Jin lab has the capabilities of mass producing such protein in the lab. The second aim is to determine the optimal concentration of streptavidin to allow the vesicle-streptavidn-biotin complex to endocytose and deliver other proteins or biomolecules to the cell following conjugation between the protein and vesicle. This aim also requires the determination of targeting ligands that are capable of attaching to the complex system and can appropriately direct the complex carrier system within the cell after endocytosis. Overall, this is perhaps is considered the most difficult aim to achieve due to the intricate balance of pH, structure and binding capacities the vesicles and proteins play with.

Despite the difficulties, the findings and results following these aims will be considered fundamental and quite interesting. Importantly, once the optimal concentrations and methods of attaching the streptavidin-biotin complex upon the liposome is established, further applications within this research can be applied. Other applications include utilizing nanoparticles such as superparamagnetic iron oxide particles (SPIO) as well as commercial quantum dots as the carrier system.

Contact info
E-mail: tsl28@cornell.edu

Last updated: 08-11-08