Protein  cages

Drug  delivery

Thrombosis, the condition that results in the clotting of an artery or vain, can lead to serious fatal conditions such as cardiac arrest and hypoxia. The difficulty in designing a drug to combat such a condition lies in the trade-off between drug efficacy and toxicity. Instead of modifying the drug's design, we have successfully been able to incorporate a thrombin protease cleavage site onto the exterior of two different protein cage nanostructures and induce disassembly of these cages upon treatment with a thrombin protease. Thus, mutations can be inserted into protein cages in order to modify them into potential targeted drug delivery vehicles.

Mutations

To design the mutant cages, specific criteria were set to optimize formation. Mainly, mutations must be designed away from any secondary and tertiary structures, ideally on the exterior of the cage, and near the linker region (only for PC Quad). Using these criteria, 10 mutant cages were designed for PC Quad, and 3 mutant cages for O3-33.    

We obtained two previously created synthetic cages. One is composed of 12 subunits forming a tetrahedral cage while the other is composed of 24 subunits forming an octahedral cage. 

Cage Assembly

​In order to verify cage formation, we attempted expression of our five best PC Quad designs and three of our O3-33 designs. Expression was done using standard expression protocols and purification was done using his -tag nickel column purification while cage formation was verified using Dynamic Light Scattering (DLS). Two PC Quad mutants and one O3-33 mutant successfully expressed and were tested for cage formation. Only two mutants, PC Quad Mutant M1 and O3-33 Mutant aa88 successfully formed. 

Thrombin Assay

To test for disassembly, PC Quad Mutant M1 and O3-33 Mutant aa88 were treated with various concentrations and durations of Thrombin protease. Disassembly of cage was verified using a combination of SDS page to see if the individual subunits cut at the locations of the cleavage site insert and DLS to see if the entire cage structure fell apart.

Results

The introduction of the thrombin assay ​demonstrate cleavage of individual subunits and cage disassembly of PCQuad Mutant M1 and O3-33 Mutant aa88. Possible further investigations include exploring the therapeutic viability of these protein cages by functionalizing the exterior of the cage for specific ligand-receptor based targeting, or functionalizing the interior of the cage with binding domains to facilitate drugs to stay within the cage until cleavage.