Development of Silk Microparticles Capable of Bioluminescence

The use of silk microparticles (µPs) as drug delivery devices has gained attention due to slow degradation properties, mild preparation conditions, and advantageous biocompatibility. However, little research has been done on where these particles go once injected. To expand these studies, the goal for this work is to create bioluminescent silk µPs that can be tracked in vivo. Here several methods are demonstrated for preparing bioluminescent silk µPs containing Nanoluciferase (NLuc) and/or its substrate furimazine (FZ). In this study, silk µPs were formed using a salting-out procedure. The first method involved non-specific adsorption of NLuc and non-specific adsorption of FZ to separate silk µPs. Light output produced by introducing these two silk µPs sample types to each other was monitored over time in buffer as well as in the presence of other proteins. This study found that stable light emission could be achieved in all scenarios tested including when NLuc was absorbed to silk µPs and then exposed to FZ in solution, when FZ was absorbed to silk µPs and then exposed to NLuc in solution, and when silk µPs containing NLuc and silk µPs containing FZ were mixed in buffer. Both types of silk µPs were also found to stabilize light emission in the presence of other proteins. For the next method, tyrosine residues of silk µPs were chemically modified to contain an azide functional group. The µPs were then soaked in dibenzocyclooctyne(DBCO)-linked NLuc solution. This resulted in the covalent linkage of silk µPs to NLuc via strain-promoted alkyne-azide cycloaddition. Light output produced by these luminescent silk µPs in the presence of FZ was monitored over extended periods of time. These two methods were compared to determine if covalently attaching the NLuc enzyme to silk µPs results in a more stable light output. This study found that both modified and unmodified a stable light output, silk µPs that had been soaked in NLuc DBCO produced indicating that covalent attachment of the NLuc enzyme to silk µPs was not necessary. The future role these BL silk µPs could play in drug delivery and cancer imaging is also discussed.