Protein Cages Engineered for Interaction-Driven Selective Encapsulation of Biomolecules.

Area of Science:

• Biotechnology
• Nanotechnology
• Biochemistry

Background:

• Protein cages offer biocompatible and tunable drug delivery platforms.
• Effective encapsulation of diverse biomolecules within protein cages remains a significant challenge.
• Existing methods lack versatility for loading various cargo types and achieving targeted delivery.

Purpose of the Study:

• To develop a novel strategy for specific interaction-driven encapsulation and cellular delivery of biomolecules using engineered protein cages.
• To create a versatile platform for loading both protein and non-protein cargos into a single nanocarrier.
• To demonstrate efficient cellular uptake and delivery of encapsulated therapeutic agents.

Main Methods:

• Computationally designed hyperstable mi3 protein cage engineered with surface-displayed RGD tripeptide for cancer targeting and internal SpyTag (ST).
• SpyCatcher (SC)-fused proteins and SC-fused monomeric avidin used for covalent encapsulation via ST/SC reaction.
• Cryo-electron microscopy (EM) used to confirm cargo protein encapsulation.
• Encapsulation of non-protein cargos (oligonucleotides, doxorubicin) via biotin-avidin interaction.

Main Results:

• Engineered mi3 protein cage successfully encapsulated SC-fused proteins of varying sizes and charges.
• Cryo-EM confirmed stable encapsulation of proteins within the nanocage.
• Bi-functional nanocage effectively encapsulated non-protein cargos like oligonucleotides and doxorubicin.
• All engineered nanocages demonstrated efficient cellular delivery of their respective cargos.

Conclusions:

• The developed ST/SC system provides a highly versatile and specific method for protein cage cargo loading.
• This engineered protein cage platform facilitates the delivery of diverse biomolecules, including therapeutic agents, into cells.
• The strategy allows for customizable targeting moieties and cargo types, paving the way for advanced nanomedicine applications.