Engineering Responsive Chemical Heterogeneity in Protein Vesicles for Simultaneous Delivery of Diverse Cargoes

Non-Technical Summary

The work proposed here creates protein vesicles, tiny hollow containers made from proteins, that can bind to specific types of cells and carry three different kinds of therapeutic drugs inside cells. The functional demonstration of this concept will target breast cancer cells and deliver a combination of protein, nucleic acid and chemotherapeutic molecules that could be uniquely effective against them.

This is significant because these vesicles have the potential to address challenges with drug resistance and metastasis by simultaneous triple drug delivery. Beyond engineering protein vesicles, the proposed work will ask both fundamental and practical questions that will build key knowledge of the vesicle assembly process and properties and extend this work into other areas and vesicles made from other types of molecules.

One of the advantages of protein vesicles is their ability to present a range of proteins on the surface, which can target different types of cells, and carry a variety of cargoes, so that many other drug delivery applications could be envisioned, such as vaccines and immunotherapies. Broad impact could also be realized by other uses including biosensors, artificial cells, and small bioreactors.

In addition to societal benefits of the research on critical diseases, the PI will implement activities to recruit and retain women, especially women from underrepresented groups, in STEM at education levels ranging from middle school to graduate. Middle school girls will visit the lab as part of summer camps and field trips. The research will provide the basis for technical training and critical personal mentoring of female graduate and undergraduate students.

It will also provide meaningful research experience, professional development, and mentoring to underrepresented Atlanta high school students. The results from this work will be shared in the chemical engineering introductory course and the PI’s protein engineering elective at Georgia Tech, and at K-12 school visits, to inform and inspire students to pursue and persist in STEM degrees.

Technical Summary

The goal of this research is to understand, engineer, and employ self-assembled protein vesicles made from globular, functional proteins for therapeutic delivery. Specifically, the vesicles will be made from full-size therapeutic or targeting proteins that also carry three different classes of therapeutic cargos in the lumen or membrane: nucleic acids, small molecules, and additional proteins.

Objective 1 will incorporate responsive elements and distinct chemical environments into protein vesicles by modifying the amino acid sequences of the vesicle building block proteins. The ability to modify the protein sequence, with both natural and non-natural amino acids, will enable tuning to ensure the cargos are released and function when and where needed.

Objective 2 will understand how these sequence changes affect vesicle self-assembly, properties and stability using techniques including dynamic light scattering, transmission electron microscopy, and absorbance and fluorescence spectroscopy. This knowledge will help researchers rationally design synthetic, recombinant, or hybrid molecules capable of forming self-assembled structures with responsive properties and cargo loading.

Objective 3 will use this knowledge to create functional protein vesicles capable of simultaneous, receptor targeted delivery of protein (YopJ), nucleic acid (anti-p-glycoprotein siRNA) and small molecule (doxorubicin) cargoes to breast cancer cells (epidermal growth factor receptor positive) in vitro as proof of concept. Delivery will be demonstrated via flow cytometry, confocal microscopy, and cell viability and function assays.

The research will provide the basis for technical training and critical personal mentoring of female graduate, undergraduate, and Atlanta high school students. The PI will also implement activities to recruit and retain women, especially women from underrepresented groups, in STEM at education levels ranging from middle school to graduate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.