A versatile method to fabricate giant vesicles that can encapsulate a range of membrane-bound proteins, luminal proteins, and cell lysates under physiologically relevant conditions

Unmet Need
Creating artificial vesicular systems that can mimic natural cells continues to be a challenge in the fields of synthetic biology as well as gene and drug delivery. This class of artificial systems serve as a powerful platform for both studying biology in a minimal, controlled system and moreover can be used as biocompatible vehicles to deliver various biological and chemical materials in a cellular environment. Currently, giant vesicles can be fabricated with geometry, membrane lamellarity, and lipid compositions to mimic real cells; however, encapsulating biological materials inside these vesicles has proven difficult. This invention allows for the biocompatible encapsulation and cellular delivery of biological materials under physiologically relevant conditions. This is especially paramount in the burgeoning field of genome editing as this vesicular platform can be used to encapsulate a relatively high concentration of both protein (e.g. Cas9) and nucleotide (e.g. guide RNA) material in these vesicles. This platform is especially effective in applications where the biomaterial of choice does not have high water solubility which is the case for most chemical therapeutics or where targeted delivery in high doses is desired.  
 
Technology Overview
Johns Hopkins researchers found that biological material (e.g., purified proteins, DNA material, and cell lysates) can be reliably encapsulated in giant unilamellar vesicles (GUVs). GUVs are cell-sized model membrane systems, which can be fabricated with different lipid compositions, in sizes ranging from 10 to 100 microns in diameter under physiologically relevant pH, buffer, and temperature conditions. Hopkins researchers invented a methodology to include proteins and cell lysates into a GUV embodiment given the physiologically-relevant platform. This unique method of encapsulation allows for anchoring of membrane-bound proteins to the inner vesicular lipid leaflet and loading of cytosolic proteins in its lumen. Moreover, these vesicles proved permeable with respect to small drug compounds such as rapamycin. The technology allows for deeper studies of cellular singling as well as transient delivery of cytosolic and membrane-bound proteins for applications such as localized delivery of drugs or genome editing constituents such as Cas9 protein and the desired guide RNA. Additionally, these fabricated GUVs are biocompatible and have great potential as a biologic drug delivery platform not limited by surface interacting proteins.
 
Stage of Development
The inventors have developed a technique for the encapsulation of various proteins in GUVs and demonstrated this in a recent publication.
 
Publications
Meier EL, et al. Molecular Microbiology. 2016 Jul;101(2):265-80 DOI: 10.1111/mmi.13388

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