Presentation Information
[4FMBS-13]Enzymatic surface modification of lipid-based microbubbles and nanodroplets enables versatile, functional protein attachment
○Johannes Buechler1, Pascal Poc1, Carina Frehen1, Ines Oberhuber1, Simone Schuerle1 (1. ETH Zürich (Switzerland))
Keywords:
Microbubbles,Nanodroplets,Ultrasound contrast agent,Sortase,Biocatalysis
[Purpose]
Microbubbles (MBs) and nanodroplets (NDs) have gained traction in medicine as ultrasound contrast agents (UCAs) and carriers for therapeutics. To enable cellular targeting and site-specific drug delivery, proteins are conjugated to the surface of MBs which presents several challenges. Common conjugation methods often require accessible surface cysteines or lysines, which may be crucial for the protein's functionality. Additionally, common tags like streptavidin, have been reported to trigger immune responses or interfere with the natural biotin pathways. The use of electrostatic interactions as an alternative non-chemical approach is prone to instability and may cause nonspecific binding to certain cell types. Finally, hydrophobic loading methods are restricted to small molecules and ill-suited for larger protein attachments. To overcome these limitations, we use an engineered sortase enzyme from S. aureus, which ligates two specific peptide sequences, a N-terminal multi glycine (GGG) and the sortag XLPXTGX (X: any amino acid).
[Method]
We synthesized a cysteine-terminated peptide with the sequence CLPETGW using solid-phase peptide synthesis and conjugated it to DSPE-PEG(5000)-maleimide through a Michael addition reaction. Concurrently, the desired cargo proteins were modified at the N-terminus with a GGG motif via primer extension. To test our approach on UCAs we created microbubbles and nanodroplets through amalgamation and microfluidics, respectively. We were able to incorporate up to 10% of the peptide-modified lipid DSPE-PEG5000-CLPETGW into standard lipid-based formulations. Following purification, the UCAs were reacted with varying concentrations of sortase and GGG-modified protein (GGG-sfGFP) for 1 hour at ambient temperature. After purifying the modified UCAs, qualitative fluorescence imaging was performed using confocal spinning disc microscopy.
[Results]
The difference in green fluorescent protein signal intensity between sortag-modified UCAs and standard UCAs qualitatively demonstrates the successful attachment of GGG-sfGFP to the peptide-modified MB surface. To rule out the possibility of non-covalent interactions, an SDS gel analysis was performed on the starting material, GGG-sfGFP and the purified sortag-MBs after the reaction. The gel showed minimal traces of the starting material and predominantly the newly formed DSPE-PEG5000-sfGFP conjugate. Beyond MBs, this sortase-mediated surface functionalization strategy has also been successfully applied to nanodroplets.
[Consideration]
The reaction conditions still require optimization to prevent MB aggregation and precipitation of the protein-lipid conjugate.[Conclusion]
We demonstrated the successful attachment of protein to MBs and NDs using an enzymatic ligation strategy. By finetuning the reaction conditions, we aim to provide a universally applicable method for the attachment of protein-based cargo, such as targeting motifs or peptide-based drugs, under conditions that allow inherent amino acid backbone to stay intact and do not require surface exposed residues.
Microbubbles (MBs) and nanodroplets (NDs) have gained traction in medicine as ultrasound contrast agents (UCAs) and carriers for therapeutics. To enable cellular targeting and site-specific drug delivery, proteins are conjugated to the surface of MBs which presents several challenges. Common conjugation methods often require accessible surface cysteines or lysines, which may be crucial for the protein's functionality. Additionally, common tags like streptavidin, have been reported to trigger immune responses or interfere with the natural biotin pathways. The use of electrostatic interactions as an alternative non-chemical approach is prone to instability and may cause nonspecific binding to certain cell types. Finally, hydrophobic loading methods are restricted to small molecules and ill-suited for larger protein attachments. To overcome these limitations, we use an engineered sortase enzyme from S. aureus, which ligates two specific peptide sequences, a N-terminal multi glycine (GGG) and the sortag XLPXTGX (X: any amino acid).
[Method]
We synthesized a cysteine-terminated peptide with the sequence CLPETGW using solid-phase peptide synthesis and conjugated it to DSPE-PEG(5000)-maleimide through a Michael addition reaction. Concurrently, the desired cargo proteins were modified at the N-terminus with a GGG motif via primer extension. To test our approach on UCAs we created microbubbles and nanodroplets through amalgamation and microfluidics, respectively. We were able to incorporate up to 10% of the peptide-modified lipid DSPE-PEG5000-CLPETGW into standard lipid-based formulations. Following purification, the UCAs were reacted with varying concentrations of sortase and GGG-modified protein (GGG-sfGFP) for 1 hour at ambient temperature. After purifying the modified UCAs, qualitative fluorescence imaging was performed using confocal spinning disc microscopy.
[Results]
The difference in green fluorescent protein signal intensity between sortag-modified UCAs and standard UCAs qualitatively demonstrates the successful attachment of GGG-sfGFP to the peptide-modified MB surface. To rule out the possibility of non-covalent interactions, an SDS gel analysis was performed on the starting material, GGG-sfGFP and the purified sortag-MBs after the reaction. The gel showed minimal traces of the starting material and predominantly the newly formed DSPE-PEG5000-sfGFP conjugate. Beyond MBs, this sortase-mediated surface functionalization strategy has also been successfully applied to nanodroplets.
[Consideration]
The reaction conditions still require optimization to prevent MB aggregation and precipitation of the protein-lipid conjugate.[Conclusion]
We demonstrated the successful attachment of protein to MBs and NDs using an enzymatic ligation strategy. By finetuning the reaction conditions, we aim to provide a universally applicable method for the attachment of protein-based cargo, such as targeting motifs or peptide-based drugs, under conditions that allow inherent amino acid backbone to stay intact and do not require surface exposed residues.
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