Poster N18 - Liquid-liquid Phase Separating Histidine-rich Beak Peptides for Therapeutic Delivery

Introduction: Peptides can undergo self-assembly through liquid-liquid phase separation (LLPS) in contrast to the classical self-assembly via monomer-by-monomer addition. Due to their unique interactions with the cell membrane, phase-separating peptides have emerged as attractive vehicles for the delivery of therapeutic agents. These peptides form condensates in physiological bufers that can interact with cell membranes, facilitating the release of cargo directly into the cytosol or via the endocytic pathway. Here we demonstrate that peptides derived from histidine-rich beak proteins (HBPs) containing the consensus sequence (GHGXY)4 (X=L/V/P) undergo phase separation and can encapsulate DNA, mRNA, and protein forms of eGFP for successful delivery to cells. Using the model antigen DQ-OVA that only fluoresces post cleavage in the lysosomal compartments, we demonstrate that these condensates can also deliver antigens for appropriate processing and presentation to T cells. Using enantiomeric versions of the peptides, we also show that chirality is not a prerequisite for condensate formation and that L- and D-peptide condensates readily undergo fusion opening up new possibilities for drug and vaccine delivery.

Materials and

Methods: Phase-separating peptides (GHGXY)4 where X=L/V/P or their D-form analogs and (GhGXy)4 (X=l/v/p) were synthesized using the Liberty Blue microwave peptide synthesizer. D-amino acids are all indicated in lower case. Live cell imaging was done on the the Lionheart FX imaging system which can capture real-time videos of peptide fusion. Using difference cargoes functionalized with different fluorescent dyes, fusion was visualized and quantified using a custom Python script for tracking interactions of different colored peptide droplets. For antigen delivery studies, dendritic cells (DCs) were treated with DQ-OVA loaded peptide condensates and fluorescence microscopy was used to track their internalization and localization within the cells. Bafilomycin A1 (BA) and ammonium chloride (NH4Cl) DCs were used as controls to interfere with endolysosomal acidification. The ability of the condensates to deliver multiple cargo types was tested by encapsulating DNA, mRNA, and protein forms of eGFP and intracellular fluorescence was monitored.

Results, Conclusions, and Discussions: Live imaging revealed rapid coacervation and fusion of the phase-separating peptide droplets (Fig. 1A). Peptides encapsulating different fluorescent dyes provided a visualization of dynamic interactions, showing effective membrane fusion capabilities. Quantitative analysis from the custom Python script confirmed the high efficiency of peptide interactions, with significant fusion events observed within 10 minutes of mixing as evidenced by a significant increase in condensate size (Fig 1B). Interestingly, condensates of (GHGLY)4 and its enantiomer (GhGly)4 also undergo fusion (Fig. 1C). Fluorescence microscopy demonstrated successful internalization of the DQ-OVA its localization to the lysosomal compartments as evidenced by green fluorescence that co-localized with lysotracker red dye (Fig 1D). Treatment with BA and NH4Cl significantly reduced the fluorescence signal from DQ-OVA underscoring that the condensates are effective at delivering antigens to endolysosomal compartments (Fig. 1E). The encapsulated DNA and mRNA within the phase-separating peptides showed promising results in facilitating gene expression. Fluorescence microscopy confirmed the expression of the introduced genes in target cells, with marked efficiency and sustainability over time compared to traditional delivery methods (Fig. 1F).

Conclusions: Our study demonstrates the potent capabilities of phase-separating peptides in drug and gene delivery applications. The ability of these peptides to access the endocytic pathway or deliver therapeutics directly into the cytosol could revolutionize treatment modalities for various diseases.

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