Co-Encapsulating the Fusogenic Peptide INF7 and Molecular Imaging Probes in Liposomes Increases Intracellular Signal and Probe Retention

Abstract

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging.

Dynamics of dual-fluorescent polymersomes with durable integrity in living cancer cells and zebrafish embryos

“…The images showed colocalization of TBP and SRB fluorescence in localized spots throughout the cytoplasm, reminiscent of endo-lysosomal compartments of A549 cells. [35,[38][39][40] Staining of lysosomes with Lysotracker Red revealed that the polymersomes indeed accumulated in lysosomes ( Figure S1), confirming uptake via the usual endocytosis pathways. [41][42][43] The colocalization of TBP and SRB fluorescence clearly indicates that the encapsulated SRB had not leaked out and thus the nanovesicles remained intact inside the cells once they have been endocytosed.…”

“…In this case, SRB was selected as hydrophilic fluorescent probe because it is known to be rapidly cleared from the cell when it is released from liposomes. [35] Also, the absorption spectra of TBP and SRB do not overlap significantly, which allows for optical distinction between the location of the two chromophores. Additionally, both of these probes are cheap and easy to integrate into the polymersome.…”

Tailoring Iron Oxide Nanoparticles for Efficient Cellular Internalization and Endosomal Escape

“…For instance, several studies have incorporated pH-responsive polymers on liposomal surfaces or inside liposomes, which facilitate cargo release by destabilizing liposomal and endosomal membranes [ 290 , 291 ]. Similar fusion events are observed when glutamate-rich fusogenic peptides (e.g., GALA, pHLIP, INF7) are incorporated, mainly because their amphipathic structures switch from random coil to α-helical upon pH-triggered glutamate protonation [ 292 , 293 , 294 ]. These membrane-destabilizing peptides usually derive from viral proteins responsible for the endosomal escape of these pathogens [ 295 ].…”

Stimuli‐responsive liposomes for drug delivery

“…Several different types of fusogenic peptides have been investigated for their ability to induce endosomal escape and facilitate the delivery of liposomal cargo into the cytosol. Burks et al demonstrated the functionalization of pH‐sensitive influenza‐derived peptide (INF7) using a liposomal carrier for delivery of molecular imaging probes to increase intracellular signal intensity and probe retention . The INF7 peptide prepared was a glutamine‐enriched analogue of HA2 that has pH‐dependent membrane‐disruptive activity.…”