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Co-encapsulating the fusogenic peptide INF7 and molecular imaging probes in liposomes increases intracellular signal and probe retention.

Burks SR, Legenzov EA, Martin EW, Li C, Lu W, Kao JP - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland, Baltimore, Maryland, 21201, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Activating endocytosed INF7 with acetic acid causes endosomal escape of SR into the cytosol.A. Liposomes containing SR with or without INF7 were incubated with CV1 cells for 1 hr, washed with Ca2+/Mg2+-free HBSS containing 1 mM EDTA, and imaged by fluorescence microscopy (Pre Acid); endosomal SR appears as fluorescent puncta. After the addition of 1% acetic acid to the extracellular solution, fluorescence in cells incubated with INF7-containing liposomes (+INF7) becomes cytosolic and more homogeneous. In contrast, in cells incubated with liposomes lacking INF7 (-INF7), the appearance of SR fluorescence is unchanged by acidification. B. Time course showing changes in SD of pixel intensity relative to mean intensity (σ/F, normalized to mean baseline value before acidification at time 0). Time of addition of acetic acid is indicated by the black arrowhead. Activated INF7 rapidly releases SR into the cytosol, and the more homogeneous distribution of SR leads to lower SD values than in the case of incubation without INF7, where SR remains confined to endosomal compartments. Also see S1 and S2 Videos in Supporting Information.
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pone.0120982.g004: Activating endocytosed INF7 with acetic acid causes endosomal escape of SR into the cytosol.A. Liposomes containing SR with or without INF7 were incubated with CV1 cells for 1 hr, washed with Ca2+/Mg2+-free HBSS containing 1 mM EDTA, and imaged by fluorescence microscopy (Pre Acid); endosomal SR appears as fluorescent puncta. After the addition of 1% acetic acid to the extracellular solution, fluorescence in cells incubated with INF7-containing liposomes (+INF7) becomes cytosolic and more homogeneous. In contrast, in cells incubated with liposomes lacking INF7 (-INF7), the appearance of SR fluorescence is unchanged by acidification. B. Time course showing changes in SD of pixel intensity relative to mean intensity (σ/F, normalized to mean baseline value before acidification at time 0). Time of addition of acetic acid is indicated by the black arrowhead. Activated INF7 rapidly releases SR into the cytosol, and the more homogeneous distribution of SR leads to lower SD values than in the case of incubation without INF7, where SR remains confined to endosomal compartments. Also see S1 and S2 Videos in Supporting Information.

Mentions: To investigate endosomal release of fluorophores by INF7, CV1 cells were incubated with liposomes bearing a negative surface charge and encapsulating SR at 90 mM, at which concentration SR fluorescence is quenched. CV1 cells avidly endocytose negatively-charged liposomes [34]; subsequent degradation of liposomes releases SR into the endosomal compartment and the consequent dilution de-quenches SR to restore its fluorescence. Since SR bears three ionic charges at physiological pH it is expected to remain trapped inside endosomes in the absence of INF7. Liposomes, with or without INF7, were incubated with cells for 1 hr. Thereafter, the cells were washed in divalent-cation-free HBSS to remove un-endocytosed liposomes, returned to normal HBSS, and then immediately imaged for SR fluorescence (Fig. 4A). After 1 hour, sufficient liposomal degradation had occurred in the endocytic pathway and SR fluorescence was visualized as intense puncta in both groups. The cells were then rapidly acidified by addition of acetic acid (1% v/v) to the extracellular solution, which attained pH ~3.5; SR in cells incubated with INF7-containing liposomes rapidly dispersed through the intracellular volume and was visualized as more homogenous fluorescence throughout the volume of the cell. Cells that were incubated with liposomes lacking INF7 showed no change in appearance after acidification—demonstrating that without INF7 to facilitate its release, SR remained inside endosomes (S1 Video and S2 Video). The visually apparent changes in intracellular localization of SR after acidification can also be quantitatively analyzed by measuring changes in the standard deviation of pixel intensity ratioed to the mean pixel intensity (σF/F; Fig. 4B). Before acidification, endosomal localization of SR is seen as highly spatially heterogeneous distribution of fluorescence in the cells, and σF/F is high for both groups of cells. In the cells incubated with INF7-containing liposomes, after acidification and consequent INF7 activation, SR fluorescence becomes more homogenously distributed in the cells, resulting in lower σF/F values. In contrast, cells incubated with liposomes containing no INF7, SR fluorescence remains spatially heterogeneous, and the σF/F value remains high.


Co-encapsulating the fusogenic peptide INF7 and molecular imaging probes in liposomes increases intracellular signal and probe retention.

Burks SR, Legenzov EA, Martin EW, Li C, Lu W, Kao JP - PLoS ONE (2015)

Activating endocytosed INF7 with acetic acid causes endosomal escape of SR into the cytosol.A. Liposomes containing SR with or without INF7 were incubated with CV1 cells for 1 hr, washed with Ca2+/Mg2+-free HBSS containing 1 mM EDTA, and imaged by fluorescence microscopy (Pre Acid); endosomal SR appears as fluorescent puncta. After the addition of 1% acetic acid to the extracellular solution, fluorescence in cells incubated with INF7-containing liposomes (+INF7) becomes cytosolic and more homogeneous. In contrast, in cells incubated with liposomes lacking INF7 (-INF7), the appearance of SR fluorescence is unchanged by acidification. B. Time course showing changes in SD of pixel intensity relative to mean intensity (σ/F, normalized to mean baseline value before acidification at time 0). Time of addition of acetic acid is indicated by the black arrowhead. Activated INF7 rapidly releases SR into the cytosol, and the more homogeneous distribution of SR leads to lower SD values than in the case of incubation without INF7, where SR remains confined to endosomal compartments. Also see S1 and S2 Videos in Supporting Information.
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Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4376389&req=5

pone.0120982.g004: Activating endocytosed INF7 with acetic acid causes endosomal escape of SR into the cytosol.A. Liposomes containing SR with or without INF7 were incubated with CV1 cells for 1 hr, washed with Ca2+/Mg2+-free HBSS containing 1 mM EDTA, and imaged by fluorescence microscopy (Pre Acid); endosomal SR appears as fluorescent puncta. After the addition of 1% acetic acid to the extracellular solution, fluorescence in cells incubated with INF7-containing liposomes (+INF7) becomes cytosolic and more homogeneous. In contrast, in cells incubated with liposomes lacking INF7 (-INF7), the appearance of SR fluorescence is unchanged by acidification. B. Time course showing changes in SD of pixel intensity relative to mean intensity (σ/F, normalized to mean baseline value before acidification at time 0). Time of addition of acetic acid is indicated by the black arrowhead. Activated INF7 rapidly releases SR into the cytosol, and the more homogeneous distribution of SR leads to lower SD values than in the case of incubation without INF7, where SR remains confined to endosomal compartments. Also see S1 and S2 Videos in Supporting Information.
Mentions: To investigate endosomal release of fluorophores by INF7, CV1 cells were incubated with liposomes bearing a negative surface charge and encapsulating SR at 90 mM, at which concentration SR fluorescence is quenched. CV1 cells avidly endocytose negatively-charged liposomes [34]; subsequent degradation of liposomes releases SR into the endosomal compartment and the consequent dilution de-quenches SR to restore its fluorescence. Since SR bears three ionic charges at physiological pH it is expected to remain trapped inside endosomes in the absence of INF7. Liposomes, with or without INF7, were incubated with cells for 1 hr. Thereafter, the cells were washed in divalent-cation-free HBSS to remove un-endocytosed liposomes, returned to normal HBSS, and then immediately imaged for SR fluorescence (Fig. 4A). After 1 hour, sufficient liposomal degradation had occurred in the endocytic pathway and SR fluorescence was visualized as intense puncta in both groups. The cells were then rapidly acidified by addition of acetic acid (1% v/v) to the extracellular solution, which attained pH ~3.5; SR in cells incubated with INF7-containing liposomes rapidly dispersed through the intracellular volume and was visualized as more homogenous fluorescence throughout the volume of the cell. Cells that were incubated with liposomes lacking INF7 showed no change in appearance after acidification—demonstrating that without INF7 to facilitate its release, SR remained inside endosomes (S1 Video and S2 Video). The visually apparent changes in intracellular localization of SR after acidification can also be quantitatively analyzed by measuring changes in the standard deviation of pixel intensity ratioed to the mean pixel intensity (σF/F; Fig. 4B). Before acidification, endosomal localization of SR is seen as highly spatially heterogeneous distribution of fluorescence in the cells, and σF/F is high for both groups of cells. In the cells incubated with INF7-containing liposomes, after acidification and consequent INF7 activation, SR fluorescence becomes more homogenously distributed in the cells, resulting in lower σF/F values. In contrast, cells incubated with liposomes containing no INF7, SR fluorescence remains spatially heterogeneous, and the σF/F value remains high.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland, Baltimore, Maryland, 21201, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus