Background and Objectives: Autophagy is an important intracellular recycling mechanism that removes toxins and provides nutrients during starvation. Dysregulation of autophagy is involved in all major categories of human diseases including infections, cancer, neurodegeneration, and cardiovascular disorders.(1) Despite its importance, the study of autophagy has been severely impeded by the lack of tools to quantify autophagic flux. Existing methods are not suitable for imaging live cells or organs, and/or require genetic transfection.(2)  

The autophagy cascade begins with the encapsulation of cytoplasmic cargo into autophagosomes which then fuse with lysosomes. The resulting autophagolysome degrades the contained cargo through the action of lysosomal cathepsins.(3) We have recently developed an Autophagy Detecting Nanoparticle (ADN) that can quantify this autophagic flux.(4) ADN  is composed of a nanoparticle decorated with arginine-rich cathepsin-cleavable peptides bound to the near-infrared fluorochrome Cy5.5 (Figure A). In the early autophagosome, ADN fluorescence is silent due to stacking of the Cy5.5 fluorophores.  Once the autophagosome fuses with the lysosome, the polyarginine peptide is cleaved by cathepsins thereby unstacking Cy5.5 and activating ADN fluorescence.(4) ADN is limited in that encapsulation of the probe within early autophagosomes is fluorescently silent, therefor early autophagosomal localization of ADN can only be achieved with immunohistochemical staining which is not feasible in live cells. The objective of this work is to improve upon the design  of ADN by modifying the nanoparticle with a second, non-cleavable fluorophore that would allow for imaging/tracking of probe distribution and encapsulation within early autophagosomes as well as probe activation during autophagy.

Methods: ADN was modified using standard click chemistry.with the non-cleavable second fluorophores AZDye546, Cy3.5 or rhodamine X (ROX) to produce ADN2, ADN3 and ADN4 respectively. ADNs were characterized by UV-Vis, fluorescence spectroscopy and Dynamic Light Scattering (DLS). Uptake and activation of ADNs in H9C2 murine cardiac cells was assessed by flow cytometry. Autophagy was induced in H9C2 cells by starvation (Stv) in nutrient-depleted cell-culture media. Mouse model of increased autophagy was achieved by fasting adult C57BL/6J mice for 24 hours (Stv).  

Results: We developed ADN2, ADN3 and ADN4  probes decorated with three different secondary fluorophores (Figure A).  Our lead candidate, ADN2, had the fastest rate for enzymatic activation in vitro (Figure B). ADN2 also resulted in the greatest extent of Cy5.5 fluorescence activation both in vitro (Figure C) and intracellularly in H9C2 cells undergoing autophagy (Figure D). We found that the anionic secondary fluorophore (AZDye546) on ADN2 interacts with the positively charged polyarginine motifs of the Cy5.5-polyArg activatable fluorophore (Figure E), but not the Cy5.5 dye alone (Figure F). This attraction between the anionic AZDye546 and the cationic polyarginine of Cy5.5-polyArg results in ADN2 being smaller in size compared to the other ADNs (Figure G, H). In contrast, the other secondary fluorophores Cy3.5 and ROX are respectively cationic and neutral, which leads to repulsion or no interaction with the Cy5.5-polyArg. Consequently, ADN3 and ADN4 are larger in size compared to ADN (Figure G,H).  We further found that ADN2 co-localizes in autophagosomes with the autophagy protein LC3 (Figure I). This co-localization confirms that ADN2 ends up in the early autophagosome. Finally, we successfully use ADN2 to image (Figure J) and quantify (Figure K) probe uptake (546 signal) and autophagy activation (Cy5.5 signal) in mouse hearts ex vivo with fluorescence reflectance imaging.

Conclusions: Modification of the Autophagy-Detecting Nanoprobe (ADN) with a secondary, non-cleavable fluorophore significantly affects the behavior of the nanoparticle as a function of the type of fluorophore used. The negatively-charged nature of the AZDye546 secondary fluorophore in ADN2 results in electrostatic attraction that improved the overall performance of the probe and allows for multichannel tracking of both early and late autophagy activation. 

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Image/Figure Caption:

A: Design of the second-generation Autophagy Detecting Nanoprobes (ADNs). ADN’s are tagged with a cleavable Cy5.5-containing polyarginine  peptide (Cy5.5-polyArg) as well as second  non-cleavable fluorophore. Cathepsins can selectively cleave the Cy5.5-polyArg  peptide leading to unstacking of Cy5.5 fluorophores thereby activating Cy5.5 fluorescence. The second fluorophore cannot undergo enzyme-mediated cleavage from the nanoparticle. B: The rate constants for enzyme-mediated cleavage of the Cy5.5-polyArg groups on ADNs as calculated from the fluorescence increase of the Cy5.5 signal as a function of time. C: The in vitro change in Cy5.5 fluorescence before and after cleavage of Cy5.5-polyArg shows the highest change for ADN2. D: The normalized percent of Cy5.5-positive H9C2 cells under autophagy-inducing conditions as assessed by flow cytometry shows that ADN2 outperforms all other ADNs in the fold Cy5.5 increase compared to baseline. E: UV-vis spectra of AZDye546, Cy5.5-polyArg and Cy5.5-DBCO either alone or mixed in solution. Th negatively charged AZDye546 fluorophore interacts with the positively charged Cy5.5-polyArg peptides causing broadening of the UV-Vis spectral signature of Cy-5.5-polyArg. This same interaction is not observed when AZDye546 is mixed with Cy5.5-DBCO alone (F). This interaction results in ADN2 being smaller in size than any other ADNs (G) as measured by dynamic light scattering. H: Schematic for the charge/electrostatic interaction effect of nanoparticle size. ADN2 contains the only negatively charged secondary fluorophore. The anionic nature of the secondary fluorophore on ADN2 attracts the Cy5.5-polyArg groups, resulting in an overall decrease in size not observed in the other particles. I: ADN2 localizes in the autophagosome, allowing monitoring of early autophagosome through red fluorescence. Fluorescence confocal microscopy images of H9C2 cells transfected with LC3-GFP plasmid after treatment with ADN2 show that punctates positive for ADN2 red fluorescence are also positive for LC3-GFP green fluorescence. J: Fluorescence reflectance imaging of axial slices of hearts of mice  that were either fed normally (Fed) or fasted (Stv) then injected with ADN2.  The 546 fluorescence images are indicative of ADN2 uptake/distribution while the Cy5.5 fluorescence signal is due to autophagy activation. Quantification of the red fluorescence intensity and Cy5.5 fluorescence intensity (K) shows that while uptake of ADN2 is similar in both Fed and Stv conditions, Cy5.5 activation is significantly higher under Stv condition where autophagy is active

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