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Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy–lysosomal pathway.
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This content will become publicly available on September 11, 2025
pH-Responsive Rhodamine Nanotube Capable of Self-Reporting the Assembly State
Nanomaterials that respond to intracellular signals, such as pH, have potential for many biomedical applications, such as drug delivery, because the assembly/disassembly process can be tailored to respond to a stimulus characteristic of a specific subcellular location. In this work, two rhodamine-peptides that form stable nanotubes at physiological pH but dissociate into highly fluorescent monomers within the acidified interior of endosomal/lysosomal cellular compartments has been developed. The rhodamine dipeptide conjugates, NH2-KK(RhB)-NH2 (RhB-KK) and NH2-EK(RhB)-NH2 (RhB-KE) with rhodamine B chromophores appended at the ε-amino position of a lysine residue, were shown to assemble into well-defined nanotubes at pH values above ~4-5 and to dissociate into a fluorescent monomer state at lower pH values. The pH-dependence of the assembly process was investigated using CD and fluorescence spectroscopy along with TEM, AFM and confocal imaging. Although the ring opening/closing transition of the rhodamine chromophore took place at pH 4.1 for both peptides, the onset of assembly began at pH 4.6 for RhB-KE and at a comparatively more basic pH (5.8) for RhB-KK. Accordingly, the rhodamine-peptides interconverted between three, pH-dependent states: an open-ring, monomeric state (max 580 nm, 𝜆ex 550 nm) at pH values at or below ~4.6; a closed-ring, nanotube form that exhibits AIEE (max 460 nm, 𝜆ex = 330 nm) at higher pH values and a closed-ring, non-emissive monomeric state that emerged below the CMC. The pH-responsive features of the peptides were evaluated by live-cell imaging in three cancer cell lines using confocal laser scanning microscopy (CLSM). Visualizing the cells after incubation with either RhB-KE or RhB-KK produced CLSM images with a punctate appearance in the Texas Red channel that colocalized with the lysosomes. These experiments indicating that the nanotubes were rapidly trafficked into the acidic lysosomal compartments within the cells, which induced dissociation into a monomeric, open state. Uptake inhibition studies suggested that cellular uptake was mediated by either or both caveolae- and clathrin-mediated endocytosis, depending on the cell line studied.
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- Award ID(s):
- 2106924
- PAR ID:
- 10542937
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Applied Materials & Interfaces
- Volume:
- 16
- Issue:
- 36
- ISSN:
- 1944-8244
- Page Range / eLocation ID:
- 47089 to 47099
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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