Ferritin nanocage-enabled detection of pathological tau in living human retinal cells

Tauopathies, including Alzheimer’s disease and Frontotemporal Dementia, are debilitating neurodegenerative disorders marked by cognitive decline. Despite extensive research, achieving effective treatments and significant symptom management remains challenging. Accurate diagnosis is crucial for developing effective therapeutic strategies, with hyperphosphorylated protein units and tau oligomers serving as reliable biomarkers for these conditions. This study introduces a novel approach using nanotechnology to enhance the diagnostic process for tauopathies. We developed humanized ferritin nanocages, a novel nanoscale delivery system, designed to encapsulate and transport a tau-specific fluorophore, BT1, into human retinal cells for detecting neurofibrillary tangles in retinal tissue, a key marker of tauopathies. The delivery of BT1 into living cells was successfully achieved through these nanocages, demonstrating efficient encapsulation and delivery into retinal cells derived from human induced pluripotent stem cells. Our experiments confirmed the colocalization of BT1 with pathological forms of tau in living retinal cells, highlighting the method’s potential in identifying tauopathies. Using ferritin nanocages for BT1 delivery represents a significant contribution to nanobiotechnology, particularly in neurodegenerative disease diagnostics. This method offers a promising tool for the early detection of tau tangles in retinal tissue, with significant implications for improving the diagnosis and management of tauopathies. This study exemplifies the integration of nanotechnology with biomedical science, expanding the frontiers of nanomedicine and diagnostic techniques.


Table of Contents:
1. Calibration curve for BT1 spectroscopic analysis 2. Preparation of functionalized HumAfFt with NPM 3. ThT fluorescence of BSA and K18 4. Retinal differentiation protocol and retinal cell characterization 5. Uptake and toxicity tests 6. NPM-HumAfFt BT1 detection of total tau in retinal cells 7. NPM-HumanAfFt-BT1 complex detection of different tau forms in Okadaic acid treated retinal cultures

NPM-HumAfFt conjugation analysis via mass spectrometry
The successful insertion of the maleimide in the internal cavity of the HumAfFt was confirmed by a mass spectrometry analysis.A sample of HumAfFt control and one linked with NPM were diluted to 1 µg/µL.The protein samples were analyzed by an UltrafleXtreme MALDI ToF/ToF, Bruker (Bruker, Bremen DE), equipped with a smartbeam-II laser, in linear and positive mode.The samples were mixed with the sinapinic acid matrix and solubilized in an aqueous solution containing 70% acetonitrile and 0.1% trifluoroacetic acid (TFA) in a ratio of 1:5 (sample: matrix).
The bioconjugation yields confirmed about 80% of cysteines modified by measuring the molar ratio between the maleimide and protein concentrations through UV-vis measurements.

ThT fluorescence analysis of fibrillated BSA and K18
K18 75 µM was kept at 37°C and different time points were selected for fluorescence analysis (0, 2, 5, 24, 48, 72, 96, 168 hours).K18 protein did not show any variation in fluorescence intensity before 72 hours (3 days).A 3-fold increase, compared to the control, was visible after 96 hours (4 days), reaching a 5-fold value after 168 hours (7 days).BSA 75 µM was kept at 63°C and different time points were selected for fluorescence analysis (0, 0.5, 1, 2, 3, 4, 5, 20 and 24 hours).It was not possible to evaluate samples over 24 hours because after that period of time under those conditions, the liquid BSA solution turned to gelatinous, and adding the fluorophore became impossible.BSA protein showed a 3-fold fluorescence intensity rise after 0.5 hours (30 min), steadily increasing up to 4-fold intensity after 5 hours.After 20 hours, the fluorescence intensity reached an intensity value 5-fold compared to the control.After 24 hours (1 day), this value remained stable, showing no increase, demonstrating that BSA had entered the lag-phase of fibrillation.

Supplementary Figure S2 .
Bioconjugation of NPM to HumAfFt.Mass Spectrometry analysis of A) HumAfFt (1 mg/mL) with a molecular mass of HumAfFt without and with the first methionine, 20254 Da and 20386 Da, respectively B) NPM-labelled HumAfFt with a molecular mass of 20565 Da and with (20700 Da) the first methionine.The peak at 20268 Da is related to HumAfFt unreacted.