Target-driven supramolecular self-assembly for selective amyloid-β photooxygenation against Alzheimer's disease

Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer's disease (AD). However, current photosensitizers cannot simultaneously achieve enhanced blood–brain barrier (BBB) permeability and selective photooxygenation of Aβ, leading to poor therapeutic efficacy, severe off-target toxicity, and substandard bioavailability. Herein, an Aβ target-driven supramolecular self-assembly (PKNPs) with enhanced BBB penetrability and switchable photoactivity is designed and demonstrated to be effective in preventing Aβ aggregation in vivo. PKNPs are prepared by the self-assembly of the Aβ-targeting peptide KLVFF and an FDA-approved porphyrin derivative (5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin). Due to the photothermal effect of PKNPs, the BBB permeability of PKNPs under irradiation is 8.5-fold higher than that of porphyrin alone. Moreover, upon selective interaction with Aβ, PKNPs undergo morphological change from the spherical to the amorphous form, resulting in a smart transformation from photothermal activity to photodynamic activity. Consequently, the disassembled PKNPs can selectively oxygenate Aβ without affecting off-target proteins (insulin, bovine serum albumin, and human serum albumin). The well-designed PKNPs exhibit not only improved BBB permeability but also highly selective Aβ photooxygenation. Furthermore, in vivo experiments demonstrate that PKNPs can alleviate Aβ-induced neurotoxicity and prolong the life span of the commonly used AD transgenic Caenorhabditis elegans CL2006. Our work may open a new path for using supramolecular self-assemblies as switchable phototheranostics for the selective and effective prevention of Aβ aggregation and related neurotoxicity in AD.

13.23mg KLVFF (0.02mmol) was dissolved in 0.5 mL distilled water and 0.28 mL triethylamine (TEA) and then added to the mixture. The reaction mixture was stirred for 12 h at room temperature. Then 4 mL distilled water was added to the reaction mixture. After acidification of the solution to pH 3 by HCl, the product was extracted by ethyl acetate (20 mL × 3) through a separating funnel. The combined organic phase was washed by distilled water (20 mL × 3) and evaporated. The crude product was collected and further purified by silica gel column chromatography to determine the product (yield 31%). Finally, the obtained PP-KLVFF was dissolved in DMSO for further use (0.5 mg mL -1 ).
Preparation of PKNPs 100 µL DMSO solution of PP-KLVFF (0.5 mg mL -1 ) was mixed with 900 µL pure water, and the obtained dispersive PKNPs were aged for 24 h and dialyzed against water. For dialysis, 1 mL PKNPs were sealed in a dialysis bag (molecular weight cut-off: 3.5 kDa) and immersed in 1 L pure water for 48 h, during which the water was replaced for 3 times. Finally, PKNPs were collected by vacuum freeze-drying .

Protein sample preparation
Aβ42 was prepared following a previous protocol. At first, Aβ was dissolved in HFIP and stored at -20 °C as the stock solution. Before use, the solvent HFIP was evaporated and Aβ was redissolved in 20 mM Tris buffer (pH 7.4). For the Aβ aggregation, the solution was incubated at 37 °C for 72 hours.

In vitro ROS generation tests
For a typical test, the PKNPs (0.2 mg mL -1 ) were mixed with the stock solution of DCFH (10 μM) in PBS buffer (pH=7.4) containing Aβ42 (30 μM), which simulated pathological Aβ accumulation in AD brains. Then, the mixture was irradiated by 450 nm laser at a power density of 0.57 W cm -2 . Immediately after the irradiation, the solutions were centrifuged, and the fluorescence of the supernatants was measured for the estimation of the produced ROS.
For the DNPH assay, the Aβ/BSA/HSA solutions (40 mM, 480 μl) in the absence or presence of PKNPs (0.2 mg mL -1 ) were precipitated with 20% TCA (trichloroacetic acid) solution in ice bath and then collected by centrifuge. Subsequently, 500 μL of 2M HCl containing 10 mM DNPH (2M HCl only for reagent blanks) was added dropwise to the protein pellet, followed by incubation under room temperature for 1h. The samples were precipitated with 20% TCA solution and then washed three times with 1 ml ethanol-ethyl acetate (1:1, v/v) solution. The sample was resuspended in a 6M guanidine hydrochloride solution (in 20 mM potassium phosphate, pH 2.3 adjusted with TCA) at 37 ˚C for 15 min. The absorbance spectrums of the samples were measured using UV-Vis absorbance spectrometer.

In vitro Photothermal activity
Aqueous solution (1.0 mL) of PKNPs (0.2 mg mL -1 ) was put in a quartz cuvette and irradiated with a 638 nm laser for 8 min. Pure water was used as a control group. A thermocouple probe with a digital thermometer was used to measure the temperature every 60 s.

Cell culture
PC12 cells (rat pheochromocytoma) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum, 10% horse serum in the CO2 (5%) environment at 37 °C. Cells were placed at a density of 1.0×10 5 cells per well on 96-well plates in fresh medium.

Cell viability assays
The cell viability was measured by MTT assays. The PC12 cells were seeded at a density of 10,000 cells cells/100 µL in a 96-well plate. To validate the inhibition of Aβmediated cellular toxicity by PKNPs. Aβ monomers (30 μM) were pretreated with or without PKNPs (0.2 mg mL -1 ), then the samples were added in each well and the cells were further cultured for 12 h. After that, cells were irradiated with laser (450nm, 0.57 W cm -2 ) for 3 min with 2 min interval and for 15 times. Cells were incubated for another 24 h after the laser excitation. Finally, 20 μL MTT (5 mg/mL) was added into the plate.
The culture medium containing MTT was replaced with DMSO (100 μL) after incubation for 4 h. The absorbance (490 nm) was measured using a microplate reader

in vitro blood-brain barrier (BBB) transportation study
The in vitro BBB model was fabricated according the literature reported method.

C. elegans experiments
C. elegans strain was fed with E. coli (OP50) and cultured at 20 °C on the nematode growth medium (NGM) after the egg synchronization. Parallel worms were cultured on solid NGM containing PKNPs (0.2 mg mL −1 ) and then irradiated with laser. For ThSstaining experiments, worms were fixed with 4% paraformaldehyde and then permeabilized with β-mercaptoethanol before immersing in ThS solution (0.125%) for 4 min. For life span experiments, when strains only moved their head or did not move at all even gently touched by a platinum loop, we classified them as paralyzed. Three independent trials were performed.

Statistical analysis
All data were expressed in this article as mean resultstandard deviation (SD). All figures shown in this article were obtained from three independent experiments with similar results.             Figure S16. PKNPs improved the motility of CL2006 strain. Quantification of the worm movement in M9 buffer (turns per minute). N2, CL2006 and PKNPs treated CL2006 strains were 3, 6 or 9 days old.