Advancing the design of artificial nano-organelles for targeted cellular detoxification of reactive oxygen species

Data underlying the figures in the publication "Advancing the design of artificial nano-organelles for targeted cellular detoxification of reactive oxygen species" published in Nano Lett., 2024, https://doi.org/10.1021/acs.nanolett.3c03888.

Table of contents:

Figure 1: Design and characterization of AnOs. (A) Schematic representation of AnOs encapsulating lactoperoxidase (LPO), self-assembled into polymersomes by mixing PMOXA29-b-PDMS10 and PDMS29-b-PMOXA10-PEG4-N3 diblock copolymers. The membrane is permeabilized by melittin insertion, while targeting is supported by external functionalization with CPP. (B) TEM micrographs of AnOs assembled by mixing PDMS29-b-PMOXA10-OH and (i) 30%, (ii) 50%, and (iii) 70% of PDMS29-b-PMOXA10-PEG4-N3. (C) TEM micrographs of AnOs assembled from a 1:1 PDMS29-b-PMOXA10-OH:PDMS29-b-PMOXA10-PEG4-N3 mixture in the presence of (i) 25 µM, (ii) 50 µM, and (iii) 100 µM melittin. (D) TEM micrographs of optimized melAnOs reacted with (i) 3.5 µM, and (ii) 7 µM CPP.

Figure 2: Morphological and functional properties of AnO membranes. (A) (i) Overview cryo-TEM micrograph of ctrlAnOs-CPP (inactive); (ii) Histogram of the membrane thickness distribution of 30 ctrlAnOs-CPP measured from cryo-TEM micrographs (after extrusion with a 100 nm membrane) by ImageJ; (iii) Histogram of the membrane thickness distribution of 30 ctrlAnOs without CPP. (B) (i) Overview cryo-TEM micrograph of melAnOs-CPP (active); (ii) Histogram of the membrane thickness distribution of 30 melAnOs-CPP; (iii) Histogram of the membrane thickness distribution of 30 melAnOs without CPP. Scale bars = 100nm. (C) Normalized FCS autocorrelation curves (open circles, raw data; filled circles, fitted curves) of free Dylight 633 (black), DyLight 633-labeled LPO (633-LPO, red), and DyLight 633-labeled LPO-encapsulating AnOs (633-LPO-AnO, blue). (D) Normalized FCS autocorrelation curves (open circles, raw data; filled circles, fitted curves) of free Cy5.5 NHS ester (black), Cy5.5-labeled CPP (Cy5.5-CPP, red) and AnOs surface-functionalized with Cy5.5-CPP (LPO-AnO-Cy5.5 CPP, blue). (E) LPO functionality of LPO-AnOs non-permeabilized and permeabilized with non-functionalized CPP and functionalized CPP was determined by monitoring the conversion of AmplexRed to resorufin-like product, RLP at 560 nm for 20 min. (F) LPO functionality of LPO-AnOs prepared without melittin (no melittin) or with 25 and 50 µM melittin for permeabilization. (G) LPO activity of non-permeabilized (ctrlAnO-CPP) and permeabilized AnOs (melAnO-CPP) surface-functionalized at 3.5 (black) and 7.0 µM CPP (red).

Figure 3: AnOs-mediated ROS detoxification in K562 myelogenous leukemia cells. (A) CMH spin trap for ESR in vitro activity of AnOs. (B) ESR spectra of CMH adducts formed in stressed (300 µM H2O2; red) and unstressed K562 cells (black) in the absence of AnOs (control cells). (C) ESR spectra of K562 cells harboring melAnOs-CPP (blue) compared to stressed (red) and unstressed (black) control cells. (D) Double integrals of the ESR signals of CMH adducts in control cells and cells loaded with melAnOs-CPP, stressed with 40 µM or 300 µM H2O2. (E) Double integrals of the ESR signals of CMH adducts in stressed (300 µM H2O2; red) and unstressed (black) control cells and cells loaded with melAnOs lacking CPP. ESR spectra are based on acquisition (60 scans).