Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment

Abstract

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.

Fig. 1

Engineering stimuli-responsive OmpF. a Schematic representation of modified OmpF acting as a gate in catalytic nanocompartments. b Molecular representation of the OmpF-M cysteine mutant. c Chemical modification of OmpF-M cysteine mutant with the spin probe bis-(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulphide. d Chemical modification of OmpF-M cysteine mutant with the fluorophore SAMSA-CF

Fig. 2

Characterisation of stimuli-responsive OmpF Panel. a EPR spectra of bis-(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulphide-labelled OmpF-M experimental (black) and simulated (blue) and b bis-(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulphide-labelled OmpF-M in 1% OG incubated with 10 mM DTT experimental (black) and simulated (blue). c Normalised FCS autocorrelation curves for SAMSA-CF in PBS (black), SAMSA-CF in 1% OG (blue) and OmpF-S-S-CF in 1% OG (Red). Dotted line—experimental autocorrelation curves, full line—fit. d SAMSA-CF release kinetics from OmpF-M in 30 mM GSH, 1% OG, as measured by FCS and analysed with a two-component fit. Error bars show standard deviations from 60 measurements

Fig. 3

Characterisation of stimuli-responsive catalytic nanocompartments. a Cryo-TEM micrographs of: (left) polymersomes loaded with HRP and equipped with OmpF-SH, (middle) polymersomes loaded with HRP and equipped with OmpF-S-S-CF, and (right) polymersomes loaded with HRP without OmpF. Scale bar = 100 nm. b Normalised FCS autocorrelation curves of SAMSA-CF in PBS (black) and OmpF-S-S-CF in the membrane of polymersomes (blue). Dotted line = experimental autocorrelation curves, solid line = fitted curve. Curves normalised to 1 to facilitate comparison. c Left panel: EPR spectrum of bis-(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulphide-labelled OmpF reconstituted in PMOXA-PDMS-PMOXA polymersomes (black line). c Right panel: bis-(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulphide-labelled OmpF reconstituted in PMOXA-PDMS-PMOXA polymersomes and incubated with 10 mM DTT experimental (black line) and simulated (blue line). d Amplex Ultra Red conversion of HRP-loaded polymersomes: immediately after addition of 30 mM GSH (left), and 1 h after addition of 30 mM GSH (right). OmpF-S-S-CF equipped HRP-loaded polymersomes (green) and OmpF-SH equipped HRP-loaded polymersomes (blue). Error bars present standard deviations in activity between three separately prepared catalytic nanocompartments (n = 3)

Fig. 4

Cellular uptake and intracellular activation of AOs. a Confocal fluorescence micrographs of HeLa cells showing cellular uptake of fluorescently labelled HRP-loaded polymersomes and AOs loaded with fluorescently labelled HRP. Scale bar: 10 µm. b Cellular uptake and intracellular activation of fluorescently labelled HRP-loaded polymersomes and fluorescently labelled HRP-loaded AOs. Blue signal: Hoechst 33342 nucleus stain. Grey signal: CellMask Deep Red Plasma membrane stain. Green signal: Atto488 HRP. Red signal: resorufin-like product (RLP). Scale bar 20 µm

Fig. 5

Internalisation and activity of AOs in macrophages in vitro and in vivo. a Localisation of AOs in ZFE. Lateral view of the ZFE injected with HRP-Atto647-loaded AOs equipped with OmpF-S-S-CF. Arrowheads: Localisation of AOs. Blue signal: ZFE melanocytes. Green signal: GFP macrophages. Red signal: Atto647-loaded AOs. b Phagocytosis of AOs by human macrophage differentiated THP-1 cells in vitro. Qualitative (inset) and quantitative analysis of macrophage differentiated THP-1 cells incubated with AOs loaded with Atto488-labelled HRP without addition of inhibitors (left), and in the presence of phagocytosis inhibitor cytochalasin B (Cyto B) (right). Blue signal: Hoechst 33342 nucleus stain. Green signal: Atto488 HRP. Scale bar inset 20 µm. c Quantification of AOs in the presence of different pharmacological pathway inhibitors by flow cytometry: polyinosinic acid (Poly(I:C)), colchicines, cytochalasin B (Cyto B) and sodium azide (NaN₃). d In vivo ZFE biodistribution and activity of AOs—lateral view of the ZFE cross-section. Blue signal: ZFE melanocytes. Green signal: HRP-Atto488. Red signal: Resazurin-like product (RZLP). Arrows show regions of enzymatic activity of AOs