A Photonastic Prototissue Capable of Photo-Mechano-Chemical Transduction

Abstract

Despite recent significant advances in the controlled assembly of protocell units into complex 3D architectures, the development of prototissues capable of mimicking the sophisticated energy transduction processes fundamental to living tissues remains a critical unmet challenge in bottom-up synthetic biology. Here a synthetic approach is described to start addressing this challenge and report the bottom-up chemical construction of a photonastic prototissue endowed with photo-mechano-chemical transduction capabilities. For this, novel protocells enclosing photothermal transducing proto-organelles based on gold nanoparticles and a thermoresponsive polymeric proto-cortex are developed. These advanced protocell units are assembled into prototissues capable of light-induced reversible contractions and complex motions, which can be exploited to reversibly switch off a coordinated internalized enzyme metabolism by blocking the access of small substrate molecules. Overall, the work provides a synthetic pathway to constructing prototissues with sophisticated energy transduction mechanisms, enabling the rational design of emergent behaviors in synthetic materials and addressing critical challenges in bottom-up synthetic biology and bioinspired materials engineering.

Keywords: energy transduction; far‐from‐equilibrium material; photonastic behaviour; protocell; prototissue.

Figure 1

Structural characterization of photonastic prototissues. a) Structures of the PNIPAM‐based copolymers (1) and (2) used to build the proteinosome proto‐cortex. b) Photograph of a prototissue composed of protocells endowed with a PNIPAM proto‐cortex and encapsulating PEG‐AuNPs showing the typical pink coloration originating from the gold colloid. c) Scheme showing the structure of a prototissue, which comprises interconnected protocells composed of a membrane of AMCA‐labeled azide‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate and of a RITC‐tagged proto‐cortex, or composed of a membrane of BDP650‐labeled BCN‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate and of an FITC‐tagged proto‐cortex. Both types of protocells enclose PEG‐AuNPs. The left scheme highlights the membrane composition, whereas the right scheme highlights the proto‐cortex architecture and composition. d,e) Confocal fluorescence microscopy images showing the inner structure of a prototissue structured like in (c), highlighting the interconnected protocell membranes (azide‐functionalized AMCA‐labeled BSA/PNIPAM‐co‐MAA nanoconjugate – blue fluorescence, and BCN‐functionalized BDP650‐labeled BSA/PNIPAM‐co‐MAA nanoconjugate – orange fluorescence) d), or the two differently tagged protocell proto‐cortexes (FITC‐labeled proto‐cortex—green fluorescence, and RITC‐labeled proto‐cortex—purple fluorescence) e). f) Scheme showing the structure of a prototissue which comprises interconnected protocells composed of a membrane of AMCA‐ or BDP650‐labeled azide‐ or BCN‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate and of a non‐fluorescently tagged proto‐cortex. Only the BDP650‐labeled protocells encapsulate PEG‐AuNPs. g) Brightfield microscopy image of the prototissue in (f). Dark areas highlight the presence of PEG‐AuNPs. h) Confocal fluorescence microscopy image of the prototissue in (f), highlighting the interconnected structure of protocell membranes (azide‐functionalized AMCA‐labeled BSA/PNIPAM‐co‐MAA nanoconjugate – blue fluorescence, and BCN‐functionalized BDP650‐labeled BSA/PNIPAM‐co‐MAA nanoconjugate – orange fluorescence). i) Merged microscopy image of channels shown in (g) and (h). The correspondence of dark areas in (g) with the orange areas in (h) indicates that the PEG‐AuNPs were specifically localized inside the BDP650‐labeled protocells.

Figure 2

Light‐induced reversible contractions in prototissues. a) Photograph of a photo‐contractile prototissue composed of protocells endowed with a PNIPAM proto‐cortex and enclosing PEG‐AuNPs. The PCM was kept in the absence of light irradiation, at 25 °C, floating on water, and held on a thermocouple (bottom right metallic object). b) Photograph of the same PCM in (a) after 90 s of green light irradiation (λ_max ≈520 nm, Irr = 1.35 W cm⁻²). c) Plot showing the material time‐dependent light‐induced contraction and concomitant localized temperature variation during a single light‐dark cycle (green area = light on, Irr = 1.35 W cm⁻², Video S1, Supporting Information). d) Plot showing the material time‐dependent light‐induced contraction and concomitant localized temperature variation during 30 light/dark cycles (green areas: light on, Irr = 1.35 W cm⁻²). e) Plot showing the temperature‐dependent area variation of photo‐contractile prototissues (no light irradiation). f) Time‐dependent reversible contraction and concomitant temperature change in photonastic prototissues upon multiple cycles of irradiation with different light intensities (light green areas: Irr = 0.3 W cm⁻², darker green areas: Irr = 1.35 W cm⁻²). Grey band in (c), (d), (e), and (f) = standard error calculated upon repeating the measurements on at least three independently prepared prototissue samples. g) Plot showing the photoinduced contraction of PCMs as a function of the amount of PEG‐AuNPs encapsulated in the protocell units that compose them (irradiation time: 120 s, λ_max ≈520 nm, Irr = 1.35 W cm⁻²). Error bars = standard error calculated upon repeating the measurements on at least three independently prepared samples. h) Comparison of Young's modulus values between prototissues composed of protocells endowed with PNIPAM proto‐cortex (red) and prototissues composed of empty proteinosomes (grey) at 25 °C (light colors) and 40 °C (dark colors). i) Comparison of storage modulus (E’) values between prototissues composed of protocells endowed with proto‐cortex (red) and prototissues composed of empty protocells (grey) at 25 °C (light colors) and 40 °C (dark colors) (data with relative errors: Figure S42, Supporting Information). j) Comparison of loss modulus values (E’’) between prototissues composed of protocells endowed with proto‐cortex (red) and prototissues composed of empty protocells (grey) at 25 °C (light colored) and 40 °C (dark colored) (data with relative errors: Figure S42, Supporting Information).

Figure 3

Structure and movement characterization of a photonastic prototissue. a) Molecular structures of the PDMAM‐based copolymers (3) and (4) used to build the non‐thermoresponsive protocell proto‐cytoskeleton. b) Scheme describing the working principle of the photonastic prototissue bilayer. The top layer was composed of photo‐contractile protocells endowed with a PNIPAM proto‐cortex and encapsulating PEG‐AuNPs light‐transducing proto‐organelles. The bottom layer was made of non‐contractile protocells endowed with a PDMAM proto‐cytoskeleton. Light irradiation induces photothermal heating, contraction and stiffening of the top layer, while causing a decrease in rigidity of the bottom layer, overall leading to a reversible bending of the prototissue. c,d) XZ orthogonal plane of a confocal fluorescence Z‐stack obtained by imaging the bilayer prototissue described in (b) at the interface between the two layers (composition: Section S4.2, Supporting Information). c) Fluorescence channels corresponding to the bio‐orthogonally reactive protein‐polymer nanoconjugates: AMCA‐labeled azide‐ or BCN‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate (blue fluorescence), and BDP650‐labeled azide‐ or BCN‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate (orange fluorescence). d) Fluorescence channels corresponding to the FITC‐labeled PNIPAM proto‐cortex (green fluorescence) and RITC‐labeled PDMAM proto‐cytoskeleton (purple fluorescence). e) Plot of the photonastic prototissue bilayer curvature over seven light‐dark cycles (λ_max = 520 nm, Irr = 1.35 W cm⁻², Video S2, Supporting Information). f) Image sequence showing the first light/dark cycle in (e) and the reversible bending of the photonastic prototissue. g) Magnitude displacement fields U (mm) on the photonastic prototissue in (f) at time 0, 5, 10, and 50 s obtained from the FEM numerical simulation of the light‐dark cycle. h) Comparison of FEM numerical results (red line) and experimental data (blue points, colors from darker to lighter correspond to 1st to 7th light‐dark cycles) of the time‐dependent curvature changes of the photonastic prototissue. The curvature from the numerical model was calculated along six radial directions and reported as mean ± standard deviation (light red band). i) Time‐dependent changes in the stored strain energy (blue line) and dissipated viscous energy (red line) evaluated by the FEM model for a single light/dark cycle (0–50 s irradiation, 51–100 s light off).

Figure 4

Prototissues capable of photo‐mechano‐chemical transduction. a) Scheme showing the structure of a prototissue which comprises interconnected protocells composed of a membrane of non‐tagged azide‐ or BCN‐functionalized BSA/PNIPAM‐co‐MAA nanoconjugate, a non‐tagged PNIPAM proto‐cortex, and PEG‐AuNPs photothermal transducing proto‐organelles. The azide‐functionalized protocells encapsulate RITC‐tagged AGx, whereas the BCN‐functionalized protocells encapsulate FITC‐tagged GOx. b) Confocal fluorescence microscopy image of the prototissue in (a), highlighting the compartmentalization of the two enzymes (RITC‐labeled AGx, purple fluorescence; FITC‐labeled GOx, green fluorescence). c) Scheme highlighting the working principles of the photo‐mechano‐chemical transduction mechanism: the light‐induced contraction of the prototissue causes the reversible impermeabilization of the membrane of the protocells that compose the prototissue, with a consequent switch‐off of the AGx/GOx enzyme cascade reaction hosted within the prototissue. d) Time‐dependent pH changes of a PBS buffer solution (1 mm, initial pH 6.2) containing 12.6 µL of 80 mg mL⁻¹ solution of dextrin in PBS (2 mm, pH 6.2) and a prototissue assembled from a 1:1 binary population of photo‐contractile proteinosomes encapsulating AGx or GOx. The black plot represents the experiment performed in the absence of light irradiation. The yellow, light blue, and green plots represent kinetic experiments performed by pre‐irradiating the prototissue for 3 min in order to contract them completely before the addition of Dex, and then by irradiating the PCM for 10, 20, and 40 min, respectively (λ_max = 520 nm, Irr = 1.35 W cm⁻²). The plots show that as soon as the light was switched off, the pH started to decrease promptly, indicating the start of the enzyme cascade reaction. e) Scheme explaining the characterization of the temperature‐dependent permeability of the prototissues. In these experiments, a prototissue was used to filter aqueous solutions of FITC‐labeled dextran of different molecular weights. If the molecular weight of the FITC‐labeled dextran was smaller than the MWCO of the PCM the polysaccharide diffused into the bulk water underneath the prototissue, otherwise it remained segregated above it. f) Reciprocal position of the maxima of fluorescence Z‐intensity profiles acquired for AMCA‐labeled prototissues and FITC‐labeled dextran as a function of the molecular weight of the dextran. Experiment performed at 25 °C (left) and 40 °C (right). The blue line represents the position of the prototissue (maximum of Z‐profile relative to blue fluorescence channel—Figure S53, Supporting Information), and the light blue bands represent the full width at half maximum (FWHM) of the blue distribution curve, providing an estimation of the thickness of the prototissue. The white points represent the position of the FITC‐labeled dextran with respect to the PCM (maximum of Z‐profile relative to green fluorescence signal—Figure S53, Supporting Information), the light green band represents the FWHM of the green distribution curve. The plots show that when the prototissue was in the relaxed state, its MWCO was ≈25 kDa, whereas when it was in the contracted state (40 °C), the membrane became completely impermeable to hydrophilic solutes.