Intracompartmental 3D Printing of Enzymatically Active Organelle Mimics

Abstract

Introducing subcellular structures in artificial cells is a key step in mimicking the structure and role of organelles, which are instrumental in compartmentalizing cellular reaction networks. Despite the variety of strategies to include subcellular features within artificial cell models, achieving spatial and morphological control over these compartments remains challenging. In this study, we engineered 3D-printed subcellular compartments within terpolymer-stabilized coacervate-based artificial cells. Coacervate-forming charged polymers were functionalized with methacrylate moieties, enabling the fabrication of a variety of architectures within droplets through photoinitiated radical polymerization. The addition of a Ni-NTA functional methacrylate monomer to the coacervates led to its sequestration upon polymerization in these subcellular regions. As a result, the compartments were able to uptake and concentrate His₆-tagged mTurquoise and β-galactosidase protein cargo molecules, despite the increase in viscosity that was induced upon polymerization. Following this affinity-based interaction approach, we demonstrated the region-specific localization of an enzymatic reaction within the artificial cells.

Keywords: 3D printing; artificial cell; artificial organelle; coacervate; compartmentalization; photopolymerization.

1

Overview of the polymers used for photopolymerizable complex coacervate formation, subsequent 3DPR printing, and the model enzymatic reaction process. (A) Chemical structures of the anionic polymer methacrylated carboxymethyl amylose (CM-Am-MA), cationic polymer methacrylated quaternary amine amylose (Q-Am-MA), methacrylated nitrilotriacetic acid (NTA-MA), photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and synthetic membrane-forming triblock copolymer (poly­(ethylene glycol)-poly­(caprolactone-gradient-trimethylene carbonate)-poly­(glutamic acid) (PEG–PCL-g-TMC-pGlu)), (B) the 3D printing of defined structures within the coacervates by the 2-axis movement of a 405 nm laser, and (C) the localized production of fluorescent molecules via the enzymatic reaction between β-galactosidase and fluorescein-di-β-d-galactopyranoside (FDG).

2

Cargo sequestration behavior of artificial cells and their cross-linked counterparts. (A) Schematic illustration for the bulk cross-linking approach. (B) Cross-linking resulted in decreased fluorescence recovery after photobleaching (FRAP), (C) the effect of cargo molecular weight and cross-linking on partitioning. The combined overview of partitioning coefficients, grouped by cargo type, across different cross-linking times, illustrates the collective effect on cargo sequestration for the higher molecular weights. Bars represent mean ± SD of individual droplet measurements. Significant differences are visualized on bar plots using asterisks to indicate p-value ranges (ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. Only statistically significant results were shown on the graph) (n = 35 per condition across 3 independent experiments).

3

Constructing 3DPRs using a confocal laser printing approach offers high structural fidelity within coacervates. (A) Schematic of CLSM based 3DPR printing (B) photopatterned structures are formed. (Scale bars: 25 μm). The uptake dynamics of (C) carboxyfluorescein (recorded for 2.5 min) and (D) FITC-Dex (40 kDa) (recorded for over 24 h) is diminished in the 3DPR compared to the (non-cross-linked) artificial cell within a 25 min measurement time. (E) Schematic of 3DPR fabrication by including a fluorescent monomer, acryloxyethyl thiocarbamoyl rhodamine B. (F) Orthogonal views of the CLSM micrographs show the distribution of the 3DPR within the droplet, while displaying high printing resolution. (Scale bar: 25 μm).

4

Localized uptake of cargo molecules can be achieved by NTA-His tag interactions. (A) Schematic of protein sequestration in printed 3DPR regions. (B) Fluorescence intensity increase of mTurquoise-His₆ being locally sequestered within the 3DPR, via the interaction between the histidine residues and the NTA moieties, and (C) localization of His₆-tagged fluorescent proteins in the 3DPR at different time points can be observed by CLSM (Scale bars: 25 μm). (D) The sequential printing and cargo addition progress in 4 steps. Upon the printing of the first 3DPRs, the localized uptake of the first cargo can be followed by CLSM. Next, the second 3DPRs are fabricated, and the subsequent second cargo addition can be seen by CLSM (scale bars: 25 μm), (dashed lines show the separate coacervates. Note the movement of each droplet due to liquid flow in between each step), and (E) the uptake behavior of both cargo molecules is quantified for both 3DPRs, respectively. The higher signal intensities showcase the localization of the cargo molecules within the 3DPRs. The decrease in the fluorescence intensity after the second print was observed due to the partial bleaching caused by the printing process. Bars represent mean ± SD of individual droplet measurements. Significant differences are visualized on bar plots using asterisks to indicate p-value ranges (ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001) (n = 5 per condition).

5

3DPR-confined enzyme activity inside artificial cells. (A) The schematic illustrates the uptake of beta-galactosidase (β-Gal-His₆) into coacervates. (B) CLSM micrographs showing the enzyme uptake within the coacervates (scale bars: 25 μm), and (C) the enzyme accumulation localized in the 3DPRs. (D) The schematic illustrates the subsequent treatment with the profluorescent substrate fluorescein di-β-d-galactopyranoside (FDG), generating fluorescent signal upon product formation. (E) CLSM micrograph showing the enzymatic formation of fluorescent molecules within the 3DPR after 60 min (scale bar: 25 μm) (F) intensity quantification of end point fluorescent signal after 60 min reaction. The analysis shows that the reaction rate is higher in NTA-containing 3DPRs than in cross-linked regions without NTA.