Combinatorial Strategy for Studying Biochemical Pathways in Double Emulsion Templated Cell‐Sized Compartments

Abstract

optimize these pathways and thus to guarantee their maximum chance of survival, cells produce enzymes at precise and constant rates. [4] However, because of cellular complexity, determination of optimal enzyme levels is still unclear, in particular because specific enzymatic functions are strongly interconnected with their cascade effects. [5] A smart manner to study the behavior of enzymes, when involved in complex reactions, consists of taking advantage of synthetic micrometer-sized compartments shaped into spherical architectural bodies. [6-8] Though these idealized models favor even partitioning of membrane proteins and simplify diffusion gradients, similarly to cells, they provide the desired membrane selectivity and permeability. [9,10] Single specific functions have been presented by combining enzymes (the catalytic compounds) with synthetic supramolecular assemblies, either intrinsically semipermeable, such as lipid-coated porous silica particles, [11] protein cages, [12] layer-by-layer capsules, [13-15] and polydopamine capsules, [16,17] or rendered permeable by the insertion of biopores/membrane proteins (acting as "gates" for the passage of molecules), as in lipid-based [7,18,19] or polymer-based [8,20-22] giant unilamellar vesicles (GUVs). At present, GUVs formed with amphiphilic block copolymers are particularly appealing because they provide enhanced structural membrane properties compared to lipids, since they possess compartments with higher chemical versatility, controlled permeability, robustness, and stability. [23] Nevertheless, common approaches to form polymer GUVs by self-assembly, as electroformation [24] and film-rehydration, [25] rely on the statistical process of encapsulation of biomolecules with a probability of finding the designed amount of one type of enzyme inside the compartments ranging from 12-57%. In the context of elementary biochemical pathways where at least two enzymes are present, this scenario is even more unsatisfactory, with co-encapsulation of two enzymes inside one compartment as low as 10-22%. [26,27] These shortcomings are worsened by the fact that these probabilities have a large uncertainty induced by specific properties of enzymes (e.g., solubility and stability). To date, scientists have struggled to work with stateof-the-art average values for number/mass of enzymes that are vastly non-representative (limited by a small sample size) Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi-step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/ metabolites in synthetic cell-sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high-precision double-emulsion microfluidics that enable: i) tight control over the absolute and...

Clustering of catalytic nanocompartments for enhancing an extracellular non-native cascade reaction

“…Similarly, such interaction was seen with the related horseradish peroxidase, 45 and recently reported for LPO in giant unilamellar vesicles as well. 44 The vesicular architecture of LPO-and GOX CNCs was conrmed by TEM micrographs revealing the deformed spherical morphology typical of this kind of polymersome (Fig. 2A, B and S1 †).…”

“…polymersomes and other cell-mimics, but never in colloidal vesicle networks. 44,56 In a previous study we had showed that in the case of un-clustered CNCs, inter-polymersome distances above 1.5 mm signicantly hinder the cascade reaction due to the slow diffusion of the "bridging" molecule (the product of the rst reaction that is released from the rst type of CNC and serves as substrate for the second reaction inside the second type of CNC). 26 On the contrary, CNC clustering averted this limitation, resulting in a considerably increased reaction rate at a constant amount of encapsulated enzymes.…”

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles

“…Microscopic GUVs were assembled from double emulsion templates created by microfluidics. Water-oil-water double emulsions were generated in a six-way junction microfluidic chip, 22 with defined flow rates using a three-module precision syringe pump (low pressure NEMESYS, Cetoni, Germany). PBS containing 20 wt/v% poly(ethylene glycol) (PEG, 3000 Da) and 300 mM sucrose was used as inner aqueous phase (IA).…”

“…Live imaging of this process was followed using a high-speed digital microscope (Meros, Dolomite). In order to evaporate the organic solvents from the double emulsions and thus form a vesicular bilayer membrane according to an established protocol, 22 198 μL of a double emulsion sample and 2 μL of a 100 μM aqueous BODIPY 630/650 solution were transferred into a Nunc Lab-Tek eight-well chamber plate (Thermo Fisher Scientific, USA) and incubated at room temperature for 20 min. Within this time, GUVs were created from double emulsions, which settled to the bottom of the well for further CLSM imaging.…”

“…[15][16][17] Advanced applications such as platforms to analyse enzymatic cascade reactions or cell mimics with controlled passage of substrates are thus considerably more difficult with (semi-) crystalline BCPs. [18][19][20][21][22][23] Polymeric self-assemblies composed of isotactic, yet noncrystalline, BCPs to create fully flexible and amorphous chiral membranes would overcome these limitations but have not been available so far. In this work, a straightforward approach to obtain small and giant unilamellar vesicles (SUVs, GUVs) composed of fully amorphous isotactic BCPs is presented.…”