Multicompartment calcium alginate microreactors to reduce substrate inhibition in enzyme cascade reactions

The formation of macromolecularly enriched condensates through associative or segregative liquid-liquid phase separation phenomena is known to play a central role in controlling various cellular functions in nature. The potential to spatially and temporally modulate multistep chemical reactions and pathways has inspired the use of phase-separated systems for the development of various synthetic colloidal micro- and nanoreactor systems. Here, we report a rational and synthetically minimal design strategy to emulate intended spatiotemporal functions in morphologically intricate and structurally defined calcium alginate hydrogel microreactors possessing multicompartmentalized internal architectures. Specifically, we implement a thermal phase separation protocol to achieve fine-control over liquid-liquid phase separation inside complex aqueous emulsion droplet templates that are loaded with hydrophilic polymer mixtures. Subsequent gelation of alginate-containing droplet templates using a novel freeze-thaw approach that can be applied to both scalable batch production or more precise microfluidic methods yields particle replicas, in which subcompartmentalized architectures can be retained. Larger active components can be enriched in the internal compartments due to their preferential solubility, and we show that selective sequestration of enzymes serves to create desired microenvironments to control and tune the reaction kinetics of a multistep enzyme cascade by reducing their mutual interference. This demonstration of mitigating substrate inhibition that is based primarily on optimizing the multicompartmentalized hydrogel particle morphology offers new opportunities for the simple and synthetically-minimal batch generation of hydrogel-based synthesis microreactors.


Figure S2 .
Figure S2.Micrographs of differently sized single phase Ca-alginate hydrogel particles prepared using droplet templates of different sizes.The different micrographs display particles at different sizes prepared using the freeze thaw approach by varying the size of the droplet precursors via controlling the flow rate in the microfluidic channels.The micrographs display large particles with a diameter of d= 208.96 µm ± 1.91 µm (a-c), medium sized particles with a diameter of d= 53.92 µm ± 1.96 µm (d-f), and small hydrogel particles with a diameter of d= 112.75 µm ± 7.19 µm (g-i) at different magnification.Scale bar: top row: 50 µm, middle row: 200 µm, bottom row: 1000 µm.

Figure S4 .
Figure S4.Investigation of the semi-permeability of synthesized hydrogel particles and determination of the molecular weight cut off (MWCO).a-e)Fluorescence micrographs of Caalginate hydrogel particles exposed to FITC or RITC-labeled dextran of different molecular weights; f) Plot of the fluorescence intensity ratio of the fluorescence from labeled dextrans in the continuous phase to the fluorescence intensity on the inside of the particles as a function of the molecular weight of the dextran.The graph reveals the molecular weight cut-off MWCO = 40 kDa, above which 80 % of the solute is retained by the hydrogel particles.

Figure S5 .
Figure S5.Test of hydrogel particle stability to variations in external conditions.Micrographs depict pristine single-phase particles, after 30 min of exposure to aqueous solutions of hydrochloric acid or sodium hydroxide at pH = 3, 5, 6.5, 8, and 10, after 30 min at elevated temperature of T = 90°C, and after 30 min exposure to aqueous NaCl solutions at concentrations of 5mM, 20 mM, and 50 mM.scale bar: 500 µm.

Figure S6 .
Figure S6.Optical micrographs of the time-dependent progress of liquid-liquid phaseseparation inside aqueous droplets consisting of PEG (MW: 35 kDa), dextran (MW: 500 kDa) and alginate (2.5 wt.%) induced via placing droplets generated at elevated temperatures at room temperature.(a) The top view optical micrographs depict the time-dependent progress of phase-separation by first forming highly multicompartmentalized droplets containing many small dextran-rich compartments that then merge into larger droplets and ultimately form Janus droplets comprised of two distinct hemispheres; scale bar: 100 µm; (b) Plot of timedependent increase in the compartment diameter of a droplet with a diameter of 130 µm; (c) Side-view optical micrograph of the droplets after complete phase separation; scale bar: 100 µm.

Figure S8 .
Figure S8.Characterization of Ca-alginate hydrogel Janus particles.a) Image of the bulk ATPS (V = 4 mL) displaying the predominant FITC-labeled alginate partitioning into the PEG-rich phase via yellow staining of the respective ATPS phase; b-c) Optical bright field (left) and fluorescence (right) micrographs of hydrogel Janus particles displaying the different crosslinking density of Ca-alginate (labeled with FITC) in the two phases due to the preferred partitioning of alginate into the PEG-rich phase of the droplet precursors; scale bars: 100 µm for b and 50 µm for c; d-e) SEM images of dried hydrogel Janus particles; scale bar: 20 µm for d and 2 µm for e.

Figure S9 .
Figure S9.Determination of the partitioning coefficient (K) of catalase.Partitioning coeffiecients of catalase inside an ATPS comprised 2.5 wt.% sodium alginate, 1.82 wt.% PEG 35K, and 3.63 wt.% dextran 500K containing different concentrations of NaCl.Enzyme concentrations in the manually separated top and bottom phases of the ATPS were determined by performing the Bradford test.

Figure S10 .
Figure S10.Oscillation frequency of catalase-functionalized hydrogel particles during the first 7 cycles.

Figure S11 .
Figure S11.Visualization of HRP and GOX enzyme cross-partitioning inside droplets and corresponding hydrogel particles.a) Visualization of preferential FITC-labeled HRP and RITClabeled GOX partitioning into the opposite phases of a bulk ATPS comprised of PEG 35k and dextran 500k through different staining of the two phases; b,c) Fluorescence micrographs showing the different partitioning of the two enzymes inside multi-compartmentalized (b,c) and Janus droplets (d,e).Droplets contained both FITC-labeled HRP and RITC-labeled GOX.Selective excitation of the two fluorophores indicates differences in the relative phase-preferential localization of the enzymes; f-i) Fluorescence micrographs of hydrogel particles obtained after gelation of the droplet precursors displayed in b-e.A direct comparison of the green vs. red fluorescence intensity distributions throughout the particles indicates differences in the phasepreferential partitioning of the two enzymes.All scale bars: 50 µm.

Figure S12 .
Figure S12.Determination of the Michaelis-Menten constant and maximum reaction velocity of the cascade reaction.a) Time-dependent increase in fluorescence intensity stemming from the cascade reaction product resorufin as a function of different added glucose concentrations inside multi-compartmentalized hydrogel particles with low phase-preferential partitioning of the two enzymes HRP and GOX; b) Same time-dependent evolution of the cascade reaction for particles containing cross-compartmentalized enzymes as controlled via modulating the salt concentration inside the respective droplet precursors; c-d) Plot of the initial reaction velocity versus glucose concentration for the determination of the maximum reaction velocity and Michaelis Menten constant for the two different systems (multicompartmentalized hydrogel particles with almost uniform enzyme distribution throughout both phases (c) and phasepreferential partitioning of the two enzymes (d)).Values for Km and Vmax were obtained by fitting the experimental data using the Micahelis-Menten equation  0 =   * []   +[]

Figure S13 .
Figure S13.Partitioning behavior of the cascade reaction substrates.The relative partitioning of the substrates of the enzyme cascade reaction was determined via addition of the substrates glucose (100 mM) or amplex red (2 mM), respectively, to a bulk ATPS solution (1 mL) prior to separating the two phases.Initiation of the enzyme cascade via addition of the enzymes HRP and GOX resulted in the formation of the fluorescent marker resorufin and the relative fluorescence intensity of the individual phases was recorded.

Figure S14 .
Figure S14.Determination of the morphology dependent reaction rate of the enzyme cascade.Determination of the pseudo-first order reaction constants for cascade reaction inside differently compartmentalized hydrogel particle structures, (a) Multi-compartment morphology, (b) few-compartment morphology, (c) Janus morphology and (d) single phase particles.

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