Heterogeneous multi-compartmental hydrogel particles as synthetic cells for incompatible tandem reactions

Abstract

In nature, individual cells contain multiple isolated compartments in which cascade enzymatic reactions occur to form essential biological products with high efficiency. Here, we report a cell-inspired design of functional hydrogel particles with multiple compartments, in which different enzymes are spatially immobilized in distinct domains that enable engineered, one-pot, tandem reactions. The dense packing of different compartments in the hydrogel particle enables effective transportation of reactants to ensure that the products are generated with high efficiency. To demonstrate the advantages of micro-environmental modifications, we employ the copolymerization of acrylic acid, which leads to the formation of heterogeneous multi-compartmental hydrogel particles with different pH microenvironments. Upon the positional assembly of glucose oxidase and magnetic nanoparticles, these hydrogel particles are able to process a glucose-triggered, incompatible, multistep tandem reaction in one pot. Furthermore, based on the high cytotoxicity of hydroxyl radicals, a glucose-powered therapeutic strategy to kill cancer cells was approached.Cells contain isolated compartments where cascade enzymatic biochemical reactions occur to form essential biological products with high efficiency. Here the authors produce functional hydrogel particles with multiple compartments via microfluidics that contain spatially immobilized natural enzymes in distinct domains for one-pot, tandem reactions.

Fig. 1

Preparation of multi-compartmental hydrogel particles using microfluidics. a The bright-field image of polyethylene glycol (PEG) multi-compartmental hydrogel particles was recorded. b The bright-field image of a single particle was recorded to observe its details. c The cores of the PEG multi-compartmental hydrogel particles were labeled with Albumin-FITC, showing green florescence signal with emission wavelength 520 nm. d The image of a single particle, whose core was labeled by Albumin-FITC was recorded. e The shells of the PEG multi-compartmental hydrogel particles were labeled with Dextran-RhB, showing red florescence signal with emission wavelength 590 nm. f The image of a single particle, whose shell was labeled by Dextran-RhB was recorded. g The image of a particle with core labeled with Albumin-FITC (green fluorescence) and shell labeled with Dextran-RhB (red fluorescence) was recorded. Scaling bars are 200 μm

Fig. 2

Encapsulation of GOX and HRP in a multi-compartmental particle. a Bright-field microscopic image of GOX@HRP. b Confocal scanning laser microscopic images of GOX@HRP in x–y, x–z and y–z planes, along with a 3D model of GOX@HRP. The GOX and HRP were labeled with rhodamine B isothiocyanate (RBITC, red) and fluorescein isothiocyanate (FITC, green), respectively. Detailed labeling procedures are discussed in Supplementary Methods (Labeling of enzymes with fluorescent dyes). c GOX and HRP release profiles from GOX@HRP in PBS (10 mM, pH 7.0). Scaling bars are 200 μm

Fig. 3

Activity characterization of GOX@HRP. a Schematic illustration and reaction equations of GOX@HRP-catalyzed Amplex Red oxidation for the production of resorufin after initiation by glucose. b Time-dependent absorbance changes caused by the oxidation product of Amplex Red catalyzed by different tandem catalytic systems: a free GOX/HRP system, a GOX@PEG/HRP@PEG system, GOX&HRP@PEG, and GOX@HRP. c Relative activities of tandem catalytic systems based on a free GOX/HRP system, a GOX@PEG/HRP@PEG system, GOX&HRP@PEG, and GOX@HRP. d Relative turnover numbers of GOX@HRP and GOX&HRP@PEG system with molar ratios of GOX to HRP from 1:1 to 10:1

Fig. 4

AA-mediated pH microenvironments of PEG hydrogels. a Schematic representation of the copolymerization of AA with PEG and the pH of the microenvironments of poly(PEG-co-AA) hydrogel particles with different AA contents (0, 5, 15, 30, and 45%). The insets are their corresponding fluorescent photographs. b Absorbance at 652 nm and visual colors of oxTMB catalyzed by MNP@PEG (black bars) and MNP@PEG-co-AA (red bars) in PBS (pH 7.0) using H₂O₂ as a substrate or coupled with the GOX-catalyzed glucose oxidation reaction. c Relative activity changes of MNPs encapsulated in different PEG-co-AA with AA contents ranging from 0 to 45%. Assay conditions: MNP@PEG-co-AA was incubated with 1.25 mM H₂O₂ and 43.75 μM TMB in PBS (pH 7.0), and the absorbance was monitored at 652 nm

Fig. 5

GOX@MNP functions as an integrated tandem catalytic system. a Microscopic image of GOX@MNP. Scale bar: 200 μm. b Absorption spectra of oxTMB (black line) and resorufin (red line) produced from the GOX@MNP-catalyzed TMB-H₂O₂ assay and glucose-triggered Amplex Red assay in the presence of GOX@MNP and HRP, respectively; both were performed in PBS (pH 7.0, 10 mM). Inset: corresponding visual colors of these two samples. c Schematic illustration of the glucose-triggered TMB oxidation reaction in the presence of GOX@MNP. d Absorption spectra and visual color changes of oxTMB obtained from the TMB oxidation reaction catalyzed by GOX@MNP in the absence (black line) and presence (red line) of glucose in PBS (pH 7.0, 10 mM)

Fig. 6

Glucose-powered anticancer therapy. a Cell viability of HeLa cells after incubation with different concentrations of GOX@MNP for 24 h. b Cell viability of HeLa cells after incubation with glucose alone (black bars) and with 5 μg ml⁻¹ GOX@MNP and different concentrations of glucose (red bars) for 24 h