Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules

Abstract

Enhancing the robustness of functional biomacromolecules is a critical challenge in biotechnology, which if addressed would enhance their use in pharmaceuticals, chemical processing and biostorage. Here we report a novel method, inspired by natural biomineralization processes, which provides unprecedented protection of biomacromolecules by encapsulating them within a class of porous materials termed metal-organic frameworks. We show that proteins, enzymes and DNA rapidly induce the formation of protective metal-organic framework coatings under physiological conditions by concentrating the framework building blocks and facilitating crystallization around the biomacromolecules. The resulting biocomposite is stable under conditions that would normally decompose many biological macromolecules. For example, urease and horseradish peroxidase protected within a metal-organic framework shell are found to retain bioactivity after being treated at 80 °C and boiled in dimethylformamide (153 °C), respectively. This rapid, low-cost biomimetic mineralization process gives rise to new possibilities for the exploitation of biomacromolecules.

Figure 1. Schematic illustration of biomimetically mineralized MOF.

(a) Schematic of a sea urchin; a hard porous protective shell that is biomineralized by soft biological tissue (b) Schematic of a MOF biocomposite showing a biomacromolecule (for example, protein, enzyme or DNA), encapsulated within the porous, crystalline shell.

Figure 2. Characterization of biomimetically mineralized biocomposite.

(a) SEM image showing the crystals obtained using BSA as a growth agent for biomimetic mineralization (scale bar, 1 μm). (b) Photograph and (c) confocal laser scanning microscopy image of the biomomimetically mineralized ZIF-8 composite obtained using BSA labelled with FITC. This biocomposite (ZIF-8/FITC-BSA) was prepared at 37 °C, washed and exposed to ultraviolet light of wavelength 365 and 495 nm, respectively (scale bar, 10 μm). (d) PXRD of the MOF-BSA biocomposite. (e) FTIR spectra of BSA (red), ZIF-8/BSA (orange), standard ZIF-8 post incubated with BSA after washing (blue), and standard ZIF-8 (black). (f) SAXS data of the ZIF-8/BSA biocomposite and a schematic showing the relative size of BSA to the mesopore. The observed Guinier knee can be fitted using the Unified model with a radius of gyration (R_g) of 35 (± 5) Å, which is 17% larger than that of BSA 29.9 Å (ref. 60). (g) Schematic proposing the biomimetically mineralized growth of ZIF-8. Each BSA molecule attracts 31 2-methylimidazole (HmIm) ligands and 22 Zn²⁺ ions, facilitating the nucleation of ZIF-8 crystals.

Figure 3. Characterization of biomimetically mineralized ZIF-8 with biomolecules.

(a) PXRD patterns of the crystals obtained using various biomacromolecules as biomimetic mineralizion agents. Protein encapsulation efficiency: BSA ∼100%, human serum albumin (HSA) ∼100%, OVA ∼100%, lysozyme ∼96%, HRP ∼100%, ribonuclease A ∼86%, haemoglobin ∼90%, trypsin ∼96%, lipase ∼88%, insulin ∼86%, glucose dehydrogenase (PQQ-GDH) ∼82%, urease ∼95%. In each case, the intensity and peak positions of the biocomposites match those of pure ZIF-8. (b–m) Scanning electron microscopy images showing crystals obtained using: (b) OVA, (c) ribonuclease A, (d) HSA, (e) pyroloquinoline quinone-dependent glucose dehydrogenase ((PQQ)GDH), (f) lipase, (g) haemoglobin, (h) lysozyme, (i) insulin, (j) HRP, (k) trypsin, (l) urease and (m) oligonucleotide. Scale bars, 1 μm. While several biomacromolecules induce the standard rhombic dodecahedral morphology for the ZIF-8 biocomposites (c,d,m), other biomacromolecules gave rise to various morphological features such as truncated cubic (e,f,h,i,l), nanoleaf (b), nanoflower (j) and nanostar (k), respectively.

Figure 4. Protective performance of ZIF-8 coatings on HRP.

Product conversion of free HRP, the biomimetically mineralized ZIF-8 using HRP (ZIF-8/HRP), HRP protected by calcium carbonate (CaCO₃/HRP) and HRP protected by mesoporous silica (SiO₂/HRP, SiO₂ with average pore size of 7, 20, 50 and 100 nm) in the presence of proteolytic agent, trypsin, after treatment in boiling water for 1 h, and after treatment in boiling dimethylformamide (DMF) for 1 h at 153 °C, respectively. Data were normalized against free HRP activity at room temperature. Error bars represent the s.d. of three independent experiments.

Figure 5. Controlled release of bioactive enzymes and proteins from ZIF-8 biocomposites.

(a) Fluorescent measurement of the PBS solution containing biomimetically mineralized zeolitic imidazolate framework-fluorogenic protein (ZIF-8/DQ-OVA) and biomimetically mineralized zeolitic imidazolate framework-trypsin (ZIF-8/trypsin) particles. At pH 7.4, the fluorescent emission (blue line) was analogous to that of free intact DQ-OVA protein (brown line). At pH 6.0, a drastic increase in the fluorescence intensity (red line) was observed, which was attributed to the proteolysis of the DQ-OVA into luminescent fragments suggesting that trypsin and DQ-OVA have been released and are free to interact in solution. (b–e) Schematics showing the release of DQ-OVA (red) and trypsin (yellow) from ZIF-8 biocomposites at pH 6.0, and degradation of DQ-OVA into fluorescent fragments as a result of proteolysis by trypsin.