Multi-layered ZIF-coated cells for the release of bioactive molecules in hostile environments

Metal–organic framework (MOF) coatings on cells enhance viability in cytotoxic environments. Here, we show how protective multi-layered MOF bio-composite shells on a model cell system (yeast) enhance the proliferation of living cells exposed to hostile protease-rich environments via the dissolution of the shells and release of a protease inhibitor (antitrypsin).


Synthetic methods
Formation of Y@ZIF-8. This protocol was modified from Liang et al. 3 Dried yeast cells (2 mg) were cultured in 2 mL of YPD, under sterile conditions. The resultant mixture was incubated under stirring (400 rpm) at 30 o C for 18 h.
The grown yeast was washed with deionized water (3 × 2 mL) and resuspended in an aqueous solution of HmIM (640 mM, 5 mL), followed by the addition of an aqueous solution of zinc acetate dihydrate (40 mM, 5 mL). The reaction mixture was shaken for 10 min (400 rpm) to yield the formation of Y@ZIF-8. The resultant powdery material was washed with deionized water (DI) (3 × 2 mL), resuspended in DI water (1 mL), and stored at 4 o C until needed.
Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2022 Formation of Y@ZIF-8@BSA. Three batches of Y@ZIF-8 samples (9 mg) were mixed and resuspended in DI water (0.9 mL). Then, an aqueous solution of BSA (1.5 mg·mL -1 , 0.1 mL) was added to the Y@ZIF-8 dispersion. The mixture was shaken for 15 min to yield the formation of Y@ZIF-8@BSA. The resultant material was isolated by centrifugation.
Formation of Y@ZIF-8@AAT. The formation of Y@ZIF-8@AAT was obtained following the same procedure as described for Y@ZIF-8@BSA, but by using an aqueous solution of AAT (1.5 mg·mL -1 , 0.1 mL) instead of BSA.
Formation of Y@ZIF-8@AAT@ZIF-8 and Y@ZIF-8@AAT@ZIF-C. The composites Y@ZIF-8@AAT@ZIF-8 and Y@ZIF-8@AAT@ZIF-C were obtained following the same synthetic procedure as described for Y@ZIF-8@BSA@ZIF-8 and Y@ZIF-8@BSA@ZIF-C; respectively. Table S1. Different synthetic conditions were explored for the formation of the second MOF coatings. The growth of different ZIF topologies was tuned by varying the concentration of the MOF precursors and the metal: ligand ratio. The conditions used for cell encapsulation were selected based on the formation of pure ZIF phases (blue represents sod-ZIF-8，cyan represents ZIF-C).

UV-Vis spectroscopy
Bradford assay and OD600 measurements were performed on a Thermo ScientificTM NanoDropTM UV-Vis spectrophotometer.

Scanning electron microscope-focused ion beam (SEM-FIB)
The SEM-FIB characterization was conducted using a ZEISS LEO 1540XB, equipped with a Ga ion FIB column (V = 30KV). The samples were previously coated with a thin conductive layer of Au-Pd (nominal thickness: 20 nm) deposited by sputter coater (Polaron SC7620). The images were acquired using a High Efficiency In-lens Detector.
The cross sectioning has been achieved in low current mode (20-50 pA) to avoid the damage of the cell.

Confocal laser scanning microscopy (CLSM)
CLSM imaging was performed using a Leica SP8 confocal microscope (Leica Microsystems Inc., Germany) with spectral detection and a HC PL APO CS 63x NA 1.2 W CORR objective. FITC was excited at 488 nm and emission detected between 500-550 nm using a noise-less hybrid photon detector. 3D data was acquired with 45 × 45 × 120 nm sampling. Deconvolution was performed using Huygens Professional (Scientific Volume Imaging Inc. The

Optical microscopy
Optical microscope images were collected on a ZEISS AxioScope A1 using 20× objective lenses.

Release tests
The cumulative release of the encapsulated protein (BSA and AAT) was assessed by UV-Vis spectroscopy. In this work, we studied two different media to trigger the release of the target protein at room temperature. The fast release was observed by exposing the cell-composites to a chelating agent EDTA (0.1 M, 1 mL). The controlled release was observed using a phosphate buffer solution (20 mM, 1 mL, pH = 6.5).
The cumulative release was recorded by suspending the MOF bio-composites (4 mg) either in EDTA or phosphate buffer. The samples were shaken using an orbital mixer. At regular intervals, the mixture was vortexed for 3 s and centrifuged for 30 s. Then, an aliquot of the supernatant (0.5 mL) was taken and replaced with the same volume of fresh EDTA or phosphate buffer. The supernatant aliquot removed from the reaction was mixed with Bradford reagent (1 mL) and analyzed by UV-Vis spectroscopy at 595 nm. All the experiments were performed in triplicates.

Adsorption efficiency optimisation using BSA as model protein
The optimisation study was carried out by mixing a stock solution of BSA (S0 BSA = 1.5 mg mL -1 , 100 µL) with different amounts of Y@ZIF-8 resuspended in DI water (900 µL). Thus, the resultant concentration of BSA was kept constant (0.15 mg mL -1 ), while the amount of Y@ZIF-8 composite varied systematically by adding incremental volumes of Y@ZIF-8 stock suspension (S0 Y@ZIF-8 =10 mg mL -1 ) (see Table S2 for details).
The resultant mixture was shaken for 15 min at room temperature. Then, the solids were collected by centrifugation, and the remaining amount of BSA in the supernatant was determined by Bradford assay. Finally, the adsorption efficiency was calculated with the following formula. Where: [BSA] i = Initial concentration of BSA (0.15 mg mL -1 ) [BSA] f = Concentration of BSA in the supernatant after the adsorption process  . S5).  From the optimization with BSA, we selected 1 mg of Y@ZIF-8 composite to adsorb ca. 17 µg of AAT.
Based on both UV-vis adsorption of the protein (280 nm) and Bradford assay from the supernatant (vide supra, e.g. S1, S2) we could confirm the 100% AAT adsorption.
Next, to examine any possibility of protein detachment from Y@ZIF-8 during the growth of the second MOF shell, we performed a micro-Bradford assay on the reaction media collected after the Y@ZIF-8@protein@ZIF synthesis (protein = BSA, AAT; ZIF=ZIF-8, ZIF-C). The calibration curve for BSA was first made using six standard solutions containing BSA or AAT at varying concentrations (0, 0.01, 0.02, 0.03, 0.04, 0.05 mg mL -1 ) ( Fig. S6 and Fig. S15). An aliquot of each sample (0.5 mL) was mixed with Bradford reagent (1.0 mL). The resultant mixture was left for 5 min at room temperature, followed by UV-vis analysis at 595 nm (Table S3 and Table S5). A similar procedure was used to assess the amount of BSA or AAT released into the reaction medium during the second coating process (Fig.   S16). All experiments were performed in triplicates. The collected data confirms that neither BSA nor AAT is released from the MOF surfaces to the reaction medium during the second coating synthesis.         The Bradford assay is typically used to determine the concentration of proteins in complex mixtures, such as cell extracts. 7,8 The protein (i.e. AAT) determination was performed following the Sigma-Aldrich standard protocol. 9 Briefly, a calibration curve for AAT was made by preparing solutions with different AAT concentrations (0, 0.06, 0.08, 0.10, 0.12, 0.15 mg·mL -1 ). An aliquot (50 µL) of each sample was mixed with Bradford reagent (1.5 mL). The resultant mixture was left for 5 min at room temperature prior to analysis by UV-Vis spectroscopy λ max = 595 nm (Table S4 and

Protease inhibitor activity of AAT released from Y@ZIF-8@AAT@ZIF (ZIF=ZIF-8, ZIF-C)
The protease inhibitor activity of AAT could be determined by exposing a solution of AAT to trypsin. Then, the enzymatic activity of trypsin was determined by using a colorimetric assay and Nα-Benzoyl-L-arginine ethyl ester (BAEE) as a substrate. 9 Briefly, the Y@ZIF-8@AAT@ZIF (ZIF = ZIF-8, ZIF-C) sample was suspended in EDTA (0.  , 17 μL). The sample was mixed by inversion and measured continuously by UV-Vis spectroscopy at 253 nm over 400 seconds. Both the activity of trypsin not exposed to AAT, and the activity of trypsin (0.25 mg·mL -1 , 25 μL) exposed to a solution of lyophilized powder of AAT (0.05 mg·mL -1 , 1 mL) were examined as experimental controls.

Fig. S23
Enzymatic activity of trypsin exposed to AAT fully released from Y@ZIF-8@AAT@ZIF-8. This plot also demonstrates how the slow release of AAT leads to the gradual inhibition of trypsin activity.

Fig. S24
Enzymatic activity of trypsin exposed to AAT fully released from Y@ZIF-8@AAT@ZIF-C. This plot also demonstrates how the slow release of AAT leads to the gradual inhibition of trypsin activity.

Cell proliferation experiment
The composite samples were suspended in an aqueous solution containing EDTA (0.1 M, 1 mL) and trypsin (0.25 mg·mL -1 , 25 μL). The mixture was shaken for 4 h, centrifuged and washed with DI water (3 × 2 mL). The released cells were dispersed in H 2 O (20 mL) and then an aliquot (2 μL) was taken and added to the growth media YPD (2 mL). The cells' growth at room temperature was monitored by Optical Density measurements at 600 nm (OD600).
As a control, the cell growth of naked cells exposed and non-exposed to trypsin was monitored by OD600. Briefly, non-encapsulated yeast cells were exposed to EDTA (0.1 M, 1 mL) with trypsin (0.25 mg·mL -1 , 25 μL) or without trypsin for 4 h. The mixture was shaken for 4 h, centrifuged and washed with DI water (3 × 2 mL). The cells were then resuspended in H 2 O (20 mL) and then an aliquot (2 μL) was taken and added to growth media YPD (2 mL). The cell growth at room temperature was monitored by OD600.