All chemicals were purchased from Sigma-Aldrich and used as received. Deionized water was used throughout. The gelator used here was synthesized following the protocols of ref. 31. Full details are provided in the Supplementary Information (p. 25). The other gelators tested (Supplementary Fig. 1) were prepared as described elsewhere32.
The pH of solutions and gels was measured using an FC200 pH probe (Hanna instruments) calibrated using pH 4.01, 7.01 and 10.01 buffer solutions. The probe was rinsed with deionized water between measurements.
Rheological measurements were carried out using Anton Paar Physica MCR301 and M101 Rheometers. A cup and vane (ST10-4V-8.8/97.5-SN42404) system was used for all frequency and strain sweeps, with a measuring gap of 1.35 mm. Gels were prepared directly in 7 ml Sterilin vials, which were loaded on to the rheometer and measured in situ to ensure that no damage was carried out to the gels by transfer from vials. Strain sweeps were performed from 0.08% to 1,000% at a frequency of 10 rad s−1. Frequency sweeps were performed from 1 to 100 rad s−1 at a constant strain of 0.1% (within the linear viscoelastic region for all gels). To test the behaviour of the materials at low frequency, frequency sweeps were further collected from 0.01 to 100 rad s−1 at a constant strain of 0.1% (within the linear viscoelastic region for all gels). To measure the yield stress of the gels, strain sweeps were performed at a frequency of 1 rad s−1 from 0.1% and 1,000%. The yield stress value was obtained by plotting the elastic stress (G′ γ0) against the strain amplitude, according to previously published methods33,34,35,36,37.
To obtain frequency sweeps of the samples at higher temperatures, gels were prepared directly in 7 ml aluminium cups and loaded on to the rheometer. The temperature was raised linearly from 25 to 60 °C with a heating rate of 2 °C min−1. Then, frequency sweeps were collected from 1 to 100 rad s−1, while keeping a constant strain of 0.1% and temperature of 60 °C.
SAXS experiments were performed at Diamond Light Source, at the I22 beamline38. The beamline operates at an energy of 12.4 keV and the camera length was set to 4.275 m to give a q range of 0.002–0.30 Å−1. The gels were prepared as described in the next section and immediately loaded in glass capillaries using a 1 ml syringe with a 21 G needle. The raw data were processed using the DAWN Science software (v.2.27)39, according to a standard I22 pipeline30. As part of the processing, the backgrounds were subtracted from the raw two-dimensional SAXS data and a full azimuthal integration was performed to reduce the data to an I versus q plot. The plots were then fitted to structural models using the SasView software (v.5.0.4).
BioSAXS experiments were performed at the B21 beamline at Diamond Light Source. The beamline operates at a fixed energy of 13 keV and a camera length of 3.600 m to obtain a q range of 0.0031–0.38 Å−1.
A total 50 μl of each sample was loaded into a 96-well plate and measured using the BioSAXS EMBL Arinax sample-handling robot. For each sample, 30 × 1 s frames were collected at 20 °C. The two-dimensional raw data were processed in the DAWN Science software (v.2.27)39 to yield the I versus q plots. The data were then averaged and the buffer background was manually subtracted using the ScÅtter software (https://bl1231.als.lbl.gov/scatter/, R. P. Rambo).
CD-005 gels were prepared either with or without the addition of CaCl2. For both, 50 mg of CD-005 was added to 20 mg of K2CO3 and dissolved in 5 ml of deionized water under overnight stirring. For the samples without CaCl2, 0.4 ml of the gelator solution was transferred to a vial, to which 1 ml of 1.5 M Tris HCl (pH 6.8) was added. For the samples formed with the calcium trigger, 5.5 µl of a 200 mg ml−1 CaCl2 solution was first added to 1 ml of 1.5 M Tris HCl (pH 6.8) in a separate vial. This was then added in one aliquot to 0.4 ml of gelator buffer. For rheological measurements, the gels were prepared in vials and left undisturbed overnight on the bench. To prepare the samples in syringes, the gels were prepared as above and immediately transferred (less than 1 min) to a syringe through a 21 G needle. All the samples needed to be prepared at 19 °C to ensure optimal gelation.
Micellar solutions were prepared as previously described at concentrations of 5 mg ml−1 (ref. 32). Gel samples were prepared in 7 ml Sterilin vials by addition of 2 ml of stock solution (adjusted to pH 10.5) to 16 mg of solid glucono-δ-lactone (GdL) for 5 mg ml−1 of solutions. The vials were swirled briefly by hand to ensure complete dissolution of GdL then left to stand overnight undisturbed.
For the CD-005 gels, dextran was encapsulated in the gel by dissolution of dextran powder in 1.5 M Tris HCl, pH 6.8 at a range of concentrations (0.14–14 wt%). A total 1 ml of this solution was transferred to 0.4 ml of the gelator solution described above to achieve the desired final concentrations of dextran (0.1–10 wt%). For the gels obtained by reduction in pH, the dextran was dissolved at the various required concentrations in deionized H2O under stirring. These solutions were then used to prepare the micellar solutions of the gelators at 5 mg ml−1.
To release the gel with dextran inclusion, the gel prepared in the syringe was passed through a 2.7 μm filter. Figure 1g exemplifies the methodology of release through the filter: the sample is first allowed to gel overnight in a syringe, then the gel is gently passed through the 2.7 μm syringe filter, which releases a clear solution. Generally, about 80% of the liquid is recovered from the procedure.
For the quantification of released insulin and dextran, samples were prepared as follows. Insulin was dissolved in 1.5 M Tris HCl (pH 6.8) at concentrations of 0.28 and 4.48 mg ml−1. To avoid aggregation, the samples were left overnight on a roller at 76 rpm to ensure complete dissolution. To form gels in the syringes, 0.4 ml of the CD-005 gelator solution prepared as described above was transferred to a 12 ml syringe. Then, 1 ml of the insulin solution in Tris was transferred using a 5 ml syringe and a 21 G needle directly in the 12 ml syringe. This resulted in quick formation of the gels at final concentrations of 0.2 and 3.2 mg ml−1. The gels were left overnight to stabilize and then gently passed through a 2.7 μm filter, releasing a clear solution. Ultraviolet–visible (UV–Vis) light was measured directly on the obtained solution. To quantify the amount of insulin released, insulin solutions were prepared at a range of concentrations (0.2–4.48 mg ml−1) in a similar way in 1.5 M Tris HCl.
For the quantification of released dextran, 1.4 wt% (14 mg ml−1) solution of fluorescein isothiocyanate dextran was prepared in 1.5 M Tris HCl buffer by dissolving 14 mg of the dextran in 1 ml of buffer. A total of 5.5 μl of 200 mg ml−1 of CaCl2 solution was then added to 1 ml of this solution and swirled briefly. To form gels in syringes, a similar method was used as described above, achieving a final concentration of 1 wt% (10 mg ml−1). The gels were left overnight to stabilize and then gently passed through a 2.7 μm filter. The resulting dextran solution was too concentrated to study by means of UV–Vis and fluorescence. For UV–Vis, a dilution of a factor of 10 was carried out using buffer, reaching a final concentration of 1 mg ml−1. For fluorescence, a factor of 100 was needed (0.1 mg ml−1) to avoid self-quenching at higher concentrations. To quantify the amount of released dextran, fluorescein isothiocyanate dextran was dissolved in 1.5 M Tris HCl buffer at desired concentrations (1–0.05 mg ml−1), ensuring the same pH throughout.
Circular dichroism data were acquired on a Chirascan VX spectrometer (Applied Photophysics) using a quartz cuvette with a 0.01 mm path length. The spectra were collected in the range 180–400 nm with a scanning step size of 1.0 nm and scanning rate of 0.25 s at room temperature. The samples were prepared in Sterilin vials as described, keeping the same volumes of the components. Small amounts of the gels were then transferred to the cuvette before measurement.
Data were recorded using an Agilent Cary 630 FTIR spectrometer (with ATR attachment). The filter paper from the 2.7 μm filter was removed by carefully opening the syringe filters. Then, the background of the empty ATR crystal was taken. Small amounts of a clean filter and one after extrusion were deposited on the ATR crystal to record the spectra.
Absorption spectra were recorded on an Agilent Cary 60 UV–Vis spectrophotometer using a quartz cuvette with 0.1 mm path length. Samples were prepared as above and 300 μl of the solution was transferred to the cuvette using a 200 μl pipette.
Fluorescence data were collected using an Agilent Technologies Cary Eclipse fluorescence spectrometer. Samples were prepared as described above at a 2 ml volume and transferred to a quartz cuvette with a 1 cm path length. For the fluorescein isothiocyanate dextran, the excitation wavelength was 470 nm. For the 2NapIF release studies, the excitation length was 320 nm. In all cases, the excitation and emission slit widths were 5 nm and 5 nm. To quantify the amount of 2NapIF in the extrudate, a known volume of the extruded sample was freeze-dried (1 ml) and then fully redissolved in DMSO.
Water suppression and STD experiments (Supplementary Fig. 17) were recorded on a Bruker spectrometer operating at 499.31 MHz and equipped with a Neo console and Bruker 5 mm SmartProbe. The 1H experiments were recorded using the perfect echo WATERGATE sequence of ref. 40 incorporating the double echo W5 sequence of ref. 41. The delay between successive pulses in the selective pulse train was set at 333 μs, corresponding to 3,000 Hz between the null points. The 1H spectra were acquired in four dummy scans and 128 scans with a relaxation delay of 1 s and signal acquisition time of 4.2 s. STD spectra were obtained using the same sequence but with an overall relaxation delay of 5 s. Presaturation was applied during the final 4 s of the relaxation delay using a train of 100 Gaussian pulses (40 ms) with peak powers of 243 Hz at 100 ppm (off resonance) and −3.8 ppm (on resonance) in separate experiments, which were recorded with 16 dummy scans and 16 scans. Spectra were processed with an exponential line broadening factor of 1 Hz and referenced to the CH3 triplet of ethanol (1.2 ppm) present as an impurity in our commercial insulin sample42.
The 3.2 mg ml−1 of insulin solution and the CD-005 gel were prepared and aged in 5 mm NMR tubes (Wilmad 528-PP) for 20 h at 22 °C.
High-resolution mass spectrometry was recorded using a ThermoScientific Exactive Plus Orbi-Trap with ESI ionization at the University of Strathclyde, Glasgow. For the analysis, the sample is directly injected by means of the UHPLC of 10 μl of sample into the solvent flow (0.1% formic acid in methanol). The sample is injected for 1.8 min at a flow rate of 0.1 ml min−1. The mass spectrometer uses positive/negative polarity switching to obtain both the positive and negative mass spectrum co-currently, with a scan range of 400–6,000 Da at a resolution of 70,000. On the basis of the predicted mass, the interest molecular peak is mass matched in the spectrum with 4 decimal point accuracy (isotopic discrimination levels). For the insulin-containing samples, the mass was best detected at 3+ charge.
First, 1.1 ml of 0.1 M NaOH was added to 8.9 ml of deionized H2O. A total of 50 mg of NapIF was then added to this solution and left to dissolve overnight on a rotary shaker. Before gelation, insulin is added to a final concentration of 0.2 mg ml−1. Gelation is initiated by adding 80 mg of GdL to this solution. The sample is then pipetted into 5 × 2 ml syringes before being sealed with parafilm and left overnight.
First, 1 ml of 0.1 M NaOH was added to 9 ml of deionized H2O. A total of 50 mg of NapFF was then added to this solution and left to dissolve overnight on a rotary shaker. Before gelation, insulin is added to a final concentration of 0.2 mg ml−1. Gelation is initiated by adding 20 mg of GdL to this solution. The sample is then pipetted into 5 × 2 ml syringes before being sealed with parafilm and left overnight.
A total of 20 mg of potassium carbonate was dissolved in 5 ml of deionized H2O. A total of 50 mg of CD-005 was added to this solution and left to dissolve overnight on a rotary shaker. A total of 4 ml of this solution was added to 10 ml of 1.5 M Tris HCl at pH 6.8. Immediately after, insulin was added to a final concentration of 0.2 mg ml−1. The sample was then pipetted into 5 × 2 ml syringes before being sealed with parafilm and left overnight.
Insulin in solution, along with insulin in gel (in syringe), were agitated on an Eppendorf Smartblock at 600 rpm, 25 °C for 6 h. Before analysis, the samples in syringes was passed through a 0.22 μm filter to separate protein from the gelators and then used in the assays.
Dynamic light scattering was used to measure the hydrodynamic radius on a Zetasizer ZS (Malvern Panalytical). Measurements were carried out using a 4 mW He-Ne 633 nm laser module operating at 25 °C at an angle of 173° (back scattering) and results were analysed using Malvern DTS 7.03 software. There were ten replications for each of the samples with at least 12 measurements recorded for each run.
A 1 mM stock solution of thioflavin T was prepared in H2O. This thioflavin T was diluted in PBS (pH 7.4) so that the final thioflavin T concentration in each well was 25 μM in 100 μl. Another 100 μl of insulin solution from each of the gelators after passing through 0.22 μm filter was added to the wells. Thioflavin T fluorescence was measured using a fluorescence microplate reader (excitation 450 nm, emission 485 nm).
Insulin in solution and in gel were prepared as described above. In brief, 50 mg of CD-005 (gelator) was dissolved in 5 ml of K2CO3 (20 mg) solution overnight the day before experiment. Insulin was dissolved in 1.5 M Tris HCl buffer (pH 6.8) at protein concentration of 0.2 mg ml−1 and then added into 0.4 ml of gelator solution. The mixture was immediately transferred into a 3 ml syringe. All the syringes, including insulin solution only, gelator solution only, Tris HCl buffer only and insulin in gel, were agitated on Eppendorf Smartblock at 600 rpm, 25 °C for 6 h. Before insulin activity cell assay, all the samples were passed through a 0.22 µm filter to separate protein from gelator.
iLite Insulin Assay Ready Cells (purchased from Svar Life Sciences) was used as received to test insulin activity and assay was performed according to manufacturer’s instructions. Cells were quickly thawed in 37 °C water bath, 250 µl of which was diluted to 6 ml using full culture media (RPMI supplied with 10% FBS and 1% PSA). A total of 40 µl of cell diluent was then mixed with insulin solution at equal volume and incubated in white tissue culture plate at 37 °C for a further 5 h. Firefly luciferase substrate of 80 µl was then added into cells and incubated at room temperature for 15 min. The whole plate was read on plate reader for firefly luciferase luminescence intensity. Specifically, fresh insulin solution was prepared to make a calibration curve with insulin stock concentration ranging from 2,000 to 0 ng ml−1, where 1,000 ng ml−1 was selected as insulin activity test concentration. Concentration of separated insulin sample was determined by A280 on NanoDrop and calculated on the basis of protein sequence. Protein was diluted to indicated concentrations for activity test. Dilution of gel only and buffer only was identical to protein dilution.
Stock solutions of the gelator and protein were freshly prepared before mixing. A total of 20 mg of KCO3 was dissolved in 5 ml of distilled water, to which 50 mg of CD-005 (gelator) was added. This mixture was stirred overnight to dissolution. In a separate vial, 5.5 µl of 200 mg ml−1 of CaCl2 solution was added to 1 ml of 1.5 M Tris HCl, pH 6.8. The β-galactosidase was added to give a 10 mg ml−1 solution. A total of 0.4 ml of the gelator in buffer was transfered to a vial, to which 1 ml of the protein solution was added. These were agitated and rapidly (less than 1 min) transferred into the syringe.
Gel-loaded syringes were stored in a thermostated incubator for the indicated amount of time, before being retrieved and assayed for function. Protein was recovered from the syringe by passing through a syringe filter (0.22 mm). A total of 50 µl of 100 µg ml−1 of β-galactosidase solution (this was adjusted using PBS as TRIS can inhibit the activity of this protein) was added into the wells of a 96-well plate, containing 100 µl of 16 mM oNPG (4.82 mg ml−1). The absorbance was then measured at 420 nm each minute for 10 min. Activity was normalized to total protein mass using a standard BCA assay, as the non-optimized calcium stabilized gels did not give 100% recovery from a single extrusion.
Gels containing β-galactosidase were prepared as described above. Five gels and one solution of β-galactosidase (that is, in the absence of gel) were triple-bagged and put in a parcel. They were posted using Royal Mail signed delivery. A temperature logger was included in the parcel. In total, the package was in delivery for 3 days. A readout of the temperature logger is shown in Supplementary Fig. 12 (the range was 17.6–24.2 °C). On receipt, samples were visually inspected for leaks (none seen) and to confirm that the gels were intact. Samples were recovered and assayed as above.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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