Structural and mechanical properties of folded protein hydrogels with embedded microbubbles

Globular folded proteins are powerful building blocks to create biomaterials with mechanical robustness and inherent biological functionality. Here we explore their potential as advanced drug delivery scaffolds, by embedding microbubbles (MBs) within a photo-activated, chemically cross-linked bovine serum albumin (BSA) protein network. Using a combination of circular dichroism (CD), rheology, small angle neutron scattering (SANS) and microscopy we determine the nanoscale and mesoscale structure and mechanics of this novel multi-composite system. Optical and confocal microscopy confirms the presence of MBs within the protein hydrogel, their reduced diffusion and their effective rupture using ultrasound, a requirement for burst drug release. CD confirms that the inclusion of MBs does not impact the proportion of folded proteins within the cross-linked protein network. Rheological characterisation demonstrates that the mechanics of the BSA hydrogels is reduced in the presence of MBs. Furthermore, SANS reveals that embedding MBs in the protein hydrogel network results in a smaller number of clusters that are larger in size (∼16.6% reduction in number of clusters, 17.4% increase in cluster size). Taken together, we show that MBs can be successfully embedded within a folded protein network and ruptured upon application of ultrasound. The fundamental insight into the impact of embedded MBs in protein scaffolds at the nanoscale and mesoscale is important in the development of future platforms for targeted and controlled drug delivery applications.


Diffusion of MBs in buffer and in the BSA hydrogel
SI Figure 3: The path of 10 trajectories for A) MBs in buffer solution (sodium phosphate with 1% glycerol) compared to B) MBs in the BSA hydrogel. Optical images for both were taken with an inverted microscope (Nikon 90i, Nikon, Japan) in 30 ms time steps, for a total 570 ms (19 time steps). In A) the mean squared displacement (MSD) was determined to be 3 ± 1 x10 -11 m 2 in X direction and 9 ± 3 x10 -11 m 2 in the Y direction. The difference in X and Y direction arises due to a drift in the velocity. The resultant diffusion coefficients (D) were 2 ± 1 x10 -11 m 2 s -1 and 8 ± 3 x10 -11 m 2 s -1 in the X and Y directions. In B) the MSD in the X and Y direction are 6 ± 2 x10 -13 and 7 ± 2 x10 -13 m 2 , and D in the X and Y direction are 1.1 ± 0.3 x10 -13 m 2 s -1 and 1.2 ± 0.4 x10 -13 m 2 s -1 . The movement of MBs is significantly reduced when embedded in the BSA hydrogel.    Figure 4: Examples of the raw gelation curves characterised with rheology (as described in the main text) for BSA (red) and BSA:MB (blue) hydrogels. Different fitting parameters using SI Equation 1 were compared where A) fits the gelation curve with one exponential component and B) fits the gelation curve with two exponential components. The results of the fits are summarised in SI Table 1.
C 624 ± 6 x10 -4 629 ± 6 x10 -4 60 ± 5 E-4 72.92 ± 0.001 t 0 (s) 127.2 ± 0.2 131.6 ± 0.6 127.9 ± 0.2 116.0 ± 0.5 τ 1 (s) 1112 ± 9 1113 ± 9 741 ± 14 33.9 ± 0.5 τ 2 (s) 1 ± 0 1 ± 0 3974181 ± 9.69 9E7 1061 ± 7 G 0 (Pa) -67 ± 6 -49 ± 5 -74 ± 6 40 ± 7 Adj. R-Square 0.99625 0.99616 0.99708 0.99763 Table 1: Fitting values with SI Equation 1 compared for one exponential constant to two exponential constants for the gelation curves in SI Figure 4. The success of the fitting can be quantified by comparing the resultant values from the fit to the data. For example, the sum of constants B 1 and B 2 is theoretically equivalent to the difference between the peak in G' and G' ∞ . When fitting with two exponential constants in this example, the BSA hydrogel results in a negative value for G' ∞ and the BSA:MB hydrogel results in a negative value for B 1 , which suggests that use of two exponential constants is unsuitable for fitting the gelation curves for BSA and BSA:MB hydrogels Time constant for relaxation of the gelation curves for BSA and BSA:MB hydrogels SI Figure 5: Time constant, τ, was determined from the average gelation curve fits of the data shown in Figure 5 in the main manuscript, using Equation 1 in the main manuscript, shown in the absence (red) and presence of MBs (blue).

Linear fits of the gelation curves.
BSA hydrogel BSA:MB hydrogel K max (Pa s -1 ) 55.8 ± 66.9 38.0 ± 0.9 C (Pa) -5320 ± 70 -3700 ± 100 Adj. R-Square 0.9989 0.99158 Table 2: Linear fit results for the BSA and BSA:MB hydrogel from the application of a linear fit. The linear fit used was G' = k max t + C, where C is where linear fit would be if extrapolated to t = 0 s.

Efficiency of photo-activated cross-linking reaction for BSA proteins
To quantify the number of dityrosine bonds formed in the chemically crosslinked in the hydrogel is broken down with acid and the fluorescence emission of dityrosine bonds assessed. Firstly, 4 mL of the pre-gel solution is pipetted into a 2 mL centrifuge tube with Pipetman Diamond (D10) pipette tips (Gilson, USA) to ensure accurate pipetting of low volumes. The pre-gel solution is irradiated under lamp for 5 mins to gel the sample. To digest the hydrogel, 2 mL of 6 M HCl is added and boiled at 105°C for 2 hours. The acid hydrolysis product was neutralised by adding 120 mL of 5 M NaOH to 100 mL of the acid, and diluted in Na 2 CO 3 -NaHCO 3 buffer. 1 SI Figure 6: Gelation curves for BSA (red) and BSA:MB hydrogels (blue), showing only the first 300 s, where the LED lamp that initiates the cross-linking procedure is turned on at t = 60s. and turned off at t = 660 s. Example of the linear fits after the lamp is turned on, to determine the gradient of the linear region, k max , for both hydrogel systems. SI Table 2 summarises the fitting values from the linear fit.
Cuvette-based steady-state fluorescence emission spectroscopy measurements were performed on an Edinburgh Instruments FLS980 fluorescence spectrophotometer. All samples were temperature controlled to 20°C during the measurements. Quartz cuvettes were used to hold 3 mL of the sample. Samples were excited with a 450 W Xenon lamp at 315 nm, the excitation wavelength for dityrosine bonds. An emission spectrum was produced from collecting the emission from 330 -600 nm with a red-sensitive photomultiplier tube (PMT, Hamamatsu R928 PMT) used for detection of light. A 4 nm bandwidth was used for both excitation and emission slits, which controls the amount of light passing through from the Xe lamp and to the PMT. The emission data was collected in steps of 1 nm, with a dwell time of 0.1 s, with 5 scans averaged for each sample to improve the signal to noise ratio. The data was compared at an emission of 415 nm.

Stress-Strain Curves
SI Figure 9: A) Stress strain curve in the absence (red) and presence of MBs (blue). B) A storage modulus (G') was calculated from the linear region of the stress strain curve. C) The energy dissipated when loading and unloading a shear strain, and efficiency of the loading and unloading cycle in the absence and presence of MBs, where the efficiency was determined with SI Equation 2.
.. Where Eff is the efficiency, strain max is the maximum shear strain that is applied to the hydrogel, stress max is the shear stress recorded at the maximum shear strain. 2