Frog nest foams exhibit pharmaceutical foam-like properties

Foams have frequently been used as systems for the delivery of cosmetic and therapeutic molecules; however, there is high variability in the foamability and long-term stability of synthetic foams. The development of pharmaceutical foams that exhibit desirable foaming properties, delivering appropriate amounts of the active pharmaceutical ingredient (API) and that have excellent biocompatibility is of great interest. The production of stable foams is rare in the natural world; however, certain species of frogs have adopted foam production as a means of providing a protective environment for their eggs and larvae from a predators and parasites, to prevent desiccation, to control gaseous exchange, temperature extremes, and to reduce UV damage. These foams show great stability (up to 10 days in tropical environments) and are highly biocompatible due to the sensitive nature of amphibian skin. This work demonstrates for the first time, that nests of the Túngara frog (Engystomops pustulosus) is stable ex situ with useful physiochemical and biocompatible properties and is capable of encapsulating a range of compounds, including antibiotics. These protein foam mixtures share some properties with pharmaceutical foams and may find utility in a range of pharmaceutical applications such as a topical drug delivery systems (DDS).All data is either images or data files with collection parameters included. Funding provided by: Engineering and Physical Sciences Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000266Award Number: University of Strathclyde DTPCircular Dichroism Spectroscopy (CD)(Figure 1A) CD was used to investigate the overall secondary structure content of the proteins. Spectra were acquired using a Chirascan Plus (Applied Photophysics) instrument using a 0.1mm quartz cuvette (Hellma) at 20⁰C. All samples (10 mg/ml protein) were measured in the far-UV in a wavelength range of 180 nm to 280 nm range, with step size of 1 nm, bandwidth of 1 nm, and reading time of 1 s per nm. Triplicate measurements were taken for each sample run, baseline peak, PBS control and foam sample spectra, with triplicate spectra then averaged. Baseline and PBS traces where subtracted from the sample spectra before secondary structure predictions were made. All data analysis was performed using Global3 software and Excel. Fourier transform infrared spectroscopy (Figure 1B & C) Fourier transform infrared (FTIR) spectroscopy was carried out using a Nicolet iS10 Smart iTR spectrophotometer (Thermo Scientific). Solid and liquid foam spectra were recorded in the range on 4000 cm-¹ and 500 cm-¹, over 128 scans at a resolution of 4 cm-¹ and an interval of 1 cm-¹. Background spectra were measured and the foam spectra were corrected accordingly. Rheology (Figure 2A & B) Rheology measurements were determined using a HAAKE MARS Rotational Rheometer (Thermo Scientific). Foam samples were subjected to oscillation sweeps and time sweeps. All experiments were carried out using P20 upper plate and TM20 lower plate. The oscillation sweeps were completed with a 1 mm gap and 0.1 Pa to 200 Pa range. Time sweep experiments were run for 1 h, at 100 Pa and 3 Hz using a 0.5 mm gap. Data points were collected in triplicate and averaged before analysis was carried out. MTT cell viability assay (Figure 3) HaCaT cells (CLS, Eppelheim, Germany), a model human keratinocyte cell line were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine and 50 units/ml penicillin/streptomycin (cDMEM; Lonza, Slough, UK). Soluble foam proteins were prepared as above, with the supernatant being passed the a 0.22 μm filter (Millex 33 mm) and subsequently concentrated using an Amicon 10 kDa spin filter. The protein concentration was determined by Bradford assay (BioRad). The HaCaT cells were plated onto 96 well plates (~1x103 cells per well and grown to 80% confluence) and were treated with buffer containing foam proteins (concentrations indicated in the figures) prior to incubation at 37 ⁰C for 24 hours. After 24 hours the media was removed from the cells, and replaced with 50 μl of fresh media and 50 μl of MTT (5mg/ml) and incubated for 1 h at 37 ⁰C. This was followed by replacing the media with 100 μl DMSO and further incubation in the dark at room temperature for 30 minutes prior to reading the absorbance at 570nm[1]. Results were expressed as the % viability compared to non-treated cells ± SEM. Microscopy (Figure 4) Optical microscopy (Figure 4A): Whole foam was defrosted at room temperature before use. All foam images were taken using transmitted light on a Nikon SMZ1500 stereomicroscope with images acquired using a DFK 33UX264 CMOS camera (The Imaging Source Europe GmbH, Germany) using NIS-Elements AR.3.2 software. Fiji software (https://fiji.sc/) from the ImageJ (https://imagej.net) package was used for image analysis. Images and measurements are provided in the raw data. Atomic force microscopy (AFM)(Figure 4B): Samples (5 μl) of foam were deposited onto a freshly cleaved mica surface (1.5 cm x 1.5 cm; G250-2 Mica sheets 25 mm x 25 mm x 0.15 mm; Agar Scientific Ltd, Essex, UK) and left to dry at room temperature for 1h before imaging. The images were obtained by scanning the mica surface in air under ambient conditions using a Scanning Probe Microscope (MultiMode® 8, Digital Instruments, Santa Barbara, CA, USA; Bruker Nanoscope analysis software Version 1.40), operating using the PeakForce QNM mode. The AFM measurements were obtained using ScanAsyst-air probes, for which the spring constant (0.58 N/m; Nominal 0.4 N/m) and deflection sensitivity had been calibrated, but not the tip radius (the nominal value used was 2 nm). In vitro release of model compounds (Figure 5A, B) Aliquots (500 mg) of whole foam were loaded with dye by mixing with either 400 μl of Nile Red (NR; hydrophobic; 1 mg/ml in ethanol) or calcein (hydrophilic; 1 mg/ml in ethanol). The mixture was the placed in dialysis tubing and sealed before being submerged in 10 ml PBS at 37 ⁰C (pH 7; for NR-based release experiments, a 1:1 mixture of ethanol and PBS was used). The release experiments were carried out at 37 ⁰C, over 168 hours. To satisfy the perfect-sink conditions, which allow for the determination of the diffusion parameters, the supernatant was replaced with fresh PBS at 37 °C at each time point (indicated in the graphs). The concentration of model compound in each sample was determined spectrophotometrically at 490 nm (calcein) or 590 nm (NR) and the concentration determined with reference to standard control calibration curves. Experiments were performed in triplicate. In vitro Antibiotic release (Figure 5C) Two in vitro techniques were used to investigate the release of the antibiotic rifampicin. Dialysis: Aliquots (400 mg) of foam were mixed with 400 ml of rifampicin (25 mg/ml). The loaded foam was placed into dialysis tubing, sealed and submerged in 10 ml of PBS. This was incubated at 37 ⁰C for 48 hours. Samples (1 ml) were taken and fresh media added to maintain sink conditions. Samples where measured by spectrophotometrically at 475 nm[35] against a calibration curve. Transwell(Figure 5D): Aliquots of foam (100 mg) were loaded with 100 ml of rifampicin (25mg/ml). Loaded foam was the placed into a transwell collagen-coated permeable support (0.4 mm; Nunc). Each support was inserted into 24 well plate well containing 600ul of PBS. The plate was then incubated for 48 hours at 37 ⁰C. PBS (600ml) was collected from a well for each time point, and the absorbance measured at 475nm, in triplicate. References [1] Niles AL, Moravec RA, Worzella TJ, Evans NJ, Riss TL. 2013 High-Throughput Screening Methods in Toxicity Testing. , 107–127. (doi:10.1002/9781118538203.ch5