Ultra-strong bio-glue from genetically engineered polypeptides

The development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

. Phase diagrams of SUP glue. Here, K72-SDBS complexes are shown as an example. (A) Dependence of the coacervate formation on the lysine:SDBS molar ratio, which is the molar ratio of lysine in K72 protein to sulfonate group in SDBS. The concentration on the x-axis represents the total concentration of protein and SDBS. The weight ratio on the y-axis shows the weight ratio of complex, water content and supernatant of the whole system. The coacervates are the sum of K72-SDBS complex and water content. (B) The weight ratio of SUP-SDBS complex in the coacervates, i.e. complex/(complex+water content), as a function of the total concentration of protein and SDBS. The lysine:SDBS molar ratio was 1:1. (C) Dependence of the coacervate formation on pH and ionic strength.
The lysine:SDBS molar ratio was 1:1. The total concentration of protein and SDBS was 100 mg•mL -1 . The red, blue and gray columns represent the weight ratios (wt%) of SUP-SDBS complex, water content and supernatant in the whole system, respectively. The broad diffraction peak at q ≈ 4 nm -1 is due to the Kapton, which was used for sealing of the SUP-SDBS fluid sample. SAXS profile showed one broad diffraction peak corresponding to a d spacing of 40.0 Å. Based on a rough estimation of volumes and comparison between TGA and SAXS experimental data, the complex is composed of hydrated SUP units of ~2.2 nm thickness separated by regions containing disordered SDBS surfactant molecules of ~1.8 nm thickness. Scale bar: 100 µm.

Mechanical characterization of the SUP glue
Supplementary Figure 9. TGA investigation of the K72-SDBS glue before lap shear testing. The measurement shows that ca.14% water are remaining in the SUP glue system. Inset represents the sticky behavior of SUP glue applied on two glass surfaces.

Biodegradation behavior of the SUP glue
Supplementary Figure 21. Degradation experiment of the SUP glue (here K144-SDBS was taken as an example). Images on the left side show that the SUP glue prior to enzymatic treatment formed a gel with opaque appearance within theEppendorf vial. After the proteinase K digest, the SUP gluedispersion became transparent and formed a liquid as can be recognized when inverting the vial.

Water cleanability of the SUP glue
Supplementary Figure 23. The SUP glue (here GFP-K72-SDBS was taken as an example) can be easily cleanedby applying external forces such as harsh tap water or sonication treatment. The photographon the left represents a PE substrate pasted with GFP-K72-SDBS glue.After cleaning with water, the substrate shows a glue-free surface (right photograph).

Recyclability of the SUP glue
Supplementary Figure 24. The recyclability experiments of SUP glues. (A) The photographs show the SUP glue (GFP-K72-SDBS) on polyethylene (PE) that can be recovered by washing with excessive amount of H2O and application of external force. Pure water is firstly applied on the surface of a fractured glue substrate. Subsequently, the glue can be collected when applying shearing force, as indicated in the middle. Thereafter, the recovered glue is reapplied on the surface of substrates for a second test (lap shear characterization). (B) Comparison between the original and recovered K72-SDBS glues on PE surface. The lap shear measurements indicate that the recovered glue sample is as strong as the original one. All presented data are mean values ± SD from the mean from N = 3 independent measurements on independent samples. All p-values were calculated using two-sided Student's t-test.

Cytotoxicity Evaluation of the SUP Glue
Supplementary Figure 25. Cell viability measurements carried out with different concentrations of K72-SDBS complex ranging from 1 to 200 μM using HeLa cells. The control group was cells treated with culture medium. All presented data are mean values ± SD from the mean from N = 3 independent measurements on independent samples. Figure 26. Evaluation of cytotoxicity of the SUPs with HeLa cells. The measurements indicate that the cell viability is not affected by the addition of SUPs at a concentration as high as 100 µg•mL -1 (blue columns), which is also consistent with our previous investigation. 27 All presented data are mean values ± SD from the mean from N = 3 independent measurements on independent samples. Figure 27. (A) Encapsulation of various cells in the SUP glue, SUP-DNA glue or alginate hydrogel for 24 h in DMEM medium. The live/dead stain images were captured by confocal laser scanning microscopy. Alginate 3D culture was performed and the cells were loaded with the same densities as for the other groups. Three times each experiment were repeated independently with similar results. (B) Quantification of cell viability of the HeLa cells embedded in SUP glue and SUP-DNA glue via MTT. Important to note is that the PI dye also stains the non-cellular DNA. Thus, the background in SUP-DNA glue group appears in red to some extent due to the dye staining the salmon sperm DNA component of the glue. All presented data are mean values ± SD from the mean from N = 3 independent measurements on independent samples. In contrast, conventional cosmetic glue products always require specific cleansing lotion, which typically contains organic solvents. (B) The adhesion performance of SUP glue persisted for as long as two days without any discernible reduction.

In vivo linear wound hemostasis and healing
Supplementary

2.
There is a small amount of inflammatory cell infiltration near the wound, but the degree of infiltration is higher than the distal skin tissue.
3. There is no difference in the degree of inflammatory cell infiltration around the wound and the distal skin tissue

Supplementary Figure 31. (A)
In vivo adhesion demonstration of SUP glue and commercial adhesive Histoacryl® on pig liver and heart models. The major functional component of Histoacryl® is cyanoacrylate. SUP glue shows excellent hemostasis effect due to robust adhesion performance and flexible deformation behavior. In contrast, the Histoacryl® solidified very fast when applied on the wounds, leading to leaking behavior on certain organ surfaces particularly the liver as shown here. (B) In vivo adhesion demonstration of SUP glue and commercial adhesive Histoacryl® on different pig liver, kidney and heart models (N = 3 biologically independent samples). (C) The quantification of hemostatic performance in terms of coagulation time in wounded areasapplied with SUP glue and Histoacryl® on different tissues. Statistical analysis was carried out employing two-sided student's t-test (N = 3 independent biological samples); *, p =0.0220. Data are presented as mean values ± SD from the mean. The results showed a significant enhancement of the performance for the liver tissue sealed with SUP glue compared to Histoacryl®. Due to various treatments on heart and kidney (artery occlusion and pre-clotting) before glue application, there is no observable difference between SUP glue and Histoacryl® in heart and kidney models.  , but also suggestits good biodegradability due to almost non GFP signal detected in the SUP glue test groups. Three times each experiment was repeated independently with similar results. Scale bar: 100 µm.