Next Generation Salivary Lubrication Enhancer Derived from Recombinant Supercharged Polypeptides for Xerostomia

Abstract

Insufficient retention of water in adsorbed salivary conditioning films (SCFs) because of altered saliva secretion can lead to oral dryness (xerostomia). Patients with xerostomia sometimes are given artificial saliva, which often lacks efficacy because of the presence of exogenous molecules with limited lubrication properties. Recombinant supercharged polypeptides (SUPs) improve salivary lubrication by enhancing the functionality of endogenously available salivary proteins, which is in stark contrast to administration of exogenous lubrication enhancers. This novel approach is based on establishing a layered architecture enabled by electrostatic bond formation to stabilize and produce robust SCFs in vitro. Here, we first determined the optimal molecular weight of SUPs to achieve the best lubrication performance employing biophysical and in vitro friction measurements. Next, in an ex vivo tongue-enamel friction system, stimulated whole saliva from patients with Sjögren syndrome was tested to transfer this strategy to a preclinical situation. Out of a library of genetically engineered cationic polypeptides, the variant SUP K108cys that contains 108 positive charges and two cysteine residues at each terminus was identified as the best SUP to restore oral lubrication. Employing this SUP, the duration of lubrication (Relief Period) for SCFs from healthy and patient saliva was significantly extended. For patient saliva, the lubrication duration was increased from 3.8 to 21 min with SUP K108cys treatment. Investigation of the tribochemical mechanism revealed that lubrication enhancement is because of the electrostatic stabilization of SCFs and mucin recruitment, which is accompanied by strong water fixation and reduced water evaporation.

Keywords: Sjögren’s syndrome; biolubrication; dry mouth; ex vivo oral lubrication system; mucins; protein adsorption; recombinant supercharged polypeptides; saliva; salivary substitutes.

Scheme 1. Schematic Representation of SUP Fabrication via Recombinant Protein Expression and Interaction with Naturally Occurring Saliva from Healthy Volunteers and Patients Suffering from Sjögren’s Syndrome

Figure 1

Kinetics of SCF formation and SUP induced viscoelastic modification. The quartz crystal microbalance with a dissipation (QCM-D) response to adsorption of salivary proteins forming a SCF, and the effect of intermediate SUP adsorption and renewed exposure to saliva to form the secondary SCF (S-SCF). (a–f) The control with intermediate buffer/no SUP adsorption, with SUP K72, K108, K144, K108cys, and K144cys, respectively. (g) Structural softness of the SCF after intermediate exposure to buffer or SUP (black columns) and after renewed exposure to saliva (S-SCF, red columns). (h) The full spectrum XPS scans, showing the chemical element of each surface. (i) The amount of glyco group on each surface. The error bars represent the SD over three independent measurements. *Statistically significant (p < 0.05) differences in structural softness with respect to control. ^#Significant differences (P < 0.05) in structural softness and glycosylation of S-SCF treated with K108cys with respect to K72, K108, and K144. ^&Significant difference in structural softness and glycosylation of S-SCF treated with K144cys with respect to K72.

Figure 2

In vitro, nanoscale lubrication properties of the SUP-modified S-SCFs for different SUP molecular weights. The friction force vs normal force measured using a colloid probe atomic force microscope, plots (a,b) used to calculate the COF as a slope of the linear fits presented in (c). (d) The correlation between structural softness of the S-SCF after interaction with SUPs and the resulting COF. Reconstituted human whole saliva (RWS) was used for these measurements. *Statistically significant (p < 0.05) differences in the COF of S-SCFs with respect to bare crystals. ^#Significant differences (p < 0.05) in the COF of all S-SCF’s treated with SUPs with respect to the S-SCF treated with buffer. ^&Significant difference in the COF of K108cys- and K144cys-treated S-SCFs with respect to the S-SCFs fabricated with K72 or K108. ^@Significant difference in the COF between films generated by K144 and K108cys.

Figure 3

Ex vivo, macroscale lubrication properties of the SUP-modified S-SCFs involving healthy and patient saliva. Relief and relief period of the S-SCF measured with healthy saliva (HSCF) and saliva from patient individuals (HSCF) in an ex vivo tongue-enamel friction system. (a) Healthy S-SCF with intermediate buffer treatment. (b) Healthy S-SCF with intermediate K108cys treatment. (c) Patient S-SCF with intermediate buffer treatment. (d) Patient S-SCF with intermediate K108cys treatment. (e) Relief of the SCF and S-SCF involving healthy saliva and saliva from patient individuals. (f) Relief period for patient saliva and healthy saliva. Error bars represent the SD over three independent measurements. Stimulated human whole saliva (SWS) and Sjögren patient saliva was used for the HSCF and the PSCF, respectively. (g) Schematic representation of the SUP restoring the oral lubrication.*Statistically significant (P < 0.05) differences in the relief period of the S-SCF with intermediate K108cys treatment with respect to the S-SCF with intermediate buffer treatment both for healthy and patient saliva. ^#Statistically significant (P < 0.05) differences between healthy and patient S-SCF, respectively, either for intermediate buffer treatment or K108cys treatment.

Figure 4

In vitro, macroscale lubrication properties of the SUP-modified SCFs from healthy and patient saliva. Relief and the relief period of the S-SCF with patient saliva (PSCF) and healthy saliva (HSCF) at the silicon rubber-germanium sliding interface. (a) Healthy S-SCF with intermediate buffer treatment. (b) Healthy S-SCF with intermediate K108cys treatment. (c) Patient S-SCF with intermediate buffer treatment and (d) patient S-SCF with intermediate K108cys treatment. (e) Relief of the SCF and the S-SCF in patient saliva and healthy saliva. (f) Relief period for patient saliva and healthy saliva. Error bars represent the SD over three independent measurements. SWS and Sjögren patient saliva was used for HSCF and PSCF, respectively. *Statistically significant (P < 0.05) differences in the relief period of the S-SCF with intermediate K108cys treatment with respect to the S-SCF with intermediate buffer treatment both for healthy and patient saliva. ^#Statistically significant (P < 0.05) differences between the healthy and the patient S-SCF, respectively, either for intermediate buffer treatment or K108cys treatment.

Figure 5

Tribochemistry of the SUP-modified S-SCFs from healthy and patient saliva. Typical FTIR adsorption bands for the S-SCF with patient saliva (PSCF) and healthy saliva (HSCF) treated with K108cys or buffer on a Ge crystal surface during sliding with PDMS pin (1 mm/s; loading force 450 mN) as a function of time. Clearly visible are the polysaccharide peaks (950–1200 cm^–1), the amide I peaks indicative of proteins (1600 and 1700 cm^–1), and the peaks between 2500 and 4000 cm^–1 are indicative of water. (a) HSCF treated with buffer. (b) PSCF treated with buffer. (c) HSCF treated with K108cys. (d) PSCF treated with K108cys. (e) The ratio between the saccharide and water peak area for the HSCF and PSCF treated with K108cys and buffer, respectively. (f) The absorbance of water on the HSCF and PSCF with K108cys or buffer treatment in function of time. Each data point and error bar on HSCF is an average and SD from triplicate measurements performed with healthy saliva and saliva from Sjögren’s syndrome patient.

Scheme 2. Schematic Illustration Showing the Strong Water Immobilization of the Layered S-SCF by Introduction of SUP