Engineering antimicrobial coating of archaeal poly-γ-glutamate-based materials using non-covalent crosslinkages

We are now entering a new age of intelligent material development using fine, sustainable polymers from extremophiles. Herein we present an innovative (but simple) means of transforming archaeal poly-γ-glutamate (PGA) into extremely durable polyionic complexes with potent antimicrobial performance. This new supra-polymer material (called PGA/DEQ) was subjected to nuclear magnetic resonance and X-ray diffraction spectroscopies to characterize in structural chemistry. Calorimetric measurements revealed its peculiar thermal properties; to the best of our knowledge, it is one of the most heat-resistant biopolymer-based polyionic complexes developed to date. PGA/DEQ is particularly useful in applications where surface functionalization is important, e.g., antimicrobial coatings. The spontaneously assembled PGA/DEQ coatings (without any additional treatments) were remarkably resistant to certain organic solvents (including chloroform), even at high salt concentrations (theoretically greater than those found in sea water), and various pH values. However, the pH-response tests also implied that the PGA/DEQ coatings could be removed only when concentrated citrate di-salts were used, whereas most crosslinked polymer composites (e.g., thermoset matrices) are difficult to recycle and treat downstream. We also discuss PGA/DEQ-immobilized surfaces that exhibit enigmatic microbicidal mechanisms.


Supplementary Figure 3. Electron microscopy of PGAIC-coated microfibers
on plastic surfaces. SEM images, from (a) a non-coated HIYEX non-woven plastic cloth (or sheet) (from Kuraray, Japan); the PGA/HDP-coated sheets (b) before and (c) after the EtOH (> 99.5 wt %)-soaking process (see Fig. 5); and the PGA/DEQ-coated sheets (d) before and (e) after the same severe treatment. The length of the black bar is 10 μm. In particular, the image e indicates the excellent durability of the PGA/DEQ coatings against alcohols.

Supplementary Figure 4. Growth curves of E. coli.
The viable cells (~ 1.7 × 10 5 CFU) were first inoculated into Luria-Bertani (LB) media (5 mL), each carrying a disk (12 mm dia.) formed from PGA/DEQ-coated sheets (open symbols) and PGA/HDP-coated sheets (closed symbols) treated in the following ways: (a) soaking in EtOH (circles) or CHCl 3 (triangles); and (b) soaking in 1.5% NaCl (diamonds), 3.0% NaCl (squares), or 5.0% NaCl (squares with crosses (or ballet boxes with an x)). The (net) growth rates of the colonies were then estimated by monitoring the culture turbidity at 600 nm using a spectrophotometer (n = 3). The standard deviations observed in the latter treatment were actually < 5% (0 to a maximum of 0.04). Symbols in parentheses represent the images of the cultures (top) acquired at the end of 36-h cultivation; the BPB-stained disks (bottom) were essentially the same as the PGAIC-coated sheets used in the experiments, the darkness of which briefly corresponded to the quantity of PGAICs retained on the surfaces.

Supplementary
(a) Viable cell counts in the liquid culture media (n = 3), indicating the expression of a fast elimination (or killing) mechanism (within 10 min), followed by sustainable antimicrobial performance (after 180 min). Particularly, it is noteworthy to be significant in the durable (e.g., extraordinary water-resistant) PGA/DEQ coatings from the viewpoint of improved contact-killing surfaces 4 . (b) Counts of viable cells adhered in the disk samples after cultivation (n = 3). The moisture of samples was gently drained, and their weights were calculated to be 28 mg averagely (n = 15), the scores of which were virtually constant regardless of the incubation times, presumably owing to the size stability of HIYEX non-woven plastic cloth. Each drained sample was then soaked into 1 mL of 100 mM citrate di-salts at 25°C for 10 min, and the resulting suspensions were subjected to the counting experiment of viable cells (see the Method section). On the PGA/DEQcoated disks, the viable cells (though their numbers are surely not large) were counted even under the circumstances where E. coli cells have disappeared from the liquid media (e.g., after the 540-min incubation), providing insight into a functional surface actively involved in bacteria elimination.

Supplementary Figure 7. Sustainable antimicrobial performance of PGAIC coatings.
Abbreviations: E. c, Escherichia coli; S. a, Staphylococcus aureus; B. s, Bacillus subtilis. Cells of microorganisms (~ 5.5 × 10 5 CFU) were inoculated into LB media (5 mL), each carrying a disk (12 mm dia.) from the non-coated (a), PGA/HDPcoated (b, before; c, after the EtOH soaking), and PGA/DEQ-coated (d, before; e, after the EtOH soaking) (see Fig. 5), and then cultured at 37°C for 5 days. The cultures of images a and c actually reached to their stationary phase after 24-h incubation, whereas the use of PGA/DEQ coatings (images d and e) brought about the long-term suppression against cell growth of Gram-positive bacteria (e.g., S. a and B. s) in addition to Gram-negative bacteria (e.g., E. c). c Cooperative PGAIC formation was first demonstrated and then kinetically characterized using the NICA model. The cooperativity (n)/affinity (K d , mM) scores of PGA for DEQ 2+ and HDP + can be found in the table-type inset. Interestingly, the composition analysis using NMR proved that the carboxyl groups of all the PGAICs in a were constantly and completely transformed with QA moieties, presumably owing to their (potent) cooperative bindings.

Supplementary Figure 10. Schematic diagrams of (A) the onsite synthesis of the PGA/DEQ coatings and (B) their quantitative colorimetric assay.
Steps (a), first coating of a PGA solution on the surfaces of base materials; (b), surface functionalization via the spontaneous coating of PGA as a widely applicable adhesive 8 ; (c), second coating with a DEQ 2+ solution on the PGA-mounting surfaces to briefly form PGA/DEQ onsite; (d), 30-min soaking in methanol (1 mL/disk; repeated a total of three times per treatment process) with gently shaking to wash out excess (unbound) DEQ 2+ and to leave only durable PGA/DEQ coatings onsite; (e), 10-min immersion of PGA/DEQ-coated materials in a BPB concentration (1 mL/disk) to form BPB/DEQs (see Supplementary Fig.  11, panel a); (f), 5-min soaking in water (5 mL/disk; repeated five times) to remove unbound BPB anions and remain water-insoluble BPBICs; (g), 24-h soaking in methanol (1 mL/disk) to extract BPBIC molecules from the dried surfaces of the resulting disks and ultimately determine the amount of PGAICs thereby immobilized as PGA/DEQ coatings; and (h), quantitative analysis of BPBICs (see Supplementary Fig. 12).