A Protease-Responsive Polymer/Peptide Conjugate and Reversible Assembly of Silver Clusters for the Detection of Porphyromonas gingivalis Enzymatic Activity

Abstract

We report the reversible aggregation of silver nanoparticle (AgNP) assemblies using the combination of a cationic arginine-based peptide and sulfur-capped polyethylene glycol (PEG). The formation and dissociation of the aggregates were studied by optical methods and electron microscopy. The dissociation of silver clusters depends on the peptide sequence and PEG size. A molecular weight of 1 kDa for PEG was optimal for the dissociation. The most important feature of this dissociation method is that it can operate in complex biofluids such as plasma, saliva, bile, urine, cell media, or even seawater without a significant decrease in performance. Moreover, the peptide–particle assemblies are highly stable and do not degrade (or express of loss of signal upon dissociation) when dried and resolubilized, frozen and thawed, or left in daylight for a month. Importantly, the dissociation capacity of PEG can be reduced via the conjugation of a peptide-cleavable substrate. The dissociation capacity is restored in the presence of an enzyme. Based on these findings, we designed a PEG-peptide hybrid molecule specific to the Porphyromonas gingivalis protease RgpB. Our motivation was that this bacterium is a key pathogen in periodontitis, and RgpB activity has been correlated with chronic diseases including Alzheimer’s disease. The RgpB limit of detection was 100 pM RgpB in vitro. This system was used to measure RgpB in gingival crevicular fluid (GCF) samples with a detection rate of 40% with 0% false negatives versus PCR for P. gingivalis (n = 37). The combination of PEG-peptide and nanoparticles dissociation method allows the development of convenient protease sensing that can operate independently of the media composition.

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed in Candida albicans and Porphyromonas gingivalis

“…The system was, again, more responsive when both proteases were present (LOD = 0.3 nM) and the color change followed in a more dramatic change from yellow to purple-blue ( Figure F, iii ). The tenfold increase in sensitivity of the silver system is consistent with previous work 35 . This data further suggests that due to their ability to produce a stronger plasmon resonance than gold, silver is able to absorb light more readily making it more sensitive to peptide assembly.…”

“…P-113, S1, and G1 were synthesized through standard solid-phase Fmoc synthesis, purified using high-performance liquid chromatography (HPLC), and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (see Methods). The cleavage of S1 by SAP9 (300 nM) and G1 by RgpB (200 nM) was first investigated in vitro using recombinant proteases via a 3-hour incubation at 37 °C in activity buffer as recommended by previous work (9.5 mM MES, 2.7 mM KCl, 140 mM NaCl, pH 5.5 for SAP9 and 0.2 M Tris HCl pH 7.6, 5 mM CaCl 2 , 100 mM NaCl and 40 mM TCEP for RgpB) 31 , 35 . MALDI-TOF showed obvious mass peaks corresponding to the cleaved fragments ( Figure 1 B-C ).…”

“…Citrate-capped AuNPs (15 nm diameter) were synthesized 62 , and AgNPs (20 nm diameter) were coated with bis(p-sulfonatophenyl)phenylphosphine (BSPP) via a seed-mediated growth procedure 61 . We previously demonstrated that the BSPP coating on silver, unlike citrate, can desorb from the AgNPs during positively charged peptide-mediated assembly, thus releasing Ag+ into the solution and permitting spontaneous assembly of the particles 35 . Both AuNP-citrate and AgNP-BSPP possess a distinct wavelength of light absorption as well as an associated extinction coefficient.…”

“…Finally, we used the zwitterionic peptide-prodrugs to produce a colorimetric signal that reports the presence of C. albicans or P. gingivalis proteases 36 , 37 . This color change is based on precious metal nanoparticles and plasmonic coupling leading to a change in optical behavior in the visible region 35 , 38 - 40 . In the work below, we characterize the prodrugs, their toxicity profiles, their selectivity to fungi and bacteria in vitro , and finally, show the diagnostic performance of the sensor.…”

Silver Nanoparticle Sensor Array for the Detection of SARS-CoV-2

“…The decrease in the ratiometric absorbance was fitted to a sigmoidal dose–response curve using GraphPad Prism 10 because the shift in absorbance often yields a sigmoidal curve as the concentration becomes diluted. ,, The sensitivity of each peptide to the viral particles was compared via the viral concentration, signifying the inflection point of the sigmoidal curve. (RRK) 2 was the most sensitive peptide because it had the lowest inflection point for all of the virus types except for the Delta variant.…”