Retout, M. | bioRxiv Science

bioRxiv2023

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

We report the controlled release of an antimicrobial peptide using enzyme-activatable prodrugs to treat and detect Candida albicans and Porphyromonas gingivalis. Our motivation lies in the prevalence of these microorganisms in the subgingival area where the frequency of fungal colonization increases with periodontal disease. This work is based on an antimicrobial peptide that is both therapeutic and induces a color change in a nanoparticle reporter. This antimicrobial peptide was then built into a zwitterionic prodrug that quenches its activity until activation by a protease inherent to these pathogens of interest: SAP9 or RgpB for C. albicans and P. gingivalis, respectively. We first confirmed that the intact zwitterionic prodrug has negligible toxicity to fungal, bacterial, and mammalian cells absent a protease trigger. Next, the therapeutic impact was assessed via disk diffusion and viability assays and showed a minimum inhibitory concentration of 3.1 - 16 {micro}g/mL, which is comparable to the antimicrobial peptide alone (absent integration into prodrug). Finally, the zwitterionic design was exploited for colorimetric detection of C. albicans and P. gingivalis proteases. When the prodrugs were cleaved, the plasmonic nanoparticles aggregated causing a color change with a limit of detection of 10 nM with gold nanoparticles and 3 nM with silver nanoparticles. This approach has value as a convenient and selective protease sensing and protease-induced treatment mechanism based on bioinspired antimicrobial peptides. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/568833v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e9cf5corg.highwire.dtl.DTLVardef@12cb36forg.highwire.dtl.DTLVardef@1b862f7org.highwire.dtl.DTLVardef@697946_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

A di-arginine additive for dissociation of gold nanoparticle aggregates: A matrix-insensitive approach with applications in protease detection

We report the reversible aggregation of gold nanoparticle (AuNPs) assemblies via a diarginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that HS-PEG thiol group has higher affinity for the AuNPs surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNPs suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates or even sea water. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favorized. Moreover, starting with AuNPs already aggregated ensured the absence of background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNPs assemblies and to solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluid. Trypsin was chosen as model enzyme and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrate. The increase of positive charge of the HS-PEG-peptide conjugate quenched the dissociation capacity of the HS-PEG molecules which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/508454v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@13a6f3org.highwire.dtl.DTLVardef@35b42dorg.highwire.dtl.DTLVardef@1c8249forg.highwire.dtl.DTLVardef@dc8236_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗