Dual-sensitive antibacterial peptide nanoparticles prevent dental caries

Background: Dental caries is the most prevalent bacterial biofilm-induced disease. Current clinical prevention and treatment agents often suffer from adverse effects on oral microbiota diversity and normal tissues, predominately arising from the poor biofilm-targeting property of the agents. Methods: To address this concern, we herein report dual-sensitive antibacterial peptide nanoparticles pHly-1 NPs upon acid and lipid-binding for treatment of dental caries. Amino acid substitutions were performed to design the peptide pHly-1. The potential, morphology and secondary structure of pHly-1 were characterized to elucidate the mechanisms of its pH and lipid sensitivity. Bacterial membrane integrity assay and RNA-seq were applied to uncover the antimicrobial mechanism of peptides under acidic condition. The in vitro and ex vivo antibiofilm assays were used to determine the antibiofilm performance of pHly-1 NPs. We also carried out the in vivo anti-caries treatment by pHly-1 NPs on dental caries animal model. Oral microbiome and histopathological analyses were performed to assess the in vivo safety of pHly-1 NPs. Results: The pHly-1 peptide underwent the coil-helix conformational transition upon binding to bacterial membranes in the acidic cariogenic biofilm microenvironment, thereby killing cariogenic bacteria. Under normal physiological conditions, pHly-1 adopted a β-sheet conformation and formed nanofibers, resulting in negligible cytotoxicity towards oral microbes. However, in acidic solution, pHly-1 NPs displayed reliable antibacterial activity against Streptococcus mutans, including standard and clinically isolated strains, mainly via cell membrane disruption, and also suppressed in vitro and human-derived ex vivo biofilm development. Compared to the clinical agent chlorhexidine, in vivo topical treatment with pHly-1 NPs showed an advanced effect on inhibiting rat dental caries development without adverse effects on oral microbiota diversity and normal oral or gastric tissues. Conclusion: Our results demonstrated the high efficacy of dual-sensitive antimicrobial peptides for the selective damage of bacterial biofilms, providing an efficient strategy for preventing and treating dental caries.

. The pH of BHI medium and the bacterial killing activity of CHX and pHly-1. A, The determination of the pH of BHI medium. The initial pH of BHI medium was 7.0. After 48 hours of culture, the pH of the medium did not change; but the pH of medium contained S.mutans UA159 (1× 10 6 ) decreased, which might be related to the acid-producing property of S.mutans UA159. B, The MIC was also performed when acidic BHI medium is modulated with different acids including HCl, H 2 SO 4 and HNO 3 . C, The MIC values of CHX against S.mutans UA159. The bacteria were incubated with series concentrations of CHX at pH 5.5 or 7.0 for 48 h. D, The time-killing kinetics of CHX against S.mutans UA159. The bacteria were treated by CHX at the concentration of 22 μM (4 MIC). E, The killing activity of 22 μM pHly-1 NPs was measured against S.mutans UA159 at pH 6.8 and 7.0, and the killing activity of 550 μM pHly-1 NPs was measured against S.mutans UA159 at pH 4.5 and 5.5, which all as revealed by counting the CFU of alive bacteria after 10 min incubation.F, The killing activity of 22 and 550 μM CHX was measured against S.mutans UA159 at different pHs as revealed by counting the CFU of alive bacteria after 10 min incubation. G, In this in vitro experiment only, the stock solutions of pHly-1 was PBS (4.5). The bacteria were treated with 22 (4 MIC) or 550 μM (100 MIC) of pHly-1 for 10 min under acidic conditions (4.5 and 5.5) and neutral conditions (6.8 and 7.0), respectively. All of the data are presented as average± SD. Each experiment consists of 3 repetitions.

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Supplementary Fig.4. The overall intermolecular forces analysis depending on the protonation status of histidine residues. The colors represent different types of forces provided by the side chains of amino acids. When the histidine residues were protonated, the overall intermolecular forces could become repulsive, leading to unstructured random coiled conformation. When the histidine residues were deprotonated, the overall intermolecular forces could become attractive due to intra-nanofiber hydrogen bonding and hydrophobicity interaction, which may cause fiber to tend to aggregate.

Supplementary Fig. 5. Experimental design for isolation, identification and treatment of clinical S.mutans strains.
Plaque biofilm samples were collected from 20 patients. After isolation, the S.mutans were preliminarily selected according to their morphological and further confirmed by the mass spectrometry and microbiochemical reaction. The isolated S.mutans were used as subjects to test the antimicrobial activity of pHly-1 NPs and CHX. Moreover, samples from 4 children were also used to establish the human-derived ex vivo biofilm model. repetitions.  The experimental procedure was similar to that to the MBIC 50 determination except for the addition of bacteria to glass slides in 24 well plates. The treatment concentration of pHly-1 NPs was 11 μM and after washing with PBS the adherent bacteria on the glass slide surface were characterized by SEM. Supplementary Fig.9. A, The effect of CHX on S.mutans UA159 biofilm development at different pHs as determined by scanned electronical microscopy (SEM). The red arrows and blue arrows represent EPS and S.mutans UA159, respectively. B and C, Quantitative analysis of the total viable cell count after the biofilms on glass slides were treated with control, CHX and pHly-1 NPs, respectively at pH 4.5(B) and 7.0 (C). The biofilm was removed and homogenized by sonication. The biofilm suspension was diluted, plated, and incubated for 48 h at 37 o C. All of the data are presented as average± SD. Each experiment consists of 3 repetitions.

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Supplementary Fig.10 A and B, The effect of CHX on S.mutans UA159 biofilms development at pH 4.5 (A) and pH 7.0 (B) as determined by Laser scanning confocal microscopy (LSCM). The red fluorescence represented the EPS and green fluorescence represented S.mutans.

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Supplementary Fig.13. The toxicity of pHly-1 NPs to human gingival fibroblast (HGF-1) was assessment by CCK-8 assay. DMEM medium(10 % FBS) was used for cell culture and DMEM medium was used for drug preparation in toxicity tests. The All of the data are represented as average±SD. Each experiment consists of 3 repetitions.