Nanoceria-Based Phospholipase-Mimetic Cell Membrane Disruptive Antibiofilm Agents

Abstract

Table of Contents Chemicals and Methods S3 Characterisation of nanoparticles S4 POPG hydrolysis by MALDI-Mass Analysis S7 Phosphatidylcholine hydrolysis UV-Vis assay scheme S8 UV-Visible analysis of p-Nitrophenylphosphatidyl choline (NPPC) hydrolysis S9 Kinetic parameters of NPPC hydrolysis S10 31 P NMR analysis of NPPC hydrolysis S11 XPS and HRTEM analysis of nanoparticles S12 Phosphatidylcholine hydrolysis by nanoceria S13 Confocal study of bacterial cell membrane disruption S13 CFU analysis of Salmonella growth and death curve S15 Confocal microscopy analysis of antibacterial activity S17 Study of pre-attachment phase of biofilm formation S18 Synthesis and characterization of FITC-tagged PAA-Cnp S18 Localization of PAA-Cnp inside biofilm S21 Dispersibility of nanozymes in solution S21 SEM images of biofilm formation/disruption S22 Comparison of biofilm inhibition using oxidase-mimetic S22 Antibacterial activity on various pathogenic bacteria S23 Antibiofouling studies on urinary catheters S28 MIC quantification and comparison S29 Cytotoxicity study against HeLa cells S30 S3 1. Chemicals Cerium chloride heptahydrate (CeCl3.7H2O) was purchased from Avra synthesis Pvt. Ltd. Cerric ammonium nitrate ((NH4)2Ce(NO3)6), ethylenediamine, hydrazine hydrate and ammonium hydroxide, 3-aminopropyltriethoxysilane used for various synthesis were purchased from Sisco Research Laboratories. Sodium polyacrylate and flouresceine isothiocynate used for preparing PAA-Cnp and FITC-tagged PAA-Cnp nanoparticles respectively were purchased from Sigma Aldrich. Trizma base and phosphotidyl choline (lecithin) used for hydrolysis assays were also purchased from Sigma Aldrich. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol and pnitrophenyl phosphorylcholine used for other hydrolysis assays were purchased from TCI Co. Ltd. and Alfa Aesar Fischer Scientific respectively. 2. Characterization Methods Scanning electron microscopy (SEM) and EDX spectrawere performed on a Carl-Zeiss Ultra 55, FEI Sirion UHR SEM and ESEM-Quanta instruments,respectively. Transmission electron microscopy (TEM), high resolution TEM (HRTEM), electron diffraction X-Ray (EDX) analysis and selected area electron diffraction (SAED) were recorded on JEOL transmission electron microscope operating at 200 kV after casting a drop of nanoparticle dispersion in acetone over Cu grid. Powder XRDwas recordedon Philips PANalytical X-ray diffractometer by using a Cu-Kα (1.5406 Å)radiation. X-ray photoelectron spectroscopy (XPS) was acquired on AXIS ULTRA, KRATOS ANALYTICAL, SHIMADAZU. Raman spectroscopy was performed on HORIBA JOBIN YVON LabRAM HR Raman spectrometer (532 nm laser. FT-IR spectra were recorded on aPerkinElmer FT-IR spectrometer. Zeta potential was measured usingMalvern Zetasizer Nano UK.UV-Vis absorption spectra were acquired onSHIMADAZU UV-2600 spectrophotometer. Fluorescence spectroscopy was measured by using HORIBA JOBIN YVON (Fluoromax-4 Spectrofluorometer) instrument. All the 31 P NMR spectra were recorded using AV400 and AV500 MHz Avance Bruker High Resolution...

New facets of nanozyme activity of ceria: lipo- and phospholipoperoxidase-like behaviour of CeO2 nanoparticles

“…Khulbe et al focused their attention on the phospholipase-like (phospholipase C) mimetic activity of polymer-coated CeO 2 nanoparticles. 16 CeO 2 nanoparticles demonstrated bactericidal activity, causing membrane disruption in a wide range of pathogens, including respiratory pathogens ( Klebsiella pneumoniae ) and biofilm-forming bacteria. 16 Important data on the molecular mechanisms of CeO 2 nanoparticles' interaction with the cell membrane model were obtained by Liu et al , 53 who showed that the interaction of CeO 2 nanoparticles with phosphocholine liposomes composed of two different types of phospholipid, DOPC and DPPC, proceeds mainly via the phosphate groups in the phospholipids.…”

“…16 CeO 2 nanoparticles demonstrated bactericidal activity, causing membrane disruption in a wide range of pathogens, including respiratory pathogens (Klebsiella pneumoniae) and biolm-forming bacteria. 16 Important data on the molecular mechanisms of CeO 2 nanoparticles' interaction with the cell membrane model were obtained by Liu et al, 53 who showed that the interaction of CeO 2 nanoparticles with phosphocholine liposomes composed of two different types of phospholipid, DOPC and DPPC, proceeds mainly via the phosphate groups in the phospholipids. 53 The present study demonstrates that citrate-stabilised CeO 2 sol exhibits prooxidant activity towards organic hydroperoxides that were different in nature.…”

“…[3][4][5][6][7] Cerium dioxide is a striking representative of a new generation of nanozymes. The enzyme-like properties of nanocrystalline CeO 2 , mimicking the functions of some natural enzymes, superoxide dismutase (SOD), 8-10 catalase 11 and oxidase, 12,13 have recently been supplemented with the discovery of new types of biochemical activity, including phosphatase-, 14 photolyase-, 15 phospholipase- 16 and nuclease-like 17 properties. This makes cerium dioxide a unique multifunctional nanozyme and expands the scope of its future biomedical applications.…”

Recent Trends in Composite Nanozymes and Their Pro-Oxidative Role in Therapeutics

“…Nanomaterials mimic natural enzymes in action and not only display pro- or antioxidative catalytic activity but also mimic hydrolase-like activity ( Jiang et al,2019 ). For instance, nanomaterials exhibiting DNAse ( Chen et al, 2016 ) and phospholipase activity ( Khulbe et al, 2020 ) cause hydrolytic cleavage of extracellular DNA (eDNA) and phospholipids, respectively, and display antibiofilm activity. Hence, a brief understanding of the mechanism of different enzyme mimetic actions of nanozymes becomes important toward designing novel nanomaterials.…”

“…A major component of the bacterial cell membrane is phospholipid, which plays a key role in biofilm formation, and its cleavage could disrupt biofilms. Phospholipase-mimetic ceria-based nanozyme could hydrolyze the phospholipids in bacterial cell membranes and bacterial biofilms efficiently ( Khulbe et al, 2020 ). The study also demonstrated the potential of nanozyme in preventing bacterial colonization on the surface of urinary catheters.…”