Butnarasu, C

Caron, G

Vallaro, M

Visentin, S

English

Institutional Research Information System University of Turin

16 October 2023AperTO - Archivio Istituzionale Open Access dell'Università di TorinoOriginal Citation:A mucus model to evaluate the diffusion of drugs for more efficient cystic fibrosis therapiesTerms of use:Open Access(Article begins on next page)Anyone can freely access the full text of works made available as "Open Access". Works made availableunder a Creative Commons license can be used according to the terms and conditions of said license. Useof all other works requires consent of the right holder (author or publisher) if not exempted from copyrightprotection by the applicable law.Availability:This is the author's manuscriptThis version is available http://hdl.handle.net/2318/1732254 since 2020-02-28T14:14:04ZA MUCUS MODEL TO EVALUATE THE DIFFUSIONOF DRUGS FOR MORE EFFICIENT CYSTIC FIBROSIS THERAPIESPacheco DP1, Butnarasu C5, Bertoglio F2,3,4, Suarez Vargas N1, Briatico-Vangosa F1, van Uden S1, Visai L3, Petrini P1,Vallaro M5, Caron G5, Visentin S51 Department of Chemistry, Materials and Chemical Engineering "Giulio Natta" at Politecnico di Milano, Italy; 2 Molecular Medicine Department (DMM), Center for Health Technologies (CHT), UdRINSTM, University of Pavia, Italy; 3 Department of Occupational Medicine, Toxicology and Environmental Risks, Istituti Clinici Scientifici Maugeri S.p.A., IRCCS, Italy; 4 School of Advanced StudiesIUSS Pavia, Italy; 5 Molecular Biotechnology and Health Sciences Department, University of Torino, ItalyMaterials &MethodsCystic fibrosis (CF) mucus exhibits altered chemical and viscoelastic features, limiting its clearance and leading to chronic bacterialinfections. Current bacterial culture fails to recreate bacteria communities and microenvironments of lung microbiota. Additionally, it isdifficult to induce representative human multi-bacterial infections in animals.AIM Engineering a mucus model of pathological CF mucus (Bac3Gel) able to model chemical, structural gradient and viscoelastic properties,while supporting CF lung microbiota to be applied as a screening platform for antimicrobial studies in the pharmaceutical fieldIntroductionConsecutive infections exacerbate disease progressionStaphylococcus aureus Pseudomonas aeruginosaCF ChildrenFunctional LungsCF AdultsLung Failure leads to patient deathMucus presents altered chemical and viscoelastic propertiesFig. 3 Viscoelastic properties of both Bac3Gel and Mucin-Bac3Gel in comparison to those reported forpathological CF mucus1. Fig. 4 Estimated mesh size of both Bac3Gel and Mucin- Bac3Gel in comparisonto that reported for pathological CF mucus2. The developed mucus models appear to display mucus-likebehaviour. The scale bar corresponds to 25 mm2. A 3D Mucus model: Bac3Gel50 µm27.6 µm17.5 µm16.1 µm15.8 µmFig. 5 CFU count after infecting both Bac3Gel and Mucin-Bac3Gel with 1000: (A) S. aureus; and (B) P. aeruginosa for 24hours depicting physiological ranges3. (C) Confocal laserscanning microscope images of Bac3Gel colonized by P.aeruginosa, with a close-up view of bacterial aggregatesresembling those (D) observed in CF sputum4.DFig. 6 Antimicrobial treatment of P. aeruginosa infections: (A) Ciprofloxacinand (B) Tobramycin. The Miniminal Inibitory Concentration (MIC) of ColonyForming bacteria was determined following the EUCAST guidelines.Afterwards, both infected Bac3Gel and Mucin-Bac3Gel were treated withthree different concentrations: 0.1, 1 and 10 MIC.The mismatch determined between planktonic cultures and both infectedBac3Gel and Mucin-Bac3Gel was three-orders of magnitude higher,confirming what is extensively stressed by clinics5.ReferencesImportantIntestineEyesVagina1Interaction of CF drugs  with mucinPreparation and rheological analysis of the 3D mucus modelP. aeruginosaInfectionsAntimicrobial TreatmentConfocal microscopy to determine bacterial migrationP. aeruginosacolonization2 3 54PAMPA/cells & mucus modelDrug diffusion3. PAMPA & mucus model4. Recreating Lung Microbiota of CF PatientsAlginate-based mucus model(Patent Pending)5.Antimicrobial Treatment of P. aeruginosa infections3D model for HTSis needed18th PhysChem FORUM , Frankfurt , May  2019 1. Interaction with mucin1.Butnarasu et al.doi.org/10.1016/j.ijpharm.2019.04.0322; 2. Yuan S, et al. DOI: 10.1126/scitranslmed.3010525; 2. Suk JS, et al. DOI: 10.1016/j.biomaterials.2008.12.076; 3. Wong K, et al. DOI: 10.1099/00222615-17-2-113; 4. Bjarnsholt T, et al. DOI: 10.1016/j.tim.2013.06.002; 5. Macià MD, et al. DOI: 10.1111/1469-0691.12651 Fig1. Selected drugs and mucin interaction Fig2. PAMPA and Mucus model testHR-MS LTQ Orbitrap!"#"$%&&'()"$*+,-."/.0.12$,03&3/4+,!$35,/"13)."#Buffer63&3/,127'$832#).9&2)."#%$).9&.21,0'0:$2#',127'$!"#"$%&&'()"$*+,-."/.0.12$,03&3/4+,!$35,/"13)."#Buffer63&3/,127'$832#).9&2)."#%$).9&.21,0'0:$2#',127'$Ampicillin(PENICILLIN)Levofloxacin(QUINOLONE)NSOH2NHO OHO CH3OSSOO OOOOHONHNNNNNH2H3CCH3HNSOOOOCH3HNONSNOOOHH3CH3CH3NSOHOOONNHNH2CH3CH3HN SSOFFFOOHOHOHONH2OHONH2NH2HOO ONH2OHH2NFOOOHONNNCH3H3CNNNCH3NHOOOHOHH3COOCH3H3COH3COH3COH3CHOCH3OHCH3CH3OH7-ACA(CEPHALOSPORIN)Ceftazidime(CEPHALOSPORIN)Aztreonam(MONOBACTAM)CFTR(inh)-172(INHIBITOR OF CFTR PROTEIN)Tobramycin(AMINOGLYCOSIDE)Rifampicin(ANSAMYCIN)200 250 300 350 400 4500.00.20.40.6λ (nm)Abs (a.u.)ag300 350 400 4500255075100λ (nm)Fluorescence Intensity (a.u.)ah325 350 3758095110λ (nm)Fluorescence Intensity (a.u.)ah320 360 400 440 4800.000.050.100.1502×1064×1066×1068×1061×107λ (nm)AbsIntensity0 1 2 30.91.01.11.21.3[CFTR(inh)-172] (10-6 M)F 0/F296 K303 K310 KHowever, the study of the only mucin-drug interaction is incomplete.UV-Vis Fluorescence FRET Stern-VolmerCFTR(inh)-172Levofloxacin Aztreonam Ceftazidime0246KSV (104 M)1.4-0.65-6.5 -7.6LogD7.4Propranolol CFTR Ampicillin Ceftazidime0.01.5×10-53.0×10-54.5×10-5Permeability (cm/s)Mucus freeMucus 

A mucus model to evaluate the diffusion of drugs for more efficient cystic fibrosis therapies

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A mucus model to evaluate the diffusion of drugs for more efficient cystic fibrosis therapies

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