27 July 2021
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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
Pacheco DP
1
, Butnarasu C
5
, Bertoglio F
2,3,4
, Suarez Vargas N
1
, Briatico-Vangosa F
1
, van Uden S
1
, Visai L
3
, Petrini P
1
,
Vallaro M
5
, Caron G
5
, Visentin S
5
1 Department of Chemistry, Materials and Chemical Engineering "Giulio Natta" at Politecnico di Milano, Italy; 2 Molecular Medicine Department (DMM), Center for Health Technologies (CHT), UdR
INSTM, 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 Studies
IUSS Pavia, Italy; 5 Molecular Biotechnology and Health Sciences Department, University of Torino, Italy
Materials
&
Methods
Cystic fibrosis (CF) mucus exhibits altered chemical and viscoelastic features, limiting its clearance and leading to chronic bacterial
infections. Current bacterial culture fails to recreate bacteria communities and microenvironments of lung microbiota. Additionally, it is
difficult to induce representative human multi-bacterial infections in animals.
AIM
Engineering a mucus model of pathological CF mucus (Bac
3
Gel) 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 field
Introduction
Consecutive infections exacerbate disease
progression
Staphylococcus aureus Pseudomonas aeruginosa
CF Children
Functional Lungs
Lung Failure leads to patient death
CF Adults
Mucus presents
altered chemical and
viscoelastic
properties
Fig. 3 Viscoelastic properties of both Bac3Gel and Mucin-Bac3Gel in comparison to those reported for
pathological CF mucus1. Fig. 4 Estimated mesh size of both Bac3Gel and Mucin- Bac3Gel in comparison
to that reported for pathological CF mucus2. The developed mucus models appear to display mucus-like
behaviour. The scale bar corresponds to 25 mm
2. A 3D Mucus model: Bac
3
Gel
50 µm
27.6 µm
17.5 µm 16.1 µm
15.8 µm
Fig. 5 CFU count after infecting both Bac3Gel and
Mucin-Bac3Gel with 1000: (A) S. aureus; and (B) P. aeruginosa for 24
hours depicting physiological ranges3. (C) Confocal laser
scanning microscope images of Bac3Gel colonized by P.
aeruginosa, with a close-up view of bacterial aggregates
resembling those (D) observed in CF sputum4.
D
Fig. 6 Antimicrobial treatment of P. aeruginosa infections: (A) Ciprofloxacin
and (B) Tobramycin. The Miniminal Inibitory Concentration (MIC) of Colony Forming bacteria was determined following the EUCAST guidelines. Afterwards, both infected Bac3Gel and Mucin-Bac3Gel were treated with
three different concentrations: 0.1, 1 and 10 MIC.
The mismatch determined between planktonic cultures and both infected Bac3Gel and Mucin-Bac3Gel was three-orders of magnitude higher,
confirming what is extensively stressed by clinics5.
References
Important
Intestine
Eyes
Vagina
1
Interaction of CF drugs
with mucin
Preparation and rheological
analysis of the 3D mucus model
P. aeruginosa
Infections
Antimicrobial Treatment
Confocal microscopy to
determine bacterial migration
P. aeruginosa
colonization
2
3
4
5
PAMPA/cells &
mucus model
Drug diffusion
3. PAMPA & mucus model
4. Recreating Lung Microbiota
of CF Patients
Alginate-based mucus model
(Patent Pending)
5.Antimicrobial Treatment of
P. aeruginosa infections
3D model for HTS
is needed
18
th
PhysChem FORUM , Frankfurt , May 2019
1. Interaction with mucin
1.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 test
HR-MS LTQ Orbitrap !"#"$ %&&'()"$ *+,-."/.0.12$, 03&3/ /"13)."#4+,!$35, Buffer Buffer 63&3/,127'$ 832#).9&2)."# %$).9&.21, 0'0:$2#', 127'$ !"#"$ %&&'()"$ *+,-."/.0.12$, 03&3/ /"13)."#4+,!$35, Buffer Buffer 63&3/,127'$ 832#).9&2)."# %$).9&.21, 0'0:$2#', 127'$ Ampicillin (PENICILLIN) Levofloxacin (QUINOLONE) N S O H2N H O OH O CH3 O S S O O O O O OH O N HN N N N NH2 H3C CH3 H N S O O O O CH3 H N O N S N O O OH H3C H3C H3N S O HO O O N NH NH2 CH3 CH3 H N S S O F F F O OH O HO HO NH2 OH O NH2 NH2 HO O O NH2 OH H2N F O O OH O N N N CH3 H3C N N N CH3 NH O O OH OH H3C O O CH3 H3C O H3C O H3C O H3C HO CH3 OH CH3 CH3 OH 7-ACA (CEPHALOSPORIN) Ceftazidime (CEPHALOSPORIN) Aztreonam (MONOBACTAM) CFTR(inh)-172 (INHIBITOR OF CFTR PROTEIN) Tobramycin (AMINOGLYCOSIDE) Rifampicin (ANSAMYCIN) 200 250 300 350 400 450 0.0 0.2 0.4 0.6 λ (nm) Abs (a.u.) a g 300 350 400 450 0 25 50 75 100 λ (nm)
Fluorescence Intensity (a.u.)
a h 325 350 375 80 95 110 λ (nm)
Fluorescence Intensity (a.u.)
a h 320 360 400 440 480 0.00 0.05 0.10 0.15 0 2×106 4×106 6×106 8×106 1×107 λ (nm) Abs Intensity 0 1 2 3 0.9 1.0 1.1 1.2 1.3 [CFTR(inh)-172] (10-6 M) F0 /F 296 K 303 K 310 K However, the study of the only mucin-drug
interaction is incomplete.
UV-Vis Fluorescence FRET Stern-Volmer
CFTR(inh)-172Levofloxacin Aztreonam Ceftazidime
0 2 4 6 K SV (10 4 M) 1.4 -0.65 -6.5 -7.6 LogD7.4
Propranolol CFTR Ampicillin Ceftazidime
0.0 1.5×10-5 3.0×10-5 4.5×10-5 Permeability (cm/s) Mucus free Mucus
