Paula Bellés Sancho - UAB Barcelona · THE BATTLE THAT SEEMED LOST Nanoparticles against...
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THE BATTLE THAT SEEMED LOST Nanoparticles against multiresistant Pseudomonas aeruginosa Paula Bellés Sancho EXPECTED RESULTS BIBLIOGRAPHY Pseudomonas aeruginosa is a gramnegative bacteria which causes most of the nosocomial infections in humans. It is frequently associated with respiratory diseases, being the principal cause of mortality (1) . These infections are difficult to treat with antibiotics due to three main factors (2) , shown in the following figure (Figure 1): The combination of these factors and the low bioavailability that, in most cases, traditional drugs present, make the treatment ineffective. For this reason, new strategies based on nanocarriers carrying antimicrobial drugs, such as polymeric nanoparticles (NPs), have been studied to due to their potential to encapsulate and deliver the drug. These nanocarriers-based strategies are expected to increase bactericidal effectiveness, avoiding the side effects reported on traditional drugs (2) . OBJECTIVES INTRODUCTION AND BACKGROUND MATERIAL AND METHODS Synthesis of PLGA-PEI diblock copolymers Precipitation Micelle- like aggregates with CFZM Covalent grafting of DNase I 1 4 3 2 Size and surface charge Morphological characterization Drug encapsulation and in vitro release Quantification of DNase I activity Sterilization 1 2 3 4 5 Flowchart 1. Diagram of the specific objective. Each objective is considered as a work-package. DIFUSION PLAN Suspension of P. aeruginosa + LB medium + NPs Increasing CFZM-loaded NPs concentration Recover biofilm forming cells and make serially dilutions in LB agar plates 3.1. Inhibition of biofilm formation 3.2. Activity against established biofilm Peg lid 24 h 37ºC Peg lid with a 48 h established biofilm LB medium + NPs Increasing CFZM-loaded NPs concentration 2. Minimal inhibitory concentration (MIC) assay Artificial mucus SILF Mammalian cytotoxicity Preparation of the PLGA/PEI–CFZM–DNase I NPs DNase I Figure 4. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium (MTT) colorimetric assay after 24 h and 48 h of exposure to different NPsconcentrations, tested in murine macrophage cells. Based on A. Baelo et al. 2015 (4) . Figure 2. NPs preparation diagram based and modified from Y. S. Nam et al. 2003 (3) (1, 2 and 3) and A. Baelo et al. 2015 (4) (4). The NPs resulting is an amphiphilic NP based on PLGA- PEI polymer and modified with Dnase I and CFZM. P. aeruginosa PAO1 CFZM resistant P. aeruginosa strain PLGA CFZM PEI Increasing NPs concentration Murine macrophage cell culture 24h 48 h Figure 3. Flowchart on NPs characterization, baseded and modified from A. Baelo et al. 2015 (4) (1, 2, 3 and 4). Step 5 is based on M. A. Vetten et al. 2014 (5) . In vitro assays Figure 5. In vitro assays adapted from I. d’Angelo et al. 2015 (6) (1) and A. Baelo et al. 2015 (4) (2, 3.1. and 3.2.) 1. Artificial mucus assay NPs dispersion Polycarbonate membrane SILF Artificial CF mucus Preparation of NPs NP characterization Determination of mammalian cytotoxicity In vitro assays Protocol optimization Physical features Quantification of drug encapsulation and DNase I activity Sterilization of the NPs Mucolitic assay Minimal inhibitory concentration assay Antibiofilm assay Characterization 4 μg/ml 0 μg/ml CFZM-loaded NPs DNase I PEI Antibiofilm capacity PEI + PLGA Synergyc propierties with CFZM Mucolytic propierties Protection of CFZM against β-lactamases Biodegradable Burst release of CFZM Figure 6. Scheme of expected results. To evaluate and valorize results by UAB Valorization and Patents Office Publications in high-impact scientific journals Generation of scientific reports every 4 months and after finishing a work-package Divulgation of results by national and international seminars Results of in vitro assays are represented in Figure 6. Each expected result is related to a component of the NPs, based on previous studies (2, 4) . This project is designed for 3 years. Results are expected to be obtained in the last stage of the third year. A. Thicker mucus layer of patients B. Biofilm formed by the pathogen C. Resistance of the microorganism A C B Figure 1. Anatomical and biological barriers in respiratory infection diseases (Image modified from I. d’Angelo et al. 2014 (2) ). Bronchial cross-section Airway inflammation Human airway epithelial cells Antibiotic Nucleic acid Antinflammatory drugs Airway-hydrating agents Mucus layer Macrophages Local therapy Neutrophil infiltration Non-mucoid bacteria Mucoid bacteria 1. Lyczak, J. B., Cannon, C. L., & Pier, G. B. (2002). Lung infections associated with cystic fibrosis. Clinical microbiology reviews, 15(2), 194-222. 2. d'Angelo, I., Conte, C., La Rotonda, M. I., Miro, A., Quaglia, F., & Ungaro, F. (2014). Improving the efficacy of inhaled drugs in cystic fibrosis: challenges and emerging drug delivery strategies. Advanced drug delivery reviews, 75, 92-111. 3. Nam, Y. S., Kang, H. S., Park, J. Y., Park, T. G., Han, S. H., & Chang, I. S. (2003). New micelle-like polymer aggregates made from PEI–PLGA diblock copolymers: micellar characteristics and cellular uptake. Biomaterials, 24(12), 2053-2059 4. Baelo, A., Levato, R., Julián, E., Crespo, A., Astola, J., Gavaldà, J., ... & Torrents, E. (2015). Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections. Journal of Controlled Release, 209, 150-158. 5. Vetten, M. A., Yah, C. S., Singh, T., & Gulumian, M. (2014). Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine, 10(7), 1391-1399 6. d’Angelo, I., Casciaro, B., Miro, A., Quaglia, F., Mangoni, M. L., & Ungaro, F. (2015). Overcoming barriers in Pseudomonas aeruginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide. Colloids and Surfaces B: Biointerfaces, 135, 717-725. The aim of this project is: To develop a polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA) and polyethylenimine (PEI). They will be modified by covalent grafting of deoxyribonuclease I (DNase I) and will encapsulate ceftazidime (CFZM). To assess the NP bactericidal potential against P. aeruginosa infection with three different in vitro assay (Flowchart 1).
Transcript of Paula Bellés Sancho - UAB Barcelona · THE BATTLE THAT SEEMED LOST Nanoparticles against...
THE BATTLE THAT SEEMED LOSTNanoparticles against multiresistant Pseudomonas aeruginosa
Paula Bellés Sancho
EXPECTED RESULTS
B I B L I O G R A P H Y
Pseudomonas aeruginosa is a gramnegative bacteria which causes most of the nosocomialinfections in humans. It is frequently associated with respiratory diseases, being the principal causeof mortality(1). These infections are difficult to treat with antibiotics due to three main factors(2),shown in the following figure (Figure 1):
The combination of these factors and the low bioavailability that, in most cases, traditional drugspresent, make the treatment ineffective. For this reason, new strategies based on nanocarrierscarrying antimicrobial drugs, such as polymeric nanoparticles (NPs), have been studied to due totheir potential to encapsulate and deliver the drug. These nanocarriers-based strategies are expectedto increase bactericidal effectiveness, avoiding the side effects reported on traditional drugs(2).
OBJECTIVES INTRODUCTION AND BACKGROUND
MATERIAL AND METHODS
Synthesis of PLGA-PEI diblock
copolymers
Precipitation
Micelle-like
aggregates with CFZM
Covalentgrafting of
DNase I
1
4
3
2
Size and surface charge
Morphological characterization
Drug encapsulation and in vitro release
Quantification of
DNase I activity
Sterilization
1
2
3
4
5
Flowchart 1. Diagram of the specific objective. Each objective is considered as a work-package.
DIFUSION PLAN
Suspension of P. aeruginosa + LB medium + NPs
Increasing CFZM-loaded NPs concentration
Recover biofilm forming cells and make serially dilutions in LB agar
plates
3.1. Inhibition of biofilm formation 3.2. Activity against established biofilm
Peg lid
24 h37ºC
Peg lid with a 48 h established biofilm
LB medium + NPs
Increasing CFZM-loaded NPs concentration
2. Minimal inhibitory concentration (MIC) assay
Artificial mucus
SILF
Mammalian cytotoxicityPreparation of the PLGA/PEI–CFZM–DNase I NPs
DNase I
Figure 4. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) colorimetric assayafter 24 h and 48 h of exposure to differentNPsconcentrations, tested in murine macrophagecells. Based on A. Baelo et al. 2015(4).
Figure 2. NPs preparation diagram based and modified fromY. S. Nam et al. 2003(3) (1, 2 and 3) and A. Baelo et al. 2015(4)
(4). The NPs resulting is an amphiphilic NP based on PLGA-PEI polymer and modified with Dnase I and CFZM.
P. aeruginosa PAO1
CFZM resistant P. aeruginosa strain
PLGA
CFZMPEI
Increasing NPs concentration
Murine macrophage cell culture
24h 48 h
Figure 3. Flowchart on NPs characterization,baseded and modified from A. Baelo et al.2015(4) (1, 2, 3 and 4). Step 5 is based on M.A. Vetten et al. 2014(5).
In vitro assays
Figure 5. In vitro assaysadapted from I. d’Angeloet al. 2015(6) (1) and A.Baelo et al. 2015(4) (2,3.1. and 3.2.)
1. Artificial mucus assay
NPs dispersion
Polycarbonate membrane SILF
Artificial CF mucus
Preparation of NPs
NP characterization
Determination of mammaliancytotoxicity
In vitro assaysProtocol
optimization
Physical features
Quantification of drug encapsulation and
DNase I activity
Sterilization of the NPs
Mucolitic assay
Minimal inhibitory concentration assay
Antibiofilm assay
Characterization
4 µg/ml 0 µg/ml CFZM-loaded NPs
DNase I
PEI
Antibiofilmcapacity
PEI + PLGA
Synergycpropiertieswith CFZM
Mucolyticpropierties
Protection of CFZM againstβ-lactamases
Biodegradable
Burst release of CFZM Figure 6. Scheme
of expected results.
To evaluate and valorize results by UAB Valorization and Patents Office
Publications in high-impact scientific journals
Generation of scientific reports every 4 months and after finishing a work-package
Divulgation of results by national and international seminars
Results of in vitro assays are representedin Figure 6. Each expected result is relatedto a component of the NPs, based onprevious studies(2, 4).
This project is designed for 3 years.Results are expected to be obtained in thelast stage of the third year.
A. Thicker mucus layer of patients B. Biofilm formed by the pathogen C. Resistance of the microorganism
A
C
B
Figure 1. Anatomical and biological barriers in respiratory infection diseases (Image modified from I. d’Angelo et al. 2014(2)).
Bronchial cross-sectionAirway
inflammation
Human airway epithelial cells
Antibiotic
Nucleic acid
Antinflammatory drugs
Airway-hydrating agents
Mucus layer
Macrophages
Local therapy
Neutrophil infiltration
Non-mucoid bacteria
Mucoid bacteria
1. Lyczak, J. B., Cannon, C. L., & Pier, G. B. (2002). Lung infections associated with cystic fibrosis. Clinical microbiology reviews, 15(2), 194-222.
2. d'Angelo, I., Conte, C., La Rotonda, M. I., Miro, A., Quaglia, F., & Ungaro, F. (2014). Improving the efficacy of inhaled drugs in cystic fibrosis:
challenges and emerging drug delivery strategies. Advanced drug delivery reviews, 75, 92-111.
3. Nam, Y. S., Kang, H. S., Park, J. Y., Park, T. G., Han, S. H., & Chang, I. S. (2003). New micelle-like polymer aggregates made from PEI–PLGA
diblock copolymers: micellar characteristics and cellular uptake. Biomaterials, 24(12), 2053-2059
4. Baelo, A., Levato, R., Julián, E., Crespo, A., Astola, J., Gavaldà, J., ... & Torrents, E. (2015). Disassembling bacterial extracellular matrix withDNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections. Journal of Controlled Release, 209, 150-158.
5. Vetten, M. A., Yah, C. S., Singh, T., & Gulumian, M. (2014). Challenges facing sterilization and depyrogenation of nanoparticles: effects onstructural stability and biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine, 10(7), 1391-1399
6. d’Angelo, I., Casciaro, B., Miro, A., Quaglia, F., Mangoni, M. L., & Ungaro, F. (2015). Overcoming barriers in Pseudomonas aeruginosa lunginfections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide. Colloids and Surfaces B: Biointerfaces, 135,717-725.
The aim of this project is:
To develop a polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA) andpolyethylenimine (PEI). They will be modified by covalent grafting ofdeoxyribonuclease I (DNase I) and will encapsulate ceftazidime (CFZM).
To assess the NP bactericidal potential against P. aeruginosa infection with threedifferent in vitro assay (Flowchart 1).
