microparticles composed of nanoparticles for pulmonary application of antibiotics in cystic fibrosis

116 Pages·2017·6.47 MB·English

M N
ICROPARTICLES COMPOSED OF ANOPARTICLES
P A A
FOR ULMONARY PPLICATION OF NTIBIOTICS
C F
IN YSTIC IBROSIS

Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät
der Universität des Saarlandes

von
Afra Torge

Saarbrücken
2017
Tag des Kolloquiums: 14.07.2017
Dekan: Prof. Dr. Guido Kickelbick
Berichterstatter: Prof. Dr. Marc Schneider
Prof. Dr. Claus-Michael Lehr
Vorsitz: Prof. Dr. Rolf W. Hartmann
Akad. Mitarbeiter: Dr. Matthias Engel
Die vorliegende Arbeit wurde von Mai 2013 bis Januar 2017
an der Philipps-Universität Marburg, der Universidade Federal do Rio Grande do Sul
(Porto Alegre, Brasilien) sowie der Universität des Saarlandes unter Betreuung von
Prof. Dr. Marc Schneider angefertigt.

Sie entstand im Rahmen des FiDel-Projekts (“Cystic Fibrosis Delivery”, Förderkennzeichen
N° 13N12530), gefördert durch das Bundesministerium für Bildung und Forschung
(BMBF).
C
ONTENTS
1. Summary .................................................................................................................................. - 1 -
2. Kurzzusammenfassung .......................................................................................................... - 2 -
3. Introduction ............................................................................................................................. - 3 -
3.1. Scientific background ..................................................................................................................................... - 5 -
3.1.1. Cystic fibrosis ........................................................................................................................................ - 5 -
3.1.2. Limitations of antibacterial therapy – Biological barriers in CF lungs ............................ - 6 -
3.1.3. Overcoming biological barriers by nanoparticles ................................................................... - 8 -
3.1.4. The challenge of delivering nanoparticles to the lungs ..................................................... - 10 -
3.1.5. Microparticles as pulmonary delivery system for nanoparticles .................................... - 14 -
3.2. Techniques of interest .................................................................................................................................. - 16 -
3.2.1. Nanoparticle preparation ............................................................................................................... - 16 -
3.2.2. Microparticle preparation .............................................................................................................. - 17 -
3.2.3. Characterization of aerodynamic properties .......................................................................... - 21 -
4. Aim of this work .................................................................................................................... - 23 -
5. Visualization of mucus ......................................................................................................... - 25 -
5.1. Introduction ...................................................................................................................................................... - 25 -
5.2. Experimental details ...................................................................................................................................... - 26 -
5.2.1. Materials ................................................................................................................................................ - 26 -
5.2.2. CSEM Imaging .................................................................................................................................... - 26 -
5.2.3. Image analysis..................................................................................................................................... - 27 -
5.3. Results and discussion .................................................................................................................................. - 28 -
5.3.1. Influence of freeze drying on porcine tracheal mucus ....................................................... - 28 -
5.3.2. Suitability of freezing for preservation of human tracheal mucus ................................ - 30 -
5.3.3. Visualization of equine respiratory mucus .............................................................................. - 33 -
5.4. Conclusion ......................................................................................................................................................... - 34 -
6. Ciprofloxacin-loaded lipid-core nanocapsules ................................................................. - 35 -
6.1. Introduction ...................................................................................................................................................... - 35 -
6.2. Experimental details ...................................................................................................................................... - 36 -
6.2.1. Preparation of nanocapsules ........................................................................................................ - 36 -
6.2.2. Testing of interaction between polymer and lipid ............................................................... - 37 -
6.2.3. Physicochemical characterization of nanocapsules ............................................................. - 37 -
6.2.4. HPLC method for quantification of ciprofloxacin ................................................................. - 37 -
6.2.5. Encapsulation efficiency and drug loading ............................................................................. - 38 -
6.2.6. Determination of the presence of drug crystals ................................................................... - 39 -
6.2.7. Visualization of nanocapsules ...................................................................................................... - 39 -
6.2.8. Mucus permeation assay ................................................................................................................ - 40 -
6.2.9. In vitro drug release study ............................................................................................................. - 40 -
6.2.10. Antibacterial growth assays ........................................................................................................... - 41 -
6.2.11. SEM analysis of S. aureus aggregates ....................................................................................... - 41 -
6.3. Results and discussion .................................................................................................................................. - 42 -
6.3.1. Interaction testing between polymer and lipid ..................................................................... - 42 -
6.3.2. Physicochemical characterization ............................................................................................... - 43 -
6.3.3. Mucus permeation assay ................................................................................................................ - 46 -
6.3.4. In vitro drug release ......................................................................................................................... - 49 -
6.3.5. Antibacterial activity ......................................................................................................................... - 52 -
6.4. Conclusion ......................................................................................................................................................... - 55 -
7. Spray-dried nano-embedded microparticles .................................................................... - 56 -
7.1. Introduction ...................................................................................................................................................... - 56 -
7.2. Experimental details ...................................................................................................................................... - 57 -
7.2.1. Nanoparticle preparation and characterization .................................................................... - 57 -
7.2.2. Preparation of nano-embedded microparticles .................................................................... - 58 -
7.2.3. Morphology analysis by SEM and CLSM.................................................................................. - 58 -
7.2.4. Evaluation of aerodynamic properties ...................................................................................... - 59 -
7.2.5. Evaluation of redispersibility in simulated lung fluid .......................................................... - 60 -
7.2.6. Disintegration under conditions similar to the lungs ......................................................... - 60 -
7.3. Results and discussion .................................................................................................................................. - 61 -
7.3.1. Nanoparticle characterization ...................................................................................................... - 61 -
7.3.2. Morphology of spray-dried particles ......................................................................................... - 61 -
7.3.3. Aerodynamic properties ................................................................................................................. - 66 -
7.3.4. Quantitative evaluation of redispersibility ............................................................................... - 67 -
7.3.5. Disintegration under conditions similar to the lungs ......................................................... - 68 -
7.4. Conclusion ......................................................................................................................................................... - 72 -
8. Comparison of cylindrical and spherical microparticles composed of nanoparticles - 73 -
8.1. Introduction ...................................................................................................................................................... - 73 -
8.2. Experimental details ...................................................................................................................................... - 74 -
8.2.1. Nanoparticle preparation and characterization .................................................................... - 74 -
8.2.2. Preparation of cylindrical microparticles .................................................................................. - 74 -
8.2.3. Preparation of spherical microparticles .................................................................................... - 75 -
8.2.4. SEM imaging and size analysis..................................................................................................... - 75 -
8.2.5. CLSM imaging ..................................................................................................................................... - 76 -
8.2.6. Determination of nanoparticle content .................................................................................... - 76 -
8.2.7. Evaluation of aerodynamic properties ...................................................................................... - 76 -
8.2.8. Redispersibility behavior ................................................................................................................ - 76 -
8.3. Results and discussion .................................................................................................................................. - 77 -
8.3.1. Nanoparticle characterization ...................................................................................................... - 77 -
8.3.2. Microparticle morphology ............................................................................................................. - 77 -
8.3.3. Nanoparticle content ....................................................................................................................... - 81 -
8.3.4. Aerodynamic properties ................................................................................................................. - 83 -
8.3.5. Disintegration behavior .................................................................................................................. - 85 -
8.3.6. Comparison of the preparation methods ................................................................................ - 86 -
8.4. Conclusion ......................................................................................................................................................... - 86 -
9. Conclusion and outlook ....................................................................................................... - 87 -
10. Annex ...................................................................................................................................... - 92 -
10.1. List of abbreviations ...................................................................................................................................... - 92 -
10.2. List of materials ............................................................................................................................................... - 93 -
10.3. List of devices ................................................................................................................................................... - 95 -
10.4. List of used software ..................................................................................................................................... - 96 -
10.5. References ......................................................................................................................................................... - 97 -
10.6. Curriculum vitae ........................................................................................................................................... - 105 -
10.7. Scientific output ........................................................................................................................................... - 106 -
10.8. Acknowledgements .................................................................................................................................... - 109 -
Summary
1. S
UMMARY
The eradication of bacteria in cystic fibrosis lungs is often unsuccessful due to biological
barriers such as respiratory mucus and bacterial biofilms. Nanoparticles are suitable
carriers to transport drugs through these barriers. As nanoparticles show low lung
deposition, they need to be converted into microparticles for an inhalation as dry powder
formulation.
In this work, different mucus types were evaluated towards pore size and structure.
Antibiotic nanoparticles were prepared by loading lipid-core nanocapsules with
ciprofloxacin. The nanocarriers showed an improved transport of the drug over the
mucus barrier and efficacy against pathogens found in cystic fibrosis lungs. Furthermore,
nano-embedded microparticles were prepared as delivery systems for nanoparticles to
reach a good lung deposition. Subsequent disintegration of microparticles enables a
release of nanoparticles. The influence of a matrix excipient on properties of spray-dried
nano-embedded microparticles was investigated. Finally, cylindrical microparticles
composed of nanoparticles were prepared by a template-assisted technique and
compared with spherical particles.
Concluding, this work elucidates unanswered questions concerning the mucus barrier
and presents a promising approach to improve treatment of pulmonary infections in
cystic fibrosis.

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Kurzzusammenfassung
2. K
URZZUSAMMENFASSUNG
Die Eradikation von Erregern bei Mukoviszidose ist aufgrund von biologischen Barrieren
wie Mukus und bakteriellem Biofilm oft erfolglos. Nanopartikel sind geeignete
Trägersysteme, um Arzneistoffe über diese Barrieren zu transportieren. Da sie jedoch nur
unzureichende Lungendeposition aufweisen, müssen sie in Mikropartikel eingebettet
werden, um eine Inhalation als Trockenpulver zu ermöglichen.
In dieser Arbeit wurden verschiedene Mukusarten in Hinblick auf Porengröße und
Struktur charakterisiert. Antibiotische Nanopartikel wurden durch Einkapselung von
Ciprofloxacin in Nanokapseln mit Lipidkern hergestellt. Die Nanopartikel verbesserten
den Transport des Arzneistoffs durch den Mukus und zeigten Wirksamkeit gegen Erreger,
die Lungeninfektionen bei Mukoviszidose-Patienten hervorrufen. Des Weiteren wurden
Nanopartikel in Mikropartikel als Trägersystem eingebettet, um eine gute
Lungendeposition zu erreichen. Durch den anschließenden Zerfall der Mikropartikel wird
die Freisetzung der Nanopartikel ermöglicht. Der Einfluss eines Matrixbildners auf die
Eigenschaften der sprühgetrockneten Nano-in-Mikropartikel wurde untersucht. Zudem
wurden zylindrische Mikropartikel bestehend aus Nanopartikeln mittels Templatmethode
hergestellt und mit sphärischen Partikeln verglichen.
In dieser Arbeit wurden unbeantwortete Fragen bezüglich Mukus als Barriere geklärt und
ein vielversprechender Ansatz für die Behandlung bakterieller Infektionen bei
Mukoviszidose-Patienten vorgestellt.

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Introduction
3. I
NTRODUCTION
Tremendous progress has been made in the last decades in the treatment of cystic
fibrosis, a genetic disorder characterized mainly by pulmonary symptoms. The survival
age could be significantly increased; nevertheless, there is no cure available yet and
patients’ lives are still rather short. The main cause of death is pulmonary insufficiency
resulting from chronic and recurrent lung infections with Pseudomonas aeruginosa [1].
Despite a broad range of different antibiotics with several application routes, efficacy of
antibacterial treatment is still limited as the pathogens are forming biofilms inside a thick
layer of immobile and viscous mucus covering the airways. Both mucus and biofilm
represent biological barriers for antibiotic drugs, impairing the diffusion to their target [2,
3]. A recent approach for overcoming these barriers is the use of nanocarriers that are
able to transport drugs through mucus and biofilm [3]. In this thesis, antibiotic-loaded
nanoparticles with mucus-permeating properties will be developed. While nanoparticles
are well suited delivery systems for drugs, their size is not appropriate for an inhalation as
dry powder formulation. Sizes in the lower micrometer range are required for an efficient
deposition in the lungs. In this work, nanoparticles shall be converted into microparticles
to enable an application of nanoparticles by dry powder inhaler. By spray drying,
nanoparticles will be embedded into an excipient matrix. Furthermore, cylindrical nano-
embedded microparticles will be prepared by template-assisted technique. The
microparticles serve only as delivery system to transport the nanocarriers into the lungs
and are supposed to disintegrate after deposition to release the nanoparticles (Fig. 3.1).
These nano-in-micro drug delivery systems combine the advantages of two size ranges,
the applicability of microparticles as dry powder formulation as well as the improved
drug transport by nanoparticles. Due to higher drug amounts reaching the site of action
and a sustained release provided by the nanoparticles, both the overall dose and the
dosing frequency might be reduced. The efficacy of the antibacterial therapy may be
enhanced with at the same time decreased systemic side effects. Nano-embedded
microparticles hence feature the perspective to improve the therapy of bacterial
infections in cystic fibrosis and in consequence also to increase survival and quality of life
of patients.
In the following chapter the scientific background will be presented more detailed.
- 3 -
Introduction

Fig. 3.1. The principle of nano-embedded microparticles: Microparticles are inhaled as
dry powder formulation and transport the embedded nanoparticles into the lungs. After
deposition in the airways, the microparticles disintegrate and release the nanoparticles.
By permeating through the mucus network, the nanoparticles can transport the drug to

the site of action, the bacterial biofilms.

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