Engineering a Dynamic Model of the Alveolar Interface for the Study of Aerosol Deposition

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UNIVERSITÁ DI PISA

PHD PROGRAM IN INFORMATION ENGINEERING

Engineering a Dynamic Model of the Alveolar

Interface for the Study of Aerosol Deposition

DOCTORAL THESIS

Author

Roberta Nossa

Pisa, October 2019

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UNIVERSITÁ DI PISA

PHD PROGRAM IN INFORMATION ENGINEERING

Engineering a Dynamic Model of the Alveolar

Interface for the Study of Aerosol Deposition

DOCTORAL THESIS

Author

Roberta Nossa

Tutor

Prof. Arti Ahluwalia

Reviewers

Prof. Barbara Rothen-Rutishauser Dr.Diana Massai

The Coordinator of the PhD Program

Prof. Fulvio Gini

Pisa, October 2019

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A mio papà, per avermi regalato

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"The pursuit of truth and beauty is a sphere of activity in which we are permitted to remain children all our lives.”

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I

Acknowledgements

RRIVATA alla fine di questo percorso, mi sembra giusto e doveroso soffermarmi a ringraziare coloro i quali hanno reso possibile il raggiungimento di tale traguardo.

In primo luogo, vorrei esprimere la mia gratitudine nei confronti della Professoressa Arti Ahluwalia, la quale ha creduto in me accogliendomi nel suo gruppo di ricerca del centro

E. Piaggio. La ringrazio per avermi dato la possibilità di entrare nel vivo di un ambiente

creativo e stimolante qual è il mondo della ricerca. Un mondo in continua evoluzione, che non conosce la parola fine, ma solo traguardi che segnano nuovi inizi. La ringrazio per avermi dato la possibilità di partecipare a conferenze e meeting internazionali, permettendomi di conoscere nuove culture, luoghi e persone. Persone così diverse, ma in fondo accumunate da una stessa passione, la passione per la ricerca. Infine, le sono grata per avermi fatto crescere sia dal punto di vista personale che professionale, ponendomi di fronte ad ostacoli, ma fornendomi i giusti mezzi per poterli superare.

Un altro ringraziamento va sicuramente al Professor Marco Luise, che per tre anni è stato il coordinatore del programma di dottorato e fino all’ultimo giorno di carica ci ha seguito con passione e aiutato nel nostro percorso di formazione, fungendo da riferimento per tutti noi dottorandi.

Since my research could not happened without financial support, I would also like to thank the PATROLS network. Among all the partners, I express my deepest appreciation to Professor Barbara Rothen for hosting me in her amazing research institute, the Adolphe

Merkle Institute, where I spent three months of my PhD life. This experience offered me

the possibility to acquire new skills and join a team who helped me a lot. I am particularly grateful to Hana, my patient tutor during my Swiss experience.

Vorrei quindi ringraziare il mio gruppo di ricerca, quello in cui sono “cresciuta” e con cui ho vinto e perso battaglie: l’IVM Group. Grazie a Chiara, che è un po’ la mamma di noi del CP: si preoccupa sempre di tutti, offrendoci aiuto senza volere nulla in cambio. Grazie a Chiara anche perché prepara dei dolci molto buoni. Grazie a Ludo, emblema di rigore e professionalità, da lei avrai sicuramente franchezza, sincerità e aiuto, soprattutto se si tratta di Arduino e di sopravvivere ad alberghi di dubbio gusto. Grazie a Jo, per la sua simpatia e ottimismo, invidio molto la sua calma e positività nell’affrontare la vita.

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Spalla durante gli sfoghi, fedele compagna di avventure, chiacchierate e risate, soprattutto di risate. Grazie ad Ermes, ultimo “acquisto” del gruppo: ultimo ad arrivare, ma non nel supportarti. Come si dice? Gli ultimi saranno i primi! E lui sarà tra i primi, nonostante i suoi tempi. Infine, un sentito pensiero va a Giorgio, Daniele ed Alejandro, un tempo parte del gruppo, ora alla scoperta di nuovi sentieri.

Un altro team che è stato fondamentale in questi anni di dottorato è sicuramente quello di Biofabrication. Presente nei momenti di difficoltà e non, perché la ricerca non è fatta solo di scoperte, ma anche di persone che lavorano al tuo fianco indipendentemente dal gruppo di appartenenza. Grazie quindi al Professor Giovanni Vozzi, Francesca (la paziente e ironica Francesca) e Carmelo. Grazie anche a tutti i ragazzi del team: Gabriele, Irene, Biagio, Amedeo e Aurora. Un pensiero speciale va poi ai membri di uno dei gruppi più illustri dell’intera Università di Pisa: il Gruppo Cibo. Tra di loro ci tengo a menzionare Lucia, che con le sue ricette mi ha fatto scoprire sensi che non sapevo di avere. Grazie anche per i consigli, le chiacchierate e per essere stata una compagna di sfogo, non solo di mangiate.

Vorrei inoltre esprimere la mia gratitudine nei confronti di Carla e Salvatore, che da dietro le quinte sono importanti figure a sostegno della nostra ricerca, e con loro tutti i tecnici e amministrativi del centro. Ci tengo anche a ringraziare tutti gli altri studenti, dottorandi, post-doc e ricercatori del centro Piaggio, in particolare i miei compagni della XXXIIesima avventura: Anna, Giuse, Franco, Simo, Andrea e Vincenzo. Grazie per aver reso divertente un percorso che, per quanto stimolate e produttivo, è stato seminato anche di ostacoli e difficoltà.

Tra i membri del centro, uno dei ringraziamenti più sentiti va sicuramente a Licia ed Anna, che prima di essere colleghe sono diventate tra le mie più care amiche. Gli anni di dottorato ci hanno permesso di coltivare un bellissimo rapporto, ed è forse questo uno dei principali motivi per cui rifarei questo percorso altre mille volte. Sono state la mia gioia e la mia forza, spettatrici e protagoniste di cambiamenti che ci hanno fatto diventare quel che siamo oggi. Migliori? Chi lo sa! Sicuramente più forti e consapevoli.

Ringrazio inoltre la Dottoressa Diana Massai, per la cura con cui ha letto il mio manoscritto e l’interesse che ha mostrato nei confronti del mio lavoro. Le sono grata per gli importanti suggerimenti che mi ha fornito, molto utili per il miglioramento del mio elaborato. Vorrei esprimere la mia gratitudine anche nei confronti della Professoressa Serena Danti e la Dottoressa Luisa Trombi, per avermi ospitata nel loro laboratorio, insegnandomi a prendermi cura delle tanto amate/odiate cellule.

Infine, non potevano mancare alla lista le mie amiche di sempre: Stefi, Gaia e Mary. Quelle che, nonostante cambi di città, vita e abitudini, sai sempre dove trovare. Il porto sicuro su cui fare affidamento, coloro a cui penso quando penso a Casa. E se loro sono la certezza, vorrei ringraziare anche chi di certezze non me ne ha date. È quando non hai certezze che trovi forze recondite, è quando ti poni domande che trovi le risposte, è quando scopri il diverso che riesci a guardare il mondo da un’altra prospettiva. Per tutti questi motivi, grazie anche a te.

E da ultimo, il ringraziamento più grande va alla mia famiglia. Va a mia mamma, in eterno equilibrio tra estrema forza e fragilità di bambina. Un esempio di indipendenza, coraggio e costanza. La mia valvola di sfogo e il mio ring da combattimento. Colei che,

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III

nonostante questo, non mi volterà mai le spalle. E infine va al mio papà, la cui assenza è la più viva presenza. Il cui esempio è stato quasi un peso da sopportare, per la paura di non essere all’altezza. Peso che negli anni si è trasformato in sfida: sfida nel diventare la grande donna che potesse renderlo più orgoglioso di quanto già non fosse. Ringrazio mio papà per essere tornato quando non avevo le forze, per avermi fatto nascere un’inspiegata felicità che partiva dalla pancia. Ringrazio mio papà perché so che ci sarà sempre, con me, in me e in tutti i sorrisi che nasceranno da quel piccolo aiutino rosa.

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V

Summary

ANOPARTICLES are widely used in industrial, household and medicinal applications. However, these dispersed particles can cause inflammation and stress in lung tissue, leading to the development of disease, such as asthma or chronic obstructive pulmonary disease. Moreover, their effect on human tissue is complex and not completely understood, since it is mediated by different factors, such as the humidity in the alveolar environment and the rhythmic contraction of the diaphragm. This rhythmic contraction generates a periodic change in the alveolar volume and the displacement of the alveolar wall, influencing nanoparticles (NPs) deposition, substances uptake and inflammatory response.

To understand and study the complex interaction between inhaled particles and lung tissue, both in vitro and in vivo models can be used. However, traditional in vitro models cannot reproduce the entire complexity of the alveolar environment, since they are not able to apply a mechanical cyclic strain to the cultured cells. On the other hand, in vivo models do not consider interspecies differences, which lead to a different response to drugs or nanoparticles due to a different physiology, resulting a non-predictive model of the human alveolar microenvironment. Moreover, animal tests are expensive and pose ethical problems.

To overcome these issues, an air-liquid interface bioreactor was developed, provided with a mobile elastic membrane to simulate physiological lung muscle stretching. This system, named DALI (Dynamic model for ALveolar Interface), consists of an aerosol generator and a bioreactor with a moving membrane placed between an air-liquid interface to study drug and nanoparticles deposition and passage.

A biohybrid electrospun membrane made of 1:1 Bionate®:gelatin was selected as support of the alveolar barrier, since it was shown to be porous, biocompatible, cell adhesive and highly elastic. Flexibility is an essential property, because it allows cyclic stretching of the membrane, mimicking the movements of the alveolar barrier during breathing.

To mimic natural breathing, an external compressed air system was used to stretch the elastic membrane where alveolar epithelial cells are seeded. Finally, a Quartz Crystal Microbalance (QCM) was integrated to quantify the amount of aerosolized

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VI nanoparticles on the cell layer.

The bioreactor was validated in terms of liquid and air tightness, biocompatibility, and capability of applying a cyclic strain to the seeded membrane. The stretching system was calibrated in order to correlate the membrane displacement to the pressure applied with the compressed air system. Moreover, the lower limit of the linearity range of the QCM was calculated, and deposition measurements were performed to verify its capacity to quantify the nebulized nanoparticles.

After validating the device from an engineering point of view, preliminary biological studies were performed to investigate the effects of flow and mechanical stretch on the growing cells. To this end, A549 cells (adenocarcinomic human alveolar basal epithelial cells) were cultivated within the bioreactor under fluidic dynamic conditions, applying a cyclic mechanical strain on the cells. In particular, the stretching magnitude was set to a 5% linear strain, corresponding to the normal breathing. After 5 days of culture, morphological analysis with confocal images were used to evaluate the effect of the stretching. Cells cultivated under fluidic flow were able to proliferate on the membrane retaining their typical morphology and forming a homogeneous cell layer. Thus, biological experiments suggest the suitability of the system forthe culture of epithelial cell at air-liquid conditions under flow. Further studies will be performed to optimise cell culture under cyclic stretch conditions.

Therefore, the DALI system can be collocated within the 4th_{of the Technology}

Readiness Levels (TRLs), which corresponds to the validation in laboratory environment. Indeed, the system has already been validated from an engineering point of view, but biological studies are still ongoing.

To conclude, the designed system could help in bridging the gap between in vivo and

in vitro models, overcoming some of the shortcomings of traditional in vitro models,

and paving the way towards the development of a device for physiologically relevant studies of aerosol and drug delivery and toxicology.

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VII

List of publications

International Journals

1. Nossa, R., Costa, J., Cacopardo, L., Ahluwalia, A.: Characterization of new biohybrid membranes for in vitro models of physiological barriers. In preparation.

2. Nossa, R., Costa, J., Cacopardo, L., Ahluwalia, A.: Breathing in vitro: designs and applications of engineered lung models. Submitting.

3. Cacopardo, L., Guazzelli, N., Nossa, R., Mattei, G., Ahluwalia, A.: Engineering hydrogel viscoelasticity, Journal of the Mechanical Behavior of Biomedical

Materials. 89:162-167, September 2018. DOI: 10.1016/j.jmbbm.2018.09.031

4. A. Remuzzi, A., Figliuzzi, M., Bonandrini, B., Silvani, S., Azzollini, N., Nossa, R., Benigni, A., Remuzzi, G.: Experimental Evaluation of Kidney Regeneration by Organ Scaffold Recellularization, Scientific Reports. 7:43502, March 2017. DOI: 10.1038/srep43502.

International Conferences/Workshops with Peer Review

1. Nossa, R.*, Cacopardo, L., Costa, J., and Ahluwalia, A.: Engineering of a dynamic model of the alveolar interface for the study of aerosol deposition.

European Society for Biomaterials (ESB 2019). Dresden, Germany, September

2019. Oral presentation.

2. Nossa, R.*, Cacopardo, L., Costa, J., and Ahluwalia, A.: Characterization of new biohybrid membranes for in vitro models of physiological barriers. European

Society for Biomaterials (ESB 2019). Dresden, Germany, September 2019. Oral

presentation.

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by the PATROLS and CITYCARE consortia, Fribourg, Switzerland, July 2019. Oral presentation.

Second “Centro 3R” Annual Meeting. Genova, Italy, June 2019. Poster

presentation. Winner of the 4th_{-place poster prize.}

5. Nossa, R.*, Domina, R., Cei, D., and Ahluwalia, A.: Engineering a dynamic model of the alveolar interface for the study of aerosol deposition. European

Society for Alternatives to Animal Testing (EUSAAT 2018), Linz, Austria,

September 2018. Winner of the Young Scientist Travel Award. Oral presentation.

6. Cacopardo L., Guazzelli N., Nossa R., Mattei G., Ahluwalia A*.: Engineering Viscoelasticity in Biomaterials. World Congress of Biomechanics 2018, Dublin, Ireland. July 2018. Oral presentation.

7. Nossa, R.*, Domina, R., and Ahluwalia, A.: A new

poly(carbonate)urethane/gelatin electrospun membrane for physiological barriers in in vitro models. Nanoengineering for Mechanobiology, Camogli, Italy, March 2018. Poster presentation

8. Cacopardo L.*, Guazzelli N., Nossa R., Mattei G., Ahluwalia A.: Engineering Viscoelasticity in Biomaterials. Nanoengineering for Mechanobiology, Camogli, Italy, March 2018. Oral presentation.

9. Cacopardo, L.*, Guazzelli, N., Nossa, R., Mattei, G., Ahluwalia, A.: Engineering viscoelasticity in biomaterials, Italian European Society of Biomechanics

(ESB-ITA), Rome, Italy, September 2017. Oral presentation.

10. Mattei, G., Cacopardo, L., Guazzelli, N., Nossa, R., Ahluwalia, A.*: Effect of the testing and sample geometry on biomaterial mechanical properties. European

Society of Biomechanics Conference, ESB Seville 2017, Spain, July 2017. Oral

presentation.

11. Nossa, R.*, Cei, D., Di Ciolo, C., Ahluwalia, A.: Engineering a dynamic model of the alveolar interface for the study of aerosol deposition, Advances in Cell and

Tissue Culture (ACTC), KIRK-STALL, Manchester, UK, May 2017. Poster

presentation.

Others

1. Nossa, R*. Domina, R., Cei, D., and Ahluwalia, A.: Engineering a dynamic model of the alveolar interface for the study of aerosol deposition, Gruppo

Nazionale di Bioingegneria, GNB 2018, Milan, Italy, June 2018. Poster

presentation. * Presenting author

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IX

List of Abbreviations

A

AC Alternating Current AEC Alveolar Epithelial Cell AFE Analog Front-End ALI Air/Liquid Interface ATP Adenosine Triphosphate

ATCC American Type Culture Collection B

BSA Bovine Serum Albumin C

CFTR Cystic Fibrosis Transmembrane Conductance Regulator COPD Chronic Obstructive Pulmonary Disease

D

DALI Dynamic Model for the ALveolar Interface DAPI 4’6-diamidino-2-phenylindole

DC Direct Current dH2O Distilled Water

DMSO Dimethyl Sulfoxide E

ECM Extracellular Matrix ENM Engineered Nanomaterial EtOH Ethanol

EVOM Epithelial Volt-Ohm-meter F

FBS Fetal Bovine Serum FEM Finite Element Method FRC Functional Residual Capacity FSI Fluid Structure Interaction G

GMP Good Manufacturing Practice H

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X

hPSCs Human Pluripotent Stem Cells I

I/O Input/Output

iPSCs Induced Pluripotent Stem Cells IR-LED Infrared Emitting Diode M

MEM Minimum Essential Medium Eagle N

NHBE Normal Human Bronchial Epithelial NPs Nanoparticles

P

PAMP Pathogen-Associated Molecular Patterns PBS Phosphate Buffer Saline

PC Polycarbonate PCB Printed Circuit Board PDMS Polydimethylsiloxane PET Polyester

PFA Paraformaldehyde PLA Polylactide Acid

PRR Pattern Recognition Receptors PSD Position Sensitive Detector PTFE Polytetrafluoroethylene PVC Polyvinyl Chloride Q

QCM Quartz Crystal Microbalance R

RPMI Roswell Park Memorial Institute Medium RV Residual Volume

RWV Rotating-Wall Vessel S

SAECs Small Airway Epithelial Cells SEM Scanning Electron Microscope T

TLC Total Lung Capacity

TRL Technology Readiness Level TSM Thickness-Shear Mode TV Tidal Volume

V

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XI

Summary

Acknowledgements ... I Summary... V List of publications ... VII

International Journals ... VII International Conferences/Workshops with Peer Review ... VII Others ... VIII

List of Abbreviations ... IX Contents ... XI

Motivation and Outline ... 1

Introduction ... 5

2.1 The respiratory system ... 5

2.2 Alveolar architecture ... 8

2.3 Stress and strain transmission in the lung ... 10

2.4 Pathological conditions: acute lung injury ... 11

2.5 The importance of studies on the alveolar barrier ... 14

2.6 Nanoparticles and their interaction with lung tissue ... 15

State of Art: Modelling the Alveolar Barrier ... 17

3.1 Introduction ... 17

3.2 In vivo models for the alveolar barrier ... 18

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3.4 Aim of the thesis ... 37

DALI System ... 39

4.1 Introduction to Bioreactors ... 39

4.2 The DALI system and its specifications ... 41

4.3 An analytical model of the membrane displacement when applying a radial stretching ... 44

4.4 Bioreactor geometry and its working principle ... 45

4.5 FEM Model of the bioreactor ... 47

4.6 Elements of the DALI System ... 51

4.7 Calibration of the membrane stretching system ... 68

4.8 Liquid and air tightness of the bioreactor... 71

4.9 QCM characterization and validation ... 71

4.10 Conclusions ... 75

An Elastic Membrane as Support for the Alveolar Barrier ... 79

5.1 Introduction ... 79

5.2 A porous and flexible membrane made of PDMS to support the alveolar barrier ... 80

5.3 A biohybrid membrane to support the alveolar barrier ... 86

5.4 Conclusions ... 96

Biological Studies ... 99

6.1 Introduction ... 99

6.2 Membrane biocompatibility evaluation ... 100

6.3 Bioreactor biocompatibility evaluation ... 103

6.4 Dynamic cell culture experiments with DALI system... 105

6.5 Conclusions ... 108

Conclusions and Future Developments ... 109

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CHAPTER

Motivation and Outline

This study was performed within the PATROLS project (Physiologically Anchored Tools for Realistic nanO-materiaL hazard aSsessmen), which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 760813. The goal of the PATROLS project is to deliver advanced and realistic tools and methods for nanomaterial safety assessment. PATROLS aims to providing an innovative and effective set of laboratory techniques and computational tools to more reliably predict potential human and environmental hazards resulting from engineered nanomaterial (ENM) exposures. The goal is to minimise the necessity of animal testing and stimulate future categorisation of ENMs, in order to support safety assessment frameworks. In particular, the aim of my thesis was to design an in vitro lung model for physiologically relevant studies of aerosol and drug delivery and toxicology, promoting possible alternatives to animal testing. Therefore, this work has been developed following the 3R’s principles [1] (Figure 1.1): ‘Replacement, Reduction and Refinement’ as laid out in Directive 2010/63/EU on the protection of animals used for scientific purposes (see section 3.2.2 in Chapter 3). It foresees to ‘Replace’ animals used in experiments with non-sentient alternatives; to ‘Reduce’ the number of animals employed; and to ‘Refine’ animal experiments so that they cause minimum pain and distress.

Figure 1. 1: Schematization of the 3R’s principles.

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Chapter 1. Motivation and Outline

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Currently, most of the studies in life sciences are performed on traditional simple two-dimensional (2D) static human in vitro cell cultures, or on animals, mostly rodents [2]. However, both the methodologies are not completely predictive of the human physiology. In fact, even if “traditional” in vitro models are directly scalable and robust, their lack of biological functionality makes them over-reductive. On the other hand, animal models replicate organ- and multi-organ-level function; however, this can be viewed also as a “negative” aspect, since the multifactorial systems are extremely complex to investigate, due the fact that multiple factors play synergistically and it is difficult to investigate the effect of a single event. Moreover, they do not consider interspecies physiology differences and are more expensive than in vitro tests [3]. Finally, there are the mentioned ethical problematic related to them. Hence, the success key relies on developing in vitro models that can provide robust data, replicating at the same time organ-level function and dynamics, leading to more predictive results. In this context, my research has focused on designing a physiologically relevant in vitro model of the alveolar barrier – the gas exchanging region of the lungs. The alveolar barrier is formed by the epithelial cells of the alveolar wall, the endothelial cells of the capillaries and the basement membrane between the two cell types. Figure 1.2 shows its organization and schematisation: it can be viewed as a double compartment container (air and liquid compartment), divided by a support where cells are seeded. One compartment is intended for the air flow, the other for the blood flow.

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The goal was to advance the traditional existing in vitro models, starting from an analysis of the state of art and assessing which aspects could be improved through the application of engineering approaches. From there, I have addressed some of the ‘fundamental engineering issues’ that must be resolved to obtain the “ideal” model of the alveolar barrier. As a first step, the key elements of an ideal model that are missing in traditional static 2D models were identified (Figure 1.3):

• The presence of an air-liquid interface (ALI), which simulates the interface between capillary and alveolus walls (alveolar barrier). In fact, the ALI reproduces the in vivo condition, where the alveolar barrier is in contact with both the air that reaches the lungs, and with the blood that flows through the capillaries that surround the alveoli.

• The presence of an air and fluid flow on the two sides of the alveolar barrier, which simulate, respectively, the airflow that reaches the alveolus during the inhalation, and the blood flow which carries carbon dioxide and oxygen between the lungs and tissues.

• The cyclic mechanical stretching of the alveolar barrier that occurs during breathing (during inhalation the alveolus inflates, during exhalation it deflates). • An aerosol generating system directly integrated to the system, for studying the

effect of drugs and nanoparticles on lung tissues. • Tools for real-time monitoring of NPs deposition.

Figure 1. 3: Schematization of the key elements of an ideal model of the alveolar barrier.

From there, a systematic research was performed in order to address the missing elements and design a new cell culture device.

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The introduction to the present thesis comprises Chapter 2, where the characteristics of the organ under investigation relevant for this work are described. Chapter 3 presents the state of art of the in vivo and in vitro methods currently used to model the alveolar barrier. Chapter 4 describes each component of the new designed device, as well as the methodologies and tools employed. Since one of the most crucial elements of the system is the support for the alveolar cells, Chapter 5 focuses on the methodologies used to obtain the support, and on its mechanical and structural characterisation. Finally, Chapter 6 presents and examines the results concerning cell cultivation using the designed system, whileChapter 7 summarizes the main conclusions of the thesis work. To better elucidate the outline of the thesis, Figure 1.4 illustrates the working flow of its contents.

Figure 1. 4: Working flow presented in this thesis work.

Finally, Figure 1.5 summarizes the engineering solutions for addressing the key elements of the fabricated in vitro model of the alveolar barrier, which are described in detail in the rest of this thesis.

Figure 1. 5: Engineering solutions for the addressing the key elements of an in vitro model of the alveolar barrier.

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CHAPTER

Introduction

2.1

The respiratory system

The respiratory system consists in a series of organs and structures involved in breathing (Figure 2.1), the gas exchange that occurs at two different levels, the external and the internal (or cellular) respiration [5]. With cellular respiration, we refer to the use of oxygen inside the mitochondria to generate ATP (Adenosine triphosphate). External respiration is the oxygen and carbon dioxide exchange between the atmosphere and tissues. It consists of four processes [5]:

1. Pulmonary ventilation, the movement of air into (inhalation) and out (exhalation) from the lungs.

2. Diffusion of oxygen and carbon dioxide between the air cavities and blood. 3. Oxygen and carbon dioxide transport between the lungs and tissues, carried by

the blood.

4. Diffusion of oxygen and carbon dioxide between the blood and tissues.

Figure 2. 1: Schematic representation of the respiratory system [6].

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Chapter 2. Introduction

6

Lungs have evolved to serve the essential function of extracting oxygen from the air for use in aerobic metabolism and to remove the gaseous waste of this process from the body [7]. Humans require an enormous quantity of oxygen to maintain baseline energy levels needed for survival [7]. To meet these energy demands, lungs maximize the surface area available for gas exchange by forming a complex network of tube-like epithelial branches known as the conducting airway, which consists of the trachea, bronchi, and bronchioles [7] (Figure 2.2). The tubes in this branched network get progressively smaller until they terminate with thin distal air sacs, called alveoli, which are closely associated with the capillary network to allow diffusion of oxygen into the bloodstream and removal of carbon dioxide [7]. Due to the subsequent subdivision of the respiratory network, the cross-sectional area of each alveolus is small; however, the total surface area is equal to 70 m2, meaning there is low resistance at the terminal bronchioles and alveoli [8]. This condition is important and necessary to permit fresh air to reach and diffuse into the alveolar bed, allowing gas exchange with the blood stream.

Figure 2. 2: Representation of the respiratory ducts [9].

The human conducting airway is divided into three regions: upper (nasal and oral cavities, pharynx, and larynx), lower (trachea and primary bronchi), and distal (small airways) [9]. During inhalation, fresh air enters the mouth, where it is warmed and moistened; then, air moves from the upper respiratory tract, through the trachea and bronchi, eventually reaching the lungs (Figure 2.3). The lymphatic and immune system protect the human body against antigens dispersed in the air that could deposit on the respiratory duct [10]. Moreover, the mucous secreted by nasal ciliated cells filters particles bigger than 30 µm, such as dust, pollen, smoke, and a small part of fine particles

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7 [11] (Figure 2.3).

Figure 2. 3: Flow chart showing the passage of air in and out of the lungs: from the nose to the lungs, fresh air is prepared and cleaned to reach the alveoli passing through a series of different tubes

The entire respiratory system is protected and supported by the rib cage, spinal cord and sternum bone [12]. Moreover, a double layer of serous membrane called pleura encloses each lung (Figure 2.4). The parietal pleura adheres to the thoracic cavity, while the visceral one adheres to the surface of the lung: this membrane produces a serous fluid that holds the two pleural layers together by surface tension, and therefore the lungs must follow the movement of the thorax when breathing occurs [13].

A network of different muscles drives the inhalation and exhalation during the breathing (Figure 2.4). While inhalation, the diaphragm contracts and drags down the rib cage and the pleura that adhere on it. This event generates a decrease in pleural and alveolar pressure: the difference between environmental pressure and internal pressure allows fresh air to enter the respiratory ducts. Once the inhalation phase ends, the exhalation takes place: the diaphragm and the other muscles relax and the thoracic cavity recovers the initial dimension, generating a positive pressure in the alveolar bed which drags the air (now charged with carbon dioxide) to the mouth and then out of the body [14].

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Figure 2. 4: Anatomical distribution of the muscles involved in breathing process inside the rib cage. The position of the pleura is also shown [15].

2.2

Alveolar architecture

The alveolus is a complex three-dimension structure where all the gas exchanges take place [12]. In the lungs, more than 300 million alveoli are present, giving a total cross sectional area of 50-80 m2 [16]. A huge mesh of blood capillaries surrounds the alveoli, carrying oxygen and carbon dioxide. There are more than one billion capillaries in the lungs, almost three for each alveolus. Gas exchange between the blood and external atmosphere presents a short diffusion time thanks to the thin layer (≈ 0.2 µm) that separates the two compartments (alveolus and capillary) [5]. The interface between capillary and alveolus walls is called alveolar barrier. It is composed of a thin basement membrane and an interstitial space lined with epithelial cells (exposed to the air space) and endothelial cells (exposed to the capillary side) [17] (Figure 2.5).

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The small airway epithelium consists of type I (ATI) and type II (ATII) pneumocytes (Figure 2.6): the squamous ATI covers approximately 96 % of the alveolar surface and is responsible for gas exchange, while the cuboidal ATII cells secrete surfactants, metabolize drugs, and differentiate into ATI cells when required [19].

Figure 2. 6: Cell population in the alveolus [9].

Another cell population that lies on the alveolar airside are the macrophages. They are responsible for the first defence mechanism to external compounds and micro-organisms that reach the alveolar tissue [20]. In fact, the alveolar epithelium has an important role as barrier function: the small airway epithelium possesses protein complexes (i.e. tight junctions, adherens junctions and gap junctions) that form a barrier between the circulation and the external environment. These complexes separate the apical and basolateral surfaces of the epithelium and establish polarity [21]. In addition to acting as a physical barrier to pathogens, the small airway epithelium also generates a strong immune response. The alveolar epithelial cells (AECs) express multiple pattern recognition receptors (PRRs), which recognize bacterial components and viruses. They also express cytoplasmic nucleotide-binding oligomerization domain-containing (NOD) proteins, which recognize the pathogen-associated molecular patterns (PAMPs). After recognition of PAMPs, the AECs activate signaling cascades, which further activate transcription factors, leading to the expression and release of chemokine/cytokines, and recruitment of inflammatory cells [22]. These cells also produce several lipid mediators of inflammation, such as prostaglandins, leukotrienes, and hepoxilins, which are important for immune cell recruitment and the maintenance of alveolar barrier integrity [23]. Interestingly, ATI and ATII cells play almost complimentary roles in immunity. ATI cells are pro-inflammatory, express a number pro-inflammatory cytokines, and enjoy an intimate cross-talk with alveolar macrophages and neutrophils [24]. On the other hand, ATII cells are predominantly anti-inflammatory, via their secretion of surfactant protein (SP)-A, D, lysozyme, and defensins [25].

The support for the alveolar epithelial cells is the basement membrane, composed of collagen and elastin, which are extracellular matrix (ECM) proteins [26]. It is a pressure-supported structure [27], such as the entire lung tissue, which means that it does not

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possess a stress free condition in the relaxed state. In fact, during inflation, the muscular architecture preserves the lungs in a state of pre-stress, providing an external distending stress that results in an internal positive transpulmonary pressure (between the pleura and air sacs). This condition affects mechanical behaviour of the whole tissue as well as the microstructural mechanical behaviour [28]–[30], as the tissue acts like a tensile structure at every instant. Despite the high content of elastic components in the basement membrane, the walls of the alveoli are very thin and susceptible to damage in the presence of high or intense mechanical stretching [31]. This aspect justifies the increasing interest in studying stress and strain transmission in lungs during breathing.

2.3

Stress and strain transmission in the lung

During spontaneous breathing or mechanical ventilation, lung tissues are continuously subjected to cyclic stretch, varying breathing frequency and volume amplitude to match lung ventilation to the metabolic state of the subject [32]. At rest, human lungs expand and recoil above functional residual capacity (FRC) with a rate of about 12 cycles/min (0.2 Hz) and a tidal volume (TV, air volume that enters in the lung during a normal respiratory act) equal to 500 mL, which approaches 10% of total lung capacity (TLC) (Figure 2.7) [32]. Breathing frequency and TV increase during exercise to fulfill the rise in O2 consumption and CO2 production. Lung volume (also called vital capacity, VC)

ranges from 1200 to 5700 mL, where 1200 mL is the residual volume (RV, air volume that remains in the lung after a maximal expiration), and 5700 mL is the TLC at maximum inspiratory effort [32].

Figure 2. 7: Pulmonary volumes and capacities in a healthy man of ≈ 70 Kg.

Measurement of bronchial dimensions of excised dog lungs with stereoscopic radiography has shown that bronchial length and diameter are proportional to changes

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in the cube root of absolute lung volume [32]. Assuming that linear dimensions of lung tissue scale isotropically with the cube root of lung volume, normal tidal breathing at rest has been estimated to correspond to an average tissue strain of about 4% [32]. Accordingly, tissue strain increases to 12% in a deep inspiration and up to 25% in a vital capacity manoeuvre from TLC to RV [32]. Lung physiological functions strongly depend on the mechanical properties of the tissue [33]. Stress-strain characteristics, together with transpulmonary pressure generated by the muscular architecture, determine how far lungs can expand during inhalation, the pulmonary compliance, the frequency range, and the maximal amount of air that can reach the alveolar tissue and exchange gases with blood circuit [34]–[39]. In an injured lung, tissue structures and mechanical properties can change the magnitude of stretch experienced by the alveolus compared with a healthy lung [17], leading to cell apoptosis, which is a form of programmed cell death to preserve physiologic and homeostatic functions. In particular, AEII cells undergo apoptosis both during normal lung development and maturation, and as a consequence of disease like acute lung injury [40].

2.4

Pathological conditions: acute lung injury

Acute lung injury is a nonspecific response of the lung to a wide variety of insults that cause a disruption of the epithelial and endothelial barriers [41]. The early stage of the disease is characterized by extensive lung damage, comprising edema formation and focal destruction of the pulmonary endothelium and epithelium [41]. In this situation a fibrotic layer deposits on the alveolar barrier, the alveolar sac collapses and several lung functions such as gas exchange are compromised (Figure 2.8). Acute lung injury is also one of the consequences of medical assistance after lung dysfunction. In fact, forced ventilation at low lung volumes and pressures promotes a repetitive collapse and reopening of the airways [42], [43]. In particular, the airflow through collapsed ducts generates high shear-stresses on airway walls, potentially damaging the tissues [30], [44]–[46]. Airway collapse and reopening usually does not occur during normal breathing lung because of the presence of surfactant that stabilizes the airways, preventing collapse. Acute lung injury can also occur in the presence of lung disease that changes tissue properties and characteristics. In the next sections, the main pathological conditions that affects the respiratory system will be presented.

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12 2.4.1 Pneumonia

Pneumonia is an infection of the lungs caused by several viruses or bacteria (i.e. influenza), in which terminal airways fill with thick fluid. In this condition, gas exchange is reduced and breathing frequency increase to compensate. High fever and chills, associated with headache and chest pain, are symptoms of pneumonia. Pneumonia can be caused by a bacterium that is usually present in the lung, but increases its virulence due to stress and/or reduced immunity [13] (Figure 2.9).

Figure 2. 9: Lobules under normal condition and in pneumonia (adjusted from Harvard Medical School [47]).

2.4.2 Chronic Obstructive Pulmonary Disease (COPD)

Severe irritation of lung tissue can lead to cystic fibrosis, emphysema, and asthma. These diseases together are known as chronic obstructive pulmonary disease (COPD). Cigarette smoke, cystic fibrosis or exposure to pollution are common causes of COPD.

Cystic fibrosis is a genetic disorder caused by the mutation of two genes for the cystic

fibrosis transmembrane conductance regulator (CFTR), a transmembrane protein needed for the transport of Cl- and HCO3- ions through the plasma membrane of epithelial cells.

In healthy lungs, secretion of Cl-_{regulates the fluid lining on the cell with the osmosis}

of water. In cystic fibrosis, insufficient secretion of Cl- and the resulting lack of water produces a thick, sticky mucus layer that covers the epithelia and reduces the ability of ciliated cells to expel it from the lungs (Figure 2.10). In addition, diminished secretion of HCO3- lowers the pH of mucus, making it more comfortable for inhaled bacteria [16].

Figure 2. 10: The cystic fibrosis lead to a decrease of the diameter of the airways, mucus production and reduced airflow [48].

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Emphysema is a chronic and incurable disorder in which the alveolar walls are damaged,

resulting in an over-distention of the tissue. The elastic recoil of the lung is reduced, as well as the expiration driving force. Moreover, the surface area available for gas exchange is reduced due the decrease of the distension range [13] (Figure 2.11). In this pathology, airways present cell proliferation, while alveolar lung tissue disappears: recent studies pointed out that tissue destruction in emphysema rests on the imbalance of protease and antiprotease activities, leading to enzymatic degradation of elastin and collagen matrix due the presence of inflammatory cells, including macrophages, T lymphocytes, B lymphocytes, and neutrophils [49].

Figure 2. 11: Alveolus under normal and emphysema conditions: the reduction of the alveolar basement membrane leads up to alveolar sac collapse [50].

Asthma is a periodic constriction of the bronchi and bronchioles, which induces

difficulties in breathing, and it is associated with cough and expectoration of mucus. Airborne irritant compounds, such as chemical fumes, pollen, cigarette smoke, or airborne particles to which the patient is allergic [16] are the principal causes of asthma attacks. Most asthma patients show some degree of bronchial inflammation that reduces the diameter of the airways and contributes to the aggravation of an attack [13] (Figure 2.12). From the molecular point of view, prolonged inflammation results in a thickening of the airway wall, which can be found throughout the bronchial tree, including airways less than 2 mm in diameter, with collagen deposition in the subepithelial space and increased tissue elastance and resistance [49].

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14 2.4.3 Lung Cancer

Another common disease that affects lung tissue and change dramatically its properties and functionality is lung cancer, in which occurs an uncontrolled proliferation of cells. There are several forms of lung cancer, but the most common (and most rapidly increasing) involves the epithelial cells lining the bronchi and bronchioles. Ordinarily, the lining of these airways consists of two layers of cells. Chronic exposure to irritants causes the number of layers to increase, especially close to the bronchioles’ branches. The first event appears to be thickening and callusing of the cells lining the bronchi. The ciliated and mucus-secreting cells disappear, replaced by a disorganized mass of cells with atypical nuclei. If the process continues, some of these cells break through the membrane and penetrate the underlying basement generating a metastasis [52]. At this point, malignant cells can be carried in lymph and blood to other parts of the body where they may lodge and continue to proliferate. When a lung cancer spreads, it may develop and close a bronchus: in this situation the entire lung linked to that branch collapses, the secretions trapped in the lung spaces become infected, and pneumonia or a lung abscess (localized area of pus) results. The only treatment that offers a possibility of cure is to remove a lobe or the whole lung before metastasis has had time to occur (pneumonectomy).

2.5

The importance of studies on the alveolar barrier

In recent years, the interest in studying the respiratory tract has increased, due the great impact that lung diseases have on the life quality of a person. Moreover, the oral route is an attractive non-invasive alternative for drug delivery [53], as it offers attractive features. These include its large absorptive area, extensive vasculature and low extracellular and intracellular enzymatic activity. Different studies recognize the permeability of the lung to a variety of drugs, including peptides and proteins [54], [55]. Moreover, the direct targeting that occurs with this route results in an immediate onset of drug action and reduced side effects. Therefore, a detailed study of the barrier properties, transport processes and permeability characteristics are crucial to a better understanding of drug absorption in the respiratory tract.

In addition to drugs testing, in vitro models of the alveolar barrier are important tools in toxicity studies. Several chemical agents can be found in urban air: carbon monoxide (CO2), nitrogen oxide (NO and NO2), sulphur oxide (SO2 and SO3) and nitric and nitrous

acid (HNO3 and HNO2). Moreover, there are diesel exhaust and small volatile particles

(PM10) that contain several toxic heavy metal particles, such as lead, cadmium and quicksilver. Since atmospheric pollution represents a severe risk for human health, research has focused on the study of the biological effect related to pollution agents’ interaction with the human body. Recent epidemiologic studies show a correlation between the increase of toxicity due to exposure to pollutants and the decrease of particles size, especially if inhaled [56], [57], even if the mechanisms of action are not well understood [58]. Hence, the growing interest in studying the interaction between lung tissues and nano-sized particles.

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2.6

Nanoparticles and their interaction with lung tissue

Nanoparticles are defined as particles whose length ranges from 1 to 100 nm in two or three dimensions [59]. Last years have been shown an increase in manufacturing of nanoparticle-containing merchandise along with the constant discovery of new applications of nanoparticles [59]. Therefore, the number of efforts aimed at determining the health risks associated with nanoparticle exposure continues to grow. On the other side, nanoparticles are becoming more and more important in emerging biomedical applications as drug-delivery agents, biosensors, or imaging contrast agents [59]. For biomedical purposes, toxicity is a critical factor to consider when evaluating their potential. Nanoparticles for imaging and drug delivery are often purposely coated and engineered to interact with cells, and it is important to ensure that these enhancements are not causing any adverse effects. Moreover, it is crucial to investigate their biodegradation processes, and how they affect cellular responses [59].

One of the most common routes of airborne nanoparticles exposure is through the lungs. During inhalation, nanoparticles enter the respiratory tract through the mouth and nose and deposit along the respiratory ducts. This deposition depends on their size/volume [60]: bigger particles (> 5 µm) deposit on the first part of the higher respiratory tract (nose, pharynx and tracheobronchial duct), due to the interaction with mucus and cilia; on the other hand, smaller particles (< 5 µm) are able to reach the alveoli, interacting with the alveolar interface via diffusion [60]. Ferin et al. [61] showed how nanometric dimensions allow the particles to access and pass through biological membranes, once they deposit on or between cells. This passage and internalization boost of ultrafine particles can induce an acute macrophage activity, followed by an acute pulmonary inflammatory response.

Inhalation is also an increasingly important route for drug delivery [62]. Mostly of the drug delivery products sold worldwide are aimed at the central regions of the lung for the treatment of asthma, chronic obstructive pulmonary disease (COPD) and other bronchial-related disease [63]. Although delivery to the deep lung is technically more challenging, the alveolar regions are an attractive site for absorption into the systemic circulation, since the alveolar epithelium is extremely thin with an extensive surface area [64]. Therefore, experimental studies are focusing their attention to maximize drug deposition efficiency and passage through the epithelial lining, in order to avoid huge dose treatment and drug waste [63]. On the other hand, in the last fifty years, several studies have been published regarding the potentially toxic effects of inhaled nanoparticles on lung cells [60], [62], [65]–[69]. Therefore, additional investigations are necessary and both in vivo and in vitro models can be used for toxicity studies and drug testing. In vitro models are replicates of an organ or a tissue, mimicking their topology (spatial distribution) and function, while in vivo models refer to tests that are conducted on whole, living organism, usually animals. In Chapter 3, the state of art of in vivo and

in vitro models used to study the respiratory tract will be presented, underling the

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CHAPTER

State of Art: Modelling the Alveolar Barrier

3.1

Introduction

Epithelial barriers regulate the passage from one domain to another, resulting as the body’s natural defense against external substances [70]. In particular, the alveolar epithelium of the lung is the most permeable epithelial barrier of the human body [60], and it is object of different studies regarding drugs and nanoparticles delivery and toxicology. Recent developments in delivering drugs to the lung are driving the need for studies to evaluate the fate of inhaled medicines [71]. In particular, inhalation of aerosolized drugs is a promising route for noninvasive targeted drug delivery to the lung [72]. On the other hand, different researchers are focusing their attention on the adverse effects caused by inhaled nanoparticles and chemical compounds, which depend on their hazard (negative action on cells and organism) and on exposure (concentration in the inhaled air and pattern of deposition in the lung) [60]. To understand what can and cannot cross the alveolar barrier and their effects on the human tissues, models have emerged as a reductionist approach to rigorously study and investigate these questions. Both in

vivo and in vitro models are used: with the development of advanced in vitro models,

not only in vivo, but also cellular studies can be used for toxicological testing [60]. Advanced in vitro studies use combinations of cells cultured in the air-liquid interface for investigating particle uptake and mechanistic studies. Whole-body, nose-only, and lung-only exposures of animals could help to determine retention of NPs in the body [60]. However, both approaches have their limitations: cellular studies cannot mimic the entire complexity of the organism, but, on the other side, data obtained by inhalation exposure from in vivo models are not completely predictive due to differences in the respiratory system of humans and animals. Combination of biological data generated in

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Chapter 3. State of Art: Modelling the Alveolar Barrier

18

different biological models and in vitro modelling appears suitable for a realistic evaluation of results regarding drugs and NPs delivery and toxicology.

In this chapter, the state of the art regarding in vivo and in vitro models of the alveolar barrier will be presented, showing their advantages and limitations. It will conclude with a short description of the device designed during my PhD (systematically presented in the following chapters), which could help in bridging the gap between in vivo and in

vitro models.

3.2

In vivo models for the alveolar barrier

In vivo models are commonly used for the evaluation of drug deposition efficiency, or

to study the effects of nanomaterials and inhaled chemicals on lungs and peripheral tissue. In particular, animals such as rats [53], [60], [73]–[75], mice [53], [60], [75], rabbits [53], [60], [75] and dogs [53], [60], [75] are commonly used for toxicity testing. The effects of nanoparticles can be studied by oral and dermal route exposure [60]. For inhalation, three different exposure methods exist: the whole-body exposure, nose/head-only exposure or lung-nose/head-only exposure (intratracheal instillation/inhalation) [60].

In the whole-body exposure, aerosol is generated from filtered compressed air; then, it is heated and added to dry filtered room air. A charge neutralizer decreases the electrostatic interaction with the chamber. At one side of the exposure chamber, the aerosol is collected for size determination and composition [76]. The chamber housing the animals should have a steady concentration of aerosol over the entire exposure time. Moreover, the aerosol should be contaminants-free and present a stable size distribution. The latter requirement is particularly challenging for NP-containing aerosols, because NPs tend to form large agglomerates that cannot be broken up [77]. Another negative aspect is that the doses can be highly variable. This variety can be due to the contribution of other routes of exposure (e.g., mouth, eyes); for instance, 60%–80% of the material deposited on the pelts during the exposure is ingested [78]. In addition, the animals can avoid exposure by huddling together or burying their noses in corners of cages or in the fur of another animal [60].

Compared to whole-body exposure, in the nose/head only exposure the housing chamber is very small. Aerosols are usually generated in one chamber for all exposed animals, and then transfer to the single animal. For rodents, they are placed in a tube connected the aerosol chamber (Figure 3.1a). At the back end of the tube, a restraint prevents the animal from backing out and avoids air leakages. To prevent overproduction of moisture and heat in the tube, the restraint can also only partially close the back end of the tube [79]. For exposure times longer than one hour, additional cooling is usually advised [80]. Even if with this exposure technique the dose variability is not as high as the whole-body exposure, the small chamber hinders animal movement and may cause discomfort. Moreover, younger animals may attempt to turn to escape from the tube, which bears the danger of suffocation [75]. Another problem is ventilation: if the flow through each port approaches the minute ventilation of the animal, the animal will rebreathe its exhaled atmosphere, carbon dioxide concentrations may increase and oxygen supply decrease, leading to animal suffocation. To prevent this, minimum flow through the

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nose-only chamber of 2.5 times the animal’s minute volume is recommended [60].

Figure 3. 1: Exposure of rodents to aerosols. (a) nose-only exposure. The restraint (R) prevents loss of aerosol by leakage around the animal. The small opening at the bottom allows temperature regulation of

the animal through the tail; Intratracheal instillation (b) and oropharyngeal aspiration (c) uses commercial or self-designed syringes for manual application of aerosol [60].

Finally, intratracheal instillation is performed by inserting a delivering device into the trachea and projecting its tip close to the bifurcation of the trachea (Figure 3.1b). Alternatively, a less invasive technique is to deliver the aerosol by oropharyngeal intubation (Figure 3.1c), where small animal laryngoscopes enable correct insertion of the delivery device [60]. Devices in standard length and in custom sizes (Penn Century Inc., Glenside, PA, USA) are available for delivery of NP-loaded liquid aerosols [81] and from powders [82]. Although the applied dose is well defined, non-physiological distribution within the lung may occur after initial placement [79]. Moreover, lung-only exposure may lead to artificial results by bypassing nose and defensive reflexes and may cause organ damage by dehydration of the trachea. Historically, partial lung exposure was also used, where the test substance was injected in one lobe, while another lobe served as control. When using this exposure technique, great technical skills are necessary for anaesthesia and precise placement of the catheters [60]. Different aspects determine the choice of the exposure method [60]:

• The availability of the testing material: whole body exposure needs high amounts of material.

• The technical expertise of the personnel: intratracheal instillation is technically demanding.

• The duration of the exposure: except for whole body exposure, anaesthesia/sedation is needed, which is not tolerated by the animal for prolonged times.

• Dose-control is best with intratracheal instillation, but this technique can cause local tissue damage and uneven distribution of the test substance in the lung. • Dose per animal can be less well determined in chambers, where animals are

housed together.

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3.2.1 Advantages and limitations of in Vivo Systems

Many topics, such as retention of inhaled particles, necessitate long-term studies and cannot be performed in vitro [60]. Inhaled NPs (≤100 nm) are retained in the body for long periods: for instance, 75% of 100 nm carbon NPs were retained for more than 48 h in hamster lungs [83]. Reasons for the prolonged retention include deep penetration into the mucus or deposition in areas with reduced lung lining layer [60]. In both cases, interaction with airway cells and likelihood of transmigration is increased. Surprisingly, not all particles retained in the lungs translocate to lymph nodes or enter the systemic circulation [84]. In inhalation studies with dogs, iridium NPs re-appeared on the epithelium, where macrophage-mediated clearance occurred and 90% of the inhaled NPs were recovered in the brochioalveolar lavage [85].

Despite the established role of animal experimentation and advantages related to this kind of experiments, specific limitations apply for the testing of inhaled NPs. Rodents are the animals most commonly used for toxicology testing, but they are obligatory nose breathers and, therefore, not representative models for human inhalation exposure. Even when this limitation is accepted, it is financially and technically not possible to assess all currently known NPs in vivo. According to estimates, comprehensive long-term testing would cause costs of $1.18 billion and require 34–53 years [86].

Another negative aspect of animal experiments, obviously, is interspecies differences regarding kinetics, efficacy, and reaction to particulates [60]. Dogs and primates are the animals that show the highest similarity to the human respiratory system. For example, in the canine respiratory system, exposure to cigarette smoke inhalation produces clinical and histopathological changes similar to COPD and emphysema in humans [87]. Canine thoracic deposition for Co3O4 particles is not significantly different from that

observed in the human respiratory tract, rising from 12% to 35% of the inhaled particles of 0.7 to 3.7 μm aerodynamic diameter [88]. A percentage of 32% of 0.02 μm large particles and 25% of 0.1 μm large particles were deposited in canine lungs [89]. Based on the similarity between human and beagle lungs shown for larger particles, similar deposition rates are also expected in humans [60]. However, due to ethical issues and experimental costs, these species are rarely used for toxicity studies. Rats are the most studied animals for NPs and chemical toxicity testing, while mice, with their 2000 different strains with carefully controlled genetics, results particularly interesting to study the influence on genetic variations on pathologies. Syrian hamster lungs have been mainly used for carcinogenesis and chronic respiratory studies. Other small mammals are mostly used for specific research topics: Guinea pigs were used for sensitization to inhaled antigens since their airways show similar sensitivity to mediators as human airways [60]. However, there are great differences in lung parameters between laboratory species and humans (Table 3.1) [60].

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Table 3.1: Comparison of physiological lung parameters between laboratory animals and humans [60]

Species Breath rate (resting, per minute)

Tidal volume (mL) Total lung capacity (mL) Rat 85 1 10 Mouse 163 0.15 1 Hamster 30 1 7 Guinea pig 84 1.7 23 Human 15 500 6000

For instance, rodents have a breath rate much higher than humans, while the tidal volume is extremely lower. In addition, even if rodents and human lungs contain most of the same cell types, the anatomy of the lung varies [7]. For example, human lungs contain basal cells throughout the trachea and bronchi, whereas mouse basal cells are restricted to the trachea. There is no evidence that human lungs possess a BASC population, and the majority of cells in the human proximal airway are multiciliated cells, whereas club cells are more abundant in rodents [90].

Moreover, rat lungs present prominent differences to human airways, which are particularly relevant when studying the effects of particles [60]. In fact, while terminal bronchioles in rat measure 0.2 mm in diameter and 0.35 mm in length, they are 0.6 mm wide and 1.68 mm long in humans [91]. Particle deposition is also different: for both humans and rats results minimal when the sizes is lower than 0.5 μm, while is maximal for 1 μm in rats and for 2-4 μm in humans [92]. Pulmonary deposition is, therefore, much higher for particles between 2–3 μm in humans than in rats [60]. In contrast to the extrathoracic (nasopharyngeal) deposition, the tracheobronchial and pulmonary (lobar) deposition fractions are practically insensitive to the change in aerodynamic diameter in the range of 1–5 μm [60]. Therefore, in animal models, lung-regional distribution is little altered by changes in aerodynamic diameter, a situation different from human inhalation. In addition, mucociliary clearance velocities are higher in rats than in humans: 10%– 15% of particles (0.1–7 μm) deposited in the human bronchial tree are still detectable after 24 h, while particles deposited in the rat bronchial tree are cleared after 6–8 h [93]. The delayed clearance appears to be due to the preferentially more peripheral deposition of particles in the human lung compared to a more central deposition in the rat lung [60]. Mucus velocities decrease with decreasing diameter of the airways in both species and, consequently, small airways have a slower clearance. Concerning the mice, pulmonary studies are problematic also because the measurement of lung function (flow, volume, and transpulmonary pressure) is technically challenging, and they are less suitable than rats due to more pronounced differences in lung anatomy to humans. In all these species, the right lung has more lobes than the left lung, but this aspect results particularly evident in the laboratory rodents, where the right lung has four lobes, while the left only one. In fact, the left lung of all small laboratory animals (mice, rats, hamsters) is not divided into lobes: only the larger laboratory animals, such as guinea pigs and rabbits, have two lobes [60]. Additional differences are in the anatomy of peripheral airways and the interdigitation of conducting airways and gas-exchange regions [60]. Respiratory bronchioles are extensive in cat, dog, sheep, monkey, and humans, and minimal in mouse, rat, hamster, rabbit, pig, cow, and horse [60]. The combination of differences in

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the extent of respiratory bronchioles, in acinar size, and in air-blood barrier thickness may account for the different sensitivity to inhaled toxicants between species [60]. For instance, murine alveoli are very small with mean linear intercept of 80 μm, compared to 100 μm for rats and 210 μm in humans, and the air-blood barrier measures 0.32 μm in mice, 0.38 μm in rats, and 0.62 μm in humans [94]. Hamster lungs are similar to rodent lungs.

Concerning the reaction to particulates, many studies identified differences between humans and rats. For example, the autophagy inhibitor 3-methyladenine, which reduced acute lung injury triggered by polyamidoamine dendrimers in mice, lacks clinical efficacy in humans due to reduced stability [95]. Different sensitivity to toxicants in rodent and human lungs is often explained by the much higher expression of metabolizing enzymes (mostly belonging to the CYP superfamily) in rodent lungs compared to human ones [96]. As reaction to dust exposure, human AT-II cells proliferate more than rat AT-II cells; as reaction to smoke and mineral dusts, smooth muscle hyperplasia is more pronounced in humans [97]. Silicates induce granuloma formation in both species, but rodent lesions are more cellular and less fibrotic than those in humans [60]. Only humans show a profound remodeling as a reaction to asbestos and other fibrous minerals [60]. Some features developing after chronic exposure, such as emphysema, are difficult to detect in rats due to their short life span [60]. Accumulation of dust is in the interstitial tissue in humans, while in the intraluminal one in rats [98]. Probably, the different accumulation patterns are the dominant reason for the observed differences in cellular responses [60]. While granuloma formation was common in rats, fibrosis was the predominant response in humans. After the exposure to fibrogenic and non-fibrogenic dust, acute intraluminal inflammatory and degenerative changes were more severe in rats than in humans. Moreover, while in rats all dusts induced epithelial neoplasia, in humans only dusts that initiated epithelial hyperplasia were associated with lung cancer. The different reaction pattern can explain these differences in carcinogenicity [99].

3.2.2 The 3Rs Principles

In addition to all these problems related to differences in lung physiology and responses to particles, animal testing is also a sensitive topic from an ethical point of view. The first to face this argument in a systematic way were Russel and Birch, which published The Principles of Humane Experimental Technique in 1959 [1]. In this essay, they proposed a new applied science aimed to improving animal treatment in laboratories, and to promote research quality in studies where animal testing is used. They presented and defined the 3Rs principles - Replacement, Reduction and Refinement - nowadays known as alternative methods, which aim to minimize animal sufferance in biomedical studies. More in detail, the definition of the principles is as follow:

• Replacement indicates every scientific method that uses a no sentient material instead of alive and conscious vertebrates. These no sentient materials include higher plants, microorganisms and endoparasites with an atrophied nervous and