Emulsion loaded with active agents allowing controlled delivery by ultrasound

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delivery by ultrasound

Nour Alrifai

To cite this version:

Nour Alrifai. Emulsion loaded with active agents allowing controlled delivery by ultrasound. Hu-man health and pathology. Sorbonne Université; Université libanaise, 2020. English. �NNT : 2020SORUS007�. �tel-03240163�

THESE de doctorat en Cotutelle

Pour obtenir le grade de Docteur délivré par

Sorbonne Université

L’Ecole Doctorale Pierre Louis de santé publique, ED393

L’Université Libanaise

L’Ecole Doctorale des Sciences et Technologie

Spécialité : Imagerie Biomédicale

Présentée par

Mlle. ALRIFAI Nour

Soutenue le 26 Mai 2020

Emulsion chargée en principe actifs permettant une libération

controlée par ultrasons

Membres du Jury :

M. Taulier Nicolas, Chercheur, CNRS, Sorbonne Université

Directeur de thèse

M. Charara Jamal, Professeur, Université Libanaise

Directeur de thèse

M. Nassereddine Mohamad, Professeur associé, Université Libanaise

Encadrant

Mme. Mignet Nathalie, Directrice de recherche CNRS

Présidente du jury

M. Inserra Claude, McF, Université Lyon 1

Rapporteur

M. Postema Michiel, Professeur, Université de Witwatersand

Rapporteur

M. Coulouvrat François, Directeur de recherche CNRS

Examinateur

Sorbonne Université

Bureau d’accueil, inscription des doctorants et base de données

étage

ème

Esc G, 2

15 rue de l’école de médecine 75270-PARIS CEDEX 06

Tél. Secrétariat : 01 42 34 68 35 Fax : 01 42 34 68 40 Tél. pour les étudiants de A à EL : 01 42 34 69 54 Tél. pour les étudiants de EM à MON : 01 42 34 68 41 Tél. pour les étudiants de MOO à Z : 01 42 34 68 51 E-mail : scolarite.doctorat@upmc.fr

Mme. Esteve Marie Anne, MCU-PH, Aix Marseille Université

Examinatrice

iii

Abstract

Drug encapsulation is a thriving field where potential therapeutic improvement are significant. In this regard, the ability to control the drug release using focused ultra-sound is appealing since this approach allows to precisely decide on the localization of the drug delivery as well as to manage the amount of delivered drug.

The most efficient drug carriers compatible with this approach are thermosen-sitive carriers or carriers comprising a perfluorocarbon liquid to take advantage of the acoustic droplet vaporization (ADV) mechanism to induce drug delivery. Ex-cept for Thermodox (thermosensitive liposomes encapsulating doxorubicin), none of these carriers have been considered for clinical trials. The reason is that the acous-tic energy needed to induce the drug release remains important and may induce side effects that have not been documented so far, in addition to the fact that many drug carriers used non-biocompatible compounds as stabilizer agents.

To overcome these difficulties, my research investigates the possibility to use low intensities ultrasound to control drug release. To this end emulsions were used as drug carrier to encapsulate two kinds of drug: Paclitaxel, as anti-cancerous drug, and "Levofloxacin", as antibiotics.

Paclitaxel was encapsulated in an emulsion made of nanodroplets comprising a core made of oil (5% v/v) and Perfluorooctyl bromide (PFOB) (95%) core which was stabilized by recently patented fluorinated and biocompatible surfactant called Dendritac. The presence of PFOB served to provide a detection by19_{F MRI.}

Levofloxacin was more water soluble than Paclitaxel, thus an important passive release was expected. To ensure that a sufficient quantity of Levofloxacin remains encapsulated into the droplets, we first get rid of PFOB so that the whole volume participate in the drug loading, then we hope to reduce the diffusion of Levofloxacin out of the droplet by adding triglyceride esters. The nanodroplets was this time sta-bilized by a biocompatible surfactant made of a hydrocarbon chain.

We investigated the ultrasound-triggered delivery of Paclitaxel in the presence of colorectal carcinoma cell line (CT-26) and of Levofloxacine in the presence of Es-cherichia coli (E. coli).

We showed that ultrasound triggers the drug delivery for acoustic pressures as small as 0.4 MPa when using ultrasound at a frequency of 1 MHz with a duty cycle of 5 % and a pulse repetition frequency of 200 Hz.

Our results showed that cavitation or ADV were absent during this release and suggested that the drug release is probably due to an enhanced diffusion of the drug out of the droplet. This mechanism requires a long pulse (of 275 periods in our case) which is to the contrary to what is used for ADV for instance. Moreover, this

mechanism induces a slow release rate, but sufficient enough so that the effect of the delivered drug can be important. More importantly, our data showed that the use of perfluorocarbon is not a prerequisite in ultrasound drug delivery which open way to the design of new kind of drug carriers.

v

Résumé

L’encapsulation de médicaments est un domaine florissant où les améliorations thé-rapeutiques potentielles sont importantes. À cet égard, la capacité de contrôler la libération du médicament à l’aide des ultrasons est séduisante car cette approche permet de localiser l’administration du médicament ainsi que de gérer la quantité de médicament délivrée.

Les vecteurs de médicaments les plus efficaces compatibles avec cette approche sont les vecteurs thermosensibles ou les vecteurs comprenant un liquide perfluoro-carboné qui utilise le mécanisme de vaporisation acoustique des gouttelettes (ADV) pour induire l’administration de médicaments. À part le thermodox (liposomes ther-mosensibles encapsulant la doxorubicine), aucun de ces vecteurs ont abouti à des essais cliniques. La raison en est que l’énergie acoustique nécessaire pour induire la libération du médicament reste importante et peut induire des effets qui n’ont pas été documentés jusqu’à présent, sans compter que de nombreux vecteurs utilisent un composé non biocompatible comme agents stabilisants.

Pour atténuer ces difficultés, j’ai étudié la possibilité d’utiliser de faibles intensi-tés ultrasonores pour contrôler la libération du médicament. Pour ce faire, j’ai utilisé des émulsions pour encapsuler deux types de médicaments : le «paclitaxel», un mé-dicament anticancéreux, et la «lévofloxacine», un antibiotique.

Le paclitaxel a été encapsulé dans une émulsion composée de nanogouttelettes comprenant un noyau composé d’huile (5% v / v) et de bromure de perfluorooctyl (PFOB) (95%) stabilisé par un tensioactif fluoré et biocompatible récemment breveté appelé Dendritac. La présence de PFOB doit permettre une détection par IRM19F.

La lévofloxacine est plus soluble dans l’eau que le paclitaxel, donc une libération passive plus importante était attendue. Pour s’assurer qu’une quantité suffisante de lévofloxacine reste encapsulée dans les gouttelettes, d’abord on s’est débarrassé du PFOB pour que tout le volume participe à l’encapsulation du médicament, nous avons aussi tenté de réduire la diffusion de la lévofloxacine hors de la gouttelette en ajoutant des esters de triglycérides. Les nanogouttelettes furent cette fois stabilisées par un tensioactif biocompatible constitué d’une chaîne hydrocarbonée.

Nous avons étudié la délivrance déclenchée par ultrasons du paclitaxel en sence de lignée cellulaire de carcinome colorectal (CT-26) et du lévofloxacine en pré-sence d’Escherichia coli (E. coli). Nous avons montré que les ultrasons déclenchent la délivrance de médicaments pour des pressions acoustiques aussi petites que 0.4 MPa lors de l’utilisation d’ultrasons à une fréquence de 1 MHz avec un rapport cy-clique de 5% et une fréquence de répétition des impulsions de 200 Hz.

Nos résultats ont montré que la cavitation ou ADV sont absents lors de cette li-bération et suggèrent que la lili-bération du médicament est probablement due à une

diffusion accrue du médicament hors de la gouttelette. Ce mécanisme nécessite une longue impulsion (de 275 périodes dans notre cas), ce qui est différent de ce qui est utilisé pour l’ADV par exemple. De plus, ce mécanisme induit une vitesse de libé-ration lente, mais suffisante pour que si l’effet du médicament délivré soit important. De plus, nos données montrent que l’utilisation du perfluorocarbone n’est pas une condition nécessaire à la délivrance de médicaments par ultrasons ce qui ouvre la voie à la conception de nouveaux types des vecteurs de médicaments.

vii

Acknowledgements

This work has been performed at the Laboratory of Biomedical Imaging of the Sor-bonne University. I would like to acknowledge the following people who have streng-thened me in my journey of pursuing a PhD cotutelle between Sorbonne University and Lebanese University and writing this thesis :

First and foremost, I would like to thank my thesis reviewers, Michiel Postema and Claude Inserra, and the thesis examiners : François Coulouvrat, Nathalie Mi-gnet, and Marie-Anne Esteve. It is not easy task, reviewing an interdisciplinary the-sis, and I am grateful for their patience, interest and thoughtful comments.

I express again my sincere gratitude to Nicolas Taulier, my first advisor (Sor-bonne University) for giving me the opportunity to do my Ph.D. in his group. I thank him for giving me a lot of opportunities to present our results at conferences and workshops and for his patience in explaining the acoustics basics and for sha-ring wonderful chocolate, which really stimulated me to work. I would like to sin-cerely thank Wladimir Urbach, for his endless support and explanations, and for his decision to work with me. And I would like to thank both Nicolas Taulier and Wladimir Urbach for the pleasant and comfortable environment in the office, that helped me a lot to be so confident and to express me at all levels.

I would like to thank my first and second advisor (Lebanese University) Jamal Charara and Mohamad Nassereddine for their confidence in me to apply to the CNRS-L / UL scholarship, that allowed me to do this Ph.D. thesis.

I am very grateful to all them for attention and all the time spent revising our ar-ticles and this thesis, also for complete freedom they gave me in a scientific research and at the same time readiness to discuss all issues and proposals.

I am thankful to all my colleagues who contributed to the work of this thesis through theoretical discussions and allowing me to perform experiments using their instruments. I thank Houssain Benabdelhak, Christine Contino Pepin, José Quintas, and Delphine Le Guillou Buffello for providing materials to investigate and fruitful discussions during our project meetings.

I am grateful to the Laboratory of Biomedical Imaging lab members for good mood due to small talks with a lot of jokes and discussions about cultural and eve-ryday life of France.

Thanks to my parents for their endless love, support and encouragement. To all my friends Sarah AL Gharib, Sara AL Homsi, Fatima EL Hajj, Jawad Ayoubi, Issaad Kacem, Choayb Omar, Abed Taleb and Mohamad Ibrahim. I thank you for your understanding and supporting me in many moments of crisis and illness, and, espe-cially, for being there when I need someone who care about me.

This thesis is only a beginning of my journey that, for sure, will be very interes-ting.

viii

Table of Contents

Chapter 1 General Introduction 1

1.1 Motivation . . . 1

1.2 Objectives . . . 1

Chapter 2 Literature Review on Vectorization 3 2.1 Introduction . . . 3

2.1.1 Importance of nanoparticles systems for delivery of active agents 3 2.2 Encapsulation and nanovectors . . . 4

2.2.1 Immune system . . . 6

2.2.2 Tumor accumulation of nanoparticles thanks to the "EPR" effect 6 2.2.3 Bioavailability and improved solubility . . . 8

2.2.4 Controlled delivery . . . 9

2.2.5 Size and elimination . . . 9

2.3 Nanoparticles Vectors . . . 9 2.3.1 The micelles . . . 10 2.3.2 Nanospheres . . . 12 2.3.3 Nanocapsules . . . 13 2.3.4 Polymersomes . . . 14 2.3.5 Emulsions . . . 15 2.3.5.1 Emulsion systems . . . 15 2.3.5.2 Emulsification . . . 16

2.3.5.2.1 Steps to manufacture an emulsion . . . 17

2.3.5.3 Aging mechanisms . . . 18

2.3.5.3.1 Sedimentation and creaming . . . 19

2.3.5.3.2 Flocculation . . . 20

2.3.5.3.3 Coalescence . . . 20

2.3.5.3.4 Ostwald ripening or molecular diffusion . . . 21

2.3.5.4 Medical applications . . . 22

2.3.5.4.1 Drug delivery . . . 22

2.3.5.4.2 Contrast agent . . . 25

2.3.5.4.3 Temporary blood substitutes . . . 25

2.4 Potential mechanisms for ultrasound delivery of encapsulated drugs . 25 2.4.1 Acoustic cavitation . . . 26

2.4.1.2 Inertial cavitation . . . 26

2.4.2 Heat generation . . . 28

2.4.3 Thermal Ablation . . . 29

2.4.4 Radiation forces . . . 29

2.4.5 Sonoporation . . . 29

2.5 Delivery of encapsulated medication triggered by ultrasound . . . 30

2.5.1 Microbubbles . . . 31

2.5.2 Perfluorocarbon particles or perfluorinated hydrocarbons . . . 32

Chapter 3 Materials and Methods 35 3.1 Materials . . . 36

3.2 Description of the droplet constituents . . . 36

3.2.1 Fluids . . . 36 3.2.1.1 Perfluorocarbon . . . 36 3.2.1.2 SuppocirerA . . . 38 3.2.1.3 Tributyl O-acetylcitrate . . . 39 3.2.1.4 Surfactants . . . 39 3.3 Paclitaxel . . . 40 3.4 Levofloxacin . . . 41 3.5 Nile red . . . 41 3.6 Production of emulsions . . . 42 3.6.1 Preparation method . . . 42

3.6.2 Paclitaxel-loaded emulsion preparation . . . 43

3.6.3 Levofloxacin-loaded emulsion preparation . . . 44

3.7 Emulsions size . . . 45

3.7.1 Dynamic Light Scattering . . . 45

3.7.2 Basic Principles . . . 46

3.7.3 Droplet size measurements . . . 46

3.8 Cell and bacteria cultures . . . 48

3.8.1 CT-26 tumor cells . . . 49

3.8.2 Gram negative escherichia coli . . . 49

3.8.2.1 Bacterial strains preparations . . . 50

3.8.2.2 Viability assessment . . . 50

3.9 Ultrasound-triggered drug delivery system . . . 50

3.9.1 Ultrasonic setup . . . 50

3.9.2 Transducer H101 1.1 MHz . . . 52

3.9.3 Focalised Hydrophone Y107 . . . 52

3.9.4 Fiber optic Hydrophone . . . 56

3.9.5 Chemical validation of the cavitation dose . . . 56

3.9.6 Ultrasonic measurements . . . 57

3.9.6.1 Experiments using tumor cells . . . 58

3.9.6.2 FUS exposure on planktonic suspensions . . . 58

3.9.6.3 Release experiments using encapsulated Nile red . . . 58

3.10 Statistical analysis . . . 58 Chapter 4 Ultrasound-triggered delivery of paclitaxel encapsulated in

an emulsion at low acoustic pressures 60

Chapter 5 Ultrasound-triggered release of levofloxacin encapsulated in emulsion and in vitro effect on planktonic Escherichia coli 70

x

Chapter 6 Conclusion and perspectives 80

6.1 Conclusion . . . 80

6.2 Perspectives . . . 81

Annexe A Résumé en Francais 83 A.1 Introduction . . . 83

A.2 Importance des systèmes nanoparticulaires pour la délivrance de prin-cipes actifs . . . 83

A.2.1 Encapsulation et nanovecteurs . . . 84

A.3 Utilisation des ultrasons comme élément déclencheur . . . 85

A.3.1 Ultrasons thérapeutique . . . 86

A.3.2 Libération ultrasonique de médicaments dans des sites ciblés . 86 A.3.3 Mécanismes d’administration de médicaments . . . 87

A.3.3.1 Déclenchement thermique . . . 88

A.3.3.2 Déclenchement mécanique . . . 88

A.4 Objectifs . . . 89

A.4.1 Nanovecteur : Émulsion chargée en médicament . . . 89

A.4.2 Préparation des émulsions . . . 89

A.4.3 1er objectif : Émulsion chargée en Paclitaxel . . . 90

A.4.3.1 Résultats . . . 90

A.4.4 2ème objectif : Émulsion chargée en lévofloxacine . . . 92

A.4.4.1 Résultats . . . 92

A.5 Conclusion générale . . . 93

A.6 Perspectives . . . 94

List of Figures

2.1 Size range of nanoparticles . . . 4

2.2 Nanoparticle active and passive targeting . . . 7

2.3 systems and strategies . . . 9

2.4 Schematic illustrations (not to scale) of various nanoparticles . . . 10

2.5 Surfactant molecules and micelles formation . . . 11

2.6 Representation of isolated and . . . 11

2.7 Representation of polymeric nanosphere . . . 12

2.8 Nanoparticles prepared by nanoprecipitation . . . 13

2.9 Schematic highlighting the advantages of polymersomes for drug de-livery . . . 14

2.10 Different types of emulsions . . . 16

2.11 Overview of high energy and low energy methods for preparing O/W nanoemulsions. . . 17

2.12 Schematic of various nanoemulsion . . . 19

2.13 Creaming and sedimentation phenomena . . . 19

2.14 Flocculation phenomena . . . 20

2.15 Coalescence phenomena . . . 21

2.16 Ostwald ripening phenomena . . . 21

2.17 Nanoemulsions for applications in drug delivery and the food industry. 23 2.18 Schematic diagram illustrating the effects of acoustic fields of identical frequency but differing pressure on microbubble behaviour . . . 27

2.19 Schematic diagram illustrating the effects of acoustic fields of identical frequency but differing pressure on microbubble behaviour. . . 27

2.20 Schematic diagram illustrating the effects of acoustic fields of identical frequency but differing pressure on microbubble behaviour. . . 30

2.21 Schematic of various nanoemulsion . . . 33

3.1 Schematic of a nanodroplet . . . 36

3.2 Mechanism showing the principle of the high-pressure microfluidizer (copied from Microfluidics documentation). . . 42

3.3 Paclitaxel calibration curve obtained by measuring the absorbance (Abs) of paclitaxel solubilized in tributyl O-acetyl citrate (ATBC) by using the spectrophotometer at λ = 227 nm. This curve was used to quantify the encapsulated quantity of paclitaxel in our emulsion. . . . 43

3.4 Levofloxacin calibration curve. The absorbance of Levofloxacin was measured using the spectrophotomter at λ = 298 nm. This curve was used to quantify the encapsulated quantity of Levofloxacin in our emulsion. . . 44

3.5 ALV / CGS-3 Compact Goniometer setup . . . 45

3.6 ALV / CGS-3 Compact Goniometer setup . . . 46

3.8 CT26 . . . 49

xii

3.10 Illustration of the ultrasonic setup used to insonify the samples . . . . 51

3.11 Illustration of part of the emitted electrical signal measured by the focalized hydrophone, having a period od repetition Trepetition= 5 ms and the effective sonication (While ultrasound is ON) time t = 0.25 ms. 52 3.12 Beamplots H-101G SN: 140 Fundamental Realtive Pressure, Linear Scale. . . 53

3.13 Recorded ultrasonic signal at 0.4 MPa and 3.5 MPa . . . 54

3.14 Recorded ultrasonic signal at 7 MPa . . . 55

3.15 Size range of nanoparticles . . . 56

3.16 Nile red calibration curve measured by spectrofluorometer at λex = 530 nm using different concentration of Nile red and leading to have a regression equation that allow us to quantify the released quantity of Nile red. . . 59

4.1 Graphical abstract (TOC). . . 60

5.1 Graphical abstract (TOC). . . 70

A.1 Plusieurs vecteurs sensibles aux ultrasons ont été conçus . . . 87 A.2 Viabilités des cellules CT-26 mesurées à 24 h (cercles) et 48 h (carrés) . 91

List of Tables

2.1 Comparison of macroemulsions, nanoemulsions (also referred to as miniemulsions) and microemulsions . . . 15 2.2 Commercialized emulsions based on the formulation of Intralipidr. . 24 3.1 Properties of perfluorooctyl bromide (PFOB) liquids . . . 37 3.2 Chemical structure of all used components of emulsion. . . 38 3.3 Chemical structure of the drug or dye that was encapsulated into the

emulsions. . . 40 3.4 Pictures of the transducer and hydrophones during experiments. . . . 50

xiv

Abbreviations

APD : Avalanche photo diode

ADV : Acoustic Droplet Vaporization ATBC : Acetyl tributyl citrate

CFU : Colony forming units

CHEL : Chinese hamster epithelial liver CMC : Critical micellar concentration CT-26 : Colorectal carcinoma cell line CT scan : computed tomography scan DC : Duty cycle

DLS : Dynamic light scattering

DMEM : Dulbecco/Vogt modified Eagle’s minimal essential medium DMSO : Dimethyl sulfoxide

E.coli : Escherichia coli

EIP : Emulsion inversion point

EPR : Enhanced permeability and retention FBS : Fetal bovine serum

FDA : Food and drug administration FPI : Fabry-Perot interferometer F6diTAC12: Dendritac surfactant

FUS : Focused ultrasound F6TAC12: FTAC surfactant

19_{F MRI :}19_{Fluor Magnetic Resonance Imaging}

HIFU : High intensity focused ultrasound HIV : Human immunodeficiency virus HPH : High pressure homogenization HTA : Hydroxytherytephtalate

MHA : Muller hinton agar

MH : Muller-hinton broth medium MPS : Mononuclear phagocytic system OD : Optical density

ODN : Oligonucleotides O / W : Oil-in-water

PCD : Passive cavitation detecting PEG : Polyethylene glycol

PFCE : Perfluoro-15-crown-ether PFP : Perfluoropentane

PFOB : Perfluorooctyl bromide PIT : Phase inversion temperature PLA : Polylactic acid or polylactide PRF : Pulse repetition frequency RES : Reticuloendothelial system SLS : Static light scattering TA : Terephtalate

THLB: Phase inversion temperature

US : Ultrasound

xvi

To my family,

My only and one inspiration

For their advice, their patience, and their faith,

Because they always understood.

يمأ ىلإ

يبأو

،

يتوق ىلإ

يمزعو

يفعضو

،

محتل اركش

بعاصملا هذه لك امكل

.مويلا هيلع انأ امل اببس متنك مكنلأ اركش ،يعم

،ديحولا يماهلإ ردصم متنك مكنلأ اركش

يهتني لا يذلا مكبحل اركش

.

.

1

Chapter 1

General Introduction

1.1

Motivation

Spectacular progress has been made in recent decades concerning the treatment of several pathologies. Nevertheless, certain treatments, in particular in the case of can-cerous diseases and bacterial infections, have a limited therapeutic efficacy and are still accompanied by considerable side effects. These side effects result on the one hand in the unwanted accumulation of the active ingredient in healthy tissues that causes some toxicity, and on the second hand in the nature of the excipients of the pharmaceutical formulation, such as Cremophor for Taxolrused as anticancerous drug, or antibiotics used for treating bacterial infections.

Targeting of therapeutic molecules to act on a specific site (a diseased tissue or cell) is currently a major challenge for the treatment of cancerous and infectious diseases. This concept is similar to that of "Magic bullet" imagined a hundred years ago by the scholar Paul Ehrlich, whose goal was to deliver a drug to its site of action in a specific way [1]. This concept comes closer and closer to reality with the develop-ment of nanotechnology which offers interesting opportunities for the targeting of active ingredients or contrast agents. Several types of nanoparticles have been de-veloped to protect these agents from degradation and to route them to their site of action. This vectorization thus makes possible to increase the therapeutic efficacy of the active ingredient and to reduce side effects.

Active methods of triggering encapsulated drugs were then implemented. Among these methods, ultrasound targeting is a very promising technique which has been the subject of numerous studies. Potentially, the application of ultrasound on a tu-mor, after accumulation of droplets inside it, not only frees the drug but also pro-motes its absorption in tumor cells.

The main mechanisms are acoustic droplet vaporization, cavitation and hypother-mia. All these mechanisms require the use of important acoustic pressure that can lead to bioeffects.

1.2

Objectives

This work represents a first step towards the use of focused ultrasound at low pres-sure with a cargo capacity and understanding of interactions between such agents and ultrasound.

we started by using an emulsion made with a Perfluorooctyl bromide (PFOB) core and oil. These droplets appear to be efficient as contrast agent for Fluor MRI [2, 3, 4]. Due to their stealth, they passively target solid tumors by EPR effect (En-hanced Permeability and Retention) [5, 6]. Our work will consist in encapsulating hydrophobic drugs (anticancer drug ’Paclitaxel’, antibiotic "Levofloxacin") into this droplets to accomplish their pharmacokinetic and biodistribution, and to develop an ultrasonic method inducing the release of the active agent under the effect of fo-cused ultrasound (FUS). The efficiency of droplets composed of an oily and PFOB core was tested in vitro on colorectal cell line CT-26 and the second type of droplets made only with oily core were tested on Escherichia coli (E. coli).

This thesis is divided into 2 parts, according to the methodology usually used. The first is a bibliographic section devoted to the study of the various publications showing the importance of nanoparticles systems for encapsulating active agents, and mentioning different types of nanoparticles vectors. We are interested in emul-sions systems, and their use in pharmaceutical domain and the influence of emulsion components on their response to ultrasound. Summary of the various nanoparticles systems triggered by focused ultrasound and the potential mechanisms for ultra-sound on the deliverance of encapsulated drugs will allow a rapid and global vision of what has been studied in this area.

The second part is reserved for the experimental part with a first part devoted to the formulation of nanoparticles and the measurements of droplet size. The focused ultrasound system is also presented in this part to study their effectiveness in vitro. These studies show the effectiveness of the technique of targeted release of active ingredient under the action of ultrasound. These results are presented in form of scientific articles.

Finally, a general conclusion takes up the various experimental results obtained. It is followed by showing the perspectives of the study.

3

Chapter 2

Literature Review on Vectorization

2.1

Introduction

2.1.1 Importance of nanoparticles systems for delivery of active agents

The combination of nanotechnology and molecular biology has developed an emerg-ing research area known as nanobiotechnology [7], whereas the similar term of " biomedical nanotechnology" is given to the use of nanotechnology in the medical sector.

The development of biomedical nanotechnology research has focused on the de-tection of molecules associated with diseases such as cancer, diabetes, mellitus and neurodegenerative diseases, along with the detection of microorganisms and viruses associated with infections (e.g. pathogenic bacteria, fungi and HIV viruses) [8].

Since 30 years, nanotechnologies has become more and more important in the biomedical field. This discipline offers unique perspectives for targeted delivery in Medical Imaging (Diagnosis), Gene Therapy and Agent Administration [9, 10]. The development of nanotechnology has made it possible to propose the concept of vec-torization of drugs using nanoscale tools that are capable to deliver active substances (intended for therapy) or contrast agents (intended for diagnosis) to their site of ac-tion.

A nanoparticle is an assembly of atoms or molecules, forming an object whose three dimensions are at the nanoscale, that is to say a particle whose nominal di-ameter is between 1 and 100 nm. In comparison with organic natural structures, nanoparticles are mainly in the corresponding size range of proteins (see figure 2.1) [11].

A nanoparticle is also defined as having "a diameter small enough so that their physical and chemical properties differ measurably from those of bulk materials [11]". The small size of the nanoparticles gives them interesting properties. It of-fers a large surface area and high stability. These particles have the advantage of forming dispersions that do not sediment.

New submicron delivery systems (nanoparticles), with new physicochemical prop-erties (size, surface property, stability in the biological environment ...) and new materials (polymers, lipids, metals ...), are regularly developed and tested. These systems provide an excellent opportunity for the administration of active ingredi-ents with high toxicity or low solubility, or even in in vivo noticeable instability. The toxicity of these active ingredients is generally due to their undesirable accumulation

FIGURE 2.1 – Size range of nanoparticles, in comparison with the

main chemical and biological structures (copied from [11]).

in healthy tissues, resulting in many cases in the abandonment of treatment. Sub-micron systems are able not only to protect active molecules (chemical molecules, peptides, proteins, DNA, RNA) from degradation but also to control their libera-tion in time and space. In the same way, the guidance of a contrast agent towards a pathological region makes it possible to improve diagnosis through better medical imaging quality.

The design of submicron tools for biomedical applications (nanomedicine) is a very complex process that requires understanding and mastering of different aspects related to nanoparticles: chemical, physicochemical, biopharmaceutical aspects and pharmacological.

The design of nanovectors must necessarily meet two main criteria:

1. The materials used must be biodegradable and biocompatible. They must be able to be eliminated by the body and not induce toxic and / or inflamma-tory reactions. Indeed, the accumulation and storage of certain materials in the body could lead to toxic effects for cells (a toxicity by thesaurismosis), in particularly after repeated administration of these nanosystems.

2. They must ensure the encapsulation of active agents, their transport and their release at the level of the biological target.

In addition, the biodistribution of nanoparticles in the body is affected by phys-ical characteristics of nanovectors, including their size, shape and flexibility, as well as by their chemical characteristics. These nanoparticles collide in vivo with different biological barriers. The challenge is certainly to succeed in designing nanoparticles that are able to cross these barriers and route the active agent to its site of action [11].

2.2

Encapsulation and nanovectors

For a beneficial therapeutic effect, the active agents, in their free state or encapsu-lated within nanoparticles, must reach their site of action. However, there are several biological barriers to overcome by these nanoparticles. These barriers can be classi-fied into three categories: the external barriers (skin and mucous membrane), the

2.2. Encapsulation and nanovectors 5

blood barrier and the cell barrier [12]. External barriers can be avoided by opting for intravenously injection, which is consistent with the nanoscale size of most nanopar-ticles. On the other hand, with this type of administration, nanoparticles encounter with several problems, such as renal and hepatic elimination, destabilization, aggre-gation, opsonization and elimination by the mononuclear phagocytic system (MPS) [13]. MPS belongs to the immune system composed of phagocytic cells, such as monocytes in blood and macrophages accumulated in lymphatic nodes, spleen and liver (Küpffer cells).

Among the barriers to the treatment of serious illnesses, the main factors are low proportion of actually administered drug particles and effective on medication-site due to non-specificity of biodistribution, and low retention time at the cellular level, which prevents a long-term action without regular administration of new doses.

Indeed, after the administration of the drug, the molecule, usually created with as the sole purpose of therapeutic activity passively spreads in the body due to the circulatory systems. Only a small percentage of the molecules, those present at the site of interest, have the desired effect. Also, besides, many drugs present unwanted side effects, caused by their presence and action in other parts of the body. The "needed dose" is often several times greater than the "effective dose".

For more than twenty years, a new field of research has been developed in order to solve this problem: "nanomedicine". This field includes encapsulation of drugs in supramolecular assemblies called nanovectors. These nanovectors can be com-pounds of polymers, lipids, peptides, carbohydrates, organometallic comcom-pounds, or a combination of these materials, and generally measure between ten and more hun-dreds of nanometers.

The ideal nanovector is expected to protect the drug from degradation or prema-ture interaction, which improves the absorption of the drug by the targeted tissues, in order to control the pharmacokinetics and tissue distribution (biodistribution) of the drug, and to improve cell penetration. It is also necessary that its constituents be biocompatible and easily functionalizable, whether soluble or suspended colloidal in aqueous conditions, whether they have reasonable circulation and conservation time, and a low rate of aggregation [14].

The term "nanovector" covers an incredibly wide range of supramolecular molecu-les assemblies used to carry other molecumolecu-les and isolate them from the external en-vironment. The first examples are liposomes, known since the 60s [15] and studied for this type of applications since the 90s [16]. Other nanovectors rely on the use of viral capsules emptied of their contents, carbon nanotubes or mesoporous silica nanoparticles.

The nanovectors can be roughly divided into three categories.

1. Nanocapsules and liposomes, hollow shells that may contain solution or sus-pension isolated from the outside environment. It is often an aqueous suspen-sion for a hydrophilic drug [17, 18].

2. Nanospheres, nanoparticles and micelles, without interior compartments, hav-ing spherical shape. The interstices of the matrix can accommodate molecules,

often hydrophobic, because the heart of the nanosphere is often more hydropho-bic than its shell [19, 20].

3. Dendrimers: repetitive branched spherical molecules with tree structure. The interest of dendrimers comes from their rigorous monodispersity, their ex-tremely predictable symmetry, and the properties of outer (surface) and inter-nal shells (between the dendrimer core and the exterinter-nal shell): as each "level" can theoretically be constituted of different monomers, customization is infi-nite [21].

With drug encapsulation, an exceptionally wide range of possibilities are open up for doctors and researchers, offering them tools to tailor a drug and adapt a de-livery mechanism to the patient. Passive or active targeting controlled dede-livery, pro-tection and solubilization of the drug, system avoidance immunity, extended circu-lation time, here is summarized a good part of the benefits that encapsucircu-lation can bring compared to the free drug.

2.2.1 Immune system

The surface charge of nanoparticles is an important parameter that contributes to non-specific interaction of nanoparticles with cells and plasma proteins. Positively charged nanoparticles are rapidly removed by MPS cells, because of their interaction with plasma proteins (by electrostatic interactions) which induces complement ac-tivation [22]. Cationic surfaces also interact with phospholipids and the negatively charged proteins present on the surface of membranes cell. This interaction pro-motes the cellular capture of nanoparticles by endocytosis or by direct penetration through the cell membrane [23].

Opsonization is also influenced by the hydrophilic / hydrophobic balance of nanomaterials. This process is faster when the nanoparticles are more hydrophobic. Indeed, plasma proteins adsorb more easily on the surface of hydrophobic particles through hydrophobic interactions [24, 25]. Modification of the surface properties of nanoparticles surrounded by certain hydrophilic and neutral polymers (e.g. PEG) allows in certain cases to minimize the opsonization phenomenon [26, 27]. This al-lows nanoparticles to circulate longer in the blood and reach more efficiently the tumor tissue. Hydrophilic nanoparticles with a neutral surface appear to be the best candidates to avoid capture by macrophages of MPS [22]. However, PEG polymers with a molecular weight of 200 have a genotoxic effect after metabolic activation as evaluated by induction of chromosome abberrations in Chinese hamster epithelial liver (CHEL) cells. These findings suggest a potential mutagenic risk also for PEG derivatives of similar size [28].

2.2.2 Tumor accumulation of nanoparticles thanks to the "EPR" effect

Nanoparticles designed to avoid their capture by MPS are able to reach the tumor site after prolonged circulation in the blood. Theoretically, the prolonged circula-tion time of nanoparticles allows them to accumulate in preference in some sites with vascular abnormalities (tumors and inflammations), thanks to the enhanced permeability and retention "EPR" effect. Maeda et al. [29] have well described this phenomenon.

2.2. Encapsulation and nanovectors 7

Indeed, in inflammatory conditions, the permeability of blood vessels increase by the action of different agents (commonly called "factors") acting on endothelial cells and opening intercellular tight junctions. Among these factors, we cite bradykinin, histamine, prostaglandins, and tumor necrosis factor. This phenomenon has also been demonstrated in the case of microbial infections. In the case of tumor tissue (cancer), and infected tissue (biofilms, prostheses) there are at least two factors that modulate vascular permeability :

1. Vascular permeability factor (VPF) and 2. Bradykinin

Both factors induce hypervascularisation of the tumor tissue, with vessels pre-senting large fenestrations (or pores) [29]. Macromolecules larger than 40 kDa and nanoparticles, having a prolonged blood circulation show significant tumor accu-mulation [29].

FIGURE2.2 – Nanoparticle active and passive targeting (schematic, not drawn to scale). The fact that nanoparticles passively accumulate in tumors via the EPR effect is a result of differences between tumors and healthy tissue. Cancer cell proliferation requires a constant sup-ply of nutrients and oxygen, which is why tumors form a rich vascu-lar network. Tumor vessels are structurally irreguvascu-lar, heterogeneous, and leaky (i.e., they have more and larger endothelial pores / fenes-trations) compared to normal vessels. Tumor vessel endothelial cells do not form a normal monolayer, may overlap, may form projections, and are irregularly spaced. In the EPR effect, nanoparticles enter a tumor through gaps (fenestrations / pores) in the tumor vasculature

endothelium (copied from [30]).

At the tumor site, small particles diffuse significantly outside of the tumor to rejoin the bloodstream, hence a higher low tumor accumulation compared to large molecules. In addition to tumor tissue permeability, there is a second factor that in-fluences the passive accumulation of macromolecules and nanoparticles at the level of the tumor. The Tumor tissues are characterized by lower lymphatic drainage, in comparison with healthy tissues [29], retained for a prolonged period of time in the tumor interstitium. The combination of this weak drainage with the increase of the vascular permeability at the level of the tumor gives rise to the EPR effect (see fig-ure 2.2). This EPR effect is a general phenomenon that encourages the development of macromolecular therapeutic agents and nanomedicine. It is therefore essential

to design nanoparticles capable of avoiding the opsonization phenomenon and ac-quire prolonged circulation in the blood, in order to achieve effectively the tumor tissue. The observed extravasation of macromolecules within specific tumor tissue was found to be very similar to that noted within the inflamed tissue resulting from bacterial infection (or inflammation) and factors affecting the inflammation of in-fected tissue were almost the same as cancer tissue [31]. Several strategies have been developed to prepare this nanoparticles type called "stealth". The most commonly used method is the coverage of nanoparticles by a crown of PEG which, by a steric repulsion effect, will prevent the adsorption of plasma proteins on the surface of the nanoparticles. The addition of ligands to the surface of the nanoparticles would, in turn, to recognize specific receptors in order to deliver the therapeutic agent within the target cell )see figure 2.2). All these aspects will be discussed in more details in the case of polymeric nanoparticles section.

Within the last couple of years, scientists have increasingly realized that the EPR effect is highly heterogeneous, changing over time during development and possi-bly also being transient. This pathophysiological phenomenon does not only vary between mouse models and patients, but also among tumor types of the same ori-gin, and among tumors and metastases within the same patient [32, 33].

Pancreatic, colon, breat, and stomach cancers showed the highest accumulation of nanomedicines. Tumor size also had an effect on the accumulation of nanomedicines, with large-size tumors having higher accumulation than both medium- and very large-sized tumors. However, medium tumors had the highest percentage cases (of 100% of patients) with evidence of the EPR effect [34].

As a consequence, the clinical outcome of nanomedicine treatments is also highly heterogeneous, and not as good as anticipated on the basis of preclinical results [35]. The notion that the EPR effect strongly varies [36] between individuals is of high importance, and may lead to misunderstandings and to a too pessimistic view on EPR-mediated passive tumor targeting (see e.g. [37], claiming that EPR is absent in patients, which is not the case and cannot be generalized [32, 34]).

2.2.3 Bioavailability and improved solubility

However, some drugs whose having an interesting therapeutic activity, are not us-able, because they are accompanied by heavy side effects, and due to their sensitivity to enzymes and proteins present in the circulatory system, or their low solubility in aqueous media including blood [38].

It is possible to increase the solubility of drugs, using a nanovector whose am-phiphilic character is sufficiently marked to favor the solubilization of the drug within the nanovector, and the solubility of the nanovector in the blood. These two solubilities (drug in the nanovector, nanovector in the blood) can be sensitive to various parameters such as temperature or pH. We can also open new routes of ad-ministration that are not feasible for nude drug, such as transcutaneous distribution [38].

2.3. Nanoparticles Vectors 9

2.2.4 Controlled delivery

With active targeting, controlled delivery is the second biggest goal in nanomedicine: being able to dictate place, duration, and release rate which limits side effects by lim-iting the circulating dose, but also to decrease the number of injections or tablets, so to increase the comfort of the patient.

We can even think of a modular remote release that we can activate and disable non-invasively. The use of light or magnetic field are two examples in development [39, 40] to control drugs delivery.

FIGURE2.3 – Systems and strategies used for drug targeting tumors (copied from [41]).

2.2.5 Size and elimination

The choice of the particle size is determined by the chosen site of action, which has a great influence on the elimination of nanoparticle in the body. The site of action can be intravascular, intratumoral or intracellular. Each one has its constraints and for a better understanding of the choice of size, a cut-off of the different biological barriers is detailed below. The particles having a diameter of the order of 100-150 nm usually have the time of longest circulation. Micron-sized particles can accumu-late in capillaries of the lungs. There is retention of nanoparticles with a diameter greater than 200-250 nm in the kidneys, because of interendothelial clefts measuring between 200 and 500 nm [42]. Nanoparticles having the size between 50-100 nm are eliminated by the liver [43]. Finally, the nanoparticles whose diameter are less than 10 nm are rapidly cleared by renal glomeruli after intravenous administration [44, 45].

2.3

Nanoparticles Vectors

There is a wide variety of vectors or "vehicles" capable of encapsulating drugs like chemotherapy. Some are already marketed, others are in pre-clinical or clinical tri-als, or in the experimental phase in laboratories. Their size can vary from a few nanometers (nanoparticles) to a few micrometers (microparticles). It is difficult to name them all and even to classify them as they are diverse.

FIGURE2.4 – Schematic illustrations (not to scale) of various nanopar-ticles that may be used in ultrasonic-enhanced drug and gene deliv-ery. A: Micelle (non-polymeric) composed of amphiphilic surfactants. B: Polymeric micelle composed of amphiphilic block copolymers. C: Nanoemulsion consisting of a hydrophobic liquid core stabilized by surfactant. D: Crystalline nanoparticles. E: Amorphous polymeric nanoparticle. F: Condensed ionic oligomers, such as DNA condensed with polyethyleneimine (PEI) or cationic lipids. G: Single-walled li-posome consisting of an amphiphilic bilayer surrounding an aqueous

core (copied from [46]).

Thus, Cho et al. [47] classifies nanovectors into: vectors of drugs based on polymers (polymeric nanoparticles, micelles, dendrimers), based on lipids, viral nanoparticles, carbon nanotubes. Soussan et al. [48] classifies them into two broad categories: matrices (micelles, emulsions, dendrimers, hydrogels, nanospheres, solid lipid nanoparticles) and vesicles (polymersomes, liposomes, niosomes, cationic vesi-cles). These particles can be qualified as solid, liquid or gaseous according to the macroscopic phase in where they are at ambient temperature [46]. Conventional vectors have a membrane and usually contain drugs in liquid or precipitated form. Other vehicles have been developed for specific applications. For example, mi-crobubbles whose surface are stabilized by a surfactant, are used for imaging and to release their contents under the action of ultrasound. In this section we will present only the most classic and current "vehicles". We will now present the major features of the different forms of polymeric nanoparticles, namely micelles, nanospheres, nanocapsules and polymersomes.

2.3.1 The micelles

Micelles are aggregates of spherical morphology of amphiphilic molecules, gener-ally surfactants whose hydrophilic polar head is in contact with water while the hydrophobic parts are grouped and directed inwards to minimize their contact with water (see figure 2.5).

Polymeric micelles are formed from amphiphilic block copolymers possessing one hydrophilic part and another hydrophobic part. These copolymers can self-assemble in an aqueous medium, such as surfactants, to form nanoparticulate struc-tures with a hydrophobic core and a hydrophilic crown. Indeed, amphiphilic macro-molecules are oriented to move the hydrophobic part of the environment to reach a state of minimal free energy. When the concentration of amphiphilic copolymers in-creases, the free energy of the system begins to increase due to adverse interactions between the water molecules and the hydrophobic region of the amphiphilic. This results in a structuring of the surrounding water, followed by a decrease in entropy [21]. At a certain specific concentration, called the critical micellar concentration

2.3. Nanoparticles Vectors 11

FIGURE2.5 – Surfactant molecules and micelles formation above crit-ical micelle concentration (CMC).

(CMC), several amphiphilic copolymers will self-assemble into colloidal particles called micelles (see figure 2.5). It should be noted that these objects are in dynamic balance with free amphiphilic copolymers ("unimers").

FIGURE 2.6 – Representation of isolated and self-associated am-phiphilic copolymers in aqueous media and general characteristics

of a polymeric micelle.

In comparison with micellar surfactant, polymeric micelles are generally much more stable and form at CMCs remarkably inferior to those of conventional surfac-tants (10−4−10−3 mM vs 0.1-1 mM) [49]. Moreover, in some dilution cases, these micelles resist disassembly because of the physical interactions between chains in the heart of the micelle [50].

The micelles typically have diameters between 10 and 100 nm and are character-ized by a "heart-crown" architecture. They are generally used for the solubilization of lipophilic therapeutic molecules (2.6). The conformation of the hydrophilic crown sterically removes the opsonization phenomenon, therefore micelles resist phago-cytosis by macrophages and decreases their elimination by the reticuloendothelial system (RES), allowing prolonged circulation time [51, 52]. The PEG is the most widely used hydrophilic polymer for the preparation of polymeric micelles [53, 54]. Genexol-PMr is an example of a micellar formulation of mPEG2000-PLA1750 en-capsulating paclitaxel. This formulation avoids the use of CremophorEL surfac-tant and ethanol for administration of paclitaxel and thereby reduce the resulting toxicity [54]. Other types of micelles, sensitive to the environment, have been de-veloped. For example, copolymers pH sensitive were used for the preparation of micelles. Among these copolymers, there is mPEG-Poly (2-vinyl pyridine) (mPEG-PVP), which has a critical pH, which corresponds to the pKa of the PVP chain, of the

order of 5 [55]. Indeed, at pH less than 5, the micelles are destabilized in "unimers", because of the protonation of the amine functions of the chain of PVP that becomes soluble in water. This type of micelles can therefore be used to deliver drugs at the level of tumors and release them into the intracellular medium that has an acid en-vironment (pH = 5). Other light-sensitive micelles attract currently a big interest due their use for the delivery of active ingredients. This photosensitivity is made possible thanks to the incorporation of different photochromes, such as azobenzene, spiropyran, dithienylethene, diazonaphthoquinone and stilbene [56].

However, the major disadvantage of micelles is their sensitivity to dilution, in addition to their limited capacity to load drugs. They are also easily destabilized in vivo. In several cases, after intravenous injection, the active ingredient encapsulated in the micelles is found to be released too quickly in the blood (in a few minutes) [53].

2.3.2 Nanospheres

FIGURE2.7 – Representation of polymeric nanosphere (copied from [57]).

A polymeric nanosphere can be defined as a solid colloidal particle in which the therapeutic molecules are dissolved, trapped, encapsulated, bound chemically or adsorbed to the polymeric matrix (see figure 2.7) [58]. These nanoparticles are gen-erally larger than micelles, with diameters ranging between 100 and 200 nm. The solid nature of the polymeric matrix gives these nanoparticles a great stability, com-pared with polymeric micelles. The polymers used for the nanospheres can be nat-ural polymers (biopolymers) or degradable or non-degradable synthetic polymers.

The preparation of polymeric nanospheres can be done according to two general methods, either by direct polymerization of the monomers in emulsion [ex. poly (methylmethacrylate) and poly (ethyl cyanoacrylate)], starting from a preformed polymer. If the polymer is preformed, as in the case of polyesters, the nanospheres can be prepared by the following methods: emulsion / solvent evaporation, salt ef-fect "Salting-out" and emulsion / solvent diffusion. But, the most used method is the nanoprecipitation (see figure 2.8) [59, 19].

The latter method consists in dissolving the polymer and the active ingredient in an organic solvent miscible with water, then mixing by adding drop by drop with an aqueous medium (containing or not surfactants). The organic solvent diffuse then in

2.3. Nanoparticles Vectors 13

all the aqueous solution, which leads to a precipitation of the polymer, and therefore to the formation of nanospheres.

FIGURE2.8 – Nanoparticles prepared by nanoprecipitation method leading to submicron particles.

It is also important to note that a clear distinction between micelles and nanospheres formed from amphiphilic copolymers is not always possible. Davis et al. have ex-plored, using mPEG-PLA copolymers, the effects of increasing the length of the hy-drophobic chain (PLA) on the physico-chemical properties of formed nanoparticles [60, 61]. They could show that increasing chain length PLA makes the central core of nanoparticles more and more "solid", giving rise to nanospheres, while shorter PLA blocks provide micelle type assemblies [60]. The capture of nanospheres by the mononuclear phagocytic system (MPS) is unavoidable. They therefore accumulate mainly in the liver and spleen.

2.3.3 Nanocapsules

Nanocapsules are composed of a thin monolayer of polymer, surrounding an oily cavity in the majority of cases. The active ingredient is confined in the internal cav-ity surrounded by a thin polymer wall (see figure 2.7) [62].

The most widely used method for producing nanocapsules is the interfacial de-position of preformed polymers [63]. In this procedure, a solution of active ingredi-ent and polymer in an organic solvingredi-ent (e.g acetone) miscible with water is prepared. For this solution, an oil (eg triglycerides) miscible with organic solvent but not mis-cible with mixture is added, and the solution is dispersed in an aqueous phase con-taining surfactants (ex. Poloxamer). Under moderate agitation, the organic solvent diffuses into the aqueous phase and the polymer aggregates around oily droplets, leading to the formation of nanocapsules [63]. The polymers used in the preparation of the nanocapsules include the homopolymers of polyesters such as PLA, PLGA and PCL. Copolymers of mPEG-PLA have also been used in recent years to avoid the phenomenon of opsonization [64]. These systems allow the encapsulation and delivery of hydrophobic active ingredients, such as the anti-estrogen RU58668 [64]. Nanocapsules with an aqueous cavity have also been developed for the delivery of Oligonucleotides (ODN) [65].

2.3.4 Polymersomes

Polymersomes are vesicular systems with colloidal size. The size of the polymer-somes can vary between 5 nm and 5 µm [66, 67].

FIGURE 2.9 – Schematic highlighting the advantages of polymer-somes for drug delivery. Surface functionalization of polymerpolymer-somes with carbohydrates, proteins, or small molecules allows for polymer-some targeting to specific locations within the body (green box). Stim-uliresponsive drug release allows for controlled drug delivery (blue box) of hydrophilic drug molecules, loaded within the polymersomes core (pink box) and hydrophobic drug molecules, loaded within the polymersome membrane bilayer (orange box) (copied from ([68]))

.

The polymersomes consist of an aqueous cavity surrounded by a bilayer of am-phiphilic polymer, thus resembling to liposome structure (see figure 2.9) [67]. These Nanoparticles are usually obtained by the method of "film rehydration". The am-phiphilic copolymer is dissolved in a volatile organic solvent which is evaporated to get a polymer film. The film is then rehydrated with a strong aqueous phase agitation, sonication or extrusion, leading to the formation of polymersomes with a narrow distribution [69, 70]. The most widely used copolymers for the prepara-tion of polymersomes are the diblocks mPEG-PBD (polybutadiene) and mPEG-PEE (Polyethylethylene) [69]. These materials are bioinert but not biodegradable, so al-ternative, research has focused on the use of "pegylated" polyesters, such as m-PLA and mPEG-PCL, each as single blister or blended with mPEG-PBD [70, 71].

Unlike nanocapsules, it is possible to encapsulate two types of active ingredients in the polymersomes: the hydrophobic active ingredients in the polymeric bilayer and the hydrophilic active ingredients in the aqueous cavity. This property was im-plemented by Discher et al. to co-encapsulate paclitaxel (hydrophobic) in the bilayer and the doxorubicin (hydrophilic) in the aqueous cavity, in order to increase the ef-fectiveness of anti-cancer [72]. The major disadvantage of polymersomes remains their instability. Moreover, the passive encapsulation requires a large quantity of active molecules.

2.3. Nanoparticles Vectors 15

2.3.5 Emulsions

An emulsion is a system including at least two immiscible liquids, of which one is dispersed in the other, in a more or less stable form. An emulsion is often described as a dispersion of droplets from one of the phases into the other. We distinguish therefore a dispersed phase and a continuous phase. For the emulsion to be durable ( that is, the dispersed state remains when mechanical agitation ceases), it is neces-sary to use an emulsifier. Its role is to stabilize the dispersed system by inhibiting degradation phenomena. The surfactants, polymers and divided solids are consid-ered as emulsifiers. Those most widely used are surfactants.

2.3.5.1 Emulsion systems

TABLE 2.1 – Comparison of macroemulsions, nanoemulsions (also

referred to as miniemulsions) and microemulsions with respect to size, shape, stability, method of preparation, and polydispersity. Na-noemulsions and microemulsions have a larger surface area per unit volume than do macroemulsions because of their size. In addition, due to a strong kinetic stability, nanoemulsions are less sensitive to

physical and chemical changes. Copied from [73].

macroemulsions nanoemulsions microemulsions

size 1-100 µm 20-500 nm 10-100 nm

shape spherical spherical spherical,

hexagonal lamellar stability thermodynamically thermodynamically thermodynamically

unstable, weakly unstable, stable

kinetically stable kinetically stable

method of high & low energy high & low energy low energy

preparation methods methods method

polydispersity often high typically low typically low

(>40 %) (< 10-20 %) (< 10 %)

Macroemulsion or emulsions: these are non-equilibrium dispersed systems com-prising two immiscible liquid phases. Emulsions are thermodynamically unstable systems because the separation of two phases leads to a decrease in free energy. However, the kinetics of droplet magnification can be sufficiently delayed for the emulsion to remain stable for a predetermined period of time. The average diam-eter of these conventional emulsions is greater than or equal to one micromdiam-eter.

Given their size, and depending on the viscosity of the continuous phase, emul-sion droplets sediment (cream) under the effect of gravity.

There are several types of emulsions according to the dispersion of aqueous and oily phases (see figure 2.10). Single emulsions are called water-in-oil (W / O) when water droplets are dispersed in the oily phase, and oil-in-water (O / W) for con-versely. Multiple emulsions are symbolized by o / W / O or w / O / W; o (respec-tively w) indicates the innermost phase and W (respec(respec-tively O) indicates the outer-most. The phases w and W or o and O may be the same or different. Bi-emulsions are emulsions containing two different internal phases of droplets, either of the same nature (but of different size), or of different nature (whatever the size).

FIGURE2.10 – Different types of emulsions (copied from [74]).

Nano/mini emulsions: these terms are used to name biphasic systems, of droplets size between 20 and 200 nm [75]. Because of the size of the droplets, nanoemulsions are transparent or translucent to the eye and are stable to sedimentation or creaming. The preparation of nanoemulsions requires either the use of highly energetic meth-ods, such as microfluidization, or the use of unconventional and complex methmeth-ods, but low energy consumption, such as phase inversion. The advantage of miniemul-sions is their extraordinary stability in aging and dilution [73].

Microemulsions: this term is used today to designate a monophasic system in which a particularly powerful surfactant makes possible the coexistence, on a quasi-molecular scale, of the water and oil phases. Unlike macro or nanoemulsions, they are thermodynamically stable.

2.3.5.2 Emulsification

The energy required for the emulsification operation can be supplied to the system in different ways, which leads to the existence of numerous processes. It can be mechanical (most commonly used) but also sonic, electrical, etc. One way to classify the most common emulsification processes according to the mechanism they involve is to distinguish the systems that generate shear and those that use the cavitation

2.3. Nanoparticles Vectors 17

phenomenon. The first group brings together, among others, emulsification specific mobiles (turbines, propellers, etc.), rotor-stator devices and colloid mills. The second group includes ultrasonic techniques and high pressure homogenizers [76].

2.3.5.2.1 Steps to manufacture an emulsion

FIGURE2.11 – Overview of high energy and low energy methods for preparing O/W nanoemulsions. (a) High energy such as high pres-sure homogenization (HPH) and ultrasonication break macroemul-sion droplets into smaller droplets. (b) Low energy methods start with W/O macroemulsions and break coarse emulsions into smaller droplets as they pass through a state of low interfacial tension dur-ing phase inversion. The emulsion inversion point (EIP) technique induces a phase inversion by water dilution whereas the Phase Inver-sion Temperature (PIT) approach induces a phase inverInver-sion on cool-ing of the mixture. To prepare W/O nanoemulsions, one can simply

reverse the continuous and dispersed phases (copied from [73]).

Generally, the emulsification is broken down into two successive stages:

• first a dispersion-mixing stage, which is called pre-emulsification and which will lead to a simple suspension of the droplets from the dispersed phase to the continuous phase (droplets of the order of 100 µm),

• then a so-called homogenization step aiming to reduce the size of the droplets so as to give the emulsion the required properties and to stabilize it. These two operations are carried out in agitated tanks or in pipes equipped with tools called respectively dispersers and homogenizers.

The preparation methods for O/W nanoemulsions are High pressure homoge-nization (HPH), ultrasonication, Emulsion inversion point (EIP) and Phase inversion temperature (PIT) routes. W/O nanoemulsions can also be prepared in a similar

fashion by reversing the dispersed and continuous phase.

As figure 2.11. shows, the first step in preparing an O/W nanoemulsion through a high energy method is to prepare an O/W macroemulsion, which is usually ac-complished by mixing oil, water and surfactant in a simple batch stirrer system for a sufficient period of time; in the second step, the macroemulsion is converted into a nanoemulsion. In a microfluidizer, a high pressure pump pushes the macroemul-sion through a narrow gap (gap height is on the order of a few microns [77]) where the large droplets break into smaller droplets as they are subjected to extreme elon-gational and shear stress [78, 77]. The homogenization process is typically repeated multiple times (referred to as the number of passages) until the droplet size becomes constant [79]. In an ultrasonicator, high energy shock waves create turbulence (due to cavitation) which ruptures the droplets. As with HPH, ultrasonication is contin-ued until the droplet size becomes constant [80].

In contrast to high energy methods, low energy methods begin with a W/O macroemulsion which is then transformed into an O/W nanoemulsion following changes either in composition or temperature (see figure 2.11.(b)). In EIP, a W/O macroemulsion is prepared at room temperature and is then diluted slowly with water. During this dilution process, the system passes through an inversion point where the transformation from W/O to O/W emulsion takes place. At this inver-sion point, the interfacial teninver-sion of the oil-water interface is very low and thus small droplets can be formed without a significant energy loss [81]. In PIT, on the other hand, the W/O macroemulsion is prepared at a temperature higher than the phase inversion temperature (THLB) of the mixture. When the oil-water surfactant mixture

is cooled down to room temperature, it passes through the inversion temperature at which the transformation of the mixture from a W/O to an O/W emulsion takes place. As with EIP, the interfacial tension of the oil-water interface near the inversion point is very low, and small droplets with high specific surface area can be generated with low energy requirements [82].

A similar dependence of droplet size in HPH was reported by Mason et al., [79] and Gupta et al [83, 84], where the droplet size was observed to reach a constant value with an increased number of passes. In all cases, the droplet size decayed exponentially with increasing ultrasonication time or, in the case of HPH, with the number of passes, a clear sign that droplet breakage dominates over coalesence dur-ing nanoemulsion formation [84, 85].

2.3.5.3 Aging mechanisms

Despite emulsions are not thermodynamically stable, such systems can exist for a long time: from days to months and years due to kinetic stabilization. In an emul-sion, four destabilization process befall: creaming, flocculation, coalescence, and molecular diffusion (or Ostwald ripening).

A scheme representing these mechanisms is shown on figure (2.12). The first two phenomena provoke particles adhesion but are generally reversible. Only coa-lescence and molecular diffusion degradation are responsible for droplets size evo-lution, thus are irreversible and lead to destabilization and subsequent of phase sep-aration.

2.3. Nanoparticles Vectors 19

FIGURE 2.12 – Schematic of various nanoemulsion destabilization mechanisms. Though nanoemulsions can be destabilized through any of the possible routes, initial growth of droplets generally oc-curs through Ostwald ripening. However, coalescence and floccu-lation become more important as the droplet size increases (copied

from [73]).

Different rupture mechanisms exist, they can be reversible or irreversible [86]. 2.3.5.3.1 Sedimentation and creaming

FIGURE2.13 – Creaming and sedimentation phenomena.

This mechanism results from the difference in density between dispersed phase and continuous phase. We speak of creaming when it comes to an ascent of the dis-persed phase and sedimentation when the disdis-persed phase falls (see figure 2.13). It

is a reversible phenomenon: the droplets still exist, a simple shaking let resuspen-sion of the droplet solution [87, 88].

To limit this phenomenon, we have several possibilities: • reduce the size of the dispersed phase droplets,

• add an agent which increases the viscosity of the continuous phase, • decrease the density difference between the two phases,

• avoid the aggregation of the droplets. 2.3.5.3.2 Flocculation

FIGURE2.14 – Flocculation phenomena. The small spheres of oil join together to form clumps or flocks which rise or settle in the emulsion

more rapidly than individual particles.

This mechanism results from the aggregation of the droplets due to the attrac-tive interactions. The interaction energy between the particles is due to the sum of the electrostatic repulsion forces and the Van der Waals attraction potential. This phenomenon can be reversible when the attraction is weak or irreversible when it is very strong [89].

To avoid this phenomenon, it is necessary to:

• avoid creaming and sedimentation (because these phenomena put the droplets in contact),

• Increase steric and electrostatic repulsions (using ionic surfactants for exam-ple).

2.3.5.3.3 Coalescence

This irreversible mechanism (see figure 2.15) results from the rupture of the interfa-cial film between the droplets of the dispersed phase. Two or more droplets merge to form a larger drop [87, 88]. The process is repeated, the total interfacial area be-comes smaller and the dispersed phase dissolves, and eventually we return to the two-phase system.

2.3. Nanoparticles Vectors 21

FIGURE2.15 – Coalescence phenomena. Emulsified particles merge with each to form large particles.

• prevent flocculation,

• Strengthen the resistance of the film by the choice of surfactant. 2.3.5.3.4 Ostwald ripening or molecular diffusion

FIGURE2.16 – Ostwald ripening phenomena. Larger emulsions grow at the expense of smaller ones due to the higher solubility of the smaller particles and to molecular diffusion through the continuous

phase.

There is still partial solubility of the dispersed phase in the continuous phase. At the end of the emulsification stage, the droplet population is not homogeneous in size. There is a flow of material from the small to the large droplets, through the continuous phase. The small droplets are emptied in favor of the big ones, and the granulometry is modified since the classes of small size disappear. This irreversible phenomenon constitutes the ripening of Ostwald [90, 91].

This phenomenon isn’t a problem in most manufactured emulsions. This phenomenon can be avoided by: