Optimized ultrafast laser cell poration

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Thesis

Reference

Optimized ultrafast laser cell poration

COURVOISIER, Sébastien

Abstract

Improvement of methods to modify and control the genetic expression of cells in terms of efficiency, cell survival, and throughput benefits widely to biology and medicine. Within the field of ultrafast laser induced transfection, we investigated two approaches: One is based on a plasmonic substrate that induces the local and transient poration of cell membrane for a gentle and high-throughput transfection by a purely optically induced ultrafast process. The second approach investigates the effect of temporal envelope modification of femtosecond laser pulse producing temporal Airy pulses for a direct and optimized laser-cell interaction.

These pulses are effective for reducing the energy and peak intensity thresholds required for cell poration with single pulse in the nJ range as well as controlling the morphology of the induced pores, with prospective applications from cellular to tissue opto-surgery and transfection.

COURVOISIER, Sébastien. Optimized ultrafast laser cell poration. Thèse de doctorat : Univ. Genève, 2016, no. Sc. 5027

URN : urn:nbn:ch:unige-912651

DOI : 10.13097/archive-ouverte/unige:91265

Available at:

http://archive-ouverte.unige.ch/unige:91265

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE Section de physique

Groupe de physique appliquée (GAP)

FACULTÉ DES SCIENCES Professeur Jean-Pierre Wolf Docteur Luigi Bonacina

Optimized Ultrafast Laser Cell Poration

THÈSE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention physique

par

Sébastien Courvoisier

de Sonvilier (BE)

Thèse N^◦ 5027

GENÈVE 9 décembre 2016

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Sébastien Courvoisier: Optimized Ultrafast Laser Cell Poration,A PhD Thesis on two ultrafast laser cell poration methods:

substrate-based plasmonics and phase pulse shaping, © Friday the 9th of December,2016

d i r e c t o r:

Professor Jean-Pierre Wolf

s u p e r v i s o r:

Doctor Luigi Bonacina j u r y m e m b e r s:

Professor Thomas Baumert, University of Kassel, Germany

Professor Alexander Heisterkamp, Leibniz Universität Hannover, Ger- many

l o c at i o n:

Geneva, Switzerland t i m e f r a m e:

Friday the 9th of December,2016 p u b l i c at i o n s:

— Courvoisier, S., Saklayen, N., Huber, M., Chen, J., Diebold, E.

D., Bonacina, L., Wolf, J.P., Mazur, E. (2015). Plasmonic Tip- less Pyramid Arrays for Cell Poration. Nano Letters, 15(7), 4461–4466.[28]

— Courvoisier, S., Götte, N., Zielinski, B., Winkler, T., Sarpe, C., Senftleben, A., Bonacina, L. Wolf, J. P., Baumert, T. (2016). Tem- poral Airy pulses control cell poration. APL Photonics, 1(4), 46102.[27]

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R É S U M É

L’amélioration des méthodes de modification de l’expression géné- tique des cellules bénéfice grandement à la médecine et aux sciences du vivant. Ces améliorations ciblent l’efficacité, la survie cellulaire ainsi que le débit de ces méthodes de transfection. Plus particuliè- rement, dans le domaine de la transfection induite par laser à impulsions laser ultra brèves, nous avons investigué deux approches.

La première méthode utilise les propriétés optiques d’un substrat plasmonique qui permet de concentrer l’énergie des impulsions laser en de multiples volumes nanoscopiques. Cette concentration permet d’induire la poration transitoire de la membrane plasmique des cellules pour des applications à haut débit tout en ménageant les cellules. La seconde investigation exploite des impulsions laser dont l’enveloppe temporelle a été altérée pour une interaction laser-cellule optimale.

Dans la méthode plasmonique, nous avons exploré numériquement et expérimentalement la réponse optique d’un substrat composé d’un réseau de pyramides nanostructurées transparentes couvertes d’une mince couche d’or, excepté sur leur pointe. La réponse optique par imagerie multiphotonique et par microscopie optique en champs proche a été comparée aux simulations numériques. En optimisant les paramètres géométriques pour une excitation à 800nm, nous avons obtenu une multiplication par 100 de l’intensité du champ proche électrique dans la région de contact entre les cellules et le substrat tout en conservant une faible section efficace moyenne d’absorption par le substrat. Enfin, nous avons montré expérimentalement qu’un tel système permet d’induire la poration transitoire de cellules euca- ryotes sur une large étendue permettant potentiellement de réaliser une technique de transfection purement optique fiable et à haut débit.

La seconde étude démontre que le façonnage de la phase spectrale d’impulsion laser femtoseconde peut-être utilisée afin d’optimiser l’interaction laser-cellule en milieu aqueux. Les seuils d’énergie et d’intensité crête requises pour la poration cellulaire avec une seule impulsion a pu être sensiblement réduite (25% de réduction en éner- gie et88% de réduction en intensité crête) en utilisant des impulsions d’Airy temporelles, contrôlée par une dispersion de troisième ordre, comparé à des impulsions de durée minimale. Les impulsions d’Airy temporelles sont aussi efficaces pour contrôler la morphologie des pores créés dans les cellules. Cette technique ouvre des possibilités d’application dans les domaines de l’opto-chirurgie tissulaire ou cellulaire ainsi que la transfection.

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A B S T R A C T

Improvement of methods to modify and control the genetic expression of cells in terms of efficiency, cell survival, and throughput benefits widely to biology and medicine. Within the field of ultrafast laser induced transfection, we investigated two approaches: One is based on a plasmonic substrate that induces the local poration of cell membrane for high-throuput application, while the second investigates the effect of temporal envelope modification of femtosecond laser pulse for a direct and optimized laser-cell interaction.

In the former study, we investigated computationally and experimentally by nonlinear scanning- and near-field microscopy a nanostructured plasmonic substrate made of tipless pyramids. By optimiz- ing the geometrical parameters for 800 nm excitation, our findings show a100-fold intensity enhancement of the electric near-field at the cell-substrate contact area while keeping the low absorption typical for gold at this wavelength. We demonstrate that such a substrate can induce transient poration of cells over a large surface and potentially high-throughput transfection by a purely optically induced ultrafast process.

The second study demonstrates that spectral phase shaping of fs- laser pulses can be used to optimize laser-cell membrane interactions in water. The energy and peak intensity thresholds required for cell poration with single pulse in the nJ range can be substantially reduced (25% reduction in energy and 88% reduction in peak intensity) by using temporal Airy pulses, controlled by positive third order dispersion, as compared to bandwidth limited pulses. Temporal Airy pulses are also effective for controlling the morphology of the induced pores, with prospective applications from cellular to tissue opto-surgery and transfection.

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A C K N O W L E D G M E N T S

The successful conclusion of this long-term work has been made possible only through the conjunction and interaction of a multitude of people. All these advice, support and shared expertise allowed me to reach the goal of this amazing experience. The technical resources made available by the various institutions involved were no less incredible. Before going further in my acknowledgements, I would like to thank Jean-Pierre Wolf for revealing my taste for physics research in my early years at the University and Luigi Bonacina for being my day after day advisor in my researches.

For clarity, the acknowledgements are divided in two parts which correspond to the two main chapters of my thesis and then a general section concerning all people not directly involved in this research.

p l a s m o n-a s s i s t e d c e l l p o r at i o n

This thesis with an unusual timeline began abroad, in the United States, with the project of cell poration using plasmonic substrate.

I thank Eric Mazur from Harvard University for welcoming me so warmly in his research group and Jean-Pierre Wolf for making this possible by his strong recommendation. Eric Mazur, who is always very supportive, has built a dynamic research group with many human qualities. My interactions with the Mazur Group will always remain engraved in my memory. I thank Eric for the confidence he showed by putting me on this project while the bio-subgroup under- went serious rearrangements. In addition, I thank him for having renewed his support by giving me a year of research assistantship.

Although I did not have the chance to collaborate directly with him, I would like to thank Eric Diebold who laid the first steps of this research during his doctoral thesis in the group of Eric Mazur with the notable contribution of Alexander Heisterkamp. I thank Oliver Hauser and Valeria Nuzzo for having patiently transmitted the fun- damentals of the project while I was battling with learning English.

I thank Jun Chen for her invaluable help on simulations, nano- fabrication as well as his unfailing availability. I thank Nabiha Sak- layen for her effective contribution through the project and to the cell poration experiment. I warmly thank Marinus Huber for our friendly transatlantic collaboration about our numerical simulations. I would of course like to thank Luigi Bonacina who contributed greatly to the success of this project as soon as I returned to the University of Geneva.

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Funding and equipment support

This academic travel has been greatly facilitated by the Ernest Bon- inchi Foundation, which I would like to thank here. Similarly, this research was supported by the National Science Foundation (US) under contract PHY-1219334 and PHY-1205465 as well as by the Swiss National foundation. Key facilities have been made available by the Harvard Center for Nano Scale, by NCCR Molecular Ultrafast Sci- ence and Technology (NCCR MUST), a research instrument of the Swiss National Science Foundation (SNSF), by the Aizenberg laboratory Harvard and by DPMC at the University of Geneva.

c e l l p o r at i o n c o n t r o l l e d b y t e m p o r a l a i r y p u l s e s This section cover the cell poration experiment made in collaboration with the Thomas Baumert’s group in Kassel University, Germany.

I would like to thank Thomas Baumert and his research team at for making this experience possible. Being able to share their collective expertise embodied in the state of the art and reliable ultrafast laser processing setup was an incredible experience. I would like to name specifically here, Nadine Götte and Bastian Zielinsky for building with me this experiment and working during late night cell poration experiments and finally sharing well earned Glühwein in cold winter nights at Weihnachtsmarkt. I thank Cristian Sarpe for applying his

"magic touch" on the laser system that, for sure, knows his master. I thank also Arne Senftleben and Thomas Winkler for their advice and reviews.

Back at the University of Geneva I would like to thank Michel Moret who helped me each time I needed reliable and stainless solu- tions. At the bioimaging center of the faculty of sciences, Christoph Bauer and Jérôme Bosset were always welcoming and available for advice on imaging. Furthermore, I would like to thank Miwa Ume- bayashi for arranging access to the nice cell culturing facility at Riez- man laboratory. Thanks also to Cameron Scott who responded to my message in a bottle and provided the Hela-MZ strain I used. At the physics department, I would like to thank Geraldine Cravotto-Barnois and Sébastien Müller for their technical support.

Funding and equipment support

This work was supported by the NCCR MUST. The main experiment was done at Thomas Baumert laboratory at the University of Kassel. Equipments were provided by DPMC UNIGE, the Riezman laboratory in biochemistry department and the bioimaging center of the Faculty of sciences at the University of Geneva.

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o t h e r s w h o s h a l l b e a c k n o w l e d g e d

I would also like to thank those who are not officially involved in this project: In the group of Eric Mazur, I wish to thank Orad Reshef for his wise advice on nanofabrication and his enthousiastic attitude.

Similarly, Philip Muñoz was of invaluable help with my simulations and my research queries, always available to provide his unconven- tional and enlightening vision. In addition, I would like to thank in a general way all the team of Eric Mazur’s group not named explicitly because they provided an incredible human and scientific framework to the accomplishment of this research. I would like to thank all the Thomas Baumert’s group members also not directly named here for their kind and very professional welcome.

On the GAP Biophotonics group side, I thank our dear Heavy- musician, Nicolas Berti for the friendly and noisy atmosphere he created in our vast office; as well as Julien Gateau for putting the cat among the pigeons during our lunchtime debates. I thank also Elise Schubert who showed me that it is possible to defend your funda- mentals no matter who is your opponent. Denis Mongin for allowing me to peep through the keyhole of the Care Bears Wonderland. I was really glad to meet Debbie Eeltink because she is the most badass of us all. I thank Mary Matthews for all the language debates and our bike races uphill. Also I would like to thank the GAP-bio Run- ner Team (Geoffrey Gaulier, Denis Mongin, Gustavo Souza, Julien Gateau, Luigi Bonacina) who trained every possible day around Pin- chat and provided a dynamic ambiance to my weeks, even in misty winter days. I thank Jérôme Kasparian for his kind availability providing always useful advice; Andrea Armaroli for his subtle touch to our not always subtle lunchtime debates; Jean-Gabriel Brisset for his epicforêt noirecake and hisbourrin touch; Cedric Schmidt for the good surfer type vibes he provided to the lab. Andrii Rogov and his calm advice on microscopy and lab as well as his respectful attitude.

Luigi, I acknowledge you can say "no" to coffee and chocolate: bravo!

Furthermore, I thank Sylvain Hermelin and his always useful advice tainted with high open-source standards.

Finally, I would like to thank all my familly and friends for their kind support in all circumstances; especially Delphine for her academic and strategic advice as well as her patience during my grumpy days; Tancred and Valérian because we moved abroad and they adapted without complaining. Geneviève because she was always helpful and available. With her help our academic travel was made possible and we could focus on our researches.

Thank you.

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C O N T E N T S

1 i n t r o d u c t i o n 1

1.1 Laser opto-poration . . . 1 1.1.1 Plasmon-assisted cell poration . . . 2 1.2 Improving laser-based poration methods . . . 3 1.2.1 Substrate-based ultrafast plasmonic poration . . 4 1.2.2 Temporal Airy pulses and optimized cell-poration 4 1.3 Overview . . . 5 2 m a i n t h e o r e t i c a l a n d e x p e r i m e n ta l b a c k g r o u n d 7

2.1 The goal: cellular surgery, cell poration and molecular intake . . . 7 2.2 The target: cells and the plasma membrane . . . 7 2.2.1 Eukaryotic cells: brief structural description . . 7 2.2.2 Cell membranes and the plasma membrane . . 9 2.2.3 Cell poration and transfection . . . 11 2.2.3.1 Cell membranes resealing after poration 12 2.2.3.2 Poration resealing timeline . . . 16 2.3 The means: ultrafast near-infrared laser . . . 17 2.3.1 Using ultrafast NIR laser in biological media . . 17 2.4 Ultrafast opto-poration . . . 18 2.4.1 Plasma formation in water . . . 19 2.4.2 Bubble formation and relaxation . . . 19 2.4.3 Size of holes, volume exchanged and molecular

delivery during laser-assisted poration . . . 20 2.5 Summary and general considerations . . . 21 2.5.1 Selecting an adapted cell line . . . 21 3 p l a s m o n-a s s i s t e d c e l l p o r at i o n 23 3.1 Introduction . . . 23 3.1.1 Cell poration using plasmonics nanostructures 24 3.1.2 Surface plasmon . . . 26

3.1.2.1 Non-resonant or resonant plasmonic light concentrators . . . 26 3.1.3 Energy transfer pathways in plasmonics follow-

ing laser excitiation . . . 27 3.1.3.1 Consequences of varying laser pump

intensity or duration on gold nanostructures . . . 28 3.1.4 Choice of substrate for plasmonic poration . . . 29 3.2 Micro- and nano-fabrication . . . 30 3.2.1 Summary of tipless pyramid array fabrication . 30 3.2.1.1 Fabrication control . . . 30 3.2.2 Fabrication side-project around Si templates for

optical application . . . 31

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xii c o n t e n t s

3.3 Optical response . . . 32

3.3.1 Scanning Near-field Optical Microscope examination . . . 32

3.3.2 Second harmonic scanning imaging . . . 35

3.3.3 Simulation: FDTD and FEM . . . 35

3.3.4 Optical response: comparing simulation and experimental results . . . 35

3.4 Poration experiment . . . 40

3.5 Discussion . . . 44

4 c e l l p o r at i o n c o n t r o l l e d b y t e m p o r a l a i r y p u l s e s 47 4.1 Introduction . . . 47

4.2 Pulse phase shaping and material processing . . . 48

4.2.1 Spectral phase properties of ultrashort light pulses 48 4.2.1.1 Second and third order dispersion . . 49

4.2.2 Pulses temporal shaping in the frequency domain 52 4.2.3 Pulses used in this study . . . 55

4.2.3.1 BWL pulses . . . 55

4.2.3.2 GDD modulated pulses . . . 55

4.2.3.3 Temporal Airy pulses . . . 56

4.2.4 TAPs in high band-gap materials . . . 57

4.2.4.1 Laser processing of fused silica with TAPs . . . 57

4.2.4.2 TAPs and the free electron density production in water . . . 61

4.3 Cell poration with BWL pulses, GDD pulses and TAPs 64 4.3.1 Material and method summary . . . 64

4.4 Results and discussion . . . 65

4.5 Conclusion . . . 72

5 m at e r i a l a n d m e t h o d s, c o m p l e m e n t 75 5.1 Cell lines . . . 75

5.2 Plasmonic poration . . . 78

5.2.1 Tipless pyramid array fabrication . . . 78

5.2.1.1 Summary of tipless pyramid array fabrication . . . 78

5.2.1.2 Step-by-step cleanroom fabrication . . 78

5.2.1.3 Deposition variants . . . 83

5.2.2 Diagnostics, labelling and characterization . . . 84

5.2.3 Simulation . . . 85

5.2.3.1 Finite-difference time-domain method 86 5.2.4 Sample and cells preparation for plasmonics poration . . . 87

5.3 Temporal Airy pulse cell poration . . . 88

5.3.1 Shaped pulse poration and micromachining setup at sample . . . 88

5.3.2 Sample protocol preparation for shaped pulse experiment . . . 88

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c o n t e n t s xiii

5.3.2.1 Sample reference marks . . . 90 5.3.3 Post-mortem sample examination . . . 91

6 d i s c u s s i o n 93

6.1 Result overview . . . 93 6.2 Tipless pyramid array for ultrafast plasmon-assited cell-

poration . . . 93 6.2.0.1 Tipless pyramid poration: what comes

next? . . . 94 6.3 Temporal Airy pulse and controlled poration . . . 94 6.3.0.1 TAP poration: what comes next? . . . 95 6.4 General conclusion and prospective . . . 96

i au t h o r c o n t r i b u t i o n s 99

a c o n t r i b u t i o n o f s. c o u r v o i s i e r t o t h e p r e s e n t e d

r e s e a r c h e s 101

a.1 Substrate-based plasmonic ultrafast poration . . . 101 a.2 Cell poration controlled by temporal Airy pulses . . . 102

ii a p p e n d i x 103

b a p p e n d i x: c e l l c l u s t e r s a m p l e p r e pa r at i o n p r o-

t o c o l s 105

b.1 Cell clusters fixation protocol . . . 105 b.2 Cell dehydration protocol prior to SEM imaging . . . . 105

c a p p e n d i x: l e n s e s a r r ay 107

c.1 Array of pyramidal microlenses . . . 107 c.2 Anti-adhesion layer formation protocol . . . 107 c.3 Microlenses array fabrication . . . 108

b i b l i o g r a p h y 111

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A C R O N Y M S

AFM atomic-force microscope AM acetoxymethyl

AuNP gold nanoparticle BWL bandwidth-limited CPD critical point drying DNA deoxyribonucleic acid EM electron microscopy

FDTD finite-difference time-domain FEM finite element method

FIB focused ion beam

FWHM full width half maximum GDD group delay dispersion HMDS hexamethyldisilazane IPA isopropanol

NA numerical aperture NIR near-infrared NP nanoparticle

PBS phosphate-buffered saline PDMS polydimethylsiloxane PMMA Poly(methyl methacrylate) RNA ribonucleic acid

ROS reactive oxygen species SEM scanning electron microscope SH second harmonic

SNOM scanning near-field optical microscope SPP surface plasmon polariton

TAP temporal Airy pulse Ti:Sa titanium-sapphire TOD third order dispersion

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1

I N T R O D U C T I O N

The field of cellular and subcellular surgery and related applications such as molecular delivery and genetic modification are topics of interest in biology and medicine. Indeed, the cell genetic vector delivery and expression, namely the transfection of cells, is a important process in the customization of cell lines especially in the age of personnalized medicine and regenerative medicine.[24] Current DNA delivery techniques can be divided into two major categories: viral- and non-viral-mediated.[98] In viral transfection, the genes of interest replace viral genetic material without eliminating the ability of the virus to target specific cells and deliver the vector efficiently to the nucleus.[19] The use of viral methods has been limited so far to clinical trials and laboratory assays because of the risk of devel- oping cellular-specific immune response and of inducing genotoxicity after insertional mutagenesis.[44, 69, 77, 80, 123] Electroporation was introduced as a non-viral technique for a direct entry of the DNA into the cell. It involves pulsing or cycling large gradients of the electric potential that induce transient permeability of the membrane.[78] While applicable to many systems, such harsh conditions often result in high mortality.[89] Electroporation and its successful recent improvements[100, 130] in transfection efficiency and cell viability are currently the most widely used physical gene-delivery methods. However, the spatial selectivity of electroporation is limited by the electrode shape and size.[127] When it comes to fragile cells such as human pluripotent stem cells that tend to go into apoptosis (cell programmed death) when electroporated,[51] gentle poration and transfection methods that optimize viability and efficiency while limiting the induced stress are required. Furthermore, the low transient-transfection efficiency in many non-integrative transfection methods (when gene are not integrated and are expressed transiently) may need multiple transfection events for sufficient sucess.[16]

1.1 l a s e r o p t o-p o r at i o n

Starting in1984, a first article by Tsukakoshi et al.[104] reports on a laser assisted gene delivery method relying on the focusing of a 5 ns laser pulses at 355 nm onto the cell membrane to porate the plasma barrier. With improvement in pulse duration in the 1990’s, the new transfection technique perfected. Generation of a transient pore allows uptake of a vector with efficiencies up to 50–100% for

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2 i n t r o d u c t i o n

transfecting cells with plasmids encoding enhanced green fluorescent protein.[99,102] In the case of stem cells however, yields of only25% have been achieved with opto-transfection.[31,105] A large range of laser sources, from UV to near-infrared (NIR) and from the nanosec- ond to the femtosecond range, were tested with various outcomes (for reviews, see Yao et al.[124] or Chung et al. [23]).

More generally, Vogel et al.[106] investigated physical processes involved in cellular and subcellular laser ablation. The laser treatement in the UV and visible light raised concern about the absorption of cellular constituents and their related toxicity[124] as well as the same damage coming from multiphoton absorption.[106] Indeed, UV light can be highly deleterious to cells as it causes DNA damage and gene alteration.[53]

For the direct laser-based approaches, the most efficient and most gentle poration and transfection method seems to be focusing an ultrafast (below 250 fs) NIR (700-1300 nm) laser on the cell membrane.[7, 99, 102, 124] The ultrafast 800 nm laser poration efficiency in mammalian cells was tested for pulse duration, pulse energy and length of pulse train (0.5 to 7.5·10⁶ pulses) to find optimal conditions.[7, 87, 99] The comparison was further extended by Davis and al.[30] comparing a train of100fs800nm pulses (3.8·10⁶pulses) at76 MHz repetition rate to two amplified pulses at1 kHz repetition rate.

Both approaches allowed succesful poration and comparable results even though they relied on different processes. The former approach produces a low-density plasma. The poration is produced by cumula- tive free-electron-mediated chemical effects. The latter, at irradiance above the optical breakdown in water, can produce a cavitation bubble and needs only one to a few shots to disrupt the membrane.[30, 106]

1.1.1 Plasmon-assisted cell poration

During the last decade, inspired by the direct laser subcellular opto- poration, scientists experimented with using nanoparticles. In particular, metallic such as gold nanoparticles (AuNPs) have the ability to focus the light energy at the nanoscale.[45] Indeed, the renewed interest in plasmonics starting in the1990’s was triggered by a conjunction of (a) a multidisciplinary interest to explore the nanoscale and (b) technical improvements.[45] Nanoplasmonics started to permeate in- terdisciplinary fields of research in particular in applications such as light guiding and emitting devices, enhanced spectroscopy, nanoscale imaging systems and bio-sensors. These applications were made possible by the improvement of nanoscale fabrication techniques com- bined with affordable simulation tools running on increasingly cost-

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1.2 i m p r ov i n g l a s e r-b a s e d p o r at i o n m e t h o d s 3

effective calculation power, and facilitated by the wide range of laser sources available. [45] For cellular dissection applications aiming at targetting and killing cancer cells, experimental successes were re- ported with plasmon assisted photothermal bubble generation with functionnalized AuNP.[50,52,68, 131] During the same period, plasmon assisted cell poration was experimentally tested with selectively bound AuNP to plasma membrane in the quest to find better methods for molecular delivery.[125] Plasmonic poration became a candidate of choice for improvement of molecular delivery and transfection.

Compared to direct laser poration that rely on single cell targeting, the throughput of plasmon assisted method poration is much higher because it can scan large cell-covered areas with a weakly focused laser.[94]

An ultrafast NIR off-resonance regime was proven to be an efficient and gentle way to porate and transfect cells.[95] This method relies on the same physical processes (multiphoton ionization followed by multiple inverse bremsstrahlung processes) as the pure laser-based method but with a lower irradiance for the plasmon enhanced method. The process doesn’t need precise laser focusing because the multiphoton ionization occurs only at the near-field hot spots created by the laser-nanostructure-dielectric interaction. Com- pared to direct laser poration, the irradiance can be divided by the intensity enhancement factor. When the local enhanced optical field is sufficient to create nanoplasma by multiphoton absorption, the remaining photon bath crossing the biological medium is absorbed efficiently only at the nanoscale hot spots (inverse Bremstrahlung processes). Outside the gold nanostructure, the photon bath will not induce significant damage to the cell sample.[12] Compared to poration with laser excitation matching the plasmon resonance of a nanostructure[75] (for example, wavelength of ≈540nm for 100 nm diameter NP), the off-resonance regime also porates the cell effectively while avoiding overheating, melting and fragmentation of the nanoparticles.[9] The fragmentation of AuNPs should be avoided because small NP sizes around 20 nm were proven toxic by inducing oxydative stress and very small AuNPs with diameters of1-2nm could be toxic because of the possibility of irreversible binding to important biopoly- mers.[20,60,72] Hopefully, in ultrafast NIR off-resonance plasmonic poration, melting and fragmentation of the AuNP occur only at flu- ence high enough to kill the cells and is not used in typical ultrafast laser plasmon assisted poration.[9]

1.2 i m p r ov i n g l a s e r-b a s e d p o r at i o n m e t h o d s

Aiming at improving the ultrafast laser assisted poration, two main ways are explored and presented in this work. First, a substrate-based method for plasmon-assisted poration. Second, a spectral phase shap-

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4 i n t r o d u c t i o n

ing method that can modify the temporal intensity distribution of ultrafast laser pulses for direct laser poration.

1.2.1 Substrate-based ultrafast plasmonic poration

The substrate based plasmonic poration method is related to the AuNP method but it presents some notable differences. 1) The enhancement factor can be higher at hot-spot compared to spherical NP while keeping the off-resonance asorption regime. 2) The substrate can be designed to produce hot spot at specific locations. While NPs can be functionalized with antibodies, for exemple, to target specific cells[60], substrate based method create spatial selectivity of cells ex- clusively by selecting the scanned area. 3) The number of poration events per cell can be controlled by design. 4) The substrate scheme can only be used on cells in close contact with the substrate (with sedimented detached cells or adherent cells). In contrast, the AuNPs scheme could be potentially used in thin biological tissue providing the AuNPs delivery to the target cells. Previous work done by Diebold [32] suggested an efficient near-field enhancement using a gold pyramid array, with a good cell adhesion and a likely efficient cell poration. His work served as a staring point for our investigation and application.

1.2.2 Temporal Airy pulses and optimized cell-poration

The second part of this work explores the effect of single pulse spectral phase shaping on cell-laser interaction for a prospective use for poration, transfection and cell surgery. In particular, we focused on temporal Airy pulses (TAPs) that were extensively studied in fused silica and water by Baumert research group.[35, 36, 42, 90, 114, 116, 117] The temporal intensity distrubution induced by positive cubic spectral phase to the spectrum leads to an intense initial pulse followed by a sequence of decaying pulses.[117] In fused silica, the modified temporal flux of energy going through the material leads to some control over the confinement and the extent of the electronic density within the focal volume. The modified free electron density can result in much deeper and narrower remodeling of the material compared to that produced by BWL pulses.[42] Similarly, in water, TAPs+ (with positive cubic spectral phase) lead also to a higher excitation deeper inside the sample compared to ultrashort Gaussian pulses.[114] Our experimental investigation on cancerous human cells focus on the poration conditions and type of poration induced around damage threshold.

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1.3 ov e r v i e w 5

1.3 ov e r v i e w

The manuscript is organised as follows: Chapter 2 describes the common background of the two investigated laser-based poration methods. First, cell components and structures with a focus on the plasma membrane is presented. Then, the poration, the resealing dynamics and its time-scale is overviewed. To end this theoretical background, the ultrafast laser ablation, poration, and opto-transfection process are presented. The following chapter (3) focuses on substrate- based ultrafast plasmonic poration providing first the relevant background. Our work covers the substrate design, nano-fabrication, optical charaterization and a proof-of-concept poration. The second investigation, in chapter4, is about temporally shaped pulses to control the poration. The production of TAPs is explained and experimental results of their interaction with fused silica and water is described.

Our original work focuses on the direct cell membrane ablation and poration threshold and the related type of damages induced for TAPs versus short Gaussian pulses and temporally broadened pulses. The final chapter6summarizes all main results of this work and connect it to the fabric of scientific advances in the field of transfection.

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2

M A I N T H E O R E T I C A L A N D E X P E R I M E N TA L B A C K G R O U N D

2.1 t h e g oa l: c e l l u l a r s u r g e r y,c e l l p o r at i o n a n d m o l e c- u l a r i n ta k e

In this chapter, we present the general framework and theoretical background of laser-assisted subcellular cell dissection for poration and molecular delivery applications. We begin with an overview of the cell and its major constituent with a focus on the plasma membrane. Succesively, we provide general considerations on cell poration and transfection and the time-scale involved in membrane pore formation and resealing. We continue with a presentation of the ultrafast laser poration by introducing the physical processes involved during a laser-cell interaction that result in sub-cellular ablation or a poration event.

2.2 t h e ta r g e t: c e l l s a n d t h e p l a s m a m e m b r a n e

The general presentation of cell structures described in the following section uses the well known textbook "Molecular Biology of the Cell" by Alberts et al. [2]. Other references are mentionned within the text.

Living organisms composed of eukaryotic cells are very diverse including for example small unicellular organisms such as yeast (Sac- charomyces cerevisiaefor ex.) as well as pluricellular organisms such as humans. Pluricellular organisms can be composed of a large variety of specialized cells with various functions, and shapes. All eukaryotic cell types share similar fundamental structures (plasma membrane, compartment membranes, nucleus, aminoacids, DNA, RNA) as well as hereditary and functioning structures and processes. Figure1 is a schematic overview of an eukaryotic cell. If we exclude cells whose structure is deeply altered by functional specilization such as final stage keratinized epithelium, all cell types are mostly composed of water and are similar in fundamental structure and physical properties.

2.2.1 Eukaryotic cells: brief structural description

7

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8 m a i n t h e o r e t i c a l a n d e x p e r i m e n ta l b a c k g r o u n d

Microtubule Centrosome Chromatin (DNA) Nuclear pore Nuclear membrane Vesicles

Extracellular matrix Lysosome Mitochondrion

Endoplasmic reticulum

Nucleus

Nucleolus

Plasma membr aneIntermediate filamentsGolgi apparatusCytosolic ribosome

Peroxisome

Actin filament

Figure1– Schematic of a typical eukaryotic cell and its subunits (organelles). The figure is reproduced from reference [2] (fig.1-31).

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2.2 t h e ta r g e t: c e l l s a n d t h e p l a s m a m e m b r a n e 9

An eukaryotic cell is mostly composed of water (≈70%) and can be seen as a structured gel of proteins divided into well-defined compartments separated by lipidic bilayer membranes. The three main structures of a cell are the plasma membrane, the cytoplasm and the nucleus. The volume of the cell is enclosed by the plasma membrane, which defines its outer barrier and maintain osmotic and composition differences between the extracellular environment and the cytosol. The cytoplasm is composed of the cytosol and specialized compartments or organelles such as the endoplasmic reticulum, the Golgi apparatus and mitochondria (see figure 1). These specialized compartments are also delimited by their membranes allowing different chemical environment to be created and tailored for their specific function. As an example, mitochondria and its electron transport chain need two layers of bilipidic membranes to create the proton gradient required to produce ATP with the help of the ATP-synthase (an inner mitochondria transmembrane enzymatic complex that act like a nano-motor). The nucleus can be seen as the control center of the cell as well as the main repository and processing unit of the genetic material. It is not considered part of the cytoplasm and is delimited by two membranes of lipid bilayer. The nucleus membrane is covered by nuclear pores responsible of the translocation of molecule in or out of the nucleus.

2.2.2 Cell membranes and the plasma membrane

Cell membranes are composed essentially of a lipidic bilayer with inclusion of proteic molecules (figure 2). These lipidic layers allow the creation and the upkeep of differential chemical environment between the outside versus the inside of the cell and also between the various internal compartments of the cell. The membranes are composed mainly of phospholipids, cholesterol and glycolipids. Four phospholipids types are dominant (>60% of lipidic membranes mass) in most mammalian cell membranes: the phosphatidylethanolamines, phosphatidylserine, phosphatidylcholine and sphingomyelin. The relative amount of these lipidic molecules in cell membranes vary as a function of its localization (internal versus external side and or- ganelle) and is related to its functions as it influence the physical parameters (permeability, fluidity). Cells have an active regulation of the lipidic composition of their membranes. This composition varies not only between cell types but also as a function of the environmen- tal parameters. For example, to maintain a constant fluidity some cells synthetize more unsaturated phospholipids (with double bonds in their lipidic carbon chain), and thus, change their membrane composition. Figure 2 shows in (A) a cross section of a red blood cell at the plasma membrane. Figure 2 (B) and (C) depict schematically the lipidic bilayer. One can see the membrane is approximately 5- to

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8-nm-thick considering the distance hydrophilic head to hydrophilic head. However, the plasma membrane, as the molecular barrier between the extra-cellular and the intracellular part, is thicker than 8- mn. Indeed, we can consider this barrier extending from the actin matrix anchored under the membrane to the part of the glycolipids that decorate the cell outmost side. This covering is called the glycocalyx and is composed of glucidic molecules that are covalently bound to lipids (glycolipids) and proteins (glycoproteins and proteoglycans).

The boundary between the glycocalyx and the extracellular matrix is not always clear. In some cell types (epithelium), the glycocalyx can be really thick (up to 10 micrometer) but usually is below 500- nm-thick.[33] The likely functions of the glycocalyx are (a) to protect cells from chemical or mechanical stress or injuries, (b) to maintain exogenous molecules or bodies away from the cell surface (protection versus viruses for example), (c) to protect from unwanted protein- protein interactions, (d) to act as a mechanical sensor and regulator, and (e) to repel negatively charged molecules such as nucleic acid molecules. Thus, the migration of genetic material in or out of the cell is prevented by the anionic nature of this covering, ensuring the genetic stability of the cell.[81] Above the cell, if we consider the tissue structure, the space between the cells is called the extracellular matrix and is composed mostly of collagen and elastin. This matrix is also a barrier to overcome for poration but is not investigated further in our application as its composition and structure is highly dependent on the tissue (for a detailed description see reference [38]).

lipidic molecule

lipidic molecule proteic molecule

proteic molecule lipicic bilayer (5nm)

(A)

(B) (C)

Figure2– Lipidic bilayer membrane: (A) Electron microscopy of a cell focusing on the smooth plasma membrane of a red blood cell. (B) bidimensional and (C) tridimensional schematic close-up view of a cell membrane with inclusion of membrane proteins. The figure is reproduced from reference [2] (fig.10-1).

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Flexion

Flip-flop

Rotation Lateral diffusion

Figure3– Phospholipids mobility: Possible movements of phospholipids within the lipidic bilayer. Flip-flop event is a less probable event compared to lateral diffusion. The figure is reproduced from reference [2] (fig. 10-8).

2.2.3 Cell poration and transfection

The cell poration is the process of disturbing or making holes in the plasma membrane of cells that allows the crossing of molecules in and out. A local perturbation of the membrane structure is sufficient for allowing Ca²⁺ ions to cross this barrier.[126,132] Evidently, larger molecules need larger holes to pass through. For example, Dex- tran chains (polysaccharide composed of many glucose molecules) with various length (up to 2000 kDalton) are used to assess the intake capability of poration techniques.[66, 83, 95] These uncharged molecules, can however, cross the porated membrane more easily compared to DNA molecules that are usually very large and negatively charged.[95]

The transfection is the delivery of exogenous genetic vector into cells resulting in the modification of its genetic expression. Trans- fection methods can be categorized into physical, chemical and biological methods. Physical transfection methods rely on direct physical process to deliver the genetic material into the cell. It includes microinjection, optical transfection[102, 104], ballistic gene delivery, electroporation and sonoporation.[57] One major advantage of physical transfection is that genetic vector intake does not depend on cell biological or chemical specificities. Thus, cells that are difficult to transfect by chemical or biological methods, T-cells, for example, can be successfully transfected by physical methods.[57] If the transfection is successful, the genetic modification can be transient or long- term. The transient transfection occurs when the injected genetic vector is not integrated in the chromosomes of the host cell and will fade out in the cell line after several cell divisions because this vec-

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Glycocalyx Cytosol Nucleus Plasma membrane

200 nm

Figure4– Glycolyx at cell membrane. TEM of a cell (lymphocyte) cross- section with glycocalyx stained by ruthenium red. The figure is reproduced from reference [2] (fig. 10-44).

tor is not replicated. Long-term transfection is achieved only if the gene is integrated and is replicated during cell-division (for example a transposon DNA sequence).[16]

2.2.3.1 Cell membranes resealing after poration

Plasma membrane injury repair mechanisms are truly fundamental to eukaryotic cell survival. Very small plasma membrane holes can spontaneously repair.[43]. Larger hole need active resealing mechanisms as the cytoskeleton opposing forces widen the opening.[25] Figure8schematizes this process. The wounded cell can survive only if a quick repair response is engaged in order to restore the boundary integrity between intra- and the extracellular medium. Studies on many cell models showed that all cell type were prepared to deal with membrane disruption through various resealing mechanisms. These mechanisms including cytoskeleton reorganization, trafficking, vesicle patching and recruitment of dedicated proteins at the wounded sites are diverse and seem to be peculiar to the cell type. The molecular mechanisms involved in the repair are contractile rings, vesicle fusion and cytoskeletal remodeling as well as cell mechanisms shared with phagocytosis, vesicles transport and secretion, or cell division.

Apparently eukaryotic cells reuse molecular pathway and machinery well established at an early stage during the eukaryotic cell evolution to repair the damaged membranes.[25, 54] Figure 7 schematizes the repair mechanisms involved in large membrane disruption.

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Figure5– Glycolyx at cell membrane. Schematic cross-section of a cell membrane with transmembrane proteins with glucidic molecules that are covalently bound to proteins and lipids. The figure is reproduced from reference [2] (fig.10-45).

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Membrane Disruption

B1

Cytoskeleton

B2

H2O

B3 Lipid Disorder

Membrane Tension

B4

Membrane Disruption

A1

A2

H2O

A3 Lipid Disorder

A4

Figure6– Spontaneous phospholipid bilayer resealing (A) versus no spontaneous resealing in real plasma membranes due to the opposing forces produced by the tethered cytoskeleton (B) (figure 1 from ref [25])

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Large membrane injuries result in rapid influx of Ca²⁺.

Vesicular delivery of extra membrane reduces membrane tension and facilitates

resealing driven by lipid disorder.

Exocytosis also provides small membrane patches to repair lesions.

Ca²⁺ influx triggers exocytosis.

Membrane integrity must be rapidly restored for cell survival

Hastily-repaired large injuries may require remodeling via exosomal shedding or endocytosis. These pathways are used for removal of bacterial pores,

though their role in repair of larger lesions is not yet clear.

Figure7– Schematic of a large plasma membrane disruption resealing process (figure3from ref [25])

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2.2.3.2 Poration resealing timeline

In the following section we present an overview of the membrane repair timeline[25]

t =0: Poration, membrane injury

Here we consider holes in cells membrane produced by a physical approach (pipette tip poking, particle impact, laser ablation, cavitation bubble, electroporation and sonoporation) and excluding bacterial pore-forming proteins poration. The hole diameter range from several nanometers to micrometers.

t =0-10s: Calcium-signaling activation and fast local vesicle fusion.

Extracellular calcium flood locally into the cell through the plama membrane openning, creating a high concentration of calcium ions that trigger calcium-activated signaling molecules. Local cytoskeletal and plasma membrane substrate are cleaved by calcium-dependent proteases (calpains). The signaling trigger the fast fusion of local vesicles. The filamentous cortical actin is depolymerized and proteic cleavage is reduced and the opening widens. Dysferin is recruited from distal regions of the cells and targeted to the damage site through cytoplasmic vesicles.

t = 10-30s: Active and slower phase of vesicle fusion. Cytoskele- tal network removal.

Calcium influx continues to promote exocytic fusion of lysosomes in the area surrounding the injury. A slower phase of vesicle fusion be- gins with the help of the active transport along microtubule and actin- myosin molecular motors. Allowing specific proteins to be recruited to the wounded site (Annexins VI and XI, ALG-2). The plasma membrane opening still expands.

t = 30-60s: Polymerization of resealing cytoskeleton network and lesion contraction

Actin accumulates around the injured area. The interplay between local actin filaments and polymerizing microtubules pull the microtubules and anchor them in position for a quick transport of signaling proteins and vesicles. Microtubules act on the polymerizing actin to ease the formation of an acto-myosin contractile ring. Endocytosis is triggered to remove damaged plasma membrane. Overall, the damaged area reduces in size.

t = 60-240s: Closing of plasma membrane opening, reconstitution of the hastily repaired plasma membrane. Back to normal

The actine-myosin contraction ring closes the plasma membrane opening. The cytoskeletal network reforms beneath the membrane. The quickly repaired area is remodeled by exocytosis, endocytosis, and ex-

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2.3 t h e m e a n s: u lt r a f a s t n e a r-i n f r a r e d l a s e r 17

osomal shedding. This process reconstructs the regular plasma membrane composition replacing membrane proteins, receptors, lipids and lipidic rafts.

2.3 t h e m e a n s: u lt r a f a s t n e a r-i n f r a r e d l a s e r

In our work we use titanium-sapphire (Ti:Sa) lasers as ultrafast pulsed light source. This technology was developped in the 1990’s.

It is nowadays stadard to produce ultrashort laser pulses in the femtosecond range (10⁻¹⁴−10⁻¹³s) around800nm (700-1100nm). These systems typically provide two regimes of pulses: one directly from the laser oscillator at high frequency (10⁸ Hz) with a energy per pulse in the nJ range. The second uses a chirped-pulse amplification stage that amplifies selected pulses from the oscillator and operates in the kHz repetition rate range. Usually, an amplified system can provide energy per pulse in the mJ range. By focusing with microscope objec- tives these amplified ultrashort pulses we can reach very high peak intensities (> 10²⁰W·cm⁻²).[6]

Femtosecond laser pulses can be seen as a set of monochromatic waves added coherently together. A given laser cavity allows only specific sets of waves to exist in it. These allowed spectral bandwidth

∆ν determines the shortest duration of the pulse ∆t, which can be calculated by the time-bandwidth product∆t∆ν≈0.44(for Gaussian pulses). For example, a spectrum of47nm full width half maximum (FWHM) centered at800nm is capable of producing pulses as short as18fs. The shortest pulses are achievable only if all spectral components are in phase, these kind of pulses are called Fourier-limited or bandwidth-limited (BWL) pulses.[6,116]

2.3.1 Using ultrafast NIR laser in biological media

UV and visible light can be significantly absorbed in biological media leading to the deposition of energy outside the tageted volume.

This energy deposited out-of-focus usually leads to unwanted damages. Thus, one major advantage of using ultrashort NIR laser is that biological media is mostly transparent at these wavelengths. Signif- icant light absorption occurs in biological media only when several photon absorption events can occur simultaneously, i.e. in the focal region which is characterized by high optical field intensities. This process is called multiphoton absorption and requires both the pulse duration to be short and focused.[120]

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2.4 u lt r a f a s t o p t o-p o r at i o n

Vogel et al. [106] studied theoretically the mechanisms of ultrafast laser surgery in biological substrate. They used water solution as a model system for their calculations. The model in water solution is a good approximation for laser-cell interaction helping nar- rowing down parameters in experiments and understanding under- ling processes. When tightly focused into biological media in water, ultrashort NIR pulses can locally ablate or modify matter even at irradiance below the breakdown threshold in pure water. At this breakdown threshold (≈ 10¹³W cm⁻² for a 100-fs, 800nm pulse) a free-electron density of 10²¹cm⁻³ is generated. Above threshold, the energy deposited in the focal region is sufficient to generate a bubble.[107] We should keep in mind that the pure water model from Vogel et al. neglected other molecular component that can make up to 40-20 % of the remaining matter in cells. For example, the multiphoton and cascade ionization rates can be influenced by dye and biomolecules. Thus, the free-electron density as a function of the laser pulse energy may differ substantially in a real cell compared to water solution only.[67]

The origin of laser induced damages range from mostly photochemical damages at low irradiance, well below the threshold, to a combination of photochemical damages, thermal and thermoelas- tic stresses at breakdown threshold irradiance. Around and above threshold, the induced cavitation bubble can affect strongly the spatial confinement of damages. There is no clear separation between these regimes and the induced damages result from the interplay of these mechanisms. Indeed, the free-electrons production precedes any thermal or thermomechanical effects. But the thermomechanical effects are never independent from free-electron induced chemistry or multiphoton-induced chemical effects.[106] However, applications involving molecular intake, like transfection, seem to require a bubble formation to be successful.[3,9,13,118] Depending on the target application, laser poration/transfection or sub-cellular dissection different type of irradiance may be chosen.

One can note that the production of reactive oxygen species (ROS) can follow the ionization and dissociation of water molecules and other biomolecules. In water species such as OH* and H2O2 can be produced.[40] ROS can be produced by ultrafast NIR pulses[8, 46, 103] and can be deleterious to cells and lead them to apoptosis-like death (programmed cell death).[103]

Our applications aim to use mostly high irradiance (plasmon enhanced or not) compared to the breakdown threshold. At this regime, the free-electron mediated effects become dominant compared to pho-

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2.4 u lt r a f a s t o p t o-p o r at i o n 19

tochemical reactions because the high-order nonlinearity of the free- electron generation.

2.4.1 Plasma formation in water

The laser induced plasma is essentially formed by the production of quasi-free electrons. An interplay of processes starting with multiphoton ionization or tunnel ionization followed by inverse Brems- strahlung processes in the plasma and impact ionization that can continue through avalanche ionization contribute to the free-electrons density. First, two processes start the free-electron production: multiphoton ionization and tunnel ionization. The multiphoton ionization dominates the low irradiance regime. At field strength around 1.3–2.6x10¹³ W cm² a transition from multiphoton to tunnel ionization occur in the ionization mechanism. Indeed, the high optical field induces a sufficient potential distortion allowing the tunneling of electron from water molecules potential well. For water molecules, multiphoton ionization occurs when the effective bandgap energy is overcome. The effective bandgap is composed by both the band-gap energy and the electron oscillation energy induced by the high optical field added together. This oscillation energy increases with the optical intensity linearly and is usually not negligible at the femtosecond pulse regime. These strong-field ionizations are almost instan- taneous and produce the "seed" free-electrons that will feed the following non-resonant process of inverse Bremsstrahlung. During this latter, a sequence of photon absorptions, involving a third particle, occur increasing the electron kinetic energy by successive steps. Af- ter several invers Bremsstrahlung processes, the electron reaches a critical kinetic energy that is sufficient to ionize one or several other water molecule by impact ionization. This process can start again with the free electrons available in the so-called avalanche ionization.

The inverse Bremsstrahlung processes take some time. Indeed the average time between collision in condensed matter was estimated to be roughly1fs. The minimum doubling time of free-electron density was estimated to be at least13.6fs.[106] With very short pulses, this value can have a significant impact on the final contribution of each ionization process to the free-electron density.

2.4.2 Bubble formation and relaxation

The pulse energy transferred to the plasma then thermalizes in the focal volume within a few picosecond. If the energy density is sufficient, a transient bubble can be produced that expand within the focal volume for low irradiance and can expand at a micrometer scale for irradiance around breakdown threshold.[108] The bubble expansion dynamic involving its fast expansion, its collapse and shock wave for-

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Figure8– Interplay of the Multiphoton ionization, inverse Bremsstrahlung processes and impact ionization involved in the free-electron plama generation. Avalanche ionization is the reccuring sequence of inverse Bremsstrahlung processes and impact ionization. (fig- ure1from reference [106])

mation contribute to the material dissection. A large range of bubble size can be produced depending on both the method and the irradiance.[106] For example, 20-nm radius bubble were observed by X- Ray scattering using AuNP plasmon-enhanced method with 400-nm 50-fs pulses. These nanoscale bubbles grow and collapse at a sub- picosecond time scale.[65] At the opposite side of the energy density range, experimental results with100-fs NIR pulses in water at intensi- ties10times above the optical breakdown threshold showed that from 200fs to20 ps the produced plasma doesn’t expand and is confined in the focal volume. From30 to 200ps, the plasma expands rapidly out of the focal region. At approximately 800ps a pressure wave is launched. After approximately 10 ns, the plasma is mostly recom- bined and converted to heat, and the vapor bubble can expand more at this point.[93] One can note that 10 ns is typically the interpulse duration of a typical Ti:Sa oscillator.

2.4.3 Size of holes, volume exchanged and molecular delivery during laser- assisted poration

As we saw before, the delivery of large charged molecules like DNA to the cytosol is not trivial as it needs to cross the plasma membrane. Indeed the negatively charged glycocalyx is repelent to the DNA. Large opening are necessary for the entry of large molecules.

This consideration is important because it was shown that large volume exchange between extra- and intra-cellular content is necessary for successful laser transfection. Indeed the relative cellular water and ions volume exchange during fs-laser induced poration was experimentally evaluated by patch clamp assay and calculated to be0.4

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2.5 s u m m a r y a n d g e n e r a l c o n s i d e r at i o n s 21

times the total cell volume for successful large molecule intake and transfection.[7] Hopefully, Davis et al. gathered evidences on the resealing dynamics and found that larger laser-induced holes resealed faster compared to smaller holes.[30] As we saw before in section 2.2.3.1, larger holes (µm range) can trigger different resealing mechanisms than smaller ones. Thus, we cannot directly connect the extent of damages on the cell plasma membrane to cell survival.

2.5 s u m m a r y a n d g e n e r a l c o n s i d e r at i o n s

Our application focuses on the local disruption of the main barrier of the cell, the plasma membrane as this process allows a molecular intake into the cell. Membrane poration should be done without damaging the cell in a way it could compromise its long-term survival. For example, the nucleus should be avoided because it con- tain almost all the genetic instructions of a cell. Thus any random damage to the nucleus may compromise cell survival and should be avoided.[29,64,86]

The processes involved in ultra-fast laser surgery are the local disruption of chemical bonds, formation of low-density plasma through multi-photon ionization, and the subsequent absorption within the plasma by inverse Bremsstrahlung processes followed by the generation of new free-electrons by impact ionization. Plasma relaxation can lead to the formation of a cavitation bubble, followed by its collapse, and then the full relaxation of the system through thermal diffusion. The timescale of the physical processes involved from the first moments of the laser-cell interaction to the full relaxation of the bubble (usually tens of nanoseconds) is several orders of magnitude shorter than the biological process of resealing. Indeed, the timescale for resealing after electroporation can range from 50 millisecond for the shortest to hours but usually are dcompleted within a few min- utes.[91] These first instants can be considered fully cell-type independent, and therefore, almost any cell line could be used as a model.

2.5.1 Selecting an adapted cell line

In the present study we choose cell type mainly based on their growth behavior, size, morphology and ease of handling. On can note, the cell response over time to the local disruption of the cell is dependent on the type of damage induced as well as the cell type and can range from microsecond to days. On the other hand, for experiments focused on the cell response and survival (from microsecond to days or cell generations) to transfection, we should keep in mind that the resealing process, the survival rate, the success of the genetic expression may vary greatly depending on the chosen species and cell types.

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3

P L A S M O N - A S S I S T E D C E L L P O R AT I O N

Figure9– Artist view of the tipless pyramid substrate with adherent cells, laser illumination and the plasmonic enhacement.

This chapter is based on, and extended from our published work:

Courvoisier, S., Saklayen, N., Huber, M., Chen, J., Diebold, E. D., Bonacina, L., Wolf, J.P., Mazur, E. (2015). Plasmonic Tipless Pyramid Arrays for Cell Poration. Nano Letters,15(7),4461–4466.

http://doi.org/10.1021/acs.nanolett.5b01697[28]

3.1 i n t r o d u c t i o n

With the advent and improvement of nanoscale fabrication tech- nologies and simulation capabilities, the field of plasmonics became in the last decade a flourishing field of research opening new pos- sibilities in terms of applications. Plasmonics study the physics of collective electron displacement in a nanostructured conductor at the interface with a dielectric. It allows the routing and the manipula- tion of light at a nanometer scale. One of the interesting advantages of these nanostructured conductors is the ability to overcome the diffration limit of light when focused with lenses (focal spot limited to ∼ λ/2). The plasmonic light concentrators are used, for example, to improve spatial resolution of imaging techniques, to conceive com- pact and ultrafast photodetectors, or to improve solar cell efficiency.

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24 p l a s m o n-a s s i s t e d c e l l p o r at i o n

[96] Plasmonic nanostructured substrates have also recently emerged as an attractive alternative to deposited nanoparticles for applications such as label-free bio sensing, Surface Enhanced Raman Scattering (SERS) spectroscopy, and nanometric sized particle trapping.[55, 76] In particular, nanopyramids have attracted much attention because they provide large field enhancement in a very confined and well defined volume at the tip [21, 73] enabling, among other applications, near-field imaging and spectroscopy.[56] Moreover, their relative ease of fabrication using a template-stripping process opens up the possibility of large-scale production.

3.1.1 Cell poration using plasmonics nanostructures

Plasmonic nanostructures, such as gold nanoparticles, have been used for inducing transient poration of cell membranes, allowing neighboring molecules to enter the cell. The first experiments on laser induced plasmonic transfection were conducted using gold nanoparticles as strong near-field light scattering centers.[94] The material en- tering the cell can be genetic material in the case of transfection but it can also be other biological or non-biological material. For instance, harmonic nanoparticles could be introduced as a non-bleaching high- contrast agent to follow a cell while it travels and lives within a host.[10] Compared to other methods (table 1), such as lipid-based transfection, plasmonic transfection is spatially selective and shows a better cell viability.[75] A transfection efficiency of about25% and cell viability of 80% was achieved using an off-resonance nano-particles plasmonic setup.[9] Likewise, transfection efficacy (75%) and viability (84% after48h) was achieved by plasmon-induced cavitation bubbles.[75] The plasmonic bubbles are created by irradiating gold nanoparticle clusters selectively attached on cell membranes by covalently bound antibodies. However, plasmonic transfection using nanoparticles raises the issue of possible nanoparticle toxicity.[60] Gold nanoparticles and nanorods showed no visible cytotoxicity in HeLa cells [84] while significant size-dependent toxicity was observed in fibroblast, epithelial cells, and melanoma cells.[129] Asharani et al.[4], [5] exam- ined the cyto- and genotoxicity from silver nanoparticles that enter cells without any additional interventions, when, for instance, they are used as antimicrobial agents. Oxidative stress seems to play a major role in nanoparticle toxicity.[82, 119, 121, 129] It has specific effects in the cells, including oxidative damage to protein and DNA and can cause autophagy as a cell survival response behavior.[72] Ox- idative attack on the DNA results in mutagenic structures such as 8- hydroxyadenine and 8-hydroxyguanine, which induces instability of repetitive sequences that can result in mismatched pairing. Baumgart et al. [9] also evaluated toxicity by assessing cell viability using cell survival. They report a >90% cell viability with only gold nanopar-