universita' degli studi di verona

0 downloads 0 Views 6MB Size Report
All the reagents and solvents used for the microparticle preparation and ...... Muthuswamy, S.M. Kauzlarich, R.D. Tilley, J.G. Veinot, ACS Nano, 7 (2013) 2676 ...... were determined by adding excess potassium cyanide to the solution to convert.

UNIVERSITA’ DEGLI STUDI DI VERONA

DEPARTMENT OF BIOTECHNOLOGY

GRADUATE SCHOOL OF SCIENCE ENGINEERING AND MEDICINE

DOCTORAL PROGRAM IN NANOTECHNOLOGY AND NANOMATERIALS FOR BIOMEDICAL APPLICATIONS

Cycle / Year

XXVIII/2013

LUMINESCENT POROUS SILICON FOR NANOTHERANOSTICS

FIS 01 - EXPERIMENTAL PHYSICS

Coordinator: Prof. Adolfo Speghini Signature Tutor: Prof. Gino Mariotto Signature Doctoral Student: Dott. Ali Ghafarinazari Signature

Declaration of Authorship

I, Ali Ghafarinazari, hereby certify that this thesis has been composed by me and is based on my own work, unless stated otherwise. No other person’s work has been used without due acknowledgement in this thesis. All references and verbatim extracts have been quoted, and all sources of information, including graphs and data sets, have been specifically acknowledged.

Date:

Signature:

ii

Dedication

To My Beloved Wife

Talieh Rajabloo

iii

Executive Summary The application of micro-to-nanosized fragments of porous silicon (pSi) as a vehicle for delivery and controlled release of drugs or nanoparticles is a promising strategy. In fact, the pSi morphology offers a large loading capacity and pSi in biological environments undergo dissolution, producing non-toxic and harmlessly removed wastes. pSi shows also an intrinsic visible photoluminescence (PL) that is derived from the combination of quantum confinement and surface effects. After a brief introduction to nanotheranostics and biomedical applications of Si in Chapter I, Chapter II presents experimental data about the fabrication of light emitting pSi. In this regard, we utilized metal-assisted chemical etching and then anodization. However, the optical properties of pSi quenched with time based on oxidation processes, thus surface stabilization is necessary in order to get optical stability (Chapters III to V). Organic functionalizations have been utilized to stabilize PL properties in atmospheric conditions. These modifications with related characterizations demonstrated in Chapter III. Further modifications for long-term optical stability in biological conditions have been reported in Chapter IV. The other approach of surface modification of Si is thermal oxidation as an effective way to passivate its surface. Chapter V demonstrates experimental and analytical results concerning kinetics of thermal oxidation reaction of pSi. As for application of the pSi, Chapters VI and VII report results of interaction with cells and drugs, respectively, in in vitro conditions. In the Chapter VI, pSi incubated with human dendritic cells to check toxicity and immune response. In addition, bioimaging properties of luminescent pSi monitored by a conventional confocal microscopy inside the cells and with two photon absorption microscopy. On the other side, the main challenge for usage of pSi in drug delivery is the redox activity of pSi on drugs. In Chapter VII this phenomenon was investigated comprehensively combine with loading capacity and release profile. Indeed, Nanotheranostics is a big challenging topic in Nanomedicine. In this research I would like to modify pSi as a candidate for nanotheranostics. iv

In this dissertation, Chapters two, three, four, five, six, and seven are, in part, reprints of the following publications:

1.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V.

Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, G. Mariotto, and M. Scarpa, Orange and blue luminescence emission to track functionalized porous silicon microparticles inside the cells of the human immune system, Journal of Materials Chemistry B, 2 (37) (2014) 6345 - 6353. 2.

A. Ghafarinazari, and M. Mozafari, A systematic study on metal-assisted

chemical etching of high aspect ratio silicon nanostructures, Journal of Alloys and Compounds, 616 (2014) 442 - 448. 3.

A. Ghafarinazari, E. Zera, A. Lion, M. Scarpa, G.D. Soraru, and N. Daldosso,

Isoconversional

kinetics

of

thermal

oxidation

of

mesoporous

silicon,

Thermochimica Acta, 623 (2016) 65 - 71. 4.

A. Ghafarinazari, V. Paterlini, P. Cortelletti, P. Bettotti, M. Scarpa, and N.

Daldosso, Optical study of diamine coupling on carboxyl functionalized mesoporous silicon, accepted at Journal of Nanoscience and Nanotechnology. 5.

N. Daldosso, A. Ghafarinazari, E. Locatelli, C. Laperchia, F. Boschi, P.

Bettotti, M. Scarpa, and M.C. Franchini, Long-lasting optical properties of mesoporous silicon functionalized with PEG and chitosan in physiological medium, Submitted. 6.

A. Ghafarinazari, G. Zoccatelli, M. Scarpa, and N. Daldosso, Comprehensive

study on redox activity of functionalized mesoporous silicon on drug delivery and bioimaging, Manuscript in preparation.

v

Moreover, the outcomes have been represented at International Conferences:

A. Ghafarinazari, Silicon Nanostructures for Biomedicine, Meeting on Nanowires, Amsterdam, Netherlands, 5.2016 (invited speaker) A. Ghafarinazari, M. Franchini, M. Scarpa, N. Daldosso, Luminescent Mesoporous Silicon, NANOMED, Manchester, UK, 11.2015 (Oral) A. Ghafarinazari, E. Zera, A. Lion, M. Scarpa, G.D. Soraru, N. Daldosso, Thermal Oxidation Mechanism of Mesoporous Silicon, The 11th Conference for Young Scientists in Ceramics, Novi Sad, Serbia, 10.2015 (Oral) A. Ghafarinazari, E. Locatelli, M.C. Franchini, M. Scarpa, N. Daldosso, Biopolymers functionalization of Mesoporous Silicon, International Conference on Nanotheranostics, Limassol, Cyprus, 10.2015 (Oral) A. Ghafarinazari, N. Daldosso, E. Locatelli, M.C. Franchini, M. Scarpa, Photoluminescent Mesoporous Silicon for Nanomedicine, The 16th Nanoscience and Nanotechnology Conference, Frascati, Roma, 9.2015 (Invited speaker) A. Ghafarinazari, N. Daldosso, P. Cortelletti, M. Scarpa, Mesoporous Silicon as a for Nanotheranostics, The 11th International Conference on Nanosciences Nanotechnologies (NN14), Thessaloniki, Greece, 7.2014 (best poster award) A. Ghafarinazari, N. Daldosso, P. Cortelletti, M. Donini, V. Paterlini, M. Scarpa, S. Dusi, Light Emitting Silicon Micro-particles as Biocompatible and Traceable Drug Delivery System, The European Materials Research Society (EMRS), Lille, France, 5.2014 (Oral) A. Ghafarinazari, P. Cortelletti, M. Donini, V. Paterlini, N. Daldosso, S. Dusi, M. Scarpa, Luminescent Porous Silicon Micro-particles as Biomaterial, The Nanotech Italy, Venice, Italy, 11.2013 (Poster)

vi

Table of Contents

Declaration of Authorship................................................................................ ii Dedication ....................................................................................................... iii Executive Summary ........................................................................................ iv Table of Contents ........................................................................................... vii Acknowledgements .......................................................................................... x Vita ................................................................................................................. xii

Introduction ................................................................................... 1 1.1 Motivation .............................................................................................. 2 1.2 Nanotheranostics .................................................................................... 2 1.3 Porous Silicon ........................................................................................ 4 1.4 References .............................................................................................. 6

Photoluminescent Silicon............................................................. 9 2.1 Summary .............................................................................................. 10 2.2 Introduction .......................................................................................... 10 2.3 Experimental fabrication ...................................................................... 12 2.3.1 Sample Preparation by Metal-Assisted Chemical Etching ........... 12 2.3.2 Sample Preparation by Anodization ............................................. 14 2.4 Morphological and Chemical Characterization ................................... 14 2.4.1 MACE Method.............................................................................. 14 2.4.2 Anodization Method ..................................................................... 16 vii

2.5 Conclusion ........................................................................................... 19 2.6 References ............................................................................................ 20

Functionalization of pSi............................................................ 23 3.1 Summary .............................................................................................. 24 3.2 Introduction .......................................................................................... 24 3.3 Experimental Procedure ....................................................................... 25 3.4 Morphological and Chemical Characterization ................................... 29 3.5 Conclusion ........................................................................................... 40 3.6 References ............................................................................................ 40

Biological Stabilization of pSi ................................................. 46 4.1 Summary .............................................................................................. 47 4.2 Introduction .......................................................................................... 47 4.3 Chemical functionalization procedure and experimental details ......... 49 4.4 Morphological and Chemical Characterization ................................... 52 4.5 Optical Characterization ...................................................................... 55 4.5.1 Two-Photon Absorption Imaging ................................................. 62 4.5 Conclusions .......................................................................................... 63 4.6 References ............................................................................................ 63

Thermal Oxidation of pSi .......................................................... 69 5.1 Summary .............................................................................................. 70 5.2 Introduction .......................................................................................... 70 5.3 Experimental Procedure ....................................................................... 71 viii

5.4 Structural and morphological characterization .................................... 74 5.5 Conclusions .......................................................................................... 86 5.6 References ............................................................................................ 86

Interaction of pSi with Human Dendritic Cells ........................ 92 6.1 Summary .............................................................................................. 93 6.2 Introduction .......................................................................................... 93 6.3 Experimental Procedure ....................................................................... 94 6.4 Results and Discussion ........................................................................ 96 6.5 Conclusions ........................................................................................ 104 6.6 References .......................................................................................... 104

Interaction of pSi with Cobinamide ...................................... 109 7.1 Summary ............................................................................................ 110 7.2 Introduction ........................................................................................ 110 7.3 Cobinamide synthesis and experiments ............................................. 112 7.4 Results and Discussion ...................................................................... 115 7.5 Conclusions ........................................................................................ 120 7.6 References .......................................................................................... 121 List of Figures .............................................................................................. 125 List of Tables ............................................................................................... 131 Conclusions of the Dissertation ................................................................... 132

ix

Acknowledgements

Working toward a Ph.D. for over three years is a great challenge, and certainly not a singular effort. I have met a variety of people in academic and non-academic settings that have contributed to this thesis and to the overall graduate student experience. I value all for the help I’ve received, and I would like to acknowledge the various sources of support hereafter. I thank our all the collaborators on the different projects. First, I want to thank my Ph.D. advisor Doctor Nicola Daldosso, an outstanding scientist, a respectable leader, and a great mentor. His guidance, encouragement, and support through the years helped me grow as a scientist in the field that I did not have any background on it. Also I appreciate Professor Gino Mariotto, my main supervisor, which provides conditions for me to research without any pressure. I’d like to thank Dr. Valentina Adolli, Dr. Marco Giarola, and Arun Kumar for helping me with my experiments and sharing Italian and Indian cultures. I would like to thank Prof. Marina Scarpa and her group (Laboratory of Nanoscience in Department of Physics, University of Trento). They taught me how to do synthesis and functionalizaztion of mesoporous silicon microparticles; particularly, Paolo Cortelletti who first explained to me how to do them. I’d like to thank Dr. Elena Froner, Dr. Paolo Bettotti, Anna Lion and everybody else from the Marina lab who helped me over the years. In addition, I’d like to thank Prof. Gian Domenico Sorarù and Emanuele Zera, from Department of Industrial Engineering at University of Trento, for their help and suggestions on thermal analyses. I would like thank form Prof. Mauro Comes Franchini and Dr. Erica Locatelli, Department of Industrial Chemistry at University of Bologna, for their efforts on surface coating by biopolymers and related characterizations. I would like to foreordain Prof. Stefano Dusi, Dr. Marta Donini, and Dr. Laura Marongiu for providing lab space and facility for cell experiments in general pathology division. Their insightful perspective on clinical research has helped me x

tremendously. Moreover, I should thank Dr. Federico Boschi and Dr. Claudia Laperchia from Department of Neurological and Movement Sciences for their help and suggestions on bioimaging experiments. Furthermore, I thank Dr. Gianni Zoccatelli from Department of Biotechnology at University of Verona, particularly for the preparation of cobinamide by HPLC. Finally, I thank my family, particularly my wife. Their support has been unwavering over the years, and I cherish all of their kind thoughts.

xi

Vita Ali was born in Tehran (Iran) at 21.3.1985. After completing his study at the Sama high school in June 2003, he entered University of Semnan. He received BSc in Sep. 2007. During these years, he was employed as a math teacher; and then, as an engineer of R&D at Loabiran Co. in Shiraz. After, he graduated on MSc at Azad University of Tehran. In 2012, he started research on silicon nanostructures with Prof. Ivano Alessandri at University of Brescia. In Jan. 2013, he entered the PhD School of Sciences Engineering Medicine at the University of Verona at Italy.

Publications (since 2013) A. Ghafarinazari, E. Zera, A. Lion, M. Scarpa, G. D. Soraru, N. Daldosso, Isoconversional

kinetics

of

thermal

oxidation

of

mesoporous

silicon,

Thermochimica Acta, 623 (2016) 65 - 71. G. Sargazi, D. Afzali, N. Daldosso, H. Kazemian, T. Tajerian, A. Ghafarinazari, M. Mozafari, A systematic study on the use of ultrasound energy for the synthesis of nickel-metal organic framework compounds, Ultrasonics Sonochemistry, 27 (2015) 395 - 402. M. Abbasi, S. Reddy, A. Ghafarinazari, M. Fard, Multiobjective crashworthiness optimization of multi-cornered thin-walled sheet metal members, Thin-Walled Structures, 89 (2015) 31 - 41. T. Rajabloo, A. Ghafarinazari, S. Sharifi-Asl, J.C. Caicedo, M. Mozafari, Multi-objective optimization of reaction parameters and kinetic studies of cobalt disulfide nanoparticles, Powder Technology, 269 (2015) 488 - 494. N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi and M. Scarpa, Orange and blue luminescence emission to track functionalized porous silicon microparticles inside the cells of the human immune system, J. Materials Chemistry B, 2 (37) (2014) 6345 - 6353. xii

A. Ghafarinazari, M. Mozafari, A systematic study on metal-assisted chemical etching of high aspect ratio silicon nanostructures, J. Alloys and Compounds, 616 (2014) 442 - 448. T. Rajabloo, A. Ghafarinazari, L. Seyed Faraji, M. Mozafari, Taguchi based fuzzy logic optimization of multiple quality characteristics of cobalt disulfide nanostructures, Journal of Alloys and Compounds, 607 (2014) 61 - 66. G. Sargazi, A. Ghafainazari, H. Saravani, Rapid synthesis of cobalt metal organic framework, J. Inorganic and Organometallic Polymers and Materials, 24 (2014) 786 - 790. A. Ghafarinazari, E. Amiri, M. Karbassi, Natural zeolite, a cost-effective Anatase stabilizer in glass–ceramic glaze, J. Powder Metallurgy & Mining, 3 (2014) 125 - 129. M. Abbasi, A. Ghafarinazari, S. Reddy, A new approach for optimizing automotive crashworthiness, concurrent usage of ANFIS and Taguchi method, Structural and Multidisciplinary Optimization, 49 (3) (2014) 485 - 499. M. Rezakazemi, A. Ghafarinazari, S. Shirazian, A. Khoshsima, Numerical Modeling and optimization of wastewater treatment using porous polymeric membranes, Polymer Engineering and Science, 53 (6) (2013) 1272 - 1278. A. Alidoosti, A. Ghafarinazari, F. Moztarzadeh, N. Jalali, M. Mozafari, Electrical discharge machining characteristics of nickel–titanium shape memory alloy based on full factorial design, J. Intelligent Material Systems and Structures, 24 (13)

(2013) 1546 - 1556.

xiii

Introduction

Introduction

2

1.1 Motivation The war on cancer was declared in the United States with the passing of the National Cancer Act in 1971 [1]. The European Union, through its Association of European Cancer Leagues, has recently developed the third version of its European Code Against Cancer: a list of eleven commandments on general lifestyle choices that can be adopted by all individuals to reduce the number of cancer associated deaths [2]. According to the recent World Health Organization’s Global status report cancers accounted for 7.6 million deaths worldwide [3]. It is no surprise that many resources over the last 40 years have been dedicated toward finding new manners to diagnose and treat the various types of cancers. With an ever-increasing and aging population, the number of cancer cases is anticipated to rise in the years ahead [4]. Therefore, it is essential that new types of diagnostics and therapeutics are explored in cancer research to enable earlier detection and improve patient treatment. Through employing newly developed nanotechnological tools, Nanomedicine may offer such alternatives.

1.2 Nanotheranostics In the vast field of Nanomedicine, “Nanotheranostics” combine therapeutics and diagnostics, aiming to provide a comprehensive platform for diagnosis, therapy and monitoring of the patient, leading to customized approaches and personalized treatment [5]. Figure I-1 shows Nanotheranostics steps in detail [6].

3

Introduction

Figure I-1. Scheme of nanotheranostics functionality [6].

Emerging nanotechnology discoveries provide a unique opportunity to design and develop such combination agents, permitting the delivery of therapeutics and concurrently allowing the detection modality to be used not only before or after but also throughout the entire treatment regimen, defining new supra-disciplinary fields in major clinical specialties such as Radiology, Surgery, Neurology and Oncology, to mention few. The

current

nanotechnology-based

Theranostics

systems

engineered

applications are not yet sufficient [7]. The main reason is Nanotoxicity, which is toxicity based on high aspect ratio of a material that in the bulk state is biocompatible [8]. Nanotoxicity has been observed recently for many nanomaterials such as Quantum Dots [9], Iron oxides [10], and Titan [11]. Then, there is a big challenge in the Nanotheranostics and in order to achieve this aim, it has to be mentioned not only material selection but also a deep characterization of their structural and functional properties.

4

Introduction

1.3 Porous Silicon A porous material can be divided into three main categories depending on the pore size: microporous is in the range of size less than 2 nm, mesoporous is in the range of 2 – 50 nm size, and finally macroporous which is more than 50 nm [12]. One of the major properties of a nanobiomaterial could be porosity; because, pore area provides a place for loading drugs based on capillary force. By decomposition or degradation of the pore walls, drug releases in a controlled rate. Among them, the optimum size is mesoporous for drug delivery [13]. Porous silicon (pSi) is a sponge-like material with a surface area that can be as large as 800 m2/cm3. About 25 years ago, L. Canham reported that pSi emits bright light in the visible range, and supposed that the reason of this emission was the remaining sponge-like silicon nanostructure that emits light thanks to a quantum confinement effect [14]. This photoluminescent (PL) property provided implementation of pSi in bioimaging techniques [15]. Due to these properties (i.e. PL and porosity) and biocompatibility [16], which is the most interesting characteristic, pSi became a promising candidate for Nanotheranostics and leads to increase number of publications on porous silicon (Figure I-2).

1500

Recourd Count

1200 900 600 300 0 1990

1995

2000

2005

2010

2015

Year

Figure I-2. Number of publications on Porous Silicon, from 1990 to 2015 (Web of Science).

Introduction

5

There are several methodologies to obtain silicon nanostructures based on corrosion of silicon wafer in HF solutions, among them anodization is the more promising leading to luminescent pSi (Chapter II). In general, this freshly produced pSi surface is SiySiHx (x+y=4) terminated and reacts with ambient air which affecting both its structural and optoelectronic properties [17]. It has been established that hydride terminated pSi is converted to native oxide, where oxide growth is dependent on atmospheric conditions [18]. To prevent native oxide growth, surface modification has been investigated (Chapter III). Moreover, surface modification can also be used to add functionalities to the pSi surface to enable use in specific applications. Surface modification of pSi can be divided into two broad categories: chemical functionalization and oxidation [19]. Functionalization is generally regarded as the attachment of carbon chains to the surface via various mechanisms, where both the Si - H and Si - Si bonds are reactive. Addition of biopolymers after surface functionalization leads to increase stability of pSi for months in biological conditions (Chapter IV). This component is promising not only for bioimaging but also for drug delivery and even for photothermal therapy. On the other hand, oxidation occurs via the controlled exposure of pSi to various oxidizing agents to induce the formation of oxide species on the surface [20]. As a part of this project, thermal oxidation has been investigated to assess the mechanism of oxidation (Chapter V). By knowing the mechanism in details, we can control the degree of oxidation of pSi in order to optimize the surface passivation, which is useful for protein delivery [21]. It must be noticed that among silicon wafers, pSi structures that are produced from p-type wafers, they are more stable against oxidation [22]. Then, n-type wafers have been used for thermal oxidation analysis, and p-types have been utilized for the other aspects, which we need less oxidized structure of pSi. After structural modifications and investigations by different methodologies, the modified pSi must be investigated in practical situations particularly in view of Nanotoxicity. For this purpose, interaction of the pSi with human dendritic cells has been investigated in standard conditions (Chapter VI). It has been demonstrated

Introduction

6

that pSi particles are uptaken by the cells without decreasing cell viability. Also, it was not observed any stimulation of the secretion of pro-inflammatory cytokines, suggesting particles do not activate these immune cells. The major concern on pSi for loading and then releasing of drugs is redox activity of pSi during degradation. This phenomenon leads to decomposition of drugs, mainly anticancer drugs that are sensitive to redox. Therefore, finally interaction of pSi with drug was investigated in Chapter VII. These outcomes indicate that these pSi particles are interesting candidates as delivery vehicles to the immune cell system of drugs and anticancer vaccines. Moreover, it was demonstrated by conventional microscopies that the optical properties of the pSi studied here can be used to monitor the intracellular localization of the particles. As a conclusion, we can propose pSi as a promising candidate for the challenging field of Nanotheranostics.

1.4 References

1.

The National Cancer Act of 1971, Journal of the National Cancer Institute, 48 (3) (1972) 577.

2.

V. Karagkiozaki, S. Logothetidis, Horizons in Clinical Nanomedicine, CRC Press, 2014.

3.

World Health Organization, Global status report on noncommunicable diseases, World Health Organization, Geneva, Switzerland, 2011.

4.

K. W. Jung, S. Park, Y. J. Won, H. J. Kong, J. Y. Lee, H. G. Seo and J. S. Lee, Cancer Research and Treatment, 44 (1) (2012) 25 - 31.

5.

S. Kunjachan, J. Ehling, G. Storm, F. Kiessling, T. Lammers, Chemical reviews, 115 (19), (2015) 10907 – 10937.

6.

en.wikipedia.org/wiki/Nanomedicine

7

Introduction

T.H. Kim, S. Lee, and X. Chen, Expert Review of Molecular Diagnostics, 13 (3)

7.

(2013) 257 269. –

274.

8.

A.S. Barnard, Nature nanotechnology, 5 (4) (2010) 271

9.

T.C. King Heiden, and P.N. Wiecinski, Environmental science & technology, 43 (5)



-

(2009) 1605 1611. –

10

.

T.R. Pisanic, J.D. Blackwell, V.I. Shubayev, and R.R. Fiñones, BioMaterial, 28 (16) (2007) 2572 2581. –

11.

L.K. Braydich- Stolle, and N.M. Schaeublin, Journal of Nanoparticle Research, 11 (6) (2009) 1361 1374. –

12.

N.K. Park, Y.B. Seong, M.J. Kim, Y.S. Kim, T.J. Lee, International Journal of Precision Engineering and Manufacturing, 16 (7) (2015) 1239



1244.

13.

P. Ghosh, Adv Drug Deliv Rev, 60 (2008) 1307

14.

A. Cullis, and L. Canham, Nature, 353 (1991) 335

15.

J. Rytkönen, R. Miettinen, M. Kaasalainen, Journal of NanoMaterial, 2012 (2012) 1 –

16

.



1315. –

338.

9.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, M. Scarpa, Journal of Material Chemistry B, 2 (2014) 6345

17.

6353.

L. J. Karyn, J.B. Timothy, A. P. Clive, Advances in Colloid and Interface Science, 175 (2012) 25

18.





38

.

AG. Cullis, LT. Canham, PDJ . Calcott. Journal of Applied Physics, 82 (1997) 909



914. 19.

K.L. Jarvis, T.J. Barnes, and C.A. Prestidge, Advances in colloid and interface science, 175 ( 2012) 25

20

.



38 .

A. Halimaoui, in Properties of porous silicon, ed. L. T. Canham, INSPEC The Institution of Electrical Engineers, London, 1997, 12 22. –

Introduction

8

21.

K.L. Jarvis, T.J. Barnes, and C.A. Prestidge, Langmuir, 26 (2010) 14316 – 14322.

22.

J. Salonen, M. Bjorkqvist, E. Laine, L. Niinisto, Phys Status Solidi A, 182 (2000) 249

Photoluminescent Silicon

Photoluminescent Silicon

10

Chapter 3: Functionalization of pSi surface

2.1 Summary Silicon nanostructures have been widely used as a highly promising semiconductor material in different applications. This study describes experiments on light emitting porous silicon (pSi) production by corrosion of silicon wafer with HF solutions. First, we utilized metal-assisted chemical etching and then anodization to inject election to silicon. By the metal-assisted chemical etching methodology, we obtained silicon mesoporous and nanowire structures. However, these structures did not have emission properties, then we used anodization to produce light emitting mesoporous silicon. Finally, morphology, optical and surface properties of the optimized structure of pSi have been investigated.

2.2 Introduction Semiconductor nanostructures are attracting substantial research, since they are promising candidates for improving the performance and cost of several types of devices. In particular, silicon nanostructures with high aspect ratios are continuously explored for the applications in biomedicine due to biocompatibility particularly for bioimaging. By engineering of silicon structure, it can be photoluminescence (PL) in the visible range. The most commonly accepted model for PL of pSi in the visible range is Quantum Confinement [1]. The reduction in the size provides a widening of the wave function of the HOMO and LUMO orbitals. It means actually that the bandgap does not become a direct band gap, but the widening of HOMO and LUMO orbitals increases the probability of radiative recombination because the transition of an electron from valence to conduction band and vice versa does not necessarily need be phonon-assisted. For this reason the transition is not really direct, and it can be called “pseudo-direct” band-gap [2]. The increase in the band-gap energy is a direct consequence of the Heisenberg Principle (∆P·∆x  h/4), so it means that the reduction of the dimensions forces the electrons to have a higher kinetics energy. The increase in the kinetics energy

Photoluminescent Silicon

11

Chapter 3: Functionalization of pSi surface is expressed by the equation ∆E = (∆P)2/2m = h2/[32m·2·(∆x)2], so it means that a larger band-gap is necessary, and that the increase in the bang-gap energy is inversely proportional to (∆x)2 [1]. Quantum confinement provides an increase in electrons energy and this also increases electrons lifetime in the excited state, so the probability of a radiative relaxation is higher [2]. Other mechanisms have been proposed to explain porous silicon photoluminescence, but the more complete is Quantum Confinement that also explains in a clear way the PL emission by quantum nanocrystals. Several approaches have developed for fabrication of pSi nanostructures. First, we chose Metal-Assisted Chemical Etching (MACE) method which is a simple and low-cost technique, offering controllability of structural parameters without electrical bias [3, 4]. Briefly, under appropriate etching conditions, the metal ions are reduced, and the ions inter the holes into the valence band of the Si substrate. As it is shown in Figure II.1, This localized microscopic electrochemical processes leads to self-assemble nanowire or mesoporous structures, via formation of an intermediate Si oxide formation [5].

Figure II-1. Schematic of Silicon wafer etching by increasing the corrosion conditions in MACE method.

Another common methodology to obtain pSi is electrochemical anodic etching of Si wafer in an HF aqueous solution. Mechanism of anodization is quite similar with MACE. In anodization a power supply provides electron, instead of a metal, for corrosion of silicon wafer. Following an electrochemical reaction, at the silicon

Photoluminescent Silicon

12

Chapter 3: Functionalization of pSi surface surface occurs a partial dissolution of Si itself. Due to the hydrophobic character of the clean silicon surface, ethanol is usually added to the aqueous solution to increase the wettability of the pSi surface. In fact, ethanol solutions infiltrate the pores, while purely aqueous HF solutions do not. This is very important for homogeneity and uniformity of the pSi layer. In addition, ethanol helps the removal of hydrogen released during the reaction [1].

2.3 Experimental fabrication All the reagents and solvents used for the microparticle preparation and functionalization were purchased by Sigma-Aldrich (Milan) and were of the highest available purity. Moreover, silicon wafer was provided from University wafers, Boston MA. Hydrofluoric acid (HF 48%) was diluted in Teflon vessels and used under a fume cupboard conforming to the required standards. As for the type of silicon wafer, the production of pSi can be achieved using p or n-type silicon wafers. P-type wafers are boron doped while antimony (Sb) is used to dope n-type wafers. Doping is used to achieve specific electrical properties. Heavily doped p-type wafers are the most commonly used to produce pSi. Although p-type wafers produce pSi with lower surface areas than other types, they are more stable against oxidation [6]. At this research, we used both types based on the aim; i.e. when we want to oxidize the pSi, n-type has been selected (Chapter V) but in the other cases we utilized p-type. Therefore, in this chapter we focus on p-type wafer that used for all of this project except the Chapter V. In the Chapter V, the optimized method for n-type wafer has been implemented based on the doctoral thesis of Neeraj Kumar [7]. 2.3.1 Sample Preparation by Metal-Assisted Chemical Etching The Si nanostructure was synthesized on single-crystalline [100] p-type wafer (15-25 Ω cm, 79.2 mg). Initially, the wafer immersed in a mixture of 5 M HF aqueous and 0.02 M noble metals, Ag and/or Pt, from AgNO3 and/or hexachloroplatinic (IV) acid (H2PtCl6) respectively, along with H2O2 aqueous solution, in different time durations and temperatures. All of these parameters have

13

Photoluminescent Silicon

Chapter 3: Functionalization of pSi surface been designed by Taguchi method. The Taguchi method, established by Genichi Taguchi (1924 – 2012), has been adopted to optimize the design variables because this systematic approach can minimize the overall testing time and cost [8]. By selection of 4 parameters with 3 levels, we have to do 9 different tests based on Taguchi method. These experiments were repeated three times in order to reduction of experimental errors and sample weight monitored to obtain amount of corrosion. Table II-1 shows experimental design in details and correspond results. With analysis of variance, effect of each parameter on corrosion estimated and finally a statistical model for this reaction validated.

Table II-1. Experimental design of Si nanostructure synthesis by MACE and corresponded mass of samples (mi). Tests

Parameters Noble metal

1

mi (mg) Time (min) Temp. (°C) H2O2 (M) m1

m2

m3

10

27

0

75.8 76.3 76

60

45

0.15

64.8 66.9 65.5

3

300

90

0.3

8.3

4

10

45

0.3

72.5 71.9 72.6

60

90

0

75.3 74.9 75.2

300

27

0.15

72.2 71.9 72.7

10

90

0.15

70.7 70.3 70.5

60

27

0.3

73.0 72.4 72.6

300

45

0

78.4 78.7 78.6

2

5

0.02 M Ag

0.02 M Pt

6 7 8 9

0.01 M Ag + 0.01 M Pt

8.1

7.8

Following the wet electroless etching process, the wafer wrapped with a thick film of noble metals. The as-prepared samples were dipped into a nitro-hydrochloric acid aqueous solution to remove the capped metals. Finally, the samples were cleaned with deionized water and blown dry with nitrogen. Mass of sample was recorded to measure corrosion of silicon for each condition [9].

Photoluminescent Silicon

14

Chapter 3: Functionalization of pSi surface 2.3.2 Sample Preparation by Anodization At this step, to produce pSi with the desired light emission property, a slice of [100] p-type silicon alluminated on the back side, with resistivity 10 – 20 Ω·cm has been etched in a PTFE anodization cell with a metal anode and a Platinum cathode in an ethanol-HF solution 2:1, using a current of 80 mA·cm-2 for 5 min [10]. Therefore, the pSi particle is produced by top-down approach, by sonication in ethanol or toluene for 15 min.

2.4 Morphological and Chemical Characterization Scanning Electron Microscopy (SEM) images were obtained by using a Zeiss SUPRA 40, with a thermal field emission source, operating at an accelerating voltage in a range of 1.5 – 5 kV. Furthermore, the analysis of the particle size distribution was done by optical microscopy using an Olympus microscope equipped with a 100× magnification objective (MPlan, NA 0.9; Olympus) and 10× oculars. Images were analysed using Fiji software [11]. The porosity was determined by gravimetric analysis [12] and the specific surface area was calculated according to Halimaoui [13]. The chemical groups present on the pSi were investigated by Fourier Transform Infra-Red (FTIR) spectroscopy. The pSi film, before sonication, left to dry under gentle nitrogen flow. The spectra were acquired by using a micro-FTIR Nicolet iN10 instrument, in the spectral range of 500 - 4000 cm−1 with 4 cm−1 resolutions by the mode of reflection. Also, The Photoluminescent (PL) properties of the pSi has been investigated by a Varian Cary Eclipse Fluorescence Spectrophotometer. Sample was excited at 350 nm, with 10 nm silt. 2.4.1 MACE Method Experimental conditions and mass samples represented in the Table II-1. Interestingly, Si nanostructures produced in different morphologies by MACE method. After reaction at HF, surface of silicon wafer covered by metals that induced electron during corrosion of Si. Silver formed dendritic shape (Figure II.2.a) and platinum precipitated as a thick layer (Figure II.2.b) on the surface of

Photoluminescent Silicon

15

Chapter 3: Functionalization of pSi surface silicon wafers. After acid washing, morphology of Si was observed in the form of nanowire (Figure II.2.c) or mesoporous (Figure II.2.d).

Figure II-2. SEM image from Si nanostructures production by MACE of p-type silicon wafer. Usage of Ag (a) or Pt (b) as novel metal, before acid washing. Production of nanowire (c) and mesoporous (d) structures.

Final results of variance analysis on the results of experiments (Table II-1) shows at Figure II-3. It is clear that temperature, time, and H2O2 concentration are significant parameters for having corrosion and by increasing them, rate of corrosion would be increased; however, as it can be seen by the Figure II-3, the effectiveness of novel metal is the highest amount based on Taguchi method and silver is better than Pt due to higher electron injection and easier mass transport.

Photoluminescent Silicon

16

Chapter 3: Functionalization of pSi surface

Figure II-3. Effiency for each factor in corrosion of silicon by MACE method. A correspond to novel metal (1: Ag; 2: Pt; and 3: Ag + Pt); B related time duration (1: 10; 2: 60; and 3: 300 min); C related to reaction temprature (1: 27; 2: 45 and 3: 90 ℃) and D conrespond to H2O2 concentration (1: 0; 0.15; and 0.3 M).

However, after a systematic study on MACE for Si nanostructure by Taguchi method, I could not succeed to obtain a significant light emitting structure combine with porosity as a candidate for nanotheranostics. Therefore, we shifted to another well-known production of pSi which is anodization and for further details related to MACE method refer to the published article [14]. Indeed, anodization is a common methodology to obtain luminescent pSi. Therefore, by some experiments, luminescent pSi has been achieved and the optimized structure disclosed here. Besides of luminescent activity, morphology and surface chemistry of structure have been investigated. 2.4.2 Anodization Method The SEM image shows that the particles are of irregular cylindrical shape is in the range 1–10 μm (Figure II-4a). The pores can be clearly visible as black spots in grey background of Figure II-4b, with an average diameter of about 30 nm which is related to mesoporous materials. The average porosity has been determined by gravimetric measurements and it is about 85%. This value corresponds to a specific

Photoluminescent Silicon

17

Chapter 3: Functionalization of pSi surface surface area of 370 m2·cm−3. Native pSi particles are hydride terminated and well dispersible in toluene.

Figure II-4. SEM of pSi microparticles produced by anodization method (a). Image of the surface porosity (b).

For the sake of clarity, size distribution of this cylinder shape material was investigated by image analysis. As it can be seen in the Figure II-5, the size distribution peaked at 1.5 μm for length of this cylinder shape. The lower size range (about 1 μm) is determined under mild sonication conditions followed by a centrifugation step at 500g. In fact, we can estimate that the pSi with size below the micrometre range do not sediment at 500g [15] and are discarded with the supernatant.

Figure II-5. Size distribution of pSi based on image analysis.

18

Photoluminescent Silicon

Chapter 3: Functionalization of pSi surface The upper size range (about 10 μm) is determined by the thickness of the pSi film. In fact, the mild sonication preferentially cleaves the pSi along the pores which are rather directional, propagating predominantly in the 〈1 0 0〉 crystallographic direction, perpendicular to the (100) face of the wafer [16]. And in view of the diameter, is in the range of 500 nm. This morphological property is useful for drug delivery and crossing brain blood barrier. FTIR has been used extensively to characterize the surface chemistry of pSi with individual peaks for a number of surface species. The FTIR spectrum of the pSi, Figure II-6, exhibits several features, including three peaks present together at approximately 2140, 2100 and 2090 cm−1 [17]. These peaks are assigned to SiSiH3, Si2SiH2 and Si3SiH stretching, respectively. In general, the native pSi surface is terminated by this groups, and their relative concentrations are dependent on fabrication parameters. Also, the other broad peak at 790 - 800 cm-1 is ascribable to the SixSiHy groups (x + y = 4) [18]. The sonication step was performed in organic solvents (Toluene), based upon the pSi is completely hydrophobic due to bearing Si-H groups on the surface. On the other hand, a peak at 1100 cm−1 is due to bulk Si-O-Si stretching [17].

15

Int. (a.u.)

Si-O-Si

SiHx 0 1000

1500

2000

2500

3000

3500

-1

wavelength (cm )

Figure II-6. FTIR spectrum of pSi just after electrochemical etching. The pSi layer was still attached to the bulk silicon, for this reason the FTIR spectrum was acquired in reflectance mode.

Photoluminescent Silicon

19

Chapter 3: Functionalization of pSi surface Fracture by sonication in anhydrous toluene of the pSi layer obtained by electrochemical etching produces a particle suspension. This sample has emission in visible light regime. Results of emission under an ultraviolet lamp and by spectra photometer have been exhibited at Figure II.7. As we mentioned before, mechanism of this emission is quantum confinement. Based on high reactivity of pSi to oxidation [19], this structure rapidly quenched and reduced significantly porosity [20]. Due to this structure has short time stability, other optical analyses will be carried out after passivation by chemical functionalization in next chapters in details.

Figure II-7. PL properties of pSi dispersed in ethanol under UV radiation (a) and by excitation at 350 nm with spectra photometer.

2.5 Conclusion The first step of the project is production of PL silicon nanostructure, particularly mesoporous morphology in regard to drug delivery. Then, MetalAssisted Chemical Etching initially chose due to a simple method of Silicon nanostructure synthesis. Even by using Taguchi method, as a systematic investigation, I could not succeed to obtain a PL structure. Then, anodization was implemented to achieve the aim. Finally, we are able to set-up a simple and wellassessed fabrication procedure to obtain luminescent pSi with anodization method,

20

Photoluminescent Silicon

Chapter 3: Functionalization of pSi surface as the first aim of the project. The optimized product has been investigated in view of morphology, surface chemistry, and PL property.

2.6 References

1

Bisi, O., S. Ossicini, and L. Pavesi, Surface Science Reports, 38 (1) (2000) 1 - 126.

2.

John, G.C. and V.A. Singh, Physics reports, 263 (2) (1995 ) 93 - 151.

3.

S. Yae, Y. Morii, Y. Fukumuro, N. Matsuda, H. Catalytic, Nanoscale research letters,

.

7 ( 2012) 352 360. -

4.

H. Han, Z. Huang, W. Lee, Nano Today, 9 (2014 ) 271 - 304

5.

C. Chartier, S . Bastide, C. Lévy Clément, Electrochimica Acta, 53 (2008) 5509 -

-

5516. 6.

J. Salonen, M. Bjorkqvist, E. Laine, L. Niinisto, Phys Status Solidi A, 182 (2000), 249

7.

P. Bettotti, Neeraj Kumar, fabrication of n -type porous silicon membranes for sensing applications, 2013, University of Trento, Italy.

8.

A. Ghafarinazari, A. Tahari, F. Moztarzadeh, Micro Nano Lett., 6 (2011) 713 717.

9

Z.P. Huang, N. Geyer, P. Werner, J. de Boor, and U. Gosele, Advanced Material, 23

.



(2011) 285 308. -

10.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, M . Scarpa, Journal of Material Chemistry B, 2 (2014) 6345 - 6353 .

11.

J. Schindelin, I. Arganda Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. -

Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.- Y. Tinevez, D. J. White, V.

21

Photoluminescent Silicon Chapter

Functionalization of pSi

Hartenstein, K. Eliceiri, P. Tomancak and A. Cardona, Nature Methods, 9 (2012 ) 676 682. –

12.

R. Herino, G. Bomchil, K. Barla, C. Bertrand and J. L. Ginoux, The Electrochemical Society, 134 (1987) 1994 2000. –

13.

A. Halimaoui, in Properties of porous silicon, ed. L. T. Canham, INSPEC The Institution of Electrical Engineers, London, 1997, 12 22. –

14.

A. Ghafarinazari, M. Mozafari, Journal of Alloys and Compounds, 616 (2014) 442 -

15

.

448.

O. Akbulut, C. R. Mace, R. V. Martinez, A. A. Kumar, Z. Nie, M. R. Patton and G. M. Whitesides, Nano Letter, 12 (2012 ) 40 - 60.

16.

Z. Qin, J. Joo, L. Gu and M. J. Sailor, Part. Part. Syst. Charact., 31 (2014 ) 252

17.

K.L. Jarvis, T.J. Barnes, C.A. Prestidge, Advances in colloid and interface science, 175 (2012) 25

18.



38.

Y. Ogata, H. Niki, T. Sakka, M. Iwasaki, Journal of The Electrochemical Society, 142 (1) (1995) 195 - 201.

19.

N. Daldosso, L. Pavesi, Laser & Photonics Reviews, 3 (2009) 508 534.

20.

A. Najar, H. Ajlani, J. Charrier, N. Lorrain, Physica B: Condensed Matter, 396 (1)

-

(2007) 145 149. –

Photoluminescent Silicon

22

Chapter two, in part of full, is a reprint (with co-author permission) of the material as it appears in the following publications: A. Ghafarinazari, M. Mozafari, A systematic study on metal-assisted chemical etching of high aspect ratio silicon nanostructures, Journal of Alloys and Compounds, 616 (2014) 442 – 448; furthermore, N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, M. Scarpa, Orange and Blue Luminescence Emission to track Functionalized Porous Silicon Microparticles inside the cells of the Human Immune System, Journal of Materials Chemistry B, 2 (2014) 6345 - 6353. The author of this dissertation is the co-author of these manuscripts.

Functionalization of pSi

Functionalization of pSi

24

Chapter 3: Functionalization of pSi surface

3.1 Summary As described in Chapter II, luminescent porous silicon (pSi) was obtained by anodization methodology. However, the optical properties of pSi quenched with time based on oxidation processes, thus chemical surface functionalization was necessary in order to get optical stability. According to this goal, we have optimized a derivatization protocol that permits the grafting of negative (carboxyl) or positive (amino) functionalities on pSi surface. This protocol preserves the intrinsic PL of pSi, and also though the grafting of the amino groups introduces different light emission pathways.

3.2 Introduction The discovery of visible photoluminescence (PL) at room temperature of porous Si (pSi) [1] and the lack of evidence of toxicity for this nanostructured material [2] has stimulated a huge research of effective methods to provide silicon micro and nanoparticles [3, 4]. In regard to biomedical applications, silicon is a promising substitute of quantum dots, which mainly contain heavy metals that often present cytotoxicity [5] or systemic toxicity [6]. In addition, degradation of silicon in biological systems leads to bone regeneration [7]. Apart from the absence of toxicity, pSi possesses several properties that make it advantageous for its use in living organisms, such as the large surface area, the tuneable pore sizes and volumes, and the ability to support cell growth. Chemical surface modification provides a mean to adjust the degradation rate of the material as well as allows the loading inside the pores of drugs or sensory molecules. As an example, the native pSi surface, rapidly after synthesis, can be covered by an organic layer bearing carboxyl groups by silylation. This chemical modification improves the chemical and optical properties of pSi. In fact, the organic layer capping the pSi surface protects it from the oxidation occurring in aqueous environments, slows down the rate of PL disappearance and introduces negatively charged groups for electrostatic attraction of positively charged drugs at physiological pH [8].

Functionalization of pSi

25

Chapter 3: Functionalization of pSi surface In the present chapter, we focus on the stabilization of pSi by carboxyl and amine functions by chemical functionalizations (Figure III-1). Moreover, their effects on optical properties and on some specific aspects of the coating procedure, which were demonstrated to play a fundamental role on the peculiar luminescence emission. In particular, we monitored the PL changes induced by a polar aliphatic amine on the orange and blue emission and we measured the emission lifetimes and the quantum yields. The obtained results permit the optimization of the coating procedure in order to obtain pSi microparticles with two bright emission bands. Moreover, we provided insights into the emission mechanisms and into the interaction of nitrogen containing molecules with surface trap states.

Figure III-1. Scheme of pSi functionalization by carboxyl and amine groups.

3.3 Experimental Procedure The pSi samples were prepared by anodic electrochemical etching as described in the Chapter II. As a summary, a boron doped p-type Si wafers ( orientation, 10–20 Ω·cm resistivity) in 2:1 (v:v) 48% aqueous HF:ethanol. Samples were etched at constant current density of 80 mA·cm-2 for 5 min. After electrochemical etching, fracture by sonication in anhydrous toluene produces a microparticle suspension (native pSi). The morphology of the microparticles have been obtained by scanning electron microscopy (SEM, Zeiss SUPRA 40). An organic layer bearing carboxyl groups was introduced on the native pSi surface by light-driven hydrosylilation [9]. Indeed, the just etched porous silicon was sonicated in toluene containing NHS ester of acrylic acid (5 mM). The pSi powder suspension obtained after sonication was illuminated with white light (250

Functionalization of pSi

26

Chapter 3: Functionalization of pSi surface W) for two hours, under Argon atmosphere. Then, the pSi powder was centrifuged and rinsed at least ten times with toluene. To obtain microparticles exposing the free carboxylic group (COOH-pSi), the powder was suspended in ethanol and then rinsed several times with the same solvent, since the NHS ester slowly hydrolysis in ethanol. In order to obtain microparticles exposing amino groups (NH2-pSi), 30 mg of the powder functionalized by acrylic acid-NHS ester were suspended in 25 mL toluene and left to react for a time in the range of 1 - 70 h with a variable concentration of 4, 7, 10 Trioxa 1, 13 tridecanediamine, under gentle shacking. Moreover, Dicyclohexyl-carbodiimide (200 μM) was added to increase the reaction yield, as a catalyser. The surface chemistry was investigated by Fourier Transform Infra-Red (FTIR) spectroscopy. The suspension was deposited on a ZnSe slab and left to dry under gentle nitrogen flow, to avoid further oxidation. The spectra were acquired by a micro-FTIR Nicolet iN10 instrument, in the spectral range of 500–4000 cm-1 with 4 cm-1 resolution. Optical properties were investigated by using Horiba Jobin-Yvon Nanolog instrument. As for PL spectra, the setup was: 2 nm slit size, density grating of 1200 g/mm (blazed at 500 nm), integration time of 0.1 second, and cut-off filtration at 370 nm. Lifetime analysis was performed by time-correlated single-photon counting method (TCSPC) using a 450 W Xenon pulsed lamp for the orange band and a pulsed nano-LED excitation source (375 nm, 1.2 ns) for the blue band. The 𝑡 𝛽

PL decays were fitted by a stretched exponential function, 𝐼(𝑡) = 𝐼0 ∙ exp[− (𝜏) ], where τ is the lifetime, and β is the stretched parameter. Measurement of optical density (OD) is always a two-step procedure. First the radiation source intensity I0, also called the blank, has to be recorded, then the sample is put into the beam path and the radiation intensity I that hits the detector in this condition is collected. The I / I0 ratio is the sample transmittance, and it is dependent on the radiation wavelength. Usually the spectrum intensity is measured

27

Functionalization of pSi

Chapter 3: Functionalization of pSi surface in absorbance, that is defined as a simple logarithmic equation that is 𝑂𝐷 = 𝐼

−𝐿𝑜𝑔10 (𝐼 ). 0

The efficiency of the conversion of absorbed photons into emitted photons by a chromophore is the photoluminescence quantum yield (Φ). There are two conventional methods to determine it: comparative method [10], and De Mello method [11]. Initially for quantum yield measurements, the comparative method was employed through a properly chosen fluorescent standard which ought to have photo physical properties similar to the sample, with a known quantum yield. The experimental conditions should be as similar as possible. Indeed, the quantum yield of a sample was calculated with equation III.1. 𝛷s = 𝛷r ×

𝑚s 𝑛s × ( )2 𝑚r 𝑛𝑟

(III. 1)

where m is slope of the integrated PL intensity vs the absorbance and n is the refractive index of the buffer. The subscripts S and R refer to the sample and the reference solutions, respectively. As all materials are suspended in ethanol, then index ratio (ns/nr) is equal to one. The samples were diluted to keep the absorbance less than 0.1 in order to minimize non-uniform irradiation. However, the absolute quantum yield can be determined by De Mello's method [12] without any comparison with a reference dye. This value is given by equation: 𝛷s =

𝐼𝑠𝑎𝑚𝑝𝑙𝑒,𝑖𝑛 − (1 − 𝐴) × 𝐼𝑠𝑎𝑚𝑝𝑙𝑒,𝑜𝑢𝑡 𝑋𝑏𝑙𝑎𝑛𝑘,𝑖𝑛 × 𝐴

(III. 2)

with: 𝐴=1−

𝑋𝑠𝑎𝑚𝑝𝑙𝑒,𝑖𝑛 𝑋𝑠𝑎𝑚𝑝𝑙𝑒,𝑜𝑢𝑡

(III. 3)

where Isample,in and Isample,out are respectively the integrated emission intensities of the sample when the laser beam directly hits the sample and when it strikes the inner wall of the integrating sphere and the secondary diffuse light hits the sample. Xblank,in

Functionalization of pSi

28

Chapter 3: Functionalization of pSi surface is the integrated excitation profile of blank that directly hits blank (blank is the solvent filled cuvette). Xsample,in and Xsample,out are respectively the integrated excitation profile of sample as a result of direct and secondary light excitation. Moreover, A is the fraction of light absorbed, expressed as one minus the transmittance. Figure III-2 shows different states of excitation and emission for sample inside or outside of the laser beam with relative emission spectra in Figure III-3.

Figure III-2. Description for the determination of the absolute emission quantum yield proposed by De Mello method.

Figure III-3. Example of the determination of the emission quantum yield of a sample using the De Mello’s method.

Functionalization of pSi

29

Chapter 3: Functionalization of pSi surface Absolute Φ values were obtained with an integrating sphere setup from Horiba housed in Nanologe. The device is based up a Labsphere® integrating sphere (diameter of 100 mm) with a coating in optical Spectralon®, which provides a reflectance over 99% over the 400 – 1500 nm range (more than 95% within 250 – 2500 nm). The sample holder accessory is in Teflon. The sample holder for solution measurement is a cylindrical micro cuvette of optical quartz with a diameter of 8 mm, and is equipped with a Teflon cap. The sample holder was tilted of 30° left with respect to the excitation beam to avoid losing of the incident radiation. Because of the high intensity of the excitation profiles, the spectra were recorded through a gauze neutral density (ND) filter of 2% transmittance in the 400 – 750 nm range, positioned on the output port of the sphere, in order to reduce the light intensity without changing the spectral profiles. The spectra were recorded with a photomultiplier. All fluorescence spectra have also been corrected for the instrumental sphere response. The fluorescence absolute quantum yields were calculated on the basis of de Mello’s method following the procedure previously described by Porres et al. [13].

3.4 Morphological and Chemical Characterization The suspension of pSi microparticles obtained by electrochemical etching according to the protocol reported in the Chapter II and in Ref. [14] are irregular, though approximately cylindrical shaped and their average length is 2 µm as shown by a statistical analysis of the optical images of the microparticles deposited on a glass slide. Figure III-4a is a representative SEM image of the native pSi particles. The magnification of the microparticle surface (Figure III-4b) shows a mesoporous structure, as expected for pSi obtained under the etching conditions we utilized [15]. The SEM images of the microparticles after functionalization with the diamine reveal an average decrease of the particle size and an enhancement in the surface roughness (Figures III-4c and d).

Functionalization of pSi

30

Chapter 3: Functionalization of pSi surface

Figure III-4. SEM images of pSi. A representative native pSi just after sonication in anhydrous toluene (a) and a magnification of the porous structure (b). A pSi carrying amino groups on the surface (c) and details of its surface roughness (d).

Conversely, the average diameter of the pore is almost unaffected. The carboxyl or amine groups not only make the pSi capable of interacting electrostatically with other molecules penetrating inside the pores but also can be used for the binding of a protective polymer shell to the external surface. The NH2pSi particles are well dispersed in polar solvents, but they agglomerate in toluene. The light-driven silylation by the N-hydroxysuccinimide (NHS) ester of acrylic acid [16] followed by hydrolysis of the NHS groups produces pSi microparticles with carboxyl groups at the surfaces which are negatively charged at pH 7 (COOHpSi). On the other hand, positive microparticles (NH2-pSi) have been produced by binding a diamine to the NHS-activated carboxyl groups. The efficiency of the functionalization procedure is confirmed by FTIR analysis. In Figure III-5, we show the spectra of pSi after both functionalization steps (i.e. sylilation and coupling with the diamine). The absorbance FTIR spectra of

31

Functionalization of pSi

Chapter 3: Functionalization of pSi surface functionalized pSi (both continuous and dashed lines) show the Si-O-Si (1038 cm1

) and OSiHx (2252 cm-1) bands together with the broad band of O-H at 3300 cm-1

indicating that some oxidation has occurred.

1

Absorbance (a.u.)

COOH-pSi NH2-pSi

C=O, in NHS amide II

OSiHx

Si-O-Si 0 1000

SiH3 amide I 1500 2000 2500

O-H

3000

3500

-1

Wavenumber (cm ) Figure III-5. FTIR measurements of the pSi microparticles after both the functionalization steps: pSi after the functionalization with the NHS ester of acrylic acid (COOH-pSi, continuous trace), and diamine (NH2-pSi, dashed trace). In this experiment, the coupling of the diamine to obtain the NH2-pSi was performed with 200 M diamine concentration and 12 h reaction time.

The functionalization reactions were successful, since the characteristic stretching vibration bands of NHS carboxyl-ester at 1735, 1788, and 1817 cm-1 [17] are present in the pSi spectrum after the silylation reaction with the NHS ester of acrylic acid. Moreover, the amide I and II bands (1644 and 1550 cm-1), overlapping a broad unresolved signal probably due to the bending of N-H [18], are noticeable in the NH2-pSi spectrum. The fast oxidation confirms that native pSi is vulnerable to attack by different compounds, in particular by oxygen and nitrogen containing molecules.

Functionalization of pSi

32

Chapter 3: Functionalization of pSi surface The PL of the pSi after the first and second functionalization steps (resulting in COOH- and NH2-pSi, respectively) was investigated and monitored over time. Figure III-6a reports the photoluminescence excitation (PLE) spectrum (detection wavelength 600 nm) of the COOH-pSi (continuous trace) and the NH2-pSi (dashed trace). The PLE spectrum of COOH-pSi is broader and centred at about 350 nm. The NH2-pSi PLE spectrum is shifted toward the blue region, probably because of partial oxidation induced by the amino groups [19].

Figure III-6. (a) Solid trace: PLE of COOH-pSi, the emission was 600 nm; dashed trace: PLE of NH2-pSi, the emission was 600 nm; dotted trace: PLE of NH2-pSi, the emission was 420 nm. (b) PL spectra by excitation at 350 nm of COOH-pSi (solid trace) and NH2-pSi (dash trace). (c) Optical density of COOHpSi a diluted sample. (d) PL of the NH2-pSi sample as a function of the excitation wavelength.

Functionalization of pSi

33

Chapter 3: Functionalization of pSi surface From the data of Figure III-6a, where we showed also the PLE spectrum of the NH2-pSi at a detection wavelength of 420 nm (dotted trace), it appears that the emission at 600 of the NH2-pSi (dashed trace) is excited with wavelengths in the 260 – 500 nm range, with a maximum at about 300 nm. Conversely, the emission at 420 nm (Figure III-6a, dotted trace) has two main excitation peaks at about 275 and 350 nm. The corresponding emission spectra are shown in Figure III-6b, for the COOHpSi (continuous trace) and NH2-pSi (dashed trace) for excitation at 350 nm. The different peak feature and intensity of the PLE spectra acquired by looking at the two maximum emission peaks suggest that different mechanisms are responsible for the orange and the blue emission. The origin of the orange emission was attributed to quantum confinement effects in the silicon cores and to interfacial defects [20]. It must be mentioned that the PL of the native pSi (refer to the Figure II-7) is quite the same with the COOH-pSi. The blue one has been described for amine capped nanocrystals and attributed to the nitrogen impurities introduced at the silicon, silicon oxide interface [21]. As for functionalized samples, in the both spectra, the typical broad emission characteristic of nanostructured silicon [22, 23] with a maximum around 590 – 610 nm is present. This peak is blue shifted, and it is about 20 nm narrower in the case of the NH2-pSi. However, the PL spectrum of NH2-pSi displays another significant emission at 420 nm, which is neither typical of the Si nanocrystals obtained by pSi sonication [24] nor is observed in the spectrum of the COOH-pSi. This band is characterized by a larger energy band-width with respect to that in the 590 – 610 nm range (the half-height band-width is 563 meV for the emission at 420 nm and about 450 meV for that of the COOH- and NH2-pSi at 590 – 610 nm). For the sake of clarity on PL and PLE of samples, a 3-dimensional map for excitation and emission correlation of COOH-pSi and NH2-pSi have been shown in the Figure III-7. Optical density (OD) of carboxyl functionalization exhibited at Figure III-6c. It must be note that optical density of the NH2-pSi is the same as the COOH-pSi.

Functionalization of pSi

34

Chapter 3: Functionalization of pSi surface This outcome is the main evidence to show that quantum confinement carried out in these samples which attributed to PL at the orange regime [4].

Figure III-7. 3D map for correlation of emission and excitation of COOH-pSi (a) and NH2-pSi (b) samples.

Figure III-6d reports the PL of the NH2-pSi by changing the excitation wavelength from 350 to 420 nm. As for the orange peak at about 610 nm, there is no significant modification in shape and position, only an intensity reduction is observable, in agreement with the PLE spectrum (Figure III-6b). As for the blue peak (at about 420 nm for excitation wavelength at 350 nm), a clear red-shift up to about 460 nm is evident with increasing the excitation wavelength from 350 to 420 nm. This is due to quasi direct intra-band excitation and it is not consistent with quantum confinement in agreement to lifetime measurements (see later on) that allow us to confirm that this emission is attributed to nitrogen impurities introduced at silicon/silicon oxide interface. It is worth noting that the sharp peak shift with the excitation wavelength (at about 430 nm for excitation at 350 nm) is due to the Raman effect of the ethanol buffer. In order to further understanding the origin of the blue emission band, PL intensities have been monitored as a function of the diamine concentration (Figure III-8a) and the reaction time (Figure III-8b). The orange peak shows a blue-shift from 615 to 600 nm with increasing the diamine concentration (Figure III-8a), and

35

Functionalization of pSi

Chapter 3: Functionalization of pSi surface a decrease in PL intensity with increasing the reaction time (Figure III-8b). The blue-shift of the orange band is consistent with a reduction of the silicon core size due to a progressive nitrogen diffusion and/or oxidation, thus confirming that the emission mechanism is based on quantum effects]. According to the quantum confinement model, the relationship between the PL energy and the size of Si crystals can be written as [25]: 𝐸𝑃𝐿 = 𝐸0 +

3.73 0.881 + − 0.245 𝑑1.39 𝑑

(III. 4)

Where d is the diameter of silicon crystals in nano meters, Eo is the band gap energy of bulk silicon (1.17 eV) and EPL is the size-dependent band gap energy of Silicon with diameter that correlates to the position of the PL intensity maximum.

Figure III-8. (a) Blue-shift of the maximum value of the orange band as a function of diamine concentration (after 70 hours from mixing); (b) Variation of the maximum PL intensity (orange and blue peak) as a function of the time after addition of 300 μM diamine. Insets: PL spectra of sample without diamine (continuous trace) and after 70 h incubation with 300 μM diamine.

Based upon the equation 1, the crystal size of pSi should be around 3.06 nm to emission at orange. For obtaining bellow 444 nm emission, we need crystalline size less than 2 nm, which is in line with the other theoretical model [26]. On the other hand, it is also reported that the defects in the silica matrix, such as non-bridging

36

Functionalization of pSi

Chapter 3: Functionalization of pSi surface oxygen hole centres and twice-coordinated silicon can also be responsible for blue emission. Indeed, the orange peak intensity reduction could be explained by the increase of newly formed non-radiative defect centres upon modification of the pSi [27, 28]. On the contrary, the blue peak increases in PL intensity with reaction time (Figure III-8b). There is no significant wavelength shift of the blue band with increasing the concentration of diamine or the reaction time. This result is in line with the data of Gupta et al. [29], and provides further evidence that the blue band is size independent. Based on these experiments, 200 µM of diamine and 12 h reaction time have been selected as the best conditions to get both orange and blue emission for the NH2-pSi samples. Time degradation of the optical properties of pSi is a key point to be investigated for their future in vivo applications. PL reduction with time (i.e. aging effect) of COOH-pSi and NH2-pSi samples in ethanol is quite negligible: no significant PL reduction or wavelength shift even after two years has been observed [30]. However, the PL half-life is about 2 days in water solution, as shown in Figure III-9 and in agreement with the other report [31]. This is accompanied by the oxidation and dissolution of pSi; indeed, the orange band shifts toward shorter wavelengths before completely disappearing.

a)

Int. (a.u.)

b) 200000

Initial 7h Day 1 Day 2 Day 3 Day 7 Day 8 Day 9 Day 11 Day 14 Day 15 Day 16

150000

100000

50000

0

400 450 500 550 600 650 700

Wavelength (nm)

Figure III-9. a) Time degradation of the PL for NH2-pSi (9 µg/mL) in water and in PBS solution for the orange and blue emission band. b) PL spectra of NH2pSi into PBS in different times.

Functionalization of pSi

37

Chapter 3: Functionalization of pSi surface This degradation with time has been also checked in PBS buffer for possible in vitro applications. In such a case, the orange band is completely reduced after one week; conversely, the blue band is persistent up to two weeks, even showing a slight increase in the first six days. We can argue that the ions present in PBS induce a faster silicon oxidation which reduces the crystallite size till to complete dissolution. We measured PL decay curves of the NH2-pSi sample as a function of the wavelength within the emission band for both peaks, the blue (Figure III-10a) and the orange (Figure III-10b) emission decay, respectively. The lifetime of the blue band is about 4 ns with β value equal to one (i.e. not stretched decay). This short lifetime is comparable with previous reports on blue emission from defects in silicon nanostructures upon exposure to nitrogen containing reagents [32]. The negligible dependence of the lifetime on the emission wavelength (refer to inset of Figure III-10a) clearly indicates that quantum confinement is not responsible for blue light emission in such samples [33, 34]. Moreover, based on the constant and close to unit value of the stretching parameter β, we can suggest that the emission sites (i.e. the surface defects) are isolated and no inter-dot hopping occurs [35].

Figure III-10. PL decays of the NH2-pSi sample for (a) the blue emission band (excitation wavelength is 375 nm) and (b) the orange emission band (excitation wavelength is 375 nm) as a function of the emission wavelength. Inset: lifetimes () and stretching parameter (β) obtained by fitting the experimental PL decay curves with a stretched exponential function.

Functionalization of pSi

38

Chapter 3: Functionalization of pSi surface The time decay of the functionalized pSi in the orange emission band was fitted by a stretched exponential function and a lifetime of about 18 µs was found with β values of 0.85, in agreement with the literature about quantum confinement [3]. Moreover, the quantum confinement was confirmed by the wavelength linear dependence of the lifetime as a function of the emission wavelength, which increased from 10 to 32 µs in the interval 550 - 650 nm (Figure III-10b). This behaviour is consistent with the increment of the radiative rates as a function of pSi size reduction [36, 37]. This observation also implies a high crystalline quality [38]. Very similar results (not reported here) have been obtained for COOH-pSi samples as regard to the orange emission band. To estimate the light emission efficiency of the pSi, quantum yield measurements have been performed by the comparative method [13]. To evaluate the method and our experimental setup, the quantum yield of Fluorescein was determined to be 75.5% (Figure III-11) by using Rhodamine 101 in ethanol (Rh 101, Φ = 100% [31]) as a standard, in line with the published data for Fluorescein in ethanol (Φ = 79%) [39]. Similarly, the quantum yield of the pSi samples was determined, by taking Rh 101 as a reference. The quantum yield values obtained by integrating all the optical emission in the range 360 - 800 nm, were about 1.7 and 1.2% for the COOH-pSi and NH2-pSi, respectively. The quantum yield reduction induced by diamine functionalization can be ascribed to nitrogen penetration [40, 41]. Indeed, carboxyl modification acts as a barrier to penetrate nitrogen and quenching PL property. In general, these quantum yield values for pSi microparticles are in line with other findings and in some cases considerable larger: for instance, Sugimoto et al. [42] reported 0.3% quantum yield for Si quantum dots. However, there are specific procedures to obtain Si nanoparticles with more than 10.2% light efficiency [31]. Nevertheless, we have to consider that the thermal relaxation of the light absorbed should play an important role [43, 44]. This pathway would be quite promising for photo-thermal therapy applications [45].

39

Functionalization of pSi Chapter 3: Functionalization of pSi surface Integrated PL (×107 a.u.)

50 40 30

Rh 101 Fluorescien COOH-pSi (×20) NH2-pSi (×20) NH2-pSi, Orange (×20)

20 10 0 0.00

0.05

0.10

Absorbance Figure III-11. Integrated PL of Rhodamine 101 (Rh 101), Fluorescein, used as standard, and different pSi microparticles to determine quantum yield by comparative method.

In order to determine light quantum efficiency of each band for the amine pSi, we neglected the blue peak by appropriate cut-off filter at 550 nm. These measurements are shown as “NH2-pSi orange” in Figure III-11. Quantum yield of the orange peak resulted about 0.6%, and consequently the light efficiency of the blue band is around 0.6% because light efficiency of the NH2-pSi is 1.2% in the whole emission range. To sum up, this amount of efficiency with compering with published data suggest that COOH- and NH2-pSi can be monitored with conventional PL detection systems, such as biological confocal microscopes [31]. The values of Φ found by us for the samples through integrating sphere are summarized in Table III-1 together with the Φ mean value deviations derived from the measurement related uncertainties, the results found it for Rhodamine 101 and Fluorescein are matching with literatures [31, 39]. As it can be seen, the fluorescence quantum yields for all of the samples obtained with the two independent methods are in good agreement among them and the maximum deviations between the results are almost covered by the derived uncertainties.

40

Functionalization of pSi Chapter 3: Functionalization of pSi surface Table III-1. Φ values determined by de Mello method.

Sample

Absolute Φ (%)

Rhodamine

95 ±9

Fluorescein

75 ±7

COOH-pSi

1.6 ±0.2

NH2-pSi, orange band

0.5 ±0.1

NH2-pSi, blue band

0.8 ±0.2

3.5 Conclusion We obtained mesoporous silicon microparticles brightly luminescent after silylation with acrylic acid and coupling with a diamine by amide bond. The PL emission is changed by the reaction with the diamine: the orange PL emission is partially quenched and blue shifted, while a blue PL band appears. The quenching induced by the diamine was controlled by optimizing the amine concentration and the reaction time. The lifetime of the blue band is about 4 ns with no dependence on the emission wavelength, while the lifetime of the orange emission band is about 18 µs with strong wavelength dependence. Quantum yields of the order of 1 - 2 % are in line with previous results obtained with silicon microstructures.

3.6 References

1.

L.T. Canham, Applied Physics Letters, 57 (1990) 1046 - 1048.

2.

L.T. Canham, C.L. Reeves, D.O. King, P.J. Branfield, J.G. Crabb, M.C. Ward, Advanced Material, 8 (1996) 850 - 852.

41

Functionalization of pSi

Chapter 3: Functionalization of pSi

3.

O. Bisi, S. Ossicini, L. Pavesi, Surface Science Reports, 38 (2000) 1 - 126.

4.

N. Daldosso, L. Pavesi, Laser & Photonics Reviews, 3 (2009) 508 534.

5.

A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Nano Letters, 4 (2004) 11 - 18.

6.

J. Geys, A. Nemmar, E. Verbeken, E. Smolders, M. Ratoi, M.F. Hoylaerts, B.

-

Nemery, P.H. Hoet, Environmental Health Perspectives, 116 (2008) 1607 - 1613. S.H.C. Anderson, H. Elliott, D.J. Wallis, L.T. Canham, J.J. Powell, Physica Status

7.

Solidi (A) Applied Research, 197 (2003) 331 335. -

O. Tabasi, C. Falamaki, Z. Khalaj, Colloids and Surfaces B: Biointerfaces, 98 (2012)

8.

18 25. -

9.

M. P. Stewart and J. M. Buriak, Journal of the American Chemical Society, 123 (2001) 7821

10.

J.H. Ahire, Q. Wang, P.R. Coxon, G. Malhotra, R. Brydson, R. Chen, Y. Chao, ACS Applied Material & Interfaces, 4 (2012) 3285 - 3292.

11.

A. Alessi, M. Salvalaggio, G. Ruzzon, Journal of Luminescence, 134 (2013) 385



389. 12

J.C. de Mello, H.F. Wittmann, R.H. Friend, Advanced Materials, 9 (1997) 230.

13.

L. Porres, A. Holland, L.O. Palsson, A.P. Monkman, C. Kemp, A. Beeby, J.

.

Fluoresc. 16 (2006) 267. 14.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, M. Scarpa, Journal of Material Chemistry B, 2 (2014) 6345 6353 . -

15

.

J. Riikonen, M. Salomaki, J. van Wonderen, M. Kemell, Xu, W. Korhonen, O. Ritala, M. MacMillan, F. Salonen, J. Lehto, Langmuir, 28 (2012) 10573 10583. -

16.

M.P. Stewart, J.M. Buriak, Journal of the American Chemical Society, 123 (2001) 7821 - 7830.

42

Functionalization of pSi

17.

X. Liu, H. Han, H. Liu, S. Xiao, Surface Science, 604 (2010) 1315 - 1319.

18.

T.D. James, G. Parish, C.A. Musca, Electrochemical and Solid State Letters, 13 -

(2010) 428 431. –

19.

R. R. Chandler Henderson, B. Sweryda Krawiec and J. L. Coffer, The Journal of -

-

Physical Chemistry, 99 (1995) 8851.

3: Functionalization pSi A. Bsiesy, F. Gaspard, R. Herino, M. Ligeon 20Chapter A. Halimaoui, C. Oules, G. of Bomchil, .

and F. Muller, Applied Physics Letter, 59 (1991) 304 21.

J. Fuzell, A. Thibert, T. M. Atkins, M. Dasog, E. Busby, J. G. C. Veinot, S. M. Kauzlarich and D. S. Larsen, Journal of Physical Chemistry Letters, 4 (2013 ) 3806

22.

C. Delerue, G. Allan and M. Lannoo, Physical Review B: Condens. Matter Material Phys., 48 (1993) 11024.

23.

G. B. Li, L.-S. Liao, X. -B. Liu, X.-Y. Hou and X. Wang, Applied Physics Letter, 70 (1997) 1284

24.

E. Froner, R. Adamo, Z. Gaburro, B. Margesin, L. Pavesi, A. Rigo and M. Scarpa, Journal of Nanopart. Res., 8 ( 2006) 1071.

25.

G. Ledoux, O. Guillois, D. Porterat, C. Reynaud, F. Huisken, B. Kohn, V. Paillard, Physical Review B, 62 (2000) 15942 - 15951.

26.

C. Delerue, G. Allan, and M. Lannoo, Physical Review B, 48 (15) (1993) 11024



11036. 27

.

S.M. Prokes, W.E. Carlos, and V.M. Bermudez, Applied Physics Letters, 61 (1992) 1447 - 1449.

28.

X.D. Pi, L. Mangolini, S.A. Campbell, U. Kortshagen, Physical Review B, 75 (2007) 085423.

29.

A. Gupta, H. Wiggers, Nanotechnology, 22 (2011) 55707.

43

Functionalization of pSi

30.

N. Prtljaga, E. D’Amato, A. Pitanti, R. Guider, E. Froner, S. Larcheri, M. Scarpa, L. Pavesi, Nanotechnology, 22 (2011) 215704.

31.

J.H. Park, L. Gu, G. Von Maltzahn, E. Ruoslahti, S.N. Bhatia, M.J. Sailor, Nature Material, 8 (2009) 331 - 336.

32.

V. Švrcek, T. Sasaki, R. Katoh, Y. Shimizu, N. Koshizaki, Applied Physics B, 94 (2009) 133 - 139.

33.

J. Kistner, X. Chen, Y. Weng, H.P. Strunk, M.B. Schubert, J.H. Werner, Journal of Applied Physics, 110 (2011) 23520 23524. -

34

.

M. Dasog, Z. Yang, S. Regli, T.M. Atkins, A. Faramus, M.P. Singh, E. Muthuswamy, S.M. Kauzlarich, R.D. Tilley, J.G. Veinot, ACS Nano, 7 (2013) 2676 -

2685.

35.

L. Pavesi, M. Ceschini, Physical Review B, 48 (1993) 17625 - 17628.

36.

M.S. Hybertsen, Physical Review Letters, 72 (1994) 1514 1517.

37.

T. Takagahara, K. Takeda, Physical Review B, 46 (1992) 15578 - 15581.

38.

M. Fujii, K. Imakita, K. Watanabe, S. Hayashi, Journal of Applied Physics, 95

-

(2004) 272 280. -

39.

G. Weber, F.W.J. Teale, Transactions of the Faraday Society, 53 (1957) 646 - 655.

40.

M.T. Swihart, X. Li, Y. He, W.D. Kirkey, A.N. Cartwright, Y. Sahoo, P.N. Prasad, SPIE's 48th Annual Meeting, International Society for Optics and Photonics, 48 (2003) 108 - 117.

41.

R.R. Chandler Henderson, B. Sweryda Krawiec, J.L. Coffer, The Journal of -

-

Physical Chemistry, 99 (1995) 8851 - 8855. 42.

H. Sugimoto, M. Fujii, Y. Fukuda, K. Imakita, K. Akamatsu, Nanoscale, 6 (2014) 122 126. -

44

Functionalization of pSi

43.

W. de Boer, D. Timmerman, K. Dohnalová, I.N. Yassievich, H. Zhang, W.J. Buma, T. Gregorkiewicz, Nature Nanotechnology, 5 (2010) 878 884. -

44.

A. Ghafarinazari, M. Mo zafari, Journal of Alloys and Compounds, 616 (2014) 442 - 448.

45.

P. Haro González, W.T. Ramsay, L.M. Maestro, B. del Rosal, K. Santacruz Gomez, ‐



F. Sanz‐Rodríguez, J.Y. Chooi, P.R. Sevilla, M. Bettinelli, D. Choudhury, Small, 9 (2013) 2162 2170. -

Functionalization of pSi

45

Chapter III, in part of full, is a reprint (with co-author permission) of the material as it appears in the following publication: A. Ghafarinazari, V. Paterlini, P. Cortelletti, P. Bettotti, M. Scarpa, N. Daldosso, Optical study of diamine coupling on carboxyl functionalized mesoporous silicon, accepted at Journal of Nanoscience and Nanotechnology. The author of this dissertation is the primary author of this manuscript.

Biological Stabilization of pSi

Biological Stabilization of pSi

47

4.1 Summary Meso-porous silicon (pSi) nanoparticles combine the great promise to be versatile drug delivery vectors (i.e. large and sizable porosity) with the intrinsic luminescence properties (i.e. visible light emission). Unfortunately, till now the applications of this system have been severely limited due to its incompatibility with water solutions, which leads to immediate degradation of the material and to the loss of its optical properties. This chapter reports about pSi with carboxyl functionalization and its optical properties that have been investigated before and after surface modification with both a polyethylene glycol and a biocompatible natural polymer, the carbohydrate chitosan. The herein presented functionalization of pSi maintained the optical properties of the native core in physiological solutions for prolonged period and represents an important step towards Nanomedicine’s applications.

4.2 Introduction Space and time controlled delivery of drugs is a pharmacological issue since it would reduce the waste and side effects of drugs, thus improving their therapeutic properties [1, 2, 3]. Drug delivery systems (DDS) should offer large room for drug loading, provide internal surface with affinity for the cargo and penetrate homogeneously and efficiently the target tissue. In this regard, meso-porous silicon (pSi) appears a suitable material for the fabrication of DDS, because of the large pore volume [4] the tuneable surface chemistry [5] and the low toxicity associated to safe biodegradation pathways [6, 7]. Moreover, well assessed fabrication protocols produce pSi with intense photoluminescence (PL) in the visible region, which can be tracked in cells by fluorescence imaging in real time [8, 9, 10]. However, un-resolved questions which still limit the effective utilization of the pSi as delivery system, are the fast degradation of pSi in vitro [11] and in harsh biological environments [12], which is not compatible with the kinetics of drug release, and the irreversible quenching of the pSi luminescence due to interaction with water or biomolecules [13].

Biological Stabilization of pSi

48

It must be noticed that in order to use light emission for bio-imaging, low rate of degradation is not enough, but also the pSi must be stable to avoid PL ageing based on its reaction with other components. pSi covered by a shell of acrylic groups introduced by a hydrosylilation reaction (COOH-pSi) has PL stability in ethanol for years at room temperature [14]. On the other hand, optical stability of porous silicon in phosphate-buffered saline (PBS) is a big challenge [11]. Some protocols have been proposed in order to delay the pSi degradation up to hours [13, 15, 16]; and then, researchers succeeded in stabilization for few days by using particular procedures such as rapid thermal oxidation [12, 17]. However, PL decay of oxidized particles was reported to be in the range of few hours [18], comparable to that of COOH-pSi [14]. Stability in the desired media frequently remains an issue for application in biomedicine of many nanomaterials, not only for pSi. Due to their high surface to volume ratio, the surface is highly reactive and can interfere with the solvents. Modification of the surface with compounds able to limit this effect, meanwhile conferring desired properties to the final product, has been largely researched in the last decade [19, 20]. Among all the possibilities, the use of polyethylene glycol (PEG) has been showed appealing for applications in biomedical field. Indeed PEG can ensure water stabilization to the nano systems being also biocompatible and able to reduce, once in vivo, opsonisation phenomena, which usually are the main factor affecting particles circulation and earlier elimination from the bloodstream [21]. PEG is not the only polymer used for this purpose, recently biopolymers derived from natural sources [22], thus considered extremely biocompatible, have showed promising and interesting capacity. Chitosan is the product resulting from de-acetylation of chitin, the second most abundant biopolymer in the world generally found in crustacean shells [23]. More interestingly, chitosan can be used to form nano or micro watersoluble particles able to incorporate in their core drugs or active agents. The possibility to use it for stabilization of particles is surely appealing also because it presents several free amino groups that can be further exploited for conjugation reactions and synthesis of more complex architectures as DDS [24]. Finally, both PEG and Chitosan due to their carboxylic acids and amino groups respectively, offer the possibility of being linked to biomolecules (peptide, proteins, monoclonal

Biological Stabilization of pSi

49

antibody and aptamers) thus providing to the final DDS potential targetable features. The optical properties of the COOH-pSi particles allow their detection also with two-photon absorption (TPA) [10]. TPA allows 3D imaging of the sample and permits a more depth penetration with respect to confocal microscopy [14]. It also reduces photo-bleaching and photo-toxic effects at the same time [25]. In this chapter, we report a procedure to obtain water-soluble pSi particles by surface-functionalization and coverage through either PEG or chitosan. We demonstrated that the PL of these microparticles is stable for long time also in PBS and can be efficiently excited by two photon absorption.

4.3 Chemical functionalization procedure and experimental details All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. All aqueous solutions were prepared with ultrapure water obtained using an ultrafiltration system (Milli-Q, Millipore) with a measured resistivity above 18 MΩ·cm-1. Polyethylene glycol with amino and carboxylic acid end groups (NH2-PEG-COOH, MW ~3 kDa) was purchased from Rapp Polymere GmbH (Tübingen, Germany). Synthesis of COOH-pSi: As it was described in Chapter III, pSi was obtained by anodic etching of single crystal (100) p- silicon wafers, boron doped, with a resistivity of 10-20 Ω·cm (University wafers, Boston MA). The etching was performed by applying a constant current of 80 mA·cm-2 for 5 min in a solution containing absolute ethanol:HF (48%) 2:1. The porous silicon layer was sonicated in anhydrous toluene (80 mL) containing acrylic acid (5 mM) under nitrogen atmosphere. The sonication was performed in bath, for 15 min, delivering a power of 350 W. The particle suspension was collected and refluxed at 50 ℃ for two hours under illumination with white light (250 W). The powder (COOH-pSi) was then collected and rinsed ten times with ethanol. Morphological and optical properties of this sample were already studied in detail [14].

Biological Stabilization of pSi

50

To test the drug loading capacity, 3.5 mg of dried microparticles have been incubated for 2 h at pH 6.0, in 2.0 mL of 1 mM citrate buffer containing 0.36 mM amino methyl fluorescein, under weak shaking. After several washings, the powder was dried and re-suspended in 10 mL of 10 mM Hepes, pH 6.8, to monitor the payload release. Synthesis of PEG-pSi (A in Figure IV-1): Ethanol was removed in nitrogen flow and particles (1 mL, 0.3 mg) were redispersed in anhydrous tetrahydrofuran (THF, 1 mL). Carbonyldiimidazole (CDI, 0.8 µmol) was added to the reaction mixture under nitrogen. After 1.5 hours at room temperature, HCl*H2N-PEGCOOH (50 mg, 1.6 µmol) dissolved in 1 mL of THF and diisopropylethylamine (DIPEA, 10 µL, 50 µmol) were added. The reaction was stirred in vortex for 24 hours at room temperature. After that, particles were collected in centrifuge (13100 rpm, 4 min.), washed twice with 2 mL of a mixture 1:1 THF:water and centrifuged again before redispersion in ultrapure water (1 mL). In order to better understand the effect of PEG on pSi, the ratio of PEG/pSi was increased twice (sample PEG2pSi).

Figure IV-1. Schematic of the synthesis and functionalization procedures.

Biological Stabilization of pSi

51

Synthesis of Chitosan-pSi (B in Figure IV-1): To a solution of the COOH-pSi in ethanol (1 mL, 0.3 mg), chitosan dissolved in acetate buffer (pH = 4.3, 0.25 mg·mL-1, 1.25 mg) was added drop wise. The system was let to incubate with mechanical stirring for 24 hours. After that, particles were collected in centrifuge (13100 rpm, 4 min), washed twice with 2 mL of water and centrifuged again before re-dispersion in ultrapure water (1 mL). Table IV-1 summarizes the different samples investigated in this work.

Table IV-1. Summary of samples functionalization. COOH-pSi

pSi functionalized with carboxyl group, used as reference

PEG-pSi

COOH-pSi sample chemically functionalized with PEG

PEG2-pSi

COOH-pSi sample functionalized with double amount of PEG

Chitosan-pSi

COOH-pSi sample functionalized with chitosan

Final concentration of the obtained particles was determined by gravimetric analysis: briefly, 100 µL of solution were dried at 130 ℃ for 24 hours then accurately weighted thus determining the amount of dry matter. Dynamic light scattering (DLS) analysis of the ζ-potential values were conducted with a Zetasizer Nano-S (Malvern) instrument, working with a 532 nm laser beam at 25 ℃, using Clear Disposable Z cells and the results expressed as average of three measurements. Furthermore, scanning electron microscopy images were acquired with a SEM Zeiss instrument EVO 50' EP. The surface chemistry was investigated by Fourier Transform Infra-Red (FTIR) spectroscopy. The spectra were acquired by a micro-FTIR Nicolet iN10 instrument, in the spectral range of 700 – 4000 cm-1 with about 4 cm-1 resolution. Photoluminescence measurements were performed by Horiba Jobin-Yvon Nanolog instrument. The configuration

Biological Stabilization of pSi

52

setup was as, excitation at 350 nm, 2 nm slit size, 1200 g·mm-1 density grating (blazed at 500 nm), cut-off filtration at 370 nm, and 0.1 second integration time. In order to estimate the light efficiency of the samples, quantum yield (Φ) measurements have been carried out by the comparative method [26, 27]. In this regard, Rhodamine 101 was selected as reference which has about 100% quantum yield [15]. The quantum yield of a sample was calculated via Φs = Φr·Is/Ir·Ar/As·(ns/nr)2, where I is the integrated emission intensity, A is the absorbance, and n is the refractive index of the buffer solution. The subscript S and R refer to the sample and the reference solutions, respectively. The samples were further, diluted to keep the absorbance less than 0.1 in order to minimize nonuniform irradiation [28]. PBS is used as simulator of biological fluids. For this, about 30 µg from each dried sample was suspended in 3 mL PBS. Then, PL spectrum of the samples was monitored at the selected time. TMP images were acquired by a two-photon microscope DM-6000 CS (Leica) using a Chameleon ULTRA II laser (Coherent) and a 20X objective, water immersed, numerical aperture of 1 [29, 30]. Excitation was set at 700 nm and the emission in the range 550-650 nm.

4.4 Morphological and Chemical Characterization The COOH-pSi samples were characterized by ζ-potential analysis for the determination of the surface charge and by gravimetric analysis for the dry matter concentration. Particles were analysed in the native solvent, ethanol, with a 1:100 dilution. The results showed COOH-pSi microparticles with a negative ζ-potential value of -26.2 mV, due to the presence of carboxylic acids onto the surface, and a concentration of 0.3 - 0.5 mg/mL. SEM analysis was performed to better evaluate the morphology of the sample (Figure IV-2). As it can be seen in Figure IV-2a, COOH-pSi particles have a rough surface and rod shape, and the size of the major axis is in the range 1-10 μm with a size distribution peaked at 1-2 μm as reported in Ref. [14]. By increasing the

Biological Stabilization of pSi

53

magnification of SEM, the diameter of the pores has been observed about 30 nm (Figure IV-2b). The pore network visible in the SEM image suggests a large drug loading capacity for this material, which indeed was found as high as 2 × 1014 molecules per mg of COOH-pSi by using aminomethyl fluorescein as drug model.

Figure IV-2. SEM images of COOH-pSi sample in one-micron scale range to show particle dimension (a) and in submicron scale to better point out porosity at the surface (b), PEG-pSi sample in ten-micron scale range (c) with a higher magnification (1-micron scale range) in the inset, and Chitosan-pSi sample in 20-micron scale range (d).

In order to allow structural stability and optical properties preservation in water and/or in other biological buffers, modifications of the surface were experimented. Firstly, PEG through covalent attachment to the microparticle surface was tried. A PEG functionalized at the two ends with an amino and a carboxyl group (H2N-PEGCOOH) was selected, in order to create an amide bond between the amino group of PEG and the carboxylic acids present on the pSi surface, thus leaving the same

Biological Stabilization of pSi

54

functionality (COOH) available at the end of PEG for further conjugations (i.e. DDS). The conservation of a highly negative ζ-potential value (-37.9 mV) suggested the covalent attachment of several moieties of PEG-COOH. SEM analysis showed the functionalized sample with rod like particles clearly visible (Figure IV-2c). In case of functionalization with a double amount of PEG (PEG2pSi), the results in terms of ζ-potential value were consistent with those found for PEG-pSi: this suggests also in this case the attachment of several PEG-COOH moieties. Finally, the surface modification with a biopolymer, chitosan, through physical adsorption and electrostatic interaction was also considered. Thanks to its abundance of positively charged amino groups, chitosan can electrostatically interact with the negatively charged COOH-pSi, thus keeping protected the particles between its chains meanwhile leaving free amino groups for further conjugation. Correspondingly, the so obtained hybrid microparticles are well dispersible in water. Indeed, ζ-potential analysis showed a highly positive value (+48.3 mV) due to the amino groups of chitosan, meaning that an efficient coating of the native pSi particles has been obtained. SEM analysis revealed the presence of spheres-like shaped microparticles, homogeneously dispersed in the sample (Figure IV-2d) with an average dimensions of about 2-5 µm. In fact, based on the chemical reaction during the functionalization, homogeneity of COOH-pSi sample morphology gets increased. The FTIR spectral features are in agreement with the expected surface modification (Figure IV-3). For the sake of clarity, all spectra have been normalized and shifted along the y axis. The native pSi particles present the characteristic vibration mode at 2100 - 2300 cm-1, and also the peak at 790 - 800 cm-1, which are ascribable to SiHx [31]. On the contrary, COOH-pSi did not present this signal but a strong peak at 1060-1110 cm-1; this is due to the presence of asymmetric stretching of Si-O-Si bond [32]. Interestingly, also a signal at 1715 cm-1 appears, which is the typical stretching vibration of –C=O containing compounds thus confirming the presence of COOH groups onto the surface [32]. The other peak at 873 cm-1 is related to Si-O bending.

Biological Stabilization of pSi

55

Figure IV-3. FTIR measurements of the pSi microparticles before and after surface functionalization and successive coating: native psi (a), COOH-pSi functionalized (b) and surface modified by PEG (c) and Chitosan (d). The baselines of the middle and top spectra are offset from the x-axis for comparison.

In the case of PEG functionalization, it is possible to observe a strong peak at 1046 cm-1 as typical stretching vibration in ethers, the presence of the amide bond vibrations at about 1395 cm-1, of –C=O at 1644 cm-1 (amide I band) and of -NH stretching at 3200 - 3400 cm-1 which overlaps with Si-OH peak. After the functionalization with Chitosan, the sharp peak at around 1070 cm-1 was retained and a new band is found at 1550 - 1600 cm-1 due to the presence of primary amides, which are abundant in Chitosan; also Si-OH vibrations appeared at 3200 - 3600 cm-1 as a broad band, which is typical of hydrogen bonded –OH, while alkyl -CH stretching modes appeared at 2850 - 2950 cm-1 [33].

4.5 Optical Characterization Figure IV-4 reports normalized PL spectra excited at 350 nm of all the samples dispersed in PBS. Reporting normalized PL is a common practice when dealing with different samples [34, 35]. Indeed, the emission quantum yield (see Table IV-2

Biological Stabilization of pSi

56

and relative discussion) is the right parameter for the comparison of the relative intensities among the various compounds rather than the PL itself. As it can be seen, COOH-pSi has a peak in the red region (about 665 nm) [36]. It is worth noting that the sharp peak at 390 nm is due to the Raman effect of water, as basic solvent of PBS [37].

Figure IV-4. Normalized PL spectra (exc. wavelength: 350 nm) of samples dispersed in PBS: COOH-pSi, PEG-pSi, PEG2-pSi and Chitosan-pSi. Chitosan (x10) signal is reported for comparison.

After functionalization, the PEG-pSi and the Chitosan-pSi PL bands are very similar in shape, just blue-shifted of about 30 nm with respect to COOH-pSi sample. The blue-shift of the red band is consistent with a reduction of the silicon core size, which is not detectable by SEM analysis, as suggested by quantum confinement effects [38, 39, 40]. PL of PEG2-pSi has been shifted even more, about 180 nm. It must be considered that even though the microparticles functionalized with both PEG concentrations present similar molecular structure and size, the larger amount of PEG (i.e.: HCl*H2N-PEG-COOH) used during functionalization leads to a stronger surface modification. The observed blue shift of PL is probably due to surface

57

Biological Stabilization of pSi

oxidation that induces a size reduction of the light emitting nanostructures, which form the microporous particles. In fact, it has been reported that amines catalyse the oxidation of nanostructured silicon. Moreover, this oxidative process is often accompanied by a reversible loss of PL due to surface adducts formation [41, 42]. In our case, the coating by acrylic acid should prevent the formation of large adducts. Conversely, the amine-induced silicon oxidation is mediated by reactive oxygen species which diffuse across the organic coating [7, 43, 44]. Our data are in agreement also with the findings of Sweryda-Krawiec et al. [45], who reported that amine-pSi interaction is concentration dependent, since by doubling the PEG concentration the PL shift is strongly increased. The emission quantum yield (Φ) of all samples dispersed in PBS has been estimated by comparative method (Table IV-2). The obtained values (between 0.8 % and 1.1%) are not significantly reduced if compared to that of the COOH-pSi that is about 1.7%. Unlike the blue shift, the Φ reduction is not the same for both kinds of functionalization. As for the PEG-pSi sample, Φ is reduced of about 33% [14]. A similar decrease of the quantum yield is observed after the binding of a diamine to the carboxyl group of COOH-pSi.

Table IV-2. Optical quantum yields obtained by comparative method. Sample

Φ (%)

COOH-pSi

1.7

PEG-pSi

1.1

PEG2-pSi

0.8

Chitosan-pSi

0.8

58

Biological Stabilization of pSi

However, quantum yield gets further reduced by increasing PEG amount (down to 0.8 for the PEG2-pSi sample). As for Chitosan-pSi sample, the quantum yield is about 0.8, which is roughly one half with respect to that of COOH-pSi sample. The reduction with respect to PEG-pSi sample can be attributed to the chitosan matrix that acts as optical absorber. In fact, it appears more opaque due to the larger passivation upon the COOH-pSi structures. These quantum yield reductions are in line with recent experimental data for surface functionalization of pSi [46]. The degradation over time of the spectral features (that is of the optical intensity and the emission wavelength) of functionalized pSi samples have been studied and compared. All the tested samples showed the typical red emission band, apart from the chitosan functionalized sample which presents a second emission feature at 400 nm (Figure IV-5b). Figure IV-5a shows change over time of the emission intensity of the red band (solid lines) of the differently functionalized samples. The intensities have been normalized by Equation 1, in order to let them appreciable in the same figure.

  Sample I  I   d     I i  Sample   pSi COOH

   

(IV.1)

Herein, Ii and Id are the PL intensities of the sample in PBS immediately after suspension and after “d” days. ΦSample and ΦCOOH-pSi are the quantum yield of the sample and COOH-pSi, respectively. Indeed, the initial point (at time zero) is just related to reduction of Φ value due to functionalization in comparison with COOHpSi. The dashed line of Figure IV-5a shows the emission intensity in the violet band (at about 400 nm) of the Chitosan-pSi sample. In the same figure, it appears that the half-life of the COOH-pSi in PBS is about 6 h, which is longer than what reported (about 3 h) for this kind of functionalization in PBS buffer [15, 47]. The half-life increases to six days after suspension in PBS for the PEG-pSi, without any blue shift (Figure IV-6a). Moreover, hydrodynamic radios of sample, i.e. DLS outcomes,

Biological Stabilization of pSi

59

confirms that this sample does not change in one week. It is worth noting that oneweek optical stability is more than required for bio-imaging applications and it is also promising for Photo-Thermal therapy [48].

Figure IV-5. a) PL intensity vs. time (days) in PBS buffer (“pSi-Chi. Red” and “pSi-Chi. Viol” are the intensity of red and violet band of Chitosan-pSi sample, respectively). b) Optical emission spectra of Chitosan-pSi sample after suspension in PBS as a function of time (after 1 day and up to 90 days).

Interestingly, by the time this sample started to polymerization and grafting of the pSi self-assembly. As you can see in the Figure IV-6b shows photograph of the sample after 4 days suspension in PBS. In this figure polymerisation is clear. However, as we mentioned before, in the first week PL properties (carves “1 d” to “7 d” from Figure IV-6a) and DLS value were quite the same. After 9 days, there is a polymer structure with encapsulation of pSi particles (Figure IV-6c). In such a way, obtaining PL spectra by the spectrophotometer is not possible based on fast participation of the sample (Figure IV-6a) however this sample is clearly light emission under a conventional UV lamp radiation (Figure IV-6c). There are so many systems for obtaining grafted pSi structures [49, 50, 51]. But, to the best of our knowledge mechanism of this self-assembly grafting is not yet reported.

60

Biological Stabilization of pSi

Int. (a.u.)

a) 120000

PEG-pSi 60 min 1d 2d 5d 7d 9d 9d+S 15 d 15 d + S

635 nm

615

600

0 400

500

600

700

800

Wavelength (nm)

Figure IV-6. a) PL spectra of PEG-pSi sample after suspension in PBS as a function of time up to 9 days, then sonication (9 d + S), PL after 6 more days (15 d) and then finally after for the second time sonication (15 d + S). b) photograph of sample after 4 days of suspension. c) photograph of sample under UV radiation after 15 days of suspension in PBS.

In order to restore PL by spectroscopy and checking properties of this hybrid component, sample after 9-days suspension was sonicated. As it can be seen in the Figure IV-6a, PL intensity about 50% restored but after pSi started to reactions based on shift of red band about 30 nm within 6 days and increasing blue band intensity. These variations on PL is similar to reactions for attaching diamine with COOH-pSi sample as discussed in the Chapter III which could be related to amine bands on the PEG precursor. By increasing the amount of PEG (PEG2-pSi sample), PL intensity reduces of about 30% (Table IV-2) and PL peak shifts up to about 480 nm from red to blue spectral region (Figure IV-4). After this initial shift, the PL band is stable up to three months (90 days). To the best of our knowledge, this optical stability (both in intensity and shape) for Si-based nano or micro-structures in PBS has not been reported till now. Moreover, these data may indicate that the PL band could be further tuned by varying the PEG amount thus having an additional post-processing tool for wavelength shifting without influencing the intensity stability over the time. Interestingly, in this duration, the PEG2-pSi sample does not polymerize in opposite of PEG-pSi which means that polymerisation of COOH-pSi with PEG is sensitive

Biological Stabilization of pSi

61

to aspect ratio of them and there is a limited range. This mechanism of selfassembly polymerisation needs more investigation and attention but at the moment is out of our aims. Very similar behaviour was observed for Chitosan-pSi microparticles showing a long-term stability; accordingly, Figure IV-5b reports the emission spectra of the chitosan functionalized pSi after the suspension in PBS at various days: the red emission (about 635 nm) decreases, while the violet one (at 400 nm) appears and increases with time. The decrease of the red emission can be due to the slow but progressive oxidation of luminescent silicon particles that reduce the dimension of the nano crystallites and introduce oxygen defects [52]. For this reason, the red emission (related to quantum confinement) progressively decreases in intensity and slightly shifts to shorter wavelength. Conversely, the optical emission at 400 nm (violet band) is attributed to the surface defects at the nanocrystallites induced by nitrogen containing compounds derived from the amino groups [14, 53, 54], which are progressively released because of the dissolution of chitosan [55]. We demonstrated that thanks to the coverage of pSi surface by an organic layer we obtain a long-lasting PL stabilization in organic solvents (toluene and ethanol) but the PL half-life in aqueous environment is of the order of few hours. By using the carboxylic group present on the organic layer to graft chitosan or PEG chains we were able to stabilize the PL intensity and feature up to months even in a high ionic strength aqueous buffer such as PBS. To the best of our knowledge this is the first functionalization protocol which stabilizes the PL of pSi particles for such a long period of time. Both PEG and chitosan functionalized pSi microparticles seem to be very suitable for future medical applications and offer orthogonal potential functionalization since they bear in the outer shell carboxylic and amino functional groups, respectively.

Biological Stabilization of pSi

62

4.5.1 Two-Photon Absorption Imaging Emission of the pSi is in biological window but unfortunately its absorption (and thus excitation) occurs at lower wavelengths thus strongly limiting the range of applications [10]. To overcome this, TPA technique has been considered and investigated. The results clearly showed that by exciting at 700 nm (exactly the double of the 350 nm used in PL measurements) the optical emission are the same and clear images can be easily obtained (Figure IV-7a).

Figure IV-7. Two-photon absorption microscopy images of COOH-pSi sample by excitation at 700 nm (a) and 810 nm (b) then looking at emission of 550-650 nm.

The images show COOH-pSi particles with size of about 3 µm confirming the results obtained with SEM and the presence of large cluster of particles up to 10 µm. The optical emission comes from specific sites in the particles as revealed by the heterogeneous distribution of the light signal. Excited at 700 nm, they show light emission in the 550 - 650 nm range. Also excitation wavelength at 810 nm has been used (Figure IV-7b) according to the needs for photo-thermal therapy applications [56]. In both cases, particles are bright, and then these pSi components are promising for many medical applications based on long-term stability and PL properties at biological window.

Biological Stabilization of pSi

63

4.5 Conclusions We have developed a simple and straightforward process to stabilize photoluminescent meso-porous silicon particles by PEG and chitosan. We demonstrated that the PL of the functionalized microparticles is stable for long time in phosphatebuffered saline. This extremely long optical stability combined to the preserved quantum efficiency in vitro conditions is very promising for any kind of biomedical applications. Moreover, we demonstrated by TPA that the system can be excited at biological window needed for any possible application either in drug delivery or bio-imaging. All these findings, together with the well assessed masking effect of PEG and chitosan and their amino or carboxylic functional groups, propose these particles as efficient targetable DDS for long-lasting systemic delivery of drugs.

4.6 References

1.

L. T. Canham, Advanced Materials, 7 (1995) 1033.

2.

J. Salonen, A. M. Kaukonen, J. Hirvonen and P. V. Lehto, Journal of pharmaceutical sciences, 97 (2008) 632.

3.

L. Canham, Tunable Properties of Porous Silicon, in Handbook of Porous silicon, Springer International Publishing, Switzerland, 2014, 201 - 207

4.

O. Bisi, S. Ossicini and L. Pavesi, Surface Science Reports, 38 (2000) 1.

5.

M. L. Sailor, Chemical reactivity and surface chemistry of porous silicon, in Handbook of Porous silicon, Springer International Publishing, Switzerland, 2014, 335 - 380.

6.

S. P. Low and N. H. Voelcker, Biodegradability of Porous Silicon, in Handbook of Porous silicon, Springer International Publishing, Switzerland, 2014, 381.

7.

E. J. Anglin, L. Cheng, W. R. Freeman and M. J. Sailor, Advanced Drug Delivery Reviews, 60 (2008) 1266.

Biological Stabilization of pSi

8.

64

L. Gu, J-H Park, K. H. Duong, E. Ruoslahti and M. J. Sailor, Small, 6 (22) (2010 ) 2546

9.



2552.

R. E. Serda, B. Godin, E. Blanco, C. Chaippini and M. Ferrari, Biochimica et Biophysica Acta, 1810 (2011) 317.

10.

E. Secret, M. Maynadier, A. Gallud, A. Chaix, V. Hugues, M. Auffan, Céline Frochot, A. Morère, P. h. Maillard, M. Blanchard -Desce, M. J. Sailor, M. Garcia, J. Durand and F. Cunin, Advanced Materials, 26 (2014 ) 7643.

11.

S. Näkki, J. Rytkönen, T. Nissinen, C. Florea and J. Riikonen, Acta Biomaterialia, 13 (2015) 207.

12.

B. Godin, J.Gu, R. E. Serda, R. Bhavane, E. Tasciotti, C. Chiapinni, X. Lu, T. Tanaka, P. Decuzzi and M. Ferrari, Journal of Biomedical Materials Research Part A, 94 (4) (2010) 1236.

13

.

R. R. Chandler -Henderson, B. Sweryda- Krawiec and J. L. Coffer, Journal of Physical Chemistry Letters , 99 (1995) 8851.

14.

N. Daldosso, A. Ghafarinaza ri, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi and M. Scarpa, Journal of Materials Chemistry B, 2 (37) (2014) 6345.

15.

J. H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti and S. N. Bhatia, M. J. Sailor, Nature Material, 8 (4) (2009) 331.

16.

N. K. Hon, Z. Shaposhnik, E. D. Diebold, F. Tamanoi and B. Jalali, ECS Trans., 45 (2013) 7.

17

.

A. Tzur -Balter, A. Rubinskia and E. Segal, Journal of Materials Research, 28 (2) (2013) 231.

18.

L. Gu, D. J. Hall, Z. Qin, E . Anglin, J. Joo, D. J. Mooney, S. B. Howell and M. J. Sailor, Nature Commun., 4 (2013) 2326.

65

Biological Stabilization of pSi

19.

C. A. Prestidge, T. J. Barnes, C. H. Lau, C. Barnett, A. Loni and L. Canham, Expert Opin. Drug Delivery, 4 (2007) 101.

20.

E. Locatelli, I. Monaco and M. Comes Franchini, RSC Advances, 5 ( 2015) 21681.

21.

K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert, Angew. Chem. Int. Ed., 49 ( 2010) 6288.

22

S. K. Nitta and K. Numata, Int. Journal of Molecular Sciences, 14 (2013) 1629.

23.

M. N. V. R. Kumar, Reactive and functional polymers, 46 (2000) 1.

24.

K. Nagpal, S. K. Singh and D. N. Mishra. Chemical and Pharmaceutical Bulletin, 58

.

(11) (2010) 1423. 25.

A. A. Khan, S. K. Fullerton-Shirey and S. S. Howard, RSC Advances, 5 (2015) 291.

26.

D. Timmerman, J. Valenta, K. Dohnalová, W. De Boer and T. Gregorkiewicz, Nature Nanotech., 6 (11) (2011) 710.

27

.

A. Ogunsipe, J.-Y. Chen and T. Nyokong, New Journal of Chemistry, 28 (7) (2004) 822.

28.

J. H. Ahire, Q. Wang, P. R. Coxon, G. Malhotra, R. Brydson, R. Chen and Y. Chao, ACS Applied Materials & Interfaces, 4 (2012) 3285.

29.

C. Laperchia, A. L. Allegra Mascaro, L. Sacconi, A. Andrioli and A. Matté, PLoS ONE, 8 (2) (2013) 56144.

30.

E. Rampazzo, F. Boschi, S. Bonacchi, R. Juris, M. Montalti, N. Z accheroni, L. Prodi, L. Calderan, B. Rossi, S. Becchi, A. Sbarbati, Nanoscale, 4 (3) (2012) 824.

31.

Y Ogata, H Niki, T Sakka and M Iwasaki, Journal of The Electrochemical Society, 142 (1995) 195 201. –

32.

G. Korotcenkov, B.K. Cho, Silicon Porosification: state of the art, Critical Reviews in Solid State and Material Sciences, 35(3) (2010) 153 260. –

66

Biological Stabilization of pSi

33.

ML. Mastronardi, F. Maier -Flaig, D. Faulkner, Nano letters, 12(1) (2011) 337 342.

34.

X. Li, Y. He, S. S. Talukdar, M. T. Swihart, Langmuir, 19 (20) (2003) 8490 8496.

35.

Z. F. Li, E. Ruckenstein, Nano Letters, 4(8) (2004) 1463 - 1467.

36.

G. B. Li, L. S. Liao, X. B. Liu, X.-Y. Hou and X. Wang, Applied Physics Letter, 70



-

(1997) 1284. 37.

I. R. RAO, Nature, 125 (1930) 600.

38

A. G. Cullis, L. T. Canham and P. D. J. Calcott, Journal of Applied Physics, 82

.

(1997) 909. 39.

J. Rytkönen, R. Miettinen, M. Kaasalainen, V. P. Lehto, J. Salonen and A. Närvänen, -

Journal of NanoMaterial, 2012 (2012) 896562. 40.

G. Ledoux, O. Guillois, D. Porterat, C. Reynaud, F. Huisken, B. Kohn and V. Paillard, Physical Review B, 62 (23) (2000) 15942.

41

.

W. D. Kirkey, Y. Sahoo, X. Li, Y. He, M. T. Swihart, A. N. Cartwright, S. Bruckenstein and P. N. Prasad, Journal of Materials Chemistry, 15 (2005) 2028.

42.

R. R. Chandler -Henderson, B. Sweryda- Krawiec and J. L. Coffer, The Journal of Physical Chemistry, 99 (1995) 8851.

43.

W. Xu, J. Riikonen, T. Nissinen and M. Suvanto, The Journal of Physical Chemistry C, 116 (2012) 22307.

44.

A. Imhof, D. J. Pine, Nature, 389 (1997) 948.

45.

B. Sweryda- Krawiec, R. R. Chandler- Henderson , J. L. Coffer, Y. G. Rho and R. F. Pinizz otto, The Journal of Physical Chemistry, 100 (1996) 13776.

46.

K. Dohnalová, A. N. Poddubny and A. A. Prokofiev, Light. Sci. Appl., 2 (2013) 47.

47.

N. K. Hon, Z. Shaposhnik, E.D. Diebold, F. Tamanoi and B. Jalali, J. Biomed. Material Res., 100 (12) (2012) 3416.

67

Biological Stabilization of pSi

48.

M. A. Avidad, A. Nieto, A. Salinas -Castillo, L. Capitan-Vallvey, G. Miskelly and M. J. Sailor, Nano. Res. Lett., 9 (1) (2014) 1.

49.

F. Hua, M.T. Swihart, E. Ruckenstein, Langmuir, 21 (13) (2005) 6054

50.

A. Nieto, H. Hou, S.W. Moon, M.J. S ailor, Investigative ophthalmology & visual



6062.

science, 56 (2) (2015) 1070 1080. –

51

.

52.

Z.F. Li, E. Ruckenstein, Nano Letters, 4 (8) (2004) 1463



1467.

B. Gelloz, Biodegradability of Porous Silicon, in Handbook of Porous silicon, Springer International Publishing, Switzerland, 2014, 307.

53.

J. Fuzell, A. Thibert, T. M. Atkins, M. Dasog, E. Busby, J. G. Veinot, S. M. Kauzlarich and D. S. Larsen, Journal of Physical Chemistry Letters, 4 (21) (2013) 3806.

54.

W. De Benedetti, S. Chiu, R. Radlinger, J. Ellison, B. A. Manhat, J. Z. Zhang and J. Shi, The Journal of Physical Chemistry C, 119 (17) (2015) 9595



9608.

55.

Y. Cai and Y. Lapitsky, Colloids and Surfaces B: Biointerfaces, 115 (2014) 100.

56.

D. Jaque, L. M. Maestro, B. Del Rosal, P. Haro Gonzalez, E. M. Rod ríguez and J. -

G. Solé, Nanoscale, 6 (2014) 9494.

68

Biological Stabilization of pSi

Chapter IV, in part of full, is a reprint (with co-author permissions) of the material as it appears in the following publication: N.

Daldosso,

A.

Ghafarinazari, E. Locatelli, C. Laperchia, F. Boschi, P. Bettotti, M. Scarpa, M.C. Franchini, long-term optical stability of functionalized meso-porous silicon, Submitted. The author of this dissertation is a co-author of this manuscript.

Thermal Oxidation of pSi

70

Thermal Oxidation of pSi

5.1 Summary Mesoporous silicon (pSi) has several interesting features that makes it suitable for various biomedical applications as discussed in the previous Chapters. In particular, the large surface area makes it very sensitive to environment changes. Among organic functionalization (such as amine, Chitosan), thermal oxidation is an effective way to passivate its surface specially to avoid redox of biological systems. Herein, we present experimental and modelling results concerning thermal oxidation of pSi particles. The experiments were conducted on pSi powders produced from silicon wafer by anodization and sonicated. Oxidation experiments were carried out at different heating rates. Structure and morphology of the samples have been investigated by XRD and SEM before and after oxidation. The modelfree kinetics proposed by Ozawa–Flynn–Wall was used to determine the kinetic triplet of the Arrhenius relationship for the pSi thermal oxidation to get insight about the oxidation mechanism. These outcomes have been confirmed by Starink method and model fitting methodology.

5.2 Introduction Mesoporous

silicon

(pSi)

possesses

some

specific

properties

like

biocompatibility, photoluminescence and non-immune response, which makes it a promising material for biomedical applications [1]. Due to its large surface area and highly reactive surface functional groups, pSi is particularly susceptible to air, water, or chemical oxidation [2]. This oxidation has side effects of redox in the biological systems such as tissues [3], proteins [4], and drugs [5]. Nowadays, partial thermal oxidation of pSi has been suggested to solve this issue [6]. Thermal oxidation has been extensively employed by the microelectronic industry to produce high-quality oxides on silicon, and this approach also works with pSi [7]. Noticeably, the rate of oxidation of pSi is greater than that of flat bulk silicon due to the porous structure. The oxidation conditions can be tuned to obtain materials with different properties. Up to now, a variety of effects of oxidation on properties of pSi has been investigated, such as photoluminescence [8, 9], biodegradability

Thermal Oxidation of pSi

71

[10], phonon thermal conductivity [11], and surface reactivity [12]. However, there are only few preliminary reports about thermal oxidation mechanism of pSi [13, 14, 15]. For instance, A.E. Pep et al. [13] reported that thermal oxidation of pSi consists in two separated steps as a function of the temperature but without any further information and discussion about the kinetic mechanism and the modelling. In this work, we performed a comprehensive study, both experimental and theoretical, on thermal oxidation of pSi by model-free kinetic (MFK) and model fitting to determine oxidation mechanism. In the case of MFK, OFW (OzawaFlynn-Wall) methodology has been used to estimate the kinetic triplet (E, A, and f(α)); the obtained E values were confirmed by Starink method. Moreover, model fitting has been applied to find the best kinetic model to disclose the mechanism of thermal oxidation in pSi. The understanding of the oxidation process and the relative mechanism is an important result for basic research on porous materials and also for applicative purposes, mainly in microelectronic and biomaterials fields.

5.3 Experimental Procedure The porous silicon samples have been prepared by anodizing n-type Si (100) wafers with a resistivity of 0.01-0.02 Ω·cm, Sb doped, in a HF (48%), water and Triton X-100 solution (25:200:1). The current density was kept constant at 15 mA·cm-2 and the etching time was 10 min. The porosity of the samples obtained under these experimental conditions was determined by gravimetric analysis [16] and was found 74 ± 8 %. The porous layer was detached from the bulk silicon and fragmented into micro particles by 20 min sonication at 400 W delivered in 80 mL hexane sample volume. Then, the micro particle suspension was centrifuged at 500 xg for 5 min, the surnatant was discarded, the micro particles were dried under gentle nitrogen flow and used within few hours. X-ray diffraction (XRD) patterns were measured with a D8 Advance Bruker diffractometer, equipped with a Göbel mirror using Cu-Kα1 radiation at 1.5406 Å. The patterns were collected with a scan rate of 0.04 °/s in the 8°–90° 2θ range. In order to enhance both background and small peak signals, square root of intensity has been illustrated. The crystallite phases were then identified using the Joint

72

Thermal Oxidation of pSi

Compounds Powder Diffraction Standards (JCPDS) database. Morphological characteristics have been investigated by scanning electron microscopy with a FESEM Zeiss supra 60. The accelerating voltage was 2 kV. The samples were sputtered with gold to assure sufficient conductance. The simultaneous thermal analyses (STA; i.e. differential thermal analysis (DTA) and Thermogravimetry (TG) analysis) were done in synthetic air atmosphere from room temperature to 1273 K with STA 409 (Netzsch–Gerӓtebau GmbH, Selb, Germany). The STA were conducted at three different heating rate values: 3, 6, and 10 K/min with a flow of 50 mL/min, under ambient atmospheric pressure up to 1273 K. Then, the sample was cooled to room temperature by the rate of 10 K/min. The powder samples, about 20 mg, were kept in alumina crucibles.A direct way to monitor oxidation phenomenon is TG measurement [13]. The mass variation from TG analysis is correlated to a conversion value by Equation (V.1): 𝛼𝑖 =

𝑚𝑠 − 𝑚𝑖 𝑚𝑠 − 𝑚𝑓

(V. 1)

where ms is the starting mass and mf is the mass after the oxidation. Reaction kinetic can be evaluated by the kinetic triplet (A, E, and f(α)) in the kinetic equation expressed in the form of the Arrhenius relationship: 𝑑𝛼 𝐸 = 𝐴 · 𝑒 − ⁄𝑅𝑇 · 𝑓(𝛼) 𝑑𝑡

(V. 2)

Values of the Arrhenius parameters (E and A) are accepted as providing the height of the energy barrier for reaction to occur (the activation energy, E) and the frequency of occurrence of reaction configuration that may lead to product formation (the frequency factor, A). Hence, the reaction model, f(α), represents the dependence on the conversion extent. The best methodology to evaluate the kinetic triplet (E, A and f(α)) is the MFK approach for all kind of reactions [17, 18]. Ozawa, Flynn and Wall (OFW) adapted this method to the TG analysis [19, 20] and the obtained procedure was validated by the American Society for Testing and Materials (ASTM) for differential

73

Thermal Oxidation of pSi

scanning calorimetry (DSC) analysis [21]. In summary, integration of Equation V.2 and Doyle approximation [19] leads to: 𝛼 𝐴∙𝐸 𝑑𝛼 𝐸 Ln(𝛽) = Ln ( ) − Ln(∫ ) − 5.330 − 1.052 𝑅 𝑅𝑇 0 𝑓(𝛼)

(V. 3)

where β is the heating rate. According to Equation 3, plotting of Ln (β) as function of 1/T at different α led to straight lines with slope equal to -1.052 E/R. The apparent activation energy at the different conversion degree can be calculated 𝛼 𝑑𝛼

from these slopes. Moreover, under the condition of constant α (Ln(∫0

) = 0),

𝑓(𝛼)

by introducing in Equation 3 the calculated E value, and plotting Ln (β) versus 1/T, we can estimate the value of Ln (A) [22]. Moreover, Starink [23] proposed another equation (Equation V.4) to estimate the oxidation kinetic in a more accurate way. α A∙E dα E Ln ( 1.92 ) = Ln ( ) − Ln(∫ ) − 1.0008 T R RT 0 f(α)

β

(V. 4)

In this case, plotting Ln(β/T1.92) vs. 1/T leads to the apparent activation energy. We implemented this method and compared the results with those obtained by OFW method. Therefore, f(α) can be estimated by inserting E and A values in Equation 2. The kinetic triplet can be derived without any assumption or approximation. By these outcomes, mechanism and kinetics of the oxidation mechanism can be investigated. Beside to MFK, Model-fitting method is the conventional way to estimate the reaction model (f(α)) [24]. In this method, initially, the conventional models (Table V-1) were implemented to obtain the best fit model [17]. For each model, the goodness of the fit is customarily estimated by a coefficient of linear correlation (r). A single pair of E and A is then commonly chosen as that corresponding to a reaction model that gives rise to the maximum absolute value of the correlation coefficient [25]. However, this pair of E and A does not have physical meaning. At the end, the obtained model (f(α)) has been compared with f(α) estimated by OFW. Finally, differential thermal analysis (DTA) is performed with identical thermal cycles to obtain an indication of the enthalpy change

74

Thermal Oxidation of pSi

associated to the reactions (exothermic or endothermic) [25]. This technique helps to define the critical temperatures of the oxidation steps.

Table V-1. Reaction types and corresponding type of f(α). Model Notation

Reaction type

f(α)

Fn

nth order (n= 1-3)

(1-α)n

Cn

nth order Autocatalysis (n= 1-3)

(1+Kcatα)×(1-α)n

An

Avrami-Erofeev (n-D nucleation; n= 1-3)

n×(1-α)×[-ln(1-α)](1 - 1/n)

D1

1D diffusion

0.5α

D2

2D diffusion

[-ln(1-α)]-1

D3

Jander 3D diffusion

1.5(1-α)2/3×[1-(1-α)1/3]-1

D4

Ginstling–Brounshtein 3D diffusion

1.5[(1-α)-1/3-1]-1

Rn

Reaction on the n-D interface (n= 2, 3)

n×(1-α)(1 - 1/n)

Bna

Prout–Tompkins nth order autocatalysis

(1-α)n×αa

5.4 Structural and morphological characterization The structure and the morphology of pSi samples were investigated by both XRD and SEM. XRD patterns of the as-anodized sample (pSi) and of the sample after heating at 3 K/min up to 1273 K (pSiO2) are shown in Figure V-1, top and bottom panel, respectively. Formation of single crystalline silicon nanostructure in pSi sample was confirmed (fit to the JCPDS file 27-1402). Indeed, the Bragg condition is only satisfied for reflection peaked at 2θ = 69.13°, according to Ogata et al. [26]. The broad peak at low angles (i.e. 15° – 30°) is associated to the amorphous silica [27], probably due to surface oxidation of the silicon crystallites

75

Thermal Oxidation of pSi

during the sonication. The interface between the oxidized layer and the silicon core is amorphous silicon in the order of ppm, thus not appreciable [28, 29]. Moreover, on the pSi surface there are functional groups such as SiHx or SiOH, formed during the fabrication [1], which make phase composition not quantitatively assessable [7]. Oxidized pSi (pSiO2) XRD pattern is shown in Figure V-1 (bottom panel). After heating treatment, the Si peak at 69.13° disappeared and a crystalline phase was detected, as shown by the sharp peaks at 2θ = 20.76° and 36.60°. This new crystalline phase corresponds to α-quartz (JCPDS 02-0458). It is worth noting that the broad signal at low angle (15° – 30°) did not change by heating up to 1273 K, which confirms that the main part of oxidized pSi remains amorphous silicon oxide.

Sqrt Intensity (Counts1/2)

15000

pSi

*

0 500

#

pSiO2

#

0

10

20

30

#

#

40

50

60

70

80

90

2 (Degree) Figure V-1. XRD diffraction of the freshly anodized pSi powder; and after oxidation by heating rate of 3 K/min up to 1273 K (pSiO2). The sharp peak at 69.1° (“” at upper panel) is due to the < 4 0 0 > reflection of crystalline Si, while the sharps reflection peaks () at bottom panel suggest the occurrence of α-quartz phase.

Morphological characterization was carried out by SEM analysis (Figure V-2). Figure V-2a shows pSi particles in the micrometre scale. This uniform porous

Thermal Oxidation of pSi

76

structure can be better appreciated in Figure V-2b with pore diameter about 50 nm, layer thickness about 6 µm. The thickness of pSi layers, pore walls, is around 20 nm, which is one of the most critical parameters for oxygen diffusion at the pSi to get efficient thermal oxidation [30]. Figure V-2c and d report SEM images after thermal oxidation by heating the sample at 3 K/min. Particle size and morphology did not change significantly while the porous surface is rather changed (Figure V-2d), the silica formed upon oxidation led to a closure of the pores.

Figure V-2. SEM images of pSi samples before and after thermal oxidation. a) pSi particles as anodized; b) surface of pSi particles with higher magnification; c) thermally oxidized pSi particles with rate of 3 K/min; and d) surface of thermally oxidized pSi particles.

The final value of the mass variation from TG analysis, according to the Equation V.1, is 94.6% for heating to 1273 K with rate of 3 K/min. Taking into account the reaction stoichiometry, complete oxidation of pure silicon would cause 114.3% of mass increase [27]. Owing to the fact that oxidation was completed by heating the sample under the reported condition, based on XRD analyses, reaction

Thermal Oxidation of pSi

77

conversion (α) should be around 100%. Therefore, initial value of α is estimated to be 17.2% (i.e.

(114.3−94.6) 114.3

× 100), which is related to the reactive surface groups

that can be oxidized during the sample preparation [1]. Taking into consideration this effect, α value was calculated from TG analyses by Equation 1 as reported in Figure V-3. Note that during the cooling from 1273 K to room temperature, there is less than 4% weight reduction, which could be related to the oxygen absorption and desorption [27, 31]. Since this value was lower than 4% of the total mass, it was neglected in our analysis, in which we considered the other correction methods as recommended by the International Kinetics Committee, such as baseline corrections, buoyancy effect, sample mass, heating rates [32].

Figure V-3. Conversion vs. temperature at heating rates of 3, 6, and 10 K/min.

According to Figure V-3, it is possible to infer that no plateau is reached in the case of oxidation with heating rate of 10 K/min. This rate is the maximum acceptable one for kinetic analysis [17] and it is also the most common rate for thermal analysis studies [26, 33]. This sample after reaching the maximum temperature (1273 K) was isothermally heated for 10 min at the same temperature to be sure about saturation state. However, the TG value increased from 90% to 93.7% suggesting that oxidation was not complete. It is also worth noting that oxidation increased remarkably with a reduction of the heating rates to 6 and 3

Thermal Oxidation of pSi

78

K/min. At these lower rates, saturation state is reached and the onset temperature was dependent on the heating rate. In the isothermal analysis, the type of reaction (f(α)) can be pointed out by looking α-T curves. But based on initial heat up, isothermal has fundamental problems. Sigmoidal shape of the isoconversional curves is a common form for all reactions [17]. Then we need further calculations to obtain f(α). Furthermore, sudden increase of α above 500 K is an evidence that the oxidation process is a multistep reaction, therefore being unsuitable to propose just one kinetics model [34]. Based on Equation V.3, Ln(β) versus 1/T was plotted in Figure V-4a. According to OFW methodology, for a series of measurements with different heating rates (β), the slope and linear coefficient of plotting Ln (β) versus 1/T at different and constant α gives the E and A values (Figure V-4b). In order to validate E values obtained by OFW method, also Starink method has been implemented as described in the experimental section. In general, E is considered as the threshold or energy barrier that must be overcome to permit the bond redistribution steps that are essential for the transformation of reactants into products. The variation of E with α indicates that this reaction is a complex (multi-step) process [35]. At the beginning of the conversion, E value is just about 10 KJ/mol since pSi is easily oxidized in air [36]. This value is in line with published results [13]. In the initial stage, α  37%, there is a positive convexity of the E versus α curve (Figure V-4b), which indicates that this process is irreversible [37]. This outcome is in agreement with enthalpy of SiO that is higher than other Si bonds such as Si-Si, Si-H, and Si-C [7]. The second stage, at α  37%, accounts for a continuous linearly increment of E value; this stage seems to be driven by parallel processes [38, 39]. The final value is in accordance with energy of oxygen diffusion into fused silica, which has been reported to be 104.6 KJ/mol [40]. As it can be seen, E values estimated by Starink method (Est) have no significant difference from those obtained by OFW method. This similarity has been also found by other experimental data sets [41, 42].

79

Thermal Oxidation of pSi

Figure V-4. (a) Natural logarithm of heating rate versus 1000/T of the pSi thermal oxidation at different reaction degree. (b) Variation of E and Ln(A) by conversion obtained from Eq. 3, Est.() is the E value obtained by the Starink method (c) The dependence of Ln(A) with E.

The pre-exponential term, A, is a measure of the frequency of occurrence of the reaction and it is regarded to include the vibration frequency in the reaction coordinate. Ln(A) and E show a similar dependence on α (Figure V-4b). Figure V-4c shows a linear dependence of A versus E for the two ranges considered (after and before α=37%): {

Ln(A) = 0.27E − 9.7 α ≤ 37% (I) Ln(A) = 0.12E − 7.9 α > 37% (II)

(V. 4)

This linear behaviour is described by the compensation equation [43]. Although many theoretical explanations for compensation behaviour have been

80

Thermal Oxidation of pSi

proposed, none of them has received general acceptance. Aspects of the compensation phenomena in thermo-analytical data are reviewed by Koga [44]. These two lines (Lines I and II in Figure V-4c) are another evidence of having two consecutive reactions. Finally, the f(α) shape can be matched to the theoretical models describing the oxidation mechanism [45]. In fact, once both E and A have been evaluated, it is possible to numerically reconstruct the reaction model, f(α), derived from Equation 2 as shown in Figure V-5 [46]. As described earlier, there are two separate stages: after and before α = 37%. In the initial stage, the shape of f(α) is compatible with the Avrami-Erofeev model. This model is generally used to describe nucleation and growth phenomena in random places [47]. On the other hand, the final step agrees with the diffusion mechanism. Clearly, the initial silicon dioxide layer acts as a barrier and oxygen must diffuse through this layer to react with silicon at the interface silicon/silicon dioxide [48, 49]. Two different mechanisms depending on the conversion degree were already observed by Leppavuori et al. by isothermal

f ()

measurements [13].

20

30

40

50

60

70

80

90

(%)

Figure V-5. F(α) derived from the Equation 2 for thermal oxidation of pSi.

Thermal Oxidation of pSi

81

In order to get more information about the mechanism of pSi oxidation and also to check the values obtained by OFW method, model fitting has been used. We could not obtain acceptable correlation coefficient (r  0.95) of Arrhenius parameters by implementation of this method by assumption of having a single stage reaction. Then, experimental data was separated to two stages based on MFK method, and in this case, the model fitting was satisfied. The fitted values of Arrhenius parameters evaluated for the experimental data by the model-fitting method are presented in Table V-2 and 3 for comparison. As it can be seen in the Table V-2, the best fitted model for the first stage (α  37%) is Avrami-Erofeev n-dimensional nucleation model. Recently, the AvramiErofeev kinetic model was applied for surface reactions at mesoporous chitosan [50]. This model was also applied to describe the adsorption of anionic dyes on a functionalized silica [51]. After this assumption, there are several articles for application of this model for surface reactions of mesoporous materials, mainly about absorption on silica/silicon [52, 53]. Therefore, the Avrami-Erofeev kinetic model has high potential to describe the first step of pSi oxidation based on absorption and reaction of oxygen at the surface. As for the second stage of the oxidation (α  37%), the best models to interpret the data are diffusion ones, particularly three dimensional (3D) models ( Table V-3). This model is in agreement with oxygen diffusion across silica, produced by the first step of oxidation, through the network of pSi as described before. In order to better identify and separate the oxidation steps, particularly at α  37%, Derivative Thermogravimetry curve (DTG) has been calculated (dashed line, Figure V-6). It turns out in good agreement with DTA trace. To this aim, the reaction has been investigated as a function of temperature. Figure V-6 shows the results obtained at 10 K/min heating rate; similar results have been obtained with other heating rates with a small shift to the lower temperature. All of the reaction steps are exothermic (based on DTA) and bring to mass increment (based on TG).

82

Thermal Oxidation of pSi

As it was mentioned [54, 55], by heating of pSi rapidly surface functions decomposed and evaporated. But, these reactions have no significant effect on enthalpy and mass loss in comparison to oxidation as it can be seen. Also, due to the broadness of the DTA peaks, no information can be extracted regarding possible physical or morphological transformations. The plot resulting from the superimposition of the DTA and DTG traces is in line with Salonen results [56], with the difference that the second and third steps are more separated and evident. This is due to the lower aging of the pSi and mainly the use of lower heating rate; since these authors utilized 20 K/min heating rate.

Table V-2. Arrhenius parameters by model fitting for stage 1 (α  37%).

Model

E (kJ.mol-1)

Ln A (s-1)

R

F1

93.65

13.79

0.651

F2

7.50

-7.81

0.841

Fn

21.17

-2.85

0.939

C1

7.12

-7.98

0.936

Cn

21.17

-2.85

0.939

A2

7.91

-6.69

0.932

A3

8.08

-6.45

0.961

An

17.56

-6.14

0.985

D1

19.46

-6.80

0.922

D2

19.69

-7.40

0.922

D3

19.86

-8.82

0.921

83

Thermal Oxidation of pSi

D4

19.73

-8.88

0.932

R2

10.72

-7.48

0.945

R3

10.83

-7.84

0.946

Bna

21.17

-2.84

0.929

Table V-3. Arrhenius parameters by model fitting for stage 2 (α  37%).

Model E (kJ.mol-1) Ln A (s-1) R F1

20.30

-5.59

0.911

F2

26.94

-4.07

0.924

Fn

25.08

-4.48

0.924

C1

20.30

-5.59

0.911

Cn

25.08

-4.48

0.924

A2

14.28

-6.43

0.714

A3

16.43

-6.06

0.508

An

43.24

-2.42

0.948

D1

29.43

-5.33

0.955

D2

33.18

-5.19

0.955

D3

38.75

-5.57

0.964

D4

35.07

-6.30

0.966

R2

18.05

-6.89

0.873

84

Thermal Oxidation of pSi

R3

18.54

-7.14

0.890

Bna

35.79

-2.89

0.912

Owing to the fact that oxygen at around 350 K will begin to remove Si–H and Si–Si species present on the surface, generating Si–O [57], the first stage of oxidation started at about this temperature up to 600 K. As we mentioned before, this stage of oxidation is in accordance with the Avrami-Erofeev model. At this stage, the main enthalpy variation (DTA) is at 500 K [56]. As mentioned before, pSi leads to reduction in biological systems during protein or drug delivery and also bioimaging. Previous studies confirm that partial oxidation (up to 600 K) would be sufficient to avoid redox reaction with proteins (such as Albumin) [4]. On the other hand, this degree of oxidation is not enough to avoid redox reactions in other biological systems (tissue and also drugs) so further oxidation of pSi is needed [3, 5, 6].

Figure V-6. DTA, DTG, and E derived from heating rate of 10 K/min.

Thermal Oxidation of pSi

85

In the second oxidation step, which is controlled by diffusion mechanism, there are two peaks at about 850 and 1150 K on DTA curve that are in agreement with phase transition of silicon dioxide. In the bulk silicon dioxide, trigonal α-quartz (with density of 2.65 g/cm3) at 846 K transforms reversibly and easily into hexagonal β-quartz (2.53 g/cm3), and upon further temperature increase the βquartz will transform into hexagonal β-tridymite (2.25 g/cm3) at 1143 K [58]. Differently from α to β-quartz transition, transformation of β-quartz to β-tridymite is hard and irreversible for a bulk silica, but probably based on existence of Sb, as dopant n type silicon wafer, and nanostructural size effect this phase transition simplified [59]. It should be reminded that XRD results showed a small amount of crystalline SiO2 at the end of thermal oxidation under the minimum heating rate (Figure V-1). Then, by expansion of the crystalline structure of SiO2, disordering in the interface of silicon dioxide, amorphous oxides, and silicon of core structure increased. This disordered layer by heating has been observed by XPS analysis [60]. Diffusion of oxygen from the atmosphere to core silicon across silica is completely controlled by silica morphology [61]. And finally, maximum value of E is in line with published data of oxygen diffusion in fused silica as discussed before [40]. Apparent activation energy is represented as a function of temperature in the range 20%  α  90% and each point represents 1% of conversion. For the sake of clarity, the value of ∆α is less than 4% until about 500 K; therefore, we cannot calculate E value at temperature lower than 500 K. As it can be seen, enthalpy of reaction (DTA) shows correlation with E value. Initially, E value is low because the reaction is exothermic and releases high energy based on peak of DTA. Indeed, this release of energy provides power for further oxidation till the maximum value of the first DTA peak. Next, apparent activation energy increased up to 600 K. Then, E decreased according to the Avrami-Erofeev model. At the second stage of oxidation based on oxygen diffusion in silicon dioxide layer more energy is needed, so the E increased dramatically. The slope of this increment was increased by phase transformation of silicon dioxide.

Thermal Oxidation of pSi

86

5.5 Conclusions The mechanism of thermal oxidation of mesoporous silicon (pSi) particles has been systemically investigated. For this purpose, pSi was synthesised by anodization of silicon wafer and then fractured to particles by sonication. XRD confirmed single crystallinity of pSi and also complete oxidation after thermal oxidation. SEM investigations disclosed morphological behaviour of oxidation in details. The kinetic triplet for the pSi thermal oxidation was obtained by model-free kinetics. These results were validated by Starink method and model fitting kinetics for E and f(α), respectively. The results confirm that there are two separate oxidation steps. The first one is oxidation at low temperature of surface functional groups which can be modelled with the Avrami-Erofeev. The second appears to be oxidation according to 3-dimensional diffusion mechanism controlled by the diffusion of oxygen through the silica layer on the surface of the pSi. Phase transformation of silica influenced the second oxidation step according to differential thermal analysis.

5.6 References

1.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi, M. Scarpa, Journal of Material Chemistry B. 2 (37) (2014) 6345-6353.

2.

L. J. Karyn, J.B. Timothy, A. P. Clive, Advances in Colloid and Interface Science, 175 (2012) 25-38.

3.

S. Low, L. Canham, N. Voelcker, Biomaterials, 27 (26) (2006) 4538-4546

4.

K.L. Jarvis, T.J. Barnes, C.A. Prestidge, Langmuir, 26 (2010) 14316-14322

5.

E.C. Wu, J.S. Andrew, A. Buyanin, J.M. Kinsella, M.J. Sailor, Biomaterials, 32 (2011) 1957-1966

6.

N.L. Fry, G.R. Boss, M.J. Sailor, Chemical Material, 26 (2014) 2758 − 2764.

87

Thermal Oxidation of pSi

M. L. Sailor, Chemical reactivity and surface chemistry of porous silicon, in

7.

Hand book of Porous silicon, Springer International Publishing, Switzerland, 2014, 335 380. -

8

S.M. Prokes, Applied physics letters, 62 (1993) 25

9.

G. Aggarwal, P. Mishra, B. Joshi, S.S. Islam, Journal of Porous Material, 21 (2014)

.

.

23 . 10.

N.K. Hon, Z. Shapo shnik, E.D. Diebold, F. Tamanoi, B. Jalali, Journal of Biomedical Material Research Part A, 100 (2012) 3416 .

11

.

W. Park, A. M. Marconnet, T. Kodama, IEEE Intersociety Conference on Thermal and Thermomechanical Phenomena in Electronic Systems (ITherm), 637 (2014).

12.

J. Riikonen, M. Salomaki, J. van Wonderen, M. Kemell, Langmuir, 28 (2012) 10573.

13.

A.E. Pap, K. Kordas, T.F. George, S. Leppavuori, The Journal of Physical Chemistry

̈

B, 108 (34) (2004) 12744 12747. –

14.

B.E. Deal, A.S. Grove, Journal of Applied Physics, 36 (12) (1965) 3770 3778.

15.

A.S. Grove, O. Leistiko, C.T. Sah, Journal of Applied Physics, 35 (9) (1964) 2695





2701. 16.

R . Herino, G. Bomchil, K. Barla, C. Bertrand, J.L. Ginoux, Journal of The Electrochemical Society, 134 (1987 ) 1994.

17.

S. Vyazovkin, A.K. Burnham, J.M. Criado, L.A. Pérez-Maqueda, C. Popescu, N. Sbirrazzuoli, Thermochimica Acta, 520 (1) (2011) 1



19

.

18.

S. Vyazovkin, Journal of Thermal Analysis and Calorimetry, 83 (1) (2006) 45

19

T. Ozawa, Bulletin of the chemical society of Japan 38 (1965) 1881 1886

20.

J.H. Flynn, L.A. Wall. A Quick, Journal of Polym. Sci. Polym. Lett. 4 (1966) 323

.



51.



328.

.



88

Thermal Oxidation of pSi

21.

ASTM E698 -11 Standard Test Method for Arrhenius Kinetic Constants for Thermally Unstable Material Using Differential Scanning Calorimetry and the Flynn/Wall/Ozawa Method .

22

.

C.A. Ribeiro, W.R. De Souza, M. Spirandeli Crespi, J.A. Gomes Neto, F.L. Fertonani, Journal of thermal Analysis and Calorimetry, 90 (2007) 801



805.

23.

M.J. Starink, Thermochimica Acta, 404 (2003) 163

24.

S. Vyazovkin, C.A. Wight, Thermochimica Acta, 340 (1999) 53

25.

P. Budrugeac, E. Segal, Journal of Therm. Anal. 49 (1997) 183.

26.

Y.H. Ogata, N. Yoshimi, R. Yasuda, T. Tsuboi, T. Sakka, A. Otsuki, Journal of Applied Physics 90 (2001) 6487





176. –

68 .

6492.

27

R. Cisneros, H. Pfeiffer, C. Wang, Nanoscale Res. Lett, 5 (2010) 686

28.

F.M. Ross, J.M. Gibson, Structural Evolution of the Silicon/Oxide Interface During

.



691

.

Passive and Active Oxidation, Fundamental Aspects of Silicon Oxidati on, Springer, 2001, 35 29.

60.

A. Ghafarinazari, M. Mozafari, Journal of Alloys and Compounds, 616 (2014) 442 –

30.



448.

YL . Lai, TY. Chang, KM. Chang, CF . Chen, Japanese Journal of Applied Physics, 54 (2015) 06FG12

31.

F. Rouquerol, Journal of Rouquerol, K. Sing, Adsorption by Powders and Porous Solids: Principles, Methodology and Applications, Academic Press, London, 1999, p. 9

32.

S. Vyazovkin, K. Chrissafis, M.L. Lorenzo, N. Koga, M. Pijolat, B. Roduit, N. Sbirrazzuoli, J.J. Suñol, Thermochimica Acta, 590 (2014) 1 23 . –

33.

F. Baghbani, F. Moztarzadeh, A.H. Razavi Kamran, F. Tondnevis, N. Nezafati, M. Gholipourmalekabadi, M. Mozafari, Advanced Composites Letters, 21 (1) (2012) 16 –

24.

89

Thermal Oxidation of pSi

34.

D.R. Wolters, A.T.A. Zegers -Van Duijnhoven, Microelectron. Reliab. 38 (2) (1998) 259 264. -

35.

V. Marcu and E. Segal, Thermochimica Acta, 35 (1980) 43.

36.

G.F. Cerofolini, L. Meda, Journal of Non Crystalline Solids, 216 (1997) 140

37.

A. K. Galwey, M. E. Brown, Kinetic background to thermal analysis and

-



147.

calorimetry, Ed. M. E. Brown, in Handbook of Thermal Analysis and Calorimetry, Vol. 1: Principles and Practice, Amsterdam 1998, 147



225.

38.

S.V. Vyazovkin, A.I. Lesnikovich, Thermochimica Acta, 165 (1990) 273 .

39.

P. Gallagher, Handbook of Thermal Analysis and Cal orimetry: Principles and Practice. Volume 1. 1998. Second impression 2003, p 204 .

40

F. Journal of Norton, Nature, 191 (1961) 701

41.

R.M. Braga, J.M.F. Barros, D.M.A. Melo, M.A.F. Melo. Journal of thermal analysis

.

.

and calorimetry, 111 (2013) 1013 1018. –

42.

M.A. El -Oyoun. Journal of Alloys and Compounds 486 (2009) 1 8

43.

J. Perez, J.Y. Cavaille, Journal de Physique III, 5 (1995) 791 805.

44.

N. Koga, Thermochimica acta, 244 (1994) 1

45.

M.E. Brown, D. Dollimore and A. K. Galwey, in Comprehensive Ch emical Kinetics,







20.

Vol. 22, Reactions in Solid State, ed. C. H. Bamford and C. F. Tipper, Amsterdam 1980, p. 57 46.

S.V. Vyazovkin, Thermochim Acta, 211 (1992) 181 187.

47.

T.J.W. Bruijn., W.A. Jong, P.J. Van Den Berg, Thermochimica Acta, 45 (3) (1981)



315 325 . –

48.

M. Y. Bashouti, K. Sardashti, J. Ristein, Nanoscale research letters, 8 (1) (2013) 1 5.



90

Thermal Oxidation of pSi

49.

V Depauw, O Richard, H Bender, I Gordon, G Beaucarne, J Poortmans, R Mertens, J P Celis, Thin Solid Films, 516 (2008) 6934 -

50.



6938.

E.C.N. Lopes, F.S.C. dos Anjos, E.F.S. Vieira, A.R. Cestar, Journal of Colloid Interface Science, 263 (2003) 542 547 –

51.

A.R. Cestari, E.F.S. Vieira, G.S. Vieira, L.E. Almeida, Journal of Hazardous Material, 138 (1) (2006) 133

52.



141 .

R. George, S. Sugunan, Journal of Molecular Catalysis B: Enzymatic 105 (2014) 26 –

53

.

32.

R. Serna -Guerrero, A. Sayari, Chemical Engineering Journal, 161 (2010) 182 190 –

.

.

54.

A. Loni, L.T. Canham, Journal of Applied Physics, 113 (2013) 173505 .

55

K.L. Jarvis, T.J. Barnes, C.A. Prestidge, Langmuir, 24 (2008) 14222

.



14226

.

56.

J. Salonen, V.P. Lehto, E. Laine, Applied Physics Letters, 70 (1997) 637

57

Y. Ogata, H. Niki, T. Sakka, M. Iwasaki, Journal of The Electrochemical Society,

.

142 (1995) 1595





639.

1601.

58.

P.J. Heaney, D.R. Veblen, J.E. Post, American Mineralogist, 79 (1994) 452 - 460.

59.

H. Ozawa, A Yamakage, M Sato, Y Tanaka, Physical Review B, 90 (4) (2014) 45309.

60

.

Li. GB, LS Liao, XB L iu, XY Hou, X Wang, Applied physics letters, 70 (10) (1997) 1284 - 1286.

61.

K Kajihara, T Miura, H Kamioka, A Aiba, Journal of Non-Crystalline Solids, 354 (2) (2008) 224 232 . –

91

Chapter five, in part of full, is a reprint (with co-author permission) of the material as it appears in the following publication: A. Ghafarinazari, E. Zera, A. Lion, M. Scarpa, G. D. Soraru, N. Daldosso, Isoconversional Kinetics of Thermal Oxidation of Mesoporous Silicon. Thermochimica Acta, 623 (2016) 65 - 71. The author of this dissertation is the primary author and corresponding of this article.

Interaction of pSi with Human Dendritic Cells

Interaction of pSi with Human Dendritic Cells

93

6.1 Summary In this Chapter, the interaction of porous silicon (pSi) with human dendritic cell has been investigated. The pSi is efficiently internalized by human dendritic cells and do not show any toxic effect even at a concentration of 1 mg·mL−1. The intrinsic luminescence of the differently functionalized pSi is preserved inside the cells and permits the selective and efficient tracking of the microparticles without using molecular tags and thus leaving the organic coating available for the interaction with the drug. The results obtained suggest that the functionalized pSi is an efficient platform for simultaneous imaging and delivery of therapeutic agents to the disease site.

6.2 Introduction The use of micro-to-nano sized fragments of pSi is a promising strategy as a vehicle for delivery and controlled release of drugs or nanoparticles [1, 2, 3, 4]. In fact, the pSi morphology offers a large loading capacity and pSi in biological environments undergo dissolution, producing non-toxic and harmlessly removed wastes [5, 6]. It has been shown also an intrinsic visible photoluminescence (PL) that is derived from the combination of quantum confinement [7, 8] and surface effects [9]. The intrinsic PL of pSi could allow the monitoring of the kinetics of the carrier distribution and the localization of the delivery site of the loaded molecules in cell cultures and in vivo, without the need for a molecular tag such as fluorophores. Recently, Sailor et al. [10] reported about the use of oxidized and luminescent pSi particles in cells and in living animals; however, the PL of pSibased drug delivery vehicles has been scarcely exploited so far. The reason is probably due to the loss of PL during the vehicle fabrication. In fact, the PL emission of pSi depends not only on the nanocrystaline size [11] but also on surface states [12]. Morphological properties determine also the performances of pSi as a drug delivery vehicle and must be optimized to carry the load and reach the delivery site [13]. The pore size, porosity and even the fragment size and shape can be changed by adjusting the fabrication protocols [14]. The pSi particles with dimensions

Interaction of pSi with Human Dendritic Cells

94

spanning from micrometres to few hundreds of nanometres have been recently obtained [15, 16] and different surface functionalization strategies have been suggested [17]. However, many chemical procedures require high temperatures [15] or exposition to nitrogen containing compounds [18], which irreversibly quench the PL [19]. Stabilization of the PL of nanostructured silicon is obtained by mild surface oxidation [20], however the surface coating by organic molecules is desirable since it improves the drug delivery properties. In particular, though the grafting of amino groups (that bear positive charge and have good coupling properties) is attractive [21], it must be taken into account that amines are quenchers of the PL of pSi [19, 22, 23]. The control of surface chemistry is also important from the perspective of safe use of nanomaterials [24]. In this regard, a relevant limit for the medical use of inorganic nano- or micro-particles is their ability to stimulate the release of proinflammatory cytokines by immune cells, such as dendritic cells (DCs) [25, 26]. DCs possess a huge and diverse functional repertoire [27], including the capacity of presenting the antigens to T lymphocytes in order to stimulate an immune response [28]. This last function makes DCs the preferential target of nano vaccines for cancer immunotherapy [29]. In this section, the pSi interaction with cells has been tested on human DCs. We found that the luminescent pSi was efficiently internalized by DCs preserving their optical properties without inducing apoptosis, and without causing any proinflammatory response. These results suggest the safe use of these materials in biological environments and the efficient delivery of specific antigens to the immune system by pSi.

6.3 Experimental Procedure Protocols for pSi synthesis and surface functionalizations with carboxyl and amine have been explained in Chapter III in details. Briefly, porous silicon layer was formed by electrochemical etches of p-type Si wafer, 〈100〉oriented, 10–20 Ω·cm resistivity. Then, the porous layer was removed and fragmented into microparticles (pSi) by 15 min sonication at 400 W in toluene.

Interaction of pSi with Human Dendritic Cells

95

The pristine pSi was suspended in toluene saturated with argon and containing acrylic acid N-hydroxysuccinimide ester. Reaction carried out by two-hours illumination. The powder was collected and washed with ethanol or toluene. Ethanol was used to obtain free carboxylic groups on the powder surface. In fact, the reactive N-hydroxysuccinimide group (NHS) slowly hydrolyses in this solvent [30]; then, the final product is carboxyl functionalized pSi (COOH-pSi). Conversely, the washing steps were performed in toluene to further modification by coupling with a diamine. N, N'-Dicyclohexylcarbodiimide (200 μM) and 4, 7, 10-trioxa-1, 13-tridecanediamine (200 μM) were added to COOH-pSi (30 mg) suspended in toluene and left to react for a night under gentle shaking. Then, the suspension was centrifuged and the powder (NH2-pSi) was purified from byproducts. Fluorescence characterization was performed by Horiba Jobin-Yvon Nanolog. The configuration was: 2 nm slit size, 0.1 second integration time, and cut-off filtration at 370 nm. PL of a single microparticle was obtained on an inverted Olympus Microscope iX70 by using a 488 nm solid state laser as an excitation source and an Avantes ULS2048XL - spectrometer. As for effects of pSi on immune cell response, after written informed consent and upon approval of the ethical committee, buffy coats from the venous blood of normal healthy volunteers were obtained from the Blood Transfusion Centre of the University of Verona. Monocytes were isolated from buffy coats by Ficoll-Hypaque and Percoll (GE Healthcare Life Science) density gradients and purified using the human monocyte isolation kit II (Miltenyi Biotec). The final monocyte population was 99% pure, as measured by FACS (Fluorescence-Activated Cell Sorting applied in flow cytometry) analysis. To generate dendritic cells (DCs), monocytes were incubated at 37 ℃ in CO2 (5%) for 5–6 days at 1 × 106 mL−1 in 6-well tissue culture plates (Greiner, Nürtingen, Germany) in RPMI 1640, supplemented with heat-inactivated low endotoxin FBS (10%), L-glutamine (2 mM), GM-CSF (50 ng·mL−1), and IL-4 (20 ng·mL−1). The final DC population was 98% CD1a+, as measured by FACS analysis.

Interaction of pSi with Human Dendritic Cells

96

The toxicity of the pSi was tested using an Apoptosis Detection Kit (Miltenyi Biotec) according to the manufacturer's protocol. The percentages of live cells, dead cells and cells in the early apoptotic process were determined by Annexin V FITC conjugate and propidium iodide staining. Cells were acquired on a seven-colour MACS Quant Analyser (Miltenyi Biotec) and FlowJo software (Tree Star, Ashland, OR, USA) was used for data analysis. Cytokine production in culture supernatants was determined using Ready-Set-Go ELISA kits purchased from Bioscience (San Diego, CA). The protein amount of IL-12 (range 4–500 pg.mL−1), IL-23 (range 15– 2000 pg mL−1), TNF-α (range 4–500 pg.mL−1), IL-1β (range 4–500 pg.mL−1) and IL-6 (range 2–200 pg.mL−1) was analysed according to the manufacturer's protocol. For confocal microscopy analysis the DCs were seeded on 13 mm poly-Llysine-coated cover slips and treated for 24 hours with the pSi. The cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature and quenched with NH4Cl (50 mM). The cells were then permeabilized with PBS–Triton X-100 (0.1%) and blocked with BSA (1%) for 30 minutes. After washing, the cover slips were incubated for 30 minutes with Phalloidin-Rhodamine (Cytoskeleton, Denver, CO, USA) to visualize F-actin. The cells were washed and mounted in glycerol based anti-fading medium. The images were acquired by using a confocal microscope (LeicaMicrosystems, Wetzlar, Germany) at 400× magnification by using the 63× oil immersion objective (1.25 NA). Z-stacks were acquired and the maximum intensity projections (MIPs) were obtained by using the LAS-AF software (LeicaMicrosystems).

6.4 Results and Discussion As explained before, surface of the native Si nano crystallites is vulnerable to attack by different compounds, such as amines which have been shown to quench the PL of free Si nanoparticles by opening non-radiative pathways to carrier relaxation [22]. The PL of the native pSi was stabilized by suitable surface modifications (refer to Chapter IV). Results confirm that carboxyl or amine groups not only make the pSi capable of interacting electrostatically with other molecules

97

Interaction of pSi with Human Dendritic Cells

penetrating inside the pores but also can be used for the binding of a protective polymer shell to the external surface. PL emission of the COOH-pSi and the NH2-pSi (continuous and dashed spectra, respectively) is reported in Figure VI.1, left panel, for excitation at 350 nm. In both spectra, the typical broad emission characteristic of nanostructured silicon with a maximum around 590–610 nm is present based on quantum confinement mechanism.

Exc. 350 nm

b)

1

Exc. 405 nm

PL (a.u.)

1

PL (a.u.)

a)

0

400

500

600

700

Wavelength (nm)

800

0

COOH-pSi NH2-pSi

450 500 550 600 650 700

Wavelength (nm)

Figure VI-1. Emission spectra of functionalized pSi. The solid trace is for the COOH-pSi and the dashed trace for the NH2-pSi. Left panel: excitation 350 nm. The sharp peak at 700 nm wavelength in the NH2-pSi sample is related to the second order of the spectrometer grating. Right panel: excitation 405 nm.

Moreover, NH2-pSi has another emission at about 420 nm due to nitrogen diffusion. It appears also that both the COOH-pSi and the NH2-pSi can be excited at 405 nm (which is the excitation wavelengths used in confocal microscopy). The corresponding emission spectra are shown in Figure VI.1 for the COOH-pSi (continuous trace) and NH2-pSi (dashed trace). From this Figure it appears that the emission in the blue region of the NH2-pSi by exciting at 405 nm is still intense and red-shifted, due to the proximity between excitation and emission wavelengths. The cell uptake of pSi was tested on human DCs since these cells mediate the immune response and are the preferential target of nano vaccines [29]. Experiments

Interaction of pSi with Human Dendritic Cells

98

were performed to analyse whether the pSi affect human DC viability and/or cytokine release. The analysed cytokines include IL-12 that stimulates natural killer cells and T lymphocytes, as well as IL-23, which induces the secretion of proinflammatory mediators by T cells [31, 32]. Moreover, IL-6, IL-1β, and TNF-α were tested which elicit the systemic acute phase reaction, characterized by fever, headache, anorexia, nausea, emesis, and changes in the sleep–wake cycle [26, 33, 34]. Human blood monocytes were cultured for 5 days with GM-CSF and IL-4 to obtain DCs, which were then challenged with various doses of pSi or with lipopolysaccharide (LPS), a well-known bacterial immune cell stimulator, as a positive control. Figure VI.2 shows the results of Annexin V staining experiments indicating that 24 h exposure to different concentrations of COOH- and NH2-pSi did not induce DC apoptosis, even when a dose of 1 mg·mL−1 was added to the cells. This finding is relevant considering that this dose is extremely high for the standard in vitro DC stimulation protocols. A 24 h treatment of DCs with LPS did not affect cell viability (data not shown). We then analysed whether NH2- or COOH-pSi stimulate the release of pro-inflammatory cytokines by DCs.

Figure VI-2. Cell viability of human dendritic cells. DCs were treated with the indicated doses of COOH- and NH2-pSi for 24 hours. The incubation volume was 1 mL. The results are the mean ± SD of three experiments.

Interaction of pSi with Human Dendritic Cells

99

Figure VI.3 illustrates an ELISA assay performed on DC culture supernatants showing that the NH2-pSi did not stimulate the release of IL-12, IL-23, IL-1β, IL-6 or TNF-α by DCs. Similar results have been obtained upon DC treatment with COOH-pSi. This lack of toxicity is in line with the findings of Fisichella et al. [35] who showed that amine derivatized mesoporous silica microparticles elicit no toxic effect on cells and with the emerging opinion that the interaction of nanomaterials with the immune system necessitates careful assessment of nanomaterial toxicity [36].

Figure VI-3. Analysis of cytokine production by DCs treated for 24 hours with different doses of NH2-pSi or LPS used as the positive control. The results are the mean ± SD of three experiments.

These results also demonstrate that pSi suspensions are not contaminated by microorganisms or their derivatives capable of stimulating the immune cells [37]. For the purpose of exploiting the intrinsic luminescence of these microparticles, DCs were incubated with 0.5 mg·mL−1 of COOH- or NH2-pSi. The particles were ingested by the cells by an endocytosis mechanism as reported by Serda et al. for discoidal pSi [38].

Interaction of pSi with Human Dendritic Cells

100

After 24 hours, the particle cellular uptake was visualized by confocal microscopy using different excitation wavelengths and detection channels. Figure VI.4 (panel A) shows a confocal image from which it appears that NH2-pSi is efficiently internalized by human DCs and preserves a bright emission in the red region. The panel B is the zoom-in of the panel A field area enclosed in dashed lines, highlighting a cell showing an ingested particle. Similar results have been obtained using COOH-pSi, indicating that the presence of amine or carboxyl functionalities on the pSi surface did not change the particle uptake by DCs. In average 1.5 pSi per cell were observed, corresponding to an ingested pSi volume of about 0.3 μm3 (0.05 μm3 of silicon).

Figure VI-4. Confocal microscopy images of DCs treated with 500 μg of NH2pSi (in green). After 24 h incubation the cells were fixed and stained with Phalloidin to label filamentous F-actin (red). One representative experiment of three is shown. (A) Internal z-stack of a general field and (B) a zoom-in of (A).

Figure VI-5 shows the excitation at 405 nm, which corresponds to the highest photon energy usually available for conventional confocal microscopes, and acquiring the images with the red and green detection channels. This Figure shows that the NH2- and COOH-pSi luminescence can be detected both at 500–550 nm (green) and 600–670 nm (red).

Interaction of pSi with Human Dendritic Cells

101

Figure VI-5. Confocal microscopy analysis of NH2- and COOH-pSi ingested by DCs. The excitation is set at 405 nm and the emission collected in the green (left panel) and red (right panel) region.

Furthermore, we mention to the PL outcomes from combination of pSi with DCs at spectroscopy instrument. For this purpose, 0.5 mL from functionalized pSi poured on cuvette with concentration of 30 mL/mg. Then, 1.5 mL PBS added to the cuvette. After that 5 million DCs subjoined. At the end, solution centrifuged at 2000 rpm for 2 min. PL data shows in Figure VI-6 for each step. Taking spectra from supernatant (liquid part) is clear, but in order to analyse precipitated materials at the bottom (solid), it diluted by 1.5 mL ethanol. For both kinds of functionalizations, there is no emission at 600 nm in the supernatant in opposite to the solid part which means there is attachment between DCs and pSi as showed before (refer to Figure VI-5). Indeed, we can estimate attachment of cells with particles in a fast and simple technique. In Figure VI-7 we show the image of a single NH2-pSi particle obtained by excitation at 405 nm and acquisition in the green and red regions. The merged image is shown in the bottom right panel.

Interaction of pSi with Human Dendritic Cells

102

Figure VI-6. Emission spectrum of COOH–pSi (left) or NH2-pSi (right) with DCs, in PBS before and after centrifuge.

Figure VI-7. Confocal microscopy images of an NH2-pSi. Excitation at 405 nm. Left top: emission collected in the range 500–550 nm; right top: emission collected in the 600–650 nm; left bottom: bright field image; right bottom: merged image.

The corresponding emission spectrum in the red region acquired by a singleparticle detection set-up (excitation at 488 nm) is shown in Figure VI-8. These data

103

Interaction of pSi with Human Dendritic Cells

demonstrate that single NH2-pSi can be imaged and analysed both by a spectral or an imaging apparatus. Moreover, the broad PL of these microparticles provides a signal traceable by both green and red channels. This property can help the intracellular localization of the pSi or the tracking of the labelled cell, since the spectral region of detection can be chosen to be the one with lower interference from other fluorescence signals or from the background.

PL Int. (a.u.)

1500

1000

500

0 500

550

600

650

700

750

800

850

Wavelength (nm) Figure VI-8. PL emission from an NH2-pSi particle. Excitation at 488 nm.

These outcomes contribute significantly to the understanding and the successful exploitation of different emission mechanisms of pSi to perform a biomedical experiment with a standard confocal microscope. The highly porous structure, in conjunction to the optical features and the absence of toxic effects, proposes these silicon microparticles as superior delivery vehicles traceable by conventional optical microscopes in cell cultures or in the tissue surface. A further improvement will be the use of the emerging multiphoton near-infrared microscopy techniques [39, 40] which are expected to allow the microparticle localization at deeper (up to 500 μm) imaging depth.

Interaction of pSi with Human Dendritic Cells

104

6.5 Conclusions It has been demonstrated that the pSi particles are uptaken by primary human DCs and this process is not associated with a decrease in cell viability, even when the cells are incubated with very high concentrations of both carboxyl and amine functionalization. Furthermore, it was not observed any stimulation of the secretion of pro-inflammatory cytokines by DCs, suggesting that the particles do not activate these immune cells. The organic coating layer introduced by functionalization allows the microparticles to be effectively swallowed by DCs, indicating that these particles are good candidates as delivery vehicles to the immune cell system of drugs and anticancer vaccines. Finally, it was clearly demonstrated by confocal microscopy that the optical properties of the pSi studied here can be used to monitor the intracellular localization of the particles.

6.6 References

1.

E. J. Anglin, L. Cheng, W. R. Freeman and M. J. Sailor, Advanced Drug Delivery Reviews, 6 (2008) 1266.

2.

R. E. Serda, B. Godin, E. Blanco, C. Chaippini and M. Ferrari, Biochimica et Biophysica Acta, 1810 (2011) 317.

3.

J. Salonen, A. M. Kaukonen, J. Hirvonen and P. V. Lehto, Journal of Pharm. Sci., 97 (2008) 632.

4.

F. Peng, Y. Su, Y. Zhong, C. Fan, S.-T. Lee and Y. He, Acc. Chem. Res., 47 (2014) 612.

5.

L. T. Canham, Advanced Materials, 7 (1995) 1033.

6.

D. M. Reffitt, R. Jogdaohsingh, R. P. Thompson and J. J. Powell, J. Inorg. Biochem., 76 (1999) 141.

Interaction of pSi with Human Dendritic Cells

105

G. Ledoux, O. Guillois, D. Porterat and C. Reynaud, Physical Review B: Condens.

7.

Matter Material Phys., 62 (2000 ) 15942. 8.

C. Delerue, G. Allan and M. Lannoo, Physical Review B: Condens. Matter Material Phys., 48 (1993) 11024. J. L. Gole, F. P. Dudel, D. Grantier and D. A. Di xon, Physical Review B: Condens.

9.

Matter Material Phys., 56 (1997 ) 2137 . 10.

L. Gu, D. J. Hall, Z. Qin, E. J. Anglin Joo, D. J. Mooney, S. B. Howell and M. J. Sailor, Nature Commun., 4 (2013) 2326.

11

.

D. S. English, L. E. Pell, Z. Yu, P. F. Barbara and B. A. Korgel, Nano Lett., 2 (2002 ) 681.

12.

A. Saar, Journal of Nanophotonics, 3 (2009) 032501.

13.

M.A. Shahbazi, M. Hamidi, E. M. Mäkilä, H. Zhang, P. V. Almeida, M. Kaasalainen, J. J. Salonen, J. T. Hirvonen and H. A. Santos, BioMaterial, 34 (2013) 7776 .

14.

E. Tasciotti, X. Liu, R. Bhavane, K. Plant, A. D. Leonard, B. K. Price, M. M.-C. Cheng, P. Decuzzi, J. M. Tour, F. Robertson and M. Ferrari, Nature Nanotechnol., 3 (2008) 151.

15.

L. M. Bimbo, M. Sarparanta, H. A. Santos, A. J. Airaksinen, E. Mäkilä, T. Laaks onen, L. Peltonen, V.-P. Letho, J. Hirvonen and J. Salonen, ACS Nano, 4 (2010) 3023 .

16.

P. Decuzzi, B. Godin, T. Tanaka, S. -Y. Lee, C. Chiappini, X. Liu and M. Ferrari, Journal of Controlled Release, 141 (2010) 320.

17.

J. Rytkönen, R. Miettinen, M. Kaasalainen, V. P. Lehto, J. Salonen and A. Närvänen, -

Journal of NanoMaterial, 2012 (2012) 896562. 18.

B. Godin, J. Gu, R. E. Serda, R. Bhavane, E. Tasciotti, C. Chiappini, X. Liu, T. Tanaka, P. Decuzzi and M. Ferrari, J. Biomed. Material Res., Part A, 94 ( 2010) 1236.

106

Interaction of pSi with Human Dendritic Cells

19.

W. D. Kirkey, Y. Sahoo, X. G. Li, Y. Q. He, M. T. Swihart, A. N. Cartwright, S. Bruckenstein and P. N. Prasad, Journal of Materials Chemistry, 15 (2005 ) 2028.

20.

L. Gu, J. -H. Park, K. H. Duong, E. Ruoslahti and M. J. Sailor, Small, 6 ( 2010) 2546.

21.

W. Xu, J. Riikonen, T. Nissinen, M. Suvanto, K. Rilla, B. Li, Q. Wang, F. Deng and V. P. Lehto, The Journal of Physical Chemistry C, 116 (2012) 22307. -

22

.

B. Sweryda -Krawiec, R. R. Chandler-Henderson, J. L. Coffer, Y. G. Rho and R. F. Pinizzotto, The Journal of Physical Chemistry, 100 (1996) 13776.

23.

R. R. Chandler -Henderson, B. Sweryda-Krawiec and J. L. Coffer, Journal of Phys. Chem., 99 (1995 ) 8851

.

24.

A. Verma and F. Stellacci, Small, 6 (2010) 12.

25

C. Chang, Journal of Autoimmun., 34 (2010 ) J234

26.

M. Di Gioacchino, C. Tetrarca, F. Lazzarin, L. Di Giampaolo, E. Sabbioni, P.

.

.

Boscolo, R. Mariani-Costantini and G. Bernardini, Int. Journal of Immunopathol. Pharmacol., 24 (suppl. 1), (2011) 65S. 27.

K. Schäkel, Exp. D ermatol., 18 ( 2009) 264.

28

F. Granucci, I. Zanoni and P. Ricciardi-Castagnoli, Cell. Mol. Life Sci., 65 ( 2008)

.

1683. 29.

L. E. Paulis, S. Mandal, M. Kreutz and C. G. Figdor, Curr. Opin. Immunol., 25 (2013) 389.

30.

S. Kalkhof and A. Sinz, Anal. Bioanal. Chem., 392 (2008) 305.

31.

D. A. Vignali and V. K. Kuchroo, Nature Immunol., 13 ( 2012) 722.

32.

M. Xu, I. Mizoguchi, N. Morishima, Y. Chiba, J. Mizuguchi and T. Yoshimoto, Clin. Dev. Immunol., 2010 (2010) 832454 .

33.

J. K. Elmquist, T. E. Scammell and C. B. S aper, Trends Neurosci., 20 ( 1997) 565.

107

Interaction of pSi with Human Dendritic Cells

34.

A. F. Suffredini, G. Fantuzzi, R. Badolato, J. J. Oppenheim and N. P. O'Grady, Journal of Clin. Immunol., 19 (1999) 203 .

35.

M. Fisichella, H. Dabboue, S. Bhattacharyya, M. L. Saboungi, J. P. Salvetat, T. Hervor and M. Guerin, Toxicol. In Vitro, 23 (2009 ) 697

.

36.

B. Fadeel, Swiss Med. Wkly. 142 ( 2012) w13609 .

37

B. Fadeel and A. E. Garcia -Bennett, Advanced Drug Delivery Reviews, 62 (2010 )

.

362. 38.

R. E. Serda, S. Ferrati, B. Godin, E. Tasciotti, X. W. Xu and M. Ferrari, Nanoscale, 1 ( 2009) 250

.

39.

J. H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti, S. N. Bhatia and M. J. Sailor, -

Nature Material, 8 (2009) 331. 40.

P. Sarder, S. Yazdanfar, W. J. Akers, R. Tang, G. P. Sudlow, C. Egbulefu and S. Achilefu, Journal of Bio med. Opt., 18 (2013) 106012.

Interaction of pSi with Human Dendritic Cells

108

Chapter six, in part of full, is a reprint (with co-author permission) of the material as it appears in the following publication: N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi and M. Scarpa, Orange and blue luminescence emission to track functionalized porous silicon microparticles inside the cells of the human immune system, Journal of Materials Chemistry B, 2014, 2 (37) 6345 - 6353. The author of this dissertation is a co-author of this manuscript.

Interaction of pSi with Cobinamide

Interaction of pSi with Cobinamide

110

7.1 Summary In the previous chapters, the biocompatibility of light emitting mesoporous silicon showed the possibility to use it for bioimaging applications. Herein, we would like to explore the chance to use pSi for drug delivery. Therefore, first drug stability has been checked during and after grafting to pSi particles. After that, drug loading capacity and release profile has been tested. Finally, workability of drug loaded pSi for bioimaging has been evaluated. The promising results showed that pSi has high potential for Theranostics applications.

7.2 Introduction Porous materials that can load and slowly release therapeutics are of interest because they offer the potential to reduce systemic toxicity and increase the amount of drug at the target site [1, 2, 3]. Among them, mesoporous silicon (pSi) is a promising candidate based on its biocompatibility [4], highly tuneable structure [5], chemically modifiable surface [6], and biodegradability [7]. In addition, luminescent pSi can be used to monitor the loading and release of biomolecules, allowing pSi to be used as a self-reporting drug carrier [4]. Moreover, the large surface area and surface charge of pSi provides a substantial drug loading capacity [6]. However, interaction between pSi and the drug must be carefully considered to design an effective delivery system without any side effect on the drug. Loaded molecules will quickly diffuse out of porous materials because of local concentration gradients unless additional interactions between the porous matrix and drug are generated. The native pSi contains highly reactive surface SixSiHy (x + y = 4) species with underlying elemental silicon, both of them are good reducing agents [8, 9]. As already reported, pSi in biological systems leads to redox drugs [10, 11], proteins [12], and cells [13]. Thus, modification of pSi is needed to both limit redox degradation of drugs and increase the affinity of the drug for the pSi surface. At the moment, the conventional modification of pSi to avoid drug reduction is oxidation. For instance, Sailor et al. [10, 11, 14] proposed oxidation at high

Interaction of pSi with Cobinamide

111

temperature (800 ℃) to avoid redox of drugs. In his recent paper [14], he concluded “it is important that the silicon skeleton be completely oxidized to ensure the drug is not reduced or degraded by contact with elemental silicon during the particle dissolution−drug release phase”. On the other hand, Barnes et al. [12] proposed a mild oxidation at 400 ℃ to avoid redox of proteins. In this regard, we recently proposed a mechanism for pSi oxidation[15]. However, oxidation leads to quench light emission of pSi [16, 17], which is mandatory for bioimaging [18] and photothermal therapy [19]. Moreover, oxidation results in reduction of the pores volume because of swelling of the pore walls as oxygen is incorporated into the silicon skeleton, which leads to significantly reduction of drug loading amount. As an example, oxidation at 400 ℃ and 800 ℃ led to 3-fold and 10-fold reduction of loading capacity of proteins [12] and drugs [14], respectively, in comparison with the native pSi. The redox activity of the pSi matrix with the drug is an additional constraint: to prevent the drug from being reduced, the silicon skeleton must be stabilized by something other than the drug being loaded. In the present study, we have addressed the possibility of using surface functionalization to control redox acting of pSi in biological systems thus maintaining the light emission properties. In this chapter, we present a comprehensive study on the interaction of a drug with pSi and the monitoring effects of pSi functionalization on redox and also loading amount, release profile, and PL properties. As drug candidate, we chose cobinamide (Cbi), vitamin B12 analogue, that is being developed as a cyanide antidote [20, 21, 22]. Recently, Sailor et al. [14] proposed Cbi for checking redox activity of the oxidized pSi because its hydrophilicity, small size, and redox-active cobalt centre (Figure VII-1) make it difficult to load into porous matrices in its active oxidized state.

Interaction of pSi with Cobinamide

112

Figure VII-1. Structure of Cobinamide (C48H72CoN11O8) when it is dissolved in neutral aqueous solutions, with hydroxide and water molecules bound in the axial positions (aquohydroxocobinamide).

Thus, it presented a greater challenge than most drugs, and if the method worked for Cbi, it would likely work for other drugs. Moreover, the distinctive UV– visible absorbance spectra of Cbi are very useful for quick and easy detection and redox state assessment. We also used Cbi as a model to check redox activates of our functionalized pSi during drug delivery.

7.3 Experimental Procedure All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received (all ACS grade). All aqueous solutions were prepared with ultrapure water obtained using an ultrafiltration system (Milli-Q, Millipore) with a measured resistivity above 20 MΩ·cm-1. Porous silicon layer was formed by electrochemical etches of Boron-doped p-type Si wafer, 〈1 0 0〉 oriented, 10–20 Ω·cm resistivity. Then, the porous layer was removed and fragmented into microparticles (pSi) by 15 min sonication at 400 W in toluene as it was described in the Chapter II. Oxidized porous silicon (pSiO2) obtained by oxidation of pSi at 1000 ℃ for 10 min by heating rate of 3 min/K. The protocols for surface functionalizations of pSi have been

Interaction of pSi with Cobinamide

113

explained in Chapters III and IV in details. As for reminder, Table VII-1 summarizes the different samples investigated in this work.

Table VII-1. Different functionalization of pSi. COOH-pSi

pSi functionalized with carboxyl group

NH2-pSi

pSi functionalized with amine group

PEG-pSi

COOH-pSi sample functionalized with PEG

PEG2-pSi

COOH-pSi sample functionalized with double amount of PEG

Chitosan-pSi

COOH-pSi sample functionalized with chitosan

Cobinamide was synthesized from hydroxocobalamin (OH-Cbi, purchased from Sigma-Aldrich) by acid hydrolysis using HCl proposed by K. E. Broderick et al. [23]. Purity of Cbi was confirmed both spectrophotometrically by comparison with published spectra [20, 21, 24] and by high performance liquid chromatography (HPLC) by using a C18 reversed phase column eluted isocratically in 100 mM NaH2PO, pH 4.0, and 15% methanol (v/v) [25]. The column effluent was monitored at multiple wavelengths at 168 – 351 nm by a diode array detector. Figure VII-2 shows HPLC outcomes, which confirmed at 6.34 min Cbi obtained and has similar optical density with the references. When stored under 20 ℃, Cbi was spectroscopically stable for at least one month. To load Cbi onto pSi, we slightly modified the procedures proposed before [14, 26, 27]. Small amount of dried pSi (in the range 0.5 - 1 mg), containing the same amount of Cbi, has been incubated for 2 h in 1.5 mL of deionized water at pH 6 by HCl under weak shaking. The pH of the solution was kept around 6.0 since at this pH value, the carboxyl groups on the surface of COOH-pSi and PEG-pSi (COOHPEG-NH2-COOH-pSi) are negative. Indeed, logarithmic constant of acid dissociation constant (pKa) of the free carboxyl is around 5 [28]. Conversely, the

Interaction of pSi with Cobinamide

114

amine groups of Cbi are positive since their pKa is around 10. In this way, the electrostatic interactions favour the absorption for these two type samples.

Figure VII-2. HPLC trace of compounds released from hydroxocobalamin (OHCbi) by UV detection at 168 – 351 nm (a). Optical density at times of 4.82 min (b) and 6.34 min (c).

Additionally, to distinguish effects of surface charge absorption and capillary force on loading capacity of Cbi by pSi, native pSi (without electrostatic charge from primary functionalization) and samples with amine groups, i.e. NH2-pSi and Chitosan-pSi (the same surface charge with Cbi) have been used. The Cbi loaded particles were then washed three times each with water and ethanol by centrifugation and dried in freeze-dryer. Then, the powder was immersed in 1 mL of aqueous phosphate-buffered saline (PBS, pH 7). The supernatant containing released cobinamide was collected at different times and replaced with fresh buffer.

Interaction of pSi with Cobinamide

115

As for the loading capacity and release rate studies, pSi microparticles loaded with cobinamide were immersed in 1 mL of aqueous PBS (pH 7.4) at a particle concentration of 100 µg/mL and agitated at room temperature for 2 h under mild shaking. The supernatant containing released cobinamide was collected at set times and replaced with fresh water or buffer. Concentrations of released cobinamide were determined by adding excess potassium cyanide to the solution to convert cobinamide to the dicyano form and then measuring the absorbance value at 370 nm (e = 30,000 M-1.cm-1). Optical density (absorbance) spectra were recorded on a Thermo Scientific Evolution 201 UV-Visible Double Beam spectrophotometer (Thermo Fisher Scientific Inc.) with 1 cm matched Quartz cells, in a range of wavelengths between 250 and 800 nm. Optical density of samples was analysed for one week, as a reasonable time for drug delivery implement [29]. Finally, in order to check workability of the samples as bioimaging agent, Photoluminescence (PL) measurements were performed by Horiba Jobin-Yvon Nanolog instrument. The configuration setup was as, excitation at 350 nm, 2 nm slit size, 1200 g·mm-1 density grating (blazed at 500 nm), cut-off filtration at 370 nm, and 0.1 second integration time.

7.4 Results and Discussion The surface chemistry of pSi plays a large role in the adsorption of drugs within the porous matrix. Therefore, we first studied the interaction between Cbi and the pSi surface of samples by using UV-Visible spectrophotometer (Figure VII-3). The redox interaction between cobinamide and the silicon matrixes was investigated by this simple method. In fact, the absorbance spectrum of suspensions was utilized to determine whether any redox degradation of cobinamide had occurred. The spectra of the cobinamide solution after it had been exposed to pSi for 3 h revealed significant changes compared to that of the original cobinamide solution. The characteristic peaks for cobinamide are at about 500 and 525 nm (cobalt is in Co3+ state). The cobalt centre of Cbi was in the Co3+ oxidation state initially; however, the appearance of new absorption peak at 450 nm (in the dot curve)

116

Interaction of pSi with Cobinamide

suggested the Co3+ centre was reduced to Co2+ by the pSi. The absorbance of the cobinamide supernatant obtained after the sample had been exposed to pSi for 3 h revealed a complete loss of the lowest-energy absorption band. Such changes suggest substantial alterations, if not complete loss of the cobalt−corrin ring coordination [24].

1.0

ABS (a.u.)

Cbi pre-loading Cbi 3h loading Cbi 24h loading

0.5

0.0 300

400

500

600

700

800

Wavelength (nm)

Figure VII-3. Optical density of Cbi based on combination with the native pSi.

On the other hand, Cbi is stable in contact with pSiO2 in PBS solution. These outcomes are in line with previous observations [14]. Therefore, to protect the loaded Cbi from redox degradation, like pSiO2, the efficiency of different added functional groups (described in the previous Chapters) has been evaluated. Table VII-2 listed reaction times of the different functionalized pSi samples during Cbi decomposition. In this table, the duration to get the maximum intensity of the λ = 470 nm peak recorded as Co3+ → Co2+ transformation (step 1 of decomposition) is reported [21]. Whole Cbi decomposition is relate to the disappearing of the absorption peak. It is worth noting that for all of the samples investigated, there is no Cbi absorption peak at 525 nm after 24 h. In the case of functionalized samples, we can divide experimental results based on the surface charge. Samples with negative charge are interesting; in fact,

117

Interaction of pSi with Cobinamide

carboxyl functionalization leads to significantly reduce the decomposition rates for both steps. More attractively, the PEG functionalization has no effect on the structure of Cbi, such as pSiO2. To the best of our knowledge, redox stability by functionalization has not been reported before.

Table VII-2. Observed times for reaction of Cbi with pSi samples as a function of the different surface functionalization. Step 1: Co3+ → Co2+ reaction; step 2: complete decomposition of Cbi. Samples

Step 1

Step 2

pSi

3h

24 h

pSiO2

NO*

NO

COOH-pSi

10 h

3d

PEG-pSi

NO

NO

PEG2-pSi

NO

NO

NH2-pSi

~ s+

1h

Chitosan-pSi

~s

1h

* No reaction carried out during one week. +

In the order of few seconds.

On the other hand, the reactions with the positively charged pSi samples, NH2pSi and Chitosan-pSi, were similar. We could not determine reaction time of the first step because it was too fast, and with a weak peak at about 470 nm related to the Co2+. Weakness of this peak could be related to the low amount of Cbi loaded. The fast reaction (few seconds) of Cbi with samples containing amine functional groups could be related to the reduction of Cbi by nitrogen impurities as already reported [21]. In fact, based on this report, kinetic rate of this redox is in order of 109 M-1S-1. Afterward, within just about one hour, this small peak was completely

Interaction of pSi with Cobinamide

118

disappeared (reaction Step 2). This suggests that functionalization by amine groups led to a decomposition faster than without any modification. After stability check of the drug itself, one of the major parameter for a candidate drug delivery system is the loading capacity. Different types of functionalization were compared to determine which one provides the larger loading efficiency and more sustained release of cobinamide. The cobinamide loading efficiency was measured by extracting the loaded cobinamide from the pSi microparticle matrix via treatment with a sodium hydroxide solution (pH 9). The basic solution dissolved the Si and SiO2 components of the microparticle, releasing the drug payload into solution. We quantified the cobinamide by converting it into the dicyano form (by adding excess potassium cyanide) and measuring the optical absorbance at 370 nm (e = 30,000 M-1.cm-1). The average cobinamide loadings were 110 ± 15 and 32 ± 2 µg of cobinamide/mg of microparticle for the pSi and pSiO2, respectively. Amount of loading dramatically reduced by oxidation based on less surface area and native surface charge of oxidized pSi. Loading capacity for PEG-pSi and COOH-pSi is 225 ± 18 and 380 ± 32 µg of cobinamide/mg of microparticle, respectively. Indeed, by surface functionalization with PEG and carboxyl loading capacity increased significantly (2 and 5 fold). PEG has less capacity than Carboxyl due to longer chain of PEG, which leads to reduction of pore volume. The loading capacity for PEG2pSi is around native pSi (120 ± 10). Such a huge loading capacity based on electrostatic attraction, particularly for COOH-pSi which is near to 40 %, have not seen before by the oxidation trapping [14] or other methodologies [29]. On the other hand, positive surface charge leads to reduction of loading capacity even less than pSiO2: about 12 ± 2 and 18 ± 1 of cobinamide per mg of microparticle for NH2-pSi and Chitosan-pSi, respectively. Therefore, surface charge, tuneable by functional groups, has huge effect on the loading capacity. We next tested the release of Cbi from the pSi microparticles with different surface functionalization. Results of this experiment are shown in Figure VII-4. If cobinamide was not physically attached, it would be free to diffuse out of the pores even if the silicon matrix was not completely degraded. Consistent with this

119

Interaction of pSi with Cobinamide

argument, we can categorize the release of cobinamide in three different types. The medium release rate related to the native pSi and pSiO2 particles. However, rate of pSi is less based on oxidation trapping of Cbi during oxidation of Si in PBS.

100

pSi pSiO2 COOH-pSi NH2-pSi

Cbi Release (%)

80

PEG-pSi PEG2-pSi Chitosan-pSi

60

40

20

0 0

1

2

3

4

5

6

7

Time (days)

Figure VII-4. First week release profile of cobinamide into PBS from pSi particles with different surface chemistry, that are native pSi (filled cubics); pSiO2 (empty cubics); COOH-pSi (); NH2-pSi (◊); PEG-pSi (); PEG2-pSi (⋆); and Chitosan-pSi (Δ).

The fastest rate is related to negatively charge samples. As it can be seen, release rate of Cbi from pSiO2 was significantly less than from these functionalized particles which are NH2-pSi and Chitosan-pSi. This fast release might be attributed to the electrically desorption of two similar charge transfers. Finally, lowest rate is related to the positively charge samples. Rate of PEG-pSi and PEG2-pSi is slightly less than COOH-pSi, which could be related to the trapping of Cbi in the longer chains of PEG. samples. Cbi has no PL properties in the visible range. Then, PL of loaded samples is just based on pSi. Additionally, we have to avoid native pSi and pSiO2 samples. Because, from the beginning pSiO2 does not have PL properties. In addition, native

120

Interaction of pSi with Cobinamide

pSi quenched during loading of Cbi. On the other side, PL properties of Cbi loaded NH2-pSi and Chitosan-pSi samples were similar with unloaded ones which it could be few amount of loading (less than 2%). In the case of COOH-pSi, this sample has fast quenching in PBS, Figure VII-4.a, which is explained before at description of Figure IV-5.a. As it can be seen in Figure VII-5.b, PL intensity was only slightly reduced by loading Cbi. This stability is attributed to the bonding of Cbi on the surface of COOH-pSi, that works as a barrier for corrosion of silicon of the core structure. Therefore, loading of Cbi has no modification also in view of bioimaging application.

a) 1

b) 1 Initial 3h Int. (a.u.)

Int. (a.u.)

Initial 3h

0 400 450 500 550 600 650 700 750

Wavelength (nm)

0 400 450 500 550 600 650 700 750

Wavelength (nm)

Figure VII-5. a) Variation of PL spectrum of COOH-pSi into PBS in 3 h. b) Variation of PL spectrum of COOH-pSi loaded Cbi into PBS in 3 h.

As for PEG functionalized samples, we could not observe any variation due to Cbi loading in one week. The reason could be the electrostatically attraction of Cbi with PEG-pSi or PEG2-pSi without any combination with silicon of core structure.

7.5 Conclusions In this chapter, the interaction of mesoporous silicon (pSi) with different surface chemistry, with cobinamide has been investigated. Cobinamide has been

121

Interaction of pSi with Cobinamide

selected as a drug for loading into the pSi pores based on high redox sensitively with simple redox detection. Results confirmed that loading capacity and release profile is completely controlled by the surface chemistry. Functionalization with opposite surface charge leads to controlling of the redox in Cbi significantly, for instance: Cbi was stabile into PEG-pSi. Moreover, this functionalization leads to significantly modification on loading capacity and time of release. On the other side, functionalization by amine functions, like diamine and chitosan, leads to faster decomposition and reducing loading capability and faster release. Finally, Cbi loading into carboxyl functionalization leads to improving workability of pSi for bioimaging. On the other hand, Cbi does not have side-effect for emission properties of PEG-pSi. To sum up, functionalized pSi would be a promising candidate for nanotheranostics by having high loading capacity and also bioimaging properties.

7.6 References

1.

M.

Arruebo,

Wiley

Interdisciplinary

Reviews:

Nanomedicine

and

Nanobiotechnology, 4 (1) (2012) 16 – 30. 2.

P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, Nature Material, 9 (2) (2010) 172 – 178.

3.

Q. Fang, J. Wang, S. Gu, R.B. Kaspar, Journal of the American Chemical Society, 137 (26) (2015) 8352 – 8355.

4.

N. Daldosso, A. Ghafarinazari, P. Cortelletti, L. Marongiu, M. Donini, V. Paterlini, P. Bettotti, R. Guider, E. Froner, S. Dusi and M. Scarpa, Journal of Material Chemistry B, 2014, 2 (37) 6345 – 6353.

5.

R. Herino, Pore size distribution in porous silicon, chapter 2.2. In: L.T. Canham (ed) Properties of porous silicon. EMIS DATAREVIEWS series no 18. INSPEC/IEE, London. ISBN: 0 85296 932 5.

122

Interaction of pSi with Cobinamide

6.

A. Correia, M.A. Shahbazi, EM Mäkilä, S. Almeida, J. Salonen, J. Hirvonen, and H. A. Santos, ACS applied Material & interfaces, 7 (41) 23197 23204. –

7.

L. Gu, J. -H. Park, K. H. Duong, E. Ruoslahti and M. J. Sailor, Small, 6 ( 2010) 2546.

8.

T. Laaksonen, H. Santos, H. Vihola, J. Salonen, J. Riikonen, T. Heikkila, L. Peltonen, N. Kumar, D. Y. Murzin, V. P. Lehto, J. Hirvonen. Chem. Res. Toxicol., -

20 (2007) 1913. T. F. Harper, M. J. Sailor, Journal of the American Chemical Society, 119 (1997)

9.

6943. 10

.

E.C. W u, J.S. Andrew, A. Buyanin, J.M. Kinsella, M.J. Sailor, Bio materials, 32 (2011) 1957 1966. –

11.

E. C. Wu, J. S. Andrew, A. Buyanin, J. M. Kinsella, M. J. Sailor , Chemical Communications, 47 (20) (2011) 5699 5701. –

12.

K.L. Jarvis, T.J. Barnes, C.A. Prestid ge, Langmuir, 26 (2010) 14316 14322.

13.

S. Low, L. Canham, N. Voelcker, BioMaterial, 27 (26) (2006) 4538 4546.

14.

N.L. Fry, G.R. Boss, M.J. Sailor, Chemical Material, 26 (2014) 2758 − 2764.

15

A. Ghafarinazari, E. Zera, A. Lion, M. Scarpa, G. D. Sorar u, N. Daldosso,

.





Thermochimica Acta, 623 (2016) 65 - 71. 16.

S.M. Prokes, Applied physics letters, 62 (1993) 25 .

17.

G. Aggarwal, P. Mishra, B. Joshi, S.S. Islam, Journal of Porous Material, 21 (2014) 23 .

18.

F. Peng, Y. Su, Y. Zhong, C. Fan, S.T. Lee, Accounts of chemical research, 47 (2) (2014) 612 623 –

19.

.

M. A. Avidad, A. Nieto, A. Salinas Castillo, L. Capitan Vallvey, G. Miskelly and -

-

M. J. Sailor, Nano. Res. Lett., 9 (1) (2014) 1. 20.

K. Proinsias, M. Giedyk, D. Gryko, Chemical Society Reviews, 42 (2013) 6605 6619.

-

123

Interaction of pSi with Cobinamide

21.

V.S. Sharma, R.B. Pilz, G.R. Boss, D. Magde, Biochemistry, 42 (29) (2003) 8900



8908. 22.

K. E. Broderick, P. Potluri, S. Zhuang, Experimental Biology and Medicine, 231 (5) (2006) 641 649. –

23.

K. E. Broderick, V. Singh, S. Zhuang, A. Kambo, J. C. Chen, V. S. Sharma, R. B. Pilz, G. R. Boss, The Journal of Biological Chemistry, 208 (10) (2005) 8678 8685. –

24.

I. A. Dereven'kov, D. S. Salnikov, S. V. Makarov, R. Silaghi Dumitrescu, G. Boss, -

Journal of Inorganic Biochemistry, 125 (2013) 32 39. –

244.

25

S. H. Ford, A. Nichols, M. Shambee, J. Inorg. Biochem. 41 (1991) 235

26.

J. Lu, M. Liong, Z. Li, J. I. Zink, F. Tamanoi, F. Tamanoi, Small, 6 (16) (2010) 1794

.



27.



1805.

T. Tanaka, L. S. Mangala, P. E. Vivas-Mejia, R. Nieves- Alicea, A. P. Mann, E. Mora, H. D. Han, M. K. Shahzad, X. Liu, R. Bhavane, Cancer research, 70 (9) (2010) 3687

28.



3696.

C. E. Housecroft, A. G. Sharpe, Inorganic Chemistry (3rd ed.). Prentice Hall. ISBN 978 0131755536. Chapter 6: Acids, Bases and Ions in Aqueous Solution. 2008 -

29.

J. Salonen , Drug Delivery with Porous Silicon, in Handbook of Porous silicon, Springer International Publishing, Switzerland, 2014, 909 - 919.

124

Interaction of pSi with Cobinamide

Chapter VII, in part of full, is a reprint (with co-author permission) of the material as it appears in the following publication: A.

Ghafarinazari,

G.

Zoccatelli, M. Scarpa, and N. Daldosso, Comprehensive study on redox activity of functionalized mesoporous silicon on drug delivery and bioimaging, manuscript in preparation.

List of Figures Chapter I Figure I-1. Scheme of nanotheranostics functionality [6].

3

Figure I-2. Number of publications on Porous Silicon, 1990–2015 (Web of Science).

4

Chapter II Figure II-1. Schematic of Silicon wafer etching by increasing the corrosion conditions in MACE method.

11

Figure II-2. SEM image from Si nanostructures production by MACE of p-type silicon wafer. Usage of Ag (a) or Pt (b) as novel metal, before acid washing. Production of nanowire (c) and mesoporous (d) structures.

15

Figure II-3. Effiency for each factor in corrosion of silicon by MACE method. A correspond to novel metal (1: Ag; 2: Pt; and 3: Ag + Pt); B related time duration (1: 10; 2: 60; and 3: 300 min); C related to reaction temprature (1: 27; 2: 45 and 3: 90 ℃) and

D

conrespond

to

H2O2

concentration

M).

(1:

0;

0.15;

and

0.3 16

Figure II-4. SEM of pSi microparticles produced by anodization method (a). Image of the surface porosity (b).

17

Figure II-5. Size distribution of pSi based on image analysis.

17

Figure II-6. FTIR spectrum of pSi just after electrochemical etching. The pSi layer was still attached to the bulk silicon, for this reason the FTIR spectrum was acquired in reflectance mode.

18

Figure II-7. PL properties of pSi dispersed in ethanol under UV radiation (a) and by excitation at 350 nm with spectra photometer.

125

19

List of Figures

126

Chapter III Figure III-1. Scheme of pSi functionalization by carboxyl and amine groups.

25

Figure III-2. Description for the determination of the absolute emission quantum yield proposed by De Mello method.

28

Figure III-3. Example of the determination of the emission quantum yield of a sample using the De Mello’s method.

28

Figure III-4. SEM images of pSi. A representative native pSi just after sonication in anhydrous toluene (a) and a magnification of the porous structure (b). A pSi carrying amino groups on the surface (c) and details of its surface roughness (d).

30

Figure III-5. FTIR measurements of the pSi microparticles after both the functionalization steps: pSi after the functionalization with the NHS ester of acrylic acid (COOH-pSi, continuous trace), and diamine (NH2-pSi, dashed trace). In this experiment, the coupling of the diamine to obtain the NH2-pSi was performed with 200 M diamine concentration and 12 h reaction time.

31

Figure III-6. (a) Solid trace: PLE of COOH-pSi, the emission was 600 nm; dashed trace: PLE of NH2-pSi, the emission was 600 nm; dotted trace: PLE of NH2-pSi, the emission was 420 nm. (b) PL spectra by excitation at 350 nm of COOH-pSi (solid trace) and NH2-pSi (dash trace). (c) Optical density of COOH-pSi a diluted sample. (d) PL of the NH2-pSi sample as a function of the excitation wavelength.

32

Figure III-7. 3D map for correlation of emission and excitation of COOH-pSi (a) and NH2pSi (b) samples.

34

Figure III-8. (a) Blue-shift of the maximum value of the orange band as a function of diamine concentration (after 70 hours from mixing); (b) Variation of the maximum PL intensity (orange and blue peak) as a function of the time after addition of 300 μM diamine. Insets: PL spectra of sample without diamine (continuous trace) and after 70 h incubation with 300 μM diamine.

35

127

List of Figures

Figure III-9. a) Time degradation of the PL for NH2-pSi (9 µg/mL) in water and in PBS solution for the orange and blue emission band. b) PL spectra of NH2-pSi into PBS in different times.

36

Figure III-10. PL decays of the NH2-pSi sample for (a) the blue emission band (excitation wavelength is 375 nm) and (b) the orange emission band (excitation wavelength is 375 37

nm) as a function of the emission wavelength.

Figure III-11. Integrated PL of Rhodamine 101 (Rh 101), Fluorescein, used as standard, and different pSi microparticles to determine quantum yield by comparative method.

39

Chapter IV Figure IV-1. Schematic of the synthesis and functionalization procedures.

50

Figure IV-2. SEM images of COOH-pSi sample in one-micron scale range to show particle dimension (a) and in submicron scale to better point out porosity at the surface (b), PEG-pSi sample in ten-micron scale range (c) with a higher magnification (1-micron scale

range)

in

the

inset,

Chitosan-pSi

in

20-micron

scale(d).

53

Figure IV-3. FTIR measurements of the pSi microparticles before and after surface functionalization and successive coating: native psi (a), COOH-pSi functionalized (b) and surface modified by PEG (c) and Chitosan (d). The baselines of the middle and top

spectra

are

offset

comparison.

from

the

x-axis

for 55

Figure IV-4. Normalized PL spectra (exc. wavelength: 350 nm) of samples dispersed in PBS: COOH-pSi, PEG-pSi, PEG2-pSi and Chitosan-pSi. Chitosan (x10) signal is reported for comparison.

56

Figure IV-5. a) PL intensity vs. time (days) in PBS buffer (“pSi-Chi. Red” and “pSi-Chi. Viol” are the intensity of red and violet band of Chitosan-pSi sample, respectively). b) Optical emission spectra of Chitosan-pSi sample after suspension in PBS as a function of time (after 1 day and up to 90 days).

59

List of Figures

128

Figure IV-6. a) PL spectra of PEG-pSi sample after suspension in PBS as a function of time up to 9 days, then sonication (9 d + S), PL after 6 more days (15 d) and then finally after for the second time sonication (15 d + S). b) photograph of sample after 4 days of suspension. c) photograph of sample under UV radiation after 15 days of suspension in PBS.

60

Figure IV-7. Two-photon absorption microscopy images of COOH-pSi sample by excitation at 700 nm (a) and 810 nm (b) then looking at emission of 550-650 nm.

62

Chapter V Figure V-1. XRD diffraction of the freshly anodized pSi powder; and after oxidation by heating rate of 3 K/min up to 1273 K (pSiO2). The sharp peak at 69.1° (“” at upper panel) is due to the < 4 0 0 > reflection of crystalline Si, while the sharps reflection peaks () at bottom panel suggest the occurrence of α-quartz phase.

75

Figure V-2. SEM images of pSi samples before and after thermal oxidation. a) pSi particles as anodized; b) surface of pSi particles with higher magnification; c) thermally oxidized pSi particles with rate of 3 K/min; and d) surface of thermally oxidized pSi particles. Figure V-3. Conversion vs. temperature at heating rates of 3, 6, and 10 K/min.

76 77

Figure V-4. (a) Natural logarithm of heating rate versus 1000/T of the pSi thermal oxidation at different reaction degree. (b) Variation of E and Ln(A) by conversion obtained from Eq. 3, Est.() is the E value obtained by the Starink method (c) The dependence of Ln(A) with E.

79

Figure V-5. F(α) derived from the Equation 2 for thermal oxidation of pSi.

80

Figure V-6. DTA, DTG, and E derived from heating rate of 10 K/min.

84

129

List of Figures Chapter VI

Figure VI-1. Emission spectra of functionalized pSi. The solid trace is for the COOH-pSi and the dashed trace for the NH2-pSi. Left panel: excitation 350 nm. The sharp peak at 700 nm wavelength in the NH2-pSi sample is related to the second order of the spectrometer

grating.

Right

panel:

excitation

nm.

405 97

Figure VI-2. Cell viability of human dendritic cells. DCs were treated with the indicated doses of COOH- and NH2-pSi for 24 hours. The incubation volume was 1 mL. The results

are

the

mean

±

SD

of

experiments.

three 98

Figure VI-3. Analysis of cytokine production by DCs treated for 24 hours with different doses of NH2-pSi or LPS used as the positive control. The results are the mean ± SD of three experiments.

99

Figure VI-4. Confocal microscopy images of DCs treated with 500 μg of NH2-pSi (in green). After 24 h incubation the cells were fixed and stained with Phalloidin to label filamentous F-actin (red). One representative experiment of three is shown. (A) Internal

z-stack

of

general

field

and

(B)

a

(A).

zoom-in

of 100

Figure VI-5. Confocal microscopy analysis of NH2- and COOH-pSi ingested by DCs. The excitation is set at 405 nm and the emission collected in the green (left panel) and red (right panel) region.

101

Figure VI-6. Emission spectrum of COOH–pSi (left) or NH2-pSi (right) with DCs, in PBS before and after centrifuge.

102

Figure VI-7. Confocal microscopy images of an NH2-pSi. Excitation at 405 nm. Left top: emission collected in the range 500–550 nm; right top: emission collected in the 600– 650 nm; left bottom: bright field image; right bottom: merged image.

102

Figure VI-8. PL emission from an NH2-pSi particle. Excitation at 488 nm.

103

130

List of Figures Chapter VII

Figure VII-1. Structure of Cobinamide (C48H72CoN11O8) when it is dissolved in neutral aqueous solutions, with hydroxide and water molecules bound in the axial positions (aquohydroxocobinamide).

112

Figure VII-2. HPLC of compounds from hydroxocobalamin (OH-Cbi) by UV at 168– 351nm (a) at times of 4.82 (b) 6.34 min (c). Figure VII-3. Optical density of Cbi based on combination with the native pSi.

114 116

Figure VII-4. First week release profile of cobinamide into PBS from pSi particles with different surface chemistry, that are native pSi (filled cubics); pSiO2 (empty cubics); COOH-pSi ();

NH2-pSi (◊); PEG-pSi (); PEG2-pSi (⋆); and Chitosan-pSi

(Δ).

119

Figure VII-5. a) Variation of PL spectrum of COOH-pSi into PBS in 3 h. b) Variation of PL spectrum of COOH-pSi loaded Cbi into PBS in 3 h.

120

List of Tables

Chapter II Table II-1. Experimental design of Si nanostructure synthesis by MACE and corresponded mass of samples (mi).

13

Chapter III Table III-1. Φ values determined by de Mello method.

40

Chapter VI Table IV-1. Summary of samples functionalization.

51

Table IV-2. Optical quantum yields obtained by comparative method.

57

Chapter V Table V-1. Reaction types and corresponding type of f(α).

74

Table V-2. Arrhenius parameters by model fitting for stage 1 (α  37%).

82

Table V-3. Arrhenius parameters by model fitting for stage 2 (α  37%).

83

Chapter VII Table VII-1. Different functionalization of pSi.

113

Table VII-2. Observed times for reactions of Cbi with pSi samples. Step 1: Co3+ → Co2+ reaction; step 2: whole decomposition of Cbi.

131

117

Conclusions of the Dissertation

First, we focused to obtain a photoluminescent (PL) pSi from silicon wafer. Therefore, metal assistant chemical etching and anodization methods have been used; which anodization had promising results to get a light emitting nanostructure. Then, the fabricated luminescent pSi structures were modified by both thermal oxidation and organic functionalization. In the case of thermal oxidation, kinetics of reaction established in details, by model free kinetics, for the first time. But it did not lead to PL stability; t hen, organic functionalization has been utilized for further activities. Carboxyl function leads to stability in ethanol for years, and also amine addition leads to different surface charge and blue emission. Further modification with biopolymers, such as PEG, leads to PL stability in biological solutions for months. To the best of my knowledge, this long stability has not yet reported before. In view of biomedical applications, first, interaction of pSi with human cells confirms that the pSi is inert without immune response. Interestingly, the cells uptake pSi and PL of pSi observed from inside the cell by a conventional confocal microscopy. On the other side, the main challenge for usage of pSi in drug delivery is the redox activity of pSi. This phenomenon was investigated comprehensively. Polymeric functionalization works as a barrier for reduction of drug; moreover, increased loading amount and release time significantly. To sum up, pSi is a promising material for nanotheranostics based on having biocompatibility, bioimaging, and drug delivery. Particularly, by a suitable functionalization, these properties could be improved.

132