Quantum Dots-capped hybrid materials for pH triggered release / Author Carolin Hörhager

Author

Carolin H¨orhager Submission Institut f¨ur

Chemie der Polymere Thesis supervisor Univ.-Prof. Dr. Oliver Br¨uggemann Assistant thesis supervisor Dr. Yolanda Salinas Soler October 2016 JOHANNES KEPLER UNIVERSITY LINZ Altenbergerstraße 69 4040 Linz, ¨Osterreich www.jku.at

Quantum

Dots-capped

hybrid materials for pH

triggered release

Master’s Thesis

to confer the academic degree of

Diplom-Ingenieurin

im in the Masters Program

STATUTORY DECLARATION

I hereby declare that the thesis submitted is my own unaided work, that I have not used other than the sources indicated, and that all direct and indirect sources are acknowledged as refer-ences.

This printed thesis is identical with the electronic version submitted.

Linz, October 2016

Carolin Hörhager This work was developed in the period from February to September 2016 at the Institute of Poly-mer Chemistry at the Johannes Kepler University Linz under the supervision of Dr. Yolanda Salinas Soler and Univ.-Prof. Dr. Oliver Brüggemann.

EIDESSTATTLICHE ERKLÄRUNG

Ich erkläre an Eides statt, dass ich die vorliegende Masterarbeit selbstständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt bzw. die wörtlich oder sinngemäß entnommenen Stellen als solche kenntlich gemacht habe.

Die vorliegende Masterarbeit ist mit dem elektronisch übermittelten Textdokument identisch.

Linz, Oktober 2016

Carolin Hörhager Diese Arbeit entstand in der Zeit von Februar bis September 2016 am Institut für Chemie der Polymere der Johannes Kepler Universität Linz unter der Betreuung von Dr. Yolanda Salinas Soler und Univ.-Prof. Dr. Oliver Brüggemann.

CURRICULUM VITAE

PERSONAL INFORMATION

Date of Birth: 01 September 1991 Place of Birth: Innsbruck

Nationality: Austria

Parents: Waltraud and Gerhard H¨orhager E-Mail: h carolin@hotmail.com

EDUCATION

Since 2014: Master studies

”Wirtschaftsingenieurwesen Technische Chemie“, JKU Linz 2010 to 2014: Bachelor studies

”Technische Chemie“, JKU Linz 2002 to 2010: Bisch¨ofliches Gymnasium Paulinum

1998 to 2002: Primary school

OCCUPATIONS

Since January 2016: Swarovski Optik KG, Absam March 2016 to June 2016: Tutor at

”Praktikum aus Anorganischer Chemie“ November 2015 to January 2016: Tutor at

”Praktikum aus Allgemeiner Chemie“ 01 September to 30 September 2015: Swarovski Optik KG, Absam

04 November 2014 to 23 January 2015: Tutor at

”Praktikum aus Allgemeiner Chemie“ 15 July to 15 September 2014: Institute IMDEA for Materials, Madrid

22 July to 30 August 2013: Papierfabrik Wattens GmbH & Co KG

30 July to 03 August 2012: DSM Fine Chemicals Austria Nfg GmbH & CoKG, Linz 04 July to 28 August 2011: Sandoz GmbH, Kundl

05 July to 27 August 2010: Papierfabrik Wattens GmbH & Co KG 13 July to 07 August 2009: Papierfabrik Wattens GmbH & Co KG 07 July to 01 August 2008: Papierfabrik Wattens GmbH & Co KG 09 July to 03 August 2007: Papierfabrik Wattens GmbH & Co KG

Danksagung

Meinen Dank richte ich ganz besonders an Dr. Yolanda Salinas Soler für die Möglichkeit der Ausführung meiner Masterarbeit, die ausgezeichnete Betreuung, das tolle Arbeitsklima und die Expertise. ¡ Muchas Gracias! Darüber hinaus geht mein Dank an Univ.-Prof. Dr. Oliver Brügge-mann, Institutsvorstand des Instituts für Chemie der Polymere (ICP), für die Unterstützung und sehr gute Betreuung während der Masterarbeit. Auch bei Mag. Martin Ertl, DI Daniela Otte und Assoz. Univ.-Prof. Dr. Uwe Monkowius , Institut für Anorganische Chemie (CNPS), möchte ich mich ganz herzlich für die Verwendung des Fluoreszenzspektrometers, die Geduld und die fachliche Unterstützung bedanken. Weiters danke ich Ramona Kiss für die BET Messungen, Andreas Schnölzer für die Betreuung und Instandhaltung der verwendeten Geräte, ZONA für die TEM Analysen und Assoc. Univ.-Prof. Dr. Wolfgang Schöfberger, Institut für Organische Chemie, für die NMR Messungen. Auch ein großer DANK gilt den ICP Mitarbeitern für die praktischen Tips und Tricks, die vielen lustigen Momente und Ausflüge und dafür, dass ich jeden Tag gerne zur Arbeit ging! :)

Nun ist es aber auch einmal an der Zeit mich bei all denjenigen zu bedanken, die mich während der Studienzeit unterstützt und begleitet haben.

Daher gilt zunächst mein ganz besonderer Dank an meine Eltern, die mir mein Studium er-möglicht haben und mich in jeder Hinsicht immer unterstützt haben.

Dann möchte ich mich bei meinen Grömer‘s bedanken, die immer aber ganz besonders in den schlechten Zeiten in Linz für mich da waren und mich in ihrer herzlichen Familie wie ihr eigenes Kind aufgenommen haben.

Meinen besten Freunden Anna Seidl, Magdalena Schindl, Melanie Lubinger, Thomas Wieselthaler, Christoph Steinlechner, Verena Hagn, Anna Zemann und Maria Hitzenberger, meiner Bibliotheks-Mami Christina Keferböck und meinen Mitbewohnern danke ich für die innige Freundschaft in den letzten 6 Jahren, die Gespräche und ihr Einfühlungsvermögen. Meiner Schwester Claudia Hörhager und meinem Freund Christoph Strassmayer danke ich besonders für den allgegenwärti-gen starken emotionalen Rückhalt. Ich danke euch allen ausdrücklich für die aufbauenden Worte in den schwachen Tagen, eure Aufmerksamkeit und eure Zeit, in denen wir unzählige schönen Momente in den letzten Jahren nicht nur an der Uni sondern ganz besonders in meinem geliebten Studentenheim und abends an der USI erlebt haben.

An Christoph Steinlechner und Stefan Humer richte ich meinen Dank vor allem in fachlicher und freundschaftlicher Hinsicht. Natürlich richtet sich dies auch an alle anderen Studienkollegen mit denen ich die letzten Jahre den Weg gemeinsam ging.

Abstract

This master thesis proposes the preparation of a hybrid "gate-like" system based on MCM-41 nanoparticles as inorganic scaffold. The pores of the material serve as host for guest molecules, such as dye or anticancer drugs, and the surface of MCM-41 is capable of a chemical reaction by surface functionalization with Quantum Dots, closing the pores.

The initial idea was to functionalize the silica nanoparticles, previously uploaded on the pores with a dye, via grafting with a linker unit (N -(3-Triethoxysilylpropyl)gluconamide) between the silica surface and the Quantum Dots. The dye used in this thesis was Safranin O. Release Studies at different pHs of this first material showed unsatisfactory results. Therefore, a con-trol material was prepared by only uploading the MCM-41 silica nanoparticles with the dye. After extensive characterization of this control material, delivery studies of the Safranin O (at pH3, 5 and 7) showed expected good results, where pH does not affect the release kinetics. Consequently, the silica surface was functionalized directly with Quantum Dots. For this second hybrid material different ZnS Quantum Dots were prepared and characterized (manganese doped and not doped ones). These Quantum Dots were capped with both, 1-thioglycerol (TG) and 4-mercaptophenylboronic-acid (B) in order to increase their photostability and photochemical properties. Only ZnS Quantum Dots capped with 4-mercaptophenylboronic-acid (ZnS@B) were selected for the preparation of the final "gate-like" system. The gate mechanism involves the reaction between the alcohol groups of the silica surface scaffold via reversible boronate ester bond with the boronic acid from the selected Quantum Dots (ZnS@B). The release studies for this final solid were performed in aqueous solution triggered by the external stimulus, pH. The exposition of the system to acidic pH, such as pH3, induces the hydrolysis of the boroester groups resulting in a rapid release of the guest molecule, Safranin O, from the pores to the sur-rounding aqueous solution. In less acidic environment, the guest molecule delivery is inhibited. At pH7 it was observed a negligible release, showing the promising behavior of the system. As characterization methods standard techniques were applied, such as TGA, nitrogen adsorption-desorption isotherms, TEM, FTIR, UV-Vis, DLS and fluorescence spectroscopy for the prepared materials.

This kind of drug delivery system was shown to provide excellent new platforms driven by easy to apply pH triggered response theranostic tool for future application in nanomedicine, as the general idea was to prove this concept for detection and treatment of cancer. The possibility to use manganese doped Quantum Dots instead of basic ZnS based ones could enhance the applicability for in vivo imaging.

Zusammenfassung

Diese Diplomarbeit beschäftigt sich mit der Herstellung eines hybriden „Schranken-ähnlichen“ Systems basierend auf MCM-41-Nanopartikel als anorganisches Gerüst. Die Poren des Materials dienen als Gastgeber für die Gastmoleküle, wie zum Beispiel Farbstoffe oder Krebsmedikamente, und an der Oberfläche von MCM-41 besteht die Möglichkeit durch chemische Reaktionen eine Funktionalisierung mit Quantum Dots durchzuführen, welche die Poren schließen.

Die Ausgangsidee ist eine Funktionalisierung der Silika Nanopartikeln über Veredelung mit einer Verknüpfungseinheit (N -(3-Triethoxysilylpropyl)gluconamide) zwischen der silikatischen Ober-fläche und den Quantum Dots, wobei die Poren zuvor mit Farbstoff beladen werden. Der verwen-dete Farbstoff ist Safranin O. Freisetzungsuntersuchungen von diesem ersten Material bei unter-schiedlichen pH Werten lieferten nicht zufriedenstellende Ergebnisse. Dies macht die Herstellung eines Kontrollmaterials notwendig, bestehend aus MCM-41 Silika Nanopartikel beladen mit den Gastmolekülen Safranin O. Nach umfangreicher Charakterisierung des Kontrollmaterials, zeigen Freisetzungsuntersuchungen von Safranin O (bei pH3, 5 und 7) gute Resultate, denn der pH Wert der Lösung beeinflusst die Kinetik der Freisetzung nicht. Resultierend daraus, wird die Silika Oberfläche direkt mit Quantum Dots funktionalisiert. Für das zweite hybride System wer-den verschiewer-dene ZnS Quantum Dots vorbereitet und charakterisiert (Mangan dotierte und nicht dotierte). Die Quantum Dots werden umhüllt von 1-thioglycerol und 4-mercaptophenylboronic-acid, um die Fotostabilität zu erhöhen und die photochemischen Eigenschaften zu verbessern. Nur ZnS Quantum Dots umhüllt mit 4-mercaptophenylboronic-acid (ZnS@B) werden für die Herstellung des finalen „Schranken-ähnlichen“ Systems verwendet. Der Schranken-Mechanismus beruht auf der reversiblen Reaktion zwischen den Alkoholgruppen der silikatischen Oberflächen des Grundgerüsts und einer Boronatester Bindung mit der Borsäure des ausgewählten Quan-tum Dots (ZnS@B). Freisetzungsuntersuchungen des finalen Systems wurden in wässriger Lö-sung durchgeführt und durch pH als externen Stimulus gesteuert. Die Aussetzung des Systems in saure pH Umgebung, beispielsweise pH3, induziert die Hydrolyse der Boroestersgruppen. Dadurch ergibt sich eine schnelle Freisetzung des Gastmoleküls, welches aus den Poren in die umgebende wässrige Lösung austritt. In weniger saurer Umgebung wird die Freisetzung des Gast-moleküls gehemmt. Bei pH7 wird eine vernachlässigbare Freisetzung beobachtet wodurch sich eine vielversprechende Eigenschaft des Systems andeutet. Verwendete Charakterisierungsmetho-den für die hergestellten Materialien sind Standardtechniken, wie beispielsweise TGA, Stickstoff Adsorptions- Desorptions-Isothermen, TEM, FTIR, UV-Vis, DLS und Fluoreszenzspektroskopie. Dieser Typus eines Wirkstoffabgabesystems stellt sich als hervorragende neue Plattform her-aus, zumal pH Reaktionen mühelos anwendbar sind und als gut kontrollierbares theranostis-ches Werkszeug für zukünftige Anwendungen im Bereich der Nanomedizin dienen könnten. Die Möglichkeit, Mangan dotierte Quanten Dots anstelle von einfachen ZnS QDs zu verwenden, würde die Anwendbarkeit für in-vivo-Bildgebung verbessern.

Abbreviations and Acronyms

B 4-mercaptophenylboronic-acid BET Brunauer–Emmett–Teller BJH Barrett-Joyner-Halenda

CNPS Institut für Anorganische Chemie- Center for Nanobionics and Photochemical Sciences CTABr N -Cetyltrimethylammonium-bromide

d-QD doped Quantum Dot DLS Dynamic Light Scattering DMF Dimethylformamide DNA deoxyribonucleic acid

ESI-MS Electrospray ionization mass spectrometry FRET Förster Resonance Energy Transfer

FTIR Fourier Transform Infrared Spectroscopy f.fl. for fluorescence

gluconamide N -(3-Triethoxysilylpropyl)gluconamide GSH gluthathione

IC Internal conversion

ICP Institute of Polymer Chemistry ISC Intersystem crossing

IUPAC International Union of Pure and Applied Chemistry MCM-41 Mobil Composition of Matter No. 41

MCM-48 Mobil Composition of Matter No. 48 MCM-50 Mobil Composition of Matter No. 50 NaOH sodium hydroxide

NMR Nuclear Magnetic Resonance Spectroscopy NPs nanoparticles

p.a. pro analysis

PBS phosphate-buffered saline

PMOs Periodic mesoporous organosilicas QD Quantum Dot

QY Quantum Yield R Organic Residue S singlet

TEM Transmission Electron Microscopy TEOS tetraethylorthosilicate

TGA Thermogravimetric analysis TG 1-thioglycerol

T triplet

Overview of prepared materials

Table 1 – Overview of shortcuts of prepared materials.

shortcut type product characterization

CH01A MCM-41 silica mesoporous NPs TGA, Ads-Des. Isoth. CH01B MCM-41 silica mesoporous NPs Ads-Des. Isoth., DLS CH01C MCM-41 silica mesoporous NPs Ads-Des. Isoth., TEM, DLS CH01D MCM-41 silica mesoporous NPs Ads-Des. Isoth., DLS CH01G MCM-41 silica mesoporous NPs TGA, Ads-Des. Isoth., DLS

CH02 d-QD Mn:ZnS@TG FTIR, UV-Vis, DLS, fluorescence

CH03 QD ZnS@TG FTIR, UV-Vis, DLS, fluorescence

CH05 d-QD Mn:ZnS@TG:B (9:1) FTIR, UV-Vis, DLS, Fl. CH06 d-QD Mn:ZnS@TG:B (7:3) FTIR, UV-Vis, DLS, Fl. CH010 d-QD Mn:ZnS@TG:B (5:5) FTIR, UV-Vis, DLS, Fl. CH011 d-QD Mn:ZnS@TG:B (3:7) FTIR, UV-Vis, DLS, Fl. CH013 QD ZnS:B in DMF TGA, FTIR, DLS CH014 d-QD ZnS:Mn@B in DMF FTIR CH016 QD ZnS FTIR

CH017A S1 MCM-41+ Safranin O+ Glucon. TGA, Ads-Des. Isoth., UV-Vis CH017B S1 MCM-41+ Safranin O+ Glucon. TGA, UV-Vis

CH017C S1 MCM-41+ Safranin O+ Glucon. TGA, UV-Vis

CH017D control MCM-41+ Safranin O+ Glucon. TGA, Ads-Des. Isoth.,UV-Vis

CH017E S1 MCM-41+ gluc TGA, MS, NMR

CH017F S1 MCM-41+ Safranin O+ Glucon. TGA, Ads-Des. Isoth., UV-Vis CH017G S1 MCM-41+ Safranin O+ Glucon. TGA, Ads-Des. Isoth., UV-Vis CH017H control MCM-41+ Safranin O TGA, Ads-Des. Isoth.

CH017I S1 MCM-41+ Safranin O+ Glucon. TGA, Ads-Des. Isoth., UV-Vis CH018A S2 CH017D+ ZnS:B (100:1) TGA, Ads-Des. Isoth., FTIR, UV-Vis, DLS CH018B S2 CH017D+ ZnS:B (100:2) TGA, Ads-Des. Isoth., FTIR, UV-Vis, DLS CH018C S2 CH017D+ ZnS:B (100:4) TGA, Ads-Des. Isoth., FTIR, UV-Vis

Contents

I

General Introduction

1

1 Aim of the project 1

2 Porous materials 2

2.1 Mesoporous materials . . . 4

2.1.1 General concepts about mesoporous silica material . . . 5

2.1.2 Synthesis of mesoporous materials . . . 5

2.1.3 The incorporation of functionalities . . . 7

2.1.4 Mobil Composition of Matter No. 41 (MCM-41) and its characterization 9 2.1.5 Molecular gates and triggered release . . . 9

3 Quantum Dot (QD) 12 3.1 ZnS QDs and their Characterization . . . 12

3.2 Doped semiconductor nanocrystals Quantum Dots (d-QDs) . . . 13

3.2.1 Synthesis of doped Quantum Dots . . . 14

4 Fluorescence Spectroscopy 15 4.1 Fluorescence and Phosphorescence . . . 15

4.2 Quantum Yield (QY) . . . 16

4.3 Fluorescence Quenching effect . . . 16

II

Experimental section

18

6.2 Nitrogen adsorption-desorption isotherms (BET and BJH model) . . . 20

6.3 Transmission Electron Microscopy (TEM) . . . 20

6.4 Fourier Transform Infrared Spectroscopy (FTIR) . . . 20

6.5 Ultraviolet–Visible spectroscopy (UV-Vis) . . . 20

6.6 Dynamic Light Scattering (DLS) . . . 20

6.7 Fluorescence spectroscopy . . . 20

6.8 Release Studies . . . 21

7 Design and preparation of the hybrid materials 21 7.1 Preparation and characterization of mesoporous MCM-41 NPs . . . 21

7.2 Preparation and characterization of Quantum Dots . . . 22

7.2.1 Undoped QDs preparation . . . 22

7.2.2 doped QDs preparation . . . 24

7.3 Preparation and characterization of the proposed controlled release „gate- like“ sys-tem . . . 26

III

Results and Discussion

30

8.2 Nitrogen adsorption-desorption isotherms . . . 30

8.3 TEM . . . 33

8.4 DLS . . . 33

9 Characterization of Quantum Dots 34 9.1 TGA . . . 34

9.2 FTIR . . . 34

9.3 UV-Vis . . . 38

9.4 DLS . . . 38

9.5 Fluorescence spectroscopy . . . 39

10 MCM-41 NPs functionalized with gluconamide and uploaded with Safranin O (S1) 44 10.1 TGA . . . 44

10.2 Nitrogen adsorption-desorption isotherms . . . 45

10.3 UV- Vis calibration curve . . . 47

10.4 Release Studies . . . 48

11 MCM-41 NPs uploaded with Safranin O (control material) 52 11.1 TGA . . . 52

11.2 Nitrogen adsorption-desorption isotherms . . . 52

11.3 Release Studies . . . 54

12 MCM-41 NPs uploaded with Safranin O and functionalized with QD (S2) 55 12.1 TGA . . . 55

12.2 Nitrogen adsorption-desorption isotherms . . . 55

12.3 FTIR . . . 58

12.4 DLS . . . 58

13 Conclusion und Prospects for further work 61

1 Aim of the project

Part I

General Introduction

1

Aim of the project

In the last century, interest for detection and treatment of diseases increased, especially great effort in the approach of efficient cancer treatment is made. Combining detection and treatment, known as theranostic, is a fairly new concept of highly innovated nanomedicine enabling the drug release in the area of the body where it is required. In this sense inorganic silica nanoparticles have been selected as a drug delivery scaffold as they provide excellent characteristics such as high thermal stability, easy functionalization of the external surface and biocompatibility. Fur-thermore the no-chemical-modification of the drug brings big advantages, as it can be uploaded directly into the pores of the silica scaffold, and the easy surface modification with "molecular gates" gives the possibility to control the release of the drug. Therefore the scientific challenge of this project is to develop a new diagnostic system based on pH triggered response.

The objectives of this project are:

• To prepare and characterize different Quantum Dots coated with suitable capping agents. • To synthesis and characterize mesoporous silica nanoparticles MCM-41, selected as

inor-ganic scaffold.

• To design, to prepare and to characterize the hybrid organic- inorganic "gate-like" system. • To study the potential release of Safranin O (selected dye) at different pH (pH3, 5 and 7)

from the previous prepared materials

Schematic representation of the "gate-like" system is shown in Figure 1.

2 Porous materials

2

Porous materials

A solid material that contains holes, gaps and channels is defined as porous. A lot of natural materials like wood, cork and bone consist of different porous structures. These materials are built up by complex compositions but they are formed like that due to stability reasons. Porous material guarantees high stability by using small amount of solid material. The materials can either be characterized by its pores or by the walls of the pores.[1]

Figure 2 – Difference in pore types.[1]

A general distinction is made between open and close pores. Open pores like (b), (c), (d), (e) and (f) do have access to its surrounding environment, whereas closed ones like (a) are completely isolated (Figure 2). Although closed pores do not show any effect in chemical reactions, they do have influence on the macroscopic characteristics, such as weight, stability and ductility of a material. Open pores are called "blind" if they are open at just one end (case (b)). "Through pores" is the name for pores that offer different ways to be passed (case (e)). The classification of a pore can also be done by its shape, for example: (c) and (f) have a cylindrical shape, (d) is a funnel shaped pore and (b) is called ink-bottle shaped pore. Furthermore, a solid material can be characterized by its roughness (case (g)). Additional characteristics of a solid porous material are the pore diameter, high porosity and surface area.[1]

Nowadays, techniques are developed to foam not only polymers but also ceramics, glasses and also metals. Materials like that are used in industrial applications for insulation, catalysis or for attenuation. The applicability of porous solid materials is highly depending on its physical properties (e.g. density, strength and size) and in chemical applications the pore size, the surface area and the reactivity is very important.[1]

The pore size is the most influencing factor for most applications, and it was defined by Inter-national Union of Pure and Applied Chemistry (IUPAC)[2] as Micro-, Meso- or Macropores. According with the pore size, a different transport mechanism occurs (Figure 3).[1]

2 Porous materials

a) Micropores: Pores with a diameter less than 2 nm. Molecules that pass these pores are about the size of the pore itself. Therefore permselectivity due to molecular size and a specific interaction is possible. The main mechanism of transport through this pore is the activated one.[1]

b) Mesopores: Pore diameters between 2 to 50 nm. The pores are generally the same or higher order of the mean free path. Due to that, the main transport mechanisms follow Knudsen diffusion and surface diffusion. Capillary transport can also occur.[1]

c) Macropores: Pore diameter of more than 50 nm. When transport through macropores is mediated by viscous flow and bulk diffusion, in case of applied pressure gradients. The permselectivities is very low due to its large pore diameter.[1]

Figure 3 – Different mechanisms of transport through the pores.[1] a) Micropores, b) Mesopores and c) Macropores.

2 Porous materials

2.1

Mesoporous materials

Mesoporous materials are porous solids with an ordered porosity, refers to a regular arrangement of the pores with a narrow distribution of the pore size.[1]

Mesoporous silicate materials called M41S were discovered in 1992 by the Mobil Research and Development Corporation[3] by their Scientists, based on the knowledge of zeolites, due to grow-ing demand and higher expectations towards this kind of materials in industrial and research applications. The synthesis of these materials is done with supramolecular arrangements of surfactant micelles in order to reach the highly ordered structure with the large pore diameter that offers the mesoporous silica material (see Figure 4(a)). The surfactant presents amphiphilic behavior and determines the resulting pore size. The modification of the surfactant in the syn-thesis preparation represented a major evolution from zeolites, as they are synthesized through use of existing templates of single solvated compounds with an organic structure, or with shape determining metal ions.[1]

(a) (b)

Figure 4 – (a) General structure of an amphiphilic surfactant.[1] (b) Phase diagram of surfactant concentration in water and resulting shape.[1]

2 Porous materials

2.1.1 General concepts about mesoporous silica material

Generally mesoporous silica materials offer: highly ordered pore structure[4], high temperature stability[4], clear pore radius distribution[4], amorphous pore walls[5] and a high surface area with a homogeneous distribution of functional groups and good functionalization abilities[4]. The material is biocompatible[4], it can be produced at low cost and it further belongs just like metal oxides (e.g. SiO2, T iO2), layered silicates, semiconductors and metallic nanoparticles

like gold and copper to the group of inorganic nanofillers[6]. These materials present very large specific surface areas (between 700 to 1000 cm3_g−1_{) and the pore volume reaches up to}

1 cm3_g−1_{.[7] Their application is of great interest in different areas like ion exchange, catalysts or}

its support material, sensing and adsorption.[7, 8] Furthermore, they provide opportunities for various biomedical usages, such as bioimaging for diagnostics, scaffold engineering, drug delivery and bone repair.[9]

2.1.2 Synthesis of mesoporous materials

The synthesis of the mesoporous silica material by using a template is carried out in three steps: the formation of the template; followed by the fixation of the template; and finally the organic template elimination.

First, the arrangement of the surfactant takes place, where the correct surfactant selection and its concentration determine the shape and size of the formed template. N -Cetyltrimethylammonium-bromide (CTABr) for example, forms liquid crystalline micelles minimizing the number of un-wanted interactions from the hydrophobic tail in water. However, as shown in Figure 4(b), at higher concentration it can form lamellar or hexagonal cylindrical shaped packing liquid crystals.[1, 10]

In the second step called sol-gel-Method, the inorganic siliceous precursor (for example tetraethy-lorthosilicate (TEOS)) is polymerized around the surfactant micelles, in aqueous solution[11], and due to hydrolysis and condensation, it frames the shaped surfactant. Traditionally sol-gel processing is used to form aluminosilicates and mesoporous silica materials. The process graph-ically shown in Figure 5, starts with the sol formation, followed by sol gelation and ending up in the solvent removing step:[12] A colloidal suspension consisting solid precursor components in solution is called "sol".[13] Commonly, the precursors used are metal- and organometallic com-pounds, for example Zr(IV)-Propoxide, Ti(IV)-Butoxide or TEOS.[12, 13] The hydrolysis step (Reaction 1) and water or alcohol condensation step (Reaction 2, 3) form the shape, followed by clusters of the surfactant.[12]

2 Porous materials

M−O−R + H₂O −−→ M−OH + R−OH (1)

M−OH + HO−M −−→ M−O−M + H₂O (2)

M−O−R + HO−M −−→ M−O−M + R−OH (3)

M represents Si, Zr or Ti.[12] The polymerization of the compounds to clusters succeeding to hydrolysis and, if one big cluster is formed out of all the smaller ones, a gel is produced. At this stage the material is an elastic solid without fluidity. However, the chemical reactions carry on (after the gel point) increasing stiffness and strength of the gel, known as "aging". The product, after drying, can either result as xerogel or aerogel, depending on the conditions and the resulting interactions. Xerogels develop during drying process under normal conditions. In this process, capillary forces raise, causing shrinking effects of the clusters. So the initial volume of the wet material is about a factor of 5 to 10 bigger than the resulting solid. During drying step in an autoclave under critical conditions, the so called supercritical drying process is performed and the aerogels are formed. In this step, no capillary pressure arises, thus shrinking behavior does not take place. Aerogels and xerogels are interesting as catalysts due to their high surface area and high porosity.[13]

Figure 5 – Sol-Gel Process.[13]

The last part of the synthesis is the removal of the surfactant by solvent extraction, calcination, oxygen plasma treatment or supercritical drying. For electrostatically templated materials, sol-vent extraction is typically used. But if covalently bonded material must be removed, calcination is currently the most applied procedure.[1, 10] During calcination processes, the samples are heated up under aerobic conditions (500 to 600◦C) for at least 4 h, the organic template is oxi-dized and turns into CO2 and water, and so surfactant is eliminated, letting the pores empty.[14]

2 Porous materials

MCM-41 silica material belongs to the M41S mesoporous family.[3] The material is classified as a xerogel.[15] The whole pathway synthesis is shown in Figure 6. Depending on the mesoporous silicate reaction conditions, three different phases were obtained: Mobil Composition of Matter No. 41 (MCM-41) has a hexagonal pore arrangement, Mobil Composition of Matter No. 48 (MCM-48) is a cubic structure and Mobil Composition of Matter No. 50 (MCM-50) is built up in lamellar silica panels filled with surfactant.[11]

Figure 6 – Pathway of the synthesis of mesoporous silica materials. Reprinted with mission from „The discovery of mesoporous molecular sieves from the twenty year per-spective“, from Charles T. Kresge and Wieslaw J. Roth. Copyright(2013) Royal Society of Chemistry.

2.1.3 The incorporation of functionalities

Functionalities can be incorporated into the mesoporous materials following three approaches (see Figure 7). These techniques enable the combination of the variety of organic functionaliza-tion in the same system and the stability of inorganic material.[14]

a) Grafting or post-synthetic functionalization: This process functionalizes the inner surface of the mesoporous silica material with organic functional groups subsequently to calcina-tion. Generally organosilanes (RO)3SiR, chlorosilanes ClSiR3 and silazanes HN (SiR3)3

react with the existing silanol groups on the surface. A wide range of functionalities can be attached by varying the Organic Residue (R), keeping the original silica phase, whereas the porosity of the cell.[14]

b) Co-Condensation or direct functionalization: This method refers to both the condensation of the silica and the functionalization taking place at the same time. Therefore, this type of functionalization is also called "one-pot" synthesis and the organic residues are embedded

2 Porous materials

and more evenly distributed on the surface. A disadvantage is that the mesoscopic order or the resulting product decreases in case of using a high concentration of organosilanol groups.[14]

c) Periodic mesoporous organosilicas (PMOs): Organic groups are incorporated as bridging compounds into the inorganic system of the silica matrix and the organic component is homogeneously spread in cell walls. These systems are obtained as aerogels and reach surface areas up to 1800 m2_g−1_{. PMOs have a high thermal stability but a drawback of}

this system is the large pore radius distribution.[14]

(a) Grafting. (b) Co-Condensation.

(c) PMOs.

Figure 7 – Functionalization approaches of mesoporous silica material. (a) Grafting, (b) Co-Condensation and (c) PMOs. R presents the organic functional group in (a) and (b) and the organic bridge in (c).[14]

2 Porous materials

2.1.4 MCM-41 and its characterization

Mesoporous silica materials are selected as the inorganic scaffolds, since the characteristics of this material has been investigated most extensively and its properties are adequate for molecular gate system development. These materials are easy to functionalize with alcoxysi-lane chemistry, they are biocompatibility, high thermal stability, homogeneous porosity, inert-ness, high loading capacity and tunable pore size (2 to 10 nm).[4] Standard techniques can be used for the characterization of MCM-41: X-ray diffraction, for the crystallography control; Brunauer–Emmett–Teller (BET) model for the specific surface area calculation and Barrett-Joyner-Halenda (BJH) method for the determination of the pore volume and pore size, by nitrogen adsorption-desorption isotherms; TEM is applied to check the porous structure and together with DLS to determine the size of the particles.[4, 16, 17]

2.1.5 Molecular gates and triggered release

Mesoporous materials serving as scaffold can also be employed for example to attach antibodies, proteins, nucleic acids, peptides, saccharides, or even small molecules like folic acid. As it can be seen in Figure 8, molecules can also be attached by electrostatic and hydrophobic interactions or even by host guest interactions.[7] Moreover, scientists inspired from the thought of easy functionalization of molecules into the surface of materials that could act like "gates" for a better control mass transport, created the concept of "molecular gates". The idea is to trigger the opening and closing process of a pore by external stimuli response unit. Therefore, the material is supposed to be composed of two units: a suitable inorganic scaffold, and an organic functionality to control the system release (molecular gate). The organic linker should respond to physical or chemical changes like certain enzymes, pH variations, temperature, key molecules, redox conditions or light.[4] Thus, the entrapped molecules cannot escape until the external stimuli is initiated that trigger the release.[7]

Applications such as drug delivery is the main purpose of the majority of the described systems.[4, 18] Possible control mechanisms are shown in Figure 9, and pH, redox and temperature are the most common stimuli.[7]

• pH- responsive systems: This system has a wide range of possibilities in the human body, considering that there are a lot of pH variations in organs, tissues and on the cellular level. Additionally, pathological concepts are reflected in pH gradients.[7] For example, the pH value outside a tumor cell is between 6.4 and 7.0 [19], in case of an inflamed tissue and a wound between the pH is between 5.4 and 7 [20] whereas a normal tissue and the pH of blood reaches a value of 7.4 [19]. Due to its high applicability, many systems are already developed, for example grafting of polymer chains as possible pH-responsive units.[7] Under neutral conditions the polymer is charged neutrally and is in

2 Porous materials

Figure 8 – Grafting methods of mesoporous materials. Reprinted with permission from „Mesoporous Silica Functionalized Nanoparticles for Drug Delivery“, from Simon Giret, Michel Wong Chi Man and Carole Carcel. Copyright(2015) Chemistry - A European Journal.

2 Porous materials

Figure 9 – Triggered release by different stimuli. Reprinted with permission from „Meso-porous Silica Functionalized Nanoparticles for Drug Delivery“, from Simon Giret, Michel Wong Chi Man and Carole Carcel. Copyright(2015) Chemistry - A European Journal.

a collapsed or crumbled conformation that closes the pore. But under a pH value of 5 it is charged, expands itself and opens the gate towards the surrounding environment.[21] Another concept is a host-guest interaction that immobilizes the molecule covalently, or in other cases the surface being functionalized with pH decomposable material uncaps the gate upon exposition to mild acidic pH conditions.[22] Gan et al. [23] created a system based on iron oxide nanoparticles capped with boronic acid acting as caps. The boronic acid was attached covalently to the polyalcohols, the N -(3-Triethoxysilylpropyl)gluconamide acting as gates, functionalized to the surface of the mesoporous material, closing the pores. Exposing the system to acidic conditions, the boronate ester linker was cleaved.[23] Other authors, Aznar et al. used boronic acid to functionalize gold nanoparticles[17] reacting in a pH driven reversible gate mechanism similarly with polyalcohols, due to boronated ester formation.[17]

• Redox- responsive systems: In tumor tissues the tripeptide gluthathione (GSH)[24], which is a reducing agent, exists in extremely high concentration. GSH cleaves easily disul-fide groups which leads to the idea of the researchers to functionalize the mesoporous nanoparticles with a disulfide link (S-S).[24] Therefore, one concept is that silylated hold-ing disulfide bonds is attached to the silica scaffold where different inorganic materials like Au nanoparticles[25] or CdS[26] nanoparticles or also organic polymers like collagen[27] are used as gatekeeper. After the exposure to the reductive environment, the disulfide bond is cleaved and the gatekeeper releases the encapsulated molecule.[7, 24]

• Thermo- responsive systems: Thermo responsive systems are a type of anticancer treat-ment system that makes use of temperature differences in cancer cells. By heating body tissues, up to about 45◦C, cancer cells get more sensitive to radiation or specific drugs. Furthermore, thermosensitive polymers like poly-N -vinyl-caprolactam[28] can be

intro-3 Quantum Dot (QD)

duced in the body carrying anticancer drugs. [7] Another concept is the hybridization of single strand deoxyribonucleic acid (DNA) with embedded thermosensitive caps, like iron oxid nanoparticles[29]. Dissociation of the DNA strings is forced by increasing the temperature leading to a drug release.[7]

3

Quantum Dot (QD)

QDs are semiconductors nanocrystaline of II-VI and III-V groups.[30] Their size affects its pro-perties significantly, concerning emission and extinction. Both, electrical and optical propro-perties arise because of strong confinement effects, determined by the size of the crystallites.[30, 31] Hence, their size gives them the power to convert light into almost every color of the visible spectrum. So, the bigger the size of the QD, the longer the wavelength it emits and vice versa (e.g. long wavelength refers to the color red and short one to blue/green, see Figure 10). Each Quantum Dot absorbs energy from a 450 nm source and converts the high energy (short wavelength) into lower energy (higher wavelength). The smallest QD has a size of 2 nm and emits green light at 500 nm, whereas the biggest QD has a diameter of 6 nm that emits at 630 nm in the red light.[32]

QDs present very important characteristics: narrow emission peaks, resistance to photobleaching and high fluorescent quantum yields.[33] This characteristic is the reason of their great potential in optoelectronic devices and in the biological area for example for cell labeling, optical sensors and genomic detection.[30, 34] By influencing the reaction parameters like temperature, time or solvent, wide range of different particle sizes can be synthesized.[30, 32] Furthermore, the use of capping agent bonded covalently to the surface of the nanocrystalline material prevent photo corrosion, increase fluorescence characteristics, avoid bulk formation, increase the solubility and to stabilize the particles under atmospheric conditions.[30, 33]

3.1

ZnS QDs and their Characterization

ZnS nanocrystals are a well-known II-VI semiconductor with widespread applications applied in solar cells, electroluminescent and optoelectronic devices.[35] In addition, ZnS is a proper host for many dopants, such as different transition metal ions, due to its wide band.[30, 35] It can for example be doped with copper, copper and lead, or manganese. For instance, it is known that ZnS doped with manganese decreases the lifetime compared to the ZnS bulk, but it increases the efficiency of luminescence.[35] This is explained by the energy transfer that results from the electron-hole pairs being delocalized inside the ZnS nanocrystal.[36] ZnS Quantum Dots doped with manganese can be characterized by UV-Vis, photoluminescence, X-ray diffraction, electron diffraction and high resolution transmission electron microscopy.[30, 35] The reason for

3 Quantum Dot (QD)

Figure 10 – Emission wavelength according to the QDs size.[32]

the application in biomedicine is because it is biocompatible, is efficient in luminescence and the elements Zn and S are present in the human body.[36–38] Traditionally, QD research has been focused on CdSe, however, its solubility is limitated to non-polar solvents and the core cell is highly toxic for human beings. Bearing in mind the application in drug delivery systems in the human body, the material has to avoid those drawbacks.[38]

3.2

Doped semiconductor nanocrystals Quantum Dots (d-QDs)

QD can be doped in order to tailor the nanocrystals for its applications, increasing the efficiency of fluorescence, the thermal and the photochemical stability [31, 39] and the Stoke shift that prevents self-absorption.[31] Doping of semiconductors consist of the introduction of impurity atoms for changing and improving optical, magnetically and electronic characteristics. Impurities can either added an additional valence electron (n- type doping) compared to the hosting atom or offering one valence electron less (p- type doping). Concerning the additional valence electron that is called n-type doping it can give its electron to the semiconductor and be ionized thermally. Generally, the holes and electrons serve for the electric current as carriers[39]. To clarify this concept, Figure 11 shows the Jablonksi diagram of the fluorescence of a II-VI QD where is represented the emitting signal produced after excitation at one to three photon (1P, 2P, 3P). Commonly, imaging by excitation in the ultraviolet (UV) area, at 300 nm, is applied due to the easy availability of excitation sources. The use of the three-photon imaging process (3P) in the near infrared (NIR) area, at 900 nm, provides a deepest penetration, at high resolution

3 Quantum Dot (QD)

(see Figure 11(b)) and lowest photodamage is provided, what is very interesting for biological molecules, tissues and cells in vivo imaging, application (avoiding photodamaging).[37]

(a) (b)

Figure 11 – (a) Jablonski diagram of ZnS Quantum dots. The emission of the fluorescence of the ZnS QD is about 430 nm and of the manganese doped ZnS 580 nm. (b) Fluorescence microscopy comparing one photon imaging to three photon imaging.[37]

3.2.1 Synthesis of doped Quantum Dots

For the synthesis of nanocrystals, the components are combined in either solid-, liquid- or gas-phase reactor. In the process the precursors are decomposed thermally and this results in nucleation and growth of the nanocrystals. The most successful syntheses of homogeneous crystallites are obtained by applying colloidal chemistry, and the usual approach is adding a precursor comprising the impurity directly to the synthesis for doped Quantum Dot (d-QD).[39] If the crystallites get doped for example with a metal ion, the ion gets incorporated in the matrix.[30] But to be sure that the QD was doped successfully an extense characterization has to be done.[39] The characterization of the d-QDs can be done by X-ray diffraction, transmission electron microscopy, photoluminescence and UV-Vis absorption.[30, 35, 36] Furthermore, DLS and FTIR can be used to characterize the samples.

A schematic representation fo a doped QD is shown graphically in Figure 12. The core (orange part) is the QD itself doped with manganese (yellow dots) and the blue surrounding represents the capping agents, such as TG or B, commonly used.

4 Fluorescence Spectroscopy

Figure 12 – ZnS doped with manganese surrounded by TG as a capping agent (Mn:ZnS@TG).

4

Fluorescence Spectroscopy

4.1

Fluorescence and Phosphorescence

Excited states are classified by their angular momentum as singlet (S) or triplet (T). In case of a zero net spin (diamagnetic) singlet and triplet appears when two electrons with the same spin occupy different orbitals (paramagnetic). Although most of the molecules do have their ground state as singlet, it is important to know if a molecule is singlet or triplet because the excitation always takes places within the origin multiplicity.[40] So if a molecule is excited and raised from the S0 state to a higher electronic state S1, there are various processes to reach the electronic

ground state again. In Figure 13 a Jablonski diagram shows those different possibilities.

Figure 13 – Jablonski diagram showing fluorescence and phosphorescence.[40]

Internal conversion (IC) refers to the process when a molecule falls from an excited to a lower state, releasing energy in form of heat (relaxation) and maintaining its multiplicity. Fluorescence happens when it is returned to the ground state within its multiplicity (e.g. from S1 to S0) by

4 Fluorescence Spectroscopy

emitting photons. If the multiplicity is changed to a lower energy level, it is described as Intersystem crossing (ISC). However, if the spin state is changed, reaching the ground state (e.g. from T1 to S0), the process it is known as phosphorescence.[40]

Generally it can be distinguished between three types of fluorophores, used in biological research already for more than 100 years:[41]

• Organic dyes: This type was the first one applied in research. Synthetic organic dyes like Fluorescein (FTIC) and its derivates, were improved constantly to increase photostability and guarantee solubility. Dyes are further used for bioconjunction because their small size, and their easy functionalization to macromolecules, like antibodies. Important advantages of organic dye molecules are the wide range of emission and excitation and possible selected quantum yield.[41]

• Biological fluorophores: In this case biological materials, such as jellyfish aequorea victora are used as biological expression systems, but one drawback of using biological fluorophores is the lack of photostability.[41]

• Quantum dots: As previously explained in Section 3, these semiconductors can turn the fluorescence in a specific wavelength depending on their particle size. They can additionally be coated depending on the future applications and present higher photostability than classical organic fluorophores.[41]

4.2

Quantum Yield (QY)

The measurable fluorescence Quantum Yield is not only defined due to intrinsic molecular prop-erties, but also because of external factors. The QY indicates the probability that one absorbed quantum is emitted as light quantum, even though the energy (for example: longer wavelength) is different.[42] Equation 4 describes mathematically the QY, where F is the area of the emission peak, Abs is the absorbance at the wavelength of the determined excitation and n represents the refractive index of the solvent used.[31] The highest possible QY is 100 %.[42]

QY = Fsample FRef · AbsRef Abssample ·n 2 sample n2 Ref · QYRef (4)

4.3

Fluorescence Quenching effect

The quenching is defined as the decrease or non-radiative transition of the fluorescence intensity of a fluorescing substance, called fluorophore, induced by a mechanism or substance (quencher) from both S1 and T1 returning to the ground state.[42] Thus, this effect depends on the quencher

4 Fluorescence Spectroscopy

molecule, the emitted wavelength of the fluorophore and the interaction class. Different kinds of quenching are shown in Figure 14. Förster Resonance Energy Transfer (FRET) is the mechanism that describes the transference of energy between two molecules, where a light source excites the donor fluorophore which is raised to an excited state.[41] If another molecule is close (fluo-rophore or non-fluo(fluo-rophore) acting as acceptor, this receives the excitation energy via dipol-dipol interactions[41, 43], and the energy is quenched. When the acceptor molecule is excited, reaching the excited state, then, it can release photons, that will emit at the wavelength of fluorescent acceptor molecule. Contact quenching arises when the fluorophore is complexed with a second molecule (or contact molecule) while the excitation process takes place, and the excitation energy is directly delivered to the contact molecule (which at that moment acts as a quencher). The quencher transforms the energy into heat, and collision quenching takes place when the excited fluorophore reacts with a molecule while being excited. Then, energy is transferred and hence quenching occurs.[41]

5 Chemicals

Part II

Experimental section

5

Chemicals

All chemicals used in the following experiments are registered in Table 2 and Table 3.

Table 2 – List of chemicals, part 1.

chemical molecular formula molar mass purity company g mol−1 %

Acetonitrile C2H3N 41.05 ≥ 99.9 Fluka

Diethylether C4H10O 74.12 - VWR

Dimethylformamide

di-sodium hydrogen phos-phate dihydrate

N a2HP O4∗ 2H2O 177.99 99 J.T. Baker

Ethanol C2H6O 46.07 p.a. Merck

Hydrochloric acid HCl 36.46 - J.T. Baker

Manganese acetate tetrahy-drate C4H6M nO4∗ 4H20 245.09 ≥ 99 Sigma Aldrich 4-mercaptophenylboronic acid C6H7BO2S 153.99 95 FluoroChem N -cetyltrimethyl-ammoniumbromide C19H42BrN 364.45 98 Sigma Aldrich N -(3-triethoxysilylpropyl)-gluconamide C15H33N O9Si 399.51 50 % in EtOH abcr

Potassium chloride KCl 74.55 - J.T. Baker

Potassium dihydrogen phosphate

KH2P O4 136.09 99 J.T. Baker

Rhodamine B C28H31ClN2O3 442.02 f.fl. Sigma Aldrich

Safranin O C20H19ClN4 350.84 ≥ 85 Sigma Aldrich

Sodium chloride NaCl 58.44 - J.T. Baker

Sodium hydroxide NaOH 39.99 97 J.T. Baker

Sodium phosphate monoba-sic, monohydrate

N aH2P O4∗ H2O 137.98 - J.T. Baker

6 Characterization of the solids

Table 3 – List of chemicals, part 2.

chemical molecular formula molar mass purity company g mol−1 %

Tetraethylorthosilicate SiC8H20O4 208.33 98 Sigma Aldrich

1-thioglycerol C3H8O2S 108.16 97 Sigma Aldrich

Toluene C7H8 92.14 p.a. VWR

Zinc acetate dihydrate C4H6O4Zn ∗ 2H20 219.51 ≥ 99 Sigma Aldrich

6

Characterization of the solids

The methods and the measurement details are listed in this chapter. The device characteristics used and extra information are listed in Table 4.

Table 4 – Instruments used for the characterization of the solids.

Device Company additional Information Institute

TGA TA Q5000 ICP

BET micromeritics Flow/ Vac Prep 060 ICP

TEM Jeol JEM-2011 ZONA

FTIR Spectrometer Perkin Elmer Spectrum 100 ICP UV-Vis Spectrometer Perkin Elmer Lambda 35 ICP

DLS Malvern Zetasizer nano ZSP ICP

Centrifuge Heraeus Multifuge 1s, Thermo ICP

Muffle Furnace ICP

Drying oven 37◦C, 60◦C ICP

MilliQ Millipore ReferenceA+, 0.22 µm ICP

Fluorolog Horiba Jobin Yvon CNPS

UV-Vis Spectrometer Varian Cary 300 Bio CNPS

6.1

Thermogravimetric analysis (TGA)

Thermogravimetric analysis is a technic to determine the organic content in the prepared mate-rials, though a change in weight of the material by increasing the temperature constantly. The samples (from 5 to 10 mg) were tested under nitrogen gas atmosphere, with a heating program ramp of 10◦C min−1 from 40 to 900◦C, in a platinum pan.

6 Characterization of the solids

6.2

Nitrogen adsorption-desorption isotherms (BET and BJH model)

Brunauer–Emmett–Teller (BET) model is applied to determine specific surface area and Barrett-Joyner-Halenda (BJH) is used to evaluate the mesopore volume and mesopore size through adsorption of nitrogen molecules on a porous solid nanoparticles (NPs). A samples mass is used in the analysis between 20 to 400 mg. Analysis bath temperature of 77.30 K was used and around 110 measurement points recorded.

6.3

Transmission Electron Microscopy (TEM)

Transmission Electron Microscopy is a microscopic technique used to observe and determine size and shape of the material. Images of the samples are captured due to interactions between the electron beam and the atoms of the sample.

6.4

Fourier Transform Infrared Spectroscopy (FTIR)

Fourier Transform Infrared Spectroscopy is a special method of spectroscopy used to identify chemical groups in the material. The samples were measured in the range between 600 and 4000 cm−1.

6.5

Ultraviolet–Visible spectroscopy (UV-Vis)

The absorbance measurements of the materials were performed with the spectrometer. The Quantum Dots absorb at around 270 to 300 nm and Safranin O shows its maximum at 520 nm.

6.6

Dynamic Light Scattering (DLS)

Dynamic Light Scattering is used to determine the size of an inorganic particle or a polymer in solution. MilliQ water was used as dispersant at 25◦C and Disposable cuvette (DTS 0012) were used to measure the materials samples. Samples were measured in a concentration of about 1 mg mL−1 and previous any measurement, sonification treatment for around 15 min was performed to break up formed agglomerations.

6.7

Fluorescence spectroscopy

To determine the emission and calculate the Quantum Yield (QY) for the manganese doped QDs, Rhodamine B was used as reference standard. The settings defined in the Fluorolog

7 Design and preparation of the hybrid materials

instrument were: Excitation at 300 nm, Filter at 475 nm, emission measurements from 500 to 800 nm, detector S1c/R1c.

6.8

Release Studies

25 mL of phosphate-buffered saline (PBS) solution with a certain pH is prepared and poured with continuous stirring to 10 mg of the testing sample. Samples of 1.5 mL were collected at different times, between 0 s and 5 h, and filtered with a Fisher 0.2 µm nylon filter. The dye release is followed with the UV-Vis spectrometer. The evaluation of release studies from Safranin O is done by plotting the cumulative dye release at 520 nm against the time when the respective sample was taken. The maximum cumulative dye release (100 %) refers to the sample taken at 5 h from solution pH 3.

7

Design and preparation of the hybrid materials

7.1

Preparation and characterization of mesoporous MCM-41 NPs

The first step was the preparation and the characterization of the support material which is sup-posed to serve, in the following steps, as carrier for dye molecules and in the future for entrapped anticancer drugs. The synthesis was preceded according to previous procedure reported[44] and the material was characterized by standard techniques.

Synthesis of MCM-41 (CH01)

N -Cetyltrimethylammoniumbromide (1.00 g, 2.77 mmol) were dissolved in 480 mL MilliQ water

by stirring slowly in a 1000 mL beaker. Then 3.5 mL of 2 M sodium hydroxide were added. The mixture was heated up until 80◦C while covering with an watch glass, then tetraethylorthosil-icate (5.00 mL, 25.7 mmol) was added dropwise to the solution and stirred fast for 2 h. The obtained white precipitate was centrifuged (15 min, 5000 rpm) and washed 3 times with 30 mL MilliQ water till the pH was neutral. The sample was dried over night at 60◦C. After using a mortar to grind the material, it is calcinated at 550◦C for 5 h and the final porous material, MCM-41, was obtained.[44] The various charges are listed in Table 5.

7 Design and preparation of the hybrid materials

Table 5 – MCM-41 Synthesis.

Charge resulting before calcination after calcination

g g g A 1.88 1.73 0.86 B 1.55 1.53 0.66 C 1.47 1.46 0.61 D 1.67 1.66 0.78 G 1.70 1.67 0.83

7.2

Preparation and characterization of Quantum Dots

Different Quantum Dots were synthesized and characterized by TGA, FTIR, UV-Vis, DLS and fluorescence spectroscopy. After the analytical part, selected QDs were applied in the hybrid solid S2 preparation.

It was started with the synthesis of simple Quantum Dots, like they have already been published[30, 31]. First it was intended to synthesize QDs just with zinc acetate and a well-known capping agent, 1-thioglycerol (TG). As good results were obtained, 4-mercaptophenylboronic-acid (B) was also used as capping agent. Furthermore, manganese doped and not doped Quantum Dots were prepared.

7.2.1 Undoped QDs preparation

Synthesis of QDs in water capped with 1-thioglycerol (ZnS@TG, CH03)

Under nitrogen, 1 M zinc acetate dihydrate (5.0 mL, 5.1 mmol) and 1 M 1-thioglycerol (1.5 mL, 20 mmol) were mixed and the pH was adjusted to 10 with 2 N sodium hydroxide. The solution was stirred for 30 min, then 1 M sodium sulfide nonahydrate (4.5 mL, 4.5 mmol) were added quickly and the solution was refluxed at 100◦C for 20 h. After cooling the QD’s were precipi-tated, separated and washed three times with 25 mL ethanol and the resulting solid was dried at 37◦C (see Figure 15).[31]

Characterization: FTIR, UV-Vis, DLS, fluorescence spectroscopy

7 Design and preparation of the hybrid materials

Synthesis of QDs in DMF capped with 4-mercaptophenylboronic-acid (ZnS@B, CH013)

Under inert conditions and in a 2-neck round- bottom flask zinc acetate dihydrate and 4-mercaptophenylboronic-acid were stirred in dimethylformamide. Dimethylformamide was cho-sen as solvent and not water, because of non-solubility of 4-mercaptophenylboronic-acid in water (Figure 16). Sodium sulfide nonahydrate (dissolved in case of A in 1 mL and in case of B and C in 4 mL water) was added dropwise and the pH was adjusted with 0.1 M sodium hydroxide. The reaction was heated up slowly to 150◦C and refluxed overnight. The next day the mixture was cooled down, poured in a one neck flask, the dimethylformamide removed with the rotary evaporator and the precipitating particles were washed by centrifugation three times with 30 mL of ethanol. At the end, diethylether was added and the particles were dried under atmospheric conditions.[30] The different charges produced are listed in Table 6

Characterization: TGA, FTIR, DLS

Figure 16 – Reaction scheme of CH013 preparation.

Table 6 – Overview QD Synthesis of CH013

Zn(OAc)2 Zn(OAc)2 B B DMF N a2S N a2S NaOH Received Product

mg mmol mg mmol mL mg mmol mL mg

A 29.61 0.135 50.35 0.33 10 4.02 0.017 0.123 1.62 B 240.50 1.096 401.30 2.61 25 33.43 0.139 1.000 8.42 C 240.69 1.096 399.90 2.60 25 33.60 0.140 1.000 6.32

Synthesis of QDs in DMF without capping agent (ZnS, CH016)

Zinc acetate dihydrate (60.91 mg, 0.28 mmol) was added under inert conditions to 15 mL dimethyl-formamide and sodium sulfide nonahydrate (8.21 mg, 0.034 mmol) was dissolved in 2 mL water and was added dropwise. The pH was adjusted with 30 µL 0.1 M of sodium hydroxide. The temperature was increased slowly to 150◦C and the solution was refluxed overnight. Afterwards, the mixture reaction was cooled and the solvent was removed by the rotary evaporator. The resulting particles were washed three times by centrifugation with 30 mL ethanol and finally diethyl ether was added. The particles were dried under air.[30] 4.59 mg of the final material

7 Design and preparation of the hybrid materials

were obtained (see Figure 17). Characterization: FTIR

Figure 17 – Reaction scheme of CH016 preparation.

7.2.2 doped QDs preparation

Synthesis of d-QDs in water capped with 1-thioglycerol (Mn:ZnS@TG, CH02)

To achieve doping, 1 M zinc acetate dihydrate (5.0 mL, 5.1 mmol), 1 M 1-thioglycerol (1.5 mL, 20 mmol) and 0.1 M manganese acetate tetrahydrate (0.15 mmol) under nitrogen gas were mixed. The pH was adjusted to 10 by using 2 N sodium hydroxide. The solution was stirred for 30 min and afterwards 1 M sodium sulfide nonahydrate (4.5 mL, 4.5 mmol) was added quickly. The solution was refluxed at 100◦C for 20 h and after cooling to room temperature precipitated, separated and washed three times with 25 mL ethanol. The solid was dried at 37◦_{C (see}

Fig-ure 18).[31]

Characterization: FTIR, UV-Vis, DLS, fluorescence spectroscopy

Figure 18 – Reaction scheme of CH02 preparation.

Synthesis of d-QDs in water capped with 1-thioglycerol and 4-mercaptophenylboronic-acid (Mn:ZnS@ TG:B, CH05-06,10-11)

Under nitrogen gas 0.1 M zinc acetate dihydrate (5 mL, 0.5 mmol), 0.01 M manganese acetate tetrahydrate (1.5 mL, 0.015 mmol) and different proportions of 1-thioglycerol and

4-mercaptophenylboronic-acid (Table 7, in total 2 mmol) were added. The pH was adjusted to 10 by using 0.2 N sodium hydroxide, stirred under nitrogen for 30 min and 0.1 M sodium sulfide nonahydrate (1.5 mL, 0.45 mmol) was added quickly. The solution was refluxed at 100◦C for 20 h and after cooling to room temperature the product was precipitated with 15 mL ethanol, separated and washed three times with 20 mL with ethanol. The solid was dried at 37◦C (see Figure 19).[31].

7 Design and preparation of the hybrid materials

Figure 19 – Reaction scheme of preparation.

Table 7 – Overview TG:B- Ratios

shortcut Ratio 1-thioglycerol 4-mercaptophenylboronic-acid additional information

TG:B mmol mmol

CH05 9:1 1.75 0.19

CH06 7:3 1.39 0.56

CH10 5:5 0.99 0.99

CH11 3:7 0.60 1.39 B did not dissolve

Synthesis of d-QDs in DMF capped with 4-mercaptophenylboronic-acid (Mn:ZnS@B, CH014)

In a round bottom flask, inc acetate dihydrate (29.63 mg, 0.135 mmol), manganese acetate tetrahydrate (1.03 mg, 0.004 mmol) and 4-mercaptophenylboronic-acid (50 mg, 0.33 mmol) were stirred under inert conditions in 50 mL dimethylformamide. Sodium sulfide nonahydrate (4.10 mg, 0.017 mmol) was dissolved in 1 mL water and was added by dripping. The adjustment of the pH was done by adding 123 µL 0.1 M sodium hydroxide. It was heated up slowly till 150◦C and re-fluxed overnight. Then, the solution was cooled to room temperature and it was tried to remove the solvent by vacuum in a rotary evaporator, but it could not be done completely. Due to that it was dragged with toluene (two times with 15 mL) and ethanol (two times with 10 mL). The particles started to precipitate after the first evaporation of toluene. The precipitated particles were washed three times being centrifuged with 30 mL ethanol. Diethyl ether was added in the last step and the particles were air-dried.[30] The product obtained was 0.34 mg (see Figure 20). Characterization: FTIR

7 Design and preparation of the hybrid materials

7.3

Preparation and characterization of the proposed controlled

re-lease „ gate- like“ system

The MCM-41 synthesized previously (CH01) was uploaded with Safranin O, selected as model molecule. This material will be called control material. Furthermore, the external surface of the mesoporous silica material will be functionalized with N -(3-Triethoxysilylpropyl)gluconamide (gluconamide) as molecular gate like in previous report [4, 17]. The resulting material is called S1 and was prepared according to a grafting type of functionalization (see Figure 7(a)). gluconamide was chosen where polyalcohol groups will react with boronic acid to form boronate esters. In addition, it is further supposed to build a dense network on the surface preventing the entrapped molecules to be release.[4] The same material was prepared without gluconamide, as control to prove the diffusion behavior of the dye molecules from the pores without presence of any molecular gate. The materials were characterized by TGA and nitrogen adsorption-desorption isotherms and release studies at different pH were carried out.

The final material preparation step is known as S2. In this step, MCM-41 NPs uploaded with Safranin O were functionalized with ZnS@B QDs directly. The characterization was done by standard techniques.

Synthesis of materials S1 and control material (CH017)

MCM-41 NPs (CH01) and Safranin O were mixed in acetonitrile. To remove water from the pores of the MCM-41 material 10 mL of the solvent were removed via azeotropic distillation. The suspension was stirred for approximately 24 h and N -(3-Triethoxysilylpropyl)gluconamide (50 % in ethanol) was added under nitrogen and stirring the suspension for 5.5 h more. The precipitation was removed and washed with 15 mL acetonitrile by centrifugation. The solid was dried at 37◦C.[17] The different charges were summarized in Table 8, whereas material A and I were synthesized exactly according to literature [17] and in B, C, F and G the amount of gluconamide was varied. In charge E no dye was used at all. In charge D and H no gate was attached. The preparation (uploading and functionalization) of S1 and control materials are presented in Figure 21.

7 Design and preparation of the hybrid materials

(a) Reaction scheme of CH017A, B, C, F, G and I.

(b) Reaction scheme of CH017E.

(c) Reaction scheme of CH017D and H (control material).

7 Design and preparation of the hybrid materials

Table 8 – Preparation data.

MCM-41 MCM-41 Safranin O Safranin O gluconamide gluconamide Product

CH017 CH01 mg mg mmol µL mmol mg A C 400.64 113.60 0.323 420.00 0.5253 532.74 B B 50.37 14.71 0.042 13.00 0.0163 55.30 C B 50.15 14.79 0.042 40.00 0.050 60.31 D D 201.40 57.97 0.165 0 0 236.17 E D 50.18 0 0 52.50 0.0656 47.50 F G 100.19 28.48 0.081 0.13 0.0002 112.01 G G 100.61 28.41 0.081 0.26 0.0003 115.24 H G 400.84 113.46 0.323 0 0 482.10 I D 400.50 114.03 0.325 420.00 0.5250 528.16

Synthesis of S2 hybrid material (without gluconamide) capped with QDs (CH018)

S1 material (CH017D or CH017H), 2 N sodium hydroxide (NaOH) and ZnS@B QDs (CH013) dispersed in in 25mL ethanol were stirred at room temperature for about 16 h. The ratio, listed in Table 9, refers to the ratio of mg S1 to mg Safranin O. Note that in CH018A and CH018C additionally Safranin O was added. The precipitate was removed and washed by centrifugation with 10 mL ethanol. The obtained product was dried at 37◦C overnight (see Figure 22).[31] Characterization: TGA, nitrogen adsorption-desorption isotherms, FTIR, DLS, fluorescence spectroscopy, Release Studies

7 Design and preparation of the hybrid materials

Table 9 – Overview S2 Synthesis (CH018).

Charge S1 S1 QD QD Ratio NaOH Safranin O Safranin O Product

CH018 CH017 mg CH013 mg µL mg mmol mg

A D 100.56 B 1.05 100:1 40 29.08 0.083 87.88

B D 76.91 B 2.30 100:3 40 0 0 68.42

C H 50.27 B 2.06 100:4 20 17.08 0.049 87.88

Preparation of the PBS solutions used for Release Studies

PBS 1: Sodium chloride (8.08 g, 138 mmol), potassium chloride (0.21 g, 2.8 mmol), di-sodium hydrogen phosphate dihydrate (1.49 g, 8.4 mmol) and potassium dihydrogen phosphate (0.24 g, 1.8 mmol) was poured into 1 L MilliQ water and completely dissolved. The pH of 3, 5 and 8 was adjusted by 0.1 M hydrochloric acid and 0.1 M sodium hydroxide.

PBS 2: di-sodium hydrogen phosphate dihydrate (12.48 mg, 70 mmol), sodium phosphate monoba-sic, monohydrate (1.39 mg, 10 mmol) and sodium chloride (8.47 mg, 145 mmol) were dissolved in 1 L MilliQ water. The desired pH of 3, 5 and 7 was adjusted by 0.1 M hydrochloric acid and 0.1 M sodium hydroxide.

8 Characterization of mesoporous MCM-41 NPs

Part III

Results and Discussion

8

Characterization of mesoporous MCM-41 NPs

8.1

TGA

TGA of MCM-41 was made as a control, to see if there is any affect caused by the material itself. No alteration of the material was detected and thus no further evaluations were made. The graph of MCM-41, CH01E, is attached in the Appendix, Figure 51. The first decrease in weight is the loss of water, the second peak is the molecular gate, the third the entrapped guest molecule or dye and the fourth the condensation of silanols.

8.2

Nitrogen adsorption-desorption isotherms

Specific surface area, pore volume and pore size of the synthesized materials were determined via the Brunauer–Emmett–Teller (BET) and Barrett-Joyner-Halenda (BJH) method. The results are listed in Table 10. The analysis of the sample CH01A was made two times because the sample was calcined on two different days. The adsorption-desorption isotherms of the calcinated MCM-41 materials are shown in Figure 24. The curves of the MCM-41 material show a typical behavior of mesoporous material. Only one adsorption step can be observed between 0.1 and 0.3 at the intermediate p/p0 _{and according to literature [4], nitrogen condensation takes place}

inside the pores. Comparing the resulting values to literature [4, 17, 23], the surface area is supposed to be between 963.3 and 1293.4 m2_g−1_{, whereas all samples can be found in this}

area but CH01A is below this values. Concerning pore volume all the materials presented the expected values, comparing with the literature (0.50 and 1.15 cm3_g−1_{) [4, 17, 23]. The pore size}

presents also expected values compared to literature (2.1 and 3.6 nm) [4, 17, 23]. The narrow BJH pore distribution (Figure 23) and the absence of a hysteresis loop in Figure 24 lead to the conclusion that the resulting pores do have a uniform cylindrical shape. The pore diameter is shown representative by CH01G and it is about 3.2 nm with a pore volume of approximately 0.96 cm3_g−1_.

8 Characterization of mesoporous MCM-41 NPs

Table 10 – Nitrogen adsorption-desorption isotherms of mesoporous silica nanoparticles.

shortcut BET BJH BJH

of calcinated material surface area pore volume pore size m2 _g−1 _cm3 _g−1 _nm MCM-41 CH01A 1 873.7 0.97 2.34 MCM-41 CH01A 2 697.9 0.79 2.47 MCM-41 CH01B 1203.2 0.69 3.53 MCM-41 CH01C 1178.6 0.62 3.67 MCM-41 CH01D 971.9 0.79 3.06 MCM-41 CH01G 1001.3 0.96 3.23

8 Characterization of mesoporous MCM-41 NPs

(a) CH01A, calcination 1 (b) CH01A, calcination 2

(c) CH01B (d) CH01C

(e) CH01D (f) CH01G