ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1363

Mesoporous magnesium carbonate

Synthesis, characterization and biocompatibility

SARA FRYKSTRAND ÅNGSTRÖM

ISSN 1651-6214ISBN 978-91-554-9540-4urn:nbn:se:uu:diva-281522

Dissertation presented at Uppsala University to be publicly examined in Polhemssalen,Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, Friday, 20 May 2016 at 09:30 for thedegree of Doctor of Philosophy. The examination will be conducted in English. Facultyexaminer: Professor Mika Lindén (University of Ulm).

AbstractFrykstrand Ångström, S. 2016. Mesoporous magnesium carbonate. Synthesis, characterizationand biocompatibility. Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1363. 75 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-9540-4.

Mesoporous materials constitute a promising class of nanomaterials for a number of applicationsdue to their tunable pore structure. The synthesis of most mesoporous materials involves asurfactant liquid crystal structure to form the pores. As well as the many advantages associatedwith this method of synthesis, there are disadvantages such as high production costs anda substantial environmental impact which limit the possibilities for large scale production.Therefore there is a need for other synthesis routes.

The aim of the work described herein was to contribute to this field by developing a synthesisroute that does not rely on surfactants for pore formation. A mesoporous magnesium carbonatematerial was therefore formed by self-assemblage of the particles around carbon dioxide gasbubbles, which functioned as pore templates. It was also possible to vary the pore diameterbetween 3 and 20 nm.

The biocompatibility of the formed magnesium carbonate material was evaluated in terms ofin vitro cytotoxicity and hemocompatibility, in vivo skin irritation and acute systemic toxicity.The results from the in vitro cytotoxicity, in vivo skin irritation and acute systemic toxicity testusing a polar extraction vehicle showed that the material was non-toxic. While signs of toxicitywere observed in the acute systemic toxicity test using a non-polar solvent, this was attributedto injection of particles rather than toxic leachables. In the in vitro hemocompatibility test, nohemolytic activity was found with material concentrations of up to 1 mg/ml. It was furthershown that the material had anticoagulant properties and induced moderate activation of thecomplement system. The anticoagulant properties were ascribed to uptake of Ca2+.

Finally, the ability of the material to increase the dissolution rate of the poorly soluble drugitraconazole was analyzed. Itraconazole was dissolved up to 23 times faster from the magnesiumcarbonate pores than when the free drug was used. The release rate from the delivery vehiclewas dependent on the pore diameter.

The work presented herein is expected to be useful for the development of alternativesynthesis routes for mesoporous materials and also for encouraging the development ofbiomedical applications for these materials.

Keywords: mesoporous, magnesium carbonate, pore size control, cytotoxicity, in vivo, skinirritation, acute systemic toxicity, hemocompatibility, Ca2+ uptake, solubility enhancement

Sara Frykstrand Ångström, Department of Engineering Sciences, Nanotechnology andFunctional Materials, Box 534, Uppsala University, SE-75121 Uppsala, Sweden.

© Sara Frykstrand Ångström 2016

ISSN 1651-6214ISBN 978-91-554-9540-4urn:nbn:se:uu:diva-281522 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-281522)

Till mamma och pappa

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I. A template-free, ultra-adsorbing, high surface area carbonate

nanostructure J. Forsgren*, S. Frykstrand*, K. Grandfield, A. Mihranyan and M. Strømme, PLoS ONE, 2013, 8 (7), e68486

II. On the pore-forming mechanism of Upsalite, a micro- and mesopo-rous magnesium carbonate S. Frykstrand, J. Forsgren, A. Mihranyan and M. Strømme, Mi-croporous and Mesoporous Materials, 2014, 190, 99-104

III. Nanostructure and pore size control of template-free synthesized mesoporous magnesium carbonate Upsalite O. Cheung, P. Zhang, S. Frykstrand, H. Zheng, M. Sommariva, X. Zhou and M. Strømme, submitted

IV. Cytotoxicity, in vivo skin irritation and acute systemic toxicity of the mesoporous magnesium carbonate Upsalite® S. Frykstrand, J. Forsgren, P. Zhang, M. Strømme and N. Ferraz, Journal of Biomaterials and Nanobiotechnology, 2015, 6, 257-266

V. Study of mesoporous magnesium carbonate in contact with whole human blood S. Frykstrand, P. Zhang, O. Cheung, J. Forsgren, J. Hong, M. Strømme and N. Ferraz, submitted

*In Paper I the first authorship is shared with J. Forsgren

All published papers are published under the terms of Creative Commons Attribution-NonCommercial-No Derivatives License (CC BY NC ND).

The author’s contribution to the included papers

I. I participated in the planning of the study, performed major parts of the experimental work (except the TEM studies), wrote parts of the initial manuscript and contributed to the remainder of the writing process.

II. I had a major part in planning the study, performed all experimental work (except the TEM studies), wrote parts of the initial manuscript and contributed to the remainder of the writing process.

III. I participated in planning the study and the analysis, performed the

XPS studies and wrote parts of the manuscript.

IV. I had a major part in planning the study and writing the manuscript, and performed all of the experimental work (except synthesis of the material) and the analysis.

V. I had a major part in planning the study and writing the manuscript, and performed all of the experimental work (except synthesis of the material and ion adsorption) and the analysis.

Also published

Journal articles

i. Synthesis, electron microscopy and X-ray characterization of ox-ymagnesite, MgO·2MgCO3, formed from amorphous magnesium carbonate S. Frykstrand, C. Strietzel, J. Forsgren, J. Ångström, V. Potin and M. Strømme, CrystEngComm, 2014, 16, 10837-10844

ii. Water and ion transport in ultra-adsorbing porous magnesium car-bonate studied by dielectric spectroscopy I. Pochard, S. Frykstrand, O. Ahlström, J. Forsgren and M. Strømme, Journal of Applied Physics, 2014, 115, 044306

iii. Dielectric spectroscopy study of water behavior in calcined Upsalite®; a mesoporous magnesium carbonate without organic surface groups I. Pochard, S. Frykstrand, J. Eriksson, S. Gustafsson, K. Welch and M. Strømme, The Journal of Physical Chemistry C, 2015, 119, 15680-15688

iv. Asymmetric supercapacitors based on carbon nanofibre and

polypyrrole/nanocellulose composite electrodes P. Tammela, Z. Wang, S. Frykstrand, P. Zhang, I-M. Sintorn, L. Nyholm and M. Strømme, RCS Advances, 2015, 5, 16405-16413

Patent applications

Anhydrous, amorphous, and porous magnesium carbonates and methods of production thereof M. Strømme, A. Mihranyan, J. Gómez de la Torre and S. Frykstrand, U.S. Patent Application 14/648,780 Highly porous magnesium carbonate and method of production thereof O. Cheung, P. Zhang, S. Gustafsson, S. Frykstrand Ångström, M. Strømme, Patent Application submitted

Abbreviations

AMC Amorphous Magnesium Carbonate AP Alternative Pathway API Active Pharmaceutical Ingredient ATR Attenuated Total Reflection BET Brunauer-Emmett-Teller CP Classical Pathway DFT Density Functional Theory DLS Dynamic Light Scattering DMSO Dimethyl sulfoxide DSC Differential Scanning Calorimetry EDTA Ethylenediaminetetraacetic acid EGA Evolved Gas Analysis ELISA Enzyme-Linked Immunosorbent Assay FFT Fast Fourier Transform FTIR Fourier Transform Infrared hDf human Dermal fibroblasts HRTEM High Resolution Transmission Electron Microscopy ICP-OES Inductively Coupled Plasma – Optical Emission Spectroscopy IP Intraperitoneal IR Infrared ISO International Organization for Standardization ITZ Itraconazole IUPAC International Union of Pure and Applied Chemistry MAC Membrane Attack Complex MMC Methyl Magnesium Carbonate MOF Metallic Organic Framework NMC Nanoporous Magnesium Composite P/P0 Relative pressure PDF Pair Distribution Function SEM Scanning Electron Microscopy TAT Thrombin-Antithrombin TEM Transmission Electron Microscopy TGA Thermogravimetric Analysis XPS X-ray Photoelectron Spectroscopy XRD X-ray Diffraction

Contents

1. Introduction ............................................................................................... 11 

2. Aims of the thesis...................................................................................... 12 

3. Background ............................................................................................... 13 3.1 Nanotechnology ................................................................................. 13 3.2 Mesoporous materials ........................................................................ 14 

3.2.1 Biomedical applications of mesoporous materials ..................... 16 3.3 Magnesium carbonates ....................................................................... 16 3.4 Sol-gel synthesis ................................................................................. 18 3.5 Biocompability ................................................................................... 19 

3.5.1 Hemocompatibility ..................................................................... 20 3.5.2 Biocompatibility testing .............................................................. 24 

4. Surfactant-free formation of mesoporous materials .................................. 25 4.1 Materials and Methods ....................................................................... 25 

4.1.1 Synthesis of Nanoporous Magnesium Composite (NMC) Materials .............................................................................................. 25 4.1.2 Material characterization ............................................................ 27 

4.2 Results and discussion ........................................................................ 30 4.2.1 Synthesis and nanostructure of NMC ......................................... 30 4.2.2 Formation of NMC ..................................................................... 34 4.2.3 Pores in NMC ............................................................................. 36 4.2.4 Stability of NMC ........................................................................ 41 

5. Biocompatibility of NMC ......................................................................... 43 5.1 Materials and Methods ....................................................................... 43 

5.1.1 In vitro cytotoxicity .................................................................... 44 5.1.2 In vitro hemocompatibility ......................................................... 45 5.1.3 In vivo studies ............................................................................. 46 

5.2 Results and discussion ........................................................................ 47 5.2.1 In vitro cytotoxicity .................................................................... 47 5.2.2 In vitro hemocompatibility ......................................................... 48 5.2.3 In vivo tests ................................................................................. 52 

6. Increasing drug dissolution rate with NMC .............................................. 54 6.1 Materials and Methods ....................................................................... 54 

6.1.1 Materials ..................................................................................... 54 6.1.2 Drug loading and dissolution testing .......................................... 54 

6.2 Results and discussion ........................................................................ 55 6.2.1 Loading of ITZ ........................................................................... 55 6.2.3 Release of ITZ ............................................................................ 56 

7. Summary and conclusions ........................................................................ 58 

8. Future work ............................................................................................... 60 

9. Svensk sammanfattning ............................................................................ 61 

10. Acknowledgments ................................................................................... 63 

Appendix: Analytical techniques .................................................................. 65 Nitrogen gas sorption ............................................................................... 65 Infrared Spectroscopy .............................................................................. 66 Thermogravimetric analysis ..................................................................... 67 Scanning electron microscopy .................................................................. 67 Transmission electron microscopy ........................................................... 67 X-ray Scattering ....................................................................................... 68 X-ray photoelectron spectroscopy ............................................................ 69 Enzyme-linked immunosorbent assay ...................................................... 70 

References ..................................................................................................... 71 

11

1. Introduction

Wood, rocks and biological tissues such as lungs, cancellous bone and kidneys are all examples of porous materials found in nature. A porous material is a material with empty spaces (pores) within its structure; this results in a greater surface area than in a nonporous material. This property allows more opportunities for external compounds to come in contact with the surface of the material, which could result in the material having a greater reactivity. Porous materials are abundant in nature, and researchers have started to show increased interest in them, especially in nanoporous materials. Nanoporous materials contain pores sized in the nanoscale. One of the main goals of this research is to produce materials with large surface areas and controllable pore sizes.

Mesoporous silica is a family of nanoporous material with ordered pores in the mesoporous size range, i.e. pore diameter between 2 and 50 nm [1], that was discovered in the early 1990s [2, 3]. The interest of the scientific community in nanoporous materials has increased remarkably since then. At the beginning of 2016, according to Web of Science 1, there were more than 150,000 published scientific papers on nanoporous materials (prior to 1990 the number of articles was insignificant). One of the reasons for the increased research interest in nanoporous materials is their many industrial applications. They are often found in applications such as catalysis, adsorption and separation [4-6].

Mesoporous materials have many interesting properties, one of which is that active molecules can be loaded and released from their pores. This property has recently inspired researchers to start investigating these materials for biomedical applications as well, such as drug delivery and bone regeneration [7-9].

Today, the synthesis of mesoporous materials mainly involves the use of organic surfactants as pore-forming agents. In this thesis an alternative synthesis route was explored for mesoporous materials, without the use of organic surfactants. Instead, gas bubbles were used to form the pores. This offers the advantages of reducing both the cost and the production time. Since the material synthesised in this fashion could be suitable for biomedical applications, its biocompatibility was also examined herein.

1 www.webofscience.com, combining the results using the search words: mesoporous silica, zeolites and metal-organic frameworks (obtained Jan 2016).

12

2. Aims of the thesis

The overall aim of this thesis was to develop a mesoporous material with well defined pores diameters where the pores are formed without the use of organic surfactants as pore-forming templates. The possibility of controlling and varying the diameter of the pores was also explored. Because our group is currently evaluating the formed material in a number of biomedical applications, an examination of its biocompatibility was also included in this thesis.

The specific aims of the included papers were as follows:

To synthesize and characterize a mesoporous magnesium

carbonate material (Papers I and III);

To study the formation of the mesopores and the possibility of controlling the diameter of the pores in the mesoporous magnesium carbonate material (Papers II and III);

To study drug release from the mesoporous magnesium carbonate material (Paper III);

To study the biocompatibility of the mesoporous magnesium carbonate material, specifically its in vitro cytotoxicity and hemocompatibility, and its risk of inducing in vivo skin irritations or acute systemic toxicity (Papers IV and V).

13

3. Background

3.1 Nanotechnology The Lycurgus cup, dated to the fourth century, is one of the first known examples of an artefact made using nanotechnology. It was made of glass containing gold nanoparticles; these nanoparticles give the cup a greenish tint in reflected light, e.g. daylight, but make it red when light is transmitted through the glass [10]. This optical effect is due to the size of the gold particles; when the particle diameter is smaller than the wavelength of the visible light spectrum, i.e. 400-700 nm, the color is size dependent. When the cup is illuminated with reflected light, the nanoparticles back-scatter green light and the cup appears green. When the cup is illuminated from the inside, the same properties result in backscattered green light and only the red light is transmitted through the glass.

The more recent talk entitled “There’s plenty of room at the bottom” given by Richard P. Feynman in 1959 at the California Institute of Technology [11] is often considered as the origin of modern nanotechnology. Even though he never used the term himself, Feynman described a future in which scientists would be able to control and manipulate individual atoms.

Today, nanotechnology is defined as “the understanding and control of materials and systems at dimensions between approximately 1 and 100 nanometers”2. At this scale, materials can exhibit alternative and/or significantly improved properties (physical, chemical and biological) [12]. Consequently, nanomaterials are materials that are or have certain structural features that are between 1 and 100 nm in at least one dimension; this includes nanoparticles, nanofibers, nanolayers, nanoporous materials, etc., which exhibit properties/behavior that are different from those of their bulk, non-nano counterparts.

A nanoporous material has a considerably greater surface area than a nonporous material, which is desirable for, for example, catalysis. The pores can also be used for adsorption and desorption of compounds of interest. Nanoporous materials have been the subject of extensive research in recent decades. Types of nanoporous materials include aerogels, nanoporous carbon, nanoporous oxides, zeolites and metal-organic frameworks (MOFs).

2 US National Nanotechnology Initiative, 2000.

14

Figure 1. Schematic image of A) a zeolite (zeolite-A) and B) a metal-organic framework (MOF-5). The green and grey tetrahedrals represent MO4 entities, for zeolites M=Si/Al and for MOFs M= metal ion e.g. Zn2+ for MOF-5 (image courtesy of Dr. Andrew-Kentaro Inge).

Aerogels are highly porous materials derived from gels where the liquid has been replaced with air. The result is a set of materials with very low density and excellent insulating properties. Aerogels generally possess pores in all three pore size classes defined by the International Union of Pure and Applied Chemistry (IUPAC) [1]: macropores, diameter > 50 nm; mesopores, diameter 2-50 nm; and micropores, diameter < 2 nm.

Zeolites are naturally occurring or synthetically made framework materials, composed of aluminasilicate that typically contain micropores. Zeolites have been used for decades for catalysis. MOFs, on the other hand, are a relatively new class of inorganic-organic hybrid materials which are made by linking organic molecules to metal ions to form a porous three-dimensional network. Highly porous MOFs were first reported by Li et al. in 1999 [13]. A schematic image of a zeolite and MOF can be found in Figure 1.

The diameter of the pore suggests the applications for which the material might be used. The narrow pores of zeolites, and to some extent also MOFs, make these materials highly desirable in applications such as gas adsorption. However, they are less appealing for applications using larger molecules, such as catalysts for larger reactants and drug delivery. The larger pores of a mesoporous material make them more suitable for those applications [4].

3.2 Mesoporous materials Mesoporous materials have pores between 2 and 50 nm in diameter [1]; the pores can be either ordered or disordered. Typical mesoporous materials include amorphous silica and alumina but mesoporous oxides of titanium, zirconium, niobium, tantalum, tungsten, hafnium and tin have been reported as well [14]. In the early 1990s, two independent research groups, one in Japan and one in the US, first reported the synthesis of mesoporous silica. In

15

both cases the material was synthesized using surfactant micelles that self-assembled. The inorganic silica condensed around the organic template to form amorphous silica. The organic template was then removed either by calcination or solvent extraction, leaving open pores in the material [2, 3, 15-18], Figure 2. The final material had highly ordered pores and a large surface area [2, 3]. This discovery was followed by intense research aiming to tailor the properties of the mesoporous silica using different synthesis routes.

The silica in mesoporous silica materials is formed in a sol-gel reaction from precursors that form amorphous silica via hydrolysis and condensation. Alkoxides are common silica precursors; the hydrolysis reaction replaces the alkoxide groups (-OR) with hydroxyl groups (-OH) which subsequently, via condensation reactions, form the amorphous silica (≡Si-O-Si≡) [19].

The use of surfactants in the synthesis has many advantages: it offers the opportunity to tailor the pore diameter [2, 3, 15], the shape of the particles and the pores, and the pore connectivity. This synthesis route is also the most common one, although there are examples in the literature where mesoporous silica has been synthesized without the use of surfactants [20, 21].

The use of surfactants often results in mesoporous material with ordered, unidirectional pores. In these materials there is a risk of pore blockage during adsorption. They are, therefore, not ideal for applications such as catalysis

or

the adsorption of larger molecules, where mesoporous materials might

Figure 2. Schematic description of the synthesis of mesoporous silica material. Organic surfactants co-assemble with inorganic silica precursors. The self-assembly of organic surfactants is essential for formation of the pores within the material. The mesoporous particles are

16

be used. A material with a disordered, interconnected network of pores of uniform size would work better [21, 22]. Additionally, using surfactants in the synthesis includes a step where the surfactant has to be removed which increases the time, costs and environmental impact of the synthesis, all of which are important aspects in terms of up-scaling potential. Moreover, the alkoxides and the surfactants used for the production of the mesoporous silica materials are generally expensive and require strict control of the temperature and pH during the reaction. All of these aspects provide obstacles for large scale production, which generally makes these materials very expensive.

In addition, using organic surfactants rules out the possibility of in situ loading of an active pharmaceutical ingredient, API, into the pores during the synthesis of the mesoporous particles, unless the API itself works as a pore forming agent [16, 20].

3.2.1 Biomedical applications of mesoporous materials The use of mesoporous materials in biomedical applications such as diagnostics [23], vaccine adjuvants [24-26], regeneration of bone tissue [27], implant coatings [28], and drug delivery vehicles [8, 9, 29-32] has received a lot of attention in recent years.

One of the main reasons for this interest is the possibility of loading APIs into and then releasing them from the pores. This is highly interesting, not least from a commercial view point, since up to 90 % of the compounds in the drug discovery pipeline of big pharmaceutical companies are estimated to have poor aqueous solubility and limited bioavailability [33]. Mesoporous materials have provided the opportunity to increase the bioavailability of such drugs by suppressing their crystallization. This is achieved by incorporation of the drug molecules into the mesopores [34]. This concept has been proven viable for several drugs such as ibuprofen [9], atazanavir [30] and celecoxib [29].

3.3 Magnesium carbonates In its pure form, magnesium (Mg) is a light metal used, for example, as an alloying compound in aluminum (Al) alloys. It is the eighth most abundant element in the earth’s crust and it is found in most living organisms. In nature, magnesium is often found in silicates or double salt carbonates, such as dolomite CaMg(CO3)2 [35]. It can also form several hydrated and basic carbonates on its own, such as nesquehonite (Mg(HCO3)(OH)·2H2O), lansfordite (MgCO3·5H2O), hydromagnesite (Mg5(CO3)4(OH)2·4H2O), and dypingite (Mg5(CO3)4(OH)2·5H2O) as well as the anhydrous magnesite

17

(MgCO3) [36]. These carbonates are industrially important and are used in products such as paper and pharmaceutical formulations.

Magnesite is the most stable of the magnesium carbonates [37, 38]. How-ever, in contrast to the hydrous magnesium carbonates which are relatively easily synthesized, magnesite is difficult to synthesize in the laboratory using aqueous solutions under ambient conditions and has usually been formed using elevated pressures or temperatures [38-42]. The strong hydration status of the magnesium ion (Mg2+) is often blamed for the difficulty in precipitat-ing magnesite from aqueous solutions under ambient conditions. Its small ionic radius gives rise to a high charge density, resulting in a strong water association for the ion [38, 43-47]. However, a recent study has suggested that, in addition to the hydration status of Mg2+, the difficulties in precipitat-ing magnesite under ambient conditions are also the result of an intrinsic crystallization barrier that prevents magnesium and carbonate from forming long-range ordered structures. The relative ease with which hydrous magne-sium carbonates are nucleated, in comparison to magnesite, may be due to water facilitating the nucleation process by direct incorporation into the structure [48]. While there are some recent reports of magnesite being formed under ambient conditions in aqueous solution, in those cases the activity of the water had been decreased be the addition of either inorganic salts or organic molecules [37-39, 49, 50].

Prior to the studies where the activity of the water was lowered, research-ers had tried to use organic solvents, in particular alcohols, to circumvent the problems associated with synthesizing magnesite from aqueous solutions. For example, in 1908 Neuberg and Rewald tried to synthesize magnesite from an alcoholic suspension containing magnesium oxide (MgO) [51]. This was, however, unsuccessful, as confirmed by subsequent researchers who reattempted this synthesis [52, 53]. It was therefore concluded that magne-site cannot be synthesized by passing carbon dioxide (CO2) gas through such a suspension. This was assumed to be the result of MgO preferentially form-ing methyl magnesium carbonate (MMC), (Mg(OCO)(OCH3)2), when it is reacted with methanol and CO2 [51-53].

MMC has recently been used to produce a mesoporous amorphous mag-nesium carbonate (AMC) aerogel [54]. The magnesium carbonate sol was formed by hydrolysis of MMC into a magnesium carbonate alcogel which was then dried under supercritical conditions to form a mesoporous AMC aerogel with a large surface area (~400 m2/g). It has also been reported that AMCs can be formed, for example, from decomposition of hydrated crystal-line magnesium carbonates [55, 56].

Because of their use in pharmaceutical formulations and as food addi-tives, magnesium carbonate and magnesium oxide are listed as being Gener-ally recognized as safe [57] and have so-called E-numbers (magnesium ox-ide E530 and magnesium carbonate E504).

18

3.4 Sol-gel synthesis In a sol-gel synthesis, Figure 3, the precursors are mixed in solution and a sol (a colloidal suspension of particles or polymeric clusters) is formed by condensation and/or hydrolysis reactions. The sol is further condensed to form a gel, which will be particulate or polymeric in nature depending on the reaction. The final stage is removal of the solvent, which can be done by evaporation or extraction. When the liquid in the pores of the gel is evapo-rated, the network of the gel shrinks and becomes denser as a result of capil-lary forces to form a xerogel3. Therefore, supercritical drying can be used to prepare a highly porous material, i.e. an aerogel. In this process capillary pressure is avoided or at least minimized and the pore liquid is extracted from the gel without the structure cracking or losing its structural integrity upon drying. If the sol is dried directly, uniform particles, films or fibers can also be obtained [58].

A sol-gel synthesis is often used for the preparation of ceramic materials. The even distribution of the precursors in the liquid phase allows for lower temperatures than those used in solid-state synthesis [58]. Sol-gel syntheses have, for example, been used to prepare a calcium carbonate aerogel [59] and the magnesium carbonate aerogel described in Section 3.3 [54].

Figure 3. Illustration of sol-gel synthesis. In the first step the precursors are reacted so that a sol is formed. The sol subsequently forms a gel: a xerogel or an aerogel is formed, depending on the drying conditions.

3 Xero = dry.

19

3.5 Biocompability Mesoporous materials are of interest for use in a number of biomedical ap-plications, as described in Section 3.2.1. For these applications it is essential that the material is biocompatible. Biocompability is defined as “the ability of a material to perform with an appropriate host response in a specific ap-plication” [60]. This includes in principle two things: the absence of cyto-toxic effects and the presence of functionality. The absence of cytotoxicity means that there are no toxic effects on the survival of cells and that cellular function is maintained under the influence of the material and its degradation products. Functionality includes, for example, the integration of a biomateri-al into a biological tissue or organ and requires that the mechanical, chemical and physical features of the material are sufficient to allow cell-specific functions to be carried out [61]. In recent years the term biocompatibility has come to include in vivo toxicity and changes of cells and bodies at both mo-lecular and histological levels as well [62].

Both nanoparticles and the pores in mesoporous materials are in the same size range as several biological entities, Figure 4. Properties such as surface area, pore diameter, particle size and shape, surface charge, etc., play an important role in the biocompability of the nanomaterial [62]. It is therefore important to understand that, although the same material in bulk form may be considered safe for humans, new studies are required to ensure the bio-compability of the nano version of the material. For example, although mes-oporous silica has the same chemical composition and atomic structure as nonporous amorphous silica, which is an approved food additive, its high surface area and capacity for extensive adsorption of substances within the pores demand de novo evaluation of its biocompability [7].

Figure 4. Size of nanomaterials and pore diameter of mesopores in relation to different bio-logical entities.

20

3.5.1 Hemocompatibility Blood circulates within the body and is responsible for immune responses, tissue repair, nutrient supply, etc. It is a complex fluid composed of a large number of interacting cell types and proteins. It contains red blood cells (erythrocytes), white blood cells (leukocytes), platelets4 (thrombocytes) and plasma5. Because of its many specific functions, blood is very sensitive, and damage to the blood or inappropriate activation of the cells or protein factors in the blood can have a large impact on the human body. There-fore, the examination of hemocompatibility is an important part of gen-eral biomaterial biocompability testing [63].

A material is hemocompatible if, upon contact with blood, it does not cause inappropriate activation of any of the blood components or cause lysis of the blood cells.

Hemolysis, the breakdown of red blood cells, directly impairs the ability of blood to provide oxygen to the tissues and may be induced by the pres-ence of a foreign material [61]. Furthermore, in vitro hemolysis induced by materials such as different silica [64], alumina and magnesia nanoparticles, has been shown to be a good indicator for an in vivo inflammatory response to those particles and has been recommended as a reliable way of assessing the biocompatibility of particles [65].

Blood participates in the body’s defence against foreign substances and microorganisms via the complement system [61]. The complement system, Figure 5, is part of the innate immune system and affects the recruitment and adhesion of inflammatory cells. It consists of over 20 plasma proteins, which are involved in a system that causes the release of inflammatory mediators and ultimately the lysis of foreign cells through the formation of pores in their membranes. Lysis is caused by the membrane attack complex (MAC), which forms in the final stage of the cascade and disrupts the membranes of pathogens6 [63, 66, 67]. Activation of the complement system can be evalu-ated by measuring the levels of sC5b-9 [68] and C3a [67] in plasma. The sC5b-9 complex is the fluid-phase analog of MAC, and C3a is formed when the protein C3 is cleaved into two subunits: C3a and C3b. This cleavage is the central event in the complement system. The C3b protein continues to react and a subsequent series of reactions and protein assemblage finally results in the formation of MAC [63, 66].

4 Non-nucleated cell fragments in blood. 5 The acellular part of blood. 6 Infectious agents.

21

Figure 5. Schematic view of the complement system. It consists of three pathways: the classi-cal pathway, the alternative pathway and the lectin pathway. All three pathways result in the formation of the C3-convertases, which cleave C3 into C3a and C3b. C3b forms the C5 con-vertases which cleave C5 into C5a and C5b. C5b binds to the cell membrane and together with C6-C9 forms the membrane attack complex (MAC), or sC5b-9 in the absence of a bio-logical membrane. The activation of the complement system can be assessed by measuring levels of sC5b-9 and C3a in plasma.

22

After an injury, the body has a mechanism designed to arrest bleeding. This involves an interaction between platelets, the vascular system and the blood coagulation cascade. The coagulation cascade, Figure 6, comprises a series of reactions that ultimately results in the formation of a fibrin clot, which is the main constituent of a blood clot.

The coagulation cascade can occur via either the extrinsic or the intrinsic pathway. The extrinsic pathway is initiated by the release of tissue factor, a membrane-associated protein that is released from damaged cells or ex-pressed on the surfaces of monocytes7 or endothelial cells8. As in the com-plement system, the two pathways of the coagulation cascade end up in a common pathway where fibrinogen (a monomer) is converted to fibrin (a polymer). The proteins involved in the coagulation cascade are designated as factors and an attached Roman number [63]. Activation of the coagulation cascade can be assessed by measuring the levels of thrombin-antithrombin (TAT) complexes in plasma [69]. Thrombin is responsible for the formation of fibrin [63], and antithrombin is a small protein that is a natural anticoagu-lant.

Calcium ions (Ca2+) are required for almost all reactions in the coagula-tion cascade [70]. This is why calcium chelators, e.g. ethylenediaminetet-raacetic acid (EDTA), are effective anticoagulants [63].

Assessing the activation of the coagulation cascade and the complement system, and the level of hemolysis after blood comes into contact with a material gives an indication of the hemocompatibility of the tested material.

7 Type of white blood cell. 8 Cells that make up the inner surfaces of blood vessels.

23

Figure 6. Schematic view of the coagulation cascade. It consists of the intrinsic and extrinsic pathways. The intrinsic pathway begins with the surface activation of Factor ΧΙΙ while the extrinsic pathway is triggered by tissue factor. Both pathways converge into a common path-way with the conversion of Factor X to Factor Xa. Factor Xa forms a complex with Factor Va (prothrombinase complex) which converts prothrombin into thrombin. Thrombin is responsi-ble for the formation of fibrin and Factor ΧΙΙΙa promotes the formation of a stable fibrin clot. Activation of the coagulation cascade can be measured by measuring levels of Thrombin-Antithrombin (TAT) complexes in plasma.

24

3.5.2 Biocompatibility testing The International Organization for Standardization (ISO) has set guidelines for testing the biocompatibility of new materials intended for biomedical applications. The guidelines include tests for cytotoxicity, hemocompatibil-ity, skin irritation and sensitization, genotoxicity, etc. These tests are per-formed in vitro9 as well as in vivo10.

One of the basic requirements of biocompability testing is to keep animal studies to a minimum whenever possible. Therefore, in vitro tests are rec-ommended for screening novel materials prior to in vivo tests [63]. The cyto-toxicity of a material is tested in vitro using cell lines that are selected ac-cording to the intended application of the material. In a direct cytotoxicity test, the cells are in direct contact with the tested material. In this way the effects of the material properties (such as porosity, topography, surface charge, etc.) on the cells can be tested. The cell response is generally as-sessed in terms of cell viability and morphology [63]. It is important to re-member that the growth properties of cell lines have been modified and they may have lost some of their normal specific cell functions and consequently they may be less sensitive than living tissues [61]. Therefore, the results from an in vitro cytotoxicity test might not translate directly to in vivo condi-tions.

In vitro tests can also be used to test hemocompatibility using fresh whole blood or plasma from humans [69, 71]. In these models the material comes into contact with blood or plasma under controlled conditions. Thereafter, the plasma samples are analyzed for the activation of the blood cascades, release of activation factors [69, 71], or hemolysis.

In vivo tests are those where the effects of a material, pharmaceutical compound, etc. are tested on whole, living organisms. Consequently animal testing and clinical trials are major parts of in vivo research.

9 In vitro = in glass, i.e. in an artificial environment. 10 In vivo = in the living, i.e. in a living organism.

25

4. Surfactant-free formation of mesoporous materials

As described in Section 3.2, a mesoporous material is generally formed by using organic surfactants as pore-forming agents. However, this work de-scribes an alternative synthesis route for a mesoporous material, which does not rely on the use of surfactants for formation of the pores. As will be shown in this section, the mesoporous material is composed of MgCO3/MgO nanometer-sized composite particles. The MgCO3/MgO composite particles self-assemble around CO2 gas bubbles, which function as templates for the pores. The results presented in this section are described in Papers I, II and III.

4.1 Materials and Methods

4.1.1 Synthesis of Nanoporous Magnesium Composite (NMC) Materials A series of NMC materials was synthesized in a sol-gel synthesis that in-cludes three steps: i) sol-gel synthesis, ii) gel/powder formation and iii) dry-ing, as described below. i) Sol-gel synthesis: In a typical synthesis, MgO (Sigma-Aldrich) was mixed with methanol (Sigma-Aldrich) using a 1:15 solid:liquid ratio, in a reaction vessel. The vessel was sealed and a CO2 (AirLiquide) pressure of 1-4 bar was applied. The reaction mixture was left stirring for 24-72 hours at 25-50 °C, and after this the CO2 pressure was released. The mixture was now a cloudy, off-white solution.

Since some MgO particles did not react during this synthesis procedure, the solution was centrifuged at 5000 rpm (4696g) for 60 min to obtain an optically clear, yellow-colored liquid (Paper III). The separated solid parti-cles were identified as MgO using powder X-ray Diffraction (XRD) and were discarded.

26

ii) Gel/powder formation: Upon solvent evaporation in a ventilated area, the solution formed an alcogel. The alcogel subsequently started to break up into small, wet particles. The particles were dried in air at 70 °C (Papers I and II) or under a flow of nitrogen (N2) gas at 85 °C (Paper III) for a mini-mum of 6 hours. iii) Drying: The powder was then dried at 300 °C for 10 hours to form a dry, opaque, white powder (Paper II). In Paper III the dried powder was first kept at 150 °C for 3 hours and then at 250 °C overnight, both under N2 flow. The resulting powder in Paper III was optically transparent. The samples are summarized in Table 1 and the results from the different synthesis steps are shown in Figure 7.

Table 1. Summary of the NMC samples.

Sample

name

Description Paper

NMC-h70 Synthesized according to steps i and ii, i.e. excluding the drying

step. The sample particles were formed from evaporation of the

synthesis liquid at 70 °C.

I

NMC-h300

Synthesized according to all three synthesis steps (i, ii and iii). The

sample particles were formed by evaporation of the synthesis liquid

at 70 °C. The formed particles were dried at 300 °C in air, using a 10

hour ramping time from ~25 °C, and a 10 hour hold time.

II

NMC-c Synthesized according to all three synthesis steps (i, ii and iii). The

synthesis liquid was centrifuged in order to remove the unreacted

MgO particles. The transparent particles were formed by evapora-

tion of the synthesis liquid at 85 °C under N2 flow. The formed

particles were first dried at 150 °C for 3 hours and then finally at

250 °C over night, both under N2 flow.

III

27

Figure 7. The appearance of A) the NMC-c reaction mixture at the different synthesis steps (1: reaction liquid after centrifugation; 2 and 3: gel formed after removal of CO2 pressure and start of evaporation; 4: particles dried at ~25 °C, and 5: dried transparent particles); B) the resulting transparent particles; and C) a higher magnification of the resulting transparent particles. Adapted from Paper III.

4.1.2 Material characterization The NMC samples synthesized in this thesis were characterized using a number of analytical techniques. See the Appendix: Analytical techniques for a more detailed description of some of these techniques.

X-ray Photoelectron spectroscopy (XPS), Raman spectroscopy and Atten-uated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectrosco-py were used to identify the chemical groups in the NMC samples. XPS was performed using a Phi Quantum 2000 Instrument from Physical Instruments equipped with a monochromated Al-Kα1 source. The spectra were calibrated against the O1s peak of MgO (531.0 eV) [72]. The Raman analyses were performed on a Reinshaw Ramanscope equipped with an Ar-laser, calibrated using the Si band at 521 cm-1. A Bruker IFS 66v/S spectrometer and an ATR cell from SENSIR, with 4 cm-1 resolution, were used for the IR spectrosco-py.

The synthesis of NMC-c was followed by IR spectroscopy using a Varian 610-IR FTIR instrument equipped with a Specac Goldengate ATR accesso-ry, with 4 cm-1 resolution.

28

Powder XRD was used to study the crystallinity of the NMC samples. The XRD patterns were recorded with a Siemens/Bruker D5000 instrument using Cu-Kα radiation (λ = 1.54 Å). Samples were ground and put on a zero back-ground silicon sample holder prior to analysis.

N2 gas sorption was used to study the textural properties of the NMC samples, i.e. to calculate Branauer-Emmet-Teller (BET) surface area [73], total pore volume and pore diameter distribution. The pore diameter distribu-tions were calculated using the Density Functional Theory (DFT) model for slit-shaped pores [74, 75]. An ASAP 2020 volumetric surface area analyzer from Micromeritics was used for the porosity measurements.

Scanning Electron Microscopy (SEM) was used to study the morphology of the NMC samples. The SEM images were obtained using a LEO 1550 SEM microscope from Zeiss, equipped with a Schottky field emission gun, operated with an accelerating voltage of 5-10 kV. All samples were coated with a thin layer of gold/palladium prior to analysis. An in-lens detector was used to collect the secondary electrons.

Transmission Electron Microscopy (TEM) was used to obtain high reso-lution (HR) images of the NMC samples and structural information about NMC-c. The HRTEM images were recorded using a JEOL-3010 microscope operating at 300 kV, and a JEOL JEM-2100 operating at 200 kV.

Thermogravimetric analysis (TGA) was used to study the decomposition of the NMC samples, and to calculate the amount of MgCO3 and MgO in the NMC-c samples. An SDTA851e TGA instrument from Mettler Toledo was used for the analyses. The samples were heated from room temperature us-ing a constant heating rate of 5 or 10 ºC/min in air, up to 750 or 850 °C.

Evolved Gas Analysis (EGA) was used to identify the decomposition products of NMC-h70. For this, a TGA Q5000 instrument coupled to an FTIR Nicolet iS10, with a TGA/FTIR interface, was used. During the analy-sis the temperature of the gas tube between the two instruments was set at 220 ºC. The analysis was performed in N2 atmosphere.

Dynamic Light Scattering (DLS) experiments were performed on the syn-thesis liquid for NMC-c in order to study the growth in particle size during the reaction. A Malvern Instruments ZetaSizer NanoZS instrument was used for the analysis. The reported values are the means and standard deviations of measurements from triplicate samples.

Total X-ray Scattering and Pair Distribution Function (PDF) analysis was performed in order to study the short range order in NMC-c. The total scattering measurements were performed on a PANalytical Empyrean multi-purpose diffractometer equipped with a sealed high resolution X-ray tube with a silver anode (λ = 0.5609 Å for Ag Kα) and a solid state GaliPIX3D detector. The sample was loaded into a glass capillary with a 2.0 mm exter-nal diameter. An empty capillary of the same type was measured in the same way subtraction of background noise.

29

Elemental analysis of NMC-c was performed using Carbon, Hydrogen, Nitrogen (CHN) analysis and Inductive Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Both analyses were performed by MEDAC Ltd, UK. For the ICP-OES experiments, solid samples were digested using nitric acid (HNO3).

30

4.2 Results and discussion

4.2.1 Synthesis and nanostructure of NMC Mixing MgO with methanol under CO2 pressure and drying the synthesis liquid at 70 °C, resulted in a white, coarse powder, called NMC-h70 herein, Figures 8A and B. The material was composed of amorphous MgCO3 and crystalline MgO, as evidenced by powder XRD, Raman and IR spectroscopy and XPS Figure 9. The X-ray diffractogram showed a hump at ~ 30 degrees which probably stemmed from amorphous MgCO3 while the peaks corre-spond to those expected for MgO, Figure 9A. One band in the Raman spec-trum and three bands in the IR spectrum all indicated the presence of CO3 groups [76], Figures 9B and C. In the XPS Mg2p spectrum the peak was de-termined to be caused by MgCO3, Figure 9D, and in the O1s spectrum the three peaks were determined to be caused by MgO, MgCO3 [77] and ad-sorbed H2O [78, 79], respectively, Figure 9E (Paper I).

Figure 8. A) Photograph and B) SEM micrograph of NMC-h70. Reprinted from Papers II and IV.

31

Figure 9. Characterization of NMC-h70. A) Powder XRD pattern: the halo at 2ϴ ~ 30º was caused by amorphous MgCO3; the peaks at 2ϴ ~ 43º and 62º were cuased by MgO. B) Raman spectrum: the band at ~1100 cm-1 was determined to be caused by the CO3 group. C) IR spec-trum: the three bands at 850, 1100 and 1440 cm-1 were all determined to be caused by CO3

group. D) XPS Mg2p spectrum: the peak at 52.1 eV was explained by the presence of MgCO3 (red line, squares). E) XPS O1s spectrum: the peak at 531.0 eV was explained by MgO (yel-low line, triangles), the peak at 533.5 eV was explained by MgCO3 (red line, squares) and the peak at 535.6 eV was explained by adsorbed H2O (green line, tilted squares). Reprinted from Paper I.

NMC-c (the material formed after centrifugation) was X-ray amorphous and optically transparent, Figure 7, Section 4.1, in contrast to the opaque, non-centrifuged, NMC-h70, Figure 8A; this indicated that the MgO had been removed. However, NMC-c still contained around 14-18 % (w/w) of MgO as assessed by CHN, ICP-OES and TGA analyses, Table 2. This 14-18 % (w/w) gives a stoichiometric MgO content of about 32-46 % (n/n) (Paper III).

Table 2. MgO content of NCM-c, calculated from CHN, ICP and TGA analyses. Adapted from Paper III.

Analysis technique CHN ICP TGA

MgO content (% (w/w)) 17.6 17.6 13.8

32

TEM micrographs of NMC-c showed that it was a composite material that consisted of nanometer-sized, randomly oriented, MgO crystals sur-rounded by an amorphous layer, most likely of MgCO3. HRTEM, Figure 10A, showed that MgO was present in the material as single or aggregated nanocrystals (with diameters < 5 nm); these are seen at higher magnification in Figure 10B, featured by a ring-like Fast Fourier Transform (FFT) electron diffraction pattern, Figure 10C. The d-spacing of the crystals was in agree-ment with values reported in the literature for MgO: 0.219 and 0.153 nm, corresponding to the {200} and {220} planes, respectively (Paper III).

Figure 10. A) HRTEM image of an NMC-c particle; crystalline grains marked red. B) Higher magnification of the crystalline region in the green square in A and C) FFT electron diffrac-tion pattern for the crystalline part of the particle. Adapted from Paper III.

33

PDF analyses using data from X-ray total scattering were used to study the X-ray amorphous NMC-c material, Figure 11. Most of the PDF fit was explained by amorphous MgCO3 [80] but the fit was slightly improved by including MgO. The PDF further shows that the material has very little long range order, since the atom-atom correlations significantly diminished at r > 5 Å. Peaks below r ~ 1 Å were artifacts from data termination or imperfect corrections [81] (Paper III).

Figure 11. PDF of NMC-c. A) No structural features were visible at distances > 5 Å, indicat-ing lack of long-range order. B) Higher magnification of the PDF. The nearest neighbor dis-tances related to MgCO3 were assigned to C-O (at 1.27 Å), Mg-O (at 2.10 Å), O-O (at 2.77 Å), Mg-C (at 3.10 Å) and Mg-O (at 4.14 Å). MgO nearest neighbor distances were assigned to Mg-O (2.14 Å), Mg-Mg (3.02 Å), Mg-O (3.70 Å) and Mg-Mg (4.27 Å). Adapted from Paper III.

34

4.2.2 Formation of NMC When MgO and methanol were mixed together to synthesize the NMC sam-ples, the synthesis liquid first appeared as a cloudy, white mixture. This was not surprising since MgO is almost insoluble in methanol (solubility ~ 0.1 %) [82]. However, when the same mixture was subjected to CO2 at ~1-4 bar, it turned less cloudy and slightly yellow after ~ 24 hours, possibly indicating part dissolution of MgO (Papers I and III). When centrifuged, the solution became optically transparent and slightly yellow, Figure 7, Section 4.1 (Pa-per III). It should be noted that MMC is yellow (in methanol and dimethyl-formamide) and might be present in the reaction mixture at this stage. MMC has been shown to form in the reaction between magnesium methoxide (Mg(OCH3)2) and CO2 [52, 54, 83]. MMC is also known to form MgCO3, methanol and CO2 upon decomposition [52, 84, 85]. This supports the sug-gestion that MMC could be part of the reaction mixture.

During solvent evaporation of NMC-c, the solution first formed a gel and then formed transparent, solid, particles, Figure 7, Section 4.1. This transi-tion was followed by IR spectroscopy, Figure 12, but no chemical reactions could be identified using this technique. It was, however, noted that IR bands related to the CO3 group were present in all the spectra. The presence of these bands suggests that either the amorphous MgCO3 in NMC or MMC are formed during the early stages of the synthesis, since it is not possible to distinguish the two in a methanol solution using IR (Paper III).

35

Figure 12. IR spectra of NMC-c during the different reaction stages: A) reaction solution, B) gel, C) wet powder (dried at room temperature, ~25 °C and D) powder dried at 250 °C. Adapted from Paper III.

36

4.2.3 Pores in NMC The textural properties of NMC-h70 were analyzed using TEM and N2 sorp-tion. A TEM micrograph of the powder, Figure 13, showed spots of light contrast which indicated that the material was nanoporous. The N2 sorption isotherm for the as-synthesized material, Figure 14A, confirmed that the material was indeed nanoporous. The shape of the isotherm was indicative of a microporous material [1]. The corresponding pore diameter distribution, obtained using the DFT-based method for pore diameter determinations, gave two maxima at 1.5 and 2.5 nm, Figure 14B (Paper II).

After being stored in air at ~25 °C for approximately 1 month, the BET surface area of the material was decreased and the isotherm had a typical Type IV shape according to the IUPAC classification, which is an indication of mesopores [1], Figure 14C. As expected from the shape of the isotherm, the calculated pore diameter distribution for the material was shifted towards larger pores and the total pore volume was increased, Figure 14D and Table 3 (Paper II).

An even longer storage time had only a marginal effect on the calculated surface area, pore diameter and total pore volume. However, as shown by the EGA analysis of the NMC-h70 material stored for three months, discussed in Section 4.2.4, organic groups from the synthesis still remained in the NMC-h70 samples. After removal of these groups, by heat treatment at above 250 °C, the BET surface area was decreased and the calculated total pore volume and pore diameter slightly increased, Figure 14E, 14F and Table 3. After this heat treatment, the material was stable in terms of pore diameter, BET surface area and total pore volume (Paper II).

Figure 13. TEM micrograph of NMC-h70 shown in dark contrast due to the structure of the material.

37

Table 3. BET surface area, total pore volume and pore diameter for NMC-h70. Data ob-

tained from Paper II.

Surface area

[m2 g-1]a Total pore volume

[cm3g-1]b

Pore diameter

[nm]c

After synthesis, dried at 70

°C 638 0.36 2.5

1 month at 70 °C 397 0.50 4.7

Heat treated at 300 °C 265 0.42 5.5

a Calculated with the BET equation [73]. b Single point adsorption at P/P0 ̴ 0.99. c Established by the DFT method using the N2 isotherm, pore diameter value from the mode in the incremental pore diameter plot (Figure 14B, 14D, 14F).

Figure 14. N2 isotherms and corresponding DFT-based incremental pore diameterdistribution for NMCh-70: A) as-synthesized, B) stored at 70 °C for 1 month and E) heat-treated at 300 °C. Data obtained from Paper II.

38

Mechanism for pore formation The pores in NMC formed upon solvent evaporation when the CO2 dissolved in the reaction mixture left as gas bubbles. If depressurized at temperatures > 50 °C, this could be seen as a swelling of the gel. The gel consisted of na-nometer-sized particles, as evidenced by Tyndall scattering11, which was observed when a red laser was shone through the reaction solution for NMC-c, Figures 15A and 15B. Nanometer-sized aggregates of around 50-100 nm were detected in the reaction solution using DLS. DLS also showed that these aggregates increased in size with time when the solution was left standing slightly covered (i.e. without active evaporation), at ~25 °C, Figure 15C (Paper III).

Two types of pores were commonly found in the NMC samples, as evi-denced by the pore diameter distributions in Figures 14B, 14D and 14F. The first type were micropores (diameter <~2 nm); these pores are believed to be spaces between the agglomerated nanometer-sized particles. It should, how-ever, be noted that the DFT method for pore diameter calculations might have resulted in some errors in this pore diameter region since the relative pressure (P/P0) for filling micropores coincides with the P/P0 for monolayer formation, which may have resulted in underestimation of the pore diameter in this size range [86].

The second type were mesopores formed by CO2 gas bubbles. The aver-age diameter of these mesopores can be varied by adjusting the gel for-mation/evaporation rate. To be eliminated from the reaction liquid, CO2 molecules have to travel to the liquid/air interface; prior to reaching the air they aggregate and form gas bubbles. The nanometer-sized particles aggre-gate around these gas bubbles during the evaporation process, Figure 16. In this way the average size of the bubble renders the average pore diameter in

Figure 15. Tyndall scattering observed in the synthesis mixture of A) NMC-c and B) methanol used as a reference. C) Growth of nanometer-sized particles during evaporation of said syn-thesis mixture. Data points show the means from three measurements and the variance (error bars). Adapted from Paper III.

11 Light scattered by particles in a colloid or suspension.

39

the NMC material. A low energy input, e.g. a low temperature, during the gel formation allowed the CO2 molecules to aggregate together more than occurred with high temperatures, and hence to form larger bubbles. In this way it was possible to control the average diameter of the mesopores from ~3 nm to ~20 nm, Table 4. Stirring during the gel formation is another type of energy input; using stirring during the evaporation resulted in smaller pores than when no stirring was used, at the same temperature, see Samples D and E, Table 4 (Paper III).

Figure 16. Schematic description of the mechanism and energy input, e.g. temperature de-pendence, related to the pore formation in NMC. Adapted from Paper III.

40

Table 4. Gel/powder formation temperature, BET surface area, pore diameter and total pore

volume of NMC samples formed using different gel formation temperatures. Mechanical

stirring was used during the gel formation for all samples except D. Adapted from Paper III.

Sample Gel/powder for-

mation temperature

[°C]

Surface area

[m2/g]a

Pore

diameter [nm]b

Total pore

volume [cm3/g]c

A -20 263 ~13 1.27

B 10 297 ~10 1.15

C 20 499 ~6.5 0.97

D* 20 173 ~20 1.00

E 30 618 ~5.1 0.76

F 50 661 ~3.4 0.60

G 80 690 ~2.9 0.50

* Without stirring. a Calculated with the BET equation [73]. b Established by the DFT method using the N2 isotherm, pore diameter value from the mmode in the incremental pore size plot c Single point adsorption at P/P0 ̴ 0.99

41

4.2.4 Stability of NMC The stability of NMC was analyzed in terms of pore diameter and crystalli-zation. As described in Section 4.2.3 above, the pore diameter of NMC-h70 was not stable; this was due to remaining hydroxyl (-OH) and methoxyl (-OCH3) groups in the sample, as detected by EGA. EGA, Figure 17, showed that the material released H2O and CO2 upon decomposition at temperatures below 250 ºC. No changes in the XRD pattern for NMC-h70 were seen be-fore or after heat treatment at 300 °C. Hence, it was clear that the decompo-sition products formed prior to that were from organic groups that remained in the material from the synthesis. H2O and CO2 are formed upon decompo-sition of -OCH3 and -OH groups in N2 atmosphere, Reaction 1 [87]. It could therefore be concluded that these groups remained in the material after syn-thesis (Paper II).

-OCH3 + 2 –OH → H2O + CO2 + H2 (1)

Figure 17. EGA spectra for NMC-h70 stored for 3 months showing the decomposition prod-ucts (CO2 and H2O) from the material as a function of temperature. Reprinted from Paper II.

42

NMC-h70 with its remaining organic groups was metastable; it did not crystallize after 11 weeks of storage in an atmosphere saturated with H2O vapor. In contrast, NMC-h300 crystallized after 2 weeks when subjected to the same conditions, Figure 18. This indicated that the organic groups in NMC-h70 protected the surface of the amorphous MgCO3 from H2O mole-cules, which induce crystallization; this has also been shown in papers not included in this thesis [88, 89] (Paper II).

Figure 18. Powder XRD pattern for NMC-h300, stored in an atmosphere saturated with water vapor. The peaks correspond to those expected for nesquehonite, (MgCO3)(OH)·2H2O. Re-printed from Paper II.

43

5. Biocompatibility of NMC

The many possible biomedical applications suggested for mesoporous mate-rials [7, 28-31, 62, 90-96] demand evaluation of their biocompatibility. It is foreseen that the synthesized mesoporous material presented in this thesis might be suitable for biomedical applications. NMC has, actually, already shown promising results in vitro in this area [95, 97]. As mentioned in Sec-tion 3.5, the biocompatibility of a nanomaterial cannot be predicted just from the chemical nature of the material and new studies are needed to ensure the safety of the nanoform. For all of these reasons it was important to evaluate the biocompatibility of NMC. The biocompatibility of NMC was analyzed by means of in vitro cytotoxicity and hemocompatibility as well as in vivo skin irritation and acute systemic toxicity. The results presented in this sec-tion are described in Papers IV and V.

5.1 Materials and Methods Three NMC samples were synthesized for the biocompatibility studies as described in Section 4.1.1 (excluding the centrifugation step); these materials were denoted NMC-bc1, NMC-bc2, and NMC-bc3, Table 5. After a final heat treatment at 250 °C, the particles were ground to reduce their size. Thereafter, two sieves were used to obtain samples with a controlled particle size (between 50 and 200 µm for NMC-bc1 and NMC-bc2 and between 50 and 100 µm for NMC-bc3). NMC-bc1 and NMC-bc3 were sterilized at 180 ºC for 30 minutes prior to the experiments [98]. The materials were analyzed using N2 sorption, SEM and IR spectroscopy as described in Section 4.1.2, and helium (He) pycnometry was used to measure the true density of the NMC-bc samples using an AccuPyc 1340 instrument from Micromertics. Six measurements were taken for each sample.

44

5.1.1 In vitro cytotoxicity Human dermal fibroblasts (hDf) were chosen as model cells for the in vitro cytotoxicity test. Dermal fibroblasts are cells within the dermis layer of the skin which are responsible for generating connective tissue and allow the skin to recover from injuries [63]. The test was performed according to the ISO guidelines for evaluation of biomaterials in vitro [99]. The negative control was culture medium and the positive control was 5 % (v/v) dimethyl-sulfoxide (DMSO) in culture medium.

The cell viability was evaluated after 24 and 48 hours of exposure to NMC-bc1 based on changes in morphology and proliferation using light microscopy and the alamar blue assay, respectively. Alamar blue is a non-toxic metabolic indicator of cell growth. On uptake into the cell, the alamar blue dye is reduced and changes from a non-fluorescent to a fluorescent form. The change in fluorescence correlates approximately with cell prolif-eration in the sample [100]. The fluorescence was measured at 560 nm exci-tation and 590 nm emission using a spectrofluorometer (Tecan infinite® M200).

Table 5. Material characteristics of the samples used for the biocompatibility studies. Obtained from Papers IV and V.

Sample Study Pore volume

[cm3/g]a

Surface area

[m2/g]b

Pore diameter

[nm]c

Density [g/cm3]d

Particle size [µm]e

NMC-bc1 Cytotoxicity and in vivo toxicity

0.34 207 5.3 2.28 50-200

NMC-bc2 In vitro he-mocompati-bility

0.90 322 9 2.21 50-100

NMC-bc3 IR of sesame oil extract

0.48 284 5.4 2.31 50-200

a Single point adsorption at P/P0 ~ 0.99. b Calculated with the BET equation [73]. c Calculated with the DFT method using the N2 isotherm, pore diameter value from the mode in the incremental pore size plot. d True density, calculated with He pyconometry. e Measured from SEM images.

45

5.1.2 In vitro hemocompatibility The hemocompatibility of NMC-bc2 was evaluated in vitro using a modified version of the loop model recently described by B. Ekstrand-Hammarström et al. [71], Figure 19, which uses non-anticoagulated human whole blood from healthy donors. The effect of NMC-bc2 on blood was assessed in terms of coagulation and complement activation as well as red blood cell lysis (hemolysis).

Fresh blood samples were drawn from seven healthy volunteers. The blood was collected in an open system with no soluble anticoagulant and was placed in falcon tubes internally coated with heparin. Any material that came in contact with the blood during the sampling was coated with heparin using the Corline method [101].

NMC-bc2 particles were added to the loops together with 2.0 ml of the freshly drawn blood, giving final concentrations of 0.05 mg/ml, 0.25 mg/ml, 1 mg/ml and 10 mg/ml. Control loops without particles were included in each experiment. All samples were run in duplicate.

Figure 19. Illustration of the blood model used for the hemocompatibility study: The model consists of loops that are internally coated with heparin. Freshly drawn human blood was added to the loops together with the NMC-bc2 particles. Adapted from Paper V.

46

The loops were closed and vertically rotated for 60 min at 37 ºC. After rota-tion, the blood was collected and mixed with EDTA-K3 or citrate. The blood was subsequently centrifuged and the plasma was collected and stored for analysis. Levels of TAT, C3a and sC5b-9 were analyzed using Enzyme-linked Immunosorbent Assays (ELISAs) using the EDTA-K3 treated blood. A more detailed description of the ELISA technique can be found in the Appendix: Analytical Techniques. The citrate-treated plasma was analyzed for hemolysis.

C3a was detected according to the technique described by Nilsson Ekdahl et al. [102]. Zymosan-activated serum, calibrated against a solution of puri-fied C3a, served as standard.

sC5b-9 was detected according to the method described by Mollnes et al. [68], using zymosan activated serum containing 4000 AU/ml as standard.

TAT was detected using wells coated with anti-human thrombin antibody (Enzyme Research Laboratories). Horseradish peroxidase-conjugated anti-human antithrombin antibody (Enzyme Research Laboratories) was used for detection. Pooled human serum diluted in normal citrate–phosphate–dextrose plasma was used as standard.

Hemolysis was measured spectrophotometrically by reading the absorb-ance at 540 nm [103]. The absorbance value for the plasma that had been in contact with the material was compared with the value for 1 % (v/v) Triton-X-treated blood which served as positive control. The positive control was assumed to yield 100 % hemolysis.

Calcium ion (Ca2+) exchange with NMC-bc2 In order to gain further understanding of the effects of NMC on blood, Ca2+ exchange with the NMC-bc2 sample was measured using the same particle concentrations used in the whole blood test. Standard solutions containing 0.05 mg/ml and 0.10 mg/ml of Ca2+, representing the concentrations of Ca2+ and total calcium in blood, respectively [104], were prepared using CaCl2. The levels of magnesium and calcium in the solution were measured using ICP-OES after 60 min incubation with the particles. ICP-OES was per-formed in the same way as described in Section 4.1.2.

5.1.3 In vivo studies Two in vivo toxicity studies were performed using NMC-bc1: skin irritation and acute systemic toxicity. Both studies were conducted at NAMSA® con-tract research laboratory, France, and followed the National Institute of Health guidelines for care and use of laboratory animals.

A skin irritation study was performed according to the ISO guidelines de-scribing skin irritation evaluation of biomaterials [105]. In short, two 2.5 cm ͯ 2.5 cm squares of sterile gauze, and two 2.5 cm ͯ 2.5 cm squares of sterile gauze containing NMC-bc1 were topically applied to the skin of three male

47

rabbits (New Zealand White) and left in place for 24 hours. The skin was observed 1, 24, 48 and 72 hours after removal of the gauze for signs of irrita-tion (erythema or edema).

An acute systemic toxicity study was performed according to the ISO guidelines describing acute systemic toxicity evaluation of biomaterials [106]. For the study two extracts of NMC-bc1 were prepared, one in sesame oil and one in 0.9 % NaCl, using 0.2 mg material / ml extraction vehicle. Five female mice (OF1 Ico) were injected with each extract, via the intraper-itoneal (IP) route. Five additional mice were injected with each extraction vehicle as controls. The mice were weighed 24, 48 and 72 hours after injec-tion and observed for adverse reactions immediately and 4, 24, 48 and 72 hours after injection. In addition to the in vivo study, the sesame oil extract was analyzed for the presence of leachables using IR spectroscopy. A spec-trum of the as-received sesame oil was compared with a spectrum of the sesame oil after sample extraction. The IR study was performed on a Bruker Tensor27 instrument using a Platinum ATR diamond cell, with 4 cm-1 reso-lution.

5.2 Results and discussion

5.2.1 In vitro cytotoxicity Direct cell tests were used to assess the cytotoxicity of NMC by evaluating the viability of dermal fibroblasts in contact with NMC-bc1 at four concen-trations: 1000, 500, 200 and 50 µg/ml, and two time points: 24 and 48 hours, Figures 20A and 20B. The cell viability was well above the 70 % toxicity limit defined in the ISO guidelines [105] after both 24 and 48 hours’

Figure 20. Cell viability of hDf cells exposed to NMC-bc1 particles after A) 24 and B) 48 hours. Data represent mean ± 95 % confidence interval for n = 5. Cell viability values larger than 70 % of the negative control indicate a non-cytotoxic effect. Reprinted from Paper IV.

48

Figure 21. Representative light microscopy images of hDf cells exposed to NMC-bc1 particle suspensions for 48 hours together with a positive control (5 % (v/v) DMSO) and negative control (cell culture medium). Scale bars 100 µm. Reprinted from Paper IV.

for all tested concentrations. Further, light microscopy images of the cells after 48 hours exposure to the particles, Figure 21, showed that the hDf cells adhered in large numbers and showed typical fibroblast morphology, compa-rable with the cell adhesion and morphology observed for the negative con-trol (cell culture medium). These results suggested that NMC-bc1 was non-toxic to hDf at concentrations up to 1000 µg/ml (Paper IV).

5.2.2 In vitro hemocompatibility The biocompatibility of NMC was further assessed in terms of hemocompat-ibility, specifically by looking at the effect of NMC-bc2 on blood coagula-tion and the complement system as well as its hemolytic activity. An in vitro model using fresh human whole blood from healthy donors, was used for the studies [71], Figure 19.

Activation of the coagulation cascade by NMC Generation of the TAT complex in plasma was used to monitor the coagula-tion system after blood contact with NMC-bc2, Figure 22A. Levels of TAT were measured after incubation of NMC-bc2 particles (concentrations 0.05, 0.25, 1.0, and 10 mg/ml) with human whole blood for 60 min at 37 °C in the in vitro blood model.

NMC-bc2 particles at concentrations between 0.25 and 10 mg/l did not promote significant generation of TAT. In fact, all NCM-bc2 particle con-centrations except 0.05 mg/ml induced a significant reduction of the levels of TAT complexes compared to the negative control, Figure 22A: from ~160 µg/l for the negative control to between 25 and 10 µg/l for the three highest

49

NMC-bc2 concentrations. Moreover, the three highest NMC-bc2 concentra-tions produced TAT levels comparable to the value found for the initial blood sample (0 min control). This anticoagulant effect was in contrast to the coagulant effect seen for certain inorganic and clay materials which has been attributed to their large surface area and extensive water sorption [90, 107-109]. The anticoagulant effect observed for NMC-bc2 was, however, in agreement with what has been seen for magnesium and its alloys, where none of the studied materials showed thrombogenic properties [110] (Paper V).

Activation of the complement system by NMC Generation of sC5b-9 and C3a in plasma was used to assess complement activation by NMC-bc2, Figures 22C and 22D. Levels of C3a and sC5b-9 were measured after incubation of NMC-bc2 particles (concentrations 0.05, 0.25, 1.0, and 10 mg/ml) with human whole blood for 60 min at 37 °C in the in vitro blood model.

Figure 22. The effect of NMC-bc2 particles on A) blood coagulation, measured as generation of thrombin-antithrombin (TAT) complexes, B) hemolytic activity of NMC-bc2 particles, measured as absorbance at 540 nm and presented as % of the positive control (1 % (v/v) Triton-X), and the effect of the particles on the complement system measured as generation of C) sC5b-9 and D) C3a. All measurements were performed in plasma after 60 min particle incubation with whole human blood at 37 °C. Data represent mean ± standard error of mean for n = 7. Lines illustrate the groups between which there is a significant difference (* p < 0.05). Adapted from Paper V.

50

All NMC-bc2 concentrations promoted significant generation of sC5b-9 compared with the initial sample, Figure 22C. However, although these val-ues tended to be higher than the negative control, only the value for 1 mg/ml was significantly different. The C3a values, Figure 22D, showed the same pattern, i.e. all studied NMC-bc2 concentrations significantly differed from the initial sample. Again, although NMC-bc2 concentrations between 0.05 and 1.0 mg/ml tended to promote higher values of C3a than the negative control, only 0.25 mg/ml was statistically significantly different. Other stud-ies of the effects of magnesium and its alloys on the complement system have shown an increase in sC5b-9 levels, i.e. indicating an activation of the complement system [110] (Paper V).

The absence of statistically significant differences between the C3a and sC5b-9 values found for NMC-bc2 and the negative control may be due to the large variation within the groups, which is common in biological sam-ples.

Red blood cell lysis by NMC particles The hemolytic activity of NMC-bc2 was measured as absorbance at 540 nm of plasma from blood incubated with NMC-bc2 particles for 60 min. The absorbance values were compared with a positive control, blood treated with 1 % (v/v) Triton-X, assumed to yield 100 % hemolysis, Figure 22B. Only 10 mg/ml, i.e. the highest concentration, of NMC-bc2 particles, led to signifi-cantly higher hemolytic activity than seen with the negative control, ~13 % against ~4 % (Paper V).

These results are in contrast to previous results, where pure magnesium was shown to significantly induce hemolysis [110], and where mesoporous silica particles had up to 90 % hemolytic activity [111]. The hemolytic ac-tivity of the tested silica particles in the study by Shi et al. [111] was signifi-cantly reduced when the pH was increased from 7.4 to 8.0, and was therefore attributed to the surface chemistry of the particles. At higher pHs the parti-cles had an increased negative charge. This led to increased electrostatic repulsion between the particles and red blood cells. The carbonate surface groups of NMC heat treated to 250 °C (Paper II) might explain the absence of hemolysis seen for the NMC-bc2 particles.

Ca2+ uptake by NMC Cations of calcium and magnesium play an important role in the cascades of blood. While the role of Ca2+ in the coagulation cascade is clear, the effects of Mg2+ are more debatable [112-114]. However, most studies indicate that Mg2+ has an anticoagulant effect since it competes with Ca2+ for the clotting factors. In line with this, the uptake of Ca2+ by NMC-bc2 and the simultane-ous release and/or exchange of Mg2+ were measured.

The Ca2+ uptake study, Figure 23, showed that the removal of Ca2+ from the standard solutions increased with increasing NMC-bc2 concentrations

51

and seemed to reach a plateau at 1 mg/ml of NMC-bc2. The plateau repre-sents ~90 % Ca2+ uptake. The levels of magnesium were measured in the same solution; however, the results were higher than the starting Ca2+ con-centrations for NMC-bc2 concentrations of 1 and 10 mg/ml. Nonetheless, Mg2+ and Ca2+ are exchangeable cations when adsorbed at different calcium- and/or magnesium-containing minerals, e.g. magnesite [115]. It was there-fore assumed that the uptake of Ca2+ was partially due to ion exchange with Mg2+, although another mechanism must have been involved as well. There-fore, some amounts of Mg2+ were present in the blood after contact with NMC-bc2 (Paper V).

The low levels of Ca2+ in the blood were hypothesized to be the reason behind the observed anticoagulant effect, Figure 22A. The Mg2+ levels pre-sent in blood could have further contributed to the anticoagulant effect by competing with Ca2+ for coagulation factors. The levels of Ca2+ and Mg2+ in blood influence not only the blood coagulation but also the complement system. The complement system may be activated via either the classical pathway (CP) or the alternative pathway (AP) by contact with biomaterials [116]. While the CP requires both Mg2+ and Ca2+, only Mg2+ is needed for activation via the AP [117]. Thus, the observed activation of the complement system by NMC-bc2 was most likely via the AP (Paper V).

Figure 23. Fractions of Ca2+ taken up by NMC-bc2 after incubation with Ca2+ solutions for 60 min under agitation. Two concentrations of Ca2+ were tested: 0.05 g/l (■) and 0.1 g/l (▲). Adapted from Paper V.

52

5.2.3 In vivo tests Two in vivo studies were conducted for NMC using NMC-bc1: skin irrita-tion and acute systemic toxicity.

Skin irritation In the skin irritation study there were no signs of erythema12 or edema13 on the skin of the rabbits after NMC-bc1 application for up to 48 hours. The scoring from the observations, Table 6, indicated that the cutaneous reaction was negligible after application of NMC-bc1 powder (Paper IV).

Acute systemic toxicity Mice were used for the in vivo acute systemic toxicity study; all animals appeared normal during the study and there was no mortality. The body weight data from the study can be found in Table 7. The weights of the ani-mals injected with the polar 0.9 % NaCl extract increased similarly to those of the control group; there were no significant differences between the two groups. Hence, there was no evidence of systemic toxicity after injection using the polar extract. In contrast to this, the mice that had been injected with the non-polar sesame oil extract showed significant weight loss 24 hours after injection. Consequently, significant systemic toxicity was ob-served using the non-polar extract. However, it was noted that all the mice had recovered their initial body weight 72 hours after injection with the non-polar extract (Paper IV).

At first the acute systemic toxicity results seemed to suggest that the ma-terial released toxic substances in the non-polar extraction medium. Howev-er, IR spectroscopy was used to study the presence of possible leachables in the sesame oil extract of NMC-bc3. The spectrum of the extract appeared identical to the spectrum of the as-received sesame oil. Although IR spec-troscopy cannot detect all possible leachables, it was assumed that the ex-

12 Redness of skin. 13 Swelling.

Table 6. Dermal observations for the treatment and control (gauze) groups after NMC-bc1 powder application. Three rabbits, with two sites/rabbits were observed during the study; all sites on all animals resulted in scores of 0a for all time points. Reprinted from Paper IV.

Dermal observations scoring

Observations Sample 1h 24h 48h Erythema NMC-bc1 0 0 0 Control (gauze) 0 0 0 Edema NMC-bc1 0 0 0 Control (gauze) 0 0 0 a 0 stands for no signs of erythema or edema

53

tract medium was basically the same as the control medium. However, in contrast to the polar extract, the sesame oil extract appeared cloudy with many small particles in the solution. The particles caused the needles to clog and therefore the mice had to be injected several times using the sesame oil extract. Moreover, the two extraction vehicles differed not only in polarity but also in viscosity. The higher viscosity for the sesame oil extract most likely explained the presence of particles in that extract, i.e. the particles remained in suspension and therefore clogged the needles. It is likely that several particles were injected into the mice using the sesame oil extract. A likely explanation for the weight loss therefore seems to be the injection of particles (mechanical irritant after injection) rather than toxic leachables (Paper IV).

The injection of certain mesoporous silica particles (SBA-15, MCM-41 and MFC) via the IP route was fatal in one study, while injection of filtered extraction medium appeared non-toxic [93]. In our study, the mice regained their initial weight 72 hours after injection of the non-polar extract, further suggesting that the injection of particles caused the initial weight loss rather than the injection of toxic leachables. If centrifugation or decantation prior to injection were to be included in this experiment, it would be possible to check whether the particles were responsible for the systemic toxicity. This is not included in the ISO guidelines, however, so it was not carried out (Pa-per IV).

Table 7. Body weight data for the mice used in the acute systemic toxicity test. The treatment group was injected with extracts from NMC-bc1 while the control group was injected only with the extraction vehicle. Five animals were used in the study; the weight data represent means ± standard deviations for all animals. Reprinted from Paper IV.

Weight (g)

Extraction route T0a 24 h 48 h 72 h

NMC-bc1 powder 0.9 % NaCl 19.0 ± 0.6 19.4 ± 0.6 21.0 ± 0.5 22.3 ± 0.6

Control 0.9 % NaCl 18.3 ± 0.8 19.0 ± 0.8 19.9 ± 0.9 21.0 ± 0.9

NMC-bc1 powder Sesame oilb 19.1 ± 0.5 17.0 ± 0.4 18.8 ± 0.3 20.6 ± 0.5

Control Sesame oil 17.9 ± 0.3 18.6 ± 0.4 19.9 ± 0.4 21.2 ± 0.6 a Prior to injection. b The sesame oil extract had to be injected several times because of presence of particles.

54

6. Increasing drug dissolution rate with NMC

NMC has recently been shown to increase the dissolution rate of ibuprofen loaded into the mesopores [95, 97]. The dissolution rate was enhanced by stabilizing the drug in its amorphous state; the NMC particles completely suppressed crystallization. Furthermore, the release profile could be tuned by changing the particle size [97]. For the studies with ibuprofen, NMC heat treated to 250 °C with small pore diameters (5-10 nm) were used.

In this work, NMC-c with different pore diameters was used to stabilize the drug itraconazole (ITZ) in its amorphous state, and enhance its dissolu-tion. ITZ is an anti-fungal drug that is known for its low aqueous solubility (1 ng/ml at neutral pH and 4 µg/ml in gastric fluid, pH 1.2). It is also a rela-tively large molecule with a molecular weight of 705.6 g/mol [118]. The results presented in this section are described in Paper III.

6.1 Materials and Methods

6.1.1 Materials The loading and release of ITZ were tested using three NMC-c samples: A, D, and E, Table 4 Section 4.2.3, with pore diameters: small <5 nm, medium ~13 nm, and large ~20 nm. The samples were synthesized as described in Section 4.1.1 including the centrifugation step.

6.1.2 Drug loading and dissolution testing Dissolution testing is widely used in the pharmaceutical industry to provide in vitro kinetic release information; herein it was used to study the dissolu-tion of IZT as it was released from the NMC-c samples versus the dissolu-tion of the free drug.

IZT (3Pharmaceutica, China) was loaded into the sample by solvent evaporation for the dissolution test. Three NMC-c samples with different pore diameters were loaded with three different amounts of IZT from a solu-tion of methylene chloride (CH2Cl2) containing 10 mg/ml of IZT. To allow time for the loading to take place, the mixture was left shaking for 24 hours

55

at ~25 °C, after which the solvent was evaporated at 45 °C, and the loaded NMC-c samples were dried at 85 °C.

To mimic the in vivo milieu in the tests, simulated gastric fluid (pH 1.3) was used as dissolution medium. The dissolution was measured in a standard apparatus defined by the United States Pharmacopeial Convention (USP): the USP Dissolution Apparatus 2 – Paddle (37 °C) which is the most com-monly used apparatus for solid oral dosage forms [119]. UV/vis absorption of the fluid at λ= 263.1 nm was used to calculate the amount of released ITZ. The UV/vis spectra were recorded using a 1650PC UV/vis spectrophotome-ter.

Differential scanning calorimetry (DSC) was used to estimate the extent of crystallinity of the drug inside the pores of NMC-c. The DSC analysis was performed on a DSC Q2000 instrument from TA Instruments. Samples of 4.5-5.5 mg were weighed into 5 mm aluminium pans and sealed by a press. The samples were first cooled to -35 °C and then heated to 250 °C, using a heating rate of 3 °C/min. TGA was used to calculate the amount of drug loaded into the sample; the same instrument and analysis conditions as described in Section 4.1.2 were used.

6.2 Results and discussion

6.2.1 Loading of ITZ

DSC was used to study the structure of ITZ loaded into the pores of the NMC-c samples, Figures 24A, 24B, and 24C, and TGA was used to calcu-late the amount of drug loaded into the different NMC-c samples. The TGA analyses indicated that the percentages of drug inside the NMC-c samples were 30, 45 and 60 % (w/w), respectively. The endothermic peak at 169 °C in the DSC curves corresponds to the melting point of crystalline ITZ. This

Figure 24. DSC curves of ITZ loaded in NMC-c samples with A) 5 nm pores, B) 13 nm pores and C) 20 nm pores, at three different loading concentrations, as well as the DSC curve for the free drug. Adapted from Paper III.

56

peak was absent in all NMC-c samples loaded to 30 % (w/w), which con-firmed that crystallization of ITZ was completely suppressed. The results also indicated that the drug was inside the pores with only minor amounts present on the surface of the material [29].

Crystallization was not completely suppressed with higher loading in the NMC-c samples. However, crystallization was completely suppressed in the NMC-c samples with small and medium pore diameters loaded up to 45 % (w/w). A small hump, possibly indicating melting of the crystalline phase of the drug, was, however, present in the large pore sample for this concentra-tion and for all NMC-c samples when loaded to 60 % (w/w).

This indicated that there was some room for the drug to rearrange and crystallize in the pores with larger diameters, which emphasizes the im-portance of matching the pore diameter of the drug carrier with the size of the drug molecule. Since ITZ remained amorphous in all samples at 30 % (w/w) loading, these samples were used for the release/dissolution measure-ments (Paper III).

6.2.3 Release of ITZ

The dissolution of ITZ from NMC-c particles was measured in simulated gastric fluid, pH = 1.3, Figure 25. As evidenced by the figure, the dissolu-tion rate was enhanced by a factor of 23 when ITZ was released from the NMC-c sample with 20 nm diameter pores, as compared to the free drug, i.e. without any delivery vehicle. For the medium and small pore NMC-c sam-ples, the dissolution rate was enhanced by a factor of 17 and 13, respective-ly. The large pore NMC-c released all of the loaded drug molecules within 60 min; this was noticeably faster than the corresponding time for the small

Figure 25. Time-related release of IZT loaded into NMC-c samples, in simulated gastric fluid. The dissolution of ITZ as it was released from three averages pore diameters of NMC-c (5 nm, blue curve; 13 nm, green curve; and 20 nm, red curve) was compared with the dissolution of the free drug (black curve). The error bars represent variations over three measurements. The lines are drawn as guides for the eye. Adapted from Paper III.

57

and medium pore NMC-c samples, which reached 100 % release after 180 and 90 min, respectively. Because of the acidic conditions of the release medium, NMC-c started to dissolve noticably after a few hours, and after 24, hours all the powder was visibly dissolved (Paper III).

58

7. Summary and conclusions

The overall aim of the work presented herein was to develop a mesoporous material with well-defined pore diameters in which the pores were formed without the use of organic surfactants as pore-forming templates. The possi-bility of controlling and varying the pore diameters of the material was also an important aim of the work.

My work has resulted in the creation of a mesoporous magnesium car-bonate composite material called NMC herein which was formed in a low temperature process using methanol, MgO and CO2. It is composed of na-nometer-sized composite particles that consist of MgO crystals surrounded by amorphous MgCO3. These particles aggregate around CO2 gas bubbles during the evaporation of the solvent while drying the material. When the CO2 is removed, open pores are formed in the material. NMC has a narrow pore diameter distribution that till now has been almost exclusively found when surfactants are used as pore-forming templates. Furthermore, the pore diameter can be adjusted between ~3 nm and ~20 nm by controlling the en-ergy input (e.g. temperature and stirring) during the evaporation/drying step in the synthesis.

The material was predicted to be suitable for biomedical applications. Therefore, the biocompatibility of NMC was evaluated in terms of in vitro cytotoxicity and hemocompatibility, in vivo skin irritation and acute system-ic toxicity. NMC was found to be non-toxic in the in vitro cytotoxicity and in vivo skin irritation tests as well as in the acute systemic toxicity test using a polar extraction vehicle. Although signs of toxicity were observed in the acute systemic toxicity test with a non-polar extraction vehicle, this toxicity was attributed to injection of particles rather than toxic leachables from the material. The hemocompatibility of NMC was analyzed in an in vitro loop model, using whole human blood, which investigated complement and coag-ulation activation as well as hemolysis. It was found that the material pos-sesses anticoagulant properties, most likely due to the uptake of Ca2+. Acti-vation of the complement system was evaluated by measuring the levels of C3a and sC5b-9 in plasma. Moderate to high levels of C3a and sC5b-9 were observed after contact with NMC; however, they were only significantly different from the negative control with NMC concentrations of 0.25 mg/ml for C3a, and 1.0 mg/ml for sC5b-9. Moreover, no hemolytic activity was found for the material at concentrations up to 1 mg/ml and higher concentra-tions (10 mg/ml) were associated with only ~13 % hemolysis.

59

As evidenced by the work presented herein, NMC provides great potential for the development of new drug formulations of APIs with poor aqueous solubility. NMC suppressed crystallization of the poorly soluble antifungal drug molecule ITZ, resulting in faster dissolution from NMC than seen with the free drug. Further, by tuning the pore diameter of NMC it was possible to tailor the drug release rate from the material.

60

8. Future work

Since NMC is new in the mesoporous field, there are still many properties and possible applications to be investigated. For example, post-synthesis modification could increase the stability with regard to crystallization of the material, and further tailor the release properties of APIs from the pores. In this work, only ITZ was investigated and there are of course a number of other interesting drugs and active compounds eligible for future studies.

The fast release of ITZ formulated with NMC is indicative of an im-proved apparent solubility of the substance, how this translates into a higher bioavailability or faster onset of action will be evaluated in vivo in coming studies. The increase in apparent solubility will also be analyzed more in detail in in vitro studies. However, the results presented herein using the model substance ITZ shows promising data for the applicability of using NMC as solubility enhancer of different APIs.

For NMC to be used in biomedical applications, such as drug delivery, it is essential to know the biocompatibility of the material. This work merely presents the start of these evaluations. There are a number of additional stud-ies required; e.g. investigation of oral toxicity. It is also important to evaluate whether the different NMC materials with different pore diameters differ in biocompatibility.

It was interesting to discover in this work that Ca2+ is taken up by NMC. This should be investigated for other ions as well, and might be useful for applications such as waste water cleaning.

As for the synthesis protocol for NMC, it could be of interest to vary the starting materials (MgO, methanol and CO2) to investigate if it is possible to form other types of mesoporous materials using the method described in this work.

61

9. Svensk sammanfattning

Denna avhandling handlar om ett alternativt sätt att tillverka mesoporösa material. Ett mesoporöst material innebär ett material med porer, eller hål-rum, mellan 2-50 nm i diameter. Mesoporösa material har många använd-ningsområden, inte minst i det biomedicinska fältet där de t.ex. kan användas för att öka upplösningshastigheten och lösligheten för läkemedel. Materialet som har tillverkats i denna avhandling väntas lämpa sig väl för vissa biome-dicinska tillämpningar.

Idag tillverkas merparten av mesoporösa material genom att man blandar beståndsdelarna av det ämne man vill ha i mesoporös form med ett, så kallat, porbildande ämne. Det porbildande ämnet består av en typ av organiska mo-lekyler som kallas för surfaktanter. Dessa surfaktanter bildar en tredimens-ionell struktur i en lösning som sedan materialets molekyler kan ordna sig kring. Slutligen så bränns surfaktanterna bort och det bildas ett material med porer inuti. Detta tillverkningssätt innebär att storleken på hålrumen precist kan styras, men även ett flertal syntessteg som är svåra att skala upp, samt att många av kemikalierna som används är både dyra och skadliga för miljön. Målet med denna avhandling var därför att komma fram till ett alternativt sätt att tillverka mesoporösa material, som även det innebar möjligheten att styra porstorleken, men där man inte använder de typer av kemikalier som beskrivits ovan.

I avhandlingen presenteras ett sätt att tillverka mesoporös magnesium-karbonat. Magnesiumkarbonat är ett ämne som idag används i t.ex. livsme-delsindustrin och därför anses vara ofarligt för människor. Materialet bildas från en lösning med startmaterialen, som i nästa steg bildar en gel och som slutligen torkas till ett torrt material. Porerna bildas i gelen runt gasbubblor av koldioxid som används vid tillverkningen. Detta illustreras i Figur 26. Storleken på gasbubblorna kan styras med t.ex. temperaturen, vilket innebär att pordiametern på det slutgiltiga materialet kan styras.

62

Figur 26. Schematisk beskrivning av tillverkning av mesoporös magnesiumkarbonat. Start-materialen blandas och regerar med varandra under koldioxidtryck. När koldioxidtrycket släpps och temperaturen höjs något blir vätskan till en gel, här bildas porerna kring koldioxid-gasbubblorna som frigörs när trycket släpps. Slutligen så torkas materialet och blir torr meso-porös magnesiumkarbonat.

Eftersom materialet antogs kunna användas för biomedicinska tillämpningar testades dess påverkan på hudceller, blod och även på djur i två in vivo stu-dier. In vivo studierna undersökte hudirritation och akut systemisk toxicitet. Materialet fanns vara ofarligt för celler, så som det testades här, och orsa-kade ingen hudirritation. I det akuta systemiska toxicitetstestet var materialet ofarligt när ett polärt lösningsmedel används, men toxiskt då ett opolärt lös-ningsmedel användes. Det senare antogs dock vara en effekt av att partiklar injicerades snarare än av materialet självt var toxiskt.

När materialet testades i kontakt med blod visade det sig ha anti-koagulanta egenskaper. Detta antogs vara en effekt av att materialet tog upp nästan alla kalciumjoner tillgängliga i blod, kalciumjonerna är nödvändiga för att blodet ska koagulera. Det visade sig även ha en viss påverkan på blo-dets komplementsystem som är en del av kroppens immunförsvar. Slutligen visade det sig att materialet inte förorsakade celldöd av röda blodceller, upp till koncentrationer så höga som 1 mg material/ml blod.

Avslutningsvis studerades materialets förmåga att öka upplösningshastig-heten av läkemedlet itraconazole, som är ett svårlösligt läkemedel mot svampinfektioner. Upplösningen mättes in vitro i ett lösningsmedium som liknar vätskan i magsäcken. I studien visade det sig att materialet avsevärt ökade upplösningshastigheten av läkemedelet, när det jämfördes med upp-lösningen av rent läkemedel som inte var inkorporerat i något läkemedels-bärarämne.

Denna avhandling kommer förhoppningsvis att vara till nytta för alla som vill hitta alternativa sätt att tillverka mesoporösa material utan användning av surfaktanter, men även för de som jobbar med mesoporös magnesiumkar-bonat för biomedicinska tillämpningar.

63

10. Acknowledgments

First and foremost, I would like to thank my supervisor Maria Strømme; thank you for the opportunity to perform my PhD studies under your guid-ance and for your confidence in me to design my own projects as well as your support and encouragement throughout these years. Your enthusiasm for this project has been truly inspiring!

I would also like to thank my co-supervisor Johan Gómez de la Torre. Thank you for teaching me how to be a researcher, both when it comes to working in the lab and how to write scientific documents. I would also like to thank you for not abandoning me when you left the building.

My co-supervisor, Natalia Ferraz, thank you for introducing me into the fascinating world of cells and blood. I am truly happy to have had the oppor-tunity to work with you, and I learned a lot!

Ocean Cheung, I am glad that you came to our group and that I got to work with you. Thanks for sharing your knowledge about porous materials with me and for proofreading this thesis. I also know now why one needs a “summer car” and that people actually drive to UK. I would also especially like to thank you and Andrew Kentaro-Inge for the image of porous materi-als used in this thesis.

My partner in crime Peng Zhang, thank you so much for so much!!! Thank you, in particular, for your help with synthesizing materials, proof-reading parts of this thesis, and for teaching me about Chinese traditions and recipes.

Albert Mihranyan, thank you for sharing some of your knowledge with me in our joint projects. Especially thank you for finding old, interesting Russian papers =).

Alfonso Garcia-Bennet thank you for teaching my about porous materials in general and gas adsorption in particular and also thanks for the TEM im-ages of my samples.

I like to thank my collaborators outside this group: Marco Sommariva, Kathryn Grandfield, Jaan Hong and Isabelle Pochard for our work together. I would also like to thank Lillemor Stenbeck-Funke, Jan Bohlin and Jan-Åke Gustafsson for invaluable technical support in the lab. And of course Jonatan Bagge for all computer-related help!

My super-master-students Christian Strietzel, Joakim Eriksson and Simon Gustafsson, thank you for being such good help in the lab and also for good times outside of the lab.

64

I would also like to thank all other past and present members of the NFM group, especially my office roommates for constructive and not so construc-tive conversations. Thank you Henrik for keeping me on my fingers and for decorating our office; Igor and I are trying to bring the plant back to life! Thank you Danne for lunch trips, especially to the outstanding restaurant Heat and for maintaining the tea assortment in the kitchen, and of course for proofreading the thesis. Christoffer, thank you for showing me infinite dif-ferent ways to tie a tie and for occasional Origin help. Petter, thanks for many nice chats, afterworks, nollningevents etc. both during our PhD studies and before (and hopefully after =) ). Hao, thank you for keeping track of Jonas, in case he wanders of I know who to call! Thank you Teresa for nice talks during lunches and coffee breaks and for always making Harald feel special when he visits the office, Jonas L thanks for fun discussions about “Kaffemannens” events in New Zealand, thank you Rikard (+ Etna) for keeping me updated about the NFM group during my parental leave, Martin thanks you for interesting scientific and food-related lunch discussions, and thank you Lisa and Mia for nice lunches at other places than Heat =)

I am also grateful to the DM-team, especially Mattias, Malin and Björn, that was there from the beginning, for being so interested in this research =)

My friends in the neighbouring departments also deserve be acknowl-edged: David for always having an arm chair ready when I need to escape the first floor, Adam for nice chats and help in the basement, Torbjörn and Ingrid for great fun at the Bioceramics conference in Barcelona and occa-sional coffee breaks, and Maryam for nice chats in the cell lab.

Jag vill tacka mina bästa och mest energiska supportrar mamma, pappa och Jens. Framförallt mamma och pappa för att ni alltid tror på mig och har fått mig att tro att jag kan göra allt jag vill. Den här avhandlingen är till er!

Men mest av allt tack världens bästa Jonas, som lär mig så mycket om det mesta och uppmuntrar mig att göra det jag vill och framför allt får mig att tro att jag kan. Och såklart tack för att du har läst och kommit med feed-back till hela den här avhandlingen. Du och Harald är det bästa jag har, jag älskar er!

65

Appendix: Analytical techniques

Nitrogen gas sorption Porous materials are often characterized using N2 gas sorption, in order to calculate specific surface area, total pore volume and pore diameter distribu-tion. This can be assessed from a plot of the amount of adsorbed gas against the P/P0, i.e. an isotherm, at the temperature when the gas condenses at P/P0 = 1, i.e. at 77 K for N2. In mesoporous materials, gas adsorption occurs via monolayer formation followed by multilayer adsorption and finally capillary condensation. Capillary condensation and evaporation do not occur at the same pressure in mesoporous materials, giving rise to a hysteresis. Mesopo-rous materials generally exhibit a Type IV isotherm according to the IUPAC classification [1]. A Type IV isotherm is recognized by the hysteresis and the round knee at low P/P0 indicative of monolayer coverage, Figure 27A.

The total pore volume (Vtot) is calculated from the amount of gas ad-sorbed close to saturation, i.e. P/P0 ~ 0.99. The BET surface area is calculat-ed from the amount of gas molecules needed to form a monolayer on the material, this is typically achieved between 0.05 and 0.3 P/P0 [73]. Finally, the isotherm is used to assess the pore diameter distribution of the material. There are a number of models available to calculate the pore diameter distri-bution of a material from the isotherm. In this thesis the DFT model [74, 75] was mainly used, and the pore diameter distribution was calculated from the adsorption branch of the isotherm. The DFT model is a complex mathemati-cal model of fluid interactions and pore geometry based on thermodynamic properties. Like most models of pore diameter distributions, it assumes that the gas condenses in the pores and that the volume of the pores is therefore the volume of the gas, converted to a liquid volume. In contrast to other pore size models, the DFT model assumes that the experimental isotherm consists of a number of individual “single pore isotherms” multiplied by their relative distribution. In order to obtain the pore diameter distribution for the sample a mathematical fit is made of the theoretical isotherms to the experimental one, Figure 27B.

66

Figure 27. A) N2 sorption isotherm and B) pore diameter distribution obtained from the iso-therm, for a NMC material. In the nitrogen isotherm (A) a marks the round knee seen at mon-olayer coverage and b marks the hysteresis typically seen for mesoporous materials.

Infrared Spectroscopy Chemical bonds within molecules and functional groups can vibrate and rotate. These vibrations and rotations can be excited by absorption of IR light if the light carries the exact amount of energy needed to induce a transi-tion from one vibrational level to another, and if the absorption induces a change in the dipole moment of the chemical bond. This absorption is often characteristic of specific functional groups and can therefore be used to iden-tify them.

In an FTIR spectrometer, all frequencies in the spectrum can be collected simultaneously, making it a quick analysis technique. The IR radiation, pro-duced by a light source, e.g. Globar, passes through an interferometer and then through the sample. The radiation is converted in the interferometer so that it is a function of time instead of frequencies. This allows the detector, e.g. deuterated triglycine sulphate, to detect all signals at once in a so-called interferogram, which is converted back to a spectrum using Fourier trans-formation.

IR transmission spectroscopy requires thin samples; thicker samples can be studied with an ATR accessory coupled to the FTIR spectrometer. With an ATR, the sample is pressed against the surface of an IR inert single crys-tal (e.g. diamond or ZnSe). The IR beam is reflected in the crystal and inter-acts with the sample at the interface with the crystal, and the sample absorbs its characteristic frequencies.

67

Thermogravimetric analysis TGA is an analytical technique that measures the weight loss of a material as a function of temperature. If the chemical composition of the material is known, the technique can be used to monitor the decomposition of the mate-rial and to calculate the relative amounts of different chemical groups. A TGA instrument can also be coupled to an IR spectrometer to identify the decomposition products from the material, in an EGA. Studying the decom-position products of a material allow the relative amounts of different chem-ical groups in the material to be calculated.

Scanning electron microscopy A SEM microscope is often used to study the morphology and topography of dry samples, as it gives high resolution images of surfaces. In a SEM micro-scope electrons are used to create the image of the sample, instead of pho-tons as in optical microscopy. A beam of electrons interacts with the surface of the sample in a manner so that electrons are emitted from the sample. The emitted electrons give information about the topography and the elemental composition of the surface, depending on the type of electrons emitted. Sec-ondary electrons mainly give topographical contrast, while back-scattered electrons primarily give compositional information.

In order for the technique to work well, the sample surface has to be elec-trically conductive; if this is not the case the surface can be covered by a thin layer of a conducting material, e.g. gold or palladium.

Transmission electron microscopy Just as in SEM, TEM uses an electron beam to study a material. In a TEM microscope, the high-energy electron beam is transmitted through the sample and focused into an image. The image is either projected onto a fluorescent screen or detected using a charged coupled device camera. In a TEM micro-scope it is possible to get information about the sample down to the atomic scale. In a TEM image of a sample, dark contrast corresponds to high elec-tron density areas and light contrast corresponds to areas of less electron density [120]. Hence, for a mesoporous material, the lighter parts in the im-age likely correspond to the pores.

In addition to providing an image of the sample, TEM can provide crys-tallographic information. In crystalline samples, i.e. long-range ordered samples, some electrons will be diffracted when they hit the sample; these can then provide a diffraction pattern for the sample. In this way TEM can be used to identify crystal structures.

68

X-ray Scattering Materials with short and/or long range order can be analyzed by elastic X-ray scattering. When scattered with X-rays, the order in the material will cause constructive interference and a diffractogram can be obtained by plot-ting the intensity of the scattered X-rays as a function of the angle of diffrac-tion, 2ϴ. If there is long range order in the sample this will result in peaks in a powder diffractogram. In contrast, if there is only short range order this will be seen as humps in the diffractogram instead, Figure 28. Standard XRD techniques can be used to study the former and PDF techniques can be used to study the latter. Powder XRD is typically used to identify crystalline phases in a sample, since each crystalline phase gives rise to a characteristic diffraction pattern.

PDF analysis of the diffractogram can be used to study materials with lit-tle or no low range order. PDF gives information about the interatomic dis-tances in a sample. The analysis describes the probability of finding two atoms at a given interatomic distance, d. The main principles of PDF are schematically described in Figure 29.

Figure 28. Powder X-ray diffractogram for a NMC sample. The two sharp peaks are due to the crystalline parts in the sample while the hump is from the amorphous part.

69

Figure 29. Principle of total scattering PDF. Interatomic distances d cause maxima in the PDF. The area below the peaks corresponds to the number of neighbours, scaled by the scat-tering power of respective atoms.

X-ray photoelectron spectroscopy XPS is used to study the elemental composition and chemical state of a sam-ple; it is both a qualitative and quantitative characterization technique. The technique is highly surface sensitive; when using standard Al Kα radiation, only the outermost surface layers of the sample are analyzed.

XPS makes use of the photoelectric effect: when X-rays are irradiated against a surface photoelectrons are ejected from the material. If the energy of the incoming X-ray photons (hν) and the work function (Ф) (constant for the instrument) are known and the kinetic energy of the emitted photoelec-trons (Ekinetic) is measured, the binding energy of the electrons (Ebinding) can be calculated. The equation can be written as:

Ф

The output of an XPS measurement is a spectrum with intensity as a function of energy, generally Ebinding , where the peak positions correspond to energy levels in the sample. Each element has a characteristic binding energy range, and the number of detected electrons can be used to quantify a specific ele-ment in the sample. All elements with Z ≥ 3 (Li) can be detected by the technique. The exact binding energy will depend on the chemical environ-ment of the atom, giving rise to chemical shifts. The chemical shifts can be used for identification of the chemical state of the studied sample. When

70

studying MgCO3, the spectra were calibrated against the O1s peak for MgO (531.0 eV) [72].

Enzyme-linked immunosorbent assay An ELISA is a highly sensitive immunoassay for qualitative and quantitative measurement of an analyte, usually proteins or other substances with anti-genic properties. The technique is based on the interaction between an anti-body and its antigen. The sandwich ELISA measures the amount of antigen between two layers of antibodies: one antibody is immobilized on a solid support, this one is used to capture the antigen, and the second antibody is in solution and is used to detect the antigen. The detecting antibody is generally conjugated with an enzyme, giving rise to a color change in the presence of an enzyme substrate; the color change is proportional to the concentration of antigen in the solution [121]. The method is illustrated in Figure 30. Another set-up is to use detecting antibodies that are biotinylated, followed by en-zyme-conjugated streptavidin.

Figure 30. Illustration of the ELISA method for detection of antigens: A capturing antibody is used to capture the protein of interest (sample). In the next step an enzyme-conjugated detect-ing antibody binds to the protein and when the enzyme substrate is added a detectable color change is induced.

71

References

[1] J. Rouquerol, D. Avnir, C. Fairbridge, D. Everett, J. Haynes, N. Pernicone, J. Ramsay, K. Sing and K. Unger, Pure and Applied Chemistry, 66 (1994) 1739-1758.

[2] C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli and J. S. Beck, Nature, 359 (1992) 710-712.

[3] T. Yanagisawa, T. Shimizu, K. Kuroda and C. Kato, Bulletin of the Chemical Society of Japan, 63 (1990) 988-992.

[4] A. Corma, Chemical Reviews, 97 (1997) 2373-2420. [5] Z. Wu and D. Zhao, Chemical Communications, 47 (2011) 3332-3338. [6] R. Brady, B. Woonton, M. L. Gee and A. J. O'Connor, Innovative Food Science

& Emerging Technologies, 9 (2008) 243-248. [7] A. E. Garcia-Bennett, Nanomedicine, 6 (2011) 867-877. [8] M. Vallet-Regí, F. Balas and D. Arcos, Angewandte Chemie International

Edition, 46 (2007) 7548-7558. [9] M. Vallet-Regí, Journal of Internal Medicine, 267 (2010) 22-43. [10] U. Leonhardt, Nature Photonics, 1 (2007) 207-208. [11] R. P. Feynman, Journal of Microelectromechanical Systems, 1 (1992) 60-66. [12] National Nanotechnology Initiative: Leading to the Next Industrial Revolution,

Washington, D.C., 2000. [13] H. Li, M. Eddaoudi, M. O'Keeffe and O. M. Yaghi, Nature, 402 (1999) 276-

279. [14] P. Yang, D. Zhao, D. I. Margolese, B. F. Chmelka and G. D. Stucky, Nature,

396 (1998) 152-155. [15] J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D.

Schmitt, C. T.-W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins and J. L. Schlenker, Journal of the American Chemical Society, 27 (1992) 10834-10843

[16] Y. Wan and D. Zhao, Chemical Reviews, 107 (2007) 2821-2860. [17] S. Inagaki, Y. Fukushima and K. Kuroda, Journal of the Chemical Society,

Chemical Communications, 0 (1993) 680-682. [18] S. Inagaki, A. Koiwai, N. Suzuki, Y. Fukushima and K. Kuroda, Bulletin of the

Chemical Society of Japan, 69 (1996) 1449-1457. [19] S. Y. Chang and T. A. Ring, Journal of Non-Crystalline Solids, 147–148 (1992)

56-61. [20] R. Atluri, N. Hedin and A. E. Garcia-Bennett, Journal of the American

Chemical Society, 131 (2009) 3189-3191. [21] L. Baù, B. Bártová, M. Arduini and F. Mancin, Chemical Communications,

(2009) 7584-7586. [22] R. Ryoo, J. M. Kim, C. H. Ko and C. Shin, The Journal of Physical Chemistry,

100 (1996) 17718-17721. [23] K. M. Taylor, J. S. Kim, W. J. Rieter, H. An, W. Lin and W. Lin, Journal of the

American Chemical Society, 130 (2008) 2154-2155.

72

[24] H. Vallhov, N. Kupferschmidt, S. Gabrielsson, S. Paulie, M. Strømme, A. E. Garcia-Bennett and A. Scheynius, Small, 8 (2012) 2116-2124.

[25] N. Kupferschmidt, K. R. Qazi, C. Kemi, H. Vallhov, A. E. Garcia-Bennett, S. Gabrielsson and A. Scheynius, Nanomedicine, 9 (2014) 1835-1846.

[26] H. Vallhov, S. Gabrielsson, M. Strømme, A. Scheynius and A. E. Garcia-Bennett, Nano Letters, 7 (2007) 3576-3582.

[27] A. López-Noriega, D. Arcos, I. Izquierdo-Barba, Y. Sakamoto, O. Terasaki and M. Vallet-Regí, Chemistry of Materials, 18 (2006) 3137-3144.

[28] W. Xia, K. Grandfield, A. Hoess, A. Ballo, Y. Cai and H. Engqvist, Journal of Biomedical Materials Research Part B: Applied Biomaterials, 100 (2012) 82-93.

[29] J. Forsgren, M. Andersson, P. Nilsson and A. Mihranyan, Advanced Healthcare Materials, 2 (2013) 1469-1476.

[30] X. Xia, C. Zhou, L. Ballell and A. E. Garcia-Bennett, ChemMedChem, 7 (2012) 43-48.

[31] M. Strømme, U. Brohede, R. Atluri and A. E. Garcia-Bennett, Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology, 1 (2009) 140-148.

[32] U. Brohede, R. Atluri, A. E. Garcia-Bennett and M. Strømme, Current Drug Delivery, 5 (2008) 177-185.

[33] L. Jia, Current nanoscience, 1 (2005) 237. [34] Y. Nakai, K. Yamamoto and S. Izumikawa, Chemical and Pharmaceutical

Bulletin, 37 (1989) 435-438. [35] G. Hägg, Allmän och oorganisk kemi, 6 ed., Almqvist & Wiksell Förlag AB,

Stockholm, 1963. [36] R. Rowe, P. Sheskey and M. Quinn, Magnesium Carbonate., in: B. Truitt (Ed.)

Handbook of Pharmaceutical Excipients, Pharmaceutical Press, London, 2009, pp. 397-400.

[37] C. L. Christ and P. B. Hostetler, American Journal of Science, 268 (1970) 439-453.

[38] J. C. Deelman, Neues Jahrbuch für Mineralogie - Abhandlungen, 7 (1999) 289-302.

[39] F. L. Sayles and W. S. Fyfe, Geochimica et Cosmochimica Acta, 37 (1973) 87-99.

[40] G. H. Wolf, A. V. Chizmeshya, J. Diefenbacher and M. J. McKelvy, Environmental science & technology, 38 (2004) 932-936.

[41] D. E. Giammar, R. G. Bruant Jr and C. A. Peters, Chemical Geology, 217 (2005) 257-276.

[42] M. Hänchen, V. Prigiobbe, R. Baciocchi and M. Mazzotti, Chemical Engineering Science, 63 (2008) 1012-1028.

[43] Y. Yang, N. Sahai, C. S. Romanek and S. Chakraborty, Geochimica et Cosmochimica Acta, 88 (2012) 77-87.

[44] C. Berg, M. Beyer, U. Achatz, S. Joos, G. Niedner-Schatteburg and V. E. Bondybey, Chemical Physics, 239 (1998) 379-392.

[45] P. E. Barran, N. R. Walker and A. J. Stace, Journal of Chemical Physics, 112 (2000) 6173-6177.

[46] F. C. Lightstone, E. Schwegler, R. Q. Hood, F. Gygi and G. Galli, Chemical Physics Letters, 343 (2001) 549-555.

[47] D. D. Tommaso and N. H. de Leeuw, Phyical Chemistry Chemical Physics, 12 (2010) 894-901.

[48] J. Xu, C. Yan, F. Zhang, H. Konishi, H. Xu and H. H. Teng, Proceedings of the National Academy of Sciences, 110 (2013) 17750-17755.

73

[49] K. Sandengen, L. O. Josang and B. Kaasa, Industrial & Engineering Chemistry Research, 47 (2008) 1002-1004.

[50] A. P. A. Anjos, A. Sifeddine, C. J. Sanders and S. R. Patchineelam, Carbonates and Evaporites, 26 (2011) 213-215.

[51] C. Neuberg and B. Rewald, Zeitschrift für Chemie und Industrie der Kolloide, 2 (1908) 321-324.

[52] V. I. Kurov, Russian Jounral of General Chemistry, 31 (1961) 9-11. [53] A. Buzágh, Kolloid Zeitschrift, 38 (1926). [54] T. Kornprobst and J. Plank, Journal of Non-Crystalline Solids, 361 (2013) 100-

105. [55] R. M. Dell and S. W. Weller, Transactions of the Faraday Society, 55 (1959)

2203-2220. [56] A. Botha and C. Strydom, Journal of Thermal Analysis and Calorimetry, 71

(2003) 987-996. [57] U. S. Food and Drug Administration, Food Additive Status List, 2015. [58] C. J. Brinker and G. W. Scherer, Sol-gel science: the physics and chemistry of

sol-gel processing, Academic Press, 1990. [59] J. Plank, H. Hoffmann, J. Schö, W. Seidl, I. Zeitler and Z. Zhang, Advances in

Materials Science and Engineering, (2009). [60] D. F. Williams, Definitions in biomaterials, Elsevier, Amsterdam, 1987, pp. 4. [61] K. Peters, R. Unger and C. Kirkpatrick, Biocompatibility testing, in: R. Narayan

(Ed.) Biomedical materials, Springer Science & Business Media 2009, pp. 261-292.

[62] F. Tang, L. Li and D. Chen, Advanced Materials, 24 (2012) 1504-1534. [63] J. S. Temenoff; and A. G. Mikos, Biomaterials: The Intersection of Biology and

Materials Science, Prentice Hall2008. [64] D. B. Warheit, T. R. Webb, V. L. Colvin, K. L. Reed and C. M. Sayes,

Toxicological Sciences, 95 (2007) 270-280. [65] S. Lu, R. Duffin, C. Poland, P. Daly, F. Murphy, E. Drost, W. MacNee, V.

Stone and K. Donaldson, Environmental Health Perspectives, 117 (2009) 241-247.

[66] B. Nilsson, K. N. Ekdahl, T. E. Mollnes and J. D. Lambris, Molecular Immunology, 44 (2007) 82-94.

[67] O. Götze and H. J. Müller-Eberhard, The Journal of Experimental Medicine, 134 (1971) 90-108.

[68] T. E. Mollnes, T. Lea, S. S. Froland and M. Harboe, Scandinavian Journal of Immunology, 22 (1985) 197-202.

[69] J. Hong, K. Nilsson Ekdahl, H. Reynolds, R. Larsson and B. Nilsson, Biomaterials, 20 (1999) 603-611.

[70] E. W. Davie, K. Fujikawa and W. Kisiel, Biochemistry, 30 (1991) 10363-10370.

[71] B. Ekstrand-Hammarström, J. Hong, P. Davoodpour, K. Sandholm, K. N. Ekdahl, A. Bucht and B. Nilsson, Biomaterials, 51 (2015) 58-68.

[72] S. Ardizzone, C. Bianchi, M. Fadoni and B. Vercelli, Applied surface science, 119 (1997) 253-259.

[73] S. Brunauer, P. H. Emmett and E. Teller, Journal of the American Chemical Society, 60 (1938) 309-319.

[74] P. I. Ravikovitch, A. Vishnyakov, R. Russo and A. V. Neimark, Langmuir, 16 (2000) 2311-2320.

[75] A. V. Neimark, P. I. Ravikovitch, M. Grün, F. Schüth and K. K. Unger, Journal of Colloid and Interface Science, 207 (1998) 159-169.

[76] G. Raade, American Mineralogist, 55 (1970) 1457-1465.

74

[77] M. Santamaria, F. Di Quarto, S. Zanna and P. Marcus, Electrochimica Acta, 53 (2007) 1314-1324.

[78] S. D. Gardner, C. S. Singamsetty, G. L. Booth, G.-R. He and C. U. Pittman, Carbon, 33 (1995) 587-595.

[79] Y. Xie and P. M. Sherwood, Chemistry of Materials, 2 (1990) 293-299. [80] C. E. White, N. J. Henson, L. L. Daemen, M. Hartl and K. Page, Chemistry of

Materials, 26 (2014) 2693-2702. [81] T. Egami and S. J. Billinge, Underneath the Bragg peaks: structural analysis of

complex materials, Elsevier 2003. [82] S. Gryglewicz, Applied Catalysis A: General, 192 (2000) 23-28. [83] H. L. Finkbeiner and M. Stiles, Journal of the American Chemical Society, 85

(1963) 616-622. [84] L. R. Green and M. Lesse, Restaurator, 12 (1991). [85] F. Daniel, F. Flieder and F. Leclerc, The Effects of Pollution on Deacidified

Paper, 1990, pp. 179. [86] J. P. Olivier, Carbon, 36 (1998) 1469-1472. [87] J. Suuronen, I. Pitkänen, H. Halttunen and R. Moilanen, Journal of Thermal

Analysis and Calorimetry, 69 (2002) 359-369. [88] I. Pochard, S. Frykstrand, O. Ahlström, J. Forsgren and M. Strømme, Journal of

Applied Physics, 115 (2014) 044306-044306-044310. [89] I. Pochard, S. Frykstrand, J. Eriksson, S. Gustafsson, K. Welch and M.

Strømme, The Journal of Physical Chemistry C, 119 (2015) 15680-15688. [90] S. E. Baker, A. M. Sawvel, J. Fan, Q. Shi, N. Strandwitz and G. D. Stucky,

Langmuir, 24 (2008) 14254-14260. [91] C. Dai, Y. Yuan, C. Liu, J. Wei, H. Hong, X. Li and X. Pan, Biomaterials, 30

(2009) 5364-5375. [92] M. E. Davis, Nature, 417 (2002) 813-821. [93] S. P. Hudson, R. F. Padera, R. Langer and D. S. Kohane, Biomaterials, 29

(2008) 4045-4055. [94] M. Liong, J. Lu, M. Kovochich, T. Xia, S. G. Ruehm, A. E. Nel, F. Tamanoi

and J. I. Zink, ACS Nano, 2 (2008) 889-896. [95] P. Zhang, J. Forsgren and M. Strømme, International Journal of

Pharmaceutics, 472 (2014) 185-191. [96] J. M. Rosenholm, J. Zhang, M. Linden and C. Sahlgren, Nanomedicine (Lond),

11 (2016) 391-402. [97] P. Zhang, T. Zardán Gómez De La Torre, J. Forsgren, C. Bergström and M.

Strømme, Journal of pharmaceutical sciences, (2015). [98] The International Pharmacopedia, Fourth edition 2014 (incl. First, Second,

Third and Fourth Supplements). [99] ISO 10993-10: Biological evaluation of medical devices. Part 10: Tests for

Irritation and skin sensitization 2009 [100] B. Pagé, M. Pagé and C. Noël, International journal of oncology, 3 (1993)

473-476. [101] J. Andersson, J. Sanchez, K. N. Ekdahl, G. Elgue, B. Nilsson and R. Larsson,

Journal of Biomedical Materials Research Part A, 67A (2003) 458-466. [102] K. N. Ekdahl, B. Nilsson, M. Pekna and U. R. Nilsson, Scandinavian Journal

of Immunology, 35 (1992) 85-91. [103] W. Hadnagy, B. Marsetz and H. Idel, International journal of hygiene and

environmental health, 206 (2003) 103-107. [104] L. Larsson and S. Ohman, Clinical chemistry, 24 (1978) 1962-1965. [105] ISO 10993-5: Biological evaluation of medical devices. Part 5: Tests for in

vitro cytotoxicity, 2009.

75

[106] ISO 10993-11: Biological evaluation of medical devices. Part 11: Tests for systemic toxicity 2006, last updated 2010

[107] S. E. Baker, A. M. Sawvel, N. Zheng and G. D. Stucky, Chemistry of Materials, 19 (2007) 4390-4392.

[108] A. E. Pusateri, A. V. Delgado, E. J. Dick Jr, R. S. Martinez, J. B. Holcomb and K. L. Ryan, Journal of Trauma-Injury, Infection, and Critical Care, 57 (2004) 555-562.

[109] Y. Li, A. M. Sawvel, Y.-S. Jun, S. Nownes, M. Ni, D. Kudela, G. D. Stucky and D. Zink, Toxicology Research, 2 (2013) 136-144.

[110] F. Feyerabend, H.-P. Wendel, B. Mihailova, S. Heidrich, N. A. Agha, U. Bismayer and R. Willumeit-Römer, Acta biomaterialia, 25 (2015) 384-394.

[111] J. Shi, Y. Hedberg, M. Lundin, I. Odnevall Wallinder, H. L. Karlsson and L. Möller, Acta Biomaterialia, 8 (2012) 3478-3490.

[112] W. A. Ames, N. McDonnell and D. Potter, Anaesthesia, 54 (1999) 999-1001. [113] T. Sacha and A. B. Skotnicki, Przegl Lek, 54 (1997) 122-125. [114] J. Jankun, E. Skrzypczak-Jankun and B. Lipinski, Central European Journal

of Immunology, 38 (2013) 149-153. [115] C. Neal, Clays and Clay minerals, 25 (1977) 253-258. [116] A. E. Engberg, P. H. Nilsson, S. Huang, K. Fromell, O. A. Hamad, T. E.

Mollnes, J. P. Rosengren-Holmberg, K. Sandholm, Y. Teramura, I. A. Nicholls, B. Nilsson and K. N. Ekdahl, Biomaterials, 36 (2015) 55-65.

[117] A. L. Sandberg and A. G. Osler, The Journal of Immunology, 107 (1971) 1268-1273.

[118] R. Mellaerts, J. A. G. Jammaer, M. Van Speybroeck, H. Chen, J. V. Humbeeck, P. Augustijns, G. Van den Mooter and J. A. Martens, Langmuir, 24 (2008) 8651-8659.

[119] The United States Pharmaceutical (USP) Convnetion <711> Dissolution, 2011.

[120] D. B. Williams and C. B. Carter, Transmission electron microscopy: a textbook for materials science 2nd ed., Springer, New York, 2008.

[121] D. C. Harris, Quantitative Chemical Analysis, 7th ed., W. H. Freeman and Company, Michelson Laboratory China Lake, California, 2007.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1363

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science andTechnology, Uppsala University, is usually a summary of anumber of papers. A few copies of the complete dissertationare kept at major Swedish research libraries, while thesummary alone is distributed internationally throughthe series Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology.(Prior to January, 2005, the series was published under thetitle “Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-281522

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016