Thesis
Reference
Chitosan-grafted-ssDNA copolmer self-assembly and morphological study of crstallization on surfaces
SAFIR, Ilyes
Abstract
Les macromolécules amphiphiles à base d'ADN se structurent à des échelles variant du nano au micromètre. La compréhension du mécanisme d'auto-assemblage et de l'organisation de structures fonctionnelles est l'objectif premier de cette thèse décrivant pour la première fois le greffage du chitosan à un simple brin d'ADN (chitosan-g-ssDNA), l'auto-assemblage sous la forme de structures sphérique de diamètres submicrométriques en solution et la cristallisation en surface. Les études menées au cours de ce travail de thèse ont été effectuées en induisant l'auto-assemblage du chitosan-g-ssDNA en milieu aqueux et en surface, et déterminer le mode de structuration gouverné par les interactions macromoléculaires engagées. La fonctionnalité des structures résultantes pourrait être d'une grande importance pour l'encapsulation de médicaments et en nanotechnologie.
SAFIR, Ilyes. Chitosan-grafted-ssDNA copolmer self-assembly and morphological study of crstallization on surfaces. Thèse de doctorat : Univ. Genève, 2015, no. Sc. 4816
URN : urn:nbn:ch:unige-760880
DOI : 10.13097/archive-ouverte/unige:76088
Available at:
http://archive-ouverte.unige.ch/unige:76088
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie
Département de chimie minérale
et analytique Professeur Corinne Nardin
CHITOSAN-GRAFTED-ssDNA COPOLMER
SELF-ASSEMBLY AND MORPHOLOGICAL STUDY OF CRSTALLIZATION ON SURFACES
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention chimie
par Ilyès SAFIR
de Oran (Algeria) Thèse N° 4816
GENÈVE Atelier Repromail
2015
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3
Acknowledgements
First of all, I would like to express my profound gratitude to my supervisor Professor Corinne Nardin who allowed me to do this PhD work in her group. She gave me huge freedom and confidence, trust, help, sympathy and advice during my PhD research.
I am grateful to Dr. Kien Ngo who helped me at the start of this work, as well as Jancy Nixon Abraham for helping me with the synthesis of the used compounds.
Also, I thank all the former and present members of the group for the warm atmosphere in the laboratory.
I would like to thank Professor Michal Borkovec for granting me access to the instruments required during this work. I am also grateful to Dr. Plinio Maroni, for his great help for all matters related to AFM measurements.
Concerning light scattering measurements I would like to express my thankfulness to Dr. Istvan Szilagyi for his support.
I express my immense gratitude to Magali Cissokho, Anne-Marie Loup and Sandra Salinas for their help, to solve the bureaucratic issues as well as to Olivier Vassalli for his great sense of humour and his help to solve technical problems.
I would like to thank my parents and my family for their deep love and their encouragements in pursuing a scientific career.
4
Abstract
Nowadays, self-assembly of materials into ordered structures is an important challenge in biology and materials science. As an example, the self-assembly of proteins is essential for their function as enzymes or carrier systems. Deoxyribonucleic acid (DNA), the molecule carrying the genetic information in biological systems is currently one of the building blocks of self-assembling systems, which are becoming one of the promising fields of research in bioactive materials. DNA nanotechnology is a common term to describe the use of DNA as a structural material rather than as a medium to support genetic information. Amphiphilic macromolecules based on DNA are known to self-assemble into well-ordered structures at the nanometer and micrometer length scales. The scope of this thesis was to describe for the first time the grafting of chitosan to the hydrophilic single stranded DNA (chitosan-g-ssDNA), and study the self-assembly of the resulting material into submicrometer size spherical structures in solution. The studies of structure formation were conducted considering the hydrophobic weight fraction of the chitosan as the key factor directing the self-assembly. In addition crystallization on different substrates such as silica and gold was further investigated. The resulting structures are of high interest for drug delivery and nanotechnology.
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Résumé
Actuellement, le mécanisme d’auto-assemblage est un mode de structuration autant en biologie qu’en science des matériaux. Par exemple, les interactions intermoléculaires et intramoléculaires au cœur de l’auto-assemblage des protéines sont essentielles à leurs fonctions comme enzyme ou comme système de transport. Le double brin d’ADN, lui permet la transmission de l’information génétique dans tous les systèmes biologiques. Cette macromolécule est aussi de nos jours un des constituants majeur dans les domaines prometteurs de recherche tel que celui des bioactifs qui sont par définition des molécules ou des macromolécules qui ont une activité biologique à entreprendre dans l’organisme.
L’ADN est en effet dans ces domaines un bloc structurel. Les macromolécules amphiphiles à base d’ADN se structurent à des échelles variant du nano au micromètre. La compréhension du mécanisme d’auto-assemblage et de l’organisation de structures fonctionnelles est l’objectif premier de cette thèse décrivant pour la première fois le greffage du chitosan à un simple brin d’ADN (chitosan-g-ssDNA), l’auto-assemblage sous la forme de structures sphérique de diamètres submicrométriques en solution et la cristallisation en surface.
Les études menées au cours de ce travail de thèse ont été effectuées en induisant l’auto- assemblage du chitosan-g-ssDNA en milieu aqueux et en surface, et déterminer le mode de structuration gouverné par les interactions macromoléculaires engagées. La fonctionnalité des structures résultantes pourrait être d’une grande importance pour l’encapsulation de médicaments et en nanotechnologie.
6 Table des matières
1 INTRODUCTION ... 8
1.1 GENERAL INTRODUCTION ... 8
1.2 POLYMERS AND COPOLYMERS ... 8
1.3 BLOCK COPOLYMERS STRUCTURE FORMATION ... 12
1.4 DEOXYRIBONUCLEIC ACID (DNA) ... 15
1.5 SELF-ASSEMBLY OF AMPHIPHILIC COPOLYMERS ... 22
1.6 CHITOSAN ... 26
1.7 CRYSTALLIZATION OF BLOCK COPOLYMERS (BCPS) ... 28
1.8 SCOPE OF THE THESIS ... 34
2 MATERIALS ... 35
3 EXPERIMENTAL TECHNIQUES AND METHODS ... 38
3.1 MATRIX-ASSISTED LASER DESORPTION/IONIZATION-TIME-OF-FLIGHT (MALDI-TOF) MASS SPECTROMETRY ... 38
3.2 ANALYTICAL ULTRA-CENTRIFUGATION (AUC) ... 40
3.3 SIZE EXCLUSION CHROMATOGRAPHY/GEL PERMEATION CHROMATOGRAPHY (SEC/GPC) 41 3.4 DIALYSIS ... 43
3.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC) ... 44
3.6 NUCLEAR MAGNETIC RESONANCE (NMR)SPECTROSCOPY ... 45
3.7 IMAGING ... 47
3.7.1 Atomic Force Microscopy (AFM) ... 47
3.7.1.1 Modes of imaging ... 50
3.7.1.1.1 Contact mode ... 50
3.7.1.1.2 Tapping mode ... 51
3.7.1.1.3 Non-contact mode ... 51
3.7.1.2 Optimizing image quality ... 52
3.7.1.2.1 Set point ... 52
3.7.1.2.2 Scan Speed ... 52
3.7.1.2.3 Feedback loop gains ... 53
3.7.1.2.4 Open/closed loop operation ... 53
3.7.1.3 Cantilevers and tips ... 54
3.7.1.3.1 Cantilever properties ... 54
3.7.1.3.2 Tip geometry ... 56
3.7.1.4 Detector system ... 57
3.7.1.5 Applications ... 58
3.7.1.6 AFM Apparatus ... 58
3.7.1.7 Samples preparation ... 59
3.7.2 Transmission Electron Microscopy (TEM) ... 60
3.7.2.1 Negative staining TEM ... 61
7
3.7.2.2 Cryogenic TEM ... 62
3.7.2.3 Apparatus ... 63
3.8 ULTRAVIOLET-VISIBLE (UV-VIS) SPECTROSCOPY ... 64
3.9 ATTENUATED TOTAL REFLECTION INFRARED (ATR-IR) SPECTROSCOPY ... 66
4 SYNTHESIS OF CHITOSAN-GRAFTED-SSDNA ... 70
4.1 DNACOPOLYMER ... 70
4.2 CHITOSAN-BASED POLYMERS ... 75
4.3 EXPERIMENTAL ... 79
4.3.1 Enzymatic digestion of chitosan ... 79
4.3.2 Solid phase synthesis of chitosan-g-ssDNA ... 80
4.3.3 Purification by dialysis ... 82
4.4 RESULTS AND DISCUSSION ... 82
4.5 CONCLUSION ... 92
5 SELF-ASSEMBLY IN AQUEOUS SOLUTION ... 93
5.1 INTRODUCTION ... 93
5.2 RESULT AND DISCUSSION ... 100
5.2.1 Mechanism of structure formation ... 105
5.3 CONCLUSION ... 111
6 CRYSTALLIZATION OF CHITOSAN-G-SSDNA COPOLYMER IN THIN FILMS 112 6.1 INTRODUCTION ... 112
6.2 RESULTS AND DISCUSSION ... 113
6.2.1 Morphological characterization ... 114
6.3 CONCLUSION ... 125
7 CONCLUSIONS AND OUTLOOKS ... 126
8 IMPACT ... 129
TABLE OF FIGURES ... 130
REFERENCES ... 135
8
1 Introduction
1.1 General introduction
The latest advances in materials science demonstrate how the fundamental principles of supramolecular chemistry could be used to craft the size, shape, and internal structure of nanoscale objects. The main objective in this research field is to design building blocks in such a way that they contain all the necessary information to direct the self-assembly into functional materials. Self-assembled superstructures of surfactants and amphiphilic block copolymers are examples of these systems [1-6]. Self-assembly of molecular building blocks into large architectures is a central feature in the chemistry of life science. It has become a central field of research for supramolecular chemists and many current materials such as nanostructured polymers, nanostructured metals, nanoparticles, and ceramics are based on self-organized systems. The main types of amphiphilic molecules used currently are surfactants, lipids, blocks copolymers, polysaccharides, and DNA due to their high capability for molecular recognition and their potential for biomedical applications.
The following studies are dedicated to conjugating DNA to chitosan and the self-assembly in aqueous solution and crystallization in thin films. Since these occur owing to covalent bonds between the nucleotide strands and the polymer segment, in the following, a general introduction to polymers and copolymers is given.
1.2 Polymers and copolymers
Polymers (or macromolecules) are defined as large molecules made up of smaller units, called monomers or repeating units, covalently bonded together (see Figure 1-1). This specific
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molecular structure of polymeric materials is responsible for their intriguing mechanical properties [7]. Polymers exist in the natural form, and those such as DNA, RNA, proteins and polysaccharides play crucial roles in plant and animal life [7].
Figure 1-1: A polymer chain, A is a monomer unit
Depending on the configuration of monomers, polymers can be divided in the following families:
1) Homopolymer: consists of monomers of the same type
2) Copolymers: are composed of different repeating units, depending on the arrangement of the monomers along the polymer chain, four classes can be distinguished.
a) Random copolymers: consisting of two or more different repeating units, randomly distributed along the chain.
b) Alternating copolymers: alternating sequences of the different monomers
c) Block copolymers: sequences of a monomer followed by sequences of another one
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d) Graft copolymer: chain from one type of monomer with branches of another type
The simplest measure of the length of a polymer chain is the contour length. It is the length of the stretched-out molecule, i.e. for a chain of n bonds of length l the contour length is nl. This does not, however, give a realistic measure of the size of the polymer chain, which in the molten state or in a dilute solution is coiled up [8]. In the simplest model for coils, the chain is supposed to consist of n volume-less links of length l, which can rotate freely in space. This model is then called the freely jointed chain model. Since each link can adopt any orientation, the polymer coil effectively executes a random walk, (see Figure 1-2) [8].
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Figure 1-2: Representative section of a random walk configuration of a polymer coil [8].
All the polymer chains in a sample do not have exactly the same mass. This is a result of the polymerization process, in which chain growth is controlled by the probability of attachment of a given monomer. Thus, polymers are said to be polydisperse, meaning that they have a distribution of molar masses. A consequence of this is that polymers have to be characterized by an average molar mass, and also that there are different ways of defining this average [8].
The number average molar mass depends on the number of molecules ni having a molar mass Mi:
!
!=
!!!!!!
!!!!!!!!
!Equation!1 Similarly, the weight average molar mass is defined by:!
!=
!!!!!!
!!!!
Equation!2 Where wi is the fraction by weight of molecules having a molar mass Mi. The ratio !! !! is called the polydispersity index (or heterogeneity index), giving a measure of the distribution of molar masses.12
For an ideal polymer, the ratio !! !! would be equal to one. However, in practise, this ratio is always greater than one, due to the distribution of molar masses of real polymers [8]. In a dilute solvent, the conformation of a polymer chain depends on the interaction between chain segments and solvent molecules. In a good solvent, a chain expands from its unperturbed dimensions to maximize the number of segments-solvent contacts and the coil is said to be swollen. In a poor solvent, the chains will contract to minimize interactions. However, competing with this effect is the tendency for chains to expand to reduce unfavourable segment-segment interactions, which is the excluded volume effect. If these two effects are perfectly balanced the polymer molecule will adopt an unperturbed conformation.
The solvent quality affects the thermodynamics of dilute polymer solutions, because interactions between polymer chains are modified by the presence of solvent molecules. Interactions between polymer molecules in solution depend strongly on concentration. In a dilute solution, the molecules are well separated on average and do not interact with each other. Each molecule can therefore be considered as an isolated chain [8].
1.3 Block copolymers structure formation
Microphase separation in block copolymer is driven by chemical incompatibilities between the different blocks that compose block copolymer molecules. In the simplest case of AB diblock copolymer (left side of Figure 1-3), there is only the issue of compatibility between the dissimilar A and B blocks. Thus, even minor chemical or structural differences between A and B blocks are sufficient to produce excess free energy contributions that are usually unfavourable to mixing. The phase behaviour of block copolymer melts is, to a first approximation, represented in a morphology diagram in terms of χN and f [9], where f is the volume fraction of one block and χ
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is the Flory-Huggins interaction parameter (inversely proportional to temperature) that reflects the interaction energy between different segments [10]. Two additional parameters that determine the ultimate morphology of microphase separated systems are the degree of polymerization, N, and the relative composition fractions, fA and fB, where fA=NA/N and fA +fB =1 [11]. Common periodic phases for A-B diblocks, with increasing fA, include body centered cubic A spheres in a B matrix, bicontinuous gyroid, and lamellae. The Figure 1-4 depicts these morphologies with the diblock molecules represented as simplified two-color chains [1], and A-B represents the diblock copolymer. The chains self-organize such that contact between the immiscible blocks is minimized, with the structure determined primarily by the relative lengths of the two polymer blocks (fA) [1].
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Figure 1-3: Block copolymers architectures classified by number of monomer types and topology (linear versus branched sequencing). A, B and C shown as blue, red and green, respectively are different polymers blocks [9].
The molecular weights of typical block copolymers lead to periodicities in the range of 10-20 nm.
For many applications, an increase in length scale by an order of magnitude or more would be desirable. Although in principle this can be done by increasing N, it is not generally practical because molecular weight well in excess of 106 would be required, which causes both synthesis and processing problems. Consequently, blending and dilution strategies are more promising [3].
In a solvent, the block copolymer phase behaviour is controlled by the interaction between the segments of the polymers and the solvent molecules as well as the interaction between the
15
polymer segments of the two blocks. If the solvent is unfavourable for one block this can lead to micelle formation in dilute solution [10].
Figure 1-4: Schematic of thermodynamically stable A-B diblock copolymer phases [1].
1.4 Deoxyribonucleic acid (DNA)
The DNA macromolecule is incontestably one of the most intriguing known molecules, having evolved in nature for billions of years. The establishment of the molecular structure of hybridized complementary nucleic acid strands should be of value in the effort to understand the fundamental phenomena of life. The discovery of DNA and its characterization began with the Swiss scientist Friedrich Miescher (1844-1895), when he isolated the first crude of DNA from
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cells and named it nuclein, known currently as nucleic acids [12].
Later in 1889, the German scientist Richard Altmann, separated from nuclein, proteins and an acidic substance, which is the nucleic acid, and in 1886 another German scientist called Albrecht Kossel discovered the primary nucleobases into the nucleic acid, namely cytosine, guanine, thymine, adenine, (C, G, T, A respectively). Those nucleobases were divided into two groups, purines that include adenine and guanine, and the group of pyrimidines including cytosine and thymine. Later in 1928, Phoebus Levene and Walter A. Jacobs identified deoxyribose and starting from 1935, the scientific community gave the name of deoxyribonucleic acid (DNA) to nucleic acids. Because purines and pyrimidines are molecules present in DNA, they are named the DNA bases [12].
In 1953, American scientists, Linus Pauling and Robert Corey [13], proposed for the first time a structure of the nucleic acids. Unfortunately it turned out to be wrong; in the same year, two American scientists, James D. Watson and Francis Crick figured out the structure of DNA [14].
They suggested that it consists of two long twisted chains organized in a double helix, made up of nucleotides. Each nucleotide contains one base, one sugar and one phosphate group and the deoxyribose sugar molecule. Purine and pyrimidine bases maintain the twisted chains together, as shown in Figure 1-5. They found out that bases are joined together in pairs, in which a base from one chain is hydrogen-bonded to a single base from the other helix chain, so that the two lie side by side with identical z-coordinates. For the bonding to occur, one of the pair must be a purine while the other one must be a pyrimidine. These pertinent results were obtained with X-ray diffraction performed previously by. R. E. Franklin. In 1962, J. D. Watson and F. Crick got the Nobel Prize in physiology and medicine [12].
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Figure 1-5: Helical structure of the DNA molecule, two hydrogen bonds connects T to A; three hydrogen bonds connect G to C. The sugar-phosphate backbones (grey) run antiparallel to each other, so that the 3’ and 5’ ends of the two strands are aligned [15] .
DNA is known to be the hereditary material in humans and almost all other organisms, present mostly in the cell nucleus of almost all organisms. The information in DNA is stored as a code based on the four bases cited previously. The order, or sequence, of these bases determines the information available for building and maintaining an organism functioning, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences. However, DNA as a stable extended double helix is by now a concept that is familiar to all.
Interest in DNA has expanded beyond its central genetic role to applications in nanotechnology, materials science and also as a drug. This last is reached when DNA is coupled to a polymer
18
segment that results in a macromolecule that is expected to have enhanced intercellular delivery and specific targeting of biochemically active, potentially therapeutic DNA fragments [16].
The DNA secondary structure is achieved by the hybridization of two complementary single- stranded DNA sequences into double-helical strands. DNA hybridization was first proposed as an artificial strategy for complex nanoscale assembly by Seeman [17], who is the founder of the field of DNA nanotechnology. DNA self-assembly can be prepared specifically for several roles, such as to create DNA block copolymers for nanoscience and biomedicine [16] to organize into drug delivery vehicles [18] or for DNA engineering which consists in selecting specific DNA molecules with conventional nucleotides [19] to create specific topologies based on DNA. Shapes and arrangements of secondary and tertiary structures can also be used to prepare 3D macroscopic crystals or for preparing hydrogels or conductive polymer based nanomaterials [20].
The key to DNA’s biological role lies in the specificity of the base pairing holding the two strands of the double helix together: adenine pairs with thymine and guanine pairs with cytosine.
The familiar double helix that results from these complementary interactions is a linear sequence, in the topological sense, which means that its axis is not branched. However, by designing appropriate sequences, it is possible in synthetic systems to produce branched DNA molecules [17]. The functions of the structures rising from DNA are fully tuneable because of the designability and specificity of DNA assembly [20]. For example the development of efficient therapeutic agents based on nucleotide sequences delivery (i.e., fraction of DNA molecules getting into the nucleus) has been largely hampered by their reduced availability, mainly because of their characteristic polyanionic nature. Poor intracellular delivery, limited access to the specific target, and low resistance to nucleases are the major obstacles to the in vivo efficacy of synthetic, non-modified oligodeoxyribonucleotides (ODNs), which therefore limit their use as
19
gene expression regulators in antisense, antigene, or aptamer therapeutic strategies [21, 22]. The challenge of DNA delivery is to develop a system that is both highly efficient in delivery/expression (i.e., fraction of nuclear DNA molecules that undergo transcription) [23] and applicable to basic research as well as clinical setting.
Another interest in DNA polymers is due to their role in the nanotechnology field through conjugation of polymers to DNA in order to induce self-assembly for potential applications of high interest as well as for the achievement of a comprehensive understanding of the structure formation and mode of interaction of the resulting self-assembled DNA-polymer conjugates [24].
One of the most recent work concerning DNA-polymer conjugates, has involved the covalent binding between a DNA fragment and water-soluble polymer segments such as carbohydrates [22], poly(amine) [25] or a poly (ethylene glycol) (PEG) polymer segment [26]. The conjugation of DNA fragments to carbohydrate [22] improves the poor cell-or tissue-specific delivery of nucleotide sequences through cell-receptor-mediated endocytosis. DNA-carbohydrate could thus be used for the investigation of biological processes in vitro. The high selectivity of DNA- carbohydrate conjugates for cell recognition and increased specific cellular uptake due to stability against nuclease is of additional interest. Another example of binding DNA to polymer segments, is conjugation of DNA to poly(amine) [25]. It is known that, positive cationic polyamines stabilize oligonucleotide duplexes, as other cations do like Na+ or Mg++. Conjugation of poly(amine) with negatively charged nucleic acids, sequences induces the formation of complexes which have received much attention as potential enhancers of the delivery and bio distribution of therapeutic nucleotide sequences [25]. DNA polymers conjugated with poly (ethylene glycol) (PEG), which is neutral, soluble, non-toxic, and non-immunogenic, were conducted owing to PEG biocompatibility [26]. All those examples revealed that DNA-polymer
20
conjugates resulting in water-soluble macromolecules are of high potential for application in pharmacology, gene therapy and chemotherapy. The main advantage for nucleotide sequences to be used for conjugation to other polymer segments, rises from limited plasma half-life as well as limited cellular penetrability and uptake of DNA [23]. Hence, an increasing interest in designing nucleic acid-decorated nanostructures to improve cell penetrability and potential use as carriers developed. Nucleotide sequences have thus been grafted to hydrophobic polymers to induce the formation of self-assembled nanostructures [27-30]. The resulting structures are specific regarding their interaction with the complementary DNA sequence of the one involved in the self-assembly. Due to the inherent properties of hybridization of complementary nucleotide sequences upon functionalization with a ligand of a cell surface receptor, drugs could be targeted to specific cells. Thereby the development and characterization of nucleic acids based structures is of fundamental importance and of potential for application in the drug delivery, biomedicine, and technological fields [23, 24, 27, 28, 31, 32].
Chapter 4 describes the conjugation of nucleic acids to the chitosan polymer by solid phase synthesis. Structure formation in aqueous solution and on surfaces is described in chapter 5 and 6.
There are two main methods used for polymer coupling to nucleic acids, i. e. solution coupling or solid phase synthesis (SPS). The solution coupling method requires separate synthesis and purification of the nucleic acid and polymer segment, prior to the reaction, which could drive to the formation of both reversible and irreversible not desired bond [21]. This method is thus challenging for the preparation of amphiphilic DNA-copolymers and especially in achieving satisfying yields. The second method, SPS was used because of its simplicity since it avoids tedious chemistry and purification steps [29]. As a result of the synthesis, amphiphilic DNA- copolymers such a copolymers could form various supramolecular structures like monolayers,
21
micelles [33] or vesicles [34-39]. Micelle formation is described in chapter 5. The self-assembly of DNA-polymer conjugates into micelles has drawn much attention because of their potential application in biomedicine and nanoscience [16]. When a hydrophobic polymer segment is coupled to a nucleotide sequence it may self-assemble in aqueous solution to give rise to core- shell micelles composed of a hydrophobic core surrounded by a hydrophilic corona of the DNA fragment, which may act as an excellent vehicle for targeted delivery. Aside from the self- assembly of DNA-polymer conjugates into micelles, amphiphilic DNA-polymer have shown that they may self-assemble into vesicular structures as well [24]. Nardin reported for the first time the self-assembly of vesicular structures from DNA-polymer conjugates through coupling of a suitable polymer segment to a 12 long nucleotide sequence [27, 29, 40, 41]. The strongly hydrophobic poly(butadiene) (PB) or poly(isobutylene) (PIB) were coupled to a nucleotide sequence by SPS. The formation of vesicles resulting from the self-assembly of the resulting amphiphilic DNA-polymer conjugate is of major interest for potential pharmaceutical and biomedical applications because both water-soluble and hydrophobic substances could be loaded and specifically delivered to cells [24].
Another work realised by Nardin et al. was the synthesis of DNA-peptide hybrids by the conjugation of the diphenylalanine (FF) dipeptide ligand to a C6 amino modified 12 mer nucleotide sequence [27]. This conjugation has resulted into a DNA-peptide hybrid, which self- assembles into vesicular structures in aqueous solution. As FF is a common structural motif of amyloid fibrils forming peptide sequences known to play a key role in Alzheimer’s and related diseases, the conjugation with oligonucleotides is of therapeutic potential due to the fact that the coupling of nucleic acids to the FF dipeptide leads to a drastic morphological transition from fibrillar to hollow spherical structures [27]. Another work achieved by Nardin et al. was the
22
grafting of a short nucleic acid strand modified at the 5’ end by an amino group to the ditryptophan dipeptide (WW) through solid phase synthesis, which results into an amphiphilic, rod-coil peptide-DNA hybrid. Due to the chemical incompatibility between the rigid hydrophobic dipeptide fragment and the water-soluble flexible nucleotide sequence, the amphiphilic molecule organizes into spherical structures in dilute aqueous solution. They noticed that, at high concentration and under controlled conditions (time and temperature), fibrils are formed. Of high interest is the intrinsic fluorescence of the dipeptide, which enables the quantification of a critical aggregation concentration and the indirect evidence in a label-free optical mode that the WW- DNA hybrid organization is analogous to that of the nucleation polymerization of polypeptides fibrils [42].
1.5 Self-assembly of amphiphilic copolymers
In amphiphilic block copolymers hydrophobic and hydrophilic parts of the immiscible blocks can form various supramolecular structures depending on the copolymer topology, length, and solubility of the blocks. The self-assemblies of block copolymers in water give a wide range of aggregates of different morphologies such as spherical micelles, rods, lamellae, vesicles, large compound vesicles (LCVs) [39, 43-46]. Overall, more than 20 morphologies have been identified, some of which are thermodynamically induced, whereas others are kinetically controlled. At higher concentrations, self-assembled block copolymers in solution form a variety of lyotropic liquid mesophases [47]. The self-assembly of copolymers in various structures depends on the hydrophilic weight fraction of the polymeric backbone. Hydrophilic polymers are completely soluble in aqueous medium while hydrophobic polymers form only solid matrices by aggregation. Increasing hydrophilicity in the polymeric backbone pushes them toward the
23
formation of superstructures such as polymersomes, tubular, and spherical micelles structure [48]. The formation of different morphologies is attributed to two competing factors: interfacial energy between the two blocks (an enthalpic contribution), and chain stretching (an entropic contribution) [39]. In the case when the microphase separation occurs, both blocks will separate from each other in such a way as to minimize the interfacial area to lower the total interfacial energy. The control of the Flory-Huggins (χ) parameter the total degree of polymerization N and volume fraction of the constituent blocks fA, can generate a variety of microdomains (MD) morphologies such as spheres, cylinders, lamellae or gyroid [45, 49-51], (see Figure 1-6).
Figure 1-6: Amphiphilic diblock copolymer aggregates: (a) micelles, (b) vesicles, (c) inverse micelles, (d) lamellar structures, and (e) cylindrical or tubular micelles [51].
24
The χAB-parameter specifies the degree of incompatibility between the A and B blocks, which drives the phase separation. The relationship between χAB and temperature (T) is given in the following equation [52]:
!
!"=
!!!!
!
!"−
!!
(!
!!+ !
!!) !!
Equation!3Where z is the number of the nearest neighbours per repeat unit in the polymer, kB is the Boltzmann constant, kBT is the thermal energy, and εAB, εAA, and εBB are the interactions energies per repeating unit of A-B, A-A, and B-B, respectively. For a diblock copolymer, e.g.
polyisopropene-block-polystyrene (PI-b-PS) [9, 53] in which there are no strong specific interactions, such as hydrogen bonding, or electrostatic interaction, χAB is generally positive and varies inversely with temperature [4]. The degree of microphase separation of diblocks is determined by the product, χ N, which measures the degree of segregation of block copolymers.
As cited earlier, the chemical incompatibility between the two blocks induces a large difference in interaction with their environment and free behaviour in solution (selective solvent). These differences can induce microphase separation of amphiphilic block copolymers not only in aqueous media but also in organic solvent [54]. Block copolymers undergo two elementary processes in solvent media: micellization or gelation. Micellization occurs when the block copolymer is dissolved in a large amount of a selective solvent for one of the blocks. Under these terms, the polymer chains tend to organise in a diversity of structures from micelles to cylinders [51]. The soluble block will be oriented towards the continuous solvent medium and become the
‘corona’ of the micelle formed, whereas the insoluble part will be shielded from the solvent in the
‘core’ of the structure. In contrast to micellization, gelation occurs from the semidilute to the high concentration regime of block copolymer solutions and results in an arrangement of ordered
25
micelles [51]. Two extremes of micellar structures can be distinguished for diblock copolymers, depending on the relative length of the blocks. If the soluble block is larger than the insoluble one, the spherical micelles (Figure 1-7) formed will consist of a small core and a very large corona, called ‘star-micelles’. In contrast micelles having a large insoluble segment with a short soluble corona are referred to as ‘crew-cut micelles’ [33].
Figure 1-7: Extreme morphologies of micelles depending on the relative blocks lengths: (a) star micelle, (b) crew-cut micelle [51].
The process of micellization in block copolymers relies on two crucial parameters: the critical micelle temperature (CMT) and the critical micelle concentration (CMC) [44, 51]. When CMT or CMC are not reached, self-assembly will not occur, and the block copolymer will behave in the solution as a unimer, referred to as free or unassociated surfactant in the literature. On the contrary, if micelle formation is triggered, the micelles will be in thermodynamic equilibrium with unimers. Block copolymer vesicles, also called polymersomes [34], in contrast to micelles, are nanometre-sized hollow spherical structures, typically with a hydrophobic wall and hydrophilic internal and external coronas. Amphiphilic block copolymers can form various
26
architectures in solution [39]. They encompass uniform common vesicles, large polydisperse vesicles, entrapped vesicles, or hollow concentric vesicles [51]. The preparation of vesicles consists first in dissolving the amphiphilic block copolymer in a suitable solvent that is beneficial for both the core and corona blocks. Then, water is added as a precipitant for the hydrophobic block, and self-assembly takes place at some critical water content, which depends on the relative, and absolute block lengths and the nature of the polymer. Eisenberg et al. [38] have synthetized disproportional amphiphilic diblock copolymers with the aim of creating crew-cut micellar structures [55]. They used polystyrene-b-poly (acrylic acid) (PS-b-PAA) and polysterene-b-poly (ethylene oxide) (PS-b-PEO). Their idea was to use a hydrophobic block much longer than the hydrophilic segment to generate a corona much shorter than the core block.
Several amphiphilic block copolymers form vesicles. Among common core blocks used, are:
polystyrene [56], polyisoprene [57], polysiloxane [58], poly(propylene oxide) [59], poly(ethyl ethylene) [35], and poly(butadiene) [60]. Biodegradable vesicles were assembled from polylactide-block-poly(ethylene glycol) [61] with sizes ranging from 70 nm to 50 µm. Other amphiphilic systems such as macrocyclic, and hydroxyethylated β-cyclodextrins can assemble in vesicles with sizes ranging from 50 nm to 300 nm in water [36]. Other morphologies of more complex structure based on specifically designed block copolymers with a defined composition have also been observed with amphiphilic polystyrene-block-poly (ethylene oxide) (PS-b-PEO) like cylindrical structures in aqueous solution [62].
1.6 Chitosan
Chitosan is a linear polysaccharide consisting of randomly distributed β (1→4) linked residues of N-acetyl-2 amino-2-deoxy-D-glucose (glucosamine) and 2- amino-2-deoxy-D-glucose (N-acetyl-
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glucosamine) [63], (see Figure 1-8). It is a non-toxic, semicrystalline [64], biodegradable [65, 66], and biocompatible [67, 68] polymer. Commercial chitosan samples are typically prepared by deacetylation under alkaline conditions of chitin the second most abundant polysaccharide in nature after cellulose [64].
Figure 1-8: chemical structure of chitosan [63].
In nature, chitin is present in insects and crustaceans in which it represents the major component of their exoskeleton. Chitin is also present in the cell wall of some mushrooms [63]. Generally, chitosan produced from mushrooms is of narrow molecular weight distribution compared to chitosan produced from shrimps, and a non-animal source is considered to be safer for biomedical and healthcare uses [69]. Currently, chitosan is explored intensively for its application in several fields such as pharmacy, biomedicine [69], agriculture [70], food industry [71] and biotechnology [72]. Chitosan is a polycation (pKa- 6.5) whose charge density depends on the degree of acetylation and pH. This macromolecule can dissolve in diluted aqueous acidic solvents due to the protonation of –NH2 groups at the C2 position [64]. Chitosan chains are thus able to interact by electrostatic interaction with negatively charged molecules [69]. As can be seen in Figure 1-8 chitosan has a primary amino group, and a primary and secondary free hydroxyl
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groups. The advantage of chitosan and its strong functionality over other polysaccharides, (two hydroxyl groups (C3, C6) and one primary group (C2) per repeat-unit), gives several possibilities for chemical modification [73]. However, chitosan suffers from a poor solubility in water, which is a major drawback for drug formulation. Indeed, chitosan is only soluble in acidic solutions, required to insure the protonation of the primary amine. In such cases, the presence of positive charges on the chitosan backbone increases the repulsion between the different polymer chains, facilitating their solubilisation [69]. Chitosan can be depolymerized to reduce its molecular weight and viscosity, improving also it solubility in aqueous media [67]. Chitosan solubility can also be modified by deacetylation or by the introduction of hydrophobic moieties that alter chitosan hydrogen bonds among other interactions [63]. Since relevant chitosan properties (e.g.
mucoadhesive properties, absorbance enhancer) depend on the presence of a positive charge on the polymer, chitosan has been modified to introduce non-pH dependent positive charges on the chitosan backbones such as N- Alkyl-chitosans [63].
1.7 Crystallization of block copolymers (BCPs)
Crystallization of polymers is of great technological importance due to enhancing the material’s strength and toughness, disrupt optical clarity, or improve conductivity, which result from the change in molecular conformation. In semicrystalline block copolymers (BCPs), the presence of a non-crystalline block enables the modification of the mechanical and structural properties compared to a crystalline homopolymer, through introduction of a rubbery or glassy component [74]. It is well known that polymers crystallize into lamellae in either a folded-chain conformation (metastable state) or an extended chain conformation (equilibrium state) [75].
Hoffman and Lauritzen [76] demonstrated that the growth rate (G) of crystals is proportional to
29
the inverse of the supercooling temperature, ΔT, which is the distance from the equilibrium melting temperature, Tm0 [77].
∆! =!!! −!!! Equation)4
where !!! is the equilibrium melting temperature and !! is the crystallization temperature.
Crystals can be defined as materials having a three-dimensional ordered structure. Thus crystallization can also be viewed as one of the driving forces for the self-assembly into an ordered structure, along with the microphase and solvophilic/solvophobic driving forces [78].
When crystallization is undergone by a block copolymer, it may compete with the driving force of microphase separation in bulk and thin film or with the solvophilic/solvophobic driving force in a selective solvent [78]. On the other hand, crystallization of a polymer is strongly dependent on temperature and therefore the relative strength of crystallization and other driving forces can be regulated. Crystallization is usually a kinetically controlled process. When the crystalline block copolymer is cast from a homogeneous solution, the orientation of microdomains is related to the crystallization rate. Before evaporation of the solvent, both blocks in the homogeneous solution can be in contact with the substrate surface [78]. The microphase-separated structure with both blocks in contact with the substrate surface, i.e., perpendicular orientation of the microdomains, may by solidified by crystallization before the surface-phobic block is repelled to the top layer of the thin film [78]. Controlling the crystallization pathway may thus lead to various structures. Therefore, one has to distinguish clearly among several processes involved in the formation of polymer crystals: nucleation (from which the crystals start to form), growth (how fast and under which constraints they form), and relaxations (evolution and reorganization after they have been formed) [79]. These characteristics lead to abundant morphologies [78].
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However, there are two typical crystallization modes for semicrystalline block copolymers in the bulk: break-out and confined crystallizations. In break-out crystallization, driving force growth is strong and submerges the microphase separation driving force [78]. By contrast, when the strength of crystallization is weaker than that of microphase separation, confined crystallization may occur. Therefore, this involves confinement in microdomains formed by microphase separation, and the microphase-separated morphology formed in the melt is retained after crystallization. Since the segments in BCPs are covalently linked, the conformations of the blocks are affected by each neighbour and are different from those of the free counter parts. This effect is especially pronounced in short BCPs such as block oligomers [80]. In fact, the two blocks of BCPs share an interface and occupy the same area, whereas for semicrystalline BCPs of high molecular weight, the amorphous block usually adopts a coiled conformation and occupies larger interface areas [78]. As a result, chain folding may take place allowing the crystalline block to occupy the same interface area and, for this reason, chain folding can be thermodynamically stable in semicrystalline BCPs. This is in contrast to crystalline homopolymers, in which the extended chains are the most stable thermodynamically [78]. The chain folding of the crystalline block depends on both thermodynamic and kinetic factors and may be variable with the chain lengths of both blocks and the annealing conditions such as time of heat and supercooling temperature [78, 81, 82]. The number of folding chains usually increases with the length of the crystalline block and fewer chain folds are achieved at a higher crystallization or annealing temperature. This can be used to regulate the thickness of the lamellar [83] microdomains. Fewer chain folds mean a higher degree of stretching for both the crystalline and amorphous blocks [78]. When the other block has a higher transition temperature (Tg) (for an amorphous blocks), or liquid crystal transition temperature or Tm (for a crystalline block) than the Tm of this block, this
31
high degree of deformation is retained after melting, which may result in a faster process rate upon crystallization [84]. The crystallization of BCPs depends on the relationship between the following three sufficient branching to create a 3 D sphere, which is typically formed at high supercooling due to the higher growth rate [77]. Dendrites are single-crystalline structures that grow due to a concentration gradient at the growth front of the crystal. The ratio of the diffusion constant, D, to growth rate, G, is critical for dendrite formation. This limitation causes branching that leads to needle-like growth eventually leading to tree shaped crystals. In thin films, formation of both of these structures occurs. For spherulites, because of the pseudo-2D space, the branching occurs in-plane. The growing lamellae continue to branch as the material is consumed during the crystallization process. In addition to the overall morphology, the crystal orientation can vary under given conditions. Crystals can assume an “edge-on” orientation or a “flat-on”
orientation. This orientation is defined by the chain axis of the crystallized material. If the chain axis is parallel to the surface, they are “edge-on”. If it is normal to the surface, then they are “flat- on”, see Figure 1-9. Dendrites, or diffusion-limited aggregates (DLA) [85], are formed as flat-on
“single crystals” with a large degree of branching. However, there are a few different kinds of DLA’s as described by Brener and coworkers [86]. They are compact seaweeds (CS), fractal seaweeds (FS), compact dendrites (CD), and fractal dendrites (FD) where seaweeds are isotropic and dendrites are anisotropic. All of these morphologies are highly dependent on supercooling as well as substrate surface energy.
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Figure 1-9: Typical preferential orientations of crystal lamellae in the polymer films: (A) edge- on and (B) flat-on lamellae [87].
The second classification of crystals concerns single crystals [88]. They are composed of a single lamella, consisting of two folded surfaces that have a characteristic shape based on the growth planes of the translational lattice. In most cases, this shape is polygonal [77]. The chain direction is always perpendicular to the substrate (flat-on). Due to the difference in surface free energy between the folded surface and the lateral surface, the lateral dimensions are in the order of microns whereas the thickness is in the order of nanometer. In thin films, the molecular mobility and metastable crystal thickness are important in growing single crystals. The polymer molecules must crystallize in such a way that the growth of the lateral face and diffusion to that face are similar. Unlike spherulites or dendrites, there is no branching caused by secondary nucleation or disparity between diffusion and growth. The transition from a given morphology to another is either due to the degree of undercooling ΔT, or crystallization temperature, or the thickness. The changes with respect to temperature are similar to those in bulk. By confining the polymer in thinner films, the diffusion is decreased. This change in diffusion with respect to the growth rate leads to different morphologies. If the growth rate is much larger than diffusion, then 2D spherulites can be observed [77]. In most cases, spherulites grow with an “edge-on” orientation, while DLA’s and single crystals grow with a “flat-on” orientation. The orientation is determined
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at the nucleation step [77]. The surface energy of the substrate and the availability of the material play an important role in the nucleation, and therefore the resulting orientation. Cho and coworkers [89] showed with isotactic polypropylene (i-PP) that crystallinity at the polymer- substrate interface increased as the surface energy of the substrate increased. They observed that the orientation of the chains at the interface was parallel (edge-on), and as the surface energy increased, the chains took on a perpendicular orientation. As well reported by Sutton et al [90], the substrate effect plays an important role on the crystalline morphology of lamellar crystals. In general, semicrystalline polymers can crystallize on a substrate, whereby molecular chains fold back and forth into stems to form crystal lamellae.
The growth rate and kinetics are also determined by the temperature and film thickness. In general, the thickness also affects the final crystallinity of the film. The growth rate decreases with decreasing thickness as well. The effect of temperature, however, follows a similar trend as in the bulk.
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1.8 Scope of the thesis
The present work deals with the combination of straight biomedical and organic routes to graft nucleotide sequences to a chitosan polymer of low molecular weight to induce self-assembly.
The first chapter of this thesis work is a general introduction about polymers and block copolymers and how the fundamental principles of supramolecular chemistry could be used to craft the size, shape, and internal structure of nanoscale objects. Later, deoxyribonucleic acid (DNA) and chitosan polymers are described prior to introduce the basics of self-assembling amphiphilic copolymers based on DNA. Introduction to crystallization of block copolymers is described as well. In the second chapter, all materials used for this thesis work are gathered in a table. In the third chapter, all experimental techniques used for characterization are introduced. In the fourth chapter the details of the synthesis, purification and characterization of the final pure compounds of chitosan and chitosan-g-ssDNA are given.
The fifth chapter describes how self-assembly of chitosan-g-ssDNA was induced in aqueous solution and the characterization of their morphology and size. The mechanism of structure formation was investigated as well in order to understand which force is responsible for driving and maintaining the chitosan-g-ssDNA self-assembled structures. The resulting chitosan-g- ssDNA hybrid self-assembled into submicrometer size structures in dilute aqueous solution. In the last chapter, the morphological study of chitosan-g-ssDNA macromolecule crystallization on surfaces and the mechanism of structure formation are described.
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2 Materials
Chemicals used are gathered in the following table:
Names Features
Papain 23000 gmol-1 (1.3 Umg-1, pH 6.2), purchased from Sigma-Aldrich (Buchs, Switzerland)
N-hydroxysuccinimide (NHS) &
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
Chemical activators purchased from Sigma-Aldrich (Buchs, Switzerland)
2-(N-morpholino) ethanesulfonic acid (MES)
Buffer (0.5 M, pH=2.5-4.0), purchased from Sigma Aldrich (Buchs, Switzerland)
Ammonium hydroxide Solution 35%, purchased from Fisher Scientific SA (Wohlen, Switzerland)
Acetic acid Purification grade 90 to 100 %, purchased from Sigma Aldrich (Buchs, Switzerland)
Acetonitrile Analytical solvent reagent grade, purchased from Fisher Chemical Scientific (Waltham, USA)
Sephadex G-50 Gel filtration medium, beads size varying between 50- 150 µm, purchased from Sigma Aldrich GE Healthcare (Switzerland)
Bio-Gel polyacrylamide beads P-10 Gel filtration medium, fractionating range varying from Mw 1,500 to 20,000 Da, purchased from BioRad Laboratories AG (Cressier, Switzerland)
Salts NaCl, MgCl2 and other organic solvents
Purchased from Sigma Aldrich, Fluka (Cressier, Switzerland)
Potassium hydroxide (KOH) Mw 56.11 gmol-1, used for precipitating chitosan in an acidic medium, purchased from Sigma-Aldrich (Steinheim, Germany)
Chitosan Deacetylation degree (DD) above 75%, Mw between 50000 and 160000 Da, purchased from Sigma-Aldrich (Buchs, Switzerland)
36 Single strand DNA
(ssDNA)
The sequence used for these studies is a twelve nucleotide-long single stranded sequence functionalized with a carboxylic acid group through a C10-linker at the 5’ –end whereas the 3’ –terminus is bound to the control pore glass resin.
The sequence is composed of Cytosine (C) and Thymine (T); (5’-COOH- CTCTCTCTCTTT-3’) purchased on controlled pore glass (CPG) (desalted, Mw 3763.9 g.mol-1), provided by Microsynth (Balgach, Switzerland), see figure 2-1.
Complementary ssDNA
Sequence 5’-AAAGAGAGAGAG’-3, HPLC purification, purchased from Microsynth (Balgach, Switzerland) (Mw 3776.2 gmol-1)
Silica wafers Orientation (100), purchased from Silchem, (Freiburg, Germany).
Mica substrates High-grade quality V1, purchased from Plano GmbH, (Wetzlar, Germany) Gold substrate Orientation (100) on mica surface, purchased from PHASIS (Geneva,
Switzerland) AFM tips for
imaging in air
Silicon probes; Al reflex coated, spring constant Kc =2 N.m-1, resonance frequency fr= 70 kHz, purchased from Asylum research (Santa Barbara, USA)
AFM tips for imaging in liquid
BioLever mini, Sharpened silicon on nitride lever, tip radius of 10 nm, Gold reflex coating, Kc ~ 0.1 N.m-1, fr = 110 kHz, purchased from Asylum research (Santa Barbara, USA)
Deuterium oxide (D2O)
Heavy water used for NMR, 99,8 % purchased from ARMAR chemicals (Döttingen, Switzerland)
Deuterium chloride (DCl)
Deuterieum chloride 99 %, purchased from sigma-Aldrich GE Healthcare (Balgach, Switzerland)
Membrane dialysis Dialysis tubing, benzoylated, average flat width 9 mm, Mw cut off 2000 Da, purchased from Sigma-Aldrich (Steinheim, Germany)
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Figure 2-1: Nucleotide sequence (5’-(CT)5T2-3’).
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3 Experimental techniques and methods
3.1 Matrix-Assisted Laser Desorption/Ionization-Time-of- Flight (MALDI-TOF) Mass Spectrometry
Matrix-Assisted Laser Desorption/Ionization Mass spectrometry is an analytical tool introduced firstly by Hillenkamp and Karas in 1988 [91]. MALDI-TOF is a soft ionization technique used in mass spectrometry, allowing the analysis of large organic molecules (such as polymers or dendrimers) and biomolecules (biopolymers such as DNA, proteins, peptides and sugars), which tend to be fragile and fragment when ionized by more conventional ionization methods, such as electron ionization and electrospray ionization.
The principle consists in at first co-crystallizing the analyte (sample) with a large molar excess of a matrix compound. Usually the matrix is an ultraviolet (UV)-absorbing weak organic acid, after which laser radiation of this analyte-matrix mixture results in the vaporization of the matrix which carries the analyte [92]. The matrix also serves as either a proton donor or acceptor, acting as an ionization agent of the analyte in both positive and negative ionization modes, respectively [93]. Three different types of mass analysers [92] can be used in matrix-assisted laser desorption/ionization: a linear time-of-flight (TOF), a TOF reflectron, and a Fourier transform mass analyser. The linear TOF mass analyser is the simplest of the three devices [92]. TOF analysis is based on accelerating a set of ions to a detector on which all the ions are given the same amount of energy [92].
Because the ions have the same energy, yet a different mass, the ions reach the detector at different times. The smaller ions reach the detector first because of their greater velocity while
39
the larger ions take longer time owing to their larger mass [92] see Figure 3-1. Hence, the analyser is called TOF because the mass is determined from the ions’ time of flight [92]. The arrival time at the detector is dependent on the mass, charge, and kinetic energy (KE) of the ion, while the time of ions flight is used to determine the mass.
Figure 3-1: Schematic representation of MALDI-TOF spectrometer operation principle [94].
Measurements were performed on a Bruker Daltonik GmbH (Bremen, Germany), from the MALDI-TOF autoflexTM series, equipped with the smartbeam-II laser technology with adjustable repetition rates up to 2,000 Hz. The matrix chosen for the detection was 2, 5-dihydroxy benzoic acid (DHB) commonly used for glycoproteins, glycopeptides, oligosaccharide and oligonucleotides characterization [95].
Sample preparation consisted in mixing 5 µL of DHB matrix with 5 µL of prepared solution sample, chitosan or ssDNA or chitosan-g-ssDNA, and kept mixed solution to evaporate at room temperature prior to measurements.
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Mass spectrometry measurements were performed at least three times for samples of chitosan and chitosan-g-ssDNA.
3.2 Analytical Ultra-Centrifugation (AUC)
Analytical Ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution which relies on the mass and the fundamental laws of gravitation. AUC has a broad range of applications and can be used to analyze the solution behaviour of a variety of molecules in a wide range of solvents and over a large range of solute concentrations [96]. In contrast to many commonly used methods, during analytical ultracentrifugation, samples could be characterized in their native state under biologically relevant solution condition around physiological pH ∼ 7. Two complementary views of solution behaviour are available from AUC. Sedimentation velocity provides hydrodynamic information about the size and shape of molecules [97] and sedimentation equilibrium provides thermodynamic information about the solution molar masses, stoichiometry, association constants [97]. During the measurement, the application of a centrifugal force causes the depletion of macromolecules at the meniscus and the formation of a concentration boundary that moves toward the bottom of the centrifuge cell as a function of time [98]. The definition of the sedimentation coefficient of a macromolecule (s), and the molecular parameters that determine the s-value are given by the Svedberg equation:
! =
!!!!!!!
Equation 5!where u is the observed radial velocity of the macromolecule, ω is the angular velocity of the rotor, r is the radial position from the center of rotation and ω2r is the centrifugal field. For
41
samples containing many components, aggregates, low molecular weight contaminants, or high concentration samples, size distribution and average quantities may be determined [98].
Experiments were performed on an Optimal XL-I ultracentrifuge (Beckmann-Coulter, Palo Alto, CA) equipped with Rayleigh interference optics. Sedimentation-velocity experiments were performed with 1 mg mL-1 solutions of polymer in acetonitrile/water (1:1) (v/v) at a rotational speed of 50 krpm at 25 °C.
3.3 Size Exclusion Chromatography/Gel permeation chromatography (SEC/GPC)
Size exclusion chromatography (SEC), also referred to as gel chromatography, gel filtration, or gel permeation chromatography (GPC), is a technique that is used to fractionate and characterize molecules, polymers and macromolecules according to their size. The sieving medium is a porous gel. When all molecules are in the gel medium, the molecules having a much smaller size than the pore diameter will have more probability of penetrating the gel and will pass through the column more slowly. Therefore, the speed of each molecule in a mixture is dependent on the ease with which molecules can pass through the gels and be retarded [99]. Molecules with diameters much larger than the pore size will have less probability to penetrate the gel particles and will be excluded from the gel and pass through the column freely [99]. Intermediate size molecules can pass into some of the gel particles but compared to very small molecules, a greater proportion of the intermediate size molecules will be outside the gel at any time. The most widely used types of gels are cross-linked dextrans (Sephadex), cross-linked agarose (Sepharose), cross-linked polyacrylamide (Bio-gel), cross-linked allyldextran (Sephacryl) and controlled pore glass beads.
These are graded according to pore size. A wide range of molecular weights can thus be
42
fractionated. The upper limit of the fractionation range is the exclusion limit which means that the molecules with a molecular weight greater than this will have less probability of penetrating the gel and will be completely excluded [99].
Size exclusion chromatography experiments were performed on a BioLogic LP low-pressure system model from BIO-RAD (California, USA) equipped with UV optics modules including a mercury lamp and filters for fixed wavelength UV detection at 280 and 254 nm. The flow cell has a path length of 2 mm, an internal volume of 80 µL, and an illuminated volume of 3 µL. The device is equipped with a 2110 fraction collector using a stationary drop-dispensing head to collect up to 80 fractions in a carousel.
Sample preparation prior to collect chitosan fractions using SEC consisted in injecting 1 mL of solution of digested chitosan by papain into an Econo-column (Bio-Rad, 1 x 50 cm) loaded with sephadex G-50 gel medium and 1 % of acetic acid degassed, pH=3 used as eluent.
In the case of purification of chitosan-g-ssDNA from unbound DNA subsequent to synthesis, 1 mL of the solution material was injected on the Econo-column (1 x 50 cm) loaded with the Bio- Gel P-10 gel medium. 2-(N-morpholino)ethane sulfonic acid buffer (MES) (0.1 M, pH=4.9) elution buffer was used as eluent. Measurements of SEC during this research work were achieved after each chitosan digestion and repeated more than three times during this research work, as well as for purification of chitosan-g-ssDNA.
The GPC experiments were carried out with a Viscotek instrument (Malvern Instruments Ltd, UK) controlled by the OmniSec 4.7 software supplied by Viscotec. The instrument consisted of a VE 122 solvent delivery system, a manual injector equipped with a 100 µL loop, a VE 7510 GPC degasser and a multi detector system including UV (270 dual detector), refractive index (RI), right-angle light scattering (RALS) and low angle light scattering (LALS) detectors. Separations
