FUNCTINALIZATION OF CELLULAR MEMBRANE BY CHOLESTEROL-DENDRIMER CONJUGATES NGUYEN THI THUY LINH (B. Sci., VNU, Vietnam) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY, FACULTY OF SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements First, I would like to express my heartfelt thank is my direct supervisor Dr. Tan Choon Hong for his invaluable guidance, for his continuous flow of idea and source of inspiration. Although very busy with his schedule, he is always available for making helpful discussion and advice. I am also especially grateful to my co-supervisor Prof. Hanry Yu for recognizing my intent and encouraging me to pursue the difficult project presented in this thesis. His constant concern is very much appreciated. I must thank Prof. Yao Shao Quin for providing access to the equipments in his lab. He Lijuan and Ong Siew Min are acknowledged for their guidance on my first biological experiments while Kong Kien Voon is credited for his help with microscopy FT-IR experiment. I also wish to express my appreciation to staffs from NMR, MS, Analytical lab as well as Confocal unit for their technical support. Last but not least, I wish to express my gratitude to all my friends and colleagues for their help and friendship throughout these years. i Table of Contents Acknowledgements i Table of Contents ii Summary v List of Tables, Figures and Schemes vii List of Symbols ix Chapter 1 Introduction 1.1 Mammalian cellular membrane and its lipid domains 1 1.1.1 Overview of cellular membrane 1 1.1.2 Lipid domain and its charge 4 1.2 Cholesterol and cellular membrane 6 1.2.1 General feature, location of cholesterol within cellular membrane 6 1.2.2 Roles of cholesterol 8 1.3 Dendrimers in bioengineering 9 1.3.1 Chemistry of dendrimers 9 1.3.2 Biological application of dendrimers 12 1.3.3 Cytotoxicity of dendrimers 14 1.4 Chemical modification of mammalian cellular membrane 14 1.4.1 Insertion of molecules into cell surface 15 1.4.2 Reaction using exogeneous enzymes 16 1.4.3 Inhibition of biosynthetic pathways 17 ii 1.4.4 Metabolic engineering 18 1.4.5 Covalent ligation to cell surface chemical groups 19 1.4.6 Application of surface engineered mammalian cells 20 Chapter 2 The chemistry of cellular membrane functionalization 2.1 Chemoselective ligation reactions 22 2.2 Cholesterol-dendrimer conjugates 26 2.3 Preparation of conjugates with thiol functionality 28 2.4 Preparation of conjugates with ketone functionality 31 2.5 Preparation of conjugates with azide functionality 34 2.6 The roles of each conjugate component 41 Chapter 3 Delivery of non native functional groups 3.1 Labeling cellular membrane by cho-dab conjugates 44 3.1.1 Preliminary investigation 44 3.1.2 Detection of non native surface thiol 47 3.1.3 Detection of non native surface ketone 51 3.1.4 Detection of non native surface azide 54 3.2 Characterization of the labeling methods 58 3.2.1 Cytotoxicity assay 58 3.2.2 Time dependent depletion 61 3.3 Comparison with reported methods 64 3.4 Conclusion and future work 65 Chapter 4 Materials and Methods iii 4.1 Synthetic procedures and compound characterization data 68 4.2 Biological assays 78 4.2.1 Cell culture 79 4.2.2 The introduction of cell surface functional groups 80 4.2.3 The detection of displayed functional groups on cell surface 80 4.2.4 Cytotoxicity assay 82 4.2.5 Determination of cell surface functional group’s depletion 82 Appendices 84 References 88 iv Summary The prospect of chemical cellular membrane modifications allows one to exercise considerable creativity in remodeling cell surface. Non-native functional groups cell surface decoration has been achieved but the range of functional groups is rather limited. We would like to design a new cholesterol-dendrimer conjugate that might be able to deliver various non-native reactive functional groups to cell surface. In this thesis, we performed the synthesis of different cholesterol-dendrimer conjugations. These conjugations were then proved as vehicle to deliver non native functionalities to cellular membrane. The functional groups that could be delivered to the membrane were quite profuse since different conjugates-containing thiol, ketone and azide were successfully synthesized. Detection of these surface functional groups was examined by chemoselective ligation chemistry and microscopy FT-IR. Synthesis of some ligated complementary functionality compounds was also demonstrated. Besides, cholesterol containing functionality and dendrimer containing functionality were synthesized and tested on cells. This study verified the roles of cholesterol and dendrimer. Cytotoxicity test, which shows the toxicity of the chemical to cells, was carried to find out the best condition for further study. Experimental data revealed that the optimum working condition of conjugates was 0.01-0.1 mM with 30 minutes incubation time at 4 ºC. At this condition, the cell viability was acceptable whilst the v cell morphology was unchanged. The time dependent depletion of surface non native functionalities was also tested. These functionalities were found to stay within the membrane for at least 12 hours under culture condition. These biological abilities of cholesterol-dendrimer conjugates suggested the feasibility as an alternative approach for cell surface engineering. vi List of Tables, Figures and Schemes Figure 1.1 Structure of some phospholipids 5 Figure 1.2 Structure of cholesterol 7 Figure 1.3 Common commercial dendrimers 11 Table 2.1 The chemistries used for the synthesis of native proteins by chemical ligation of unprotected peptide segments 22 Figure 2.1 List of most common chemoselective ligation reactions 24-25 Scheme 2.1 The synthesis of tagged cho-dab conjugates 27 Scheme 2.2 The synthesis of membrane-anchored thiol ma3 30 Scheme 2.3 The reaction of surface-thiol and the maleimide probe 31 Scheme 2.4 The synthesis of membrane-anchored ketone 32 Scheme 2.5 The reaction of surface ketone and biotin hydrazide 33 Scheme 2.6 The synthesis of azidoacetyl chloride 34 Scheme 2.7 The synthesis of membrane-anchored azide 35 Scheme 2.8 The model of [3+2] cycloaddtion of azide and terminal alkyne 36 Scheme 2.9 The synthesis of biotinylated alkyne 37 Scheme 2.10 The classic Staudinger and modified Staudinger reaction 38 Scheme 2.11 The synthesis of the phosphorane 39 Scheme 2.12 The synthesis of phosphine ligand ma14 40 Scheme 2.13 The synthesis of non-linker vehicle 42 Figure 3.1 The confocal analysis of tagged-cho-dab treated cells 46 vii Figure 3.2 The confocal analysis of stained-thiol expressing cell 48 Figure 3.3 Role of cholesterol and linker was asserted by confocal analysis 50 Figure 3.4 Confocal analysis of ketone-expressing cells 53 Figure 3.5 Microscope FI-IR analysis of cell surface azide 56-57 Figure 3.6 Cytotoxicity test of chemical treatment to cells 60 Figure 3.7 Cells after treatment with conjugate-ketone at 0.1 mM 62 Figure 3.8 Cell after treatment with conjugate-ketone at 0.05 mM 63 viii List of Symbols 2-IT 2-iminothiolane DAB-Am-4, DAB 4 Polypropylenimine tetramine dentrimer, Generation 1.0 DAB-Am-8, DAB-8 Polypropylenimine octaamine Dendrimer, Generation 2.0 DCC 1,3-dicyclohexylcarbodiimide di Deinonized DIEA Diisopropylethylamine DMEM Dulbecco’s modified Eagle’s medium DMF (N,N-Dimethylformamide) DMSO Dimethyl Sulfoxide EA Ethyl acetate EDTA Disodium ethylenediaminetetraacetate FBS Fetal bovine serum FC Flash chromatography FCS Fetal caft serum FITC Fluorescein 5’-isothiocyanate FG Functional group HCl Hydro chloride HOBt (1-hydroxbenzotriazole) HPLC High pressure liquid chromatography HRMS High resolution mass spectroscopy/mass spectra IR Infrared LRMS Low resolution mass spectroscopy/mass spectra ManLev N-levulinoylmannosamine ManNAz N-azidoacetylmannosamine MeCN Acetonitrile ix MeOH Methanol MRI Magnetic resonance imaging MS Mass spectroscopy/ mass spectra MWCO Molecular weight cut-off NMR Nuclear magnetic resonance PAMAM Polyamides and amines PEI Poly (ethylenimine) Ppm Parts per million rt Room temperature SEM Standard error of the mean SiaLev N-levulinoyl sialic acid TEA triethylamine TLC Thin layer chromatography x Chapter 1 Introduction 1.1 Mammalian cellular membrane and its lipid domains The cell represents the fundamental unit of living matter. Every living being, with the exception of viruses, is made of cells. The simplest such beings consist of only a single cell, like bacteria and protozoa. But most living beings contain a very large number of cells. One gram of human tissue, for example, contains about a billion cells! The dimensions, shapes, and structure of cell are extremely varied, since they play very different roles in an organism. A bone cell, a liver cell, and a brain cell perform completely different functions.1-3 Cells are enclosed by a cell membrane that encapsulates all the intracellular components, organelles, of the cell which to carry out metabolic chemical processes for growth and replication. The cell membrane provides the mean for cells to separate their external environment from their internal environment. The cell membrane was at one time envisaged as only a passive barrier for diffusion and permeability, but it is now known to play an active role in chemical transport, energy transduction, and information transfer to and from the cells.4 1.1.1 Overview of cellular membrane The term ‘plasma membrane’ derives from the German Plasmamembran, a word coined by Karl Wilhelm Nägeli (1817-1891) to describe the firm film that forms when the proteinaceous sap of an injured cell comes into contact with water.3 Much knowledge concerning membrane structure and function derives from studies of red blood cells.5-8 In 1925, Gorder and Grendel,5 two Dutch biochemists, extracted lipids 1 from human erythrocyte membranes and placed them in a water trough. These lipids formed a mono layer at the air-water interface, with their hydrocarbon tails facing the air and polar head groups in the water. When the phospholipids were compressed with a movable barrier, the surface area covered by the phospholipids was twice the surface are of the erythrocyte membranes from which they were extracted. Together with data estimated by light microscopy for surface area of red cells, they concluded that the chromocytes were covered by a layer of fatty substances that is two molecules thick. In 1935, H. A. Davson and J. F. Danielli9-10 proposed that biologic membranes were made up of lipid bilayers. With the realization that biological membranes also contain proteins, they incorporated protein into their model of membrane, known as the Davson-Danielli paucimolecular membrane. Paucimolecular means that this model included just a few molecules: a bimolecular lipid leaflet with adhering proteins films on the inner and outer surfaces. However, they visualized the protein as being attached to only the periphery of the membrane by association with the polar head groups of the phospholipids. In their noteworthy review of membrane permeability,11 the two authors recognized that the ion permeability of red blood cells, together with electrical impedance measurements, lipid extraction studies and the birefringence of red-cell ghosts, are all consistent with the postulation of a bimolecular lipid membrane as first proposed in 1925. The book has greatly influenced subsequent development of cellular and membrane physiology. Nevertheless, Davson-Danielli model could not account for numerous properties of membrane proteins without doubt. The year 1972 was it superseded by fluid mosaic model of Singer and Nicholson.12 The basic principles of this model were that membrane proteins could be globular, just as could water-soluble proteins. The 2 globular membrane proteins were embedded within the bilayer, with the hydrophobic portions of the proteins buried within the hydrophobic core of the lipid bilayer and hydrophilic portions of the protein exposed to the aqueous environment. It suggested that the lipids form a viscous, two-dimesional solvent into which proteins were inserted and integrated more or less deeply. Cell membranes studied by electron microscopy and X-ray diffraction13 indicate that the cell membrane structure is a bilayer, 80-100 Å in thickness, with < 50 Å of this due to the lipid bilayer and the remainder due to other molecules. The latter comprises the glycolipids and glycol proteins extending from the exofacial side and the cytoskeleton extending from the cytofacial side of the plasma membrane. Proteins and enzymes are molecularly associated with the lipid bilayers in very unique manner. These molecules can move rather freely. The proteins on or in the lipid bilayers allow the membrane to carry out transmembrane reactions, that is, chemical transport, energy transfer, and signal transduction. Therefore, every type of membrane has a unique set of proteins and enzymes to account for its function. Non polar, non charged molecules can cross these cell membranes fairly readily by diffusion mechanism. This permits oxygen to enter cells and carbon dioxide to leave cells, and permits some very lipid-soluble substances to cross easily into cells, passing directly through the bilayer. The situation is more complex with ions or polar molecules. Since the basic structure of the plasma membrane is the phospholipid bilayer, it is impermeable to most water soluble molecules. This bilayer acts as a barrier to the diffusion of ions or charged molecules. The passage of ions and most biological membrane permeable molecules across the plasma is therefore mediated by proteins, which are responsible for selective traffic of molecules into and out of the cells.2 These particles pass into or out of cells through protein molecules which span 3 the lipid bilayer in much the same manner as bridges, tunnels, or ferries permit to access an island. Eukaryotic cells are also able to take up macromolecules and particles from the surrounding medium by a distinct process called endocytosis. In endocytosis, the material to be internalized is surrounded by an area of plasma membrane, which the buds off inside the cell to form a vesicle containing the ingested material. The term ‘endocytosis’ was coined by Christian deDuve in 1963 to include both the ingestion of large particles (such as bacteria) and the uptake of fluids or macromolecules in small vesicles. The former of these activities is known as phagocytosis (cell eating) and the latter as pinocytosis (cell drinking). 1.1.2 Lipid domain and its charge Lipids constitute approximately 50% of the mass of most cell membranes, although this proportion varies depending on the type of membrane. Mammalian plasma membranes phosphatidylcholine, are complex, containing phosphatidylserine, four major phospholipids: phosphatidylethanolamine, and sphingomyelin-which together constitute 50 to 60% of total membrane lipid. Other phospholipids, phosphatidylinositol and phosphatidylglycerols, in a quantitatively minor amount, are also localized in plasma membrane. In addition to the phospholipids, the plasma membranes of mammalian cells contain glycolipids and cholesterol. The glycolipids are found exclusively in the outer (exofacial) leaflet of the plasma membrane, with their carbohydrate portions exposed on the cell surface. They are relatively minor membrane components, constituting only about 2% of the lipids of most plasma membranes. Cholesterol, on the other hand, is a major membrane constituent of animal cells, such that the cholesterol/phospholipids molar 4 ratio ranges from 0.4-1.0. For instance, in red blood cell, the molar ratio of cholesterol/phospholipids is 0.8-1.14 The chemical structure of the polar headgroup of these phospholipids determines what charge the phospholipids as a whole may carry. Phosphatidylcholine at physiological pH value carries a full negative charge on the phosphate and a full positive charge on the quaternary ammonium. It is zwitterionic, but electrically neutral. O O R O O O R' O O O O P O R N O O Phosphatidylcholine R' O O O NH3 P O Phosphatidylethanolamine O O R O O R' O O O NH2 P O OH O phosphatidylserine Figure 1.1 The structure of some phospholipids at neutral pH. R and R’ represent for fatty acid chains. The length of the commonly found fatty acids varies from as few as 12 carbons to as many as 26 carbons. The number of double bonds per fatty acid commonly ranges from one to as many as six. The distribution of fatty acids in membrane phospholipids is peculiar to the class of phospholipid and the membrane type.13 5 Phosphatidylethanolamine is constructed similarly. It carries a positive charge on the amine (which can be deprotonated as described above) as well as negative charge on the phosphate. Phosphatidylserine contains, in addition to the negatively charge phosphate, a positively charged amino group and a negatively charged carboxyl. Therefore, this lipid exhibits an overall negative charge at neutral pH. The group of negatively charged lipids includes phosphatidylglycerols and phosphatidylinositol. These phospholipids carry a negative charge, because the sugar carries no positive charge to balance the negative charge of the phosphate. Diphosphatidylglycerol normally carries two negative charges, because of its two phosphates. The discussion explains why some phospholipids carry charges. Those charges are held at the surface by the organization of the membrane lipid bilayer. Phospholipids can therefore be important in determining the surface charge of the membrane.15-19 1.2 Cholesterol and cellular membrane 1.2.1 General feature, location of cholesterol within cellular membrane Cholesterol is a prominent constituent of mammalian cell membranes (as much as 30% of the plasma membrane lipid content in some tissues).20 Cholesterol is quite different in structure from the other membrane lipids that have been discussed. The French chemist M. E. Chevreul is credited with the initial discovery of cholesterol in 1815. The empirical formula for cholesterol (C27H46O) was not established until 1888 by F. Reinitzer. 21,22 At the beginning of the 20th century, it was known that cholesterol had an alcoholic functional group and a double bond.23 Thus, a great deal of work was required to elucidate the structure. The details of this chemistry have 6 been briefly discussed by Konrad Bloch23 and summarized in greater detail by Louis and Mary Fieser.24 H H HO H cholesterol Figure 1.2 Structure of cholesterol The structure of cholesterol appears in Figure 1.2. The structure consists of four fused rings, referred to as the A, B, C and D rings, reading from left to right. The most common conformation of this steroid ring system is planar. One face of the plane is flat; the opposite side of this sterol is not flat, due to two protruding methyl groups. This is not the only conformation available to the cholesterol, however. From crystal structures, it is evidence that the A ring can adopt an alternative conformation in the crystal, indicating some conformational flexibility in the A ring. Much more conformational flexibility is enjoyed by the tail of cholesterol.25 In addition to an approximately planar steroid ring system, and the hydrophobic tail just referred to, cholesterol possesses a 3β-hydroxyl function. All three features are important for characteristic cholesterol-like behavior. Although most of the molecule is hydrophobic, the 3β-hydroxyl is polar and gives the molecule an amphipathic character, as the phospholipids. Cholesterol is surface active, orienting in a phospholipid bilayer with its polar hydroxyl facing the aqueous phase and the hydrophobic steroid ring parallel to, and buried in, the fatty acid chains of the phospholipids and perpendicular to the membrane surfaces. X-ray diffraction and neutron diffraction data26,27 have provided a clearer picture of the location of 7 cholesterol within the membrane. The data showed that the cholesterol molecule is located so that the hydroxyl is in the immediate vicinity of the phospholipid ester carbonyl. 1.2.2 Roles of cholesterol Consideration on the behavior and consequences of cholesterol in cell membranes helps to focus attention on several important issues concerning the roles of cholesterol in mammalian cell membranes. The basic role of cholesterol is membrane lipid constituent. Membrane cholesterol level was proved to influence membrane permeability and stability properties.28 Cholesterol does this by its special ordering effect on the membrane lipids. Because of its hydrocarbon ring structure, cholesterol plays a role in determining membrane fluidity.29 Cholesterol molecules insert into the bilayer with their polar hydroxyl groups close to the hydrophilic head groups of the phospholipids. The rigid hydrocarbon rings of cholesterol therefore interact with the regions of the fatty acid chains that are adjacent to the phospholipid head groups. This interaction decreases the mobility of the outer portions of the fatty acid chains, making this part of the membrane more rigid. On the other hand, insertion of cholesterol interferes with interactions between fatty acid chains, thereby maintaining membrane fluidity at lower temperatures.2 Recent studies suggest that not all lipids diffuse freely in the plasma membrane. Instead, discrete membrane domains appear to be enriched in cholesterol and the sphingolipids (sphingomyelin and glycolipids). These clusters of sphingolipids and cholesterol are thought to form “rafts” that move laterally within the plasma membrane and may associate with specific membrane proteins. Although the functions of lipid rafts remain to be understood, they are thought to play important 8 roles in processes such as cell signaling and the uptake of extracellular molecules by endocytosis.2,30 1.3 Dendrimers in bioengineering Dendrimers constitute a unique class of polymers that are distinguished from all other synthetic macromolecules by their globular shapes resulting from their perfectly branched architecture and their monodisperse nature.31-33 In recent years dendrimers have attracted more and more attention in biomedical applications,34-36 especially as transfection agents for DNA transfer into eukaryotic cells,37-39 as contrast agents for magnetic resonance imaging (MRI),40-42 in boron neutron capture therapy (BNCT) for cancer treatment,34,43,44 and most recently as potentially selective drug delivery vehicles.45-48 1.3.1 Chemistry of dendrimers Since the first Starburst® dendrimers were reported in the 1980s,49 these aesthetically pleasing macromolecules have now reached the point of commercial development. Dendrimer is a polymeric molecule composed of multiple perfectly branched monomers that emanate radially from a central core, reminiscent of a tree, whence dendrimers derive their name (Greek, dendron, meaning tree or branch, and meros, meaning part). Despite their large molecular size, dendrimers have welldefined structure, with a low polydispersity compared with traditional polymers. The size, molecular weight, and chemical functionality of dendrimers can be easily controlled through the synthetic methods used for their preparation both by divergent49-51 and by convergent52,53 methods. In the divergent approach, the dendrimer is synthesized from the core and built up generation by generation. The 9 alternative convergent approach starts from the surface and ends up at the core, where the dendrimer segments are coupled together. In both these approaches, a branch point is inserted in the dendritic structure at each monomer unit leading to a well defined macromolecule. A number of identical fragments called dendrons are remained after the removal of the central core. The number of dendrons depends on the multiplicity of the central core (2, 3, 4 or more). A dendron can be divided into three different regions: the core, the interior (or branches) and the periphery (or end groups). The number of branch points encountered upon moving outward from the core of the dendron to its periphery defines its generation (G-1, G-2, G-3); dendrimers of higher generations are larger, more branched and have more end groups at their periphery than dendrimers of lower generation. Over 50 compositionally different families of these nanoscale macromolecules, with over 200 end-group modification, have been reported,54 their chemical and physical properties as well as their solution behaviors have been studied and well characterized.33,55 The dendrimer design can be based on a large variety of linkages, such as polyamines (PPI dendrimers),56 a mix of polyamides and amines (PAMAM dendrimers)49 and more recent designs based on carbohydrate57 or calixarene core structure,58 or containing ‘third period’ elements like silicon or phosphorus.59 Although the synthesis of dendritic system is a crucial aspect of their development, it is also important to ask where the future applications of these molecules lie, and what their unique properties are. In fact, it becomes clear that dendrimer chemistry is itself ‘branching out’ in two directions, that is towards biological and materials chemistry. 10 The most exploited property of dendrimers is their multivalency with a high number of potential reactive sites. Unlike in linear polymers, as dendrimer molecular weight and generation increase, the terminal units become more closely packed which exploited by many investigators as a means to achieve concentrated payloads of drugs or spectroscopic labels for therapeutic and imaging applications.60 Figure 1.3 Common commercially available dendrimers. Top left: Polypropylene imine dendrimer (G5). Top right: Polyamido amine dendrimer (G3). Bottom: Polyamido amine (Starburst™) dendrimer (G5). Each generation is marked with a circle. Adapted from Boas and Heegaard.46 11 1.3.2 Biological applications of dendrimers The use of dendrimers as frameworks and as carrier systems for the study and modulation of biological processes is gaining popularity. In the past years, significant advances have been made in the synthesis and study of glycodendrimers and peptide dendrimers. Application of these dendrimers to the study of carbohydrate-protein and protein-protein interactions has facilitated the understanding of these processes. The term ‘glycodendrimer’ is used to describe dendrimers that incorporate carbohydrates into their structures.59,61,62 Glycodendrimers have been used for variety of biologically relevant applications. Most notably, they have been used to study the protein-carbohydrate interactions that are implicated in many intercellular recognition events. Compared with other frameworks that have been used to study such interaction, dendrimers are appealing because of their size (between those typical of small glycoclusters and large glycopolymers) and their low polydispersity (compared with that of most large glycopolymers). Glycodendrimers are also likely to be beneficial in some other areas such as incorporation into analytical devises,63 formulation of gels,64 targeting MRI contrast agents65 and drug and gene delivery systems. Peptide dendrimer is another class of wide application dendrimers.66 Peptide dendrimers have potential applications as protein mimics, antiviral and anticancer agents, vaccines and drug and gene delivery systems. Amino acids are appealing dendrimer building blocks because peptide-coupling techniques including solid-phase synthesis can be used. Generally, the peptide dendrimers are more soluble in water, more stable to proteolysis, and less toxic to human cells than their linear polymeric analogs; comparable antimicrobial potency was demonstrated. Preliminary studies with the peptide dendrimers to evaluate peptide-protein and protein-protein 12 interactions indicate that dendrimer research will ultimately make significant contributions to understand these processes. In addition, dendrimers show great promise as drug delivery system due to their ability to increase the selectivity and stability of therapeutic agents. Dendrimer drugdelivery systems of several different types have been proposed.67-69 By attaching a drug to a suitable carrier, it is possible to enhance its aqueous solubility, increase its circulation half-life, target drug to certain tissues, and improve drug transit across biological barriers and slow drug metabolism. Interestingly, whereas the majority of dendrimer designs have been used as carriers for drugs, some dendrimers acts as drugs themselves by stimulating the removal of prion proteins present in infected cells.70 Besides, PAMAM dendrimers71,72 polypropylenimide dendrimers73 and partially hydrolyzed PAMAM dendrimers34 have been used as DNA delivery systems due to their ability to form compact polycations under physiological conditions. This can be explained by the fact that their cationic charges allow binding with negatively charged nucleic acids, resulted in submicrometer-sized water soluble particles. Moreover, these dendrimers have been showed to cross cell barriers at sufficient rates74,75 to act as potential DNA transporting agents, allow efficient transfection of a variety of established cell lines as well as primary cells.76 Dendrimers also exhibit lower cytotoxicity than the widely used lipopolymers delivery systems.77 Clearly, there are many other areas of biological chemistry where application of dendrimer systems may be helpful. 13 1.3.3 Cytotoxicity of Dendrimers In most cases, the nature of a dendrimer’s numerous end groups dictate whether or not it displays significant toxicity. Cationic dendrimers with terminal primary amino groups, such as PAMAM and polypropylenimide (PPI) dendrimers display concentration-dependent toxicity and hemolysis,78-80 whereas dendrimers containing only neutral or anionic components have been show to be much less toxic and less hemolysis.78,81-84 The toxicity of cationic PAMAM dendrimers increases with each generation78,80,85 but, cationic PPI dendrimers do not follow this trend closely. Molecular modeling and experimental data revealed that DAB 4 (G1) is more toxic than DAB 8 (G2) but they both combine a sufficient level of DNA binding with low level of cytotoxicity to give their optimum in vitro gene transfer activity.86 The mechanism of cell death for cationic dendrimers is proposed to be attributable to necrosis and/or apoptosis, although it has not been precisely determined for all dendrimer types and can differ among cell lines. 87,88 However, it should be noted that the toxicity and biological profile of a dendrimer-based delivery system (with surface modifiers and a payload of drug) is likely to be different from that of an unmodified dendrimer.89 1.4 Chemical modification of mammalian cellular membrane The boundary between biology and chemistry has eroded in recent years. The ability to modify cellular membrane both in vitro and in vivo by chemical approach has led to the development of the rapidly expanding field – cell surface engineering. 14 1.4.1 Insertion of molecules into cell surface The lipophilic nature of mammalian cell surface has been exploited by a number of research groups in order to display bioactive molecules, both naturally occurring and synthetic, on the cell surface. To achieve this insertion, a fatty moiety is attached to the biomolecule of interests and, when applied to the cell, the fatty moiety incorporates into the membrane, leaving the biomolecules exposed on the cell surface. Two main classes of compound, namely GPI-anchored proteins and cholesteroltethered compounds have been used for this strategy. GPI represents for glycosylphosphatidylinositol. As the name implies, GPIanchored proteins are posttranslationally-added structures that attach a subset of cell surface proteins to the plasma membrane.90,91 These anchor moieties are utilized by all eukaryotes. They tether a wide range of functionally diverse proteins to cells and operate ubiquitously irrespective of the extracellular structural properties of the attached proteins. A unique feature of GPI-anchored proteins is that following their extraction from cells, they are able to reintegrate into plasma membranes when added to other cells92 with their full biological functionality. Nevertheless, in recent years, evidence has been obtained that GPI-anchored proteins are not diffusely expressed on cell surface membranes and that their activities are not entirely limited to the cell exterior as initial hypothesized. Substantial data93-95 indicated that in many cell types, GPI-anchored proteins localized in ‘rafts’ domain. Under certain conditions, they are able to participate in intracellular signaling96 and possibly in cell differentiation.97 Furthermore, there is almost no variable strategy for the synthesis of different GPIanchors and the attachment of a peptide had been largely overlooked. Working in this area has almost exclusively involved the use of purified GPI-linked proteins from natural sources.98 15 An alternative method to the use of GPI anchors is the transfer of cholesteroltethered molecules into the lipid bilayer. This method has potential use for any biomolecules of interest. Recent works by Peterson et al.99-101 have shown that cholesterol conjugate molecules are useful in delivery of non-native cell surface receptors. These receptors are thought to be located in ‘rafts’ domains of the plasma membrane which is high in cholesterol level. Thus, this method has potential use as drug or gene delivery system. 1.4.2 Reaction using exogeneous enzymes Enzymes are widely employed in the formation of glycosidic bonds, particularly in carbohydrate chemistry. Such reactions can be performed on the cell surface as well, utilizing the existing surface glycoforms as acceptors for the reactions with an exogenously applied glycosyltransferase and appropriate activated sugar donor. Remodeling of cell surface glycoforms has been achieved with the use of glycosyltransferases and their sugar nucleotide substrates. Brossmer and coworkers102 decorated cells with 9-azido and 9-amino sialic acid analogs by first removing natural sialic acids enzymatically, followed by reinstalling the unnatural analogs with a sialyltranserase and the corresponding synthetic CMP-sialic acid derivatives. In other study, Hindsgaul and coworkers103 demonstrated that a fucosyltransferase could transfer fucose analogs with C6 substituents of enormous size and complexity onto cell surface glycoforms. Sialyltransferases have also been used for the chemical engineering of cell surfaces. In principle, a complex epitope of any type could be delivered to cells in one enzymatic step. However, the exogenous application of tolerant glycosyltransferases has progressed into and to a certain extent, been 16 superseded by the utilization of endogenous metabolic machinery for cell surface engineering. Notably, exogenous galactose oxidase is able to oxidize terminal galactosyl and N-acetylgalactosaminyl residues to perform unnatural aldehydes on cell surfaces. However, these sugars are often the penultimate residues in the surface glycoforms and are not easily recognized. In order to oxidize these sugars, the terminal monosaccharide residue must be cleaved and, since this is invariably sialic acid, neuraminidase treatment precedes the application of galactose oxidase.104 1.4.3 Inhibition of biosynthetic pathways Because the glycosylation of proteins and lipids is an important factor influencing the molecular complexity and functionality of the cell surface, inhibition of carbohydrate metabolism presents an alternative chemical strategy for engineering cell surface. Diverse complements of enzymes are required for a monosaccharide to be converted into an active sugar donor.105,106 This enables the inhibition of specific enzymes, which thereby makes it possible to subtly change the surface glycosylation. The development of potent and selective glycosylation inhibitors is of great interest for numerous therapeutic applications. Natural products have become the resources of some inhibitory molecules, such as carbohydrate mimetic alkaloids from plants and microorganisms.107 Specifically designed synthetic drugs are important additions to those natural occurring inhibitors. Most inhibitors exert their effects by competing with the natural enzyme substrates, which can be sugar donor or acceptor species, and acting as transition state analogues of the enzyme–substrate complex or behaving as decoys for glycoside biosynthesis.108 17 1.4.4 Metabolic engineering An alternative strategy to modify the chemical functionality of the cell surfaces is metabolic engineering. Cells were shown to take up and metabolize unnatural synthetic precursor of cell surface moieties, resulting in the incorporation of the unnatural structures on the exterior of the cells. This can be explained by the fact that some certain enzymes involved in the biosynthesis of cell surface molecules are tolerant to a degree of structural variability.109 The best known example is the incorporation of unnatural sialic acid precursors into cell surface glycoforms. This approach has been used on different cell types to alter the structure of sialic acids on cell surfaces, which has potential therapeutic applications110-112 since sialic acids play a critical role in cell recognition and adhesion events.113 Rather than focusing on unnatural sugars as altered glycoforms for the study of biological function, Bertozzi and coworkers have used oligosaccharide pathways as vehicles to introduce novel chemical reactivity onto cell surfaces. They have demonstrated that ManLev (an unnatural precursor to sialic acid that bears a ketone group) and ManNAz (an unnatural precursor to sialic acid that bears an azide) are metabolically delivered to cell surface114-116 of many different cell types with no adverse effects on cell viability. Cells treated with these compounds are showed to express the corresponding ketone-sialic acid or azide-sialic acid on cell surface glycoforms. Studies have also showed that the level of cell surface ketone expression is dependent on species. It is possibly because in different cell types, the tolerances of the sialic acid metabolic enzymes to structural variation are also different. 18 1.4.5 Covalent ligation to cell surface chemical groups Covalent ligation is the last technique that enables the chemical modification of cell surfaces has been known so far. Although it is difficult to alter the behavior of living cells by labeling cells with available reactive probes via their exposed functional groups, it is still might be a viable technique in the area of tissue engineering, where implants are particularly susceptible to recognition and destruction by host immune cells.117,118 This technique would offer a real potential applications where non-specifically altering cell surface architecture is acceptable. No specificity between cell surface functionalities with reactive molecules is the major drawback of this approach in some cases. To enable a more selective approach, the generation of unnatural reactive groups at specific sites on cell surface molecules has been developing. These reactive groups are not normally found on the cells surface, so they can be used to chemoselectively ligate suitably functionalized molecules to them. Two different types of chemical groups, reactive aldehydes and ketones, and azides are the focus of current research. Aldehyde and ketone groups on the cell surface can selectively reacted with hydrazide, aminooxy or thiosemicarbazide functionalities119 while azide can be ligated to a phosphine ligand by Staudinger ligation.120 Besides two discussed methods to generate surface ketones/aldehydes, a simple yet effective method to generate aldehyde is the oxidation of sialic acid residues with sodium periodate.121 This method is very rapid and it was found to be concentration dependent, selective for the vicinal diol present in sialic acid under mild condition.122,123 Although a relatively crude technique, it has been demonstrated that aldehyde groups can be incorporated into the cell surface of adherent cell monolayers by mild periodate oxidation which did not affect the viability, or morphology of the cells.124 19 1.4.6 Application of surface engineered mammalian cells The processes governed by cell surface molecules are fundamental to many biological phenomena. The ability to decorate cell surfaces with various bioactive molecules offers a multitude of opportunities for studying cellular adhesion, signal transduction and cell-cell recognition events. In addition, cell surface molecules are uniquely accessible to the outside world, thereby, ‘chemical restructuring of cell surface’ for tailored purposed gains a great attention in the field of drug target delivery and cell-base therapy. Chemically engineering cell surfaces has been showed to enhance specific interactions between surface molecules with drugs and drug delivery systems. For example, surface incorporated synthetic adenovirus receptor was showed to facilitate the entrance of adenovirus into cells that are normally resistant to infection by this virus.125 In addition, tumor cells could also be tagged by metabolic incorporation of unnatural sugar into the sialic acid molecules on the cell surface, which provided a strategy to specifically kill tumor cells.126 Taking advantage of metabolic engineering, Bertozzi and coworkers have demonstrated that synthetic oligosaccharides functionalized with aminooxy, hydrazide, and thiosemicarbazide groups can be attached to endogenous cell-surface glycans, affording remodeled cells with novel receptor-binding activities. Other applications include targeted gene delivery125 and antitumor diagnostic.127 This later application was prompted by reports from the last decade that correlate sialic acid expression on the cell surface with a malignant phenotype. As cell-cell interaction and cell-matrix interaction are essential factors in the development or repair of tissue, there is considerable potential use of cell surface engineering as tool in this field. Blocking the cell - cell recognition that activates 20 immune rejection of a foreign cell or tissue is one of major interest.117 Other area of great interest that surface engineering was involved is the repair of nerve damage.112 Besides, it was also found that cellular aggregation could be induced by cell surface engineering.124 This fact makes an important contribution to tissue engineering research. The manipulation of cell surface molecules and its application are still in their fancy, although significant progress has already been achieved, the idea of designing living cells with new properties is waiting to be exploited. 21 Chapter 2 The Chemistry of Cellular Membrane Functionalization 2.1 Chemoselective ligation reactions The search for highly selective reaction in watery environment at body temperature, compare to that of antibody-antigen recognition has brought a new concept “chemoselective ligation reaction”. Accurately, in the year 1990s, the principle of chemoselective reaction128,129 was adapted to enable the use of unprotected peptide segments in chemical protein synthesis.130 Chemistry Product O 130 Thioester-forming ligation R R' S O Oxime-forming ligation131 R R' N R'' O Thioether-forming ligation132 SR' R Directed disulfide formation133 O R S R' S O Thiazolidine-forming ligation134,135 HN R R' S Peptide bond-forming ligation136 Table 2.1 Chemistries used for the synthesis of native proteins by chemical ligation of unprotected peptide segments.137 22 This novel “chemical ligation” approach relied on a conceptual breakthrough, as the coupling of two mutually and uniquely reactive functional groups in an aqueous environment. As the name of the technique implies, these mutual chemoselective uniquely reactive functional groups are selective for each other and also tolerate a diverse array of other functionalities, which renders the use of protecting groups unnecessary.119 Thus, even among a multitude of potentially reactive functional groups, two chemoselective ligation partners will react only with each other. These reactions hence offer advantages similar to those of enzymatic reactions, with the potential of much broader range of substrates for use as coupling partners. Chemoselective ligation reactions designed to modify only one cellular component among all others have provided insight into cellular processes.138 The transforming concept embodied by the technique of chemoselective ligation is that of chemical orthogonality, a term that refers to the mutually exclusive reactivity of pairs of functional groups. For instance, if a non native functional group with orthogonal reactivity to native amino acid side-chains could be introduced site specifically into peptide segments, their reaction with complementary functional groups would give homogeneous, chemically defined products without interference by other components of the molecule. By now, numbers of chemoselective ligation reactions have been developed and applied in the field of chemistry and biology. 23 A O H2 N O R N H R'' R R' R' N H N R'' O B R O H2N O R'' R' R R' C N O R'' S H2N O R' R N H N H R'' R' R N H N H N R'' S D - O O SR' X R SR' R X = Cl, Br, I E O HX O R NH2 H O O O NHR' O R X = O, S NHR' N X HO O F NHR' O NHR' NH2 O R NH N H N H H N H O HN O R G R O O O O O S- S O S NHR' SH O NH2 R N H NHR' O 24 H O - X NHR'' O R XH O NH2 SR' NHR'' N H R O X = S, Se O I O O O NHR' R R N3 Ph2P O N H Ph2P O NHR' O R X J Ph2P R N3 R X = O, S H N R' O Figure 2.1 List of most common chemoselective ligation reactions.139 For years, these specific couplings have brought several achievements. As covalent bond formation to proteins is made difficult by their multiple unprotected functional groups and normally low concentrations, an appealing strategy for the synthesis of large, full length proteins is the convergent coupling of moderately sized peptide segments ([...]... surface by the organization of the membrane lipid bilayer Phospholipids can therefore be important in determining the surface charge of the membrane. 15-19 1.2 Cholesterol and cellular membrane 1.2.1 General feature, location of cholesterol within cellular membrane Cholesterol is a prominent constituent of mammalian cell membranes (as much as 30% of the plasma membrane lipid content in some tissues).20 Cholesterol. .. consequences of cholesterol in cell membranes helps to focus attention on several important issues concerning the roles of cholesterol in mammalian cell membranes The basic role of cholesterol is membrane lipid constituent Membrane cholesterol level was proved to influence membrane permeability and stability properties.28 Cholesterol does this by its special ordering effect on the membrane lipids Because of. .. plasma membranes of mammalian cells contain glycolipids and cholesterol The glycolipids are found exclusively in the outer (exofacial) leaflet of the plasma membrane, with their carbohydrate portions exposed on the cell surface They are relatively minor membrane components, constituting only about 2% of the lipids of most plasma membranes Cholesterol, on the other hand, is a major membrane constituent of. .. Heegaard.46 11 1.3.2 Biological applications of dendrimers The use of dendrimers as frameworks and as carrier systems for the study and modulation of biological processes is gaining popularity In the past years, significant advances have been made in the synthesis and study of glycodendrimers and peptide dendrimers Application of these dendrimers to the study of carbohydrate-protein and protein-protein... structure The details of this chemistry have 6 been briefly discussed by Konrad Bloch23 and summarized in greater detail by Louis and Mary Fieser.24 H H HO H cholesterol Figure 1.2 Structure of cholesterol The structure of cholesterol appears in Figure 1.2 The structure consists of four fused rings, referred to as the A, B, C and D rings, reading from left to right The most common conformation of this steroid... outer portions of the fatty acid chains, making this part of the membrane more rigid On the other hand, insertion of cholesterol interferes with interactions between fatty acid chains, thereby maintaining membrane fluidity at lower temperatures.2 Recent studies suggest that not all lipids diffuse freely in the plasma membrane Instead, discrete membrane domains appear to be enriched in cholesterol and... clusters of sphingolipids and cholesterol are thought to form “rafts” that move laterally within the plasma membrane and may associate with specific membrane proteins Although the functions of lipid rafts remain to be understood, they are thought to play important 8 roles in processes such as cell signaling and the uptake of extracellular molecules by endocytosis.2,30 1.3 Dendrimers in bioengineering Dendrimers... the fatty acid chains of the phospholipids and perpendicular to the membrane surfaces X-ray diffraction and neutron diffraction data26,27 have provided a clearer picture of the location of 7 cholesterol within the membrane The data showed that the cholesterol molecule is located so that the hydroxyl is in the immediate vicinity of the phospholipid ester carbonyl 1.2.2 Roles of cholesterol Consideration... modification of mammalian cellular membrane The boundary between biology and chemistry has eroded in recent years The ability to modify cellular membrane both in vitro and in vivo by chemical approach has led to the development of the rapidly expanding field – cell surface engineering 14 1.4.1 Insertion of molecules into cell surface The lipophilic nature of mammalian cell surface has been exploited by a number... properties of membrane proteins without doubt The year 1972 was it superseded by fluid mosaic model of Singer and Nicholson.12 The basic principles of this model were that membrane proteins could be globular, just as could water-soluble proteins The 2 globular membrane proteins were embedded within the bilayer, with the hydrophobic portions of the proteins buried within the hydrophobic core of the lipid ... Overview of cellular membrane 1.1.2 Lipid domain and its charge 1.2 Cholesterol and cellular membrane 1.2.1 General feature, location of cholesterol within cellular membrane 1.2.2 Roles of cholesterol. .. 1.3 Dendrimers in bioengineering 1.3.1 Chemistry of dendrimers 1.3.2 Biological application of dendrimers 12 1.3.3 Cytotoxicity of dendrimers 14 1.4 Chemical modification of mammalian cellular membrane. .. location of cholesterol within cellular membrane Cholesterol is a prominent constituent of mammalian cell membranes (as much as 30% of the plasma membrane lipid content in some tissues).20 Cholesterol