External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

Glycoalkaloid Content and Starch Structure in Solanum Species and Interspecific Somatic Potato Hybrids

When genome sections of wild Solanum species are bred into the cultivated potato (S. tuberosum L.) to obtain improved potato cultivars, the new cultivars must be evaluated for their beneficial and undesirable traits. Glycoalkaloids present in Solanum species are known for their toxic as well as for beneficial effects on mammals. On the other hand, glycoalkaloids in potato leaves provide natural protection against pests. Due to breeding, glycoalkaloid profile of the plant is affected. In addition, the starch properties in potato tubers can be affected as a result of breeding, because the crystalline properties are determined by the botanical source of the starch. Starch content and composition affect the texture of cooked and processed potatoes. In order to determine glycoalkaloid contents in Solanum species, simultaneous separation of glycoalkaloids and aglycones using reversed-phase high-performance liquid chromatography (HPLC) was developed. Clean-up of foliage samples was improved using a silica-based strong cation exchanger instead of octadecyl phases in solid-phase extraction. Glycoalkaloids alpha-solanine and alpha-chaconine were detected in potato tubers of cvs. Satu and Sini. The total glycoalkaloid concentration of non-peeled and immature tubers was at an acceptable level (under 20 mg/100 g of FW) in the cv. Satu, whereas concentration in cv. Sini was 23 mg/100 g FW. Solanum species (S. tuberosum, S. brevidens, S. acaule, and S. commersonii) and interspecific somatic hybrids (brd + tbr, acl + tbr, cmm + tbr) were analyzed for their glycoalkaloid contents using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The concentrations in the tubers of the brd + tbr and acl + tbr hybrids remained under 20 mg/100 g FW. Glycoalkaloid concentration in the foliage of the Solanum species was between 110 mg and 890 mg/100 g FW. However, the concentration in the foliage of S. acaule was as low as 26 mg/100 g FW. The total concentrations of brd + tbr, acl + tbr, and cmm + tbr hybrid foliages were 88 mg, 180 mg, and 685 mg/100 g FW, respectively. Glycoalkaloids of both parental plants as well as new combinations of aglycones and saccharides were detected in somatic hybrids. The hybrids contained mainly spirosolanes, and glycoalkaloid structures having no 5,6-double bond in the aglycone. Based on these results, the glycoalkaloid profiles of the hybrids may represent a safer and more beneficial spectrum of glycoalkaloids than that found in currently cultivated varieties. Starch nanostructure of three different cultivars (Satu, Saturna, and Lady Rosetta), a wild species S. acaule, and interspecific somatic hybrids were examined by wide-angle and small-angle X-ray scattering (WAXS, SAXS). For the first time, the measurements were conducted on fresh potato tuber samples. Crystallinity of starch, average crystallite size, and lamellar distance were determined from the X-ray patterns. No differences in the starch nanostructure between the three different cultivars were detected. However, tuber immaturity was detected by X-ray scattering methods when large numbers of immature and mature samples were measured and the results were compared. The present study shows that no significant changes occurred in the nanostructures of starches resulting from hybridizations of potato cultivars.

Entsymaattisen hydrolyysin vaikutuksista mikrokiteisen selluloosan rakenteeseen

Cellulose can be used as a renewable raw material for energy production. The utilization requires degradation of cellulose into glucose, which can be done with the aid of enzymatic hydrolysis. In this thesis, various x-ray methods were used to characterize sub-micrometer changes in microcrystalline cellulose during enzymatic hydrolysis to clarify the process and factors slowering it. The methods included wide-angle x-ray scattering (WAXS), small-angle x-ray scattering (SAXS) and x-ray microtomography. In addition, the samples were studied with transmission electron microscopy (TEM). The studied samples were hydrolyzed by enzymes of the Trichoderma reesei species for 6, 24, and 75 hours, which corresponded to 31 %, 58 %, and 68 % degrees of hydrolysis, respectively. Freeze-dried hydrolysis residues were measured with WAXS, SAXS and microtomography, whereas some of them were re-wetted for the wet SAXS and TEM measurements. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures similar cylindrical and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results were ambiguous and partly imprecise, but showed a change in the structure of wet samples in scale of 10-30 nm. According to the WAXS results, the degrees of crystallinity and the crystal sizes remained the same. The gained results support the assuption, that the cellulosic particles are hydrolyzed mostly on their surface, since the enzymes are unable to penetrate into the nanopores of wet cellulose. The hydrolysis therefore proceeds quickly in easily accessible particles and leaves the unaccesible particles almost untouched. The structural changes observed in the SAXS measurements might correspond to slight loosening of the microfibril aggregates, which was seen only in the wet samples because of their different pore structure.

X-ray scattering and diffraction enhanced imaging studies of in vitro breast tissues

This thesis is a study of the x-ray scattering properties of tissues and tumours of the breast. Clinical radiography is based on the absorption of the x-rays when passing right through the human body and gives information about the densities of the tissues. Besides being absorbed, x-rays may change their direction within the tissues due to elastic scattering or even to refraction. The phenomenon of scattering is a nuisance to radiography in general, and to mammography in particular, because it reduces the quality of the images. However, scattered x-rays bear very useful information about the structure of the tissues at the supra-molecular level. Some pathologies, like breast cancer, produce alterations to the structures of the tissues, being especially evident in collagen-rich tissues. On the other hand, the change of direction due to refraction of the x-rays on the tissue boundaries can be mapped. The diffraction enhanced imaging (DEI) technique uses a perfect crystal to convert the angular deviations of the x-rays into intensity variations, which can be recorded as images. This technique is of especial interest in the cases were the densities of the tissues are very similar (like in mammography) and the absorption images do not offer enough contrast. This thesis explores the structural differences existing in healthy and pathological collagen in breast tissue samples by the small-angle x-ray scattering (SAXS) technique and compares these differences with the morphological information found in the DEI images and the histo-pathology of the same samples. Several breast tissue samples were studied by SAXS technique in the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Scattering patterns of the different tissues of the breast were acquired and compared with the histology of the samples. The scattering signals from adipose tissue (fat), connective tissue (collagen) and necrotic tissue were identified. Moreover, a clear distinction could be done between the scattering signals from healthy collagen and from collagen from an invasive tumour. Scattering from collagen is very characteristic. It includes several scattering peaks and scattering features that carry information about the size and the spacing of the collagen fibrils in the tissues. It was found that the collagen fibrils in invaded tumours were thinner and had a d-spacing length 0,7% longer that fibrils from healthy tumours. The scattering signals from the breast tissues were compared with the histology by building colour-coded maps across the samples. They were also imaged with the DEI technique. There was a total agreement between the scattering maps, the morphological features seen in the images and the information of the histo- pathological examination. The thesis demonstrates that the x-ray scattering signal can be used to characterize tissues and that it carries important information about the pathological state of the breast tissues, thus showing the potential of the SAXS technique as a possible diagnostic tool for breast cancer.

Scattering and Refraction as Contrast Mechanisms in X-Ray Imaging

Differentiation of various types of soft tissues is of high importance in medical imaging, because changes in soft tissue structure are often associated with pathologies, such as cancer. However, the densities of different soft tissues may be very similar, making it difficult to distinguish them in absorption images. This is especially true when the consideration of patient dose limits the available signal-to-noise ratio. Refraction is more sensitive than absorption to changes in the density, and small angle x-ray scattering on the other hand contains information about the macromolecular structure of the tissues. Both of these can be used as potential sources of contrast when soft tissues are imaged, but little is known about the visibility of the signals in realistic imaging situations. In this work the visibility of small-angle scattering and refraction in the context of medical imaging has been studied using computational methods. The work focuses on the study of analyzer based imaging, where the information about the sample is recorded in the rocking curve of the analyzer crystal. Computational phantoms based on simple geometrical shapes with differing material properties are used. The objects have realistic dimensions and attenuation properties that could be encountered in real imaging situations. The scattering properties mimic various features of measured small-angle scattering curves. Ray-tracing methods are used to calculate the refraction and attenuation of the beam, and a scattering halo is accumulated, including the effect of multiple scattering. The changes in the shape of the rocking curve are analyzed with different methods, including diffraction enhanced imaging (DEI), extended DEI (E-DEI) and multiple image radiography (MIR). A wide angle DEI, called W-DEI, is introduced and its performance is compared with that of the established methods. The results indicate that the differences in scattered intensities from healthy and malignant breast tissues are distinguishable to some extent with reasonable dose. Especially the fraction of total scattering has large enough differences that it can serve as a useful source of contrast. The peaks related to the macromolecular structure come to angles that are rather large, and have intensities that are only a small fraction of the total scattered intensity. It is found that such peaks seem to have only limited usefulness in medical imaging. It is also found that W-DEI performs rather well when most of the intensity remains in the direct beam, indicating that dark field imaging methods may produce the best results when scattering is weak. Altogether, it is found that the analysis of scattered intensity is a viable option even in medical imaging where the patient dose is the limiting factor.

X-ray and neutron scattering studies on some nanoscale structures in molecular biology

Scattering of X-rays and neutrons has been applied to the study of nanostructures with interesting biological functions. The systems studied were the protein calmodulin and its complexes, bacterial virus bacteriophage phi6, and the photosynthetic antenna complex from green sulfur bacteria, chlorosome. Information gathered using various structure determination methods has been combined to the low resolution information obtained from solution scattering. Conformational changes in calmodulin-ligand complex were studied by combining the directional information obtained from residual dipole couplings in nuclear magnetic resonance to the size information obtained from small-angle X-ray scattering from solution. The locations of non-structural protein components in a model of bacteriophage phi6, based mainly on electron microscopy, were determined by neutron scattering, deuterium labeling and contrast variation. New data are presented on the structure of the photosynthetic antenna complex of green sulfur bacteria and filamentous anoxygenic phototrophs, also known as the chlorosome. The X-ray scattering and electron cryomicroscopy results from this system are interpreted in the context of a new structural model detailed in the third paper of this dissertation. The model is found to be consistent with the results obtained from various chlorosome containing bacteria. The effect of carotenoid synthesis on the chlorosome structure and self-assembly are studied by carotenoid extraction, biosynthesis inhibition and genetic manipulation of the enzymes involved in carotenoid biosynthesis. Carotenoid composition and content are found to have a marked effect on the structural parameters and morphology of chlorosomes.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Characterisation of cellulose- and lignin-based materials using x-ray scattering methods

Wood is an important material for the construction and pulping industries. Using x-ray diffraction the microfibril angle of Sitka spruce wood was studied in the first part of this thesis. Sitka spruce (Picea sitchensis [Bong.] Carr.) is native to the west coast of North America, but due to its fast growth rate, it has also been imported to Europe. So far, its nanometre scale properties have not been systematically characterised. In this thesis the microfibril angle of Sitka spruce was shown to depend significantly on the origin of the tree in the first annual rings near the pith. Wood can be further processed to separate lignin from cellulose and hemicelluloses. Solid cellulose can act as a reducer for metal ions and it is also a porous support for nanoparticles. By chemically reducing nickel or copper in the solid cellulose support it is possible to get small nanoparticles on the surfaces of the cellulose fibres. Cellulose supported metal nanoparticles can potentially be used as environmentally friendly catalysts in organic chemistry reactions. In this thesis the size of the nickel and copper containing nanoparticles were studied using anomalous small-angle x-ray scattering and wide-angle x-ray scattering. The anomalous small-angle x-ray scattering experiments showed that the crystallite size of the copper oxide nanoparticles was the same as the size of the nanoparticles, so the nanoparticles were single crystals. The nickel containing nanoparticles were amorphous, but crystallised upon heating. The size of the nanoparticles was observed to be smaller when the reduction of nickel was done in aqueous ammonium hydrate medium compared to reduction made in aqueous solution. Lignin is typically seen as the side-product of wood industries. Lignin is the second most abundant natural polymer on Earth, and it possesses potential to be a useful material for many purposes in addition to being an energy source for the pulp mills. In this thesis, the morphology of several lignins, which were produced by different separation methods from wood, was studied using small-angle and ultra small-angle x-ray scattering. It was shown that the fractal model previously proposed for the lignin structure does not apply to most of the extracted lignin types. The only lignin to which the fractal model could be applied was kraft lignin. In aqueous solutions the average shape of the low molar mass kraft lignin particles was observed to be elongated and flat. The average shape does not necessarily correspond to the shape of the individual particles because of the polydispersity of the fraction and due to selfassociation of the particles. Lignins, and especially lignosulfonate, have many uses as dispersants, binders and emulsion stabilisers. In this thesis work the selfassociation of low molar mass lignosulfonate macromolecules was observed using small-angle x-ray scattering. By taking into account the polydispersity of the studied lignosulfonate fraction, the shape of the lignosulfonate particles was determined to be flat by fitting an oblate ellipsoidal model to the scattering intensity.