Search

The present work concerns the development of liposomal formulations that can adsorb to the human dental enamel. The overall aim of this pharmaceutical approach is to physically protect the teeth against detrimental processes, such as tooth wear, acidic challenges and dental caries. Adsorption experiments of different liposomal formulations to hydroxyapatite (HA), a model substance for the dental hard tissue, and the human dental enamel were performed. To find which liposomal formulations are promising for the adsorption to teeth, formulation factors important for the interaction were initially mapped by the use of experimental design and multivariate analysis (Paper I). The type of surface charge became the most significant factor for the adsorption process. Positively charged liposomes adsorbed better than the negatively charged liposomes to HA in phosphate buffer, pH 6.8-7. However, the adsorption of positively charged liposomes to HA in a salivary environment was interfered as they were found to aggregate with components of saliva (Paper II). To overcome problems related to the positively charged liposomes, the surface of the liposomes was modified with the polymer pectin. Three types of pectin were investigated for the surface coating of liposomes: LM-, HM- and amidated pectin (Paper III). Pectin coating of positively charged liposomes was successfully prepared, and a reproducible method was established. Pectin coated liposomes did not seem to interact with salivary components (Paper IV), and were therefore promising for use in the oral cavity. Pectin coated liposomes adsorbed to HA in saliva, and liposomes coated with LMand HM-pectin were selected for further investigation with the dental enamel (Paper IV). Both uncoated negatively charged liposomes and pectin coated liposomes adsorbed onto enamel specimens in a salivary environment (Paper IV), indicating their potential use in the protection of the teeth. The adsorption was examined by exposing a flow on the enamel surfaces for certain time intervals. Pectin coated liposomes seemed to retain better than uncoated negatively charged liposomes at longer time intervals. It was hypothesized that pectin may help to prolong the adhesion of liposomes on the tooth surfaces.

SUMMARY In the last decade, techniques based on DNA markers have been used to detect variation at DNA level and have proven to be reliable and very effective for distinguishing between closely related genotypes. The development of the random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990; Welsh et al., 1991) allowed genome characterization in several plant groups. The RAPD technique has been useful in studying polymorphism, identifying genes of interest, and permitting the determination of phylogenetic relationships and intraspecific diversity at a molecular genetic level. In four minor studies, this thesis deals with the evolutionary relationships and genetic variation among different populations of plants in two genera of Juniperus and Calocedrus using the methodology of RAPD-polymerase chain reaction (PCR) together with morphological traits. By performing chemical analysis of leaf essential oils and comparing their oil compositions, these data are also utilized to define phylogeny and taxonomy of species in these genera. In this respect, the synthesis of data from different levels, from biochemical to genetic, attempts to increase our understanding in plant evolution and systematics. The basics of the methods employed will be described briefly and their applications also reviewed. The first study examined the varieties of the species Juniperus deppeana. Populations of J.deppeana var. deppeana, var. pachyphlaea, var. patoniana, var. robusta, var. zacatecensis, and J. gamboana were analyzed by DNA fingerprinting (RAPDs). By comparison with the closely related J. saltillensis, J. gamboana was found to conspecific with J. deppeana and recognized as a new variety: J. deppeana var. gamboana (Mart.) R. P. Adams. Juniperus deppeana var. pachyphlaea and J. d. var. zacatecensis were found to be a part of J. deppeana var. deppeana. Juniperus deppeana var. zacatecensis was reduced to a new forma: J. deppeana f. zacatecensis (Mart.) R. P. Adams. Considerable amounts of intra-population variation was present. Previous Pleistocene distributions and past gene flow seems to have contributed to the lack of populational differentiation. A key and revised distribution map of J. deppeana is also presented here. In the second study, another species of Juniperus, J. communis, was investigated to detect the genetic variation in Norwegian junipers, also using RAPD-DNA fingerprinting applications. It has been shown (Looman and Baerheim Svendsen, 1992) that the needle essential oils of J. communis from the mountains of Norway were dominated by sabinene, in contrast to the oils from the lowland characterized by having high percentage of á-pinene. RAPD data of these two populations, one from the mountains of central Norway and the other from the coastal areas, were analyzed to determine whether these differences are due to divergence in the plant genome or they are merely products of environmentally induced changes. Principal coordinate analysis did not show two distinctive groups as expected; the coastal samples (high in á-pinene) formed a uniform group but some individuals from the mountain group (high in sabinene) were more scattered and linked to the coastal group, resulting in a complex pattern. The varied oil concentration ratio of á-pinene: sabinene: in some individuals of Looman and Baerheim Svendsen report may be reflected in the intermediate juniper individuals seen here. Thus, there is not enough evidence to consider a new variety or forma of J.communis grown in Norway, so that influence of environmental factors may be the explanation and supporting Looman and Baerheim Svendsen s suggestion of the existence of several mountain juniper chemotypes. The third study discusses the leaf essential oil composition of the genus Calocedrus which consists of only three taxa. Leaf essential oils of C. decurrens, (west North America), C. macrolepis (southwest China, northern Vietnam, northern Thailand) and C. macrolepis var. formosana (Taiwan) were analyzed and compared to the oils of the closely related species, Platycladus orientalis (east, northeast China, Korea, far eastern Russia). The oils of C. macrolepis var. macrolepis and C. m. var. formosana were very similar, being high in á-pinene (57.2, 67.1%) and myrcene (11.2, 6.2), but they differed in many smaller components. The oils of C. decurrens, from two populations in Oregon and one disjunct population in south California, were high in ä-3-carene (15.2 - 20.2%), limonene (18.2 - 23.6), á-pinene (8.7 - 15.8), terpinolene (5.7 - 8.0), endo-fenchyl acetate (3.5 - 9.7), with some cedrol (0.8 - 1.2). No large differences in oil compositions were seen between the three C. decurrens populations. Platycladus orientalis (= Thuja orientalis, = Biota orientalis) was found to contain considerable amounts of ä-3-carene (29.8%), cedrol (22.2%), á-pinene (15.1) and terpinolene (4.9). Overall, the oil of C. decurrens is as different from C. macrolepis and C. m. var. formosana, as it is from Platycladus orientalis, suggesting considerable divergence between Calocedrus species but not between C. macrolepis and C. m. var. formosana The composition of the leaf oils from seven populations of Juniperus sabina L. and one population of J. sabina var. arenaria (E. H. Wilson) Farjon were examined for geographic variation in the final study. In addition, the leaf oils of J. chinensis L. and J. davurica Pall. are compared to J. sabina. In Juniperus sabina var. arenaria, the sand loving juniper, the oil was found to be very similar to that of J. davurica, Mongolia, and J. sabina, on sand dunes in Mongolia. This suggests that J. sabina var. arenaria might be conspecific with J. davurica and Farjon's move (2001) of J. sabina var. arenaria out of J. chinensis is therefore supported. Considerable differentiation was found in populations of J. sabina from the Iberian peninsula. Cedrol, citronellol, safrole, trans-sabinyl acetate, terpinen-4-ol and trans-thujone were found to be polymorphic in several populations.

Når bakterier vokser i form av biofilm er de beskyttet mot antibiotika, og i større grad resistent. Ved bruk av liposomer som et leveringssystem rettet mot biofilmer kan de gi en forbedret antimikrobiell effekt. Ladning på liposomene kan resultere i en forbedret interaksjon mellom liposomene og bakteriecelleoverflaten. Dette kan forbedre penetrasjonen av biofilmen hvor bakterier sitter i og under en matrix av ekstracellulære materiale. I denne masteroppgaven ble en nøytral hovedlipid, DPPC, kombinert med enten positiv ladet DPTAP eller negativ ladet DPPG i ulik molforhold. Totalt ble det prøvd å produsere fire ulike liposomformuleringer og en passende fremstillingsmetode ble etablert. Enkelte formuleringer som DPPC/ 10% DPTAP og DPPC/ 2.5% DPPG viste dårlig stabilitet fra første måling av deres egenskaper. Dermed ble disse to formuleringene utelukket som kandidater for innkapsling av ulike peptidbaserte antibiotika (polymyxin B, gentamicin og bacitracin). DPPC/ 10% DPPG viste seg å være en dårlig formulering for innkapsling av de ulike antibiotikaene. Alle formuleringene viste dårlig stabilitet ved karakterisering etter fremstilling med forhøyet zeta-average samt en størrelsesfordeling (ved intensitet) med flere topper. DPPC/ 2.5% DPTAP var den eneste formulering som hadde ønskede egenskaper etter produksjonen. De mikrobiologiske testene på planktoniske celler ble gjennomført ved bruk av to ulike bakterier; P. aeruginosa (DSM22644) og S. aureus (DIM2569). Testing på planktoniske tester ble gjennomført ved måling av absorbansen ved bruk av en plateleser. Volum til og med 1.25 μL ga ingen vekstrespons. Resultatene viser også at veksthemingen var stabil over testperioden på 12 timer. Dette indikerer at DPPC/ 2.5% DPTAP frigjører minst MIC under hele analyse tidsperioden. De mikrobiologiske testene på biofilm ble gjort på to ulike metoder for P. aeruginosa og S. aureus. P. aeruginosa biofilm ble etablert i 96-brønnplater av plast og ble behandlet med mindre liposomvolum. Her var det dårlig hemming av allerede etablert biofilm. S. aureus biofilm ble etablert på kammerobjektglass og ble behandlet med større liposomvolum. Resultatene fra testen mot S. aureus biofilm antyder at allerede bruk av 100 μL av liposomformuleringen gir en svak hemming av biofilmer. CFU/mL for 100 μL ble beregnet til 3.31818 x 104, 1000 μL til 8.7 x 104 og 2000 μL kontra CFU/mL på 4.22523 x 108 for bakterier som ble ikke utsatt for DPPC/ 2.5% DPTAP med innkapslet gentamicin. When bacteria grow in biofilms, they are protected from antibiotics and are to a greater extent resistant. By using liposomes as a drug delivery system directly aimed at biofilms it can enhance the antimicrobial effect. Charged liposomes can increase the interaction between the liposomes and the bacterial cell surface. This can improve the penetration of the biofilm where bacteria are in and under a matrix of extracellular material In this study a neutral lipid, DPPC, was combined with either a positively charged lipid, DPTAP, or negatively charged lipid, DPPG, in different molar ratios. In total, four different liposome formulations were made, and a suitable preparation method was established. Some formulations such as DPPC/ 10% DPTAP and DPPC/ 2.5% DPPG showed poor stability; thus, these two formulations were excluded as candidates for encapsulation of peptide-based antibiotics (polymyxin B, gentamicin and bacitracin). DPPC/ 10% DPPG was not suitable for encapsulation of the different antibiotics; the formulation showed poor stability with an elevated zeta-average and a size distribution with several peaks. Only gentamicin encapsulated in DPPC/ 2.5% DPTAP had the desired properties post-production. During storage there was a change in the zeta potential, but this formulation was nevertheless used for the microbiological tests. P. aeruginosa (DSM22644) and S. aureus (DIM2569) was used for the microbiological tests. The first test was conducted on agar plates. Testing on planktonic cells was done by measuring the absorbance of the cells using a plate reader. A minimum volume of μL gave no bacterial growth response. The results from this test shows that growth inhibition was stable over the 12-hour test period. This indicated that DPPC/ 2.5% DPTAP release at least MIC during the entire analysis. The tests on P. aeruginosa and S. aureus biofilms was conducted in two different manners. P. aeruginosa biofilm was grown in a 96-well plastic plate and was treated with lower liposomal gentamicin volume and there was poor inhibition of the already established biofilm. S. aureus biofilm was grown in 1-well chamber slides and treated with higher volume of the liposome gentamicin suspension. The results from the test against S. aureus biofilm suggested that a volume of as low as 100 μL of liposomal gentamicin gives an inhibition. CFU /mL for 100 mL was calculated to 3.31818 x 104 CFU/mL, 1000 μL to 8.7 x 104 CFU/mL and 2000 μL versus CFU /mL of 4.22523 x 108 for bacteria not exposed to DPPC / 2.5% DPTAP with encapsulated gentamicin.