The roughness and imaging characterisation of different pharmaceutical surfaces

The surface properties of solid state pharmaceutics are of critical importance. Processing modifies the surfaces and effects surface roughness, which influences the performance of the final dosage form in many different levels. Surface roughness has an effect on, e.g., the properties of powders, tablet compression and tablet coating. The overall goal of this research was to understand the surface structures of pharmaceutical surfaces. In this context the specific purpose was to compare four different analysing techniques (optical microscopy, scanning electron microscopy, laser profilometry and atomic force microscopy) in various pharmaceutical applications where the surfaces have quite different roughness scale. This was done by comparing the image and roughness analysing techniques using powder compacts, coated tablets and crystal surfaces as model surfaces. It was found that optical microscopy was still a very efficient technique, as it yielded information that SEM and AFM imaging are not able to provide. Roughness measurements complemented the image data and gave quantitative information about height differences. AFM roughness data represents the roughness of only a small part of the surface and therefore needs other methods like laser profilometer are needed to provide a larger scale description of the surface. The new developed roughness analysing method visualised surface roughness by giving detailed roughness maps, which showed local variations in surface roughness values. The method was able to provide a picture of the surface heterogeneity and the scale of the roughness. In the coating study, the laser profilometer results showed that the increase in surface roughness was largest during the first 30 minutes of coating when the surface was not yet fully covered with coating. The SEM images and the dispersive X-ray analysis results showed that the surface was fully covered with coating within 15 to 30 minutes. The combination of the different measurement techniques made it possible to follow the change of surface roughness and development of polymer coating. The optical imaging techniques gave a good overview of processes affecting the whole crystal surface, but they lacked the resolution to see small nanometer scale processes. AFM was used to visualize the nanoscale effects of cleaving and reveal the full surface heterogeneity, which underlies the optical imaging. Ethanol washing changed small (nanoscale) structure to some extent, but the effect of ethanol washing on the larger scale was small. Water washing caused total reformation of the surface structure at all levels.

Physical characterization of plastic surfaces in wearing and cleanability research

Plastic surfaces are a group of materials used for many purposes. The present study was focused on methods for investigation of surface topography, wearing and cleanability of polyvinyl chloride (PVC) model surfaces and industrial plastic surfaces. Contact profilometry, scanning electron microscopy (SEM) and atomic force microscopy (AFM) are powerful methods for studying the topography of plastic surfaces. Although they have their own limitations, they are together an effective tool providing useful information on surface topography, especially when studying laboratory-made PVC model surfaces with known chemical compositions and structures. All examined laboratory-made PVC plastic surfaces examined in this work could be considered as smooth according to both AFM and profilometer measurements because height differences are in the nanoscale on every surface. Industrial plastic surfaces are a complex group of materials because of their chemical and topographical heterogeneity, but they are nevertheless important reference materials when developing cleaning and wearing methods. According to the results of this study the Soiling and Wearing Drum and the Frick-Taber methods are very useful when simulating three-body wearing of plastic surfaces. Both the investigated wearing methods can be used to compare the wearing of different plastic materials using appropriate evaluation methods of wearing and industrial use. In this study, physical methods were developed and adapted from other fields of material research to cleanability studies. The thesis focuses on the methodology for investigating the cleanability of plastic surfaces under realistic conditions, where surface topography and the effect of wear cleanability were among the major topics. A colorimetric method proved to be suitable for examining the cleanability of the industrial plastic surfaces. The results were utilized to evaluate the relationship between cleanability and the surface properties of plastic surfaces. The devices and methods used in the work can be utilized both in material research and product development.

Movement-associated proteins of Potato virus A: attachment to virus particles and phosphorylation

The particles of Potato virus A (PVA; genus Potyvirus) are helically constructed filaments that contain multiple copies of a single type of coat-protein (CP) subunit and a single copy of genome-linked protein (VPg), attached to one end of the virion. Examination of negatively-stained virions by electron microscopy revealed flexuous, rod-shaped particles with no obvious terminal structures. It is known that particles of several filamentous plant viruses incorporate additional minor protein components, forming stable complexes that mediate particle disassembly, movement or transmission by insect vectors. The first objective of this work was to study the interaction of PVA movement-associated proteins with virus particles and how these interactions contribute to the morphology and function of the virus particles. Purified particles of PVA were examined by atomic force microscopy (AFM) and immuno-gold electron microscopy. A protrusion was found at one end of some of the potyvirus particles, associated with the 5' end of the viral RNA. The tip contained two virus-encoded proteins, the genome-linked protein (VPg) and the helper-component proteinase (HC-Pro). Both are required for cell-to-cell movement of the virus. Biochemical and electron microscopy studies of purified PVA samples also revealed the presence of another protein required for cell-to-cell movement the cylindrical inclusion protein (CI), which is also an RNA helicase/ATPase. Centrifugation through a 5-40% sucrose gradient separated virus particles with no detectable CI to a fraction that remained in the gradient, from the CI-associated particles that went to the pellet. Both types of particles were infectious. AFM and translation experiments demonstrated that when the viral CI was not present in the sample, PVA virions had a beads-on-a-string phenotype, and RNA within the virus particles was more accessible to translation. The second objective of this work was to study phosphorylation of PVA movement-associated and structural proteins (CP and VPg) in vitro and, if possible, in vivo. PVA virion structural protein CP is necessary for virus cell-to-cell movement. The tobacco protein kinase CK2 was identified as a kinase phosphorylating PVA CP. A major site of CK2 phosphorylation in PVA CP was identified as a single threonine within a CK2 consensus sequence. Amino acid substitutions affecting the CK2 consensus sequence in CP resulted in viruses that were defective in cell-to-cell and long-distance movement. The CK2 regulation of virion assembly and cell-to-cell movement by phosphorylation of CP was possibly due to the inhibition of CP binding to viral RNA. Four putative phosphorylation sites were identified from an in vitro phosphorylated recombinant VPg. All four were mutated and the spread of mutant viruses in two different host plants was studied. Two putative phosphorylation site mutants (Thr45 and Thr49) had phenotypes identical to that of a wild type (WT) virus infection in both Nicotiana benthamiana and N. tabacum plants. The other two mutant viruses (Thr132/Ser133 and Thr168) showed different phenotypes with increased or decreased accumulation rates, respectively, in inoculated and the first two systemically infected leaves of N. benthamiana. The same mutants were occasionally restricted to single cells in N. tabacum plants, suggesting the importance of these amino acids in the PVA infection cycle in N. tabacum.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Hiilen ja typen pysyvät isotoopit vesistötutkimuksissa: pohjaeläimistön ravinnonlähteet ja pohjan ravintoverkon rakenne Saaristomerellä

Saaristomeren pinnanalaisen ravintoverkon rakennetta sekä ravinteiden alkuperää ja kulkeutumista tutkittiin hiilen ja typen pysyviä isotooppeja hyväksi käyttäen. Tutkimuksen kohteena olivat suspendoitunut partikkeliaines (SPM), sedimentin pintakerros, pohjaeläimet, kalat ja makrolevät. Näytteitä otettiin toukokuussa, kesäkuussa ja lokakuussa. Ravinnelähteiden arvioinnin ohella tutkittiin voiko sisäsaariston kalankasvatuksen vaikutusta määritellä analysoimalla kalanrehupellettejä. Lisäksi arvioitiin joidenkin lajikohtaisten tai lajien välisten ominaisuuksien erojen vaikutusta (esimerkiksi eläimen koko ja kudosten C:N –suhde rasvapitoisuuden kuvaajana) /-arvojen määräytymiseen. Myös joitain tarkennuksia tehtiin lajien ravinnonkäyttöluokituksiin. Sisäsaaristossa toukokuussa pian lumien sulamisen aiheuttaman kasvaneen jokivirtaaman jälkeen SPM:stä havaittiin korkeita /13C- ja /15N-arvoja (keskimäärin -16 ‰ ja 9 ‰, vastaavasti). Ulkosaariston asemilta saatiin paljon alempia arvoja (-24 ‰ ja 4 ‰, vastaavasti). Sisäsaaristossakin arvot laskivat kesäkuuhun mennessä (-26 ‰ ja 5 ‰, vastaavasti) ollen kuitenkin hieman korkeampia kuin samaan aikaan tutkituilla ulommilla välisaariston asemilla. Kasviplanktonin lajistokoostumusta tutkittiin touko- ja kesäkuussa, mutta suuria eroja ei ajankohtien välillä havaittu. Lokakuussa sateisen loppukesän jälkeen /13C oli tasainen (-23 ‰ - -24 ‰) koko tutkimusalueella, mutta /15N oli taas huomattavasti korkeampi sisäsaaristossa (8 ‰) kuin ulkosaaristossa (4 ‰). Päinvastoin kuin yleensä tutkimuksissa on esitetty, tässä tutkimuksessa korkeat /13Cja/ tai /15N-arvot kuvannevat lisääntynyttä jokien tuomaa terrestristä alkuperää olevaa ravinnekuormaa. Lisäksi kauden aikana suuresti vaihtelevat, mutta silti voimakkaasti korreloivat /13C ja /15N indikoivat hiilen ja typen olevan pääosin samaista alkuperää. Kirjallisuudessa ravinnepulssien yhtäaikaisesti korkeat /13C ja /15N katsotaan alkuperältään ihmis- tai eläinperäisiksi ja yleensä liitetään karjanlannan käyttöön lannoitteena. Tätä ei kuitenkaan voitu Saaristomerellä osoittaa, koska terrestrisiä ravinnelähteitä ei tutkittu. Kuitenkin muista tunnetuista SPM:n /-arvoja voimakkaasti muokkaavista tekijöistä voitiin poissulkea ravinnerajoitteisuudesta johtuva fraktionaation muutos sekä synteettisten lannoitteiden käyttö ja kalankasvatus ravinnelähteinä. Pintasedimentistä saatiin paljon tasaisempia /13C- ja /15N-arvoja kaikilla näytteenotoilla (keskimäärin -24 ‰ ja 4 ‰, vastaavasti), jotka kuvannevat sedimentoituvan aineksen pitkäaikaisia keskiarvoja. Havaittavissa oli hienoinen laskeva suuntaus kohti ulompia asemia ja monista pohjan lähellä tai sedimentissä elävistä eläimistä saatiin samaan tapaan laskevia arvoja. SPM:n vaihtelevat ja eläinten /-arvot korreloivat heikosti lukuunottamatta joitakin suspensionsyöjiä (Balanus improvisus ja Mytilus edulis). /13C ja C:N korreloivat negatiivisesti joillakin lajeilla (Macoma balthica, Monoporeia affinis ja Mytilus edulis), joka johtunee muuttuvasta rasvojen osuudesta kudoksissa. Ainakin näiden lajien käyttöön ravinnelähteiden arvioinnissa kannattaa suhtautua varauksella. Makrolevien /15N oli n. 4 ‰ korkeampi sisäsaaristossa kuin ulommilla asemilla, joka myöskin heijastanee jokien tuoman terrestristä alkuperää olevien ravinteiden vaikutusta. Fucus vesiculosus, tutkimuksen ainut varsinainen monivuotinen levä, sai 6 ‰ korkeampia /13C-arvoja kuin muut levät keskimäärin. Gammarus sp:n Idothea baltican /15N seurasi makrolevien /15N:n vaihtelua, mutta /13C:n perusteella arvioituna ne suosivat rehevöitymisen seurauksena runsastuvia rihmaleviä sisäsaaristossa, kun taas F. vesiculosus näytti kasvattavan tärkeyttä ravintona ulkosaaristossa. Hiilen ja typen pysyvät isotoopit osoittautuivat tehokkaiksi työkaluiksi ravinteiden lähteiden ja kulkeutumisen tarkasteluissa erityisesti sisäsaaristossa, vaikkakin joitakin lajikohtaisia piirteitä on otettava huomioon ja joidenkin biologisten prosessien, kuten denitrifikaation osuus fraktionaation aiheuttajana pitäisi määrittää ennen kuin luotettavia arvioita ravinnelähteistä voidaan tehdä.