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Biological drugs are increasingly important for patients and industry due to their application in the treatment of common and potentially life-threatening diseases such as diabetes, cancer, and obesity. While most marketed biopharmaceuticals today are injectables, the potential of mucoadhesive delivery systems based on dendron-coated mesoporous silica nanoparticles for oral delivery of biological drugs is explored in this project. We hypothesize that specifically designed dendrons can be employed as mucoadhesive excipients and used to decorate the surface of nanoparticles with properties to embed a drug molecule. We initially tested a novel synthesis method for the preparation of dendrons, which was successfully validated by the chemical characterization of the compounds. The interaction between dendrons and mucin was studied through isothermal titration calorimetry and quartz crystal microbalance with dissipation monitoring and proved to be spontaneous and thermodynamically favorable. Dendrons were conjugated onto 244.4 nm mesoporous silica nanoparticles and characterized for chemical composition, size, and surface charge, which all showed a successful conjugation. Finally, dynamic light scattering was used to study the interaction between nanoparticles and porcine gastric mucin, whereas the interaction between nanoparticles and porcine intestinal mucus was characterized by rheological measurements. This study shows a deeper biophysical understanding of the interaction between nanoparticles and mucin or native porcine intestinal mucus, further leveraging the current understanding of how dendrons can be used as excipients to interact with mucin. This will provide knowledge for the potential development of a new generation of mucoadhesive nanoformulations for the oral delivery of biopharmaceuticals.

The effect of churning temperature (10 °C vs. 22 °C) is evaluated with respect to water content, rheology, microstructure and stability of butter produced using the batch churning method with a temperature ramp of 4 °C/min. Using pulsed-nuclear magnetic resonance, an increase in relative solid fat content from 44% to 49.5% was observed when decreasing the churning temperature. Due to lower solid fat content formed upon churning at high temperatures, average water droplet size significantly increased from 5.5 μm to 18.5 μm and less water could be incorporated into the butter during mixing. Using differential scanning calorimetry, it was observed that water addition as well as churning at low temperatures induced a transition toward more stable crystal structures, as the melting point in the high melting fraction was slightly lower for butter churned at high temperature. This did, however, not reflect in any changes in terms of crystal polymorphism, and all butters contained primarily β′-crystals with traces of α- and β-crystals. Despite the observed changes, small deformation rheology revealed no difference as a function of churning temperature or water content. During isothermal storage at 5 °C, the solid fat content increased in all butters, but only butter churned at 10 °C showed an increase in hardness during storage. However, no difference in rheological behavior was observed among the butters. Thus it can be concluded that low temperature allows more water to be incorporated in the system without inducing any changes in rheological behavior or crystal polymorphism after four weeks of storage.

Crystallization, rheological properties and microstructure of butter with varying water content and subjected to different cooling rate were studied during four weeks of storage at 5 °C. Using small and large deformation rheology, the elastic modulus (G′) and Hencky strain at fracture was followed. When comparing samples with an equal water content, samples produced from fast cooled cream (7.5 °C/min) have a higher G′ compared to butter produced from slow cooled cream (0.4 °C/min), at day 1–7. However, no difference in G′ is observed as a function of time, even though the solid fat content increases. Increasing the water content from 20% to 32% decreases G′ at day 1–14, yet X-ray scattering and differential scanning calorimetry shows no difference in crystal polymorphism or crystallinity. After 21 days of storage, no difference in G′ is observed as a function of cream cooling rate or water content. For all samples, small angle X-ray scattering shows formation of 2L (41 A) and 3L (57 A) lamellar organization, while the wide angle spectra shows mainly β′-crystals (4.2 A & 3.81 A) together with traces of β (4.6 A).

Samples of anhydrous milk fat and rapeseed oil were characterized by small amplitude oscillatory shear rheology using nine different instrumental geometrical combinations to monitor elastic modulus (G′) and relative deformation (strain) at fracture. First, G′ was continuously recorded during crystallization in a fluted cup at 5C. Second, crystallization of the blends occurred for 24 h, at 5C, in external containers. Samples were gently cut into disks or filled in the rheometer prior to analysis. Among the geometries tested, corrugated parallel plates with top and bottom temperature control are most suitable due to reproducibility and dependence on shear and strain. Similar levels for G′ were obtained for samples measured with parallel plate setup and identical samples crystallized in situ in the geometry. Samples measured with other geometries have G′ orders of magnitude lower than identical samples crystallized in situ. This emphasizes the importance of gentle sample pre handling, temperature control and preventing slip. Practical Applications Small deformation rheology is widely used to study and evaluate textural behavior of fat-based systems. As different research groups use different geometries, a systematic evaluation of data gained using different geometrical combinations is needed. By conducting a methodic evaluation, this work provides an increased knowledge of how to characterize fat-based systems using small deformation rheology and evaluates the relations between geometries. Because of the rigid nature of fat-based systems, wall slip is likely to occur. Moreover, when characterizing such systems during storage and/or produced at industrial scale, in situ crystallization is not possible. In such cases, the sample must be loaded at the geometry prior to analysis. Such loading will affect the fat crystal network to some extent and consequently the data obtained. However, as the physical conditions differ between the geometries available, they affect both the fat crystal network and the tendency of wall slip to occur differently.

In the margarine and butter industry, sustainable and more efficient refrigerants, such as CO2, are introduced to industrial scraped surface heat exchangers, allowing an increased capacity compared to conventional use of NH3. The effect of such changes in capacity and a varied rotational speed was studied in relation to the structural behavior of puff pastry butter during 4 weeks of isothermal storage at 20 °C. The physical properties of the fat crystal network were studied in detail at several length scales by combining X-ray diffraction with differential scanning calorimetry, confocal laser scanning microscopy, LR-NMR and rheology. Our data shows that a high capacity combined with high rotational speed decreases the brittleness of puff pastry butter after 7 days of storage. This effect is, however, diminished after 28 days of storage. Likewise, changes in capacity and rotational speed are shown to induce no microstructural and polymorphic differences after 28 days of storage. However, the degree of work softening is related to the manufacturing conditions: a high capacity and a high rotational speed increase the ability of the puff pastry butter to resist structural breakdown during working. With this being the only observed difference; a wide operational window exists on an industrial level to produce puff pastry butter with similar structural behavior.

Butter and other milk fat-based products are valuable products for the dairy industry due to their unique taste, their textural characteristics, and nutritional value. However, an increased consumer demand for low-fat-based products increases the need for an increased essential understanding of the effective factors governing the structure of milk fat-based products. Today, 2 manufacturing techniques are available: the churning method and the emulsification method. The first is typically used for production of butter with a globular structure, which has become increasingly popular to obtain low-fat-based products, typically without presence of milk fat globules. The microstructure of milk fat-based products is strongly related to their structural rheology, hence applications. Structural behavior is not determined by one single parameter, but by the interactions between many. This complexity is reviewed here. Parameters such as thermal treatment of cream prior to butter making, water content, and chemical composition influence not only crystal polymorphism, but also the number and sizes of fat crystals. The number of crystal-crystal interactions formed within the products is related to product hardness. During storage, however, postcrystallization increases the solid fat content and strengthens the fat crystal network. The fat crystal network is strengthened by the formation of more and stronger crystal-crystal interactions due to mechanically interlinking of fat crystals, which occurs during crystal growth. Postcrystallization is directly linked to chemical composition. The initially observed microstructural difference causing different rheological behavior will disappear during storage due to postcrystallization and formation of more crystal-crystal interactions.

There is a growing interest in the use of cell-penetrating peptides (CPPs) as carriers for transepithelial drug delivery. This chapter gives an introduction to and discussion of the commonly used production and characterization methods for CPP-cargo samples including high-throughput cell viability screening. Moreover, we describe methods for permeation and cell viability assessment in the Caco-2 cell culture model with and without implementation of biosimilar mucus. Last, a method to assess metabolic degradation in vitro is described.

There is a growing interest in the use of cell-penetrating peptides (CPPs) as carriers for transepithelial drug delivery. This chapter gives an introduction to and discussion of the commonly used production and characterization methods for CPP-cargo samples including high-throughput cell viability screening. Moreover, we describe methods for permeation and cell viability assessment in the Caco-2 cell culture model with and without implementation of biosimilar mucus. Last, a method to assess metabolic degradation in vitro is described.

Milk fat exists as globules in its natural state in milk. The potential of using globular fat to modulate the rheological properties and crystallization behavior in butter-like emulsions was studied in the present work. We conducted a comparative study of butter-like emulsions, with a fat phase consisting of 0, 10, 25, 50, or 100% anhydrous milk fat (AMF), the remaining fat being butter grains, and all samples containing 20% water, to obtain systematic variation in the ratio of globular fat. All emulsions were studied over 4wk of storage at 5°C. By combining small and large deformation rheology, we conducted a detailed characterization of the rheological behavior of butter-like emulsions. We applied differential scanning calorimetry to monitor thermal behavior, confocal laser scanning microscopy for microstructural analysis, and low-field pulsed nuclear magnetic resonance spectrometry to measure solid fat content. By combining these techniques, we determined that increasing the fraction of globular fat (by mixing with butter grains) decreases the hardness of butter-like emulsions up to an order of magnitude at d 1. However, no difference was observed in thermal behavior as a function of butter grain content, as all emulsions containing butter grains revealed 2 endothermal peaks corresponding to the high (32.7°C ± 0.6) and medium (14.6°C ± 0.1) melting fractions of fatty acids. In terms of microstructure, decreasing the amount of butter grains in the emulsions resulted in formation of a denser fat crystal network, corresponding to increased hardness. Moreover, microstructural analysis revealed that the presence of butter grains resulted in faster formation of a continuous fat crystal network compared with the 100% AMF sample, which was dominated by crystal clusters surrounded by liquid oil. During storage, hardness remained stable and no changes in thermal behavior were observed, despite an increase in solid fat content of up to 5%. After 28d of storage, we observed no difference in either microstructural or rheological properties, indicating that formation of primary bonds occurs primarily within the first day of storage. The rheological behavior of butter-like emulsions is not determined solely by hardness, but also by stiffness related to secondary bonds within the fat crystal network. The complex rheological behavior of milk fat-based emulsions is better characterized using multiple parameters.

The current study examined the reliability and validity of a Danish adaptation of "Darryl", a cartoon-based measure of DSM-IV symptoms of post-traumatic stress disorder (PTSD) in a sample of Danish children and adolescents. Exposure to potentially traumatizing events, PTSD symptoms and diagnostic status were assessed in 65 children between the ages of 7 and 17 years old. Participants were recruited from a crisis centre for battered women and a school for children with emotional and behavioural problems. The study revealed that Darryl has good internal consistency for the overall scale and adequate reliability for each DSM-IV symptom cluster. Scores from Darryl were significantly correlated with the Clinician Administered PTSD Scale for Children and Adolescents (CAPS-CA) scores and receiver operating characteristic (ROC) analysis showed that it could significantly predict the presence of a PTSD diagnosis according to the CAPS-CA. In comparison to the CAPS-CA, Darryl has comparable psychometric properties and assesses PTSD symptoms in a developmentally appropriate manner. Furthermore, Darryl is quick to administer and was easier for the children in this sample to comprehend than the more traditional measure of PTSD. Overall, Darryl is an effective instrument for screening children at risk of a PTSD diagnosis. The relatively high specificity suggests that screening positive for PTSD using Darryl merits further diagnostic assessment. Copyright © 2016 John Wiley & Sons, Ltd.

The effect of cream heat treatment prior to butter manufacturing, fluctuating temperatures during storage and presence of fat globules vs. no fat globules was examined in laboratory scale produced butter. X-ray diffraction and differential scanning calorimetry was used to study crystallization behaviour and nuclear magnetic resonance to measure solid fat content and water droplet size distribution. Furthermore, the crystal structure was linked to the rheological properties and microstructure of the butter using confocal laser scanning microscopy. Butter produced from non-matured cream mainly formed α- and β′-crystals with minor traces of β-crystals. Maturing of the cream caused a transition from α- to β′- and β-form. The rheological behaviour of slow cooled butter deviated from the matured ones by having a lower elastic modulus, caused by a weaker crystal network. Presence of fat globules did not affect the rheological properties significantly.

The d -vitamin analogue calcipotriol is commonly used for topical treatment of psoriasis, but skin penetration is required for calcipotriol to reach its pharmacological target: the keratinocytes in the lower epidermis. Liposomes can enhance the delivery of drugs into the skin, but a major challenge for the development of dosage forms containing liposomes is to maintain the colloidal stability in the formulation. The purpose of this study was to investigate the effect of stabilising liposomes with the lipopolymer poly(ethylene glycol)-distearoylphosphoethanolamine (PEG-DSPE) on the physicochemical properties of the liposomes and the ability to deliver membrane-intercalated calcipotriol into the skin. Inclusion of 0.5, l and 5 mol% PEG-DSPE in the membrane enhanced the colloidal stability of the liposomes without compromising the delivery of calcipotriol from the vehicle into excised pig skin. Calcipotriol-loaded liposomes with 1 mol% PEG-DSPE did even provide for a significantly increased deposition of calcipotriol into the stratum corneum. The size of the liposomes affected the penetration of calcipotriol into the stratum corneum since small unilamellar vesicles enhanced calcipotriol penetration as compared to large multilamellar vesicles, indicating that the liposomes to some extent migrate as intact vesicles into the stratum corneum. However, calcipotriol penetrated the skin better than the lipid component of the liposomes, suggesting that at least a fraction of the drug is released from the liposomes during skin migration. In conclusion, PEGylation is therefore a promising approach for stabilising calcipotriol-containing liposomal dispersions without compromising their favourable skin accumulation properties.

Lipid-protein interactions can function as “co-factors” that affect the properties / function of transmembrane proteins. Herein, the interaction between anionic dimyristoylphosphatidylglycerol (DMPG) and zwitterionic dimyristoylphosphatidylcholine (DMPC) with the amphiphatic membrane scaffold protein (MSP), were studied. Two 25 kDa MSP wrap around the circumference of discoidal bilayer in a belt-like manner to form a nanodisc [1,2]. The membrane-like structure of nanodiscs has been used for reconstitution of membrane proteins in a native-like environment. Differential scanning calorimetry was employed to characterize lipid-protein interactions in these particles by evaluating changes in MSP denaturation temperature and lipid gel-liquid phase transition as a function of nanodisc lipid composition and ionic strength. Small-angle X-ray scattering and size-exclusion chromatography were used to determine the overall structure of the nanodisc. We suggest the nanodisc lipid is divided into a lipid rim that interacts with the internal face of the MSP helical segments, while the centrally located nanodisc lipids maintain a more bulk-like lipid behavior. This finding is important for reconstitution of membrane proteins since the presence of a ‘lipid rim’ serves to prevent contact between the membrane protein and the MSP. Furthermore, the presence of two distinct lipid environments reduces the available area for reconstituted membrane proteins in the nanodisc. We also show that the negatively charged DMPG has a higher preference for the rim due to its negatively charged headgroup. Finally, we conclude that DMPG stabilizes the nanodisc in a twofold manner: i) DMPG ’freezes’ the MSP conformation preventing flexibility / dissociation that may lead to aggregation. ii) DMPG also contributes to prevention of aggregation due to electrostatic repulsion between the negatively charged lipids on neighboring nanodiscs.[1] T.H. Bayburt et al.: Nano Letters 2 (2002) 853.[2] N. Skar-Gislinge and J.B. Simonsen et al.: JACS 132 (2010) 13713.