A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.

Showing an extensive distribution in the nervous system, and often in co-localization with the classical neurotransmitters, neuropeptides are functioning as important modulators of neuronal signaling. Subsequently, compelling evidence has implicated a modulatory role for the neuropeptide galanin in several physiological functions. The effect of galanin is trancduced intracellularly by three different receptors, and defining the explicit effect from these receptor subtypes is of outmost interest, and likely to result in future therapeutic utilization of the galanin system. The main aim of this thesis was to improve the development of subtype selective ligands utilized to differentiate between the galanin receptor subtypes. To achieve this, we have designed and developed novel galanin receptor ligands and characterized the molecular interactions necessary for ligand bindig at the GalR2 subtype. The major findings include the introduction and characterization of two galanin receptor ligands, selectively activating GalR1 or inhibiting GalR2. Although having moderate selectivity, the two ligands have been utilized in a number of studies, pursuing their initial presentation, in order to differentiate between the galanin receptors and to establish their specific function. Further optimization is likely to improve the selectivity and utilization of these ligands. By identifying the major pharmacophores in the Gal(2-11) ligand and the residues in the GalR2 subtype participating in ligand binding, we have been able to characterize the binding site in this receptor subtype and interactions that are of significance for recognition of subtype specific ligands. Together, these findings on GalR2 and Gal(2-11) are of importance for future design of ligands acting on this receptor.

The neuropeptides galanin and neuropeptide Y (NPY) play an important role in the reproduction of rodents, e.g. by modulating the release of gonadal hormones, the nutritional status by effects on feeding behavior and also by influencing mating behavior. There are age- and gender- differences in galanin- and NPY- like immunoreactivities (LIs) in brain areas important for higher functions including the hippocampal formation (HiFo) and cortex, that are related to the concentrations of 17β-estradiol. Neuropeptides in general are currently not considered critical in normal integrative neuronal functions but are rather thought to act as slow modulators during periods of stress or injury. In the present thesis we attempted to investigate, if the normal cyclical changes in the female sex-hormone 17β-estradiol can affect neurotransmission in brain areas important for memory, cognition and mood. We studied not only ”long term” (days and weeks) but also ”short-term” (one hour) effects on galanin and NPY concentrations in 17β-estradiol-primed ovariectomized (ovx) rats and mice. Radioimmunoassay (RIA) of galanin-LI in extracts of brain tissues from ”long-term” 17β-estradiol-treated ovx rats showed that its effects on galanin are dependent on boththe dose and on duration. Galanin - and NPY-LI in brain tissues of young ovx rats and mice increased in response to 17β-estradiol treatment in the HiFo, frontal cortex and striatum already within hours. This effect was not blocked by Tamoxifen® in rats. The mechanism of the 17β-estradiol effects on galanin levels in the rat HiFo may be related to decreased release of galanin into the extracellular fluid, since galanin-LI decreased in microdialysis samples two hours after a single injection of 17β-estradiol. Species differences were observed with regards to galanin, possibly due to tissue and species differences in the distribution of estrogen receptors. In the HiFo and caudate nucleus of mice, we found an increase in NPY-transcript after two hours by means of insitu hybridization, perhaps a compensatory up-regulation of NPY mRNA after increased 17β-estradiol-induced release in these areas. Taken together with no effects of Tamoxifen® on the levels on galanin in the HiFo of rats, the short duration, and the fact that the density of classical estrogen receptors seems to be limited in the striatum, we suggest that these effects are mediated through a membrane-related mechanism perhaps not involving the classical ER route. With an antiserum raised against the C-terminal end of the first 16 aminoacids of galanin- the sequence important for binding of intact galanin to its receptor - we found a novel compound which appears to be a homologue to galanin. Chromatographical analysis revealed that it was not galanin(1-29) or the galanin related peptide, galaninlike peptide (GALP), but appeared with immunohistochemistry in the galanin systems in the brain and was further influenced by 17β-estradiol in the HiFo and frontal cortex in a similar manner as galanin(1-29). In conclusion, tissue concentrations of galanin, a putative galanin homologue and NPY can be altered already after one hour by 17β-estradiol treatment e.i. in the HiFo. These ”short-term” effects are most likely to be due to effects on estrogen-primed peptide release which might influence mechanisms important for memory, cognition and mood.

The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

<p>Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism.</p><p>In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells.</p><p>In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed.</p><p>In the third study, the cells were challenged with hydrogen peroxide (H₂O₂) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H₂O₂ than the ScN2a cells, according to the cell viability assay.</p><p>In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.</p>

Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism. In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells. In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed. In the third study, the cells were challenged with hydrogen peroxide (H2O2) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H2O2 than the ScN2a cells, according to the cell viability assay. In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.

The plasma membrane functions as a barrier, restricting entry of hydrophilic pharmaceutical agents. Cell-penetrating peptides (CPPs) are capable of transporting bioactive cargos into the cell and have consequently been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids. The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides in the form of ds DNA decoy for sequestering of transcription factors or PNAs for redirection of splicing. In addition, peptides derived from the interaction interface of a tumor suppressor protein were investigated for their potential to combine a biological effect with internalization. Peptides with or without any cargo were predominantly dependent on some form of endocytic mechanism for internalization, substantiated by using a functional assay, where all tested CPPs were associated with endocytosis for delivery of splice correcting PNAs. A new CPP, M918 proved most efficient in promoting splice correction and internalized mainly via macropinocytosis. In addition, TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.

High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics. In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10. The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders. / At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted.Peper 5: In press. / VINNOVA-SAMBIO Multidisciplinary BIO