| 1. |
- De Jong, Wim H., et al.
(författare)
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Round robin study to evaluate the reconstructed human epidermis (RhE) model as an in vitro skin irritation test for detection of irritant activity in medical device extracts
- 2018
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 50, s. 439-449
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Tidskriftsartikel (refereegranskat)abstract
- Assessment of skin irritation is an essential component of the safety evaluation of medical devices. OECD Test Guideline 439 describes the use of reconstructed human epidermis (RhE) as an in vitro test system for classification of skin irritation by neat chemicals. An international round robin study was conducted to evaluate the RhE method for determination of skin irritant potential of medical device extracts. Four irritant polymers and three non-irritant controls were obtained or developed that had demonstrated their suitability to act as positive or negative test samples. The RhE tissues (EpiDerm™ and SkinEthic™ RHE) were dosed with 100 μL aliquots of either saline or sesame oil extract. Incubation times were 18 h (EpiDerm™) and 24 h (SkinEthic™ RHE). Cell viability reduction > 50% was indicative of skin irritation. Both the EpiDerm™ and SkinEthic™ RHE tissues were able to correctly identify virtually all of the irritant polymer samples either in the saline, sesame oil or both solvent extracts. Our results indicate that RhE tissue models can detect the presence of strong skin irritants at low levels in dilute medical device polymer extracts. Therefore, these models may be suitable replacements for the rabbit skin irritation test to support the biological evaluation of medical devices.
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| 2. |
- Fant, Kristina, 1979
(författare)
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Condensation of DNA for Gene Delivery: Studies on Vector-Nucleic Acid Interactions and Transfection Efficiency
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concerns the delivery of nucleic acids into cells and tissues with the aim to change or correct gene expression. The field of gene delivery has the ultimate goal of producing genetic drugs for clinical applications, and the activity in this area is intense although so far the use is limited to research purposes. The main bottleneck is the lack of suitable delivery vectors. Viral vectors have undisputed efficiency but have shown to impose large safety risks, why non-viral methods are being increasingly explored.In this work, two classes of non-viral vectors, dendrimers and cell-penetrating peptides (CPPs), have been investigated for their interactions with DNA and their ability to mediate gene delivery into cultured cells with the aim of deducing relations between vector structure and biological activity. In addition, methods allowing biophysical characterization of vector-DNA complexes with focus on their dynamic nature have been developed. For dendrimeric vectors, the effect of surface-modifications aiming to increase their biocompatibility was studied. The results showed that although these modifications render dendrimers completely non-toxic, they also abolish the ability to mediate transfection. By biophysical studies, this could be related to a decreased DNA binding capacity of the modified dendrimers, which highlights a previously largely neclected issue in the dendrimer field. CPPs are promising vectors given their inherent ability to stimulate uptake into mammalian cells and deliver macromolecular cargo. Here, peptides with varying content of arginines and lysines were examined and it was found that arginine residues enhance the uptake efficiency compared to lysines. Arginines also showed to promote more efficient condensation of DNA, and the arginine-enriched variant of the peptide was the only one that displayed successful gene delivery. However, arginines alone were not sufficient since other CPPs with equal numbers of arginines were non-active in this respect. Hydrophobic residues in the peptide were found to be equally important, with one possible mechanism being to provide stability upon interaction with negatively charged cell surface residues. In conclusion, the thesis contributes with biophysical insights into the complicated process of gene delivery and gives clues for future development of new and more efficient vectors.
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| 3. |
- Fant, Kristina, 1979
(författare)
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Dendrimer-mediated gene delivery: Effect of polymer structure on cellular response
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The delivery of nucleic acids to cells, in vitro and in vivo, is becoming a realistic approach for intervention with gene expression at a molecular level. It can be used to repair malfunctioning genes or to introduce new functionalities, with the most important application being clinical treatment of human diseases. The main obstacle on the way to successful gene therapy is to achieve safe and efficient internalization of nucleic acids into the target cells, which requires a carrier that protects the DNA, promotes its interaction with the cell surface, and enables cellular uptake. There is intense research in this field and many new synthetic carriers have been developed. However, there is a lack of systematic studies relating their chemical structure to the resulting transfection efficiency. In this Thesis, we seek to understand factors that are important for successful gene delivery in order to enable improved carrier design. We investigate for a competent class of DNA carriers, cationic dendrimers, how small variations in size and surface structure affect their DNA binding properties and the morphology of the resulting dendrimer-DNA complexes, and in turn what effect this has on cells in vitro. Using advanced spectroscopic techniques we are able to thoroughly characterize the DNA condensates as well as their uptake and subsequent expression in mammalian cells. We have been able to show explicitly, for the first time, that dendrimers bind to DNA in a cooperative manner. We also show that exposure to cell growth media significantly alter dendrimer-DNA complex morphology. These factors are extremely important to consider when trying to relate biophysical studies of dendrimer-DNA complexes to the outcome of transfection experiments. Furthermore, we have developed methods to quantify cellular uptake and to discern different endocytotic mechanisms. These will be applied to the dendrimer library in the near future.
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| 4. |
- Fant, Kristina, 1979, et al.
(författare)
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DNA condensation by PAMAM dendrimers: Self-assembly characteristics and effect on transcription
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 47:6, s. 1732-1740
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Tidskriftsartikel (refereegranskat)abstract
- Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system.
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| 5. |
- Fant, Kristina, 1979, et al.
(författare)
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Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency
- 2010
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8392 .- 1543-8384. ; 7:5, s. 1734-1746
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Tidskriftsartikel (refereegranskat)abstract
- Poly(amidoamine) (PAMAM) dendrimers are promising multipotent gene delivery vectors, providing favourable DNA condensation properties also in combination with the possibility of conjugation of different targeting ligands to their surface. They have been used for transfection both in vitro and in vivo, but their application is currently somewhat limited due to inherent cytotoxicity. In this work we investigate how two types of surface modification, acetylation and PEGylation, affect the DNA binding characteristics, the cytotoxicity and the in vitro transfection efficiency of generation 4 and 5 PAMAM dendrimers. Particularly, we address how the morphology of DNA-dendrimer complexes, formed under low salt conditions, changes upon dilution in cell growth medium, an event that inevitably occurs before the complexes reach the cell surface in any transfection experiment. We find that acetylation and PEGylation essentially eliminates the inherent dendrimer cytotoxicity. However, the transfection efficiency of the modified dendrimers is lower than that of the corresponding unmodified dendrimers, which can be rationally understood by our observations that DNA is less condensed when complexed with these modified dendrimers. Although small DNA-dendrimer particles are formed, the availability for ethidium intercalation and nuclease degradation is significantly higher in the modified DNA-dendrimer complexes than in unmodified ones. Dilution in cell growth medium has a drastic effect on these electrostatically assembled complexes, resulting in increase in size and DNA availability. Our results strongly add to the notion that it is of importance to perform a biophysical characterization under conditions as close to the transfection situation as possible, to enable conclusions regarding structure- activity relations of gene delivery vectors.
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| 6. |
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| 7. |
- Fant, Kristina, 1979, et al.
(författare)
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Using Ethidium To Probe Nonequilibrium States of DNA Condensed for Gene Delivery
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 50:7, s. 1125-1127
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Tidskriftsartikel (refereegranskat)abstract
- Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding.
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| 8. |
- Krebs, Alice, et al.
(författare)
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Template for the Description of Cell-Based Toxicological Test Methods to Allow Evaluation and Regulatory Use of the Data
- 2019
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Ingår i: Altex. - : ALTEX Edition. - 1868-596X .- 1868-8551. ; 36:4, s. 682-699
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.
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| 9. |
- Matson Dzebo, Maria, 1985, et al.
(författare)
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Enhanced Cellular Uptake of Antisecretory Peptide AF-16 through Proteoglycan Binding
- 2014
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 53:41, s. 6566-6573
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Tidskriftsartikel (refereegranskat)abstract
- Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and antiinflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparinincreases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide−glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16.
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| 10. |
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