Signe Danielsen - Academia.edu (original) (raw)

Papers by Signe Danielsen

Polycation mediated gene delivery has emerged as a promising alternative to the use of viral vect... more Polycation mediated gene delivery has emerged as a promising alternative to the use of viral vectors for gene therapy. Although yielding significantly lower transfection efficiency than viral vectors, the use of polycations possess advantages over viruses such as reduced immune response and the possibility of carrying large DNAs at low cost. The polycations will condense DNA into a more compact form that may allow easier transport through the extracellular matrix and probably across the cell membrane, as well as protect the DNA from nuclease attacks. The non-toxic and biodegradable polycation chitosan has shown great potential as a gene delivery vehicle, as high transfection efficiencies are reported. In this study the effect of chitosan molecular parameters like charge density (determined by the fractional content of acetylated units, FA) and the degree of polymerization (DP) was evaluated for its ability to compact DNA. AFM-imaging revealed that chitosan compacted DNA into toroida...

European Journal of Gynaecological Oncology, 2021

Concepts, Applications and Perspectives, 2005

... Emin Oroudjev, Signe Danielsen, and Helen G. Hansma 24.1 Introduction ... The tip of the AFM ... more ... Emin Oroudjev, Signe Danielsen, and Helen G. Hansma 24.1 Introduction ... The tip of the AFM probe is characterized by its aspect ratio (ratio between width and height of the tip's pyramid or cone) and tip sharpness (expressed as tip radius of curva-ture). ...

... I also thank Sabina P. Strand for collaboration and discussions. I am grateful to Professor H... more ... I also thank Sabina P. Strand for collaboration and discussions. I am grateful to Professor Helen G. Hansma who gave me the opportunity to visit her lab at the Department of Physics at University of California Santa Barbara and allowed me to use her lab to perform experiments. ...

Acta Oncologica, 2010

Partial breast irradiation (IORT or brachytherapy) differ from external radiation of whole breast... more Partial breast irradiation (IORT or brachytherapy) differ from external radiation of whole breast in terms of irradiated volumes, fractionation, radiation energy and dose rate; all factors influencing the treatment outcome in a complex manner. Theoretically obtained RBE values comparing effects of radiation used in IORT and external therapy are published, but experimental studies are required to confirm these data. The aim of this study is to establish such RBE values for two breast cancer cell lines. Colony formation of breast cancer cell lines (MCF-7 and T-47D) were studied after photon irradiation with qualities and dose rates used in IORT, brachytherapy and external radiation. RBE values from survival data were used to compare effects. Increasing the photon energy (dose rate 0.2 Gy/min) from 50 kV (Intrabeam) to 380 keV (¹⁹²Ir source) and 6 MV (linear accelerator) yielded an increase in the cell survival, whereas increasing the dose rate to 6 Gy/min had minor effect. Average RBE values for 50 kV with 6 MV as reference radiation varied from about 1.4 (for doses < 5 Gy) to > 1.9 (for doses < 0.02 Gy) for MCF-7 cells and from about 1.4 to > 3.1 for T-47D cells for the same dose levels. Corresponding RBE values for 380 keV radiation were about 1.4 for MCF-7 cells and 1.3-2.3 for T-47D cells. RBE data for breast cancer cells exposed to radiation used in IORT, brachytherapy or external radiation differ among the cell lines tested. The values are in agreement with published theoretical and experimental work.

Radiotherapy and Oncology, 2019

Anticancer Research, Feb 1, 2015

Aim: To compare five inverse treatment plans with one conventional manually-optimized plan for ce... more Aim: To compare five inverse treatment plans with one conventional manually-optimized plan for cervical cancer brachytherapy (BT) using radiobiological parameters combined with dosimetric and volumetric parameters. Materials and Methods: Five inverse treatment plans were calculated using an inverse planning simulated annealing (IPSA) algorithm for each of four fractions for 12 cervical cancer patients treated with high-dose-rate (HDR) brachytherapy. The inverse treatment plans were compared to a manually-optimized plan used for the actual treatment of the patients. The comparison of the plans was performed with respect to the probability of cure without complication (P + ). Results: Overall, the manually optimized plan scored the best results; however, the probability of cure without complication is within an acceptable clinical range for all the plans. Conclusion: Although there are still considerable uncertainties in the radiobiological parameters, the radiobiological plan evaluation method presents itself as a potential complement to physical dosimetric methods.

Radiotherapy and Oncology, 2014

Radiotherapy and Oncology, 2012

Anticancer research, 2015

To compare five inverse treatment plans with one conventional manually-optimized plan for cervica... more To compare five inverse treatment plans with one conventional manually-optimized plan for cervical cancer brachytherapy (BT) using radiobiological parameters combined with dosimetric and volumetric parameters. Five inverse treatment plans were calculated using an inverse planning simulated annealing (IPSA) algorithm for each of four fractions for 12 cervical cancer patients treated with high-dose-rate (HDR) brachytherapy. The inverse treatment plans were compared to a manually-optimized plan used for the actual treatment of the patients. The comparison of the plans was performed with respect to the probability of cure without complication (P+). Overall, the manually optimized plan scored the best results; however, the probability of cure without complication is within an acceptable clinical range for all the plans. Although there are still considerable uncertainties in the radiobiological parameters, the radiobiological plan evaluation method presents itself as a potential complemen...

To compare five inverse treatment planning methods with the conventional manually optimized plann... more To compare five inverse treatment planning methods with the conventional manually optimized planning approach for brachytherapy of cervical cancer with respect to dosimetric parameters. Eighteen cervical cancer patients treated with magnetic resonance imaging (MRI)-guided high dose rate (HDR) brachytherapy were included in this study. Six plans were created for each of the 4 HDR brachytherapy fractions for each patient: 1 manually optimized and 5 inversely planned. Three of these were based on inverse planning simulated annealing (IPSA) with and without extra constraints on maximum doses of the target volume, and different constraints on doses to the organs at risk (OARs). In addition there were two plans based on dose to target surface points. The resulting dose-volume histograms were analyzed and compared from the dosimetric point of view by quantifying specific dosimetric parameters, such as clinical target volume (CTV) D₉₀, CTV D₁₀₀, conformal index (COIN), and D₂cm3 for rectum, bladder and the sigmoid colon. Manual optimization led to a mean target coverage of 78.3% compared to 87.5%, 91.7% and 82.5% with the three IPSA approaches (p < 0.001). Similar COIN values for manual and inverse optimization were found. The manual optimization led to better results with respect to the dose to the OARs expressed as D₂cm3. Overall, the best results were obtained with manual optimization and IPSA plans with volumetric constraints including maximum doses to the target volume. Dosimetric evaluation of manual and inverse optimization approaches is indicating the potential of IPSA for brachytherapy of cervical cancer. IPSA with constraints of maximum doses to the target volume is closer related to manual optimization than plans with constraints only to minimum dose to the target volume and maximum doses to OARs. IPSA plans with proper constraints performed better than those based on dose to target surface points and manually optimized plans.

The Journal of Physical Chemistry B, 2003

The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular p... more The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular plasmid DNA, and xanthan were investigated using tapping mode atomic force microscopy followed by quantitative image analysis. A shape factor was calculated for each of the observed polyelectrolyte complexes and used as a basis for dividing the structures into ensembles of morphologically linear, toroidal, and globular structures for subsequent quantitative analysis. Compaction of polyanions with chitosan yielded a small fraction of the torus morphology when the persistence length, L p , of 25 nm (acetan) was reached. For both DNA, L p ) 50 nm, and xanthan, L p ) 120 nm, it was found that the toroids make up a substantial fraction of the complexed structures formed by the given chitosan and at room temperature. Rodlike complexes were additionally observed within DNA-chitosan complexes, whereas they do not appear as a significant fraction of chitosan-complexed high-molecular-weight xanthan. The average height of the condensates was observed to be ∼2 nm for the compacted xanthan toroids, while it was determined to be ∼5 nm for compacted DNA toroids. Reducing the degree of polymerization of xanthan yielded a decrease in the fraction of toroids. Compacted xanthan at room temperature displays a number of racquets and other morphologies similar to the reported intermediate, metastable states by simulations. The reduced abundance of such structures following annealing supports the interpretation of their metastable nature. † Part of the special issue "International Symposium on Polyelectrolytes".

Naturwissenschaften, 2001

In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wi... more In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca 2+ with Na + , forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca 2+ , Sr 2+ and Ba 2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

Biopolymers, 2005

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force m... more Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.

Biophysical Journal, 2004

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly,... more Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length \20 mm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of \7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.

Biomacromolecules, 2004

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising ... more Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.

Biomacromolecules, 2005

The interactions between DNA and chitosans varying in fractional content of acetylated units (F A... more The interactions between DNA and chitosans varying in fractional content of acetylated units (F A ), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on F A , and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomerbased polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

Biomacromolecules, 2007

In this study the relative importance of valence and charge density of the polycation chitosan on... more In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.

Polycation mediated gene delivery has emerged as a promising alternative to the use of viral vect... more Polycation mediated gene delivery has emerged as a promising alternative to the use of viral vectors for gene therapy. Although yielding significantly lower transfection efficiency than viral vectors, the use of polycations possess advantages over viruses such as reduced immune response and the possibility of carrying large DNAs at low cost. The polycations will condense DNA into a more compact form that may allow easier transport through the extracellular matrix and probably across the cell membrane, as well as protect the DNA from nuclease attacks. The non-toxic and biodegradable polycation chitosan has shown great potential as a gene delivery vehicle, as high transfection efficiencies are reported. In this study the effect of chitosan molecular parameters like charge density (determined by the fractional content of acetylated units, FA) and the degree of polymerization (DP) was evaluated for its ability to compact DNA. AFM-imaging revealed that chitosan compacted DNA into toroida...

European Journal of Gynaecological Oncology, 2021

Concepts, Applications and Perspectives, 2005

... Emin Oroudjev, Signe Danielsen, and Helen G. Hansma 24.1 Introduction ... The tip of the AFM ... more ... Emin Oroudjev, Signe Danielsen, and Helen G. Hansma 24.1 Introduction ... The tip of the AFM probe is characterized by its aspect ratio (ratio between width and height of the tip's pyramid or cone) and tip sharpness (expressed as tip radius of curva-ture). ...

... I also thank Sabina P. Strand for collaboration and discussions. I am grateful to Professor H... more ... I also thank Sabina P. Strand for collaboration and discussions. I am grateful to Professor Helen G. Hansma who gave me the opportunity to visit her lab at the Department of Physics at University of California Santa Barbara and allowed me to use her lab to perform experiments. ...

Acta Oncologica, 2010

Partial breast irradiation (IORT or brachytherapy) differ from external radiation of whole breast... more Partial breast irradiation (IORT or brachytherapy) differ from external radiation of whole breast in terms of irradiated volumes, fractionation, radiation energy and dose rate; all factors influencing the treatment outcome in a complex manner. Theoretically obtained RBE values comparing effects of radiation used in IORT and external therapy are published, but experimental studies are required to confirm these data. The aim of this study is to establish such RBE values for two breast cancer cell lines. Colony formation of breast cancer cell lines (MCF-7 and T-47D) were studied after photon irradiation with qualities and dose rates used in IORT, brachytherapy and external radiation. RBE values from survival data were used to compare effects. Increasing the photon energy (dose rate 0.2 Gy/min) from 50 kV (Intrabeam) to 380 keV (¹⁹²Ir source) and 6 MV (linear accelerator) yielded an increase in the cell survival, whereas increasing the dose rate to 6 Gy/min had minor effect. Average RBE values for 50 kV with 6 MV as reference radiation varied from about 1.4 (for doses < 5 Gy) to > 1.9 (for doses < 0.02 Gy) for MCF-7 cells and from about 1.4 to > 3.1 for T-47D cells for the same dose levels. Corresponding RBE values for 380 keV radiation were about 1.4 for MCF-7 cells and 1.3-2.3 for T-47D cells. RBE data for breast cancer cells exposed to radiation used in IORT, brachytherapy or external radiation differ among the cell lines tested. The values are in agreement with published theoretical and experimental work.

Radiotherapy and Oncology, 2019

Anticancer Research, Feb 1, 2015

Aim: To compare five inverse treatment plans with one conventional manually-optimized plan for ce... more Aim: To compare five inverse treatment plans with one conventional manually-optimized plan for cervical cancer brachytherapy (BT) using radiobiological parameters combined with dosimetric and volumetric parameters. Materials and Methods: Five inverse treatment plans were calculated using an inverse planning simulated annealing (IPSA) algorithm for each of four fractions for 12 cervical cancer patients treated with high-dose-rate (HDR) brachytherapy. The inverse treatment plans were compared to a manually-optimized plan used for the actual treatment of the patients. The comparison of the plans was performed with respect to the probability of cure without complication (P + ). Results: Overall, the manually optimized plan scored the best results; however, the probability of cure without complication is within an acceptable clinical range for all the plans. Conclusion: Although there are still considerable uncertainties in the radiobiological parameters, the radiobiological plan evaluation method presents itself as a potential complement to physical dosimetric methods.

Radiotherapy and Oncology, 2014

Radiotherapy and Oncology, 2012

Anticancer research, 2015

To compare five inverse treatment plans with one conventional manually-optimized plan for cervica... more To compare five inverse treatment plans with one conventional manually-optimized plan for cervical cancer brachytherapy (BT) using radiobiological parameters combined with dosimetric and volumetric parameters. Five inverse treatment plans were calculated using an inverse planning simulated annealing (IPSA) algorithm for each of four fractions for 12 cervical cancer patients treated with high-dose-rate (HDR) brachytherapy. The inverse treatment plans were compared to a manually-optimized plan used for the actual treatment of the patients. The comparison of the plans was performed with respect to the probability of cure without complication (P+). Overall, the manually optimized plan scored the best results; however, the probability of cure without complication is within an acceptable clinical range for all the plans. Although there are still considerable uncertainties in the radiobiological parameters, the radiobiological plan evaluation method presents itself as a potential complemen...

To compare five inverse treatment planning methods with the conventional manually optimized plann... more To compare five inverse treatment planning methods with the conventional manually optimized planning approach for brachytherapy of cervical cancer with respect to dosimetric parameters. Eighteen cervical cancer patients treated with magnetic resonance imaging (MRI)-guided high dose rate (HDR) brachytherapy were included in this study. Six plans were created for each of the 4 HDR brachytherapy fractions for each patient: 1 manually optimized and 5 inversely planned. Three of these were based on inverse planning simulated annealing (IPSA) with and without extra constraints on maximum doses of the target volume, and different constraints on doses to the organs at risk (OARs). In addition there were two plans based on dose to target surface points. The resulting dose-volume histograms were analyzed and compared from the dosimetric point of view by quantifying specific dosimetric parameters, such as clinical target volume (CTV) D₉₀, CTV D₁₀₀, conformal index (COIN), and D₂cm3 for rectum, bladder and the sigmoid colon. Manual optimization led to a mean target coverage of 78.3% compared to 87.5%, 91.7% and 82.5% with the three IPSA approaches (p < 0.001). Similar COIN values for manual and inverse optimization were found. The manual optimization led to better results with respect to the dose to the OARs expressed as D₂cm3. Overall, the best results were obtained with manual optimization and IPSA plans with volumetric constraints including maximum doses to the target volume. Dosimetric evaluation of manual and inverse optimization approaches is indicating the potential of IPSA for brachytherapy of cervical cancer. IPSA with constraints of maximum doses to the target volume is closer related to manual optimization than plans with constraints only to minimum dose to the target volume and maximum doses to OARs. IPSA plans with proper constraints performed better than those based on dose to target surface points and manually optimized plans.

The Journal of Physical Chemistry B, 2003

The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular p... more The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular plasmid DNA, and xanthan were investigated using tapping mode atomic force microscopy followed by quantitative image analysis. A shape factor was calculated for each of the observed polyelectrolyte complexes and used as a basis for dividing the structures into ensembles of morphologically linear, toroidal, and globular structures for subsequent quantitative analysis. Compaction of polyanions with chitosan yielded a small fraction of the torus morphology when the persistence length, L p , of 25 nm (acetan) was reached. For both DNA, L p ) 50 nm, and xanthan, L p ) 120 nm, it was found that the toroids make up a substantial fraction of the complexed structures formed by the given chitosan and at room temperature. Rodlike complexes were additionally observed within DNA-chitosan complexes, whereas they do not appear as a significant fraction of chitosan-complexed high-molecular-weight xanthan. The average height of the condensates was observed to be ∼2 nm for the compacted xanthan toroids, while it was determined to be ∼5 nm for compacted DNA toroids. Reducing the degree of polymerization of xanthan yielded a decrease in the fraction of toroids. Compacted xanthan at room temperature displays a number of racquets and other morphologies similar to the reported intermediate, metastable states by simulations. The reduced abundance of such structures following annealing supports the interpretation of their metastable nature. † Part of the special issue "International Symposium on Polyelectrolytes".

Naturwissenschaften, 2001

In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wi... more In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca 2+ with Na + , forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca 2+ , Sr 2+ and Ba 2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

Biopolymers, 2005

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force m... more Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.

Biophysical Journal, 2004

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly,... more Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length \20 mm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of \7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.

Biomacromolecules, 2004

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising ... more Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.

Biomacromolecules, 2005

The interactions between DNA and chitosans varying in fractional content of acetylated units (F A... more The interactions between DNA and chitosans varying in fractional content of acetylated units (F A ), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on F A , and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomerbased polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

Biomacromolecules, 2007

In this study the relative importance of valence and charge density of the polycation chitosan on... more In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.