Astrid Bjørkøy

Cellular uptake of DNA-chitosan nanoparticles: the role of clathrin- and caveolae-mediated pathways.

The success of gene therapy depends on efficient delivery of DNA and requires a vector. A promising non-viral vector is chitosan. We tailored chitosan to optimize it for transfection by synthesizing self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which show superior transfection efficacy compared with linear chitosan (LCO). The aim of the work was to compare the cellular uptake and endocytic pathways of polyplexes formed by LCO and SBTCO. Both polyplexes were taken up by the majority of the cells, but the uptake of LCO was lower than SBTCO polyplexes. LCO polyplexes were internalized through both clathrin-dependent and clathrin-independent pathways, whereas SBTCO polyplexes were primarily taken up by clathrin-independent endocytosis. The different level of cellular uptake and the distinct endocytic pathways, may explain the difference in transfection efficacy. This was supported by the observation that photochemical internalization increased the transfection by LCO polyplexes considerably, whereas no effect on transfection was found for SBTCO polyplexes.

Electrooptic analysis of macromolecule dipole moments using asymmetric reversing electric pulses

The use of symmetric reversing electric field pulses in electrooptic studies of rigid macromolecules in order to determine the ratio between the permanent and the induced dipole moments is well established. Application of this method to studies of small macromolecules requires a field reversal time of only a few nanoseconds. No high current pulse generator capable of producing symmetric kV pulses with such a short reversal time is available for studies of small macromolecules in physiological salt solutions, but it has long been known how to make such reversing pulses that are asymmetric. In order to take advantage of the opportunity offered by the latter fact, we here present a theoretical analysis in the thermal domain of the electrooptic properties of solutions containing rigid macromolecules with axial symmetry when exposed to asymmetric reversing electric field pulses. The analytical expressions needed for quantitative determination of the ratio between the permanent and the induced electric dipole moments of rigid macromolecules using electrooptic data obtained employing reversing electric pulses with given asymmetry are presented. The feasibility of this new approach is demonstrated by including experimental electric birefringence data for a 12 kDa protein (segment 14 of alpha-spectrin from Drosophila brains) in near physiological salt solutions obtained using a coaxial cable pulser producing 2 microseconds long pulses with a reversal time of about 15 ns.

Electric birefringence of recombinant spectrin segments 14, 14–15, 14–16, and 14–17 from Drosophila α-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.

Ultrasound Improves the Delivery and Therapeutic Effect of Nanoparticle-Stabilized Microbubbles in Breast Cancer Xenografts

Compared with conventional chemotherapy, encapsulation of drugs in nanoparticles can improve efficacy and reduce toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous because of various biological barriers and uneven tumor perfusion. We investigated a unique multifunctional drug delivery system consisting of microbubbles stabilized by polymeric nanoparticles (NPMBs), enabling ultrasound-mediated drug delivery. The aim was to examine mechanisms of ultrasound-mediated delivery and to determine if increased tumor uptake had a therapeutic benefit. Cellular uptake and toxicity, circulation and biodistribution were characterized. After intravenous injection of NPMBs into mice, tumors were treated with ultrasound of various pressures and pulse lengths, and distribution of nanoparticles was imaged on tumor sections. No effects of low pressures were observed, whereas complete bubble destruction at higher pressures improved tumor uptake 2.3 times, without tissue damage. An enhanced therapeutic effect was illustrated in a promising proof-of-concept study, in which all tumors exhibited regression into complete remission.

Heterogeneity in GFP expression in isogenic populations of P. putida KT2440 investigated using flow cytometry and bacterial microarrays

Individual bacteria, even bacteria belonging to an isogenic population under uniform environmental conditions, display phenotypic diversity and variability in gene expression. The increased focus that is lately given to this topic within microbiology and quantitative biology has motivated a quest for new experimental approaches providing insight into phenotypic traits at the single-cell level. In the present paper we investigate the applicability of bacterial microarrays for studies of bacterial populations including their inherent heterogeneity. The results obtained from the microscopy of bacterial microarrays are compared to the results obtained using flow cytometry. Two different positive-regulated promoter systems were utilized in isogenic cultures of Pseudomonas putida, both systems leading to green fluorescent protein production upon induction. Using both experimental approaches, a shift to higher fluorescence intensities with increasing time and increasing inducer concentration was observed, as well as a broadening of the distributions. Additionally, the micrographs of the bacterial microarrays revealed heritable inter-cell variation in green fluorescent protein levels. These observations indicate that for the bacteria studied here the cell to cell heterogeneity in green fluorescent protein expression, as also detected in the flow cytometer, are due to relatively static inter-cell differences.

Transient electric birefringence of human erythroid spectrin dimers and tetramers at ionic strengths of 4 mM and 53 mM.

Abstract In conventional electrooptic studies the sample ionic strength must for technical reasons be kept below about 3 mm, which is only 2% of the ionic strength at physiological conditions. In particular for flexible polyelectrolytic macromolecules it can in general not be ruled out that both the conformational average and dynamics at ionic strength 3 mm and below may differ significantly from what it is at physiological conditions. Here we report on the first electrooptic study of human erythroid spectrin dimers and tetramers at ionic strengths higher than 3 mm. All measurements in this study were carried out at both ionic strength 4 mm (2.5 mm HEPES + 1 mm NaCl) and 53 mm (2.5 mm HEPES + 50 mm NaCl). Spectrin tetramers were studied only at 4°C whereas the dimers were studied at both 4 °C and 37°C. At 4°C there is a striking quantitative similarity between the transient electric birefringence (TEB) of spectrin dimers and tetramers. Also, the TEB of spectrin dimers at 37°C was very similar to the results at 4°C. The contour length and the molecular weight of spectrin dimers and tetramers are known. The dominating TEB relaxation time is in all cases only a fraction of what is predicted theoretically if the spectrin dimers and tetramers are assumed to be stiff and extended molecules. In sum, the new TEB data constitute strong electrooptic evidence confirming that spectrin dimers and tetramers have a highly flexible structure, and demonstrate for the first time that a major part of the intrachain dynamics of the spectrin is quite insensitive to an increase of the ionic strength from 4 mm to 53 mm. Use of the reversing electric field pulse technique for all conditions studied yields TEB data suggesting that the orientation of both spectrin dimers and tetramers in an electric field is dominated by a permanent rather than an induced electric dipole moment.

Recovering fluorophore concentration profiles from confocal images near lateral refractive index step changes

Abstract. Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration.

Abstract 5404: DNA-chitosan nanoparticles in gene delivery: Endocytotic pathways and intracellular trafficking

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The success of gene therapy depends on efficient delivery of DNA and efficient transcription of the transgene. This involves several steps, including penetration through the extracellular matrix to target cells, intracellular uptake, and trafficking to the nucleus. A DNA vector is necessary, packing DNA into smaller polyplexes and protecting the DNA from degradation. A promising non-viral vector is chitosan, a cationic polysaccharide derived from chitin. The interactions with DNA and ability to transfect cells depend strongly on intrinsic chitosan properties. We are tailoring the chitosan to optimize transfection and have synthesized self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which seem to compact DNA less tightly and have shown superior transfection efficiency compared to linear chitosan (LCO) of equivalent molecular weight (1-3). The aim of the present work was to compare the endocytotic pathways and intracellular trafficking of polyplexes formed by SBTCO and LCO, to see if such differences could explain the difference in transfection efficacy. Material and Methods: Uptake of the two polyplexes in HeLa cells was studied using flow cytometry. Clathrin- and caveolin-dependent endocytosis was compared by inhibiting the two pathways using chlorpromazine, genistein and dynasore. Intracellular trafficking was studied using confocal laser scanning microscopy. Quantitative colocalization of polyplexes with early endosomes and intracellular DNA-chitosan colocalization were performed. The impact of photochemical internalization on transfection was studied by flow cytometric analysis of GFP expression (4). Results: All three inhibitors reduced the uptake of LCO polyplexes and genistein and dynasore were somewhat more efficient than chlorpromazine. For SBTCO polyplexes chlorpromazine showed little inhibition, whereas genistein and dynasore efficiently inhibited endocytosis. Consistent with this, only a fraction of the polyplexes was found in early endosomes. 60-80% of the intracellular DNA was binding to chitosan the first 4 hrs. Polyplexes of LCO showing low transfection was increased significantly by photochemical internalization. Conclusion: LCO polyplexes are taken up by cells both by clathrin and caveolin mediated pathways, whereas SBTCO polyplexes are primarily taken up by caveolin mediated endocytosis. The difference in endocytotic pathway results in different intracellular routes and this may explain the difference in transfection efficacy. 1. Reitan et al. Biomacromolecules 10, 2009 2. Strand et al. Biomaterials 31, 2010 3. Strand et al. Biomacromolecules 9, 2008 4. Berg et al Curr. Pharma Biotech 8, 2007 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5404. doi:10.1158/1538-7445.AM2011-5404

Deconvolution can be used in electrooptic studies to correct for non-ideal electric excitation pulses only when the electric dipole moment of the studied molecules is predominantly induced

The electric field pulses used for most measurements of transient electrooptic properties such as birefringence and dichroism, are rectangular and assumed to be ideal, but in practice do all such pulses have non-zero rise and fall times. Claims have been made that this non-ideality may be taken into account by employing standard deconvolution techniques. We find that this approach yields exact results in the zero electric field limit when the electric dipole moment of the studied macromolecules is predominantly induced. However, for finite electric field strengths and/or macromolecules with partly or fully permanent electric dipole moments, we find that the deconvolution method yields erroneous estimates of the electrooptic relaxation times. When the decay time of the electric pulse and the electrooptic decay time are equal, and the system is operated in the Kerr domain, this systematic error for macromolecules with purely permanent electric dipole moment equals 37%. In a research field where the uncertainty of the reported relaxation times normally is assumed to be only a few percent this is an error that may seriously mislead unsuspecting users. We find that this systematic error can readily be avoided by employing standard numerical integration of a set of coupled first-order differential equations instead of the standard deconvolution techniques.