Nina Kristine Reitan

Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking

Chitosan is an attractive gene delivery vehicle, but the criteria and strategies for the design of efficient chitosan gene delivery systems remain unclear. The purpose of this work was to investigate how the strength of the charge-based interaction between chitosan and DNA determines the gene expression levels and to design chitosan vectors with an optimized balance between polyplex stability and polyplex unpacking. Using 21 formulations based on low molecular weight chitosans with constant charge density and a number-average degree of polymerization (DPn) in the range of 21-88 (M(w) 4.7-33kDa), we studied the relationship between the chain length and the formulation properties, cellular uptake of polyplexes and gene transfer efficacy. We were able to identify a narrow interval of DPn31-42 that mediated the maximum level of transgene expression. An increase in chain length and/or the amino-phosphate (A/P) ratio reduced and delayed transgene expression. Compared to DPn31, transfection with the same amount of DPn72 or DPn88 resulted in 10-fold-lower expression levels. The gene transfer pattern correlated with the ability of heparin to release DNA from the polyplexes. As a tool to facilitate the unpacking of the polyplexes, we substituted the chitosans with uncharged oligosaccharides that reduced the interaction with DNA. The substitution of chitosans that originally yielded too stable polyplexes, such as DPn72 and DPn88 resulted in a 5-10-fold enhancement of the expression levels. However, the substitution of chitosans shorter than DP28 completely abolished transfection. Tailoring of the chain length and the substitution of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA and to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.

Intravital microscopy in window chambers: a unique tool to study tumor angiogenesis and delivery of nanoparticles

Solid tumor growth is heavily dependant on angiogenesis. Tumor angiogenesis is the result of a complex interplay between tumor cells, endothelial cells, and other stromal cells. It has been found to be under strict control of a plethora of molecular factors that function as angiogenic up- and down-regulators; nevertheless, the identification of molecular and cellular players and their roles in angiogenesis is still ongoing. The microvasculature resulting from tumor angiogenesis lacks hierarchy and has a high permeability for macromolecules and nanoparticles, which offers significant potential for nanoparticulate tumor imaging and drug delivery platforms. However, improvements in the delivery to poorly vascularized regions and the distribution throughout the tumor interstitium are critical for nanoparticles to become more effective in the battle against cancer. A tool that has proven extremely valuable in both unraveling angiogenic pathways and characterizing in vivo nanoparticle behavior in solid tumors is intravital microscopy of tumors grown in window chamber preparations. In this review this technique is explained, several exciting examples illustrating its value in elucidating tumor angiogenesis are presented and the study of nanoparticle behavior in solid tumors using this approach is described. We conclude with a discussion of the potential value of intravital microscopy in window chambers in multimodality studies of tumor pathophysiology and nanoparticle dynamics.

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.

Evidence for age-dependent in vivo conformational rearrangement within Aβ amyloid deposits.

Deposition of aggregated Aβ peptide in the brain is one of the major hallmarks of Alzheimers disease. Using a combination of two structurally different, but related, hypersensitive fluorescent amyloid markers, LCOs, reporting on separate ultrastructural elements, we show that conformational rearrangement occurs within Aβ plaques of transgenic mouse models as the animals age. This important mechanistic insight should aid the design and evaluation of experiments currently using plaque load as readout.

Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging

Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were used as molecular tracers for CLSM and DCE-MRI, respectively. A significant correlation was found between permeability indicators. The extravasation rate K(i) as measured by CLSM correlated positively with DCE-MRI parameters, such as the volume transfer constant K(trans) and the initial slope of the contrast agent concentration-time curve. This demonstrates that these two techniques give complementary information. Extravasation was further related to microvascular structure and was found to correlate with the fractal dimension and vascular density. The structural parameter values that were obtained from CLSM images were higher for abnormal tumor vasculature than for normal vessels.

Characterizing DNA condensation by structurally different chitosans of variable gene transfer efficacy.

Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.

Macromolecular diffusion in the extracellular matrix measured by fluorescence correlation spectroscopy

Diffusion of therapeutic macromolecules through the extracellular matrix of tumor tissue is a crucial step in drug delivery. We use fluorescence correlation spectroscopy (FCS) to measure diffusion of IgG (150 kDa) and dextrans (155 kDa and 2 MDa) in solution, 5% gelatin hydrogel, and multicellular spheroids. Gel and spheroids are used as model systems for the extracellular matrix. The diffusion depends on the complexity of the environment, as well as on the size and structural shape of the diffusing molecules. The results based on one-photon FCS are in good agreement with diffusion coefficients obtained with two-photon fluorescence recovery after photobleaching (FRAP) using the same microscope (Zeiss LSM510 META/Confocor2). However, FCS reveals anomalous or multicomponent diffusion in gel and spheroids, which are not resolvable with FRAP. This study demonstrates that one-photon FCS can be used to study the extracellular transport of macromolecules in tumor tissue, and that FCS provides additional information about diffusion properties compared to FRAP.

Hyperspectral analysis using the correlation between image and reference

Abstract. We present the use of correlation analysis on spectral data in order to quantify the amount of a given spectrum present with respect to a reference spectrum. The method is shown to be useful in analyzing hyperspectral fluorescence images. It is unhindered by the linear relationship assumed in linear spectral unmixing, and in addition, it is shown to be robust with respect to noise.

Spectral correlation analysis of Amyloid β plaque inhomogeneity from double staining experiments

Abstract. A spectral correlation algorithm for the analysis of hyperspectral fluorescence images is proposed by Ellingsen et al. [J. Biomed. Opt. 18, 020501 (2013)]. Here, it is applied to the analysis of double-stained Aβ amyloid plaques being related to the Alzheimer’s disease (AD). Sections of APP/PS1 AD mice model brains are double stained with luminescent-conjugated oligothiophenes, known to bind to amyloid protein deposits. Hyperspectral fluorescence images of the brain sections are recorded and by applying the correlation algorithm the spectral inhomogeneity of the double-stained samples is mapped in terms of radial distribution and spectral content. To further investigate the progression of Aβ amyloid plaque formation, 19 AD mice of different ages up to 23 months are characterized, enabling a statistical analysis of the plaque heterogeneity. In accordance with recent findings by Nyström et al. [ACS Chem. Biol. 8, 1128–1133 (2013)], the spectral distribution within Aβ plaques is found to vary with age throughout the lifespan of the mouse. With the new correlation algorithm, it is possible to quantify the spectral abundance of the two stains depending on the relative distance from the plaque center and mouse age. Thus, we demonstrate the use of the correlation analysis approach in double-staining experiments and how it is possible to relate these to structural/spectral changes in biological samples.

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