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Acoustic radiation force (ARF) might improve the distribution of nanoparticles (NPs) in tumors. To study this, tumors growing subcutaneously in mice were exposed to focused ultrasound (FUS) either 15 min or 4 h after the injection of NPs, to investigate the effect of ARF on the transport of NPs across the vessel wall and through the extracellular matrix. Quantitative analysis of confocal microscopy images from frozen tumor sections was performed to estimate the displacement of NPs from blood vessels. Using the same experimental exposure parameters, ARF was simulated and compared to the experimental data. Enhanced interstitial transport of NPs in tumor tissues were observed when FUS (10 MHz, acoustic power 234 W/cm2, 3.3 % duty cycle) was given either 15 min or 4 h after NP administration. According to acoustic simulations, the FUS generated an ARF per unit volume of 2.0 x 106 N/m3. The displacement of NPs was larger when FUS was applied 4h after NP injection compared to after 15 min. This study shows that ARF might contribute to a modest improved distribution of NPs into the tumor interstitium. © 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumour aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behaviour remains poorly understood.Unexpectedly, we find here that fibrillar collagen-related gene expression is associated with favourable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo-like microarchitectures, we uncover that the activation of DDR1 has pro-apoptotic and integrin β1 pro-survival function, specifically in 3D rhabdomyosarcoma cell cultures. We demonstrate that rhabdomyosarcoma cell-intrinsic or extrinsic matrix remodelling promotes cell survival. Mechanistically, we find that the 3D-specific collagen-induced apoptosis results from a dual DDR1-independent and a synergistic DDR1-dependent TRPV4-mediated response to mechanical confinement. Altogether, our results indicate that dense microfibrillar collagen-rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signalling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasise to low fibrillar collagen microenvironments such as the lung.

In the present study, we expand on the understanding of hydrogels with embedded deoxyribonucleic acid (DNA) cross-links, from the overall swelling to characterization of processes that precede the swelling. The hydrogels respond to target DNA strands because of a toehold-mediated strand displacement reaction in which the target strand binds to and opens the dsDNA cross-link. The spatiotemporal evolution of the diffusing target ssDNA was determined using confocal laser scanning microscopy (CLSM). The concentration profiles revealed diverse partitioning of the target DNA inside the hydrogel as compared with the immersing solution: excluding a nonbinding DNA, while accumulating a binding target. The data show that a longer toehold results in faster cross-link opening but reduced diffusion of the target, thus resulting in only a moderate increase in the overall swelling rate. The parameters obtained by fitting the data using a reaction-diffusion model were discussed in view of the molecular parameters of the target ssDNA and hydrogels.

Therapeutic agents can benefit from encapsulation in nanoparticles, due to improved pharmacokinetics and biodistribution, protection from degradation, increased cellular uptake and sustained release. Microbubbles in combination with ultrasound have been shown to improve the delivery of nanoparticles and drugs to tumors and across the blood-brain barrier. Here, we evaluate two different microbubbles for enhancing the delivery of polymeric nanoparticles to cells in vitro: a commercially available lipid microbubble (Sonazoid) and a microbubble with a shell composed of protein and nanoparticles. Various ultrasound parameters are applied and confocal microscopy is employed to image cellular uptake. Ultrasound enhanced cellular uptake depending on the pressure and duty cycle. The responsible mechanisms are probably sonoporation and sonoprinting, followed by uptake, and to a smaller degree enhanced endocytosis. The use of commercial Sonazoid microbubbles leads to significantly lower uptake than when using nanoparticle-loaded microbubbles, suggesting that proximity between cells, nanoparticles and microbubbles is important, and that mainly nanoparticles in the shell are taken up, rather than free nanoparticles in solution.

Penetration of nanoscale therapeutic agents into the extracellular matrix (ECM) of a tumor is a limiting factor for the sufficient delivery of drugs in tumors. Ultrasound (US) in combination with microbubbles causing cavitation is reported to improve delivery of nanoparticles (NPs) and drugs to tumors. Acoustic radiation force (ARF) could also enhance the penetration of NPs in tumor ECM. In this work, a collagen gel was used as a model for tumor ECM to study the effects of ARF on the penetration of NPs as well as the deformation of collagen gels applying different US parameters (varying pressure and duty cycle). The collagen gel was characterized, and the diffusion of water and NPs was measured. The penetration of NPs into the gel was measured by confocal laser scanning microscopy and numerical simulations were performed to determine the ARF and to estimate the penetration distance and extent of deformation. ARF had no effect on the penetration of NPs into the collagen gels for the US parameters and gel used, whereas a substantial deformation was observed. The width of the deformation on the collagen gel surface corresponded to the US beam. Comparing ARF caused by attenuation within the gel and Langevin pressure caused by reflection at the gel-water surface, ARF was the prevalent mechanism for the gel deformation. The experimental and theoretical results were consistent both with respect to the NP penetration and the gel deformation. © 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

In the present study, we expand on the understanding of hydrogels with embedded deoxyribonucleic acid (DNA) cross-links, from the overall swelling to characterization of processes that precede the swelling. The hydrogels respond to target DNA strands because of a toehold-mediated strand displacement reaction in which the target strand binds to and opens the dsDNA cross-link. The spatiotemporal evolution of the diffusing target ssDNA was determined using confocal laser scanning microscopy (CLSM). The concentration profiles revealed diverse partitioning of the target DNA inside the hydrogel as compared with the immersing solution: excluding a nonbinding DNA, while accumulating a binding target. The data show that a longer toehold results in faster cross-link opening but reduced diffusion of the target, thus resulting in only a moderate increase in the overall swelling rate. The parameters obtained by fitting the data using a reaction-diffusion model were discussed in view of the molecular parameters of the target ssDNA and hydrogels.

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.

Ultrasound (US)and systemic administration of microbubbles (MBs)have been shown to improve the delivery of drugs and nanoparticles (NPs)to tumor tissue. A better understanding of the mechanisms is crucial for effective delivery of NPs and drugs. To elucidate the kinetics of extravasation events, and relate it to the vessel diameter and flow, we have performed real-time intravital multiphoton microscopy during US sonication of tumors growing in dorsal window chambers. We studied the effects of four different mechanical index (MI)levels (0.2 to 0.8)while injecting SonoVue or in-house made MBs with NPs in the shell (NPMB). We found that high MI of 0.8 induced a violent extravasation of both NPs and dextrans. Using lower MIs (0.2-0.6), less extravasation was detected and it occurred in vessels with larger diameters compared to sonication at MI 0.8. The rate of extravasation of both NPs and dextrans, and the displacement of NPs and dextrans from the vessel into the tumor tissue, correlated with MI. The observed extravasation events happened within milliseconds to minutes after the US exposure started. Moreover, we have observed a change of blood flow rate and direction with all the MIs tested © 2018 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.

The emission of an electron from a metal nanostructure under illumination and its subsequent acceleration in a plasmonic field forms a platform to extend these phenomena to deposited nanoparticles, which can be studied by state-of-the-art confocal microscopy combined with femtosecond optical excitation. The emitted and accelerated electrons leave defect tracks in the immersion oil, which can be revealed by thermoluminescence. These photographic tracks are read out with the confocal microscope and have a maximum length of about 80 μm, which corresponds to a kinetic energy of about 100 keV. This energy is consistent with the energy provided by the intense laser pulse combined with plasmonic local field enhancement. The results are discussed within the context of the rescattering model by which electrons acquire more energy. The visualization of electron tracks originating from plasmonic field enhancement around a gold nanoparticle opens a new way to study with confocal microscopy both the plasmonic properties of metal nano objects as well as high energy electron interaction with matter.

Guluronate oligomers have been shown to modify the gastrointestinal mucus barrier and improve nanoparticle penetration. Here we investigate the effect of guluronate oligomers on nanoparticle mobility in saliva, a central extracellular barrier in the oral cavity. Guluronate oligomers reduced nanoparticle aggregation in saliva and improved nanoparticle mobility over and above that which would be expected to result from the decrease in size. As such guluronate oligomers may be of interest for nanomedicine delivery in the oral cavity.

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. © 2017 The Authors. Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A major goal of cancer nanotherapy is to use nanoparticles as carriers for targeted delivery of anti-tumour agents. The drug–carrier association after intravenous administration is essential for efficient drug delivery to the tumour. However, a large number of currently available nanocarriers are self-assembled nanoparticles whose drug-loading stability is critically affected by the in vivo environment. Here we used in vivo FRET imaging to systematically investigate how drug–carrier compatibility affects drug release in a tumour mouse model. We found the drug's hydrophobicity and miscibility with the nanoparticles are two independent key parameters that determine its accumulation in the tumour. Next, we applied these findings to improve chemotherapeutic delivery by augmenting the parent drug's compatibility; as a result, we achieved better antitumour efficacy. Our results help elucidate nanomedicines' in vivo fate and provide guidelines for efficient drug delivery., The in vivo anticancer efficacy of nanoparticle-mediated drug delivery depends on the association between the drug and its carrier. Here, the authors use FRET to show that the drug hydrophobicity, and miscibility with the carrier, influence nanoparticle accumulation in murine tumour models.

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.

The composition and structure of membrane biofouling in water treatment can be studied using specific stains (fluorochromes) and confocal laser scanning microscopy (CLSM). An image analysis program (CMem) has been developed to quantify parameters describing the biofilm structure on curved membrane surfaces, such as hollow fiber membranes. Two CLSM images stacks were acquired: one of the stained biofilm and one of the membrane underneath it. The curved reference surface in a three-dimensional stack of CLSM images was located, based upon detection of membrane autofluorescence/reflection signals. The stained components identified in the biofilm stack were then related to the membrane surface. Structural parameters such as thickness, volume of biomass, porosity and roughness of biofilms on curved membrane surfaces were computed.

Photodynamic therapy (PDT) is a noninvasive treatment for solid malignant and flat tumors. Light activated sensitizers catalyze photochemical reactions that produce reactive oxygen species which can cause cancer cell death. In this work we investigated the photophysical properties of the photosensitizer ruthenium(II) porphyrin (RuP), along with its PDT efficiency onto rat bladder cancer cells (AY27). Optical spectroscopy verified that RuP is capable to activate singlet oxygen via blue and red absorption bands and inter system crossing (ISC) to the triplet state. In vitro experiments on AY27 indicated increased photo-toxicity of RuP (20μM, 18h incubation) after cell illumination (at 435nm), as a function of blue light exposure. Cell survival fraction was significantly reduced to 14% after illumination of 20μM RuP with 15.6J/cm(2), whereas the "dark toxicity" of 20μM RuP was 17%. Structural and morphological changes of cells were observed, due to RuP accumulation, as well as light-dependent cell death was recorded by confocal microscopy. Flow cytometry verified that PDT-RuP (50μM) triggered significant photo-induced cellular destruction with a photoxicity of (93%±0.9%). Interestingly, the present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis. RuP "dark toxicity" compared to the conventional chemotherapeutic drug cisplatin was higher, both evaluated by the MTT assay (24h). In conclusion, the present investigation shows that RuP with or without photoactivation induces cell death of bladder cancer cells.

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 α-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°C, but scaled to what is expected at 20°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°C. When the temperature is increased to 37°C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20°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.

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 α -spectrin from Drosophila brains) in near physiological salt solutions obtained using a coaxial cable pulser producing 2 μ s long pulses with a reversal time of about 15 ns.

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. © 2016 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.21.12.126014] CC-BY license

Multichannel microscopy is frequently used to study intermolecular interactions and spatial relationships between biomolecules and organelles or vesicles in cells. Based on multichannel images, quantitative colocalization analysis can provide valuable information about cellular internalization, vesicular transport, and the intracellular kinetics and location of biomolecules. However, such analyses should be performed carefully, because quantitative colocalization parameters have different interpretations and can be highly affected by image quality. We use quantitative three-dimensional colocalization analysis of deconvolved and chromatic-registered confocal images to study the dissociation of double-labeled pDNA-chitosan polyplexes in HeLa cells and their colocalization with early endosomes. Two chitosans that form polyplexes with highly different transfection efficacies are compared. Pearson's correlation coefficient, Manders' colocalization coefficients, and the intensity correlation quotient are estimated to determine the intracellular localization of polyplexes, free pDNA, and free chitosans. Differences are observed in the amount of uptake, and in the intracellular pathways and rates of dissociation for the two chitosans. The results support previous findings that polyplexes formed by self-branched, glycosylated chitosan oligomers are more favorable for cellular uptake and intracellular trafficking to the nucleus compared with polyplexes formed by linear chitosans. Open Acees article. Published by Society of Photo-optical Instrumentation Engineers (SPIE) 2012.

A critical step in the delivery of nanomedicines to tumour cells is transporting these particles through the extracellular matrix. Tumour-specific anticancer agents, such as encapsulated drugs, proteins, and genes, show low uptake in tumour tissue. It is not clear whether the collagen network or the glycosaminoglycan gel plays the most important role in limiting the interstitial transport of macromolecules. Therefore, we measured the effect of the collagen- and hyaluronan-degrading enzymes, collagenase and hyaluronidase, on interstitial diffusion.Human osteosarcomas were grown as multicellular spheroids and xenografts in dorsal skinfold window chambers. Diffusion of fluorescein isothiocyanate (FITC)-dextran molecules was measured by fluorescence recovery after photobleaching based on two-photon scanning laser excitation.Collagenase, hyaluronidase, and relaxin increased the diffusion coefficient of the 2-MDa FITC-dextrans in the spheroids, but 150-kDa FITC-dextran diffusion was not affected by the enzymatic treatment. In tumour tissue in vivo, collagenase and hyaluronidase increased the diffusion of the 150-kDa FITC-dextrans. In xenografts, anomalous diffusion occurred, whereas only free diffusion was seen in spheroids.The results indicate that the collagen network has a greater impact on the interstitial diffusion of macromolecules in tumour tissue than the hyaluronan gel.

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.

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 degrees C whereas the dimers were studied at both 4 degrees C and 37 degrees C. At 4 degrees C there is a striking quantitative similarity between the transient electric bire-fringence (TEB) of spectrin dimers and tetramers. Also, the TEB of spectrin dimers at 37 degrees C was very similar to the results at 4 degrees 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.

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