Noncoding DNA in lipofection of HeLa cells-a few insights.

In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.

Stimuli-responsive electrospun fibers and their applications.

Stimuli-responsive electrospun nanofibers are gaining considerable attention as highly versatile tools which offer great potential in the biomedical field. In this critical review, an overview is given on recent advances made in the development and application of stimuli-responsive fibers. The specific features of these electrospun fibers are highlighted and discussed in view of the properties required for the diverse applications. Furthermore, several novel biomedical applications are discussed and the respective advantages and shortcomings inherent to stimuli-responsive electrospun fibers are addressed (136 references).

Macrophage reprogramming into a pro-healing phenotype by siRNA delivered with LBL assembled nanocomplexes for wound healing applications.

Excessive inflammatory responses in wounds are characterized by the presence of high levels of pro-inflammatory M1 macrophages rather than pro-healing M2 macrophages, which leads to delayed wound healing. Macrophage reprogramming from the M1 to M2 phenotype through knockdown of interferon regulatory factor 5 (irf5) has emerged as a possible therapeutic strategy. While downregulation of irf5 could be achieved by siRNA, it very much depends on successful intracellular delivery by suitable siRNA carriers. Here, we report on highly stable selenium-based layer-by-layer (LBL) nanocomplexes (NCs) for siRNA delivery with polyethyleneimine (PEI-LBL-NCs) as the final polymer layer. PEI-LBL-NCs showed good protection of siRNA with only 40% siRNA release in a buffer of pH = 8.5 after 72 h or in simulated wound fluid after 4 h. PEI-LBL-NCs also proved to be able to transfect RAW 264.7 cells with irf5-siRNA, resulting in successful reprogramming to the M2 phenotype as evidenced by a 3.4 and 2.6 times decrease in NOS-2 and TNF-α mRNA expression levels, respectively. Moreover, irf5-siRNA transfected cells exhibited a 2.5 times increase of the healing mediator Arg-1 and a 64% increase in expression of the M2 cell surface marker CD206+. Incubation of fibroblast cells with conditioned medium isolated from irf5-siRNA transfected RAW 264.7 cells resulted in accelerated wound healing in an in vitro scratch assay. These results show that irf5-siRNA loaded PEI-LBL-NCs are a promising therapeutic approach to tune macrophage polarization for improved wound healing.

Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

On the biological activity of anti-ICAM-1 oligonucleotides complexed to non-viral carriers.

An important challenge in antisense technology remains the adequate delivery of the oligonucleotides (ON) to individual cells. Understanding the subcellular distribution of ONs and their carrier is essential to explain the (lack of) biological activity. The ability of several cationic carriers to efficiently deliver anti-ICAM-1 oligonucleotides to their site of action was studied using a cell-based assay. In this assay we evaluated the ability of the ONs to downregulate the expression of the ICAM-1-protein in A549 cells. To understand why some carrier/ONs combinations showed biological activity while others failed, flow cytometry and confocal laser scanning microscopy (CLSM) measurements were used to study cellular uptake and intracellular distribution of the (fluorescently labeled) ONs. We showed that free ONs (both PS-ONs and PO-ONs) and ONs complexed to pEGpEI failed to decrease the ICAM-1 protein level. This was due to the inability of the (free or complexed) ONs to enter the cell, as shown by flow cytometry and CLSM. Flow cytometry and CLSM showed cellular uptake when PO-ONs and PS-ONs were complexed to graft-pDMAEMA and Lipofectin. However, while the uptake and intracellular localization seemed similar for ONs complexed to, respectively, graft-pDMAEMA and Lipofectin, the biological activity of the ONs was clearly dependent on their carrier: both PO-ONs and PS-ONs complexed to graft-pDMAEMA reduced the ICAM-1 expression; however, when complexed to Lipofectin only PS-ONs showed biological activity. Also, PS-ONs complexed to graft-pDMAEMA were more active than PO-ONs complexed to graft-pDMAEMA which could not be explained by the results from CLSM and flow cytometry. While the ICAM-1 assay proves whether a certain pharmaceutical carrier successfully delivers ONs or not, it does not answer the important question why one carrier is successful while another one fails. Also, our study shows that flow cytometry and CLSM, although useful techniques, failed to clearly explain the difference in transfection behavior between graft-pDMAEMA and Lipofectin. As ONs become susceptible to degradation by cytosolic DNase as soon as they are released from their carrier, one could argue that a better understanding of the time and (intracellular) place at which the dissociation of the complexes occurs could be crucial to fully explain our observations.

Magnetic layer-by-layer coated particles for efficient MRI of dendritic cells and mesenchymal stem cells.

AIM: Cell detection by MRI requires high doses of contrast agent for generating image contrast. Therefore, there is a constant need to develop improved systems that further increase sensitivity, and which could be used in clinical settings. In this study, we devised layer-by-layer particles and tested their potential for cell labeling. MATERIALS & METHODS: The advantages of layer-by-layer technology were exploited to obtain magnetic particles of controllable size, surface chemistry and magnetic payload. RESULTS: Flexibility in size and surface charge enabled efficient intracellular delivery of magnetic particles in mesenchymal stem cells and dendritic cells. Owing to the high magnetic payload of the particles, high MRI contrast was generated, even for very low cell numbers. Subcutaneous injection of the particles and subsequent uptake by dendritic cells enabled clear visualization of dendritic cells homing towards nearby lymph nodes in mice. CONCLUSION: The magnetic particles offer several possibilities as efficient cellular MRI contrast agents for direct in vitro or in vivo cell labeling.

Elucidating the pre- and post-nuclear intracellular processing of 1,4-dihydropyridine based gene delivery carriers.

The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre- and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre- and the post-nuclear intracellular processes is equally essential for successful transgene expression.

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.

Can ultrasound solve the transport barrier of the neural retina?

PURPOSE: Intravitreal injection of nonviral gene complexes may be promising in the treatment of retinal diseases. This study investigates the permeation of lipoplexes and polystyrene nanospheres through the neural retina and their uptake by the retinal pigment epithelium (RPE) either with or without ultrasound application. MATERIALS AND METHODS: Anterior parts and vitreous of bovine eyes were removed. The neural retina was left intact or peeled away from the RPE. (Non)pegylated lipoplexes and pegylated nanospheres were applied. After 2 h incubation, the RPE cells were detached and analyzed for particle uptake by flow cytometry and confocal microscopy. RESULTS: The neural retina is a significant transport barrier for pegylated nanospheres and (non)pegylated lipoplexes. Applying ultrasound improved the permeation of the nanoparticles up to 130 nm. CONCLUSIONS: Delivery of liposomal DNA complexes to the RPE cells is strongly limited by the neural retina. Ultrasound energy may be a useful tool to improve the neural retina permeability, given the nucleic acid carriers are small enough. Our results underline the importance to design and develop very small carriers for the delivery of nucleic acids to the neural retina and the RPE after intravitreal injection.

New strategies for nucleic acid delivery to conquer cellular and nuclear membranes.

After administration to the body, nucleic acid containing nanoparticles (NANs) need to cross several extra- and intracellular barriers to reach the cytoplasm or nucleus of the target cells. In the last decade several groups tried to overcome these barriers by arming non-viral delivery systems with targeting moieties, polyethylene glycol chains, fusogenic peptides and so forth. However, the drawback of this upgrading strategy is that each of the encountered barriers requires a new functionality, leading to very complex multi-component NANs. Moreover, there are currently no components available that can efficiently transport genes or NANs inside the nucleus of non-dividing cells. In this article a new, ultrasound based delivery system that possesses the capacity to simultaneously overcome several key barriers in non-viral nucleic acid delivery is presented. Additionally, a small amphiphilic compound that induces nuclear uptake of plasmid DNA and enhances non-viral gene transfer is presented.

A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum.

SiRNA based therapeutics are currently under investigation for treatment of cancer and viral infections. Upon intravenous administration, the nanoscopic delivery systems which carry the siRNA need to be stable in serum, an aspect which is often overlooked in numerous publications on siRNA delivery systems. Techniques currently available for studying the dissociation of siRNA-liposome complexes are time consuming and incompatible with full serum. We therefore developed a fluorescence fluctuation spectroscopy (FFS) based method which allows to monitor the integrity of siRNA-carrier complexes. The method can very rapidly provide quantitative information on the complex integrity in biological media, like full human serum, and at very low siRNA concentrations (approximately 20 nM siRNA). Information on the integrity of intravenously injected siRNA nanoparticles in serum is crucial. Consequently, the FFS method reported in this work may find broad applicability in the field of siRNA-carrier design.

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples.

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles.

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.

In situ analysis of single-stranded and duplex siRNA integrity in living cells.

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.