Targeted liposome-loaded microbubbles for cell-specific ultrasound-triggered drug delivery.

One of the main problems in cancer treatment is disease relapse through metastatic colonization, which is caused by circulating tumor cells (CTCs). This work reports on liposome-loaded microbubbles targeted to N-cadherin, a cell-cell adhesion molecule expressed by CTCs. It is shown that such microbubbles can indeed bind to N-cadherin at the surface of HMB2 cells. Interestingly, in a mixture of cells with and without N-cadherin expression, binding of the liposome-loaded microbubbles mainly occurs to the N-cadherin-expressing cells. Importantly, applying ultrasound results in the intracellular delivery of a model drug (loaded in the liposomes) in the N-cadherin-expressing cells only. As described in this paper, such liposome-loaded microbubbles may find application as theranostics and in devices aimed for the specific killing of CTCs in blood.

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.

Efficient transfection of hepatocytes mediated by mRNA complexed to galactosylated cyclodextrins.

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.

Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles.

The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since, at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged or polyethyleneglycolated (PEGylated) ones) of the beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2- to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells postdivision but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles from being present near the chromatin at the moment of cell division.

Elucidating the mechanisms behind sonoporation with adeno-associated virus-loaded microbubbles.

Microbubbles are Food and Drug Administration (FDA) approved contrast agents for ultrasound imaging. It has been reported that applying ultrasound on drug-loaded microbubbles facilitates drug uptake by cells, due to so-named sonoporation. However, the biophysics behind sonoporation are not fully understood. It is believed that sonoporation results in a "direct" delivery of drugs in the cytoplasm of cells, though it has been suggested as well that sonoporation facilitates endocytosis which would improve the internalization of drugs by cells. To get a better understanding of sonoporation, this study reports on the ultrasound assisted delivery of adeno-associated virus (AAV) loaded on the surface of microbubbles. AAVs rely on endocytosis for efficient transduction of cells and are, consequently, an elegant tool to evaluate whether endocytosis is involved in ultrasound-induced sonoporation. Applying ultrasound on AAV-loaded microbubbles clearly improved the internalization of AAVs by cells, though transduction of the cells did not occur, indicating that by sonoporation substances become directly delivered in the cytosol of cells.

Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved.

Drug delivery with microbubbles and ultrasound is gaining more and more attention in the drug delivery field due to its noninvasiveness, local applicability, and proven safety in ultrasonic imaging techniques. In this article, we tried to improve the cytotoxicity of doxorubicin (DOX)-containing liposomes by preparing DOX-liposome-containing microbubbles for drug delivery with therapeutic ultrasound. In this way, the DOX release and uptake can be restricted to ultrasound-treated areas. Compared to DOX-liposomes, DOX-loaded microbubbles killed at least two times more melanoma cells after exposure to ultrasound. After treatment of the melanoma cells with DOX-liposome-loaded microbubbles and ultrasound, DOX was mainly present in the nuclei of the cancer cells, whereas it was mainly detected in the cytoplasm of cells treated with DOX-liposomes. Exposure of cells to DOX-liposome-loaded microbubbles and ultrasound caused an almost instantaneous cellular entry of the DOX. At least two mechanisms were identified that explain the fast uptake of DOX and the superior cell killing of DOX-liposome-loaded microbubbles and ultrasound. First, exposure of DOX-liposome-loaded microbubbles to ultrasound results in the release of free DOX that is more cytotoxic than DOX-liposomes. Second, the cellular entry of the released DOX is facilitated due to sonoporation of the cell membranes. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles could be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo.

The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.

Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

Connexin32 hemichannels contribute to the apoptotic-to-necrotic transition during Fas-mediated hepatocyte cell death.

The present study was set up to investigate the fate of connexin32 and its channels in hepatocellular apoptosis. Primary hepatocyte cultures were exposed to Fas ligand and cycloheximide, and modifications in connexin32 expression and localization, and gap junction functionality were studied. We found that gap junction functionality rapidly declined upon progression of cell death, which was associated with a decay of the gap junctional connexin32 protein pool. Simultaneously, levels of newly synthesized connexin32 protein increased and gathered in a hemichannel configuration. This became particularly evident towards the end stages of the cell death process and was not reflected at the transcriptional level. We next either silenced connexin32 expression or inhibited connexin32 hemichannel activity prior to cell death induction. Both approaches resulted in a delayed termination of the cell death response. We conclude that connexin32 hemichannels facilitate the apoptotic-to-necrotic transition, which typically occurs in the final stage of hepatocellular apoptosis.

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.

Maintaining the silence: reflections on long-term RNAi.

Since the demonstration of RNA interference (RNAi) in mammalian cells, considerable research and financial effort has gone towards implementing RNAi as a viable therapeutic platform. RNAi is, without doubt, the most promising strategy for the treatment of human genetic disorders. Because many of the targets proposed for RNAi therapy require chronic treatment, researchers agree that the emphasis must now be placed on the safe and long-term application of RNAi drugs to reap the benefits at last.

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes.

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.

Mucolytic activity of bacterial and human chitinases.

Several pulmonary pathologies, like cystic fibrosis (CF), are characterized by hypersecretion and stasis of tenacious mucus. Bacterial glycosidases are known to degrade mucins but their use as mucolytic agents is questionable. The observation that bacterial chitinases degrade mucins and the recent discovery of human chitinases, which have been proposed to be involved in the genesis of asthma, prompted us to evaluate the mucolytic properties of human derived chitinases. The effect of these human chitinases, and bacterial chitinases (positive control), on the viscoelasticity of CF sputa and on the electrophoretic mobility of human mucins was tested. Commercial bacterial chitinase drastically degraded CF sputum, while human derived chitinases did not. Accordingly, the commercial bacterial chitinase was found to degrade mucins, whereas recombinant human chitinases did not. A thorough analysis of the commercial chitinase elucidated that contaminating proteases and also nucleases assisted in the mucolytic effect. Indeed, recombinant bacterial chitinases very slightly reduced the viscoelasticity of CF sputum, but they caused a significant degradation of the CF sputum when they were combined with proteases. In conclusion, this work shows that recombinant human and recombinant bacterial chitinases have no or very low mucolytic activities, respectively. The observed mucolytic properties of commercial bacterial chitinase are due to a synergistic effect between chitinolytic and proteolytic enzymes at one hand and at the other hand also due to the presence of contaminating nucleases.

Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly(beta-amino esters).

BACKGROUND: Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(beta-amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules. METHODS AND RESULTS: We show for the first time that PbAE : siRNA complexes, containing 1,4-butanediol (PbAE1) or 1,6-hexanediol (PbAE2) diacrylate-based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics. CONCLUSIONS: From the time-dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications.

Encoding microcarriers: present and future technologies.

In answer to the ever-increasing need to carry out many assays simultaneously in drug screening and drug discovery, several microcarrier-based multiplex technologies have arisen in the past few years. The compounds to be screened are attached to the surface of microcarriers, which can be mixed together in a vessel that contains the target analyte. Each microcarrier has to be encoded to know which compound is attached to its surface. In this article, the methods that have been developed for the encoding of microcarriers are reviewed and discussed.

Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.

In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.

Vitreous: a barrier to nonviral ocular gene therapy.

PURPOSE: Intravitreal injection of therapeutic DNA, complexed to nonviral carriers such as cationic liposomes, may be promising in the treatment of many severe retinal eye diseases. However, after intravitreal injection, such DNA/cationic liposome complexes-called lipoplexes (LPXs)-which are typically hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was to elucidate whether vitreous is a barrier for the LPXs and to find strategies to overcome this barrier. METHODS: Fluorescent polystyrene nanospheres and LPXs were mixed with vitreous, and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy-based technique. The stability of LPXs and naked plasmid DNA in vitreous was studied by gel electrophoresis. RESULTS: We showed that polystyrene nanospheres, in our first experiments used as a model for the LPXs, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surfaces of the polystyrene nanospheres hydrophilic by attaching hydrophilic polyethylene glycol (PEG) chains at their surfaces circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that "pegylated" polystyrene nanospheres, as long as they are smaller than 500 nm, are indeed mobile in the vitreous. It was further demonstrated that LPXs severely aggregate in vitreous and strongly bind to biopolymers in the vitreous, which immobilizes them completely. However, as observed for the polystyrene nanospheres, coating of the LPXs with PEG averted their aggregation in the vitreous and their binding to fibrillar structures. CONCLUSIONS: Modifying the surfaces of LPXs with hydrophilic PEG chains prevents them from aggregating in vitreous. In this way, LPXs are obtained that can freely move in vitreous, an absolute criterion for reaching the retina after intravitreal injection.

Disregarded Effect of Biological Fluids in siRNA Delivery: Human Ascites Fluid Severely Restricts Cellular Uptake of Nanoparticles.

Small interfering RNA (siRNA) offers a great potential for the treatment of various diseases and disorders. Nevertheless, inefficient in vivo siRNA delivery hampers its translation into the clinic. While numerous successful in vitro siRNA delivery stories exist in reduced-protein conditions, most studies so far overlook the influence of the biological fluids present in the in vivo environment. In this study, we compared the transfection efficiency of liposomal formulations in Opti-MEM (low protein content, routinely used for in vitro screening) and human undiluted ascites fluid obtained from a peritoneal carcinomatosis patient (high protein content, representing the in vivo situation). In Opti-MEM, all formulations are biologically active. In ascites fluid, however, the biological activity of all lipoplexes is lost except for lipofectamine RNAiMAX. The drop in transfection efficiency was not correlated to the physicochemical properties of the nanoparticles, such as premature siRNA release and aggregation of the nanoparticles in the human ascites fluid. Remarkably, however, all of the formulations except for lipofectamine RNAiMAX lost their ability to be taken up by cells following incubation in ascites fluid. To take into account the possible effects of a protein corona formed around the nanoparticles, we recommend always using undiluted biological fluids for the in vitro optimization of nanosized siRNA formulations next to conventional screening in low-protein content media. This should tighten the gap between in vitro and in vivo performance of nanoparticles and ensure the optimal selection of nanoparticles for further in vivo studies.

Mobility and stability of gene complexes in biogels.

The tenacious secretions lining the conductive airways of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy to the lung. In this work, we evaluated the diffusion of nanospheres and cationic DOTAP lipoplexes through CF sputum and the influence of CF mucus components on the physicochemical properties and gene expression of cationic DOTAP lipoplexes and neutral, pegylated GL67 lipoplexes. The number of particles transported through the sputum was extremely low (<0.5%) and strongly depending on the size of the particles, with almost no transport for the largest nanospheres (560 nm). For small particles (<150 nm), the low transport was primarily due to the long distance they have to travel through the sputum, while for larger particles also sterical obstruction was responsible for the low transport. Upon exposure of the cationic DOTAP lipoplexes to albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these neutral, pegylated GL67 lipoplexes.

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.

A comparison between the use of dynamic mechanical analysis and oscillatory shear rheometry for the characterisation of hydrogels.

In this study the use of dynamic mechanical analysis (DMA) for the mechanical characterisation of pharmaceutical hydrogels was evaluated. DMA was used in two different modes, the "controlled force" and the "multi-strain" (MS). The results obtained on dextran methacrylate hydrogels of various compositions were compared to those obtained using an oscillatory shear rheometer. The best agreement was found between the MS-DMA and the rheometer results. The moduli measured in MS-DMA were extrapolated towards zero compression to obtain the modulus of the hydrogels. This procedure resulted in good agreement with the data obtained with the rheometer. Hydrogels were analysed after swelling to equilibrium with both methods, DMA and rheology. A scaling between the elastic modulus (G') and the equilibrium swollen polymer volume fraction (v(2,s)) could be found, although the best correlation between G' and v(2,s) was obtained with the rheometer.