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.

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.

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.

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.

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.

Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice.

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5h after microbubble and ultrasound treatment. An increase in tissue temperature to 41°C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.

Transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy.

AIM: The aim of this study was to evaluate the effect of the surface functionalization of model nanoparticles on their mobility in bacterial biofilms and cystic fibrosis sputum. MATERIALS & METHODS: With single-particle tracking microscopy, the mobility of 0.1- and 0.2-µm fluorescent polyethylene glycol (PEG) modified, carboxylate- and N,N-dimethylethylenediamine-modified polystyrene nanospheres were evaluated in fresh cystic fibrosis sputum, as well as Burkholderia multivorans and Pseudomonas aeruginosa biofilms. RESULTS: PEGylation increased the mobility of the particles in sputum and biofilms, while the charged nanospheres were strongly immobilized. However, the transport of the PEGylated nanoparticles was lower in sputum compared with biofilms. Furthermore, the particle transport showed heterogeneity in samples originating from different patients. CONCLUSION: This study's data suggest that for future nanocarrier design it will be essential to combine PEGylation with a targeting moiety to ensure sufficient mobility in mucus and a better accumulation of the nanoparticles in the biofilm.

The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts.

Delivery of reprogramming factor-encoding mRNAs by means of lipofection in somatic cells is a desirable method for deriving integration-free iPSCs. However, the lack of reproducibility implies there are major hurdles to overcome before this protocol becomes universally accepted. This study demonstrates the functionality of our in-house synthesized mRNAs expressing the reprogramming factors (OCT4, SOX2, KLF4, c-MYC) within the nucleus of human fibroblasts. However, upon repeated transfections, the mRNAs induced severe loss of cell viability as demonstrated by MTT cytotoxicity assays. Microarray-derived transcriptome data revealed that the poor cell survival was mainly due to the innate immune response triggered by the exogenous mRNAs. We validated the influence of mRNA transfection on key immune response-associated transcript levels, including IFNB1, RIG-I, PKR, IL12A, IRF7 and CCL5, by quantitative real-time PCR and directly compared these with the levels induced by other methods previously published to mediate reprogramming in somatic cells. Finally, we evaluated chemical compounds (B18R, chloroquine, TSA, Pepinh-TRIF, Pepinh-MYD), known for their ability to suppress cellular innate immune responses. However, none of these had the desired effect. The data presented here should provide the basis for further investigations into other immunosuppressing strategies that might facilitate efficient mRNA-mediated cellular reprogramming in human cells.

Liposome based systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design.

siRNA therapeutics are currently regarded as promising candidates to make a leap forward in the search for treatments of various hard to cure diseases. In order to exploit the full potential of siRNA based therapeutics, development of delivery systems that can efficiently guide the siRNA molecules to their target without major side effects will be the key to success. Lipid based delivery systems, originating from earlier research in the fields of gene delivery, are the most studied candidates for siRNA delivery. Here we discuss the requirements that need to be met by these siRNA delivery systems to ensure adequate stability after systemic application and subsequent deposition in the target tissue. The encountered hurdles in the blood stream and the solutions proposed in literature are discussed.

Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery.

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues.

mRNA transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers.

Messenger RNA encoding luciferase (mLUC) was complexed to the cationic lipids Lipofectamine or DOTAP/DOPE, and to the cationic polymer linear poly(ethyleneimine) (linPEI). The complexes were incubated with HeLa cells and luciferase expression was assessed. The type of non-viral carrier used determined the extent and duration of protein expression. Maximal duration of mRNA expression was about 9 days for Lipofectamine complexes, i.e. not very much shorter than with pDNA polyplexes. Interestingly, luciferase activity was already detected 30 min after adding the mRNA complexes to the cells, independent on the type of carrier. We also assessed the proportion of cells that become transfected by means of transfection with an mRNA encoding GFP. For both cationic lipids transfection with mRNA yielded a substantially larger fraction of transfected cells (more than 80%) than transfection with pDNA (40%). In addition we tested the carriers for their ability to mediate delivery of mRNA encoding CXCR4 into mesenchymal stem cells. The fraction of CXCR4-positive cells obtained with the mRNA-cationic lipid complexes was around 80%, as compared to 40% for the linPEI polyplexes. Our results demonstrate that the advantage of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period.

Monitoring the disassembly of siRNA polyplexes in serum is crucial for predicting their biological efficacy.

To exploit the full therapeutic potential of short interfering RNA (siRNA), efficient delivery vehicles are needed as siRNA fails to enter cells spontaneously. Such carriers should also protect siRNA against degradation while it is on its way to the cytosol of the target cells. Cationic polymers are widely investigated as siRNA carriers. Cationic polymers and siRNA self-assemble into siRNA polyplexes which have been shown to silence genes in cell cultures. While siRNA polyplexes will become exposed to full blood after intravenous injection, in vitro gene knockdown is mostly evaluated in serum free media or media containing only a few percent of serum. Little knowledge is currently available on the stability of siRNA polyplexes in blood, while there are no methods available which allow a quantitative measurement of the disassembly of nucleic acid containing nanoparticles in such complex biological media. This paper shows that fluorescence fluctuation spectroscopy allows us to quantitatively monitor the disassembly of siRNA containing nanoparticles in full serum. It further shows that the gene silencing efficacy of siRNA polyplexes in serum containing media can be very well explained by their disassembling behavior in these media. Our findings are important for the further development of siRNA polyplexes and also other nanoparticulate nucleic acid delivery systems.

Elucidating the encapsulation of short interfering RNA in PEGylated cationic liposomes.

Short interfering RNA (siRNA) holds great potential for the treatment of hard-to-cure diseases. One of the major challenges to translate siRNA into drugs is its efficient delivery to its site-of-action, namely the cytoplasm of the target cells. Cationic liposomes have been shown to do the trick, but their short circulation lifetime and potential aggregation in blood limit their applicability for intravenous administration. These hurdles might be overcome by attaching poly(ethylene glycol) (PEG) at the surface of the cationic liposomes through the use of PEGylated lipids. However, this paper reveals that the classical mixing of siRNA with preformed PEGylated cationic liposomes, as frequently done to load PEGylated liposomes with siRNA, prevents an efficient encapsulation of the siRNA in the liposomes. We show that only a minor fraction of the siRNA becomes encapsulated in the core of the PEGylated liposomes, whereas a major part of the siRNA becomes bound at the liposome's outer surface. In serum, the surface-bound siRNA is immediately released and becomes degraded by serum nucleases. By contrast, hydrating a lipid film (containing PEGylated and cationic lipids) directly with a concentrated solution of siRNA (so-called HYDRA protocol), instead of mixing the siRNA with preformed PEGylated liposomes, encapsulates almost 50% of the siRNA in the core of the PEGylated liposomes, which is the maximal encapsulation efficiency for this type of complexes. We show that the siRNA encapsulated in the core of the thus obtained "HYDRA siPLexes" remains fully encapsulated upon dispersing the PEGylated liposomes in human serum.

Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles.

Short interfering RNA (siRNA) attracts much attention for the treatment of various diseases. However, its delivery, especially via systemic routes, remains a challenge. Indeed, naked siRNAs are rapidly degraded, while complexed siRNAs massively aggregate in the blood or are captured by macrophages. Although this can be circumvented by PEGylation, we found that PEGylation had a strong negative effect on the gene silencing efficiency of siRNA-liposome complexes (siPlexes). Recently, ultrasound combined with microbubbles has been used to deliver naked siRNA but the gene silencing efficiency is rather low and very high amounts of siRNA are required. To overcome the negative effects of PEGylation and to enhance the efficiency of ultrasound assisted siRNA delivery, we coupled PEGylated siPlexes (PEG-siPlexes) to microbubbles. Ultrasound radiation of these microbubbles resulted in massive release of unaltered PEG-siPlexes. Interestingly, PEG-siPlexes loaded on microbubbles were able to enter cells after exposure to ultrasound, in contrast to free PEG-siPlexes, which were not able to enter cells rapidly. Furthermore, these PEG-siPlex loaded microbubbles induced, in the presence of ultrasound, much higher gene silencing than free PEG-siPlexes. Additionally, the PEG-siPlex loaded microbubbles only silenced the expression of genes in the presence of ultrasound, which allows space and time controlled gene silencing.

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.