Broes Naeye

Chitosan nanoparticles for siRNA delivery: Optimizing formulation to increase stability and efficiency

This study aims at developing chitosan-based nanoparticles suitable for an intravenous administration of small interfering RNA (siRNA) able to achieve (i) high gene silencing without cytotoxicity and (ii) stability in biological media including blood. Therefore, the influence of chitosan/tripolyphosphate ratio, chitosan physicochemical properties, PEGylation of chitosan as well as the addition of an endosomal disrupting agent and a negatively charged polymer was assessed. The gene silencing activity and cytotoxicity were evaluated on B16 melanoma cells expressing luciferase. We monitored the integrity and the size behavior of siRNA nanoparticles in human plasma using fluorescence fluctuation spectroscopy and single particle tracking respectively. The presence of PEGylated chitosan and poly(ethylene imine) was essential for high levels of gene silencing in vitro. Chitosan nanoparticles immediately released siRNA in plasma while the inclusion of hyaluronic acid and high amount of poly(ethylene glycol) in the formulation improved the stability of the particles. The developed formulations of PEGylated chitosan-based nanoparticles that achieve high gene silencing in vitro, low cytotoxicity and high stability in plasma could be promising for intravenous delivery of siRNA.

PEGylation of biodegradable dextran nanogels for siRNA delivery.

Delivering intact small interfering RNA (siRNA) into the cytoplasm of targeted cells in vivo is considered a major obstacle in the development of clinically applicable RNA interference-based therapies. Although dextran hydroxyethyl methacrylate (dex-HEMA) nanogels have been reported to be suitable carriers for siRNA delivery in vitro, and are ideally sized (approximately 180 nm) for intravenous delivery to tumors, they likely possess insufficient blood circulation times to enable an adequate extravasation and accumulation in the tumor tissue. PEGylation of these nanogels should not only improve their circulation time but also minimize their aggregation upon intravenous injection. For this reason, a new type of nanogels and three different methods of PEGylating dextran nanogels were evaluated. Covalent PEGylation of the siRNA-loaded nanogels using N-hydroxysuccinimidyl polyethylene glycol (NHS-PEG) was shown to be superior to the addition of both polyethylene glycol (PEG) and PEG grafted poly-l-glutamic acid (PGA-PEG). Flow cytometry and confocal microscopy revealed that PEGylated nanogels are still taken up efficiently by HuH-7 human hepatoma cells and A431 human epithelial carcinoma cells and that the process is cell type dependent. Moreover, PEGylated nanogels loaded with siRNA cause significant EGFP knockdown in a human hepatoma cell line (HuH-7_EGFP) and are non-toxic for these cells.

In vivo disassembly of IV administered siRNA matrix nanoparticles at the renal filtration barrier

Intravenous administration of siRNA nanocarriers may provide unique therapeutic opportunities for tissue-specific gene silencing. Although often engineered to overcome the numerous barriers that exist in the systemic circulation, many nanocarriers fail in extending the circulation time of the siRNA. A more detailed assessment of the different clearance mechanisms that are in play after intravenous injection could therefore be of value to improve siRNA nanocarrier design. In this report, the biodistribution in mice of siRNA loaded dextran nanogels was investigated in detail. Both single photon emission computed tomography (SPECT) imaging and fluorescence single particle tracking (fSPT) indicate that the particles are rapidly cleared from the circulation. PEGylation of the nanogels was not able to increase the half-life in the bloodstream. Carrier disassembly in the systemic circulation and phagocytic clearance are known to facilitate the elimination of siRNA nanoparticles. Additionally, it is demonstrated for dextran nanogels that also the kidneys play an important role in their elimination from the bloodstream. SPECT imaging revealed an accumulation of the siRNA loaded dextran nanogels in the kidneys shortly after intravenous injection and a significantly delayed transition of siRNA from kidney to bladder, as opposed to the injection of free siRNA. These data indicate that components of the glomerular filtration barrier may contribute to the dissociation of siRNA from its carrier, as was recently suggested for cationic cyclodextrin siRNA polyplexes. This clearance mechanism should therefore be taken into account when designing siRNA nanocarriers for intravenous administration.

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization.

Small interfering RNAs (siRNAs) show potential for the treatment of a wide variety of pathologies with a known genetic origin through sequence-specific gene silencing. However, siRNAs do not have favorable drug-like properties and need to be packaged into nanoscopic carriers that are designed to guide the siRNA to the cytoplasm of the target cell. In this report biodegradable cationic dextran nanogels are used to deliver siRNA across the intracellular barriers. For the majority of non-viral siRNA carriers studied so far, endosomal confinement is identified as the most prominent hurdle, limiting the full gene silencing potential. Thus, there is a major interest in methods that are able to enhance endosomal escape of siRNA to improve its intracellular bioavailability. Photochemical internalization (PCI) is a method that employs amphiphilic photosensitizers to destabilize endosomal vesicles. We show that applying PCI at a later time-point post-transfection significantly prolonged the knockdown of the target protein only in case the siRNA was carried by nanogels and not when a liposomal carrier was used. Combining siRNA nanogels and PCI creates new possibilities to prolong gene silencing by using intracellular vesicles as depots for siRNA and applying PCI at the time when maintaining the RNAi effect becomes critical.

Hemocompatibility of siRNA loaded dextran nanogels

Although the behavior of nanoscopic delivery systems in blood is an important parameter when contemplating their intravenous injection, this aspect is often poorly investigated when advancing from in vitro to in vivo experiments. In this paper, the behavior of siRNA loaded dextran nanogels in human plasma and blood is examined using fluorescence fluctuation spectroscopy, platelet aggregometry, flow cytometry and single particle tracking. Our results show that, in contrast to their negatively charged counterparts, positively charged siRNA loaded dextran nanogels cause platelet aggregation and show increased binding to human blood cells. Although PEGylating the nanogels did not have a significant effect on their interaction with blood cells, single particle tracking revealed that it is necessary to prevent their aggregation in human plasma. We therefore conclude that PEGylated negatively charged dextran nanogels are the most suited for further in vivo studies as they do not aggregate in human plasma and exhibit minimal interactions with blood cells.

Advanced fluorescence microscopy methods illuminate the transfection pathway of nucleic acid nanoparticles

A great deal of attention in biopharmacy and pharmaceutical technology is going to the development of nanoscopic particles to efficiently deliver nucleic acids to target cells. Despite the great potential of nucleic acids for treatment of various diseases, progress in the field is fairly slow. One of the causes is that development of suitable nanoscopic delivery vehicles is hampered by insufficient knowledge of their physicochemical and biophysical properties during the various phases of the transfection process. To address this issue, in the past decade we have developed and applied advanced fluorescence microscopy techniques that can provide a better insight in the transport and stability of nanoparticles in various biological media. This mini-review discusses the basic principles of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and gives an overview of studies in which we have employed these techniques to characterize the transport and stability of nucleic acid containing nanoparticles in extracellular media and in living cells.

Comparison of Polymeric siRNA Nanocarriers in a Murine LPS-Activated Macrophage Cell Line: Gene Silencing, Toxicity and Off-Target Gene Expression

ABSTRACTPurposeTumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro.MethodsTNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining.ResultsPAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration.ConclusionsDextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.

Interactions of siRNA loaded dextran nanogel with blood cells

Adsorption and internalization of siRNA loaded dextran nanogels by blood cells were determined using flow cytometry. Positively and negatively charged nanogels with various PEGylation degrees were compared in order to find a formulation showing minimal interactions with blood.

Biodegradable dextran nanogels as functional carriers for the intracellular delivery of small interfering RNA

RNA interference (RNAi) certainly is a hot topic among the scientific community, judging by the broad financial investments and the tremendous research output to date.[1] This statement is further illustrated through a PubMed search on the term ‘RNA interference’ (performed on June 15th 2009), which yielded ~16.500 entries. RNAi is a naturally conserved gene silencing mechanism functioning in eukaryotic cells and is activated by small interfering RNAs (siRNAs) that trigger the degradation of mRNA in a sequence‐specific manner. Besides its use as a laboratory tool in functional genomics and drug target discovery, the therapeutic potential of RNAi by blocking the production of disease‐causing proteins has also long been recognized. In conclusion, this thesis comprises a novel FRET based approach for the intracellular assessment of small interfering RNA (siRNA) integrity, which could aid in clarifying the correlation between intracellular siRNA fate and the eventual RNAi outcome. Besides siRNA stability, this thesis also describes the design of cationic and biodegradable nanogels for the time‐controlled delivery of siRNA. We provide evidence that these nanogels can effectively deliver active siRNA across the cellular barrier, leading to substantial and durable gene silencing. Endosomal escape is identified as the predominant barrier confining the full RNAi potential, opening up new opportunities to further improve this delivery concept. It is conceivable that, although RNAi can generally be applied to interfere with the expression of virtually any gene, several distinct in vivo delivery agents will be needed depending on the disease target and the chosen route of administration. Our nanogels may well claim a future position in this ensemble of siRNA delivery systems.

Matrix Systems for siRNA Delivery

Over the last decade, considerable effort has been put in the implementation of RNA interference (RNAi) as a treatment for various disorders. As RNAi occurs in the cytoplasm of cells, it is imperative that RNAi mediators such as small interfering RNA (siRNA) cross several extracellular and intracellular barriers to reach this site of action. Among the extensive range of proposed delivery systems for siRNA, matrix systems possess interesting properties to promote the delivery of siRNA to a target tissue. In this review, a number of recently developed matrix and hybrid systems for siRNA delivery are discussed.