Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness.

BACKGROUND: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread. METHODS: Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed. RESULTS: FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×10(4) exosomes per cell for FL3, T24 and HCV-29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively). T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. CONCLUSIONS: Malignant (metastatic and non-metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non-malignant cell line exosomes, which may explain the observed increased endothelial monolayer disruption and transendothelial transport of these vesicles.

Polycation-based nanoparticles for RNAi-mediated cancer treatment.

Cancer disorders exhibit an increasing high global incidence, in part, to an aging population with a high socio-economic burden. The cellular transition from normal to malignant state is linked to deregulated gene expression. The discovery of microRNA-mediated cellular regulation by the RNA interference (RNAi) pathway and the possibility to engage this pathway with exogenous triggers such as small interfering RNA (siRNA) could offer a new paradigm in anti-cancer intervention with RNAi-based therapeutics. The potential to silence the expression of any cancer-relevant protein with high selectivity promotes RNAi therapeutics as a more effective and safer treatment to traditional approaches. This combined with microRNA-based tumour profiling could pave the way for personalised approaches based on the genetic characteristics of the individual. Clinical translation of this technology, however, depends on the development of systems for effective delivery of the molecular medicine to the target site. Polycation-based nanoparticles (termed polyplexes) constitute an attractive platform for RNAi therapeutic delivery due to flexibility and versatility in design to overcome extracellular and intracellular barriers. In this review we focus on pre-clinical and clinical studies using polycation-based nanocarriers for RNAi mediated anti-cancer intervention after intratumoural or intravenous administration. Potential RNAi targets are highlighted and special attention is given to the enhanced permeability and retention (EPR) effect commonly cited at the predominant mechanism of delivery after systemic administration. The cyclodextrin polymer-based system now in clinical trials offers optimism that polyplexes may potentially be used for RNAi-mediated cancer intervention in the clinic.

The application of RNAi-based treatments for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic, relapsing, idiopathic inflammation of the gastrointestinal tract with no permanent cure. Present immunosuppressive and anti-inflammatory therapies are often ineffective and associated with severe side effects. An RNA interference (RNAi)-based approach in which small interfering RNA (siRNA) mediates specific downregulation of key molecular targets of the IBD inflammatory process may offer a precise, potent and safer alternative to conventional treatments. This review describes the aetiology of Crohn's disease and ulcerative colitis and the cellular and molecular basis for current treatments to highlight target candidates for an RNAi-based approach. Promising preclinical studies support an RNAi application; however, optimal siRNA designs that maximise potency and development of enabling technologies for site- and cellular-specific delivery are prerequisites for clinical translation.

Clinical translation of RNAi-based treatments for respiratory diseases.

The ability to harness the RNA interference (RNAi) mechanism as a potential potent therapeutic has attracted great interest from academia and industry. Numerous preclinical and recent clinical trials have demonstrated the effectiveness of RNAi triggers such as synthetic small interfering RNA (siRNA). Chemical modification and delivery technologies can be utilized to avoid immune stimulation and improve the bioactivity and pharmacokinetics. Local application to the respiratory epithelia allows direct access to the site of respiratory pathogens that include influenza and respiratory syncytial virus (RSV). This review outlines the essential steps required for the clinical translation of RNAi-based respiratory therapies including disease and RNA target selection, siRNA design, respiratory barriers, and delivery solutions. Attention is given to antiviral therapies and preclinical evaluation with focus on the current status of anti-RSV clinical trials.

Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions.

Small interfering RNA (siRNA) that silence genes by the process of RNA interference offers a new therapeutic modality for disease treatment. Polycation-based nanoparticles termed polyplexes have been developed to maximise extracellular and intracellular siRNA delivery, a key requirement for enabling the clinical translation of RNAi-based drugs. Medical applications are dependent on safety; therefore, detailed investigation into potential toxicity to the cell or organism is required. This review addresses potential adverse effects arising from cellular and tissue interactions, immune stimulation and altered gene expression that can be associated with the assembled polyplex or the polycation and siRNA component parts. A greater understanding of the cellular mechanisms involved allows design-based solutions for rationale development of safe, effective and clinically relevant polyplex-based RNAi drugs.

Chitosan/siRNA nanoparticles encapsulated in PLGA nanofibers for siRNA delivery.

Composite nanofibers of biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) encapsulating chitosan/siRNA nanoparticles (NPs) were prepared by electrospinning. Acidic/alkaline hydrolysis and a bulk/surface degradation mechanism were investigated in order to achieve an optimized release profile for prolonged and efficient gene silencing. Thermo-controlled AFM in situ imaging not only revealed the integrity of the encapsulated chitosan/siRNA polyplex but also shed light on the decreasing T(g) of PLGA on the fiber surfaces during release. A triphasic release profile based on bulk erosion was obtained at pH 7.4, while a triphasic release profile involving both surface erosion and bulk erosion was obtained at pH 5.5. A short alkaline pretreatment provided a homogeneous hydrolysis and consequently a nearly zero-order release profile. The interesting release profile was further investigated for siRNA transfection, where the encapsulated chitosan/siRNA NPs exhibited up to 50% EGFP gene silencing activity after 48 h post-transfection on H1299 cells.

RNAi using a chitosan/siRNA nanoparticle system: in vitro and in vivo applications.

Delivery is a key issue in development of clinically relevant RNAi therapeutics. Polymeric nanoparticles formed by self-assembly of polycations with siRNA can be used for extracellular delivery, cellular uptake and intracellular trafficking as a strategy to improve the therapeutic potential of siRNA. This chapter describes a chitosan-based nanoparticle system for in vitro and in vivo transfection of siRNA into cells. The method exploits the mucoadhesive and mucopermeable properties of this cationic polysaccharide to deliver siRNA across mucosal epithelium and provides a platform for targeting human diseases with RNAi therapeutics.

Effect of physicochemical properties on intranasal nanoparticle transit into murine olfactory epithelium.

Small molecular weight drugs, peptides, and nanoparticles have previously been shown to localize in the central nervous system after intraneural administration. A basic understanding of direct nose-to-brain drug delivery, particularly for nanoparticles with different physicochemical characteristics, remains unclear. In this study, fluorescence microscopy and stereology were used to track intranasally administered chitosan-coated polystyrene (C-PS) or polysorbate-coated polystyrene (P80-PS) nanoparticles (100 nm or 200 nm in diameter) in olfactory and respiratory nasal epithelia and olfactory bulbs in mice. Chitosan coating caused particles to adhere to the extracellular mucus which could provide useful modality for paracellular drug transport. Nanoparticle transport was exclusively transcellular. None of the nanoparticle formulations showed preference for uptake into olfactory axons over other nasal epithelial cells. Both 100 nm PS and 100 nm P80-PS were observed in olfactory epithelial cells but were absent from the olfactory bulbs; therefore, it is speculated that an optimal nanoparticle diameter for axonal transport is <100 nm in mice.

Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration.

The use of systemic siRNA therapeutics for RNA interference-mediated silencing of disease genes is limited by serum instability and inadequate biodistribution. We have previously reported on the EGFP gene silencing effect of chitosan/siRNA nanoparticles in the bronchoepithelium of mice lungs following intranasal delivery and improved serum stability and reduced off-targeting effects in vitro by incorporation of locked nucleic acid (LNA). In this study, we examine the pulmonary gene silencing effect of siLNAs targeting enhanced-green-fluorescent-protein (EGFP) in lung bronchoepithelium upon intravenous delivery of naked siLNAs and upon intranasal delivery of either naked siLNA or chitosan/siLNA nanoparticles. We show that naked siLNA administered intravenously efficiently reduces the EGFP protein expression. A similar effect is obtained with intranasal delivery of chitosan nanoparticles containing siLNA whereas intranasally instilled naked siLNA did not cause a knockdown.

The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice.

Instability and inadequate biodistribution of double-stranded RNA are major drawbacks to the clinical use of RNA interference. This work compares chemical modification and nanoparticle formulation as strategies to improve the systemic delivery of small interfering RNA (siRNA). Variable levels of chemical modified siRNA, either naked or within nanoparticle, were intravenously injected into mice to study temporal stability and biodistribution detected by direct radioactive labeling or by northern blotting. Naked siRNA showed rapid renal clearance, with circulatory half-life of <5 minutes that could be extended to >30 minutes by cholesterol conjugation. The integrity of the chemically stabilized siRNA was maintained in blood for at least 30 minutes, whereas, unmodified siRNA duplex was degraded within 1 minute. Intact chemically modified siRNA could also be detected in all analyzed organs at 30 minutes but disappeared at 24 hours, except for heavy locked nucleic acid (LNA)-modified and cholesterol-conjugated siRNA in the lungs. Chitosan, liposomal, or JetPEI formulation greatly improved the stability and biodistribution of siRNA. Interestingly, high siRNA accumulation of the chitosan/siRNA formulation within the kidney was observed 24 hours postadministration. This comparative study highlights improvements to siRNA stability and pharmacokinetics, key determinants for development of clinically relevant RNAi therapeutics.

Delivery of RNA interference therapeutics using polycation-based nanoparticles.

RNAi-based therapies are dependent on extracellular and intracellular delivery of RNA molecules for enabling target interaction. Polycation-based nanoparticles (or polyplexes) formed by self-assembly with RNA can be used to modulate pharmacokinetics and intracellular trafficking to improve the therapeutic efficacy of RNAi-based therapeutics. This review describes the application of polyplexes for extracellular and intracellular delivery of synthetic RNA molecules. Focus is given to routes of administration and silencing effects in animal disease models. The inclusion of functional components into the nanoparticle for controlling cellular trafficking and RNA release is discussed. This work highlights the versatile nature of polycation-based nanoparticles to fulfil the delivery requirements for RNA molecules with flexibility in design to evolve alongside an expanding repertoire of RNAi-based drugs.

The development of polyplex-based DNA vaccines.

DNA immunisation provides new possibilities for the development of effective vaccines for the prophylaxis and treatment of several diseases and infections. Application of such vaccines for mucosal (secretory or local) vaccination provides a powerful means to gain protection against local infection that enters and colonises the mucosa whilst inducing concomitant systemic immunity. This review examines the current and potential applications for polyplex-based mucosal vaccination strategies, notably those aimed at gaining expression of transgenes within dendritic cells, in order to gain both T-helper and cytotoxic T cell (CTL) responses. Emphasis is given to the development of polyplex-based oral vaccines in conjunction with microparticulate systems.