Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models.

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.

Intravitreal Pharmacokinetics in Mice: SPECT/CT Imaging and Scaling to Rabbits and Humans.

Preclinical in vivo tests of retinal drug responses are carried out in mice and rats, often after intravitreal injections. However, quantitative pharmacokinetics in the mouse eye is poorly understood. Ocular pharmacokinetics studies are usually done in rabbits. We investigated elimination of three compounds ([99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin with molecular weights of 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively) from mouse vitreous using imaging with single photon emission computed tomography/computed tomography (SPECT/CT). Increasing molecular weight decreased elimination of the compounds from the mouse eyes. Half-lives of [99mTc]Tc-pentetate, [111In]In-pentetreotide, and [99mTc]Tc-human serum albumin in the mouse eyes were 1.8 ± 0.5 h, 4.3 ± 1.7 h, and 30.0 ± 9.0 h, respectively. These values are 3-12-fold shorter than half-lives of similar compounds in the rabbit vitreous. Dose scaling factors were calculated for mouse-to-rabbit and mouse-to-man translation. They were 27-90 and 38-126, respectively, for intravitreal injections in rabbit and man. We show ocular pharmacokinetic parameters for mice and interspecies scaling factors that may augment ocular drug discovery and development.

Elucidating the signal responses of multi-parametric surface plasmon resonance living cell sensing: a comparison between optical modeling and drug-MDCKII cell interaction measurements.

In vitro cell-based assays are widely used during the drug discovery and development process to test the biological activity of new drugs. Most of the commonly used cell-based assays, however, lack the ability to measure in real-time or under dynamic conditions (e.g. constant flow). In this study a multi-parameter surface plasmon resonance approach in combination with living cell sensing has been utilized for monitoring drug-cell interactions in real-time, under constant flow and without labels. The multi-parameter surface plasmon resonance approach, i.e. surface plasmon resonance angle versus intensity plots, provided fully specific signal patterns for various cell behaviors when stimulating cells with drugs that use para- and transcellular absorption routes. Simulated full surface plasmon resonance angular spectra of cell monolayers were compared with actual surface plasmon resonance measurements performed with MDCKII cell monolayers in order to better understand the origin of the surface plasmon resonance signal responses during drug stimulation of cells. The comparison of the simulated and measured surface plasmon resonance responses allowed to better understand and provide plausible explanations for the type of cellular changes, e.g. morphological or mass redistribution in cells, that were induced in the MDCKII cell monolayers during drug stimulation, and consequently to differentiate between the type and modes of drug actions. The multi-parameter surface plasmon resonance approach presented in this study lays the foundation for developing new types of cell-based tools for life science research, which should contribute to an improved mechanistic understanding of the type and contribution of different drug transport routes on drug absorption.

Independent versus cooperative binding in polyethylenimine-DNA and Poly(L-lysine)-DNA polyplexes.

The mechanism of polyethylenimine-DNA and poly(L-lysine)-DNA complex formation at pH 5.2 and 7.4 was studied by a time-resolved spectroscopic method. The formation of a polyplex core was observed to be complete at approximately N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. The data were analyzed further both by an independent binding model and by a cooperative model for multivalent ligand binding to multisubunit substrate. At pH 5.2, the polyplex formation was cooperative at all N/P ratios, whereas for pH 7.4 at N/P < 0.6 the polyplex formation followed independent binding changing to cooperative binding at higher N/Ps.

Exploring the efficiency of gallic acid-based dendrimers and their block copolymers with PEG as gene carriers.

The synthesis of a new family of amino-functionalized gallic acid-triethylene glycol (GATG) dendrimers and their block copolymers with polyethylene glycol (PEG) has recently being disclosed. In addition, these dendrimers have shown potential for gene delivery applications, as they efficiently complex nucleic acids and form small and homogeneous dendriplexes. On this basis, the present study aimed to explore the interaction of the engineered dendriplexes with blood components, as well as their stability, cytotoxicity and ability to enter and transfect mammalian cells. Results show that GATG dendrimers can form stable dendriplexes, protect the associated pDNA from degradation, and are biocompatible with HEK-293T cells and erythrocytes. More importantly, dendriplexes are effectively internalized by HEK-293T cells, which are successfully transfected. Besides, PEGylation has a marked influence on the properties of the resulting dendriplexes. While PEGylated GATG dendrimers have improved biocompatibility, the long PEG chains limit their uptake by HEK-293T cells, and thus, their ability to transfect them. As a consequence, the degree of PEGylation in dendriplexes containing dendrimer/block copolymer mixtures emerges as an important parameter to be modulated in order to obtain an optimized stealth formulation able to effectively induce the expression of the encoded protein.

Hyaluronic acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an application for pDNA and siRNA delivery.

PURPOSE: To design hyaluronic acid (HA) and chitosan-g-poly(ethylene glycol) (CS-g-PEG) nanoparticles intended for a broad range of gene delivery applications. METHODS: Nanoparticles formulated at different HA/CS-g-PEG mass ratios were developed to associate either pDNA or siRNA. The physico-chemical characteristics, morphology, association efficiency and nuclease protection ability of the nanocarriers were compared for these two molecules. Their biological performance, including transfection effciency, nanoparticle cellular uptake and citotoxicity, was assesed. RESULTS: The resulting nanoparticles showed an adequate size (between 130 and 180 nm), and their surface charge could be modulated according to the nanoparticle composition (from +30 mV to -20 mV). All prototypes exhibited a greater association efficiency and nuclease protection for pDNA than for siRNA. However, cell culture experiments evidenced that HA/CS-g-PEG nanoparticles were effective carriers for the delivery of both, siRNA and pDNA, eliciting a biological response with minimal cytotoxicity. Moreover, experiments performed in the HEK-EGFP-Snail1 cell line showed the potential of the HA/CS-g-PEG nanoparticles to silence the expression of the Snail1 transcription factor, an important mediator in tumor progression. CONCLUSIONS: HA/CS-g-PEG nanoparticles can be easily modulated for the delivery of different types of gene molecules, offering great potential for gene therapy applications, as evidenced by their biological performance.

Chitosan-based nanostructures: a delivery platform for ocular therapeutics.

Nanoscience and nanotechnology has caused important breakthroughs in different therapeutic areas. In particular, the application of nanotechnology in ophthalmology has led to the development of novel strategies for the treatment of ocular disorders. Indeed, the association of an active molecule to a nanocarrier allows the molecule to intimately interact with specific ocular structures, to overcome ocular barriers and to prolong its residence in the target tissue. Over the last decade, our group has designed and developed a delivery platform based on the polysaccharide chitosan, which suits the requirements of the topical ocular route. These nanosystems have been specifically adapted for the delivery of hydrophilic and lipophilic drugs and also polynucleotides onto the eye surface. The results collected up until now suggest the potential of this delivery platform and the subsequent need of a full preclinical evaluation in order to satisfy the specific regulatory demands of this mode of administration.

Isoproterenol enhancement of I(Ks) current in amiodarone-induced long QT syndrome.

The response over time of the QT interval and the Transmural Dispersion of Repolarization (TDR) to Isoproterenol infusion (ISO) in an 85 year-old-man with severe and syntomatic bradycardia, in the setting of an Amiodarone-induced Long QT Syndrome (LQTS) is the subject of this communication. ISO shortened the QT and decreased the TDR. We propose that ISO increased the generation of signals from the beta-Adrenergic Receptor (beta-AR) restoring the components of the hKCNQ1 macromolecular complex, thereby enhancing the I(Ks) function; shortening of the QT interval, reduction of the TDR and normalization of the T wave morphology was then possible. In Amiodarone-induced LQTS, the increase in I(Ks) activity promoted by beta-AR stimulation is prominent; at the same time, during I(Kr) block, I(Ks) activation limits excessive QT prolongation. Clinically, severe and syntomatic bradycardia was the main concern: ISO activation of the beta-AR proved useful both to increase heart rate and to reduce QT prolongation. A slow heart rate, associated to a prolonged QT interval and to a big TDR, are not sufficient to develop Torsades de Pointes in Amiodarone-induced LQTS.

Adrenergic-mediated exposure of hyperkalemia in the electrocardiogram.

We document full exposure of the electrocardiographic hyperkalemia signs brought about by exogenous epinephrine in a patient on long-term beta-blockade treatment in whom spironolactone-induced hyperkalemia developed. We propose that the stimulated adrenergic receptor promoted re-expression of two main potassium channels (I(Kr), I(Ks)) involved in ventricular repolarization, modifying the ECG waveform. The effects of hyperkalemia could be aggravated in the presence of beta-adrenergic blockade.

Localized and dynamic repolarization alternans in Ajmaline accentuated Brugada syndrome.

We performed incremental atrial pacing immediately after Ajmaline infusion in an asyntomatic female patient whose basal ECG was suggestive of Brugada Syndrome. Ajmaline accentuated the BS ECG pattern. Incremental Atrial pacing induced localized and dynamic repolarization alternans and unequal diastolic intervals (TQ intervals). No ventricular rhythms were elicited (other than short-coupled, monomorphic, low-density ventricular beats.