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

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 proteinThe authors gratefully acknowledge support from the Spanish Ministry of Science and Innovation (SAF2004‐09230‐004‐01, CTQ2006‐12222/BQU, and CTQ2009‐10963) and the Xunta de Galicia (10CSA209021PR). M. Raviña and A. Sousa‐Herves also acknowledge fellowships from the Spanish Government (FPI and FPU, respectively)S

Simulated full SPR spectra for optical changes within different regions of a cell monolayer.

<p>A) Schematic representation of the sample layer used in simulating full SPR angular spectra of cell monolayers. The cell monolayer was theoretically split into three sections in order to clarify the effect of changing different optical properties in it: 1) a thin section in the magnitude of evanescent field close to the sensor surface (<i>ñ_ef</i>, <i>d_ef</i>  = 500 nm), 2) a thick section consisting of the rest of the cell (<i>ñ_cell</i>, <i>d_cell</i>  = 3000 nm), and 3) an infinite bulk medium layer (<i>ñ_bulk</i>, <i>d_bulk</i>  =  ∞). Simulated full SPR angular spectra when changing; B) the real (<i>n_ef</i>) and C) the imaginary (<i>k_ef</i>) parts of the refractive index for a cell monolayer within the evanescent field, D) the real part of the refractive index (<i>n_cell</i>) for a cell monolayer not within the evanescent field and E) the imaginary part of the refractive index (<i>k_cell</i>) for a cell monolayer not within the evanescent field. Insets in B-E are more detailed views of the TIR regions. The following parameters were used for simulations: B) <i>d_cell</i>  = 3000 nm, <i>k_cell</i>  = 0.002, <i>n_cell</i> varied from 1.340–1.345, <i>d_ef</i>  = 500 nm, <i>n_ef</i>  = 1.34 and <i>k_ef</i>  = 0.002, C) <i>d_cell</i>  = 3000 nm with <i>n_cell</i>  = 1.340, <i>k_cell</i> varied from 0–0.005, <i>d_ef</i>  = 500 nm, <i>n_ef</i>  = 1.34 and <i>k_ef</i>  = 0.002, D) <i>d_cell</i>  = 3000 nm, <i>k_cell</i>  = 0.002, <i>n_cell</i>  = 1.34, <i>d_ef</i>  = 500 nm, <i>k_ef</i>  = 0.002 and <i>n_ef</i> varied from 1.340–1.345, and E) <i>d_cell</i>  = 3000 nm, <i>k_cell</i>  = 0.002, <i>n_cell</i>  = 1.34, <i>d_ef</i>  = 500 nm, <i>n_ef</i>  = 1.34 and <i>k_ef</i> varied from 0–0.005.</p

Kretschmann configuration and key parameters obtained from the full SPR angular spectra.

<p>A) A simplified chart of the Kretschmann configuration enabling plasmon excitations and SPR measurements. The intensity of the reflected light from a monochromatic light source is measured as a function of incident light angle (θ). The light passes from a high refractive index medium (glass, ε₀) to a low refractive index medium (air or liquid, ε₁+ε_bulk). In between, the light is reflected from an interface containing a metal with a high density of free electrons and an optimal thickness for plasmon excitation (gold 50 nm, ε₂) to a photodetector. The surface plasmons on the metal surface are excited at a certain incident light angle (θ) and the evanescent field created by the plasmon extends to the adjacent low refractive index medium (ε₁) where samples are introduced to the system. B) A schematic full SPR angular spectrum showing the positions of the TIR region, the main SPR peak angular position and the main SPR peak minimum intensity.</p

SPR signal responses during drug stimulation of MDCKII cells.

<p>A) Measured full SPR angular spectra of a pure gold coated SPR sensor slide (grey line) and MDCKII cell monolayer immobilized on the SPR sensor slide (black line). B) Measured changes in the angular position of the SPR peak minimum as a function of time when a MDCKII cell monolayer was stimulated with propranolol (blue line) and D-mannitol (red line). C) Focused part of full SPR angular spectra showing the main SPR peak curves measured before (black line), during (red line) and after (blue line) stimulating a MDCKII cell monolayer with 25 µM propranolol. D) Measured changes in the SPR peak minimum intensity as a function of time when a MDCKII cell monolayer was stimulated with propranolol (blue line) and D-mannitol (red line). In figure C) and D) the downward arrows represent the time of sample injections, and upwards arrows represent the injection of buffer without sample.</p