Quantification of propidium iodide delivery with millisecond electric pulses: A model study

A model study of propidium iodide delivery with millisecond electric pulses is presented; this work is a companion of the experimental efforts by Sadik et al. [1]. Both membrane permeabilization and delivery are examined with respect to six extra-cellular conductivities. The transmembrane potential of the permeabilized regions exhibits a consistent value, which corresponds to a bifurcation point in the pore-radius-potential relation. Both the pore area density and membrane conductance increase with an increasing extra-cellular conductivity. On the other hand, the inverse correlation between propidium iodide delivery and extra-cellular conductivity as observed in the experiments is quantitatively captured by the model. This agreement confirms that this behavior is primarily mediated by electrophoretic transport during the pulse. The results suggest that electrophoresis is important even for the delivery of small molecules such as propidium iodide. The direct comparison between model prediction and experimental data presented in this work helps validate the former as a robust predictive tool for the study of electroporation

Vitamin A Transport Mechanism of the Multitransmembrane Cell-Surface Receptor STRA6.

Vitamin A has biological functions as diverse as sensing light for vision, regulating stem cell differentiation, maintaining epithelial integrity, promoting immune competency, regulating learning and memory, and acting as a key developmental morphogen. Vitamin A derivatives have also been used in treating human diseases. If vitamin A is considered a drug that everyone needs to take to survive, evolution has come up with a natural drug delivery system that combines sustained release with precise and controlled delivery to the cells or tissues that depend on it. This "drug delivery system" is mediated by plasma retinol binding protein (RBP), the principle and specific vitamin A carrier protein in the blood, and STRA6, the cell-surface receptor for RBP that mediates cellular vitamin A uptake. The mechanism by which the RBP receptor absorbs vitamin A from the blood is distinct from other known cellular uptake mechanisms. This review summarizes recent progress in elucidating the fundamental molecular mechanism mediated by the RBP receptor and multiple newly discovered catalytic activities of this receptor, and compares this transport system with retinoid transport independent of RBP/STRA6. How to target this new type of transmembrane receptor using small molecules in treating diseases is also discussed

Cellular delivery of antibodies: effective targeted subcellular imaging and new therapeutic tool

It is already more than a century since the pioneering work of the Nobel Laureate Ehrlich gave birth to the side chain theory1, which helped to define antibodies and their ability to target specific biological sites. However, the use of antibodies is still restricted to the extracellular space due to the lack of a suitable delivery vehicle for the efficient transport of antibodies into live cells without inducing toxicity. In this work, we report the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious affect on the cell metabolic activity. This delivery system is based on poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-(2-(diisopropylamino)ethyl methacrylate), (PMPC-PDPA), a pH sensitive diblock copolymer that self-assembles to form nanometer-sized vesicles, also known as polymersomes, at physiological pH. These polymersomes can successfully deliver relatively high antibody payloads within live cells. Once inside the cells, we demonstrate that these antibodies can target their epitope by immune-labelling of cytoskeleton, Golgi, and transcription factor proteins in live cells. We also demonstrate that this effective antibody delivery mechanism can be used to control specific subcellular events, as well as modulate cell activity and pro-inflammatory process

Acid-Labile Traceless Click Linker for Protein Transduction

Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system

Modelling drug coatings: A parallel cellular automata model of ethylcellulose-coated microspheres

Pharmaceutical companies today face a growing demand for more complex drug designs. In the past few decades, a number of probabilistic models have been developed, with the aim of improving insight on microscopic features of these complex designs. Of particular interest are models of controlled release systems, which can provide tools to study targeted dose delivery. Controlled release is achieved by using polymers with different dissolution characteristics. We present here an approach for parallelising a large-scale model of a drug delivery system based on Monte Carlo methods, as a framework for Cellular Automata mobility. The model simulates drug release in the gastro-intestinal tract, from coated ethylcellulose microspheres. The objective is high performance simulation of coated drugs for targeted delivery. The overall aim is to understand the importance of various molecular effects with respect to system evolution over time. Important underlying mechanisms of the process, such as erosion and diffusion, are described

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways