Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.

Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerge as versatile non-viral gene delivery systems that can help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF).

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo.

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration.

Enhancing the pharmacokinetic/pharmacodynamic properties of therapeutic nucleotides using lipid nanoparticle systems.

Although activity has been reported in vivo, free nucleic acid-based drugs are rapidly degraded and cleared following systemic administration. To address these challenges and improve the potency and bioavailability of genetic drugs, significant efforts have been made to develop effective delivery systems of which lipid nanoparticles (LNP) represent the most advanced technology currently available. In this review, we will describe and discuss the improvements to the pharmacokinetic and pharmacodynamic properties of nucleic acid-based drugs mediated by LNP delivery.

siRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer.

Purpose: Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model.

Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration.

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP-siRNA formulations in clinical trials. The aim of this work was to develop LNP-siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.

Microfluidic-based manufacture of siRNA-lipid nanoparticles for therapeutic applications.

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles.

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo.

Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain.

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity.

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA.

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility.

Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA.

Unlabelled: Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake.

Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA.

Unlabelled: The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species.

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo.

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.

Organellar (Na+, K+)/H+ exchanger NHE7 regulates cell adhesion, invasion and anchorage-independent growth of breast cancer MDA-MB-231 cells.

Na+/H+ exchangers (NHEs) are a group of secondary active antiporters that regulate cellular pH, cell volume and ion homeostasis. In humans, nine isoforms (NHE1-NHE9) were identified and characterized as functional NHEs. While a growing body of evidence indicates that NHE1 generates an acidic tumor environment and thereby contributes to tumor invasion, little is known about the role of other NHE isoforms in tumor progression.

Caveolins bind to (Na+, K+)/H+ exchanger NHE7 by a novel binding module.

NHE7 was identified as the first mammalian organelle-membrane type (Na+, K+)/H+ exchanger that may contribute to the ion homeostasis in the trans-Golgi network (TGN) and endosomes. Here we show that caveolins directly bind to the C-terminal extension of NHE7 by an unconventional binding-module. NHE7 is partly associated with caveolae/lipid raft fractions, and heterologous expression of caveolin dominant-negative mutants as well as cholesterol depriving drugs diminished such associations.

RACK1 associates with NHE5 in focal adhesions and positively regulates the transporter activity.

Na+/H+ exchanger isoform 5 (NHE5) is a brain-enriched NHE that may play important roles in ion homeostasis and cell-volume regulation. However, the regulation mechanism of NHE5 has not been fully elucidated. Here, we show that Receptor for Activated C-kinase 1 (RACK1) directly binds to NHE5 and positively regulates the transporter function.

Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7.

The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7.