Investigating histidinylated highly branched poly(lysine) for siRNA delivery.

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material.

Corrigendum to 'LC-MS metabolomics comparisons of cancer cell and macrophage responses to methotrexate and polymer-encapsulated methotrexate' [International Journal of Pharmaceutics: X Volume 1 (2019) 100036].

[This corrects the article DOI: 10.1016/j.ijpx.2019.100036.].

Investigating the intracellular effects of hyperbranched polycation-DNA complexes on lung cancer cells using LC-MS-based metabolite profiling.

Cationic polymers have emerged as a promising alternative to viral vectors in gene therapy. They are cheap to scale up, easy to functionalise and are potentially safer than viral vectors, however many are cytotoxic. The large number of polycations, designed to address the toxicity problem, raises a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a detailed and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Here, we applied metabolomics to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethyleneimine polyplexes at sub-toxic concentrations on the metabolic pathways of A459 and H1299 lung carcinoma cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The relative changes of the metabolites indicated that the polyplexes of polyethyleneimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. Our work highlights the importance of the metabolomics approach not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying any adverse effects, which could help in designing more efficient vectors.

LC-MS metabolomics comparisons of cancer cell and macrophage responses to methotrexate and polymer-encapsulated methotrexate.

Methotrexate (MTX) is a folate analogue antimetabolite widely used for the treatment of rheumatoid arthritis and cancer. A number of studies have shown that MTX delivered via nanoparticle carriers is more potent against cancer cells than free MTX, a phenomenon attributed to higher cellular uptake of the particles compared to the saturable folate receptor pathway. In this study, a cell-based global metabolic profiling approach was applied to study the effects of MTX in both free drug form and when encapsulated in -poly(lactide-co-glycolide) (PLGA) nanoparticles on a cancer cell line, A549, and also on human-like THP-1 macrophages. The results showed that MTX loaded nanoparticles had less impact on the macrophages than free MTX, and the effects on macrophages were limited to changes in nucleotide metabolism and suppression of the tricarboxylic acid cycle, whereas free MTX also led to a drop in glycolytic activity and impairment in redox homeostasis. In contrast, MTX loaded nanoparticles showed a greater impact on A549 cells than the free drug, which was in accord with studies in other cell lines in prior literature with MTX-carrier nanoparticles.

Water Solubility Enhancement of Pyrazolo[3,4-d]pyrimidine Derivatives via Miniaturized Polymer-Drug Microarrays.

A miniaturized assay was optimized to evaluate the enhanced apparent water solubility of pyrazolo[3,4-d]pyrimidine derivatives used extensively as anticancer drug scaffolds. The applied amount of drugs used in the reported strategy ranged from 5 to 10 µg per formulation which were dispensed by an inkjet 2D printer directly into a 96-well plate. The selected polymer/drug formulations with high water solubility demonstrated improved cytotoxicity against a human lung adenocarcinoma cancer cell line (A549) compared to the free drugs. We attribute the enhanced efficacy to the improved apparent-solubility of the drug molecules achieved via this methodology. This novel miniaturized method showed promising results in terms of water solubility improvement of the highly hydrophobic pyrazolo[3,4-d]pyrimidine derivatives, requiring only a few micrograms of each drug per tested polymeric formulation. In addition, the reported experimental evidence may facilitate identification of suitable polymers for combination with drug, leading to investigations on biological properties or mechanisms of action in a single formulation.

Rapid formulation of redox-responsive oligo-ß-aminoester polyplexes with siRNA via jet printing.

Here we describe a rapid inkjet formulation method for screening newly-synthesised cationic materials for siRNA delivery into cancer cells. Reduction responsive oligo-ß-aminoesters were prepared and evaluated for their ability to condense siRNA into polyplexes through a fast inkjet printing method. A direct relationship between the oligomer structures and charge densities, and the final cell response in terms of uptake rate and transfection efficacy, was found. The oligo-ß-aminoesters were well-tolerated by the cancer cells, compared to conventional cationic polymers so far employed in gene delivery, and were as active in silencing of a representative luciferase gene.