Hyperbranched polymers as non-viral vectors for gene delivery

The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyperbranched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. In the first part of this thesis, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known ‘ultra-facile’ thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 and H1299 cells, surprisingly, indicated that the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyperbranched polylysines synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer Tg, pKa and ability to form stable polyplexes with plasmid DNA. These results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyperbranched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy. It has become known that many cationic polymers are cytotoxic and although a large number of polycations have now designed to address the toxicity problem, there is still a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a high throughput and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Therefore, in the second part of this thesis, metabolomics was applied to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethylenimine polyplexes, on the metabolic pathways of A459 and H1299 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 fold changes of the metabolites indicated that the polyplexes of polyethylenimine 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. This part highlights the importance of metabolomics approaches not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying their action, which could help to design more efficient vectors. In the third part of this thesis, we investigated the ability of the hyperbranched polymers to condense and deliver siRNA. The results indicated that the higher molecular mass polymers achieved better siRNA delivery and gene silencing than the lower molecular mass form of the polymers and the lysine-only polymer was more efficient than the histidinylated one. These results can be attributed to the low charge (molecular mass) and stiffness of siRNA molecules in comparison with plasmid DNA, which in combination with the impact of histidine incorporation on the structure of the hyperbranched polymers can also explain the lower efficiency of histidinylated polymers. Overall, this thesis is highlighted the impacts of structural factors on the gene delivery applications of hyperbranched polymers and the importance of these factors to inform the design of new polymeric vectors. Also, metabolomics approaches were introduced to this area, not only to evaluate the safety of gene vectors but also to understand the molecular basis by which these vectors act. The data together suggest that the hyperbranched polymers prepared during thermal polycondensation of amino acids have some efficacy in preliminary gene delivery applications, and that these might be improved with future studies to be a candidate for clinical purposes

Rapid formulation of redox-responsive oligo-β-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-β-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-β-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

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]

© 2020 The Author(s) The authors regret that the original version of Fig. 3 incorrect. The updated figure is below for reference. [Figure presented] Fig. 3 Super-resolution microscopy image of methotrexate loaded PLGA nanoparticles (green) (92 μg/mL, 3 h) in THP-1 derived macrophages. Cells were fixed and stained for cytoskeleton (Alexafluor 647, magenta) and nucleus (Hoechst nucleus, blue). Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) The authors would like to apologise for any inconvenience caused

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. This journal i

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

Rapid formulation of redox-responsive oligo-β-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-β-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-β-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

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 also presume to be safer than the viral vectors, however many of them 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 fold 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 metabolomics approach not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanism of them which could help to design more efficient vectors

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 5-10 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 μg 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

Hyperbranched polymers as non-viral vectors for gene delivery

The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyperbranched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. In the first part of this thesis, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known ‘ultra-facile’ thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 and H1299 cells, surprisingly, indicated that the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyperbranched polylysines synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer Tg, pKa and ability to form stable polyplexes with plasmid DNA. These results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyperbranched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy. It has become known that many cationic polymers are cytotoxic and although a large number of polycations have now designed to address the toxicity problem, there is still a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a high throughput and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Therefore, in the second part of this thesis, metabolomics was applied to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethylenimine polyplexes, on the metabolic pathways of A459 and H1299 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 fold changes of the metabolites indicated that the polyplexes of polyethylenimine 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. This part highlights the importance of metabolomics approaches not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying their action, which could help to design more efficient vectors. In the third part of this thesis, we investigated the ability of the hyperbranched polymers to condense and deliver siRNA. The results indicated that the higher molecular mass polymers achieved better siRNA delivery and gene silencing than the lower molecular mass form of the polymers and the lysine-only polymer was more efficient than the histidinylated one. These results can be attributed to the low charge (molecular mass) and stiffness of siRNA molecules in comparison with plasmid DNA, which in combination with the impact of histidine incorporation on the structure of the hyperbranched polymers can also explain the lower efficiency of histidinylated polymers. Overall, this thesis is highlighted the impacts of structural factors on the gene delivery applications of hyperbranched polymers and the importance of these factors to inform the design of new polymeric vectors. Also, metabolomics approaches were introduced to this area, not only to evaluate the safety of gene vectors but also to understand the molecular basis by which these vectors act. The data together suggest that the hyperbranched polymers prepared during thermal polycondensation of amino acids have some efficacy in preliminary gene delivery applications, and that these might be improved with future studies to be a candidate for clinical purposes