Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery

Abstract

The successful clinical translation of nonviral gene delivery systems has yet to be achieved owing to the biological and technical obstacles to preparing a safe, potent, and costeffective vector. Hyperbranched polymers, compared with other polymers, have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification, which makes them more feasible for scale-up and manufacturing. Here, we compare hyperbranched poly(amino acids) synthesised by copolymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known "ultrafacile" 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 to 30 kDa. Spectroscopic, rheological, and thermal analyses 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 cells indicated that all the new hyperbranched polymers were well tolerated, but, surprisingly, the copolymers containing histidine were not more effective in transfecting a luciferase gene than were 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 we obtain structure-function information relating histidine content with polymer stiffness, pKa, and ability to form stable polyplexes with DNA. The 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 not only changes the buffering capacity of the polymer but has significant effects on the overall structure, architecture, and gene delivery efficacy.

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

“…The polyplexes were prepared at the N/P ratio of 10, where they showed the best transfection efficiency as reported in our previous work. 28 The size and zeta potential measurements indicated that the polymers efficiently condensed plasmid DNA into positively charged nanoparticles of around 150 nm in diameter and the AFM images confirmed the results (Fig. S1, ESI †).…”

“…Hyperbranched polylysine, hyperbranched polylysine-co-histidine, and their FITC-labelled forms were prepared and characterised as described previously. 28 Branched polyethyleneimine (25 kDa), methylthiazolyldiphenyl-tetrazolium bromide (MTT), the annexin V-FITC/PI apoptosis detection kit, N-acetylcysteine (NAC) and all buffers were purchased from Sigma-Aldrich. The Cytotoxicity Detection Kit PLUS (for LDH) was obtained from Roche.…”

“…The N/P ratios, the charge ratio, were calculated based on the results of a fluorescamine assay and all the physicochemical characterisations were as reported in our previous work. 28 Cell culture A549 (human adenocarcinomic alveolar basal epithelial, ATCC s CCL-185t) and H1299 (human non-small cell lung carcinoma, ATCC s CRL-5803t) cells were cultured in DMEM and RPMI media respectively, supplemented with 10% FBS and 1% L-glutamic acid, in a humidified atmosphere containing 5% CO 2 . The cells were used at a passage number between 30 and 40.…”

“…Also, thermally polymerised hyperbranched polymers have attracted our interest due to their advantages over dendritic polymers in terms of applicability and feasibility for manufacturing and scale up, and because the impact of these polymers and their polyplexes at the cellular and molecular level is not fully understood. 27,28 In the present work, a global metabolic profiling approach was employed to study the effect of hyperbranched polylysine (hb-polyK), hyperbranched polylysine-co-histidine (hb-polyKH2) and branched polyethyleneimine (PEI) polyplexes with plasmid DNA on two lung carcinoma cell lines (A549 and H1299). These two cell lines were chosen as they are initial targets for local administration and inhalation in cancer therapy.…”

“…These two cell lines were chosen as they are initial targets for local administration and inhalation in cancer therapy. In addition, these cell lines showed unexpected variations in their responses to gene delivery complexes in terms of uptake and transfection, 28 thus an evaluation of their metabolomics profiles was expected to enhance our understanding of the intracellular behaviour of the polyplexes and their potential toxicity at sub-toxic concentrations.…”