HyPlex focused on the development of a modular poly(L-lysine)-based hybrid nanogel platform for biomedical delivery applications. The work started with the preparation of a well-defined methacrylated polylysine precursor. Z-Lysine N-carboxyanhydride (Z-Lys-NCA) was synthesised from Z-Cbz-L-lysine using triphosgene in anhydrous tetrahydrofuran (THF), giving the NCA monomer in 75% yield. Controlled ring-opening polymerisation (ROP) was then performed in anhydrous dimethylformamide (DMF) using n-butylamine as initiator at an NCA-to-initiator ratio of 100:1. The targeted degree of polymerisation (DP) of 100 was closely reproduced experimentally, with a DP of 107 determined by proton nuclear magnetic resonance spectroscopy (1H NMR) and a number-average molar mass (Mn) of 13,800 g mol⁻¹. Size-exclusion chromatography (SEC) confirmed a narrow and unimodal molecular-weight distribution, with an apparent Mn of 10,100 g mol⁻¹ and dispersity (Đ) of 1.08. The polymer was subsequently deprotected using hydrobromic acid in trifluoroacetic acid (HBr/TFA), yielding the water-soluble polylysine hydrobromide in 95% yield while preserving the polymer chain and primary amine functionality. Selective methacrylation was then carried out using methacrylic anhydride. ^1H NMR analysis showed a degree of methacrylation of 22%, in close agreement with the target value of 20%, providing sufficient polymerisable groups for subsequent nanogel formation while retaining residual amine functionality. Hybrid nanogels were prepared by inverse miniemulsion photopolymerisation using acrylamide (AAm) or N-isopropylacrylamide (NIPAM) as functional comonomers. Seven formulations were produced in which the polylysine/comonomer ratio and Tween 20 concentration were systematically varied. The use of AAm and NIPAM enabled the hydrophilic/hydrophobic balance and responsiveness of the nanogels to be tuned. Mannose methacrylate was also successfully synthesised as originally planned, demonstrating the feasibility of introducing carbohydrate-based functionality. However, purification proved technically demanding and the amount of purified product obtained was low. Consequently, systematic formulation studies using this monomer were not practical within the reporting period, and the commercially available AAm and NIPAM were used for the main formulation study. The seven formulations contained either 90/10 wt.% poly(Z-Lys-MA)/AAm, 90/10 wt.% poly(Z-Lys-MA)/NIPAM, or 50/50 wt.% poly(Z-Lys-MA)/NIPAM, while Tween 20 was varied between 5 and 25 wt.%. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) demonstrated that nanogel size and morphology could be controlled through formulation parameters. Increasing Tween 20 generally reduced the hydrodynamic diameter from approximately 240 nm to approximately 125 nm, while the particle-size distributions remained relatively narrow. Incorporation of NIPAM additionally introduced a clear thermoresponsive behaviour. For example, Zeta-potential measurements further demonstrated composition-dependent surface properties. At pH 7.2 the AAm-based NG1 and NG2 formulations showed zeta potentials of +7.99 and +2.28 mV, respectively, whereas the NIPAM-containing NG4 and NG5 formulations showed higher values of +12.1 and +11.0 mV. All analysed formulations therefore retained a positive surface charge originating from the polylysine component, while incorporation of NIPAM resulted in higher positive zeta-potential values. The applicability of the platform for therapeutic cargo delivery was evaluated using indomethacin (IMC) as a model hydrophobic drug. IMC was incorporated by solvent diffusion and quantified by ultraviolet-visible spectroscopy (UV-Vis). Drug-loading content (DLC) ranged from 21% to 68%, with the highest value obtained for the NIPAM-containing NG5 formulation. Selected AAm- and NIPAM-based nanogels also displayed clearly different release profiles. Both released more than 90% of the encapsulated IMC within 300 min, but NG4 showed substantially faster release, reaching 86.3% after 30 min, whereas NG1 reached approximately 50% over the same period. Release data were further analysed using the Higuchi, Korsmeyer-Peppas and Weibull kinetic models, confirming that the release mechanism depended on nanogel composition. Overall, the project established a reproducible synthetic route from controlled polypeptide synthesis to functional hybrid nanogels and demonstrated that particle size, surface charge, thermoresponsive behaviour, drug loading and release kinetics can be tuned through nanogel composition. Selected lead formulations will subsequently undergo cytotoxicity assessment, providing the basis for further biological validation and future studies with nucleic-acid cargo.
