David Pizzi

Stereochemistry is reported as a design parameter for modulating immune interactions of polymeric nanocarriers.

Poly(cyclic imino ether)s (PCIEs) including poly(2-alkyl-2-oxazoline)s (POx), poly(2-alkyl-2-oxazine)s (POz) and poly(2,4-dialkyl-2-oxazoline)s (PdOx) are a rapidly emerging polymer class for use in biomedical and therapeutic applications due to the biocompatibility and “stealth-like” properties of their water-soluble homologues similar to poly(ethylene glycol) (PEG). The physico-chemical properties of PCIE can be easily “tuned” via appropriate monomer selection resulting for example in polymers ranging from water-soluble to water-insoluble. To date, studies focussing on the hydrophilicity of PCIEs have been limited to the well-known POx, with minimal comparison to POz and especially PdOx. In this study, the effect of degree of hydrophilicity for water-soluble POx, POz, and PdOx systems were assessed for the first time under one testing regime. Specifically, a library of 20 PCIEs was created, consisting of 10 different polymers each synthesised at two different degrees of polymerisation (DP = 20, 50). The hydrophilicity of each polymer was assessed by turbidimetry, high-performance liquid chromatography (HPLC), octanol–water partition coefficient (log K_OW), surface tension, and ¹H NMR relaxometry. Additionally, log K_OW was compared against in silico predictive techniques and hydrophilciity trends seen to correlate between the two techniques, though the predictive software utilised could not accurately predict log K_OW for long polymer chains. This investigation lead to the elucidation of hydrophilicity trends stemming from molar mass, side chain length, backbone spacing, and additional backbone functionality for the case of PdOx. More specifically, hydrophilicity followed a POz > PdOx > POx trend when comparing between structural isomers, and a POx > POz > PdOx trend when comparing between polymers with the same 2-side chain. The knowledge resulting from this study can be utilised for the future design of smart, solubility-tailored PCIE systems for a range of biomedical applications.

Radical trapping agents such as Ferrostatin-1 (Fer-1) are capable of rescuing cells from ferroptosis, an iron-dependent form of cell death. Previously, poly(2-oxazoline)-Fer-1 (POx-Fer-1) conjugates were reported, which possess increased water-solubility and remain active after covalent conjugation of Fer-1. In this study, we break down the structural and functional layers of POx-Fer-1 conjugates and reveal that drug-free POx containing arylalkylamine and benzamide motifs show anti-ferroptosis properties. Intriguingly, even the basic construct poly(2-methyl-2-oxazoline-grad-2-phenyl-2-oxazoline) P(MeOx-grad-PhOx) was found to be active. Therefore, P(MeOx-grad-PhOx) of varying compositions were prepared, characterized by ¹H NMR spectroscopy and size exclusion chromatography and investigated with regard to their self-assembly in aqueous solution and activity in an in vitro ferroptosis model. These findings were further explored for the design of defined and bioactive core-crosslinked micelles with intrinsic anti-ferroptosis behaviour. Cellular interaction studies involving C₁₁-BODIPY assays and confocal microscopy investigations revealed lysosomal processing of the nanomaterials and perturbation of ferroptotic cell death through reducing lipid-peroxidation. This study highlights new drug/cargo-free anti-ferroptotic nanomaterials as proof of concept that hold potential for therapy of ferroptosis-associated diseases and highlights the role of nanocarriers in a therapeutic context.

Poly(2-oxazoline)s (POx) have received substantial attention as poly(ethylene glycol) (PEG) alternatives in the biomedical field due to their biocompatibility, high functionality, and ease of synthesis. While POx have demonstrated strong potential as biomaterial constituents, the larger family of poly(cyclic imino ether)s (PCIE) to which POx belongs remains widely underexplored. One highly interesting sub-class of PCIE is poly(2,4-disubstituted-2-oxazoline)s (PdOx), which bear an additional substituent on the backbone of the polymers' repeating units. This allows fine-tuning of the hydrophilic/hydrophobic balance and renders the PdOx chiral when enantiopure 2-oxazoline monomers are used. Herein, we synthesize new water-soluble (R-/S-/RS-) poly(oligo(2-ethyl-4-methyl-2-oxazoline) methacrylate) (P(OEtMeOxMA)) bottlebrushes and compare them to well-established PEtOx- and PEG-based bottlebrush controls in terms of their physical properties, hydrophilicity, and biological behavior. We reveal that the P(OEtMeOxMA) bottlebrushes show a lower critical solution temperature behavior at a physiologically relevant temperature (∼44 °C) and that the enantiopure (R-/S-) variants display a chiral secondary structure. Importantly, we demonstrate the biocompatibility of the chiral P(OEtMeOxMA) bottlebrushes through cellular association and mouse biodistribution studies and show that these systems display higher immune cell association and organ accumulation than the two control polymers. These novel materials possess properties that hold promise for applications in the field of nanomedicine and may be beneficial carriers for therapeutics that require enhanced cellular association and immune cell interaction.

Phytochemical profiling was undertaken on the crude extracts of Drosera magna to determine the nature of the chemical constituents present. In total, three new flavonol diglycosides (1–3), one new flavan-3-ol glycoside (4), and 12 previously reported compounds of the flavonol (5, 9), flavan-3-ol (15), flavanone (8), 1,4-napthoquinone (6, 7, 13, 14), 2,3-dehydroxynapthalene-1,4-dione (10–12), and phenolic acid (16) structure classes were isolated and identified. Compounds 1–9, 13, 17, and 18 were assessed for antimicrobial activity, with compounds 6, 7, 8, and 9 showing significant activity. Compounds 1, 2, and 6 were also evaluated for anthelmintic activity against larval forms of Hemonchus contortus, with compound 6 being active.

In light of research reporting abnormal pharmacokinetic behavior for therapeutics and formulations containing poly(ethylene glycol) (PEG), a renewed emphasis has been placed on exploring alternative surrogate materials and tailoring specific materials to distinct nanomedicine applications. Poly(2-oxazolines) (POx) have shown great promise in this regard; however, a comparison of POx and PEG interactions with components of the immune system is needed to inform on their distinct suitability. Herein, the interaction of isolated immune cells following injection of hyperbranched polymers comprised of PEG or hydrophilic POx macromonomers was determined via flow cytometry. All materials showed similar association with all of the splenic immune cells analyzed. Interestingly, splenic CD68^hi and CD11b^hi macrophages showed similar levels of polymer association, despite CD11b^hi being a smaller population, suggesting CD68 is linked to increased recognition and phagocytosis of these nanomaterials. This is of interest given that CD68 is a scavenger receptor and directly facilitates the clearance of cellular debris and promotion of phagocytosis, as opposed to CD11b, which is associated with the mediating inflammation via the production of cytokines as well as complement-mediated uptake of foreign particles. In the liver, PEG and poly(2-methyl oxazoline) hyperbranched polymers showed no discernible differences in their cellular association, while hyperbranched poly(2-ethyl oxazoline) showed increased association with dendrocytes and CD68^hi macrophages, suggesting that this material exhibited a greater propensity to interact with components of the immune system. This work highlights the importance of how subtle changes in chemical structure can influence the immune response.

Cellular uptake and intracellular targeting to specific organelles are key events in the cellular processing of nanomaterials. Herein, we perform a detailed structure-property relationship study on carboxylic acid side chain bearing polyacrylates in order to provide design criteria for the manipulation of their cellular interactions. Redox-initiated RAFT polymerization of three tert-butyl protected N-acylated amino ester based acrylate monomers of different substitution and degree of polymerization (DP) yielded defined and pH-responsive carboxylic acid side chain polymers upon deprotection (N-acetyl, DP 1: P(M1); N-propionyl, DP 1: P(E1), DP 2: P(E2)). Flow cytometry studies revealed time-dependent cell association with P(E2) > P(E1) > P(M1) at any given time point. Importantly, the type of cyanine dye used for labeling was found to significantly influence the cellular processing of the polymers. Changing the dye from Cy5 to its sulfonated version sulfoCy5 resulted in a much lower cellular association. Moreover, Cy5-labeled polymers were targeted to mitochondria, while sulfoCy5 modification caused a significant change of the cellular fate of the polymers towards lysosome trafficking. This study highlights the importance of selecting a suitable dye but also demonstrates the possibilities for the rational design of organelle-specific targeting of carboxylated polyacrylates.