Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

Schiff-Base Cross-Linked Poly(2-oxazoline) Micelle Drug Conjugates Possess Antiferroptosis Activity in Numerous In Vitro Cell Models.

A great deal of nanocarriers have been applied to induce ferroptosis in cancer research, yet there are limited examples of nanocarrier formulations to rescue ferroptosis, which can be applied to neurodegeneration, inflammation, liver damage, kidney disease, and more. Here, we present the synthesis, characterization, and in vitro evaluation of pH-responsive, core-cross-linked micelle (CCM) ferrostatin-1 (Fer-1) conjugates with amine, valproic acid, and biotin surface chemistries. Fer-1 release from stable and defined CCM Fer-1 conjugates was quantified, highlighting the sustained release for 24 h. CCM Fer-1 conjugates demonstrated excellent ferroptosis rescue by their antilipid peroxidation activity in a diverse set of cell lines in vitro. Additionally, CCMs showed tunable cell association in SH-SY5Y and translocation across an in vitro blood-brain barrier (BBB) model, highlighting potential brain disease applications. Overall, here, we present a polymeric Fer-1 delivery system to enhance Fer-1 action, which could help in improving Fer-1 action in the treatment of ferroptosis-related diseases.

Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer-N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines.

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer-NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer-NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

Bioactive poly(2-oxazoline)-based nanomaterials bearing arylalkylamine and benzamide motifs possess intrinsic radical trapping and anti-ferroptosis properties.

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) - Ferrostatin-1 drug conjugates inhibit ferroptotic cell death.

Ferroptosis is a form of non-apoptotic iron induced cell death mechanism implicated in neurodegeneration, yet can be ameliorated with potent radical scavengers such as ferrostatin-1 (Fer-1). Currently, Fer-1 suffers from low water solubility, poor biodistribution profile and is unsuitable for clinical application. Fer-1 polymer-drug conjugates (PDCs) for testing as an anti-ferroptosis therapeutic candidate have yet to be described. Here, we report the synthesis and characterization of a library of water-soluble Fer-1 based poly(2-oxazoline)-drug conjugates. The cationic ring opening polymerization (CROP) of water-soluble 2-oxazoline monomers, and a novel protected aromatic aldehyde 2-oxazoline (DPhOx), produced defined copolymers, which after deprotection were available for modification with Fer-1 via reductive amination and Schiff base chemistry. The conjugates were tested for their activity against RSL3-induced ferroptosis in vitro, and first structure-activity relationships were established. Irreversibly conjugated Fer-1 PDCs possessing an arylamine structural motif showed a greatly increased anti-ferroptosis activity compared to reversibly (Schiff base) linked Fer-1. Overall, this work introduces the first active ferrostatin-PDCs and a new highly tuneable poly(2-oxazoline)-based PDC platform, which provides access to next generation polymeric nanomaterials for anti-ferroptosis applications.

Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity.

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition-fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

Nitrile-Functionalized Poly(2-oxazoline)s as a Versatile Platform for the Development of Polymer Therapeutics.

In recent years, polymers bearing reactive groups have received significant interest for biomedical applications. Numerous functional polymer platforms have been introduced, which allow for the preparation of materials with tailored properties via post-polymerization modifications. However, because of their reactivity, many functional groups are not compatible with the initial polymerization. The nitrile group is a highly interesting and relatively inert functionality that has mainly received attention in radical polymerizations. In this Article, a nitrile-functionalized 2-oxazoline monomer (2-(4-nitrile-butyl)-2-oxazoline, BuNiOx) is introduced, and its compatibility with the cationic ring-opening polymerization is demonstrated. Subsequently, the versatility of nitrile-functionalized poly(2-oxazoline)s (POx) is presented. To this end, diverse (co)polymers are synthesized and characterized by nuclear resonance spectroscopy, size-exclusion chromatography, and mass spectrometry. Amphiphilic block copolymers are shown to efficiently encapsulate the hydrophobic drug curcumin (CUR) in aqueous solution, and the anti-inflammatory effect of the CUR-containing nanostructures is presented in BV-2 microglia. Furthermore, the availability of the BuNiOx repeating units for post-polymerization modifications with hydroxylamine to yield amidoxime (AO)-functionalized POx is demonstrated. These AO-containing POx were successfully applied for the complexation of Fe(III) in a quantitative manner. In addition, AO-functionalized POx were shown to release nitric oxide intracellularly in BV-2 microglia. Thus nitrile-functionalized POx represent a promising and robust platform for the design of polymer therapeutics for a wide range of applications.

pH-switchable bacteria detection using zwitterionic fluorescent polymer.

A zwitterionic fluorescent polymer with high sensitivity to pH changes was constructed for the detection and imaging of both gram-positive and gram-negative pathogenic bacteria. A detection probe using the zwitterionic fluorescent polymer was synthesized with single boron dipyrromethane (BODIPY) as a hydrophobic dye and bromoethane as a cationic group for bacteria binding with conjugated poly(sulfobetaine methacrylate) (BOD/BE-PSM). The zwitterionic fluorescent polymer bound to bacteria through ionic complexes between anionic groups on the bacterial surface and cationic BOD/BE-PSM groups after 1h incubation. This finding demonstrated that the fluorescence on/off system operated via changes in the hydrophilic and hydrophobic nature of the zwitterionic fluorescent polymer, depending on the pH (6.0, 7.4, or 9.0), at a fixed 1mg/mL polymer concentration. The system showed good stability with a limit of detection of 1mg/mL. Quenching caused by interactions with the hydrophobic BODIPY dye was also observed, enabling bacteria detection, as shown by fluorescence spectroscopy and confocal microscopy images. Our results indicated that the zwitterionic fluorescent polymer could be used to detect bacteria over a wide range of pH values.