Poly(ethylene glycol) (PEG) is the standard stealth polymer to functionalize lipid nanoparticle (LNP) surfaces, requiring conjugation to a lipid tail for anchoring in the LNP’s shell. We eliminate the need for lipid conjugates by introducing a new polymer-only design principle using function-encoded statistical polymers. For that, we synthesized statistical amphiphilic copolymers in which hydrophobic units are followed by hydrophilic units, with the intention that the hydrophobic units of the polymer anchor to the LNP surface, and its hydrophilic parts stealth the particle surface. We screened the formulation space using a 42-member poly(2-oxazoline) library, yielding design rules for successful LNP formulations. Cryogenic transmission electron microscopy confirmed the intact LNP structure, and biological activity was assessed in multiple human cell types differentiated from inducible pluripotent stem cells. Intravenously administered barcoded LNPs formulated with function-encoded statistical polymers compared to the equivalent PEG-lipid LNPs showed 2.5- and 5-fold higher amounts in mice and lower bloodstream levels, leading to significantly higher accumulation in the spleen and liver. Rather than assembling LNP interfaces from multiple molecular components, this work establishes that orthogonal interfacial functions can be encoded directly into the architecture of a single statistical polymer, providing a general molecular design principle for programmable nanomedicine interfaces.

Carbon monoxide (CO) has gained increasing attention as an endogenous gasotransmitter with potential therapeutic relevance. In preclinical studies, including acute lung injury, sepsis, transplantation, inflammatory bowel, and cardiovascular diseases, CO has shown anti-inflammatory, anti-apoptotic, vasodilatory, and cytoprotective properties, suggesting its application in treating a wide range of diseases associated with cellular stress. CO impairs blood oxygen transport when systemic exposure occurs, but it is safe when blood oxygen transport is not compromised. Consequently, treatment modalities benefit from local rather than systemic CO delivery to open the therapeutic window of this physiological gasotransmitter. This review, therefore, focuses on local drug delivery strategies for generating and delivering CO, and on solutions and perspectives for various applications that leverage CO's anti-inflammatory and cytoprotective effects with an enhanced safety profile. We present the use of CO-releasing molecules (CORMs) and their incorporation into advanced drug delivery devices to control local CO exposure. Special emphasis is placed on drug vehicles featuring controlled on-target delivery, dosing, and biocompatibility. Therefore, we identify key principles and remaining obstacles in CO delivery technologies, which confluences in strategies that reduce the risk for pharmaceutical development and clinical application for safe, controlled, and targeted therapies.

Influenza has caused the deadliest pandemics in history, thereby prompting advances in our ability to ensure vigilance at all stages of future outbreaks. Quarantining patients early is crucial when it comes to preventing these outbreaks, but it is challenging with influenza due to presymptomatic transmission. Presymptomatic detection translates into massive screening needs, which necessitate cost-effective tools with access for anyone, anywhere, and at any time. We met these challenges by synthesizing sensors that respond to influenza infections with taste generation by using the tongue as an always-available detector. In doing so, we utilized the virus's need for neuraminidase cleavage of α-glycosidic bonds to detect its presence in patients. We synthesized N-acetylneuraminic acid-thymol derivatives and chemically tuned them to respond to viral but not bacterial neuraminidase. Viral selectivity was further confirmed via structural analysis and molecular docking. Influenza sensors that respond to viral presence with taste may have unmatched advantages regarding accessibility and cost-effectiveness, including the potential to first-line stratify millions of healthy individuals from flu patients, thereby enabling us to leverage our response armamentarium in future outbreaks.

l-Amino acid oxidases (LAAO) deaminate amino acids to α-keto acids and generate hydrogen peroxide, a reactive oxygen species (ROS) with potential value for cancer therapy. We recombinantly expressed the LAAO from Aplysia punctata, called APIT (Cuvier 1803). The resulting wild-type APIT (APITwt) was conjugated to polyethylene glycol (APIT-PEG). Furthermore, an APIT mutant with an affibody targeting the human epidermal growth factor receptor 2 (HER2; zHER2-APIT) was genetically engineered resulting in a binding affinity KD of ∼ 2.2 nM to the HER2 receptor ectodomain. Further, we evaluated if the APIT and tumor-targeted APIT can be used as an APIT-drug conjugate by covalently amidating the lysine residues on the protein surface. However, for the HER2-targeted APIT, the affibody contains lysines as well, and amidation of these lysines could have impaired the affibody's affinity to the HER2 receptor. Therefore, we designed a lysine-free variant of the tumor-targeting part of zHER2-APIT using an in silico mutation analysis, suggesting the replacement of the lysines of the affibody by arginine or alanine. This new variant is referred to as zHER2(K-del)-APIT. To simulate a covalent drug loading to APIT and the targeting constructs, we attached biotin by amidation. Biotin-zHER2(K-del)-APIT successfully allowed binding to HER2-positive but not HER2-negative cells in vitro. The biodistribution of these novel constructs was tested in xenografted mice with a HER2-positive and negative tumor in each animal. The zHER2(K-del)-APIT lost its ability to target HER2-positive tumors despite the in vitro data suggesting otherwise. The zHER2-APIT accumulated within the HER2-positive tumors but not in the negative tumors. APIT-PEG had increased uptake in HER2-positive and negative tumors compared to APITwt, which can be attributed to a prolonged serum half-life achieved by PEGylation, due to the absence of any tumor-targeting effect. These biodistribution studies point to HER2-targeting LAAOs for cancer therapy and PEGylation increasing tumor accumulation.

Lipid nanoparticles have gained significant attention during the COVID-19 pandemic, particularly due to their role in mRNA vaccine delivery. However, their rapid advancement has outpaced the development of established harmonized protocols for the quality control of the various excipients. In this study, we focused on the "stealth" lipopolymer 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), a critical excipient used in Moderna's Spikevax® mRNA vaccine. We investigated different commercial batches of DMG-PEG 2000 for impurities originating from both synthesis and degradation. Synthesis-related impurities include free glycerol and fatty acids of varying chain lengths, while degradation products result from single or double hydrolysis reactions. These synthetic and degradation-related impurities were primarily analyzed using an optimized high-performance liquid chromatography method with a charged aerosol detector (HPLC-CAD). Applying this validated method, a high purity of commercially available DMG-PEG 2000 was revealed, with every batch investigated exceeding a purity of 98.5%. In addition, gas chromatography (GC), HPLC with an evaporative light scattering detector (HPLC-ELSD), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed for comparative purposes and to enable further characterization. Based on these analyses, we developed a streamlined and robust impurity profiling protocol that (i) provides essential insight into the impurity profile of DMG-PEG 2000 in marketed products and (ii) may facilitate more decentralized and standardized validation processes in the future. The analytical approach presented here may also serve as a foundation for a future pharmacopeial monograph proposal for DMG-PEG 2000.

Given the extracellular matrix (ECM) essential role in tissue development, maintenance, and repair, exploiting its glycan structures offers new opportunities for dynamic drug storage and targeted delivery of peptides via injectable solutions. This study investigates the reversible, pH-driven interaction between boronic acid-functionalized compounds and diol-rich glycans within cell-derived decellularized extracellular matrices (CDMs). An N-terminally functionalized model peptide was produced using carboxy-phenyl-boronic acid (CPBA), and its pH-dependent binding to the diol-rich structures of CDMs was demonstrated. A CPBA-conjugated myostatin inhibitor maintained its full potency as a prerequisite for future therapeutic application. The findings suggest N-terminally boronic acid-modified peptides as drug candidates for targeted delivery and storage to diol-rich glycans of the natural ECM.

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

Mechanical tissue properties increasingly serve as pivotal phenotypic characteristics that are subject to change during development or pathological progression. The quantification of such material properties often relies on physical contact between a load-applying probe and an exposed sample surface. For most tissues, meeting these requirements entails an invasive preparation, which poses the risk of yielding mechanical properties that do not portray the physiological state of a tissue within a functioning organism. Brillouin microscopy has emerged as a non-invasive, optical technique that enables the assessment of mechanical cell and tissue properties with high spatio-temporal resolution. In optically transparent specimens, it does not require animal sacrifice, tissue dissection or sectioning. However, the extent to which results obtained from Brillouin microscopy allow to infer conclusions about potential results obtained with a contact-based technique, and vice versa , is unclear. Sources for discrepancies include the varying characteristic temporal and spatial scales, the directionality of measurement, environmental factors, and mechanical moduli probed. In this work, we addressed those aspects by quantifying the mechanical properties of acutely dissected murine retinae using Brillouin microscopy and atomic force microscopy (AFM)-based indentation measurements. Our results show a distinct mechanical profile of the retinal layers with respect to the Brillouin frequency shift, the Brillouin linewidth and the apparent Young’s modulus. Contrary to previous reports, our findings do not support a simple correlative relationship between Brillouin frequency shift and apparent Young’s modulus. Additionally, the divergent sensitivities of Brillouin microscopy and AFM-indentation measurements to structural features, as visualized by transmission electron microscopy, to cross-linking or changes post mortem underscore the dangers of assuming interchangeability between the two methods. In conclusion, our study advocates for viewing Brillouin microscopy and AFM-based indentation measurements as complementary tools, discouraging direct comparisons a priori and suggesting their combined use for a more comprehensive understanding of tissue mechanical properties.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

Accurately identifying tumor tissue is crucial during surgery, especially when removing head and neck squamous cell carcinomas (HNSCC). Our tumor-responsive probes are tailored for ex vivo diagnostics, streamlining today's complex surgical workflows and potentially enabling pathologists and surgeons to rapidly and objectively distinguish between healthy and tumor tissue. Designed based on insights from biological furin substrates and cleavage site screening, the probes detect HNSCC-associated protease activity. Within ten minutes of incubation, tumor tissue is differentiated from healthy tissue by visible fluorescence in biopsy supernatant. Fluorescence-emitting probes designed to detect furin, a biomarker protease that is upregulated in tumor tissue, simplifying complex diagnostic workflows and enabling tissue-conserving surgery through direct visual readout.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

Attachment of polyethylene glycol (PEG) chains is a common, well-studied, and Food and Drug Administration-approved method to address the pharmacokinetic challenges of therapeutic proteins. Occasionally, PEGylation impairs the activity of pharmacodynamics (PD). To overcome this problem, disease-relevant cleavable linkers between the polymer and the therapeutic protein can unleash full PD by de-PEGylating the protein at its target site. In this study, we engineered a matrix metalloproteinase (MMP)-responsive fibroblast growth factor 2 (FGF-2) mutant that was site-specifically extended with a PEG polymer chain. Using bioinspired strategies, the bioconjugate was designed to release the native protein at the desired structure/environment with preservation of the proliferative capacity in vitro on NIH3T3 cells. In vivo, hepatic exposure was diminished but not its renal distribution over time compared to unconjugated FGF-2. By releasing the growth factor from the PEG polymer in response to MMP cleavage, restored FGF-2 may enter hard-to-reach tissues and activate cell surface receptors or nuclear targets.

Tissue-type plasminogen activator (tPA) is the gold standard for emergency treatment of ischemic stroke, which is the third leading cause of death worldwide. Major challenges of tPA therapy are its rapid elimination by plasminogen activator inhibitor-1 (PAI-1) and hepatic clearance, leading to the use of high doses and consequent serious side effects, including internal bleeding, swelling and low blood pressure. In this regard, we developed three polyethylene glycol (PEG)ylated tPA bioconjugates based on the recombinant human tPA drug Alteplase using site-specific conjugation strategies. The first bioconjugate with PEGylation at the N-terminus of tPA performed by reductive alkylation showed a reduced proteolytic activity of 68 % compared to wild type tPA. PEGylation at the single-free cysteine of tPA with linear and branched PEG revealed similar proteolytic activities as the wild-type protein. Moreover, both bioconjugates with PEG-cysteine-modification showed 2-fold slower inhibition kinetics by PAI-1. All bioconjugates increased in hydrodynamic size as a critical requirement for half-life extension.

Increasing resistance against antimycotic drugs challenges anti-infective therapies today and contributes to the mortality of infections by drug-resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'-dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole-susceptible and fluconazole-resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.

In recent years, a novel treatment method for cancer emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the L-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata . Previously isolated from the ink, the L-amino acids oxidase was described to oxidate the essential amino acid L-lysine and L-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of amino acid oxidase Aplysia Punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG which lead to deprivation of arginine was well tolerated.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site‐specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization ( trans → cis ) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

The synthesis and detailed characterization of low-viscosity room-temperature ionic liquids (RTILs) and [BnPh3P]+ salts with the cyano(fluoro)borate anions [BF(CN)3]- (MFB), [BF2(CN)2]- (DFB), and [BF3(CN)]- as well as the new mixed-substituted anion [BFH(CN)2]- (FHB) is described. The RTILs with [EMIm]+ or [BMPL]+ as countercations were obtained in yields of up to 98% from readily available alkali metal salts and in high purities that allow application in electrochemical devices. Trends in thermal stability, melting and freezing behavior, density, electrochemical stability, dynamic viscosity, specific conductivity and ion diffusivity have been assessed and compared to those of the related tetracyanoborate- and cyano(hydrido)borate-RTILs. The crystal structure analysis of the [BnPh3P]+ salts of [BFn(CN)4-n]- (n = 0-4), [BHn(CN)4-n]- (n = 1-3) and [BFH(CN)2]- provided experimental access to anion volumina that together with ion molecular mass, electrostatic potential, shape and chemical stability have been correlated to physicochemical properties. In addition, the cytotoxicity of the [EMIm]+-ILs and potassium or sodium salts was studied.

Vascular endothelialgrowth factor A-165 (VEGF-A(165))positively modulates neointimal hyperplasia, lumen stenosis, and neovascularization.One challenge for the use of VEGF-A(165) for potential therapyis its short serum half-life. Therefore, we are designing VEGF-A(165) bioconjugates carrying polyethylene glycol (PEG). The purity of the recombinantlyexpressed human VEGF-A(165) exceeded 90%. The growth factorhad a half-maximal effective concentration of 0.9 ng/mL (EC50) andinduced tube formation of human umbilical vein endothelial cells.PEGylation was conducted by Schiff base reaction followed by reductiveamination. After purification, two species were obtained, with oneor two PEG attached per VEGF-A(165) dimer. Both resultingbioconjugates had a purity exceeding 90%, wild-type bioactivity, andincreased hydrodynamic radii as required for prolonging the half-life.