Philipp Uhl

Evaluation of blood-brain barrier (BBB) permeability is a crucial step in drug research and development, particularly for the pharmacotherapy of targets in the central nervous system. Current preclinical permeability predictions are hindered by limited transferability or BBB model inaccessibility for analysis. In order to generate relevant pharmacological data, it is necessary to establish qualified physiological, translational, and quantitative systems with validated methods. 3D BBB organoids composed of human cerebral microvascular endothelial cells (hCMEC/D3), and primary pericytes and astrocytes were generated. The BBB barrier function was evaluated using the paracellular marker ᴅ-Tyr(Me)Orn. Confocal laser scanning microscopy was used to determine the architecture of the cell arrangements, crucial BBB hallmarks and the apoptosis rate (cleaved caspase 3). An experimental setup was established to evaluate drug uptake into the organoids using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The concentration- and time-dependent intraorganoidal drug uptake was determined for 13 drugs, and used to calculate the ratio of intra- and extraorganoidal concentrations. These organoid uptake ratios were subsequently correlated with reported in vivo brain-to-plasma ratios. Additionally, the performance of four in silico BBB permeability prediction tools was assessed with the in vivo data. Optimal analysis time point was identified as day 3 post self-assembly based on organoid architecture, endothelial coverage and apoptosis rate. Modular barrier properties were shown by a reduction of intraorganoidal ᴅ-Tyr(Me)Orn after cell connection were compromised, and potential wash out cross-validated by fluorescent Cy5 in the wash solution. Deming regression demonstrated a correlation with pseudo R ² of 0.64 between the organoid uptake ratios and reported mammalian in vivo brain-to-plasma ratios. In contrast, the in silico tools evaluated displayed no correlations with these in vivo data. We established and validated a simple and rapid experimental setup for the UPLC-MS/MS drug quantification into single 3D BBB organoids. We demonstrated that the 3D BBB organoids have translational relevance with regard to human and mammalian in vivo BBB permeability. This system therefore qualifies as New Approach Methodology (NAM) and can be used for approximate evaluation of in vivo brain uptake, while outperforming state of the art in silico models.

We previously described that a non-toxic ceftazidime-hexa-arginine conjugate (CTZ-R6) surpassed the antibacterial potency of ceftazidime (CTZ) against multi-resistant bacteria by up to 1,000-fold and effectively reduced the bacterial load in murine bacteremia. However, the mechanistic understanding of its resistance-overcoming activity remained elusive. Here, we report on the mode of action of CTZ-R6 in gram-positive and gram-negative bacteria. Against a panel of multi-resistant Enterobacteriaceae and non-fermenters, CTZ-R6 outperformed ceftazidime. The conjugate synergized with avibactam in the same manner as CTZ, but its MICs were much lower than those of CTZ. CTZ-R6 crossed the outer membrane of Escherichia coli by self-promoted uptake and permeabilized this barrier to a fluorescent dye and otherwise ineffective antibiotics. Investigations in Bacillus subtilis demonstrated that the electrochemical gradient at the cytoplasmic membrane remained high, but membrane topology was perturbed, and the peptidoglycan sacculus was damaged. Lysis kinetics were β-lactam-like. CTZ-R6 showed higher affinity than CTZ for penicillin-binding protein 2a (PBP2a) of B. subtilis , lower affinity for PBP1a/b, and an overall more balanced PBP affinity than CTZ. The covalent linkage between CTZ and the hexa-arginine moiety was essential for potency. In dissecting the contributions of the individual moieties, we found that R6C destabilizes the outer membrane, enabling porin-independent uptake. The peptide also interacts with the cytoplasmic membrane, facilitating CTZ access to the PBPs in B. subtilis . More balanced targeting of PBPs and porin-independent entry is beneficial for overcoming established resistances. In conclusion, both components of the conjugate contribute to the increased antibacterial potency of CTZ-R6 in a balanced and synergistic manner. IMPORTANCE: Given the global scale of antibacterial resistance, it becomes clear that numerous infections may eventually become untreatable as former, well-established treatment regimens fail. To prevent the emergence of a post-antibiotic era, strategies are being explored to extend the usefulness of clinically important antibiotics, including the synthesis of conjugates that improve the potency of established drugs. We recently demonstrated that attaching polycationic peptides to all four β-lactam subclasses improved MICs against multi-resistant gram-positive and gram-negative pathogens. The ceftazidime-hexa-arginine conjugate showed the best overall profile and improved the potency of ceftazidime against multi-resistant bacteria by up to 1,000-fold. However, the molecular mechanism of its resistance-overcoming activity remained elusive. Here, we describe which characteristics and capabilities of the peptide and the β-lactam allow them to synergize for increased potency. Mechanistic understanding is essential for further development of such conjugation approaches.

Nanobodies (Nbs) are considered promising antibody fragments for overcoming limitations in precision oncology due to their high specificity and deep tissue penetration. However, their therapeutic potential remains limited by their rapid renal clearance. In this study, anti‐HER2 Nb‐loaded liposomes with dual functionalization combining polyethylene glycol 2000 (PEG) and cyclic cell‐penetrating peptides are developed to ameliorate their pharmacokinetic behavior while retaining binding specificity. Liposomal formulations with high encapsulation efficiencies are produced with dual centrifugation. Biophysical characterization reveals that PEGylation effectively mitigates cCPP‐induced membrane destabilization, ensuring structural integrity. In vitro assays confirm that, despite the steric shielding by PEG, the encapsulated Nbs retain their functionality and specific binding to HER2‐overexpressing cells. In vivo studies in zebrafish larvae demonstrate excellent biocompatibility and lack of immunogenicity. Crucially, liposomal encapsulation significantly modulates the pharmacokinetic profile of Nbs in rats, reducing renal accumulation compared to free Nbs. This study presents a robust liposomal platform that successfully balances the stealth properties of PEG with the functional benefits of cCPPs. Consequently, this platform offers an effective strategy to enhance the therapeutic window of low‐molecular‐weight biologics.

The growing concerns regarding the immunogenicity of polyethylene glycol (PEG) have prompted the search for alternative excipients that can prolong circulation in parenteral drug delivery. Archaeal lipids, known for their structural stability and unique membrane properties, represent a promising material in this context. In this study, we evaluated the impact of archaeal lipid‐based liposomes on the pharmacokinetics of intravenously administered vancomycin in male Wistar rats. Liposomes containing either purified caldarchaeol (GDGT) or an archaeal lipid extract (ALE), consisting of 5 lipid species, were compared to conventional PEGylated liposomes and free vancomycin regarding their effect on systemic exposure of vancomycin. All liposomal formulations significantly enhanced exposure and yielded a prolonged circulation time compared to the free drug. GDGT containing liposomes achieved pharmacokinetic effects comparable to PEGylated systems, markedly reducing clearance and central volume of distribution based on population pharmacokinetic modeling. Our findings provide the first in vivo evidence that archaeal lipids, particularly GDGT, might act as effective PEG replacement excipient for parenteral delivery, offering a promising alternative platform.

Polyethylene glycol (PEG)ylation is an established, increasingly used method to prolong the half-lives of active compounds applied intravitreally. However, the biocompatibility of high doses of high molecular weight PEG has never been systematically assessed in a translational in vivo model. The aim of this study was to evaluate the intraocular inflammatory and pro-angiogenic response as well as structural and functional retinal alterations after the intravitreal injection of PEG of varying molecular weight injected intravitreally in a large animal in vivo pig model. This cohort study includes 12 pigs divided into 4 cohorts. Each cohort received an intravitreal injection of either 0.1 mL with 400 mg/mL of PEG-400, PEG-2000, PEG-40000 or an injection of balanced salt solution (BSS), respectively. Biocompatibility measurements including fundoscopy, optical coherence tomography (OCT) and electroretinography (ERG) were performed prior and 2, 4, and 6 weeks post-injection. Six weeks post-injection, the animals were euthanized, aqueous and vitreous taps were taken for cytokine and signal protein measurements, and immunostainings were performed. All animals with intravitreal PEG-injections showed significant preretinal vitreous cells, most likely hyalocytes, as evidenced by OCT, but no signs of intraretinal inflammation were seen in the immunostainings conducted 6 weeks after injection. Untreated left control eyes and eyes injected with BSS showed no signs of inflammation throughout the whole study period. Functional and structural retinal parameters showed no significant alterations over the course of 6 weeks. Cytokine analyses showed no prolonged inflammatory reaction. The one-time intravitreal injection of PEG causes mild intravitreal inflammatory reactions, as evidenced in OCT by increased vitreous cells. However, retinal structure and function is not affected.

Methotrexate (MTX) inhibits cell proliferation, which underlies ocular diseases, including intraocular lymphoma and proliferative vitreoretinopathy. However, MTX normally requires bi‐weekly intravitreal injections due to a short half‐life, causing rapid clearance below therapeutic thresholds within 72h. To overcome these limitations, sustained‐release carriers, including poly(lactic‐co‐glycolic) acid‐based implants, were investigated in vitro previously. These systems offer prolonged drug delivery but require relatively large‐gauge surgical implantation, which increases the risk of surgical complications and limits their practical use. In this study, MTX‐loaded liposomes that can be administered via a 30‐gauge cannula are developed, obviating the need for more invasive surgical implantation. This phospholipid‐based liposomal formulation succeeded in enabling sustained methotrexate release at therapeutic levels for over six weeks following a single intravitreal injection, demonstrated in vivo in a large animal pig model. High biocompatibility of this novel liposomal formulation is confirmed through longitudinal retinal structure (optical coherence tomography) and function (electroretinography) assessments. This liposomal formulation of MTX provides a clinically and surgically optimized drug delivery system that allows improved management of intraocular lymphoma and proliferative vitreoretinopathy in the future.

Purpose: Landrace pigs are increasingly used as a large-animal model in ophthalmic research due to their cone-enriched visual streak and anatomical similarity to the human eye. However, they are commonly studied at 16–20 weeks of age, a timeframe in which the animals double their weight and development-related physiological changes may occur. This study aims to characterize retinal function and morphology and establish reference values for future translational studies. Methods: Landrace pigs (16–20 weeks old) underwent standardized examinations of the left eye at baseline (16 weeks), 18 and 20 weeks. The left eye was examined by optical coherence tomography (OCT), fundus photography, histology, and full-field electroretinography (ffERG) under light- and dark-adapted conditions, using the ISCEV-compliant 6-step Dog, Cat, Nonhuman Primate protocol. Results: A total of 30 animals were included. Retinal morphology remained stable throughout the study period, with no significant changes in retinal thickness observed by OCT (baseline: 252 ± 24 µm; week 20: 249 ± 11 µm; p = 0.17) or by histology. ffERG revealed increased amplitudes under light- and dark-adapted conditions at 20 weeks compared to baseline at 16 weeks of age (e.g. light-adapted b-wave: + 65 µV, + 18.4%, p < 0.01), while latencies remained stable without clinically relevant changes. Conclusions: During this phase of rapid development, Landrace pigs undergo significant functional retinal maturation without corresponding morphological changes emphasizing importance of functional testing in retinal assessments. This study provides reference data in a large number of animals.

Therapeutic vaccines represent a promising treatment option for (pre)cancerous lesions, such as human papillomavirus-induced malignancies. They act via administration of tumor-specific antigens, leading to induction of antigen-specific cytotoxic T cell responses. However, vaccination efficiency is often limited when the antigen is administered alone, due to antigen instability and inefficient uptake by antigen-presenting cells (APCs). To address these limitations, nanoparticle-based vaccine delivery systems are currently under investigation. Here, we present a novel silica nanoparticle (SiNP)-based vaccine delivery platform that can be applied for the treatment of various diseases and cancer types. We show that surface-functionalized SiNPs are non-cytotoxic and quickly taken up by APCs. Incorporation of a linker/solubilizer sequence N-terminal of the epitope allows attachment of peptides regardless of their solubility as well as efficient processing and surface presentation by APCs. Whole-body distribution studies confirmed retention of the antigen at the injection site and decelerated excretion when connected to SiNPs. Furthermore, treatment with SiNPs, especially when combined with the adjuvant poly(I:C), resulted in activation of dendritic cells capable of priming CD8⁺ T cells. In C57BL/6 and MHC-humanized A2.DR1 mice, the SiNP-based vaccinations induced epitope-specific CD8⁺ T cells. Moreover, they exhibited anti-tumor activity and provided a survival benefit in a tumor model using HPV16 E6/E7-expressing PAP-A2 cells. Thus, the novel SiNP platform represents a promising new vehicle for therapeutic vaccine delivery.