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360 million people are chronically infected with the human hepatitis B virus (HBV) and are consequently prone to develop liver cirrhosis and hepatocellular carcinoma 1 . As approved therapeutic regimens—which modulate patients' antiviral defenses or inhibit the viral reverse transcriptase—are generally noncurative, strategies interfering with other HBV replication steps are required. Expanding on our demonstration that acylated peptides derived from the large HBV envelope protein block virus entry in vitro 2 , 3 , 4 , 5 , we show their applicability to prevent HBV or woolly monkey hepatitis B virus infection in vivo , using immunodeficient urokinase-type plasminogen activator (uPA) mice repopulated with primary human or Tupaia belangeri hepatocytes 6 , 7 . Accumulation of the peptides in the liver, their extraordinary inhibitory potency and specific mode of action permit subcutaneous delivery at very low doses. Inhibition of hepadnavirus entry thus constitutes a therapeutic approach to prevent primary HBV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Infections caused by multidrug-resistant bacteria are a global emerging problem. New antibiotics that rely on innovative modes of action are urgently needed. Ranalexin is a potent antimicrobial peptide (AMP) produced in the skin of the American bullfrog Rana catesbeiana. Despite strong antimicrobial activity against Gram-positive bacteria, ranalexin shows disadvantages such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting exclusively of d-amino acids (named danalexin) was synthesized and compared to the original ranalexin for its antimicrobial potential and its biodistribution properties in a rat model. Danalexin showed improved biodistribution with an extended retention in the organisms of Wistar rats when compared to ranalexin. While ranalexin is rapidly cleared from the body, danalexin is retained primarily in the kidneys. Remarkably, both peptides showed strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria of the genus Acinetobacter with minimum inhibitory concentrations (MICs) between 4 and 16 mg/L (1.9–7.6 µM). Moreover, both peptides showed lower antimicrobial activities with MICs ≥32 mg/L (≥15.2 µM) against further Gram-negative bacteria. The preservation of antimicrobial activity proves that the configuration of the amino acids does not affect the anticipated mechanism of action, namely pore formation.

Multidrug-resistant bacteria represent one of the most important health care problems worldwide. While there are numerous drugs available for standard therapy, there are only a few compounds capable of serving as a last resort for severe infections. Therefore, approaches to control multidrug-resistant bacteria must be implemented. Here, a strategy of reactivating the established glycopeptide antibiotic vancomycin by structural modification with polycationic peptides and subsequent fatty acid conjugation to overcome the resistance of multidrug-resistant bacteria was followed. This study especially focuses on the structure–activity relationship, depending on the modification site and fatty acid chain length. The synthesized conjugates showed high antimicrobial potential on vancomycin-resistant enterococci. We were able to demonstrate that the antimicrobial activity of the vancomycin-lipopeptide conjugates depends on the chain length of the attached fatty acid. All conjugates showed good cytocompatibility in vitro and in vivo. Radiolabeling enabled the in vivo determination of pharmacokinetics in Wistar rats by molecular imaging and biodistribution studies. An improved biodistribution profile in comparison to unmodified vancomycin was observed. While vancomycin is rapidly excreted by the kidneys, the most potent conjugate shows a hepatobiliary excretion profile. In conclusion, these results demonstrate the potential of the structural modification of already established antibiotics to provide highly active compounds for tackling multidrug-resistant bacteria.

Lipid nanoparticles, including liposomes, have emerged as promising vehicles for the delivery of a variety of therapeutics. Several formulations have been approved and are used in medical practice—the COVID-19 mRNA vaccines represent the most recent milestone. Achieving effective oral delivery would elevate the potential of these formulations. Therefore, this study investigates the oral application of mRNA using liposomes as a nanocarrier system. A cyclic cell-penetrating peptide was coupled to the liposomal surface to allow uptake into the intestinal mucosal cells. The liposomes were loaded with mRNA (up to 112 µg/mL) and characterized in terms of their size (Z-average; 135.4 nm ± 1.1 nm), size distribution (polydispersity index (PDI); 0.213 ± 0.007 nm), surface charge (2.89 ± 0.27 mV), structure, lamellarity (multilamellar liposomes), and cargo capacity (>90%). The impact of freeze-drying and long-term storage of liposomal formulations was examined, and in vitro experiments on Caco-2 cells were conducted to evaluate the cytotoxicity of the liposomal formulations and demonstrate the uptake of the liposomes into cells. The efficiency of the formulations could be proven in vitro. When compared to control liposomes and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-liposomes, the new formulations exhibited significantly enhanced uptake in Caco-2 cells, an immortalized epithelial cell line. Moreover, the cytocompatibility of the formulations could be proven by the absence of cytotoxic effects on the viability of Caco-2 cells. Hence, this liposomal drug delivery system holds significant promise for the oral delivery of mRNA.

In this study, the potential of liposomes containing tetraether lipids (TELs) and cell‐penetrating peptides (CPPs) as a platform technology for oral delivery of peptides, using the glucagon‐like peptide 1 (GLP‐1) analogs semaglutide, exenatide, and dulaglutide as model compounds, was investigated. As this technology was proven effective in vitro and in vivo in mice and dogs for a small, hydrophilic glycopeptide drug (vancomycin) before, the focus of this study was to evaluate the applicability for peptide therapeutics with higher molecular weight (exenatide–4186.8 Da, semaglutide–4113.6 Da, and dulaglutide–59670.6 Da) and different physico‐chemical properties (semaglutide). For this purpose, liposomal formulations were prepared by dual centrifugation and characterized by size, polydispersity index, zeta potential, morphology, and encapsulation efficiency. All GLP‐1 analogs liposomal formulations revealed promising liposomal characteristics and were subsequently investigated with respect to uptake on colon‐carcinoma‐2 (CaCo‐2) cells and biocompatibility. Besides the high biocompatibility, a highly increased uptake of all three GLP‐1 analogs encapsulated into the liposomes containing TELs and CPPs in comparison to the free compound could be demonstrated on CaCo‐2 cells. These findings highlight the great potential of this liposomal technology for oral delivery of peptide therapeutics in general. Liposomes functionalized with tetraether lipids and cell‐penetrating peptides are explored as a platform technology for oral peptide delivery. Therefore, peptide drugs (GLP‐1 analogs) with different sizes and physicochemical properties are used as model drugs. The liposomes show favorable characteristics and increased cellular uptake by a simulated intestinal epithelium.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

Sepsis is the number one cause of death in intensive care units. This life-threatening condition is caused by bacterial infections and triggered by endotoxins of Gram-negative bacteria that leads to an overreaction of the immune system. The synthetic anti-lipopolysaccharide peptide Pep19-2.5 is a promising candidate for the treatment of sepsis as it binds sepsis-inducing lipopolysaccharides and thus prevents initiation of septic shock. For clinical evaluation precise quantification of the peptide in blood and tissue is required. As the peptide is not extractable from biological samples by commonly used methods there is a need for a new analysis method that does not rely on extraction of the peptide. In order to quantify the peptide by mass spectrometry, the peptide was synthesized containing 13C9,15N1-labeled phenylalanine residues. This modification offers high stability during acidic hydrolysis. Following acidic hydrolysis of the samples, the concentration of 13C9,15N1-labeled phenylalanine determined by LC-MS could be unambiguously correlated to the content of Pep19-2.5. Further experiments validated the accuracy of the data. Moreover, the quantification of Pep19-2.5 in different tissues (as studied in Wistar rats) was shown to provide comparable results to the results obtained with radioactively-labeled (14C) Pep19-2.5- Radioactive labeling is considered as the gold standard for quantification of compounds that refrain from reliable extraction methods. This novel method represents a valuable procedure for the determination of Pep19-2.5 and sticky peptides with unpredictable extraction properties in general.

Silicone oil endotamponades provide a reservoir for drugs in the eye. Following vitrectomy surgery to treat retinal detachments, extensive diabetic retinopathy or endophthalmitis, they can be used as long-term lipophilic depots. This study aimed to investigate the physicochemical properties of intravitreally applied drugs of different lipophilicity, namely vancomycin, ceftazidime and voriconazole. For this purpose, an in vitro model of the silicone-oil-filled eye compared to porcine vitreous bodies (PVBs) was used. In a glass container, either light or heavy silicone oil or PVB was set into equilibrium with an aqueous fluid. Vancomycin, voriconazole and ceftazidime were added in concentrations commonly applied in clinical practice. The time course of the concentration of the drugs was determined in the hydrophilic phase for up to 24 h. With silicone oil present, the concentrations of vancomycin, voriconazole and ceftazidime were elevated in the aqueous humor when compared to the vitreous body (p < 0.001 for all drugs). With increasing lipophilicity, higher concentrations of the drug dissolved in silicone oil after 24 h (52.7%, 49.1% and 34.3% for vancomycin, ceftazidime and voriconazole, respectively). While no difference between lighter- and heavier-than-water silicone oil was apparent for vancomycin and ceftazidime (p = 0.17 and p = 0.72), voriconazole dissolved significantly better in the heavier-than-water silicone oil (p = 0.002). A higher-than-expected percentage of the glycopeptide vancomycin dissolved in the porcine vitreous body, possibly due to protein binding. In conclusion, silicone oils influence the drug concentration and distribution of intravitreally applied drugs depending on their lipophilicity. The addition of F6H8 used to create heavy silicone oils attenuates these effects for lipophilic drugs. Knowledge of the distribution of these intravitreally applied drugs is crucial to ensure the desired anti-infectious effect.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage, which was cloned more than 20 years ago. Here we uncovered a novel function of GM-CSF in the central nervous system (CNS). We identified the GM-CSF α-receptor as an upregulated gene in a screen for ischemia-induced genes in the cortex. This receptor is broadly expressed on neurons throughout the brain together with its ligand and induced by ischemic insults. In primary cortical neurons and human neuroblastoma cells, GM-CSF counteracts programmed cell death and induces BCL-2 and BCL-Xl expression in a dose- and time-dependent manner. Of the signaling pathways studied, GM-CSF most prominently induced the PI3K-Akt pathway, and inhibition of Akt strongly decreased antiapoptotic activity. Intravenously given GM-CSF passes the blood—brain barrier, and decreases infarct damage in two different experimental stroke models (middle cerebral artery occlusion (MCAO), and combined common carotid/distal MCA occlusion) concomitant with induction of BCL-Xl expression. Thus, GM-CSF acts as a neuroprotective protein in the CNS. This finding is remarkably reminiscent of the recently discovered functionality of two other hematopoietic factors, erythropoietin and granulocyte colony-stimulating factor in the CNS. The identification of a third hematopoietic factor acting as a neurotrophic factor in the CNS suggests a common principle in the functional evolution of these factors. Clinically, GM-CSF now broadens the repertoire of hematopoietic factors available as novel drug candidates for stroke and neurodegenerative diseases.

As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity.