Electrospun α-Lactalbumin Nanofibers for Site-Specific and Fast-Onset Delivery of Nicotine in the Oral Cavity: An In Vitro, Ex Vivo, and Tissue Spatial Distribution Study

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

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Low efficiency of leucocyte plugging-based drug delivery to cancer in mice

Drug Delivery and Translational Research ◽

10.1007/s13346-021-01028-y ◽

2021 ◽

Author(s):

Baifeng Qian ◽

Andreas Termer ◽

Christof M. Sommer ◽

Arianeb Mehrabi ◽

Eduard Ryschich

Keyword(s):

Drug Delivery ◽

Blood Vessels ◽

Ex Vivo ◽

Human Neutrophils ◽

Specific Cell ◽

Specific Drug ◽

Site Specific ◽

Tumour Blood

AbstractCells of the immune system were proposed for use as Trojan horse for tumour-specific drug delivery. The efficacy of such cell-based drug delivery depends on the site-specific cell homing. This present study was aimed to investigate the potential of leucocytes for intratumoural site-specific enrichment using a locoregional application route in experimental liver tumours. Human neutrophils were isolated from peripheral blood and directly labelled with calcein AM or loaded with doxorubicin. The neutrophil loading and release of doxorubicin and the migration and adhesion to ICAM-1 were analysed in vitro. Macrophages were isolated and activated in vitro. Leucocyte plugging and the distribution pattern in the liver microvasculature were studied ex vivo, and the efficacy of leucocyte plugging in tumour blood vessels was analysed in vivo after superselective intra-arterial injection in mouse liver tumour models. Neutrophils were characterised by the high dose-dependent uptake and rapid release of doxorubicin. Doxorubicin loading did not affect neutrophil migration function. Neutrophil plugging in liver microvasculature was very high (> 90%), both after ex vivo perfusion and after injection in vivo. However, neutrophils as well as activated macrophages plugged insufficiently in tumour blood vessels and passed through the tumour microvasculture with a very low sequestration rate in vivo. Neutrophils possess several properties to function as potentially effective drug carriers; however, the tumour site-specific drug delivery after selective locoregional injection was observed to be insufficient owing to low intratumoural microvascular plugging. Graphical abstract

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Site-Specific Radiolabeling of a Human PD-L1 Nanobody via Maleimide–Cysteine Chemistry

Pharmaceuticals ◽

10.3390/ph14060550 ◽

2021 ◽

Vol 14 (6) ◽

pp. 550

Author(s):

Dora Mugoli Chigoho ◽

Quentin Lecocq ◽

Robin Maximilian Awad ◽

Karine Breckpot ◽

Nick Devoogdt ◽

...

Keyword(s):

Checkpoint Inhibitors ◽

Ex Vivo ◽

Tracer Uptake ◽

Whole Body ◽

Cancer Therapies ◽

Tumor Biopsy ◽

Efficient Treatment ◽

Site Specific ◽

Ex Vivo Biodistribution

Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 °C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 ± 5% DC-RCY with a RCP > 99%, and was stable in injection buffer and human serum up to 3 h (>99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 ± 0.67% IA/g in the positive tumors compared with 0.42 ± 0.04% IA/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.

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Effects of the Tibetan herbal preparation PADMA 28 on blood lipids and lipid oxidisability in subjects with mild hypercholesterolaemia

VASA ◽

10.1024/0301-1526.34.1.11 ◽

2005 ◽

Vol 34 (1) ◽

pp. 11-17 ◽

Cited By ~ 12

Author(s):

Brunner-La Rocca ◽

Schindler ◽

Schlumpf ◽

Saller ◽

Suter

Keyword(s):

Endothelial Dysfunction ◽

Blood Lipids ◽

Ex Vivo ◽

Hdl Cholesterol ◽

Blood Lipid ◽

Tibetan Medicine ◽

Double Blind ◽

Intraarterial Infusion ◽

Padma 28

Background: Previous studies showed an anti-atherosclerotic effect of PADMA 28, an herbal formula based on Tibetan medicine. As the mechanisms of action are not fully understood, we investigated whether PADMA 28 may lower blood lipids and lipid oxidisability, and affect early endothelial dysfunction. Patients and methods: Sixty otherwise healthy subjects with total cholesterol ≥5.2 mmol/l and < 8.0 mmol/l were randomly assigned to placebo or PADMA 28, 3 x 2 capsules daily, for 4 weeks (double-blind). Blood lipids (total, LDL-, and HDL-cholesterol, triglycerides, Apo-lipoprotein A1 and B) and ex vivo lipid oxidisability were measured before and after treatment. In a subset of 24 subjects, endothelial function was assessed using venous occlusion plethysmography with intraarterial infusion of acetylcholine. Isolated LDL and plasma both untreated and pre-treated with PADMA 28 extract were oxidised by the radical generator AAPH. Conjugated diene formation was measured at 245 nm. Results: Blood lipids did not change during the study in both groups. In contrast to previous reports in mild hypercholesterolaemia, no endothelial dysfunction was seen and, consequently, was not influenced by therapy. Ex vivo blood lipid oxidisability was significantly reduced with PADMA 28 (area under curve: 5.29 ± 1.62 to 4.99 ± 1.46, p = 0.01), and remained unchanged in the placebo group (5.33 ± 1.88 to 5.18 ± 1.78, p > 0.1). This effect persisted one week after cessation of medication. In vitro experiments confirmed the prevention of lipid peroxidation in the presence of PADMA 28 extracts. Persistent protection was also seen for LDL isolated from PADMA 28-pretreated blood after being subjected to rigorous purification. Conclusions: This study suggests that the inhibition of blood lipid oxidisability by PADMA 28 may play a role in its anti-atherosclerotic effect.

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The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000116 ◽

2012 ◽

Vol 82 (3) ◽

pp. 228-232 ◽

Cited By ~ 7

Author(s):

Mauro Serafini ◽

Giuseppa Morabito

Keyword(s):

Antioxidant Capacity ◽

Dietary Intervention ◽

Ex Vivo ◽

The Body ◽

Dietary Polyphenols ◽

Enzymatic Antioxidant ◽

Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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