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Open AccessArticle

A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin α_vβ₆ Binding Peptide

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Pharmaceutics 2022, 14(4), 745; https://doi.org/10.3390/pharmaceutics14040745 - 30 Mar 2022

Viewed by 1413

Abstract

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when [...] Read more.

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin α_vβ₆ binding peptide (α_vβ₆-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified α_vβ₆-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [⁶⁴Cu]Cu DOTA-EB-α_vβ₆-BP ([⁶⁴Cu]1) and [⁶⁴Cu]Cu DOTA-IP-α_vβ₆-BP ([⁶⁴Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroβ6 (α_vβ₆ +) and DX3puro (α_vβ₆ −), and pancreatic BxPC-3 (α_vβ₆ +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [⁶⁴Cu]1 and [⁶⁴Cu]2 was 53–63% and 42–44%, respectively, with good human serum stability (24 h: [⁶⁴Cu]1 76%, [⁶⁴Cu]2 90%). Selective α_vβ₆ cell binding was observed for both [⁶⁴Cu]1 and [⁶⁴Cu]2 (α_vβ₆ (+) cells: 30.3–55.8% and 48.5–60.2%, respectively, vs. α_vβ₆ (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([⁶⁴Cu]1 and [⁶⁴Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improvement over the non-ABM parent peptide and thereby providing improved PET images. Comparing [⁶⁴Cu]1 and [⁶⁴Cu]2, the IP-ABM-α_vβ₆-BP [⁶⁴Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the α_vβ₆ (+) tumor by PET imaging. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells

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Itzik Cooper

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Michal Schnaider-Beeri

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Mati Fridkin

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Yoram Shechter

Pharmaceutics 2022, 14(1), 71; https://doi.org/10.3390/pharmaceutics14010071 - 28 Dec 2021

Viewed by 900

Abstract

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative [...] Read more.

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Molecular-Level Release of Coumarin-3-Carboxylic Acid and Warfarin-Derivatives from BSA-Based Hydrogels

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Niuosha Sanaeifar

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Karsten Mäder

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Dariush Hinderberger

Pharmaceutics 2021, 13(10), 1661; https://doi.org/10.3390/pharmaceutics13101661 - 11 Oct 2021

Cited by 4 | Viewed by 1707

Abstract

This investigation aimed at developing BSA hydrogels as a controlled release system to study the release behavior of spin-labeled coumarin-3-carboxylic acid (SL-CCS) and warfarin (SL-WFR). The release profiles of these spin-labeled (SL-) pharmaceuticals from BSA hydrogels prepared with different procedures are compared in [...] Read more.

This investigation aimed at developing BSA hydrogels as a controlled release system to study the release behavior of spin-labeled coumarin-3-carboxylic acid (SL-CCS) and warfarin (SL-WFR). The release profiles of these spin-labeled (SL-) pharmaceuticals from BSA hydrogels prepared with different procedures are compared in detail. The mechanical properties of the gels during formation and release were studied via rheology, while a nanoscopic view on the release behavior was achieved by analyzing SL-drugs–BSA interaction using continuous wave electron paramagnetic resonance (CW EPR) spectroscopy. The influence of type of drug, drug concentration, duration of gel formation, and gelation methods on release behavior were characterized by CW EPR spectroscopy, EPR imaging (EPRI), and dynamic light scattering (DLS), which provide information on the interaction of BSA with SL-drugs, the percentage of drug inside the hydrogel and the nature and size of the released structures, respectively. We found that the release rate of SL-CCS and SL-WFR from BSA hydrogels is tunable through drug ratios, hydrogel incubation time and gelation procedures. All of the results indicate that BSA hydrogels can be potentially exploited in controlled drug delivery applications. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Albumin-EDTA-Vanadium Is a Powerful Anti-Proliferative Agent, Following Entrance into Glioma Cells via Caveolae-Mediated Endocytosis

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Pharmaceutics 2021, 13(10), 1557; https://doi.org/10.3390/pharmaceutics13101557 - 25 Sep 2021

Cited by 2 | Viewed by 1126

Abstract

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA [...] Read more.

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH₂)₂-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC₅₀ values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO⁺⁺ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices

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Meike-Kristin Abraham

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Pharmaceutics 2021, 13(9), 1504; https://doi.org/10.3390/pharmaceutics13091504 - 18 Sep 2021

Cited by 3 | Viewed by 1504

Abstract

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new [...] Read more.

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA-CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA-CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA-CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

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Byungseop Yang

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Inchan Kwon

Pharmaceutics 2021, 13(8), 1298; https://doi.org/10.3390/pharmaceutics13081298 - 19 Aug 2021

Cited by 3 | Viewed by 1678

Abstract

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter [...] Read more.

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

The Therapeutic Effect of Human Serum Albumin Dimer-Doxorubicin Complex against Human Pancreatic Tumors

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Pharmaceutics 2021, 13(8), 1209; https://doi.org/10.3390/pharmaceutics13081209 - 05 Aug 2021

Cited by 4 | Viewed by 1890

Abstract

Human serum albumin (HSA) is a versatile drug carrier with active tumor targeting capacity for an antitumor drug delivery system. Nanoparticle albumin-bound (nab)-technology, such as nab-paclitaxel (Abraxane^®), has attracted significant interest in drug delivery research. Recently, we demonstrated that HSA dimer [...] Read more.

Human serum albumin (HSA) is a versatile drug carrier with active tumor targeting capacity for an antitumor drug delivery system. Nanoparticle albumin-bound (nab)-technology, such as nab-paclitaxel (Abraxane^®), has attracted significant interest in drug delivery research. Recently, we demonstrated that HSA dimer (HSA-d) possesses a higher tumor distribution than HSA monomer (HSA-m). Therefore, HSA-d is more suitable as a drug carrier for antitumor therapy and can improve nab technology. This study investigated the efficacy of HSA-d-doxorubicin (HSA-d-DOX) as next-generation nab technology for tumor treatment. DOX conjugated to HSA-d via a tunable pH-sensitive linker for the controlled release of DOX. Lyophilization did not affect the particle size of HSA-d-DOX or the release of DOX. HSA-d-DOX showed significantly higher cytotoxicity than HSA-m-DOX in vitro. In the SUIzo Tumor-2 (SUIT2) human pancreatic tumor subcutaneous inoculation model, HSA-d-DOX could significantly inhibit tumor growth without causing serious side effects, as compared to the HSA binding DOX prodrug, which utilized endogenous HSA as a nano-drug delivery system (DDS) carrier. These results indicate that HSA-d could function as a natural solubilizer of insoluble drugs and an active targeting carrier in intractable tumors with low vascular permeability, such as pancreatic tumors. In conclusion, HSA-d can be an effective drug carrier for the antitumor drug delivery system against human pancreatic tumors. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Development of In Situ Gelling Meloxicam-Human Serum Albumin Nanoparticle Formulation for Nose-to-Brain Application

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Pharmaceutics 2021, 13(5), 646; https://doi.org/10.3390/pharmaceutics13050646 - 01 May 2021

Cited by 10 | Viewed by 2176

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The aim of this study was to develop an intranasal in situ thermo-gelling meloxicam-human serum albumin (MEL-HSA) nanoparticulate formulation applying poloxamer 407 (P407), which can be administered in liquid state into the nostril, and to increase the resistance of the formulation against mucociliary [...] Read more.

The aim of this study was to develop an intranasal in situ thermo-gelling meloxicam-human serum albumin (MEL-HSA) nanoparticulate formulation applying poloxamer 407 (P407), which can be administered in liquid state into the nostril, and to increase the resistance of the formulation against mucociliary clearance by sol-gel transition on the nasal mucosa, as well as to improve drug absorption. Nanoparticle characterization showed that formulations containing 12–15% w/w P407 met the requirements of intranasal administration. The Z-average (in the range of 180–304 nm), the narrow polydispersity index (PdI, from 0.193 to 0.328), the zeta potential (between −9.4 and −7.0 mV) and the hypotonic osmolality (200–278 mOsmol/L) of MEL-HSA nanoparticles predict enhanced drug absorption through the nasal mucosa. Based on the rheological, muco-adhesion, drug release and permeability studies, the 14% w/w P407 containing formulation (MEL-HSA-P14%) was considered as the optimized formulation, which allows enhanced permeability of MEL through blood–brain barrier-specific lipid fraction. Cell line studies showed no cell damage after 1-h treatment with MEL-HSA-P14% on RPMI 2650 human endothelial cells’ moreover, enhanced permeation (four-fold) of MEL from MEL-HSA-P14% was observed in comparison to pure MEL. Overall, MEL-HSA-P14% can be promising for overcoming the challenges of nasal drug delivery. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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Open AccessArticle

Effect of C-terminus Conjugation via Different Conjugation Chemistries on In Vivo Activity of Albumin-Conjugated Recombinant GLP-1

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Pharmaceutics 2021, 13(2), 263; https://doi.org/10.3390/pharmaceutics13020263 - 15 Feb 2021

Cited by 1 | Viewed by 1731

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Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum [...] Read more.

Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1_8G16AzF-HSA showed only moderate in vivo glucose-lowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucose-lowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1_8G37AzF-HSA and GLP1_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1_8G16AzF-HSA, while GLP1_8G37AzF-HSA did not. Another GLP-1 variant, GLP1_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucose-lowering activity and prolonged plasma half-life in vivo. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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