Temperature Responsive Polymer Conjugate Prepared by "Grafting from" Proteins toward the Adsorption and Removal of Uremic Toxin.

In this study, temperature-responsive polymer-protein conjugate was synthesized using a "grafting from" concept by introducing a chain transfer agent (CTA) into bovine serum albumin (BSA). The BSA-CTA was used as a starting point for poly(N-isopropylacrylamide) (PNIPAAm) through reversible addition-fragmentation chain transfer polymerization. The research investigations suggest that the thermally responsive behavior of PNIPAAm was controlled by the monomer ratio to CTA, as well as the amount of CTA introduced to BSA. The study further synthesized the human serum albumin (HSA)-PNIPAAm conjugate, taking the advantage that HSA can specifically adsorb indoxyl sulfate (IS) as a uremic toxin. The HSA-PNIPAAm conjugate could capture IS and decreased the concentration by about 40% by thermal precipitation. It was also revealed that the protein activity was not impaired by the conjugation with PNIPAAm. The proposed strategy is promising in not only removal of uremic toxins but also enrichment of biomarkers for early diagnostic applications.

Artificial Viral Capsid Dressed Up with Human Serum Albumin.

Capsid of tomato bushy stunt virus consists of an outer coat protein shell decorated on an internal skeleton comprising a ß-annulus motif. We mimicked this capsid structure with our artificial viral capsid dressed up with protein. We synthesized the ß-annulus peptide bearing a Cys at the C-terminal side and linked it with Cys34 of the human serum albumin (HSA) via a bismaleimide linker. The ß-annulus peptide-HSA conjugate self-assembled into spherical structures of a 50-70 nm size range in the Tris-HCl buffer, with the ζ-potential of assemblies of such conjugate revealing that HSA proteins were displayed on the outer surface of the artificial viral capsid. Interestingly, the critical aggregation concentration (CAC) of the conjugate in the Tris-HCl buffer at 25 °C was approximately 0.01 µM, or 1/2500 lower than that of the unmodified ß-annulus peptides, suggesting that the artificial viral capsids were stabilized via HSA modification. The present strategy of constructing protein nanocapsule by self-assembly of a ß-annulus peptide-protein conjugate is simpler than that of previously reported protein nanocapsules.

Synthesis and Fabrication of Surface-Active Microparticles Using a Membrane Emulsion Technique and Conjugation of Model Protein via Strain-Promoted Azide-Alkyne Click Chemistry in Physiological Conditions.

The rapid surface immobilization of protein on monodispersed polyester microcarriers is reported. A model protein, functionalized with a dibenzocyclooctyne core, immobilizes on the surface of azide-terminal polycaprolactone microcarriers within 10 min compared to 12 h for other conjugation techniques, and it is conducted in physiological conditions and in the absence of coupling reagents.

Development of 99mTc-Labeled Human Serum Albumin with Prolonged Circulation by Chelate-then-Click Approach: A Potential Blood Pool Imaging Agent.

Technetium-99m-labeled human serum albumin (99mTc-HSA) has been utilized as a blood pool imaging agent in the clinic for several decades. However, 99mTc-HSA has a short circulation time, which is a critical shortcoming for a blood pool imaging agent. Herein, we developed a novel 99mTc-labeled HSA with a long circulation time using click chemistry and a chelator, 2,2'-dipicolylamine (DPA), (99mTc-DPA-HSA). Specifically, we examined the feasibility of copper-free strain-promoted alkyne-azide cycloaddition (SPAAC) for the incorporation of HSA to the [99mTc (CO)3(H2O)3]+ system by adopting a chelate-then-click approach. In this strategy, a potent chelate system, azide-functionalized DPA, was first complexed with [99mTc (CO)3(H2O)3]+, followed by the SPAAC click reaction with azadibenzocyclooctyne-functionalized HSA (ADIBO-HSA) under biocompatible conditions. Radiolabeling efficiency of azide-functionalized DPA (99mTc-DPA) was >98%. Click conjugation efficiency of 99mTc-DPA with ADIBO-HSA was between 76 and 99% depending on the number of ADIBO moieties attached to HSA. In whole-body in vivo single photon emission computed tomography images, the blood pool uptakes of 99mTc-DPA-HSA were significantly enhanced compared to those of 99mTc-HSA at 10 min, 2, and 6 h after the injection ( P < 0.001, 0.025, and 0.003, respectively). Furthermore, the blood activities of 99mTc-DPA-HSA were 8 times higher at 30 min and 10 times higher at 3 h after the injection compared to those of conventional 99mTc-HSA in ex vivo biodistribution experiment. The results exhibit the potential of 99mTc-DPA-HSA as a blood pool imaging agent and further illustrate the promise of the pre-labeling SPAAC approach for conjugation of heat-sensitive biological targeting vectors with [99mTc (CO)3(H2O)3]+.

Protein Nanoparticle Fabrication for Optimized Reticuloendothelial System Evasion and Tumor Accumulation.

Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.

Docetaxel-loaded human serum albumin (HSA) nanoparticles: synthesis, characterization, and evaluation.

BACKGROUND: Docetaxel (DTX) is an anticancer drug that is currently formulated with polysorbate 80 and ethanol (50:50, v/v) in clinical use. Unfortunately, this formulation causes hypersensitivity reactions, leading to severe side-effects, which have been primarily attributed to polysorbate 80. METHODS: In this study, a DTX-loaded human serum albumin (HSA) nanoparticle (DTX-NP) was designed to overcome the hypersensitivity reactions that are induced by polysorbate 80. The methods of preparing the DTX-NPs have been optimized based on factors including the drug-to-HSA weight ratio, the duration of HSA incubation, and the choice of using a stabilizer. Synthesized DTX-NPs were characterized with regard to their particle diameters, drug loading capacities, and drug release kinetics. The morphology of the DTX-NPs was observed via scanning electron microscopy (SEM) and the successful preparation of DTX-NPs was confirmed via differential scanning calorimetry (DSC). The cytotoxicity and cellular uptake of DTX-NPs were investigated in the non-small cell lung cancer cell line A549 and the maximum tolerated dose (MTD) of DTX-NPs was evaluated via investigations with BALB/c mice. RESULTS: The study showed that the loading capacity and the encapsulation efficiency of DTX-NPs prepared under the optimal conditions was 11.2 wt% and 63.1 wt%, respectively and the mean diameter was less than 200 nm, resulting in higher permeability and controlled release. Similar cytotoxicity against A549 cells was exhibited by the DTX-NPs in comparison to DTX alone while higher maximum tolerated dose (MTD) with the DTX-NPs (75 mg/kg) than with DTX (30 mg/kg) was demonstrated in mice, suggesting that the DTX-NPs prepared with HSA yielded similar anti-tumor activity but were accompanied by less systemic toxicity than solvent formulated DTX. CONCLUSIONS: DTX-NPs warrant further investigation and are promising candidates for clinical applications.

Human Serum Albumin, a Suitable Candidate to Stabilize Freeze-Dried IgG in Combination with Trehalose: Central Composite Design.

Freeze-dried immunoglobulin G (IgG) incorporating trehalose and human serum albumin (HSA) was statistically evaluated regarding the existence of synergism between additives on the stability profile. The levels of HSA (X1) and trehalose (X2) were independent variables. Aggregation following the process (Y1), after 2 and 3 months at 40°C (Y2) and (Y3), respectively, along with the rate constant of aggregation (Y4) were dependent variables. Aggregation and beta-sheet conformation were quantified through size-exclusion chromatography (SEC-HPLC) and Fourier transform infrared spectroscopy (FTIR). Central composite design (CCD) suggested the best formulation. The integrity and thermodynamic stability of optimized formulation were investigated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC). The calculated responses were Y1, 0-0.90%; Y2, 0.4-4.3%; Y3, 2.10-13.46%; and Y4, 0.16-0.69 1/month. The optimized formulation had 10 mg IgG, 86 mg trehalose, and 1 mg HSA with observed responses of Y1, 0.01%; Y2, 0.51%; Y3, 3.08%; and Y4, 0.33 1/month. The models were statistically well-fitted. The optimized formulation was amorphous during freeze-drying (FD), and no fragmentation was observed. Trehalose and HSA demonstrated statistical synergism. CCD was successfully employed to recommend the best ratio of stabilizers and achieve the maximum stabilization of IgG as a model freeze-dried antibody.

Coordination-Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

Preparation and in Vitro Analysis of Human Serum Albumin Nanoparticles Loaded with Anthracycline Derivatives.

Nanoparticles prepared using human serum albumin (HSA) have emerged as versatile carriers for improving the pharmacokinetic profile of drugs. The desolvation of HSA using ethanol followed by stabilization through crosslinking with glutaraldehyde is a common technique for preparing HSA nanoparticles, but our knowledge concerning the characteristics (or functions) of HSA nanoparticles and their efficiency when loaded with drugs is limited. To address this issue in more detail, we prepared anthracycline-loaded HSA nanoparticles. Doxorubicin-loaded HSA nanoparticles with a size similar to doxorubicin-unloaded particles could be prepared by desolvating at a higher pH (8-9), and the size (100-150 nm) was optimum for delivery to tumor tissues. Using this procedure, HSA nanoparticles were loaded with other anthracycline derivatives, and all showed cytotoxicity in cancer cells. However, the efficiency of drug loading and dissolution rate were different among them possibly due to the differences in the type of association of the drugs on nanoparticles (doxorubicin and daunorubicin; covalently bound to nanoparticles, pirarubicin; both covalently bound to and adsorbed on nanoparticles, aclarubicin; adsorbed on nanoparticles). Since the formulation of such drug-loaded HSA nanoparticles should be modified for efficient delivery to tumors, the findings reported herein provide the useful information for optimizing the formulation and the production process for the HSA nanoparticles using a desolvation technique.

Albumin conjugates and assemblies as versatile bio-functional additives and carriers for biomedical applications.

As the most abundant plasma protein, serum albumin has been extensively studied and employed for therapeutic applications. Despite its direct clinical use for the maintenance of blood homeostasis in various medical conditions, this review exclusively summarizes and discusses albumin-based bio-conjugates and assemblies as versatile bio-functional additives and carriers in biomedical applications. As one of the smallest-sized proteins in the human body, albumin is physiochemically stable and biochemically inert. Moreover, albumin is also endowed with abundant specific binding sites for numerous therapeutic compounds, which also endow it with superior bioactivities. Firstly, due to its small size and binding specificity, albumin alone or its derived assemblies can be utilized as competent drug carriers, which can deliver drugs through the enhanced permeability and retention (EPR) effect or actively target lesion sites through binding with gp60 and secreted protein acidic and rich in cysteine (SPARC) in tumor sites. Furthermore, its biochemical stability and inertness make it a safe and biocompatible coating material for use in biomedical applications. Albumin-based surface modifying additives can be used to functionalize both macro substrates (e.g. surfaces of medical devices or implants) and nanoparticle surfaces (e.g. drug carriers and imaging contrast agents). In this review, we elaborate on the synthesis and applications of albumin-based bio-functional coatings and drug carriers, respectively.

Isobaric duplex based on a combination of 16O/18O enzymatic exchange and labeling with pyrylium salts.

Enzymatic 18O exchange, the well-established approach in comparative proteomics, has some disadvantages such as back exchange of labeled oxygen and overlapping the peak of a labeled peptide with isotopic peaks of an unlabeled one. Herein we demonstrated a simple procedure in which samples digested with a trypsin (with and without H218O) were reacted with unlabeled and quadrupled 13C-labeled pyrylium salt respectively which results in formation of pyridinium cations. Thus, each isobarically labeled peptide containing zero or four 13C atoms in the mass reporter group, during tandem MS/MS forms an unique reporter ion useful for a relative quantitation. Such a sample treatment improves the signal to noise ratio, reduces overlapping of the isotopic peaks and completely eliminates the back exchange problem.

An artificial metalloenzyme biosensor can detect ethylene gas in fruits and Arabidopsis leaves.

Enzyme biosensors are useful tools that can monitor rapid changes in metabolite levels in real-time. However, current approaches are largely constrained to metabolites within a limited chemical space. With the rising development of artificial metalloenzymes (ArM), a unique opportunity exists to design biosensors from the ground-up for metabolites that are difficult to detect using current technologies. Here we present the design and development of the ArM ethylene probe (AEP), where an albumin scaffold is used to solubilize and protect a quenched ruthenium catalyst. In the presence of the phytohormone ethylene, cross metathesis can occur to produce fluorescence. The probe can be used to detect both exogenous- and endogenous-induced changes to ethylene biosynthesis in fruits and leaves. Overall, this work represents an example of an ArM biosensor, designed specifically for the spatial and temporal detection of a biological metabolite previously not accessible using enzyme biosensors.

Encoding function into polypeptide-oligonucleotide precision biopolymers.

We report a novel synthesis strategy to prepare precision polymers providing exact chain lengths, molecular weights and monomer sequences that allow post modifications by convenient DNA hybridization. Two grafted single strand DNA (ssDNA) side chains serve as a versatile platform for sequence-specific attachment of chromophores, proteins, cell-targeting peptide, and a Y-shape DNA linker. This approach resembles a LEGO®-type incorporation of functionalities to create functional biopolymers of high structure definition under mild conditions.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery.

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Effect of peroxynitrite on human serum albumin: a multi technique approach.

In this study, human serum albumin (HSA), the most abundant protein of blood plasma, was modified with varying concentrations of peroxynitrite. The peroxynitrite-induced changes in HSA was monitored by spectroscopy, SDS-PAGE, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal denaturation studies, and matrix-assisted laser desorption/inonization-time of flight mass spectrometry (MALDI-TOF MS). Aggregate formation was studied by thioflavin T binding and scanning electron microscopy (SEM). The results indicated formation of 3-nitrotyrosine, 6-nitrotryptophan, dityrosine, and carbonyls in modified samples and showed retarded mobility in SDS-polyacrylamide gel. Reduction in α-helicity and surface protein hydrophobicity confirmed the secondary and tertiary structure alterations in peroxynitrite-modified-HSA. Also, attachment of nitro group and increase in melting temperature was observed in modified sample. Furthermore, significant enhancement in the fluorescence intensity of ThT upon binding with peroxynitrite-modified-HSA and images under scanning electron microscope are suggestive of protein aggregation. It is, therefore, speculated that HSA modified by endogenously formed peroxynitrite might act as a trigger for nitration/aggregation and suggested the role of peroxynitrite-modified-HSA in SLE.