Search Results (88)

This work presents the synthesis, characterisation, photophysical properties, time-resolved spectroscopic behaviour, and biological evaluation of two structurally distinct heavy-atom-free BODIPY-anthracene dyads (BDP-1) and the newly designed 2,6-bis[1-(tert-butyl) 4-(prop-2-yn-1-yl) piperazine-1,4-dicarboxylate] BODIPY-anthracene (BDP-2), incorporating 2,6-alkynyl-piperazine substituents for potential application in antimicrobial photodynamic therapy. BDP-1 exhibits absorption and emission maxima at 507 nm and 516 nm, respectively, with a Stokes shift of 344 cm⁻¹ in dichloromethane (DCM), characteristic of unsubstituted BODIPYs. In contrast, BDP-2 undergoes a red-shift in the absorption maximum to 552 nm (Stokes shift of 633 cm⁻¹), which is attributed to the extended conjugation from the introduction of the alkyne groups. Time-resolved infrared spectroscopy confirmed efficient spin-orbit charge transfer intersystem crossing, and nanosecond transient absorption studies confirmed the formation of a long-lived triplet state for BDP-2 (up to 138 µs in MeCN). A binding constant (K_b) of 9.6 × 10⁴ M⁻¹ was obtained for BDP-2 when titrated with bovine serum albumin (BSA), which is higher than comparable BODIPY derivatives. BDP-2 displayed improved hemocompatibility compared to BDP-1 (<5% haemolysis of human erythrocytes up to 200 μg·mL⁻¹). Antimicrobial activity of BDP-1 and BDP-2 was most potent when irradiated at 370 nm compared to the other wavelengths employed. However, BDP-2 did not retain the potent (6 log) and rapid (within 15 min) eradication of Staphylococcus aureus achieved by BDP-1 under irradiation at 370 nm. These findings demonstrate the rational design of BDP-2 as a biocompatible, and heavy-atom-free BODIPY offering promise for targeted antimicrobial photodynamic therapeutic applications. Full article

Background/Objectives: Paclitaxel (PTX) is a potent anticancer drug that is poorly soluble in water. To enhance its delivery efficiency in aqueous environments, amphiphilic polymer micelles are often used as nanocarriers for PTX in clinical settings. However, the hydrophilic polymer segments on the surface of these micelles may possess potential immunogenicity, posing risks in clinical applications. To address this issue, nanomicelles based on human serum albumin (HSA)–hydrophobic polymer conjugates constructed via site-specific in situ polymerization-induced self-assembly (SI-PISA) are considered a promising alternative. The HSA shell not only ensures good biocompatibility but also enhances cellular uptake because of endogenous albumin trafficking pathways. Moreover, compared to traditional methods of creating protein–hydrophobic polymer conjugates, SI-PISA demonstrates higher reaction efficiency and better preservation of protein functionality. Methods: We synthesized HSA-PMEMA nanomicelles via SI-PISA using HSA and methoxyethyl methacrylate (MEMA)—a novel hydrophobic monomer with a well-defined and stable chemical structure. The protein activity and the PTX intracellular delivery efficiency of HSA-PMEMA nanomicelles were evaluated. Results: The CD spectra of HSA and HSA-PMEMA exhibited similar shapes, and the relative esterase-like activity of HSA-PMEMA was 94% that of unmodified HSA. Flow cytometry results showed that Cy7 fluorescence intensity in cells treated with HSA-PMEMA-Cy7 was approximately 1.35 times that in cells treated with HSA-Cy7; meanwhile, HPLC results indicated that, under the same conditions, the PTX loading per unit protein mass on HSA-PMEMA was approximately 1.43 times that of HSA. These collectively contributed to a 1.78-fold overall PTX intracellular delivery efficiency of HSA-PMEMA compared to that of HSA. Conclusions: In comparison with HSA, HSA-PMEMA nanomicelles exhibit improved cellular uptake and higher loading efficiency for PTX, effectively promoting the intracellular delivery of PTX. Tremendous potential lies in these micelles for developing safer and more efficient next-generation PTX formulations for tumor treatment. Full article

Background/Objectives: The principal aim of this work was to prepare a liposomal drug delivery system based on the commercial drug doxorubicin (DOX) and a budding agent with promising anticancer activity, 9-(N-piperazinyl)-5-methyl-12(H)-quino [3,4-b][1,4]benzothiazinium chloride (9-PBThACl). Methods: A spectrophotometric methodology was used to meticulously investigate the drug entrapment and release characteristics of the new liposomal complexes (L) based on dipalmitoylphosphatidylcholine (DPPC) with human serum albumin (HSA) and its defeated analog (dHSA). Results: The impact of the operational parameters (temperature and pH) on the liposome/drug(s)/(d)HSA, namely [L_{DPPC/9-PBThACl/DOX} ]:(d)HSA] systems, as well as the polarity of the phospholipid bilayer, was examined. In order to compare the experimental findings, mathematical models were employed to specify the analytical factors controlling the process of drug release/potential drug release from liposomes. The observed variations in the drug encapsulation and release profiles were due to the combination of liposomal conjugates with human plasma protein. Conclusions: It was proven that changes in the environmental pH directly affect the percentage of drug entrapment in liposomes and the medicine release efficiency. Moreover, the grouping tendency of the liposomal combinations was investigated using a principal component analysis (PCA) and a hierarchical clustering analysis (HCA). Finally, an analysis of variance (ANOVA) confirmed the statistical impact of pH buffering and changing temperature factors on the drug release characteristics of liposomal conjugates. Full article

The formation of protein coronas around engineered nanoparticles (ENPs) in biological environments is critical in nanomedicine, as these coronas significantly influence the biological behavior of ENPs. Despite extensive research on protein coronas, understanding the in situ influence of whole (soft plus hard) protein coronas has remained challenging. In this study, we demonstrate a strategy to assess the in situ effects of whole coronas on the model biosensing of anti-IgG using IgG-conjugated gold nanoparticles (IgG-AuNPs) through fluorescence nanoparticle tracking analysis (F-NTA), which enables the selective tracking of fluorescent particles within complex media. In our approach, anti-IgG and IgG-AuNPs were labeled with distinct fluorescent dyes. The accordance in hydrodynamic diameter distributions observed at two different wavelengths verifies the successful capture of anti-IgG on the IgG-AuNPs. The counting of fluorescent anti-IgG within the size distribution allows for a quantitative assessment of biosensing efficiency. This method was applied to evaluate the effects of four protein coronas—human serum albumin, high-density lipoproteins, immunoglobulin G, and fibrinogen—as well as their mixture across varying incubation times and concentrations. The results suggest that the physical presence of whole protein coronas surrounding the IgG-AuNPs may assist the biosensing interaction in situ rather than screening it. Full article

We report the design and development of a novel multifunctional nanostructure, RB-AuSiO₂_HSA-DOX, where tri-modal cancer treatment strategies—photothermal therapy (PTT), photodynamic therapy (PDT), chemotherapy—luminescent properties and targeting are integrated into the same scaffold. It consists of a gold core with optical and thermo-plasmonic properties and is covered by a silica shell entrapping a well-known photosensitizer and luminophore, Rose Bengal (RB). The nanoparticle surface was decorated with Human Serum Albumin (HSA) through a covalent conjugation to confer its targeting abilities and as a carrier of Doxorubicin (DOX), one of the most effective anticancer drugs in clinical chemotherapy. The obtained nanostructure was fully characterized through transmission electron microscopy (TEM), dynamic light scattering (DLS) and UV-visible spectroscopy, with a homogeneous and spherical shape, an average diameter of about 60 nm and negative ζ-potential value Singlet oxygen generation and photothermal properties were explored under green light irradiation. The interaction between DOX-HSA anchored on the nanoplatform was investigated by fluorescence spectroscopy and compared to that of DOX-HSA, pointing out different accessibility of the drug molecules to the HSA binding sites, whether the protein is free or bound to the nanoparticle surface. To the best of our knowledge, there are no studies comparing a drug–HSA interaction with that of the same protein anchored to nanoparticles. Furthermore, the uptake of RB-AuSiO₂_HSA-DOX into MDA-MB-231 mammary cells was assessed by confocal imaging, highlighting—at early time of incubation and as demonstrated by the increased DOX luminescence displayed within cells—a better internalization of the carried anticancer drug compared to the free one, making the obtained nanostructure a suitable and promising platform for an anticancer multimodal approach. Full article

The objective of this study was to compare the properties of core–shell nanoparticles with a PLGA core and shells composed of different types of polymers, focusing on their structural integrity. The core PLGA nanoparticles were prepared either through a high-pressure homogenization–solvent evaporation technique or nanoprecipitation, using poloxamer 188 (P188), a copolymer of divinyl ether with maleic anhydride (DIVEMA), and human serum albumin (HSA) as the shell-forming polymers. The shells were formed through adsorption, interfacial embedding, or conjugation. For dual fluorescent labeling, the core- and shell-forming polymers were conjugated with Cyanine5, Cyanine3, and rhodamine B. The nanoparticles had negative zeta potentials and sizes ranging from 100 to 250 nm (measured using DLS) depending on the shell structure and preparation technique. The core–shell structure was confirmed using TEM and fluorescence spectroscopy, with the appearance of FRET phenomena due to the donor–acceptor properties of the labels. All of the shells enhanced the cellular uptake of the nanoparticles in Gl261 murine glioma cells. The integrity of the core–shell structures upon their incubation with the cells was evidenced by intracellular colocalization of the fluorescent labels according to the Manders’ colocalization coefficients. This comprehensive approach may be useful for the selection of the optimal preparation method even at the early stages of the core–shell nanoparticle development. Full article

This study introduces novel CD44-targeted and redox-responsive nanoparticles (FNPs), proposed as doxorubicin (DOX) delivery devices for breast cancer. A cationized and redox-responsive Human Serum Albumin derivative was synthesized by conjugating Human Serum Albumin with cystamine moieties and then ionically complexing it with HA. The suitability of FNPs for cancer therapy was assessed through physicochemical measurements of size distribution (mean diameter of 240 nm), shape, and zeta potential (15.4 mV). Nanoparticles possessed high DOX loading efficiency (90%) and were able to trigger the drug release under redox conditions of the tumor environment (55% release after 2 h incubation). The use of the carrier increased the cytotoxic effect of DOX by targeting the CD44 protein. It was shown that, upon loading, the cytotoxic effect of DOX was enhanced in relation to CD44 protein expression in both 2D and 3D models. DOX@FNPs significantly decrease cellular metabolism by reducing both oxygen consumption and extracellular acidification rates. Moreover, they decrease the expression of proteins involved in the oxidative phosphorylation pathway, consequently reducing cellular viability and motility, as well as breast cancer stem cells and spheroid formation, compared to free DOX. This new formulation could become pioneering in reducing chemoresistance phenomena and increasing the specificity of DOX in breast cancer patients. Full article

Secondary polyphenol metabolites, urolithins (UROs), have anti-oxidative, anti-inflammatory, and antidiabetic properties. Therefore, their biological activity relies on blood transport via human serum albumin (HSA) and tissue distribution. The main goal we set was to investigate the interaction between HSA and different URO (URO A, URO B, URO C, URO D, and glucuronidated URO A and B) using a combination of multi-spectroscopic instrumental and in silico approaches. The fluorescence spectroscopy revealed that URO can quench the naturally occurring fluorescence of HSA in a concentration-dependent manner. The HSA fluorescence was quenched by both a static and dynamic mechanism. The results showed that free UROs bind to HSA with higher affinity than their conjugated forms. CD spectroscopy and FTIR revealed that the alpha-helical structure of HSA is preserved. The calculated Gibbs free energy change indicates that the URO–HSA complex forms spontaneously. There is a single binding site on the HSA surface. The molecular docking results indicated that unconjugated Uro binds to Sudlow I, while their conjugation affects this binding site, so in the conjugated form, they bind to the cleft. Docking experiments indicate that all UROs are capable of binding to both thyroxine recognition sites of ligand-bound HSA proteins. Examining interactions under the following conditions (298 K, 303 K, and 310 K, pH 7.4) is of great importance for determining the pharmacokinetics of these bioactive compounds, as the obtained results can be used as a basis for modulating the potential dosing regimen. Full article

Aflatoxin M1 (AFM1) is the hydroxylated form of Aflatoxin B1 (AFB1) and is expelled in the milk of both humans and animals following the consumption of AFB1-contaminated food. AFM1 has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer. Consequently, the European Commission has established a maximum allowable concentration of 50 pg/mL for AFM1 in dairy products and milk. Here, a rapid and sensitive approach for detecting AFM1 in bovine milk is presented. The analytical setup comprises a broad-band white LED, a spectrophotometer, and a silicon photonic probe, all interconnected by a bifurcated optical fiber [¹]. Additionally, a laptop powers the system and facilitates signal monitoring through specialized software. The silicon photonic probe is equipped with two Mach–Zehnder interferometers: one functionalized with AFM1-bovine serum albumin conjugate, and the other with bovine serum albumin to serve as a blank. The analysis involves immersing the probe directly into a mixture of anti-AFM1 antibodies and the sample, followed by sequential immersion into biotinylated anti-rabbit IgG antibody and streptavidin solutions. The entire assay process takes 12 min, and the limit of detection in undiluted milk is 20 pg/mL, below the EU maximum allowable limit of 50 pg/mL. The assay demonstrates accuracy, with %recovery values ranging from 87.5 to 112%, and repeatability, with intra/inter-assay coefficients of variation below 7.6%. Given its analytical performance and compact instrumentation, the proposed immunosensor proves to be an ideal solution for precise on-site determination of AFM1 in milk samples. Full article

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water. Full article

In cancer diagnostics, magnetic resonance imaging (MRI) uses contrast agents to enhance the distinction between the target tissue and background. Several promising approaches have been developed to increase MRI sensitivity, one of which is Overhauser dynamic nuclear polarization (ODNP)-enhanced MRI (OMRI). In this study, a macromolecular construct based on human serum albumin and nitroxyl radicals (HSA-NIT) was developed using a new synthesis method that significantly increased the modification to 21 nitroxide residues per protein. This was confirmed by electron paramagnetic resonance (EPR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry. Gel electrophoresis and circular dichroism showed no significant changes in the structure of HSA-NITs, and no oligomers were formed during modification. The cytotoxicity of HSA-NITs was comparable to that of native albumin. HSA-NITs were evaluated as potential “metal-free” organic radical relaxation-based contrast agents for ¹H-MRI and as hyperpolarizing contrast agents for OMRI. Relaxivities (longitudinal and transversal relaxation rates r₁ and r₂) for HSA-NITs were measured at different magnetic field strengths (1.88, 3, 7, and 14 T). Phantoms were used to demonstrate the potential use of HSA-NIT as a T₁- and T₂-weighted relaxation-based contrast agent at 3 T and 14 T. The efficacy of ¹H Overhauser dynamic nuclear polarization (ODNP) in liquids at an ultralow magnetic field (ULF, B₀ = 92 ± 0.8 μT) was investigated for HSA-NIT conjugates. The HSA-NITs themselves did not show ODNP enhancement; however, under the proteolysis conditions simulating cancer tissue, HSA-NIT conjugates were cleaved into lower-molecular-weight (MW) protein fragments that activate ODNP capabilities, resulting in a maximum achievable enhancement |E_max| of 40–50 and a radiofrequency power required to achieve half of E_max, P_1/2, of 21–27 W. The HSA-NIT with a higher degree of modification released increased the number of spin probes upon biodegradation, which significantly enhanced the Overhauser effect. Thus, HSA-NITs may represent a new class of MRI relaxation-based contrast agents as well as novel cleavable conjugates for use as hyperpolarizing contrast agents (HCAs) in OMRI. Full article

Transthyretin binders have previously been used to improve the pharmacokinetic properties of small-molecule drug conjugates and could, thus, be utilized for radiopharmaceuticals as an alternative to the widely explored “albumin binder concept”. In this study, a novel PSMA ligand modified with a transthyretin-binding entity (TB-01) was synthesized and labeled with lutetium-177 to obtain [¹⁷⁷Lu]Lu-PSMA-TB-01. A high and specific uptake of [¹⁷⁷Lu]Lu-PSMA-TB-01 was found in PSMA-positive PC-3 PIP cells (69 ± 3% after 4 h incubation), while uptake in PSMA-negative PC-3 flu cells was negligible (<1%). In vitro binding studies showed a 174-fold stronger affinity of [¹⁷⁷Lu]Lu-PSMA-TB-01 to transthyretin than to human serum albumin. Biodistribution studies in PC-3 PIP/flu tumor-bearing mice confirmed the enhanced blood retention of [¹⁷⁷Lu]Lu-PSMA-TB-01 (16 ± 1% IA/g at 1 h p.i.), which translated to a high tumor uptake (69 ± 13% IA/g at 4 h p.i.) with only slow wash-out over time (31 ± 8% IA/g at 96 h p.i.), while accumulation in the PC-3 flu tumor and non-targeted normal tissue was reasonably low. Further optimization of the radioligand design would be necessary to fine-tune the biodistribution and enable its use for therapeutic purposes. This study was the first of this kind and could motivate the use of the “transthyretin binder concept” for the development of future radiopharmaceuticals. Full article

Human serum albumin (HSA) is a multifunctional protein, known to be a natural carrier for a number of endogenous and exogenous compounds, including drugs. HSA-based drugs formulation is a clinically validated approach to improve pharmacological properties and biodistribution (such as in Abraxane). Based on this, one might like to modify HSA in a way that its distribution is more favorable for certain therapeutic purposes. Levofloxacin (LV), a broad-spectrum antibiotic drug, could benefit from extended systemic exposure, and stronger interactions with plasma proteins could be useful for this purpose. We engrafted monomeric or polymeric cyclodextrins (CDs) on the surface of HSA molecules to strengthen the LV adsorption (the CD−LV dissociation constant is three orders of magnitude lower than that of HSA−LV). We found that (HSA−HPolS)_conj+LV exhibited the highest activity against E. coli, whereas (HSA−HPCD)_conj+LV was the most effective against B. subtilis, and both HSA conjugates were more potent than LV alone or LV with HSA. Further fine-tuning of HSA could yield an improvement in biodistribution and thus a more favorable risk/benefit ratio. Full article

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI. Full article

Peptide-protected gold nanoclusters (AuNCs), possessing exceptional biocompatibility and remarkable physicochemical properties, have demonstrated intrinsic pharmaceutical activity in immunomodulation, making them a highly attractive frontier in the field of nanomedicine exploration. Autoimmune hepatitis (AIH) is a serious autoimmune liver disease caused by the disruption of immune balance, for which effective treatment options are still lacking. In this study, we initially identified glutathione (GSH)-protected AuNCs as a promising nanodrug candidate for AIH alleviating in a Concanavalin A (Con A)-induced mice model. However, to enhance treatment efficiency, liver-targeted delivery needs to be improved. Therefore, human serum albumin (HSA)-encapsulated AuNCs were constructed to achieve enhanced liver targeting and more potent mitigation of Con A-induced elevations in plasma aspartate transaminase (AST), alanine transaminase (ALT), and liver injury in mice. In vivo and in vitro mechanism studies indicated that AuNCs could suppress the secretion of IFN-γ by Con A-stimulated T cells and subsequently inhibit the activation of the JAK2/STAT1 pathway and eventual hepatocyte apoptosis induced by IFN-γ. These actions ultimately protect the liver from immune cell infiltration and damage caused by Con A. These findings suggest that bio-protected AuNCs hold promise as nanodrugs for AIH therapy, with their liver targeting capabilities and therapeutic efficiency being further improved via rational surface ligand engineering. Full article