Fluorescent Bioconjugates Research Articles

Strategic Decoy Peptides Interfere with MSI1/AGO2 Interaction to Elicit Tumor Suppression Effects.

Simple SummaryPeptide drugs that can specifically target undesirable protein–protein interactions that lead to oncogenic developments have emerged as the next era of future medicine for cancers. To combat GBM tumor progression, our study offers an alternative therapeutic strategy via targeting the protein–protein interaction between MSI1 and AGO2 with synthetic peptides identified from the C-terminus of MSI1 in peptide arrays. Our present data revealed for the first time that peptidic disruption to the MSI1/AGO2 complex known for promoting cancer stemness and progression could lead to encouraging therapeutic efficacy at both in vitro and in vivo levels. The significantly suppressed tumor growth and prolonged survival rates in PDX tumor models by decoy peptides evidently provided a new rationale for stratifying patients with MSI1/AGO2-targeted therapeutics.Peptide drugs that target protein–protein interactions have attracted mounting research efforts towards clinical developments over the past decades. Increasing reports have indicated that expression of Musashi 1 (MSI1) is tightly correlated to high grade of cancers as well as enrichment of cancer stem cells. Treatment failure in malignant tumors glioblastoma multiform (GBM) had also been correlated to CSC-regulating properties of MSI1. It is thus imperative to develop new therapeutics that could effectively improve current regimens used in clinics. MSI1 and AGO2 are two emerging oncogenic molecules that both contribute to GBM tumorigenesis through mRNA regulation of targets involved in apoptosis and cell cycle. In this study, we designed peptide arrays covering the C-terminus of MSI1 and identified two peptides (Pep#11 and Pep#26) that could specifically interfere with the binding with AGO2. Our Biacore analyses ascertained binding between the identified peptides and AGO2. Recombinant reporter system Gaussian luciferase and fluorescent bioconjugate techniques were employed to determine biological functions and pharmacokinetic characteristics of these two peptides. Our data suggested that Pep#11 and Pep#26 could function as decoy peptides by mimicking the interaction function of MSI1 with its binding partner AGO2 in vitro and in vivo. Further experiments using GMB animal models corroborated the ability of Pep#11 and Pep#26 in disrupting MSI1/AGO2 interaction and consequently anti-tumorigenicity and prolonged survival rates. These striking therapeutic efficacies orchestrated by the synthetic peptides were attributed to the decoy function to C-terminal MSI1, especially in malignant brain tumors and glioblastoma.

Preparation and Application of Fluorescent Bioconjugates in Probing the pH and Solvent Relaxation Dynamics in ZIF-8 Cavities

Preparation and Application of Fluorescent Bioconjugates in Probing the pH and Solvent Relaxation Dynamics in ZIF-8 Cavities

Comparison of cRGDfK Peptide Probes with Appended Shielded Heptamethine Cyanine Dye (s775z) for Near Infrared Fluorescence Imaging of Cancer.

Previous work has shown that the sterically shielded near-infrared (NIR) fluorescent heptamethine cyanine dye, s775z, with a reactive carboxyl group produces fluorescent bioconjugates with an unsurpassed combination of high photostability and fluorescence brightness. This present contribution reports two new reactive homologues of s775z with either a maleimide group for reaction with a thiol or a strained alkyne group for reaction with an azide. Three cancer-targeting NIR fluorescent probes were synthesized, each with an appended cRGDfK peptide to provide selective affinity for integrin receptors that are overexpressed on the surface of many cancer cells including the A549 lung adenocarcinoma cells used in this study. A set of cancer cell microscopy and mouse tumor imaging experiments showed that all three probes were very effective at targeting cancer cells and tumors; however, the change in the linker structure produced a statistically significant difference in some aspects of the mouse biodistribution. The mouse studies included a mock surgical procedure that excised the subcutaneous tumors. A paired-agent fluorescence imaging experiment co-injected a binary mixture of targeted probe with 850 nm emission, an untargeted probe with 710 nm emission and determined the targeted probe’s binding potential in the tumor tissue. A comparison of pixelated maps of binding potential for each excised tumor indicated a tumor-to-tumor variation of integrin expression levels, and a heterogeneous spatial distribution of integrin receptors within each tumor.

Agarose Micro-Well Platform for Rapid Generation of Homogenous 3D Tumor Spheroids.

In recent years, 3D culture of tumor spheroids has managed to revolutionize cancer research and drug discovery. 2D monolayer cells grown in cell culture flasks undergo radical changes in cell behavior, structure, and function owing to varying environmental cues and are unable to provide predictive data for preclinical evaluation. 3D tumor spheroids can better recapitulate tumor architecture, cell-cell and cell-matrix connectivity, and the tissue complexity of tumors grown in animal models. However, many of the existing techniques to culture 3D spheroids are time-consuming and ineffective and produce irregular-shaped spheroids that cannot be easily incorporated in biological assays. The set of protocols described herein makes use of a commercial hair brush as a template to create concave micro-well impressions in agarose. This technique is easy, inexpensive, and adaptable and also has the ability to produce uniform, homogenous cancer spheroids, with large diameter (∼1000 μm) and thickness (∼250 μm), within 24 to 48 hr after cell seeding. The 3D spheroids produced using the agarose micro-well platform function as an excellent 3D in vitro model for understanding the extent of penetration, uptake, and distribution of targeted cargos such as a diagnostic or therapeutic agents for identification and treatment of cancer. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of agarose micro-well scaffold for growing tumor spheroids using a commercial hair brush Basic Protocol 2: Formation of homogenous tumor spheroids in agarose micro-well platform Basic Protocol 3: Assessing viability of 3D tumor spheroids grown in agarose micro-wells using confocal microscopy Basic Protocol 4: Analyzing uptake and penetration of targeted fluorescent bioconjugate in 3D tumor spheroids using two-photon imaging.

Imaging of prostate cancer: optimizing affinity to prostate specific membrane antigen by spacer modifications in a tumor spheroid model

Early diagnosis of prostate cancer (PCa) is crucial for staging, treatment and management of patients. Prostate specific membrane antigen (PSMA), highly over-expressed on PCa cells, is an excellent target for selective imaging of PCa. In recent years, various scaffolds have been explored as potential carriers to target diagnostic and therapeutic agents to PSMA+ tumour cells. Numerous fluorescent or radioisotope probes linked via a peptide linker have been developed that selectively binds to PCa cells. However, there are very few reports that examine the effects of chemical modifications in the peptide linker of an imaging probe on its affinity to PSMA protein. This report systematically investigates the impact of hydrophobic aromatic moieties in the peptide linker on PSMA affinity and in vitro performance. For this, a series of fluorescent bioconjugates 12–17 with different aromatic spacers were designed, synthesized, and their interactions within the PSMA pocket were first analysed in silico. Cell uptake studies were then performed for 12–17 in PSMA+ cell lines and 3D tumour models in vitro. Binding affinity values of 12–17 were found to be in the range of 36 to 157.9 nM, and 12 with three aromatic groups in the spacer exhibit highest affinity (KD = 36 nM) compared to 17 which is devoid of aromatic groups. These studies suggest that aromatic groups in the spacer region can significantly affect deep tissue imaging of fluorescent bioconjugates. Bioconjugate 12 can be a promising diagnostic tool, and conjugation to near-infrared agents would further its applications in deep-tissue imaging and surgery. Communicated by Ramaswamy H. Sarma

Riboflavin based conjugated biomolecule for ultrasensitive detection of nitrophenols.

Real time detection of explosive compounds in today's time is of utmost necessity due to security and severe environmental safety issues. Herein, we have synthesized a biobased conjugated molecular system from riboflavin and l-cystine utilized it for detecting picric acid in trace amount using optical sensing technique. The bioconjugate probe showed high quenching efficiency towards picric acid, which is 92.2%. In depth mechanistic study showed that ground state electrostatic interaction and inner filter effect are the factors leading to the diminishing of the probe's fluorescence intensity on addition of trace amount of the nitrophenol, picric acid. The detection limit of the conjugate is 0.37 nM which is extremely low and highly desirable for clinical applications of this system.

Tyrosine-based asymmetric urea ligand for prostate carcinoma: Tuning biological efficacy through in silico studies

In this article, we have explored the chemical interactions of tyrosine-based asymmetric urea ligands in the binding pockets of prostate specific membrane antigen (PSMA) through in silico studies. The S1 pocket of the PSMA protein offers better scope for modifications in the urea ligands to improve the binding affinity. Accordingly, tyrosine-based (S)-2-(3-((S)-1-carboxy-2-(4-(carboxymethoxy)phenyl)ethyl)ureido)pentanedioic acid (CYUE, 3) ligand was designed, synthesized and predicted to possess inhibition constant (Ki) of 55 nM with PSMA protein. The CYUE (3) ligand was further elaborated into a fluorescent diagnostic probe for detection of PSMA+ cancers. In vitro studies on human malignant cell lines such as LNCaP and PC-3 were performed to show the efficacy and specificity of the newly synthesized bio-construct. The fluorescent bio-conjugate was found to be very specific to the PSMA protein with an overall binding affinity constant (KD) of 88 nM.

Site-specific near-infrared fluorescent labelling of proteins on cysteine residues with meso-chloro-substituted heptamethine cyanine dyes.

Near-infrared (NIR) fluorescence imaging is a promising new medical imaging modality. Associated with a targeting molecule, NIR fluorophores can accumulate selectively in tissues of interest and become valuable tools for the diagnosis and therapy of various pathologies. To facilitate the design of targeted NIR imaging agents, it is important to identify simple and affordable fluorescent probes, allowing rapid labelling of biovectors such as proteins, ideally in a site-specific manner. Here, we demonstrate that heptamethine cyanine based fluorophores, such as IR-783, that contain a chloro-cyclohexyl moiety within their polymethine chain can react selectively, at neutral pH, with cysteine residues in proteins to give stable, site-specifically labelled conjugates, that emit in the NIR spectral window. This reaction is exemplified with the labelling of peptides and two protein models: albumin and a Fab' antibody fragment. The resulting fluorescent proteins are stable and suitable for in vivo NIR imaging applications, as shown on a mice model. This straightforward one-step procedure, that does not require the prior derivatisation of the fluorophore with a bioconjugatable handle, should facilitate the production and use of near-infrared labelled proteins in life sciences.

Doubly Caged Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification.

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual "click" coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual "click" coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody-imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

AbstractIn situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1, whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery.

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

Cysteine-based amphiphilic peptide-polymer conjugates via thiol-mediated radical polymerization: Synthesis, self-assembly, RNA polyplexation and N-terminus fluorescent labeling for cell imaging

A water-soluble amphiphilic peptide-poly (1-vinylimidazole) [Boc-Cys (PVim)-Trp-OMe] bioconjugate is synthesized by ‘grafting from’ technique based on thiol-mediated radical polymerization. The attachment of fluorescein isothiocyanate (FITC) at the N-terminus of the peptide moiety of the bioconjugate results in the generation of fluorescent bioconjugate [FITC-Cys (PVim)-Trp-OMe]. MALDI-TOF-MS analysis confirms the attachment dipeptide moiety as well as FITC group. Owing to the amphiphilic nature, the [Boc-Cys (PVim)-Trp-OMe] bioconjugate undergoes spontaneous self-assembly into primary micelles and their subsequent secondary aggregation into spherical micellar aggregates (nanospheres) in water. The aqueous FITC-Cys (PVim)-Trp-OMe bioconjugate solution emits green light and exhibits a fluorescence emission with a maximum at 520 nm. The amphiphilicity of the fluorescent bioconjuagte molecules also triggers their self-assembly into micelles and micellar aggregates (nanospheres) with hydrophobic FITC-tagged dipeptide core and hydrophilic PVim cell in water. Further, the highly stable and non-toxic fluorescent bioconjugates are used for imaging of Chinese Hamster Ovary (CHO) cells. Finally, the polyplex formation between the untagged cationic Boc-Cys (PVim)-Trp-OMe bioconjugate and ribonucleic acid (RNA) at pH 5.5 is studied by fluorescence spectroscopy.

Carboxymethylcellulose/ZnCdS fluorescent quantum dot nanoconjugates for cancer cell bioimaging

In this study, it is reported the use of sodium carboxymethyl cellulose (CMCel) as a multifunctional biocompatible polysaccharide for the direct synthesis of fluorescent alloyed-ZnCdS quantum dot (QD) nanoconjugates via aqueous “green” process at room temperature. The nanoconjugates were extensively characterized by spectroscopical (NMR, FTIR, UV-vis, PL) and morphological techniques (DLS, TEM) for accessing their structural and physicochemical properties associated with X-ray photoelectron spectroscopy (XPS) for surface and interface analysis. The results proved the hypothesis of formation of core-shell nanostructures composed by the semiconductor ZnCdS QD core and the organic biocompatible ligand CMCel shell. Moreover, CMCel chemical functional groups played a pivotal role for controlling the size of water-soluble colloidal nanocrystals (2r=4–5nm) and hydrodynamic diameters (<15nm) evidenced by metal complexation and interactions at the nanointerfaces. Additionally, these nanoconjugates were cytocompatible and luminescent for bioimaging human osteosarcoma cancer cells. Thus, these novel polysaccharide-based fluorescent bioconjugates offer promising perspectives as nanoplatforms for cancer cell bioimaging and diagnosis purposes.

Fluorescent Bioconjugate Based on Gold Nanoparticles for the Determination of Staphylococcus aureus

ABSTRACTA practical method to determine Staphylococcus aureus using bioconjugated gold nanoparticles is reported. The protocol uses gold nanoparticles stabilized by tetramethylrhodamine isothiocyanate-labeled streptavidin followed by functionalization with biotinylated anti-S. aureus antibodies. The streptavidin-stabilized gold nanoparticles were obtained by titration and analyzed by ultraviolet–visible spectroscopy and transmission electron microscopy. The obtained fluorescent bioconjugate selectively linked to the surface of S. aureus in samples contaminated with the microorganism, as demonstrated by confocal micrographs. The biorecognition process was performed by mixing the fluorescent bioconjugate with the sample. Bacterial dilutions from 1 × 108 to 1 × 102 cell/ml of S. aureus were determined, obtaining sensitivity values of 1 × 105 cell/ml by photoluminescence and 1 × 102 cell/ml by bioimpedance. This methodology represents a useful bioanalytical approach for the determination of S. aureus.

Fluorescent Gold Nanoparticle-Based Bioconjugate for the Detection of Salmonella

ABSTRACTAn easily prepared fluorescent bioconjugate colloidal solution based on gold nanoparticles is reported for the detection of Salmonella. This bioconjugate was prepared by conjugation of staphylococcal protein A on the surface of gold nanoparticles, followed by biofunctionalization over this layer with fluorescein isothiocyanate-labeled antibodies. The surface plasmon resonance band observed in the ultraviolet–visible spectrum of the gold nanoparticles showed a shift to low energy after covering the surface with protein A and a broadening of the band after biofunctionalization with the antibody. Surface-enhanced infrared spectroscopy showed the absorption bands of the conjugate and bioconjugate due to their proximity to the nanoparticles. In particular, vibrations at 1649, 1588, and 1400 cm−1 were from C˭O, N–H, and C–NH2 groups from the staphylococcal protein A covering the gold nanoparticles. The final bioconjugate colloidal solution was also used as probe to detecting Salmonella by confocal microscopy of solutions containing 1E3 and 1E5 CFU/ml. The total time required to detect these bacteria was less than 1 h. Fluorescent emission of the bacillus suggests that this methodology may have potential to develop a universal method for detecting different bacteria.

Fluorescence plate reader for quantum dot-protein bioconjugation analysis.

We present here a new and alternative method that uses a Fluorescence Plate Reader in a different approach, not to study protein-protein interactions, but to evaluate the efficiency of the protein bioconjugation to quantum dots (QDs). The method is based on the QDs' native fluorescence and was successfully tested by employing two different QDs-proteins conjugation methodologies, one by promoting covalent binding and other by inducing adsorption processes. For testing, we used bioconjugates between carboxyl coated CdTe QDs and bovine serum albumin, concanavalin A lectin and anti-A antibody. Flow cytometry and fluorescence spectroscopy studies corroborated the results found by the Fluorescence Plate Reader assay. This kind of analysis is important because poor bioconjugation efficiency leads to unsuccessful applications of the fluorescent bioconjugates. We believe that our method presents the possibility of performing semi-quantitative and simultaneous analysis of different samples with accuracy taking the advantage of the high sensitivity of optical based measurements.

Quantum dot conjugated S. cerevisiae as smart nanotoxicity indicators for screening the toxicity of nanomaterials.

In this study, we have evaluated the toxicity of different forms of carbon nanotubes (CNTs) using S. cerevisiae-QD (SQD) bioconjugates as a novel fluorescent biological nanotoxicity indicator. A CNT mediated effect in SQD bioconjugates was used as an indicator for the changes occurring at the cell-membrane interfaces that induced disruption of membrane bound QDs resulting in the loss of fluorescence. Single, double and multiwalled carbon nanotubes (SWCNTs, DWCNTs and MWCNTs) were tested for their toxicities imposed on SQD bioconjugates. Bioconjugates exposed to varying concentrations of different forms of CNTs exhibited different modes of toxicities on SQD bioconjugates. SQD bioconjugates were highly responsive in the 0.1-10 μg mL-1 CNT concentration range after 1 h of exposure. The toxicity of CNTs was linked to the number of CNT walls. These results were further confirmed by SEM analysis and cell-viability tests that were consistent with the toxicity assays using fluorescent bioconjugates with different types of CNTs. SWCNTs imposed more severe cellular toxicity followed by MWCNTs and DWCNTs and the order of increasing cellular-damage by CNTs followed DWCNTs < MWCNTs < SWCNTs. This study speculates that the cell-injury by CNTs depends on their physical properties, such as layers of walls, non-covalent forces and dispersion states. Our results demonstrated a facile optical strategy that enables rapid and real-time cytotoxicity screening with yeast as model living-cells for engineering nanomaterials.

Monitoring layer-by-layer self-assembly process of natural polyelectrolytes by fluorescent bioconjugate with aggregation-induced emission characteristic.

A novel chitosan-based fluorescent bioconjugate (TPE-CS) with aggregation-induced emission (AIE) characteristic is synthesized and used as a fluorescent probe for monitoring layer-by-layer self-assembly process of natural polyelectrolytes. QCM results and contact angle measurement indicate that this AIE active TPE-CS bioconjugate can be assembled with alginate (ALG) through layer-by-layer deposition. Ellipsometry and fluorescence (FL) spectroscopy show an exponential growth of the TPE-CS/ALG multilayer films. Moreover, the exponential relationship between the FL intensity and the number of bilayers, which is in accordance with the thickness variation of multilayer films, provides solid evidence for its capacity to monitor the layer-by-layer self-assembly process.

Sub-minute formation of supported nanoporous mesoscale patterns programmed by surface energy

We demonstrate an original and powerful concept for elaborating spontaneous, high fidelity patterns of nanoporosity from nanoscale building blocks using patterned surface chemistry (i.e., “surface energy gating”) to corral the growth of colloidal structures at a solid surface. Composite films consisting of polymethylsilsesquioxane nanoparticles uniformly dispersed in polypropylene glycol polymer were examined at temperatures beyond the decomposition of the polymer as a function of the substrate surface energy to clarify nanoparticulate ensemble behavior. The principle behind this colloidal assembly can be understood by taking into consideration the entropy and enthalpy dictating the mutual interactions between substrate surface, polymeric solvent, and dispersed colloids in the decomposition regime. The relevance of this research is shown by demonstrating how the principle of surface energy gating can be utilized to achieve spontaneous and controllable spatial patterns of nanoporous, high surface area thin films in a cost-effective and energy-efficient manner via brief thermal exposure. The simplicity and general nature of this methodology are further exemplified by showing the facility with which high-contrast fluorescent bioconjugate arrays can be prepared from nanoporous organosilicate patterns.

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group for neurotransmitter amino acids

Aiming at the development of new benzopyran-based photocleavable protecting groups, novel chloromethylated and hydroxymethylated 2-oxo-2H-benzo[h]benzopyran derivatives bearing a methoxy substituent were designed and used in the synthesis of a series of fluorescent bioconjugates, by linking through an ester or urethane bond to several model neurotransmitter amino acids (glycine, alanine, beta-alanine and gamma-aminobutyric acid, GABA). The resulting fluorescent bioconjugates with emission in the visible range and high fluorescent quantum yields, were subjected to photocleavage reaction in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (250, 300, 350 and 419 nm) and photocleavage kinetic data were obtained.