A lipoic acid(LA)-base hydrogel polymerization, viscosity, and relaxivity were explored by varying several different bases, additives, Gd3+ chelates, and with or without a reducing agent (tris(2-carboxyethyl)phosphine, TCEP). The polymerization of LA was greatly improved by using TCEP to convert the more inert disulfides into thiol initiators. The base deprotonation of LA was found to be essential to solubilize LA for polymer propagation. The various bases and TCEP additions had a substantial effect on viscosity, with values ranging from 1 × 108 to 1 × 103 mPa·s. This can enable targeting of many different applications that depend on viscosity, such as topical ointments or injectable hydrogels. The relaxivity effects of the LA-hydrogels were explored by varying the Gd3+ chelates' inner-sphere coordination or by adding one or two sulfur atoms, thereby allowing for a terminal or internal-linking Gd3+ chelate. It was found that the inner sphere and microenvironment dominate the relaxivity of Gd3+ chelates in the LA-hydrogel, with a high relaxivity of r1 = 49.2 ± 1.2 mM-1 s-1 at 1.4 T and 37 °C. Cellular uptake was confirmed by confocal fluorescence microscopy, and contrast was assessed in vivo at 9.4 T.

Spectroscopic detection of paramagnetically shifted 1H signals of trivalent lanthanide complexes provide a means for ratiometric detection of enzymatic activity. Herein, we report on the synthesis and characterization of TbL1, DyL1, and TbL2 complexes for ratiometric detection of the enzyme nitroreductase (NTR). Ln(III) coordination environments with distinct off- and on- probe states provide unique paramagnetic contributions to the chemical shift of a 5-(tBu)-pyridine-based reporter group for quantitative detection of enzyme activity. The paramagnetic contribution to chemical shift from Tb(III) and Dy(III) shift the 1H chemical signal beyond the background region to low frequencies (δPCS < -15) for zero background signal. Paramagnetic relaxation enhancement of reporter group protons enables the development of probe-specific pulse sequences with short acquisition and delay times. A probe-specific pulse sequence for TbL2 acquired 256 scans in under 1 min and enabled ratiometric detection of NTR activity.

Basal Cell Carcinoma (BCC) is the most frequently diagnosed cancer globally and affects about one in five Americans. Given the frequency of diagnosis, it is surprising that there are very few therapeutic options. Surgical removal is currently the most common treatment option; however, this can lead to noticeable scarring and cosmetic issues. As a result, there is a compelling interest in developing non-invasive therapeutic approaches to this disease. Here, we introduce a new transition metal-DNA derivative called CoGli-GOPEI that inhibits the migration of murine ASZ BCC cells in laboratory experiments. Notably, this complex significantly outperforms two established hedgehog-pathway inhibitors: GANT-61 (an investigational compound) and vismodegib (an FDA-approved drug). These inhibitors target the hedgehog signaling pathway-specifically the Gli family of transcription factors-to slow cancer progression. By effectively reducing cell migration, CoGli-GOPEI offers a less invasive alternative to traditional treatments like surgical resection and chemotherapy. Our results highlight how targeting the Gli transcription factors within the hedgehog pathway can create a novel therapeutic strategy against BCC. The ultimate goal of these new derivates is to reduce the spread of cancer cells while minimizing the downsides of surgery.

A new push-pull dye has been developed as a pH indicator when covalently copolymerized in a lipoic acid-based polymer (LA-Py). Furthermore, various charged forms of the dyes are investigated as antifouling diluents in self-assembled monolayers (SAMs) on gold electrodes. The dyes contain an amino donor incorporated into a seven-membered heterocyclic disulfide ring, a pyridine or pyridinium acceptor moiety, and an oligo-phenylene ethylene (OPE) conjugated bridge. A common characteristic of a push-pull dye is the high sensitivity to solvent dipoles such as the Stokes shifts of 176 and 211 nm for pyridine (Py) in acetonitrile and pyridinium (MePy) in chloroform, respectively. This is coupled with a decrease in quantum yields with increasing polarity of the solvents. The LA-Py has an apparent pKa of 3.5 and showed high sensitivity in acidic conditions with robust cycling between pH 7 and -0.08, producing an average contrast of 36% over 10 cycles. Incorporated into a SAM, the zwitterionic pyridinium sulfonate (SPy) derivative demonstrated antifouling to BSA by continuous monitoring of impedance with electrochemical impedance spectroscopy and ionic permeability of K3[FeCN6] by cyclic voltammetry. When "backfilled" with mercaptohexadecane (MHD), an enhanced SAM was formed (SPy + MHD), which showed resistance to ion penetration and improved antifouling.

Liver cancer remains one of the most lethal malignancies worldwide, primarily due to limited diagnostic and therapeutic strategies. Biological imaging agents capable of selective accumulation in cancerous liver tissue offer a promising route for earlier detection and improved patient outcomes. In this work, we synthesized and characterized alkaline phosphatase (ALP)-targeted, gadolinium-labeled gold nanoparticles (AuNPs) designed for simultaneous detection using magnetic resonance imaging (MRI), computed tomography (CT), and fluorescence (Fl) microscopy. The synthesized AuNPs feature 13 nm gold cores functionalized with ALP-binding ligands and Gd(III)-macrocycles. Characterization by ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX) confirmed successful functionalization. During the functionalization process, variations in Gd(III) loading, surface packing density, and r1 relaxivity were observed; however, high reproducibility was achieved when including methanol during the AuNP labeling protocol. In vitro studies with HepG2 liver cancer and HEK293 kidney cells demonstrated selective cellular uptake in relation to cellular ALP expression levels. Optimized uptake conditions demonstrated 10-fold increase in Gd(III) internalization into HepG2 versus HEK293 cells. Further imaging by scanning electron microscopy (SEM) and TEM on thinly sliced cell samples verified the intracellular localization of these nanoparticles. Collectively, these findings underscore the potential of ALP-targeted, gadolinium-labeled AuNPs as a versatile multimodal imaging platform for the early detection of liver cancer.

A gadolinium-based contrast agent (GBCA) disulfide homodimer (Gd3+SS) has been incorporated into a lipoic acid (LA)-based hydrogel (Gd3+Gel) for enhanced magnetic resonance imaging (MRI). This study evaluates the magnetic properties and in vitro behavior of Gd3+Gel for potential applications in tracking internal injuries. Results indicate a direct shortening in the relaxation rate with greater magnitude of LA polymerization and a 2.8-fold enhancement in relaxivity (r1) at 1.4 T when Gd3+SS was conjugated into the hydrogel. This effect is attributed to a significant increase in the rotational correlation time (τr) from 0.22 ± 0.05 ns (Gd3+SS) to 6 ± 1 ns (Gd3+Gel). Retention studies confirm that Gd3+SS remains covalently within the hydrogel, with retention of 64.7 ± 1.9% for Gd3+SS and 14.0 ± 1.4% for noncovalent binding gadoterate. The hydrogel relaxation rate (1/T1) increases from 1.1 to 3.5 s-1 at 7 T from blank gel to Gd3+Gel (0.24 mM Gd3+). Cell studies show that PC3-PIP and RAW 264.7 cells maintain high viability with Gd3+SS but exhibit reduced viability with Gd3+Gel, consistent with known lipoic acid effects on immortalized cell lines. Cellular uptake studies using ICP-MS and confocal fluorescence microscopy confirm that monomeric Gd3+SS is readily internalized, whereas Gd3+Gel significantly limits diffusion and uptake. Rheology was conducted to determine the zero-shear viscosity of the LA hydrogel at various concentrations of LA. These findings suggest that the LA hydrogel scaffold enhances MRI contrast, minimizes leaching, and is easily injectable. Gd3+Gel is a promising tool for potential targeted imaging and controlled uptake during healing processes.

Mapping the distribution of cells within a tissue using MR imaging has remained a significant challenge for the field. Cellular MRI can trace cells within tissue, but typically does not achieve the resolution necessary to define a cell's precise anatomical location. To detect cells with ultra-high resolution MRI, a high r 1 relaxivity intracellular contrast agent is required. Localizing this contrast within its biological context also necessitates an isotropic spatial resolution corresponding to the size of a cell's cytoplasm (∼20 μm) to place it within its biological context. We here demonstrate that gadolinium gold nanoparticles (GdAuNP) induce a high T1-weighted cellular MRI contrast at ultra-high magnetic fields (9.4 T, and 11.7 T) that affords in situ labelled cell detection at very high resolutions (150, 100, 50, and 20 μm). A 20 μm 3D gradient-echo image (400 minutes scan) combined with MR image denoising robustly visualized the distribution of in situ labeled cells in the rat brain. Signal averaging (NA = 5) also consistently afforded the detection of labeled cells. Positive T1-weighted contrast was confirmed to be caused by GdAuNP using histology. Immunohistochemistry confirmed the presence of GdAuNP almost entirely inside cells, primarily those of the neuronal lineage. Histology verified that the MR images accurately visualized individual cells' distribution within their anatomical context. Cellular resolution MRI of GdAuNP-labeled cells hence affords new avenues to investigate how individual cells contribute to the development, repair, and regeneration of tissues.

C-reactive protein (CRP) is rapidly upregulated during the early stages of inflammation following injury, infection, or illness and binds to damaged cells in affected tissues, making it an attractive target for imaging. Current clinical assays measure only circulating CRP levels and provide no spatial information about the location of inflammation or its structural impact. Direct visualization of CRP in tissues could enable earlier detection, precise localization of pathology, monitoring of treatment response, and identification of subclinical disease. Here, we report the development of the first CRP-targeted magnetic resonance (MR) imaging contrast agent for molecular imaging of inflammation. We synthesized a gadolinium-labeled gold nanoparticle (AuNP) targeted to CRP through a phosphocholine (PC) ligand, termed PC-Gd@AuNP. The resulting particles were highly uniform (∼2 nm) with a tunable Gd:PC surface ratio. Biophysical characterization demonstrated strong binding affinity for CRP (KD = 135 ± 63.96 nM) and specificity in a complex biological fluid model. PC-Gd@AuNPs displayed high longitudinal relaxivity (8.5 mM-1 s-1 per Gd) and cytotoxicity thresholds (LC₅₀) of 1 μM in HeLa cells and 0.284 μM in HepG2 cells. These findings establish PC-Gd@AuNPs as a promising molecular MRI contrast agent with the potential to directly image CRP deposition in inflamed tissues and advance noninvasive detection and monitoring of inflammatory diseases.

Contrast agents capable of labeling cells in situ are essential for tracking individual cells as they migrate through tissues during dynamic biological processes. Gold nanoparticles (AuNPs) conjugated with gadolinium (Gd) and fluorochromes offer multimodal detection via magnetic resonance imaging (MRI), computed tomography (CT), and fluorescence microscopy. In this study, a systematic strategy was employed to incrementally increase the complexity of Gd-labeled AuNPs (GdAuNPs) and evaluate four distinct surface chemistries for in situ cell labeling. Comprehensive characterization of GdAuNP synthesis and stability-using inductively coupled plasma mass spectrometry, UV/visible spectroscopy, transmission electron microscopy, and MR relaxometry-demonstrated high reproducibility and a long shelf-life. Following intracerebroventricular or intrastriatal injection, histological analyses revealed that GdAuNPs labeled over 80 % of neurons in the striatum and approximately 20 % of neural stem cells (NSCs) in the subventricular zone. Only GdAuNPs functionalized with single-stranded DNA (ssDNA) were efficiently internalized by cells; GdAuNPs lacking ssDNA remained extracellular and were removed during immunohistochemical processing. ssDNA-labeled GdAuNPs localized peri-nuclearly following endocytosis. In microglia, GdAuNP also accumulated near the nucleus, whereas in macrophages, all GdAuNP formulations-including those with ssDNA-were mostly sequestered within phagosomes, indicating uptake via phagocytosis. The most effective design, termed type D GdAuNP, featured Gd chelates conjugated both to ssDNA and directly to the AuNP surface. These nanoparticles exhibited the highest MR sensitivity and contrast-to-noise ratio in MRI after in situ labeling and were also robustly detected by μCT. This stepwise approach to nanoparticle optimization demonstrates the potential to enhance multimodal imaging sensitivity, supporting the feasibility of a noninvasive visualization of in situ labeled neurons and NSCs.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Four β-carbonylphosphine oxide compounds were complexed with four Ln(NO 3 ) 3 salts (Ln = Sm, Eu, Tb, Dy). The Ln–ligand complexes were characterized in the solid state (IR, CHN) and as solutions in acetonitrile (NMR, LR-MS, photophysical properties).

Two cryptates of trivalent uranium are compared with respect to luminescence, solid-state structure, and electrochemistry.

Ferrocene (Fc)-based disulfide molecules of various lengths with amino acid scaffolds and alkane or oligo(phenylene-ethynylene) (OPE) bridges are used in a mixed SAM with a di-(ethylene oxide) terminal mercaptoundecanol diluent (PEG2). The relative height of the Fc redox reporter in the SAM is compared to determine if there are protective effects like antifouling and specific detection. The HaloTag-binding motif is used as a proof-of-concept to investigate the electrochemical response to the HaloTag protein due to its known covalent and fast linkage. When the Fc-SAMs are exposed to the HaloTag protein, there are an antifouling nature and more specific detection for the engulfed Fc-based molecules (C6tBu/Halo). The further out the Fc is from the SAM layer, the more nonspecific adsorption is detected. The double layer capacitance (CDL) has the smallest change for the C6tBu control (ΔCDL = -0.1 μF cm-2) showing antifouling properties and produces a large change (ΔCDL = 0.9 μF cm-2) as well as a shift in oxidation potential when the active C6Halo is exposed to the HaloTag protein (ΔE1/2 = 50 ± 10 mV). The remaining Fc molecules are partially in or outside the PEG2 layer, allowing more ion penetration/mobility even when the HaloTag protein is bound. Generally, a more disordered environment was observed for the Fc-based molecules when adding the HaloTag ligand, which is evident from a larger Efwhm and higher CDL. Desorption of the SAMs with sodium iodide (NaI) showed retention of the HaloTag protein bound with the corresponding ligand, whereas negative controls did not. Self-assembled monolayers for MALDI mass spectrometry (SAMDI-MS) were used as an orthogonal detection technique to show the qualitative binding of the HaloTag protein to the electrode. Together, these results provide insight into the antifouling and detection methods of engulfing the redox molecules in the SAM diluent.

The complexes described here serve as contrast agents for magnetic resonance imaging thermometry. The complexes differentially enhance contrast between 275 and 325 K. The basis of the temperature response of the fluorinated contrast complex is the modulation of water exchange caused by trifluoromethyl groups that can be chemically controlled.

Metal-based contrast agents for magnetic resonance imaging present a promising avenue to image hypoxia. Eu-II-based contrast agents have a unique biologically relevant redox couple, Eu-II/III, that distinguishes this metal for use in hypoxia imaging. To that end, we investigated a strategy to enhance the contrast-enhancing capabilities of Eu-II-based cryptates in magnetic resonance imaging by controlling the rotational dynamics. Two dimetallic, Eu-II-containing cryptates were synthesized to test the efficacy of rigid versus flexible coupling strategies. A flexible strategy to dimerization led to a modest (114 %) increase in contrast enhancement per Eu ion (60 MHz, 298 K), but a rigid linking strategy led to an excellent (186 %) increase in contrast enhancement despite this compound ' s having the smaller molecular mass of the two dimetallic complexes. We envision the rigid linking strategy to be useful in the future design of potent Eu-II-based contrast agents for magnetic resonance imaging.

We describe a systematic study of the influence of halides and solvents on the optical properties of EuII-containing complexes in solution starting from well-defined crystalline precursors. Anionic halides, chloride and bromide, blue-shift the spectroscopic properties of EuII, whereas neutral ligands, methanol and acetonitrile, cause a red shift. This system provides evidence that EuII has a stronger affinity for chloride, and to some extent bromide, relative to acetonitrile but not methanol. We also describe a simple procedure using an ion-exchange resin for the exchange of iodide counterions to hexafluorophosphate. These findings are a step toward designing ligands that can tune the optical properties of EuII-containing complexes for solution-based applications.

Due to the importance of both visible-light luminescence and lanthanides in modern society, the influence of the ligand environment on complexes of YbII were studied and compared with analogous complexes of EuII. Four ligands with systematically varied electronic and steric characteristics were used to probe the coordination environment and electronic and redox properties of the corresponding YbII-containing complexes. Strong-field nitrogenous donors gave rise to bathochromic shifts, leading to visible-light absorption by YbII. Trends in properties across the series of YbII-containing complexes were compared to trends reported for the analogous EuII-containing complexes, revealing the translatability of coordination environment effects across the divalent lanthanide series. These studies provide valuable information regarding the behavior of small and medium-sized divalent lanthanides outside of the solid state.

We report a new luminescent EuII-containing complex. The complex is excited with visible light, leading to emission centered at 447 nm with a lifetime of 1.25 μs. Computational studies suggest that the steric bulk of the ligand is a major factor influencing the wavelength of emission.