TEMPO-conjugated tobacco mosaic virus as a magnetic resonance imaging contrast agent for detection of superoxide production in the inflamed liver.

Superoxide, an anionic dioxygen molecule, plays a crucial role in redox regulation within the body but is implicated in various pathological conditions when produced excessively. Efforts to develop superoxide detection strategies have led to the exploration of organic-based contrast agents for magnetic resonance imaging (MRI). This study compares the effectiveness of two such agents, nTMV-TEMPO and kTMV-TEMPO, for detecting superoxide in a mouse liver model with lipopolysaccharide (LPS)-induced inflammation. The study demonstrates that kTMV-TEMPO, with a strategically positioned lysine residue for TEMPO attachment, outperforms nTMV-TEMPO as an MRI contrast agent. The enhanced sensitivity of kTMV-TEMPO is attributed to its more exposed TEMPO attachment site, facilitating stronger interactions with water protons and superoxide radicals. EPR kinetics experiments confirm kTMV-TEMPO's faster oxidation and reduction rates, making it a promising sensor for superoxide in inflamed liver tissue. In vivo experiments using healthy and LPS-induced inflamed mice reveal that reduced kTMV-TEMPO remains MRI-inactive in healthy mice but becomes MRI-active in inflamed livers. The contrast enhancement in inflamed livers is substantial, validating the potential of kTMV-TEMPO for detecting superoxide in vivo. This research underscores the importance of optimizing contrast agents for in vivo imaging applications. The enhanced sensitivity and biocompatibility of kTMV-TEMPO make it a promising candidate for further studies in the realm of medical imaging, particularly in the context of monitoring oxidative stress-related diseases.

A scalable synthesis of adjuvanting antigen depots based on metal-organic frameworks.

Vaccines have saved countless lives by preventing and even irradicating infectious diseases. Commonly used subunit vaccines comprising one or multiple recombinant proteins isolated from a pathogen demonstrate a better safety profile than live or attenuated vaccines. However, the immunogenicity of these vaccines is weak, and therefore, subunit vaccines require a series of doses to achieve sufficient immunity against the pathogen. Here, we show that the biomimetic mineralization of the inert model antigen, ovalbumin (OVA), in zeolitic imidazolate framework-8 (ZIF-8) significantly improves the humoral immune response over three bolus doses of OVA (OVA 3×). Encapsulation of OVA in ZIF-8 (OVA@ZIF) demonstrated higher serum antibody titers against OVA than OVA 3×. OVA@ZIF vaccinated mice displayed higher populations of germinal center (GC) B cells and IgG1+ GC B cells as opposed to OVA 3×, indicative of class-switching recombination. We show that the mechanism of this phenomenon is at least partly owed to the metalloimmunological effects of the zinc metal as well as the sustained release of OVA from the ZIF-8 composite. The system acts as an antigen reservoir for antigen-presenting cells to traffic into the draining lymph node, enhancing the humoral response. Lastly, our model system OVA@ZIF is produced quickly at the gram scale in a laboratory setting, sufficient for up to 20 000 vaccine doses.

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies.

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Intracellular delivery of virus-like particles using a sheddable linker.

Intracellular targeting is essential for the efficient delivery of drugs and nanotherapeutics. Transporting nanomaterials into cells' cytoplasm for therapeutic purposes can be challenging due to the endosomal trap and lysosomal degradation of cargo. To overcome this issue, we utilized chemical synthesis to design a functional carrier that can escape the endosome and deliver biological materials into the cytoplasm. We synthesized a thiol-sensitive maleimide linker that connects the well-known mitochondria targeting lipophilic triphenylphosphonium cation (TPP) to the surface of a proteinaceous nanoparticle based on the engineered virus-like particle (VLP) Qß. TPP facilitates endosomal escape by its lipophilic and cationic nature, which disrupts the endosomal membrane. Once in the cytosol, glutathione reacts with the thiol-sensitive maleimide linkers, severs the TPP from the nanoparticle, halting its trafficking to the mitochondria, and marooning it in the cytosol. We successfully demonstrated cytosolic delivery of a VLP loaded with Green Fluorescent Protein (GFP) in vitro and small-ultrared fluorescent protein (smURFP) in vivo, where evenly distributed fluorescence is observed in A549 human lung adenocarcinoma cells and the epithelial cells of BALB/c mice lungs. As a proof of concept, we encapsulated luciferase-targeted siRNA (siLuc) inside the VLP decorated with the maleimide-TPP (M-TPP) linker. We observed enhanced luminescence silencing in luciferase-expressing HeLa cells using our sheddable TPP linker compared to control VLPs.

Identification and physical characterization of a spontaneous mutation of the tobacco mosaic virus in the laboratory environment.

Virus-like particles are an emerging class of nano-biotechnology with the Tobacco Mosaic Virus (TMV) having found a wide range of applications in imaging, drug delivery, and vaccine development. TMV is typically produced in planta, and, as an RNA virus, is highly susceptible to natural mutation that may impact its properties. Over the course of 2 years, from 2018 until 2020, our laboratory followed a spontaneous point mutation in the TMV coat protein-first observed as a 30 Da difference in electrospray ionization mass spectrometry (ESI-MS). The mutation would have been difficult to notice by electrophoretic mobility in agarose or SDS-PAGE and does not alter viral morphology as assessed by transmission electron microscopy. The mutation responsible for the 30 Da difference between the wild-type (wTMV) and mutant (mTMV) coat proteins was identified by a bottom-up proteomic approach as a change from glycine to serine at position 155 based on collision-induced dissociation data. Since residue 155 is located on the outer surface of the TMV rod, it is feasible that the mutation alters TMV surface chemistry. However, enzyme-linked immunosorbent assays found no difference in binding between mTMV and wTMV. Functionalization of a nearby residue, tyrosine 139, with diazonium salt, also appears unaffected. Overall, this study highlights the necessity of standard workflows to quality-control viral stocks. We suggest that ESI-MS is a straightforward and low-cost way to identify emerging mutants in coat proteins.

Supramolecular and biomacromolecular enhancement of metal-free magnetic resonance imaging contrast agents.

Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further overcame the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.