The lipidome, encompassing the comprehensive lipid fingerprint of a biological system, includes thousands of unique isomeric and isobaric lipid species. Mass spectrometry (MS) is an effective technique for characterizing the lipidome, although the resolution of isomeric lipid species through MS typically requires specialized or modified equipment. In this study, we introduce a novel matrix derivatization technique that leverages the unique photoreactive properties of unsaturated lipids to reveal the double-bond location in conventional matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) experiments. The principle mechanistic framework of this technique is type II photosensitization, where the MALDI matrix norharmane acts as an organic photosensitizer to generate singlet oxygen upon light exposure. The singlet oxygen then reacts with unsaturated lipid species, forming hydroperoxide derivatives at acyl group carbon double bonds, facilitating their identification. The labile nature of these hydroperoxide-functionalized lipids allows for further decomposition under normal MALDI laser exposure, enhancing the analytical resolution of isomeric lipids without additional experiments. With this approach, we were able to distinguish the 18:1 (Δ6-cis) and 18:1 (Δ9-cis) PC lipid isomers. We also demonstrated that the approach works in an imaging context, mapping lipid species in both mouse tissue and 3D cell cultures.

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-d-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-d-lysine approach was then utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell-level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found 12 lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.

Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.

Synthetic polymeric nanoparticles (NPs) have been proven to be essential drug delivery systems and cancer therapy tools. However, NPs derived from traditional linear polymers often fail to provide the stability, biocompatibility, and loading efficiencies needed for clinical application. The present work explores strategically designed amino acid-based NPs formed from self-assembling amphiphilic Y and H-shaped polymers consisting of a tryptophan-based hydrophobic segment coupled to a hydrophilic block of polyethylene glycol (PEG). This study employs tryptophan due to its natural availability and abilities as a cancer immunotherapeutic agent. These amphiphilic polymers (SM_Trp2P and SM_Trp4P) were synthesized via a combination of Steglich esterification and "click" chemistry (azide-alkyne cycloaddition). Their resulting NPs were characterized using electron microscopy and dynamic light scattering (DLS), by which the morphologies of the NPs were confirmed. Spherical NPs possessing a tryptophan core enabled the loading of hydrophobic cargo, such as drug molecules and near-infrared dyes, demonstrating the potential for dual imaging and delivery applications. Cytotoxicity and spectroscopic analysis of the NPs supported comparative studies between the two polymers and the biological relevancy of the nanomaterials. Employing matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-MSI), full penetration and metabolism of irinotecan-loaded NPs were observed in three-dimensional cell cultures. Results indicate that these strategically designed polymer frameworks and their resulting NPs are exceptional candidates for biomedical applications.

Mass spectrometry imaging (MSI) has become increasingly utilized in the analysis of biological molecules. MSI grants the ability to spatially map thousands of molecules within one experimental run in a label-free manner. While MSI is considered by most to be a qualitative method, recent advancements in instrumentation, sample preparation, and development of standards has made quantitative MSI (qMSI) more common. In this feature article, we present a tailored review of recent advancements in qMSI of therapeutics and biomolecules such as lipids and peptides/proteins. We also provide detailed experimental considerations for conducting qMSI studies on biological samples, aiming to advance the methodology.

Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat) is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes a decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.

We herein describe the preparation, assembly, recognition characteristics, and biocompatibility of novel covalent basket cage CBC-11, composed of four molecular baskets linked to four trivalent aromatic amines through amide groups. The cage is tetrahedral in shape and similar in size to small proteins (Mw =8637 g/mol) with a spacious nonpolar interior for accommodating multiple guests. While 24 carboxylates at the outer surface of CBC-11 render it soluble in aqueous phosphate buffer (PBS) at pH=7.0, the amphiphilic nature prompts its assembly into nanoparticles (d=250 nm, DLS). Cryo-TEM examination of nanoparticles revealed their crystalline nature with wafer-like shapes and hexagonally arranged cages. Nanoparticulate CBC-11 traps anticancer drugs irinotecan and doxorubicin, with each cage binding up to four drug molecules in a non-cooperative manner. The inclusion complexation resulted in nanoparticles growing in size and precipitating. In media containing mammalian cells (HCT 116, human colon carcinoma), the IC50 value of CBC-11 was above 100 μM. While this work presents the first example of a large covalent organic cage operating in water at the physiological pH and forming crystalline nanoparticles, it also demonstrates its biocompatibility and potential to act as a polyvalent binder of drugs for their sequestration or delivery.

Lipids are essential macromolecules that play a crucial role in numerous biological events. Lipids are structurally diverse which allows them to fulfill multiple functional roles. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool to understand the spatial localization of lipids within biological systems. Herein, we report the use of ammonium fluoride (NH4F) as a comatrix additive to enhance lipid detection in biological samples, with signal increase up to 200%. Emphasis was placed on anionic lipid enhancement with negative polarity measurements, with some preliminary work on cationic lipids detailed. We observed lipid signal enhancement of [M-H]- ions with the addition of 500 uM NH4F additive attributed to a proton transfer reaction in several different lipid classes. Overall, our study demon-strates that the use of NH4F comatrix additive substantially improves sensitivity for lipid detection in a MALDI system.

The rapid, point-of-care detection of copper in plasma can greatly aid in a large number of diseases where copper has been implicated to be an important factor, such as cancer, Alzheimer's and Diabetes mellitus. Localized surface plasmon resonance (LSPR) technologies show promise in the inexpensive detection of copper, whereas previous platforms are plagued with selectivity and sensitivity issues. Herein, we have created a sensitive and selective on-chip copper sensor which can produce a colorimetric reading in 60 minutes. The selectivity of the assay is based on 'Click' chemistry and is shown to have little interference with other metal ions present in plasma. The sensitivity of the assay is generated from the coupling of the molecular resonance of a dye and the LSPR of the gold nanoparticles. The assay is capable of measuring copper concentrations in human plasma as low as 4 μM and the linear range of sensitivity, 4 to 20 μM, is in the physiologically relevant range. This robust, colorimetric assay should prove useful in a point-of-care setting.