Opto-Lipidomics of Tissues.

Lipid metabolism and signaling play pivotal functions in biology and disease development. Despite this, currently available optical techniques are limited in their ability to directly visualize the lipidome in tissues. In this study, opto-lipidomics, a new approach to optical molecular tissue imaging is introduced. The capability of vibrational Raman spectroscopy is expanded to identify individual lipids in complex tissue matrices through correlation with desorption electrospray ionization (DESI) - mass spectrometry (MS) imaging in an integrated instrument. A computational pipeline of inter-modality analysis is established to infer lipidomic information from optical vibrational spectra. Opto-lipidomic imaging of transient cerebral ischemia-reperfusion injury in a murine model of ischemic stroke demonstrates the visualization and identification of lipids in disease with high molecular specificity using Raman scattered light. Furthermore, opto-lipidomics in a handheld fiber-optic Raman probe is deployed and demonstrates real-time classification of bulk brain tissues based on specific lipid abundances. Opto-lipidomics opens a host of new opportunities to study lipid biomarkers for diagnostics, prognostics, and novel therapeutic targets.

Non-salt based co-amorphous formulation produced by freeze-drying.

Amino acids-based co-amorphous system (CAM) has shown to be a promising approach to overcome the dissolution challenge of biopharmaceutics classification system class II drugs. To date, most CAM formulations are based on salt formation at a 1:1 M ratio and are prepared by mechanical activation. However, its use in medicinal products is still limited due to the lack of in-depth understanding of non-ionic based molecular interactions. There are also limited studies on the effect of drug-to-co-former ratio, the development of more scalable, less aggressive, manufacturing processes such as freeze drying and its dissolution benefits. This work aims to investigate the effect of the ratio of tryptophan (a model non-ionic amino acid) to indomethacin (a model drug) on a non-salt-based CAM prepared via freeze-drying with the tert-butyl alcohol-water cosolvent system. The CAM material was systemically characterized at various stages of the freeze-drying process using DSC, UV-Vis, FT-IR, NMR, TGA and XRPD. Dissolution performance and physical stability upon storage were also investigated. Freeze-drying using the cosolvent system has been successfully shown to produce CAMs. The molecular interactions involving H-bonding, H/π and π-π between compounds have been confirmed by FT-IR and NMR. The drug release rate for formulations with a 1.5:1 drug: amino acid molar ratio (or 1:0.42 wt ratio) or below is found to be significantly improved compared to the pure crystalline drug. Furthermore, formulation with a 2.3:1 drug:amino acid molar ratio (or 1:0.25 wt ratio) or below have shown to be physically stable for at least 9 months when stored at dry condition (5% relative humidity, 25 °C) compared to the pure amorphous indomethacin. We have demonstrated the potential of freeze-drying using tert-butyl alcohol-water cosolvent system to produce an optimal non-salt-based class II drug-amino acid CAM.

Mechanistic study of the solubilization effect of basic amino acids on a poorly water-soluble drug.

Amino acids have shown promising abilities to form complexes with poorly water-soluble drugs and improve their physicochemical properties for a better dissolution profile through molecular interactions. Salt formation via ionization between acidic drugs and basic amino acids is known as the major contributor to solubility enhancement. However, the mechanism of solubility enhancement due to non-ionic interactions, which is less pH-dependent, remains unclear. The aim of this study is to evaluate non-ionic interactions between a model acidic drug, indomethacin (IND), and basic amino acids, arginine, lysine and histidine, in water. At low concentrations of amino acids, IND-arginine and IND-lysine complexes have shown a linear relationship (AL-type phase solubility diagram) between IND solubility and amino acid concentration, producing ∼1 : 1 stoichiometry of drug-amino acid complexes as expected due to the strong electrostatic interactions. However, IND-histidine complexes have shown a nonlinear relationship with lower improvement in IND solubility due to the weaker electrostatic interactions when compared to arginine and lysine. Interestingly, the results have also shown that at high arginine concentrations, the linearity was lost between IND solubility and amino acid concentration with a negative diversion from linearity, following the type-AN phase solubility. This is indicative that the electrostatic interaction is being interrupted by non-electrostatic interactions, as seen with histidine. The IND-lysine complex, on the other hand, showed a complex curvature phase solubility diagram (type BS) as lysine self-assembles and polymerizes at higher concentrations. The freeze-dried drug-amino acid solids were further characterized using thermal analysis and infrared spectroscopy, with results showing the involvement of weak non-ionic interactions. This study shows that the solubility improvement of an insoluble drug in the presence of basic amino acids was due to both non-ionic and ionic interactions.

Illuminating Host-Parasite Interaction at the Cellular and Subcellular Levels with Infrared Microspectroscopy.

Toxoplasma gondii (T. gondii) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by T. gondii (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of T. gondii-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to T. gondii infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during T. gondii infection.

Inexpensive Portable Infrared Device to Detect and Quantify Alcohols in Hand Sanitizers for Public Health and Safety.

The onset of Covid-19 pandemic has resulted in the exponential growth of alcohol-based hand rub (ABHR)/hand sanitizer use. Reports have emerged of ABHR products containing methanol, a highly toxic compound to humans, exposing users to acute and chronic medical illnesses. While gas chromatography-mass spectrometry (GC-MS) remains the gold-standard method for the detection and identification of impurities in ABHRs, there exist limitations at widespread volume testing. This paper demonstrates the capability of an inexpensive portable pyroelectric linear array infrared spectrometer to rapidly test ABHR and compare the performance with a benchtop Fourier transform infrared spectrometer and HS-GC-MS. Multicomponent partial least square quantification models were built with performance found to be comparable between the two spectrometers and with the HS-GC-MS. Furthermore, the portable spectrometer was field-tested with real-world samples in Malaysia on both retail products (Group A) and freely deployed public dispensers (Group B) between May and November 2020. A total of 386 samples were tested. Only 75.2% of Group A met the criteria of safe and effective ABHR [no detectable methanol and alcohol concentration above 60% (v/v)], while <50% of Group B did. In addition, 7.4 and 18.8% of Group A and Group B, respectively, were found to contain methanol above permissible limits. The high percentage of sub-standard and methanol-containing samples combined with the frequent use of ABHR by the public highlights the need for and importance of a portable and rapid testing device for widespread screening of ABHR against falsified products and protects the general public.

In Vitro Growth- and Encystation-Inhibitory Efficacies of Matcha Green Tea and Epigallocatechin Gallate Against Acanthameoba Castellanii.

We examined the inhibitory effect of matcha green tea (Camellia sinensis) and epigallocatechin gallate (EGCg; the most abundant catechin in tea) on the vegetative growth and encystation of Acanthamoeba castellanii T4 genotype. The sulforhodamine B (SRB) stain-based colorimetric assay and hemocytometer counting were used to determine the reduction in A. castellanii trophozoite proliferation and encystation, in response to treatment with C. sinensis or EGCg. Fourier transform infrared (FTIR) microscopy was used to analyze chemical changes in the trophozoites and cysts due to C. sinensis treatment. Hot brewed and cold brewed matcha inhibited the growth of trophozoites by >40% at a 100 % concentration. EGCg at concentrations of 50 to 500 µM significantly inhibited the trophozoite growth compared to control. Hot brewed matcha (100% concentration) also showed an 87% reduction in the rate of encystation compared to untreated control. Although 500 µM of EGCg increased the rate of encystation by 36.3%, 1000 µM reduced it by 27.7%. Both percentages were not significant compared to control. C. sinensis induced more cytotoxicity to Madin Darby canine kidney cells compared to EGCg. FTIR chemical fingerprinting analysis showed that treatment with brewed matcha significantly increased the levels of glycogen and carbohydrate in trophozoites and cysts.

Identifying the Responses from the Estrogen Receptor-Expressed MCF7 Cells Treated in Anticancer Drugs of Different Modes of Action Using Live-Cell FTIR Spectroscopy.

Recently, we have shown that changes in Fourier transform infrared (FTIR) spectra of living MDA-MB-231 cells (a triple negative cell line) upon exposure to anticancer drugs reflect the changes in the cellular compositions which are correlated to the modes of action of drugs. In the present study, MCF7 cells (an estrogen receptor expressing breast cancer cell line) were exposed to three anticancer drugs belonging to two well-characterized anticancer classes: selective estrogen receptor modulators (SERMs) and DNA-intercalating agent. First, we evaluated if the changes in the spectrum of cells are according to the modes of action of drugs and the characteristics of the MCF7 cell line in the same way as the MDA-MB-231 cell. Living MCF7 cells were treated in the three drugs at half maximal inhibitory concentration (IC50), and the difference spectra were analyzed using principal component analysis (PCA). The results demonstrated clear separation between tamoxifen/toremifene (SERM)-treated cells from the doxorubicin (DNA-intercalator)-treated and untreated cells (control). Tamoxifen and toremifene induced similar spectral changes in the cellular compositions of MCF7 cells and lead to the clustering of these two drugs in the same quadrant of the principal component 1 (PC1) versus PC2 score plots. The separation is mostly attributed to their similar modes of actions. However, doxorubicin-treated MCF7 cells highlighted spectral changes that mainly occur in bands at 1085 and 1200-1240 cm-1, which could be associated with the DNA-intercalation effects of the drug. Second, the pairwise PCA at various individual time points was employed to investigate whether the spectral changes of MCF7 and MDA-MB-231 cells in response to the IC50 of tamoxifen/toremifene and doxorubicin are dependent on the characteristics of the cell lines. The estrogen-expressing MCF7 cells demonstrated significant differences in response to the SERMs in comparison to the triple negative MDA-MB-231 cells, suggesting that different modes of action have taken place in the two tested cell lines. In contrast, the doxorubicin-treated MDA-MB-231 and MCF7 cells show similar changes in 1150-950 cm-1, which indicates that the DNA intercalation effect of doxorubicin is found in both cell lines. The results have demonstrated that live-cell FTIR analysis is sensitive to the different modes of action from the same drugs on cells with different characteristics.

Synchrotron Photothermal Infrared Nanospectroscopy of Drug-Induced Phospholipidosis in Macrophages.

Synchrotron resonance-enhanced infrared atomic force microscopy (RE-AFM-IR) is a near-field photothermal vibrational nanoprobe developed at Diamond Light Source (DLS), capable of measuring mid-infrared absorption spectra with spatial resolution around 100 nm. The present study reports a first application of synchrotron RE-AFM-IR to interrogate biological soft matter at the subcellular level, in this case, on a cellular model of drug-induced phospholipidosis (DIPL). J774A-1 macrophages were exposed to amiodarone (10 µM) or medium for 24 h and chemically fixed. AFM topography maps revealed amiodarone-treated cells with enlarged cytoplasm and very thin regions corresponding to collapsed vesicles. IR maps of the whole cell were analyzed by exploiting the RE-AFM-IR overall signal, i.e., the integrated RE-AFM-IR signal amplitude versus AFM-derived cell thickness, also on lateral resolution around 100 nm. Results show that vibrational band assignment was possible, and all characteristic peaks for lipids, proteins, and DNA/RNA were identified. Both peak ratio and unsupervised chemometric analysis of RE-AFM-IR nanospectra generated from the nuclear and perinuclear regions of untreated and amiodarone-treated cells showed that the perinuclear region (i.e., cytoplasm) of amiodarone-treated cells had significantly elevated band intensities in the regions corresponding to phosphate and carbonyl groups, indicating detection of phospholipid-rich inclusion bodies typical for cells with DIPL. The results of this study are of importance to demonstrate not only the applicability of Synchrotron RE-AFM-IR to soft biological matters with subcellular spatial resolution but also that the spectral information gathered from an individual submicron sample volume enables chemometric identification of treatment and biochemical differences between mammalian cells.

Transmission Fourier Transform Infrared Spectroscopic Imaging, Mapping, and Synchrotron Scanning Microscopy with Zinc Sulfide Hemispheres on Living Mammalian Cells at Sub-Cellular Resolution.

Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by infrared (IR) wavelength making subcellular study challenging. Recently, we have reported, with the use of a prototype zinc sulfide (ZnS) transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron-IR microscopy with a 36× objective with numerical aperture of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron radiation single-point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time, while measurements from the scanning methods produced a smoother image. Synchrotron radiation single-point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.

Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide.

Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol-ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.