Fluorinated Isoindolinone-Based Glucosylceramide Synthase Inhibitors with Low Human Dose Projections.

Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.

Pyrazole Ureas as Low Dose, CNS Penetrant Glucosylceramide Synthase Inhibitors for the Treatment of Parkinson's Disease.

Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.

Split-Dose Administration Enhances Immune Responses Elicited by a mRNA/Lipid Nanoparticle Vaccine Expressing Respiratory Syncytial Virus F Protein.

mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates.

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.

Plasmonic Nanoparticle-Based Digital Cytometry to Quantify MUC16 Binding on the Surface of Leukocytes in Ovarian Cancer.

Although levels of the circulating ovarian cancer marker (CA125) can distinguish ovarian masses that are likely to be malignant and correlate with severity of disease, serum CA125 has not proved useful in general population screening. Recently, cell culture studies have indicated that MUC16 may bind to the Siglec-9 receptor on natural killer (NK) cells where it downregulates the cytotoxicity of NK cells, allowing ovarian cancer cells to evade immune surveillance. We present evidence that the presence of MUC16 can be locally visualized and imaged on the surface of peripheral blood mononuclear cells (PBMCs) in ovarian cancer via a novel "digital" cytometry technique that incorporates: (i) OC125 monoclonal antibody-conjugated gold nanoparticles as optical nanoprobes, (ii) a high contrast dark-field microscopy system to detect PBMC-bound gold nanoparticles, and (iii) a computational algorithm for automatic counting of these nanoparticles to estimate the quantity of surface-bound MUC16. The quantitative detection of our technique was successfully demonstrated by discriminating clones of the ovarian cancer cell line, OVCAR3, based on low, intermediate, and high expression levels of MUC16. Additionally, PBMC surface-bound MUC16 was tracked in an ovarian cancer patient over a 17 month period; the results suggest that the binding of MUC16 on the surface of immune cells may play an early indicator for recurrent metastasis 6 months before computational tomography-based clinical diagnosis. We also demonstrate that the levels of surface-bound MUC16 on PBMCs from five ovarian cancer patients were greater than those from five healthy controls.

Longitudinal monitoring of cancer cell subpopulations in monolayers, 3D spheroids, and xenografts using the photoconvertible dye DiR.

A central challenge in cancer biology is the identification, longitudinal tracking, and -omics analysis of specific cells in vivo. To this aim, photoconvertible fluorescent dyes are reporters that are characterized by a set of excitation and emission spectra that can be predictably altered, resulting in a distinct optical signature following irradiation with a specific light source. One such dye, DiR, is an infrared fluorescent membrane probe that can irreversibly undergo such a switch. Here, we demonstrate a method using DiR for the spatiotemporal labeling of specific cells in the context of cancer cell monolayer cultures, 3D tumor spheroids, and in vivo melanoma xenograft models to monitor the proliferation of cellular subpopulations of interest over time. Importantly, the photoconversion process is performed in situ, supporting the pursuit of novel avenues of research in molecular pathology.

Raman technologies in cancer diagnostics.

Despite significant effort, cancer still remains a leading cause of death worldwide. In order to reduce its burden, the development and improvement of noninvasive strategies for early detection and diagnosis of cancer are urgently needed. Raman spectroscopy, an optical technique that relies on inelastic light scattering arising from molecular vibrations, is one such strategy, as it can noninvasively probe cancerous markers using only endogenous contrast. In this review, spontaneous, coherent and surface enhanced Raman spectroscopies and imaging, as well as the fundamental principles governing the successful use of these techniques, are discussed. Methods for spectral data analysis are also highlighted. Utilization of the discussed Raman techniques for the detection and diagnosis of cancer in vitro, ex vivo and in vivo is described. The review concludes with a discussion of the future directions of Raman technologies, with particular emphasis on their clinical translation.

Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells--a mechanistic study.

Nanoparticles (NPs), in particular noble metal nanoparticles, have been incorporated into many therapeutic and biodiagnostic applications. While these particles have many advantageous physical and optical properties, little is known about their intrinsic intracellular effects in biological environments. Here, we report the possible cell death mechanisms triggered in human oral squamous cell carcinoma (HSC-3) cells after exposure to extracellular, cytoplasm, and nuclear localized AuNPs and AgNPs. NP uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation, mitochondrial depolarization, and the levels and/or translocation of caspase-dependent and caspase-independent proteins were assessed under control and localized metal nanoparticle exposure. Exposure to AuNPs resulted the adoption of a quiescent cellular state, as AuNPs caused a decrease in intracellular ATP, but no change in viability or cell death populations. However, AgNP exposure significantly reduced HSC-3 cell viability and increased apoptotic populations, especially when localized at the cytoplasm and nucleus. Increased cell death populations were linked to an increase in intracellular ROS generation. Western blot analysis indicated cytoplasm localized AgNPs and nuclear localized AgNPs utilized a caspase-independent apoptotic pathway that involved the nuclear translocation of AIF and p38 MAPK proteins. These results demonstrate that the degree of cytotoxicity increases as AgNPs move from extracellular localization to nuclear localization, whereas changing AuNP localization does not trigger any significant cytotoxicity.

Determining Drug Efficacy Using Plasmonically Enhanced Imaging of the Morphological Changes of Cells upon Death.

Recently, we utilized the optical properties of gold nanoparticles (AuNPs) for plasmonically enhanced Rayleigh scattering imaging spectroscopy (PERSIS), a new technique that enabled the direct observation of AuNP localization. In this study, we employ PERSIS by using AuNPs as light-scattering probes to compare the relative efficacy of three chemotherapeutic drugs on human oral squamous carcinoma cells. Although the drugs induced apoptotic cell death through differing mechanisms, morphological changes including cell membrane blebbing and shrinkage, accompanied by an increase in white light scattering, were visually evident. By utilizing the AuNPs to increase the cells' inherent Rayleigh scattering, we have obtained the time profile of cell death from the anticancer drugs using a single sample of cells in real time, using inexpensive equipment available in any lab. From this time profile, we calculated cell death enhancement factors to compare the relative efficacies of the different drugs using our technique, which corresponded to those calculated from the commonly used XTT cell viability assay. Although this technique does not impart molecular insights into cell death, the ability to quantitatively correlate cell death to morphological changes suggests the potential use of this technique for the rapid screening of drug analogues to determine the most effective structure against a disease or cell line.

The optical, photothermal, and facile surface chemical properties of gold and silver nanoparticles in biodiagnostics, therapy, and drug delivery.

Nanotechnology is a rapidly growing area of research in part due to its integration into many biomedical applications. Within nanotechnology, gold and silver nanostructures are some of the most heavily utilized nanomaterial due to their unique optical, photothermal, and facile surface chemical properties. In this review, common colloid synthesis methods and biofunctionalization strategies of gold and silver nanostructures are highlighted. Their unique properties are also discussed in terms of their use in biodiagnostic, imaging, therapeutic, and drug delivery applications. Furthermore, relevant clinical applications utilizing gold and silver nanostructures are also presented. We also provide a table with reviews covering related topics.

Observing real-time molecular event dynamics of apoptosis in living cancer cells using nuclear-targeted plasmonically enhanced Raman nanoprobes.

Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.

The most effective gold nanorod size for plasmonic photothermal therapy: theory and in vitro experiments.

The development of new and improved photothermal contrast agents for the successful treatment of cancer (or other diseases) via plasmonic photothermal therapy (PPTT) is a crucial part of the application of nanotechnology in medicine. Gold nanorods (AuNRs) have been found to be the most effective photothermal contrast agents, both in vitro and in vivo. Therefore, determining the optimum AuNR size needed for applications in PPTT is of great interest. In the present work, we utilized theoretical calculations as well as experimental techniques in vitro to determine this optimum AuNR size by comparing plasmonic properties and the efficacy as photothermal contrast agents of three different sizes of AuNRs. Our theoretical calculations showed that the contribution of absorbance to the total extinction, the electric field, and the distance at which this field extends away from the nanoparticle surface all govern the effectiveness of the amount of heat these particles generate upon NIR laser irradiation. Comparing between three different AuNRs (38 × 11, 28 × 8, and 17 × 5 nm), we determined that the 28 × 8 nm AuNR is the most effective in plasmonic photothermal heat generation. These results encouraged us to carry out in vitro experiments to compare the PPTT efficacy of the different sized AuNRs. The 28 × 8 nm AuNR was found to be the most effective photothermal contrast agent for PPTT of human oral squamous cell carcinoma. This size AuNR has the best compromise between the total amount of light absorbed and the fraction of which is converted to heat. In addition, the distance at which the electric field extends from the particle surface is most ideal for this size AuNR, as it is sufficient to allow for coupling between the fields of adjacent particles in solution (i.e., particle aggregates), resulting in effective heating in solution.

XAV939: from a small inhibitor to a potent drug bioconjugate when delivered by gold nanoparticles.

Nanoparticles as potential drug delivery vectors are drawing more attention every day. Here, we used gold nanopspheres (AuNSs) to selectively target the Wnt signaling pathway in human oral squamous cell carcinoma (HSC-3) cells. In a previously conducted study, XAV939, a small inhibiter, was found to strongly regulate the Wnt pathway by inhibiting the tankyrase enzyme and subsequent stabilization of cytoplasmic axin levels. In the present study, conjugating XAV939 molecules to AuNSs is found to enhance its potency by at least 100 times over its free form in killing HSC-3 cancer cells. Additionally, XAV 939 uptake studies have demonstrated an enhanced XAV939 bioconjugate delivery to the targeted cells compared to the passive cellular diffusion of the free drug at the same concentration. Furthermore, our study revealed that drug delivery and cytotoxicity are directly related to the size of the functionalized nanoparticles.

Exploiting the nanoparticle plasmon effect: observing drug delivery dynamics in single cells via Raman/fluorescence imaging spectroscopy.

Drug delivery systems (DDSs) offer efficient and localized drug transportation as well as reduce associated side effects. In order to better understand DDSs, precise observation of drug release and delivery is required. Here, we present a strategy, plasmonic-tunable Raman/fluorescence imaging spectroscopy, to track the release and delivery of an anticancer drug (doxorubicin) from gold nanoparticle carriers in real time at a single living cell level. A pH-responsive drug release profile was attained through the conjugation of doxorubicin (DOX) to the nanoparticle surface via a pH-sensitive hydrazone linkage. When DOX is bound to the surface of the gold nanoparticle, its surface-enhanced Raman spectrum can be seen, but its fluorescence is quenched. When released, due to the lysosomes' acidic pH, its Raman enhancement is greatly reduced, changing the acquired Raman spectrum and in turn allowing for the visualization of its fluorescence signal. The plasmonic-tunable Raman/fluorescence properties enabled the tracking of the DOX release and delivery process from the gold nanoparticle surface to the lysosomes of single living cells under the acidic pH change of their microenvironments. This technique offers great potential to follow the molecular mechanisms of drug delivery and release in living cells, as well as the cellular response to drug action.

A new nanotechnology technique for determining drug efficacy using targeted plasmonically enhanced single cell imaging spectroscopy.

Recently, we described a new technique, targeted plasmonically enhanced single cell imaging spectroscopy (T-PESCIS), which exploits the plasmonic properties of gold nanoparticles, e.g. gold nanospheres, to simultaneously obtain enhanced intracellular Raman molecular spectra and enhanced Rayleigh cell scattering images throughout the entire span of a single cell cycle. In the present work, we demonstrate the use of T-PESCIS in evaluating the relative efficacy and dynamics of two popular chemotherapy drugs on human oral squamous carcinoma (HSC-3) cells. T-PESCIS revealed three plasmonically enhanced Raman scattering vibration bands, 500, 1000, and 1585 cm(-1), associated with the cellular death dynamics. Detailed analysis indicated that the decrease in the 500 cm(-1) band did not correlate well with drug efficacy but could indicate death initiation. The time it takes for the relative intensity of either the 1000 or 1585 cm(-1) band ("SERS death" bands) to appear and increase to its maximum value after the injection of a known concentration of the drug can be related to the drug's efficacy. The inverse ratio, termed cell death enhancement factor, of these characteristic death times when using either band, especially the spectrally sharp band at 1000 cm(-1), gave the correct drug efficacy ratio as determined by the commonly used XTT cell viability assay method. These results strongly suggest the potential future use of this technique in determining the efficacy, dynamics, and molecular mechanisms of various drugs against different diseases.

Real-time molecular imaging throughout the entire cell cycle by targeted plasmonic-enhanced Rayleigh/Raman spectroscopy.

Due to their strong enhancement of scattered light, plasmonic nanoparticles have been utilized for various biological and medical applications. Here, we describe a new technique, Targeted Plasmonic-Enhanced Single-Cell Rayleigh/Raman Spectroscopy, to monitor the molecular changes of any cell-component, such as the nucleus, during the different phases of its full cell cycle by simultaneously recording its Rayleigh images and Raman vibration spectra in real-time. The analysis of the observed Raman DNA and protein peaks allowed the different phases of the cell cycle to be identified. This technique could be used for disease diagnostics and potentially improve our understanding of the molecular mechanisms of cellular functions such as division, death, signaling, and drug action.

Small molecule-gold nanorod conjugates selectively target and induce macrophage cytotoxicity towards breast cancer cells.

Gold nanoparticles are known to activate anti-tumor potential in macrophage immune cells; however, the subsequent effects of these cells on others nearby are poorly understood. A novel gold-nanoparticle conjugate that selectively targets and induces cytotoxic activity of tumor-associated macrophages towards breast cancer cells in co-culture is synthesized. These constructs are promising new tools for studying fundamental biological interactions with nanoscale materials and candidates for emerging macrophage-mediated delivery applications.

Antiandrogen gold nanoparticles dual-target and overcome treatment resistance in hormone-insensitive prostate cancer cells.

Prostate cancer is the most commonly diagnosed cancer among men in the developed countries.(1) One in six males in the U.S.(2) and one in nine males in the U.K.(3) will develop the disease at some point during their lifetime. Despite advances in prostate cancer screening, more than a quarter of a million men die from the disease every year(1) due primarily to treatment-resistance and metastasis. Colloidal nanotechnologies can provide tremendous enhancements to existing targeting/treatment strategies for prostate cancer to which malignant cells are less sensitive. Here, we show that antiandrogen gold nanoparticles--multivalent analogues of antiandrogens currently used in clinical therapy for prostate cancer--selectively engage two distinct receptors, androgen receptor (AR), a target for the treatment of prostate cancer, as well as a novel G-protein coupled receptor, GPRC6A, that is also upregulated in prostate cancer. These nanoparticles selectively accumulated in hormone-insensitive and chemotherapy-resistant prostate cancer cells, bound androgen receptor with multivalent affinity, and exhibited greatly enhanced drug potency versus monovalent antiandrogens currently in clinical use. Further, antiandrogen gold nanoparticles selectively stimulated GPRC6A with multivalent affinity, demonstrating that the delivery of nanoscale antiandrogens can also be facilitated by the transmembrane receptor in order to realize increasingly selective, increasingly potent therapy for treatment-resistant prostate cancers.

Size matters: gold nanoparticles in targeted cancer drug delivery.

Cancer is the current leading cause of death worldwide, responsible for approximately one quarter of all deaths in the USA and UK. Nanotechnologies provide tremendous opportunities for multimodal, site-specific drug delivery to these disease sites and Au nanoparticles further offer a particularly unique set of physical, chemical and photonic properties with which to do so. This review will highlight some recent advances, by our laboratory and others, in the use of Au nanoparticles for systemic drug delivery to these malignancies and will also provide insights into their rational design, synthesis, physiological properties and clinical/preclinical applications, as well as strategies and challenges toward the clinical implementation of these constructs moving forward.