Diarylidene-N-Methyl-4-Piperidones and Spirobibenzopyrans as Antioxidant and Anti-Inflammatory Agents.

Curcumin has antioxidant properties resulting from its radical scavenging ability and inhibition of inflammation-associated factors. However, its lack of solubility, instability, and poor bioavailability are impediments to its therapeutic use. As potential alternatives, we synthesized and performed chemical analysis of thirty diarylidene-N-methyl-4-piperidone (DANMP), diheteroarylidene-N-methyl-4-piperidone (DHANMP), and spirobibenzopyran (SBP) derivatives, one of which was also characterized by single crystal X-ray diffraction. All compounds were evaluated for antioxidant activity via 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and for drug-like properties in silico. A subset of five compounds was investigated in terms of aqueous solubilities, which were significantly improved compared to that of curcumin. In vitro assessments of cellular and anti-inflammatory effects were conducted via real time polymerase chain reaction (RT-PCR) and Griess assays to evaluate the presence of inflammatory/activated (M1) markers and production of nitric oxide (NO) species, which are associated with inflammation. The five compounds reduced levels of markers and NO to extents similar to or better than curcumin in inflamed cells, and showed no adverse effects on cell viability. We show that these compounds possess anti-inflammatory properties and may be used as curcumin-substitutes with improved characteristics.

Dual Mass Spectrometric Tissue Imaging of Nanocarrier Distributions and Their Biochemical Effects.

Nanomaterial-based drug delivery vehicles are able to deliver therapeutics in a controlled, targeted manner. Currently, however, there are limited analytical methods that can detect both nanomaterial distributions and their biochemical effects concurrently. In this study, we demonstrate that matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) can be used together to obtain nanomaterial distributions and biochemical consequences. These studies employ nanoparticle-stabilized capsules (NPSCs) loaded with siRNA as a testbed. MALDI-MSI experiments on spleen tissues from intravenously injected mice indicate that NPSCs loaded with anti-TNF-α siRNA cause changes to the lipid composition in white pulp regions of the spleen, as anticipated, based on pathways known to be affected by TNF-α, whereas NPSCs loaded with scrambled siRNA do not cause the predicted changes. Interestingly, LA-ICP-MSI experiments reveal that the NPSCs primarily localize in the red pulp, suggesting that the observed changes in lipid composition are due to diffusive rather than localized effects on TNF-α production. Such information is only accessible by combining data from the two modalities, which we accomplish by using the heme signals from MALDI-MSI and iron signals from LA-ICP-MSI to overlay the images. Several unexpected changes in lipid composition also occur in regions where the NPSCs are found, suggesting that the NPSCs themselves can influence tissue biochemistry as well.

Macrophage activation by a substituted pyrimido[5,4-b]indole increases anti-cancer activity.

Immunotherapy has become a promising new approach for cancer treatment due to the immune system's ability to remove tumors in a safe and specific manner. Many tumors express anti-inflammatory factors that deactivate the local immune response or recruit peripheral macrophages into pro-tumor roles. Because of this, effective and specific ways of activating macrophages into anti-tumor phenotypes is highly desirable for immunotherapy purposes. Here, the use of a small molecule TLR agonist as a macrophage activator for anti-cancer therapy is reported. This compound, referred to as PBI1, demonstrated unique activation characteristics and expression patterns compared to treatment with LPS, through activation of TLR4. Furthermore, PBI1 treatment resulted in anti-tumor immune behavior, enhancing macrophage phagocytic efficiency five-fold versus non-treated macrophages. Additive effects were observed via use of a complementary strategy (anti-CD47 antibody), resulting in ∼10-fold enhancement of phagocytosis, suggesting this small molecule approach could be used in conjunction with other therapeutics.

CRISPRed Macrophages for Cell-Based Cancer Immunotherapy.

We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the "don't eat me signal" in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of "weaponized" macrophages for cancer immunotherapy.

Harnessing Biology to Deliver Therapeutic and Imaging Entities via Cell-Based Methods.

The accumulation of therapeutic and imaging agents at sites of interest is critical to their efficacy. Similarly, off-target effects (especially toxicity) are a major liability for these entities. For this reason, the use of delivery vehicles to improve the distribution characteristics of bio-active agents has become ubiquitous in the field. However, the majority of traditionally employed, cargo-bearing platforms rely on passive accumulation. Even in cases where "targeting" functionalities are used, the agents must first reach the site in order for the ligand-receptor interaction to occur. The next stage of vehicle development is the use of "recruited" entities, which respond to biological signals produced in the tissues to be targeted, resulting in improved specificities. Recently, many advances have been made in the utilization of cells as delivery agents. They are biocompatible, exhibit excellent circulation lifetimes and tissue penetration capabilities, and respond to chemotactic signals. In this Minireview, we will explore various cell types, modifications, and applications where cell-based delivery agents are used.

Biodistribution of Antibody-MS2 Viral Capsid Conjugates in Breast Cancer Models.

A variety of nanoscale scaffolds, including virus-like particles (VLPs), are being developed for biomedical applications; however, little information is available about their in vivo behavior. Targeted nanoparticles are particularly valuable as diagnostic and therapeutic carriers because they can increase the signal-to-background ratio of imaging agents, improve the efficacy of drugs, and reduce adverse effects by concentrating the therapeutic molecule in the region of interest. The genome-free capsid of bacteriophage MS2 has several features that make it well-suited for use in delivery applications, such as facile production and modification, the ability to display multiple copies of targeting ligands, and the capacity to deliver large payloads. Anti-EGFR antibodies were conjugated to MS2 capsids to construct nanoparticles targeted toward receptors overexpressed on breast cancer cells. The MS2 agents showed good stability in physiological conditions up to 2 days and specific binding to the targeted receptors in in vitro experiments. Capsids radiolabeled with 64Cu isotopes were injected into mice possessing tumor xenografts, and both positron emission tomography-computed tomography (PET/CT) and scintillation counting of the organs ex vivo were used to determine the localization of the agents. The capsids exhibit surprisingly long circulation times (10-15% ID/g in blood at 24 h) and moderate tumor uptake (2-5% ID/g). However, the targeting antibodies did not lead to increased uptake in vivo despite in vitro enhancements, suggesting that extravasation is a limiting factor for delivery to tumors by these particles.

Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules.

We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death.

Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting.

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

Promises and pitfalls of intracellular delivery of proteins.

The direct delivery of functional proteins into the cell cytosol is a key issue for protein therapy, with many current strategies resulting in endosomal entrapment. Protein delivery to the cytosol is challenging due to the high molecular weight and the polarity of therapeutic proteins. Here we review strategies for the delivery of proteins into cells, including cell-penetrating peptides, virus-like particles, supercharged proteins, nanocarriers, polymers, and nanoparticle-stabilized nanocapsules. The advantages and disadvantages of these approaches including cytosolar delivery are compared and contrasted, with promising pathways forward identified.

Polarization of macrophages to an anti-cancer phenotype through in situ uncaging of a TLR 7/8 agonist using bioorthogonal nanozymes.

Macrophages are plastic cells of the immune system that can be broadly classified as having pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. M2-like macrophages are often associated with cancers and can promote cancer growth and create an immune-suppressive tumor microenvironment. Repolarizing macrophages from M2-like to M1-like phenotype provides a crucial strategy for anticancer immunotherapy. Imiquimod is an FDA-approved small molecule that can polarize macrophages by activating toll-like receptor 7/8 (TLR 7/8) located inside lysosomes. However, the non-specific inflammation that results from the drug has limited its systemic application. To overcome this issue, we report the use of gold nanoparticle-based bioorthogonal nanozymes for the conversion of an inactive, imiquimod-based prodrug to an active compound for macrophage re-education from anti- to pro-inflammatory phenotypes. The nanozymes were delivered to macrophages through endocytosis, where they uncaged pro-imiquimod in situ. The generation of imiquimod resulted in the expression of pro-inflammatory cytokines. The re-educated M1-like macrophages feature enhanced phagocytosis of cancer cells, leading to efficient macrophage-based tumor cell killing.

PET Imaging and biodistribution of chemically modified bacteriophage MS2.

The fields of nanotechnology and medicine have merged in the development of new imaging and drug delivery agents based on nanoparticle platforms. As one example, a mutant of bacteriophage MS2 can be differentially modified on the exterior and interior surfaces for the concurrent display of targeting functionalities and payloads, respectively. In order to realize their potential for use in in vivo applications, the biodistribution and circulation properties of this class of agents must first be investigated. A means of modulating and potentially improving the characteristics of nanoparticle agents is the appendage of PEG chains. Both MS2 and MS2-PEG capsids possessing interior DOTA chelators were labeled with (64)Cu and injected intravenously into mice possessing tumor xenografts. Dynamic imaging of the agents was performed using PET-CT on a single animal per sample, and the biodistribution at the terminal time point (24 h) was assessed by gamma counting of the organs ex vivo for 3 animals per agent. Compared to other viral capsids of similar size, the MS2 agents showed longer circulation times. Both MS2 and MS2-PEG bacteriophage behaved similarly, although the latter agent showed significantly less uptake in the spleen. This effect may be attributed to the ability of the PEG chains to mask the capsid charge. Although the tumor uptake of the agents may result from the enhanced permeation and retention (EPR) effect, selective tumor imaging may be achieved in the future by using exterior targeting groups.

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer.

As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Macrophage circadian rhythms are differentially affected based on stimuli.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

Macrophage-Encapsulated Bioorthogonal Nanozymes for Targeting Cancer Cells.

Macrophages migrate to tumor sites by following chemoattractant gradients secreted by tumor cells, providing a truly active targeting strategy for cancer therapy. However, macrophage-based delivery faces challenges of cargo loading, control of release, and effects of the payload on the macrophage vehicle. We present a strategy that employs bioorthogonal "nanozymes" featuring transition metal catalysts (TMCs) to provide intracellular "factories" for the conversion of prodyes and prodrugs into imaging agents and chemotherapeutics. These nanozymes solubilize and stabilize the TMCs by embedding them into self-assembled monolayer coating gold nanoparticles. Nanozymes delivered into macrophages were intracellularly localized and retained activity even after prolonged (72 h) incubation. Significantly, nanozyme-loaded macrophages maintained their inherent migratory ability toward tumor cell chemoattractants, efficiently killing cancer cells in cocultures. This work establishes the potential of nanozyme-loaded macrophages for tumor site activation of prodrugs, providing readily tunable dosages and delivery rates while minimizing off-target toxicity of chemotherapeutics.

Selective treatment of intracellular bacterial infections using host cell-targeted bioorthogonal nanozymes.

Intracellular bacterial infections are difficult to treat, and in the case of Salmonella and related infections, can be life threatening. Antibiotic treatments for intracellular infections face challenges including cell penetration and intracellular degradation that both reduce antibiotic efficacy. Even when treatable, the increased dose of antibiotics required to counter infections can strongly impact the microbiome, compromising the native roles of beneficial non-pathogenic species. Bioorthogonal catalysis provides a new tool to combat intracellular infections. Catalysts embedded in the monolayers of gold nanoparticles (nanozymes) bioorthogonally convert inert antibiotic prodrugs (pro-antibiotics) into active species within resident macrophages. Targeted nanozyme delivery to macrophages was achieved through mannose conjugation and subsequent uptake VIA the mannose receptor (CD206). These nanozymes efficiently converted pro-ciprofloxacin to ciprofloxacin inside the macrophages, selectively killing pathogenic Salmonella enterica subsp. enterica serovar Typhimurium relative to non-pathogenic Lactobacillus sp. in a transwell co-culture model. Overall, this targeted bioorthogonal nanozyme strategy presents an effective treatment for intracellular infections, including typhoid and tuberculosis.

Circadian Alterations Increase with Progression in a Patient-Derived Cell Culture Model of Breast Cancer.

Circadian rhythm disruption can elicit the development of various diseases, including breast cancer. While studies have used cell lines to study correlations between altered circadian rhythms and cancer, these models have different genetic backgrounds and do not mirror the changes that occur with disease development. Isogenic cell models can recapitulate changes across cancer progression. Hence, in this study, a patient-derived breast cancer model, the 21T series, was used to evaluate changes to circadian oscillations of core clock protein transcription as cells progress from normal to malignant states. Three cell lines were used: H16N2 (normal breast epithelium), 21PT (atypical ductal hyperplasia), and 21MT-1 (invasive metastatic carcinoma). The cancerous cells are both HER2+. We assessed the transcriptional profiles of two core clock proteins, BMAL1 and PER2, which represent a positive and negative component of the molecular oscillator. In the normal H16N2 cells, both genes possessed rhythmic mRNA oscillations with close to standard periods and phases. However, in the cancerous cells, consistent changes were observed: both genes had periods that deviated farther from normal and did not have an anti-phase relationship. In the future, mechanistic studies should be undertaken to determine the oncogenic changes responsible for the circadian alterations found.

Nanodelivery vehicles induce remote biochemical changes in vivo.

Nanomaterial-based platforms are promising vehicles for the controlled delivery of therapeutics. For these systems to be both efficacious and safe, it is essential to understand where the carriers accumulate and to reveal the site-specific biochemical effects they produce in vivo. Here, a dual-mode mass spectrometry imaging (MSI) method is used to evaluate the distributions and biochemical effects of anti-TNF-α nanoparticle stabilized capsules (NPSCs) in mice. It is found that most of the anticipated biochemical changes occur in sub-organ regions that are separate from where the nanomaterials accumulate. In particular, TNF-α-specific lipid biomarker levels change in immune cell-rich regions of organs, while the NPSCs accumulate in spatially isolated filtration regions. Biochemical changes that are associated with the nanomaterials themselves are also observed, demonstrating the power of matrix-assisted laser desorption/ionization (MALDI) MSI to reveal markers indicating possible off-target effects of the delivery agent. This comprehensive assessment using MSI provides spatial context of nanomaterial distributions and efficacy that cannot be easily achieved with other imaging methods, demonstrating the power of MSI to evaluate both expected and unexpected outcomes associated with complex therapeutic delivery systems.

Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock.

Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression was driven via Bmal1- or Per2-promoters, were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules increase or decrease the circadian periods of Bmal1 and Per2 in a dose-dependent manner, but period length does not correlate with the extent of cell migration or proliferation. Nonetheless, molecules that affected circadian oscillations to a greater degree resulted in substantial influence on cellular behaviors (ie, motility and colony formation), which may also be attributable to noncircadian targets. Furthermore, we find that the ability and extent to which the molecules are able to affect oscillations is independent of whether they are direct or indirect modulators. Because of the numerous connections and feedback between the circadian clock and other pathways, it is important to consider the effects of both in assessing these and other compounds.

Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cancer cell lines. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also varied in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's effects on cell proliferation and migration have been studied previously were evaluated: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that circadian, migration, and proliferative effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity and substantially reduced oncogenic features, specifically two-dimensional cell motility and anchorage-independent growth. Based on these results and those previously described, we posit that the effects of nobiletin are indeed cell-type dependent, and that a positive correlation may exist between nobiletin's circadian and therapeutic effects.