Multicolor Raman Beads for Multiplexed Tumor Cell and Tissue Imaging and in Vivo Tumor Spectral Detection.

Developing new nanomaterials with strong and distinctive Raman vibrations in the biological Raman-silent region (1800-2800 cm-1) were highly desirable for Raman hyperspectral detection and imaging in living cells and animals. Herein, polymeric nanoparticles with monomers containing alkyne, cyanide, azide, and carbon-deuterate were prepared as Raman-active nanomaterials (Raman beads) for bioimaging applications. Intense Raman signals were obtained due to the high density of alkyne, cyanide, azide, and carbon-deuterate in single nanoparticles, in absence of metal (such as Au or Ag) as Raman enhancers. We have developed a library of Raman beads for frequency multiplexing through the end-capping substitutions of monomers and demonstrated five-color SRS imaging of mixed nanoparticles with distinct Raman frequencies. In addition, with further surface functionalization of targeting moieties (such as nucleic acid aptamers and targeting peptides), targetable Raman beads were successfully used as probes for tumor targeting and Raman spectroscopic detection, including multicolor SRS imaging in living tumor cells and tissues with high specificity. Further in vivo studies indicated that Raman beads anchored with targeting moieties were successfully employed to target tumors in living mice after tail intravenous injection, and Raman spectral detection of tumor in live mice was achieved only through spontaneous Raman signal at the biological Raman-silent region without any signal enhancement due to a high density of Raman reporters in Raman beads. With further copolymerization of these monomers, Raman beads with supermultiplex barcoding could be readily achieved.

Dextran-Conjugated Caged siRNA Nanoparticles for Photochemical Regulation of RNAi-Induced Gene Silencing in Cells and Mice.

RNA interference (RNAi)-based gene therapy is a precision therapeutic approach for highly efficient sequence-specific gene silencing in vivo or in vitro. Caged RNAs featuring dextran conjugation of antisense and sense RNA strands using photolabile linker were rationally designed and self-assembled to form caged siRNA nanoparticles (Dex- p-siRNA) for photoregulation of target gene expression. The dextran-conjugated caged siRNA nanoparticles showed significant serum nuclease-resistance due to the formation of dextran-siRNA nanoparticles. Photomodulation of exogenous GFP and endogenous mitotic kinesin-5 ( Eg5) gene expression in cells was achieved using the prepared caged Dex- p-siRNA nanoparticles. The caged Dex- p-siRNA nanoparticles targeting GFP successfully photoregulated GFP expression in tumor-bearing mice via intratumoral injection. Caged siRNA nanoparticles with high serum stability not only show great promise for photoregulation of exogenous and endogenous gene expression for both in vitro and in vivo applications, but also provide a novel and convenient way to spatiotemporally control RNAi-induced gene silencing.

Chemical modifications of nucleic acid drugs and their delivery systems for gene-based therapy.

Gene-based therapy is one of essential therapeutic strategies for precision medicine through targeting specific genes in specific cells of target tissues. However, there still exist many problems that need to be solved, such as safety, stability, selectivity, delivery, as well as immunity. Currently, the key challenges of gene-based therapy for clinical potential applications are the safe and effective nucleic acid drugs as well as their safe and efficient gene delivery systems. In this review, we first focus on current nucleic acid drugs and their formulation in clinical trials and on the market, including antisense oligonucleotide, siRNA, aptamer, and plasmid nucleic acid drugs. Subsequently, we summarize different chemical modifications of nucleic acid drugs as well as their delivery systems for gene-based therapeutics in vivo based on nucleic acid chemistry and nanotechnology methods.

Photomodulating Gene Expression by Using Caged siRNAs with Single-Aptamer Modification.

Caged siRNAs incorporating terminal modification were rationally designed for photochemical regulation of gene silencing induced by RNA interference (RNAi). Through the conjugation of a single oligonucleotide aptamer at the 5' terminus of the antisense RNA strand, enhancement of the blocking effect for RNA-induced silencing complex (RISC) formation/processing was expected, due both/either to the aptamers themselves and/or to their interaction with large binding proteins. Two oligonucleotide aptamers (AS1411 and MUC-1) were chosen for aptamer-siRNA conjugation through a photolabile linker. This caging strategy was successfully used to photoregulate gene expression both of firefly luciferase and of green fluorescent protein (GFP) in cells. Further patterning experiments revealed that spatial regulation of GFP expression was successfully achieved by using the aptamer-modified caged siRNA and light activation. We expect that further optimized caged siRNAs featuring aptamer conjugation will be promising for practical applications to spatiotemporal photoregulation of gene expression in the future.

Cholesterol-Modified Caged siRNAs for Photoregulating Exogenous and Endogenous Gene Expression.

siRNA has been widely applied in research and drug development due to its sequence-specific gene silencing ability. However, how to spatiotemporally control its function is still one of its challenges. Light, a fast and noninvasive trigger, is a promising tool for spatiotemporal control of gene expression. Here, we designed and synthesized a new series of caged siRNAs modified with single cholesterol at the 5' terminal of antisense strand RNA through a photolabile linker (Chol-PL-siRNAs). We demonstrated that these caged siRNAs were successfully used to photochemically regulate both exogenous ( firefly luciferase and gfp) and endogenous gene expression (mitotic kinesin-5, Eg5) in cells.

Caged siRNAs with Single cRGD Modification for Photoregulation of Exogenous and Endogenous Gene Expression in Cells and Mice.

RNA interference (RNAi) mediated gene silencing holds significant promise in gene therapy. It is very important to manually regulate the activity of small interference RNAs (siRNAs) in the controllable mode. Here, we designed and synthesized a series of caged siRNAs through bioconjugation of cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptide to the 5' end of siRNA through a photolabile linker. These cRGD modified caged siRNAs allowed for precise light-regulation of gene expression of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and an endogenous gene (the mitosis motor protein, Eg5) in the integrin αvß3 positive cells. This kind of bioconjugate further enabled photochemical activation of siRNA activity, and the target gene silencing was successfully achieved in tumor-bearing mice by intratumoral injection. This study also suggested that photomodulation of target gene expression using single cRGD caged siRNA at the 5' end of antisense strand RNA inhibited siRNA activity probably due to three factors: (1) trapping of cRGD modified siRNA in endosome and lysosome, (2) the steric hindrance of cRGD, (3) the binding of cRGD to its corresponding receptor.

Suppression of IRG-1 Reduces Inflammatory Cell Infiltration and Lung Injury in Respiratory Syncytial Virus Infection by Reducing Production of Reactive Oxygen Species.

UNLABELLED: Respiratory syncytial virus (RSV) infection is a common cause of lower respiratory tract illness in infants and children. RSV is a negative-sense, single-strand RNA (ssRNA) virus that mainly infects airway epithelial cells. Accumulating evidence indicates that reactive oxygen species (ROS) production is a major factor for pulmonary inflammation and tissue damage of RSV disease. We investigated immune-responsive gene-1 (IRG1) expression during RSV infection, since IRG1 has been shown to mediate innate immune response to intracellular bacterial pathogens by modulating ROS and itaconic acid production. We found that RSV infection induced IRG1 expression in human A549 cells and in the lung tissues of RSV-infected mice. RSV infection or IRG1 overexpression promoted ROS production. Accordingly, knockdown of IRG1 induction blocked RSV-induced ROS production and proinflammatory cytokine gene expression. Finally, we showed that suppression of IRG1 induction reduced immune cell infiltration and prevented lung injury in RSV-infected mice. These results therefore link IRG1 induction to ROS production and immune lung injury after RSV infection. IMPORTANCE: RSV infection is among the most common causes of childhood diseases. Recent studies identify ROS production as a factor contributing to RSV disease. We investigated the cause of ROS production and identified IRG1 as a critical factor linking ROS production to immune lung injury after RSV infection. We found that IRG1 was induced in A549 alveolar epithelial cells and in mouse lungs after RSV infection. Importantly, suppression of IRG1 induction reduced inflammatory cell infiltration and lung injury in mice. This study links IRG1 induction to oxidative damage and RSV disease. It also uncovers a potential therapeutic target in reducing RSV-caused lung injury.

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

Defective Lamtor5 Leads to Autoimmunity by Deregulating v-ATPase and Lysosomal Acidification.

Despite accumulating evidence linking defective lysosome function with autoimmune diseases, how the catabolic machinery is regulated to maintain immune homeostasis remains unknown. Late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (Lamtor5) is a subunit of the Ragulator mediating mechanistic target of rapamycin complex 1 (mTORC1) activation in response to amino acids, but its action mode and physiological role are still unclear. Here it is demonstrated that Lamtor5 level is markedly decreased in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). In parallel, the mice with myeloid Lamtor5 ablation developed SLE-like manifestation. Impaired lysosomal function and aberrant activation of mTORC1 are evidenced in Lamtor5 deficient macrophages and PBMCs of SLE patients, accompanied by blunted autolysosomal pathway and undesirable inflammatory responses. Mechanistically, it is shown that Lamtor5 is physically associated with ATP6V1A, an essential subunit of vacuolar H+-ATPase (v-ATPase), and promoted the V0/V1 holoenzyme assembly to facilitate lysosome acidification. The binding of Lamtor5 to v-ATPase affected the lysosomal tethering of Rag GTPase and weakened its interaction with mTORC1 for activation. Overall, Lamtor5 is identified as a critical factor for immune homeostasis by intergrading v-ATPase activity, lysosome function, and mTOR pathway. The findings provide a potential therapeutic target for SLE and/or other autoimmune diseases.

Programmable Tetrahedral DNA-RNA Nanocages Woven with Stimuli-Responsive siRNA for Enhancing Therapeutic Efficacy of Multidrug-Resistant Tumors.

Multidrug resistance (MDR) is a major obstacle limiting the effectiveness of chemotherapy against cancer. The combination strategy of chemotherapeutic agents and siRNA targeting drug efflux has emerged as an effective cancer treatment to overcome MDR. Herein, stimuli-responsive programmable tetrahedral DNA-RNA nanocages (TDRN) have been rationally designed and developed for dynamic co-delivery of the chemotherapeutic drug doxorubicin and P-glycoprotein (P-gp) siRNA. Specifically, the sense and antisense strand sequences of the P-gp siRNA, which are programmable bricks with terminal disulfide bond conjugation, are precisely embedded in one edge of the DNA tetrahedron. TDRN provides a stimuli-responsive release element for dynamic control of functional cargo P-gp siRNA that is significantly more stable than the "tail-like" TDN nanostructures. The stable and highly rigid 3D nanostructure of the siRNA-organized TDRN nanocages demonstrated a notable improvement in the stability of RNase A and mouse serum, as well as long-term storage stability for up to 4 weeks, as evidenced by this study. These biocompatible and multifunctional TDRN nanocarriers with gold nanocluster-assisted delivery (TDRN@Dox@AuNCp) are successfully used to achieve synergistic RNAi/Chemo-therapy in vitro and in vivo. This programmable TDRN drug delivery system, which integrates RNAi therapy and chemotherapy, offers a promising approach for treating multidrug-resistant tumors.

Salsalate reverses metabolic disorders in a mouse model of non-alcoholic fatty liver disease through AMPK activation and caspase-6 activity inhibition.

Salsalate, an ester formed by 2 salicylic acid molecules, has beneficial effect against metabolic disorders in clinical trials and in animal studies. This study focused on the mechanistic aspects of salsalate activity against non-alcoholic fatty liver disease (NAFLD). Using high-fat diet (HFD) fed mice, we showed that salsalate treatment decreased body-weight gains, reduced white adipose tissue mass and improved glycaemic control. Mice in salsalate-treated group also had reduced obese adipose tissue and hepatic macrophage infiltration and inflammation and adipogenesis gene expression. Histology analysis revealed predominant decreases in hepatosteatosis, including both macrovesicular and microvesicular steatoses. The treatment reversed AMPK activity repression that was accompanied by reduced caspase-6 activity and cleavage. Enzymatic assay and cell culture studies showed that salsalate promoted AMPK activation by directly activating AMPK. This study links salsalate effect against metabolic disorders to its activity on reversion of AMPK repression in NAFLD mice and on suppression of adipogenic gene induction.

Rosmarinic acid prevents refractory bacterial pneumonia through regulating Keap1/Nrf2-mediated autophagic pathway and mitochondrial oxidative stress.

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of bacterial pneumonia, featured with exuberant inflammatory cytokine production, extensive oxidative stress and tissue injury. The Keap1/Nrf2 system is the major apparatus essential for host defense against oxidative and electrophilic stresses of both exogenous and endogenous origins, representing a logical target for host-directed strategy to treat severe inflammatory diseases including MRSA-induced pneumonia. In an effort to search therapeutics for bacterial pneumonia, we identify rosmarinic acid (RA) as a covalent modifier of Keap1 and hence an activator of Nrf2. Specifically, RA forms a covalent bond with the cysteine 151 of Keap1 in BTB domain, and blocks its association with Nrf2 for proteasome-mediated degradation. Consequently, RA treatment caused the increased Nrf2 nuclear translocation to initiate antioxidant and mitochondrial biogenic programs, as well as macrophage bactericidal activity through inducing autophagic pathway, which eventually led to expedited bacterial eradication, inflammation resolution, and disease recovery. Collectively, our findings establish RA as a specific inducer of Nrf2 and show its potential to prevent MRSA pneumonia.

Synthesis and Evaluation of Caged siRNAs with Single cRGD Modification for Photoregulating RNA Interference.

We designed and synthesized caged siRNAs with photolabile linker and single cRGD peptide modifications for the photoregulation of gene expression. Photolabile linker and cRGD were inserted at 5' terminus of siRNAs to obtain cRGD-modified caged siRNAs. All these caged siRNAs could be activated through light activation to release the native siRNAs and further achieve the photoregulation of gene silencing of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and one endogenous gene (the mitosis motor protein, Eg5). The intracellular distribution and cellular uptake pathways of these caged siRNAs were also investigated. Tumor-bearing mice were further used to demonstrate the photoregulation of gene silencing with cRGD-modified caged siRNAs in vivo. Overall, the data support the use of this new generation of caged siRNAs in cancer therapy.

Multimerized self-assembled caged two-in-one siRNA nanoparticles for photomodulation of RNAi-induced gene silencing.

We rationally designed and developed caged siRNA nanoparticles (Multi-Chol-siRNA) self-assembled with cholesterol-modified multimerized caged siRNAs for photomodulation of siRNA gene silencing activity. Strong resistance to serum nuclease and RNase A was observed for these cholesterol-modified caged siRNA nanoparticles due to the formation of nanostructures with high intensity of siRNA. These caged Multi-Chol-siRNA self-assembled nanoparticles were successfully used to achieve photochemical regulation of both exogenous GFP and endogenous Eg5 gene expressions with a GFP/RFP transient transfection system and Eg5-associated assays, respectively. Further, Two-in-One caged Multi-Chol-siGFP/siEg5 self-assembled nanoparticles simultaneously targeting GFP and Eg5 genes were also developed. The caged Multi-Chol-siRNA self-assembled nanoparticles have demonstrated the effectiveness of enhancing photomodulation of multiple RNAi-induced gene silencing activities in cells.

Decisive role of pH in synthesis of high purity fluorescent BSA-Au20 nanoclusters.

Various types of bovine serum albumin (BSA)-protected fluorescent gold nanoclusters (BSA-AuNCs) have been fabricated and applied in various fields. However, the conventional synthesis methods for BSA-AuNCs usually yield a low photoluminescence quantum yield (PLQY) in solution. In this study, we systematically examined the influences of incubation time, temperature, and pH on the formation process of BSA-AuNCs and then developed a novel strategy to synthesize BSA-AuNCs with PLQY (26%), far exceeding that of existing counterparts. Of the three important factors, pH, temperature, and time, pH plays a key role in the formation of BSA-AuNCs with different compositions and fluorescence properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) results showed that BSA-Au20NCs with high purity can be produced at a pH value of 10 and the correct combination of incubation temperature and reaction time. The advantages of the obtained BSA-Au20NCs, including small size, high PLQY, long lifetime, high purity, as well as facile modification, make them ideal candidates for luminescent probes in imaging and sensing applications.

Circular siRNAs for Reducing Off-Target Effects and Enhancing Long-Term Gene Silencing in Cells and Mice.

Circular non-coding RNAs are found to play important roles in biology but are still relatively unexplored as a structural motif for chemically regulating gene function. Here, we investigated whether small interfering RNA (siRNA) with a circular structure can circumvent off-target gene silencing, a problem often observed with standard linear duplex siRNA. In the present work, we, for the first time, synthesized a series of circular siRNAs by cyclizing two ends of a single-stranded RNA (sense or antisense strand) to construct circular siRNAs that were more resistant to enzymatic degradation. Gene silencing of GFP and luciferase was successfully achieved using these circular siRNAs with circular sense strand RNAs and their complementary linear antisense strand RNAs. The off-target effect of sense strand RNAs was evaluated and no cross off-target effects were observed. In addition, we successfully achieved longer gene-silencing efficiency in mice with circular siRNAs than with linear siRNAs. These results indicate the promise of circular siRNAs for overcoming off-target effects of siRNAs and enhancing the possible long-term effect of siRNA gene silencing in basic research and drug development.

Curcumin enhances cisplatin sensitivity of human NSCLC cell lines through influencing Cu-Sp1-CTR1 regulatory loop.

BACKGROUND: Curcumin is a naturally occurring polyphenol which has been demonstrated to possess diverse biological activities. We previously reported that curcumin is a biologically active copper chelator with antitumor activity. Copper transporter 1 (CTR1) on the plasma membrane of eukaryotic cells mediates both copper as well as anticancer drug cisplatin uptake. PURPOSE: This study aims to investigate whether curcumin enhances cisplatin sensitivity of human non-small cell lung cancer (NSCLC) through influencing Cu-Sp1-CTR1 regulatory loop. METHODS: The combination effect of curcumin and cisplatin on cell proliferation and apoptosis was determined in vitro and in vivo. Platinum level in A549 cells and tumor tissue was measured by atomic absorption spectrometry (AAS). The binding ability of specificity protein 1 (Sp1) to CTR1 and Sp1 promoters was detected by ChIP assay and luciferase reporter assay system. RESULTS: Here we show that combined curcumin and cisplatin treatment markedly inhibited A549 cells proliferation and induced its apoptosis. Using a mouse model of A549 xenograft, we demonstrated that curcumin inhibits copper influx and increases uptake of platinum ion in tumor. Curcumin treatment enhances the binding of Sp1 to CTR1 and Sp1 promoters, thus induces CTR1 expression and chemosensitization to cisplatin treatment. This process is regulated by the Cu-Sp1-CTR1 regulatory loop. Moreover, the enhancement mediated by curcumin on cisplatin therapeutic efficacy in cultured human NSCLC cell lines (A549, H460, H1299) was dependent on CTR1. CONCLUSIONS: Our results demonstrated copper chelator curcumin enhances the benefits of platinum-containing chemotherapeutic agents and CTR1 could be a promising therapeutic target for non-small cell lung cancer treatment.

Curcumin is a biologically active copper chelator with antitumor activity.

BACKGROUND: Curcumin is a natural product with antitumor activity. The compound targets multiple cell signaling pathways, including cell survival and proliferation, caspase activation and oncogene expression. As a ß-diketone, curcumin also exists as a keto-enol tautomer that chelates transition metal ions with high affinity. PURPOSE: Copper has an integral role in promoting tumor growth and angiogenesis. This study aims to investigate whether curcumin exerts its antitumor activity through copper chelation. METHODS: Copper chelation ability of curcumin was validated by measuring US/VIS spectrum. The antitumor activity and in vivo copper removal ability of curcumin was determined in a murine xenograft model. The effect of curcumin on copper-induced MAPK activation and cell proliferation was determined in cell culture system. RESULTS: Administration of curcumin to tumor-bearing animals resulted in suppression of A549 xenograft growth, an effect that was also observed in animals treated with ammonium tetrathiomolybdate (TM), a metal chelator used for copper storage disorders clinically. The inhibition on tumor growth was associated with reduction of copper concentrations in the serum of treated groups. In cell culture studies, we showed that copper promoted cell proliferation through Erk/MAPK activation. Treatment with curcumin or U0126, a specific MAPK inhibitor, or suppression of cellular uptake of copper by siRNA knockdown of copper transporter protein 1 (CTR1) blocked copper-induced cell proliferation. CONCLUSIONS: This study therefore demonstrates curcumin antitumor effect to its copper chelation capability. These results also implicate copper chelation as a general mechanism for their action of some biologically active polyphenols like flavonoids.

A novel benzenediamine derivative FC98 reduces insulin resistance in high fat diet-induced obese mice by suppression of metaflammation.

Chronic low-grade metabolic inflammation (metaflammation) is a hallmark of metabolic diseases. The aim of this study was to determine the effectiveness of a newly identified benzenediamine derivative (FC98, PubChem CID: 14989837) against metaflammation and insulin resistance using a high fat diet-induced obesity (DIO) murine model. LPS and free fatty acids (FFAs)-induced gene expression and signaling was determined in cell culture systems. Inflammasome activation was determined by measuring IL-1ß release with ELISA. The in vivo activity was assayed in C57BL/6J mice fed with a high fat diet (HFD) by measuring body weight gains, glucose tolerance and insulin sensitivity. The effect was also evaluated by H&E and IHC staining, by measuring gene expression and cytokine production, and by analysis of F4/80(+)CD11b(+) macrophage infiltration. FC98 exhibited anti-inflammatory activity against LPS- and FFAs-induced IL-1ß, IL-6, and TNF-α gene expression and JNK and p38 activation. The IC50 for FC98 to inhibit NO production was determined at 6.8µM. FC98 also dose-dependently inhibited IL-1ß secretion. In DIO mice, FC98 at 10 and 20mg/kg significantly improved metabolic parameters, including body weight, fat mass, glucose disposal and insulin sensitivity. The reduction in adipocyte area, F4/80(+)CD11b(+) macrophage infiltration, proinflammatory gene expression, along with JNK activation, was also significant in those groups. Additionally, FC98-treated animals had increased AKT phosphorylation in response to insulin stimulation. FC98 inhibits metaflammation and ameliorates insulin resistance mainly by inhibiting signaling pathways of proinflammatory response in DIO animals. This study highlights the significance of targeting metaflammation for obesity-attributive metabolic syndrome.