Probing the structural evolution on the surface of cardiolipin vesicles with an amphiphilic second harmonic generation and fluorescence probe.

Investigating the influence of the ambient chemical environment on molecular behaviors in liposomes is crucial for understanding and manipulating cellular vitality as well as the capabilities of lipid drug carriers in various environments. Here, we designed and synthesized a second harmonic generation (SHG) and fluorescence probe molecule called Pyr-Py+-N+ (PPN), which possesses membrane-targeting capability. We employed PPN to investigate the response of lipid vesicles composed of cardiolipin to the presence of exogenous salt. The kinetic behaviors, including the adsorption and embedding of PPN on the surface of small unilamellar vesicles (SUVs) composed of cardiolipin, were analyzed. The response of the SUVs to the addition of NaCl was also monitored. A rapid decrease in vesicle size can be evidenced through the rapid drop in SHG emission originating from PPN located on the vesicle surface.

Monitoring the response of a model protocell to dye and surfactant molecules through second harmonic generation and fluorescence imaging.

Probing the interaction between molecules and protocells is crucial for understanding the passive transport of functional molecules in and out of artificial and real cells. Second-harmonic generation (SHG) has been proven to be a powerful method for analyzing the adsorption and cross-membrane transport of molecules on lipid bilayers. In this study, we used SHG and two-photon fluorescence (TPF) imaging to study the interaction of charged dye molecules (D289) with a lipid vesicle. Unexpectedly, it was observed that the transport of D289 at a relatively high concentration is not as efficient as that at a lower dye concentration. Periodic shrinking of the model protocell and discharging of D289 out from the vesicle were revealed by combined analyses of SHG and TPF images. The response of the vesicle to a surfactant was also analyzed with D289 as a probe. This work demonstrates that the combined SHG and TPF imaging method is a unique approach that can provide detailed information on the interaction of molecules and lipids (both morphology and molecular kinetics). Determining these subtle interfacial kinetics in molecules is important for understanding the mechanism of many biophysical processes occurring on lipids.

A NIR-Activated and Mild-Temperature-Sensitive Nanoplatform with an HSP90 Inhibitor for Combinatory Chemotherapy and Mild Photothermal Therapy in Cancel Cells.

Mild photothermal therapy (PTT) shows great potential to treat cancers while avoiding unwanted damage to surrounding normal cells. However, the efficacy of mild PTT is normally moderate because of the low hyperthermia temperature and limited light penetration depth. Chemotherapy has unlimited penetration but often suffers from unsatisfactory efficacy in view of the occurrence of drug resistance, suboptimal drug delivery and release profile. As a result, the combinatory of chemotherapy and mild PTT would integrate their advantages and overcome the shortcomings. Herein, we synthesized an NIR-activatable and mild-temperature-sensitive nanoplatform (BDPII-gel@TSL) composed of temperature-sensitive liposomes (TSL), heat shock protein 90 (HSP90) inhibitor (geldanamycin) and photothermal agent (BDPII), for dual chemotherapy and mild PTT in cancer cells. BDPII, constructed with donor-acceptor moieties, acts as an excellent near-infrared (NIR) photothermal agent (PTA) with a high photothermal conversion efficiency (80.75%). BDPII-containing TSLs efficiently produce a mild hyperthermia effect (42 °C) under laser irradiation (808 nm, 0.5 W cm-2). Importantly, the phase transformation of TSL leads to burst release of geldanamycin from BDPII-gel@TSL, and this contributes to down-regulation of the overexpression of HSP90, ensuring efficient inhibition of cancer cell growth. This research provides a dual-sensitive synergistic therapeutic strategy for cancer cell treatment.

Combined Second Harmonic Generation and Fluorescence Analyses of the Structures and Dynamics of Molecules on Lipids Using Dual-Probes: A Review.

Revealing the structures and dynamic behaviors of molecules on lipids is crucial for understanding the mechanism behind the biophysical processes, such as the preparation and application of drug delivery vesicles. Second harmonic generation (SHG) has been developed as a powerful tool to investigate the molecules on various lipid membranes, benefiting from its natural property of interface selectivity, which comes from the principle of even order nonlinear optics. Fluorescence emission, which is in principle not interface selective but varies with the chemical environment where the chromophores locate, can reveal the dynamics of molecules on lipids. In this contribution, we review some examples, which are mainly from our recent works focusing on the application of combined spectroscopic methods, i.e., SHG and two-photon fluorescence (TPF), in studying the dynamic behaviors of several dyes or drugs on lipids and surfactants. This review demonstrates that molecules with both SHG and TPF efficiencies may be used as intrinsic dual-probes in plotting a clear physical picture of their own behaviors, as well as the dynamics of other molecules, on lipid membranes.

Identification of Core Genes and Pathways in Melanoma Metastasis via Bioinformatics Analysis.

Metastasis is the leading cause of melanoma-related mortality. Current therapies are rarely curative for metastatic melanoma, revealing the urgent need to identify more effective preventive and therapeutic targets. This study aimed to screen the core genes and molecular mechanisms related to melanoma metastasis. A gene expression profile, GSE8401, including 31 primary melanoma and 52 metastatic melanoma clinical samples, was downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between melanoma metastases and primary melanoma were screened using GEO2R tool. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses of DEGs were performed using the Database for Annotation Visualization and Integrated Discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape with Molecular Complex Detection (MCODE) plug-in tools were utilized to detect the protein-protein interaction (PPI) network among DEGs. The top 10 genes with the highest degrees of the PPI network were defined as hub genes. In the results, 425 DEGs, including 60 upregulated genes and 365 downregulated genes, were identified. The upregulated genes were enriched in ECM-receptor interactions and the regulation of actin cytoskeleton, while 365 downregulated genes were enriched in amoebiasis, melanogenesis, and ECM-receptor interactions. The defined hub genes included CDK1, COL17A1, EGFR, DSG1, KRT14, FLG, CDH1, DSP, IVL, and KRT5. In addition, the mRNA and protein levels of the hub genes during melanoma metastasis were verified in the TCGA database and paired post- and premetastatic melanoma cells, respectively. Finally, KRT5-specific siRNAs were utilized to reduce the KRT5 expression in melanoma A375 cells. An MTT assay and a colony formation assay showed that KRT5 knockdown significantly promoted the proliferation of A375 cells. A Transwell assay further suggested that KRT5 knockdown significantly increased the cell migration and cell invasion of A375 cells. This bioinformatics study provided a deeper understanding of the molecular mechanisms of melanoma metastasis. The in vitro experiments showed that KRT5 played the inhibitory effects on melanoma metastasis. Therefore, KRT5 may serve important roles in melanoma metastasis.

Evaluating the cross-membrane dynamics of a charged molecule on lipid films with different surface curvature.

Does the curvature of a phospholipid membrane influence the permeability of the lipid bilayers? This is a question of great importance yet hard to answer. In this work the permeability of a positively charged rod like probing molecule (D289 dye) on the bilayers of DOPG lipid vesicles was investigated using angle resolved second harmonic generation method. It was revealed that the permeability of D289 on the surface of small vesicles with âˆ¼ 100 nm diameter was notably lower than that on giant vesicles with âˆ¼ 1000 nm diameter. With the increasing of temperature or the introducing of dimethyl sulfoxide (DMSO) in the solutions, the D289 permeability of the lipid bilayers was notably enhanced as expected, on both the small and the giant vesicles. Still, the D289 permeability of the lipid film with more curvature is lower than the relatively flat film in all these cases. This work demonstrated a general protocol for the investigating of surface permeability of lipid films with various curvature.

Unveiling the Molecular Dynamics in a Living Cell to the Subcellular Organelle Level Using Second-Harmonic Generation Spectroscopy and Microscopy.

Second-harmonic generation (SHG) microscopy has been proved to be a powerful method for investigating the structures of biomaterials. SHG spectra were also generally used to probe the adsorption and cross-membrane transport of molecules on lipid bilayers in situ and in real time. In this work, we applied SHG and two-photon fluorescence (TPF) spectra to investigate the dynamics of an amphiphilic ion with an SHG and TPF chromophore, D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), on the surface of human chronic myelogenous leukemia (K562) cells and the subcellular structures inside the cells. The adsorption and cross-membrane transport of D289 into the cells and then into the organelles such as mitochondria were revealed. SHG images were also recorded and used to demonstrate their capability of probing molecular dynamics in organelles in K562 cells. This work demonstrated the first SHG investigation of the cross-membrane transport dynamics on the surface of subcellular organelles. It may also shed light on the differentiation of different types of subcellular structures in cells.

Tumor-derived Exosome Promotes Metastasis via Altering its Phenotype and Inclusions.

Although tumor-derived exosomes play an important role in the process of metastasis, differences in exosomes secreted by the same cells at different stages or conditions have not been noticed by most of the relevant researchers. Here we developed a lung cancer model in nude mice, and the phenotype and inclusions of exosomes secreted by early and advanced tumors were analysed. The size distribution and surface topography of these two exosomes were not significantly different, but the expression of CD63 in early tumor exosome (E-exosome) was significantly lower than that in advanced tumor exosome (A-exosome). α-SMA expression on HELF cells treated with A-exosome was significantly higher than that treated with E-exosome. The ability of A-exosome to promote the migration of A549 cells was better than E-exosome. Furthermore, small RNA sequence showed that only 3 of the 171 detected-small RNAs were expressed simultaneously in both exosomes. These findings proved that there are significant differences in inclusions and functions between the early and late exosomes of the same tumor. The study highlights the importance of exosomes in cancer metastasis, and might suggest exosomes can be used as biomarkers and therapeutic targets for cancer metastasis.

Programmably tiling rigidified DNA brick on gold nanoparticle as multi-functional shell for cancer-targeted delivery of siRNAs.

Small interfering RNA (siRNA) is an effective therapeutic to regulate the expression of target genes in vitro and in vivo. Constructing a siRNA delivery system with high serum stability, especially responsive to endogenous stimuli, remains technically challenging. Herein we develop anti-degradation Y-shaped backbone-rigidified triangular DNA bricks with sticky ends (sticky-YTDBs) and tile them onto a siRNA-packaged gold nanoparticle in a programmed fashion, forming a multi-functional three-dimensional (3D) DNA shell. After aptamers are arranged on the exterior surface, a biocompatible siRNA-encapsulated core/shell nanoparticle, siRNA/Ap-CS, is achieved. SiRNAs are internally encapsulated in a 3D DNA shell and are thus protected from enzymatic degradation by the outermost layer of YTDB. The siRNAs can be released by endogenous miRNA and execute gene silencing within tumor cells, causing cell apoptosis higher than Lipo3000/siRNA formulation. In vivo treatment shows that tumor growth is completely (100%) inhibited, demonstrating unique opportunities for next-generation anticancer-drug carriers for targeted cancer therapies.

Tumor-derived exosomes can specifically prevent cancer metastatic organotropism.

Each type of cancer has its own specific metastatic route developed by disseminating circulating tumor cells (CTCs) and related extracellular vesicles to the target organ, i.e., metastasis organotropism. Tumor-derived small extracellular vesicles (herein exosomes, EXO) play an important role in determining cancer organotropic metastases to pre-metastasis niches. We therefore hypothesized that drug-loaded EXO may mix well with their companion small extracellular vesicles to specifically target the aimed metastatic organ via organotropism. Here, we demonstrate that the circulating breast-cancer-derived EXO loaded with doxorubicin (EXO-DOX) can mingled with their original companion EXO and inhibit breast cancer metastasis to lungs. The CD47 on the EXO-DOX prevented EXO-DOX from immune attack and prolonged their circulation in blood. The tissue distribution ratio of EXO-DOX is identical to the ratio of their companion EXO due to the specific affinity of EXO to integrins in targeted tissues. Quantitative accumulation of EXO-DOX in the mouse lungs is proportional to the organotropism of the circulating breast cancer cells that disseminate from subcutaneously-implanted human breast cancer cells in mice. EXO-DOX inhibited angiogenesis and cancer cell proliferation, resulting in prevention of breast cancer metastasis to the lungs. This study opens a novel path to use Trojan small extracellular vesicles for specifically controlled release of active components by small extracellular vesicles organotropism mechanism to the targeted organ for disease chemoprevention.

Near-infrared/pH dual-responsive nanocomplexes for targeted imaging and chemo/gene/photothermal tri-therapies of non-small cell lung cancer.

Combination therapy offers promising opportunities for treating advanced non-small cell lung cancer (NSCLC). Here, we established a chitosan-based nanocomplex CE7Q/CQ/S to deliver molecular-targeted drug erlotinib (Er), Survivin shRNA-expressing plasmid (SV), and photothermal agent heptamethine cyanine dye (Cy7) in one platform for simultaneous near-infrared (NIR) fluorescence imaging and triple-combination therapy of NSCLC bearing epidermal growth factor receptor (EGFR) mutations. The obtained CE7Q/CQ/S exhibited favorable photothermal effects, good DNA binding ability, and pH/NIR dual-responsive release behaviors. The conjugated Er could mediate specific delivery of Cy7 to EGFR-mutated NSCLC cells to enable targeted NIR fluorescence imaging and photothermal therapy (PTT). The in vitro and in vivo results showed that downregulation of Survivin expression and the photothermal effects could act synergistically with Er to induce satisfactory anticancer effects in either Er-sensitive or Er-resistant EGFR-mutated NSCLC cells. By integrating chemo/gene/photothermal therapies into one theranostic nanoplatform, CE7Q/CQ/S could significantly suppress EGFR-mutated NSCLC, indicating its potential use in treating NSCLC. STATEMENT OF SIGNIFICANCE: The development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has improved overall survival in patients with NSCLC driven by EGFR mutations. Unfortunately, the emergence of acquired resistance of EGFR-TKIs is almost inevitable after treatment. Here, we constructed a NIR/pH dual-responsive nanocomplex CE7Q/CQ/S based on chitosan which could integrate targeted near-infrared fluorescence imaging and chemo/gene/phototheramal tri-therapies together. We found that CE7Q/CQ/S possessed a promising outcome in fighting against EGFR-mutated NSCLC. The inhibition of Survivin expression and the application of photothermal therapy could act synergistically with erlotinib and reverse erlotinib resistance. The results of this work suggested that this chitosan-based combination therapeutic nanoplatform could be a promising candidate for NSCLC treatment.

WDR74 modulates melanoma tumorigenesis and metastasis through the RPL5-MDM2-p53 pathway.

The key molecules and underlying mechanisms of melanoma metastasis remain poorly understood. Using isobaric tag for relative and absolute quantitation (iTRAQ) proteomic screening, probing of patients' samples, functional verification, and mechanistic validation, we identified the important role of the WD repeat-containing protein 74 (WDR74) in melanoma progression and metastasis. Through gain- and loss-of-function approaches, WDR74 was found to promote cell proliferation, apoptosis resistance, and aggressive behavior in vitro. Moreover, WDR74 contributed to melanoma growth and metastasis in vivo. Mechanistically, WDR74 modulates RPL5 protein levels and consequently regulates MDM2 and insulates the ubiquitination degradation of p53 by MDM2. Our study is the first to reveal the oncogenic role of WDR74 in melanoma progression and the regulatory effect of WDR74 on the RPL5-MDM2-p53 pathway. Collectively, WDR74 can serve as a candidate target for the prevention and treatment of melanoma in the clinic.

Publisher Correction: Eliminating blood oncogenic exosomes into the small intestine with aptamer-functionalized nanoparticles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Use of lung-specific exosomes for miRNA-126 delivery in non-small cell lung cancer.

Engineered exosomes have become popular drug delivery carriers for cancer treatment. This is partially due to the interesting property, i.e. exosome organotropism, which plays an important role in organ distribution post systemic administration. Here, we demonstrated that breast cancer (MDA-MB-231) cell-derived exosomes (231-Exo) could be specifically internalized by non-small cell lung cancer cells via a specific interaction between overexpressed integrin ß4 (on exosomes) and surfactant protein C (SPC) on the cancer cells. We showed that 231-Exo was capable of recognizing A549 cells in blood and effectively escaping from the immune surveillance system in vitro. Once loaded with microRNA molecules in the exosome carriers, the resulting, miRNA-126 loaded 231-Exo (miRNA-231-Exo) strongly suppressed A549 lung cancer cell proliferation and migration through the interruption of the PTEN/PI3K/AKT signaling pathway. Intravenous administration of the miRNA-126 laden exosomes led to an effective lung homing effect in mice. When tested in a lung metastasis model, miRNA-231-Exo resulted in an efficacious effect in inhibiting the formulation of lung metastasis in vivo. Collectively, our data demonstrated the possibility of using the organotropism feature of exosomes in exosome carrier design, generating a potent anti-metastasis effect in a mouse model.

WDR74 induces nuclear ß-catenin accumulation and activates Wnt-responsive genes to promote lung cancer growth and metastasis.

Lung cancer has been notorious for its lack of advance in clinical therapy, urging for effective therapeutic targets. WD repeat-containing protein 74 (WDR74) has previously been implicated in tumorigenesis, but its mechanistic functions remain not well understood. Herein, WDR74 expression was observed to be increased upon lung cancer progression from healthy normal tissues to the primary cancer and further to the metastatic cancer. Through gain- and loss-of-function approaches, we found that WDR74 regulated lung cancer cell proliferation, cell cycle progression, chemoresistance and cell aggressiveness in vitro. Moreover, a xenograft mouse model disclosed that WDR74 knockout inhibited lung cancer growth and metastasis, whereas WDR74 overexpression reciprocally enhanced these characteristics. Mechanistically, WDR74 promoted nuclear ß-catenin accumulation and drove downstream Wnt-responsive genes, thus revealing that WDR74 activated the Wnt/ß-catenin signaling pathway. Collectively, WDR74 inducing nuclear ß-catenin accumulation and driving the downstream Wnt-responsive genes expression facilitates lung cancer growth and metastasis. WDR74 can serve as a candidate target for the prevention and treatment of lung cancer in clinic.

Eliminating blood oncogenic exosomes into the small intestine with aptamer-functionalized nanoparticles.

There are disease-causing biohazards in the blood that cannot be treated with modern medicines. Here we show that an intelligently designed safe biomaterial can precisely identify, tow and dump a targeted biohazard from the blood into the small intestine. Positively charged mesoporous silica nanoparticles (MSNs) functionalized with EGFR-targeting aptamers (MSN-AP) specifically recognize and bind blood-borne negatively charged oncogenic exosomes (A-Exo), and tow A-Exo across hepatobiliary layers and Oddi's sphincter into the small intestine. MSN-AP specifically distinguish and bind A-Exo from interfering exosomes in cell culture and rat and patient blood to form MSN-AP and A-Exo conjugates (MSN-Exo) that transverse hepatocytes, cholangiocytes, and endothelial monolayers via endocytosis and exocytosis mechanisms, although Kupffer cells have been shown to engulf some MSN-Exo. Blood MSN-AP significantly decreased circulating A-Exo levels, sequentially increased intestinal A-Exo and attenuated A-Exo-induced lung metastasis in mice. This study opens an innovative avenue to relocate blood-borne life-threatening biohazards to the intestine.

Downregulation of KCTD12 contributes to melanoma stemness by modulating CD271.

OBJECTIVE: Cancer metastasis remains the primary cause of cancer-related death worldwide. In a previous study, we found that levels of BTB/POZ domain-containing protein KCTD12 are lower in metastatic melanoma cells than in parental melanoma cells. The purpose of this study was to identify the roles of KCTD12 in cancer metastasis. METHODS: The Cancer Genome Atlas (TCGA) datasets were used to evaluate the relationship between KCTD12 and skin cutaneous melanoma (SKCM) prognosis. The effects of endogenous KCTD12 on biological behaviors were examined using the MTT assay. The impacts of KCTD12 on melanoma stemness were explored using spheroid formation assay. KCTD12 knockout A375 cells were generated to confirm the inhibitory effect of KCTD12 on CD271, and a mouse metastatic model was used to determine the impact of KCTD12 on melanoma metastasis in vivo. RESULTS: KCTD12 levels were lower in lung metastatic cells than in paired parental melanoma cells, and low KCTD12 expression indicated a poor prognosis in SKCM. Cancer metastasis-related capacities were higher in lung metastatic cells than in parental melanoma cells. Moreover, KCTD12 knockdown enhanced tumor growth and metastasis both in vitro and in vivo. Mechanistically, the interaction between KCTD12 and CD271 might be responsible for the stemness transformation after KCTD12 knockdown. CONCLUSIONS: This study identifies for the first time the role of the interaction between KCTD12 and CD271 in inducing melanoma cell stemness transformation. Moreover, KCTD12 repression enhances melanoma cell growth, adhesion, migration and invasion.

Redox/NIR dual-responsive MoS2 for synergetic chemo-photothermal therapy of cancer.

BACKGROUND: The construction of a multifunctional drug delivery system with a variety of advantageous features, including targeted delivery, controlled release and combined therapy, is highly attractive but remains a challenge. RESULTS: In this study, we developed a MoS2-based hyaluronic acid (HA)-functionalized nanoplatform capable of achieving targeted delivery of camptothecin (CPT) and dual-stimuli-responsive drug release. HA was connected to MoS2 via a disulfide linkage, forming a sheddable HA shell on the surface of MoS2. This unique design not only effectively prevented the encapsulated CPT from randomly leaking during blood circulation but also significantly accelerated the drug release in response to tumor-associated glutathione (GSH). Moreover, the MoS2-based generated heat upon near-infrared (NIR) irradiation could further increase the drug release rate as well as induce photothermal ablation of cancer cells. The results of in vitro and in vivo experiments revealed that MoS2-SS-HA-CPT effectively suppressed cell proliferation and inhibited tumor growth in lung cancer cell-bearing mice under NIR irradiation via synergetic chemo-photothermal therapy. CONCLUSIONS: The as-prepared MoS2-SS-HA-CPT with high targeting ability, dual-stimuli-responsive drug release, and synergistic chemo-photothermal therapy may provide a new strategy for cancer therapy.

Functionalized MoS2-erlotinib produces hyperthermia under NIR.

BACKGROUND: Molybdenum disulfide (MoS2) has been widely explored for biomedical applications due to its brilliant photothermal conversion ability. In this paper, we report a novel multifunctional MoS2-based drug delivery system (MoS2-SS-HA). By decorating MoS2 nanosheets with hyaluronic acid (HA), these functionalized MoS2 nanosheets have been developed as a tumor-targeting chemotherapeutic nanocarrier for near-infrared (NIR) photothermal-triggered drug delivery, facilitating the combination of chemotherapy and photothermal therapy into one system for cancer therapy. RESULTS: The nanocomposites (MoS2-SS-HA) generated a uniform diameter (ca. 125 nm), exhibited great biocompatibility as well as high stability in physiological solutions, and could be loaded with the insoluble anti-cancer drug erlotinib (Er). The release of Er was greatly accelerated under near infrared laser (NIR) irradiation, showing that the composites can be used as responsive systems, with Er release controllable through NIR irradiation. MTT assays and confocal imaging results showed that the MoS2-based nanoplatform could selectively target and kill CD44-positive lung cancer cells, especially drug resistant cells (A549 and H1975). In vivo tumor ablation studies prove a better synergistic therapeutic effect of the joint treatment, compared with either chemotherapy or photothermal therapy alone. CONCLUSION: The functionalized MoS2 nanoplatform developed in this work could be a potent system for targeted drug delivery and synergistic chemo-photothermal cancer therapy.