"One suction and one extrusion" mode-based wash-free platform for determination of breast cancer cell-derived exosomes.

A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particles⋅mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.

One-step multiplex analysis of breast cancer exosomes using an electrochemical strategy assisted by gold nanoparticles.

Exosomes, as a non-invasive biomarker, perform an important role in breast cancer screening and prognosis monitoring. However, establishing a simple, sensitive, and reliable exosome analysis technique remains challenging. Herein, a one-step multiplex analysis electrochemical aptasensor based on a multi-probe recognition strategy was constructed to analyze breast cancer exosomes. HER2-positive breast cancer cell (SK-BR-3) exosomes were selected as the model targets and three aptamers including CD63, HER2 and EpCAM aptamers were used as the capture units. Methylene blue (MB) functionalized HER2 aptamer and ferrocene (Fc) functionalized EpCAM aptamer, which were modified on gold nanoparticles (Au NPs), i.e. MB-HER2-Au NPs and Fc-EpCAM-Au NPs, were used as signal units. When the mixture of target exosomes, MB-HER2-Au NPs and Fc-EpCAM-Au NPs were added on the CD63 aptamer modified gold electrode, two Au NPs modified by MB and Fc could be specifically captured on the electrode by the recognition of three aptamers with target exosomes. Then one-step multiplex analysis of exosomes was achieved by detecting two independent electrochemical signals. This strategy can not only distinguish breast cancer exosomes from other exosomes (including normal exosomes and other tumor exosomes) but also HER2-positive breast cancer exosomes and HER2-negative breast cancer exosomes. Besides, it had high sensitivity and can detect SK-BR-3 exosomes with a concentration as low as 3.4 × 103 particles mL-1. Crucially, this method can be applicable to the examination of exosomes in complicated samples, which is anticipated to afford assistance for the screening and prognosis of breast cancer.

Investigation on protein dimerization and evaluation of medicine effects by single molecule force spectroscopy.

Monitoring the dimerization state of the mesenchymal-epithelial transition factor (Met) was essential for in-depth understanding of the tumor signal transduction network. At present, the dimerization activation pathway of Met protein was mainly studied at the macro level, while the research at the single molecule level was far from comprehensive. Herein, the dimerization activation of Met protein's extracellular domain induced by ligand hepatocyte growth factor (HGF) was dynamically studied by single-molecule force spectroscopy. Met protein was immobilized on a biomimetic lipid membrane for ensuring its physiological environment, and then the Met dimers were recognized by bivalent probe which was formed by two Met-binding aptamers. Then the dimeric state of Met protein could be distinguished from monomeric state of Met protein through some parameters, (such as unimodal ratio, bimodal ratio and separation work). The unimodal indicates the occurrence of single molecule binding event, and the bimodal represents the occurrence of double binding event (also represents the presence of Met dimer). Before HGF treatment, most of the Met protein on the lipid membrane was still in the form of monomer, so the unimodal ratio in the force curve was larger (78.8 ± 5.2%), and the bimodal ratio was smaller (17.0 ± 4.1%). After HGF treatment, the unimodal ratio decreased to 54.0 ± 7.4%, and the bimodal ratio increased to 43.2 ± 7.3%. It was due to the formation of dimers after the binding of Met protein on the fluidity lipid membrane with HGF. In addition, the average separation work increased to about 2 times after HGF treatment. Given that studies of Met protein dimerization inhibitors have contributed to the development of more potent and safe inhibitors to significantly inhibit tumor metastasis, the effects of different medicines (including anticoagulant medicines, different antibiotics and anti-cancer medicines) on the dimerization activation of Met protein were then explored by the platform described above. The results showed that anticoagulant medicines heparin and its analogs can significantly inhibit HGF-mediated Met protein activation, while different antibiotics and anticancer medicines had no significant effect on the dimerization of Met protein. This work provided a platform for studying protein dimerization as well as for screening Met protein dimerization inhibitors at the single-molecule level.

Moroidin, a Cyclopeptide from the Seeds of Celosia cristata That Induces Apoptosis in A549 Human Lung Cancer Cells.

Interference of microtubule dynamics with tubulin-targeted drugs is a validated approach for cancer chemotherapy. Moroidin (1) is an Urticaceae-type cyclopeptide having a potent inhibitory effect on purified tubulin polymerization. So far, moroidin has not been chemically synthesized, and its effect on cancer cells remains unknown. Herein, the cyclopeptide moroidin was isolated and identified from the seeds of Celosia cristata, and a revised assignment of its NMR data was presented. For the first time, moroidin (1) was demonstrated as having cytotoxic effects for several cancer cells, especially A549 lung cancer cells. The cellular evidence obtained showed that moroidin disrupts microtubule polymerization and decreases ß-tubulin protein levels, but is not as potent as colchicine. Molecular docking indicated that 1 has a high binding potential to the vinca alkaloid site on tubulin. Moreover, moroidin arrested A549 cells in the G2/M phase and induced cell apoptosis. The intrinsic mitochondrial pathway and AKT were involved in the moroidin-induced cell apoptosis. In addition, moroidin (1) inhibited the migration and invasion of A549 cells at sublethal concentrations.

Enhancing ZnO nanowire gas sensors using Au/Fe2O3 hybrid nanoparticle decoration.

Heterojunctions are an important strategy for designing high performance electrical sensor materials and related devices. Herein, a new type of metal-semiconductor hybrid nanoparticle has been successfully used to remarkably sensitize the surface of ZnO nanowires for detecting NO2 with high responses over a broad temperature window ranging from room temperature to 600 °C. These hybrid nanoparticles are comprised of iron oxide nanowires with well dispersed single crystalline Au nanoparticles. The hybrid nanoparticle decorated ZnO nanowires have achieved a giant response, as high as 74 500 toward NO2 gas, about 42 times that of Au decorated ZnO nanowire sensors. This dramatic enhancement may be attributed to the efficient charge transfer across the Au-Fe2O3 Schottky and Fe2O3-ZnO semiconductor heterojunction interfaces. Due to the incorporation of thermally-stable Fe2O3 nanoparticles as the support of Au nanoparticles, the working temperature of nanowire sensors was successfully extended to higher temperatures, with an increase of 200 °C, from 400 °C to 600 °C. Such a combination of semiconductor heterojunction and semiconductor-metal Schottky contact presents a new strategy for designing high performance electrical sensors with high sensitivity, stability, selectivity, and wide operation temperature window, which are potentially suitable for advanced energy systems such as automotive engines and power plants.

H7K(R2)2-modified pH-sensitive self-assembled nanoparticles delivering small interfering RNA targeting hepatoma-derived growth factor for malignant glioma treatment.

The lack of effective glioma therapeutics mandates the development of novel treatment strategies. Hepatoma-derived growth factor (HDGF) has been considered as a potential glioma therapeutic target, and its expression level in gliomas is positively related to the malignant grade. Although there are no effective and specific inhibitors against this target, small interfering RNA targeting HDGF (siHDGF)-mediated RNA interference (RNAi) can inhibit the target protein function by knockdown of HDGF expression. However, the application of siHDGF in glioma research and therapy is hampered by the challenge to safe and effective in vivo systemic delivery of siHDGF to gliomas. To address this question, we develop the peptide H7K(R2)2-modified pH-sensitive self-assembled hybrid nanoparticles encapsulating siHDGF (H7K(R2)2-PSNPs (siHDGF)). The acidic glioma microenvironment is beneficial to the membrane penetration of H7K(R2)2-PSNPs and the encapsulated siHDGF. Following systemic administration, H7K(R2)2-PSNPs (siHDGF) can effectively deliver siHDGF into the brain and malignant glioma cells, and therefore can significantly downregulate HDGF expression, inhibit malignant phenotypes of glioma cells, result in reduced tumor volumes and prolonged survival times in nude mice bearing U251 human glioblastoma. Thus, systemic administration of H7K(R2)2-PSNPs (siHDGF) offers an effective way for the targeted delivery of siHDGF and may serve as a practical malignant glioma therapy.

Targeted delivery of intranasally administered nanoparticles-mediated neuroprotective peptide NR2B9c to brain and neuron for treatment of ischemic stroke.

The lack of effective therapies mandates the development of new treatment strategies for ischemic stroke. The NR2B9c peptide can prevent N-Methyl-D-aspartate receptor (NMDAR)-mediated neurotoxicity induced by ischemia without affecting essential NMDAR activity and brings hope for stroke therapy. However, it is very difficult for NR2B9c to cross by itself the blood-brain barrier (BBB) and the neuron membrane. To provide a suitable delivery for unleashing the therapeutic potential of NR2B9c, in consideration of a high affinity of wheat germ agglutinin (WGA) for WGA receptors abundantly present on olfactory epithelium and neuronal surface, we developed WGA-modified nanoparticles carrying NR2B9c (NR2B9c-WGA-NPs). Following intranasal administration, NR2B9c-WGA-NPs are able to bypass the BBB and effectively transport NR2B9c into the brain and neuron, and therefore can protect neurons against excitotoxicity, reduce ischemic brain injury in rats and ameliorate their neurological function deficits. The intranasal administration of NR2B9c-WGA-NPs may serve as a practical stroke therapy.

[Establishment of a Xenografted Acute Myeloid Leukemia Model by using Zebrafish].

Objectve: To investigate the feasibility of establishing xenografted leukemia model by zebrafish, so as to provide the more direct model in vitro and experimental evidence for study of acute myeloid leukemia and screening of the drugs for targeting therapy. METHODS: Acute myeloid leukemia cell line KG-1a was labeled with red fluorescent dye-MitoRed, then the labeled cells were injected into the yolk sac of zebrafish embryos. Morphological observation, cell count and histopathological detection were used to analyse the infiltration and metastasis of KG-1a cells in zebrafish. RESULTS: KG1a cells could proliferate and gradually spread to the entire abdominal cavity of the zebrafish after KG-1a cells were injected into the yolk sac during 1-7, the results of cell counting in vitro also proved a significant proliferation of KG-1a cells in zebrafish, suggesting that the implanted leukemia stem cells could survive, proliferate and spread in zebrafish. Further study showed that the implanted cells could be transfered to the liver of zebrafish, these cells displayed the signature of KG-1a cells by hematoxylin-eosin(HE) staining. CONCLUSIONS: Human acute myeloid leukemia cells KG1a can survive, proliferate and migrate in zebrafish, suggesting xenografted leukemia model of zebrafish has been successfully established. This model may be benefitcial for the study of acute myeloid leukemia and the screening of the drugs for targeting therapy of acute myeloid leukemia.

Enhanced Antiglioma Efficacy of Ultrahigh Loading Capacity Paclitaxel Prodrug Conjugate Self-Assembled Targeted Nanoparticles.

Glioblastoma multiforme (GBM) presents one of the most lethal brain tumor with a dismal prognosis. And nanodrug delivery system (nano-DDS) have raised a lot of concern, while the conventional nanoformulations addressed many limitations, especially the low drug loading capacity and poor stability in vivo. Herein, we proposed PTX prodrug (PTX-SS-C18) conjugate self-assembled nanoparticles (PSNPs) functionalized with Pep-1, glioma homing peptide, to overcome the blood brain tumor barrier (BBTB) via interleukin 13 receptor α2 (IL-13Rα2)-mediated endocytosis for targeting GMB. This nanocarrier was with ultrahigh drug loading capacity (56.03%) and redox-sensitivity to the up-expression of glutathione in glioma tumors. And compared with PEG-PSNPs, Pep-PSNPs could significantly enhance cellular uptake in U87MG cells via IL-13Rα2-mediated endocytosis. Enhanced cytotoxicity of Pep-PSNPs against U87MG cells and BCEC cells pretreated with glutathione monoester (GSH-OEt) confirmed that this nanosystem was sensitive to reduction environment, and there was significant difference between targeting and nontargeting groups in MTT assay. Real-time fluorescence image of intracranialU87MG glioma-bearing mice revealed that Pep-PSNPs could more efficiently accumulate at tumor site and improve the penetration. Furthermore, the ex vivo fluorescence imaging and corresponding semiquantitative results displayed that the glioma fluorescence intensity of Pep-PSNPs group was 1.74-fold higher than that of nontargeting group. Pep-PSNPs exhibited remarkable antiglioblastoma efficacy with an extended median survival time. In conclusion, Pep-PSNPs had a promising perspective as a targeting drug delivery system of PTX for glioma treatment.

Conditioning with Fludarabine-Busulfan versus Busulfan-Cyclophosphamide Is Associated with Lower aGVHD and Higher Survival but More Extensive and Long Standing Bone Marrow Damage.

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a major cause of nonrelapse mortality after allo-HSCT. A conditioning regimen plays a pivotal role in the development of aGVHD. To provide a platform for studying aGVHD and evaluating the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (Bu-Cy) and Fludarabine-Busulfan (Flu-Bu). In our study, BALB/c mice were conditioned with Bu-Cy or Flu-Bu and transplanted with 2 × 107 bone marrow cells and 2 × 107 splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with Bu-Cy had shorter survivals (P < 0.05), more severe clinical manifestations, and higher hepatic and intestinal pathology scores, associated with increased INF-γ expression and diminished IL-4 expression in serum, compared to allogeneic recipients conditioned with Flu-Bu. Moreover, higher donor-derived T-cell infiltration and severely impaired B-cell development were seen in the bone marrow of mice, exhibiting aGVHD and conditioned with Flu-Bu. Our study showed that the conditioning regimen with Bu-Cy resulted in more severe aGVHD while the Flu-Bu regimen was associated with more extensive and long standing bone marrow damage.

Low-dose arsenic trioxide combined with aclacinomycin A synergistically enhances the cytotoxic effect on human acute myelogenous leukemia cell lines by induction of apoptosis.

Acute myeloid leukemia (AML) is a common disorder in the elderly. Although remarkable progress has been made over recent decades, the outcome remains poor. Thus, the development of a more effective method to overcome this problem is necessary. In this study, we aimed to investigate the synergistic cytotoxic effect of low-dose arsenic trioxide (As2O3) combined with aclacinomycin A (ACM) on the human AML cell lines KG-1a and HL-60, and to clarify the underlying mechanism. Results showed that As2O3 combined with ACM exerted a synergistic cytotoxic effect by activation of the apoptosis pathway. Additionally, we found that the combination treatment decreased Bcl-2, c-IAP and XIAP expression but increased SMAC and caspase-3 expression more significantly than the single drug treatments. Furthermore, combination index (CI) values were < 1 in all matched combination groups. Additional evaluation of As2O3 combined with ACM as a potential therapeutic benefit for AML seems warranted.

[Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis].

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.