A dual responsive nitric oxide / ß-lapachone co-delivery platform for redox imbalance-enhanced tumor therapy.

Nitric oxide (NO) / ß-Lapachone (Lap) combined therapy by causing oxidative stress is an effective tumor therapy strategy. Herein, a dual-responsive lipid nanoparticles (LNPs) LSNO for NO / Lap co-delivery were constructed from the zinc-coordinated lipid (DSNO(Zn)) and the hydrophobic drug Lap in the presence of helper lipids (DOPE and DSPE-PEG2000). The zinc-coordinated structure in LSNO might elevate the Zn2+ content in tumor cells, contributing to antioxidant imbalance. The fluorescent assays proved the light-triggered NO release and fluorescent self-reporting abilities of LSNO. In addition, the LNPs had good drug release behavior under high concentration of GSH, indicating the NO / drug co-delivery capacity. In vitro antitumor assays showed that the NO / Lap combination treatment group could induce more significant tumor cell growth inhibition and cell apoptosis than individual NO or Lap treatment. The following mechanism studies revealed that NO / Lap combination treatment led to distinct oxidative stress by producing reactive oxygen species (ROS) and peroxynitrite anion (ONOO-). On the other hand, the intracellular redox balance could be further disrupted by Lap-induced NADPH consumption and Zn2+ / NO-induced reductase activities downregulation, thus promoting the degree of cell damage. Besides, it was also found that NO and Lap could directly damage nuclear DNA and induce mitochondrial dysfunction, thereby leading to caspase-3 activation and tumor cell death. These results proved that LSNO could serve as a promising multifunctional tumor therapy platform.

Cationic lipids from multi-component Passerini reaction for non-viral gene delivery: A structure-activity relationship study.

Although many types of cationic lipids have been developed as efficient gene vectors, the construction of lipid molecules with simple procedures remains challenging. Passerini reaction, as a classic multicomponent reaction, could directly give the α-acyloxycarboxamide products with biodegradable ester and amide bonds. Herein, two series of novel cationic lipids with heterocyclic pyrrolidine and piperidine as headgroups were synthesized through Passerini reaction (P-series) and amide condensation (A-series), and relevant structure-activity relationships on their gene delivery capability was studied. It was found that although both of the two series of lipids could form lipid nanoparticles (LNPs) which could effectively condense DNA, the LNP derived from P-series lipids showed higher transfection efficiency, serum tolerance, cellular uptake, and lower cytotoxicity. Unlike the A-series LNPs, the P-series LNPs showed quite different structure-activity relationship, in which the relative site of the secondary amine had significant effect on the transfection performance. The othro-isomers of the P-series lipids had lower cytotoxicity, but poor transfection efficiency, which was probably due to their unstable nature. Taken together, this study not only validated the feasibility of Passerini reaction for the construction of cationic lipids for gene delivery, but also afforded some clues for the rational design of effective non-viral lipidic gene vectors.

Fluorescent Self-Reporting Lipid Nanoparticles for Nitric Oxide/Gene Co-Delivery and Combination Therapy.

The combination cancer therapy of nitric oxide (NO) with gene therapy is a promising method for tumor treatment. However, efficient co-delivery of gas and therapeutic genes to tumor cells remains a challenge. Herein, we designed a nano-sized ultraviolet (UV) light-responsive cationic lipid vector DPNO(Zn). Fluorescence spectroscopy and confocal imaging experiments revealed that DPNO(Zn) lipid nanoparticles (LNPs) could rapidly release NO under low-power UV light irradiation. Moreover, the fluorescence turn-on might take place along with the release of NO, indicating the self-reporting ability. Gene delivery experiments showed that DPNO(Zn) LNPs had good gene transfection ability, making such materials a good candidate for gas/gene combination therapy. In vitro antitumor assay demonstrated that the co-delivery system was more effective in inhibiting tumor cell proliferation than individual NO or pTrail treatment. Studies on the mechanism of tumor cell apoptosis induced by NO/pTrail co-delivery showed that NO could not only effectively increase the accumulation of p53 protein in tumor cells, thereby promoting the activation of caspase-3, but also induce mitochondrial damage. On the other hand, the Trail protein expressed by pTrail gene could enhance the degree of NO-induced caspase-3 activation, indicating the synergistic effect. These results proved that DPNO(Zn) LNP may serve as a multifunctional nanocarrier for potential tumor therapy.

Zn-dipicolylamine-based reactive oxygen species-responsive lipids for siRNA delivery and in vivo colitis treatment.

The reduction of reactive oxygen species (ROS) and inflammatory factor levels plays an important role in the treatment of colitis. A series of ROS-responsive lipids (ZnDPA-R) based on the thioketal structure were designed and synthesized. It was expected that the lipidic materials could combine ROS consumption and siRNA delivery capacity to achieve synergistic treatment of colitis. The target liposomes could combine with the phosphate backbone of siRNA to form lipoplexes of size ∼100 nm and could deliver siRNA cargo into the cell. The results of in vitro anti-inflammatory experiments showed that the lipids may effectively consume ROS in the cells. The lipoplexes significantly reduced the expression levels of TNF-α mRNA and related inflammatory factors in macrophages. After PEGylation, the lipoplex was used for treating mouse colitis, and the biodistribution results proved that the lipoplexes effectively aggregated in the intestine. The delivery system could not only respond to the high ROS level in colitis through breaking of thioketal structure, but it could also assist in treating inflammation by ROS consumption. The treatment results revealed that the levels of TNF-α mRNA and related inflammatory factors in the colon lesion were largely reduced, and the inflammatory symptoms were significantly relieved. Hematology test results indicated that the treatment was safe and induced no side effects in mice. The present study may shed light on the synergistic treatment of colitis through anti-inflammatory siRNA delivery and ROS depletion strategies. STATEMENT OF SIGNIFICANCE: Downregulation of inflammatory factors and reactive oxygen species (ROS) levels is critical in treating colitis. In the present study, a series of ROS-responsive lipid molecules based on the Zn-DPA headgroup and thioketal linkage were synthesized for delivering TNF-α siRNA and for treating colitis. In addition to silencing the expression of TNF-α mRNA and the related inflammatory factors, the material also achieved synergistic treatment by simultaneous consumption of ROS in the colon lesion. In vitro cell experiments and in vivo colitis treatment in mice showed that the lipoplex exerted a satisfactory therapeutic effect on colitis, and the symptoms of colitis in mice were significantly alleviated. The present study may shed light on the synergistic treatment for colitis through anti-inflammatory siRNA delivery and ROS depletion strategies.

Study on metal binding capacity of the freshwater crab Sinopotamon henanense's recombinant copper specific binding metallothionein expressed in Escherichia coli.

The copper specific binding metallothionein (CuMT) is a type of cysteine-rich, metal-binding, small protein which plays an important role in Cu2+ metabolism in vertebrates. In this study, we investigated the metal tolerance and removing ability of recombinant strains harboring CuMT obtained in vivo from the freshwater crab Sinopotamon henanense (ShCuMT) in order to study its physiological functions and metal binding capacity. We performed a 3D modeling of ShCuMT and created its structural and functional models using the I-TASSER program. The shCumt gene was inserted into a pGEX-4t-1 vector and recombinant soluble ShCuMT was expressed in Escherichia coli. In addition, in order to characterize the tolerance and removing ability of heavy metals in E. coli with ShCuMT expression, the recombinant strains harboring ShCuMT were exposed to various concentrations of Cd2+, Cu2+ and Zn2+, respectively. The results showed that ShCuMT contains transition metal binding sites. In addition, E. coli cells expressing ShCuMT exhibited enhanced metal tolerance and higher removing ability of metal ions than control cells. However, compared with Cd2+ and Zn2+, E. coli cells expressing ShCuMT have stronger tolerance and higher removing ability of Cu2+. In general, ShCuMT contains multiple transition metal binding sites, and it could enhance tolerance and removing ability of metal ions. Therefore, ShCuMT can provide potential candidates for heavy metal bioremediation. This research on the metal binding properties of ShCuMT provides a scientific basis for bioremediation of heavy metal pollution by the recombinant strains.

A Fluorescent Self-Reporting Vector with GSH Reduction Responsiveness for Nucleic Acid Delivery.

Nonviral gene vectors with stimulus responsiveness and self-reporting properties have broad application prospects in gene therapy. Herein, we developed a nanosized reduction-responsive cationic liposomal vector formed from DNS(Zn), in which a naphthalimide-sulfonamide group was used as the glutathione (GSH)-responsive group to generate a blue fluorescence signal at 458 nm. Macrocyclic polyamine (cyclen) was used as a cationic headgroup to facilitate Zn(II) coordination, which may reduce the cytotoxicity and improve transfection efficiency. The Zn-free and nonresponsive analogues were used for comparison. Fluorescent assays revealed that the GSH response of DNS(Zn) could increase the blue fluorescence signal and improve the DNA release in cells. The title material also showed higher positive ζ-potential than its nonresponsive analogue, resulting in stronger DNA binding ability and better cellular uptake. These advantages made DNS(Zn) a good candidate for nonviral gene delivery, and the transfection efficiency in HeLa cells was distinctly higher than that of its analogue and the commercially available transfection reagent. Besides plasmid DNA, DNS(Zn) could also deliver small interfering RNA (siRNA) with good gene silencing efficiency, extending the application of the liposomes. These results suggest that DNS(Zn) can serve as a highly efficient nucleic acid delivery vector with reduction-responsive fluorescence self-reporting ability in tumor cells.

Analysis of spectrometry and thermodynamics of the metallothionein in freshwater crab Sinopotamon henanense for its binding ability with different metals.

The metal binding nature of heterologously expressed metallothionein of Sinopotamon henanense (ShMT) had been demonstrated previously. In this study, we analysed the stoichiometry of ShMT yielded in vivo and exchange reactions of the Zn-ShMT with Cd2+, Pb2+ and Cu2+in vitro via electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), and isothermal titration calorimetry (ITC). The results of ESI-TOF-MS analyses showed that metal-ShMT synthesized in vivo had three major forms, namely Zn15-, Cd9-, and Pb5-ShMT. The ITC analyses of exchange reactions demonstrated that Zn-ShMT exhibited up to 6, 6, and 7 binding sites for Cd2+, Pb2+ and Cu2+. By the analyses of the UV and CD spectra in the substitution experiments showed that the geometric structural stability of metal-ShMT could be influenced when excess of over 6, 6, or 7 equivalents of Cd2+, Pb2+, or Cu2+ were added into Zn-ShMT. Although both the reconstructed apo-ShMT and substituted Zn-ShMT with three metal ions fitted the same M6Ⅱ- and M7Ⅰ-ShMT binding models for divalent and monovalent metals, the differences in their thermodynamic data suggested that discrepancies exit in their physiological functions. These results suggested that ShMT yielded in vivo had a higher storage capability for Zn2+ and a uptake ability for Cd2+, and Zn-ShMT was more easy to release Zn2+ as well as to uptake Cd2+, Cu2+, or Pb2+. The results presented in this work will be very valuable to understand the function(s) of ShMT not only in a normal physiological condition but also in the presence of non-essential metals in crabs.

Metal binding characterization of heterologously expressed metallothionein of the freshwater crab Sinopotamon henanense.

We characterized the metal tolerance of recombinant strains harboring metallothionein from the freshwater crab Sinopotamon henanense (ShMT) in vivo and metal binding properties of ShMT purified in vitro. The recombinant strains harboring ShMT were exposed to 0.1 mM Cd2+, 0.3 mM Cu2+, 0.5 mM Pb2+, and 0.8 mM Zn2+. The growth curves and spot assays of recombinant strains and the contents of heavy metal ions were analysed in the media supplemented with above metal ions provided to recombinant E. coli synthesis. The structural characteristics of the Cd-, Cu-, Pb-, and Zn-ShMT were determined through ultraviolet spectroscopy (UV-vis), circular dichroism (CD), and isothermal titration calorimetry (ITC). The in vivo results showed that, compared to control strains, recombinant strains tolerated Cd2+, Cu2+, Pb2+, and Zn2+. Furthermore, the contents of Cd2+ and Pb2+ in media decreased substantially. In vitro and the Cd-ShMT had a higher degree of folding compactness in solution. 5,5'-Dithiobis-(2-nitrobenzoic) acid (DTNB) reaction and ITC results demonstrated that ShMT yielded Cd6-, Cu7-, and Pb6-ShMT. The binding stability order was Cu-ShMT > Cd-ShMT > Pb-ShMT > Zn-ShMT. Overall, ShMT is a canonical crustacean MT and is defined as a Cd-specific MT isoform that functions mainly in a detoxifying Cd2+ and Pb2+ and in regulating Zn2+ homeostasis in S. henanense. This research on the metal binding properties of ShMT provides a better understanding of the physiological function of ShMT reducing heavy metal bioavailability and by regulating essential trace metals.

Biodegradable Gene Carriers Containing Rigid Aromatic Linkage with Enhanced DNA Binding and Cell Uptake.

The linking and modification of low molecular weight cationic polymers (oligomers) has become an attracted strategy to construct non-viral gene carriers with good transfection efficiency and much reduced cytotoxicity. In this study, PEI 600 Da was linked by biodegradable bridges containing rigid aromatic rings. The introduction of aromatic rings enhanced the DNA-binding ability of the target polymers and also improved the stability of the formed polymer/DNA complexes. The biodegradable property and resulted DNA release were verified by enzyme stimulated gel electrophoresis experiment. These materials have lower molecular weights compared to PEI 25 kDa, but exhibited higher transfection efficiency, especially in the presence of serum. Flow cytometry and confocal laser scanning microscopy results indicate that the polymers with aromatic rings could induce higher cellular uptake. This strategy for the construction of non-viral gene vectors may be applied as an efficient and promising method for gene delivery.

[Resource investigation of wild Codonopsis pilosula in Tanchang county of Gansu].

Tanchang county is the distribution of wild medicinal plant resource-rich region, in order to ascertain Tanchang county Codonopsis pilosula wild resources and reserves of the status quo, according to the fourth national Chinese medicine resources survey technology solutions, using sets of plots and investigating combined route survey method, the county wild C. pilosula var. modesta and C. pilosula resources were investigated by a comprehensive survey designed to reveal the distribution of the county's wildlife resources and herbs C. pilosula reserves. The results showed that in Tanchang county seven ecological zones 53 plots, wild C. pilosula distributed in there were 6 ecological zones 11 plots, accounting for 85.71% of the survey area, wild C. pilosula var. modesta was found only in an ecoregions plots, overlapping with C. pilosula region, accounting for 14.29% of the survey area. C. pilosula herbs reserves were calculated as about 461.85 t, economic capacity of 254.02 t, annual amount of acceptance 25.40 t. C. pilosula var. modesta herbs reserves were calculated as 67.75 t, economic capacity of 36.16 t, acceptance annual amount 3.62 t. The total ash C. pilosula was 3.25%, alcohol-soluble extract was 63.86%, while the C. pilosula var. modesta total ash was 3.69%, alcohol-soluble extract was 68.32%. C. pilosula is suitable for broad range, but wild resource is scarce, C. pilosula var. modesta is suitable for relatively narrow scope, and wild resource is scarce, it is recommended to strengthen the protection of wild resources and the rational development and utilization.

[Endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of intrapulmonary lesions].

OBJECTIVE: To evaluate real-time endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis of intrapulmonary lesions. METHODS: From October 2009 to November 2011, EBUS-TBNA was performed in 78 patients with parabrachial or parabronchial intrapulmonary lesions proved by CT scan. On-site cytological evaluation was not performed. Immunohistochemistry was applied to distinguish the type of malignant tumor when necessary. RESULTS: Sixty-five malignancies and 13 benign diseases were finally diagnosed in 78 intrapulmonary lesions, of which 62 malignancies and 13 benign diseases were distinguished by EBUS-TBNA, including 61 primary lung cancer (adenocarcinoma 36, squamous carcinoma 8, poorly-differentiated carcinoma 5, unknown type carcinoma 3, small cell carcinoma 9), one metastatic lung cancer, 7 pulmonary inflammation, 5 pulmonary tuberculosis and one fibrosis. There were 3 false negative cases which were diagnosed as pulmonary poorly-differentiated carcinoma, pulmonary sarcomatoid carcinoma and pulmonary lymphoma, respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of EBUS-TBNA in distinguishing malignant from benign thoracic lesions was 95%, 100%, 81%, 100%, 96%, respectively. Immunohistochemistry was performed in 8 malignant tumors without definite type or origin, 5 primary lung cancer and one metastatic lung adenocarcinoma were further confirmed. Moderate bleeding from the puncture site during needle aspiration forming blood clot and obstructing the central airway was noted in 1 hypercoagulable subject. CONCLUSIONS: EBUS-TBNA is a minimally invasive, safe procedure with high sensitivity for distinguishing malignant from benign lesions. Immunohistochemistry can provide evidence for the definitive diagnosis of malignant lesions.