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Nanocarrier-aided drug delivery techniques have improved the absorption and permeability of drugs in nose-to-brain delivery. However, the molecular properties of nanocarriers during the delivery process are of great interest; in particular, the characteristics when penetrating barriers in vivo are crucial for the screening and optimization of materials for nasal inhalation. In this study, we have focused on two types of delivery systems: mucoadhesive nanoparticles (MAPs) and mucopenetrating nanoparticles (MPPs); both have been widely used for mucosal delivery, although a method for selecting the more effective type of drug carriers for mucosal delivery has not been established. Molecular dynamics (MD) simulations were used to reveal the all-atom dynamic characteristics of the interaction between different delivery systems and the nasal mucus protein MUC5AC. Among the systems tested, hydroxypropyltrimethyl ammonium chloride chitosan (HTCC) had the strongest interaction with mucin, suggesting it had better mucoadhesive performance, and that it interacted with MUC5AC more strongly than unmodified chitosan. In contrast, the mucus-penetrating material polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA), had almost no interaction with MUC5AC. The results of the MD simulations were verified by in vitro experiments on nanoparticles (NPs) and mucin binding. The drug delivery performance of the four types of NPs, analyzed by in vitro and ex vivo mucosal penetration, were all generally consistent with the properties of the material predicted from the MD simulation. These clues to the molecular mechanism of MAPs and MPPs may provide useful insight into the screening and optimization of nanomaterials suitable for nasal inhalation.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.

Co-delivery nanoparticles with characteristics of intracellular precision release drug have been generally accepted as an effective therapeutic strategy for eye diseases. In this study, we designed a new co-delivery system (miRNA/NP-BRZ) as a lasting therapeutic approach to prevent the neuro-destructive after the long-term treatment of glaucoma. Neuroprotective and intraocular pressure (IOP) response were assessed in in vivo and in vitro models of glaucoma. At the meaning time, we describe the preparation of miRNA/NP-BRZ, drug release characteristics, intraocular tracing, pharmacokinetic and pharmacodynamics study and toxicity test. We found that miRNA/NP-BRZ could remarkably decrease IOP and significantly prevent retinal ganglion cell (RGC) damages. The new formula of miRNA-124 encapsulated in PEG-PSA-BRZ nanoparticles exhibits high encapsulation efficiency (EE), drug-loading capacity (DC), and stable controlled-release efficacy (EC). Moreover, we also verified that the miRNA/NP-BRZ system is significantly neuroprotective and nontoxic as well as lowering IOP. This study shows our co-delivery drug system would have a wide potential on social and economic benefits for glaucoma.

Stability of real-time hybrid simulation (RTHS) has attracted considerable attention given that actuator delay might destabilize the real-time test, especially when the compensation is not ...

With the development of modern biomedicine, research on the molecular mechanism of tumors has developed gradually. The CD147 gene has been applied to tumor molecular targeted therapy, and significant differences were found in the expression of the CD147 gene in different tumor tissues and normal tissues. Many previous studies have also shown that the expression of the CD147 gene plays a crucial role in the development of tumors. To understand whether CD147 can be used as a therapeutic target for oral cancer, CRISPR/Cas9 gene-editing technology was used to knock out the CD147 gene in cal27 cells to obtain knockout cell lines. Using CCK-8, Transwell, RT-PCR, and Western blotting, the proliferation and invasion abilities of the knockout cell lines were decreased significantly, and the expression of matrix metalloproteinase was also inhibited. Next, a subcutaneously transplanted tumor model in nude mice was constructed to detect the effect of the CD147 gene on tumors. Subcutaneous tumor growth and immunohistochemistry results showed that the proliferation and doxorubicin resistance of knockout cell line were significantly inhibited compared with those in the wild-type group. These results indicated that knocking out CD147 significantly reduced the proliferation and invasion of cal27 cells, and CD147 may be a potential therapeutic target for oral cancer.

To prepare a binary formulation delivering miRNA-146 and evaluate a nucleic acid nasal delivery system by investigating its pharmacodynamic effects in allergic rhinitis. The gel/NPs/miR-146a thermosensitive in situ chitosan hydrogel carrying a nucleic acid was prepared and evaluated for its characteristics, including temperature sensitivity, gel strength, mucosal adhesion and drug release profile. After nasal administration of the formulation to ovalbumin-sensitized rats, the treatment of allergic rhinitis was verified by assessing nasal symptoms, hematology, hematoxylin-eosin (HE) staining and immunohistochemistry. Western Blot(WB) was used to analyze nasal inflammatory factors as well as miRNA-146-related factors, and the miR146 expression level was measured by PCR. Subsequently, the effects of the gel/NPs/miR-146a binary formulation were evaluated for the nasal delivery of nucleic acids in rhinitis therapy. The prepared binary formulation quickly formed a gel in the nasal cavity at a temperature of 34 &deg, C with good mucosal adhesion, which delivered nucleic acids into the nasal mucosa stably and continuously. Gel/NPs/miR-146a was able to sustain the delivery of miRNA into the mucosa after nasal administration. When compared with the monolithic formulations, the gel/NPs/miR-146a binary formulation performed better regarding its nucleic acid delivery ability and pharmacodynamic effects. The gel/NPs/miR-146a binary preparation has a suitable nasal mucosal drug delivery ability and has a positive pharmacodynamic effect for the treatment of ovalbumin-induced rhinitis in rats. It can serve as a potential nucleic acid delivery platform for the treatment of allergic rhinitis.

Effective treatments for neurodegenerative diseases need to be developed. MiR132 is abundantly expressed in the brain, and it modulates neuron morphology and plays a key role in maintaining neuron survival. Regulating miR132 can effectively improve the symptoms of Alzheimer's disease. It can also reduce cell death after cerebral hemorrhage, improve the microenvironment of hematoma lesions and provide a certain protective effect from brain damage after cerebral ischemia. MiR132 has great potential in the treatment of cerebral ischemia and Alzheimer's disease. To prevent the decline of miR132 of miR132 levels in the blood, we used mouse and rat models of Alzheimer's disease with ischemic brain injury, and then delivered Wheat germ agglutinin (WGA)-NPs-miR132 intranasally to treat neurological damage after cerebral ischemia. Synaptic protein expression levels in Alzheimer's mouse models increased significantly after administration. We propose that, nasal delivery of WGA-NPs-miR132 is an interesting novel therapeutic approach for the treatment of neurodegenerative diseases.

Mitochondrial abnormalities accelerate the progression of ischemic brain damage. Sirtuin 3 (SIRT3) is mainly found in mitochondria and affects almost all major aspects of mitochondrial function. Luteolin, a flavonoid with diverse biological properties, including antioxidant activity, inhibition of cell apoptosis and regulation of autophagy. It also modulates the activity of AMP activated kinase and/or sirtuin 1 (SIRT 1) by regulating the expression of sirtuins. We investigated the protective effects of luteolin on cerebral ischemia-reperfusion. It was found through experiments that luteolin reduced the infarcted area of MCAO rat model, and based on the experimental results, it was inferred that luteolin affected the AMPK, mTOR and SIRT3 pathways, thereby protecting brain cells. As expected, we found that luteolin can reduce the neurological function score, the degree of cerebral edema, the cerebral infarction volume, alleviate morphological changes in the cortex and hippocampus, increase neuron survival and decrease the number of apoptotic neurons. At the same time, luteolin significantly reduced the number of GFAP and Iba-1 positive glial cells in the hippocampus while enhanced the scavenging of oxygen free radicals and the activity of SOD in mitochondria. Addtionally, it can also enhance antioxidant capacity via the reversal of mitochondrial swelling and the mitochondrial transmembrane potential. Moreover, luteolin can increase SIRT3-targeted expression in mitochondria, decrease the phosphorylation of AMPK, and increase phosphor-mTOR (p-mTOR) levels, which may have occurred specifically through activation of the SIRT3/AMPK/mTOR pathway. We speculate that luteolin reduces the pathological progression of CIRI by increasing SIRT3 expression and enhancing mitochondrial function. Therefore, the results indicate that luteolin can increase the transduction of SIRT3, providing a potential mechanism for neuroprotective effects in patients with cerebral ischemia.

Structural assessment for collapse is commonly approached by observing the failure or collapse of systems fully incorporating degradation. Challenges however exist in the performance indicator or damage measure due to compound impacts of uncertainties of external (seismic excitation) and internal (structural properties) characteristics with degradation behavior. To account for the impacts of uncertainties, the state-of-the-art kriging nonlinear autoregressive with exogenous (NARX) model is explored in this study to replicate the response of nonlinear single-degree-of-freedom systems. The generalized hysteretic Bouc-Wen model with internal uncertainties is selected to emulate the stiffness and strength degradation. A probabilistic stochastic ground motion model is introduced to represent the external uncertainties. The global terms of NARX model are selected by least-angle regression algorithm and the kriging model is utilized to surrogate uncertain parameters into corresponding NARX model coefficients. The predictions of kriging NARX models are further compared with that of the polynomial chaos nonlinear autoregressive with exogenous input form model as well as Monte Carlo simulation. The comparisons show that kriging NARX model presents an effective and efficient meta-model technique for uncertainty quantification of systems with degradation.

Three vacuolar cation/H+ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na+/H+ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T-DNA insertion mutant and 35S-driven AtNHX3 over-expression Arabidopsis plants. RT-PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre-vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K+-deficient conditions. Expression of AtNHX3 complemented the sensitivity to K+ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over-expressing AtNHX3 displayed significantly higher K+/H+ exchange rates than those isolated from wild-type plants. Furthermore, nhx3 seeds accumulated less K+ and more Na+ when both wild-type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K+/H+ antiporter required for low-potassium tolerance during germination and early seedling development, and may function in K+ utilization and ion homeostasis in Arabidopsis.

An electroporation procedure has been described for introducing plasmid DNA into Dunaliella salina cells. By this procedure, a bulk of plasmid DNA was delivered into the cells and retained for at least 3 d. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing analyses indicated that the transcription and pre-mRNA splicing of ble gene (contributing the Zeocin resistance) were detected in the cells as early as 1 h after the electroporation. Individual colonies could retain the resistance to 10 mg/L Zeocin for at least 6 mo. Subsequent Southern blot analysis showed the existence of introduced plasmid DNA inside these colonies. However, most of the cells (approx 90%) lost the resistance in the presence of 5 mg/L Zeocin during subculturing, which was consistent with the observations of both rearranged and episomal plasmid DNA existed in the cells. Nevertheless, the electroporation procedure allows introducing a gene of interest and studying its expression and function in D. salina cells.