Chain hybridization-based CRISPR-lateral flow assay enables accurate gene visual detection.

Visualized gene detection based on the CRISPR-Cas12/CRISPR-Cas13 technology and lateral flow assay device (CRISPR-LFA) has shown great potential in point-of-care testing sector. Current CRISPR-LFA methodology mainly utilizes conventional immuno-based LFA test strips, which could visualize whether the reporter probe is trans-cleaved by Cas protein, indicating the target positive detection. However, conventional CRISPR-LFA usually produces false-positive results in target negative assay. Herein, a nucleic acid Chain Hybridization-based Lateral Flow Assay platform, named CHLFA, has been developed to achieve the CRISPR-CHLFA concept. Different from the conventional CRISPR-LFA, the proposed CRISPR-CHLFA system was established based on the nucleic acid hybridization between the GNP-probe embedded in test strips and ssDNA (or ssRNA) reporter from CRISPR (LbaCas12a or LbuCas13a) reaction, which eliminated the requirement of immunoreaction in conventional immuno-based LFA. The assay realized the detection of 1-10 copy of target gene per reaction within 50 min. The CRISPR-CHLFA system achieved highly accurate visual detection of target negative samples, thus overcoming the false-positive problem that often produced in assays using conventional CRISPR-LFA. The CRISPR-CHLFA platform was further adopted for the visual detection of marker gene from SASR-CoV-2 Omicron variant and Mycobacterium tuberculosis (MTB), respectively, and 100% accuracy for the analysis of clinical specimens (45 SASR-CoV-2 specimens and 20 MTB specimens) was obtained. The proposed CRISPR-CHLFA system could provide an alternative platform for the development of POCT biosensors and can be widely adopted in accurate and visualized gene detection.

Immunological and inflammatory profiles during acute and convalescent phases of severe/ critically ill COVID-19 patients.

BACKGROUND: The 2019 Coronavirus (COVID-19) pandemic poses a huge threat internationally; however, the role of the host immune system in the pathogenesis of COVID-19 is not well understood. METHODS: Cytokine and chemokine levels and characterisation of immune cell subsets from 20 COVID-19 cases after hospital admission (17 critically ill and 3 severe patients) and 16 convalescent patients were determined using a multiplex immunoassay and flow cytometry, respectively. RESULTS: IP-10, MCP-1, MIG, IL-6, and IL-10 levels were significantly higher in acute severe/critically ill patients with COVID-19, whereas were normal in patients who had reached convalescence. CD8 T cells in severe and critically ill COVID-19 patients expressed high levels of cytotoxic granules (granzyme B and perforin)and was hyperactivated as evidenced by the high proportions of CD38. Furthermore, the cytotoxic potential of natural killer (NK) cells, and the frequencies of myeloid dendritic cells and plasmacytoid dendritic cells was reduced in patients with severe and critical COVID-19; however, these dysregulations were found to be restored in convalescent phases. CONCLUSION: Thus, elicitation of the hyperactive cytokine-mediated inflammatory response, dysregulation of CD8 T and NK cells, and deficiency of host myeloid and plasmacytoid DCs, may contribute to COVID-19 pathogenesis and provide insights into potential therapeutic targets and strategies.

Recombinant SARS-CoV-2 RBD with a built in T helper epitope induces strong neutralization antibody response.

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate.

A pH-responsive glycolipid-like nanocarrier for optimising the time-dependent distribution of free chemical drugs in focal cells.

Though Drug delivery systems have achieved accumulation at tumor sites via passive targeting and active targeting, the therapeutic effects are far from perfect. The unsatisfactory results are mainly due to limited drug release from the nanocarriers at tumor sites, while the pharmacological activities of the drug are attributed to the concentration of the free drug and the time maintained at the pharmacological targets. A pH-responsive chitosan based glycolipid-like nanocarrier (CSO-FBA-SA) was fabricated by conjugating stearyl alcohol (SA) to chitosan oligosaccharide (CSO) with the linkage of 4-formylbenzoic acid (FBA). FBA was a kind of aromatic aldehyde carbonyl compounds, which can form the benzoic-imine bond. In the presence of a Schiff's base structure, the carrier showed improved properties and could be quickly degraded in an acidic environment. In order to explore the process and mechanism of the nanocarriers in focal cells, the method for determining the intracellular concentration of released free doxorubicin was established, and the time-dependent change of the DOX-loaded micelles was revealed. The sight of drug release was also obtained with CLSM. The cytotoxicity of the CSO-FBA-SA/DOX against human breast cancer MCF-7 cells increased by 2.75-fold and 3.77-fold in comparison with the CSO-SA/DOX and DOX, respectively. Furthermore, the CSO-FBA-SA/DOX showed a 2.12-fold higher cytotoxicity against the MCF-7 cells than that treated against human ovarian cancer SKOV-3 cells with lower intracellular pH value, which indicated that the cellular inhibition positively correlated with the intracellular pH value. High tumor accumulation and fast drug release of the CSO-FBA-SA/DOX in tumor was responsible for the remarkable tumor growth inhibitory effect. Moreover, the CSO-FBA-SA/DOX could selectively respond to the acidic environment and release DOX in tumor only, which had relatively minimal cytotoxicity towards normal tissues. The results showed that this newly developed glycolipid-like nanocarrier could act as a potential vector for delivering the drug effectively with a low systemic toxicity.

MicroRNA-200c delivered by solid lipid nanoparticles enhances the effect of paclitaxel on breast cancer stem cell.

BACKGROUND: One of the major obstacles in the treatment of breast cancer is breast cancer stem cells (BCSC) which are resistant to standard chemotherapeutic drugs. It has been proven that microRNA-200c (miR-200c) can restore sensitivity to microtubule-targeting chemotherapeutic drugs by reducing the expression of class III ß-tubulin. In this study, combination therapy with miR-200c and paclitaxel (PTX) mediated by lipid nanoparticles was investigated as an alternative strategy against BCSC. MATERIALS AND METHODS: A cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane was strategically selected to formulate solid lipid nanoparticles (SLN) for miR-200c delivery. Nanostructured lipid carriers (NLC) with 20 wt% oleic acid were prepared for PTX delivery. Mammospheres, which gained the characteristics of BCSC, were used as a cell model to evaluate the efficiency of combination therapy. RESULTS: The cationic SLN could condense anionic miRNA to form SLN/miRNA complexes via charge interactions and could protect miRNA from degradation by ribonuclease. SLN/miR-200c complexes achieved 11.6-fold expression of miR-200c after incubation for 24 hours, compared with that of Lipofectamine™ 2000/miR-200c complexes (*P<0.05). Intracellular drug release assay proved that miRNA can be released from SLN/miRNA complexes efficiently in 12 hours after cellular uptake. After BCSC were transfected with SLN/miR-200c, the expression of class III ß-tubulin was effectively downregulated and the cellular cytotoxicity of PTX-loaded NLC (NLC/PTX) against BCSC was enhanced significantly (**P<0.01). CONCLUSION: The results indicated that the cationic SLN could serve as a promising carrier for miRNA delivery. In addition, the combination therapy of miR-200c and PTX revealed a novel therapeutic strategy for the treatment of BCSC.

A54 Peptide Modified and Redox-Responsive Glucolipid Conjugate Micelles for Intracellular Delivery of Doxorubicin in Hepatocarcinoma Therapy.

Redox-responsive nanomaterials applied in drug delivery systems (DDS) have attracted an increasing attention in pharmaceutical research as a carrier for antitumor therapy. However, there would be unwanted drug release from a redox-responsive DDS with no selection at nontarget sites, leading to undesirable toxicities in normal tissues and cells. Here, an A54 peptide modified and PEGylated reduction cleavable glucolipid conjugate (A54-PEG-CSO-ss-SA, abbreviated to APCssA) was designed for intracellular delivery of doxorubicin (DOX). The synthesized APCssA could be assembled via micellization self-assembly in aqueous water above the critical micelle concentration (54.9 µg/mL) and exhibited a high drug encapsulation efficiency (77.92%). The APCssA micelles showed an enhanced redox sensitivity in that the disulfide bond could be degraded quickly and the drug would be released from micelles in 10 mM levels of glutathione (GSH). The cellular uptake studies highlighted the affinity of APCssA micelles toward the hepatoma cells (BEL-7402) compared to that toward HepG2 cells. In contrast with the nonresponsive conjugate, the drug was released from APCssA micelles more quickly in 10 mM level of GSH concentration (tumor cells). Moreover, the DOX-loaded APCssA micelles displayed an increased cytotoxicity which was 1.6- to 2.0-fold that of unmodified and nonresponsive micelles. In vivo, the APCssA micelles had stronger distribution to liver and hepatoma tissue and prolonged the circulation and retention time, while the drug release only occurred in the tumor tissue. The APCssA/DOX showed the tumor inhibition rate equal to that of commercial doxorubicin hydrochloric without negative consequence. This study suggested that the APCssA/DOX showed promising potential to treat the tumor for its special tumor targeting, selective intracellular drug release, enhanced antitumor activity, and reduced toxicity on normal tissues.

Sequential delivery of therapeutic agents using a rationally designed disulfide-linked glycolipid-like nanocarrier.

Usage of combination therapies to deliver multiple therapeutics to increase treatment efficacy has shown promising results in the clinic. In an effort to maximize the synergistic effect of co-delivery of a drug and siRNA, we have developed a time-dependent sequential drug delivery system (DDS) based on a disulfide-linked chitosan-based nanocarrier (CS-ss-SA) for the co-delivery of paclitaxel (PTX) and Bcl-2 specific siRNA (siBcl-2). This CS-ss-SA nanocarrier is able to transport both drug and siRNA by entrapment of PTX and adsorption of siRNA on the shell by electrostatic attraction. We show that this nanocarrier transports siRNA into tumor cells via its glycolipid-like spatial structure and releases a hydrophobic model drug, Nile Red 8-11 h later. Next, when siRNA and the hydrophobic drug PTX were co-delivered to tumor cells, a synergistic effect was observed in both cell cycle arrest and cell viability. Ultimately, the co-delivery of PTX and siBcl-2 by CS-ss-SA may prove to be more efficacious and may even help overcome drug resistance.

Multi-cycle chemotherapy with the glycolipid-like polymeric micelles evade cancer stem cell enrichment in breast cancer therapy.

Multi-cycle chemotherapy is commonly used in the clinic, while the phenomena of enrichment of cancer stem cells (CSCs) and enhanced multi-drug resistance (MDR) are commonly involved. This research was designed for evaluating this successive administration. Chitosan oligosaccharide-g-stearic acid (CSOSA) polymer was used as the drug delivery system (DDS) to perform tri-cycle chemotherapy on a new tumor model induced by mammosphere cells. In vitro, on CSCs enriched mammospheres model, the doxorubicin-loaded CSOSA (CSOSA/DOX) displayed an improved growth inhibition effect measured by acid phosphatase assay (APH). While in vivo, the CSOSA/DOX micelles blocked tumor progression and led to a marked decrease of CSCs proportion as well as MDR capacity. What's more, the CSOSA/DOX helped decay the microenvironment and attenuate systemic side effects. We concluded that the CSOSA polymer could be a potential DDS for long-term multi-cycle chemotherapy in antitumor research.

Transport Mechanisms of Solid Lipid Nanoparticles across Caco-2 Cell Monolayers and their Related Cytotoxicology.

Solid lipid nanoparticles (SLNs) have been extensively investigated and demonstrated to be a potential nanocarriers for improving oral bioavailability of many drugs. However, the molecular mechanisms related to this discovery are not yet understood. Here, the molecular transport mechanisms of the SLNs crossing simulative intestinal epithelial cell monolayers (Caco-2 cell monolayers) were studied. The cytotoxicology results of the SLNs in Caco-2 cells demonstrated that the nanoparticles had low cytotoxicity, had no effect on the integrity of the cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. The endocytosis of the SLNs was time-dependent, and their delivery was energy-dependent. For the first time, the transport of the SLNs was directly verified to be a vesicle-mediated process. The internalization of the SLNs was mediated by macropinocytosis pathway and clathrin- and caveolae (or lipid raft)-related routes. Transferrin-related endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus were confirmed to be the main destinations of the SLNs in Caco-2 cells. As for the transport of the SLNs in Caco-2 cell monolayers, the results demonstrated that the SLNs transported to the basolateral side were intact, and the transport of the nanoparticles did not destroy the structure of tight junctions. The transcytosis of the SLNs across the Caco-2 cell monolayer was demonstrated to be mediated by the same routes as that in the endocytosis study. The ER, Golgi apparatus, and microtubules were confirmed to be important for the transport of the SLNs to both the basolateral and apical membrane sides. This study provides a more thoroughly understand of SLNs transportation crossing intestinal epithelial cell monolayers and could be beneficial for the fabrication of SLNs.

Effects of Fuzheng Paidu tablet on peripheral blood T lymphocytes, intestinal mucosa T lymphocytes, and immune organs in cyclophosphamide-induced immunosuppressed mice.

OBJECTIVE: The aim of this study is to investigate the effects of a Fuzheng Paidu tablet on peripheral blood T lymphocytes, intestinal mucosa T lymphocytes, and immune organs in cyclophosphamide (CY)-induced immunosuppressed mice. METHODS: The experimental mice (but not the control mice) were intraperitoneally injected with 80 mg/kg of CY solution every day for 3 consecutive days. Meanwhile, each mouse was administered with corresponding drugs for 7 continuous days. Then, 1 h after the last administration, each index was detected. RESULTS: The Fuzheng Paidu tablet significantly increases the CD4+/CD8+ ratio (P < 0.01) and the number of CD3+ and CD4+ cells in immunosuppressed mice (P < 0.01). In addition, the tablet apparently enhances the CD3+, CD4+, and CD8+ levels in the intestinal mucosal immune system (P < 0.01) as well as reverses the reduction of spleen lymphoid nodules and lymphocytes (P < 0.01). It also significantly improves intestinal inflammation, thymic atrophy, and sparse thymocytes in immunosuppressed mice (P < 0.01). DISCUSSION: The Fuzheng Paidu tablet greatly increases the levels of peripheral blood T lymphocytes and intestinal mucosal T lymphocytes as well as improves atrophied thymuses and spleens in CY-induced immunosuppressed mice.

Selective redox-responsive drug release in tumor cells mediated by chitosan based glycolipid-like nanocarrier.

The redox responsive nanocarriers have made a considerable progress in achieving triggered drug release by responding to the endogenous occurring difference between the extra- and intra- cellular redox environments. Despite the promises, this redox difference exists both in normal and tumor tissue. So a non-selective redox responsive drug delivery system may result in an undesired drug release in normal cells and relevant side-effects. To overcome these limitations, we have developed a chitosan based glycolipid-like nanocarrier (CSO-ss-SA) which selectively responded to the reducing environment in tumor cells. The CSO-ss-SA showed an improved reduction-sensitivity which only fast degraded and released drug in 10mM levels of glutathione (GSH). The CSO-ss-SA could transport the drug fast into the human ovarian cancer SKOV-3 cells and human normal liver L-02 cells by internalization, but only fast release drug in SKOV-3 cells. By regulating the intracellular GSH concentration in SKOV-3 cells, it indicated that the cellular inhibition of the PTX-loaded CSO-ss-SA showed a positive correlation with the GSH concentration. The CSO-ss-SA was mainly located in the liver, spleen and tumor in vivo, which evidenced the passive tumor targeting ability. Despite the high uptake of liver and spleen, drug release was mainly occurred in tumor. PTX-loaded CSO-ss-SA achieved a remarkable tumor growth inhibition effect with rather low dose of PTX. This study demonstrates that a smartly designed glycolipid-like nanocarrier with selective redox sensitivity could serve as an excellent platform to achieve minimal toxicity and rapid intracellular drug release in tumor cells.

Effect of curcumin on diabetic rat model of cerebral ischemia.

To investigate the effect of curcumin on cerebral ischemia in diabetic rats the effects and features. intravenous injection alloxan diabetes model, to give alloxan first seven days the tail measured blood glucose value, the election successful model rats were fed with large, medium and small doses of curcumin suspension, Shenqijiangtang suspension and the same volume of saline, administered once daily. The first 10 days after administration 2h (fasting 12h) rat tail vein blood glucose values measured in the first 20 days after administration of 2h (fasting 12h), do cerebral ischemia surgery; rapid carotid artery blood after 30min rats were decapitated, blood serum, blood glucose and glycated serum protein levels; take part of the brain homogenates plus nine times the amount of normal saline, made 10 percent of brain homogenates. Another part of the brain tissue, in the light microscope observation of pathological tissue. Compared with model group, large, medium and small doses of curcumin can significantly lower blood sugar and glycated serum protein levels, significantly reduced brain homogenates lactic acid content and lactate dehydrogenase activity; large, medium-dose curcumin can significantly increase brain homogenates Na(+)-K(+)-ATP activity, dose curcumin can significantly improve brain homogenates Ca(+)-Mg(+)- ATP activity. Curcumin can reduce blood sugar in diabetic rat model of cerebral ischemia and improve brain energy metabolism, improve their brain tissue resistance to ischemia and hypoxia, cerebral ischemia in diabetic rats have a good drop the role of sugar and protect brain tissue.

D-α-tocopherol polyethylene glycol succinate-based redox-sensitive paclitaxel prodrug for overcoming multidrug resistance in cancer cells.

To overcome the multidrug resistance (MDR) of P-glycoprotein (P-gp) substrate anticancer drugs, such as paclitaxel (PTX), a novel dual-functional prodrug, D-α-tocopherol polyethylene glycol succinate (TPGS) based PTX prodrug (TPGS-S-S-PTX), was synthesized here to fulfill the synergistic effect of P-gp inhibiting and intracellular redox-sensitive release. The prodrug could self-assemble into stable micelles in physiological environment with a diameter of ∼140 nm, while it disassociated in reductive condition and released PTX and TPGS active derivatives rapidly. High cell cytotoxicity in PTX-resistant human ovarian cell line A2780/T was observed with enhanced PTX accumulation due to the P-gp inhibition by the TPGS moiety. The IC50 of TPGS-S-S-PTX was 55% and 91% more effective than that of Taxol (clinical formulation of PTX) and uncleavable TPGS-C-C-PTX prodrug, respectively. This was found to be related with the increased apoptosis/necrosis and cell arrest in G2/M phase. In vivo evaluation of the TPGS-S-S-PTX prodrug exhibited an extended half-life, increased AUC (area under the concentration-time curve), enhanced tumor distribution and significant tumor growth inhibition with reduced side effects as compared to Taxol and TPGS-C-C-PTX. This prodrug has great potential in improving efficiency in the treatment of MDR tumors.

Influence of pH upon surface-enhanced enzyme-catalyzed luminol chemiluminescence at vicinity of nanoscale-corrugated gold and silver films.

Au and Ag biochips were fabricated to investigate the influence of pH upon the chemiluminescence (CL) of luminol at vicinity of surface-adsorbed peroxidase. A nanoscaled-corrugation of the metal induces an enhancement of the luminol CL which is maximal in the pH range favoring peroxidase catalysis and greater for gold than for silver. This is the proof that, in the CL process, the reactions involving peroxidase are surface-enhanced near corrugated surfaces.

Enhancement of optical nonlinearity in periodic gold nanoparticle arrays.

Linear and nonlinear optical properties of periodic triangular Au nanoparticle arrays were investigated. We compared the optical nonlinearity of periodic Au nanoparticle arrays with that of the ultra-thin gold film consisting of randomly distributed spheroidal clusters. A pronounced enhancement of the third-order nonlinear optical susceptibility χ((3)) in Au arrays was observed, and the figure of merit, χ((3))/α, of the periodic nanoparticle arrays is one order of magnitude larger than that of the ultra-thin film. Such an enhancement of the optical nonlinearity could be due to the strong local field near the triangular nanoparticles.