[Research progress on processing history evolution, chemical constituents, and pharmacological action of Scorpio].

Scorpio is a valuable Chinese animal medicine commonly used in clinical practice in China. It is the main drug in the treatment of liver wind internal movement caused by various reasons throughout the history of traditional Chinese medicine(TCM), with the effects of relieving wind and spasm, dredging collaterals, relieving pain, and eliminating toxin and mass. Scorpio is poisonous and often used as medicine after processing. There are records of its processing as early as the Song Dynasty. Afterward, there were more than 15 processing methods, including frying with vinegar, neat processing, and stir-frying. After processing, the fishy smell could be removed to correct the taste, and the toxicity could be reduced, which was beneficial to clinical application. At present, the main reported components in Scorpio are protein polypeptides, alkaloids, and lipids, with many pharmacological effects, such as anti-cancer, anti-coagulation, anti-thrombosis, anti-atherosclerosis, and anti-bacteria. In this study, the historical evolution of processing, chemical constituents, and pharmacological action of Scorpio were discussed in order to provide references for the related research on Scorpio.

Development and evaluation of a Chinese herbal gel for analgesic and anti-inflammatory effects.

This study aimed to develop a film-forming gel containing three Chinese herbal extracts, an extracted mixture of Corydalis yanhusuo, Cynanchum paniculatum and Armadillidium vulgare (Latreille) (MCCA) and evaluate its analgesic and anti-inflammatory activities. Using the Box Behnken Design, the optimal prescription for the MCCA gel was determined. The analgesic effects were tested through acid writhing and formalin pain models. while the rheumatoid arthritis model assessed the pain and anti-inflammatory effects. For the evaluation of the effect of MCCA gels on pro-inflammatory cytokines, as well, Elisa was used. Results showed that the MCCA Gel with 2% mint oil had the highest transdermal volume of 32.57±0.92µg/cm2. High doses of MCCA gel were effective in suppressing pain, with a pain inhibition rate of 54.37% during the acetic acid peristaltic test that showed pronounced inhibition in the second phase of the formalin-induced analgesia test. In the rheumatoid arthritis model, the MCCA gel reduced inflammation scores in rat feet and decreased the expressions of four inflammatory factors in serum. Generally, The MCCA gel exhibits noticeable pain-relieving and anti-inflammatory properties with high penetration with the skin.

[Research progress on processing history evolution, chemical constituents, and pharmacological effects of Hirudo].

As a traditional animal drug, Hirudo is slightly toxic and has the effects of breaking blood stasis, dredging meridians, expelling stasis, and resolving mass. It has a long history of processing, and the early boiling records can be traced back to the Han Dynasty. More than ten processing methods such as frying, roasting, and lime processing appeared later. After processing, Hirudo is deodorized and modified in taste and becomes crispy, which is conducive to crushing and clinical application. At present, the reported components in Hirudo mainly include protein polypeptides, pteridines, and lipids, which have anti-coagulant, anti-thrombotic, anti-atherosclerotic, anti-tumor, and other pharmacological effects. This study reviewed the processing history evolution, chemical consti-tuents, and pharmacological effects of Hirudo to provide a reference for the related research on Hirudo.

The prebiotic properties of polysaccharides obtained by differentiated deproteinization methods from Flos Sophorae Immaturus on Lactobacillus fermentum.

The polysaccharides derived from various deproteinization methods were prepared from Flos Sophorae Immaturus (FSI) to investigate the prebiotic efficacy of Lactobacillus fermentum (L.f ). The implications of polysaccharides from FSI (PFSI) gained after purification performed by non-deproteinization and different deproteinization processes (Savage method, papain method, and TCA method) via one-factor optimization were firstly investigated for the influences on the growth of L.f. The utilization of carbohydrate sources and the synthesis of protein and lactate during its growth were analyzed, as well as the variations of LDH, SOD, and GSH- Px enzyme dynamics. The results showed that the one-factor optimization of the deproteinization process with the protein removal rate and polysaccharide retention rate as the indexes led to the optimal methods of the Sevage method with 5 elution times, papain method with 80 U/mL concentration, and TCA method with 2.5 ratio, respectively. In addition, the PFSI obtained with or without deproteinization purification had a certain effect on promoting L.f proliferation. Moreover, the PFSI gained by the third deproteinization purification, at a concentration of 10 g/L, significantly elevated L.f biomass and growth rate compared with the blank control, and the utilization of reducing sugars and the synthesis of protein and lactic acid were higher than the control (P < 0.05); improved LDH, SOD, and GSH-Px activity in L.f (P < 0.05), and the TCA method could be effectively applied to eliminate the proteins affecting FSI in probiotics, and PFSI may be a potentially beneficial prebiotic and intestinal reinforcer.

Antibacterial effect and mechanism of anthocyanin from Lycium ruthenicum Murr.

The inhibitory effects of the anthocyanin obtained from Lycium ruthenicum Murr were tested against several food-borne pathogens were evaluated, such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Aspergillus niger and Penicillium sp. In general, anthocyanin had different antibacterial effect on different bacteria, and the best antibacterial effect on S. aureus, with minimal inhibitory concentration (MIC) of 3.125 mg/mL. Anthocyanin increased the surface hydrophobicity of S. aureus, discharged the intracellular K+, and reduced the total soluble protein, affecting protein synthesis. Fluorescent inverted microscope and flow cytometry (FCM) found a significant increase in fluorescence intensity and lethality relative to the control group, and the dead P3 region to 77.21%. The above suggested a correlation between the antibacterial mechanism of anthocyanin and cell membrane permeability integrity. Biofilm formation was evaluated by the crystal violet assay (CV), silver staining method and methyl thiazolyl tetrazolium (MTT). Scanning electron microscopy (SEM) showed that anthocyanins could change the structure of biofilm.

The Chemical Profiling and Anticancer Potential of Functional Polysaccharides from Flos Sophorae Immaturus.

Polysaccharides from Flos Sophorae Immaturus (FSI) are one of its pharmacological compounds that can perform effective activities. Aiming to extract the most effective polysaccharides against hepatocellular carcinoma (HCC), the polysaccharides were separated from FSI through ultrasonic microwave extraction, and the first comparison was carried out on the characterization of the structure and its cytotoxic properties on HCC SMMC 7721 cells of undeproteinized purified polysaccharides (PFSI-1) and papain-deproteinized polysaccharides (PFSI-2) from FSI. The findings indicated that PFSI-1 and PFSI-2 had characteristic absorption peaks of polysaccharides; PFSI-1 contained three monosaccharides and PFSI-2 contained ten; and SEM, AFM, and NMR were consistent with the verification of IR polysaccharide characteristics, suggesting probable additional latent activities. The pharmacotoxic effects of both PFSI-1 and PFSI-2 on SMMC 7721 cells (p < 0.05), attenuated the migration ability of SMMC 7721 cells (p < 0.05) and promoted apoptosis (p < 0.05), with an increase in G0/G1-phase cells and decrease in S-phase cells in the PFSI-1 as well as a decrease in G0/G1-phase cells, increase in S-phase cells, and decrease in apoptosis in the PFSI-2 (p < 0.05). The significant cytotoxic effect of PFSI-2 on SMMC 7721 cells (p < 0.05) and its protective effect on human hepatic L02 cells (HL-7702) at low concentrations (p > 0.05) could indicate its potential as a new drug for the treatment of HCC.

A PBPK Model of Ternary Cyclodextrin Complex of ST-246 Was Built to Achieve a Reasonable IV Infusion Regimen for the Treatment of Human Severe Smallpox.

ST-246 is an oral drug against pathogenic orthopoxvirus infections. An intravenous formulation is required for some critical patients. A ternary complex of ST-246/meglumine/hydroxypropyl-ß-cyclodextrin with well-improved solubility was successfully developed in our institute. The aim of this study was to achieve a reasonable intravenous infusion regimen of this novel formulation by a robust PBPK model based on preclinical pharmacokinetic studies. The pharmacokinetics of ST-246 after intravenous injection at different doses in rats, dogs, and monkeys were conducted to obtain clearances. The clearance of humans was generated by using the allometric scaling approach. Tissue distribution of ST-246 was conducted in rats to obtain tissue partition coefficients (K p ). The PBPK model of the rat was first built using in vivo clearance and K p combined with in vitro physicochemical properties, unbound fraction, and cyclodextrin effect parameters of ST-246. Then the PBPK model was transferred to a dog and monkey and validated simultaneously. Finally, pharmacokinetic profiles after IV infusion at different dosages utilizing the human PBPK model were compared to the observed oral PK profile of ST-246 at therapeutic dosage (600 mg). The mechanistic PBPK model described the animal PK behaviors of ST-246 via intravenous injection and infusion with fold errors within 1.2. It appeared that 6h-IV infusion at 5 mg/kg BID produced similar Cmax and AUC as oral administration at 600 mg. A PBPK model of ST-246 was built to achieve a reasonable regimen of IV infusion for the treatment of severe smallpox, which will facilitate the clinical translation of this novel formulation.

Comparative Analysis of Physiological, Enzymatic, and Transcriptomic Responses Revealed Mechanisms of Salt Tolerance and Recovery in Tritipyrum.

Salt stress results in the severe decline of yield and quality in wheat. In the present study, salt-tolerant Tritipyrum ("Y1805") and salt-sensitive wheat "Chinese Spring" ("CS") were selected from 121 wheat germplasms to test their physiological, antioxidant enzyme, and transcriptomic responses and mechanisms against salt stress and recovery. 56 chromosomes were identified in "Y1805" that comprised A, B, and D chromosomes from wheat parent and E chromosomes from Thinopyrum elongatum, adding to salt-tolerant trait. Salt stress had a greater inhibitory effect on roots than on shoots, and "Y1805" demonstrated stronger salt tolerance than "CS." Compared with "CS," the activities of superoxide dismutase and catalase in "Y1805" significantly increased under salt stress. "Y1805" could synthesize more proline and soluble sugars than "CS." Both the net photosynthetic rate and chlorophyll a/b were affected by salt stress, though the level of damage in "Y1805" was significantly less than in "CS." Transcriptome analysis showed that the differences in the transcriptional regulatory networks of "Y1805" were not only in response to salt stress but also in recovery. The functions of many salt-responsive differentially expressed genes were correlated closely with the pathways "peroxisome," "arginine and proline metabolism," "starch and sucrose metabolism," "chlorophyll and porphyrin metabolism," and "photosynthesis."

Comparable Intestinal and Hepatic First-Pass Effect of YL-IPA08 on the Bioavailability and Effective Brain Exposure, a Rapid Anti-PTSD and Anti-Depression Compound.

YL-IPA08, exerting rapid antidepressant-like and anxiolytic-like effects on behaviors by translocator protein (TSPO) mediation, is a novel compound that has been discovered and developed at our institute. Fit-for-purpose pharmacokinetic properties is urgently needed to be discovered as early as possible for a new compound. YL-IPA08 exhibited low bioavailability (∼6%) during the preliminary pharmacokinetics study in rats after oral administration. Our aim was to determine how metabolic disposition by microsomal P450 enzymes in liver and intestine limited YL-IPA08's bioavailability and further affected brain penetration to the target. Studies of in vitro metabolic stability and permeability combined with in vivo oral bioavailability, panel CYP inhibitor co-administration via different routes, and double cannulation rats were conducted to elucidate the intestinal and hepatic first-pass effect of YL-IPA08 on bioavailability. Unbound brain-to-plasma ratio (K p,uu ) in rats was determined at steady state. Results indicated that P450-mediated elimination appeared to be important for its extensive first-pass effect with comparative contribution of gut (35%) and liver (17%), and no significant species difference was observed. The unbound concentration of YL-IPA08 in rat brain (6.5 pg/ml) was estimated based on K p,uu (0.18) and was slightly higher than in vitro TSPO-binding activity (4.9 pg/ml). Based on the onset efficacy of YL-IPA08 toward TPSO in brain and K p,uu , therapeutic human plasma concentration was predicted to be ∼27.2 ng/ml would easily be reached even with unfavorable bioavailability.

Procyanidin B2 mitigates endothelial endoplasmic reticulum stress through a PPARδ-Dependent mechanism.

Hyperglycemia-induced endothelial endoplasmic reticulum (ER) stress is implicated in the pathophysiology of diabetes and its vascular complications. Procyanidins are enriched in many plant foods and have been demonstrated to exert several beneficial effects on diabetes, cardiovascular and other metabolic diseases. In the present study, we investigated the effect of procyanidin B2 (PCB2), the most widely distributed natural procyanidin, on ER stress evoked by high glucose in endothelial cells (ECs) and the underlying mechanisms. We showed that PCB2 mitigated the high glucose-activated ER stress pathways (PERK, IRE1α and ATF6) in human vascular ECs. In addition, we found that PCB2 attenuated endothelial ER stress via the activation of peroxisome proliferator-activated receptor δ (PPARδ). We demonstrated that PCB2 directly bound to and activated PPARδ. Conversely, GSK0660, a selective PPARδ antagonist, attenuated the suppressive effect of PCB2 on the ER stress signal pathway. Functionally, PCB2 ameliorated the high glucose-impaired endothelium-dependent relaxation in mouse aortas. The protective effect of PCB2 on vasodilation was abolished in the aortas pretreated with GSK0660 or those from the EC-specific PPARδ knockout mice. Moreover, the protective effects of PCB2 on ER stress and endothelial dysfunction required the inter-dependent actions of PPARδ and AMPK. Collectively, we demonstrated that PCB2 mitigated ER stress and ameliorated vasodilation via a PPARδ-mediated mechanism beyond its classic action as a scavenger of free radicals. These findings further highlighted the novel roles of procyanidins in intervening the ER stress and metabolic disorders related to endothelial dysfunction.

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.

Assessment and Confirmation of Species Difference in Nonlinear Pharmacokinetics of Atipamezole with Physiologically Based Pharmacokinetic Modeling.

Atipamezole, an α 2-adrenoceptor antagonist, displayed nonlinear pharmacokinetics (PK) in rats. The aim of this study was to understand the underlying mechanisms of nonlinear PK in rats and linear PK in humans and develop physiologically based PK models (PBPK) to capture and validate this phenomenon. In vitro and in vivo data were generated to show that metabolism is the main clearance pathway of atipamezole and species differences exist. Where cytochrome P450 (P450) was responsible for the metabolism in rats with a low Michaelis constant, human-specific UDP-glucuronosyltransferase 2B10- and 1A4-mediated N-glucuronidation was identified as the leading contributor to metabolism in humans with a high V max capacity. Saturation of metabolism was observed in rats at pharmacologically relevant doses, but not in humans at clinically relevant doses. PBPK models were developed using GastroPlus software to predict the PK profile of atipamezole in rats after intravenous or intramuscular administration of 0.1 to 3 mg/kg doses. The model predicted the nonlinear PK of atipamezole in rats and predicted observed exposures within 2-fold across dose levels. Under the same model structure, a human PBPK model was developed using human in vitro metabolism data. The PBPK model well described human concentration-time profiles at 10-100 mg doses showing dose-proportional increases in exposure. This study demonstrated that PBPK is a useful tool to predict human PK when interspecies extrapolation is not applicable. The nonlinear PK in rat and linear PK in human were characterized in vitro and allowed the prospective human PK via intramuscular dosing to be predicted at the preclinical stage. SIGNIFICANCE STATEMENT: This study demonstrated that PBPK is a useful tool for predicting human PK when interspecies extrapolation is not applicable due to species unique metabolism. Atipamezole, for example, is metabolized by P450 in rats and by N-glucuronidation in humans that were hypothesized to be the underlying reasons for a nonlinear PK in rats and linear PK in humans. This was testified by PBPK simulation in this study.

Procyanidin B2 Activates PPARγ to Induce M2 Polarization in Mouse Macrophages.

Procyanidins, a subclass of flavonoids found in commonly consumed foods, possess potential anti-inflammatory activity. Manipulation of M1/M2 macrophage homeostasis is an effective strategy for the treatment of metabolic inflammatory diseases. The objective of this study was to determine the effect of procyanidins on macrophage polarization. Procyanidin B2 (PCB2), the most widely distributed natural procyanidins, enhanced the expressions of M2 macrophage markers (Arg1, Ym1, and Fizz1). PCB2 activated peroxisome proliferator-activated receptor γ (PPARγ) activity and increased the expressions of PPARγ target genes (CD36 and ABCG1) in macrophages. Inhibition of PPARγ using siRNA or antagonist GW9662 attenuated the PCB2-induced expressions of M2 macrophage markers. In addition, we identified cognate PPAR-responsive elements (PPREs) within the 5'-flanking regions of the mouse Arg1, Ym1, and Fizz1 genes. Furthermore, macrophages isolated from db/db diabetic mice showed lower expressions of M2 markers. PCB2 effectively restored the Arg1, Ym1, and Fizz1 expressions in a PPARγ-dependent manner. These findings support the notion that PCB2 regulated macrophage M2 polarization via the activation of PPARγ. Our results provide a new mechanism by which procyanidins exert their beneficial anti-inflammatory effects.

Hybrid micelles containing methotrexate-conjugated polymer and co-loaded with microRNA-124 for rheumatoid arthritis therapy.

Purpose: Methotrexate (MTX) is a first-line drug for rheumatoid arthritis (RA)therapy. However, MTX monotherapy often results in irreversible joint damage due to its slow onset of action and long duration. microRNA-124 (miR-124) has shown direct bone protection activity against RA. A co-delivery system for MTX and microRNA combination may provide therapeutic synergy. Methods: Methotrexate-conjugated polymer hybrid micelles (M-PHMs) were prepared by self-assembly of two functional amphiphilic polymers (MTX-PEI-LA and mPEG-LA) at an optimized weight ratio. Incorporation of microRNA was achieved through electrostatic interactions between microRNA and cationic polymer MTX-PEI-LA. Cellular uptake, endosome escape, biodistribution, and therapeutic efficacy of M-PHMs/miR-124 complexes were investigated and evaluated in RAW264.7 cells and a rat adjuvant-induced arthritis (AIA) model. Results: M-PHMs/miR-124 complexes exhibited folate receptor-mediated uptake in activated RAW264.7 cells. miR-124 was able to escape from the endosome and down-regulate nuclear factor of activated T cells cytoplasmic1 (NFATc1). M-PHMs/miR-124 complexes accumulated in inflamed joints of AIA rats and showed superior therapeutic efficacy through both anti-inflammatory effect and direct bone protective effect. Combination of miR-124 and MTX in these micelles induced disease remission. Conclusions: M-PHMs/miR-124 was highly effective against RA through therapeutic synergy. Additional studies are warranted to further investigate its therapeutic potential and delineate its mechanisms of action.

Stachydrine Mediates Rapid Vascular Relaxation: Activation of Endothelial Nitric Oxide Synthase Involving AMP-Activated Protein Kinase and Akt Phosphorylation in Vascular Endothelial Cells.

Stachydrine (STA) is a constituent of citrus fruits and Leonurus heterophyllus Sweet. In the present study, we established that STA caused acute endothelium-dependent relaxation. The vascular action of STA was mediated by nitric oxide (NO) via cyclic guanosine monophosphate. Mechanistically, STA activated AMP-activated protein kinase (AMPK), protein kinase B/Akt, and endothelial NO synthase (eNOS) in vascular endothelial cells (ECs). AMPK inhibition by compound C blocked STA-induced Akt/eNOS phosphorylation, suggesting that AMPK is the upstream of Akt and eNOS. Inhibition of Akt by MK2206 blocked STA-stimulated eNOS phosphorylation without altering AMPK phosphorylation. Furthermore, we showed that STA activated AMPK via the induction of liver kinase B1 phosphorylation. These results indicated that STA can induce eNOS phosphorylation and vasorelaxation by regulating the interplay between AMPK and Akt pathways in ECs. These findings further highlighted the potential of STA as a nutritional factor in the beneficial effects of fruit intake in preventing the endothelial dysfunction-related metabolic cardiovascular diseases.

PPARδ agonist prevents endothelial dysfunction via induction of dihydrofolate reductase gene and activation of tetrahydrobiopterin salvage pathway.

BACKGROUND AND PURPOSE: Impaired endothelium-dependent relaxation (EDR) is a hallmark of endothelial dysfunction. A deficiency of tetrahydrobiopterin (BH4 ) causes endothelial NOS to produce ROS rather than NO. PPARδ is an emerging target for pharmacological intervention of endothelial dysfunction. Thus, the present study examined the role of PPARδ in the regulation of dihydrofolate reductase (DHFR), a key enzyme in the BH4 salvage pathway. EXPERIMENTAL APPROACH: Gene expression was measured by using qRT-PCR and western blotting. Biopterins and ROS were determined by using HPLC. NO was measured with fluorescent dye and electron paramagnetic resonance spectroscopy. Vasorelaxation was measured by Multi Myograph System. KEY RESULTS: The PPARδ agonist GW501516 increased DHFR and BH4 levels in endothelial cells (ECs). The effect was blocked by PPARδ antagonist GSK0660. Chromatin immunoprecipitation identified PPAR-responsive elements within the 5'-flanking region of the human DHFR gene. The promoter activity was examined with luciferase assays using deletion reporters. Importantly, DHFR expression was suppressed by palmitic acid (PA, a saturated fatty acid) but increased by docosahexaenoic acid (DHA, a polyunsaturated fatty acid). GSK0660 prevented DHA-induced increased DHFR expression. Conversely, the suppressive effect of PA was mitigated by GW501516. In mouse aortae, GW501516 ameliorated the PA-impaired EDR. However, this vasoprotective effect was attenuated by DHFR siRNA or methotrexate. In EC-specific Ppard knockout mice, GW501516 failed to improve vasorelaxation. CONCLUSION AND IMPLICATIONS: PPARδ prevented endothelial dysfunction by increasing DHFR and activating the BH4 salvage pathway. These results provide a novel mechanism for the protective roles of PPARδ against vascular diseases.

Targeted and Efficient Delivery of siRNA Using Tunable Polymeric Hybrid Micelles for Tumor Therapy.

BACKGROUND/AIM: Effective and targeted delivery of siRNA to tumor cells is a prerequisite to achieving their therapeutic effects. Survivin is up-regulated in tumor cells and is associated with resistance to therapy. Therefore, siRNA-mediated silencing of survivin is a potential therapeutic strategy for cancer. The aim of the study was to examine whether polymeric hybrid micelles can be used to effectively deliver siRNAs into cells. MATERIALS AND METHODS: First, linoleic acid (LA) was conjugated to polyethylenimine (PEI) and methoxy-polyethyleneglycol (mPEG) and two amphiphilic polymers (PEI-LA and mPEG-LA) were obtained. Polymeric hybrid micelle (PHM) was then prepared and characterized by self-assembly of PEI-LA and mPEG-LA at different percentages of the two amphiphilic polymers. A PHM/siRNA complex with optimized composition and good biocompatibility was then prepared and its cellular uptake, biodistribution, and antitumor effects were investigated. RESULTS: Survivin siRNA was efficiently delivered to the cells. It reduced survivin protein expression and greatly suppressed tumor growth. Moreover, siRNA loaded in PHM gathered in a solid tumor in mice and achieved an improved anticancer effect compared to naked siRNA. CONCLUSION: PHM is a promising and safe vehicle for siRNA delivery and may find utility in cancer therapy.

Targeted Co-Delivery of siRNA and Methotrexate for Tumor Therapy via Mixed Micelles.

A combination of chemotherapeutic drugs and siRNA is emerging as a new modality for cancer therapy. A safe and effective carrier platform is needed for combination drug delivery. Here, a functionalized mixed micelle-based delivery system was developed for targeted co-delivery of methotrexate (MTX) and survivin siRNA. Linolenic acid (LA) was separately conjugated to branched polyethlenimine (b-PEI) and methoxy-polyethyleneglycol (mPEG). MTX was then conjugated to LA-modified b-PEI (MTX-bPEI-LA) to form a functionalized polymer-drug conjugate. Functionalized mixed micelles (M-MTX) were obtained by the self-assembly of MTX-bPEI-LA and LA-modified mPEG (mPEG-LA). M-MTX had a narrow particle size distribution and could successfully condense siRNA at an N/P ratio of 16/1. M-MTX/siRNA was selectively taken up by HeLa cells overexpressing the folate receptor (FR) and facilitated the release of the siRNA into the cytoplasm. In vitro, M-MTX/siRNA produced a synergy between MTX and survivin siRNA and markedly suppressed survivin protein expression. In tumor-bearing mice, M-MTX/Cy5-siRNA showed an elevated tumor uptake. In addition, M-MTX/siRNA inhibited tumor growth. Immunohistochemistry and a western blot analysis showed a significant target gene downregulation. In conclusion, M-MTX/siRNA was highly effective as a delivery system and may serve as a model for the targeted co-delivery of therapeutic agents.

Influences of graphene oxide on biofilm formation of gram-negative and gram-positive bacteria.

In this study, we evaluated the influences of graphene oxide (GO) on biofilm formation. Escherichia coli MG1655 and Bacillus subtilis 168 were used as models for Gram-negative and Gram-positive bacteria. The growth profiles and viability assays indicated that GO exhibited a high antibacterial activity, of which the negative effects on bacteria growth raised with the increasing GO concentration. The antibacterial activity of GO was mainly attributed to the membrane stress and ROS-independent oxidative stress. Moreover, it was worthy to note that the biofilm formation was enhanced in the presence of GO at low dosage whereas inhibited in the high-concentration GO environment. These results could be explained by the roles of the dead cells, which were inactivated by GO. When the concentration of GO was limited, only a part of the cells would be inactivated, which may then serve as a protection barrier as well as the necessary nutrient to the remaining living cells for the formation of biofilm. In contrast, with a sufficient presence of GO, almost all cells can be inactivated completely and thus the formation of biofilm could no longer be triggered. Overall, the present work provides significant new insights on the influence of carbon nanomaterials towards biofilm formation, which has far-reaching implications in the field of biofouling and membrane bioreactor. Graphical abstract ᅟ.