Gint4.T-siHDGF chimera-capped mesoporous silica nanoparticles encapsulating temozolomide for synergistic glioblastoma therapy.

Due to glioblastoma (GBM) being the most intractable brain tumor, the continuous improvement of effective treatment methods is indispensable. The combination of siRNA-based gene therapy and chemotherapy for GBM treatment has now manifested great promise. Herein, Gint4.T-siHDGF chimera-capped mesoporous silica nanoparticles (MSN) encapsulating chemotherapy drug temozolomide (TMZ), termed as TMSN@siHDGF-Gint4.T, is developed to co-deliver gene-drug siHDGF and TMZ for synergistic GBM therapy. TMSN@siHDGF-Gint4.T possesses spherical nucleic acid-like architecture that can improve the enzyme resistance of siHDGF and increase the blood-brain barrier (BBB) permeability of the nanovehicle. The aptamer Gint4.T of chimera endows the nanovehicle with GBM cell-specific binding ability. When administered systemically, TMSN@siHDGF-Gint4.T can traverse BBB and enter GBM cells. In the acidic lysosome environment, the cleavage of benzoic-imine bond on MSN surface leads to an initial rapid release of chimera, followed by a slow release of TMZ encapsulated in MSN. The sequential release of siHDGF and TMZ first allows siHDGF to exert its gene-silencing effect, and the downregulation of HDGF expression further enhances the cytotoxicity of TMZ. In vivo experimental results have demonstrated that TMSN@siHDGF-Gint4.T significantly inhibits tumor growth and extends the survival time of GBM-bearing mice. Thus, the as-developed TMSN@siHDGF-Gint4.T affords a potential approach for the combination treatment of GBM.

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.

A multimer-based SERS aptasensor for highly sensitive and homogeneous assay of carcinoembryonic antigens.

Carcinoembryonic antigens (CEAs) are known as one of the most common tumor markers. Their facile and affordable detection is critical for early diagnosis of malignant tumors, especially in resource-constrained settings. Here, we report a novel multimer-based surface-enhanced Raman scattering (SERS) aptasensor for a specific CEA assay. The aptasensor is fabricated through aptamer-assisted self-assembly of silver-coated gold nanoparticles (Au@Ag NPs), and the self-assembled multimeric structure possesses abundant hot-spots to provide high SERS response. When CEA is introduced, the specific recognition of CEA by aptamers will lead to the disassembly of Au@Ag multimers due to the lack of a bridging aptamer between Au@Ag NPs. As a result, the number of hot-spots in the multimeric system is decreased, and the intensity at 1585 cm-1 of the SERS reporter (4-mercaptobenzoic acid, 4-MBA) on the surface of NPs will also be decreased. The Raman intensity is proportional to the logarithm of the concentration of CEA. The detection sensitivity can be down to the pg mL-1 level. The analytical method only needs a droplet of 2 µL of sample, and the detection time is less than 20 min. The multimer-based SERS aptasensor can be applied in sensitive and inexpensive detection of CEA in serum samples.

Base amount-dependent fluorescence enhancement for the assay of vascular endothelial growth factor 165 in human serum using hairpin DNA-silver nanoclusters and oxidized carbon nanoparticles.

A base amount-dependent fluorescence enhancement-based strategy is put forward to determine vascular endothelial growth factor 165 (VEGF165) in human serum by the use of hairpin DNA-silver nanoclusters (hDNA-AgNCs) and oxidized carbon nanoparticles (CNPs). The hDNA-AgNCs aptasensing probe consists of AgNCs-contained hairpin loop (that generates a fluorescence signal), hairpin stem (that makes the structure stable), and the terminal aptamer 1 (that recognizes the target together with aptamer 2). It has been demonstrated that the fluorescence intensity of hDNA-AgNCs is ~ 3-fold stronger than that of single-stranded DNA-AgNCs (ssDNA-AgNCs), and hDNA-AgNCs have a strong dependence of fluorescence enhancement on the base amount in hairpin stem and loop. Upon the addition of oxidized CNPs, the terminal aptamer 1 of hDNA-AgNCs can adsorb onto the surface of oxidized CNPs via π-π stacking, and the fluorescence of hDNA-AgNCs (with excitation/emission maxima at 490/567 nm) is quenched via fluorescence resonance energy transfer (FRET). When aptamer 2 and VEGF165 are subsequently added, aptamer 1, VEGF165, and aptamer 2 reassemble into an intact tertiary structure, and the fluorescence is recovered because hDNA-AgNCs are far away from the surface of oxidized CNPs and the FRET efficiency decreases. Under the optimized conditions, the aptasensing probe can selectively assay VEGF165 with a detection limit of 14 pM. The results provide a label-free and sensitive method to monitor VEGF165 in human serum. Schematic representation of the strong dependence of fluorescence enhancement on base amount in stem and loop of hairpin DNA-silver nanoclusters. The probe can be used to assay vascular endothelial growth factor 165 (VEGF165) and give a judgment whether human serum VEGF165 is at a normal or abnormal level for clinical diagnosis.

Metformin promotes the osseointegration of titanium implants under osteoporotic conditions by regulating BMSCs autophagy, and osteogenic differentiation.

Osteoporosis is a common bone disorder with adverse effects on oral osseointegration, and the effects of metformin on bone metabolism have received increasing attention. The aim of the present study was to test the hypothesis that metformin promoted osteogenesis of bone mesenchymal stem cells (BMSCs) and osseointegration of titanium implants. BMSCs were treated with metformin to assess autophagic capacity, reactive oxygen species (ROS) production, anti-aging ability, and osteogenic differentiation. To determine its potential application in peri-implant of the maxilla, metformin was injected around the implant each day, immediately after the implant was embedded into the tooth socket. The results showed that metformin increased the autophagic capacity and decreased ROS production of osteoporotic BMSCs under hypoxia and serum deprivation (H/SD) culturing conditions. Metformin treatment significantly enhanced stemness properties and mineralized nodule formation, and increased the expression of osteogenic markers, including runt related transcription factor 2 (Runx2), osteocalcin (OCN), and alkaline phosphatase (ALP). Moreover, metformin substantially accelerated the formation of new bone, ameliorated the bone microarchitecture and promoted osseointegration of the dental implant. Collectively, metformin induces an osteogenic effect around the implant. Considering the widespread use of metformin, the results of the present study might promote a novel understanding of the positive effects of local metformin delivery on alveolar ridge defect, and have potential clinical application for the acceleration of osseointegration.

Salt-induced gold nanoparticles aggregation lights up fluorescence of DNA-silver nanoclusters to monitor dual cancer markers carcinoembryonic antigen and carbohydrate antigen 125.

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA1-AgNCs-apta1) and red-emitting DNA-AgNCs with CA125 aptamer (rDNA2-AgNCs-apta2) in the ratio of 1:1 in volume. Upon addition of AuNPs, gDNA1-AgNCs-apta1 and/or rDNA2-AgNCs-apta2 are flexibly adsorbed onto the surface of AuNPs by terminal aptamer(s), which prevents salt-induced AuNPs aggregation under high salt condition and results in fluorescence quenching based on surface plasmon enhanced energy transfer (SPEET). With the addition of CEA and/or CA125, the target(s) and corresponding aptamer(s) coordinate to form the complex, keeping DNA-AgNCs-apta(s) far away from the surface of AuNPs and making AuNPs aggregated in high salt medium. The AuNPs aggregation leads to the recovery of fluorescence signals of DNA-AgNCs-apta(s) due to weakened SPEET. Utilizing the fluorescence aptasensor system, the limit of detection of CEA and CA125 are as low as 7.5 pg·mL-1 and 0.015 U·mL-1, respectively. The proposed method can be applied to the selective and simultaneous determination of CEA and CA125 in human serum.

A split aptamer-labeled ratiometric fluorescent biosensor for specific detection of adenosine in human urine.

A dual-emission ratiometric fluorometric aptasensor is presented for highly specific detection of adenosine. An adenosine binding aptamer (ABA) was split into two halves (termed as ABA1 and ABA2). ABA1 was covalently bound to blue-emitting carbon dots (with excitation/emission maxima at 365/440 nm) as responsive fluorophore (referred to as ABA1-CDs). ABA2 was linked to red-emitting silica-coated CdTe quantum dots (with excitation/emission maxima at 365/613 nm) acting as internal reference and referred to as ABA2-QDs@SiO2. Upon addition of graphene oxide, the fluorescence of ABA1-CDs is quenched. After subsequent addition of ABA2-QDs@SiO2 and different amounts of adenosine, the blue fluorescence is recovered and causes a color change from red to royal blue. The method represents a ratiometric turn-on assay for visual, colorimetric and fluorometric determination of adenosine. The limit of detection is as low as 2.4 nM in case of ratiometric fluorometry. The method was successfully applied to the determination of adenosine in (spiked) human urine. Recoveries range from 98.8% to 102%. Graphical abstract Adenosine binding aptamer1-carbon dots (ABA1-CDs) can absorb on graphene oxide (GO) via π stacking. This causes fluorescence to be quenched by fluorescence resonance energy transfer (FRET). After addition of ABA2-silica-coated quantum dots (ABA2-QDs@SiO2) and adenosine, binding of adenosine to two pieces of aptamers forms a complex (ABA1-CD/adenosine/ABA2-QD@SiO2) which dissociates from GO. As a result, fluorescence is recovered.

Silver nanoparticles/activated carbon composite as a facile SERS substrate for highly sensitive detection of endogenous formaldehyde in human urine by catalytic reaction.

Most of efforts have been made to prepare high performance surface-enhanced Raman scattering (SERS) substrate for amplifying Raman signals. It still remains a grand challenging task in building a simple, conventional and low-cost SERS substrate with highly dense hotspots for improved sensitivity of the target analytes. Here, we report a very dexterous strategy to fabricate a distinctive SERS substrate with high density hotspots, using common adsorbent activated carbon (AC) as template to assemble silver nanoparticles (Ag NPs). It can be estimated that the enhancement effect of Ag NPs/AC composite is about 6.5-fold that of bare Ag NPs. Different from the resonant dyes, however, formaldehyde (FA) is a Raman-inactive molecule even though enhanced. Considering that, a novel method for quantitative analysis of FA using the Ag NPs/AC composite as SERS sensor has been developed, based on the catalytic effect of trace FA on the oxidation of malachite green (MG) through bromate under acidic condition. The change of MG from reduced form into oxidized leucomalachite green (LMG) results in the quench of Raman signals of MG, responding to 0.07 ppb FA that is about 2 orders of magnitude lower than the limit defined by the Nash's method as a standard procedure recommended in Europe, Japan and China. Moreover, SERS examinations of endogenous FA in human urine signify that the proposed method has high selectivity, reliability and accuracy. Thus, as-fabricated Ag NPs/AC composite is adequate as inexpensive and versatile SERS sensor utilized in the quantification of trace targets in various complicated matrices.

Toward rapid analysis, forecast and discovery of bioactive compounds from herbs by jointly using thin layer chromatography and ratiometric surface-enhanced Raman spectroscopy technique.

Conventional isolation and identification of active compounds from herbs have been extensively reported by using various chromatographic and spectroscopic techniques. However, how to quickly discover new bioactive ingredients from natural sources still remains a challenging task due to the interference of their similar structures or matrices. Here, we present a grand approach for rapid analysis, forecast and discovery of bioactive compounds from herbs based on a hyphenated strategy of thin layer chromatography and ratiometric surface-enhanced Raman spectroscopy. The performance of the hyphenated strategy is first evaluated by analyzing four protoberberine alkaloids, berberine (BER), coptisine (COP), palmatine (PAT) and jatrorrhizine (JAT), from a typical herb Coptidis Rhizoma as an example. It has been demonstrated that this coupling method can identify the four compounds by characteristic peaks at 728, 708, 736 and 732 cm-1, and especially discriminate BER and COP (with similar migration distances) by ratiometric Raman intensity (I708/I728). The corresponding limits of detection are 0.1, 0.05, 0.1 and 0.5 µM, respectively, which are about 1-2 orders of magnitude lower than those of direct observation method under 254 nm UV lamp. Based on these findings, the proposed method further guides forecast and discovery of unknown compounds from traditional Chinese herb Typhonii Rhizoma. Results infer that two trace alkaloids (BER and COP) from the n-butanol extract of Typhonii Rhizoma are found for the first time. Moreover, in vitro experiments manifest that BER can effectively decrease the viability of human glioma U87 cells by inducing cell cycle arrest in a concentration-dependent manner.

Neuroprotective Activity of Cerebrosides from Typhonium giganteum by Regulating Caspase-3 and Bax/Bcl-2 Signaling Pathways in PC12 Cells.

An investigation of the potential neuroprotective natural product constituents of the rhizomes of Typhonium giganteum led to the isolation of two new cerebrosides, typhonosides E (1) and F (2), along with 11 known analogues (3-13). The structures of compounds 1 and 2 were elucidated by spectroscopic data interpretation. The activity of these compounds against glutamate-induced cell apoptosis was investigated in PC12 cells. All compounds exhibited such activity, which was related to the length of the fatty acyl chain. Among them, longan cerebroside II (11), with the longest fatty acyl chain, showed the most potent protective effect in PC12 cells from glutamate injury, with an EC50 value of 2.5 µM. Moreover, at the molecular level, longan cerebroside II (11) downregulated the expression of caspase-9, caspase-3, and Bax, upregulated the expression of Bcl-2, and decreased the level of cytosolic cytochrome c in a concentration-dependent manner.

A surface-grafted ligand functionalization strategy for coordinate binding of doxorubicin at surface of PEGylated mesoporous silica nanoparticles: Toward pH-responsive drug delivery.

To achieve drug targeting and on-demand releasing, surface functionalization plays a critical role in fabricating potential mesoporous silica nanoparticles (MSNs) toward tumor chemotherapy. Here, we prepared a size-controllable ligand-functionalized MSNs delivery system via coordinate bonding, which can release doxorubicin (DOX) in response to pH and prolong the circulation time of drug in vivo. After modifying the external surface of MSNs with polyethylene glycol (PEG), iminodiacetic acid (IDA) as a ligand was mainly grafted on the surface of mesopores to chelate cupric iron and DOX in sequence via coordinate bonds. The modified MSNs exhibited a uniform size of about 72nm and could be stably dispersed in saline. After DOX loading, the drug loading content and encapsulation efficiency were calculated to be 9.3±0.1% and 92.8±0.6%, respectively. Moreover, the resultant MSNs showed a pH-responsive release property, which could avoid the premature leakage of drug in circulation and achieve on-demand release within the tumor cells. Additionally, the pharmacokinetic study in healthy rats demonstrated that DOX loaded in functionalized MSNs presented the longer circulation time and lower plasma clearance rate compared with DOX solution. These results indicated that PEG/IDA modified MSNs with pH-responsive release capacity possessed great promising as an anticancer drug delivery system.

A general method to regenerate arrayed gold microelectrodes for label-free cell assay.

We developed a method to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes efficient treatment of the gold surface with trypsin (0.25%, v/v) digestion, rinsing with ethanol and deionized water and spinning steps. The proposed method ensured complete regeneration and repeated usage of gold microchips up to 4 times for the real-time electric impedance measurement of anti-cancer drug cytotoxicity.

Label-free surface-enhanced Raman scattering imaging to monitor the metabolism of antitumor drug 6-mercaptopurine in living cells.

The molecular processes of drugs from cellular uptake to intracellular distribution as well as the intracellular interaction with the target molecule are critically important for the development of new antitumor drugs. In this work, we have successfully developed a label-free surface-enhanced Raman scattering (SERS) technique to monitor and visualize the metabolism of antitumor drug 6-mercaptopurine in living cells. It has been clearly demonstrated that Au@Ag NPs exhibit an excellent Raman enhancement effect to both 6-mercaptopurine and its metabolic product 6-mercaptopurine-ribose. Their different ways to absorb at the surface of Au@Ag NPs lead to the obvious spectral difference for distinguishing the antitumor drug and its metabolite by SERS spectra. The Au@Ag NPs can easily pass through cell membranes in a large amount and sensitively respond to the biological conversion of 6-mercaptopurine in tumor cells. The Raman imaging can visualize the real-time distribution of 6-mercaptopurine and its biotransformation with the concentrations in tumor cells. The SERS-based method reported here is simple and efficient for the assessments of drug efficacy and the understanding of the molecular therapeutic mechanism of antitumor drugs at the cellular level.

Coordinate bonding strategy for molecularly imprinted hydrogels: toward pH-responsive doxorubicin delivery.

Molecularly imprinted hydrogel (MIH) as drug delivery system has been studied. It still remains a challenge to construct the stimuli-responsive MIH. Here, we report a coordinate bond strategy for imprinting doxorubicin (Dox) in hydrogel capable of pH-responsive and sustained drug delivery. The imprinting condition such as template-monomer interactions induced by metal ion was carefully investigated by spectroscopic methods. The obtained Dox-MIH was evaluated by absorption and in vitro release experiments. It has been demonstrated that the cupric ion mediated interaction between Dox and 4-vinyl pyridine via coordination and the optimal coordinate ratio of Dox/Cu(2+) was 2:1. The rebinding amount of MIH to Dox was 2.7-fold that of nonimprinted hydrogel and the Dox-loaded MIH showed a pH-responsive release property. Not more than 10% of loaded drug was released from Dox-MIH at pH 7.2 during a time course of 7 days. However, near to 60% of loaded drug was sustainedly released at pH 5.0 during the same period. These results indicated that Dox-MIH with pH-responsive behavior possessed great promising as sustained-release delivery system of anticancer drug.

Self-assembly molecularly imprinted polymers of 17ß-estradiol on the surface of magnetic nanoparticles for selective separation and detection of estrogenic hormones in feeds.

This paper reports a surface molecular self-assembly strategy for molecular imprinting on magnetic nanoparticles for selective separation and detection of estrogens in feeds. First, γ-methacryloxypropyltrimethoxysilane (MEMO) was successfully assembled at the surface magnetic nanoparticles through simple free radical polymerization, and subsequently, the copolymerization was further assembled between methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) in the presence of templates 17ß-estradiol (E2). The synthesized magnetic molecularly imprinted polymers for E2 (E2-MMIPs) showed quick separation, large adsorption capacity, high selectivity and fast binding kinetics for E2. Meanwhile, a dispersive solid-phase extraction (DSPE) based on E2-MMIPs has been established for efficient separation and fast enrichment of estrogens from the feeds. The assay exhibited a linear range of 0.1-4 µM for E2 and estriol (E3) with the correlation coefficient above 0.9996 and 0.9994, respectively. Recoveries of E2 from three kinds of feeds spiked at different concentration levels ranged from 92.7% to 97.0% with RSD<4.7%, and recoveries of E3 ranged from 71.9% to 83.1% with RSD<4.9%, respectively. The method is simple and sensitive, and can be used as an alternative tool to effectively separate and enrich the trace of estrogens in agricultural products by HPLC-UV.

The study of core-shell molecularly imprinted polymers of 17ß-estradiol on the surface of silica nanoparticles.

In this paper, highly selective core-shell molecularly imprinted polymers of 17ß-estradiol on the surface of silica nanoparticles (SiO(2)@E2-MIPs) were prepared. The SiO(2)@E2-MIPs were characterized by Fourier transform infrared spectrometer (FT-IR), transmission electron microscope (TEM), dynamic adsorption and static adsorption tests. The sorption capacity of the SiO(2)@E2-MIPs were nearly 5 times that of the non-imprinted polymers (NIPs), and it only took 25 min to achieve the sorption equilibrium. It indicated that the SiO(2)@E2-MIPs exhibited a high selectivity, large adsorption capacity and fast kinetics. When the SiO(2)@E2-MIPs were used as dispersive solid-phase extraction (dSPE) absorbents to selectively enrich and determine estrogens in duck feed, the average recoveries of E2 and estriol (E3) were higher than 96.74% and 72.07%, respectively, and the relative standard deviations (RSD) of E2 and E3 were less than 1.61% and 3.28%, respectively. The study provides an effective method for the separation and enrichment of estrogens in the complex matrix samples by the SiO(2)@E2-MIPs.