Specific Binding Ability Research Articles

Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus.

Norovirus infections are the leading cause of non-bacterial gastroenteritis and result in about 21 million new cases and $2 billion in costs per year in the United States. Existing diagnostics have limited feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools in order to contain outbreaks and prevent inappropriate treatments. In this study, a combination of phage display technology, deep sequencing and computational analysis was used to identify 12-mer peptides with specific binding to norovirus genotype GI.1 virus-like particles (VLPs). After biopanning, phage populations were sequenced and analyzed to identify a consensus peptide motif-YRSWXP. Two 12-mer peptides containing this sequence, NV-O-R5-3 and NV-O-R5-6, were further characterized to evaluate the motif's functional ability to detect VLPs and virus. Results indicated that these peptides effectively detect GI.1 VLPs in solid-phase peptide arrays, ELISAs and dot blots. Further, their specificity for the S-domain of the major capsid protein enables them to detect a wide range of GI and GII norovirus genotypes. Both peptides were able to detect virus in norovirus-positive clinical stool samples. Overall, the work reported here demonstrates the application of phage display coupled with next generation sequencing and computational analysis to uncover peptides with specific binding ability to a target protein for diagnostic applications. Further, the reagents characterized here can be integrated into existing diagnostic formats to detect clinically relevant genotypes of norovirus in stool.

A novel electrochemical sensor based on a molecularly imprinted polymer for the determination of epigallocatechin gallate.

A novel electrochemical sensor based on the molecularly imprinted polymer (MIP) was fabricated by electrochemical polymerization of β-cyclodextrins (β-CD) and epigallocatechin-gallate (EGCG) on the graphene oxide (GO) modified glassy carbon (GO/GC) electrode for the first time. The MIP/GO/GC electrode exhibits an excellent ability of specific binding of EGCG and a rapid electrochemical response, high sensitivity and selectivity for determination of EGCG. This prepared MIP sensor presents distinct advantages over conventional electrochemical methods for EGCG determination because it is a one-step preparation and the template molecule can be easily removed by cyclic voltammetry scans, and no elution reagent is required. Under the optimal experimental conditions, the linear response range for EGCG concentrations by the sensor was 3×10-8mol/L to 1×10-5mol/L and the detection limit was 8.78×10-9mol/L(S/N=3). The results demonstrate that the proposed MIP sensor can be a potential alternative for the determination of EGCG in tea samples.

Multifunctional SPIO/DOX-loaded A54 Homing Peptide Functionalized Dextran-g-PLGA Micelles for Tumor Therapy and MR Imaging.

Specific delivery of chemotherapy drugs and magnetic resonance imaging (MRI) contrast agent into tumor cells is one of the issues to highly efficient tumor targeting therapy and magnetic resonance imaging. Here, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran (A54-Dex-PLGA) was synthesized. The synthesized A54-Dex-PLGA could self-assemble to form micelles with a low critical micelle concentration of 22.51 μg. mL−1 and diameter of about 50 nm. The synthetic A54-Dex-PLGA micelles can encapsulate doxorubicin (DOX) as a model anti-tumor drug and superparamagnetic iron oxide (SPIO) as a contrast agent for MRI. The drug-encapsulation efficiency was about 80% and the in vitro DOX release was prolonged to 72 hours. The DOX/SPIO-loaded micelles could specifically target BEL-7402 cell line. In vitro MRI results also proved the specific binding ability of A54-Dex-PLGA/DOX/SPIO micelles to hepatoma cell BEL-7402. The in vivo MR imaging experiments using a BEL-7402 orthotopic implantation model further validated the targeting effect of DOX/SPIO-loaded micelles. In vitro and in vivo anti-tumor activities results showed that A54-Dex-PLGA/DOX/SPIO micelles revealed better therapeutic effects compared with Dex-PLGA/DOX/SPIO micelles and reduced toxicity compared with commercial adriamycin injection.

Highly luminescent tetra(biphenyl-4-yl)ethene-grafted molecularly imprinted mesoporous silica nanoparticles for fluorescent sensing of diethylstilbestrol

A molecularly imprinted mesoporous silica-based fluorescent sensor was prepared using a tetraphenylethene derivative (TBPE) as a signal transducer. Diethylstilbestrol (DES) was chosen as a target molecule and was connected to two triethoxysilane groups via thermally reversible urethane bonds. DES imprinted cavities with two-point binding sites were formed between the pores of the mesoporous silica by the sol-gel reaction and subsequent removal of DES. TBPE was selectively introduced to the pore surface. TBPE-grafted, DES imprinted mesoporous silica nanoparticle (TBPE-DIMSNP) showed a specific binding ability for the target template. The degree of fluorescence quenching of TBPE-DIMSNP was proportional to the concentration of DES. The sensitivity and selectivity of TBPE-DIMSNP were estimated by the Stern-Volmer equation. TBPE-DIMSNP displayed a much larger Stern-Volmer quenching constant for DES than for DES analogs with a high molecular imprinting factor. TBPE-DIMSNP also exhibited a great recovery of its initial fluorescence intensity even after several extraction and rebinding cycles.

Plasmons nanoparticles assembly and biological applications

Surface plasmon resonance (SPR) effect of nanoparticles, is an emerging subfield of nanophontonics, and it attracts increasing attention because of its potential applications in controlling and manipulating light at nanoscale dimensions. This review focuses on the molecules mediated nanoparticle assemblies and their biological applications, highlights several molecules mediated nanoparticle assembly technique and their recent applications in biomedical, and prospects the future development. Based on the specific binding ability of these functional mediate molecules (including DNA, antigen-antibody, aptamar, biotin-avidin, small organic molecules and polymers), plasmonic nanoparticles could be synthesized by self-assemble methods. Noble metal nanoparticle assemblies have broad applications in biological sensing, imaging and treatment due to their exclusive plasmon optical properties. With the rapid development of self-assembly technology, molecule-mediated self-assembly technology can be used well to control the spatial arrangement structure of plasmonic nanoparticles. Individual nanoparticle has been used to controllable assembled into one-dimensional, two- dimensional or three-dimensional conformation of the novel composite materials. SPR effect of noble metal nanoparticles, is an emerging subfield of nanophontonics, and it attracts increasing attention because of its potential applications in controlling and manipulating light at nanoscale dimensions. Compared with the individual nanoparticle, nanoparticle assemblies have a lot of novel or excellent physical properties which exhibite convenient adjustable ability in SPR spectra due to noble metal nanoparticles with irregular shape. They show notable antenna effect at the edge of nanoparticles and the coupling effect between adjacent two nanoparticles. All of these unique optical properties on plasmonic nanoparticles will be further enhanced by the self-assemble nanotechnology, which exhibits many promising applications in the field of biosensors (including photocolorimetric method, SPR, surface-enhanced Raman scattering (SERS), circular dichroism etc.), photoacoustic imaging and photothermal therapy in recently decades. This review focuses on the molecules mediated nanoparticle assemblies and their biological applications, highlights several molecules mediated nanoparticle assembly technique and their recent applications in biomedical, and prospects the future development. Based on the specific binding ability of these functional mediate molecules (including DNA, antigen- antibody, aptamar, biotin-avidin, small organic molecules and polymers etc.) plasmonic nanoparticles clusters could be synthesized to molecules mediated nanoparticle assemblies (MMNAs) by self-assemble methods following the well-designed linker. MMNAs could be widely used in bionanotechnology and biomedicine. The MMNAs would exhibit more sensitivity to the imperceptible changes of micro-environment around the assembles. There would be larger shifts in this resonance due to changes in the local index of refraction upon adsorption to the nanoparticles which could be used to detect biopolymers such as DNA or proteins. For example, the satellite gold nanoparticles linked on the surface of core particle by DEVD peptide would be cleaned by caspase-3 from the core. This method has been used directly for real-time monitoring the enzymatic reaction by MMNAs’ scattering spectra shifts. In addition, there would be abundant hot-spots between the satellite nanoparticles and the core, which is emerging as a powerful tool for SERS spectroscopic detection of trace molecular, cellular, and in vivo targets. The enhancement factors could achieve up to 1014−1015 by properly adjusting the LSPR spectroscopic profile and structural features of the MMNAs. What’s interesting is that when noble metal nanoparticles aggregate, there has been a red-shift in the light absorption spectrum, which dramatically enhances the light absorbance in the NIR region. And the MMNAs would show great adjustable ability in plasmon resonance absorption which opening up new opportunities for plasmonic nanoparticles to be utilized as photosensitizers in the NIR photothermal therapy photoacoustic imaging applications. Based on the further comprehensive researches and developments of MMNAs in recent decades, we believe that the continued development of MMNAs will be applied well in the fields of chemistry, physics, optics and biomedicine in the future.

Discovery of Fluorescence Polarization Probe for the ELISA-Based Antagonist Screening of α1-Adrenergic Receptors.

High-throughput screening (HTS) of ligand library to find new active molecules for G protein-coupled receptors is still a major interest, as well as an actual challenge. Fluorescence polarization (FP) assay portrays an essential role in HTS; however, in many cases, it was restricted by the absence of FP probes, the narrow measurement window, and low signal-to-noise (S/N) ratio. Herein, based on the modification of our previous probe 1 (QFL), we discovered an FP probe 3 (QGGFL) for α1-adrenergic receptors (α1-ARs), which has satisfactory fluorescence intensity, specific binding ability to receptors, and suitable fluorescence properties that were compatible with the filters in the FP system. Meanwhile, an "ELISA-like" strategy was designed for FP-based HTS assay in which proteins were adhered into a solid phase to improve the measurement window and S/N ratio. With fluorescent antagonist QGGFL and the ELISA strategy, we succeeded in establishing the first competitive binding FP assay for α1-AR antagonists as the alternative of the radioligand binding assay.

MiR-181a promotes the proliferation and metastasis of osteosarcoma cells by targeting RASSF1A

To investigate the role of miR-181a in promoting the proliferation and metastasis of osteosarcoma cells by targeting RASSF1A. The level of miR-181a in 30 human osteosarcoma tissues and corresponding bone tissues was detected by real-time PCR, and the correlation between the level of miR-181a and clinicopathological characteristics of osteosarcoma was analyzed. Osteosarcoma cells MG-63 were transfected with chemically-synthesized miR-181a mimics and inhibitors, and the proliferation, migration and invasion of MG-63 cells were detected by MTT and Transwell assay. The specific binding ability of miR-181a to RASSF1A 3'-UTR was theoretically predicted and detected by the dual luciferase reporter gene assay. The level of miR-181a in osteosarcoma tissues was statistically higher than that in the corresponding bone tissues (P<0.001). However, the level of miR-181a was not correlated with gender, age, tumor size and stage (P>0.05). MTT and Transwell assays showed that the growth rate, migration and invasion ability of MG-63 cells with up-regulation of miR-181a was significantly increased compared with negative control (P<0.05), while the growth rate, migration and invasion ability of MG-63 with down-regulated miR-181a was significantly decreased compared with negative control (P<0.05). Luciferase reporter gene assay showed that miR-181a targeted the 3'-UTR of RASSF1A and regulated the expression of RASSF1A. MiR-181a promotes the proliferation and metastasis of osteosarcoma cells through specifically binding to RASSF1A 3'-UTR and subsequent down-regulation of RASSF1A.

Preparation and characterization of a novel Al18F–NOTA–BZA conjugate for melanin-targeted imaging of malignant melanoma

Melanin is an attractive target for the diagnosis and treatment of malignant melanoma. Previous studies have demonstrated the specific binding ability of benzamide moiety to melanin. In this study, we developed a novel 18F-labeled NOTA–benzamide conjugate, Al18F–NOTA–BZA, which can be synthesized in 30min with a radiochemical yield of 20–35% and a radiochemical purity of >95%. Al18F–NOTA–BZA is highly hydrophilic (logP=−1.96) and shows good in vitro stability. Intravenous administration of Al18F–NOTA–BZA in two melanoma-bearing mouse models revealed highly specific uptake in B16F0 melanotic melanoma (6.67±0.91 and 1.50±0.26%ID/g at 15 and 120min p.i., respectively), but not in A375 amelanotic melanoma (0.87±0.21 and 0.24±0.09%ID/g at 15 and 120min p.i., respectively). The clearance from most normal tissues was fast. A microPET scan of Al18F–NOTA–BZA-injected mice also displayed high-contrast tumor images as compared with normal organs. Owing to the favorable in vivo distribution of Al18F–NOTA–BZA after intravenous administration, the estimated absorption dose was low in all normal organs and tissues. The melanin-specific binding ability, sustained tumor retention, fast normal tissues clearance and thelow projected human dosimetry supported that Al18F–NOTA–BZA is a very promising melanin-specific PET probe for melanin-positive melanoma.

Preparation of hydrophilic molecularly imprinted polymers via bulk polymerization combined with hydrolysis of ester groups for selective recognition of iridoid glycosides.

Hydrophilic molecularly imprinted polymers (H-MIP) with molecular recognition ability for iridoid glycosides (IGs) have been obtained via bulk polymerization combined with hydrolysis of ester groups. H-MIP were characterized by Fourier transform infrared spectroscopy (FT-IR). The hydrophilcity was measured by the contact angle measurement and the water dispersion stability. The obtained H-MIP demonstrated high selectivity and specific binding ability to five IGs in aqueous media. The group extraction efficiency of molecular imprinted solid-phase extraction (MISPE) for five IGs was investigated, including loading sample, breakthrough volume, washing solvent, and elution solvent. Compared with non-imprinted solid-phase extraction (NISPE), the higher average recovery (95.5%) of five IGs with lower relative standard deviations values (below 6.1%) using MISPE combined with high-performance liquid chromatography (HPLC) were achieved at three spiked levels in three blank samples. Under the optimum MISPE conditions, the wide linear range with the correlation coefficient of R (2) ≥ 0.9950 for five IGs with low limits of detection (LOD) and quantification (LOQ) (0.01-0.08 and 0.03-0.27μgmL(-1), respectively) were obtained. Chromatograms obtained using MISPE columns demonstrated that the matrix interference has been minimized and great interferences around IGs were also eliminated efficiently. These results indicated that the developed MISPE-HPLC method was selective, accurate, and applicable for the determination of IGs in water media. Graphical Abstract Preparation of hydrophilic molecularly imprinted polymers via bulk polymerization combined with hydrolysis of ester groups.

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis.

The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 (-) were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis.

A Novel CYP11B2-Specific Imaging Agent for Detection of Unilateral Subtypes of Primary Aldosteronism.

Although adrenal vein sampling is the standard method to distinguish unilateral from bilateral forms of primary aldosteronism, it is an invasive and technically difficult procedure. (11)C-metomidate (MTO)-positron emission tomography was reported as a potential replacement for adrenal vein sampling. However, MTO has low selectivity for CYP11B2 over CYP11B1. This study aimed to determine the selectivity of (18)F-CDP2230, a new imaging agent, for CYP11B2 over CYP11B1 and determine whether the biodistribution profile of (18)F-CDP2230 is favorable for imaging CYP11B2. The IC50 of CDP2230 for the enzymatic activities of CYP11B2 and CYP11B1 was determined using cells with stable expression of either enzyme. In vitro autoradiography of human adrenal sections with aldosterone-producing adenomas was performed to confirm the specific binding ability of (18)F-CDP2230 to CYP11B2-expressing regions. Furthermore, positron emission tomography and magnetic resonance imaging were performed to evaluate the biodistribution of (18)F-CDP2230 in rats. Although CDP2230 showed a significantly lower affinity for CYP11B2 and CYP11B1 than did MTO analogues, its selectivity for CYP11B2 over CYP11B1 was higher than that of MTO analogues. In vitro autoradiography revealed that the binding of (18)F-CDP2230 to CYP11B2-expressing regions in the adrenal gland was more specific than that of (123)I-IMTO. Moreover, the biodistribution study showed that (18)F-CDP2230 accumulated in adrenal glands with low background uptake. Our study showed a high selectivity of (18)F-CDP2230 for CYP11B2 over CYP11B1 with a favorable biodistribution for imaging CYP11B2. (18)F-CDP2230 is a promising imaging agent for detecting unilateral subtypes of primary aldosteronism.

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia.

Toward targeted therapy in chemotherapy-resistant pancreatic cancer with a smart triptolide nanomedicine.

Chemoresistance is the major impediment for treating pancreatic cancer. Herb-derived compound triptolide (TP) can inhibit proliferation of chemo-resistant pancreatic cancer (CPC) cell lines through multiple mechanisms, which exhibited superior anticancer efficacy compared with gemcitabine. However, toxicity due to non-specific exposure to healthy tissues hindered its clinical translation. Herein we successfully achieved targeting CPC cells and avoiding exposure to healthy tissues for TP by nucleolin-specific aptamer (AS1411) mediated polymeric nanocarrier. We conjugated AS1411 aptamer to carboxy terminated poly(ethylene glycol)–block–poly(d, l-lactide) (HOOC-PEG-PDLLA), then prepared AS1411-PEG-PDLLA micelle loading TP (AS-PPT) through solid dispersion technique. AS-PPT showed more antitumor activity than TP and equivalent specific binding ability with gemcitabine-resistant human pancreatic cancer cell (MIA PaCa-2) to AS1411 aptamer in vitro. Furthermore, we studied the distribution of AS-PPT (Cy3-labed TP) at tissue and cellular levels using biophotonic imaging technology. The results showed AS1411 facilitated TP selectively accumulating in tumor tissues and targeting CPC cells. The lifetime of the MIA PaCa-2 cell-bearing mice administrated with AS-PPT was efficiently prolonged than that of the mice subjected to the clinical anticancer drug Gemzar® in vivo. Such work provides a new strategy for overcoming the drug resistance of pancreatic cancer.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Acid-responsive PEGylated doxorubicin prodrug nanoparticles for neuropilin-1 receptor-mediated targeted drug delivery

Self-assembled prodrug nanoparticles have demonstrated great promise in cancer chemotherapy. In the present study, we developed a new kind of prodrug nanoparticles for targeted drug delivery. PEGylated doxorubicin conjugate with an acid-cleavable cis-aconityl spacer was prepared. Then it was functionalized with a tumor-penetrating peptide, Cys–Arg–Gly–Asp–Lys (CRGDK), providing the prodrug nanoparticles with the specific binding ability to neurophilin-1 receptor. In acid mediums, doxorubicin could be released from the prodrug nanoparticles with an accumulative release around 60% through the acid-triggered hydrolysis of cis-aconityl bond and nanoparticle disassembly. Whereas, drug release was slow under a neutral pH and the accumulative drug release was less than 16%. In the cell culture tests, our prodrug nanoparticles showed enhanced endocytosis and cytotoxicity in cancer cells including HepG2, MCF-7 and MDA-MB-231 cells, but lower cytotoxicity in human cardiomyocyte H2C9. In the animal experiments, the prodrug nanoparticles were intravenously injected into Balb/c nude mice bearing MDA-MB-231 tumors. Enhanced drug penetration and accumulation in tumors, accompanying with a rapid early tumor-binding behavior, was observed after intravenous injection of the peptide modified prodrug nanoparticles. These data suggests that the acid-sensitive and tumor-targeting PEGylated doxorubicin prodrug nanoparticle may be an efficient drug delivery system for cancer chemotherapy.

A mannose-recognizable chemosensor using gold nanoparticles functionalized with pradimicin, a nonpeptidic mannose-binding natural product

By making use of the distance-dependent optical properties of gold nanoparticles as well as the mannose (Man)-specific binding ability of pradimicin (PRM), we have successfully developed a Man-recognizable chemosensor. The dissociation constant of the chemosensor for Man was estimated to be approximately 10 μM, which was ten times lower than that of PRM itself. This result may be attributed to a cluster effect. Therefore, the chemosensor was capable of recognizing Man residues that have not yet been identified as the binding sites for PRM.

RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

Aptamers: active targeting ligands for cancer diagnosis and therapy.

Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment.

Active targeting docetaxel-PLA nanoparticles eradicate circulating lung cancer stem-like cells and inhibit liver metastasis.

Lung cancer is the major cause of cancer related lethality worldwide, and metastasis to distant organs is the pivotal cause of death for the vast majority of lung cancer patients. Accumulated evidence indicates that lung cancer stem-like cells (CSLCs) play important roles in metastagenesis, and these circulating CSLCs may be important targets to inhibit the subsequent metastasis. The present study was aimed at establishing CSLC-targeting polylactic acid (PLA) encapsulated docetaxel nanoparticles for antimetastatic therapy. Cyclic binding peptides were screened on CSLCs in vitro and the peptide CVKTPAQSC exhibiting high specific binding ability to pulmonary adenocarcinoma tissue was subsequently conjugated to the nanoparticles loaded with docetaxel (NDTX). Antimetastatic effect of CSLC-targeting nanoparticles loaded with docetaxel (TNDTX) was evaluated in a nude mouse model of liver metastasis. Results showed that, in the absence of targeting peptide, NDTX hardly exhibited any antimetastatic effect. However, TNDTX treatment significantly decreased the metastatic tumor area in the nude mouse liver. Histopathological and serological results also confirmed the antimetastatic efficacy of TNDTX. To our knowledge, this is the first report on establishing a CSLC-based strategy for lung cancer metastatic treatment, and we hope this will offer a potential therapeutic approach for management of metastatic lung cancer.

DNA aptamers against the Cry j 2 allergen of Japanese cedar pollen for biosensing applications

Sensing pollen allergens is required to prevent allergic disorders such as pollinosis. Aptamers, which bind to specific molecules, offer great potential as useful tools for detecting pollen allergens as measures against allergic disorders. Here, we report the identification of DNA aptamers binding to Cry j 2, one of the major allergens in Japanese cedar pollen, and the histochemical sensing of Cry j 2 in ruptured Japanese cedar pollen. DNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using nitrocellulose membranes. Through four rounds of SELEX, we identified aptamers binding to Cry j 2. The aptamers generated staining in ruptured Japanese cedar pollen on glass slides without extraction, similar to anti-Cry j 2 antibodies. The staining was compatible with starch localization, in which Cry j 2 is present. An aptamer, CJ2-06, which had high and specific binding ability to Cry j 2 (Kd=24nM), detected an amount of Cry j 2 equivalent to that in several tens of micrograms of pollen. Cry j 2 contained in house dust was detected in a spike test. The aptamers identified in this study can be powerful tools for allergen recognition in the practical biosensing of Cry j 2, leading to preventive measures against allergic disorders caused by Japanese cedar pollen.