Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers

AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.

Metal-Organic Framework Nanocarriers for Drug Delivery in Biomedical Applications

Investigation of metal–organic frameworks(MOFs)for biomedical applications has attracted much attention in recent years.MOFs are regarded as a promising class of nanocarriers for drug delivery owing to well-defined structure,ultrahigh surface area and porosity,tunable pore size,and easy chemical functionalization.In this review,the unique properties of MOFs and their advantages as nanocarriers for drug delivery in biomedical applications were discussed in the first section.Then,state-ofthe-art strategies to functionalize MOFs with therapeutic agents were summarized,including surface adsorption,pore encapsulation,covalent binding,and functional molecules as building blocks.In the third section,the most recent biological applications of MOFs for intracellular delivery of drugs,proteins,and nucleic acids,especially aptamers,were presented.Finally,challenges and prospects were comprehensively discussed to provide context for future development of MOFs as efficient drug delivery systems.

A process insight into production of ethyl levulinate via a stepwise fractionation

Ethyl levulinate(EL)is a key biomass-derived compounds due to its socio-economic benefits for the synthesis of commodity chemicals.Herein,we proposed an efficient one-step bamboo conversion to EL in ethanol,and a novel stepwise fractionation to purify EL and lignocellulose degradation products.A proton acid,due to its high catalytic efficiency,yielded 26.65%EL in 120 min at 200℃.The productions of ethyl glucoside and 5-ethoxymethylfurfural were analyzed in terms of by-products formation.To the best of our knowledge,there is no single report on catalyst for one step synthesis of EL directly from bamboo,as well as a stepwise fractionation to purify EL.Due to similar physiochemical properties in each fraction,the platform molecules could broaden a new paradigm of bamboo biomass utilization for renewable energy and value-added biochemicals.In addition,glucose,ethyl glucoside,corn starch,and microcrystalline cellulose were also investigated as substrates,so that the reaction intermediates of this one-pot procedure were identified and a possible reaction mechanism was proposed.

Self-assembled amphiphilic NIR-Ⅱ emissive nano-micelles for imaging-guided photothermal therapy of colorectal cancer

Colorectal cancer(CRC) is one of the major causes of cancer-related mortality worldwide. Most near-infrared(NIR) agents used in clinical CRC treatment are at NIR-I(700–900 nm) window, which has limitations on deep tissue, and fluorescent probes in the second NIR(1,000–1,700 nm) allow high-resolution bioimaging with deep tissue penetration. However, existing NIR-II fluorophores used in clinical are still rare. Herein, based on shielding-donor-acceptor-donor-shielding(S-D-A-D-S) scaffold, we developed an organic small-molecule fluorophore IR-BTGP with NIR-II emission for imaging-guided photothermal therapy(PTT) in CRC mice model. Amphiphilic IR-BTGP can be self-assembled into spherical nano-micelles, which presents reliable water solubility and photothermal conversion efficiency(30.2%). In vitro experiments indicate that cancer cells treated with IRBTGP were significantly killed upon 808 nm light irradiation. Furthermore, in vivo NIR-II fluorescence imaging confirms that IR-BTGP accumulates in the tumor region. Remarkably, a significant tumor inhibition rate(78.5%) was observed in tumorbearing mice when treated with IR-BTGP plus 808 nm irradiation. Therefore, this work shows that IR-BTGP holds great promise as an NIR-II fluorescence imaging-guided PTT platform for CRC in the future.

Aptamer-Based Cell-Surface Profiling with Single-Cell Resolution Enables Precise Cancer Characterization

Molecular profiling of cell-surface proteins is a powerful strategy for precise cancer diagnosis.While mass cytometry(MC)enables synchronous detection of over 40 cellular parameters,its full potential in disease classification is challenged by the limited types of recognition probes currently available.In this work,we synthesize a panel of heavy isotopeconjugated aptamers to profile cancer-associated signatures on the surface of hematological malignancy(HM)cells.Based on 15 molecular signatures,we performed cell-surface profiling that allowed the precise classification of 8 HM cell lines.Combined with machine-learning technology,this aptamer-based MC platform also achieved multiclass identification of HM subtypes in clinical sampleswith 100%accuracy in the training cohort and 80%accuracy in the test cohort.Therefore,we report an effective and practical strategy for precise cancer classification at the singlecell level,paving the way for its clinical use in the near future.

Molecular Engineering of Aptamer to Solubilize Hydrophobic Near-Infrared Photosensitizer for Enhanced Cancer Photodynamic Therapy

Photodynamic therapy(PDT)is a clinically representative treatment strategy for cancer.However,conventional photosensitizers(PSs)are usually hampered by poor water solubility and low targeting capacity.Here,we report the precise molecular engineering of aptamer oligonucleotides to solubilize hydrophobic near-infrared(NIR)PSs for enhanced cancer PDT.Hydrophobic pyropheophorbide A(PA)is precisely conjugated to aptamer oligonucleotides by combing DNA solid-phase synthesis technology and Cu-free click chemistry.Precise coupling of insoluble PA to hydrophilic aptamer oligonucleotides vastly improves its solubility to as high as 750μM in water without any cosolvent,resulting in an enhanced NIR fluorescence property(quantum yield=23%)and photoactivity.Moreover,the targeting ability of aptamer oligonucleotides is not affected by our molecular engineering strategy.Abundant reactive oxygen species(ROS)are produced intracellularly with 660 nm laser irradiation,eliciting mitochondria damage and cell death.Tumor growth is effectively inhibited with a single dose of aptamer-PA conjugates without in vivo toxicity.Their therapeutic effect is more than 20-fold higher than that of PA.Compared with traditional formulation,using aptamer oligonucleotides as functional carriers to solubilize hydrophobic NIR PSs is more precise and operable.Our DNA molecular engineering strategy paves a way for the rational design of molecularly targeted medicine for future clinical cancer therapy.

The First-in-Human Whole-Body Dynamic Pharmacokinetics Study of Aptamer

Serving as targeting ligands,aptamers have shown promise in precision medicine.However,the lack of knowledge of the biosafety and metabolism patterns in the human body largely impeded aptamers’clinical translation.To bridge this gap,here we report the first-in-human pharmacokinetics study of protein tyrosine kinase 7 targeted SGC8 aptamer via in vivo PET tracking of gallium-68(^(68)Ga)radiolabeled aptamers.The specificity and binding affinity of a radiolabeled aptamer,named ^(68)Ga[Ga]-NOTA-SGC8,were maintained as proven in vitro.Further preclinical biosafety and biodistribution evaluation confirmed that aptamers have no biotoxicity,potential mutation risks,or genotoxicity at high dosage(40 mg/kg).Based on this result,a first-in-human clinical trial was approved and carried out to evaluate the circulation and metabolism profiles,as well as biosafety,of the radiolabeled SGC8 aptamer in the human body.Taking advantage of the cutting-edge total-body PET,the aptamers’distribution pattern in the human body was acquired in a dynamic fashion.

Self-assembled multifunctional DNA nanoflowers for the circumvention of multidrug resistance in targeted anticancer drug delivery

Cancer chemotherapy has been limited by its side effects and multidrug resistance （MDR）, the latter of which is partially caused by drug efflux from cancer cells. Thus, targeted drug delivery systems that can circumvent MDR are needed. Here, we report multifunctional DNA nanoflowers （NFs） for targeted drug delivery to both chemosensitive and MDR cancer cells that circumvented MDR in both leukemia and breast cancer cell models. NFs are self-assembled via potential co-precipitation of DNA and magnesium pyrophosphate generated by rolling circle replication, during which NFs are incorporated using aptamers for specific cancer cell recognition, fluorophores for bioimaging, and doxorubicin （Dox）- binding DNA for drug delivery. NF sizes are tunable （down to N200 nm in diameter）, and the densely packed drug-binding motifs and porous intrastructures endow NFs with a high drug-loading capacity （71.4%, wt/wt）. Although the Dox- loaded NFs （NF-Dox） are stable at physiological pH, drug release is facilitated under acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not in nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising tools for circumventing MDR in targeted cancer therapy.

Zinc-substituted hemoglobin with specific drug binding sites and fatty acid resistance ability for enhanced photodynamic therapy

Precisely designed protein-based nanodrugs, as a kind of colloidal drug system, have attracted significant attention in tumor therapy because of their refined drug loading ratio, controlled delivery efficacy and natural biocompatibility. However, most drugs are conjugated to the protein carriers randomly without specific binding sites. Moreover, such sites could easily be replaced by lipophilic molecules in the physiological environment and result in low delivery efficiency. With strong and specific binding locations especially comparatively narrow spatial binding sites and nonflexible structure, hemin (FePPIX)-free hemoglobin or apohemoglobin (apoHb), as a natural metalloporphyrin protein carrier, represents great potential in bioapplication. Therefore, we herein introduce a folate acid (FA) modified, zinc-substituted hemoglobin (ZnPHb-FA) as a naturally occurring protein matrix-based photosensitizer for cancer photodynamic therapy (PDT). Noncovalent inserted ZnPPIX molecules in apoHb possess an extremely stable property and significant recovered photoproperties with superior biocompatibility and phototoxicity, both in vitro and in vivo. This stability was verified by molecular docking analysis and calculation of binding constant, representing a total of five drug binding sites of apoHb for ZnPPIX molecules, four of which are energetically favorable (△G value of -11.9 kcal/mol), and one which is energetically acceptable (△G value of -9 kcal/mol). Folate acid modification has been shown to efficiently enhance the internalization and retention time of ZnPHb nanodrug. ZnPHb-FA is also an efficient depressor of hemin oxygenase-1 (HO-1), which could, in turn, lower the antioxidant ability of cancer cells by decreasing the production of biiirublin. Results in vitro and in vivo both indicated that the firmly combination of apoHb and ZnPPIX described here represents a novel and efficient protein nanodrug systems for cancer therapy.

Stable and unique graphitic Raman internal standard nanocapsules for surface-enhanced Raman spectroscopy quantitative analysis

Graphitic nanomaterials have unique, strong, and stable Raman vibrations that have been widely applied in chemistry and biomedicine. However, utilizing them as internal standards （ISs） to improve the accuracy of surface-enhanced Raman spectroscopy （SERS） analysis has not been attempted. Herein, we report the design of a unique IS nanostructure consisting of a large number of gold nanoparticles （AuNPs） decorated on multilayered graphitic magnetic nanocapsules （AGNs） to quantify the analyte and eliminate the problems associated with traditional ISs. The AGNs demonstrated a unique Raman band from the graphitic component, which was localized in the Raman silent region of the biomolecules, making them an ideal IS for quantitative Raman analysis without any background interference. The IS signal from the AGNs also indicated superior stability, even under harsh conditions. With the enhancement of the decorated AuNPs, the AGN nanostructures greatly improved the quantitative accuracy of SERS, in particular the exclusion of quantitative errors resulting from collection loss and non-uniform distribution of the analytes. The AGNs were further utilized for cell staining and Raman imaging, and they showed great promise for applications in biomedicine.

Nanocarrier based on the assembly of protein and antisense oligonucleotide to combat multidrug resistance in tumor cells

Chemotherapy-induced multi-drug resistance(MDR) in tumors poses a huge challenge for clinical treatment of tumors. The downregulation of the multi-drug resistance relative protein, represented by P-glycoprotein(P-gp), can reverse MDR of cancer cells. In this study, we developed doxorubicin-loading nanocarrier based on the assembly of protein and antisense oligonucleotide(ASO) to combat MDR of cancer cells. The data demonstrate that the nanocarrier can efficiently deliver ASO to cytoplasm and downregulate the P-glycoprotein expression, subsequently improving the therapeutic effects of Dox in doxorubicin-resistant MCF-7/ADR cancer cells. The preparation is simple and effective, providing a powerful tool for gene delivery. Therefore, our nanocarrier shows high promise in cancer treatment.

Recent progress of aptamer-drug conjugates in cancer therapy

Aptamers are single-stranded DNA or RNA sequences that can specifically bind with the target protein or molecule via specific secondary structures.Compared to antibody-drug conjugates(ADC),aptamer-drug conjugate(ApDC)is also an efficient,targeted drug for cancer therapy with a smaller size,higher chemical stability,lower immunogenicity,faster tissue penetration,and facile engineering.Despite all these advantages,several key factors have delayed the clinical translation of ApDC,such as in vivo off-target effects and potential safety issues.In this review,we highlight the most recent progress in the development of ApDC and discuss solutions to the problems noted above.

Gold nanoparticle-decorated MoSe_(2) nanosheets as highly effective peroxidase-like nanozymes for total antioxidant capacity assay

A visual colorimetric detection strategy is reported for total antioxidant capacity(TAC)assay by using 3,3',5,5'-tetramethylbenzidine(TMB)oxidation as chromogenic substrate based on gold nanoparticle-decorated MoSe_(2) nanosheets(Au@MoSe_(2)).Au@MoSe_(2) nanostructures exhibit high peroxidase-like activity and can catalyze H_(2)O_(2)to oxidize TMB.Based on inhibition effect of ascorbic acid(AA)on TMB oxidation,a facile and sensitive colorimetric method was developed for AA detection.Under optimal conditions,the proposed method showed a sensitivity for AA in a concentration range from 2 to 120μM and limit of detection was 0.41μM.Furthermore,the method was employed for TAC assay in actual samples,including commercial beverages and vitamin C tablets.This work represents a model in nanostructure design and will lead to further development of TAC assay in evaluation of antioxidant food quality.

Molecular imaging:design mechanism and bioapplications

Molecular imaging is a non-invasive method to image and analyze the concentration and activity of functional biomolecules in cells or in vivo at molecular level,and plays an increasing role in deep understanding of biological processes,early and accurate diagnosis of diseases,and evaluation of treatment.Nowadays,numerous novel molecular imaging probes have been developed,involving every biomedical imaging modality,such as optical imaging,photoacoustic imaging,magnetic resonance imaging,single-photon-emission computed tomography,and positron emission tomography.In this review,we summarize the development of current state-of-the-art molecular imaging probes.We introduce the design strategies of molecular probes and detailed imaging modalities,and highlight the properties of probes and biomedical imaging applications in cells and in vivo,including disease diagnosis,drug tracking,and imaging-guided surgery.Then we discuss the perspectives and challenges in this emerging field.We expect this review could inspire more effective molecular imaging probes to be developed,achieving the goal towards clinical practices.

Activatable and Self-Monitoring Hydrogen Sulfide-Based Molecular Senomorphics for Visualized Regulation of Cellular Senescence

Specific regulation of the senescence-associated secretory phenotype(SASP)is vital to block senescence-induced detrimental cellular plasticity.Recently,some chemical compounds called senomorphics have demonstrated such potential,but it remains challenging to achieve site-specific activation and real-time monitoring of the action of senomorphics,posing great obstacles for transformable applications.Here,we report a tailor-made hydrogen sulfide(H_(2)S)donor(Lyso-FH_(2)S-Gal)as a new class of molecule senomorphics for spatially controlled delivery of H_(2)S for visualization of regulation of cellular senescence.It comprises four functional moieties in a single molecular structure,including a lysosome-targeting group for cell recognition,a lysosomal enzyme-cleaved scaffold for site-specific activation,thiocarbamate as the H_(2)S precursor,and a switchable fluorophore for concurrent selfreporting of H_(2)S release and senescence imaging.Lyso-FH_(2)S-Gal exhibited remarkable response selectivity,sustained H_(2)S release,and 141-fold fluorescence enhancement.In cellular models,Lyso-FH_(2) S-Gal preferentially enriched in senescent cells over nonsenescent cells,and alleviated the levels of SASP and reactive oxygen species(ROS)in senescent cells,while remaining inert in nonsenescent cells.More impressively,it efficiently inhibited the SASPmediated crosstalk between senescent cells and surrounding nonsenescent cells,thereby preventing senescence propagation.This work offers a useful molecular tool with the hope for controlled intervention of senescence-related important biological processes.

Precise nanomedicine for intelligent therapy of cancer

Precise nanomedicine has been extensively explored for efficient cancer imaging and targeted cancer therapy, as evidenced by a few breakthroughs in their preclinical and clinical explorations. Here, we demonstrate the recent advances of intelligent cancer nanomedicine, and discuss the comprehensive understanding of their structure-function relationship for smart and efficient cancer nanomedicine including various imaging and therapeutic applications, as well as nanotoxicity. In particular, a few emerging strategies that have advanced cancer nanomedicine are also highlighted as the emerging focus such as tumor imprisonment, supramolecular chemotherapy, and DNA nanorobot. The challenge and outlook of some scientific and engineering issues are also discussed in future development. We wish to highlight these new progress of precise nanomedicine with the ultimate goal to inspire more successful explorations of intelligent nanoparticles for future clinical translations.

Recent progress in engineering near-infrared persistent luminescence nanoprobes for time-resolved biosensing/bioimaging

Persistent luminescence nanoprobes (PLNPs) can remain luminescent after ceasing excitation.Due to the ultra-long decay time of persistent luminescence (PersL),autofluorescence interference can be efficiently eliminated by collecting PersL signal after autofluorescence decays completely,thus the imaging contrast and sensing sensitivity can be significantly improved.Since near-infrared (NIR) light shows reduced scattering and absorption coefficient in penetrating biological organs or tissues,near-infrared persistent luminescence nanoprobes (NIR PLNPs) possess deep tissue penetration and offer a bright prospect in the areas of in vivo biosensing/bioimaging.In this review,we firstly summarize the design of different types of NIR PLNPs for biosensing/bioimaging,such as transition metal ions-doped NIR PLNPs,lanthanide ions-doped NIR PLNPs,organic molecules-based NIR PLNPs,and semiconducting polymer self-assembled NIR PLNPs.Notably,organic molecules-based NIR PLNPs and semiconductor self-assembled NIR PLNPs,for the first time,were introduced to the review of PLNPs.Secondly,the effects of different types of charge carriers on NIR PersL and luminescence decay of NIR PLNPs are significantly emphasized so as to build up an in-depth understanding of their luminescence mechanism.It includes the regulation of valence band and conduction band of different host materials,alteration of defect types,depth and concentration changes caused by ion doping,effective radiation transitions and energy transfer generated by different luminescence centers.Given the design and potential of NIR PLNPs as long-lived luminescent materials,the current challenges and future perspective in this rapidly growing field are also discussed.

Bioconjugated Silica Nanoparticles: Development and Applications

Advanced bioanalysis,including accurate quantitation,has driven the need to understand biology and medicine at the molecular level.Bioconjugated silica nanoparticles have the potential to address this emerging challenge.Particularly intriguing diagnostic and therapeutic applications in cancer and infectious disease as well as uses in gene and drug delivery,have also been found for silica nanoparticles.In this review,we describe the synthesis,bioconjugation,and applications of silica nanoparticles in different bioanalysis formats,such as selective tagging,barcoding,and separation of a wide range of biomedically important targets.Overall,we envisage that further development of these nanoparticles will provide a variety of advanced tools for molecular biology,genomics,proteomics and medicine.

Gold nanorod-photosensitizer conjugate with extracellular pH-driven tumor targeting ability for photothermal/photodynamic therapy

Chlorin e6-pHLIPss-AuNRs, a gold nanorod-photosensitizer conjugate containing a pH （low） insertion peptide （pHLIP） with a disulfide bond which imparts extracellular pH （pHe）-driven tumor targeting ability, has been successfully developed for bimodal photodynamic and photothermal therapy. In this bimodal therapy, chlorin e6 （Ce6）, a second-generation photosensitizer （PS）, is used for photodynamic therapy （PDT）. Gold nanorods （AuNRs） are used as a hyperthermia agent for photothermal therapy （PTT） and also as a nanocarrier and quencher of Ce6. pHLIPss is designed as a pile-driven targeting probe to enhance accumulation of Ce6 and AuNRs in cancer cells at low pH. In Ce6- pHLIPss-AuNRs, Ce6 is close to and quenched by AuNRs, causing little PDT effect. When exposed to normal physiological pH 7.4, Ce6-pHLIPs^-AuNRs loosely associate with the cell membrane. However, once exposed to acidic pH 6.2, pHLIP actively inserts into the cell membrane, and the conjugates are translocated into cells. When this occurs, Ce6 separates from the AuNRs as a result of disulfide bond cleavage caused by intracellular glutathione （GSH）, and singlet oxygen is produced for PDT upon light irradiation. In addition, as individual PTT agent, AuNRs can enhance the accumulation of PSs in the tumor by the enhanced permeation and retention （EPR） effect. Therefore, as indicated by our data, when exposed to acidic pH, Ce6-pHLIPss-AuNRs can achieve synergistic PTT/PDT bimodality for cancer treatment.

A novel fluorescent label based on biological fluores-cent nanoparticles and its application in cell recognition

Uniform-sized fluorescent nanoparticles have been prepared by employing silica as the shell and a highly luminescent dye complex of ruthenium ion and bipyridyl, tris(2,2 ’-bipyridyl) dichlororuthenium( II) hexahydrate as the core of the nanoparticles. A novel fluorescent label method is proposed, which is based on the biological fluorescent nanoparticles on the foundation of nanotechnology, biotechnology and fluorescent label technology. In comparison with the conventional fluorophores as fluorescent labels such as fluorescein isothiocyanate (FITC) label, this new label shows more superiority in photochemical stability, detection sensitivity and application scope for the biomedicine research. SmIgG+ B lymphocytes isolated from the circulating blood of human beings can be easily recognized by using this new fluorescent label.