Visually evaluating drug efficacy in living cells using COF-based fluorescent nanoprobe via CHA amplified detection of miRNA and simultaneous apoptosis imaging.

BACKGROUNDS: Cancer is a highly fatal disease which is close relative of miRNA aberrant expression and apoptosis disorders. Elucidation of the therapeutic efficacy through investigating the changes in miRNA and apoptosis holds immense importance in advancing the development of miRNA-based precision therapy. However, it remains a challenge as how to visually evaluate the efficacy during protocol optimization of miRNA-based anticancer drugs at the cellular level. Therefore, exploring effective and noninvasive methods for real-time monitoring of therapeutic efficacy in living cells is of great significance. RESULTS: Herein, we reported a novel fluorescent nanoprobe COF-H1/H2-Peptide for visually evaluating drug efficacy in living cells through amplified imaging of low-abundant miRNA-221 with catalytic hairpin assembly (CHA) circle amplification, as well as simultaneous caspase-3 imaging. With strong stability and good biocompatibility, this newly fabricated amplified nanoprobe showed high sensitivity and specificity for the detection of miRNA-221 and caspase-3, and the limit of detection (LOD) of miRNA-221 was as low as 2.79 pM. The fluorescent imaging results showed that this amplified nanoprobe could not only detect caspase-3 in living cells, but also effectively detect low levels of miRNA-221 with increasing anticancer drug concentration and treatment time. The smart nanoprobe had effective performance for optimizing miRNA-based drug treatment schedules by dual-color fluorescence imaging. SIGNIFICANCE: This nanoprobe combined CHA amplified detection of intracellular miRNA-221 and synchronous apoptosis imaging, with excellent sensitivity for the detection of cellular low-level miRNA, enabling the realization of real-time assessment of the efficacy of miRNA-based therapy in living cells. This work presents a promising approach for revealing the regulatory mechanisms between miRNAs and apoptosis in cancer occurrence, development, and treatment.

A novel nanoprobe for visually investigating the controversial role of miRNA-34a as an oncogene or tumor suppressor in cancer cells.

MiRNA-targeted therapy has become a hot topic in current cancer research. The key to this treatment strategy is to clarify the specific role of miRNA in cancer. However, the roles of some miRNAs acting as oncogenic or tumor suppressors are still controversial, which are influenced by different tumor types, even in the same cancer type. Hence, we designed a novel fluorescent nanoprobe based on polydopamine nanoparticles (PDA NPs) for simultaneously detecting caspase-3 and miRNA-34a within living cells. The specific role of miRNA-34a in different cancer cells could be further identified by studying the expression alterations of caspase-3 and miRNA-34a. Confocal imaging indicated that miRNA-34a indeed acted as a tumor suppressor in anticancer drug-treated MCF-7 and HeLa cells, where the effect of miRNA-34a remains controversial. The designed nanoprobe can offer a promising approach to ascertain the oncogenic or tumor-suppressing role of miRNA in different cancer cells with a simple visualization method, which has valuable implications for exploring the practicability of precision therapy focused on miRNA and evaluating the efficacy of new miRNA-targeted anticancer medications.

Preparation of Gelatin-Quaternary Ammonium Salt Coating on Titanium Surface for Antibacterial/Osteogenic Properties.

Titanium (Ti) and its alloys are widely used in medical treatment, engineering, and other fields because of their excellent properties including biological activity, an elastic modulus similar to that of human bones, and corrosion resistance. However, there are still many defects in the surface properties of Ti in practical applications. For example, the biocompatibility of Ti with bone tissue can be greatly reduced in implants due to a lack of osseointegration as well as antibacterial properties, which may lead to osseointegration failure. To address these problems and to take advantage of the amphoteric polyelectrolyte properties of gelatin, a thin layer of gelatin was prepared by electrostatic self-assembly technology. Diepoxide quaternary ammonium salt (DEQAS) and maleopimaric acid quaternary ammonium salt (MPA-N+) were then synthesized and grafted onto the thin layer. The cell adhesion and migration experiments demonstrated that the coating has excellent biocompatibility, and those grafted with MPA-N+ promoted cell migration. The bacteriostatic experiment showed that the mixed grafting with two ammonium salts had excellent bacteriostatic performance against Escherichia coli and Staphylococcus aureus, with bacteriostasis rates of 98.1 ± 1.0% and 99.2 ± 0.5%, respectively.

Redox-Enhanced Photoelectrochemical Activity in PHV/CdS Hybrid Film.

Semiconductive photocatalytic materials have received increasing attention recently due to their ability to transform solar energy into chemical fuels and photodegrade a wide range of pollutants. Among them, cadmium sulfide (CdS) nanoparticles have been extensively studied as semiconductive photocatalysts in previous studies on hydrogen generation and environmental purification due to their suitable bandgap and sensitive light response. However, the practical applications of CdS are limited by its low charge separation, which is caused by its weak ability to separate photo-generated electron-hole pairs. In order to enhance the photoelectrochemical activity of CdS, a polymer based on viologen (PHV) was utilized to create a series of PHV/CdS hybrid films so that the viologen unit could work as the electron acceptor to increase the charge separation. In this work, various electrochemical, spectroscopic, and microscopic methods were utilized to analyze the hybrid films, and the results indicated that introducing PHV can significantly improve the performance of CdS. The photoelectrochemical activities of the hybrid films were also evaluated at various ratios, and it was discovered that a PHV-to-CdS ratio of 2:1 was the ideal ratio for the hybrid films. In comparison with CdS nanoparticles, the PHV/CdS hybrid film has a relatively lower band gap, and it can inhibit the recombination of electrons and holes, enhancing its photoelectrochemical activities. All of these merits make the PHV/CdS hybrid film as a strong candidate for photocatalysis applications in the future.

Influence of a Composite Polylysine-Polydopamine-Quaternary Ammonium Salt Coating on Titanium on Its Ostogenic and Antibacterial Performance.

Thin oxide layers form easily on the surfaces of titanium (Ti) components, with thicknesses of <100 nm. These layers have excellent corrosion resistance and good biocompatibility. Ti is susceptible to bacterial development on its surface when used as an implant material, which reduces the biocompatibility between the implant and the bone tissue, resulting in reduced osseointegration. In the present study, Ti specimens were surface-negatively ionized using a hot alkali activation method, after which polylysine and polydopamine layers were deposited on them using a layer-by-layer self-assembly method, then a quaternary ammonium salt (QAS) (EPTAC, DEQAS, MPA-N+) was grafted onto the surface of the coating. In all, 17 such composite coatings were prepared. Against Escherichia coli and Staphylococcus aureus, the bacteriostatic rates of the coated specimens were 97.6 ± 2.0% and 98.4 ± 1.0%, respectively. Thus, this composite coating has the potential to increase the osseointegration and antibacterial performance of implantable Ti devices.

Simultaneous Visualization of MiRNA-221 and Caspase-3 in Cancer Cells for Investigating the Feasibility of MiRNA-Targeted Therapy with a Dual-Color Fluorescent Nanosensor.

MiRNA-targeted therapy holds great promise for precision cancer therapy. It is important to investigate the effect of changes in miRNA expression on apoptosis in order to evaluate miRNA-targeted therapy and achieve personalized therapy. In this study, we designed a dual-color fluorescent nanosensor consisting of grapheme oxide modified with a molecular beacon and peptide. The nanosensor can simultaneously detect and image miRNA-221 and apoptotic protein caspase-3 in living cells. Intracellular experiments showed that the nanosensor could be successfully applied for in situ monitoring of the effect of miRNA-221 expression changes on apoptosis by dual-color imaging. The current strategy could provide new avenues for investigating the feasibility of miRNA-targeted therapy, screening new anti-cancer drugs targeting miRNA and developing personalized treatment plans.

Visualizing MiRNA Regulation of Apoptosis for Investigating the Feasibility of MiRNA-Targeted Therapy Using a Fluorescent Nanoprobe.

MiRNA-targeted therapy is an active research field in precision cancer therapy. Studying the effect of miRNA expression changes on apoptosis is important for evaluating miRNA-targeted therapy and realizing personalized precision therapy for cancer patients. Here, a new fluorescent nanoprobe was designed for the simultaneous imaging of miRNA-21 and apoptotic protein caspase-3 in cancer cells by using gold nanoparticles as the core and polydopamine as the shell. Confocal imaging indicated that the nanoprobe could be successfully applied for in situ monitoring of miRNA regulation of apoptosis. This design strategy is critical for investigating the feasibility of miRNA-targeted therapy, screening new anti-cancer drugs targeting miRNA, and developing personalized treatment plans.

A fluorescence nanoprobe for detecting the effect of different oxygen and nutrient conditions on breast cancer cells' migration and invasion.

Cancer cell migration and invasion are initial steps for tumor metastasis, which increases patient mortality. The tumor microenvironment is characterized as being hypoxic and nutrient deficient. Previous studies have suggested that hypoxia induces tumor metastasis, while the low nutrient content is not beneficial for tumor metastasis. Thus, it is necessary to develop a simple probe to study the changes in cancer migration and invasion. Herein, we designed a nanoprobe based on gold nanoparticles (Au NPs) to monitor the effect of different oxygen and nutrient conditions on the migration and invasion of breast cancer cells through detecting the changes in levels of RAB-22a and MMP-2 mRNA in living cells. After incubating MCF-7 and MDA-MB-231 cells with different concentrations of oxygen and nutrients, fluorescence imaging assays were used to evaluate migration and invasion. This work provides a new insight in the effect of different tumor microenvironments on cell migration and invasion.

Avoiding False Positive Signals: A Powerful and Reliable Au-Se Dual-Color Probe.

Nucleic acids as the important tumor markers play a crucial role in the identification of cancer. Various kinds of probes such as gold nanoparticles and graphene oxide have been explored to detect different nucleic acid markers. However, the existing probes are mostly used to detect a single tumor marker and susceptible to harsh conditions in the complex and dynamic physiological environment, which may lead to false positive results and greatly limit the sensing performance of the probe. Herein, a powerful and reliable Au-Se probe was developed for high-fidelity imaging of two cancer markers simultaneously in living cells. Compared with the traditional nucleic acid probe based on the Au-S bond, this probe was more stable against biological thiols and could effectively distinguish normal cells and cancer cells to avoid false positive results, which is more suitable for imaging in a complex physiological environment. This strategy will provide more valuable insights into designing and exploring novel biosensors in the future.

Real-time in situ monitoring of signal molecules' evolution in apoptotic pathway via Au-Se bond constructed nanoprobe.

In cell signal transduction pathways, a series of biochemical reactions and interactions between proteins guarantee physiological responses indicating cell functionality. However, there are a variety of upstream and downstream signal molecules in these pathways with multiple levels of cross-regulation, making it difficult to sequential visualize their relationships in living organisms in complex environments. To investigate the interrelationships among intracellular signaling pathways, a Au-Se bonded nanoprobe with extraordinary stability and strong anti-interference ability was designed and prepared to monitor the evolution of two kinds of apoptosis biomarkers in real time. Two different peptide chains decorated with two dyes were functionalized on the surface of Au nanoparticles (NPs) via Au-Se bonds. These peptide chains can be respectively cleaved by upstream cathepsin B proteins and downstream caspase-3 proteins to trigger fluorescence recovery. Moreover, when the living cells were stimulated to induce the apoptotic pathway, cathepsin B and caspase-3 were activated in turn with signals sequentially recovered at 2 and 4 h, respectively. This fluorescent nanoprobe can be used in complicated biological systems to achieve real-time in situ monitoring of the sequential activation of signal molecules in intracellular pathways and provides a novel approach for the future investigation of protein interactions in vivo.

A gold-selenium-bonded nanoprobe for real-time in situ imaging of the upstream and downstream relationship between uPA and MMP-9 in cancer cells.

A novel Au-Se nanoprobe with remarkable anti-interference ability for glutathione was developed for real-time in situ monitoring of the upstream and downstream regulatory relationship between uPA and MMP-9 proteins in the pathway.

Simultaneous Fluorescence Visualization of Epithelial-Mesenchymal Transition and Apoptosis Processes in Tumor Cells for Evaluating the Impact of Epithelial-Mesenchymal Transition on Drug Efficacy.

The epithelial-mesenchymal transition (EMT) process plays a pivotal role in acquiring invasive and metastatic properties and has been recognized as a crucial driver of epithelial-derived tumor malignancies. It is necessary to determine the role of EMT in promoting or suppressing carcinoma progression through investigating the relationship between EMT and apoptosis. We designed a multicolor fluorescent nanoprobe for simultaneously imaging the epithelial biomarker E-cadherin mRNA, the mesenchymal marker vimentin mRNA, and the apoptotic marker caspase-3. EMT and apoptosis progresses could be visually detected, which were used to study the effect of EMT on apoptosis and further assess the influence of EMT on drug efficacy in different cancer cells. We believe the designed nanoprobe can offer a new strategy for visualizing EMT and apoptosis in tumor cells and will be a promising tool to investigate the efficiency of drugs targeting EMT-related therapies in living cells.

Visualizing Breast Cancer Cell Proliferation and Invasion for Assessing Drug Efficacy with a Fluorescent Nanoprobe.

Hard-to-treat cancers are closely relative to uncontrolled cell proliferation, invasion, and metastasis. Assessing proliferation and invasion properties of tumor cells both in vitro and in vivo is especially important for acquiring reliable information for cancer pathogenesis, drug screening, and therapeutic effect evaluation. Herein, we developed a multicolor fluorescent nanoprobe for simultaneously monitoring breast cancer cells' proliferation marker Ki-67 and invasion marker urokinase plasminogen activator (uPA). After treated with the anticancer drugs tamoxifen and curcumin, the changes in cancer cell proliferation and invasion properties were visually detected and therapeutic effects of corresponding drugs were further assessed in vitro and in vivo. The design of the fluorescent nanoprobe opens up an avenue for investigating unscheduled proliferation, invasion, and metastasis in living cells and in vivo and as such will be a promising tool to screen antitumor drugs and evaluate drug efficiency in an extremely efficient manner.

Simultaneous detection of multiple targets involved in the PI3K/AKT pathway for investigating cellular migration and invasion with a multicolor fluorescent nanoprobe.

We develop a multicolor fluorescent nanoprobe for assessing cellular migration and invasion by simultaneously imaging miRNA-221, PTEN mRNA and MMP-9 involved in the PI3K/AKT pathway which can regulate cellular mobility and invasiveness.

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads.

Silica gel bead coated with macroporous chitosan layer (CTS-SiO(2)) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu(2+), Zn(2+), Ni(2+) ions, respectively on CTS-SiO(2), and trypsin could be adsorbed on the IMAC adsorbent through metal-protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu(2+)-CTS-SiO(2) with the value of 4980+/-125 IUg(-1) of the adsorbent, while the maximal adsorption capacity for trypsin on Zn(2+) and Ni(2+) loaded adsorbent was 3762+/-68 IUg(-1) and 2636+/-53 IUg(-1), respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5-10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO(2) beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 degrees C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO(2) could effectively digest BSA revealed by HPLC peptide mapping.