Dual Functional Ultrasensitive Point-of-Care Clinical Diagnosis Using Metal-Organic Frameworks-Based Immunobeads.

Sepsis is an acute systemic infectious syndrome with high fatality. Fast and accurate diagnosis, monitoring, and medication of sepsis are essential. We exploited the fluorescent metal-AIEgen frameworks (MAFs) and demonstrated the dual functions of protein detection and bacteria identification: (i) ultrasensitive point-of-care (POC) detection of sepsis biomarkers (100 times enhanced sensitivity); (ii) rapid POC identification of Gram-negative/positive bacteria (selective aggregation within 20 min). Fluorescent lateral flow immunoassays (LFAs) are convenient and inexpensive for POC tests. MAFs possess a large surface area, excellent photostability, high quantum yield (∼80%), and multiple active sites serving as protein binding domains for ultrasensitive detection of sepsis biomarkers (IL-6/PCT) on LFAs. The limit of detection (LOD) for IL-6/PCT is 0.252/0.333 pg/mL. Rapid appraisal of infectious bacteria is vital to guide the use of medicines. The dual-functional fluorescent MAFs have great potential in POC tests for the clinical diagnosis of bacterial infections.

A Coupling-Induced Assembly Strategy for Constructing Artificial Shell on Mitochondria in Living Cells.

The strategy of in vivo self-assembly has been developed for improved enrichment and long-term retention of anticancer drug in tumor tissues. However, most self-assemblies with non-covalent bonding interactions are susceptible to complex physiological environments, leading to weak stability and loss of biological function. Here, we develop a coupling-induced assembly (CIA) strategy to generate covalently crosslinked nanofibers, which is applied for in situ constructing artificial shell on mitochondria. The oxidation-responsive peptide-porphyrin conjugate P1 is synthesized, which self-assemble into nanoparticles. Under the oxidative microenvironment of mitochondria, the coupling of thiols in P1 causes the formation of dimers, which is further ordered and stacked into crosslinked nanofibers. As a result, the artificial shell is constructed on the mitochondria efficiently through multivalent cooperative interactions due to the increased binding sites. Under ultrasound (US) irradiation, the porphyrin molecules in the shell produce a large amount of reactive oxygen species (ROS) that act on the adjacent mitochondrial membrane, exhibiting ~2-fold higher antitumor activity than nanoparticles in vitro and in vivo. Therefore, the mitochondria-targeted CIA strategy provides a novel perspective on improved sonodynamic therapy (SDT) and shows potential applications in antitumor therapies.

Aminophenol-Decorated Gold Nanoparticles for Curing Bacterial Infections.

Nanomaterials usually kill bacteria via multiple mechanisms which are not explicit to the same degree as those of conventional antibiotics. This situation may hinder the development of novel nanoscale antibiotics. Here, we present aminophenol (AP) to modify gold nanoparticles (AP_Au NPs) which show a broad antibacterial spectrum and potent antibacterial effects against multidrug-resistant (MDR) bacteria with clear antibacterial mechanisms. AP_Au NPs can not only damage bacterial cell walls but also bind to the 16S rRNA to block bacterial protein synthesis. Moreover, AP_Au NPs show excellent performance in curing abdominal bacterial infections in an in vivo model. AP_Au NPs thus have the potential to become a novel antibacterial agent for clinical applications.

Dual Gold Nanoparticle/Chemiluminescent Immunoassay for Sensitive Detection of Multiple Analytes.

Multiple antibiotics and mycotoxins usually simultaneously exist in foods, which poses a serious threat to human health. How to detect them in one test with high sensitivity and fidelity is challenging. In this study, we develop a dual readout lateral flow immunodetection platform that can quantitatively detect five kinds of antibiotics and five kinds of mycotoxins within one sample. The platform is composed of a chip and a portable readout instrument where gold nanoparticle (AuNP)-based and chemiluminescence immunoassays could be performed to reach a maximum throughput of 220 analytes in one setting. For a rapid screen, qualitative analysis by detecting the color change of the deposited AuNPs on the chip could be realized. For quantitative results, chemiluminescence imaging and analysis can be completed within 15 min. Apart from the high throughput and high efficiency, this platform has a high detection sensitivity. For instance, the limit of detection (LOD) for thiamphenicol (a representative antibiotic) and fumonisins B1 (a representative mycotoxin) is 8 times and 40 times lower than those of the previously reported methods, respectively. Thus, this dual readout immunodetection platform is promising as a universal device for rapid and quantitative detection of multiple analytes with high throughput, high sensitivity, and high fidelity.

Oral Administration of Starting Materials for In Vivo Synthesis of Antibacterial Gold Nanoparticles for Curing Remote Infections.

Oral administration is a facile and safe way for medication. However, most of the reported nanomedicines could not be taken orally, partially due to their unsatisfied stability, poor absorbance, or toxicity in the gastrointestinal tract. Here, we demonstrate that we could robustly synthesize gold nanoparticles (GNPs) in vivo by orally administering two starting materials, tetrachloroauric acid and aminophenyl boronic acid (ABA). The ABA-activated GNPs (A-GNPs) synthesized in vivo could be absorbed by the gastrointestinal tract and reach the remote infection lesions such as peritonitis caused by multidrug resistant (MDR) bacteria in mice. The A-GNPs exhibit excellent antibacterial efficacy (MIC, 3 µg/mL), long half-life (16-17 h), effective clearance (residual concentration is near 0 within 72 h), and high biosafety (safe dose/effective dose, 8 times). Our study is a pioneering attempt for synthesizing and taking nanomedicines orally just like preparing and drinking a cocktail.

The Density of Surface Coating Can Contribute to Different Antibacterial Activities of Gold Nanoparticles.

With the widespread use of antibiotics, the number of complex infection cases caused by unknown pathogens is increasing and novel antibiotics with tunable antibacterial spectra and low toxicity are highly desirable. Herein, we report that, by selecting thiol or amine, two groups with different binding affinities with gold, as anchoring groups, phenylboronic acid can be decorated on gold nanoparticles (AuNPs) with different densities, which contributes to Gram-selective antibacterial activities of the AuNPs. The AuNPs modified with amine- or thiol-tethered phenylboronic acids specifically bind to lipopolysaccharide (LPS, Gram-negative) or lipoteichoic acid (LTA, Gram-positive), respectively. By modifying AuNPs with different ratios of thiol- and amine-tethered phenylboronic acids, the resulting AuNPs show potent and tunable antibacterial activity. The AuNP-based antibacterial agents with optional Gram selectivity are promising for applications in personalized therapy.

Integrating a Concentration Gradient Generator and a Single-Cell Trapper Array for High-Throughput Screening the Bioeffects of Nanomaterials.

We herein develop a concentration gradient generator (CGG) on a microfluidic chip for diluting different nanoparticles. Specifically designed compact disk (CD)-shaped microchannels in the CGG module could thoroughly mix the flowing solutions and generate a linear concentration gradient of nanoparticles without aggregation. We combine the CGG with a single-cell trapper array (SCA) on microfluidics to evaluate the concentration-dependent bioeffects of the nanoparticles. The precise control of the spatiotemporal generation of nanoparticle concentration on the CGG module and the single-cell-level monitoring of the cell behaviors on the SCA module by a high-content system in real time, render the CGG-SCA system a highly precise platform, which can exclude the average effect of cell population and reflect the response of individual cells to the gradient concentrations accurately. In addition, the CGG-SCA system provides an automated platform for high-throughput screening of nanomedicines with high precision and low sample consumption.

Activating the Antibacterial Effect of 4,6-Diamino-2-pyrimidinethiol-Modified Gold Nanoparticles by Reducing their Sizes.

Adequately decorated gold nanoparticles (GNPs) have excellent antibiotic activities against multidrug-resistant (MDR) bacteria. Nanoparticles exhibiting Gram selective antibacterial actions are beneficial to precise therapy. Here, we present a strategy to tune the antibacterial spectrum of a small molecule (4,6-diamino-2-pyrimidinethiol, DAPT)-modified GNPs (DAPT-GNPs, DGNPs) by adjusting their sizes. Compared to large (ca. 14 nm diameter) DGNPs (lDGNPs) and medium-sized (3-4 nm diameter) DGNPs (mDGNPs), which have no antibacterial effect or only target Gram-negative (G-) bacteria, ultrasmall DGNPs (uDGNPs, <2 nm) have a broad antibacterial spectrum, especially showing an over 60-fold increase in antibacterial efficacy against Gram-positive (G+) bacteria. Moreover, the uDGNPs-functionalized scaffolds (agarose gel) can serve as general wound dressings for healing burnt infections. Our strategy is insightful for exploring properties of the nanomaterials and their applications.

Construction of Dopamine-Releasing Gold Surfaces Mimicking Presynaptic Membrane by On-Chip Electrochemistry.

We report a strategy to construct a dopamine-releasing gold surface mimicking a presynaptic membrane on a microfluidic chip to simulate in vivo neural signaling. We constructed dopamine self-assembled monolayers (DA SAMs) by electrochemical deprotection of methyl group-protected DA SAMs on a gold surface. Electrochemically controllable release of DA SAMs can be realized by applying nonhydrolytic negative potential on the gold surface. Our method in constructing DA SAMs avoids the polymerization and protonation of DA molecules which may lead to the failure of the DA SAM formation. By combining microfluidics, we realized spatial and temporal controllable release of DA by electrochemistry from the gold surface. Furthermore, by culturing neurons on the patterned DA SAMs, the interface between the DA SAMs and the neurons could serve as a presynaptic membrane, and the spatiotemporal release of DA could modulate the neuron activity with high precision. Our study holds great promise in the fields of neurobiology research and drug screening.

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy.

Precise editing of the genome of a living body is a goal pursued by scientists in many fields. In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome-editing systems have become a revolutionary toolbox for gene editing across various species. However, the low transfection efficiency of the CRISPR/Cas9 system to mammalian cells in vitro and in vivo is a big obstacle hindering wide and deep application. In this review, recently developed delivery strategies for various CRISPR/Cas9 formulations and their applications in treating gene-related diseases are briefly summarized. This review should inspire others to explore more efficient strategies for CRISPR system delivery and gene therapy.

Synthesizing Living Tissues with Microfluidics.

In native tissues, various cell types organize and spatiotemporally function and communicate with neighboring or remote cells in a highly regulated way. How can we replicate these amazing functional structures in vitro? From the view of a chemist, the heterogeneous cells and extracellular matrix (ECM) could be regarded as various chemical substrate materials for "synthetic" reactions during tissue engineering. But how can we accelerate these reactions? Microfluidics provides ideal solutions. Microfluidics could be metaphorically regarded as a miniature "biofactory", whereas the on-chip critical chemical cues such as biomolecule gradients and physical cues such as geometrical confinement, topological guidance, and mechanical stimulations, along with the external stimulations such as light, electricity, acoustics, and magnetics, could be regarded as "catalytic cues" which can accelerate the "synthetic reactions" by precisely and effectively manipulating a series of cell behaviors including cell adhesion, migration, growth, proliferation, differentiation, cell-cell interaction, and cell-matrix interaction to reduce activation energy of the "synthetic reactions". Thus, on the microfluidics platform, the "biofactory", various "synthetic" reactions take place to change the substrate materials (cells and ECM) into products (tissues) in a nonlinear way, which is a typical feature of a biological process. By precisely organizing the substrate materials and spatiotemporally controlling the activity of the products, as a "biofactory", the microfluidics system can not only "synthesize" living tissues but also recreate physiological or pathophysiological processes such as immune responses, angiogenesis, wound healing, and tumor metastasis in vitro to bring insights into the mechanisms underlying these processes taking place in vivo. In this Account, we borrow the concept of chemical "synthesis" to describe how to "synthesize" artificial tissues using microfluidics from a chemist's view. Accelerated by the built-in physiochemical cues on microfluidics and external stimulations, various tissues could be "synthesized" on a microfluidics platform. We summarize that there are "step-by-step synthesis" and "one-step synthesis" on microfluidics for creating desired tissues with unprecedented precision, accuracy, and speed. In recent years, researchers developed various microfluidic techniques including creating adhesive domains for mediating reverse and precise adhesion, chemical gradients for directing cell growth, geometrical confinements and topological cues for manipulating cell migration, and mechanics for stimulating cell differentiation. By employing and orchestrating these on-chip tissue "synthetic" conditions, "step-by-step synthesis" could be realized on chips to develop multilayered tissues such as blood vessels. "One-step synthesis" on chips could develop functional three-dimensional tissue structures such as neural networks or nephron-like structures. Based on these on-chip studies, many critical physiological and pathophysiological processes such as wound healing, tumor metastasis, and atherosclerosis could be deeply investigated, and the drugs or therapeutic approaches could also be evaluated or screened conveniently. The "synthetic tissues on microfluidics" system would pave an avenue for precise creation of artificial tissues for not only fundamental research but also biomedical applications such as tissue engineering.

Triple-Targeting Delivery of CRISPR/Cas9 To Reduce the Risk of Cardiovascular Diseases.

A high level of low-density lipoprotein cholesterol (LDL-C) in the blood is a major risk factor for coronary heart disease. Herein, we present a triple-targeting strategy to generate a loss-of-function mutation in Pcsk9, which regulates plasma cholesterol levels, using a nanocarrier-delivered CRISPR/Cas9 system. Nuclear localization signal (NLS)-tagged Cas9 and Pcsk9-targeted single guide RNA (sgPcsk9) were complexed with gold nanoclusters (GNCs) modified with cationic HIV-1-transactivating transcriptor (TAT) peptide and further encapsulated in a galactose-modified lipid layer to target the nanoclusters to the liver. The resulting nanoclusters had an in vitro Pcsk9-editing efficiency of about 60 % and resulting in a decrease in plasma LDL-C in mice of approximately 30%. No off-target mutagenesis was detected in 10 sites with high similarity. This approach may have therapeutic potential for the prevention and treatment of cardiovascular disease without side effects.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy.

CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.

Composites of Bacterial Cellulose and Small Molecule-Decorated Gold Nanoparticles for Treating Gram-Negative Bacteria-Infected Wounds.

Bacterial infections, especially multidrug-resistant bacterial infections, are an increasingly serious problem in the field of wound healing. Herein, bacterial cellulose (BC) decorated by 4,6-diamino-2-pyrimidinethiol (DAPT)-modified gold nanoparticles (Au-DAPT NPs) is presented as a dressing (BC-Au-DAPT nanocomposites) for treating bacterially infected wounds. BC-Au-DAPT nanocomposites have better efficacy (measured in terms of reduced minimum inhibition concentration) than most of the antibiotics (cefazolin/sulfamethoxazole) against Gram-negative bacteria, while maintaining excellent physicochemical properties including water uptake capability, mechanical strain, and biocompatibility. On Escherichia coli- or Pseudomonas aeruginosa-infected full-thickness skin wounds on rats, the BC-Au-DAPT nanocomposites inhibit bacterial growth and promote wound repair. Thus, the BC-Au-DAPT nanocomposite system is a promising platform for treating superbug-infected wounds.

Point-of-Care Detection of ß-Lactamase in Milk with a Universal Fluorogenic Probe.

The illegal addition of ß-lactamase (Bla) in milk to disguise ß-lactam antibiotics has been a serious issue in the milk industry worldwide. Herein, we report a method for point-of-care detection of Bla based on a probe, Tokyo Green-tethered ß-lactam (CDG-1), as a common substrate of various Blas (Bla A, B...) which can enzymatically convert CDG-1 (low fluorescence) to Tokyo Green (high fluorescence). This approach allows rapid screening of a broad spectrum of Blas in real milk samples within 15 min without any pretreatment. Combined with the immuno-magnetic separation, we achieved sensitive and quantitative detection of Bla (10(-5) U/mL), which provides a universal platform for screening and determining Blas in complex samples with high efficiency and accuracy.

An Early-Stage Atherosclerosis Research Model Based on Microfluidics.

The arterial microenvironment plays a vital role in the pathology of atherosclerosis (AS). However, the interplay between the arterial microenvironment and atherogenesis remains unclear, partially due to the gap between cell culture and animal experiments. Addressing this problem, the present study reports a microfluidic AS model reconstituting early-stage AS. Physiological or AS-prone hemodynamic conditions are recapitulated on the model. The on-chip model recaptures the atherogenic responses of endothelial cells (ECs) in ways that the Petri dish could not. Significant cytotoxicity of a clinical anti-atherosclerotic drug probucol is discovered on the model, which does not appear on Petri dish but is supported by previous clinical evidence. Moreover, the anti-AS efficiency of platinum-nanoparticles (Pt-NPs) on the model shows excellent consistency with animal experiments. The early-stage AS model shows an excellent connection between Petri dish and animal experiments and highlights its promising role in bridging fundamental AS research, drug screening, and clinical trials.

Evaluation of the effect of the structure of bacterial cellulose on full thickness skin wound repair on a microfluidic chip.

Bacterial cellulose (BC) is a kind of nanobiomaterial for tissue engineering. How the nanoscale structure of BC affects skin wound repair is unexplored. Here, the hierarchical structure of BC films and their different effects on skin wound healing were studied both in vitro and in vivo. The bottom side of the BC film had a larger pore size, and a looser and rougher structure than that of the top side. By using a microfluidics-based in vitro wound healing model, we revealed that the bottom side of the BC film can better promote the migration of cells to facilitate wound healing. Furthermore, the full-thickness skin wounds on Wistar rats demonstrated that, compared with gauze and the top side of the BC film, the wound covered by the bottom side of the BC film showed faster recovery rate and less inflammatory response. The results indicate that the platform based on the microfluidic chip provide a rapid, reliable, and repeatable method for wound dressing screening. As an excellent biomaterial for wound healing, the BC film displays different properties on different sides, which not only provides a method to optimize the biocompatibility of wound dressings but also paves a new way to building heterogeneous BC-based biomaterials for complex tissue engineering.

A peptide-based nanofibrous hydrogel as a promising DNA nanovector for optimizing the efficacy of HIV vaccine.

This report shows that a nanovector composed of peptide-based nanofibrous hydrogel can condense DNA to result in strong immune responses against HIV. This nanovector can strongly activate both humoral and cellular immune responses to a balanced level rarely reported in previous studies, which is crucial for HIV prevention and therapy. In addition, this nanovector shows good biosafety in vitro and in vivo. Detailed characterizations show that the nanofibrous structure of the hydrogel is critical for the dramatically improved immune responses compared to existing materials. This peptide-based nanofibrous hydrogel shows great potential for efficacious HIV DNA vaccines and can be potentially used for delivering other vaccines and drugs.

Gold nanoclusters cure implant infections by targeting biofilm.

The biofilm-mediated implant infections pose a huge threat to human health. It is urgent to explore strategies to reverse this situation. Herein, we design 3-amino-1,2,4-triazole-5-thiol (ATT)-modified gold nanoclusters (AGNCs) to realize biofilm-targeting and near-infrared (NIR)-II light-responsive antibiofilm therapy. The AGNCs can interact with the bacterial extracellular DNA through the formation of hydrogen bonds between the amine groups on the ATT and the hydroxyl groups on the DNA. The AGNCs show photothermal properties even at a low power density (0.5 W/cm2) for a short-time (5 min) irradiation, making them highly effective in eradicating the biofilm with a dispersion rate up to 90 %. In vivo infected catheter implantation model demonstrates an exceptional high ability of the AGNCs to eradicate approximately 90 % of the bacteria encased within the biofilms. Moreover, the AGNCs show no detectable toxicity or systemic effects in mice. Our study suggests the great potential of the AGNCs for long-term prevention and elimination of the biofilm-mediated infections.