All Roads Lead to Rome: Comparing Nanoparticle- and Small Molecule-Driven Cell Autophagy.

Autophagy, vital for removing cellular waste, is triggered differently by small molecules and nanoparticles. Small molecules, like rapamycin, non-selectively activate autophagy by inhibiting the mTOR pathway, which is essential for cell regulation. This can clear damaged components but may cause cytotoxicity with prolonged use. Nanoparticles, however, induce autophagy, often causing oxidative stress, through broader cellular interactions and can lead to a targeted form known as "xenophagy." Their impact varies with their properties but can be harnessed therapeutically. In this review, the autophagy induced by nanoparticles is explored and small molecules across four dimensions: the mechanisms behind autophagy induction, the outcomes of such induction, the toxicological effects on cellular autophagy, and the therapeutic potential of employing autophagy triggered by nanoparticles or small molecules. Although small molecules and nanoparticles each induce autophagy through different pathways and lead to diverse effects, both represent invaluable tools in cell biology, nanomedicine, and drug discovery, offering unique insights and therapeutic opportunities.

Split G-quadruplex based PfAgo sensing platform for nucleotide mutation discrimination and human genotyping.

A PfAgo-G4 sensing platform exploiting G4 as a signal reporter was proposed, validated, and optimized. By introducing two mismatches at the Link strand, a universal nucleotide design rule was established for accurate single nucleotide polymorphism discrimination with PfAgo-G4. The FUT2 gene was then successfully and accurately genotyped using human buccal swab samples.

CRISPR-Cas12a-Based Aptasensor for On-Site and Highly Sensitive Detection of Microcystin-LR in Freshwater.

On-site monitoring of trace organic pollutants with facile methods is critical to environmental pollutant prevention and control. Herein, we proposed a CRISPR-Cas12a-based aptasensor platform (named as MC-LR-Casor) for on-site and sensitive detection of microcystin-LR (MC-LR). After hybridization with blocker DNA, the MC-LR aptamers were conjugated to magnetic beads (MBs) to get the MB aptasensor. In the presence of MC-LR, their interactions with aptamers were triggered and the specific binding caused the release of blocker DNA. Using the programmability of the CRISPR-Cas system, the released blocker DNA was designed to activate a Cas12a-crRNA complex. Single strand DNA reporters were rapidly cleaved by the complex. Signal readout could be achieved by fluorometer or lateral flow strips, which were positively correlated to MC-LR concentration. Benefiting from the CRISPR-Cas12a amplification system, the proposed sensing platform exhibited high sensitivity and reached the limit of detection of ∼3 × 10-6 µg/L (fluorescence method) or 1 × 10-3 µg/L (lateral flow assay). In addition, the MC-LR-Casor showed excellent selectivity and good recovery rates, demonstrating their good applicability for real water sample analysis. During the whole assay, only two steps of incubation at a constant temperature were required and the results could be visualized when employing flow strips. Therefore, the proposed assay offered a simple and convenient alternative for in situ MC-LR monitoring, which may hold great promise for future environmental surveillance.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection.

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 µL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.

Pretreatment with metformin prevents microcystin-LR-induced tau hyperphosphorylation via mTOR-dependent PP2A and GSK-3ß activation.

Microcystin-leucine-arginine (MC-LR) is a toxin secreted by freshwater cyanobacteria that is considered a potential environmental risk factor for Alzheimer's disease (AD). A previous study indicated that tau protein hyperphosphorylation via protein phosphatase 2A (PP2A) and GSK-3ß inhibition was the mechanism by which MC-LR induces neurotoxicity; however, how MC-LR-induced neurotoxicity can be effectively prevented remains unclear. In this study, the reversal effect of metformin on MC-LR-induced neurotoxicity was investigated. The results showed that metformin effectively prevented tau hyperphosphorylation at Ser202 caused by MC-LR through PP2A and GSK-3b activity. The effect of metformin on PP2A activity was dependent on the inhibition of mTOR in MC-LR-treated SH-SY5Y cells. Metformin prevented spatial memory deficits in rats caused by intrahippocampal MC-LR administration. In sum, the results suggested that metformin can ameliorate the MC-LR-induced AD-like phenotype by preventing tau phosphorylation at Ser202, which was mainly mediated by mTOR-dependent PP2A and GSK-3ß activation.

Cytotoxicity Induction by the Oxidative Reactivity of Nanoparticles Revealed by a Combinatorial GNP Library with Diverse Redox Properties.

It is crucial to establish relationship between nanoparticle structures (or properties) and nanotoxicity. Previous investigations have shown that a nanoparticle's size, shape, surface and core materials all impact its toxicity. However, the relationship between the redox property of nanoparticles and their toxicity has not been established when all other nanoparticle properties are identical. Here, by synthesizing an 80-membered combinatorial gold nanoparticle (GNP) library with diverse redox properties, we systematically explored this causal relationship. The compelling results revealed that the oxidative reactivity of GNPs, rather than their other physicochemical properties, directly caused cytotoxicity via induction of cellular oxidative stress. Our results show that the redox diversity of nanoparticles is regulated by GNPs modified with redox reactive ligands.

Crossing Biological Barriers by Engineered Nanoparticles.

Engineered nanoparticles (ENPs) may cause toxicity if they cross various biological barriers and are accumulated in vital organs. Which factors affect barrier crossing efficiency of ENPs are crucial to understand. Here, we present strong data showing that various nanoparticles crossed biological barriers to enter vital animal organs and cause toxicity. We also point out that physicochemical properties of ENPs, modifications of ENPs in biofluid, and physiological and pathological conditions of the body all affect barrier crossing efficiency. We also summarized our limited understanding of the related mechanisms. On the basis of this summary, major research gaps and direction of further efforts are then discussed.

Facile Construction of i-Motif DNA-Conjugated Gold Nanostars as Near-Infrared and pH Dual-Responsive Targeted Drug Delivery Systems for Combined Cancer Therapy.

Stimuli-responsive DNA-based nanostructures have emerged as promising vehicles for intelligent drug delivery. In this study, i-motif DNA-conjugated gold nanostars (GNSs) were fabricated in a facile manner as stimuli-responsive drug delivery systems (denoted as A-GNS/DNA/DOX) for the treatment of cancer via combined chemo-photothermal therapy. The i-motif DNA is sensitive to the environmental pH and can switch from a single-stranded structure to a C-tetrad (i-motif) structure as the environmental pH decreases from neutral (∼7.4) to acidic (<6.0). The loaded drug can then be released along with the conformational changes. To enhance cellular uptake and improve cancer cell selectivity, the aptamer AS1411, which recognizes nucleolins, was employed as a targeting moiety. The A-GNS/DNA/DOX nanocomposites were found to be highly capable of photothermal conversion and exhibited photostability under near-infrared (NIR) irradiation, and the pH and NIR irradiation effectively triggered the drug-release behaviors. In addition, the A-GNS/DNA/DOX nanocomposites exhibited good biocompatibility. The targeting recognition enabled the A-GNS/DNA/DOX to exhibit higher cellular uptake and therapeutic efficiency than the GNS/DNA/DOX. Notably, under NIR irradiation, a synergistic effect between chemotherapy and photothermal therapy can be achieved with the proposed delivery system, which exhibits much higher therapeutic efficiency both in monolayer cancer cells and tumor spheroids as compared with a single therapeutic method. This study highlights the potential of GNS/DNA nanoassemblies for intelligent anticancer drug delivery and combined cancer therapy.

Rattle-Type Gold Nanorods/Porous-SiO2 Nanocomposites as Near-Infrared Light-Activated Drug Delivery Systems for Cancer Combined Chemo-Photothermal Therapy.

Rattle-type nanostructures with movable cores, porous shells, and hollow interiors have become attractive nanoplatforms in the field of nanomedicine, especially for targeted and stimuli-responsive drug delivery. In this work, rattle-type gold nanorods@void@porous-SiO2 (GVPS) nanocomposites were fabricated facilely using the surface-protecting etching method and exhibited high photothermal conversion efficiency. Taking advantage of the porous shell and hollow interior, the nanocomposites have abundant space for drug loading and successfully improved the drug loading capacity up to ∼19.6%. To construct a multifunctional drug delivery system, GVPS was further functionalized with polyethylene glycol (PEG) and cyclic RGD peptides to improve biocompatibility as well as selectivity toward the targeted cancer cells. Besides, to achieve precise regulation and near-infrared laser activation of the drug release, a phase-changing material, 1-tetradecanol (1-TD, Tm: 39 °C), was employed as gatekeepers in this system. After incubation with an αVß3 integrin receptor-overexpressed cell line, the as-prepared GVPSPR-DOX/TD nanocomposites exhibited great performances in combined photothermal therapy and chemotherapy. It is worth noting that the combined therapy showed superior efficiency in cancer cell killing to chemotherapy or photothermal therapy alone.

Engineering of Porous Silica Coated Gold Nanorods by Surface-Protected Etching and Their Applications in Drug Loading and Combined Cancer Therapy.

Core-shell nanostructures, specifically gold nanorods coated with porous silica (GNR@p-SiO2), were successfully fabricated by surface-protected etching. The nanostructures, photothermal effects, drug loading and drug release behaviors, cellular uptake, and combined chemo-photothermal therapy were investigated. The results showed that the as-prepared GNR@p-SiO2 had a uniform porous silica outer layer. Etching process could be modulated by adjusting the etching time, concentrations of etching agents, and concentrations of protective agents. With doxorubicin (DOX) as the model drug, the drug loading capacity reached 18.9%, which was dependent on the DOX concentrations. The drug release profiles were dual stimulus-responsive to pH and laser irradiation. In addition, the GNR@p-SiO2 nanoparticles were biocompatible and effectively internalized by cancer cells. Compared with chemotherapy or photothermal therapy administered individually, combined chemo-photothermal therapy using GNR@p-SiO2 exhibited higher efficiency in killing cancer cells both in vitro and in vivo. Therefore, surface-protected etching is a powerful method for preparing core-shell nanostructures capped with mesoporous silica for combined cancer chemo-photothermal therapy.

Effects of Protein Corona on Active and Passive Targeting of Cyclic RGD Peptide-Functionalized PEGylation Nanoparticles.

Protein corona can alter the physiochemical properties of targeting nanoparticles (NPs), as well as their physiological responses and targeting functionality. Herein, we synthesized 20 types of NPs with diverse surface chemistry in order to study the impacts of protein corona on targeting functionality of NPs functionalized with cyclic RGD peptides and their relationships to the polyethylene glycol (PEG) length and grafting density of targeting ligands. After protein adsorption, cyclic RGD on the surface of NP was still able to bind its receptors with increased targeted cellular uptake, even at a relatively low density. However, the cellular uptake was reduced from 26 to 76% when compared with protein nonbound NPs, which was caused by the shielding effect of the outer layer adsorbed proteins. NPs functionalized with short PEG molecules and moderate cyclic RGD density performed a better targeting efficiency. Due to PEG conjugation, the protein corona was demonstrated to be beneficial for passive targeting by decreasing macrophage cellular uptake. These relationships between surface chemistry and targeting functionality will provide guidelines for the design of targeting nanoformulations in nanomedicine.

Ultrafine particle libraries for exploring mechanisms of PM2.5-induced toxicity in human cells.

Air pollution worldwide, especially in China and India, has caused serious health issues. Because PM2.5 particles consist of solid particles of diverse properties with payloads of inorganic, organic and biological pollutants, it is still not known what the major toxic components are and how these components induce toxicities. To explore this complex issue, we apply reductionism principle and an ultrafine particle library approach in this work. From investigation of 63 diversely functionalized ultrafine particles (FUPs) with adsorbed key pollutants, our findings indicate that 1) only certain pollutants in the payloads of PM2.5 are responsible for causing cellular oxidative stress, cell apoptosis, and cytotoxicity while the particle carriers are much less toxic; 2) pollutant-induced cellular oxidative stress and oxidative stress-triggered apoptosis are identified as one of the dominant mechanisms for PM2.5-induced cytotoxicity; 3) each specific toxic component on PM2.5 (such as As, Pb, Cr or BaP) mainly affects its specific target organ(s) and, adding together, these pollutants may cause synergistic or just additive effects. Our findings demonstrate that reductionism concept and model PM2.5 particle library approach are very effective in our endeavor to search for a better understanding of PM2.5-induced health effects.

Elucidation of the Molecular Determinants for Optimal Perfluorooctanesulfonate Adsorption Using a Combinatorial Nanoparticle Library Approach.

Perfluorooctanesulfonate (PFOS) persistently accumulates in the environment and in humans, causing various toxicities. To determine the key molecular determinants for optimal PFOS specificity and efficiency, we designed and synthesized a combinatorial gold nanoparticle (GNP) library consisting of 18 members with rationally diversified hydrophobic, electrostatic, and fluorine-fluorine interaction components for PFOS bindings. According to our findings, the electrostatic and F-F interactions between PFOS and nanoparticles are complementary. When F-F attractions are relatively weak, the electrostatic interactions are dominant. As F-F interactions increase, the electrostatic contributions are reduced to as low as 20%, demonstrating that F-F binding may overpower even electrostatic interactions. Furthermore, F-F interactions (28-79% binding efficiency) are 2-fold stronger than regular hydrophobic interactions (15-39% binding efficiency) for PFOS adsorption, explaining why these novel PFOS-binding nanoparticles are superior to other conventional materials based on either hydrophobic or electrostatic binding. The PFOS adsorption by the optimized nanoparticles performs well in the presence of ionic interferences and in environmental wastewater. This library mapping approach can potentially be applied to recognition mechanism investigation of other pollutants and facilitate the discovery of effective monitoring probes and matrices for their removal.

Targeted Delivery of siRNA with pH-Responsive Hybrid Gold Nanostars for Cancer Treatment.

In this work, we report the engineering of gold nanostars (GNS) to deliver small interfering RNA (siRNA) into HepG2 cells. The ligand DG-PEG-Lipoic acid (LA)-Lys-9R (hydrazone) was designed to functionalize GNS, and create the nanoparticles named as 9R/DG-GNS (hydrazone). In the ligand, 2-deoxyglucose (DG) is the targeting molecule, polyethylene glycol (PEG) helps to improve the dispersity and biocompatibility, 9-poly-d-arginine (9R) is employed to provide a positive surface charge and adsorb negative siRNA, and hydrazone bonds are pH-responsive and can avoid receptor-mediated endosomal recycling. Compared to GNS alone, 9R/DG-GNS (hydrazone) showed superior transfection efficiency. The expressions of cyclooxygenase-2 (COX-2) in HepG2 and SGC7901 cells were significantly suppressed by siRNA/9R/DG-GNS (hydrazone) complex. Notably, 9R/DG-GNS (hydrazone) possessed low cytotoxicity even at high concentrations in both normal cells and tumor cells. The combination treatment of siRNA/9R/DG-GNS (hydrazone) complex inhibited the cell growth rate by more than 75%. These results verified that the pH-responsive GNS complex is a promising siRNA delivery system for cancer therapy, and it is anticipated that near-infrared absorbing GNS with good photothermal conversion efficiency can be potentially used for photothermal therapy of tumors.

Fabrication of gold nanorods with tunable longitudinal surface plasmon resonance peaks by reductive dopamine.

Hydroxyphenol compounds are often used as reductants in controlling the growth of nanoparticles. Herein, dopamine was used as an effective reductant in seed-mediated synthesis of gold nanorods (GNRs). The as-prepared GNRs (83 × 16 nm) were monodisperse and had a high degree of purity. The conversion ratio from gold ions to GNRs was around 80%. In addition, dopamine worked as an additive. At a very low concentration of hexadecyltrimethylammonium bromide (CTAB; 0.025 M), thinner and shorter GNRs (60 × 9 nm) were successfully prepared. By regulating the concentration of silver ions, CTAB, seeds, and reductant, GNRs with longitudinal surface plasmon resonance (LSPR) peaks ranging from 680 to 1030 nm were synthesized. The growth process was tracked using UV-vis-NIR spectroscopy, and it was found that a slow growth rate was beneficial to the formation of GNRs.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

Simultaneous and multiplexed phenotyping of circulating exosomes with the orthogonal CRISPR-Cas platform.

Simultaneous and multiplexed exosome protein profiling via an orthogonal CRISPR-Cas platform was achieved in this work. Aptamers were recruited to translate exosome surface protein information into Cas12a/Cas13a cleavage activity. The established multiplexed platform performed robustly with biological matrixes and could profile exosome proteins in clinical serum samples.

Modulating the toxicity of engineered nanoparticles by controlling protein corona formation: Recent advances and future prospects.

With the rapid development and widespread application of engineered nanoparticles (ENPs), understanding the fundamental interactions between ENPs and biological systems is essential to assess and predict the fate of ENPs in vivo. When ENPs are exposed to complex physiological environments, biomolecules quickly and inevitably adsorb to ENPs to form a biomolecule corona, such as a protein corona (PC). The formed PC has a significant effect on the physicochemical properties of ENPs and gives them a brand new identity in the biological environment, which determines the subsequent ENP-cell/tissue/organ interactions. Controlling the formation of PCs is therefore of utmost importance to accurately predict and optimize the behavior of ENPs within living organisms, as well as ensure the safety of their applications. In this review, we provide an overview of the fundamental aspects of the PC, including the formation mechanism, composition, and frequently used characterization techniques. We comprehensively discuss the potential impact of the PC on ENP toxicity, including cytotoxicity, immune response, and so on. Additionally, we summarize recent advancements in manipulating PC formation on ENPs to achieve the desired biological outcomes. We further discuss the challenges and prospects, aiming to provide valuable insights for a better understanding and prediction of ENP behaviors in vivo, as well as the development of low-toxicity ENPs.

Threats of per- and poly-fluoroalkyl pollutants to susceptible populations.

Environmental exposure to per- and poly-fluoroalkyl substances (PFAS) has raised significant global health concerns due to potential hazards in healthy adults. However, the impact of PFAS on susceptible populations, including pregnant individuals, newborns, the older people, and those with underlying health conditions, has been overlooked. These susceptible groups often have physiological changes that make them less resilient to the same exposures. Consequently, there is an urgent need for a comprehensive understanding of the health risks posed by PFAS exposure to these populations. In this review, we delve into the potential health risks of PFAS exposure in these susceptible populations. Equally important, we also examine and discuss the molecular mechanisms that underlie this susceptibility. These mechanisms include the induction of oxidative stress, disruption of the immune system, impairment of cellular metabolism, and alterations in gut microbiota, all of which contribute to the enhanced toxicity of PFAS in susceptible populations. Finally, we address the primary research challenges and unresolved issues that require further investigation. This discussion aims to foster research for a better understanding of how PFAS affect susceptible populations and to pave the way for strategies to minimize their adverse effects.