Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery ...vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXO
EAA-PMO
). EXO
EAA-PMO
elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXO
EAA-PMO
elicited therapeutic levels of dystrophin restoration and functional improvements in
mdx
mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.
Synopsis
This study identifies an exosome-binding DNA aptamer (Exosomal Anchor Aptamer—EAA) and demonstrates that EAA binds to exosomes of different origins effectively and enables efficient loading of different nucleic acid drugs on exosomes. This study provides an easy generalizable strategy for loading nucleic acid therapeutics on exosomes orthogonal to CD63-binding peptides, which are better suited for protein and peptide loading.
EAA showed high binding affinity to exosomes irrespective of origin.
EAA enabled loading of different nucleic acid drugs on exosomes, with thrombin DNA aptamer inhibitor NU172 loaded on exosomes extended the serum stability.
Duchenne Muscular Dystrophy (DMD) phosphorodiamidate morpholino oligomers (PMOs) were efficiently loaded on exosomes via EAA to form EXO
EAA-PMO
and systemic administration EXO
EAA-PMO
at low doses improved muscle function and pathologies in dystrophic mice.
This study identifies an exosome-binding DNA aptamer (Exosomal Anchor Aptamer - EAA) and demonstrates that EAA binds to exosomes of different origins effectively and enables efficient loading of different nucleic acid drugs on exosomes. This study provides an easy generalizable strategy for loading nucleic acid therapeutics on exosomes orthogonal to CD63-binding peptides, which are better suited for protein and peptide loading.
Vascular smooth muscle cell (VSMC) phenotype transition plays an essential role in vascular remodeling. PGD
(Prostaglandin D
) is involved in cardiovascular inflammation. In this study, we aimed to ...investigates the role of DP1 (PGD
receptor 1) on VSMC phenotype transition in vascular remodeling after Ang II (angiotensin II) infusion in mice.
VSMC-specific DP1 knockout mice and DP1
mice were infused with Ang II for 28 days and systolic blood pressure was measured by noninvasive tail-cuff system. The arterial samples were applied to an unbiased proteome analysis. DP1
Myh11 (myosin heavy chain 11) CREERT2 R26
mice were generated for VSMC lineage tracing. Multiple genetic and pharmacological approaches were used to investigate DP1-mediated signaling in phenotypic transition of VSMCs in response to Ang II administration.
DP1 knockout promoted vascular media thickness and increased systolic blood pressure after Ang II infusion by impairing Epac (exchange protein directly activated by cAMP)-1-mediated Rap-1 (Ras-related protein 1) activation. The DP1 agonist facilitated the interaction of myocardin-related transcription factor A and G-actin, which subsequently inhibited the VSMC transition to myofibroblasts through the suppression of RhoA (Ras homolog family member A)/ROCK-1 (Rho associated coiled-coil containing protein kinase 1) activity. Moreover, Epac-1 overexpression by lentivirus blocked the progression of vascular fibrosis in DP1 deficient mice in response to Ang II infusion.
Our finding revealed a protective role of DP1 in VSMC switch to myofibroblasts by impairing the phosphorylation of MRTF (myocardin-related transcription factor)-A by ROCK-1 through Epac-1/Rap-1/RhoA pathway and thus inhibited the expression of collagen I, fibronectin, ED-A (extra domain A) fibronectin, and vinculin. Thus, DP1 activation has therapeutic potential for vascular fibrosis in hypertension.
Objective: Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims ...at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms. Methods: Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384. Results: MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells in vitro and in vivo, whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB. Conclusions: MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells via targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.
OBJECTIVESTo validate that dexlansoprazole, an anti-acid drug, can prevent pulmonary artery hypertension (PAH) in preclinical animal models and find the possible mechanism of action of ...dexlansoprazole for this new indication. METHODSThe efficacy of dexlansoprazole to attenuate PAH in vivo was evaluated in PAH animal models. Plasma guanosine 3', 5'-cyclic phosphate (cGMP) in PAH rats was measured by enzyme linked immunosorbent assay (ELISA). To investigate the anti-PAH effect of dexlansoprazole in vitro, proliferation and migration assays of primary cultured pulmonary artery smooth muscle cells (PASMCs) were performed. Furthermore, dexlansoprazole's function on fibroblast transition of vascular smooth muscle cells (VSMC) was explored by single cell ribonucleic acid (RNA) sequencing and RNAscope. RESULTSDexlansoprazole could attenuate the pathologic process in monocrotaline (MCT)-, hypoxia-induced PAH rats and SU5416/hypoxia (SuHy)-induced PAH mice. The intervention with dexlansoprazole significantly inhibited elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular wall thickness. Furthermore, plasma cGMP in MCT-induced PAH rats was restored after receiving dexlansoprazole. In vitro, dexlansoprazole could inhibit PASMCs' proliferation and migration stimulated by platelet derived growth factor-BB (PDGF-BB). Moreover, dexlansoprazole significantly ameliorated pulmonary vascular remodeling by inhibiting VSMC phenotypic transition to fibroblast-like cells in a VSMC-specific multispectral lineage-tracing mouse. CONCLUSIONSDexlansoprazole can prevent PAH through promoting cGMP generation and inhibiting pulmonary vascular remodeling through restraining PASMCs' proliferation, migration, and phenotypic transition to fibroblast-like cells. Consequently, PAH might be a new indication for dexlansoprazole.
In this work, a novel environment-friendly dual-emission Rhodamine B modified sulfur quantum dots (RhB-SQDs) sensing platform was established to economically monitor organochlorine pesticide ...2,4-dichlorophenoxyacetic acid (2,4-D) through regulating the activity of alkaline phosphatase (ALP). This dual emission RhB-SQDs exhibited excellent fluorescence and high photostability with emission wavelengths of 455 nm and 580 nm. ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate to p-nitrophenol, which quenched RhB-SQDs fluorescence at 455 nm due to the internal filtration effect, but had no effect the fluorescence intensity of RhB-SQDs at 580 nm. When 2,4-D was present, the activity of ALP was specifically inhibited and enzymatic reaction was interrupted, leading to the reduction of p-nitrophenol production, so the fluorescence of RhB-SQDs at 455 nm was restored. It demonstrated a good linear relationship between the concentration of 2,4-D and F455/F580 in the range of 0.050–0.500 μg mL−1, with a detection limit of 17.3 ng mL−1. The dual-emission fluorescent probe was successfully realized in the identification of 2,4-D in natural water samples and vegetables with the advantages of exceptional accuracy, immunity to interference, and selectivity. The platform offers a fresh look at pesticide monitoring and has the potential to prevent pesticide-related health issues.
Display omitted
•Dual emission sulfur quantum dots (RhB-SQDs) were synthesized for the first time.•Pesticide 2,4-D inhibits the catalytic activity of alkaline phosphatase.•The system exhibited specificity for 2,4-D detection due to the enzyme selectivity.•The ratio fluorescence probe was satisfactorily used to detect 2,4-D in real samples.
Display omitted
•A label-free fluorescent biosensor was built for the determination of T4 PNK.•The poly-T ssDNA was utilized as the substrate of Cas12a instead of F-Q probes.•Cas12a combined with T40 ...can be utilized as a universal signal output tool.•There is a high speed of signal output by using poly-T templated CuNPs.•Red-emitting CuNPs can avoid the background fluorescence in biological matrix.
With CRISPR/Cas12a and poly T-templated copper nanoparticles (CuNPs), we built an efficient and label-free fluorescent biosensor for determinating T4 polynucleotide kinase (T4 PNK). Single-stranded DNA (ssDNA) of poly T containing 40 bases (T40) was employed as the template for the synthesis of CuNPs. As a result of the Cas12a/crRNA combining with dsDNA, the Cas12a was activated and capable of cleaving T40 and prevented the synthesis of CuNPs. However, the target T4 PNK could phosphorylate the 5′-OH termini of dsDNA, then dsDNA was further degraded by λ exo, so the Cas12a could not be activated. Therefore, the CuNPs were formed, achieving the detection of T4 PNK in an isothermal and label-free way. As expected, the sensing platform with a low limit of detection of 0.089 U mL−1 was proved to be effective for screening and evaluating the inhibitors of T4 PNK. It is very economic to use T40 as the substrate of Cas12a instead of ssDNA probes labeled with fluorophores and quenchers. And the CuNPs can be synthesized in only five minutes. Considering the flexibility of CRISPR/Cas12a, this universal signal output strategy offers great potential to broaden its application scope for other targets assay, such as DNA, proteins, and small molecules.
Human 8-oxoguanine DNA glycosylase (hOGG1) and flap endonuclease 1 (FEN1) are recognized as potential biomarkers in lung cancer investigations. Developing analytical platforms of simultaneously ...targeting hOGG1 and FEN1 with high selectivity, sensitivity, especially programmability and universality is highly valuable for clinical research. Herein, we established a signal-amplified platform for simultaneously detecting hOGG1 and FEN1 on the basis of cleavage-induced ligation of DNA dumbbell probes, rolling circle transcription (RCT) and CRISPR-Cas12a. A hOGG1 cleavable site and FEN1 cleavable flap were dexterously designed at the 5’ end of DNA flapped dumbbell probes (FDP) for hOGG1 and FEN1. After cleavage, the resulting nick sites with juxtaposition of 5′ phosphate and 3′ hydroxyl terminus could be linked to closed DNA dumbbell probes (CDP) by DNA ligase. The CDP served as a template for RCT, producing plentiful crRNA repeats to activate the trans-cleavage activity of CRISPR-Cas12a which could cleave fluorophores (TAMRA and FAM) and quenchers (BHQ2 and BHQ1) double-labeled ssDNA reporters. Then, hOGG1 and FEN1 could be detected by the recovered fluorescence signal, allowing for the highly sensitive calculated detection limits of 0.0013 and 0.0052 U/mL, respectively. Additionally, this method made it possible to evaluate the inhibitory effects, even to measure hOGG1 and FEN1 activities at the single-cell level. This novel target enzyme-initiated, circles-transcription without promoters, real-time generation, and self-assembly features of FDP-RCT-Cas12a system suppressed nonspecific background remarkably and relieved rigorous requirement of protospacer adjacent motif site. Hence, the universality of FDP-RCT-Cas12a system toward various disease-related non-nucleic acid targets which are tested without using aptamers was extremely improved.
Display omitted
•Target enzymes induced dual-amplification (RCT/Cas12a) to realize sensitive analysis.•Simultaneously detecting hOGG1 and FEN1 in human serum and A549 cells was achieved.•The work enabled transcription without promoters and refrain of nonspecific signal.•The multiplex strategy can avoid false positive or negative results.
