An efficient in vitro organogenesis protocol for the endangered relic tree species Bretschneidera sinensis and genetic fidelity assessment using DNA markers.

Bretschneidera sinensis is a monotypic species of rare and tertiary relic trees mainly distributed in China. B. sinensis is a potentially valuable horticultural plant, which has significant ornamental and research value, and is a crucial tool for the study of phylogeography. The artificial cultivation of B. sinensis is of great scientific value and practical significance. In this study, we developed a direct organogenesis process of B. sinensis using mature zygotic embryos as initial materials. The highest sterile germination induction (54.5%) from the mature zygotic embryo was obtained in a Murashige and Skoog (MS) medium with 2.0 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg·L-1 α-naphthaleneacetic acid (NAA). The highest percentage of shoot regeneration (90.37%) was attained using 1.0 mg·L-1 6-BA and 0.01 mg·L-1 NAA in the MS medium. The Woody Plant Medium (WPM) had the greatest adventitious shoot elongation rate of 93.33%. The most optimized rooting rate was 88.89% in a half-strength MS medium containing 2.0 mg·L-1 indole-3-butyric acid (IBA) and 1.0 mg·L-1 NAA. The genetic fidelity of in vitro regenerated plantlets was assessed using inter-simple sequence repeats and random amplified polymorphic DNA molecular markers, confirming the genetic uniformity and stability of regenerated B. sinensis plantlets. Our research presents an effective in vitro propagation system for B. sinensis, laying the groundwork for its germplasm conservation and large-scale production while maintaining high genetic integrity.

Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics.

Development of ionizable lipid nanoparticles and a lyophilized formulation for potent CRISPR-Cas9 delivery and genome editing.

CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.

Chitosan functionalized gold nanostars as a theranostic platform for intracellular microRNA detection and photothermal therapy.

The development of a theranostic platform that integrates both diagnostic and therapeutic capabilities is in great need for precise and personalized medicine. Here, we present a novel nanoplatform (AuNS@CS-hpDNA) formulated by chitosan functionalized gold nanostar composites and further complexed with fluorescent hairpin DNA (hpDNA) probes for tumor-related miRNA imaging and photothermal therapy (PTT). The optimized AuNS@CS-hpDNA nanoplatform mediated efficient hpDNA probe loading and intracellular delivery. Subsequently, the cytosol transfer of the hpDNA probe enabled specific hybridization using the targeted miRNA, which triggered the recovery of fluorescence for the precise detection of biomarker miR21 in living cells and realized the distinguishing cancer cell line MCF-7 and normal cells. Meanwhile, the AuNS@CS-hpDNA nanoplatform exhibited excellent photothermal conversion properties, which induced efficient cancer cell killing under laser irradiation. Thus, the developed AuNS@CS-hpDNA nanoplatform could simultaneously realize the precise detection of cancer cells and accurately initiate efficient PTT, which represents a promising strategy for precise cancer therapy.

Engineered ionizable lipid nanoparticles mediated efficient siRNA delivery to macrophages for anti-inflammatory treatment of acute liver injury.

Small interfering RNA (siRNA) mediating specific gene silencing provides a promising strategy for anti-inflammatory therapy. However, the development of potent carriers for anti-inflammatory siRNA to macrophages remains challenging. With the aim of realizing potent delivery of siRNA to macrophages, we engineered ionizable lipid nanoparticles (LNPs) with the key component of synthetic lipid-like materials. By varying the amine molecules in the structure of synthetic lipid-like materials, a potent LNP (1O14-LNP) was identified, which exhibited efficient transfection of macrophages by facilitating efficient internalization and endosomal escape. The 1O14-LNP successfully delivered anti-inflammatory siRNA against interleukin-1ß (siIL-1ß) with more than 90% downregulation of IL-1ß expression in LPS-activated macrophages. From in vivo studies, systemic administrated 1O14-LNP/siRNA mainly distributed in liver and efficiently captured by hepatic macrophages without notable sign of toxicity. Furthermore, LPS/d-GalN-induced acute liver injury model treated with 1O14-LNP/siIL-1ß resulted in significant suppression of IL-1ß expression and amelioration of liver tissue damage. These results demonstrate that the engineered ionizable LNP provides a powerful tool for siRNA delivery to macrophages and that the strategy of silencing of pro-inflammatory cytokines holds great potential for treating inflammatory diseases.

Niosome-Assisted Delivery of DNA Fluorescent Probe with Optimized Strand Displacement for Intracellular MicroRNA21 Imaging.

MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system to achieve fluorescence imaging of miRNA21 (miR21) in cancer cells. This system consists of two components: an optimized "off-on" double-stranded DNA (dsDNA) fluorescent for miR21 sensing by efficient strand-displacement reaction and a potent carrier vesicle, termed niosome (SPN), to facilitate the efficient intracellular delivery of the dsDNA probe. A series of dsDNA probes based on fluorescence energy resonance transfer (FRET) was assembled to target miR21. By optimizing the appropriate length of the reporter strand in the dsDNA probe, high accuracy and sensitivity for miR21 recognition are ensured. To overcome the cellular barrier, we synthesized SPN with the main components of a nonionic surfactant Span 80 and a cationic lipid DOTAP, which could efficiently load dsDNA probes via electrostatic interactions and potently deliver the dsDNA probes into cells with good biosafety. The SPN/dsDNA achieved efficient miR21 fluorescent imaging in living cells, and could discriminate cancer cells (MCF-7) from normal cells (L-02). Therefore, the proposed SPN/dsDNA system provides a powerful tool for intracellular miRNA biosensing, which holds great promise for early cancer diagnosis.

Spark-Based Parallel Genetic Algorithm for Simulating a Solution of Optimal Deployment of an Underwater Sensor Network.

Underwater sensor networks have wide application prospects, but the large-scale sensing node deployment is severely hindered by problems like energy constraints, long delays, local disconnections, and heavy energy consumption. These problems can be solved effectively by optimizing sensing node deployment with a genetic algorithm. However, the genetic algorithm (GA) needs many iterations in solving the best location of underwater sensor deployment, which results in long running time delays and limited practical application when dealing with large-scale data. The classical parallel framework Hadoop can improve the GA running efficiency to some extent while the state-of-the-art parallel framework Spark can release much more parallel potential of GA by realizing parallel crossover, mutation, and other operations on each computing node. Giving full allowance for the working environment of the underwater sensor network and the characteristics of sensors, this paper proposes a Spark-based parallel GA to calculate the extremum of the Shubert multi-peak function, through which the optimal deployment of the underwater sensor network can be obtained. Experimental results show that while faced with a large-scale underwater sensor network, compared with single node and Hadoop framework, the Spark-based implementation not only significantly reduces the running time but also effectively avoids the problem of premature convergence because of its powerful randomness.

Ultrasensitive immunosensor for prostate specific antigen using biomimetic polydopamine nanospheres as an electrochemiluminescence superquencher and antibody carriers.

In this work, biomimetic polydopamine nanospheres (PDANSs) were easily prepared and were firstly exploited as an electrochemiluminescence (ECL) nanoquencher for the development of a sandwich type immunosensor. The PDANSs with abundant active functional groups can facilely label the detection antibody and show remarkable quenching effect towards the ECL of the tris-(2,2'-bipyridine)ruthenium (Ru(bpy)32+). The amino-modified multiwall carbon nanotubes/Nafion (MWCNTs-NH2@N) composite-film was adopted as a matrix to incorporate the luminophor Ru(bpy)32+ and immobilize the capture antibody. The prominent decrease of ECL signal intensity was obtained on account of the unique quenching ability of the labeled PDANSs. The quenching mechanism is believed that the excited states of Ru(bpy)32+ can be annihilated by quinone units in PDANSs via energy transfer. The ECL quenching efficiency was logarithmically related to the concentration of the prostate specific antigen (PSA) in the range from 0.1 pg mL-1 to 20 ng mL-1 with a detection limit of 35 fg mL-1. Furthermore, the proposed ECL immunosensor presented good stability, repeatability and selectivity.