Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), into cell-secreted exosomes leads to low yields. Here, we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs, and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 10 3 -fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumourgrowth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Oligonucleotide (ON) drugs, including small interfering RNA (siRNA), microRNA (miRNA) and antisense oligonucleotides, are promising therapeutic agents. However, their low membrane permeability and sensitivity to nucleases present challenges to in vivo delivery. Chemical modifications of the ON offer a potential solution to improve the stability and efficacy of ON drugs. Combined with nanoparticle encapsulation, delivery at the site of action and gene silencing activity of chemically modified ON drugs can be further enhanced. In the present review, several types of ON drugs, selection of chemical modification, and nanoparticle-based delivery systems to deliver these ON drugs are discussed.

BackgroundWe previously developed cabazitaxel (CTX)-loaded human serum albumin nanoparticles (NPs-CTX) via a self-assembly method, and these NPs showed efficacy in prostate cancer therapy. Many studies have shown that the levels of folic acid (FA) receptor on the surface of various tumor cells are high. Therefore, FA-modified NPs-CTX may have enhanced antitumor effects compared with unmodified NPs-CTX.MethodsNPs-CTX were first prepared via self-assembly, and FA was conjugated on the surface of NPs-CTX through the -NH2 groups of the NPs to produce FA-NPs-CTX. The FA-NPs-CTX were evaluated in tumor cells with high FA receptor (FR) expression in vitro and in vivo.ResultsBoth NPs-CTX and FA-NPs-CTX exhibited good stability and morphology. Drug release from the NPs was not affected by FA conjugation. Compared with CTX dissolved in a mixture of Tween 80 and 13% ethanol (w/w) at a ratio of 1:4 (v/v) (Tween-CTX), the two nanoformulations had lower lytic activity against normal red blood cells. However, FA-NPs-CTX showed greater inhibition of tumor cells with overexpressed FR, compared with NPs-CTX, in the cytotoxicity experiments. Moreover, the cellular uptake of FA-NPs-CTX was enhanced through FR-mediated endocytosis in HeLa cells in vitro and HeLa xenograft tumors in vivo. Although Tween-CTX exhibited tumor growth inhibition similar to FA-NPs-CTX in vivo, this inhibition also caused adverse side effects; the median lethal dose (LD50) of Tween-CTX to mice was 5.68 mg/kg, while FA-NPs-CTX-treated mice survived at doses exceeding 400 mg/kg.ConclusionThe results showed that FA-NPs-CTX caused inhibition of tumor growth in a manner similar to that of Tween-CTX; however, the safety and tolerability of CTX were greatly improved by FA conjugation compared with those of Tween-CTX. In summary, FA-NPs-CTX have great potential in CTX delivery, and this formulation is a promising candidate for the treatment of cancers with high FR levels.

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Magnolia denudata (Lilytree or Yulan magnolia) is an important ornamental species of the genus Magnolia. It has considerable economical value because of its beautiful fragrant flowers and excellent tree structure (Wang et al. 2010). In Beijing, nurseries cultivate M. denudata as an ornamental plant and traditional medicine. In May 2020, patches of root rotted plants were observed in a field in Beijing, China, with an estimated incidence of approximately 31%. Early symptoms comprised leaves melanocratic shrunken, and the vascular tissue of roots turned brown. Progressively, the roots rotted and the whole plant died (Fig. 1 a-d). Infected roots tissue was surface disinfested and plated on potato dextrose agar (PDA) medium at 25±2 °C and incubated in the dark for 7 days. Pure cultures were obtained by hyphal tip excision (strain MFR1215.4). Fungal colonies were entire margins, and the aerial mycelium was copious, early white, and gradually developed into cream white. Colonies developed to 45.1 mm in 4 days at 25±2 °C on PDA media. On Spezieller Nährstoffarmer Agar (SNA) medium at 25±2 °C for 10 days. The morphological characteristics including macroconidia, microconidia, and chlamydospores were shown in Fig.1 (i-p). These morphological characteristics of the isolate corresponded to the description given for Fusarium solani sensu lato (Nelson et al. 1983, Summerell, 2003). Molecular identification was confirmed via amplification of translation elongation factor 1α (EF-1α), RNA polymerase I beta subunit gene (RPB1), and RNA polymerase II beta subunit gene (RPB2) regions using EF1/EF2, RPB1-Fa/G2R, RPB2-5f2/7cR, and RPB2-7cF/11aR primers (O'Donnell, 2010). Sequences were registered in GenBank. In the Fusarium-ID database, the EF-1α, RPB1, and RPB2 sequences showed 100% (677/677 bp), 99.8% (1568/1571 bp), and 100% (1457/1457 bp) identity with the F. solani species complex (FSSC). The same species-level identification was also found using Fusarium MLST. A best maximum likelihood tree was constructed using PhyloSuite v1.2.2 (Zhang et al. 2020), and the sequences of the MFR1215.4 isolation showed the same homology with FSSC 6. Pathogenicity tests were conducted on healthy one-year-old M. denudata potted seedlings. 200 ml spore suspension (1×106 spores/ml) was poured over the roots of twenty seedlings, and sterile distilled water was irrigated into twenty seedlings as controls in a greenhouse with 25/15°C day/night temperature and 80% relative humidity. The experiment was repeated three times. All inoculated seedlings showed similar symptoms to those in the field after 65 days, whereas the controls remained symptomless. The reisolating pathogens from symptomatic tissues were identical to the original isolates by morphology and EF-1α sequence identification. Based on morphological, molecular, and pathogenic characterization, the isolated pathogen was identified as FSSC 6. Fusarium species have been recorded in various places of the world and are known to be harmful to numerous plants (Trabelsi et al. 2017). It has been reported that FSSC has infected soybeans (Coleman, 2016, Nelson et al. 1989), oil palm (Hafizi et al. 2013), tobacco (Yang et al. 2020), resulting in sudden death syndrome, crown disease, and root rot. To our knowledge, this is the first report of FSSC-induced root rot in M. denudata in China. This research may contribute to the development of epidemiology and management strategies for root rot caused by FSSC on M. denudata.