Delivery of Apoplastic Extracellular Vesicles Encapsulating Green-Synthesized Silver Nanoparticles to Treat Citrus Canker.

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30-1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP+ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (-10.32 kcal/mol and -6.13 kcal/mol, respectively) as compared to phyllanthin (-6.42 kcal/mol and -2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv.

Inhibition of Glioma Cells' Proliferation by Doxorubicin-Loaded Exosomes via Microfluidics.

BACKGROUND: Malignant glioma is a fatal brain cancer. Accumulated evidence has demonstrated that exosomes can cross the blood-brain barrier (BBB), suggesting their potential use as drug delivery vehicles to glioma. Therefore, various loading methods of anticancer agents into exosomes have been developed. However, the loading efficiency of anticancer drugs, such as doxorubicin (DOX) and paclitaxel (PTX), into exosomes is relatively low, thus challenging to improve the drug delivery efficiency to glioma cells (GMs) via exosomes. METHODS: To improve the loading efficiency of doxorubicin into exosomes, a microfluidic device (Exo-Load) was developed. Next, to increase the exosomal delivery of doxorubicin to GMs, autologous exosomes were used for its loading via Exo-Load. Briefly, exosomes from SF7761 stem cells-like- and U251-GMs were isolated and characterized by nano-tracking analysis (NTA), transmission electron microscopy (TEM), and immunogold EM. Finally, doxorubicin was successfully loaded into exosomes with saponin by Exo-Load, and the uptake and functionality of doxorubicin-loaded exosomes for parent GMs were evaluated. RESULTS: The loading efficiency of DOX into SF7761 stem cells-like- and U251-GMs-derived-exosomes were 19.7% and 7.86% via Exo-Load at the injection flow rate of 50 µL/min, respectively. Interestingly, the loading efficiency of DOX into U251 GMs-derived exosomes was significantly improved to 31.98% by a sigmoid type of Exo-Load at the injection flow rate of 12.5 µL/min. Importantly, DOX-loaded GMs-derived exosomes via Exo-Load inhibited parent GMs' proliferation more than heterologous GMs, supporting exosomes' homing effect. CONCLUSION: This study revealed that DOX and PTX could be loaded in exosomes via Exo-Load, demonstrating that Exo-Load could be a potential drug-loading device into exosomes with further optimization. This study also demonstrated that the delivery of DOX to SF7761 GMs via their daughter exosomes was much more efficient rather than U251 GMs-derived exosomes, supporting that the use of autologous exosomes could be better for glioma drug targeting.

Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance.

Most important evaluating criteria for in vitro skeletal muscle models include the extent of differentiation and the degree of alignment in the tissue model. Substrate micropatterning is considered as an effective tool as it recreates in vivo like cellular microenvironment and helps in understanding the fundamental concepts and mechanisms underlying myogenesis. However, the influence of micropatterning based contact guidance cues over satellite cell alignment and myotube formation needs to be explored and studied further. In the present work, we demonstrate the regulation of myotube size control and alignment through the substrate micropatterning. For this purpose, primary myoblast cells (i.e., satellite cells) isolated from rat hind limb muscle were characterized and cultured for a period of 14 days on micropatterned glass substrates processed by the microchannnel flowed plasma process. Several characteristic parameters of muscle differentiation, including the fusion index, maturation index, and average width of the myotubes were quantified. The functional behavior of cultured myotubes exhibiting spontaneous contractions was assessed through kymograph to determine the twitch frequency. In addition, we evaluated the degree of alignment of myotubes on micropatterned substrates through examining orientation order parameter and two-dimensional fast Fourier transform analysis. Altogether, the outcomes reveal that the contact guidance cues arising due to micropatterning of the substrates could be a key regulator for controlling the size and degree of alignment of myotubes during the myogenesis process.