Challenges and strategies: Scalable and efficient production of mesenchymal stem cells-derived exosomes for cell-free therapy.

Exosomes are small membrane vesicles secreted by most cell types, and widely exist in cell supernatants and various body fluids. They can transmit numerous bioactive elements, such as proteins, nucleic acids, and lipids, to affect the gene expression and function of recipient cells. Mesenchymal stem cells (MSCs) have been confirmed to be a potentially promising therapy for tissue repair and regeneration. Accumulating studies demonstrated that the predominant regenerative paradigm of MSCs transplantation was the paracrine effect but not the differentiation effect. Exosomes secreted by MSCs also showed similar therapeutic effects as their parent cells and were considered to be used for cell-free regenerative medicine. However, the inefficient and limited production has hampered their development for clinical translation. In this review, we summarize potential methods to efficiently promote the yield of exosomes. We mainly focus on engineering the process of exosome biogenesis and secretion, altering the cell culture conditions, cell expansion through 3D dynamic culture and the isolation of exosomes. In addition, we also discuss the application of MSCs-derived exosomes as therapeutics in disease treatment.

Nanokit coupled electrospray ionization mass spectrometry for analysis of angiotensin converting enzyme 2 activity in single living cell.

Angiotensin-converting enzyme 2 (ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry (nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living hACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.

A Nanopore Based Molnupiravir Sensor.

Nucleoside analogues are reagents that resemble the structure of natural nucleosides and are widely applied in antiviral and anticancer therapy. Molnupiravir, a recently reported nucleoside analogue drug, has shown its inhibitory effect against SARS-CoV-2. Rapid tracing of molnupiravir and its metabolites is important in the evaluation of its pharmacology effect, but direct sensing of molnupiravir as a single molecule has not been reported to date. Here, we demonstrate a nanopore-based sensor with which direct sensing of molnupiravir and its two major metabolites ß-d-N4-hydroxycytidine and its triphosphate can be achieved simultaneously. In conjunction with a custom machine learning algorithm, an accuracy of 92% was achieved. This sensing strategy may be useful in the current pandemic and is in principle suitable for other nucleoside analogue drugs.

Programmable nano-reactors for stochastic sensing.

Chemical reactions of single molecules, caused by rapid formation or breaking of chemical bonds, are difficult to observe even with state-of-the-art instruments. A biological nanopore can be engineered into a single molecule reactor, capable of detecting the binding of a monatomic ion or the transient appearance of chemical intermediates. Pore engineering of this type is however technically challenging, which has significantly restricted further development of this technique. We propose a versatile strategy, "programmable nano-reactors for stochastic sensing" (PNRSS), by which a variety of single molecule reactions of hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines or nucleoside analogues can be observed directly. PNRSS presents a refined sensing resolution which can be further enhanced by an artificial intelligence algorithm. Remdesivir, a nucleoside analogue and an investigational anti-viral drug used to treat COVID-19, can be distinguished from its active triphosphate form by PNRSS, suggesting applications in pharmacokinetics or drug screening.