One-Step Pharmaceutical Preparation of PEG-Modified Exosomes Encapsulating Anti-Cancer Drugs by a High-Pressure Homogenization Technique.

The use of exosomes encapsulating therapeutic agents for the treatment of diseases is of increasing interest. However, some concerns such as limited efficiency and scalability of conventional drug encapsulation methods to exosomes have still remained; thus, a new approach that enables encapsulation of therapeutic agents with superior efficiency and scalability is required. Herein, we used RAW264 macrophage cell-derived exosomes (RAW-Exos) and demonstrated that high-pressure homogenization (HPH) using a microfluidizer decreased their particle size without changing their morphology, the amount of exosomal marker proteins, and cellular uptake efficiency into RAW264 and colon-26 cancer cells. Moreover, HPH allowed for modification of polyethylene glycol (PEG)-conjugated lipids onto RAW-Exos, as well as encapsulation of the anti-cancer agent doxorubicin. Importantly, the doxorubicin encapsulation efficiency became higher upon increasing the process pressure and simultaneous HPH with PEG-lipids. Moreover, treatment with PEG-modified RAW-Exos encapsulating doxorubicin significantly suppressed tumor growth in colon-26-bearing mice. Taken together, these results suggest that HPH using a microfluidizer could be useful to prepare PEG-modified Exos encapsulating anti-cancer drugs via a one-step pharmaceutical process, and that the prepared functional Exos could be applied for the treatment of cancer in vivo.

Bacillus subtilis GntR regulation modified to devise artificial transient induction systems.

We modified GntR regulation in Bacillus subtilis to devise transient induction systems. GntR is the repressor antagonized by gluconate to induce transcription of the gntRKPZ operon for gluconate catabolism. On the other hand, the gnt operon is repressed by glucose via carbon catabolite repression involving CcpA/P-ser-HPr, which binds to two cre sites: one located in the gnt promoter region and the other within the gntR coding region. We initiated gntKPZ encoding of enzymes for gluconate catabolism expressed independently from the operon; this allowed constitutive degradation of gluconate. Both cre sites were mutated to abolish catabolite repression. The mutated gnt promoter was set up to drive the expression of the lacZ reporter under the control of GntR. Even in the presence of glucose, lacZ was induced upon the addition of gluconate and shut down again as gluconate was consumed. Thus, modified GntR regulation enables artificial transient induction. This will allow us to design a flexible metabolic engineering system with genes expressed only temporarily as desired.

Bacillus subtilis 5'-nucleotidases with various functions and substrate specificities.

BACKGROUND: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified. RESULTS: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not. CONCLUSIONS: Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.