Orange-derived extracellular vesicles nanodrugs for efficient treatment of ovarian cancer assisted by transcytosis effect

Extracellular vesicles (EVs) have recently received much attention about the application of drug carriers due to their desirable properties such as nano-size, biocompatibility, and high stability. Herein, we demonstrate orange-derived extracellular vesicles (OEV) nanodrugs (DN@OEV) by modifying cRGD-targeted doxorubicin (DOX) nanoparticles (DN) onto the surface of OEV, enabling significantly enhancing tumor accumulation and penetration, thereby efficiently inhibiting the growth of ovarian cancer. The obtained DN@OEV enabled to inducement of greater transcytosis capability in ovarian cancer cells, which presented the average above 10-fold transcytosis effect compared with individual DN. It was found that DN@OEV could trigger receptor-mediated endocytosis to promote early endosome/recycling endosomes pathway for exocytosis and simultaneously reduce degradation in the early endosomes-late endosomes-lysosome pathway, thereby inducing the enhanced transcytosis. In particular, the zombie mouse model bearing orthotopic ovarian cancer further validated DN@OEV presented high accumulation and penetration in tumor tissue by the transcytosis process. Our study indicated the strategy in enhancing transcytosis has significant implications for improving the therapeutic efficacy of the drug delivery system.

Disease-specific protein corona formed in pathological intestine enhances the oral absorption of nanoparticles

Protein corona (PC) has been identified to impede the transportation of intravenously injected nanoparticles (NPs) from blood circulation to their targeted sites. However, how intestinal PC (IPC) affects the delivery of orally administered NPs are still needed to be elucidated. Here, we found that IPC exerted "positive effect" or "negative effect" depending on different pathological conditions in the gastrointestinal tract. We prepared polystyrene nanoparticles (PS) adsorbed with different IPC derived from the intestinal tract of healthy, diabetic, and colitis rats (H-IPC@PS, D-IPC@PS, C-IPC@PS). Proteomics analysis revealed that, compared with healthy IPC, the two disease-specific IPC consisted of a higher proportion of proteins that were closely correlated with transepithelial transport across the intestine. Consequently, both D-IPC@PS and C-IPC@PS mainly exploited the recycling endosome and ER-Golgi mediated secretory routes for intracellular trafficking, which increased the transcytosis from the epithelium. Together, disease-specific IPC endowed NPs with higher intestinal absorption. D-IPC@PS posed "positive effect" on intestinal absorption into blood circulation for diabetic therapy. Conversely, C-IPC@PS had "negative effect" on colitis treatment because of unfavorable absorption in the intestine before arriving colon. These results imply that different or even opposite strategies to modulate the disease-specific IPC need to be adopted for oral nanomedicine in the treatment of variable diseases.

The upregulated intestinal folate transporters direct the uptake of ligand-modified nanoparticles for enhanced oral insulin delivery

Transporters are traditionally considered to transport small molecules rather than large-sized nanoparticles due to their small pores. In this study, we demonstrate that the upregulated intestinal transporter (PCFT), which reaches a maximum of 12.3-fold expression in the intestinal epithelial cells of diabetic rats, mediates the uptake of the folic acid-grafted nanoparticles (FNP). Specifically, the upregulated PCFT could exert its function to mediate the endocytosis of FNP and efficiently stimulate the traverse of FNP across enterocytes by the lysosome-evading pathway, Golgi-targeting pathway and basolateral exocytosis, featuring a high oral insulin bioavailability of 14.4% in the diabetic rats. Conversely, in cells with relatively low PCFT expression, the positive surface charge contributes to the cellular uptake of FNP, and FNP are mainly degraded in the lysosomes. Overall, we emphasize that the upregulated intestinal transporters could direct the uptake of ligand-modified nanoparticles by mediating the endocytosis and intracellular trafficking of ligand-modified nanoparticles via the transporter-mediated pathway. This study may also theoretically provide insightful guidelines for the rational design of transporter-targeted nanoparticles to achieve efficient drug delivery in diverse diseases.

Salvianolic acid B increases recombinant adeno-associated virus transduction through redirecting trafficking pathways and stabilizing perinuclear accumulations / 中国药理学与毒理学杂志

OBJECTIVE To screen small molecule compounds from traditional Chinese medicine that can enhance recombinant adeno-associated virus (rAAV) transduction. METHODS Recombinant adeno-associated virus (rAAV) has been established as a powerful tool for in vivo gene transfer and achieved much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency.In the current study,we screened a panel of small molecule compound from traditional Chinese medicine focused on AAV intracellular trafficking process and found salvianolic acid B can significantly enhance rAAV2 transduction. RESULTS Salvianolic acid B caused a dose-depen-dent increase in rAAV2 transduction regardless of vector dose,genome architecture,and over a broad range of cell line from various cell type and species(HEK293,HeLa,HepG2,Huh-7,CHO-K1,LO-2). Salvianolic acid B treatment redirected rAAV2 particles toward large vesicles positive for late endosomal (Rab7)and lysosomal(LAMP1)markers.Furthermore,salvianolic acid B acted to increase accumulation of viral particles at the perinuclear region. CONCLUSION In summary, our results suggest that salvi-anolic acid B redirects rAAV2 toward more productive trafficking pathways and stabilizes perinuclear accumulations of vectors,facilitating productive nuclear trafficking.

Role of cellular FKBP52 protein in hydroxyurea treatment-mediated increase in transduction efficiency of recombinant adeno-associated virus 2 vectors / 中国药理学通报

Aim To explore the role of cytoplasmic FKBP52 in AAV-mediated transduction.Methods Murine embryo fibroblasts(MEFs)cultures from FKBP52 wild-type(WT),heterozygous(HE),and knockout(KO)mice were established.The role of FKBP52 in intracellular trafficking of AAV was analyzed by fluorescence-activated cell sorting(FACS)analyses,electrophoretic mobility shift assays(EMSA),southern blot,immunoprecipitations and western blot analyses.Results Conventional AAV vectors failed to transduce WT MEFs efficiently,and the transduction efficiency was not significantly increased in HE or KO MEFs.AAV vectors failed to traffick efficiently to the nucleus in these cells.Treatment with hydroxyurea(HU)increased the transduction efficiency of conventional AAV vectors by～25-fold in WT MEFs,but only by～4-fold in KO MEFs.The use of self-complementary AAV(scAAV)vectors,which bypass the requirement of viral second-strand DNA synthesis,revealed that HU treatment increased the transduction efficiency～23-fold in WT MEFs,but only～4-fold in KO MEFs,indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis.Following HU treatment,～59% of AAV genomes were present in the nuclear fraction from WT MEFs,but only ～28% in KO MEFs,indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs.When KO MEFs were stably transfected with an FKBP52 expression plasmid,HU treatment-mediated increased in the transduction efficiency was restored in these cells,which correlated directly with improved intracellular trafficking.Intact AAV particles were also shown to interact with FKBP52 as well as with dynein,a known cellular protein involved in AAV trafficking.Conclusion These studies suggest that FKBP52,being a cellular chaperone protein,facilitates intracellular trafficking of AAV,which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Immunolocalization of AQP5 in Salivary Glands of Rat / 대한해부학회지

Exocrine glands secrete large volumes of fluid in response to neural or hormonal stimulation. In the present studies, we examined the immunolocalization of AQP1-5 in salivary glands of rat to clarify the potential roles of these for fluid secretion. Immunohistochemistry using cryosections of rat submandibular glands revealed that AQP1 was located in the capillary endothelia between secretory gland cells. Secretory gland cells and ductal cells were not labeled with anti-AQP1, AQP2 and AQP3 were not present in either salivary gland cells, intercalated ductal cells or striated ducts. AQP4 was localized in the basolateral plasma membrane domains of the intercalated ductal cells in rat submandibular glands whereas either secretory gland cells or striated ducts were not labeled. AQP5 was abundant in the apical domains of the secretory gland cells as well as of the secretory canaliculi and intercalated ductal cells, consistent with RT-PCR analyses as well as previous data (Nielsen et al., 1997). Importantly, AQP5 labeling was associated with the apical part of the secretory gland cells, raising the possibility for trafficking of AQP5 from intracellular vesicles to the apical plasma membranes following neuronal or hormonal stimulation. In parotid and sublingual glands, the immunolabeling pattern of AQPs was identical with that seen in submandibular glands, while the AQP5 immunolabeling was sparse in the sublingual glands which is mainly composed of mucinous glands. In conclusion, the basolateral localization of AQP4 in the intercalated ductal cells and presence of AQP5 in the apical plasma membrane of secretory acinar cells, canaliculi and intercalated ductal cells of submandibular gland suggest that both AQP4 and AQP5 may provide a critical pathway for osmotic water flow into the secreted fluid.

Advances in the Research on the Brucella Intracellular Life / 微生物学通报

Brucella organisms are facultative intracellular bacteria capable of surviving inside professional and non-professional phagocytes.Upon cell contact the bacteria is internalized via receptor molecules.Once inside cells,Brucella localizes in early phagosomes,where it avoids fusion with late endosomes and lysosomes.Then,the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum,the replicating niche.Once inside the endoplasmic reticulum,Brucella extensively replicates without restricting basic cellular functions or inducing damage to cells.Invasion,intracellular trafficking and replication of Brucella organisms in professional and non-professional phagocytes and the molecular determinants involving Brucella intracellular life are reviewed in this article.