[Preparation and Tumor Targeting Analysis of Cell Membrane Nanovesicles Derived from Breast Cancer Cells].

Objective: To prepare cell membrane nanovesicles (NVs) derived from breast cancer cells, to explore their basic characteristics, tumor cell endocytosis, and in vivo distribution in a tumor-bearing mouse model, and to investigate their tumor targeting properties. Methods: 4T1 breast cancer cells were cultured in vitro. The cell membrane of 4T1 cells was isolated through ultracentrifugation and NVs were formulated with a liposome extruder. The size distribution of NVs was determined by way of dynamic light scattering, and the morphology properties of the NVs were examined with transmission electron microscope. The stability of NVs was analyzed by measuring the diameter changes of NVs submerged in phosphate-buffered saline (PBS). The biocompatibility of NVs was investigated by measuring the viability of dendritic cells treated with NVs at different concentrations (5, 10, 20, 50, and 100 mg·L -1) by CCK-8 assay. Fluorescence microscopy was used to analyze the cellular uptake of NVs by breast cancer cells. A mice model of breast cancer model was established with mice bearing subcutaneous xenograft of 4T1 cells. The mice were treated with Cy5.5-labeled NVs injected via the tail vein and the in vivo distribution of NVs was analyzed with an imaging system for small live animals. Results: The results showed that NVs derived from 4T1 breast cancer cells were successfully prepared. The NVs had a mean diameter of 123.2 nm and exhibited a hollow spherical structure under transmission electron microscope. No obvious change in the size of the NVs was observed after 7 days of incubation in PBS solution. CCK-8 assay results showed that the viability of dendritic cells treated with NVs at different concentrations was always higher than 90%. Fluorescence microscopic imaging showed that NVs could be efficiently internalized into breast cancer cells. in vivo biodistribution analysis revealed that breast cancer cell-derived NVs showed higher distribution in tumor tissue than the NVs prepared with normal cells did. Conclusion: We successfully prepared cell membrane NVs derived from 4T1 breast cancer cells. These NVs had efficient cellular uptake by breast cancer cells and sound tumor targeting properties.

NPA motifs play a key role in plasma membrane targeting of aquaporin-4.

The two highly conserved NPA motifs (asparagine-proline-alanine, NPA) are the most important structural domains that play a crucial role in water-selective permeation in aquaporin water channels. However, the functions of NPA motifs in aquaporin (AQP) biogenesis remain largely unknown. Few AQP members with variations in NPA motifs such as AQP11 and AQP12 do not express in the plasma membrane, suggesting an important role of NPA motifs in AQP plasma membrane targeting. In this study, we examined the role of the two NPA motifs in AQP4 plasma membrane targeting by mutagenesis. We constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in AQP4-M1 and AQP4-M23 isoforms and analyzed their expression patterns in transiently transfected FRT and COS-7 cells. Western blot analysis showed similar protein bands of all the AQP4 mutants and the wild-type AQP4. AQP4 immunofluorescence indicated that deletion of one or both NPA motifs resulted in defective plasma membrane targeting, with apparent retention in endoplasmic reticulum (ER). The A99T mutant mimicking AQP12 results in ER retention, whereas the A99C mutant mimicking AQP11 expresses normally in plasma membrane. Furthermore, the AQP4-M1 but not the M23 isoform with P98A substitution in the first NPA motif can target to the plasma membrane, indicating an interaction of N-terminal sequence of AQP4-M1 with the first NPA motif. These results suggest that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs may interact with other structural domains in the regulation of membrane trafficking during aquaporin biogenesis.

[Expression of glutathione S-transferase-aquaporin 1 carboxyl terminal domain fusion protein in Escherchia coli].

We constructed a recombinant plasmid of water channel protein Aquaporin 1 (AQP1) carboxyl terminal domain (DNA sequence from 700bp-801bp) in pGEX-4T-1 vector and express the carboxyl terminal hydrophilic peptide AQP1 in E. coli. In this study, the DNA sequence of AQP1 hydrophilic peptide was amplified by PCR and was cloned into pGEX-4T-1 expression vector. After identified by restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expression cells of E. coli BL21. The GST-AQP1 fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain a fusion protein with molecular weight of 30KD. So the fusion protein of AQP1 C-terminal hydrophilic peptide combined with GST was successfully expressed and purified. We set up important bases for the further research in AQP1 gene function.