Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy.

Rationale: Developing an effective nanoplatform to realize 'multi-in-one' is essential to broaden the therapeutic potential of combination therapy. Exosomes are ideal candidates since their intrinsic abilities of integrating multiple contents and functions. However, only limited efforts have been devoted to engineering exosomes to integrate the needed properties, also considering the safety and yield, for tumor-targeted and efficient gene/chemo combination therapy. Methods: Herein, by manipulating the exosome membrane, blood exosomes with high abundance and safety are engineered as a versatile combinatorial delivery system, where the doxorubicin (Dox) and cholesterol-modified miRNA21 inhibitor (miR-21i) are co-embedded into the lipid bilayer of exosomes, and the magnetic molecules and endosomolytic peptides L17E are bind to the exosome membrane through ligand-receptor coupling and electrostatic interactions, respectively. Results: It is proved that such engineering strategy not only preserves their intrinsic features, but also readily integrates multiple properties of tumor targeting, efficient transfection and gene/chemo combination therapy into blood exosomes. The lipid bilayer structure of exosomes allows them to co-load Dox and miR-21i with high-payloads. Moreover, profiting from the integration of magnetic molecules and L17E peptides, the engineered exosomes exhibit an enhanced tumor accumulation and an improved endosome escape ability, thereby specifically and efficiently delivering encapsulated cargos to tumor cells. As a result, a remarkable inhibition of tumor growth is observed in the tumor-bearing mice, and without noticeable side effects. Conclusions: This study demonstrates the potential of engineered blood exosomes as feasible co-delivery nanosystem for tumor-targeted and efficient combination therapy. Further development by replacing the drugs combined regimens can potentially make this engineered exosome become a general platform for the design of safe and effective combination therapy modality.

Star-branched amphiphilic PLA-b-PDMAEMA copolymers for co-delivery of miR-21 inhibitor and doxorubicin to treat glioma.

The combined treatment of chemotherapeutant and microRNA (miR) has been proven to be a viable strategy for enhancing chemosensitivity due to its synergistic effect for tumor therapy. However, the co-delivery of drugs and genes remains a major challenge as they lack efficient co-delivery carriers. In this study, three amphiphilic star-branched copolymers comprising polylactic acid (PLA) and polydimethylaminoethyl methacrylate (PDMAEMA) with AB3, (AB3)2,and (AB3)3 molecular architectures were synthesized respectively by a combination of ring-opening polymerization, atom transfer radical polymerization, and click chemistry via an "arm-first" approach. The star copolymers possessed a low critical micelle concentration (CMC) and formed nano-sized micelles with positive surface charges in water as well as exhibiting a much lower cytotoxicity than PEI 25 kDa. Nevertheless, their gene transfection efficiency and tumor inhibition ability showed a remarkable dependence on their molecular architecture. The (AB3)3 architecture micelle copolymer exhibited the highest transfection efficiency, about 2.5 times higher than PEI. In addition, after co-delivering DOX and miR-21 inhibitor (miR-21i) into LN229 glioma cells, the micelles could mediate escaping miR-21i from lysosome degradation and the release of DOX to the nucleus, which significantly decreased the miR-21 expression. Moreover, co-delivery of DOX and miR-21i surprisingly exhibited an anti-proliferative efficiency compared with DOX or the miR-21i treatment alone. These results demonstrated that amphiphilic star-branched copolymers are highly promising for their combinatorial delivery of genes and hydrophobic therapeutants.

The different role of Notch1 and Notch2 in astrocytic gliomas.

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway-EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.

The oncogenic roles of Notch1 in astrocytic gliomas in vitro and in vivo.

Notch receptors play an essential role in cellular processes during embryonic and postnatal development, including maintenance of stem cell self-renewal, proliferation, and determination of cell fate and apoptosis. Deregulation of Notch signaling has been implicated in some genetic diseases and tumorigenesis. The function of Notch signaling in a variety of tumors can be either oncogenic or tumor-suppressive, depending on the cellular context. In this study, Notch1 overexpression was observed in the majority of 45 astrocytic gliomas with different grades and in U251MG glioma cells. Transfection of siRNA targeting Notch1 into U251 cells in vitro downregulated Notch1 expression, associated with inhibition of cell growth, arrest of cell cycle, reduction of cell invasiveness, and induction of cell apoptosis. Meanwhile, tumor growth was delayed in established subcutaneous gliomas in nude mice treated with Notch1 siRNA in vivo. These results suggest that Notch1 plays an important oncogenic role in the development and progression of astrocytic gliomas. Furthermore, knockdown of Notch1 expression by siRNA simultaneously downregulated the expression of EGFR and the important components of its downstream pathways, including PI3K, p-AKT, K-Ras, cyclin D1 and MMP9, indicating the crosstalk and interaction of Notch and EGFR signaling pathways.

[Influence of SEPT7 on biological characters of glioma cell line TJ905].

OBJECTIVE: To investigate the influence of SEPT7 on biological characters of gliomas cells TJ905. METHODS: Recombinant SEPT7 constructs was transfected to human glioblastoma cell line TJ905 in which SEPT7 expression is absent. The positive clones were identified by RT-PCR and Western blot analysis. The cell proliferation was determined by MTT assay and flow cytometry, cell apoptosis was detected with Annexin V staining and cell invasion was evaluated by motility in three-dimensional culture. Moreover, the molecules regulating the cell cycle progression were examined by immunofluorescence staining and Western blot analysis. RESULTS: When SEPT7 was successfully transfected to TJ905 cells, the cell proliferation activity of TJ905 cell was inhibited, the cell cycle was arrested in G0/G1 phase and S phase fraction (SPF) was lowered, the positive regulatory molecules for cell cycle progression including cyclin D1, CDk4, cyclin E and CDk2 were downregulated while the negative modulators including p16 and p21 were upregulated, apoptotic cells were increased and cell invasive ability was attenuated. CONCLUSIONS: Transfection of SEPT7 construct into the glioma cells TJ905 is able to inhibit the proliferation activity and invasive ability of TJ905 cell and to induce cell apoptosis. These results revealed that SEPT7 exerted the suppressive effect on the glioma cell growth and invasion, and induced apoptosis, and suggested that SEPT7 as a gene of glioma suppressor.

An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor.

OBJECTIVES: To study the inhibitory effects of plasmid-based siRNA targeting human epidermal growth factor receptor (EGFR) on tumor proliferation and invasion of TJ905 glioblastoma cells. METHODS: Two siRNA expression constructs targeting human EGFR extracellular domain (516-536) and catalytic domain (2400-2420) were transfected into TJ905 cell as mediated by Lipofectamine. Immunofluorescence assay and Western blotting were used to detect EGFR expression. Cell cycle was analyzed by flow cytometry, cell proliferative activity was measured by MTT assay. The expression and enzymatic activity of MMP9 were measured by Western blotting and gelatin zymography. Cell invasive capability was evaluated by Transwell method. RESULTS: The expression of EGFR was knocked-down by 90 and 92, respectively in siRNA constructs transfected groups as indicated by immunofluorescence assay and Western blotting. The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNAs transfected cells than that in parental cells and the cells transfected with empty vector. Compared to parental cells and the cells transfected with empty vector, the survival rates of glioma cells transfected with the siRNAs dramatically dropped down from the first day after implantation (P<0.05) as indicated by MTT assay. Meanwhile, the expression and enzymatic activity of MMP9 decreased significantly in siRNAs transfected in TJ905 cells, and cell invasive potential was also greatly inhibited in the Transwell study. CONCLUSION: The siRNA expression constructs targeting EGFR could specifically suppress EGFR expression, inhibit cell growth, induce cell cycle arrest and suppress invasion. The plasmid-based siRNA targeting human EGFR approach should be a new strategy for gene therapy of malignant gliomas.