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Lung cancer is the leading cause of cancer-related mortality worldwide. Most patients have metastases at the time of diagnosis, thus demanding development of more effective and specific agents. In this study, the specific anticancer effect of hydroxyapatite nanoparticles (HAPNs) to human lung cancer cells (A549) and the underlying mechanisms were investigated, using normal bronchial epithelial cells (16HBE) as the control. Rod-shaped HAPNs (∼10 nm in width and 50 nm in length) were prepared by aqueous precipitation method. Without any further functionalization and drug loading, HAPNs selectively inhibited cancer-cell proliferation. Their efficient mitochondrial targeting correlated strongly with decreased mitochondrial membrane potential and induction of mitochondria-dependent apoptosis in A549 cells. Caveolae-mediated endocytosis via lysosome trafficking was observed to be a prominent internalization pathway for HAPNs in both A549 and 16HBE cells. However, more nanoparticles were taken up into A549 cells. HAPNs triggered a sustained elevation of intracellular calcium concentration ([Ca]) in cancer cells but only a transitory increase in normal control cells. In a nude mouse lung cancer model with xenotransplanted A549 cells, HAPN treatment demonstrated nearly 40% tumor growth inhibition without apparent side effect. These results demonstrated that the enhanced cellular uptake and mitochondrial targeting of HAPNs, together with the prolonged elevation of [Ca] in A549 cells, could result in the cancer-specific cytotoxicity of HAPNs. Thus, HAPNs might be a promising agent or mitochondria-targeted delivery system for effective lung cancer therapy.

Bone morphogenetic protein-2 (BMP-2) is considered as one of the most effective and extensively used growth factor to induce osteoblast differentiation and accelerate bone regeneration. Dexamethasone (Dex) with suitable dosage can enhance the BMP-2-induced osteoblast differentiation. To strengthen this 10 synergistic osteoinductive effect, a pH-responsive chitosan-functionalized mesoporous silica nanoparticles (chi-MSNs) ensemble was fabricated for dual-delivery of BMP-2 and Dex. The MSNs are prepared by a CTAB-templated sol-gel method, and further coated by chitosan via the crosslinking of glycidoxypropyltrimethoxysilane (GPTMS). The small Dex is encapsulated in the mesopores and the large BMP-2 is incorporated into the chitosan coating. These chi-MSNs can quickly release BMP-2 in a 15 bioactive form and then can be efficiently endocytosed and further realize a controlled release of Dex with the decreased pH value into/in cells. With the synergistic action of BMP-2 and Dex outside and inside the cell, this dual hybrid delivery system can significantly stimulate osteoblast differentiation and bone regeneration in vitro and in vivo. Together, this dual-delivery strategy for osteogenic protein delivery may enhance clinical outcomes by retaining the bioactivity and optimizing the release mode of 20 the drug/protein. 65 platform to potentiate the synergistic effect of BMP-2 and Dex.Currently, co-load and co-delivery two or more agents, such as genes, proteins or therapeutic drugs, with complementary or synergistic effect are attracting tremendously interests in nanomedicine to achieve combined therapy. 15 The key for such 70

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ϳ0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (␤-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.The creation of a library of engineered promoters of various strengths has enabled the precise control of gene expression in a broad range of activities required for detailed genotypephenotype characterization and the identification of an optimal gene expression level for the desired product yield (1,4,12,20,22,29,35). For example, a library of promoters has been used to assess the influence of phosphoenolpyruvate carboxylase levels on growth yield and deoxyxylulose-P synthase levels on lycopene production and to demonstrate that the optimal expression levels of deoxyxylulose-P synthase are different in a strain preengineered to produce lycopene and in the parental strain (1). In another example, random mutagenesis of the Pm promoter has been applied to improve production of the medically important granulocyte-macrophage colony-stimulating factor in Escherichia coli at an industrial level (4). Furthermore, promoter libraries coupled with in silico modeling have been used to guide predictable gene network construction in Saccharomyces cerevisiae (13). Therefore, the promoter library constitutes a valuable addition to the genetic toolbox for the study of metabolic engineering, functional genomics, and synthetic biology, as well as for various biotechnology applications. However, this powerful tool is implemented mainly in bacteria (2,4,12,20,35,36...

Varying cytotoxicity of HAPNs was observed in different cancer cell types. Our results suggest that possible mechanisms of cytotoxicity in various types of cancer cells could be different. The elevated calcium concentration and nuclear localization of the particles might be the main mechanism of growth inhibition by HAPNs in cancer cells.