[Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure].

OBJECTIVE: To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA). METHODS: Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco's Modified Eagle's medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope. RESULTS: After incubating protamine sulfate, negative potential was transformed into positive one ( (-1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h ( P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less. CONCLUSION: Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

Effect of tetrahedral DNA nanostructures on proliferation and osteo/odontogenic differentiation of dental pulp stem cells via activation of the notch signaling pathway.

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration.

High-Temperature Mechanical and Dynamical Properties of γ-(U,Zr) Alloys.

High-temperature body-centered cubic (BCC) γ-U is effectively stablized by γ-(U,Zr) alloys that also make it feasible to use it as a nuclear fuel. However, relatively little research has focused on γ-(U,Zr) alloys due to their instability at room temperature. The effect of Zr composition on its mechanical properties is not clear yet. Herein, we perform molecular dynamics simulations to investigate the mechanical and dynamical stabilities of γ-(U,Zr) alloys under high temperatures, and we calculate the corresponding lattice constants, various elastic moduli, Vickers hardness, Debye temperature, and dynamical structure factor. The results showed that γ-U, ß-Zr, and γ-(U,Zr) are all mechanically and dynamically stable at 1200 K, which is in good agreement with the previously reported high-temperature phase diagram of U-Zr alloys. We found that the alloying treatment on γ-U with Zr can effectively improve its mechanical strength and melting points, such as Vickers hardness and Debye temperature, making it more suitable for nuclear reactors. Furthermore, the Zr concentrations in γ-(U,Zr) alloys have an excellent effect on these properties. In addition, the dynamical structure factor reveals that γ-U shows different structural features after alloying with Zr. The present simulation data and insights could be significant for understanding the structures and properties of UZr alloy under high temperatures.

Background reduction at DTU Nutech surface gamma laboratory.

Sources of background and background variation in a BEGe type HPGe detector located in a surface laboratory were identified. Different strategies for background reduction were applied. A cosmic veto was installed, and optimised using a digital acquisition system in list-mode with time-stamped data. This resulted in the reduction of total background by a factor of 1.4. Thermal and fast neutron fluxes were also calculated. The radon induced background component and its variation were significantly reduced.

Upregulated expression of serum exosomal miR-375 and miR-1307 enhance the diagnostic power of CA125 for ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is associated with high mortality in gynecological oncology; this is mainly due to the low diagnosis rate. Exosomal miRNA has demonstrated potential as a tumor biomarker. We aimed to explore the diagnostic potential of serum exosomal miR-1307 and miR-375 for OC. METHODS: The first six candidate miRNAs were selected from the previous literature. The relative quantification of qRT-PCR was used to screen for the stability of exosomal miRNAs, followed by validation of the cohort. ROC analysis was employed to analyze the specificity and sensitivity of exosomal miRNA. RESULTS: MiR-1307 and miR-375 were confirmed stably existing in serum exosomes of OC. Moreover, miR-1307 and miR-375 were both significantly up-regulated in serum exosomes of OC compared to ovarian benign and healthy groups. The overexpressed miRNAs showed independent diagnostic power and enhanced the diagnostic accuracy of traditional biomarkers when combined with CA-125 and HE4. MiR-1307 was associated with tumor staging, and miR-375 was associated with lymph node metastasis of OC. CONCLUSION: Our results suggest that serum exosomal miR-1307 and miR-375 could serve as potential tumor biomarkers to improve diagnostic efficiency for OC.

Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug.

Liposome is a promising carrier system for delivering bioactive molecules. However, the successful delivery of pH-sensitive molecules is still limited by the intrinsic instability of payloads in physiological environment. Herein, we developed a special liposome system that possesses an acidic micro-environment in the internal aqueous chamber to improve the chemical stability of pH-sensitive payloads. Curcumin-loaded liposomes (Cur-LPs) with varied internal pH values (pH 2.5, 5.0, or 7.4) were prepared. These Cur-LPs have similar particle size of 300 nm, comparable physical stabilities and analogous in vitro release profiles. Interestingly, the chemical stability of liposomal curcumin in 50% fetal bovine serum and its anticancer efficacy in vitro are both micro-environmental pH-dependent (Cur-LP-2.5 > Cur-LP-5.0 > Cur-LP-7.4). This serum stability still has space to be further enhanced to improve the applicability of Cur-LP. In conclusion, creating an acidic micro-environment in the internal chamber of liposome is feasible and efficient to improve the chemical stability of pH-sensitive payloads.

Understanding the Biomedical Effects of the Self-Assembled Tetrahedral DNA Nanostructure on Living Cells.

Recently, much attention has been paid to DNA again due to the successful synthesis of DNA-based nanostructures that can enter cells via endocytosis and thus have great potential in biomedical fields. However, the impacts of DNA nanostructures on life activities of a living cell are unknown. Herein, the promotion effect of tetrahedral DNA nanostructure (TDN) on cell growth and the underlying molecular mechanisms are reported. Upon exposure to TDN, cell proliferation is significantly enhanced, accompanied by up-regulation of cyclin-dependent kinase like-1 gene, changes in cell cycle distribution, and up-regulation of the Wnt/ß-catenin signaling-related proteins (ß-catenin, Lef 1 and cyclin D). In contrast, single-stranded DNA (ssDNA) shows no such functions. Furthermore, TDN is able to reverse the inhibition effect of DKK1, a specific inhibitor for Wnt/ß-catenin pathway. Hence, the Wnt/ß-catenin pathway is the target for TDN to promote cell proliferation. The findings allow TDN to be a novel functional nanomaterial that has great potential in tissue repair and regeneration medicine.

The effects of interleukin-1ß in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases.

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1ß (IL-1ß)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1ß induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1ß restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1ß restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

Independent effect of polymeric nanoparticle zeta potential/surface charge, on their cytotoxicity and affinity to cells.

OBJECTIVES: Up to now, little research has been focussed on discovering how zeta potential independently affects polymeric nanoparticle (NP) cytotoxicity. METHODS: Polymeric nanoparticles of gradient zeta potential ranging from -30 mv to +40 mv were fabricated using the same poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) biopolymer. Interaction forces between nanoparticles and cells were measured by atomic force microscopy (AFM). Cytotoxicity of the nanoparticles to cells was investigated by using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. RESULTS: Four kinds of nanoparticle with similar sizes and gradient zeta potentials, were fabricated. Those with positive surface charges were found to be more toxic than those with negative surface charges. Positively charged nanoparticles or nanoparticles with higher 'like' charges, offered higher interaction force with cells. CONCLUSION: This work proposes a novel approach for investigating interaction between NPs and cells, and discloses the importance of controlling zeta potential in developing NPs-based formulations in the future.

Enhanced biostability of nanoparticle-based drug delivery systems by albumin corona.

AIMS: The long-term efficacy of nanoparticles is limited by their rapid metabolism in tissues. In this work, we aim to enhance nanoparticle biostability by preforming a bovine serum albumin (BSA) corona. MATERIALS & METHODS: A BSA corona was formed by incubating poly-3-hydroxybutyrate-co-3-hydroxyhexanoate nanoparticles with BSA solution and confirmed by SDS-PAGE and x-ray photoelectron spectroscopy. The impacts of the BSA corona on the drug release, biostability and biodistribution of nanoparticles were investigated. RESULTS: In the presence of the BSA corona, the drug release (coumarin-6 was used as the model drug) of nanoparticles was significantly slower and their stability in liver homogenate and in organs was enhanced. CONCLUSION: Preformation of a BSA corona may be a promising approach for enhancing drug biostability and for developing long-acting nanoparticle formulations.

Nanocomplex based on biocompatible phospholipids and albumin for long-circulation applications.

Achieving long circulating delivery of nanoparticles (NPs) is important for efficient drug therapy, but it is difficult due largely to proteins adsorption (opsonization) or/and nonsufficient stability of NPs. In this present work, we aimed to address the above issues by constructing a phospholipid and BSA-based nanocomplex system, namely BSA-phospholipid NPs (BSA-PL-NPs). Combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray photoelectron spectroscopy and proteins adsorption property, we confirmed that some BSA molecules were fixed on the inner surface of BSA-PL-NPs via hydrophobic interactions and the others were located in the core area. This special configuration allowed BSA-PL-NPs to not only maintain the antiadsorption and low phagocytosis properties but also have the slow zero-order drug release and the enhanced nanostructure stability. Interestingly, we found that BSA-PL-NPs had no cytotoxicity to mouse L929 fibroblasts but could stimulate the cells' growth instead. In conclusion, BSA-PL-NPs have a great potential to be developed as a long-circulation drug delivery system, and the ready availability, biocompatibility and nontoxicity of phospholipids and albumin give this system great promise for practical use.

Interaction between Schwann cells and osteoblasts in vitro.

AIM: Given the well-known properties of Schwann cells in promoting nerve regeneration, transplanting Schwann cells into implant sockets might be an effective method to promote sensory responses of osseointegrated implants. The aim of this study was to evaluate the interaction between Schwann cells and osteoblasts. METHODOLOGY: Schwann cells derived from the sciatic nerves of neonatal rat were co-culured with osteoblasts using Transwell inserts. The proliferation of Schwann cells in the co-culture system was evaluated using methylthiazol tetrazolium (MTT) colorimetric method. Moreover, the secretions and mRNA levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR, respectively. In order to test the effect of Schwann cells on osteoblasts, alkaline phosphatase (ALP) staining and Alizerin red staining were performed as well. RESULTS: Schwann cells, which were co-cultured with the osteoblasts, showed an intact proliferation during the observation period. Moreover, the gene expression and synthesis of BDNF and NGF were not impaired by the osteoblasts. Meanwhile, co-cultured osteoblasts exhibited a significant increase in the proliferation on day 3 and 6 (P< 0.05). Co-culture of these two types of cells also led to a more intense staining of ALP and an elevated number of calcified nodules. CONCLUSION: These findings demonstrate that, in the in vitro indirect co-culture environment, Schwann cells can maintain their normal ability to synthesize neurotrophins, which then enhance the proliferation and differentiation of osteoblasts.

DAPT enhances the apoptosis of human tongue carcinoma cells.

AIM: To investigate the effect of DAPT (gamma-secretase inhibitor) on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma. METHODOLOGY: Human tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence (IF) were employed to determine the intracellular expression levels. RESULTS: DAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0-G1 cell cycle arrest and apoptosis. The mRNA levels of Hairy/Enhancer of Split-1 (Hes-1), a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells. CONCLUSION: DAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch-1 and Caspase-3.