Brca1 is involved in tolerance to adefovir dipivoxil­induced DNA damage.

Nucleos(t)ide analogues (NAs) are currently the most important anti­viral treatment option for patients with chronic hepatitis B (CHB). Adefovir dipivoxil (ADV), a diester pro­drug of adefovir, has been widely used for the clinical therapy of hepatitis B virus infection. It has been previously reported that adefovir induced chromosomal aberrations (CAs) in the in vitro human peripheral blood lymphocyte assay, while the genotoxic mechanism remains elusive. To evaluate the possible mechanisms, the genotoxic effects of ADV on the TK6 and DT40 cell lines, as well as DNA repair­deficient variants of DT40 cells, were assessed in the present study. A karyotype assay revealed ADV­induced CAs, particularly chromosomal breaks, in wild­type DT40 and TK6 cells. A γ­H2AX foci formation assay confirmed the presence of DNA damage following treatment with ADV. Furthermore, Brca1­/­ DT40 cells exhibited an increased sensitivity to ADV, while the knockdown of various other DNA damage­associated genes did not markedly affect the sensitivity. These comprehensive genetic studies identified the genotoxic capacity of ADV and suggested that Brca1 may be involved in the tolerance of ADV­induced DNA damage. These results may contribute to the development of novel drugs against CHB with higher therapeutic efficacy and less genotoxicity.

Targeted and Efficient Delivery of siRNA Using Tunable Polymeric Hybrid Micelles for Tumor Therapy.

BACKGROUND/AIM: Effective and targeted delivery of siRNA to tumor cells is a prerequisite to achieving their therapeutic effects. Survivin is up-regulated in tumor cells and is associated with resistance to therapy. Therefore, siRNA-mediated silencing of survivin is a potential therapeutic strategy for cancer. The aim of the study was to examine whether polymeric hybrid micelles can be used to effectively deliver siRNAs into cells. MATERIALS AND METHODS: First, linoleic acid (LA) was conjugated to polyethylenimine (PEI) and methoxy-polyethyleneglycol (mPEG) and two amphiphilic polymers (PEI-LA and mPEG-LA) were obtained. Polymeric hybrid micelle (PHM) was then prepared and characterized by self-assembly of PEI-LA and mPEG-LA at different percentages of the two amphiphilic polymers. A PHM/siRNA complex with optimized composition and good biocompatibility was then prepared and its cellular uptake, biodistribution, and antitumor effects were investigated. RESULTS: Survivin siRNA was efficiently delivered to the cells. It reduced survivin protein expression and greatly suppressed tumor growth. Moreover, siRNA loaded in PHM gathered in a solid tumor in mice and achieved an improved anticancer effect compared to naked siRNA. CONCLUSION: PHM is a promising and safe vehicle for siRNA delivery and may find utility in cancer therapy.

Targeted Co-Delivery of siRNA and Methotrexate for Tumor Therapy via Mixed Micelles.

A combination of chemotherapeutic drugs and siRNA is emerging as a new modality for cancer therapy. A safe and effective carrier platform is needed for combination drug delivery. Here, a functionalized mixed micelle-based delivery system was developed for targeted co-delivery of methotrexate (MTX) and survivin siRNA. Linolenic acid (LA) was separately conjugated to branched polyethlenimine (b-PEI) and methoxy-polyethyleneglycol (mPEG). MTX was then conjugated to LA-modified b-PEI (MTX-bPEI-LA) to form a functionalized polymer-drug conjugate. Functionalized mixed micelles (M-MTX) were obtained by the self-assembly of MTX-bPEI-LA and LA-modified mPEG (mPEG-LA). M-MTX had a narrow particle size distribution and could successfully condense siRNA at an N/P ratio of 16/1. M-MTX/siRNA was selectively taken up by HeLa cells overexpressing the folate receptor (FR) and facilitated the release of the siRNA into the cytoplasm. In vitro, M-MTX/siRNA produced a synergy between MTX and survivin siRNA and markedly suppressed survivin protein expression. In tumor-bearing mice, M-MTX/Cy5-siRNA showed an elevated tumor uptake. In addition, M-MTX/siRNA inhibited tumor growth. Immunohistochemistry and a western blot analysis showed a significant target gene downregulation. In conclusion, M-MTX/siRNA was highly effective as a delivery system and may serve as a model for the targeted co-delivery of therapeutic agents.

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells.

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4-/- cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4-/- . These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.

Biomimetic nanocomposites to control osteogenic differentiation of human mesenchymal stem cells.

The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano-hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell-culture medium. Nanocomposites comprising type I collagen and nano-hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone-formation technologies to the clinic.

The fabrication of nano-hydroxyapatite on PLGA and PLGA/collagen nanofibrous composite scaffolds and their effects in osteoblastic behavior for bone tissue engineering.

Bone is a nanocomposite consisting of two main components, nano-hydroxyapatite (n-HA) and Type I collagen (Col). The aim is to exploit the nano-scale functional and material characteristics of natural bone in order to modulate cellular functions for optimal bone repair in bone graft systems. Here, we present an effective and novel technique in obtaining n-HA in cognate with native apatite on electrospun nanofibers within minutes without any pre-treatment. Using an alternate calcium and phosphate (Ca-P) solution dipping method, n-HA was formed on poly(lactide-co-glycolide) acid (PLGA) and blended PLGA/Col nanofibers. The presence of the functional groups of collagen significantly hastened n-HA deposition closed to nine-fold. The quantity of n-HA impinged upon the specific surface area, whereby mineralized PLGA/Col had a greater surface area than non-mineralized PLGA/Col, whereas n-HA did not significantly improve the specific surface area of mineralized PLGA compared to pure PLGA. The novelty of the process was that n-HA on PLGA had a positive modulation on early osteoblast capture (within minutes) compared to pure PLGA. Contrary, cell capture on mineralized PLGA/Col was comparable to pure PLGA/Col. Interestingly, although n-HA impeded proliferation during the culture period (days 1, 4 and 7), the cell functionality such as alkaline phosphatase (ALP) and protein expressions were ameliorated on mineralized nanofibers. The amount of n-HA appeared to have a greater effect on the early stages of osteoblast behavior (cell attachment and proliferation) rather than the immediate/late stages (proliferation and differentiation).

Fabrication of mineralized polymeric nanofibrous composites for bone graft materials.

Poly-L-lactic acid (PLLA) and PLLA/collagen (50% PLLA+50% collagen; PLLA/Col) nanofibers were fabricated using electrospinning. Mineralization of these nanofibers was processed using a modified alternating soaking method. The structural properties and morphologies of mineralized PLLA and PLLA/Col nanofibers were investigated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and contact angle measurements. Human bone-derived osteoblasts were cultured on the materials for up to 1 week to assess the biological properties of the nanofibrous composites. Cell attachment on these nanocomposites was also tested within 1 h of culture at room temperature. The mechanical properties of the cell-nanocomposite constructs were determined using tensile testing. From our results, the bone-like nano-hydroxyapatite (n-HA) was successfully deposited on the PLLA and PLLA/Col nanofibers. We observed that the formation of n-HA on PLLA/Col nanofibers was faster and significantly more uniform than on pure PLLA nanofibers. The n-HA significantly improved the hydrophilicity of PLLA/Col nanofibers. From the results of cell attachment studies, n-HA deposition enhanced the cell capture efficacy at the 20-minute time point for PLLA nanofibers. The E-modulus values for PLLA+n-HA with cells (day 1 and day 4) were significantly higher than for PLLA+n-HA without cells. Based on these observations, we have demonstrated that n-HA deposition on nanofibers is a promising strategy for early cell capture.

Surface modification of ultra thin poly (epsilon-caprolactone) films using acrylic acid and collagen.

Poly (epsilon-caprolactone) (PCL) has been used as a bioresorbable polymer in numerous medical devices as well as for tissue engineering applications. Its main advantage is its biocompatibility and slow degradation rate. PCL surface, however, is hydrophobic and cell-biomaterial interaction is not the best. We attempt for the first time to modify an ultra thin PCL surface with collagen. The PCL film was prepared using solvent casting and biaxial stretching technique developed in our laboratory. This biaxial stretching produced an ultra thin PCL 3-7 microm thick, ideal for membrane tissue engineering applications. The PCL film was pretreated using Argon plasma, and then UV polymerized with acrylic acid (AAc). Collagen immobilization was then carried out. The modified film surface was characterized by Fourier Transform Infrared (FT-IR) and X-ray Photoelectron Spectroscopy (XPS). Water contact angles were also measured to evaluate the hydrophilicity of the modified surface. Results showed that the hydrophilicity of the surface has improved significantly after surface modification. The water contact angle dropped from 66 degrees to 32 degrees. Atomic Force Microscopy (AFM) showed an increase in roughness of the film. A change from 46 to 60 nm in the surface morphology was also observed. The effect of cells attachment on the PCL film was studied. Human dermal fibroblasts and myoblasts attachment and proliferation were improved remarkably on the modified surface. The films showed excellent cell attachment and proliferation rate.