Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo studies.

BACKGROUND: Mounting evidence that oxidative stress contributes to the pathogenesis of intervertebral disc (IVD) degeneration (IDD) suggests that therapies targeting oxidative stress may slow or prevent disease progression. PURPOSE: The objective of this study was to investigate the inhibitory effects of amobarbital (Amo) on the mitochondria of nucleus pulposus (NP) cells under tert-butyl hydrogen peroxide (tBHP)-induced oxidative stress or in NP tissues under oxidative stress from tissue injury as a means of identifying therapeutic targets for IDD. STUDY DESIGN/SETTING: We tested the effects inhibiting mitochondria, a major source of oxidants, with Amo in NP cells subjected to two different forms of insult: exposure to tBHP, and physical injury induced by disc transection. N-acetylcysteine (NAC), an antioxidant known to protect NP cells, was compared to the complex I inhibitor, Amo. METHODS: NP cells were pre-treated for 2 hours with Amo, NAC, or both, and then exposed to tBHP for 1 hour. Apoptosis, necrosis, and reactive oxygen species (ROS) production were assessed using confocal microscopy and fluorescent probes (Annexin V, propidium iodide, and MitoSox Red, respectively). The activation of mitogen-activated protein kinases (MAPKs) involved in oxidative stress responses were interrogated by confocal imaging of immunofluorescence stains using phospho-specific antibodies to extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38. Mitochondrial function was assessed by imaging JC-1 staining, a probe for membrane potential. RESULTS: Amo was modestly more protective than NAC by some measures, while both agents improved mitochondrial function and lowered tBHP-induced apoptosis, necrosis, and ROS production. Activation of MAPK by tBHP was significantly suppressed by both drugs. Physically injured IVDs were treated immediately after transection with Amo or NAC for 24 hours, and then stained with dihydroethidium (DHE), a fluorescent probe for ROS production. Immunofluorescence was used to track the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that induces the expression of antioxidant genes. Amo and NAC significantly reduced ROS production and increased Nrf2 expression. CONCLUSION: These findings suggest that the progression of IDD may be forestalled by Amo via protection of NP cells from oxidative stress following IVD injury. CLINICAL SIGNIFICANCE: This study will define the extent to which a novel, minimally invasive procedure targeting oxidative stress in NP cells can augment surgical interventions intended to retard IVD degeneration.

Multi-biofunctional polymer graphene composite for bone tissue regeneration that elutes copper ions to impart angiogenic, osteogenic and bactericidal properties.

Despite several recent advances, poor vascularization in implanted scaffolds impedes complete regeneration for clinical success of bone tissue engineering. The present study aims to develop a multi-biofunctional nanocomposite for bone tissue regeneration using copper nanoparticle decorated reduced graphene oxide (RGO_Cu) hybrid particles in polycaprolactone (PCL) matrix (PCL/RGO_Cu). X-ray photoelectron spectroscopy and X-ray diffraction confirmed the presence of copper oxides (CuO and Cu2O) on RGO. Thermogravimetric analysis showed that 11.8% of copper was decorated on RGO. PCL/RGO_Cu exhibited steady release of copper ions in contrast to burst release from the composite containing copper alone (PCL/Cu). PCL/RGO_Cu exhibited highest modulus due to enhanced filler exfoliation. Endothelial cells rapidly proliferated on PCL/RGO_Cu confirming cytocompatibility. The sustained release of ions from PCL/RGO_Cu composites augmented tube formation by endothelial cells evidenced enhanced angiogenic activity. Gene expression of angiogenic markers VEGF and ANG-2 was higher on PCL/RGO_Cu compared to PCL. The osteogenic activity of PCL/RGO_Cu was confirmed by the 87% increase in mineral deposition by pre-osteoblasts compared to PCL. The bactericidal activity of PCL/RGO_Cu showed 78% reduction in viability of Escherichia coli. Thus, the multi-biofunctional PCL/RGO_Cu composite exhibits angiogenic, osteogenic and bactericidal properties, a step towards addressing some of the critical challenges in bone tissue engineering.

Multi-functional nanoparticles as theranostic agents for the treatment & imaging of pancreatic cancer.

Theranostics has received considerable attention since both therapy and imaging modalities can be integrated into a single nanocarrier. In this study, fluorescent iron oxide (FIO) nanoparticles and gemcitabine (G) encapsulated poly(lactide-co-glycolide) (PLGA) nanospheres (PGFIO) conjugated with human epidermal growth factor receptor 2, (HER-2) antibody (HER-PGFIO) were prepared and characterized. HER-PGFIO showed the magnetic moment of 10emu/g, relaxivity (r2) of 773mM-1s-1 and specific absorption rate (SAR) of 183W/g. HER-PGFIO showed a sustained release of gemcitabine for 11days in PBS (pH 7.4). In vitro cytotoxicity evaluation of HER-PGFIO in 3D MIAPaCa-2 cultures showed 50% inhibitory concentration (IC50) of 0.11mg/mL. Subcutaneous tumor xenografts of MIAPaCa-2 in SCID mice were developed and the tumor regression study at the end of 30days showed significant tumor regression (86±3%) in the HER-PGFIO with magnetic hyperthermia (MHT) treatment group compared to control group. In vivo MRI imaging showed the enhanced contrast in HER-PGFIO+MHT treated group compared to control. HER-PGFIO showed significant tumor regression and enhanced MRI in treatment groups, which could be an effective nanocarrier system for the treatment of pancreatic cancer. STATEMENT OF SIGNIFICANCE: Combination therapies are best suitable to treat pancreatic cancer. Theranostics are the next generation therapeutics with both imaging and treatment agents encapsulated in a single nanocarrier. The novelty of the present work is the development of targeted nanocarrier that provides chemotherapy, thermotherapy and MRI imaging properties. The present work is the next step in developing the nanocarriers for pancreatic cancer treatment. Different treatment modalities embedding into a single nanocarrier is the biggest challenge that was achieved without compromising the functionality of each other. The surface modification of polymeric nanocarriers for antibody binding and their multifunctional abilities will appeal to wider audience.

Gemcitabine loaded biodegradable PLGA nanospheres for in vitro pancreatic cancer therapy.

Pancreatic cancer is the fourth leading cancer with 85% mortality rate in USA alone and it is prevalent in many other developed and developing countries. Clinically, gemcitabine is prescribed as the first line chemotherapeutic drug for pancreatic cancer treatment. Gemcitabine-loaded poly(lactide-co-glycolide) (PLGA) nanospheres were synthesized and their physico-chemical properties were evaluated. The FESEM images showed that the gemcitabine loaded and blank nanospheres were 180 nm and 200 nm, respectively. The optimized encapsulation efficiency of gemcitabine was 15%. It was observed that 100% of gemcitabine was released from the PLGA nanospheres for 41 days in phosphate buffered saline (PBS) at pH7.4. The uptake of nanospheres in MiaPaCa-2 cells was studied using sulforhodamine B loaded PLGA nanospheres and our results showed that the nanospheres were taken up within 3h. Furthermore, the cytotoxicity of PLGA nanospheres loaded with gemcitabine showed a relative decrease in IC50 in MiaPaCa-2 and ASPC-1 pancreatic cancer cells in comparison to free gemcitabine. The study demonstrates that this system hold promise to improve the therapeutic efficacy of gemcitabine in vitro.

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone.

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol-gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8µM to 30×10(-5)M and 1-150µM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2µM, 20µM for PG and 0.57µM, 1.92µM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.

Fungal mediated biosynthesis of silver nanoparticles, characterization and antimicrobial activity.

Microbial assisted biosynthesis of nanoparticles is a rapidly progressing area of nanobiotechnology. In this paper Aspergillus niger assisted extracellular synthesis of silver nanoparticles is reported. The silver nanoparticles were characterized by UV-vis spectrophotometry, TEM, EDX and FTIR. TEM studies showed the size of the silver nanoparticles to be in the range of 3-30 nm. The probable mechanism for the extracellular synthesis of silver nanoparticles by Aspergillus niger was investigated. The nanoparticles showed antimicrobial activity against fungal and bacterial strains.