In vitro analyses of the toxicity, immunological, and gene expression effects of cobalt-chromium alloy wear debris and Co ions derived from metal-on-metal hip implants

Joint replacement has proven to be an extremely successful and cost-effective means of relieving arthritic pain and improving quality of life for recipients. Wear debris-induced osteolysis is, however, a major limitation and causes orthopaedic implant aseptic loosening, and various cell types including macrophages, monocytes, osteoblasts, and osteoclasts, are involved. During the last few years, there has been increasing concern about metal-on-metal (MoM) hip replacements regarding adverse reactions to metal debris associated with the MoM articulation. Even though MoM-bearing technology was initially aimed to extend the durability of hip replacements and to reduce the requirement for revision, they have been reported to release at least three times more cobalt and chromium ions than metal-on-polyethylene (MoP) hip replacements. As a result, the toxicity of metal particles and ions produced by bearing surfaces, both locally in the periprosthetic space and systemically, became a concern. Several investigations have been carried out to understand the mechanisms responsible for the adverse response to metal wear debris. This review aims at summarising in vitro analyses of the toxicity, immunological, and gene expression effects of cobalt ions and wear debris derived from MoM hip implants

The effects of hydration media on the characteristics of non-ionic surfactant vesicles (NISV) prepared by microfluidics

Non-ionic surfactant vesicles (NISV) are colloidal particles that provide a useful delivery system for drugs and vaccines. One of the methods that is used for NISV preparation is microfluidics in which the lipid components dissolved in organic phase are mixed with an aqueous medium to prepare the particles through self-assembly of the lipids. In this work, we examined the effect of using different types of aqueous media on the characteristics of the NISV prepared by microfluidics. Five aqueous media were tested: phosphate buffered saline, HEPES buffer, Tris buffer, normal saline and distilled water. The resulting particles were tested for their physical characteristics and cytotoxicity. The aqueous media were found to have significant effects on the physical characteristics of the particles, as well as their overall stability under different conditions and their cytotoxicity to different human cell lines. Careful consideration should be taken when choosing the aqueous media for preparing NISV through microfluidics. This is an important factor that will also have implications with respect to the entrapped material, but which in addition may help to design vesicles for different uses based on changing the preparation medium

Investigating cobalt toxicity in the context of joint replacement patients - cobalt uptake in primary cardiac fibroblasts and in 3T3 cells

Cobalt leaches out from cobalt/chromium metal-on-metal hip implants into patient blood, and its effects are thought to be toxic. There has been a 5% estimated incidence of adverse effects, including toxicity to the heart, in joint implant patients over the last 40 years. This was investigated by examination of the effects of CoCl2 on cell proliferation and viability performed using a range of assays. To assess effects on proliferation, MTT, neutral red and crystal violet assays were all used to compare effects of increasing concentrations of CoCl2 on the Swiss 3T3 fibroblast cell line (3T3s) and primary cardiac fibroblasts (CFs). CoCl2 induced toxicity in both 3T3s and CFs in a time- and dose-dependent manner with IC50 values for CoCl2 in the range of ~300 µM in both cells. Over 72h, increasing CoCl2 concentrations (up to 500 µM) resulted in decreased proliferation. Interestingly, in terms of proliferation, the 3T3s were more tolerant of CoCl2 than CFs. Uptake of CoCl2 into the 3T3s and CFs was measured by detecting intracellular metal content using ICP-MS. Cells were cultured and exposed to various concentrations of CoCl2 (0-72 ppm) and different exposure times (24, 48 and 72 h). Analysis of cobalt content of cells revealed that with increasing medium concentration of CoCl2 intracellular Co concentration on both 3T3s and CFs increased, to a range between 0-50 ppb and 0-120 ppb, respectively. Uptake into CFs was greater than into the 3T3s, and this at least partly explains the difference in toxicity between the two cell types

Exosomes : fighting cancer with cancer

Exosomes are nanovesicles secreted by many cells, including cancer cells. Extensive research has been carried out to validate potential applications of exosomes and to evaluate their efficiency in a wide range of diseases, including cancer. The current knowledge on the origin, biogenesis and composition of exosomes is described. This review then focuses on the use of exosomes in cancer diagnostics and therapeutics

Redox-sensitive, cholesterol-bearing PEGylated poly(propyleneimine)-based dendrimersomes for drug and gene delivery to cancer cells

Stimuli-responsive nanocarriers have attracted increased attention as materials that can facilitate drug and gene delivery in cancer therapy. The present study reports the development of redox-sensitive dendrimersomes comprising disulfide-linked cholesterol-bearing PEGylated dendrimers, which can be used as drug and gene delivery systems. Two disulfide-linked cholesterol-bearing PEGylated generation 3 diaminobutyric polypropylenimine dendrimers have been successfully synthesized via an in situ two-step reaction. They were able to spontaneously self-assemble into stable, cationic, nanosized vesicles (or dendrimersomes) with lower critical aggregation concentration values for high-cholesterol-bearing vesicles. These dendrimersomes were able to entrap both hydrophilic and hydrophobic dyes, and they also showed a redox-responsive sustained release of the entrapped guests in the presence of a glutathione concentration similar to that of a cytosolic reducing environment. The high-cholesterol-bearing dendrimersomes were found to have a higher melting enthalpy, increased adsorption tendency on mica surface, entrapping ability for a larger amount of hydrophobic drugs, and increased resistance to redox-responsive environments in comparison with their low-cholesterol counterpart. In addition, both dendrimersomes were able to condense more than 85% of the DNA at all the tested ratios for the low-cholesterol vesicles, and at dendrimer : DNA weight ratios of 1 : 1 and higher for the high-cholesterol vesicles. These vesicles resulted in an enhanced cellular uptake of DNA, by up to 15-fold when compared with naked DNA with low-cholesterol vesicles. As a result, they increased the gene transfection on the PC-3 prostate cancer cell line, with the highest transfection being obtained with low-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 5 : 1 and high-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 10 : 1. These transfection levels were about 5-fold higher than those observed when treated with naked DNA. These cholesterol-bearing PEGylated dendrimer-based vesicles are, therefore, promising as redox-sensitive drugs and gene delivery systems for potential applications in combination cancer therapies

Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse

Protein S-acylation is a widespread post-translational modification that regulates the trafficking and function of a diverse array of proteins. This modification is catalysed by a family of twenty-three zDHHC enzymes that exhibit both specific and overlapping substrate interactions. Mutations in the gene encoding zDHHC9 cause mild-to-moderate intellectual disability, seizures, speech and language impairment, hypoplasia of the corpus callosum and reduced volume of sub-cortical structures. In this study, we have undertaken behavioural phenotyping, magnetic resonance imaging (MRI) and isolation of S-acylated proteins to investigate the effect of disruption of the Zdhhc9 gene in mice in a C57BL/6 genetic background. Zdhhc9 mutant male mice exhibit a range of abnormalities compared with their wild-type littermates: altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety; deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory; and a 36% reduction in corpus callosum volume revealed by MRI. Surprisingly, membrane association and S-acylation of H-Ras was not disrupted in either whole brain or hippocampus of Zdhhc9 mutant mice, suggesting that other substrates of this enzyme are linked to the observed changes. Overall, this study highlights a key role for zDHHC9 in brain development and behaviour, and supports the utility of the Zdhhc9 mutant mouse line to investigate molecular and cellular changes linked to intellectual disability and other deficits in the human population

Cobalt administration causes reduced contractility with parallel increases in TRPC6 and TRPM7 transporter protein expression in adult rat hearts

Exposure to circulating cobalt (Co2+) in patients with metal-on-metal orthopaedic hip implants has been linked to cardiotoxicity but the underlying mechanism(s) remain undefined. The aim of the current study was to examine the effects of Co2+ on the heart in vivo and specifically on cardiac fibroblasts in vitro. Adult male rats were treated with CoCl2 (1 mg/kg) for either 7 days or 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Co2+uptake into various organs of the body. Co2+ accumulated in the heart over time with significant levels evident after only 7 days of treatment. There was no evidence of cardiac remodelling following Co2+ treatment as assessed by heart weight:body weight and left ventricular weight:body weight. However, a decrease in fractional shortening, as measured using echocardiography, was observed after 28 days of Co2+ treatment. This was accompanied by increased protein expression of the ion transient receptor potential (TRP) channels TRPC6 and TRPM7 as assessed by quantitative immunoblotting of whole cardiac homogenates. Uptake of Co2+ specifically into rat cardiac fibroblasts was measured over 72 h and was shown to dramatically increase with increasing concentrations of applied CoCl2. Expression levels of TRPC6 and TRPM7 proteins were both significantly elevated in these cells following Co2+treatment. In conclusion, Co2+ rapidly accumulates to significant levels in the heart causing compromised contractility in the absence of any overt cardiac remodelling. TRPC6 and TRPM7 expression levels are significantly altered in the heart following Co2+ treatment and this may contribute to the Co2+-induced cardiotoxicity observed over time

Comparison of the physical characteristics of monodisperse non-ionic surfactant vesicles (NISV) prepared using different manufacturing methods

Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by self-assembly of a non-ionic surfactant, often in a mixture with cholesterol and a charged chemical species. Different methods can be used to manufacture NISV, with the majority of these requiring bulk mixing of two phases. This mixing process is time-consuming and leads to the preparation of large and highly dispersed vesicles, which affects the consistency of the final product and could hinder subsequent regulatory approval. In this study, we have compared the physical characteristics of NISV prepared using two conventional methods (thin-film hydration method and heating method) with a recently introduced microfluidic method. The resulting particles from these methods were assessed for their physical characteristics and in vitro cytotoxicity. Through microfluidics, nano-sized NISV were prepared in seconds, through rapid and controlled mixing of two miscible phases (lipids dissolved in alcohol and an aqueous medium) in a microchannel, without the need of a size reduction step, as required for the conventional methods. Stability studies over two months showed the particles were stable regardless of the method of preparation and there were no differences in terms of EC50 on A375 and A2780 cell lines. However, this work demonstrates the flexibility and ease of applying lab-on-chip microfluidics for the preparation of NISV that could be used to significantly improve formulation research and development, by enabling the rapid manufacture of a consistent end-product, under controlled conditions

Camptothecin-based dendrimersomes for gene delivery and redox-responsive drug delivery to cancer cells

Combination therapy involving chemotherapeutic drugs and genes is emerging as a promising strategy to provide a synergistic therapeutic effect, to overcome drug resistance while reducing the severe side effects associated with conventional chemotherapeutic drugs. However, the lack of nanomedicines able to simultaneously carry anti-cancer drugs and nucleic acids limits the application of this therapeutic strategy. To overcome this issue, we proposed to synthesize a pro-drug dendrimer by conjugating the PEGylated, positively charged generation 3-diaminobutyric polypropylenimine dendrimer to the anti-cancer drug camptothecin with a redox-sensitive disulphide linkage, and evaluate its efficacy to co-deliver the complexed DNA and camptothecin to cancer cells. This PEGylated pro-drug dendrimer was found to spontaneously self-assemble into cationic (∼3–5 mV) vesicles at pH 7.4, at a critical aggregation concentration of about 200 μg mL−1. These vesicles (dendrimersomes) became smaller (150–200 nm) with increasing dendrimer concentration and remained stable over 7 days. They were able to release about 70% of the conjugated camptothecin in presence of 50 mM glutathione (equivalent to the intracellular environment of tumor tissue). They could also condense more than 85% of the DNA at dendrimer : DNA weight ratios of 5 : 1 and higher. DNA condensation occurred instantly and was found to be stable for at least 24 h. This led to an enhanced cellular uptake of DNA (by up to 1.6-fold) and increased gene transfection (by up to 2.4-fold) in prostate cancer cells in comparison with the unmodified dendrimer. These novel dendrimersomes are therefore promising for single carrier-based combination cancer therapy

In vivo and in vitro toxicity of cobalt in the heart

Wear debris from cobalt/chromium (Co/Cr) alloy metal-on-metal bearings in prosthetic hip replacements has created a significant internal source of cobalt exposure in these patients. Co toxicity is suspected to contribute to severe systemic adverse effects, including cardiac and CNS effects, in patients with high circulating Co concentrations. This study investigated the effects of chronic Co exposure to rats (1mg/kg i.p. CoCl2, daily, for 28 days) and Co uptake into primary adult rat cardiac fibroblasts (CFs). Co treatment was associated with accumulation into various organs of the body and significant increases in Co levels were detected in liver, kidney and heart. Echocardiography showed functional changes that correlated with compromised cardiac contractility. Fractional shortening was significantly reduced in CoCl2-treated rats following 28 days treatment compared to the control group (54.01±0.90% and 60.29±0.53% respectively), providing evidence of contractile dysfunction. Cellular studies examined uptake of CoCl2 into both CFs and 3T3 fibroblasts using inductively coupled plasma mass spectrometry (ICP-MS) to measure intracellular metal content. The range of Co uptake increased proportionally (0-50 µg/L) for the 3T3 cells, as well as for the CFs (about 0-120 µg/L) when the CoCl2 concentration in the medium was increased between 0 and 72 mg/L. Uptake of Co into CFs was significantly greater than into 3T3 cells. The greater accumulation of CoCl2 into CFs suggests Co ions in vivo could accumulate in these cells, leading to functional consequences on cardiac performance. Future work will focus on determining the underlying uptake mechanism which could have important therapeutic implications