An in vitro investigation of the hepatic toxicity of PEGylated polymeric redox responsive nanoparticles.

It can be challenging to deliver drugs to cancer cells in a targeted manner at an effective dose. Polymeric nanoparticles (NPs) are promising drug delivery systems that can be targeted to cancer cells using redox responsive elements. More specifically, intracellular and extracellular levels of the antioxidant glutathione (GSH) are elevated in cancer cells and therefore the use of NPs with a cleavable GSH-responsive element allowing these NPs to target cancer cells and trigger the release of their cargo (e.g. anticancer drugs). The aim of this study was to assess the hepatotoxicity of polymeric NP delivery systems with and without a redox sensitive element. Copolymer poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) NPs with (RR-NPs) and without (nRR-NPs) a redox responsive dithiylethanoate ester linker were synthesised and their toxicity assessed in vitro. As the liver is a primary site of NP accumulation, the C3A hepatocyte cell line was used to assess NP toxicity in vitro via investigation of cytotoxicity, cytokine production, genotoxicity, intracellular reactive oxygen species (ROS) production, intracellular calcium concentration, and hepatocyte function (albumin and urea production). The cellular uptake of NPs was also assessed as this may influence the cellular dose and, therefore, the cellular response. Both NPs had no detrimental impact on cell viability. However, both NPs stimulated an increase in cytokine (IL-1ra) and ROS production and decreased hepatocyte function, with the greatest effect observed for nRR-NPs. Only nRR-NPs caused DNA damage. Cells internalised both NPs and caused a (sub-lethal) increase in intracellular calcium levels. Therefore, whilst the NPs did not have a negative impact on cell viability, the NPs were able to elicit sub-lethal toxicity. By using a battery of tests we were able to demonstrate that RR-NPs may be less toxic than nRR-NPs. Our findings can therefore feed into the development of safer and more effective nanomedicines and into the design of testing strategies to assess polymeric NP safety based on knowledge of their mechanism of toxicity.

Risk Management Framework for Nano-Biomaterials Used in Medical Devices and Advanced Therapy Medicinal Products.

The convergence of nanotechnology and biotechnology has led to substantial advancements in nano-biomaterials (NBMs) used in medical devices (MD) and advanced therapy medicinal products (ATMP). However, there are concerns that applications of NBMs for medical diagnostics, therapeutics and regenerative medicine could also pose health and/or environmental risks since the current understanding of their safety is incomplete. A scientific strategy is therefore needed to assess all risks emerging along the life cycles of these products. To address this need, an overarching risk management framework (RMF) for NBMs used in MD and ATMP is presented in this paper, as a result of a collaborative effort of a team of experts within the EU Project BIORIMA and with relevant inputs from external stakeholders. The framework, in line with current regulatory requirements, is designed according to state-of-the-art approaches to risk assessment and management of both nanomaterials and biomaterials. The collection/generation of data for NBMs safety assessment is based on innovative integrated approaches to testing and assessment (IATA). The framework can support stakeholders (e.g., manufacturers, regulators, consultants) in systematically assessing not only patient safety but also occupational (including healthcare workers) and environmental risks along the life cycle of MD and ATMP. The outputs of the framework enable the user to identify suitable safe(r)-by-design alternatives and/or risk management measures and to compare the risks of NBMs to their (clinical) benefits, based on efficacy, quality and cost criteria, in order to inform robust risk management decision-making.

A review of hepatic nanotoxicology - summation of recent findings and considerations for the next generation of study designs.

The liver is one of the most important multi-functional organs in the human body. Amongst various crucial functions, it is the main detoxification center and predominantly implicated in the clearance of xenobiotics potentially including particulates that reach this organ. It is now well established that a significant quantity of injected, ingested or inhaled nanomaterials (NMs) translocate from primary exposure sites and accumulate in liver. This review aimed to summarize and discuss the progress made in the field of hepatic nanotoxicology, and crucially highlight knowledge gaps that still exist.Key considerations include In vivo studies clearly demonstrate that low-solubility NMs predominantly accumulate in the liver macrophages the Kupffer cells (KC), rather than hepatocytes.KCs lining the liver sinusoids are the first cell type that comes in contact with NMs in vivo. Further, these macrophages govern overall inflammatory responses in a healthy liver. Therefore, interaction with of NM with KCs in vitro appears to be very important.Many acute in vivo studies demonstrated signs of toxicity induced by a variety of NMs. However, acute studies may not be that meaningful due to liver's unique and unparalleled ability to regenerate. In almost all investigations where a recovery period was included, the healthy liver was able to recover from NM challenge. This organ's ability to regenerate cannot be reproduced in vitro. However, recommendations and evidence is offered for the design of more physiologically relevant in vitro models.Models of hepatic disease enhance the NM-induced hepatotoxicity.The review offers a number of important suggestions for the future of hepatic nanotoxicology study design. This is of great significance as its findings are highly relevant due to the development of more advanced in vitro, and in silico models aiming to improve physiologically relevant toxicological testing strategies and bridging the gap between in vitro and in vivo experimentation.

Nanodelivery systems and stabilized solid-drug nanoparticles for orally administered medicine: current landscape.

The use of nanoparticles as a means of targeted delivery of therapeutics and imaging agents could greatly enhance the transport of biologically active contents to specific target tissues, while avoiding or reducing potentially undesired side effects. Generally speaking, the oral route of administration is associated with good patient compliance, as it is convenient, economical, noninvasive, and does not require special training. Here, we review the progress of the utilization of nanodelivery-system carriers or stabilized solid-drug nanoparticles following oral administration, with particular attention on toxicological data. Mechanisms of cytotoxicity are discussed and the problem of extrapolating knowledge to human scenarios highlighted. Additionally, issues associated with administration of drugs via the oral route are underlined, while strategies utilized to overcome these are highlighted. This review aims to offer a balanced overview of strategies currently being used in the application of nanosize constructs for oral medical applications.

Synthesis, characterization and evaluation of in vitro toxicity in hepatocytes of linear polyesters with varied aromatic and aliphatic co-monomers.

Polyesters are extensively used in drug delivery because of their controllable biodegradation properties and perceived favorable cytocompatibility. However, new ester-based materials are continually being sought which can be produced from readily accessible monomers, which can be tuned for drug encapsulation and which retain good cellular compatibilities. In this study, 5 polyesters of similar molar mass were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effect of the phenyl side-chain group addition on polymer properties relevant to drug delivery was investigated. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid was selected based on its ability to encapsulate a model dye in nanoparticle (NP) formulations, and was found to be slowly degradable in phosphate buffered saline (PBS) but more rapidly degraded in the presence of a lipase. The compatibility of NP formulations of this polymer either with or without a Pluronic F68 stabilizing coating was assessed in vitro using the C3A hepatocyte cell line. Cell viability was assessed, at NP concentrations ranging from 4.68-300µgmL-1 24h post-exposure, using the Alamar Blue, CDFA and Neutral Red assays. C3A cells internalized both coated and uncoated polyester NPs to a similar extent, with uptake observed to increase over time (10-1440min). Although cell viability was >80% at the concentrations tested, in all assays, it was found that a Pluronic F68 coated poly (decanediol-phenylsuccinate-co-succinate) stimulated significant DNA damage driven by an oxidant mechanism, whereas the non-coated polyester analogue and the Pluronic F68 alone had no effect. The results obtained suggest that new polyesters can be synthesized with desirable properties from the materials perspective but formulation with additional excipients requires careful evaluation for drug delivery applications.

The WalRK (YycFG) and σ(I) RsgI regulators cooperate to control CwlO and LytE expression in exponentially growing and stressed Bacillus subtilis cells.

The WalRK (YycFG) two-component system co-ordinates cell wall metabolism with growth by regulating expression of autolysins and proteins that modulate autolysin activity. Here we extend its role in cell wall metabolism by showing that WalR binds to 22 chromosomal loci in vivo. Among the newly identified genes of the WalRK bindome are those that encode the wall-associated protein WapA, the penicillin binding proteins PbpH and Pbp5, the minor teichoic acid synthetic enzymes GgaAB and the regulators σ(I) RsgI. The putative WalR binding sequence at many newly identified binding loci deviates from the previously defined consensus. Moreover, expression of many newly identified operons is controlled by multiple regulators. An unusual feature is that WalR binds to an extended DNA region spanning multiple open reading frames at some loci. WalRK directly activates expression of the sigIrsgI operon from a newly identified σ(A) promoter and represses expression from the previously identified σ(I) promoter. We propose that this regulatory link between WalRK and σ(I) RsgI expression ensures that the endopeptidase requirement (CwlO or LytE) for cell viability is fulfilled during growth and under stress conditions. Thus the WalRK and σ(I) RsgI regulatory systems cooperate to control cell wall metabolism in growing and stressed cells.

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells.

The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (ΔphoPR) cells.

Role of androgen receptor and associated lysine-demethylase coregulators, LSD1 and JMJD2A, in localized and advanced human bladder cancer.

Bladder cancer is approximately three times more common in men as compared to women. We and others have previously investigated the contribution of androgens and the androgen receptor (AR) to bladder cancer. JMJD2A and LSD1 are recently discovered AR coregulator proteins that mediate AR-dependent transcription via recently described histone lysine-demethylation (KDM) mechanisms. We used immunohistochemistry to examine JMJD2A, LSD1, and AR expression in 72 radical cystectomy specimens, resulting in evaluation of 129 tissue samples (59 urothelial carcinoma, 70 benign). We tested levels of these proteins for statistical association with clinicopathologic variables and patient survival. Expression of these markers was also assessed in human bladder cancer cell lines. The effects of pharmacological inhibition of LSD1 on the proliferation of these bladder cancer cells was determined. JMJD2A and AR levels were significantly lower in malignant versus benign urothelium, while increased LSD1 levels were observed in malignant urothelium relative to benign. A significant reduction in all three proteins occurred with cancer stage progression, including muscle invasion (JMJD2A/LSD1/AR), extravesical extension (JMJD2A/LSD1), and lymph node metastasis (JMJD2A/AR). Lower JMJD2A intensity correlated with additional poor prognostic features, including lymphovascular invasion, concomitant carcinoma in situ and tobacco usage, and predicted significantly worse overall survival. Pharmacological inhibition of LSD1 suppressed bladder cancer cell proliferation and androgen-induced transcription. Our results support a novel role for the AR-KDM complex in bladder cancer initiation and progression, identify JMJD2A as a promising prognostic biomarker, and demonstrate targeting of the KDM activity as an effective potential approach for bladder cancer growth inhibition.