Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observe

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.JRC.I.4-Nanobioscience

Anticipation of regulatory needs for nanotechnology-enabled health products

Development of nanotechnology-based applications in health sector offer innovative therapeutic and diagnostic opportunities to address medical needs. At the moment, no specific regulatory framework exists for nano-enabled health products and the current regulatory practise might require additional guidance in order to fully cover the particularities of such products. This white paper summarizes the major challenges associated with the regulation of the nano-enabled health products. Depending on their mode of action nano-enabled health products are regulated either as medicinal products or medical devices. However, due to the increased complexity of such products and their size-related properties the selection of the regulatory path can become challenging since the primary mode of action might be difficult to determine. Due to the fast progress in the field and the lack of robust datasets, only initial guidance on regulatory information needs is currently available and the question remains whether these identified requirements are sufficient for a reliable characterisation of nano-enabled products. In relation to the need for additional information on the quality, safety and efficacy standardised methods have to be available. However, many conventional methods might not be suitable or reliable for nanomaterial testing due to the interference of nanomaterial with assays components. New state-of-art methods, instruments, approaches or tools have not yet sufficiently proven their reliability and relevance for the given purpose. As patents are expiring generic versions of the innovator products will require access to the market. Since the physicochemical characteristics can be very complex and depend on the manufacturing process, pharmacokinetic assessment might not be sufficient and more guidance is needed on how the bioequivalence can be demonstrated. For the nano-enabled health products classified as medical devices, the European Definition on nanomaterials will apply, determining its further classification and regulatory requirements. Yet, the implementation of the definition and the necessity to determine the exposure to nanomaterials may pose additional challenges. The regulatory challenges highlighted in this white paper should guide the research projects and the involved communities willing to advance the regulatory science in the area of nanomedicine.JRC.F.2-Consumer Products Safet

Modulation of surface bio-functionality by using gold nanostructures on protein repellent surfaces

The integration of gold nanoparticles (Au NPs) or nanostructures with special optical properties on solid surfaces has become a major research topic in the field of nanobiotechnology in particular for the development of new generation of multifunctional bioanalytical platforms. This has led to considerable research efforts for developing quick and direct nanofabrication methods capable of producing well-ordered 2D nanostructured arrays with tunable morphological, chemical and optical properties. In this paper, we propose a simple and fast nanofabrication method enabling the creation of Au NPs patterns on a non-adhesive and cell repellent plasma-deposited poly(ethyleneoxide) (PEO-like) coating. The immobilization of Au NPs on PEO-like coatings does not require any prior chemical modifications and is achieved by a straightforward and stable self-assembly technique. By varying the size and the concentration of the Au NPs it is possible to control the Au NPs density and spatial distribution on the PEO-like coated surface with direct effects on the bio-functionality of the surface. These nanostructured surfaces have been tested for protein bio-recognition analysis and as a cell culture platform. The developed nanostructured platform has many potential applications in the field of protein-nanoparticle and cell-nanoparticle interaction studies, nanotoxicology and bioengineering.JRC.I.4-Nanobioscience

Emerging technologies and their impact on regulatory science

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools

Mechanisms of interaction of therapeutic nanoparticles with cells

Nanoparticles (NPs) have gained a lot of interest in recent years due to their huge potential for applications in industry and medicine. Their unique properties offer a large number of attractive possibilities in the biomedical field, providing innovative tools for diagnosis of diseases and for novel therapies. Nevertheless, a deep understanding of their interactions with living tissues and the knowledge about their possible effects in the human body are necessary for the safe use of nanoparticulate formulations. The aim of this PhD project was to study in detail the interactions of therapeutic NPs with living cells, including cellular uptake and release, cellular localization and transport across the cell layers. Moreover, the effects of NPs on the cellular metabolic processes were determined using adapted in vitro assays. We evaluated the biological effect of several NPs potentially used in the biomedical field, including titanium dioxide (TiO2) NPs, 2-sized fluorescent silica NPs, ultrasmall superparamagnetic iron oxide (USPIO) NPs, either uncoated or coated with oleic acid or with polyvinylamine (aminoPVA) and poly(lactic-co-glycolic acid) - polyethylene-oxide (PLGA-PEO) NPs. We have found that the NPs were internalized by the cells, depending on their size, chemical composition, surface coating and also depending on the cell line considered. The uptake of aminoPVA-coated USPIO NPs by endothelial cells was enhanced in the presence of an external magnetic field. None of the tested USPIO NPs and silica NPs was transported across confluent kidney cell layers or brain endothelial cell layers, even in the presence of a magnetic field. However, in an original endothelium-glioblastoma barrier model which was developed, uncoated USPIO NPs were directly transferred from endothelial cells to glioblastoma cells. Following uptake, TiO2 NPs and uncoated USPIO NPs were released by the kidney cells, but not by the endothelial cells. Furthermore, these NPs induced an oxidative stress and autophagy in brain endothelial cells, possibly associated with their enhanced agglomeration in cell medium. A significant DNA damage was found in brain endothelial cells after their exposure to TiO2 NPs. Altogether these results extend the existing knowledge about the effects of NPs on living cells with regard to their physicochemical characteristics and provide interesting tools for further investigation. The development of the in vitro toxicological assays with a special consideration for risk evaluation aims to reduce the use of animal experiments

Main trends of immune effects triggered by nanomedicines in preclinical studies

The application of nanotechnology to emerging medicinal products is a crucial parameter for the implementation of personalised medicine. For example sophisticated drug delivery systems can target the diseased tissue by recognising patient specific biomarker while carrying pharmacologically active molecules. However, such nanomedicines can be recognised by the immune system as foreign and trigger unexpected biological reactions. The anticipation of the immunogenic potential of emerging nanotechnology based products in the preclinical phase is challenging due to high interspecies variations between the immune systems of laboratory animals and humans. A close monitoring of the scientific literature is required to better understand the relationship between various immune reactions and the diversity of nanomedicines currently in the development pipeline. We have reviewed the most frequent immune reactions induced by the nanomaterials in vivo and identified main effects triggered by lipid based, polymer based and inorganic based nanoparticles, as main categories of nanomaterials used in medicine. In addition, we extracted the most relevant nano-specific properties responsible for the observed biological effect. Our analysis lead to identification of the most prevalent endpoints relevant for the assessment of the immunotoxic potential of the nanotechnology based products and will support the smooth and safe translation of the new formulations to clinical applications.JRC.F.2-Consumer Products Safet

Identification of regulatory needs for nanomedicines

The application of nanotechnology in health care is widely accepted as a potential driver of biomedical innovation. By exploiting their unique physicochemical properties, nanomedicines can monitor, repair, and control biological systems in order to address diseases for which currently no or only insufficient diagnostic and therapeutic tools are available. Nevertheless, the opportunities of nanotechnologies in the health sector are accompanied by challenges in the regulation of these products. Sufficient knowledge on their quality, safety, and efficacy must be gained and standardised methods must be made available to support the regulatory decision making and allow a smooth translation towards clinical applications. We have conducted a survey among regulatory authorities with the aim to obtain a general overview on the status and regulatory needs of nanomedicines and to indicate some trends on future requirements. The outcome has demonstrated strong regional differences in the regulation of nanomedicines and confirmed the need for the harmonisation of information requirements on nano‐specific properties. In addition, a number of critical physicochemical properties that have already been proposed in the scientific literature were verified in the survey as relevant for regulatory decision making. Finally, the survey has demonstrated an interest of regulatory agencies in an independent nanomedicine characterisation facility that can support regulators in the evaluation of these systems and at the same time assess the performance of existing and new test methods for their application to the field of nanomedicine.JRC.F.2-Consumer Products Safet

Identification of regulatory needs for nanomedicines 1st EU-NCL survey with the "Nanomedicine" working group of the international pharmaceutical regulators

The application of nanotechnology in healthcare is widely accepted as a potential driver of biomedical innovation. By exploiting the unique physical and physicochemical interactions at the molecular level nanotechnology based products (also known as nanomedicines) can monitor, repair and control biological systems in order to address diseases for which currently no or only insufficient diagnostic and therapeutic tools are available. Nanotechnology will also play an enabling role in the implementation of personalised medicine as it provides tools for a better prediction and early diagnosis, the design of personalised treatment as well as a close monitoring of the therapeutic success. However, the opportunities of nanotechnologies in the health sector are coming along with challenges in the regulation of this emerging product class. Sufficient knowledge on their quality, safety and efficacy must be gained and standardised methods must be made available to support the regulatory decision making and allow a smooth translation towards clinical applications. The current survey was performed within the "nanomedicine" working group of the International Pharmaceutical Regulators Forum (IPRF) with the aim to get a general overview on the status and regulatory needs of nanomedicines. The survey demonstrated strong regional differences in the regulation of nanomedicines and demonstrated that in the last years, the European authorities have received fewer applications of nanotechnology based pharmaceuticals as e.g. the USA. However, a future increase of market applications must be anticipated in Europe as other regions which have a better ecosystem for the marketing of nanomedicines are already facing challenges in the regulation of nanotech products. These expectations are supported by the fact that the last EC's framework programmes have invested hundreds of millions of Euro into nanomedicine development in order to make these promising products available to the patient. An intensive exchange of experience in the regulation of nanodrugs between regulatory bodies is now necessary to ensure harmonised regulation of products internationally. The respondents of the survey confirmed the need for the harmonisation of information requirements on nanospecific properties. A number of critical physicochemical properties have already been proposed in the scientific literature and their relevance for regulatory decisions was verified within the survey. Some regulatory agencies also indicated the need for additional (eco)toxicological testing triggered by the nanospecific property of the product. Finally, there was consensus among the regulatory agencies for a need of an independent nanocharacterisation testing facility that can support regulators in the evaluation of sophisticated material and the performance of new test methods before entering into clinical applications.JRC.F.2-Consumer Products Safet

Mapping of the available standards against the regulatory needs for nanomedicines

Appropriate documentary standards and reference materials are crucial building blocks for the development of innovative products. In order to support the emerging sector of nanomedicine, relevant standards must be identified and/or developed before the products will enter into the regulatory approval process. The anticipation of standardization needs requires a good understanding on the regulatory information requirements that can be triggered by the particularities of nanomedicines. However, robust datasets allowing firm conclusions on regulatory demands are not yet available due to a lack of regulatory experience with innovative products. Such a catch‐22 situation can only be advanced in an iterative process by monitoring continuously the scientific evidence and by promoting intensive knowledge exchange between all involved stakeholders. In this study, we have compiled information requirements released by regulatory scientists so far and mapped it against available standards that could be of relevance for nanomedicines. Our gap analysis clearly demonstrated that for some endpoints such as drug release/loading and the interaction of nanomedicines with the immune system no standards are available so far. The emerging nanomedicine sector could benefit from cross‐sector collaboration and review the suitability of standards that have been developed for nanomaterials used for other industrial applications. Only a concerted action of all parties can lead to a smooth translation of nanomedicines to clinical application and to the market. This is in particular important because nanotechnology‐based drug delivery systems are key for the development and implementation of personalized medicine.JRC.F.2-Consumer Products Safet