Nanomedicine and epigenome. Possible health risks

Nanomedicine is an emerging field that combines kno wledge of nanotechnology and material science with pharmaceutical and biomedical sciences , aiming to develop nanodrugs with increased efficacy and safety. Compared to conventi onal therapeutics, nanodrugs manifest higher stability and circulation time, reduced toxi city and improved targeted delivery. Despite the obvious benefit, the accumulation of imaging ag ents and nanocarriers in the body following their therapeutic or diagnostic applicati on generates concerns about their safety for human health. Numerous toxicology studies have demo nstrated that exposure to nanomaterials (NMs) might pose serious risks to hum ans. Epigenetic modifications, representing a non-genotoxic mechanism of toxicantinduced health effects, are becoming recognized as playing a potential causative role in the aetiology of many diseases including cancer. This review i) provides an overview of rece nt advances in medical applications of NMs and ii) summarizes current evidence on their po ssible epigenetic toxicity. To discern potential health risks of NMs, since current data a re mostly based upon i vitro and animal models, a better understanding of functional relati onships between NM-exposure, epigenetic deregulation and phenotype is required.


Abstract
Nanomedicine is an emerging field that combines knowledge of nanotechnology and material science with pharmaceutical and biomedical sciences, aiming to develop nanodrugs with increased efficacy and safety. Compared to conventional therapeutics, nanodrugs manifest higher stability and circulation time, reduced toxicity and improved targeted delivery. Despite the obvious benefit, the accumulation of imaging agents and nanocarriers in the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Numerous toxicology studies have demonstrated that exposure to nanomaterials (NMs) might pose serious risks to humans. Epigenetic modifications, representing a non-genotoxic mechanism of toxicant-induced health effects, are becoming recognized as playing a potential causative role in the aetiology of many diseases including cancer. This review i) provides an overview of recent advances in medical applications of NMs and ii) summarizes current evidence on their possible epigenetic toxicity. To discern potential health risks of NMs, since current data are mostly based upon in vitro and animal models, a better understanding of functional relationships between NM-exposure, epigenetic deregulation and phenotype is required.

Introduction
Nanomedicine, as an interdisciplinary science, combines the knowledge of molecular biology, pharmaceutics, medicine, material science, engineering, and information technology.
The application of nanotechnology to medicine provides an opportunity to study the biological systems at a more subtle level, giving rise to better understanding of disease mechanisms. Moreover, it enables more accurate and rapid diagnosis, targeted and effective drug delivery, novel ways of organ and tissue regeneration, and follow-up of diseases (http://ec.europa.eu/environment/chemicals/nanotech/faq/definition_en.htm). Nanoparticles (NPs) are NMs that have all three dimensions between 1 and 100 nm. A material can also be considered as a NM if its volume-specific surface area is larger than 60 m 2 cm -3 (Rauscher et al. 2017). However, the term NM in nanomedicine extends this commonly accepted definition to particles with dimensions up to 1000 nm (Schutz et al. 2013).
The biocompatibility and stability of NMs in physiological solutions is imperative for their development for clinical use (Mu et al. 2014). Entering the body, the surface of NMs is rapidly covered by proteins, resulting in formation of a 'corona' (Monopoli et al. 2012) which affects the distribution, pharmacokinetics, and circulation time of NMs in the body. Although it is impossible to completely avoid the formation of this protein layer (Tenzer et al. 2011) its composition can be altered through surface modification of NMs. Targeting of current nanodrugs has relied on the enhanced permeability and retention effect (Maeda 2015).
Functionalization of NMs (binding of specific ligands, antigen, aptamer, protein etc. on the surface of NMs) increases their accumulation in, e.g. a tumor region, an ischemic tissue, or organ inflamed area (Albanese et al. 2012). A controlled release of drugs can be triggered by pH (Sato et al. 2011), redox potential (Luo et al. 2011), presence of certain enzymes , and temperature (Kim and Lee 2004). NMs are able to bypass biological barriers, such as cell membranes and the blood-brain barrier, allowing delivery of high drug concentrations in the target tissue. Particle size, shape, and surface chemistry are key factors that determine cellular uptake, biodistribution patterns, and clearance mechanisms (Nel et al.  Oxidative stress, one of the main mechanisms underlying NM-induced toxicity, is closely associated with inflammatory cell responses, immunotoxicity and also genotoxicity . For the safety assessment of NMs, genotoxicity testing is essential, as it addresses both potential mutagenicity and carcinogenicity. Apart from geno-and immunotoxicity, NMs may cause changes in epigenetic regulatory mechanisms that have been involved in the pathogenesis of several complex diseases including cancer (Stoccoro et al. 2013). Although research on NM-induced epigenetic alterations has increased significantly in recent years, the evidence on epigenetic toxicity of NMs is still mostly based on in vitro and animal models. To evaluate the impact of NMs on epigenetic deregulation is not a simple task due to multiple layers of epigenetic control mechanisms and large differences in individual susceptibility. All these and other limitations currently hamper incorporation of epigenetic toxicity endpoints in the standard battery of NM safety assessment.
This article provides a comprehensive review of current knowledge on epigenetic toxicity of NMs, with the focus on their nanomedical applications. Our aim is to summarize available data on epigenetic changes induced by NMs. To discern potential health risks, a knowledge of functional relationships between individual NM-induced epigenetic deregulation and phenotypic response is required.

Uses of NMs in nanomedicine
Application of the knowledge and tools of nanotechnology in medicine offers new possibilities in medical imaging and diagnosis, development of more powerful nanodrugs (both therapeutic and imaging agents), implantable materials, cancer treatment and tissue regeneration. More than 50 FDA-approved nanodrugs are currently in clinical use (Table 1) and more than 77 nano-based products are being evaluated in Phase I -III clinical trials (Bobo et al. 2016).

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Early and highly accurate disease diagnosis is a prerequisite for effective treatment and is an integral part of clinical medicine. Nanotechnology has introduced a number of NMs that have expanded the potential of targeted diagnostic imaging.

Imaging
NMs as a new and exciting class of imaging agents can be used for both anatomic and molecular imaging. Their small size and unique physicochemical properties offer intense, non-invasive and longitudinally stable imaging signals, high avidity (a large association constant brought about by the presence of multiple ligands per particle), multimodal signal capabilities (detection of one NP by more than one imaging modality, allowing deep tissue screening), multiplexing (detection of various molecular targets simultaneously) and moreover, theranostic capabilities (use for both diagnostic and therapeutic purposes) (Thakor et al. 2016).
Several commercially available dextran-coated superparamagnetic iron oxide NPs (SPIONs) have been approved as intravenous contrast agents for in vivo magnetic resonance imaging (MRI) (e.g. Endorem, Sinerem, Resovist) and several others are under clinical investigation (Etheridge et al. 2013, Thakor et al. 2016. Although these novel contrast agents present the advantages of suitable magnetic saturation, superparamagnetic properties, and colloidal stability, they have been taken off the market and are no longer manufactured due to concerns about their toxicity and fatal anaphylactic reactions (Mahmoudi et al. 2008;

Regenerative medicine
Regenerative medicine is a broad interdisciplinary science that attempts to restore lost, damaged, or aging cells and tissues to a state as close as possible to their native architecture and function. Regenerative strategies include stem cell-based therapies and tissue engineering applications. NMs can play an important role in nanopatterning of implant surfaces and as 3D scaffolds mimicking the natural environment of cells, facilitating their mobility, adhesion, and differentiation (Engel et al. 2008, Zhang andWebster 2009). Nano-structured poly(lacticco-glycolic acid) (PLGA) surfaces have been shown to accelerate chondrocyte adhesion and proliferation (Savaiano andWebster 2004, Park et al. 2005) and titanium surface improve endothelial cell functions (Lu et al. 2008). As the regenerative capability of the central nervous system (CNS) is limited, cellular-based therapies have emerged as a promising route of therapy for CNS-related diseases and injuries (Fischbach et al. 2013).

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Cartilage Regeneration
Cartilage is an avascular tissue composed of chondrocytes entrapped in an extracellular matrix (ECM) rich in proteoglycans and collagens; therefore, current reconstructive options for cartilage repair are limited (Swieszkowski et al. 2007, Kumar et al. 2016. Tissue engineering strategies have been based mostly on matrix seeded with either chondrocytes or MSCs (Li et al. 2005). Biomaterials, such as collagen, fibrin, alginate, chitosan, hyaluronic acid and polyesters have been incorporated into 3D exogenous ECMs for guiding cartilage regeneration (Vinatier et al. 2009). To promote cell attachment to scaffold matrices of implants, surface topography could be modified by nanolithography (Fiedler et al. 2013). A common problem with prosthesis is bacterial adhesion to implants (Costerton et al. 2005). To prevent pathogen adhesion, nanostructured titanium surfaces could be utilized (Singh et al. 2011).

Dentristy
Nanodentistry is an emerging field with significant potential to yield new generation of technologically advanced clinical tools and devices for oral healthcare (Besinis et al. 2015, Neel et al. 2015).

Dental caries management
Multiple innovative applications of nanotechnology have been postulated with the aim of attaining net remineralization of enamel as a non-invasive approach for dental caries management. (ten Cate 2012). NMs used in the field of remineralization comprised nanosilver fluoride, nanosized calcium fluoride, carbonate-HA nanocrystals, nanoparticulate HA, nanosized amorphous calcium phosphate particles, casein phosphopeptide-amorphous calcium phosphate nanocomplexes, nanoparticulate tricalcium phosphate, and bioactive glass NMs (Elkassas and Arafa 2017).

Dental implants
Nanoscale modification of dental implant surfaces to improve recruitment and migration of osteoblasts includes both surface topography as well as chemistry. (Ogawa et al. 2008, Brammer et al. 2009). Nanoscale deposits of HA and calcium phosphate create a more complex implant surface for osteogenesis (Goené et al. 2007

Cell encapsulation
Cell encapsulation, i.e. the immobilization of cells within polymeric microspheres or microcapsules, has permitted the transplantation of cells into human and animal subjects without the need for immunosuppressants, while allowing the bidirectional diffusion of nutrients, oxygen and waste (Murua et al. 2008). Microencapsulation materials have comprised natural or synthetic polymers or blends, including alginate, collagen, gelatin, fibrin, polyphosphazenes, poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(alkylene oxides), poly(vinyl acetate), polyvinylpyrrolidone, PEG, polyethersulfone, polysaccharides such as agarose, cellulose sulfate, chondroitin sulfate, chitosan, hyaluronan, and copolymers, and blends of each (Rabanel et al. 2009). The

Therapy
In the past decades, a variety of nanoscale therapeutic systems have been developed and some of them have been employed in clinical diagnosis and therapy. They have gained a growing interest because of improved half-life in blood circulation, enhanced drug bioavailability, fewer side effects and better synergistic outcomes compared to conventional chemotherapeutic agents (Peng et al. 2015, Zang et al. 2017).

Drug delivery
Nanocarriers are broadly classified into three major categories: i) polymer-based nanocarriers, ii) lipid-based nanocarriers and iii) inorganic platforms (Peng et al. 2015).
Polymer-based nanocarriers is a collective term given to any type of polymer NM, but specifically nanospheres and nanocapsules. Synthetic biodegradable polymeric NMs include Inorganic nanocarriers such as metals, metal oxides, metal sulfides etc. have gained prominent attention only in the recent few years (Huang et al. 2011). In contrast to organic NMs, inorganic NMs possess optical and magnetic properties coupled with other unique physical characteristics such as inertness, stability, and ease of functionalization that makes them superior in cancer imaging and therapy. They are relatively stable over large ranges of pH and temperatures; however, their lack of biodegradation and slow dissolution rates raise concern and uncertainty regarding their degradation and elimination from the body. Noble

Phototherapy (Photothermal and Photodynamic therapy)
Phototherapy, an emerging non-invasive technique, has recently emerged as a viable  photoacoustic and optical imaging since they have a strong optical absorbance in the NIR region that makes them ideal for NIR photothermal ablation therapy.

Immunotherapy
The use of NMs to target the immune system is an intensely active area of research and development. NM-based immunotherapy represents a novel approach for cancer treatment, autoimmune diseases and regenerative medicine therapies.

Autoimmune disease therapy
Autoimmune diseases are a chronic group of diseases that arise from an inappropriate immune response against self-antigens resulting in inflammation and destruction of healthy tissues. Nanocarriers used to deliver anti-inflammatory molecules into target tissue are lipid-

Cancer immunotherapy
Tumors are known to not only avoid immune surveillance but also exploit the immune system to continue local tumor growth and metastasis. Vaccination of dentritic cells, i.e. efficient and targeted delivery of immunomodulatory and co-stimulatory molecules to antigen-presenting cells using NMs, represents a promising strategy in the multimodal treatment for different types of cancer. The most commonly used nanocarriers are lipid-and

Adverse health effects
By directly interacting with the genetic material or disturbing the mitotic spindle and its

Histone modifications
In comparison with DNA methylation, the most diverse epigenetic modifications are those that occur on histone proteins. In nucleosomes, DNA is wrapped around octameric proteins

Epigenetic changes induced by NMs in vitro and in vivo
The metal particles constitute an important class of NMs. Traditionally, the toxicity of metals  Table   2 and Table 3

Titanium
Titanium is extensively used for implanted medical devices, such as dental implants, joint

Iron
SPIONs are widely accepted as powerful MRI diagnostic agents as well as drug carriers targeting CNS. They were rarely studied from the view of epigenetic toxicity; however, they

Zinc
The application of zinc oxide NPs (ZnONPs) with a size of 100 nm is generally recognized as safe by FDA, due to their better biocompatibility compared with smaller ZnONPs

Copper
Copper oxide NPs (CuONPs) are used as anti-microbial reagents and for manufacturing intrauterine contraceptive devices ). Lu and colleagues exposed human and murine macrophages (THP-1 and RAW264.7, respectively) and human small airway

Carbon nanotubes
Low-dose carbon-based fullerene NMs (C60) and multi-walled CNTs (MWCNTs) significantly elevated global DNA methylation in human lung epithelial A549 cells after 24hour incubation ). Single-walled CNTs (SWCNTs) induced also a slight decrease of DNA methylation in the promoter of the ATM gene in lungs. Surprisingly, more genes were epigenetically altered with AuNPs than with CNTs, in contrast with the paradigm that exposure to AuNPs does not induce an adverse biological response (Tabish et al. 2017).
Alterations in DNA methylation, corresponding with lung inflammation, induced by the exposure to MWCNTs in C57BL/6 mice were shown by another group (Brown et al. 2016).
In this study, promoter methylation of inflammatory genes (IFN-γ and TNF-α) correlated with initial cytokine production. DNA methylation of a gene involved in tissue fibrosis (THY1) was also altered in a way that matched collagen deposition. In addition, MWCNT exposure led to DNA hypomethylation in the lung and blood, which coincided with disease

Soft nanomaterials
It was hypothesised that the shape, or more precisely the topography of NMs used as scaffolds for biological and medical applications, seems to affect the cellular epigenome,

Implications for human health
Despite the international effort focused on the identification of normal tissue-specific The reversible character of epigenetic regulations and their responsiveness to external stimuli give us the opportunities for prevention, delay or treatment of complex disorders.
Elucidation of the causal link between exposure-induced epigenetic aberrations and human diseases is an inevitable step for successful application of epigenetic-based therapies and risk assessment.

Future perspectives
Despite the growing body of evidence that support a real risk resulting from NM exposure for human health, this topic remains controversial especially in the area of nanomedicine. Despite the variation in published data and challenges that epigenomic research faces, the association between NM exposure and epigenetic changes remains biologically plausible.
There is a need to distinguish between the physiological response of the cells to the exposure and pathological changes. Further investigations into the long-term effects of NMs on epigenetic modifications and subsequent recovery assays after NP exposure will be required.
Epigenetic effects of coated and soft carriers, that are usually used to prevent toxicity, should be studied.

Conflict of interest
The authors declare that there are no conflicts of interest.