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Mesoporous silica nanoparticles: a potential targeted delivery vector for reproductive biology? “The use of mesoporous silica in reproductive research clearly holds great promise. This biomedical nanomaterial has been well characterized as a robust carrier of nucleic acids, which, from the reproductive biology perspective, is one of the most attractive features of a delivery vector.
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Keywords: mesoporous silica • nanotechnology • reproductive biology
Over the last decade, biomedical nanotechnology has revolutionized existing approaches for the prevention, diagnosis and treatment of a variety of medical conditions, including cancer, microbial and viral infections, and chronic internal diseases. This innovative approach has also given rise to a number of sensitive tools to investigate the specific mechanisms underlying these conditions. Accumulation of evidence for the improved performance of nanomaterial-based techniques over conventional research and therapeutic approaches will inevitably promote the rapid dissemination of nanobiotechnological ‘vision’ to a growing number of research and clinical disciplines, including reproductive biology, which has until recently received only scant attention. Reproductive biology: new frontier for nanotechnology? Reproductive biologists aim to investigate and manipulate the delicate mechanisms involved in the successful formation, transport and fusion of male and female gametes, which ultimately result in fertilization and the induction of embryonic development. Ever since the first human live birth by in vitro fertilization (IVF) in 1978, the field of fundamental and applied reproductive biology has grown exponentially in order to provide the foundation for the safe and effective practice of clinical assisted reproductive technology (ART). Today, ART, a combination of sophisticated laboratory techniques including IVF, intracytoplasmic sperm injection (ICSI), cryopreservation, and the manipulation of gametes and reproductive
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tissues outside of the body, represents the gold standard for infertility treatment, as well as a common tool in breeding livestock and the conservation of endangered species. However, despite such advances in this exciting field, and more than 5 million babies derived from ART, it is widely recognized that overall live birth rates following ART rarely exceed 30%, and therefore exhibit significant scope for improvement. Given the fact that more than 45 million couples worldwide are infertile, and that the obvious cause of infertility cannot be established in approximately a quarter of such cases [1,2] , we are now approaching the stage where novel, less-invasive, perhaps even non-ART, strategies of reproductive treatment are required. Current challenges, to a great extent, arise from our lack of knowledge with regard to the fine molecular mechanisms underlying gamete formation, fertilization, embryogenesis and implantation. Consequently, it is highly likely that substantial scientific breakthroughs in this area will lead to the next generation of research tools, the implementation of new thinking and the development of improved clinical practice. Nanotechnology has the clear potential to become one of these solutions. Indeed, the use of engineered nanodimensional materials for biosensing enables more accurate detection of target substrates, while targeted nanovectors for biological delivery facilitate the precise transport of large amounts of any type of compound to specific destinations, followed by rapid internalization via physiological uptake mechanisms and the ultimate targeting of specific intracellular
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Natalia Barkalina Author for correspondence: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK natalia.barkalina@ obs-gyn.ox.ac.uk
Celine Jones Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK
Kevin Coward Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK
part of
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Editorial Barkalina, Jones & Coward pathways [3] . A steadily growing number of studies have explored the possibility of using a range of organic and inorganic nanomaterials in typical reproductive biology scenarios: experimental gene therapy of fetal monogenic diseases in utero, targeted labeling of sperm populations for bioimaging [4] or sorting purposes [5,6] , and the labeling of preimplantation embryos to facilitate their tracking during IVF culture [7] . Alternatively, nanomaterials have been used to deliver investigational compounds into sperm to assess their functional effects upon intracellular pathways [8] , and the facilitation of sperm-mediated gene transfer (SMGT) [9,10] – a technique based upon the sperm’s inherent ability to spontaneously bind and incorporate exogenous DNA. Such methodology permits molecular constructs to be delivered directly into the oocyte at the time of fertilization in order to create transgenic/mosaic offspring. The use of nanotechnology for reproductive applications is currently in its earliest stages. Consequently, existing publications have generally adopted a pilot design in an attempt to answer the fundamental question of whether a specific nanomaterial is available, or can be designed, that exhibits favorable features for application in reproductive biology. Mesoporous silica: favorable properties for reproductive biology Mesoporous silica is a unique class of synthetically modified colloidal silica with highly ordered mesoscale-sized pores (2–50 nm) [11] , which began to receive attention as a promising tool for targeted drug and gene delivery, bioimaging and tissue engineering over the last decade or so [12] . Today, mesoporous silica is universally recognized as a powerful biomedical nanomaterial, and a solid body of evidence now supports its low cytotoxicity across a variety of cell types [13] . Mesoporous silica is characterized by a number of features that make it particularly attractive as a targeted delivery vector for reproductive biology.
“...extensive nanotoxicology studies of all candidate nanomaterials are paramount to ensure that such revolutionary technologies are safe...
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Firstly, mesoporous silica is robust and, therefore, has an increased likelihood to persist in a targeted cell population following internalization, compared with organic biodegradable materials. Hence, fluorescent modifications of targeted mesoporous silica can be used as highly-selective cytoplasmic/surface cell tags with which to investigate patterns of cellular proliferation, differentiation and migration in cell tracking studies [14] , which represent the cornerstone of repro-
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ductive biology and stem cell research. Secondly, mesoporous silica is chemically inert, and is not prone to induce free radical formation when internalized inside cells, unlike, for example, magnetic iron oxides. This minimizes the risks of free radical-associated DNA damage in gametes and early stage embryos – a proven cause of aberrant embryo development, implantation failure and recurrent spontaneous abortion [15] . In fact, mesoporous silica has even been shown to suppress the production of reactive oxygen species in a model of malignant growth – a finding necessitating further investigation, particularly in a reproductive biology setting [16] . Thirdly, this nanomaterial is highly versatile, but, at the same time, relatively easy to manufacture. Indeed, mesoporous silica can be modified with specific features (size, pore architecture and diameter, surface functionalities/coatings and fluorescent labels) by a series of reasonably straightforward and inexpensive wet-chemistry reactions without significant implications to overall cost [17] . Finally, the unique porous structure of this nanomaterial rapidly increases its total surface area, and, therefore, loading capacity. This minimizes the dose of nanovector required to deliver required amounts of biological cargo, and thus avoid the overexposure of highly specialized and sensitive reproductive tissues and gametes. Ultimately, the primary function of these highly specialized cells is to transmit genetic material to the offspring and are widely known to be vulnerable to suboptimal culture conditions (temperature, light, oxygen tension and culture media) when handled in vitro. Although mesoporous silica has been extensively studied in a variety of somatic cell types, data regarding its biocompatibility with gametes are only just starting to emerge. In a recent publication, we reported that the exposure of mammalian sperm to mesoporous silica nanoparticles under conditions similar to those used during sperm processing for IVF results in the specific binding of mesoporous silica to sperm without acute negative effects upon the main predictors of sperm fertilization potential, such as motility, viability, acrosome morphology and levels of DNA fragmentation [18] . These observations were highly promising, since many nanomaterials, which are widely used for delivery into somatic cells, for example nanogold, can demonstrate toxicity when applied to gametes [19,20] . We have also obtained encouraging pilot data regarding the effects of mesoporous silica upon mammalian oocytes, indicating that the persistence of nanoparticles in the ooplasm following microinjection does not promote oocyte degeneration [Barkalina N et al., Unpublished Data] , unlike certain polymeric nanoparticles which, if injected into fertilized eggs, are known to interfere with embryo development [7] . In addition, mesoporous
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Mesoporous silica nanoparticles: a potential targeted delivery vector for reproductive biology?
silica has also been investigated as a compound delivery system in zebrafish embryos – a common animal model for reproductive biology studies – with highly positive outcomes in terms of safety and efficacy [21] . Future perspective The use of mesoporous silica in reproductive research clearly holds great promise. This biomedical nanomaterial has been well characterized as a robust carrier of nucleic acids, which, from the reproductive biology perspective, is one of the most attractive features of a delivery vector. Targeted transgenesis is a fundamental component of reproductive biology, and availability of an alternative to common gene transfer techniques – electroporation and the use of viral vectors, both of which are associated with contradictory safety profiles in gametes and reproductive cells – would be highly favorable. Safe and effective gene transfer techniques could be implemented to precisely manipulate gene expression to investigate the role of specific molecular pathways during gametogenesis and embryogenesis, and discover the direct causes of currently unexplained cases of infertility, associated with particular genetic polymorphisms. Similarly, nanotechnology could facilitate targeted and more controllable delivery of diagnostic agents to study ligand–receptor interactions between sperm and oocytes at the time of gamete fusion for both fertility-promoting and contraceptive purposes, or at the fetomaternal interface during embryo implantation. In addition, in the context of ART, nanoparticle-mediated delivery into gametes and embryos could be used to directly and noninvasively supplement molecular deficiencies associated with aberrant gametogenesis, fertilization and embryo development, thereby increasing chances of success of the procedure. For a comprehensive overview of the emerging potential and possible application of nanomaterials in the field of reproductive medicine, refer to our recent review [22] . However, as with any novel technology, a number of critical unresolved issues must be addressed before the whole concept of nanoparticle-medicated delivery
is introduced into reproductive applications. Reproductive tissues, gametes and embryos represent highly specialized cells with unique morphology and membrane composition, which, in their mature forms, are relatively inert to the uptake of exogenous substances for the purpose of evolutionary protection. They do not exhibit prominent endocytotic activity, lack repair mechanisms for oxidative stress and require very delicate handling in vitro. Any delivery vector applied in the context of reproductive biology should therefore preserve the viability and function of these delicate targets, the structural and spatial organization of their DNA, and not interfere with gene-expression profiles and key metabolic reactions, unless specifically required for experimental purposes. The availability of a versatile delivery vehicle with small-size, largeloading capacity and spontaneous internalization into target cells therefore holds great promise for the field of reproductive biology and may represent elegant biological tools for clinical and/or research application. However, extensive nanotoxicology studies of all candidate nanomaterials are paramount to ensure that such revolutionary technologies are safe and do not result in late-onset effects in the resulting progeny. Financial & competing interests disclosure The authors have a patent pending related to the work discussed in this article entitled ‘Delivery Method’ (PCT Patent Application Number PCT/GB13/053394 filed on the 20th December 2013). N Barkalina is funded by the Clarendon, Scatcherd European and Cyril & Phillis Long Schemes. The project is also funded by the Nuffield Department of Obstetrics and Gynaecology, and an EPSRC Pathways to Impact Award (University of Oxford). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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