WO2014102539A1

WO2014102539A1 - Delivery method using mesoporous silica nanoparticles

Info

Publication number: WO2014102539A1
Application number: PCT/GB2013/053394
Authority: WO
Inventors: Kevin COWARD; Celine JONES; Junaid KASHIR; Natalia BARKALINA; Helen Elizabeth Townley
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2012-12-31
Filing date: 2013-12-20
Publication date: 2014-07-03
Anticipated expiration: 2015-06-30

Abstract

The invention relates to a method for delivering a molecule to a reproductive cell or an embryonic cell. The invention uses mesoporous silica nanoparticles.

Description

DELIVERY METHOD USING MESOPOROUS SILICA

NANOPARTICLES

Field of the Invention

Background of the Invention

Nanoscience is a novel and rapidly developing integrative discipline that studies and manipulates physical matter at the nanometer scale. Nanoparticles are being extensively introduced into biomedicine due to their unique physicochemical properties, providing large loading capacity, stability, highly selective affinity, and potential for multiple, simultaneous applications. In particular, nanoparticles are increasingly used as experimental diagnostic and therapeutic tools to deliver molecules to cells.

The multifunctionality of nanoparticles opens wide perspectives for their reproductive applications. These may range from tools for early diagnosis and simultaneous personalised therapy of slow-progressing or resistant to conventional therapy diseases, to targeted noninvasive delivery of genes/therapeutic agents into adult reproductive tissues, gametes, and, perhaps, embryos

Data regarding the use of nanoparticles in reproductive science are, however, very scarce. Current research is stimulated mainly by concerns about potential gonadotoxicity of

nanomaterials widely applied in consumer products. Therefore, most studies evaluate the impact of acute and chronic exposure to nanoparticulate titanium oxide, silver, gold and carbon on reproductive function and embryo development in various animal species (for instance Taylor et al, (2012) Adv Exp Med Biol 733 : 125-33). Among these, titanium oxide and silver induce noticeable cytotoxic reactions and are rarely used clinically. Another significant proportion of trials assess the ability of nanoparticles to cross the placental barrier and affect foetal

development in the similar context of growing chronic exposure to nanostructures (for instance Menezes et al, (2011) Curr Pharm Biotechnol 12(5): 731-42).

Only a limited number of research groups have applied nanoparticles as experimental tools for delivery to reproductive cells. Some groups have shown sperm loading with magnetite nanoparticles (Makhluf et al. , (2008) Small 4(9): 1453-8; Kim et al, (2010) Reprod Domest Anim 45(5): e201-6), sperm exposure to nanoparticulate gold (Wiwanitkit et al. ((2009) Fertil Steril 91(1): E7-E8; Taylor et al., (2010) Reproduction in Domestic Animals 45: 60-60) and use of quantum dots to tag sperm for non-invasive investigation of sperm migration and fertilization (Feugang et al., (2012) J Nanobiotechnology 10(1): 45).

Ben-David Makhluf et al. (2008 supra) described binding of anti-protein kinase C (PKC) alpha antibody to magnetite nanoparticles coated with PVA and (3-aminopropyl)

trimethoxysilane (diameter - 11±2 nm), to function as magnetic protein carriers into sperm. Internalisation of nanoparticles was observed, however viability of loaded sperm remained unknown.

Kim et al. (2010 supra) demonstrated successful gene construct (EGFP) delivery by magnetite nanoparticles into boar sperm for subsequent sperm-mediated gene transfer into porcine embryos. In this experiment, nanoparticle-loaded sperm preserved fertilization capacity and served as a natural oocyte transfection vector, as evidenced by expression of the exogenous gene (EGFP) in the cytoplasm of morula stage embryos.

Unlike magnetite nanoparticles, which have not been reported to exert a detrimental effect upon sperm functionality and seem to penetrate sperm in a relatively straightforward manner, nanoparticulate gold has been characterized as potentially toxic to sperm and rarely internalizing into this highly specialized cell. Wiwanitkit et al. (2009 supra) reported penetration of 9-nm gold nanoparticles into the human sperm head and tail after 15 minutes of exposure, associated with a 20% decrease in progressive motility and severe morphological alterations in exposed sperm. A similar decrease in bovine sperm motility following contact with nanoparticulate gold has been described by a different research group (Taylor et al, 2010 supra). In this case, however, despite exposure to extreme concentrations of gold nanoparticles, no changes in sperm morphology or membrane integrity were observed.

A recent publication reported the possibility of tagging boar sperm with quantum dots coated with arginine-rich cell penetrating peptide R9 (diameter - 20-25 nm) for in vivo tracking purposes in sperm transport studies utilizing a fluorescence endo-microscopy approach (Feugang et al., 2012 supra). Labelling of sperm was not shown to affect sperm motility, membrane integrity and fertilization potential.

Other researchers have investigated nanoparticle delivery in embryonic cells. Several research groups have reported beneficial effects of silver (2-35 nm) and heparan sulphate- conjugated gold (2-70 nm) nanoparticles as metabolic enhancers and anabolic agents in chicken embryos (e.g. Zielinska et al, (2011) Int J Nanomedicine 6: 3163-72). No negative impact of nanoparticulate gold upon development of zebrafish and murine embryos has been reported in a series of studies, even in confirmed presence of gold nanoparticles inside the embryos (as reviewed in Barchanski et al, (2011) Reprod Domest Anim 46 Suppl 3 : 42-52). Yang et al. ((2011) J Surg Res 171(2): 691-9) successfully applied chitosan nanoparticles (100-500 nm) conjugated with enhanced green fluorescent protein (EGFP) gene for in utero gene delivery into mouse embryos to demonstrate feasibility of non- viral foetal gene therapy in animal models of human monogenic diseases. Other researchers used polysterene/polyacrylonitrile nanoparticles (40-120 nm) for external and cytoplasmic tagging of mouse embryos, and demonstrated negative impact of intracytoplasmic nanoparticle injections, but not external attachment, on embryo development (Fynewever et al., (2007) J Assist Reprod Genet 24(2-3): 61-5).

A major concern over the use of nanoparticles in reproduction is their potential toxicity to the germline, gametes and embryos, which may result in transgenerational effects. For instance, silver and gold nanoparticles have been demonstrated to alter DNA integrity by inducing strand breaks, point mutations and oxidative damage; however, such effects were largely dependent upon the particle dose and size (Taylor et al, (2012) supra). Another significant concern is that nanoparticle uptake by gametes can differ from that by somatic cells due to the unique composition of gamete cell membranes. Remarkably, sperm membranes contain significantly higher levels of phospholides with unsaturated fatty acid chains as compared to somatic cells. These chains are crucial for membrane fluidity and elasticity enabling active cell movement (Tapia et al, (2012) Reprod Domest Anim 47 Suppl 3 : 65-75). In oocytes, successful

internalisation of nanoparticles should involve passage through both the zona pellucida and oolemma, together representing a substantial mechanical barrier. Finally, the 'fate' of gamete- or embryo-internalised nanoparticles remains unknown, particularly their localisation profiles, and the possibility for degradation or expulsion at later stages.

Summary of the Invention

The invention is based on the surprising finding that mesoporous silica nanoparticles (MS Ps) may be used to deliver molecules to sperm cells, ova and embryonic cells.

The inventors have surprising shown that mesoporous silica nanoparticles comprising molecules of interest are capable of highly efficient delivery of the molecules into sperm cells, ova and embryonic cells. Accordingly the invention provides a method of delivering at least one molecule into a reproductive cell or an embryonic cell, the method comprising contacting the cell with a mesoporous silica nanoparticle (MSNP) comprising the at least one molecule and thereby delivering the at least one molecule into the cell.

The invention also provides:

- use of a MSNP comprising at least one molecule to deliver the at least one molecule into a reproductive cell or an embryonic cell; a method of treating a reproductive disease or disorder in a patient in need thereof, the method comprising delivering into a reproductive cell or an embryonic cell of the patient at least one reproduction-promoting agent by contacting the cell with a MSNP comprising the at least one agent and thereby treating the reproductive disease or disorder in the patient;

a method of diagnosing a reproductive disease or disorder in a patient, the method comprising delivering into a reproductive cell or an embryonic cell of the patient at least one diagnostic agent by contacting the cell with a MSNP comprising the at least one agent and thereby diagnosing the reproductive disease or disorder in the patient; and

a method of promoting fertilisation of an ovum by a sperm comprising the point mutations H398P or H233L, or any other mutation in phospholipase C zeta (ΡΙ ζ), or by sperm devoid of ΡΙΧζ, or by sperm exhibiting aberrant expression or localisation patterning of ΡΙΧζ), the method comprising delivering ΡΙΧζ protein or a polynucleotide encoding ΡΙ ζ into the ovum by contacting the ovum with a MSNP comprising the protein or polynucleotide and thereby promoting fertilisation of the ovum by the sperm.

Brief Description of the Figures

Figure 1 shows the diameter of synthesised MSNP as assessed by sedimentation velocity during centrifugation in a liquid gradient, a) Non-fluorescent non-coated MSNPs (~250nm and ~300nm peaks), b) Non-fluorescent PEI-coated MSNPs (~130nm and ~210nm peaks); c) FITC- labelled non-coated MSNPs (~250nm peak); d) FITC-labelled PEI-coated MSNPs (-lOOnm, ~170nm, and -250 nm peaks).

Figure 2 shows the association rate between synthesised MSNPs and boar sperm. Boar sperm were treated under various conditions, including the type and concentration of MSNPs, and exposure time. Data from replicate experiments are presented as means. Error bars correspond to SEM. Symbols (*, #) indicate significant differences (p<0.05), as estimated by factorial ANOVA followed by post-hoc comparison of the means using Bonferroni adjustment. NS = 'not significant' (p>0.05). For each time period, the first (left-hand) column is FITC- labelled non-coated MSNPs, the second column is FITC-labelled PEI-coated MSNPs

0.03mg/ml, the third column is FITC-labelled PEI-coated MSNPs 0.3mg/ml and the fourth column is fluorescent siRNA-bound PEI-coated MSNPs. Figure 3 shows the association of MSNPs with boar sperm after 3 hours of exposure (surface adherence/internalisation, 40x-objective magnification). Images are presented as experimental-control pairs. Circles indicate MSNP-sperm associations. a,b) FITC-labelled non- coated MSNPs (0.03mg/ml): MSNPs attachment to sperm head, midpiece and tail can be observed, along with a large number of free MSNP agglomerates. c,d) FITC-labelled PEI-coated MSNPs (0.03mg/ml): a discrete green dot-shaped fluorescent signal in the projection of the sperm head, is suggestive of MSNPs internalisation. e,f) FITC-labelled PEI-coated MSNPs (0.3mg/ml): large round-shaped signal, partially overlaying the sperm head, suggestive of the surface MSNPs attachment. g,h) Fluorescent siRNA-bound PEI-coated MSNPs (0.3mg/ml): a discrete dot-shaped red fluorescent signal in the projection of the sperm head, suggestive of MSNPs internalisation.

Figure 4 shows the localisation of MSNPs associated with sperm in relation to the main sperm parts (head, midpiece, and tail), a) FITC-labelled non-coated MSNPs (0.03mg/ml). b) Fluorescent siRNA-bound PEI-coated MSNPs (0.3mg/ml). c) FITC-labelled PEI-coated MSNPs (0.03mg/ml and 0.3mg/ml). Assessment performed at the 40x-objective magnification. Data are presented as percentages of the total number of sperm associated with MSNPs. Results from replicate experiments were pooled, and analysed as a single dataset. '*' indicates significant differences compared to baseline (p<0.05), as estimated by the two-tailed z-test for proportions.

Figure 5 A shows the association of MSNPs with boar sperm after 3 hours of exposure (surface adherence/internalisation, confocal microscopy, 63x-oil immersion objective magnification). White arrows indicate suspected MSNP internalisation into the sperm. FITC- labelled non-coated MSNPs attach to the sperm membrane in the head and midpiece region as clusters of particles (upper and middle row of images in the group), and as discrete structures (lower row of images in the group). FITC-labelled PEI-coated MSNPs demonstrate greater affinity to the sperm head membrane, appearing as discrete particle agglomerates with a small diameter. Figure 5B also shows the association of MSNPs with boar sperm after 3 hours of exposure (surface adherence/internalisation, confocal microscopy, 63x-oil immersion objective magnification). Black arrows indicate suspected non-fluorescent MSNPs association with thesperm. Fluorescent siRNA-bound PEI-coated MSNPs demonstrate affinity to the sperm head membrane, appearing as discrete particle agglomerates with a small diameter, similarly to FITC- labelled PEI-coated MSNPs (upper row of images in the group).

Figure 6 shows the association of FITC-labelled PEI-coated MSNPs with mouse oocytes after 2 hours of exposure (20x-objective magnification). A dose-independent attachment of MSNPs to the surface of the zona pellucida was observed after oocyte incubation with

0.03mg/ml and 0.3mg/ml MSNP solutions.

Figure 7 shows mouse oocytes 3 hours after microinjection with FITC-labelled non- coated/PEI-coated MSNPs (2 Ox-objective magnification). Arrows indicate localisation of MSNPs. Following injection, FITC-labelled PEI-coated MSNPs localised in the oocytes produce discrete green signals.

Figure 8 shows characterisation of mesoporous silica nanoparticles (MSNPs). A) Transmission electron microscopy image of unmodified MSNPs. Scalebar = 0.05μπι; B) Scanning electron microscopy image of unmodified MSNPs. Scalebar = 0.1 μπι. Synthesised MSNPs were characterised by homogenous size, slightly non-spherical shape with elongation in the direction of the pore channels, and nanometre-sized pores with hexagonal symmetry.

Figure 9 shows proportions of motile, progressively motile and viable boar sperm after 2 and 4 hours of exposure to different modifications of unloaded MSNPs in three particle/cell ratios assessed by CASA and eosin Y staining (mean±SEM from six repeats in the control samples and three repeats in the experimental samples). Mean motility and viability parameters in MSNP -treated samples remained unaltered compared to time-matched controls, regardless of the incubation time, type and dose of MSNPs (p>0.05).

Figure 10 shows motility parameters of boar sperm assessed by CASA after 2 and 4 hours of exposure to different modifications of unloaded MSNPs in three particle/cell ratios (mean±SD from six repeats in the control samples and three repeats in the experimental samples). Mean sperm kinematic parameters in MSNP -treated samples remained unaltered compared to time-matched controls, regardless of the incubation time, type and dose of MSNPs (p>0.05).

Figure 11 shows association of unloaded MSNPs with sperm. A) Control; B) Association of unloaded MSNPs with sperm. Nanoparticles associated with sperm produced discrete fluorescent signals in the projection of various sperm regions (white arrows indicate MSNP- sperm associations). Scalebar = ΙΟμπι. C-E) Association of unloaded MSNPs with sperm. MSNPs bind to the sperm head and midpiece. Scalebar = 5μπι.

Figure 12 shows association rates between boar sperm and different modifications of unloaded mesoporous silica nanoparticles (MSNPs) after 2, 3 and 4 hour of exposure in three particle/cell ratios (mean±SEM from three repeats of the experiment). Association rate after 2 hours of exposure to unmodified MSNPs in the 30μg per 10⁷ sperm ratio was significantly higher compared to lower parti cle/sperm ratios (* p<0.05 vs association rate for the 30μg per 10⁷ sperm ratio). Figure 13 shows association of loaded MS Ps with sperm. A) Lamin A/C siRNA- loaded MSNPs; B) mCherry-loaded MSNPs. The density of sperm coating with lamin A/C siRNA-loaded MSNPs was lower, compared to mCherry-loaded MSNPs. Scalebar = 5μιη.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a nanoparticle" includes two or more such nanoparticle, reference to "a cell" includes two or more such cells, reference to "a molecule" includes two or more such molecules, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method

The method of the invention concerns delivering at least one molecule to a reproductive cell or an embryonic cell. The method may be carried out in vivo, in vitro or ex vivo. The reproductive cell may be returned to the body from which is was isolated following delivery of the at least one molecule.

The invention also provides a MSNP comprising at least one molecule for use in delivering the at least one molecule into a reproductive cell or an embryonic cell, the method comprising contacting the cell with the MSNP comprising the at least one molecule and thereby delivering the at least one molecule into the cell.

Reproductive cell or embryonic cell

The method of the invention is for delivering at least one molecule into a reproductive cell or an embryonic cell The reproductive cell is preferably a sperm, an oocyte, an ovum (each of which is described herein as a "cell").

Sperm and oocytes represent highly specialised cells with unique structure and functions. There is mounting evidence, that nanoparticle uptake by cells equally depends on the surface chemistry of the particle and the structure and physiology of target cells. As such, data regarding safety and efficacy of nanoparticles obtained in a somatic cell line are virtually impossible to translate into reproductive applications.

The sperm cell membrane contains a significantly greater proportion of unsaturated fatty acids, compared to most somatic cells, to increase flexibility and elasticity of the membrane and allow sperm to swim (Tapia et al. supra). It also bears a stronger negative charge than most somatic cells, has limited permeability to exogenous molecules and a great degree of functional compartmentalisation (Partington et al., (2007) Soc Reprod Fertil Suppl 65: 469-74). A hypothesis that nanoparticle interaction with sperm follows a specific pattern has been confirmed by Taylor et al. (2010 supra), who failed to deliver gold nanoparticles, rapidly uptaken by immortalised endothelial cells, into sperm. It remains unknown, to what extent the mechanism of nanoparticle internalisation in sperm remains preserved, and what the fate of internalised nanoparticles is. For instance, since sperm lack 'traditional' lysosomes, mechanisms of nanoparticles internalisation and cellular trafficking would most likely be non-lysosome mediated. Such mechanisms have been characterised as increasing nanoparticles life in the cytoplasm, and slowing down expulsion from the cell (Rees et al., (2011) BMC SystBiol 5: 31). In summary, both nanoparticle internalisation into sperm and interaction with intracellular structures are expected to differ dramatically from somatic cells, justifying separate studies of nanoparticles in the reproductive biology/medicine context.

The oocyte and early stage embryo is surrounded by an external glycoprotein meshwork (zona pellucida), which provides protection throughout the development, representing a substantial mechanical barrier. Transport of nutrients and biologically active molecules into the oocyte before ovulation occurs through intimate contacts with processes of granulosa cells, penetrating the zona pellucida. Mechanisms of transport through the zona pellucida in post- ovulatory oocytes and mammalian preimplantational embryos have not been extensively studied. As such, spontaneous nanoparticle penetration into the oocytes represents a highly challenging task and has not been described yet.

The key difference of sperm and oocytes from somatic cells is their essential function - transmission of genetic information to progeny. Therefore, nanoparticles applied in gametes should be deprived of acute toxicity and capability to induce long-term transgenerational effects, nanoparticles should not affect viability and functionality of gametes/embryos, packaging and integrity of DNA, gene expression profiles, protein biosynthesis, energy production, cell division, or induce apoptosis, release of reactive oxygen species and associated cell breakdown. Nanoparticle-mediated delivery has never been studied from such perspective, since most current uses of nanoparticles are focused in the fields where nanotoxicity in target cells is a neutral feature or a benefit, rather than a side effect (oncology, autoimmune and infectious diseases).

A sperm is a male reproductive cell. It typically comprises a single flagellum (i.e. is a spermatozoon). The sperm is typically motile.

An oocyte is a female reproductive cell. It is an immature ovum derived from an ovarian follicle. Oocytes form ova.

An ovum is a mature female egg cell. It is a female egg cell that is capable of being fertilised to form an embryo.

Oocytes and ovums all typically have a vitelline membrane surrounding their cell membrane. The vitelline membrane is a glycoprotein protein layer which binds sperm. In mammals, the vitelline membrane is called the zona pellucida. The vitelline membrane (or zona pellucida) forms a substantial mechanical barrier to entry into the oocyte or ovum. It is surprising that the MS Ps defined herein can penetrate into oocytes and ova (i.e. penetrate the zona pellucida) without physical manipulation, such as microinjection.

An embryonic cell is a cell derived from a fertilised embryo. An embryo is a

multicellular diploid eukaryote in its earliest stage of development. The embryonic cell may be derived from an embryo at any stage of embryogenesis. The embryonic cell is preferably derived from an early stage embryo, such as less than 1, 2, 3, 4, 5 or 10 days following fertilisation. The embryonic cells is most preferably a fertilised ovum (i.e. a single cell embryo).

A skilled person is capable of determining when an embryo of a particular species become a fetus (i.e. is no longer an embryo). For instance, in humans, an embryo typically becomes a fetus at about 8 weeks post fertilisation. The embryonic cell is preferably not a human embryonic cell or an embryonic stem cell.

For the purposes of the invention, the embryonic cell is not a human embryonic kidney (HEK) cell line. The HEK293 is a cell line derived from a tissue culture of normal human embryonic kidney cells. This cell line is widely used in cell biology research and biotechnology due to fast growth, ease in maintenance and straightforward transfectability. The HEK293 cell line was established in the early 1970s by the group of Alex Van der Eb at the University of Leiden in Netherlands through transformation and culturing of normal HEK cells with sheared adenovirus 5 DNA. The transformation inserted an approximately 4.5-kilobase fragment from the viral genome into the human chromosome 19 of the HEK cells.

HEK293 is an experimentally transformed cell line, and their structure and functions are altered, compared to normal HEK cells. Therefore, these cells do not represent a very accurate model for most somatic cells, including normal and cancer, and certainly, not for highly specialized cells, such as gametes. However, they can provide preliminary details regarding cell behavior upon contact with a particular structure (nanoparticles), and are commonly used to express recombinant proteins, propagate adenoviruses and replicate retroviruses.

It is of particular worth to note, that any embryonic cell line will not be an accurate representation of cell lines in a gamete/preimplantational mammalian embryo. Embryonic cell lines, unless they are embryonic stem cells, have been committed to a specific fate, namely differentiation into a specific cell line. They are diploid cells with an entirely different morphology and physiology, and cannot serve as a model to study nanoparticle internalisation and effects in gametes/preimplantational mammalian embryos.

Following delivery of the at least one molecule using the method of the invention, the reproductive cell or the embryonic cell preferably remains viable. Preferably, the reproductive cell or the embryonic cell does not die following the delivery of the at least one molecule. This can be measured using standard techniques in the art, such as using microscopy.

Following delivery of the at least one molecule using the method of the invention, the reproductive cell preferably retains its reproductive ability. The sperm preferably retains its ability to be mobile (i.e. retains its motility). This can be measured as described in the Example. The sperm preferably retains its ability to fertilise an ovum. This can be determined by contacting the sperm with an ovum and measuring its ability to fertilise the ovum using standard techniques.

The oocyte preferably retains its ability to develop into an ovum. An ovum preferably retains its ability to be fertilised by a sperm and form an embryo.

Following delivery of the at least one molecule using the method of the invention, the embryonic cell preferably retains its developmental ability. An embryonic cell preferably retains its ability to form a fetal cell. Such abilities can be measured using techniques that are known in the art.

The reproductive cell or the embryonic cell is preferably mammalian. Typically, the cell is human in origin, but alternatively it may be from another mammal such as from commercially farmed animals, such as horses, cattle, sheep or pigs, or may alternatively be from pets, such as cats, dogs or rodents (especially rats and mice). The embryonic cell is preferably not derived from a zebra fish.

The reproductive cell or the embryonic cell can be isolated and maintained in culture using standard techniques known in the art. Suitable media are described below with reference to contacting the reproductive cell or the embryonic cell with the MSNP comprising the at least one molecule and in the Example. Molecule

Any molecule(s) may be delivered into the reproductive cell or the embryonic cell using the invention. The molecule is preferably a therapeutic agent or a diagnostic agent. A therapeutic agent is a molecule that is capable of ameliorating or abolishing one or more symptoms of a disease or disorder. A therapeutic agent is preferably a molecule that is capable of curing a disease or disorder. A diagnostic agent is a molecule that is capable of indicating the presence of a disease or disorder. The disease or disorder is preferably a reproductive disease or disorder. The disease or disorder may be any of those discussed below and is preferably infertility. Suitable therapeutic and diagnostic agents are known in the art.

Table 1 shows a variety of proteins expressed by sperm and their roles in sperm. The therapeutic agent may be any molecule that promotes the expression of and/or activity of one or more of these proteins. For instance, the therapeutic agent may be a polynucleotide encoding one of the proteins shown in Table 1. The diagnostic agent may be any molecule which is capable of identifying the presence of and/or expression of one or more of the proteins in Table 1, such as an antibody against one of the proteins or a polynucleotide which specifically hybridises to the rnRNA sequence encoding one of the proteins. The diagnostic agent may be any molecule which is capable of identifying a mutation in the gene expressing one of these proteins.

Table 1 - Proteins expressed in sperm and their roles

sperm maturation

CASPASE3 Proteolytic cleavage

Ubiquitin-conjugating enzymes Involved in recognition and ubiquitination of target El, E2, E3 proteins destined for destruction by proteosome

Ubiquitin-recycling enzyme C- terminal hydrolase PGP9.5

Anaphase promoting complex

(APC, E3 subtype)

SCF complex (E3 subtype)

Sperm thioredoxins Sptrx 1 and Small redox proteins that function as general protein 2 disulphide reductases and regulate several cellular processes including transcription binding factor activity and apoptosis.

membrane peroxidise 15- Interacts with a membrane anti-oxidant (glutathione lipoxygenase (LOX) peroxidase-4) to regulate novel redox-dependent cell death pathway

PAWP Sperm-specific WW-domain binding protein, responsible for post-acrosomal assembly of sperm head protein

PLC 54 (+ALT 1, ALT2, ALT3) Involved in calcium signalling during acrosome reaction

HAPRIN Zinc-finger and coiled-coil domain (RBCC) motif protein involved in acrosome reaction

HongrESl Epididymal secretory protein, coats sperm head and regulated capacitation

Tables 2 and 3 show a variety of genes which are mutated in disorders of human male differentiation, spermatogenesis and sperm function. The therapeutic agent may be any molecule that restores the correct function of one or more of these genes, such as the non- mutated (i.e. wild-type) protein product of one or more of these genes or a polynucleotide comprising the non- mutated (i.e. wild-type) sequence of one or more of these genes. The diagnostic agent may be any molecule which is capable of identifying the presence and/or expression of one or more of these genes and/or identifying a mutation in one or more of these genes. Tables 2 and 3 also show a variety of chromosomal defects associated with disorders of human male differentiation, spermatogenesis and sperm function. The diagnostic agent may be one which is capable of identifying one or more of these chromosomal defects.

Table 2 - Mutated genes and chromosomal defects associated with defects in human male differentiation

Ambiguous genitalia Steroid biosynthesis: CYP11A1; CYP11B1; CYP11B2; CYP17; CYP21;

CYP21A2; HSD3B2; POR; SRD5A2; StAR

Others or sex reversal:

AMH; AMHR2; AR; ARX; LHCGR; LHR; NRFA1;

NROB l; RSP01; SOX9; SRY; WT1

Gonadal dysgenesis CYP11A1; Dicentric Y chromosome; NR5Al(Sfl);

NROB l; SRY; SRY promoter; WT1

Hypospadias AR; ATF3; MAMLD1 (CXorf6); Dicentric Y; EF B2;

ESR1; ESR2; FGFR2; HOXA13; HOXD11; HOXD13; INSL3; MIDI; RXFP2 (LGR8/GREAT)

Micropenis Gene defects:

ALKBHl; AHRR; ALG12; ESR1;

GHR; RSA1; SOX2; TBX3

Structural chromosome defects:

Chromosomes 2, 4, 5, 6, 7, 8, 11,

12, 14, 15, 16, 17, 18 and 21

Numerical defects:

Ring X, Y; Trisomy 14; 49, XXXXY;

47, XYY

Cryptorchidism ARX; CYP19A1; DHH; ESR1; HOXD13; INSL3; KRAS;

NRSA1; PTPN11; PWCR; RAFl; RXFP2; SOS1;

SOX2; SPAG4L; SPATA12; ZNF214; ZNF215

Testis cancer AR; BMP; CTNNB l; DIABLO; DND1; EGFR; EEF1A;

FOXL2; GNAS; HMGA1; HMGA2; KIT; KND1; KRAS; NANOG; PATZ1; POLG; POU5F1; REG1; SMADl; SMAD5; SOX2; SOX17; SPATA12. TSPY1; WT1

Vas deferens CFTR, HNF1B

Table 3 - Mutated genes and chromosome defects affecting spermatogenesis and sperm function

Defect Mutated genes and chromosomal defects

Abnormal spermatogenesis ATM; ATMAC; DAZL; ERCC2; GTF2A1L; JUN;

NLRP14; NRBOBl; POLG; PRMl; PRM2; SDHA; SOX8; XRCC1; YBX2

Azoospermia APOB; ACSBG2; ART3; ATM; BOULE; BPY2; BRCA2;

CDY1; CFTR; CREM; DAZ; DDX25; DDX3Y; DRFFY; ERCC1; ERCC2; FASLG; FHL5; FKBP6; HNRNPC; HSFY1; KLHLIO; LAP3; MBOAT1; MEI1; MLH1;

MLH3; MTR; NLRP14; PRDM16; RBMX; RBMY1A1; RBMY1F; SPATA16; SYCP1; SYCP3; TAF7L;

TGIF2LX; TSPY; TSSK4; UBE2B; USP26; UTP14C; USP9Y; UTY; XPC; XPD; XRCC1; YBX2; ZNF230

Oligospermia MT-ATP6; EGF; FASL; H19 and MEST; KLHLIO; PIGA;

PRMl; PRM2; SHBG; SDHA; TSSK4; UBE2B; VASA

Asthenozoospermia AKAP3; AKAP4C; CATSPER2; DNMT3B; DHAH5;

DNAHl l; DNALl; PDYN; GNA12; Mitochondrial DNA; MTHFR; MT- D4; PIGA; POLG; PPM1G; PRKAR1A;

SHBG; SPAG16; TEKT1; TEKT2; TPN1; TPN2;

TXNDC3; T mt DNA haplotypes

Teratozoospermia AUPvKC; PRM1; PVRL2; SPATA16; SP1

Oligoasthenozoospermia JUND; mt-ND4; NALP14

Oligoasthenoteratozoospermia MTRR; IL1B; SABP

Acrosome or fertilization POIA3

DNA damage GSTM1

Infertility AR; GSTM1 KIT; KITLG; ILIA; OAZ3; PRM1; TSPY;

TSSK4; USP26; YBX2

Varicocele effect MT-ATP6; MT-ATP; CACNA1C; MT-COl; MT-C02;

MT-ND3

Chromosome defect Numerical sex chromosome (Klinefelter' s; XXY- XXXXY)

Structural chromosome (translocations, inversions or deletions)

Y chromosome microdeletions, XX male or XY female

Therapeutic and diagnostic agents for use in the invention may be directed towards any of the genes, proteins, mutations or defects disclosed in Matzuk and Lamb (2008) Nature Medicine 14(11): 1197-1213.

Heytens et al. (2009) Hum Reprod 2009;24:2417- 2428 describes a point mutation, H398P, in an essential sperm-derived egg activation factor phospholipase C zeta (PLCζ) in an infertile male with normal sperm morphology. They also describe cases of infertile men whose sperm exhibited reduced levels of PLCζ , or abnormal localization patterns of PLCζ in the sperm head. The diagnostic agent is preferably one which identifies this point mutation (or other identified mutations), or cases of aberrant expression or localization within the sperm pertaining to phospholipase C zeta (PLCζ). Such a diagnostic agent may be delivered to a sperm cell in accordance with the invention. Similarly, the therapeutic agent is preferably PLCζ protein, more preferably human PLCζ protein, or a polynucleotide encoding PLC^ more preferably a polynucleotide encoding human PLCζ. Such a therapeutic agent may be delivered in accordance with the invention to sperm having the mutation or an oocyte or ovum which is to be fertilised with sperm having the mutation, or exhibiting deficiency in PLCζ expression or localization.

The at least one molecule is preferably a dye or a fluorescent molecule. Once a dye or a fluorescent molecule is delivered to a reproductive cell or an embryonic cell, it may be used to identify the cell or track its movement in vivo or in vitro. Fluorescent molecules may also be used to sort and/or quantify reproductive cells or embryonic cells. For instance, cells loaded with fluorescent molecules using the invention may be sorted and/or quantified using fluorescence activated cell sorting (FACS) analysis.

Suitable fluorescent molecules include, but are not limited to, fluorescent proteins, such as Green Fluorescent Protein (GFP), Yellow FP (YFP) and Red FP (RFP), xanthene derivatives, such as fluorescein, fluorescein isothiocyanate (FITC), rhodamine, Oregon green, eosin and Texas red, cyanine derivatives, such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine, naphthalene derivatives (dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives, such as pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole, pyrene derivatives, such as cascade blue, oxazine derivatives, such as Nile red, Nile blue, cresyl violet and oxazine 170, acridine derivatives, such as proflavin, acridine orange and acridine yellow, arylmethine derivatives, such as auramine, crystal violet and malachite green, and tetrapyrrole derivatives, such as porphin, phtalocyanine, bilirubin.

The molecule is preferably a calcium ionophore. Calcium ionophores facilitate transport of calcium ions across cell membranes. Suitable calcium ionophores are known in the art.

The molecule may have any chemical composition. The molecule is preferably a polymer, amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide, polynucleotide or morpholino.

The molecule is preferably selected from amino acids, peptides, polypeptides and/or proteins. The amino acid, peptide, polypeptide or protein can be naturally-occurring or non- naturally-occurring. The polypeptide or protein can include within them synthetic or modified amino acids. A number of different types of modification to amino acids are known in the art.

The protein can be selected from enzymes, antibodies, antibody fragments, hormones, growth factors or growth regulatory proteins, such as cytokines. The cytokine may be selected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-13, interferons, preferably IL-γ, and other cytokines such as TNF-a. The protein may be a bacterial protein, a fungal protein, a viral protein or a parasite-derived protein.

As discussed above, the protein is preferably one of the proteins in Table 1. The protein is more preferably ΡΙΧζ protein, such as human ΡΙΧζ protein. The protein is preferably a non- mutated (i.e. wild-type) protein product of one of the human genes shown in Tables 2 and 3.

The molecule is preferably selected from nucleotides, oligonucleotides and/or polynucleotides. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (HDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5- methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine

monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP),

deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP),

deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2' -deoxycytidine monophosphate, 5- methyl-2' -deoxycytidine diphosphate, 5 -methyl-2' -deoxycytidine triphosphate, 5- hydroxymethyl-2' -deoxycytidine monophosphate, 5 -hydroxymethyl-2' -deoxycytidine diphosphate and 5 -hydroxymethyl-2 '-deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP. The nucleotides may be abasic (i.e. lack a nucleobase). The nucleotides may contain additional modifications.

Oligonucleotides are short nucleotide polymers which typically have 50 or fewer nucleotides, such 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer or 5 or fewer nucleotides. The oligonucleotides may comprise any of the nucleotides discussed above, including the abasic and modified nucleotides.

A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The polynucleotide may be double stranded. The polynucleotide is preferably single stranded. The polynucleotide may be one strand from a double stranded polynucleotide.

The polynucleotides can be nucleic acids, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.

The polynucleotide can be any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

As discussed above, the polynucleotide preferably encodes one of the proteins shown in Table 1, preferably a human protein shown in Table 1. The polynucleotide more preferably encodes PLC^ such as human PLCζ protein. The polynucleotide preferably comprises the non- mutated (i.e. wild-type) sequence of one of the human genes shown in Tables 2 and 3.

The polynucleotide is preferably a microRNA (or miRNA). Suitable miRNAs for use in the invention are well known in the art. For instance, suitable miRNAs are stored on publically available databases (Jiang Q., Wang Y., Hao Y., Juan L., Teng M., Zhang X., Li M., Wang G., Liu Y., (2009) miR2Disease: a manually curated database for microRNA deregulation in human disease. Nucleic Acids Res).

The polynucleotide is preferably a small interfering RNA (siRNA) (or short interfering RNA or silencing RNA). These are typically 20 to 25 nucleotides in length and interfere with the expression of specific genes with a complementary nucleotide sequence. Suitable siRNA molecules for use in the invention are known in the art. The molecule may be a siRNA directed against the expression of any of the proteins shown in Table 1 or any of the genes shown in Tables 2 and 3. Such molecules may be used to study the function of the proteins in Table 1 and the genes in Table 2 and 3.

The polynucleotide is preferably a genetic cassette. These are typically a modular polynucleotide, such as DNA, sequence encoding one or more genes for a single biochemical function. The genetic cassette preferably includes one or more genes encoding one or more of the proteins shown in Table 1. The genetic cassette preferably comprises the non-mutated (i.e. wild-type) sequence of one or more of the genes shown in Table 2 and 3.

The molecule is preferably a morpholino. These synthetic molecules are usually about 25 bases in length and bind to complementary polynucleotide sequences by standard base- pairing. Structurally, the difference between morpholinos and polynucleotides is that while morpholinos have standard bases, those bases are bound to morpholine rings (instead of ribose or deoxyribose rings) and are linked through phosphorodiamidate groups (instead of phosphates).

The method of the invention may be for delivering two or more different molecules to a reproductive cell or an embryonic cell and may comprise contacting the cell with a MS P comprising the two or more different molecules. Any number of different molecules may be delivered in accordance with the invention, such as 3 or more, 4 or more, 5 or more, 10 or more or 20 or more molecules. For instance, the method may be for delivering two or more proteins, two or more polynucleotides or two or more fluorescent molecules.

MSNPs

The MSNP comprises silica, which is also known as silicon dioxide.

The MSNP typically has a diameter of 1 to lOOOnm, preferably 10 to 500nm, more preferably 20 to 300nm, more preferably 50 to 200 nm, for example about 130nm. The MSNP is preferably spherical or substantially spherical. A skilled person can readily adjust the diameter of the MSNP via modification of the synthetic process. Particle diameters can be measured by techniques known to those skilled in the art, such as transmission electron microscopy and ultracentrifugation in a density gradient (hydrodynamic sizing) as described in the Examples below.

The MSNP comprises pores of mesoscale size (ie. mesopores). Typically, the pores have an average diameter of 2 to 50 nm, preferably 5 to 25 nm. The pores are preferably cylindrical or substantially cylindrical. A skilled person can readily adjust the pore diameter via

modification of the synthetic process. Pores diameters can be measured by techniques known to those skilled in the art, such as X-ray diffraction.

The surface of the MSNP can be unmodified or modified. It can be desirable to modify the surface of the MSNP, in order to facilitate loading of the molecule for delivery into the reproductive cell or the embryonic cell.

An unmodified MSNP comprises no functional groups which have been added to the surface after formation of the MSNP. An unmodified MSNP thus has an anionic charge due to free silyl hydroxide moieties present on the surface. A surface-modified MSNP comprises functional groups which have been added to the surface after formation of the MSNP. Thus, the MSNP typically comprises one or more functional groups bonded to the surface of the MSNP. The functional groups are preferably covalently bonded to the surface of the MSNP, either directly or via a linker. Suitable linkers are known to those skilled in the art.

Typically, the functional group is a phosphonate, amine, sulfhydryl, disulfide, carboxylic acid, epoxide, halide (eg. fluoride, chloride, bromide or iodide), azide or alkyne moiety, preferably a phosphonate or an amine moiety.

In general, any reagent capable of reacting with the silyl hydroxide surface of the MSNP may be used to covalently modify the surface. For example, the surface of the MSNP may be treated with a trialkoxysilyl compound or trihydroxysilyl compound. The compound reacts with the silyl hydroxide surface of the MSNP, forming covalent silicon-oxygen bonds. Trialkoxysilyl and trihydroxylsilyl compounds bearing the desired functional groups may be used to modify the surface of the nanoparticle in this manner.

Thus, one or more phosphonate moieties can be introduced by, for example, treating the MSNP with a phosphonate bearing trialkylsiloxane compound or phosphonate-bearing trihydroxylsilyl compound, such as (trihydroxylsilyl) propyl methylphosphonate. Similarly, one or more amine functional groups can be introduced by, for example, treating the MSNP with an amine bearing trialkoxysilane compound, such as aminopropyltriethoxysilane, 3-(2- aminoethylamino)propyl-trimethoxysilane or 3- trimethoxysilylpropyl ethylenediamine.

At least part of the surface of the MSNP is optionally coated with a polymer. Thus, the MSNP typically comprises a polymer which coats at least part of the surface of the MSNP. A polymer coating can be desirable in order to facilitate loading of the molecule for delivery into a sperm cell, oocyte, ovum or embryonic cell.

The polymer is typically a cationic polymer. Preferred cationic polymers include polyethyleneimine (PEI), polyamidoamine, polylysine and polyallylamine. PEI is particularly preferred. The weight average molecular weight of the PEI is preferably less than lOkDa, for example about 1.2 kDa.

Alternatively, the polymer may be polyethylene glycol (PEG).

The polymer may be bound covalently or electrostatically to the surface of the silica body. Labelling

The MS P and/or the at least one molecule is preferably labelled with a revealing label. Once such a label is delivered to a reproductive cell or an embryonic cell, it may be used to identify, sort or quantify the cell or track its movement in vivo or in vitro. The MSNP and/or at least one molecule is preferably labelled if the at least one molecule is not a dye or a fluorescent molecule. The revealing label may be any suitable label which may be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵1, ³⁵S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin. Any of the fluorescent molecules discussed above may be used.

Molecule loading

The method of the invention utilises a MSNP comprising at least one molecule. The MSNP is loaded with the at least one molecule. The MSNP is provided with the at least one molecule.

The MSNP may comprise or be loaded /provided with the at least one molecule in any manner. The at least one molecule is preferably embedded within the MSNP. This is typically achieved by incubating the MSNP with the at least one molecule such that the at least one molecule is absorbed into the pores of the MSNP.

The at least one molecule is preferably provided on, loaded on, coated on or attached to the surface of the MSNP. As discussed above, the surface of the MSNP may be modified, such as by coating the MSNP, to facilitate its interaction with the at least one molecule.

The at least one molecule may be provided on, loaded on, coated on or attached to the surface of the MSNP in any manner. The at least one molecule may be covalently attached to the MSNP. The at least one molecule may interact with the MSNP in a non-covalent manner. Suitable non-covalent interactions include, but are not limited to, ionic bonding, hydrophobic interactions, hydrogen bonding, Van der Waal's forces, π-cation interactions and electrostatic forces.

The at least one molecule may be directly provided on, directly loaded on, directly coated on or directly attached to the surface of the MSNP or a coating on the MSNP. Alternatively, the at least one molecule may be provided on, loaded on, coated on or attached to the surface of the MSNP or a coating on the MSNP via one or more linkers. Suitable linkers are known in the art. Contacting

Techniques for culturing reproductive cells and embryonic cells are well known to a person skilled in the art. The cells are typically cultured under standard conditions of 37°C, 5% C0₂ in medium supplemented with serum.

The method of the invention comprises contacting the reproductive cell or the embryonic cell with a MSNP comprising the at least one molecule. The contacting may be carried out in any suitable manner. The method preferably comprises exposing the reproductive cell or the embryonic cell to the MSNP comprising the at least one molecule in solution. The method preferably comprises allowing the MSNP comprising the at least one molecule to spontaneously enter the reproductive cell or the embryonic cell. The MSNP comprising the at least one molecule is preferably not manipulated into the cell. The MSNP comprising the at least one molecule is preferably not physically forced into, such as injected into, the cell. The MSNP comprising the at least one molecule is preferably not filtered before it is contacted with the cell.

If the method is carried out in vitro or ex vivo, the cell is typically contacted with the MSNP comprising the at least one molecule in a buffered solution or in cell culture medium. Suitable buffers include, but are not limited to, phosphate buffered saline and 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES). Suitable cell culture media are known in the art and may be supplemented with serum. The method may be carried out in the presence of a penetration enhancer. Any of those discussed below may be used.

The in vitro or ex vivo method is typically carried out at a pH of from 7.0 to 7.7. The pH used is preferably about 7.4.

The in vitro or ex vivo method may be carried out at any suitable temperature at which the cell will survive, such as from 15 °C to 40 °C, such as from 18.5 °C to 37 °C.

The in vitro or ex vivo method may be carried in the dark, preferably for sperm.

If the method is carried out in vitro or ex vivo, the cell is typically contacted with the MSNP comprising at least one molecule for at least one hour, such as for at least two hours, such as at least three hours or at least four hours. The cell may be contacted with the MSNP comprising at least one molecule for less that one hour, such as for 30 minutes.

If the method is carried out in vivo, the MSNP comprising at least one molecule may be administered by any suitable means. Administration to a human or animal subject is typically selected from intratesticular, intraovarian or intraembryonic. Typically the method of delivery to the testis, ovary or embryo is by injection. However, the MSNP comprising at least one molecule does not need to be injected into the target sperm, oocyte, ovum or embryo. As discussed above, the MSNP comprising at least one molecule is capable of spontaneously entering reproductive or embryonic cells without physical manipulation. Once in the testis, ovary or embryo, the MS P will deliver the at least one molecule into the relevant cells. A physician will be able to determine the required route of administration for each particular patient.

The MSNP comprising at least one molecule is delivered as a composition. The composition may be formulated to facilitate delivery of the at least one molecule. For example, uptake of nucleic acids by mammalian cells is enhanced by several known transfection techniques, for example, those that use transfection agents. The formulation that is administered may contain such agents. Examples of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™).

Compositions may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with a MSNP comprising at least one molecule in conjunction with other disease therapeutic modalities (such as those described herein) in order to increase the efficacy of the treatment.

The MSNP comprising at least one molecule may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like. The composition may comprise other active agents that are used in therapy (e.g. anti-inflammatories).

Pharmaceutical compositions may include penetration enhancers in order to enhance the delivery of the at least one molecule. Penetration enhancers may be classified as belonging to one of five broad categories, i.e. fatty acids, bile salts, chelating agents, surfactants and non- surfactants. One or more penetration enhancers from one or more of these broad categories may be included.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1 -monocaprate, l-dodecylazacycloheptan-2- one, acylcarnitines, acylcholines, mono-and di-glycerides and physiologically acceptable salts thereof (i.e. oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc).

Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations. Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g. sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines). Chelating agents have the added advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9- lauryl ether and polyoxyethylene-20-cetyl ether and perfluorochemical emulsions, such as FC-43. Non- surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-andl-alkenylazacyclo- alkanone derivatives and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone.

A "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more molecules to a subject. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency etc when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or

hydroxypropyl methylcellulose, etc); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrates (e.g. starch, sodium starch glycolate, etc); or wetting agents (e.g. sodium lauryl sulphate, etc).

The compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

Colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the MSNP comprising at least one molecule to a particular cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid: oligonucleotide complexes of uncharacterised structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration.

An effective amount of the MS P comprising at least one molecule is administered. An effective amount is the amount needed to deliver the at least one molecule to the reproductive cell or the embryonic cell. A therapeutically, prophylactically or diagnostically effective amount of the MSNP comprising at least one molecule is preferably administered. A therapeutically effective amount is an amount effective to ameliorate or abolish one or more symptoms of the disease or disorder. A prophylactically effective amount is an amount effective to prevent or reduce one or more symptoms of the disease or disorder. A diagnostically effective amount is an amount effective to indicate the presence of the disease or disorder.

The dose may be determined according to various parameters, especially according to the severity of the condition, age, and weight of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. Optimum dosages may vary depending on the relative potency of individual constructs, and can generally be estimated based on EC50s found to be effective in vitro and in in vivo animal models. In general, dosage is from 1 mg/kg to 1000 mg per kg of body weight. A typical daily dose is from about 5 to 500 mg per kg, preferably from about 10 mg/kg to lOOmg/kg of body weight, according to the potency of the specific molecule, the age, weight and condition of the subject to be treated, the severity of the disease and the frequency and route of administration. Preferably, the dose of a single injection is in the range of about 5 to 20 μg. Preferably, the dose of single or multiple injections is in the range of 10 to 100 mg/kg of body weight.

Due to clearance (and breakdown of the molecule), the patient may have to be treated repeatedly, for example once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the construct in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy, wherein the construct is administered in maintenance doses, ranging from 1 mg/kg to 1000 mg per kg of body weight, once or more daily, to once every 20 years. Uses of the method

The method of the invention has a variety of applications. It may be used to deliver any molecule into a reproductive cell or an embryonic cell. The method can be used in in vitro or ex vivo bioassays to provide information on DNA fragmentation, aneuploidy, chromosomal aberration, apoptosis, oocyte activation ability, oocyte/sperm/embryo competency, protein degradation and levels and localization patterns of important functional reproductive proteins.

The method may also be used to facilitate in vitro maturation of reproductive cells, in vitro fertilization and intracytoplasmic sperm injection.

Therapeutic and diagnostic uses of the method of the invention are discussed below.

Therapy and diagnosis

The ability of the MSNPs to deliver biologically active molecules to reproductive cells and embryonic cells results in their suitability for therapeutic treatment of reproductive diseases or disorders in a subject and diagnosis of reproductive diseases or disorders in a subject. As used herein, the term "treatment" is meant to encompass therapeutic, palliative and prophylactic uses.

The method of treatment or diagnosis is suitable for any patient that has, may have, or is suspected of having, a reproductive disease or disorder. The reproductive disease or disorder is preferably infertility and/or is associated with DNA fragmentation, aneuploidy, chromosomal aberration, apoptosis in reproductive or embryonic cells, reduced oocyte activation ability, reproductive or embryonic cell competency, protein degradation in reproductive or embryonic cells. The infertility is preferably male factor infertility, female factor infertility, idiopathic infertility, failed fertilisation, oocyte activation deficiency, globozoospermia, implantation failure, developmental failure, endometriosis or recurrent miscarriage.

If the patient is male, the reproductive disorder or disease is preferably ambiguous genitalia, gonadal dysgenesis, hypospadias, micropenis, cryptorchidism, testis cancer or a vas deferens defect. Suitable reproduction-promoting agents and diagnostic agents are discussed above with reference to Table 2.

If the patient is male, the reproductive disorder or disease is preferably abnormal spermatogenesis, azoospermia, oligospermia, asthenozoospermia, teratozoospermia,

oligoasthenozoospermia, oligoasthenoteratozoospermia, acrosome defect, sperm DNA damage or varicocele effect. Suitable reproduction-promoting agents and diagnostic agents are discussed above with reference to Table 3.

The infertility may be caused by the point mutation H398P in phospholipase C zeta (ΡΙΧζ) described by Heytens et al. (2009) Hum Reprod 2009;24:2417- 2428, or other genetic mutations identified for ΡΙΧζ, such as the H233L mutation descrbed by Kashir et al (2012). Hum Reprod 2012; 27 (1), 222 - 31, or by the abberant expression or localization of ΡΙΧζ in the sperm, or by proteins either within the sperm, or in the oocyte, which interact with ΡΙΧζ in order to cause successful oocyte activation.

The method of treatment or diagnosis can be used to treat or diagnose a subject of any reproductive age. The subject is preferably mammalian, such as human. Typically, the age of the subject to be treated is from 10 to 100 years old. More preferably, the age of the subject to be treated is from 11 to 80, from 12 to 60 or from 15 to 50.

The method of treatment comprises delivering into a reproductive cell or an embryonic cell of the patient at least one reproduction-promoting agent or fertility-promoting agents. The delivery may be in vitro, ex vivo or in vivo. A skilled person can determine which agents are suitable for particular diseases or disorders. Suitable reproduction-promoting agents include, but are not limited to, any of the therapeutic agents discussed above.

The method of diagnosis comprises delivering into a reproductive cell or an embryonic cell of the patient at least one diagnostic agent. The delivery may be in vitro, ex vivo or in vivo. A skilled person can determine which agents are suitable for particular diseases or disorders. Suitable diagnostic agents are discussed above.

The invention also provides a MS P comprising at least one reproduction-promoting agent for use in a method of treating a reproductive disease or disorder in a patient in need thereof, the method comprising delivering the at least one reproduction-promoting agent into a reproductive cell or an embryonic cell of the patient by contacting the cell with the MSNP comprising the at least one agent and thereby treating the reproductive disease or disorder in the patient.

The invention also provides a MSNP comprising at least one diagnostic agent for use in a method of diagnosing a reproductive disease or disorder in a patient, the method comprising delivering into a reproductive cell or an embryonic cell of the patient the at least one diagnostic agent by contacting the cell with the MSNP comprising the at least one agent and thereby diagnosing the reproductive disease or disorder in the patient.

The invention also provides a MSNP comprising a ΡΙΧζ protein or a polynucleotide encoding ΡΙ ζ for use in a method of promoting fertilisation of an ovum by a sperm comprising the point mutations H398P or H233L, or any other mutation in phospholipase C zeta (ΡΙ ζ), or by sperm devoid of ΡΙ ζ, or by sperm exhibiting aberrant expression or localisation patterning of ΡΙΧζ), the method comprising delivering the ΡΙΧζ protein or the polynucleotide encoding ΡΙΧζ into the ovum by contacting the ovum with the MS P comprising the protein or polynucleotide and thereby promoting fertilisation of the ovum by the sperm.

General synthetic procedures

The MSNP can be prepared by any suitable synthetic technique, such as the methods and procedures described herein, or similar methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

The MSNP is typically prepared using a surfactant-tempi ated base-catalysed sol-gel reaction, such as that described in Horn, C, Lu, J., Liong, M., Luo, H., Li, Z., Zink, J. I. and Tamanoi, F. (2010) 'Mesoporous silica nanoparticles facilitate delivery of siRNA to shutdown signaling pathways in mammalian cells', Small 6(11): 1185-90.

Generally, the surfactant-tempi ated base-catalysed sol-gel reaction involves mixing a templating agent, such as cetyltrimethylammonium bromide (CTAB), with a silica source, such as tetraethylorthosilicate (TEOS), in a basic aqueous solution, which preferably has a pH of aboutl 1.0. The template is typically then removed, for example by refluxing in acidic methanol.

The surface of the MSNP can be modified in order to aid loading of the molecule for delivery into a reproductive cell or an embryonic cell. Surface modification is typically conducted prior to removal of the template. Suitable reagent(s) for surface modification can readily be selected by a skilled person, as discussed above, so that the desired functional groups (for example amine and/or phosphonate moieties) are introduced onto the surface of MSNP.

Once the template has been removed, it can be desirable to coat the surface of MSNP with a polymer, such as polyethyleneimine (PEI), as described above. A skilled person can readily determine suitable reagents and reaction conditions for coating of the surface of the MSNP.

The molecule for delivery into a reproductive cell or an embryonic cell can be loaded onto the MSNP using any suitable technique, as described herein, such as incubation of the molecule in a solution of the MSNP until a sufficient level of loading has been attained. As a skilled person will appreciate, the specific reaction conditions and techniques will depend on the nature of the molecule to be loaded.

The invention is illustrated by the following Examples: Example 1

Materials and Methods

Synthesis and functionalisation of mesoporous silica nanoparticles (MSNPs)

Synthesis of MSNPs was performed in a surfactant-templated base-catalysed sol-gel reaction as previously described by Horn et al. ((2010) Small 6(11): 1185-90). Essentially, the reaction was based upon the mixing of a templating agent, cetyltrimethylammonium bromide (CTAB), with a silica source, tetraethylorthosilicate (TEOS), in hot basic aqueous solution (pH=l 1.0), followed by amine and phosphonate surface modification, and final removal of the template via refluxing in acidic methanol. A detailed protocol for synthesis of non-fluorescent and fluorescent MSNPs, followed by optional surface coating with PEI is presented below.

Reagents

Anhydrous sodium hydroxide (>98%), hexadecyltrimethylammonium bromide (CTAB, >99%), tetraethyl orthosilicate (TEOS, 98%), 3-(trihydroxysilyl)propyl methylphosphonate (monosodium salt, 42 wt.% solution in water), fluorescein isothiocyanate (FITC, >90%), (3- aminopropyl)triethoxysilane (APTS, >98%), polyethileneimine (PEI, 50 wt.% solution in water, MW 1.3kD), concentrated hydrochloric acid (37.2 wt.% solution in water, 12.1M) and phosphate based saline (Dulbecco's PBS) were sourced from Sigma-Aldrich (Dorset, UK). Methanol (>99.8%) was obtained from Rathburn Chemicals (Wakerburn, UK). Absolute ethanol (>99.8%) was sourced from Riedel-de Haen (Seelze, Germany).

Synthesis of non-fluorescent MSNPs

In a round bottomed flask, 0. lg of CTAB was dissolved in a mixture of 48ml of distilled water and 0.35ml of 2M sodium hydroxide. The resulting basic aqueous CTAB solution was heated in a silicone bath to 80°C with magnetic stirring. After the temperature had stabilised, 0.5ml of TEOS was added to the reaction. After 15 minutes, 0.127ml of 3- (trihydroxysilyl)propyl methylphosphonate was added, and the mixture was stirred for another 2 hours at 80°C. The solution was then cooled to room temperature, centrifuged for 5 minutes at 8,603g, and washed twice with methanol by centrifugation. The pellet was resuspended in a mixture of 40ml of methanol and 2ml of 12.1M hydrochloric acid, and refluxed for 24 hours at 80°C with magnetic stirring. After refluxing had been completed, the particles were centrifuged, washed twice in absolute ethanol by centrifugation, and vacuum dried overnight. Dry particles were ground with a mortar and pestle to make the powder homogenous, and then stored at -22°C.

Synthesis of fluorescent MS Ps

FITC was introduced into the silica framework in the form of FITC-APTS conjugate. The conjugate was prepared in advance by mixing ΙΟΟμΙ of APTS with 25mg of FITC in 5ml of absolute ethanol, and magnetic stirring for 12 hours under a dry nitrogen atmosphere. To synthesise fluorescent MSNPs, a single additional step was introduced to the standard protocol. Particularly, 50μ1 of FITC-APTS conjugate was added to the reaction 10 minutes after TEOS was introduced, followed by addition of 3-(trihydroxysilyl)propyl methylphosphonate 5 minutes later, in accordance with the standard procedure. After adding FITC-APTS, care was taken to minimise particles exposure to light.

Surface functionalisation of MSNPs with PEI

Surface polyethileneimine (PEI) functionalisation of non- fluorescent and fluorescent MSNPs was performed identically. In a round bottomed flask, 5mg of MSNP powder was resuspended in a mixture of 75μ1 of PEI (MW 1.3kD, Mn l,200g/mol) and 30ml of absolute ethanol. The suspension was stirred at room temperature for 1 hour to allow surface PEI- coating. Coated MSNPs were centrifuged for 5 minutes at 8603g, and washed by centrifugation: once in absolute ethanol, and once in distilled water sterilised by filtration through a 0.2μπι cellulose acetate syringe filter (Anachem, Luton, UK). The pellet was left to vacuum dry overnight, followed by manual grinding in aseptic environment, and storage at -22°C.

Size measurement

MSNP sizes were measured using a disc centrifuge (CPS DC24000, CPS Instruments Europe, Oosterhout, Netherlands), which assesses particle diameter in solution based on their sedimentation profiles during centrifugation in a liquid gradient. Four MSNP samples were prepared for sizing: non-fluorescent non-coated MSNPs, non-fluorescent PEI-coated MSNPs, FITC-labelled non-coated MSNPs, and FITC-labelled PEI-coated MSNPs. For each sample, particles were resuspended in 500μ1 of distilled water to achieve a slightly opalescent, but not transparent, suspension. To facilitate resuspension, samples were vortexed and sonicated for 3 minutes in a sonic water bath (U50, Ultrawave, Cardiff, UK). The procedure was repeated up to 3 times, until solutions contained no clumps. Particle size was measured in three 0.1ml aliquots for each of the four MSNP samples at disk speed 24000rpm according to a standard ' Si02' operating protocol. External calibration with 0.1ml of kit calibration standard (0.377μm-sized polyvinyl chloride (PVC) latex particles dispersion in distilled water, density 1.385g/ml; PVC calibration standard, CPS Instruments, Oosterhout, Netherlands) was performed prior to the beginning of procedure, and then every 3 measurements. Data were presented by operational software as a graph of particle size distributions (weight vs. diameter).

Zeta (ζ) potential measurement

Zeta (ζ) potential (electrokinetic potential of a colloidal system) is an electric potential at the interface of the bulk solvent and the solvent layer electrostatically attached to the particle surface and moving in solution as part of the particle ('slipping plane'). MSNP ζ potentials were measured in the Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK) utilising the dynamic light scattering (DLS) technique. In brief, colloidal solution was subjected to an electric field in a capillary cell containing two oppositely charged electrodes. The electric field caused migration of particles to the oppositely charged electrode, and the velocity of movement was proportional to their ζ potential value. This velocity was measured by recording the phase/frequency shift of a laser beam coming in contact with moving particles, and converted to ζ potential by operational software.

MSNP samples for ζ potential analysis were prepared similarly to sizing samples. A ΙΟΟμΙ aliquot of each sample was loaded into a clean plastic capillary cell (DTS 1060C, Malvern Instruments, Malvern, UK), and placed into the Zetasizer Nano ZS. Samples were equilibrated for 2 minutes, followed by 12 reading runs. Data from 12 runs were processed by operating software in quads, to produce three ζ potential measurements for each sample.

Loading of MSNPs with cargo

Non-fluorescent PEI-coated MSNPs were used to demonstrate the possibility of siRNA loading onto nanoparticles. A 1.5mg portion of particles stored at -22°C was resuspended in 1.0ml of distilled water sterilised by filtration through a 20μπι cellulose acetate syringe filter (Anachem, Luton, UK), vortexed, and sonicated in a sonic water bath for 3 minutes twice.

MSNPs were centrifuged for 5 minutes at 1800g, resuspensed in 1.0ml of sterile phosphate- based saline (Dulbecco's PBS, Sigma- Aldrich, Dorset, UK) by vortexing. To load siRNA onto nanoparticles, 1.0ml of MSNP solution was mixed with ΙΟμΙ of concentrated fluorescent lamin A/C siRNA stock (20μΜ; siGLO® Lamin A/C Control siRNA (human/mouse/rat), fluorescent label absorption max: 557nm; emission max: 570nm; Dharmacon RNAi Technologies, Thermo Scientific, Epsom, UK). The mixture was incubated for 24 hours at 4°C. After 24 hours, the solution was centrifuged for 2 minutes at 1800g, and MSNPs were resuspended in 1.0ml of sterile PBS. At all steps of the procedure, care was taken to avoid excessive light exposure.

Preparation of MSNP working solutions

Working solutions of various MSNPs were prepared by resuspending particles in PBS to final concentrations of 0.03mg/ml for FITC-labelled non-coated MSNPs, 0.03mg/ml and 0.3mg/ml for FITC-labelled PEI-coated MSNPs, and 0.3mg/ml for fluorescent siRNA-bound PEI-coated MSNPs. Solutions were stored at 4°C. Prior to applying to gametes, solutions were vortexed and sonicated in a sonic water bath (VWR International, Leuven, Germany), for 3 minutes for up to 3 times until no clumps were seen, except for fluorescent siRNA-bound MSNPs. Additionally, portions of FITC-labelled PEI-coated MSNP solutions (0.03mg/ml and 0.3mg/ml) were filtered through 0.2μπι and 0.45μπι cellulose acetate syringe filters (VWR International, Leuven, Germany) as an attempt to remove particle agglomerates.

Sperm preparation

Boar semen was obtained in an extender/diluent from JSR Genetics (Southburn, UK). Prior to experiments, sperm motility was activated by incubation for 30 minutes at 18.5°C. Semen was centrifuged for 10 minutes at 532g, and sperm washed from seminal plasma twice with PBS (Oxoid, Basingstoke, UK).

Sperm motility assessment

Sperm motility was assessed at baseline and all incubation time points in experimental- control pairs. Evaluation was performed in 'wet-prep' slides under a microscope (Nikon Eclipse E200, Nikon Instruments UK) with a 40x objective on a conventional microscope stage. The number of sperm of each motility category was counted manually. The motility of each sperm was graded according to the scale: 'a+b' = progressive motility; 'c' = non-progressive motility, and 'd' = immotility. At least 200 sperm in at least 5 fields of view were assessed. The process was repeated in a second drop, and the values averaged.

Application of MSNPs to sperm and preparation of microscopy slides

In paired experimental-control samples, washed sperm were exposed to selected MSNPs solution or PBS, respectively. Samples were incubated in the dark at 18.5°C for one (fluorescent siRNA-bound PEI-coated MSNPs only), two, three and four hours. After incubation, two drops of each sample were transferred to a microscopy slide for motility assessment ('wet-preps'). Samples were then fixed with 10% formalin solution (Sigma- Aldrich, Dorset, UK) for 10 minutes, centrifuged for 15 minutes at 532g, and resuspended in PBS. Fixed sperm were transferred to a microscopy slide pre-coated with 0.01% w/v poly-L-lysine (Sigma-Aldrich, Dorset, UK). The slide was incubated in a dark humidifying chamber for 30 minutes at room temperature, washed twice with PBS, and mounted with 4',6-diamidino-2-phenylindole (DAPI)- containing mounting media (DAPI concentration: l ^g/ml; Vectashield H-1200, Vector Laboratories, Peterborough, UK). Slides were stored in the dark at 4°C. In each experiment, a baseline control slide was identically prepared at the 0-hour incubation time point.

For transmission electron microscopy (TEM), washed sperm were incubated with FITC- labelled non-coated and PEI-coated MSNPs (0.3mg/ml) according to the same procedure, fixed, centrifuged and resuspended in absolute ethanol to decrease sample concentration to 30 sperm per 50μ1. Fifty-microliter drops were loaded onto TEM grids (VWR international, Leuven, Germany), left to air-dry on the bench, and stored in the dark at -22°C.

Experiments with sperm exposure to FITC-labelled non-coated MSNPs were performed three times to provide 3 sets of slides: one for basic fluorescent microscopy, one for confocal, and one (grids) for TEM. Experiments with FITC-labelled PEI-coated MSNPs were carried out four times (two sets of slides for basic fluorescent, one set for confocal microscopy, and one set of grids for TEM), and experiments with fluorescent siRNA-bound PEI-coated MSNPs - three times (two sets of slides for basic fluorescent, one set for confocal microscopy).

Oocyte exposure

Oocytes from superovulated C57BL/6 mice were obtained from Biomedical Services (The University of Oxford) in culture media droplets overlaid with mineral oil (FertiCult, Microm UK, Bicester, UK). Mouse oocytes (n=29) were split into 4 groups, and incubated for 2 hours at 37°C in M16 media (Sigma-Aldrich, Dorset, UK) containing FITC-labelled PEI-coated MSNPs at the concentration of 0.003mg/ml (Group 1, n=8), 0.03mg/ml (Group 2, n=8), and 0.3mg/ml (Group 3, n=8). Oocytes in Group 4 (control, n=5) were incubated with PBS. After 2 hours, oocytes were washed in PBS, fixed with 10% formalin solution, and washed in PBS again (twice), followed by transfer into PBS drops on 0.01 % w/v poly-L-lysine pre-coated slides. Slides were incubated in a dark humidifying chamber for 40 minutes at room temperature, mounted with DAPI-containing medium, and stored in the dark at 4°C.

Oocyte microinjections with MSNPs and sperm exposed to MSNPs Mouse oocytes were injected with FITC-labelled MS Ps to investigate feasibility of assisted nanoparticles delivery. ICSI of mouse oocytes with boar sperm incubated with FITC- labelled MSNPs, was performed to assess the oocyte activation capacity of MSNPs-exposed sperm. Sperm were washed and incubated with FITC-labelled non-coated and PEI-coated MSNPs (0.3mg/ml) similarly to the previously described procedure. Microinjections were performed with a Nikon Narishige micromanipulator mounted on the Nikon Eclipse Ti microscope using micro-injection pipettes with an internal diameter of 5μιη (Cook Medical, PA, USA). ICSI with boar sperm was performed by experienced clinical embryologists.

Mouse oocytes (n=29) were split into 6 groups. Oocytes in Group 1 (n =5) were incubated overnight in Ml 6 media droplets covered with mineral oil (FertiCult, Microm UK, Bicester, UK) at 37°C. In Group 2 (n=5), oocytes were microinjected with PBS. In Group 3 and 4 (n=5 and n=6), ICSI with boar sperm exposed to FITC-labelled non-coated and PEI-coated MSNPs respectively, was carried out. After microinjections, oocytes were cultured overnight in fresh M16 culture media droplets overlaid with mineral oil at 37°C. On the next day, oocytes were fixed, washed, spread on the slides and stained with DAPI, as described above. In Group 5, oocytes (n=4) were injected with FITC-labelled non-coated MSNPs (0.3mg/ml), and in Group 6 (n=4) with FITC-labelled PEI-coated MSNPs (0.3mg/ml), followed by 2 hour incubation in fresh Ml 6 media at 37°C, fixation and transfer to the slides.

Imaging

Basic fluorescent microscopy was performed using the Nikon Eclipse 80i microscope with a Nikon DS-Ril camera and a set of filters with the following excitation wavelenghts: 330- 380nm (DAPI), 445-495nm (FITC), and 540-588nm (Rhodamine). For each sperm slide, images of two random fields at 20x-objective magnification and 3 random fields at 40x-objective magnification were captured. Additionally, precise representative images of sperm-MSNPs association were acquired (40x objective). Oocytes were imaged with the 20x objective. Images were acquired using the NIS Elements v3.00 software (Nikon UK, Surrey). Confocal microscopy of sperm slides for precise assessment of MSNP-sperm association was performed in the microscopy unit at the John Radcliffe hospital using a Zeiss-LSM510 microscope with a 63x oil immersion objective and the following excitation wavelengths: 365nm (DAPI), 450-490nm (FITC), and 546/12nm (Rhodamine). Images were acquired and processed using the LSM Image Browser software (Zeiss UK, Cambridge).

Image processing and data analysis Image processing and analysis was performed using the Image J vl .43 software (National Institute of Health, USA). The number of sperm associated with MSNPs and types of association were determined manually at 40x-objective magnification images obtained during the conventional fluorescent microscopy, and expressed as percentages of total sperm and total MSNP-associated sperm, respectively. Continuous variables in two independent samples were compared using a two-tailed independent t-test. Differences in percentages between two samples were compared using a two-tailed z-test for proportions. MSNP-sperm association rates were compared using factorial analysis of variance (ANOVA) followed by post-hoc comparisons of the means with Bonferroni adjustment. Statistical analysis was performed using Statistica vlO.O (StatSoft Inc., OK, USA), and representative graphs were built in GraphPad Prism v5.04

(GraphPad Software, CA, USA). Data are presented as mean±standard error of the mean (SEM), unless specified otherwise. Differences between the groups were considered statistically significant at p<0.05.

Results

Characterisation of MSNPs

Sizing of MSNPs demonstrated that the hydrodynamic diameter of synthesised particles fell within a range from approximately 130nm to 300nm (Figure 1). The smallest average diameter was observed for non-fluorescent PEI-coated MSNPs (~130nm), and the largest for non- fluorescent non-coated MSNPs (~300nm).

Measurement of ζ potential of synthesised MSNPs confirmed efficacy of PEI-coating for cationic functionalisation of the particles (Table 4). Non-coated MSNPs were characterised by an inherently negative ζ potential of unmodified silica. FITC integration into the silica scaffold provided additional functional groups capable to dissociate in water and decrease ζ potential even further. Cationic coating of particles with PEI consistently reversed ζ potential of MSNPs. In all four samples of analysed MSNPs, ζ potential was narrowly distributed, and differed significantly between the groups, which allowed to expect distinctive profiles of various MSNPs interaction with cells in vitro. Table 4 - ζ potential values of synthesised MSNPs (mV; pH=7.0, t~25.0°C)

Basic structure Non- PEI-coated

coated

Non-fluorescent MSNPs -24.03±0.48* 24.40±0.30**

FITC-labelled MSNPs -31.50±0.60* 37.73±0.27**

* p<0.05 (t-test for independent samples)

Mammalian gametes exposure to MSNPs

MSNP-associated fluorescence was detected by fluorescent microscopy in all exposed sperm samples, except those incubated with filtered solutions of FITC-labelled PEI-coated MSNPs (0.03mg/ml and 0.3mg/ml; 0.2 and 0.45μπι filters), suggesting absorption of the particles on the filter membrane. For this reason, outcomes of sperm exposure to filtered MSNPs will not be addressed in the following subsections.

Impact of MSNPs on sperm motility

Motility assessment demonstrated that synthesised MSNPs did not exert negative impact upon boar sperm, regardless of exposure duration, MSNP surface chemistry, and concentration. Progressive sperm motility fractions in samples incubated with MSNPs were not significantly different from paired controls at all time points studied (Table 5). Conventionally, sperm motility represents the absolute indicator of sperm viability. In this respect, these findings suggest that the exposure of boar sperm to various concentrations of MSNPs with different hydrodynamic diameters and surface chemistry has no negative impact upon sperm viability, at least immediately after acute contact.

Table 5 - Progressive sperm motility after exposure to different concentrations of MSNPs with various surface functionalities (% 'a+b'/total number of sperm)*

* p values indicate significance of difference compared to paired controls (data from single experiments were compared using the two-tailed z-test for proportions; data from replicate experiments were averaged and compared using the two-tailed t-test for independent samples).

Quantification of MSNP-sperm association rate

Interaction of MSNPs with boar sperm was profoundly dependent on physicochemical properties of the particles, particularly ζ potential. For the purpose of initial quantification of the MSNP-sperm exposure results, no discrimination was made between surface adherence and cell internalisation, which were collectively described as 'MSNP-sperm association' . FITC-labelled non-coated MSNPs (negative ζ potential) demonstrated significantly higher sperm association rates across all studied time points, compared to FITC-labelled PEI-coated (positive ζ potential) and fluorescent siRNA-bound PEI-coated MSNPs (balanced positive ζ potential), even despite lower concentrations (0.03mg/ml) being used (Figure 2). MSNP-sperm association could be improved by a 10-fold increase of particle concentration for FITC-labelled PEI-coated MSNPs (positive ζ potential), but not for siRNA-bound PEI-coated MSNPs (balanced positive ζ potential). Among most samples, the highest MSNP-sperm association rate was observed after 3 hours of incubation, however within-group differences did not reach statistical significance. No significant differences in the MSNP-sperm interaction profiles were detected between FITC- labelled PEI-coated and siRNA-bound PEI-coated MSNPs, regardless of the particle

concentration. These findings, however, should be interpreted with caution due to a small number of experiment replications and variability of the results obtained from individual samples.

Investigation of MSNP-sperm association using fluorescent microscopy

Under the fluorescent microscope, MSNPs associated with sperm produced discrete fluorescent signals localised on the surface of the sperm membrane or in the projection of the sperm head or midpiece, which was suggestive, but not definitive evidence of particle internalisation (Figure 3). Accurate discrimination between the adherence and internalisation of MSNPs was complicated by the small size and discrete nature of MSNP-associated fluorescent signals. Generally, surface signals were expected to have a larger size, less regular shape, and greater intensity, compared to signals localised inside the borders of the sperm head/midpiece. A considerable amount of free MSNP agglomerates was present in all samples analysed, with the greatest density observed in FITC-labelled non-coated MSNPs, despite low concentration of working solution.

Dynamics of sperm-associated MSNP localisation

A provisional assessment of localisation of sperm-associated MSNPs in relation to the main sperm segments (head, midpiece and tail) at various time points of the experiment was also carried out. The discrimination between MSNP adherence and internalisation into various sperm structures was based mainly on the strength and surface area of the signal, as well as signal relation to the sperm nucleus plane (see Figure 3 legend). Results showed a consistent trend towards an increase in the proportion of MSNPs internalised into the sperm head during the incubation period in all samples analysed (Figure 4).

Remarkably, a distinctive behaviour of FITC-labelled non-coated MSNPs was observed again, with a greater diversity of localisation profiles, including tail and midpiece region involvement, not equally common in other cases. Nevertheless, it should be noted that these results were obtained from a small dataset produced by fluorescent microscopy, and statistical tests failed to detect significance of the observed trend in most cases. Hence, critical

interpretation of these outcomes is crucial until additional evidence is recruited, preferably using higher resolution imaging techniques.

Confocal microscopy

Confocal microscopy of boar sperm samples exposed to synthesised MSNPs was performed for detailed evaluation of MSNP interaction with sperm. A single working concentration (0.3mg/ml) was chosen for all MSNP solutions to minimise potential confounders. Confocal microscopy findings supported preliminary data from conventional fluorescent microscopy regarding distinctive profiles of various MSNPs interaction with sperm, highly dependent on the physicochemical characteristics of the particles (Figure 5). As such, FITC- labelled non-coated MSNPs (negative ζ potential) were markedly more prone to adhere to the sperm membrane, both as discrete structures and large particle agglomerates, compared to FITC- labelled PEI-coated and fluorescent siRNA-bound PEI-coated MSNPs (positive and balanced positive ζ potential, respectively). Identification of MSNP-sperm associations for these two types of nanoparticles was challenging due to considerably smaller adherence/internalisation rate. Both FITC-labelled PEI-coated and fluorescent siRNA-bound PEI-coated MSNPs showed a tendency to attach to the sperm membrane in the form of discrete small agglomerates, primarily in the head region. MS P internalisation was suspected in a number of sperm exposed to FITC-labelled non-coated MSNPs, based on a perinuclear, rather than membrane location of the MSNP- associated signal. In contrast, such signs were rarely observed in the two remaining groups. Interestingly, confocal microscopy allowed the visualisation of non-typical vesicle-shaped structures inside some sperm, particularly after exposure to fluorescent siRNA-bound PEI-coated MSNPs.

Oocyte exposure

Mouse oocytes exposure to FITC-labelled PEI-coated MSNPs resulted in adherence of nanoparticles to the surface of the zona pellucida after 2 hours of incubation, as evidenced by fluorescent microscopy (Figure 6). No MSNP-associated fluorescence was observed in the oocytes in Group 1 (MSNPs concentration: 0.003mg/ml; n=5). Surface attachment of MSNPs was observed in all oocytes in Group 2 (0.03mg/ml; n=8), and 3 out of 4 oocytes available for imaging in Group 3 (0.3mg/ml; n=8) (p>0.05). Remarkably, the greatest density of MSNPs attachment to the zona pellucida was present in Group 2, suggesting the absence of a direct dose- dependent response.

Microinjections of oocytes with MSNPs and sperm exposed to MSNPs

Mouse oocytes were microinjected with FITC-labelled PEI-coated MSNPs to assess the feasibility of assisted nanoparticles delivery through the zona pellucida. Oocyte microinjections with boar sperm exposed to FITC-labelled non-coated and PEI-coated MSNPs were performed as part of the mouse oocyte activation test (MOAT) to assess the oocyte activation capacity of boar sperm post-exposure. Unfortunately, no conclusive data was obtained from these experiments due to overall poor egg quality and technical difficulties encountered during the procedure. Tight attachment of MSNPs to the injecting pipette interfered with the injection accuracy, and the diameter of the injecting pipette was unsuitable for the boar sperm. After 24 hours of incubation, spontaneous oocyte activation was observed in 3 out of 5 (60%) non- injected oocytes and in 4 out of 5 (80%) PBS-injected oocytes (p>0.05), with the remaining oocytes undergoing degradation. Following ICSI with boar sperm exposed to MSNPs, oocyte activation was not observed in any of the cases, and 8 out of the 10 injected oocytes (80%) demonstrated obvious degradation signs (p>0.05, both for comparisons with non-injected and PBS-injected oocytes). Since the oocytes with better quality were selected for ICSI, MSNP injections were performed mostly on the oocytes which underwent spontaneous activation.

Injections were carried out exclusively for the purpose of investigation if assisted MSNP delivery through the zona pellucida is possible at all. Three hours after the injection, MSNP- associated fluorescence was observed in 2 out of 8 oocytes injected with FITC-labelled non- coated/PEI-coated MS Ps (Figure 7).

Discussion

The aim of this project was to investigate the possibility and safety of MS P-mediated delivery into mammalian gametes by evaluating nanoparticle internalisation and gamete viability after exposure to various concentrations of MSNPs with different surface functionalities.

In this project, MSNPs with different surface functionalities were applied to boar sperm and mouse oocytes to elucidate the properties of nanoparticles, which facilitate safe and effective delivery into cells. The findings suggested highly individual profiles of MSNPs interaction with gametes, and provided preliminary data essential to optimise the conditions of gamete-MSNPs exposure to improve the efficacy of interaction.

MSNP association with boar sperm

This study for the first time assessed interaction profiles of various MSNPs with boar sperm by quantifying MSNP-sperm association rates after different exposure times. The term 'MSNP-sperm association' was introduced to refer collectively to surface attachment and suspected cell internalisation of MSNPs, since the imaging techniques used during this series of experiments could not accurately discriminate between these two positive outcomes. In fact, it is hypothesised that complete cell internalisation is not crucial for the delivery of such cargo as nucleic acids. Kim et al. (2010 supra) report successful transfection of boar sperm with the EGFP gene attached to magnetic nanoparticles after their sequestration inside the plasma membrane, but not complete cell entry.

The present results support the current opinion that nanoparticle-cell interaction is highly dependent on the physicochemical properties of the particles. Indeed, size and surface charge remain the most studied determinants of nanomaterials uptake. Size-dependant internalisation has been demonstrated for a large number of biomedically-applied nanoparticles, including noble metal, oxide, mesoporous silica, and liposomes, with the optimal diameter highly variable between different materials (as reviewed by Gan et al, (2012) Biomed Microdevices 14(2): 259- 70). Surface characterisation with positively charged ligands consistently improves

internalisation rates across the majority of nanomaterials (Xia et al., (2009) ACS Nano 3(10): 3273-86). Recently, the impact of nanoparticle shape, volume and aspect ratio on cellular uptake has also been demonstrated. For instance, the internalisation profile of non-spherical nanoparticles in vitro has been described as a function of particle orientation with respect to the cell membrane (Champion et al, Proc Natl Acad Sci USA 104(29): 11901-4).

In the current series of experiments, FITC-labelled non-coated MS Ps (negative ζ potential) showed significantly higher association rate with boar sperm, compared to FITC- labelled PEI-coated (positive ζ potential) and fluorescent siRNA-bound MSNPs (balanced positive ζ potential), even when 10-fold lower concentrations were used. The latter two types of MSNPs, on the contrary, behaved in a strikingly similar manner, delivering profoundly lower sperm association rates. MSNP-sperm association rate could be partially rescued by a 10-fold increase in concentration for FITC-labelled PEI-coated, but not for siRNA-bound PEI-coated MSNPs.

Collectively, these findings confirmed that PEI coating changes the profile of MSNPs interaction with sperm, allows to electrostatically bind nucleic acids on the surface of MSNPs (Xia et al, 2009 supra; Horn et al, 2010 supra), and finally, sperm association profiles of the resulting structures do not differ from those in unloaded vehicles. It is of particular worth to note that these results contradict the general opinion that cationic coating improves nanoparticle uptake. The main explanation for this phenomenon is that all results on beneficial effects of PEI- functionalisation have been obtained in mammalian somatic cells, which differ from gametes in terms of morphology, functionality, and cell membrane composition (Tapia et al., 2012 supra). However, further validation of observed outcomes in a larger set of less heterogenous series of sperm samples is required before drawing final conclusions.

Provisional results on MSNP-sperm association profiles obtained using basic fluorescent microscopy were entirely supported by more detailed confocal imaging. Nevertheless, despite higher resolution, confocal microscopy also failed to accurately differentiate between MSNP adherence to the plasma membrane and cell internalisation. This justifies use of more sophisticated imaging techniques, for instance transmission/scanning electron microscopy, for the investigation of post-exposure localisation of MSNPs in gametes/embryos, at least during pilot experiments. In fact, localisation of nanoparticles in the sperm following co-incubation has only been assessed by a limited number of research groups. Kim et al. (2010 supra) described predominantly plasma membrane incorporation and/or nuclear localisation of magnetic nanoparticles in boar sperm after a maximum of 2 hours of incubation, as evidenced by confocal microscopy and TEM. Wiwanitkit et al. ((2009) supra) report penetration of gold nanoparticles into the human sperm head and tail after only 15 minutes of exposure. However, critical assessment of these results is necessary, since they were obtained in a single sample, using a methodology described as a 'clinical microscopy technique under high power', without any additional details provided (Wiwanitkit et al, 2009 supra).

Interestingly, in our experiments, non-typical spherical structures were discovered inside a number of sperm during confocal microscopy, particularly after exposure to fluorescent siRNA-bound PEI-coated MSNPs. In the view of primarily endocytotic mechanism of nanoparticles internalisation, these vesicles could correspond to internalised MSNPs, which failed to absorb a fluorescent tag and could not be detected using conventional fluorescent microscopy. Perhaps, the assessment of nanoparticle uptake can be more accurate if functional approaches, namely specific changes in cell function indicative of successful cargo delivery, are used instead of search for MSNP-sperm associations.

This set of experiments also provided preliminary data regarding the temporal dynamics of sperm-associated MSNPs localisation in relation to the sperm segments. A trend towards an increase in the percentage of MSNPs internalised into the sperm head with time was observed for all types of MSNPs analysed, however these data should be interpreted with caution due to questionable accuracy of fluorescent imaging, small number of samples, and absence of statistical significance of within-group changes.

MSNP association with mouse oocytes

This part of the project aimed to confirm the effect of MSNP interaction with mouse oocyte. A high consistency of results was observed, with current outcomes supporting surface absorption of MSNPs on the zona pellucida after 2 hours of incubation as the main type of association.

In this experiment, no direct dose-dependent response for MSNP association with the oocytes was observed, however the 0.03mg/ml concentration seemed to deliver the best outcomes. Since the zona pellucida represents a substantial mechanical barrier preventing internalisation of nanoparticles into the oocytes and preimplantational embryos, an attempt was made to introduce MSNPs into the oocytes via microinjection. This seemingly straightforward technique resulted in effective delivery of MSNPs only in 2 out of 8 injected oocytes. The major obstacle observed during the procedure was tight adherence of MSNPs to the tip of injecting pipette, which rendered the tip blunt and made injections virtually impossible. As mentioned previously, in this experiment the oocytes with better quality were selected for ICSI with boar sperm to perform MOAT, and MSNP injections were carried out in poor-quality spontaneously activated oocytes, which is reflected in the presence of multiple nuclear signals on the fluorescent images. This experiment did not aim to investigate the effects of MSNPs on oocyte function, but simply evaluated the possibility of assisted MS P delivery through the zona pellucida. Perhaps, outcomes of the procedure could be optimised by using larger injecting pipettes, or preparing ICSI dishes in advance to allow equilibration of MSNPs.

Our findings correspond to previously reported by Fynewever at al. (2007 supra). This research group utilised polysterene and polyacrylonitrile nanoparticles to tag mouse

preimplantational embryos, and never observed spontaneous migration of externally applied particles through the zona pellucida. Externally applied nanoparticles had no effect on embryo development, while intracytoplasmic microinjection of the same nanostructures on Day 0 interfered with embryo development on Day 2 and reduced blastocyst formation rate (Fynewever et al, 2007 supra). These observations indicate that oocyte microinjection with nanoparticles is less advantageous compared to sperm-mediated delivery of molecular constructs into the developing embryo through sperm-egg fusion. However, results obtained with polymer nanoparticles cannot be readily translated to other nanomaterials due to the high individuality of their biological effects. Studies of oocyte exposure to any other nanoparticles have not been carried out thus far.

Potential cytotoxicity of MSNPs in mammalian gametes

Internalised nanoparticles induce both favourable and adverse biological effects. Since nanoparticles function primarily as vehicles, favourable effects are mainly related to the action of delivered cargo in target cells. On the contrary, adverse effects arise as the result of detrimental impact of the nanomaterial itself upon molecular pathways and cell functions both in target and off-target locations. Similarly to cell uptake mechanisms, the degree of nanotoxicity is critically dependent on the nature, surface chemistry and size of the nanoparticle, as well as the properties of surface functional molecules. Current evidence demonstrates that in vitro nanotoxicity assessed by production of reactive oxygen species (ROS), DNA damage, and cell death rates in exposed cultures, increases with decreasing particle size and increasing surface area

(proportionate to dose and/or shape) for most of the nanomaterials studied thus far.

This study for the first time evaluated the impact of different concentrations of MSNPs with various surface functionalities upon boar sperm viability by assessing progressive motility at various duration of exposure in experimental-control pairs. The findings suggested that MSNPs did not impart any detrimental effect upon progressive sperm motility, regardless of MSNP concentration or the duration of exposure. Such results indicate a favourable safety profile of MSNPs, at least in the short term after acute contact. As mentioned previously, reports of the nanoparticle impact upon sperm motility after co-incubation are scarce and conflicting. Several research groups have reported preserved sperm motility and capability to undergo acrosomal reaction after exposure to magnetic nanoparticles for a maximum of 2 hours (Kim et al., 2010 supra). On the contrary, Wiwanitkit et al. (2009 supra) described a 20% reduction in motile sperm fraction in a human sample after 15 minutes of contact with nanoparticulate gold. Remarkably, the considerably larger physical diameter of MS Ps (~130nm) used during the project compared to magnetic (1 lnm) and gold (9nm) nanoparticles, tested in sperm by other research groups, could improve the safety of exposure. As mentioned previously, nanomaterials with larger sizes generally exhibit lower reactive surface areas and, hence, decreased

nanotoxicity compared to smaller-scale nanostructures.

It should be critically noted that the quality of boar semen used in the current experiments was highly variable, as evidenced by large heterogeneity of average progressive motility in replicate samples. Such shortcomings, however, allowed us to obtain preliminary data that sperm exposure to any of the synthesised MSNPs does not seem to affect viability profiles both in good- and poor-quality samples. However, further validation of the findings is required in a larger and less heterogenous sample set.

Heterologous ICSI of boar sperm exposed to MSNPs into mouse oocytes was carried out as part of MOAT. MOAT is an established methodology assessing fertilization capacity of the sperm, both in an experimental and clinical setting. In our experiment, MOAT failed to produce any conclusive results. Such outcomes were related to poor egg quality, causing extremely high spontaneous activation rates in the control and PBS-injected group, and significant technical difficulties of the procedure. The use of standard-sized injection pipettes designed for human sperm with a nearly 2-times smaller diameter, prevented smooth aspiration and release of the boar sperm into the oocyte. The damaging nature of the procedure was reflected in high degeneration rate in post-ICSI oocytes compared to controls and PBS-injected, even though ICSI was performed by experienced clinical embryologists. Since successful fertilization and interspecies embryo development after boar sperm injection into mouse oocytes has been described previously (Barnetova et al, Journal of Reproduction and Development 56(6): 601- 606), adjustments in the injection technique could yield more favourable outcomes in future.

Conclusions

This project for the first time demonstrated the outcomes of mammalian gametes exposure to various concentrations of MSNPs with different surface functionalities, with particular emphasis on mammalian sperm viability and MSNP-association. Integration of MSNP synthesis, characterisation and cargo loading steps into the project design represented a major advantage. Such methodology facilitated continuity between chemical and biological experimentation, and reduced observer bias.

Our results demonstrated the simplicity of MSNPs production and functionalisation using conventional wet chemistry approaches, as well as the possibility to readily modify

physicochemical properties of the particles, resulting in production of MSNPs with distinctive cell interaction profiles. This project, for the first time, demonstrated that MSNPs did not exert detrimental effect upon boar sperm viability, regardless of the physicochemical properties and concentration of MSNPs, exposure time and overall semen sample quality.

Our results expanded knowledge on the impact of specific MSNPs properties upon sperm association profiles. In particular, the MSNP-sperm association rate, but not the temporal dynamics of sperm-associated MSNP location, was highly dependent upon the surface chemistry of the particles. Contrary to findings in mammalian somatic cells, negative ζ potential promotes MSNP-sperm association. Importantly, cargo loading onto MSNPs does not appear to alter cell interaction profiles, compared to unloaded vehicles. The outcome of mouse oocyte exposure to MSNPs also supported the previous unpublished results demonstrating that surface attachment to the zona pellucida is likely to be the main type of MSNP-oocyte association.

In summary, our results demonstrate safety of MSNPs as delivery vehicles in

reproductive applications, and highly distinctive MSNP-gamete association profiles, which are opposite to those in mammalian somatic cells, while remaining similarly dependent upon particle surface chemistry.

Example 2

Methods

A summary of types and surface modifications of MSNPs, and types of cargo used in this study is presented in Table 6 below.

Primary type Modification Cargo Name in text Rationale for use of MSNPs

FITC-labelled None 'Unmodified Primary tests of

MSNPs' nanotoxicity and

PEI 'PEI-coated association rates with

MSNPs' sperm for:

APTES 'APTES-coated • Unmodified MSNPs

MSNPs' • Common

modifications of

MSNPs used for cargo loading ('empty vehicles' : PEI- and

APTES-coated

MSNPs)

Experiments performed in a series of

MSNP/sperm ratios to develop the safe and effective

nanoparticle/sperm ratios and incubation times

Non- PEI Fluorescent 'Lamin A/C Tests of nanotoxicity and fluorescent lamin A/C siRNA-loaded association rates with

siRNA MSNPs' sperm for two most

or common candidates for

'siRNA-loaded delivery ('prototype' of

MSNPs' cargo):

• 'Nucleic acid'

• 'Protein'

APTES Fluorescent 'mCherry protein- mCherry loaded MSNPs' Both types of cargo- protein or loaded MSNPs applied in

'mCherry-loaded a single minimally

MSNPs' effective ratio

Table 6

Synthesis and functionalisation of MSNPs

Reagents for synthesis and functionalisation of MSNPs

Anhydrous sodium hydroxide (NaOH, >98%), hexadecyltrimethylammonium bromide (CTAB, >99%), tetraethyl orthosilicate (TEOS, 98%), 3-(trihydroxysilyl)propyl

methylphosphonate (3-THPMP, monosodium salt, 42 wt.% solution in water), fluorescein i sothiocyanate (FITC, >90%), (3-aminopropyl)triethoxysilane (APTES, >98%),

Polyethileneimine (PEI, 50 wt.% solution in water, MW 1.3kD, Mn l,200g/mol), concentrated hydrochloric acid (HCL; 37.2 wt.% solution in water, 12.1M) and Dulbecco's phosphate based saline (DPBS) were sourced from Sigma-Aldrich (UK). Methanol (>99.8%) was sourced from Rathburn Chemicals (UK). Absolute ethanol (>99.8%) was sourced from Riedel-de Haen (Germany).

Synthesis of non-fluorescent MSNPs

In a round bottom flask, lOOmg of CTAB was dissolved in a mixture of 48ml double distilled water (DDW) and 0.35ml of 2M NaOH. The solution was heated in a silicone oil bath to 80°C with magnetic stirring. After the temperature had stabilised, 0.5ml of TEOS was added to the reaction. After 15 minutes, 0.127ml of 3-THPMP was added, and reaction was stirred for 2 hrs at 80°C. The solution was then cooled to room temperature (RT), and MSNPs were recovered and washed twice with methanol via centrifugation. Particles were redispersed in a mixture of 40ml of methanol and 2ml of 12.1M HCL, and refluxed for 24 hrs at 80°C with magnetic stirring to remove CTAB. After refluxing, MSNPs were recovered and washed twice in absolute ethanol via centrifugation, and vacuum dried overnight.

Synthesis of fluorescent MSNPs

FITC was introduced into the silica framework in the form of FITC-APTES conjugate. The conjugate was prepared by mixing ΙΟΟμΙ of APTES with 25mg of FITC in 5ml of absolute ethanol, and stirring magnetically for 12 hrs under a dry nitrogen atmosphere. To synthesise fluorescent MSNPs, 50μ1 of FITC-APTES conjugate was added to the reaction 10 minutes after 0.5ml of TEOS had been introduced. After 5 minutes, 0.127ml of 3-THPMP was added, and the remaining steps were carried out in accordance with the standard procedure.

Surface functionalisation of MSNPs with PEI

Functionalisation of the surface of MSNPs with a cationic polymer PEI aimed to decrease particle agglomeration, improve cell interaction and provide positively charged surface for electrostatic binding of cargo²⁶. Coating of non-fluorescent and fluorescent MSNPs was performed in an identical way. In a round bottom flask, 5mg of dry MSNPs was redispersed in a mixture of 75μ1 of PEI and 30ml of absolute ethanol, and stirred at RT for 1 hour. MSNPs were recovered and washed by centrifugation in absolute ethanol and sterile DDW, and vacuum dried overnight.

Surface functionalisation of MSNPs with APTES Non-fluorescent and fluorescent MSNPs were coated with APTES to provide amine groups for covalent cross-linking with carboxyl groups of payloads²⁷. Coating of non- fluorescent and fluorescent MSNPs was performed identically. MSNPs were redispersed in DDW to a concentration of lOmg/ml, and APTES was added to 5% by volume. The reaction was stirred magnetically for 1 hour at RT. Coated particles were recovered and washed in sterile DDW by centrifugation, and vacuum dried overnight.

Characterisation of mesoporous silica nanoparticles

For transmission electron microscopy (TEM), dry unmodified MSNPs were redispersed in absolute ethanol, sonicated for 30s, and a drop of dispersion was loaded on a TEM grid coated with lacey carbon film (Agar Scientific, UK). Imaging was performed on a JEOL JEM-2010 analytical TEM (JEOL Ltd., Japan). Acquired images were processed using Digital Micrograph 3.7.4 for GMS 1.2 Build 45 (Gatan Inc., Pleasanton, CA, USA). Physical size of the particles and diameter of the pores was measured in a minimum of 100 nanoparticles in each sample.

For scanning electron microscopy (SEM), dry samples of unmodified MSNPs were dusted onto a carbon taped SEM stub, and 3nm layer coating of platinum was applied. The nanoparticle size and morphology was investigated using a JEOL JSM-840F SEM (JEOL Ltd., Japan). Images were collected in the secondary electron (SE) imaging mode.

Hydrodynamic size of synthesised MSNPs (non-coated, PEI-coated and APTES-coated) was analysed using a disc centrifuge (CPS DC24000, CPS Instruments Europe, Netherlands), which determines the diameter of particles in solution based on their sedimentation profiles during centrifugation in a liquid gradient. Particle size was measured at disk speed 24,000rpm and pH 7.0. External calibration with a kit calibration standard (0.377μm-sized polyvinyl chloride latex particles dispersed in distilled water, density 1.385g/ml; CPS Instruments, Netherlands) was performed before each test to maintain accuracy.

Electrokinetic (Q potential of synthesised MSNPs (non-coated, PEI-coated and APTES- coated) was measured using the dynamic light scattering (DLS) technique using a Zetasizer Nano ZS (Malvern Instruments, UK). The Zetasizer records the phase/frequency shift of a laser beam coming in contact with charged particles, moving in the electric field to the oppositely charged electrode, and converts their velocity to ζ potential. Measurements were performed at 25°C and pH 7.0.

Loading of MSNPs with cargo Non-fluorescent PEI- and APTES-coated MSNPs were loaded with fluorescent lamin A/C siRNA and mCherry protein, respectively. These two types of payloads were chosen as the 'prototypes' for the two classes of biological cargo, which represent a particular interest for delivery into mammalian sperm. Loading of siRNA onto PEI-coated MSNPs was achieved via electrostatic interaction between the negatively-charged nucleic acid and the positively-charged cationic surface of functionalised MSNPs. Loading of APTES-coated MSNPs with mCherry was performed via the cross-linking of amine groups on the functionalised surface of MSNPs with carboxyl groups of mCherry protein using l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) coupling agent (Fisher Scientific, UK).

Loading with siRNA

To load siRNA onto nanoparticles, non-fluorescent PEI-coated MSNPs were washed by centrifugation and redispersed in nuclease-free water (Ambion, UK). Fluorescent lamin A/C siRNA (siGLO Lamin A/C Control siRNA (human/mouse/rat), D-001620-01-05, Thermo Fisher Scientific, CO, USA) was added to the resulting MSNP dispersion in a 1 :2 ratio

(siRNA:MSNPs) by mass, and the mixture was incubated for 24 hrs at 4°C. MSNPs were recovered through centrifugation and redispersed in 1.0ml of nuclease-free water. Loading was confirmed by calculation of siRNA concentration in solution before and after the reaction, based on the measurement of absorbance at 260nm by spectrophotometry (BiophotometerPlus, Eppendorf, UK).

Loading with protein

Molecular cloning of mCherry expression construct

The DNA sequence encoding a synthetic construct of monomelic red fluorescent protein (mCherry; NCBI Accession Number AY678264) was amplified by polymerase chain reaction (PCR) using the High-Fidelity PCR Master kit (Roche Diagnostics Ltd., UK) and a set of engineered primers (Life Technologies Ltd., UK) under PCR conditions, as previously described¹. Detailed information on the restriction enzymes and primer sequences used herein, is presented in Table 7.

Table 7 - Restriction enzymes and primer sequences used during PCR amplification Construct name Restriction Primer sequences

enzymes

mCherry Open 5 ' -T ATACCGGTATGGTGAGC AAGGGCGAG- Reading Frame 3' (Forward)

5'-TATGGTACCCTTGTACAGCTCGTCCAT-3' (Reverse)

For cloning of the protein expression construct, the amplified mCherry sequence was ligated into the pHLSec vector. Extraction and purification of plasmid DNA from a clone of TOP 10 competent cells transformed through heat shock was performed using the QIAprep Spin Miniprep Kit (QIAGEN, UK). The yield of DNA was increased using the HiSPeed Plasmid Midi Kit (QIAGEN, UK). Cloned protein expression construct was verified by DNA sequencing (Source Bioscience LifeSciences, University of Oxford, UK).

Mammalian cell culture and mCherry expression

Transformed human embryonic kidney cells (HEK293T) were cultured under standard conditions and seeded into 175cm tissue culture flasks at a confluence of 30-50%. Transfection with the purified mCherry plasmid DNA was performed at 50-70% confluency using the jetPEI DNA transfection kit (Polyplus-transfection S A, France) according to the manufacturer' s protocol. Cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), l%v/v 5000U penicillin, 5mg/ml streptomycin, 2mM L-glutamine and sodium pyruvate (all reagents from Sigma-Aldrich, UK) for 24 hrs, after which the medium was changed to FBS-free.

Purification of mCherry protein

Culture media was collected on the day 6 of culture, supplemented with

phenylmethanesulfonylfluoride (PMSF; Sigma-Aldrich, UK) and ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, UK) to a final concentration of ImM, and centrifuged for 30min at 6,000g at 4°C. Supernatant was filtered through a 0.45 μπι polyethersulfone membrane filter, and passed through a column containing Protein A Sepharose beads (GE Healthcare Lifesciences, UK) to bind the Fc-tag. The column was washed with 20 column volumes of PBS, followed by 10 column volumes of TNED buffer (50mM Tris (pH 8.0), 150mM NaCl, lOmM EDTA, and ImM diothiothreitol; all reagents from Sigma-Aldrich, UK). 3C protease was added in 1 column volume of TNED buffer and left to shake overnight to cleave the Fc-tag. Protein was eluted with TNED. Purified protein structure was verified by liquid chromatography/mass spectrometry (Centre for Cellular and Molecular Physiology, Nuffield Department of Medicine, University of Oxford, UK).

Loading of MSNPs with mCherry protein

Loading of APTES-coated MSNPs was performed via cross-linking of amine groups on the functionalised surface of mesoporous silica with carboxyl groups of mCherry protein using l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) coupling agent (Fisher Scientific, UK). For EDC coupling, 2mg of APTES-functionalised MSNPs were redispersed in 200μ1 of 0.1M 2-[n-morpholino]ethane sulfonic acid (MES) buffer (Fisher Scientific, UK) and mixed with 6mg of mCherry protein dissolved in 1500μ1 0.1M MES buffer. lOmg of EDC was dissolved in 1ml of DDW, and ΙΟΟμΙ of the solution was added immediately to the MSNP- mCherry mixture. The reaction was incubated for 2 hours at RT on a shaker plate. MSNPs were recovered through centrifugation, and redispersed in PBS.

Semen preparation and application of MSNPs to sperm

All biological experimentations were carried out in the boar sperm, which represents a common animal model for human sperm in the pilot experiments in reproductive biology, due to the similarities in morphology and physiology, and overall robustness. Boar sperm was sourced from a licensed pig breeding company (JSR Genetics, UK), and delivered in a commercial extender at 17°C. Sperm motility was activated by incubation for 15 minutes at 35°C, according to the supplier's instructions. Sperm concentration was assessed in a Biirker-Turk

haemocytometer, at 200x magnification using a positive contrast phase objective (Nikon UK Ltd.). Following activation, 1ml aliquots were withdrawn from each sample, centrifuged at 500g for 10 minutes, and washed from the extender with phosphate-buffered saline (PBS, Oxoid, UK). Washed sperm were then subject to treatment with MSNPs or PBS (controls). Each experiment was repeated for a minimum of three times, using the samples obtained from different animals.

To evaluate potential nanotoxicity of MSNPs in sperm and determine the optimal doses and incubation times, washed sperm were exposed to solutions of three types of unloaded fluorescent MSNPs in PBS (unmodified, PEI-coated, and APTES-coated) in the ratios of lC^g, 15μg and 3C^g of particles per 10⁷ sperm. In the control group, washed sperm were incubated with PBS. Incubation was carried out for up to 4 hrs at 37°C under low-oxygen atmosphere, representing the conventional timeframe for sperm handling prior to IVF/ICSI. Sperm motility and viability, both representing the first-line parameters correlating with the sperm fertilisation capacity, were assessed after 2 and 4 hrs of incubation. Additionally, the second-line parameters correlating with sperm functionality, including acrosome morphology and sperm DNA fragmentation, were evaluated after 4 hrs of incubation. Association rates between sperm and unloaded MS Ps were determined after 2, 3 and 4 hrs.

After evaluation of potential nanotoxicity and identification of the minimally effective particle/cell ratios and incubation times, washed sperm were exposed to the MSNPs carrying prototypes of biological cargo: siRNA- and mCherry-loaded MSNPs in the ratio of lC^g of particles per ~10⁷ sperm, for 2 hrs. Incubation was carried out in similar conditions. After 2 hours, sperm motility, viability, and association rates with MSNPs were evaluated.

Motility assessment

Motility assessment was performed using a computer-assisted sperm analysis system (CASA; HTM-Ceros v.12.3, Hamilton Thorne, MA, USA). Systems for CASA automatically calculate proportions of specific subpopulations of sperm within a sample, depending on their patterns of motility, and provide a quick and detailed assessment of sperm motion profiles. In this study, analysed parameters included total motility (%), progressive motility (%), smoothed path velocity (VAP, μιη/sec), straight line velocity (VSL, μπι/sec), track velocity (VCL, μιη/sec), straightness (STR: ratio of VSL/VAP, %), linearity (LIN: ratio of VSL/VCL, %), amplitude of lateral head displacement (ALH, μπι), and beat cross frequency (BCF, Hz).

For CASA, samples were loaded into a 2C^m-deep Leja counting chamber, and equilibrated on a warm stage (37°C) for 2 minutes. Images were acquired at lOOx magnification using a negative contrast phase objective (Nikon UK Ltd.). A minimum of 5 fields, containing at least 200 sperm were evaluated in each sample.

Viability assessment

For viability evaluation, a 5μ1 drop of each sample was mixed with 5μ1 of eosin Y (1% w/v in saline, VitalScreen, Microm, UK) on a microscope slide, covered with a coverslip, and equilibrated for 30 seconds. The number of stained (red or dark-pink; 'dead') and unstained (white or light-pink; 'live') sperm was counted at 400x magnification, in a minimum of 200 sperm. Acrosome morphology was evaluated by examining the integrity of the acrosomal apical ridge in unstained fixed sperm samples. This straightforward technique allows the identification of structural impairments in the sperm acrosomal region, directly involved with the penetration of zona pellucida during fertilisation, which arise after an acute or prolonged contact with suboptimal environment and correlate with the sperm viability and fertilisation potential. To perform the evaluation, sperm samples were fixed with 4.5% phosphate buffered formalin solution (Sigma- Aldrich, UK) for 10 minutes. A 'wet drop' for analysis was prepared by placing ΙΟμΙ of the sample on a microscope slide and covering with a coverslip. Slides were examined at lOOOx magnification using a positive contrast phase oil-immersion objective (Leica

Microsystems UK Ltd.). A minimum of 200 sperm were assessed for acrosome morphology, and classified into 4 categories: normal apical ridge (NAR), damaged apical ridge (DAR), missing apical ridge (MAR), and loose acrosomal cap (LAC).

Calculation of sperm DNA fragmentation index

The proportion of sperm carrying fragmented DNA is being increasingly considered an independent marker of sperm quality and capacity to form viable embryos post-fertilisation, and can be affected by exposure to suboptimal environment. Sperm DNA fragmentation index was assessed using sperm chromatin dispersion technique, with a Sus-halomax kit (Halotech DNA SL, Spain) according to the manufacturer's instructions. This test is based on response variations of sperm chromatin with fragmented and non-fragmented DNA to protein depletion treatment, and has been previously characterised as a simple, reproducible and inexpensive technique to detect DNA breakages. In brief, analysed sperm samples were diluted in PBS to a concentration of 15-20xl0⁶ sperm/ml, and added into an agarose microgel in a 1 :2 ratio. Samples were then lysed for 5 minutes, washed with double distilled water (DDW), and dehydrated in a series of ethanol concentrations. After drying, processed slides were stained with 2μ1 of 4', 6- diamidino-2-phenylindole (DAPI)-containing mounting medium (Vectashield H-1200, Vector Laboratories, UK), and examined at 400x magnification under a fluorescent microscope with a 330-380nm (DAPI) excitation wavelength filter (Nikon UK Ltd.). A minimum of 300 sperm per sample were examined for the presence of a large halo of chromatin dispersion (fragmented DNA), and classified into either fragmented or non-fragmented DNA accordingly. Sperm DNA fragmentation index was calculated as a percentage of sperm with fragmented DNA from a total studied sperm population. Calculation of MSNP-syerm association rate

To quantify the number of sperm associated with MSNPs, samples were fixed with 10% formalin solution (Sigma- Aldrich, UK) for 10 minutes after 2, 3 and 4 hrs of incubation, and then washed from the fixative and unbound MSNPs in PBS via centrifugation. Sperm were transferred to poly-L-lysine coated slides, incubated in a humidifying chamber for 30 minutes at room temperature, washed twice in PBS, and mounted with glycerol-based media (Vectashield H-1000, Vector Laboratories, UK). Slides were examined at 400x magnification under a fluorescent microscope equipped with 465-495nm (green) and 540-588nm (red) excitation wavelength filters (Nikon UK Ltd.). The number of sperm associated with MSNPs was counted in a minimum of 200 cells. Higher resolution imaging was performed at 600x magnification with an oil-immersion objective under a confocal laser microscope with 488nm (green) and 559nm (red) excitation lines (Olympus UK Ltd.). Acquired images were processed with Fiji/ImageJ 1.47i (National Institute of Health, USA).

Statistical analysis

Data are presented as the mean±standard error of the mean (SEM), unless specified otherwise. Data were analysed by analysis of variance (ANOVA), followed by Dunnet post-hoc test for comparisons with a control group or Neuman-Keuls post-hoc test for comparisons between experimental groups. Differences were considered significant at p<0.05. Statistical analysis was performed using Statistica vlO.O (StatSoft Inc., OK, USA). Representative graphs were constructed in GraphPad Prism v5.04 (GraphPad Software, La Jolla, CA, USA).

Results

Characterisation of MSNPs

Synthesised MSNPs were characterised by electron microscopy. The particles were shown to be slightly non-spherical with elongation in the direction of the pore channels. The mesoporous silica comprised ordered nanometre-sized pores shown by TEM to have hexagonal symmetry when aligned with the beam (Figure 8). Unmodified MSNPs had a mean external diameter of 138.4±3.8nm with 2.1±0.1nm-sized pores.

Nanoparticles were coated with PEI or APTES, both representing established

methodologies of nanomaterial functionalisation. Efficacy of PEI-and APTES-coating was confirmed through measurement of electrokinetic (ζ) potential, which indicated an increase or complete reversal of inherently negative ζ potential of unmodified silica

(-31.50±0.60mV, -1.91±0.27mV and 37.73±0.27mV for FITC-labelled unmodified, APTES- coated and PEI-coated MSNPs, respectively) (see Tables 8 and 9).

Table 8 - Characterisation of synthesised fluorescent mesoporous silica nanoparticles (MSNPs+FITC): ζ potential and mean hydrodynamic diameter

ζ potential (mV)* Mean hydrodynamic diameter (nm)

Unmodified MSNPs -31.50±0.60 270

PEI-coated MSNPs 37.73±0.27 170

APTES-coated MSNPs -1.91±0.27 280

*Data presented as mean±SD

Table 9 - Characterisation of synthesised non-fluorescent mesoporous silica nanoparticles (MSNPs) before and after loading with cargo: ζ potential and mean hydrodynamic diameter

ζ potential (mV)* Mean hydrodynamic diameter (nm)

Unmodified MSNPs -24.03±0.48 275

PEI-coated MSNPs 44.53±0.29 147

Lamin A/C siRNA-loaded MSNPs 28.13±0.80 157

APTES-coated MSNPs -5.94±0.23 254

mCherry-loaded MSNPs 5.29±1.17 286

*Data presented as mean±SD

Reduced propensity of PEI-treated MSNPs to agglomerate in suspension was proven by reduced mean hydrodynamic diameter in PEI-coated samples (270nm vs 170nm for FITC- labelled unmodified and PEI-coated MSNPs, respectively). In contrast, APTES coating had little effect upon the agglomeration of MSNPs (270nm vs 280nm for FITC-labelled unmodified and APTES-coated MSNPs, respectively).

As anticipated, loading with negatively charged lamin A/C siRNA reduced the positive values of ζ potential of unloaded non-fluorescent PEI-coated MSNPs (44.53±0.29mV vs 28.13±0.80mV for unloaded and loaded MSNPs, respectively). Adsorption of mCherry protein increased the ζ potential of unloaded non- fluorescent APTES-coated MSNPs (-5.94±0.23mV vs 5.29±1.17mV, for unloaded and loaded MSNPs, respectively). Loading with both types of payloads resulted in a slight increase of the mean hydrodynamic diameter of the particles (147nm vs 157nm for unloaded PEI-coated and lamin A/C siRNA-loaded MSNPs; 254nm vs 286nm for unloaded APTES-coated and mCherry-loaded MSNPs, respectively). Nanotoxicity of unloaded MSNPs in sperm

Sperm motility and viability

Motility and viability were evaluated in sperm samples after 2 and 4 hours of incubation with three types of unloaded MSNPs (unmodified, PEI-coated and APTES-coated) at a ratio of 10, 15 and 30μg of particles per 10⁷ sperm. Exposure of sperm to MSNPs did not cause significant detrimental effect upon the mean proportions of motile, progressively motile and viable sperm assessed by CASA and eosin Y staining (p>0.05, compared to time-matched controls) (Figure 9). Moreover, there was no significant effect of the type and dose of the particles, and duration of incubation, upon total and progressive sperm motility and viability, and all parameters after incubation complied with the requirements for mammalian sperm used in IVF/ICSI. A trend towards a 'protective effect' of PEI-coated MSNPs upon progressive motility after 4 hours of incubation was oberved, however values did not reach the level of statistical significance.

Similarly, mean sperm kinematic parameters evaluated by CASA in MSNP -treated samples remained unaltered compared to controls at both time points irrespective of the type and dose of nanoparticles (Figure 10). A small reduction in mean ALH, and an increase in mean BCF, was observed in sperm samples treated with APTES-coated MSNPs, however these changes were not associated with alterations in other kinematic parameters and were not significant, compared to the control group.

Acrosome morphology

We examined the integrity of acrosomal apical ridge in sperm samples exposed to three types of unloaded MSNPs (unmodified, PEI-coated and APTES-coated) in a series of particle/sperm ratios after 4 hours of incubation. Exposure of sperm to MSNPs did not affect acrosome status at selected time points (see Table 10). Table 10 - Proportions of boar sperm categorised based on the appearance of the acrosomal apical ridge and DNA fragmentations after 4 hours of exposure to different modifications of unloaded mesoporous silica nanoparticles (MSNPs) in three particle/cell ratios (mean±SEM)

NAR (%) DAR (%) MAR (%) LAC (%) DNA

fragmentation index (%)

Control (PBS) (n=6) 84.4±3.0 2.1±0.4 2.3±0.5 11.2±2.9* 2.8±0.5 ί μg of particles Unmodified MSNPs 90.3±2.4 2.7±0.7 2.5±1.0 4.5±1.6 1.0±0.7 in PBS per 10⁷ PEI-coated MSNPs 89.0±2.1 4.0±2.9 2.6±1.0 4.4±1.4 1.4±1.0 sperm (n=3) APTES-coated 89.6±1.4 3.0±0.6 1.0±0.6 6.4±1.9 1.5±0.5

MSNPs

15μ of particles Unmodified MSNPs 88.3±3.3 4.3±1.8 1.5±0.5 6.0±2.0 0.7±0.1 in PBS per 10⁷ PEI-coated MSNPs 84.6±10.2 2.3±1.8 1.8±1.7 10.6±6.6 1.0±0.5 sperm (n=3) APTES-coated 85.2±2.4 6.5±2.1 2.5±0.5 5.8±0.7 0.7±0.4

MSNPs

30μg of particles Unmodified MSNPs 88.2±3.8 4.2±2.2 0.5±0.4 7.2±2.2 0.8±0.2 in PBS per 10⁷ PEI-coated MSNPs 86.1±4.8 4.3±3.3 1.8±1.3 7.9±0.3 0.7±0.4 sperm (n=3) APTES-coated 94.4±2.0 2.3±1.1 1.3±0.3 2.0±0.6* 1.2±0.2

MSNPs

*p<0.05; NAR = 'normal acrosomal ridge'; DAR = 'damaged acrosomal ridge'; MAR = 'missing

acrosomal ridge'; LAC = 'loosened acrosome cap'

Mean proportions of sperm with NAR, indicating a structurally intact acrosome, which correlates with preserved sperm capacity to penetrate the zona pellucida, consistently exceeded 80% across all experimental and control samples, regardless of the type and dose of MSNPs.

Interestingly, a significantly lower percentage of sperm with LAC, in which the acrosomal cap becomes detached from the sperm head, representing the acrosomal deterioration and loss of function, was observed in samples treated with APTES-coated MSNPs at the highest particle/cell ratio, compared to controls (LAC: 11.2±2.9% vs 2.0±0.6%; p<0.05; for experimental and control group, respectively). This finding suggests an unexpected protective role of APTES-coated

MSNPs in preserving acrosomal morphology during incubation, and justifies further research.

Sperm DNA fragmentation index

Sperm DNA fragmentations were visualised in MSNP -treated and control samples using the sperm chromatin dispersion method. Mean DNA fragmentation index in sperm treated with MSNPs for 4 hours was not significantly different from controls. Across all analysed samples, DNA fragmentations were consistently observed in fewer than 5% of sperm. There was no significant effect of MSNP type and dose upon sperm DNA fragmentation index (p>0.05) (Table 10). Association between sperm and unloaded MSNPs

Association rates between boar sperm and unloaded MSNPs were assessed after 2, 3 and 4 hours of incubation to evaluate the effects of time and MSNP dose upon particle-target cell interaction. The term 'association' was introduced to collectively describe surface attachment and suspected internalisation of MSNPs into sperm, both representing positive outcomes of interaction between nanoparticles and the targeted sperm cells.

MSNPs associated with sperm produced discrete fluorescent signals in the projection of the sperm head, midpiece or tail (Figure 11). Association rate between MSNPs and boar sperm was dependent upon the type and dose of nanoparticles, and did not change significantly throughout the incubation (Figure 12). A significantly higher mean percentage of sperm binding MSNPs was observed after 2 hours of treatment with unmodified particles in the highest particle/cell ratio (3C^g per 10⁷ sperm), compared to lower doses of the same type of MSNPs (17.4±2.9%, 20.8±5.7% and 41.0±9.2%, for 10, 15 and 3(^g per 10⁷ sperm, respectively;

p<0.05). This increase was temporary, as evidenced by a subsequent decline in binding between unmodified MSNPs and sperm in the highest parti cle/sperm ratio after 3 and 4 hours of exposure (Figure 12). This observation suggests a possibility for temporary weak interaction of sperm with unmodified mesoporous silica after 2 hours of treatment, followed by dissociation of bound particles from sperm at later stages. On the contrary, MSNP-sperm association rates in the two remaining types of unloaded MSNPs (PEI-coated and APTES-coated) did not change significantly throughout incubation, and were not markedly influenced by the dose of particles.

Nanotoxicity and association between sperm and loaded MSNPs

After confirming the biocompatibility of various mesoporous silica modifications in boar sperm, a minimally effective dose of 10 μg per 10⁷ sperm and a 2-hour incubation time was chosen for subsequent experimentations with cargo-loaded MSNPs. In this set of experiments, we applied a short panel of nanotoxicity tests, focusing mainly on the effects of exposure to loaded MSNPs upon the motility and viability of boar sperm.

After 2 hours of incubation, siRNA- and protein-loaded MSNPs did not significantly affect mean proportions of motile, progressively motile and viable sperm, compared to time- matched controls. Similarly to unloaded MSNPs, treatment with MSNPs carrying the two most common types of biological cargo did not alter the sperm motility parameters, as assessed by CASA (Table 11). Table 11 - Motility and vitality parameters in sperm after 2 hours of exposure to siRNA- and protein-loaded mesoporous silica nanoparticles (MSNPs) (mean±SEM)

Control Lamin A/C mCherry-loaded (PBS) siRNA-loaded MSNPs (n=3) (n=6) MSNPs (n=3)

Total motility 57.6±6.0 74.3±3.5 57.0±3.0

Progressive motility 25.4±4.3 42.0±4.6 30.0±5.1

Viability 74.3±5.9 89.0±3.1 71.6±2.0

Motility parameters:

VAP (μιη/sec) 68.9±5.0 79.2±5.0 58.3±3.1

VSL (μιη/sec) 44.3±2.3 52.9±3.2 38.2±2.3

VCL (μιη/sec) 139.6±11.7 157.7±11.1 134.9±14.3

ALH (μιη) 9.0±0.7 10.2±0.42 8.93±0.3

BCF (Hz) 28.5±1.8 26.6±1.8 28.4±0.5

STR (%) 62.1±4.3 64.0±2.6 66.0±5.0

LIN (%) 32.9±2.2 34.0±1.8 31.0±2.2

We also calculated association rates between loaded MSNPs and boar sperm at 2 hours of incubation and compared them with corresponding values for unloaded MSNPs in the lC^g per 10⁷ sperm ratio (PEI-coated MSNPs vs lamin A/C siRNA-loaded MSNPs and APTES-coated MSNPs vs mCherry-loaded MSNPs). Association rates between sperm and siRNA-loaded MSNPs were significantly lower than for unloaded PEI-coated MSNPs (1.8±0.6% vs 12.0±1.9%, respectively; p<0.05). In contrast, loading of APTES-coated MSNPs with mCherry had no significant effect upon association rates with sperm (25.3±4.2% vs 19.0±1.5% for mCherry- loaded and unloaded APTES-coated MSNPs). The density of sperm coating with lamin A/C siRNA-loaded MSNPs was lower, compared to mCherry-loaded MSNPs (Figure 13).

Discussion

In this Example, we report the effects of MSNPs, functionalised with two common types of surface coating (PEI and APTES) and optionally loaded with lamin A/C siRNA or mCherry protein, upon boar sperm after incubation in vitro.

Biocompatibility of MSNPs with boar sperm represents an important finding of this study. Our results show that exposure of boar sperm to unmodified, surface-functionalised and cargo-loaded MSNPs in various parti cle/sperm ratios does not exert detrimental effects upon the main parameters of sperm function, including motility, viability, kinematic features, and acrosome status, after up to 4 hours of incubation in vitro, the conventional timeframe for sperm handling prior to IVF. In this set of experiments, mean values of sperm motion characteristics assessed by CAS A after exposure to various modifications and doses of MSNPs remained within reported reference ranges for fertile boar sperm. Similarly, we did not detect a reduction in the proportion of sperm exhibiting a normal apical ridge after exposure to unmodified, PEI- and APTES-coated MSNPs using high-magnification light microscopy. In addition, we did not observe a significant increase in the proportion of sperm exhibiting fragmented DNA after incubation with various types and doses MSNPs, which suggests their low genotoxicity after short-term exposure.

Another significant finding of our study is that MSNPs spontaneously form associations with boar sperm during incubation, which, crucially, did not affect sperm functionality.

In the set of experiments with unloaded MSNPs, association rate with sperm was not markedly influenced by the type and dose of the particles, and incubation time. In contrast, loading MSNPs with cargo changed their association rate with sperm. In this study, we used lamin A/C siRNA and mCherry protein as models for typical types of molecular cargo for delivery into gametes or early-stage embryos. The reduction of association rates between siRNA- loaded MSNPs and sperm, compared to 'empty' (PEI-coated) MSNPs, represents an unexpected finding. It is most likely, that in our experiment, siRNA was primarily absorbed onto the surface of the particles, as evidenced by changes in ζ potential and hydrodynamic size following loading, External localisation of cargo could, therefore, limit binding of MSNPs only to dedicated regions of the sperm membrane, where interaction with exogenous nucleic acid occurs.

In conclusion, we present encouraging data concerning the biocompatibility of MSNPs with boar sperm and their potential for binding with these highly specialised cells after exposure in vitro, without compromising essential sperm functions. Therefore, MSNPs can represent a robust and versatile component in the arsenal of nanomaterial-based candidates for use in reproductive research, including delivery of molecular constructs into sperm for subsequent transfer into the oocytes and early-stage embryos at the time of fertilisation. This non-invasive approach can overcome the high costs and complexity of traditionally applied micromanipulation techniques, and facilitate the non-invasive genetic modification, targeted bioimaging of specific cellular structures or physiological processes in the early stage embryos, and supplementation of specific molecular deficiencies associated with aberrant fertilisation or embryo development profile.

Claims

1. A method of delivering at least one molecule into a reproductive cell or an embryonic cell, the method comprising contacting the cell with a mesoporous silica nanoparticle (MSNP) comprising the at least one molecule and thereby delivering the at least one molecule into the cell.

2. A method according to claim 1, wherein the reproductive cell is a sperm, an oocyte or an ovum.

3. A method according to claim 1 or 2 wherein the at least one molecule is a therapeutic agent, a diagnostic agent, a fluorescent molecule, a dye or a calcium ionophore.

4. A method according to any one of claims 1 to 3 wherein the at least one molecule is a polymer, amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide or

polynucleotide.

5. A method according claim 4 wherein the protein is phospholipase C zeta (ΡΙ ζ) or a polynucleotide encoding for phospholipase C zeta (ΡΙ ζ).

6. A method according to any one of the preceding claims wherein the MSNP and/or the at least one molecule is labelled with a revealing label.

7. A method according to any one of the preceding claims wherein the cell is

mammalian.

8. A method according to any one of the preceding claims wherein the cell is human.

9. A method according to any one of the preceding claims wherein the MSNP comprises one or more phosphonate moieties and/or one or more amine moieties covalently bonded to the surface of the MSNP.

10. A method according to any one of the preceding claims wherein the MSNP comprises a cationic polymer which coats at least part of the surface of the MSNP.

11. A method according to any one of the preceding claims wherein the at least one molecule is coated on the surface of the MS P.

12. A method according to any one of claims 1 to 10 wherein the at least one molecule is embedded within the MSNP.

13. A method according to any one of the preceding claims wherein the method comprises exposing the cell to the MSNP comprising the at least one molecule in solution.

14. A method according to any one of the preceding claims wherein the cell remains viable following delivery of the at least one molecule.

15. A method according to any one of the preceding claims wherein the cell retains its reproductive or developmental function following delivery of the at least one molecule.

16. Use of a MSNP comprising at least one molecule to deliver the at least one molecule into a reproductive cell or an embryonic cell.

17. A method of treating a reproductive disease or disorder in a patient in need thereof, the method comprising delivering into a reproductive cell or an embryonic cell of the patient at least one reproduction-promoting agent by contacting the cell with a MSNP comprising the at least one agent and thereby treating the reproductive disease or disorder in the patient.

18. A method according to claim 17, wherein the reproductive disease or disorder is infertility and/or is associated with DNA fragmentation, aneuploidy, chromosomal aberration, apoptosis in reproductive or embryonic cells, reduced oocyte activation ability, reproductive or embryonic cell competency or protein degradation in reproductive or embryonic cells.

19. A method according to claim 18, wherein the infertility is male factor infertility, female factor infertility, idiopathic infertility, failed fertilisation, oocyte activation deficiency, globozoospermia, implantation failure, developmental failure, endometriosis or recurrent miscarriage.

20. A method according to claim 17 or 18, wherein the infertility is caused by the point mutations H398P or H233L, or any other mutations identified in phospholipase C zeta (ΡΙ ζ), the reproductive cell is a sperm and the at least one fertility-promoting agent is ΡΙΧζ protein or a polynucleotide encoding ΡΙΧζ.

21. A method of diagnosing a reproductive disease or disorder in a patient, the method comprising delivering into a reproductive cell or an embryonic cell of the patient at least one diagnostic agent by contacting the cell with a MSNP comprising the at least one agent and thereby diagnosing the reproductive disease or disorder in the patient.

22. A method according to claim 21, wherein the reproductive disease or disorder is as defined in any one of claims 18 to 20.

23. A method of promoting fertilisation of an ovum by a sperm comprising the point mutations H398P or H233L, or any other mutation in phospholipase C zeta (ΡΙ ζ), or by sperm devoid of ΡΙΧζ, or by sperm exhibiting aberrant expression or localisation patterning of ΡΙΧζ), the method comprising delivering ΡΙΧζ protein or a polynucleotide encoding ΡΙΧζ into the ovum by contacting the ovum with a MSNP comprising the protein or polynucleotide and thereby promoting fertilisation of the ovum by the sperm.