General Discussion

In this project, several methodologies were employed for studying the compatibility of 5% SPC:citrem (1:1) LCNPs as drug carriers for placental drug delivery purposes. Initially, changes in LCNP physicochemical properties resulting from changes to the lipid composition, encapsulating drugs of interest, and association with placental homing peptides was studied. The combination of SAXS, cryoTEM, DLS and LDE allowed for the determination of shape, internal structure, size and surface charge of the particles.

Structural transitions as well as changes to the size and surface charge of LCNPs were observed upon changing the composition of the system. Non-lamellar to lamellar, lamellar to non-lamellar and non-lamellar to non-lamellar structural transitions were all observed in this study. Storage of LCNPs was significantly better at 4 °C than at 21 °C over a six month period, and SC-LCNPs, with and without peptides, were stable on storage at 4 °C for up to 4 weeks. The encapsulation of the hydrophilic compounds FAM, SNP and hydralazine favoured the transformation from cubic to lamellar structures. In addition, variable encapsulation efficiencies and release profiles were obtained with these compounds. The highest encapsulation efficiency was obtained with FAM (~96%) and was associated with a burst release followed by a slower release over 72 hours. Hydralazine was the second most highly encapsulated drug (~73%) and was released rapidly from the LCNPs indicating that most of the drug was attached to the surface of the particles rather than entrapped within the nanostructure. SNP had the lowest encapsulation efficiency (~44%) and was released in a controlled manner from the LCNPs over a 3-day period.

LCNP-encapsulated SNP was also able to effectively relax pre-constricted CPAs from human term placentas showing a rapid relaxation that was sustained over 1 hour.

Additionally, surface conjugation of the placental homing peptides CGKRK and iRGD on LCNPs resulted in increased accumulation in the syncytiotrophoblast of human term placentas, endothelium of CPAs from human term placentas, and the spiral arteries of C57 mice.

Placental dysfunction is associated with the majority of pregnancy disorders, most notably PE and FGR, with treatment approaches currently lacking (Brosens et al., 2011;

Sibley, 2017). In the short-term, a poorly functioning placenta can result in premature birth and even fetal demise. However, the consequences also extend into adulthood since growth restricted babies are at increased risk of several diseases as stated by the DOHaD hypothesis (Gillman, 2005; Wadhwa et al., 2010; Heindel & Vandenberg, 2015). Thus,

183 there is a dire need for modern treatment approaches to enhance placental function and potentially improve fetal outcomes. Treatment is generally focused on the management of maternal symptoms (e.g. reducing blood pressure in PE). Although the involvement of the placenta is unquestionable in PE and FGR (Myatt, 2002; Lewis et al., 2006; Jansson &

Powell, 2007), the root causes of these problems are not well-established and as a result, treatment becomes more challenging. Enhancing one or more of the functions of the placenta by delivering pharmacologically active agents is a relatively recent prospect that can be a promising research avenue (Sibley, 2017). However, the administration of drugs in pregnancy is complicated by a lack of understanding surrounding this topic, mainly due to a lack of research and the high risk of causing fetal harm. Pregnant women are also, understandably, excluded from clinical trials. Nonetheless, pregnant women may also need medication for a variety of conditions, and the resulting dearth of drugs that are licensed for use in pregnancy puts the health of both the mother and fetus at risk. If the main concerns regarding maternal side effects and fetal transfer of drugs can be eliminated, or at least significantly reduced, then safe and effective therapeutic intervention in pregnancy can become a reality. This leads the discussion towards NPs as a potential drug carrier to overcome the limitations of conventional pharmacotherapy. NPs can alter the biodistribution of drugs by targeting their release at specific sites, thus enhancing efficacy and reducing off-target side effects (Koo et al., 2005; Rannard & Owen, 2009; Joshi, 2014). Nanomedicine in pregnancy is a niche subject area with limited resourceful information and untapped potential, and therefore data through research is urgently needed.

LCNPs were the nanocarrier of choice in these studies due to their unique external and internal structural arrangements, which have a large surface area to volume ratio. This allows for the incorporation of a wide range of compounds (e.g. polar and non-polar, large MW), higher quantities of drug to be encapsulated, and higher surface expression.

Additionally, their biocompatibility, potential for controlled drug release and ability to preserve encapsulated compounds further supports their potential as nanocarriers (Guo et al., 2010; Attama et al., 2012; Kim et al., 2015; Singhvi et al., 2018). The SPC:citrem formulation was chosen for this project over the more traditional GMO and PHYT based formulations as a result of improved toxicity profile (Wibroe et al., 2015; Azmi et al., 2016). Nevertheless, the use of LCNPs in placental drug delivery cannot be recommended based solely on their potential and without tangible evidence. The work carried out throughout this PhD project therefore aimed to use a wide range of techniques to help understand how LCNPs can be used in placental drug delivery research.

184 NP preparation methods are frequently reported in the literature, but the choice of parameters is often not discussed sufficiently or justified. The findings here showed that sonication time and power have a significant impact on LCNP size, and therefore optimising these conditions is necessary to obtain smaller size particles. Smaller size NPs are desirable as immune recognition of these particles is less pronounced, and therefore have prolonged circulation times resulting in improved efficacy (Fang et al., 2006; Jiang et al., 2008; Moghimi & Hunter, 2011; Blanco et al., 2015). Furthermore, particle size affects the in vitro and in vivo behaviour of NPs depending on the desired mode of delivery (e.g.

IV, transdermal) and target condition (e.g. cancer, cardiovascular) (Danaei et al., 2018).

However, smaller sized NPs (< 10 nm) are also more efficient at penetrating through biological barriers and can be significantly more toxic (Sukhanova et al., 2018). Thus, optimising NP size is an essential step in the development of pharmaceutical nanocarriers.

Physicochemical characterisation of NP size, surface charge and structure is of upmost importance since these properties are vital determinants of stability and in vitro and in vivo behaviour (Mitragotri & Lahann, 2009; Gatoo et al., 2014; Manaia et al., 2017; Louro, 2018). These physicochemical properties have been shown to variably affect placental uptake and fetal transfer of NPs (Bajoria et al., 1997; Bajoria & Contractor, 1997a; Bajoria

& Contractor, 1997b; Bajoria et al., 2013). In this study, the LCNPs are below 200 nm making them less susceptible to immune recognition (Moghimi et al., 2001), and potentially having longer circulation times in vivo. The physicochemical properties of the LCNPs were affected by lipid composition, peptide attachment and drug encapsulation.

The effect of nanostructure on LCNP behaviour, and NPs in general, is particularly poorly understood. NP shape has a significant impact on their properties as drug delivery systems (Liu et al., 2012), in a similar manner to size and surface charge, but is not studied as often. Although the techniques used in NP structural elucidations are more complex and expensive than the standard techniques for assessing size and surface charge, their importance cannot be overlooked and thus there is a requirement for structural studies in LCNP-mediated drug delivery. Monitoring physicochemical changes therefore aids in the understanding of how LCNP properties relate to its interaction with the placenta.

The high surface area to volume ratio of NPs imparts desirable qualities on them as drug carriers. However, this also means that particle-particle interactions are more pronounced which can lead to instability. This is overcome by adding stabilisers to the surface of NPs to induce steric hindrance or electrostatic repulsion, thus keeping NPs dispersed. Citrem

185 used in this study acts using both mechanisms, known as electro-steric stabilisation, to stabilise the LCNP dispersions. Storage of empty LCNPs at 4 °C revealed improved colloidal stability over 6 months, compared to room temperature, and SC-LCNPs, with and without surface bound peptides, were stable for up to 4 weeks at 4 °C. The enhanced dispersion stability at lower temperatures has previously been reported in solid lipid NPs (Freitas & Müller, 1998), PLGA NPs (De & Robinson, 2004) and liposomes (Pan et al., 2018). The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) produces guidelines recommending the stability testing of pharmaceuticals (Muthu & Feng, 2009). However, the stability of nanopharmaceuticals is not frequently reported in the literature and should be addressed in future studies.

Encapsulation and release of the hydrophilic compounds FAM, SNP and hydralazine in LCNPs was achieved to different degrees. The individual drug properties, nanomaterial characteristics and nanomaterial drug interactions can have an impact on NP encapsulation and release properties. In order to help in the understanding of NP drug delivery strategies, it is important to consider both the drug and NP properties. Larger MWs of encapsulated compounds may retard their release from NPs (Brazel & Peppas, 1999), which is also dependent on the pore size of the particles. Additionally, the charge on the surface of NPs and the charge of the cargo determines the encapsulation and release of drugs (Honary &

Zahir, 2013a). Controlled release was achieved with FAM and SNP, whereas hydralazine exhibited a burst release profile. Controlled release from NPs allows for a longer duration of action and potentially reduces side effects by maintaining drug levels within the therapeutic window. SPC:citrem LCNPs can therefore be used to encapsulate and release vasodilatory drugs in a controlled manner in vitro, although their in vivo behaviour may be different due to the presence of blood corona forming components. NPs undergo physical changes once they are introduced to a biological environment which can be significantly different from the properties determined in vitro thus affecting their behaviour (Tenzer et al., 2011). Therefore, the development of in vitro - in vivo correlations (IVIVCs) may help translate in vitro studies into more clinically relevant interpretations (Emami, 2006; Jain et al., 2017).

LCNP release of the vasodilator SNP was found to induce relaxation of isolated pre-constricted CPAs using the wire myography technique in a similar manner to liposomally encapsulated SE175 (Cureton et al., 2017). This demonstrates that LCNP encapsulation of

186 vasodilatory drugs is a suitable approach to achieve vasorelaxation of placental vasculature in pregnancy. The nature of these experiments dictates that the effects are observed in isolation, and any potential clinical benefits cannot be forecast. An in vivo study on FGR and PE mouse models (Sunderland et al., 2011; Swanson & David, 2015) would be an extension of the current work.

Placental homing peptides have recently been identified and shown to improve placental drug delivery (King et al., 2016; Cureton et al., 2017). The targeted NP approach was investigated in this project using CGKRK and iRGD peptides attached to the surface of LCNPs. Uptake of targeted LCNPs by villous explants and CPAs from the human term placenta was significantly more pronounced than non-targeted formulations. The similar size and surface charge of the targeted and non-targeted LCNPs indicates that the preferential uptake of targeted LCNPs can be safely attributed to the presence of placental targeting moieties. Targeted LCNPs localised to the outer ST layer in human term placental explants and in the endothelial layer of CPAs, which is promising in terms of delivering drugs to placental vasculature and avoiding fetal crossover. In order to further support these observations, studies on the whole placenta using the dual placental perfusion ex vivo experimental set up may prove valuable (Bajoria & Contractor, 1997b;

Myllynen et al., 2008).

The in vivo experiments using the pregnant mouse model revealed that targeted LCNPs preferentially accumulate in the maternal spiral arteries. In terms of enhancing the function of the dysfunctional placenta, this is a valuable observation as vasodilatory drugs can potentially be delivered to this target region. Targeted LCNPs were also observed in fetal tissue suggesting that fetal crossover had occurred. Similarly, anionic polystyrene beads up to 240 nm were shown to cross the placenta in the perfused human placenta set up (Wick et al., 2010). However, NP size is not the only contributor to placental uptake and fetal transfer since nanomaterial, surface charge and structure also play a role (Muoth et al., 2016). Anionic silver NPs (80 nm) did not cross the placenta in the pregnant rat (Semmler-Behnke et al., 2014), whereas anionic silica NPs (70 nm) were observed in fetal tissues of the pregnant mouse (Yamashita et al., 2011). There appears to be several significant factors affecting research opinion on fetal transfer of NPs. In addition to NP physicochemical properties, the choice of in vitro methods (e.g BeWo cell line, perfused human placenta, explants) and in vivo models (e.g. mouse, rat) can all influence study outcomes. As a result of the limited nature of in vitro studies and difficult translation from

187 animal models to humans, careful consideration of NP properties and cautious interpretation of results is emphasised in targeted drug delivery studies.

In summary, pregnancy disorders remain poorly understood and the lack of effective treatments leads to a systemic failure of care for women with pregnancy complications.

Modern research methods are desperately needed to advance the discipline, with NPs showing promise in overcoming the long-standing hurdles faced in conventional therapy.

Targeted NPs are particularly appealing owing to their potential to deliver drugs to specific target sites, therefore increasing efficacy and reducing systemic side effects. Nevertheless, NP-mediated drug delivery is a relatively new field and suffers from its own limitations and gaps in knowledge. Thus, extensive testing of NP formulations using a range of methods is needed to better our understanding of the relationship between NP properties and their safety and efficacy. In addition, a more comprehensive testing approach should be undertaken when developing NPs for pregnancy disorders due to the additional challenges imposed by the developing fetus. In this thesis, a wide range of in vitro and in vivo techniques were used to demonstrate that LCNPs can be employed in placental drug delivery. However, as a result of their potential to cross the placenta and reach the fetus in the pregnant mouse model, further studies are needed to understand how these observations may translate in humans. Nonetheless, fetal transfer of LCNPs suggests that they may be used in fetal drug delivery studies.

6.2 Conclusions

• SPC:citrem LCNPs can be conjugated with placental homing peptides to produce stable dispersions of small (< 200 nm), negatively charged particles.

• Vasoactive drugs can be successfully encapsulated in LCNPs with an accompanying release rate that is affected by drug properties.

• Changes to the lipid composition, surface expression of peptides on LCNP surface, and the encapsulation of compounds can change the physicochemical properties of LCNPs and cause structural transitions at the nanoscale.

• LCNPs release encapsulated SNP to cause vasorelaxation of pre-constricted human term CPAs.

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• Targeted CGKRK and iRGD SC-LCNPs are taken up more effectively than non-targeted LCNPs by human term CPAs and the syncytiotrophoblast of human term placental villous explants.

• Targeted CGKRK and iRGD SC-LCNPs preferentially localise in the maternal spiral arteries of the placenta in the pregnant C57BL/6 mouse model.

• Targeted and non-targeted LCNPs are indiscriminately observed in the liver and spleen suggesting that these are their clearance routes.

• Targeted and non-targeted LCNPs are capable of crossing the placenta and reaching the fetus in the pregnant mouse model and must be further assessed for safety.