Design of nanomaterials for applications in maternal/fetal medicine

Abstract

Pregnancy complications are commonplace and the challenges of treatment during pregnancy with few options available pose a risk to the health of both the mother and baby. Patients suffering from conditions such as preeclampsia, placenta accreta, and intrauterine growth restriction have few treatment options apart from emergency caesarean section. Fortunately, researchers are beginning to develop nanomedicine-based therapies that could be utilized to treat conditions affecting the mother, placenta, or fetus to improve the prognosis for mothers and their unborn children. This review summarizes the field’s current understanding of nanoparticle biodistribution and therapeutic effect following systemic or vaginal administration and overviews the design parameters researchers should consider when developing nanomedicines for maternal/fetal health. It also describes safety considerations for nanomedicines to limit undesirable maternal or fetal side effects and discusses future work that should be performed to advance nanomedicine for maternal/fetal health. With additional development and implementation, the application of nanomedicine to treat pregnancy complications may mitigate the need for emergency caesarean sections and allow pregnancies to extend to term.

Graphical Abstract

This review discusses the design parameters that are important to consider when developing nanomaterials for applications in maternal/fetal health.

Introduction

Pregnancy complications affecting the mother, the fetus, or the placenta are commonplace and effective treatment options are difficult to develop. This is in part due to the safety and litigation concerns associated with evaluating new treatments in pregnant women. As a result, common but life-threatening pregnancy complications such as preeclampsia, placenta accreta, and intrauterine growth restriction (IUGR) are treated by inducing early labor or performing emergency caesarean sections that increase the risk of infant prematurity and make post-birth recovery more difficult for mothers. There is a dire need for new therapeutics that can be safely administered to mothers to treat pregnancy complications and allow the pregnancy to reach full term.

In the last two decades, the field of nanomedicine has grown substantially and shown great progress in facilitating the treatment of cancers and other diseases. However, few studies have explored the potential of using nanomedicine to treat pregnancy-related complications. The major goal of nanomedicine is to develop nanoparticles as vehicles to deliver drugs, nucleic acids, antibodies, and other small molecule therapeutics to targeted sites in the body while minimizing delivery to unintended locations. Accordingly, lessons learned from the use of nanoparticles to treat other diseases may be applied to maternal/fetal health to facilitate cargo delivery to specific sites in the reproductive tract (e.g., placenta, fetus, or uterus) and enable safe treatment for mothers and babies.

To achieve site-specific delivery, it is imperative that researchers understand the basic anatomy of reproductive organs and the pregnancy-related complications that can result when fetal/placental development goes awry. The first section of this review provides a basic overview of these topics to introduce nanomedicine researchers to the field. The subsequent sections then summarize the research that has been performed to date to understand the biodistribution and therapeutic effect of nanoparticles in pregnant mice following systemic or vaginal delivery. Design considerations for nanoparticles that may be drawn from this work are reviewed in order to guide researchers in developing novel treatment modalities. Finally, a discussion of safety considerations for nanomedicines applied to maternal/fetal health is provided. The overall goal of this review is to provide a primer for researchers who wish to apply their nanotherapeutics to applications in maternal/fetal medicine.

Overview of Reproductive Organs in Humans versus Mice and Pregnancy Complications that May Benefit from Nanomedicine

Human Reproductive Anatomy and Physiological Changes During Pregnancy.

Human reproductive anatomy experiences many changes throughout the 40-weeks of pregnancy. Key female reproductive organs that are involved in pregnancy related complications include the cervix, the uterus, and the placenta (Figure 1A). Targeting nanoparticles towards these sites and the developing fetus offers substantial opportunities for clinical impact. The maternal cervix and uterus undergo many structural, biological, and physiological changes throughout pregnancy, and an entirely new organ, the placenta, is formed by the fetus to successfully and safely ensure the baby is carried to term. If these changes do not occur properly, a number of pregnancy-related complications and birth defects can develop that put the mother or the fetus at risk (Table 1).

Figure 1.

Comparison of reproductive anatomy in humans and mice. (A) Human reproductive organs and placenta during pregnancy. (B) Mouse reproductive organs and placenta during pregnancy.

Table 1.

Pregnancy Complications Associated with Different Reproductive Organs

Pregnancy Complication	Reproductive Organ Involved	Anatomic or Physiologic Abnormality	Source
Preterm Birth	Cervix	Shortened cervix	[1]; [7]; [8]
Cervical Incompetence	Cervix	Increased cervical dilation	[4]
Uterine Fibroids	Uterus	Abnormal estrogen levels	[19]
Placenta Accreta, Placenta Increta, Placenta Percreta	Placenta and uterus	Abnormal placenta adherence to uterine wall	[25]; [26]
Placenta Previa	Placenta and cervix	Placenta covers cervical opening	[22]; [23]; [24]
Preeclampsia	Placenta and uterus	Abnormal spiral artery remodeling	[13]; [16]; [17]; [18]
Intrauterine Growth Restriction	Placenta	Multifactorial	[17]; [18]

The cervix is a structure made of dense connective stromal tissue that creates a narrow canal connecting the lumen of the vagina to the uterine cavity (Figure 1A). In non-pregnant women, the cervix is approximately 3 cm long and 3.5 cm in diameter. As pregnancy progresses, the cervical mucus plug forms to protect the uterus from the outside world. Additionally, the cervix softens, shortens, and dilates to prepare for delivery in a process called cervical remodeling.^1-3 Fetal growth induces remodeling of the cervical extracellular matrix (ECM) to decrease the cervical length and increase the cervical tissue volume. During this process, fibroblasts, smooth muscle cells, and collagen networks are reorganized to decrease tensile strength, increase compliance, and expand the cervix.² The epithelial cells that line the cervix play a significant role in protecting the fetus by both secreting cytokines to activate immune cells and producing mucus to eliminate pathogens.^1,2 If too little cervical remodeling occurs during pregnancy, cervical incompetence can result, leading to pregnancy loss.⁴ However, too much cervical shortening could lead to preterm birth.^1,5,6 The development of nanomedicines to prevent or treat cervical incompetence and or preterm birth is an unexplored area worthy of future investigation.

The uterus is the reproductive organ that protects the fetus during development (Figure 1A). Following conception, a fertilized egg undergoes cell division to form a zygote and implants as a blastocyst on the uterine wall to initiate placental and fetal development.⁷ After implantation, vascular endothelial growth factor (VEGF) and angiopoietins induce angiogenesis to facilitate growth of small uterine arteries in the first trimester.^8,9 At later gestational ages, these proteins support the remodeling of uterine and placental vessels to increase blood flow to the placenta and nutrient transport to the fetus. Both estrogen and progesterone play critical roles in uterine blood vessel maturation through cellular processes involving vascular contractility, growth and matrix deposition but the specific mechanisms are unknown.^8,10,11 During pregnancy, the uterus increases in size (approximately 1 cm in diameter per week) and the uterine wall thickens to provide structural support to the fetus.¹² By the end of normal gestation, the uterine artery diameter has doubled and blood flow to the uterus has increased exponentially such that a majority (>90%) of flow is directed to the placenta.^9,11 When uterine vasculature is not adequately formed or improperly remodeled, pregnancy complications that can result include preeclampsia^13-16 and intrauterine growth restriction (IUGR).^13,15 Another potential issue that can arise in the uterus is the presence of fibroids, which are abnormal, benign tumors. Before pregnancy, fibroid growth is minimal, however the increased estrogen level during pregnancy can enhance tumor growth, causing pain and often pregnancy loss in the second trimester.¹⁷ Management methods for preeclampsia, IUGR, and fibroids are limited but the development of nanomedicines could largely advance treatment methods for these pregnancy complications.

The placenta is the interface between the mother and fetus that facilitates nutrient transport and gas exchange during pregnancy (Figure 1A, inset). This disc shaped organ is approximately 15-20 cm in diameter, 2-3 cm thick, and has a surface area of almost 15 m² at full gestation.¹⁸ The chorionic villus is the basic structural unit of the placenta, which differentiates into several layers as gestation progresses. The extravillous trophoblasts infiltrate the maternal spiral arteries during remodeling in the first trimester,^19,20 replacing smooth muscle cells to prevent arterial contraction. Additionally, in early gestation, the extravillous trophoblasts plug the spiral arteries and prevent blood flow to ensure a hypoxic environment for the developing fetus during the first trimester.¹³ This hypoxic state is essential as it prevents early fetal damage from oxidative stress. During this time the fetus receives nutrients from the uterine glands. Over this initial 12-week period, the villous trophoblasts also differentiate into synctiotrophoblasts, which create a tight barrier between the maternal and fetal circulations and are in direct contact with the maternal blood. At the start of the second trimester, the trophoblast plug that blocks the spiral arteries is released, allowing maternal blood to enter the placenta.¹⁹ This change alters the transport of nutrients, cytokines, immune cells, and drugs from the maternal blood into the placenta and fetus. Transport of these factors across the placenta is not well understood; however, it is governed, at least in part, by concentration, molecular weight, and the mechanism of transport.^18,21 Notably, when properly coordinated placental development does not occur, complications that result include placenta previa^22-24, placenta accreta^25,26, preeclampsia and IUGR.

Finally, the fetus itself is a potential target for nanomedicines. Fetal development is outside the scope of this review, but is covered in greater detail in other excellent reviews.^27,28 Given the complexity of fetal development, it is perhaps unsurprising that over 4,000 types of birth defects exist²⁹ and 6% of live births worldwide are associated with birth/developmental defects.³⁰ Unlocking the ability to address congenital defects or treat the developing fetus in utero would transform maternal/fetal medicine. Few researchers have investigated the use of nanomedicines to manipulate fetuses directly,³¹ but it is a potentially impactful approach worthy of future development. To achieve this elusive goal, nanomedicines must be able to surpass a number of biological barriers to reach the fetus, and the design considerations highlighted in this review may aid researchers in accomplishing this task.

The Mouse as a Model: Anatomical Differences Between Human and Mouse.

As murine models are the primary system used to initially evaluate nanomedicines, it is imperative that researchers understand the differences between murine and human reproductive anatomy. The murine reproductive system is structurally and physiologically different from the human system (Figure 1B). Murine pregnancies, depending on the mouse genetic strain, deliver an average of 6-8 pups in one litter within a 21-day gestation period. The murine cervix undergoes similar remodeling processes as humans; however, due to the shortened gestation, cervical softening occurs at mid-gestation (embryonic day 12; E12).³² Additionally, mice do not maintain a cervical mucus plug barrier during the pregnancy for protection against bacterial invasion. Further, unlike humans, mice have a duplex uterus (containing two horns) that receives its blood supply from both the ovarian and uterine arteries.

The mouse placenta is similar to humans in that the maternal blood is in direct contact within the trophoblast cell layer.^15,33 However, there are subtle differences in placental organization, trophoblast invasion depth, and trophoblast differentiation. The labyrinth in the murine placenta is comparable to the chorionic villous in humans and facilitates nutrient exchange between maternal blood and the fetus (Figure 1B, inset). Trophoblast invasion depth is slightly less in mice than in humans and occurs mid-gestation to protect the fetus.³³ The trophoblasts also differentiate to spongiotrophoblasts that are organized in the labyrinth. Arterial remodeling of the uterine arteries to initiate maternal blood flow to the placenta occurs during the third trimester (E15-E16) in mice,³⁴ as opposed to the second trimester in humans. For two weeks following birth, neonatal mouse pups continue developing many tissues outside the womb (e.g. ear structure, vision, lung development, and bone formation) relative to humans that complete these processes in utero.

The differences between human and murine pregnancies are important considerations when researchers are developing and evaluating nanomedicine treatment methods for maternal/fetal health. In the following sections, this review describes current knowledge regarding the biodistribution and effect of nanomedicines in pregnant mice. As the field matures, it will be important to evaluate nanomedicines not only in mice, but also in other available pregnant animal models that more accurately mimic human reproductive anatomy and physiology.

Systemic Delivery of Nanotherapeutics to Advance Maternal/Fetal Health

Two main routes of nanoparticle delivery to reproductive organs in women or mice include systemic and vaginal administration, which are summarized in this section and the next, respectively. For delivery via systemic circulation, this review emphasizes intravenous delivery, as this is the most common method utilized in preclinical and clinical studies. First, the major barriers to systemic nanomedicine delivery are discussed, followed by a review of studies in literature that have examined the biodistribution and therapeutic effect of intravenously administered nanoparticles in pregnant mice. Finally, a synopsis of design criteria that one might conclude from these studies is provided.

Barriers to the Systemic Delivery of Nanoparticles.

To successfully reach desired sites in reproductive organs, systemically administered nanoparticles must overcome many biological barriers. They must be stable in circulation, cross blood vessel walls, penetrate the extracellular matrix (ECM) of the targeted tissue, and deliver their cargo into the intended cells. It is well established in the field that nanoparticles with diameters less than ~5 nm undergo rapid renal clearance, and those with diameters greater than ~200 nm are filtered by the spleen.^35-37 Nanoparticles with diameters ranging from 10-100 nm are the longest circulating, and also exhibit the greatest cellular uptake.^35,38 Beyond size, the type and density of surface molecules on nanoparticles and the surface charge (zeta potential) of nanoparticles can influence their biodistribution following systemic administration. Generally, positively charged nanoparticles are cleared more quickly from the bloodstream than negatively charged nanoparticles and they can induce hemolysis and platelet aggregation.^36,38 Therefore, negatively charged or relatively neutral nanoparticles are more suitable for in vivo use. However, even these materials face systemic delivery challenges due to hepatic clearance as noted below.

Studies in various disease models have shown that, regardless of nanoparticle size or charge, in the bloodstream these materials are quickly coated with a biological corona consisting primarily of opsonin proteins. These proteins make the nanoparticles recognizable by the mononuclear phagocytic system (MPS), promoting their accumulation in the liver and reducing their circulation half-life and distribution to targeted organs.³⁹ While the unique portal system in the liver enables it to efficiently process and metabolize nutrients in the blood, it renders nanomedicines at risk of being cleared or metabolized by the liver. Indeed, following intravenous injection in mice, nanoparticles are often found to accumulate in the liver at higher rates than at the intended site (tumors, reproductive organs, etc.).^40-45 Hepatic clearance is a major hurdle that must be overcome for nanomedicines to efficiently target reproductive tissues and treat pregnancy complications.

One strategy that is commonly used in nanomedicine to minimize hepatic clearance is to functionalize the surface of nanoparticles with “stealthing” agents or “targeting agents” that minimize opsonization and guide cell-specific binding, respectively. The most common stealthing agent used is poly(ethylene glycol) (PEG), which is a highly hydrophilic polymer that sterically and thermodynamically hinders blood protein-nanoparticle interactions. In non-pregnant animals, PEG has often been shown to increase circulation time and reduce clearance by liver and kidneys.^46,47 In the following section, studies that explored the use of PEGylated nanoparticles in pregnant mice are discussed. Additionally, studies that used targeting ligands to guide nanoparticle delivery to reproductive organs are summarized. Overall, while studies in non-pregnant animals have shown that parameters such as nanoparticle size and surface chemistry can control the hepatic clearance rate, it is unknown how these parameters impact biodistribution in pregnant mice. More research is required to establish how the altered anatomy and physiological blood flows during pregnancy affect nanoparticle biodistribution to maternal and fetal organs.

Systemic Delivery of Nanoparticles to Pregnant Mice: Findings from Literature.

To date, few studies have analyzed the biodistribution of nanoparticles in pregnant mice following systemic administration (Table 2). Unsurprisingly, these studies have shown that nanoparticle size is one of the major design criteria that can influence the biodistribution of systemically administered nanomedicines. Given that it is estimated that molecules approximately 500 Da or larger cannot readily diffuse across the placenta,^18,48 it is important to understand how nanoparticle size impacts accumulation in the placenta, fetus, and other reproductive tissues. Likewise, it is important to understand how the physiological changes that occur during pregnancy might affect particle distribution. One study that examined the biodistribution of PEGylated gold nanoparticles (AuNPs) concluded that there is a size-dependent distribution of nanoparticles in pregnant mice, but that nanoparticles of the same size will distribute equivalently in pregnant and non-pregnant mice.⁴⁹ This suggests that the physiological changes that occur during pregnancy have less influence on nanoparticle biodistribution in mothers than the nanoparticle size. A major limitation of this study, however, is that it only examined AuNP accumulation in major maternal organs (heart, liver, spleen, lung, and kidney), and it did not examine AuNP accumulation in reproductive tissues. Further studies are necessary to elucidate how AuNP size influences accumulation in specific reproductive organs.

Table 2.

Studies Evaluating Intravenous Delivery of Nanoparticles in Pregnant Mice

NP Material	NP Diameter, ζ Potential, Surface Coating	Administration day(s)	Sites of NP Accumulation	Application (if any)	Source
Iron oxide nanoworms; Liposomes	180 nm (nanoworms); 150 nm (liposomes); ζ not reported; CRKRK or iRGD peptides	E11.5, 13.5, and 17.5	Placental labyrinth, spiral arteries, maternal liver, maternal spleen	Targeted to placenta using CRKRK or iRGD to treat IUGR by delivering IGF-2	[44]
Lipid-polymer	110 nm; −28 mV; placental CSA binding peptide	E6.5-14.5	Trophoblasts, maternal liver	Targeted to placenta using pICSA-BP to treat ectopic pregnancy by delivering methotrexate	[57]
Liposome	124 nm; −24 mV; oxytoxin receptor antagonist	E18	Uterus, placenta, maternal liver	Targeted to uterus using oxytocin receptor antagonist to treat pre-term labor by delivering indomethacin	[58]
Liposome	164 nm; −1 mV; CNKGLRNK peptide	E11.5-17.5	Spiral arteries, placental labyrinth, maternal kidney, maternal spleen	Targeted to placenta using CNKGLRNK to treat IUGR by delivering a nitric oxide donor	[56]
Liposome	75-100 nm; ζ not reported; transferrin receptor antibodies	E17	Maternal and fetal brain, spleen, and lungs	Targeted to fetal brain with transferrin receptor antibody to deliver plasmid DNA	[43]
Polystyrene	20, 40, 100, 200, 500 nm; ζ not reported; carboxylated	E17	Many fetal organs, maternal placenta	N/A	[92]
Silica	25, 60, or 115 nm; aminated (+) or carboxylated (−) with varying ζ potentials	E5.5, 12.5, 16.5	Fetus and placenta, with levels impacted by NP size, ζ potential, and gestational stage	N/A	[93]

Other studies that have examined nanoparticle (NP) biodistribution and accumulation in reproductive organs following intravenous injection suggest that delivery to reproductive organs is size-dependent. For example, when pregnant mice were intravenously injected on gestational day 16 with fluorescently-labeled silica NPs and imaged 24 h later, researchers observed fluorescence in the placentas only for mice that received ~65 nm diameter silica NPs, but not for mice that received larger ~320 nm or ~1100 nm NPs.⁴² Further evaluation of excised tissue samples by transmission electron microscopy (TEM) revealed that the 65 nm silica NPs accumulated in placental trophoblasts, fetal liver cells, and fetal brain cells.⁴² Intriguingly, this same study reported that 217 nm diameter TiO₂ particles also accumulated in placental trophoblasts, fetal liver, and fetal brain.⁴² The fact that 217 nm diameter NPs, but not 320 nm NPs, could penetrate placentas and fetuses is surprising and suggests that the size cutoff for delivery to reproductive tissues is narrow. However, the difference in material properties could have been a factor in biodistribution and tissue accumulation. Although the NPs had different zeta potentials (−62 mV for the 320 nm silica NPs, −23 mV for the 217 nm TiO₂ NPs), it seems unlikely that this is the cause for the drastic difference in biodistribution because the researchers observed that 70 nm NPs with charges of −29 mV, −53 mV, and −76 mV all accumulated in reproductive tissues to a similar degree.⁴² Thus, size appears to play a larger role in dictating NP delivery to reproductive organs than surface charge.

Notably, the above study reported that, independent of size, silica NPs accumulated in the maternal liver at much higher levels than the placenta.⁴² This is consistent with another report that showed ~50 nm diameter silver NPs administered intravenously to mice over E7-9 exhibit the highest accumulation in maternal liver, with particles also found in the visceral yolk sac, placenta, and embryo to a much lesser degree.⁴¹ From these studies it is clear that hepatic clearance is a major barrier to systemic delivery of nanotherapeutics for pregnancy complications regardless of NP size, charge, or material type.

Coating nanoparticles with PEG is a widely utilized strategy to reduce their liver accumulation. To date, few studies have directly compared the biodistribution of bare versus PEGylated NPs in pregnant mice, likely due to the general acceptance of PEG as a beneficial surface coating. In one study, 13 nm diameter AuNPs coated with ferritin, PEG, or sodium citrate were prepared and administered to mice intravenously over E5.5-E15.5.⁴⁹ Maternal and fetal tissues were collected 5 hours later for analysis of gold content by inductively coupled plasma-mass spectrometry (ICP-MS), which revealed that PEGylated AuNPs exhibited much lower accumulation in maternal liver than the other AuNPs.⁴⁹ This same study also showed that when AuNPs were administered at E8.5 and fetuses collected 0-72 hours later, PEGylated AuNPs accumulated to a much higher degree in fetuses than ferritin-coated or citrate-coated AuNPs. The amount of Au in the fetus 48 hours post-injection was ~45 ng for PEG-AuNPs versus ~20 ng for ferritin-AuNPs and nearly 0 ng for citrate-AuNPs.⁴⁹ The decreased accumulation of PEGylated AuNPs in maternal liver and increased accumulation in the fetus confirms that PEG surface coatings may be advantageous for promoting maternal-fetal transfer.

Beyond PEG, nanoparticles can also be coated with “targeting ligands” to increase cell-specific binding and uptake. This has been widely explored in the field of cancer nanomedicine, with many positive results reported, but the overall benefits of adding targeting agents to nanoparticles is currently a topic of intense debate as some reports indicate there is little difference in tumor accumulation of targeted versus non-targeted nanoparticles.^50,51 Nevertheless, researchers have begun to explore the benefits of designing targeted nanoparticles for maternal/fetal medicine, and the results of these studies are discussed below.

In maternal/fetal health, the cells that are typically targeted are located in the placenta or uterus, as the receptors that are overexpressed by these cells have been well characterized. Interestingly, the placenta shares many overexpressed receptors with solid tumors, as both structures are highly vascularized, rapidly growing, and have the ability to produce a variety of growth factors and evade the immune system.⁵² Researchers have therefore used known tumor targeting peptide sequences in order to target nanoparticles and other therapies to the placenta.^44,53 For example, researchers showed that liposomes and iron-oxide nanoworms coated with the tumor-targeting peptide sequence CGKRK, which binds to calreticulin, could target the placental surface to a greater degree than nanoparticles coated with a control peptide.⁴⁴ Furthermore, liposomes coated with iRGD, which binds to neuropilin-1^54,55, were able to successfully treat intrauterine growth restriction (IUGR) in mice by delivering insulin-like growth factor-2 (IGF-2) to the placenta.⁴⁴ In a similar strategy, researchers developed liposomes that could treat IUGR by delivering a nitric oxide donor to the placenta and uterine spiral arteries.⁵⁶ These nanoparticles were coated with a placenta specific peptide, NKGLRNK, that was identified using phage display techniques.

Phage display has been used to identify other placenta targeting peptides as well. Chondroitin sulfate A (CSA) is upregulated on the surface of trophoblasts, and researchers identified a malaria-derived peptide that selectively binds CSA by phage screening.^57,58 When lipid-polymer nanoparticles were coated with this peptide, they could deliver methotrexate (a drug used to treat ectopic pregnancy and choriocarcinoma) specifically to placental trophoblasts.⁵⁷ The therapeutic potential of these CSA-targeted methotrexate delivery vehicles remains to be elucidated in future studies. A similar system comprised of CSA-targeted lipid-polymer nanoparticles that were loaded with the chemotherapeutic agent doxorubicin was shown to inhibit the growth of subcutaneous choriocarcinoma tumors in mice,⁵⁹ but this system remains to be evaluated in a more clinically relevant orthotopic tumor model.

Direct targeting of the pregnant or non-pregnant uterus can also be achieved using nanoparticles. The initiation of natural birth, labor, requires contraction of the uterus. Unwanted pre-term labor can be arrested by inhibiting these contractions. Oxytocin receptor is overexpressed in the pregnant uterus and can be used to target nanoparticles to the uterus.⁵⁸ For instance, liposomes containing the non-steroidal anti-inflammatory drug, indomethacin, were modified with an oxytocin receptor antagonist for uterine targeting. This strategy reduced the rate of induced pre-term labor in preclinical studies.⁵⁸ Compared to free indomethacin, the targeted nanotherapy showed a similar rate of pre-term birth, however the carrier and drug were able to accumulate at the uterus at a higher concentration and reduce transport across the placenta to the fetus. Other studies found similar results reducing pre-term labor with indomethacin when targeting liposomes to oxytocin receptor with an antibody.⁶⁰ There are several potential applications for targeting the uterus, such as treating fibroids or endometriosis. These initial studies show promise in uterine targeting, but more research needs to be done to be able to use nanomedicine for uterine conditions.

Finally, besides targeting the uterus or placenta, researchers are also beginning to investigate ways to deliver therapeutics specifically to the fetus. Transferrin receptors are overexpressed in both the blood brain barrier (BBB) and the placenta, and a number of transferrin-targeted nanoparticles have been described in literature.⁶¹ In one exciting study, it was shown that liposomes coated with antibodies targeting transferrin receptors could successfully transport luciferase plasmids to the fetal brain.⁴³ Incredibly, the DNA cargo was preferentially delivered to the fetal brain over the maternal brain. The authors attribute this to the overexpression of the transferrin receptor in the fetal brain.⁴³ In the future, this type of gene delivery system may be able to be expanded for treating conditions such as fragile X syndrome, Tay Sachs disease, or Lafora’s disease.⁴³

Overall, while targeted nanoparticle systems have shown promise in facilitating cargo delivery to desired reproductive tissues, there is room for improvement. Even targeted nanoparticles exhibit high accumulation in the maternal liver in most murine studies. ^44,45,56,58 Researchers should continue to investigate the role of nanoparticle size, surface chemistry, shape, and other factors in facilitating the delivery of intravenously administered nanoparticles to reproductive organs.

Vaginal Delivery of Nanoparticles in Maternal/Fetal Health

As an alternative to intravenous administration, nanoparticles may also be administered vaginally. This method avoids the complications of blood protein interactions and high clearance by the liver, spleen, and kidneys, but also experiences its own unique barriers to successful delivery. The biological barriers associated with vaginal delivery of nanoparticles are introduced below, followed by a discussion of representative studies from the literature. To date, vaginal delivery of nanoparticles has been explored primarily for the treatment of non-pregnancy related conditions including HIV, cervical cancer, and endometriosis.^17,62-65 The lessons learned from this work may be exploited in the future to enhance the vaginal delivery of nanoparticles for maternal/fetal health applications.

Barriers to Vaginal Delivery of Nanoparticles During Pregnancy: Cervicovaginal Mucus and the Cervical Mucus Plug.

One of the most substantial biological barriers that vaginally administered nanoparticles must overcome is the abundant presence of cervicovaginal mucus (CVM). CVM is a gel-like fluid made of glycoproteins arranged in porous fibrous networks (Figure 2A, B). Mucins, the main structural component of mucus, line the vaginal epithelium and cervix to prevent the transport of bacteria, viruses, and foreign antigens to the uterus.^66-68 Pathogens are entrapped in the CVM and cleared as discharge in both non-pregnant and pregnant women, but the rate of CVM clearance is increased in pregnant women to protect both the uterus and fetus.⁶⁹ As an added layer of protection for the fetus, at the end of the first trimester a cervical mucus plug (CMP) is formed from secretory cells, shed cells, and immune cells (Figure 2C).^70-72 Analysis of ex vivo CMPs showed they contain elevated concentrations of neutrophils, macrophages, and eosinophils as compared to mucus from non-pregnant women.⁷⁰ The overall purpose of the CMP is to provide a physical barrier that fills the cervical canal and prevents the migration of large bacteria and other pathogens to the uterus. The proper development of the CMP is critical, as improper morphology and bacterial invasion in the uterus can induce preterm labor. Indeed, electron microscopy images show that the structure of CVM is dramatically different between women with low-risk and high-risk pregnancies (Figure 2B).⁷¹

Figure 2.

Cervicovaginal mucus is a barrier to vaginal delivery. (A) Structure of mucus. (B) Electron micrographs of cervical mucus at 20 weeks of pregnancy in patients at low and high risk of preterm birth. Scale bar=200 nm. Reproduced with permission from Critchfield AS, et al. PLoS ONE. 2013; 8(8): e69528 under a Creative Commons Attribution License. (C) Scheme depicting the location of the cervical mucus plug. This scheme was produced using Servier Medical ART templates, which are licensed under a Creative Commons Attribution License (https://smart.servier.com).

The rheological properties of CVM play a critical role in nanoparticle transport and thus should be taken into consideration for nanoparticle design for vaginal delivery. CVM properties such as viscoelasticity, plasticity, and stringiness fluctuate throughout pregnancy due to changes in hormone levels.^66,69 Additionally, the rheological properties are complex and non-linear which depends on the length scale and shear stress. At the nanoscale and under low shear stress, CVM acts as a low viscosity fluid and the CMP acts as an elastic solid.⁶⁶ However, under high shear conditions, CVM and the CMP are highly viscous and experience very little deformity. These changes in viscosity can vary with external chemical signals, with gradual changes (occurring over days) induced by increased estradiol levels or immediate changes (occurring over minutes) induced by spermatozoa presence.^67,69

Since hormone levels vary throughout pregnancy, the rheology of CVM also fluctuates with gestational age. Following conception, estradiol levels are initially low and CVM viscosity increases to discourage sperm and bacteria transport to the uterus.^66,67 Estrogen levels gradually increase as the placenta develops and initiates mucin production. As pregnancy continues, progesterone levels decrease to initiate epithelial proliferation and cervical remodeling. This results in increased CVM production for physical and immune protection of the fetus.⁷⁰ At late gestations, estradiol synthesis and CVM production are increased and maintained until delivery. Then, immediately before delivery, progesterone levels drop significantly to end oxytocin production and initiate labor, thus releasing the CMP from the cervical canal.⁶⁹ These gestational differences in CVM and CMP are important for nanoparticle design because the pore size, viscosity, and other characteristics of these barriers will dictate the types of nanoparticles that can effectively penetrate these barriers to reach desired reproductive sites.

One major difference between humans and mice that makes pre-clinical evaluation of vaginally delivered nanoparticles challenging is that pregnant mice do not maintain vaginal plugs beyond the first day of gestation.⁷³ To study nanoparticle penetration through CVM, researchers have used ex vivo testing with human CVM samples and human CMPs or examined the biodistribution of nanoparticles in mice following vaginal delivery. In vivo, the majority of studies involving vaginal administration of nanoparticles have been performed in non-pregnant mice. There is a substantial need for further study in this realm to advance the field. A summary of the studies performed to date is discussed below and presented in Table 3.

Table 3.

Studies Evaluating Vaginal Delivery of Nanoparticles

Studies with Ex Vivo Cervicovaginal Mucus (CVM)
Nanoparticle Material	Nanoparticle Size & Surface Coating	CVM Source	Major Observations	Source
Polystyrene beads	100, 200, 500 nm; bare or PEG coated	Non-pregnant women	100 nm and 200 nm NPs penetrate CVM faster than 500 nm NPs PEG-coated NPs diffuse through CVM faster than uncoated NPs	[74]
Polystyrene beads	200 and 500 nm; bare or PEG coated	Pregnant women	PEG-coated 200 and 500 nm NPs diffuse readily through CVM, while uncoated NPs become trapped 200 nm and 500 nm PEG-coated NPs diffuse ~13-fold and 16-fold more slowly in CVM from pregnant women than from non-pregnant women	[6]
Studies in Mice
Nanoparticle Material	Nanoparticle Size & Surface Coating	Pregnant or non- pregnant mice	Major Observations	Source
PLGA	91, 117, & 144 nm; PEG	Non-pregnant; estrus phase	91 nm NPs exhibit greater vaginal coverage than larger NPs	[76]
PLGA	150-170 nm; bare, avidin, or PEG	Non-pregnant; estrus phase	More PEG-coated NPs were retained in the vagina at 6 and 24 hours post-delivery than bare or avidin-coated NPs	[75]
PLGA	112 nm diameter; 3% wt PEG or 25% wt PEG	Non-pregnant; estrus phase	PLGA NPs with high PEG density (25% wt) coat vaginal epithelium to greater extent than NPs with low (3% wt) PEG density	[76]
Progesterone nanosuspension	400 nm; no coating	Pregnant; RU486 model of preterm birth	Progesterone nanosuspension administered on E15-18 extended length of pregnancy versus no treatment or exposure to a progesterone gel	[6]

Vaginal Delivery of Nanoparticles: Lessons from Literature.

Because CVM is the first barrier that nanoparticles will encounter when they are administered vaginally, it is critical to understand the relationships between nanoparticle size, surface chemistry and diffusion though the CVM. Towards this goal, one study used multiple particle tracking analysis to evaluate the movement of bare and PEG-coated fluorescent polystyrene beads with diameters of 100 nm, 200 nm, or 500 nm through ex vivo samples of human CVM obtained from non-pregnant women.⁷⁴ The mean squared displacement values of the nanoparticles determined by this analysis revealed that 100 nm and 200 nm diameter nanoparticles penetrate CVM much faster than 500 nm nanoparticles.⁷⁴ Additionally, PEG-coated nanoparticles penetrated the mucus at rates 400 to 6,000-fold faster than uncoated nanoparticles.⁷⁴ This suggests that, as with intravenous delivery, PEG coatings can enhance nanoparticle delivery to reproductive organs. Because CVM changes with pregnancy, the researchers performed a follow-up study to investigate nanoparticle diffusion in CVM samples obtained from pregnant women.⁶ This revealed that PEG-coated 200 nm and 500 nm nanoparticles diffuse readily through human CVM, while uncoated nanoparticles become “adhesively trapped”.⁶ Interestingly, this study showed that the 200 nm and 500 nm PEG-coated nanoparticles diffused ~13 fold and ~16 fold more slowly in CVM from pregnant patients⁶ than in CVM from non-pregnant patients⁷⁴, which is consistent with the known changes in CVM that occur during pregnancy. In the future, researchers should expand on these findings by investigating nanoparticle diffusion through CVM obtained at different stages of pregnancy.

As noted above, whereas the biodistribution of vaginally administered nanoparticles has been relatively unstudied in pregnant mice, some studies have examined distribution in non-pregnant animals.^75,76 These studies generally indicate that smaller nanoparticles and those with PEG coatings exhibit greater accumulation in reproductive tissues following vaginal administration^75,76, which agrees with the observations from studies in ex vivo mucus. For example, when PEG-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles ranging from 91 nm to 144 nm diameter were vaginally delivered to estrus phase mice and the distribution evaluated ten minutes later, the 91 nm nanoparticles covered a larger surface area of the vagina than the larger particles (greater than 117 nm).⁷⁶ This suggests that nanoparticles with diameters of 120 nm or less may be best suited for vaginal delivery because they can penetrate the mucus barrier to reach the vaginal epithelium.

Several researchers have studied how nanoparticle surface coating affects the distribution of vaginally administered nanoparticles in non-pregnant mice, and it is feasible that the trends observed from these studies will hold true in pregnant animals as well. In one study, PEG-coated PLGA nanoparticles (PEG-NPs) were compared against avidin-coated PLGA nanoparticles (AVID-NPs) and bare NPs; the diameter of these nanoparticles varied between 150-170 nm and they were administered vaginally to mice in estrus.⁷⁵ Immediately following administration, and also 6 hours after administration, a larger number of PEG-NPs were retained in the vagina than AVID-NPs and uncoated NPs.⁷⁵ By 24 hours post-administration, only PEG coated nanoparticles were found in the vaginal tract, albeit at very low concentrations.⁷⁵ These results were corroborated by another study that showed at 6 hours post-vaginal delivery approximately 50% of 112 nm diameter fluorescent polystyrene particles coated with a dense outer layer of PEG are retained in the vagina, compared to only 10% of uncoated nanoparticles.⁷⁵ Importantly, the density of PEG on the surface of the nanoparticles is a key factor in facilitating mucus penetration, as PLGA nanoparticles with dense PEG content (25% wt.) coated the vaginal epithelium to a greater extent than nanoparticles with low PEG content (3% wt.)⁷⁶ in mice in estrus. Taken together, these studies suggest that “mucus-penetrating” PEG-coated nanoparticles are advantageous relative to “mucoadhesive” nanoparticles for vaginal delivery for maternal/fetal health.

Excitingly, one recent study did report the development and evaluation of vaginally administered nanomaterials in pregnant mice.⁶ Specifically, ~400 nm diameter mucoinert nanospensions of progesterone⁶ were prepared by a wet-ball nanomilling technique and administered to pregnant mice on gestation day 15 (E15).⁶ The progesterone concentration in the plasma, cervix, and proximal and distal uterus were examined at various times post-delivery by HPLC, with the level of progesterone in the samples compared to the level in mice that received hypertonic, micronized progesterone gels. The nanosuspension delivered more progesterone to all tissues than the gel formulation, prompting the researchers to evaluate this treatment in a murine model of pre-term birth.⁶ In this model, the progesterone antagonist RU486 was administered on E15 to induce pre-term birth. The mice then received daily vaginal doses of no treatment, the gel, or the nanosuspsension from E15-18. The median day of parturition was E16 for both the untreated and gel formulations; however, median parturition day increased to E19.5 for mice given the nanosuspension. Further, the percentage of mice reaching full term (E19.5 +/− 0.5) was 15% in the untreated group, 32% in the gel group, and 55% in the nanosuspension group.⁷⁷ These data indicate that vaginal delivery of progesterone via a nanosuspension may be a viable treatment to prevent pre-term birth, and the findings provide hope that other vaginally administered nanotherapeutics may also be effective in preclinical and, ultimately, clinical settings.

Safety Considerations for Maternal/Fetal Nanomedicine

When designing nanoparticles for applications in maternal/fetal medicine, safety to the mother and the fetus is of the utmost importance. To be implemented successfully, nanoparticles must be non-immunogenic, non-toxic, and biologically inert or degraded into safe by-products. Toxicity to the fetus may occur at a lower threshold than toxicity to maternal tissues due to the delicate balance of development. Fetal and maternal safety is vital for the success and translation of nanoparticles for maternal/fetal health. In this section, safety considerations for the mother and the fetus are introduced. Few studies have examined safety in detail (Table 4), and therefore this section provides an overview of safety evaluations that should be performed, such as quantification of particle accumulation, IUGR, and litter size, to validate the safety of nanomedicines.

Table 4.

Studies Examining Nanoparticle Safety in Pregnant Mice

NP Material	NP Diameter, ζ Potential, Coating	Dosing Regimen	Maternal Effects	Fetal Effects	Source
Silver	8 nm; ζ not reported; bare	1 dose i.v. at E6.5	Not Examined	Altered gene expression and methylation in embryo	[89]
Silver	10 nm; −21 mV; bare	1 dose i.v. per day E7-9	Not Examined	Accumulation in visceral yoke sac, some fetuses small for their age	[40]
Silver	50 nm; −29 mV; bare	1 dose i.v. per day E7-9	Not Examined	Accumulation in visceral yoke sac	[41]
Gold	13 nm; −1.6, −6.0, and −17.0 mV; ferritin nanocage, PEG, sodium citrate	1 dose i.v. at E5.5, 6.5, 8.5, 11.5, 13.5 or 14.5	Not Examined	NPs found in embryos, accumulation increased with gestational age, non-toxic	[49]
Gold	1.5 nm & 0 mV; 4.5 nm & −4.5 mV; 13 nm & −6.6 mV; 30 nm & −3.0 mV; 70 nm & 0.0 mV; PEG	1 dose i.v. at E5.5, 7.5, 9.5, 11.5, or 13.5	30 nm NPs induced emphysema like morphology in lung	None noted	[77]
Gold	40, 100 nm; ζ not reported; bare	1 dose i.p. at E10, 12, 14, or 17	None noted	100 nm NPs increased micronucleated poly-chromatic erythrocytes in liver and blood, and changed miRNA expression in fetal liver and lung	[83]
Gold	2, 15, 50 nm; −41.5, −36.7, and −34.9 mV; bare	1 dose i.v. per day E3.5 and 5.5	Not Examined	15 nm NPs suppressed expression of germ layer markers	[82]
Gold	30 nm; −3.0 mV; PEG	1 dose i.v. per day E5.5-7.5 or E11.5-13.5	Increased basophils	Early exposure decreased survival rate	[80]
Carbon Nanotubes	86 nm; ζ not reported; PEG	1 dose i.v. at E5.5 or 14.5 or 1 dose per day on E5.5, 8.5, & 11.5	Minor hepatic damage during consecutive dosing	Some fetal abnormalities with highest dose and consecutive dosing	[79]
Iron Oxide	30 nm; 51 mV or −52 mV; PEI or PAA	1 dose i.p. at E9 or 1 dose per day E9-16	Decreased maternal weight gain	Increased resorbed fetuses in consecutive injections	[78]
Silica & Titanium Oxide	Silica (bare): 65 nm & −53 mV, 322 nm & −62 mV, 1,140 nm &−67 mV; TiO2 (bare): 217 nm & −23 mV	1 dose i.v. at E16 or 1 dose per day E16-17	Smaller uteri when treated with 65 nm silica or 217 nm nano-TiO2; COOH or NH2-modified silica did not have these effects	Smaller fetuses, resorptions when treated with 65 nm silica or 217 nm nano-TiO2; COOH or NH2-modified silica did not have these effects	[42]
Titanium Oxide	25-70 nm; ζ not reported; bare	1 dose per day s.c. at E6, 9, 12, and 15	Not Examined	Altered cerebral cortex, olfactory bulb & dopamine systems in fetal brain	[91]
Titanium Oxide	25-70 nm; ζ not reported; bare	1 dose per day s.c. at E3, 7, 10, and 14	Not Examined	Reduced sperm	[90]
Titanium Oxide	5-6 nm, with aggregation 89 nm; −21.1 mV; bare	1 dose i.v. on E9	Not Examined	Behavioral impairments	[3]

There are many factors that may influence the toxicity observed from nanoparticles, including material type, dosing regimen, aggregation state, diameter, shape, and degradation products.^78-80 The mechanism of toxicity can also vary for different nanoparticle designs. For example, some nanoparticles induce toxicity by producing reactive oxygen species that cause inflammation and DNA damage.⁷⁹ Other nanoparticles can disrupt lipid membranes resulting in organelle dysfunction.⁷⁸ Since the mechanisms of toxicity are specific for each type of nanoparticle, a complete discussion of this topic is outside the scope of this review. Interested readers are directed towards source 79 for a detailed discussion of nanoparticle toxicity mechanisms. Below, considerations specific to maternal/fetal health are discussed.

Maternal Safety Considerations.

When evaluating nanoparticles in mice, researchers must closely monitor the mother for signs of toxicity and distress. As with nanoparticles designed for other applications, researchers should examine blood chemistry (i.e., counts of red blood cells, white blood cells, etc.) and serum cytokines in treated versus non-treated mice to confirm that detrimental changes are not occurring. Given that a large fraction of nanoparticles accumulate in the maternal liver,^40-43 researchers should also measure liver enzymes to ensure liver toxicity is not present. The amount of DNA strand breaks can also be measured as a sign of liver distress, as was previously demonstrated in pregnant mice that were exposed to carbon black particles by inhalation exposure.⁸¹ Other organ-specific effects may be important to monitor depending on the biodistribution and cargo of the specific nanoparticle under investigation. One study found that maternal lungs exhibited emphysema-like changes in morphology when 30 nm diameter AuNPs were delivered intravenously in pregnant mice, but this was not observed after the administration of any other sized nanoparticle, and the changes did not alter the behavior of the mice.⁸² Whether the changes were an artifact or a genuine effect of the nanoparticles remains to be validated in repeated studies. Lung toxicity has also been observed in pregnant mice following inhalation and intratracheal instillation of carbon black particles, which increased the count of both lymphocytes and neutrophils in the bronchoalveolar lavage fluid, indicating lung inflammation.⁸¹ It was important to study lung toxicity in this study given the mode of delivery; studies using other routes of administration should evaluate organ-specific toxicity in accordance with the distribution of the material.

Beyond the items mentioned above, researchers evaluating nanomedicines for maternal/fetal medicine should also monitor the weight of pregnant mice. If maternal weight gain does not occur at the proper rate, it can have negative consequences for fetal health. It is likely that the impacts of nanoparticles on pregnant animals and humans will depend on many factors, including nanoparticle size, shape, surface chemistry, and dose. In fact, maternal weight gain decreased when 30 nm diameter positively charged TiO₂ particles were administered to pregnant mice intravenously 8 days in a row, but weight gain was normal when only a single nanoparticle dose was administered.⁸³ Similarly, one study observed minor liver damage in pregnant mice that received multiple doses of carbon nanotubes, but not in mice that received a single dose.⁸⁴ These findings demonstrate that safety must be evaluated under multiple dosing regimens, ideally designed to mimic the planned administration in humans. Overall, maternal safety is a critical consideration when designing nanoparticles for use in pregnancy. To date, most studies demonstrate there are limited effects of nanoparticles on pregnant mice, but more studies need to be performed and the results validated in multiple animal species before moving to human trials.

Fetal Safety Considerations.

As a fetus is developing it is more vulnerable to disruption than a fully developed adult and the consequences of toxicity to a fetus may be dire. Fetal complications can include gross morphology changes, disrupted organ or tissue development, or spontaneous abortion and resorption of the fetus. Potential effects of nanoparticles on a fetus may depend on many factors, including the nanoparticle size, material, shape, and surface chemistry, as well as the gestational age at which the nanoparticles are delivered. When evaluating nanomedicines in pregnant mice, researchers should note the number of pups in a litter and the average weight of the pups as a measure of safety. They should also evaluate whether the pregnancy continues to full term, as premature labor is problematic and an undesired outcome in humans.

As the field of nanomedicine for maternal/fetal health progresses, carefully tailored studies must be designed and performed to mechanistically determine how various features of nanoparticles impact fetal safety. Numerous studies have reported safety findings for distinct nanoparticle systems (Table 4), but there is not enough data in the literature to draw major conclusions regarding how distinct features of nanoparticles impact fetal toxicity. In general, most studies have indicated intravenously and vaginally administered nanoparticles do not affect the number of pups in a litter,^{3,40,44,49,53,56,83-85} but there are exceptions where toxicity has been noted.^40,83,85-87 For example, 70 nm silica and 217 nm TiO₂ nanoparticles, often used in consumer products, were found in the fetal brain and liver of pregnant mice after systemic administration and increased the number of fetal resorptions compared to phosphate buffered saline (PBS) injected controls.⁴² To control for the possible effect of nanoparticle material, researchers often focus on one material at multiple sizes. One group showed that 100 nm diameter AuNPs, but not 40nm AuNPs, were genotoxic to fetal liver and blood and caused miRNA dysregulation in fetal lung and liver.⁸⁸ Another set of studies showed that 50 nm diameter silver nanoparticles (AgNPs) accumulated in the visceral yoke sac in large quantities, and while there were no recorded safety issues, the location of the nanoparticles may be cause for concern.^40,41

While it is difficult to draw conclusions about how specific nanoparticle features such as size or material impact fetal toxicity based on available literature, it does appear consistent that toxicity is elevated with increased doses.^42,83,84 For example, when 30 nm diameter iron oxide nanoparticles were dosed in pregnant mice for 8 consecutive days, there was a greater rate of fetal resorption compared to animals that received a single dose.⁸³ Moreover, in the animals that received multiple nanoparticle doses, some of the remaining fetuses had gross morphological changes, such has club foot or shortened tail.⁸³ Interestingly, the effects were influenced by nanoparticle charge, as repeated doses of positive nanoparticles caused more early stage resorptions while negative nanoparticles caused more late state resorptions. This may be attributed to changes in the distribution of the nanoparticles based on their surface charge.⁸³

Notably, the impact of nanoparticles on fetal development may also depend on how advanced the pregnancy is when they are administered. When 30 nm diameter AuNPs were delivered intravenously to pregnant mice early in gestation (E5.5-7.5) the spontaneous abortion rate was approximately 50%, whereas AuNPs delivered at mid gestation (E11.5-13.5) had no fetal consequences.⁸⁵ It appears based on qPCR analysis of mRNA levels in excised tissues that the spontaneous abortion at early gestations was a result of the nanoparticles suppressing the expression of ectodermal differentiation markers.⁸⁵ This finding was corroborated by another study that showed pregnant dams dosed with 15 nm diameter AuNPs at E3.5 and E5.5 suffered from up to 60% fetal resorption. In vitro testing confirmed that the 15 nm AuNPs altered the expression pattern of both neural ectoderm and mesendoderm lineage markers.⁸⁷

Some research has focused specifically on nanoparticle formulations used in consumer products, such as antimicrobial AgNPs or TiO₂ NPs used in paints and sunscreens. In female fetuses from dams that were intravenously injected with 8 nm diameter AgNPs, several mRNA molecules important for ovary development were significantly reduced.⁸⁹ In a separate study, 25-70 nm TiO₂ NPs were found in the testis, olfactory bulb, and cerebral cortex of pups whose mothers received multiple subcutaneous injections of the particles during pregnancy. Spermatogenesis was altered in the offspring and the number of caspase-3 positive cells, a marker for apoptosis, was increased in the in olfactory bulb.⁹⁰ In a follow up study, researchers examined how fetal exposure to TiO₂ NPs alters gene expression in the fetal and postnatal brain. Gene dysregulation was observed in the cerebral cortex, olfactory bulb, and regions related to the dopamine system.⁹¹ Together, these studies indicate that while there might not be gross morphological changes in offspring, it is still important to examine fertility and neurologic alterations in offspring to ensure fetal safety.

While this section has focused on toxicity caused by nanoparticles delivered systemically or vaginally, some materials, such as particulate pollutants, can enter pregnant mothers through pulmonary or intranasal routes to subsequently cause toxicity. A study in pregnant mice showed that maternal inhalation of carbon black nanoparticles increased the number of DNA strand breaks in postnatal offspring livers, but intratracheal instillation did not increase DNA strand breaks, indicating that route of delivery is important.⁸¹ Intranasal instillation of the same carbon black particles in pregnant mice caused offspring to have increased numbers of immune cells associated with allergy and inflammatory responses, such as CD3⁻B220⁻, CD3⁺, and CD4⁺ cells.⁹² This suggests that carbon black particles may induce an allergic or inflammatory response at an early age. While these studies directly study the effects of a common pollutant, the lessons learned may be applied to therapeutic nanoparticles as well.

Overall, there is a substantial need for more studies to assess the safety of nanoparticles in pregnant mice. Before nanomedicines will be able to move to clinical use for pregnancy complications, their safety must be thoroughly vetted. Unexpected toxicity to the mother or fetus would cause not just physical harm, but also substantial emotional damage. Therefore, it is imperative that researchers carefully examine the safety of their nanomedicines in multiple animal models and report the results in an unbiased manner.

Conclusions and Future Directions

This review introduced the need for nanomedicines to promote maternal/fetal health and discussed the biological barriers that nanoparticles must overcome to successfully reach their target in pregnant women following systemic or vaginal administration. It also discussed how various characteristics of nanoparticles may influence their ability to successfully reach their targeted sites and provided examples of nanoparticles that have been used to treat pregnancy complications found in the literature. The studies performed to date have revealed substantial amounts of information, but further studies are necessary to maximize the potential of nanomedicine to combat pregnancy complications. The field of maternal/fetal health is currently receiving a substantial amount of attention, and the next decade of study is likely to be an exciting era of growth.

Moving forward, more studies are needed to elucidate how nanoparticle size, shape, surface chemistry and charge affect biodistribution and therapeutic efficacy. Additionally, studies must be performed to determine how the timing and route of nanoparticle administration impacts distribution, efficacy, and safety. While these studies must initially be performed in mice with “normal” pregnancies, it is yet to be determined how preexisting maternal conditions (e.g. diabetes and high blood pressure) might affect the delivery and efficacy of nanoparticle-based therapeutics during pregnancy. It is also possible that patient histories such as the number of term pregnancies and miscarriages might affect nanoparticle delivery outcomes. Studies designed to reveal this information would add exciting and important new knowledge to the field.

A major challenge in developing nanoparticle-based and traditional therapies for pregnancy complications is the lack of established in vitro and ex vivo models available. In vitro models in this field typically use the BeWo trophoblast cell line to model drug transport across the placenta in early stages of pregnancy.^93-98 However, some studies have developed more intricate in vitro models requiring multiple cell lines in combination with BeWo.^98-100 None of these models are representative of the third trimester, and thus some researchers use ex vivo samples to model drug transport.^93,95,100 These better simulate the patient scenario, but are more complex to implement. Future studies should address the accuracy and reliability of both in vitro and ex vivo models to progress nanomedicine for pregnancy related complications.

In summary, nanoparticle-based delivery systems exhibit great promise for the treatment of pregnancy complications. Early studies in this field have demonstrated that nanoparticles can accumulate in reproductive organs following systemic or vaginal delivery, and they have also begun to define criteria that enhance delivery (e.g., dense PEGylation, reduced size). A few studies have gone beyond simple biodistribution analysis to demonstrate nanoparticles have potential as tools for in utero genome editing,³¹ prevention of preterm birth,⁷⁷ and more. As researchers build upon this knowledge to create new delivery systems, the application of nanomedicine to a wide range of maternal/fetal health problems is likely to grow exponentially. This is an exciting era of investigation in the field, and the advances in nanomedicine for maternal/fetal health that will be made in the next decade are likely to dramatically improve patient care during pregnancy.