Nanomedicine for Maternal and Fetal Health.

Drug formulation can enhance the efficacy of therapeutics by better controlling their release rates, pharmacokinetics, toxicity, and efficacy. Nano-formulation of therapeutics offers additional control over their tissue targeting ability, penetration, cellular uptake, and intracellular trafficking. Nanomedicine has shown unprecedented potential in addressing unmet medical needs, including the recent drug approvals for use in vaccination and gene therapies ( 10 , 11 ). In addition to viral vectors used for gene delivery, a plethora of natural and synthetic materials have been used to deliver drugs and genes, each having pros and cons. Liposomes and lipid-based nanoparticles (NPs) have shown great promise in the last few years as means for effective delivery of nucleic acids ( 12 ). Polymeric particles and micelles serve as a biodegradable platform to tether or encapsulate various drugs ( 13 ). Surface functionalization expands the utility of nanomedicine by rendering particles water soluble and reduces uptake by the mononuclear phagocyte system (MPS). Polyethylene glycol (PEG) is one of the most studied polymers used in the making of stealth NPs ( 14 ). Controlling the size of drug-loaded NPs is another important factor to potentiate the diffusion and crossing of therapeutics NPs across cellular and tissue barriers ( 15 ). While the field has made great progress in developing and translating nanomedicine-based therapies for a range of diseases, the application of nanomedicine to unmet needs in women’s health has been relatively understudied. However, the potential for improving safety and enabling the development of effective treatments where there are currently no clinically available options is highly attractive. In order to develop nanomedicines for applications in conditions related to fertility and obstetrics, we must first study how the unique physiology affects the pharmacokinetics and pharmacodynamics of nanomedicines ( 3 , 16 ). First, we discuss the anatomical and physiological changes that occur during the menstrual cycle and pregnancy that impact the development of nanomedicines, considerations for utilizing different routes of administration, and the potential for nanomaterials to mitigate the risk of toxicity to both mother and fetus. Significant hormonal and physiologic changes occur throughout each menstrual cycle. After ovulation, the corpus luteum undergoes fast tissue growth and angiogenesis, leading to enhanced permeability of blood vessels ( 17 ). In a study comparing delivery of intravenous (IV)-injected gadolinium liposomes during different phases of the mouse estrous cycle, there was 2-fold increased accumulation in the uterus and ovaries during the estrus phase (ovulatory), compared to the diestrus phase (nonovulatory) ( Figure 1a – g ) ( 16 ). The shift in NP accumulation may have either negative or positive implications, depending on the intended site of treatment. For example, increased accumulation of doxorubicin-loaded liposomes in the ovaries when the treatment was administered in the estrus phase led to decreased tumor size ( Figure 1h , i ). In contrast, in an orthotopic breast cancer model, liposome administration in the estrus phase was associated with decreased delivery to the tumor and decreased efficacy ( Figure 1j , k ) ( 16 ). This suggests that timing of injecting NPs or gene therapies targeting the ovaries should be taken into consideration when delivering therapeutics to treat infertility. Further, a myriad of factors, including hormonal dysregulation, can affect a woman’s ability to conceive, and these changes must also be considered in timing administration ( 18 ). During pregnancy, significant physiological and anatomical adaptations affecting every organ of the body occur to accommodate the fetus, some of which can last for many years after delivery ( 2 , 3 ). These changes include increases in plasma volume, cardiac output, renal plasma flow, and glomerular filtration, which all can impact the pharmacokinetics of administered drugs, as they can affect the distribution, absorption, metabolism, and excretion processes ( 19 ). It is also known that plasma protein concentrations, including albumin and alpha 1-acid glycoprotein, decrease during pregnancy, leading to decreased protein drug binding ( 20 ). Changes in blood proteins composition can also affect the protein corona that forms on systemically administered NPs, potentially impacting pharmacokinetics, and targeting ( 21 ). Therefore, in addition to the need of adjusting the dose to account for changes in body composition, additional research is required to fully understand how the menstrual cycle, exogenous hormone treatments, and pregnancy impact pharmacokinetics, biodistribution, and ultimately, efficacy of nanomedicines. Many of the gynecologic disorders, including hormonal imbalances, benefit from systemic drug administration, which includes oral administration, subcutaneous, intramuscular, or IV injection, among other routes. The oral route remains one of the most preferred routes for drug administration because of its convenience, often associated with improved patient adherence ( 22 ). Many researchers have focused on developing delivery systems to increase the solubility of poorly-water soluble drugs, reduce degradation in the gastrointestinal tract, and ultimately improve bioavailability ( 23 ). However, a major issue with oral administration of some drugs is first-pass metabolism in the liver ( 24 ). Many of the oral drugs frequently prescribed in gynecology and obstetrics clinics, such as the infertility drugs clomiphene citrate and bromocriptine and the tocolytic nifedipine, suffer from extensive hepatic first-pass metabolism ( 25 – 27 ). Hence, nano-formulation approaches to enhance the oral absorption of these drugs will fail to produce significant enhancement in their bioavailability. Novel nanomedicines that target the tissue of interest, while minimizing systemic metabolism, may be a better alternative. While oral is often preferred for convenience, injections may be used to bypass limitations in oral bioavailability ( 28 ). Many of the hormonal drug therapies prescribed in the management of infertility, including gonadotropins prescribed for women undergoing in vitro fertilization (IVF), are given by subcutaneous injection. Formulating drugs in sustained-release nano- or micro-particles can facilitate the development of sustained release depots for hormone and peptide delivery ( 29 ). This can simulate the pulsatile release of natural hormones, and potentially reduce the doses required to sustain drug concentrations within the therapeutic window. Controlling the release of small drugs or macromolecules can be achieved by designing different types of materials with various porosity, rates of erosion, and sizes ( 30 ). Upon reaching the systemic circulation through oral administration or injection, the next major challenge is targeting therapeutics to the intended cells and tissues. Particularly in obstetrics, maximizing targeting and limiting off-target side effects is of paramount importance. However, it is technically challenging to design nanomedicines to efficiently cross multiple biological barriers to accumulate specifically in target tissues following systemic administration. NPs are commonly administered by IV injection and may be functionalized with targeting ligands for specific tissues. For example, liposomes were preferentially delivered to the uterus by surface functionalization with oxytocin receptor targeting ligands, potentially decreasing the required dose for preventing myometrial contractions in preclinical animal models ( Figure 2a ) ( 31 , 32 ). Targeting the estrogen receptor has also been used to enhance drug delivery to tissues expressing the receptor such as ovarian or breast tumors ( 33 , 34 ). Systemic administration has also been explored for delivering NPs to the placenta, and targeting nucleic acids using dendrimers or liposomes to knock down specific genes in the placenta has been shown to ameliorate preeclampsia ( 35 , 36 ). Further, the particle size and properties can be tailored to prevent crossing to the fetal side and reduce concerns about fetal safety ( 37 ). Although the size cut-off for reducing nanoparticle transport to the fetal side is highly dependent of the type of material used, larger particles or cationic in surface charge tend to show limited crossing ( Figure 2b ) ( 38 , 39 ) Local drug delivery can be used to increase targeting to specific cells and tissue relative to the systemic levels. Vaginal administration can be employed for drug and nucleic delivery to the female reproductive tract (FRT) ( 40 – 42 ). While easily accessible and often a convenient route of administration, there are barriers to achieving effective drug absorption and transport across the vaginal epithelium, including the cervicovaginal mucus (CVM) barrier ( 43 – 45 ). CVM is a sticky, mesh-like collection of mucins, lipids, proteins, and ions that protects the underlying epithelium from foreign pathogens ( Figure 2c ) ( 46 , 47 ). Prior studies have characterized the distribution of pore sizes in human CVM (average 340 ± 70 nm), and NPs that are engineered to be small enough to pass through the pores without adhesive interactions have shown improved drug and nucleic acid delivery in various animal models ( 41 , 48 – 54 ). It was demonstrated that the vaginal delivery of PEGylated mucoinert drug nanocrystals led to increased drug concentrations in the cervix and uterus in pregnant mice, and thus, was more effective for preventing preterm birth (PTB) in murine models ( 55 ). The ovaries, often Implicated in infertility, harbor a wide range of cells and membranes to control the entry of nutrients to developing follicles, excluding foreign substances and toxins ( 56 ). The blood-follicle barrier (BFB) is composed of the vascular endothelium, sub-endothelial basement membrane, the thecal interstitial layer, the follicular basement membrane, and the membrane granulosa ( Figure 2d ). The BFB serves as a molecular sieve which is permeable to only macromolecules and particulates <500 kDa in size ( 57 ). Comparative studies showed that the presence of an ovulatory signal such as a surge in luteinizing hormone concentrations, results in the flux of proteins in the intermediate size range, such as a 220 kD inter-alpha-inhibitor ( 58 ). However, the cationization of the same protein increased its permeability across the BFB without an ovulatory signal ( 58 ). Additionally, agents capable of transcytosis such as some serotypes of viral vectors can penetrate the BFB, although their sizes might be bigger than the permeability threshold into the follicle ( 59 ). Local delivery to the uterus has also been employed in many preclinical animal studies for maternal and fetal health. Drugs, such as progesterone, absorbed through the vagina first travel through the vasculature surrounding and perfusing through the upper FRT before entering the systemic circulation, a process known as uterine first pass effect ( Figure 2a ) ( 40 , 41 ). Intrauterine nanomedicine has been explored to correct genetic mutations in the fetus before the development of phenotypic abnormalities. Since many of the biological barriers, such the blood brain barrier (BBB), are not fully developed in the fetus, in utero delivery of therapeutics can potentiate their accumulation to the target tissues and cells ( 60 ). The vitelline vein, which drains the yolk sac, has been used as a site of delivery for gene editing tools and mRNA encapsulated nanoparticles. Intravitelline vein injections drain directly into the fetal portal circulation, which represents a mid-gestation umbilical vein injection in a human fetus ( 61 ). Umbilical vein injection is used clinically to deliver hematopoietic cells or blood transfusion in utero in 16–18-week gestation fetuses with minimal risk of complications ( Figure 2b ) ( 62 ). Accessing the amniotic sac is performed in clinical settings to test a small amount of amniotic fluid when there is a suspected risk for genetic disorders, including Down’s syndrome and Patau’s syndrome ( 63 ). Intra-amniotic, or injections to the amniotic sac from outside the mother, is another injection method to access the fetus ( Figure 2b ). This method resembles oral administration, since the fetus ingests or swallows the amniotic fluids present in the sac. Intra-amniotic injection of non-viral gene therapies distributed mainly to the gut, skin and lungs of fetuses ( 64 , 65 ). While nanomedicine can be used to access the fetus in some instances, the maternal side and excluding the fetus in other instances, a large body of research has unequivocally demonstrated a potential for the treatment of a wide range of gynecologic and obstetric disorders. In the following sections, we discuss different applications of nanomedicines for maternal and fetal health, focusing on the different targeting and delivery strategies and the characteristics of each NPs needed to achieve specific therapeutic effect in infertility, pregnancy, and fetal tissues

Nanomedicine holds great potential in navigating barriers for the effective delivery to the female reproductive system, delivering therapeutic payloads to the tissues of interest, enhancing potency, and minimizing off-target toxicity. However, the heterogeneity of data available on the characteristics of NPs needed to cross barriers, such as the placenta and the BFB, impedes the clinical translation of these therapeutics. More specific and comprehensive consensus on the safety and efficacy is needed for each type of NP, because the differences can be significant depending on the type of the material. Local therapeutic delivery such as vaginal, intra-ovarian, and intrauterine can be more suitable routes to deliver emerging nanomedicines to treat conditions affecting both the mother and fetus. In addition to the direct tissue targeting ability of local or systemic nanomedicines, many conventional drugs prescribed for gynecologic or obstetric conditions may benefit from nano-formulation approaches to control drug release rates, systemic absorption and pharmacokinetics, and enhance local drug concentrations. By doing so, nanomedicine approaches can also be applied to increase compliance and convenience, with the aim to reduce dosing and injection frequency, the use of suitable administration routes and reduce side effects. The choice of type of nanomedicine for use in obstetric or gynecologic disorders highly depends on the type of drug, target organ, and intended application. Drug delivery materials that demonstrated safety and efficacy in human clinical trials could be advantageous when testing new therapies in maternal and fetal health. Of these, LNPs showed great promise in the delivery of siRNA and mRNA, but effective extrahepatic targeting remains to be achieved. Polymeric particles including micelles and dendrimers are promising long-circulating platforms with cell-specific targeting ability and clinical potential. Viral vectors and prolonged gene expression might raise fetal safety concerns. Reaching the placenta or fetus during pregnancy could be achieved using various delivery strategies by targeting specialized cells and molecules that are uniquely expressed in the transient organ. From a safety perspective, clinical trials of nanomedicines in fertility conditions may provide an avenue for higher benefit to risk ratios as a first step in development compared to pregnancy. Treating conditions during pregnancy and potential serious effects in the fetus and mother remain a major drawback for designing clinical trials in obstetric conditions. Finally, there is an urgent need for developing more predictive animal models and/or in vitro models such as organ-on-a-chip to bridge the gap between preclinical research and clinical trials. Further, in utero drug treatment comes with additional ethical considerations. In particular, in utero gene editing will also have unique ethical issues that must be considered during therapeutic development. Discussions and effective communication between people in the basic sciences, clinical research, the pharmaceutical industry, and regulatory affairs should be emphasized as effective means to maximize the potential of in utero gene editing using nanomedicine. Ultimately, nanomedicine has the potential to address a wide array of unmet needs in maternal and fetal health.

Fetal and maternal health are closely interconnected and regulated by immune, hormonal, and epigenetic interactions ( 1 ). Related physiological changes start from ovulation and may last for many years after delivery ( 2 , 3 ). Despite the paramount importance of reproduction, there are numerous areas of unmet need. In the U.S., nearly 11% of women are infertile ( 4 ), and it is estimated that ~17% of pregnancies result in fetal loss ( 5 ). In 2018, 17.3 pregnancy-related maternal deaths per 100,000 live births were reported, largely due to cardiovascular complications, hemorrhage, and infections ( 6 ). Despite the rapid rise in diagnostic and therapeutic products developed each year, there is a scarcity of effective therapies specific to pregnant women or the fetus ( 7 , 8 ). Most drugs are subject to contraindications and warnings in pregnant women because of lack of data regarding safety and efficacy during pregnancy ( 9 ). Increasing infertility rates, as well as maternal and fetal morbidity and mortality rates, generate significant emotional, societal, and economic burdens. There is a distinct need for therapeutic and delivery strategies to specifically target obstetric and fetal conditions to minimize health risk to both the mother and fetus during pregnancy. The potential for enhancing targeting and reducing toxicity using drug delivery systems may provide many unique benefits in therapeutic development for obstetric applications. In this review, we highlight the potential advantages of employing drug delivery systems, and in particular, nano-sized drug and gene carriers, in treating conditions related to female infertility and in treating and preventing complications during pregnancy that affect the mother and the fetus. It is noteworthy to emphasize that our use of the words mother, maternal, and woman applies to people of the female sex and persons who are pregnant, as is reflected in the literature cited. We are not intentionally omitting more broadly representative language or consideration of people who do not fall into traditional categories, and hopefully increasing attention will be given to representing persons who do not fall into this categorization.

With the advancement DNA sequencing and prenatal diagnostics, early detection of fetal genetic mutations became possible ( 177 ). Intrauterine interventions could open possibilities to correct genetic mutation in the fetus before the development of phenotypic abnormalities. While the focus of therapeutic development in obstetrics is often on preventing the crossing of medications through the placenta to reach the fetus, in utero drug targeting could provide new therapeutic potentials for incurable conditions by targeting various organs that are commonly inaccessible after fetal development. Crossing of the placenta, or more direct local approaches for administration to reach the uterus, while avoiding vital organs of the mother, may be necessary in these cases. Safety remains to be of paramount importance when considering such therapeutic avenues, which provides another unique opportunity for nanomedicine approaches to address unmet needs. The improvements in targeting and controlled release provided by nanomedicine may address some of the challenges and limitations associated with safety. Exposure to intrauterine inflammation is linked to a wide range of neurobehavioral disorders in preterm and term infants ( 178 ). Inflammation of the chorion and amnion results in the production of proinflammatory cytokines in fetal and maternal tissues, which may lead to fetal sepsis and cerebral white matter injury resulting in short- and long-term neurological conditions ( 179 ). Magnesium sulfate has shown to be highly effective at preventing cerebral palsy that may be associated with PTB regardless of the underlying reasons ( 180 ). The optimal regimen of magnesium sulfate for fetal neuroprotection has been described as an IV loading dose starting 4 h prior to iatrogenic PTB, or before 24 h if expected, followed by a maintenance dose that should be stopped after 24 h if undelivered ( 181 ). In addition to this inexpensive and easy to perform clinical practice, ongoing preclinical research is focused on developing more effective and targeted strategies for the management neuroinflammation in preterm infants. Entry into the fetal environment via placental crossing or local administration from the uterus is desired rather than avoided to deliver drugs to the fetal brain. A series of investigations using dendrimers that specifically accumulate in inflamed and injured regions of the fetal and neonatal brain have been reported. Those strategies rely on delivering anti-inflammatory therapies across the fetal blood brain barrier (BBB) to reach specific inflammatory cells. Activated microglial cells in the brain play a key role in the negative effects of neuroinflammation on development ( 182 ). In one example, the fetal biodistribution of G4-hydroxyl PAMAM dendrimers (around 4 nm in size) after intra-amniotic delivery of dendrimers was characterized ( 183 ). Fluorescently-labeled dendrimers were intra-amniotically injected into each gestational sac on the right side of the uterus in mice immediately following intrauterine injection of LPS or vehicle on E17. The left side of the uterus served as an internal control. Intra-amniotic administration resembles oral administration to the fetus, and thus the dendrimers accumulated at quantifiable levels in fetal organs including the brain, liver and gut in the dams with induced intrauterine inflammation. In a microscopic analysis of brain sections, the dendrimers showed accumulation in the activated microglial cells of LPS-induced inflammation animals, as well as the parenchyma ( Figure 5a – d ) ( 183 ). This was an extension to a previous finding, which demonstrated that IV injection of N-acetylcysteine-conjugated PAMAM dendrimers in newborn rabbits of intrauterine inflammation model alleviates the cerebral palsy phenotype ( 184 ). Specifically, this postnatal treatment with the conjugated dendrimers led to improved motor function and reduced neuronal injury ( 184 ). Thus, intra-amniotic injection of dendrimer-based therapies may provide greater efficacy since it allows intervention at earlier time points. Similar studies were performed in a rabbit model to assess how dendrimer surface functionality would affect the fetal biodistribution of dendrimers after intra-amniotic injection ( 185 ). Rabbits received intrauterine injections of LPS or saline vehicle into each gestational sac on E28, followed by intra-amniotic injection of fluorescently-labeled dendrimers terminated with carboxyl or hydroxyl groups. They quantified the amount of both dendrimers at 24 hours post administration and found that both had a similar extent of fetal tissue accumulation in the case of the endo toxin treated fetus. However, hydroxyl dendrimers had a higher efficiency of crossing the BBB, increased amount of uptake in the brain, and targeting the activated microglia compared carboxyl dendrimers. Using confocal microscopy, it was observed that hydroxyl dendrimers were more widely distributed into the brain parenchyma with less signal retained within the vasculature, while the carboxyl modification retained the particles mostly within the blood vessels ( 185 ). intra-amniotic delivery approaches are encouraging; however, less invasive methods of administration would be preferred. A mouse model of LPS-induced intrauterine inflammation was used to characterize the biodistribution and fetal and therapeutic effect of N-acetylcysteine-conjugated PAMAM dendrimers administered IP to pregnant mice ( 186 ). Fluorescently labeled dendrimers, ~4 nm in size, were found to localized to both the uterus and placenta. In addition, the N-acetylcysteine conjugated to the dendrimer by a disulfide bonding didn’t show any release at extracellular and plasma GSH levels (2 μM), but was readily released from the conjugates (80% in 100 min) at intracellular GSH concentrations (2 – 10 mM). Focusing on the pharmacodynamic effects in the fetus, the conjugated dendrimers were able to prevent LPS-induced placental CD8+ T-cell infiltration, reduce LPS-induced microglial activation, and improved neuromotor development ( 186 ). Fetal lung development is a critical aspect of fetal health in preparation for delivery. Disorders associated with the underdevelopment of the fetal lung, such as congenital diaphragmatic hernia and surfactant protein syndromes, have an increased risk of severe respiratory distress at birth, long-term morbidities, and death ( 187 ). Despite the advancement in prenatal diagnosis of congenital fetal lung disorders in the past years ( 188 ), there is a lack of treatment options to treat fetal lung conditions in utero. Polymeric NPs were screened for their ability to target specific cell populations in the fetus. Poly(lactic-co-glycolic acid) (PLGA) NPs ~200 nm in size and −25 mV in charge were administered via 2 injection routes: intravitelline vein injection or intraamniotic injections ( 65 ). While most of the particles accumulated in the liver and lung following intravitelline vein injection, the lung and gut were the major organs of distribution after intraamniotic injection ( Figure 5e ). Further studies showed that the intravitelline injection at E15 was more effective than intraamniotic injection for targeting epithelial, endothelial and hematopoietic cells in the fetal lung ( 189 ). The particle characteristics, such as surface modifications, were studied to show their ability to target a certain cell population in the fetal lung. Poly(amine-co-esters) (PACE) based NPs of 250 nm in size with a 31 mV surface charge were delivered more efficiently than the 250 nm PLGA NPs to lung epithelial cells by two-fold and endothelial cells by nearly four folds ( 189 ). The effect of PEG was also tested by comparing PLA-PEG, PACE-PEG, and PLGA NP 150 nm in size. There was no significant difference in NP delivery when considering the whole lung mass, but accumulation of PLA-PEG NP in epithelial cells was about 2-fold less compared to the other groups. Increased uptake of both PEGylated NPs was observed in the endothelial cells ( 189 ). Of note, a similar phenomenon was also seen in adult mice, where PEGylated NPs were delivered more effectively to lung endothelial cells and less to epithelial cells ( 190 ). mRNA delivery provides an alternative to gene editing when the genetic disease arises from protein/enzyme deficiencies ( 191 ). mRNA formulated in ionizable-lipid LNP was delivered in-utero by intraamniotic injection ( Figure f,g ) ( 64 ). As particle stability influences delivery efficiency, a library of mRNA-LNPs was prepared by microfluidic mixing and their size and size polydispersity index were measured in amniotic fluid from various species, including mouse, sheep, pig, and human samples. This screen identified the most stable LNPs that showed the least change in size and morphology after incubation. Surprisingly, the most stable LNPs developed a dense protein corona on their surfaces compared to the less stable LNPs. This suggested that some protein coronas might support the structure of the particles and make them less resistant to conformational changes. The most stable LNPs from the ex-utero screen provided mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model ( 64 ). Intravitelline injection was found to enable targeting of NPs to the fetal liver ( 65 ). PLGA NPs encapsulating peptide nucleic acids (PNAs) and donor DNAs were developed to correct a fetal genetic mutation ( 65 ). The PNAs contained nucleobases to bind to a specific genomic target site via both Watson–Crick and Hoogsteen base-pairing, inducing endogenous DNA repair mechanisms to mediate the recombination of the donor DNA. The 250 nm particles were administered via the intravitelline route to accumulate in the liver to correct a disease-causing mutation in the β-globin gene in a mouse model of human β-thalassemia. The treatment significantly increased and sustained hemoglobin levels to the normal range for up to 10 weeks, and reduced splenomegaly and peripheral blood reticulocytes. Importantly, fetal plasma cytokine levels in mice treated on E15.5 via intravitelline NP administration revealed no significant difference compared to PBS injection ( 65 ). The intravitelline route was also used to deliver mRNA-LNPs to fetal livers in mice ( Figure 5h , i ) ( 61 ). A library of ionizable cationic lipids was synthesized by Michael addition chemistry, and the LNPs were formulated by microfluidic mixing. The optimized LNP showed strong protein expression in the liver, which surpassed the level of expression obtained by the standard FDA ionizable cationic lipid (MC3), or a polyethylene imine polymer-based delivery system. Delivery of mRNA encoding erythropoietin (EPO) produced high levels of EPO levels in fetal livers 4 hours after injection, without any notable induction of liver injury or proinflammatory markers in fetuses or dams ( 61 ).